CN117098776A - Use of anti-TGF-beta antibodies and other therapeutic agents for the treatment of proliferative diseases - Google Patents

Abstract

Therapies using tgfβ inhibitors, PD-1 inhibitors and/or chemotherapeutic agents are disclosed. These drugs are useful in the treatment or prevention of proliferative diseases, such as solid tumors, including pancreatic or colorectal cancer, at dosages (including smooth administration) and regimens. Additional combinations and uses thereof are also disclosed.

Description

Use of anti-TGF-beta antibodies and other therapeutic agents for the treatment of proliferative diseases

Incorporated by reference

All publications, patents, patent applications, and other documents cited in this disclosure are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document was specifically and individually indicated to be incorporated by reference for all purposes. In the event of a discrepancy between one or more of the references incorporated herein and the teachings of the present disclosure, the teachings of the present specification control.

Sequence listing

The present application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. An ASCII copy created at 2021, 12, 17 is named PAT059084_sl.txt and is 350,107 bytes in size.

Background

The transforming growth factor beta (tgfβ) protein family consists of three different isoforms (tgfβ1, tgfβ2 and tgfβ3) found in mammals. Tgfβ proteins activate and regulate a variety of gene responses that affect disease states including cell proliferative disorders, inflammatory disorders, and cardiovascular disorders. Tgfβ is a multifunctional cytokine, originally named for its ability to convert normal fibroblasts into cells that can anchor independent growth. TGF-beta molecules are produced primarily by hematopoietic and neoplastic cells and can regulate (i.e., stimulate or inhibit) the growth and differentiation of cells from a variety of normal and neoplastic tissue sources (Sporn et al, science, 233:532 (1986)), and stimulate the formation and expansion of various stromal cells.

Tgfβ is known to be involved in many proliferative and non-proliferative cellular processes, such as cell proliferation and differentiation, embryonic development, extracellular matrix formation, bone development, wound healing, hematopoiesis, and immune and inflammatory responses. See, e.g., pircher et al, biochem. Biophys. Res. Commun. [ communication of biochemistry and biophysics studies ],136:30-37 (1986); wakefield et al, growth Factors [ Growth Factors ],1:203-218 (1989); roberts and Sport, edited Handbook of Experimental Pharmacology [ handbook of Experimental pharmacology ], pages 419-472, M.B. Sport and A.B. Roberts (Springer, heidelberg, 1990); massaguee et al, annu Rev.cell Biol [ Annual. Biol.6:597-646 (1990); singer and Clark, new Eng.J.Med. [ J.New England medical journal ],341:738-745 (1999). In addition, TGF-beta is also useful in the treatment and prevention of intestinal mucosal diseases (WO 2001/24813). TGF beta is also known to have strong immunosuppressive effects on various immune cell types, including Cytotoxic T Lymphocyte (CTL) inhibition (Ranges et al, J.Exp.Med. [ J.Endoc., 166:991,1987; espevik et al, J.Immunol. [ J.Immunol., 140:2312, 1988), reduction of B cell lymphopoiesis and kappa light chain expression (Lee et al, J.Exp.Med. [ J.Endoc., 166:1290, 1987), down-regulation of hematopoiesis (Sing et al, blood, 72:1504, 1988), down-regulation of HLA-DR expression on tumor cells (Czarniecki et al, J.Immunol. [ J.Immunol., 140:4217, 1988), and inhibition of antigen activation of B lymphocyte proliferation in response to B cell growth factors (Petit-Kosket al, eur.J.Immunol. [ J.Immunol., 18:1988). See also U.S. patent 7,527,791.

There is an unmet need for the use of tgfβ inhibitors (e.g., anti-tgfβ antibodies) to target a variety of diseases and medical conditions. Furthermore, there is a need to administer these tgfβ inhibitors in a manner effective to treat a variety of diseases and medical conditions, including proliferative diseases, while maintaining ease of administration.

Disclosure of Invention

Disclosed herein is a method of treating a proliferative disease in a subject in need thereof, comprising administering to the subject a tgfβ antibody and at least one additional therapeutic agent, wherein the tgfβ antibody is administered from about 1400mg to about 2100mg once every two, three or four weeks. In some embodiments, the tgfβ antibody is administered at about 1400mg once every two weeks. In some embodiments, the tgfβ antibody is administered at about 2100mg once every two weeks. In some embodiments, the tgfβ antibody is administered at about 2100mg once every three weeks. In some embodiments, the TGF-beta antibody is administered at about 45mg/kg once every three weeks. In some embodiments, the TGF-beta antibody is administered at about 30mg/kg once every three weeks. In some embodiments, the TGF-beta antibody is administered at about 20mg/kg once every three weeks.

The TGF-beta antibody may comprise heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively. The TGF-beta antibody may comprise a heavy chain variable region and a light chain variable region shown in amino acid sequences SEQ ID NO. 7 and 8, respectively. The TGF-beta antibody may comprise heavy and light chains as shown in amino acid sequences SEQ ID NO. 9 and 10, respectively.

In some embodiments, the tgfβ antibody is a monoclonal antibody. In some embodiments, the tgfβ antibody is a fully human antibody.

In some embodiments, the tgfβ inhibitor is administered over a period of about 30 minutes.

In some embodiments, the additional therapeutic agent is one or more of the following: PD-1 inhibitors, gemcitabine (gemcitabine), albumin-bound paclitaxel (nab-paclitaxel), leucovorin (folinic acid) (calcium folinate (leucovorin calcium) or levofolinate (levoleucovorin)), fluorouracil (5-FU), oxaliplatin (oxaliplatin (eloxatin)), bevacizumab, or irinotecan (irinotecan).

For example, in some embodiments, the additional therapeutic agent comprises a PD-1 inhibitor. The PD-1 inhibitor may be an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is PDR001 (spartamizumab), BGB-A317 (tirelizumab), or BGB-108. For example, the anti-PD-1 antibody is tirelimumab. Tiril bead mab may have the heavy chain of SEQ ID NO. 321. Tiril bead mab may have a light chain of SEQ ID NO. 322. In some embodiments, the anti-PD-1 antibody is administered at 100mg weekly. In some embodiments, the anti-PD-1 antibody is tirelimumab and is administered at 300mg every 28 day (i.e., four week) period (Q4W). In some embodiments, the anti-PD-1 antibody is tirelimumab and is administered at 200mg every 21 day (i.e., three weeks) period (Q3W).

In some embodiments, the additional therapeutic agent comprises gemcitabine. If gemcitabine is used, it may be used at about 1000mg/m² And (5) administration. In some embodiments, gemcitabine is administered on days 1, 8, and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent comprises albumin-bound paclitaxel. If albumin-bound paclitaxel is used, it can be used at about 125mg/m² And (5) administration. In some embodiments, the albumin-bound paclitaxel is administered on days 1, 8, and 15 of a 28-day cycle.

In some embodiments, the additional therapeutic agent comprises gemcitabine and albumin-bound paclitaxel. In some embodiments, the additional therapeutic agent comprises a PD-inhibitor, gemcitabine, and albumin-bound paclitaxel.

In some embodiments, the additional therapeutic agent comprises bevacizumab. If bevacizumab is used, it can be administered at about 5 mg/kg. In some embodiments, bevacizumab is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent comprises 5-fluorouracil. If 5-fluorouracil is used, it can be used at about 400mg/m² And (5) administration. In some embodiments, 5-fluorouracil can be present at about 2400mg/m² And (5) administration. In some embodiments, 5-fluorouracil is administered on days 1 and 15 of a 28 day cycle. In some embodiments, 5-fluorourinePyrimidine is administered as an Intravenous (IV) bolus. For example, 5-fluorouracil can be present at 400mg/m on days 1 and 15 of each 28-day cycle² Intravenous bolus injection followed by 2400mg/m² Administration was by continuous intravenous infusion over 46 hours.

In some embodiments, the additional therapeutic agent comprises folinic acid. If folinic acid is used, it can be used at about 400mg/m² And (5) administration. In some embodiments, the folinic acid is administered on days 1 and 15 of a 28 day cycle. In some cases, folinic acid may be replaced with levofolinic acid. If L-folinic acid is used, it can be used at 200mg/m² And (5) administration. In some embodiments, levofolinic acid is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent comprises oxaliplatin. Oxaliplatin, if used, can be present in an amount of about 85mg/m² And (5) administration. In some embodiments, oxaliplatin is administered on days 1 and 15 of a 28 day cycle.

In some embodiments, the additional therapeutic agent comprises irinotecan. Irinotecan, when used, can be present at about 180mg/m² And (5) administration. In some embodiments, irinotecan is to be administered on days 1 and 15 of the 28 day cycle.

In some embodiments, the additional therapeutic agent comprises bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin. In some embodiments, the additional therapeutic agent comprises a PD-1 inhibitor, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin. In some embodiments, the additional therapeutic agent comprises bevacizumab, 5-fluorouracil, folinic acid, and irinotecan. In some embodiments, the additional therapeutic agent comprises a PD-1 inhibitor, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan.

In some embodiments, the tgfβ antibody and the one or more additional therapeutic agents are administered to the subject over 2 or more cycles. In some embodiments, the period may be 28 days long. In some cases, the period may be 21 days long.

The proliferative disease that may be treated with the tgfβ antibody and the one or more additional therapeutic agents may be pancreatic cancer or colorectal cancer.

Further disclosed herein is a method of treating pancreatic adenocarcinoma in a subject in need thereof, the method comprising administering 2100mg of TGF-beta antibody, 1000mg/m to the subject² Gemcitabine, and 125mg/m² Wherein the TGF-beta antibody is administered on days 1 and 15 of a 28 day cycle and gemcitabine and albumin-bound paclitaxel are administered on days 1, 8 and 15 of the cycle, and wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO:1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO:4, 5 and 6, respectively.

In some embodiments, the method comprises administering a PD-1 inhibitor to the patient. The PD-1 inhibitor may be swabber or tirelimumab. If the Stbadizumab is administered, it can be administered at 400mg once every four (4) weeks. If tirelimumab is used, it can be administered at 100 mg/week of treatment. For example, tirelimumab can be administered at 400mg once every four (4) weeks. In some embodiments, the tgfβ antibody, gemcitabine, and albumin-bound paclitaxel (and optionally PD-1 inhibitor) are administered to the subject over 2 or more cycles. In some embodiments, the tgfβ antibody, gemcitabine, and albumin-bound paclitaxel (and optionally a PD-1 inhibitor) are administered intravenously to the subject.

Disclosed herein is a method of treating colorectal cancer in a subject in need thereof, the method comprising administering to the subject 2100mg of TGF-beta antibody, 5mg/kg of bevacizumab, 400 to 2400mg/m² 5-fluorouracil, 400mg/m² And 85mg/m² Wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin are administered on days 1 and 15 of a 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs 4, 5, and 6, respectively.

In some embodiments, the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin are administered to the subject in 2 or more cycles. In some embodiments, the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin are administered to the subject intravenously. In some embodiments, 5-fluorouracil is administered as a 400mg/m2 intravenous bolus followed by a 2400mg/m2 continuous intravenous infusion over 46 hours.

In some embodiments, a method of treating colorectal cancer in a subject in need thereof comprises administering to the subject 2100mg of tgfβ antibody, 5mg/kg bevacizumab, 400 to 2400mg/m² 5-fluorouracil, 400mg/m² And 180mg/m² Wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan are administered on days 1 and 15 of a 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs 4, 5, and 6, respectively.

In some embodiments, the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan are administered to the subject in 2 or more cycles. In some embodiments, the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan Kang Jingmai are administered to the subject internally. In some embodiments, 5-fluorouracil is administered as a 400mg/m2 intravenous bolus followed by a 2400mg/m2 continuous intravenous infusion over 46 hours.

In some embodiments, the tgfβ inhibitor is administered concurrently with the one or more additional therapeutic agents. In some embodiments, the tgfβ inhibitor is administered prior to the one or more additional therapeutic agents. In some embodiments, the tgfβ inhibitor is administered after the one or more additional therapeutic agents.

In some embodiments, the tgfβ inhibitor and/or the one or more additional therapeutic agents are administered until remission.

In some embodiments, the additional therapeutic agent comprises cyclophosphamide or topotecan. When cyclophosphamide is used, it can be used at 250mg/m² And (5) administration. In some embodiments, the administration of cyclophosphamideFor 5 days, for example five consecutive days. In some embodiments, the additional therapeutic agent comprises at 0.75mg/m² Topotecan is administered. When topotecan is used, its administration can last for 5 days, for example five consecutive days. In some embodiments, the proliferative disease is neuroblastoma.

In some embodiments, the additional therapeutic agent comprises gemcitabine. When gemcitabine is used, it may be used at 675mg/m² And (5) administration. In some embodiments, the administration of gemcitabine is continued for 2 days, for example, on days 1 and 8. In some embodiments, the proliferative disease is osteosarcoma.

In some embodiments, various combinations and dosages may be used with subjects considered pediatric patients (e.g., under 18 years of age).

Disclosed herein is a method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising administering to the subject (a) 2100mg of a TGF-beta antibody, (b) at 500mg/m² Intravenous bolus injection followed by 2400mg/m² 5-fluorouracil infused continuously over 46 hours, (c) at 400mg/m² Folic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of L-folinic acid (d) at 180mg/m² Irinotecan administered intravenously, and (e) 5mg/kg bevacizumab, wherein the tgfβ antibody is administered on day 1 of a 28 day cycle (and optionally day 15), and wherein the 5-fluorouracil, folinic acid (or levofolinic acid), irinotecan, and bevacizumab are administered on days 1 and 15 of the 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs 4, 5, and 6, respectively.

Disclosed herein is a method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising administering to the subject (a) 2100mg of a TGF-beta antibody, (b) at 400mg/m² Intravenous bolus injection followed by 2400mg/m² 5-fluorouracil infused continuously over 46 hours, (c) at 400mg/m² Folinic acid (leucovorin) orAt 200mg/m² Intravenous administration of L-folinic acid (d) at 180mg/m² Irinotecan administered intravenously, and (e) 5mg/kg bevacizumab, wherein the tgfβ antibody is administered on day 1 of a 28 day cycle (and optionally day 15), and wherein the 5-fluorouracil, folinic acid (or levofolinic acid), irinotecan, and bevacizumab are administered on days 1 and 15 of the 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs 4, 5, and 6, respectively.

Also disclosed herein is a method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising administering to the subject (a) 2100mg of a TGF-beta antibody, (b) at 500mg/m² Intravenous bolus injection followed by 2400mg/m² 5-fluorouracil infused continuously over 46 hours, (c) at 400mg/m² Folinic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of L-folinic acid, (d) at 85mg/m² Oxaliplatin for intravenous administration, and (e) 5mg/kg bevacizumab, wherein the tgfβ antibody is administered on day 1 (and optionally day 15) of a 28 day cycle, and wherein the 5-fluorouracil, folinic acid (or levofolinic acid), oxaliplatin, and bevacizumab are administered on days 1 and 15 of the 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively.

Also disclosed herein is a method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising administering to the subject (a) 2100mg of a TGF-beta antibody, (b) at 400mg/m² Intravenous bolus injection followed by 2400mg/m² 5-fluorouracil infused continuously over 46 hours, (c) at 400mg/m² Folinic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of L-folinic acid, (d) at 85mg/m² Intravenous administration of oxaliplatin, and (e) 5mg/kg bevacizumab, wherein the tgfβ antibody is administered on day 1 (and optionally day 15) of a 28 day cycle, and wherein the 5-fluorouracil, folinic acid (or levofolinic acid), oxaliplatin, and bevacizumab are in theDay 1 and day 15 of the 28 day cycle, and wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively.

The methods may further comprise administering a PD-1 inhibitor to the patient. In some embodiments, the PD-1 inhibitor is tirelizumab. Tiril bead mab may have a heavy chain of SEQ ID NO. 321 and a light chain of SEQ ID NO. 322. In some embodiments, tirelimumab is administered intravenously at 300mg on day 1 of each 28 day cycle.

In some embodiments, the tgfβ antibody, 5-fluorouracil, folinic acid (or levofolinic acid), oxaliplatin, irinotecan, bevacizumab, or PD-1 inhibitor is administered to a subject over 2 or more cycles.

Also disclosed herein is a method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject (a) 2100mg of a TGF-beta antibody, and (b) 200mg of a PD-1 inhibitor administered in a Q3W cycle, in combination with (c) 130mg/m on day 1² Oxaliplatin for intravenous administration, (d) at a Q3W cycle of 1000mg/m² Capecitabine is administered orally twice daily (days 1-14), wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO:1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO:4, 5 and 6, respectively. The PD-1 inhibitor may be tirelimumab. In some embodiments, the tgfβ antibody is administered at a dose of 1400mg or 2100mg at the Q2W cycle. In other embodiments, the PD-1 inhibitor is administered at a dose of 300mg in the Q4W cycle.

Also disclosed herein is a method of treating gastric cancer in a subject in need thereof, comprising administering to the subject (a) 2100mg of a TGF-beta antibody administered in a Q3W cycle, and (b) 200mg of a PD-1 inhibitor administered in a Q3W cycle, in combination with (c) 85mg/m² Oxaliplatin administered intravenously (on day 1), (d) folinic acid administered intravenously at 400mg/m2 or at 200mg/m² Intravenous administration of L-folinic acid (on day 1), (e) at 400mg/m² Intravenously administered 5-fluorouracil (on day 1) and (f) at 1200mg/m at Q2W cycle² Daily intravenous administration (on days 1-2), wherein the TGF-beta antibody comprisesHeavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively. The PD-1 inhibitor may be tirelimumab. In some embodiments, the tgfβ antibody is administered at a dose of 1400mg or 2100mg at the Q2W cycle. In other embodiments, the PD-1 inhibitor is administered at a dose of 300mg in the Q4W cycle.

Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Drawings

Figure 1 shows the average concentration versus time curve for each dose group of NIS793 in cycle 1.

Figure 2 shows the average concentration versus time curve for each dose group of NIS793 in cycle 3.

Fig. 3 shows dosages and administration of different combinations of NIS793, swabber or tirelimumab, gemcitabine, and/or albumin-bound paclitaxel. NIS793 (2100 mg) was administered to the patient every 2 weeks, stadalimumab or tirelimumab (400 mg) was administered every 4 weeks, and gemcitabine (1000 mg/m) was administered on days 1, 8 and 15² ) And albumin-bound paclitaxel (125 mg/m) was administered on day 1, day 8 and day 15² ) (FIG. 2). One cycle was 28 days.

Detailed Description

The immune system is responsible for early detection and destruction of cancer cells. Cancer cells can escape immune surveillance through various mechanisms, such as reduced immune recognition, increased resistance to immune cell attack, or due to immunosuppressive tumor microenvironment (Mittal et al, 2014). Some cancers produce tgfβ (potent immunosuppressive cytokines), which antagonizes cytotoxic lymphocytes and promotes recruitment of suppressive immune cells, thereby favoring tumor growth and progression (Wojtowicz-Praga-2003, teicher,2007, yang et al, 2010).

Tgfβ belongs to a large family of structurally related cytokines, including: bone Morphogenic Proteins (BMP), growth and differentiation factors, activins, and inhibins. TGF-beta ligands expressed in mammals have 3 isoforms, TGF-beta 1, 2 and 3. Under normal conditions, tgfβ maintains homeostasis and restricts growth of epithelial, endothelial, neuronal and hematopoietic lineages (by inducing anti-proliferative and apoptotic responses). Thus, alterations in the tgfβ signaling pathway are believed to be associated with human diseases including cardiovascular disease, fibrosis, reproductive disorders, wound healing, and cancer (Wakefield and Hill, 2013).

NIS793 is a fully human IgG2, human/mouse cross-reactive, tgfβ neutralizing antibody. NIS793 functions at the ligand-receptor level. NIS793 antagonizes tgfβ1 and 2 more specifically and to a lesser extent tgfβ3 than fresolimumab, which is a pan tgfβ inhibitor that neutralizes all tgfβ isoforms.

To escape immune surveillance, cancer cells may additionally utilize immune checkpoint pathways that tightly regulate T cell activation, such as the PD-1/PD-L1 axis (Pardoll, 2012). Thus, antagonism of tgfβ alone or in combination with immune checkpoint blockade may stimulate more effective anti-tumor immunity.

Definition of the definition

Additional terms are defined below and throughout the application.

As used herein, the article "a" or "an" refers to one or more than one (e.g., at least one) grammatical object of the article.

The term "or" means and is used interchangeably with the term "and/or" herein unless the context clearly indicates otherwise.

"about" and "approximately" mean an acceptable degree of error in the measured quantity given the nature or accuracy of the measurement. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value or range of values. In some embodiments, when a number references the term "about," the number is intended to also include the exact value of the number. For example, "about 10" includes, but is not limited to, a value of 10. It also includes 10+ -2, 10+ -1 or 10+ -0.5.

The terms "combination" or "in combination with … …" are not intended to imply that therapies or therapeutic agents must be administered simultaneously and/or formulated for delivery together, although such delivery methods are also within the scope of the disclosure. The therapeutic agents in the combination may be administered simultaneously with, before or after one or more other additional therapies or therapeutic agents. These therapeutic agents or regimens may be administered in any order. Typically, each agent will be administered at a dosage and/or schedule determined for that agent. It will also be appreciated that the additional therapeutic agents used in the combination may be administered together in a single composition or separately in different compositions. The administration of the therapeutic agents may be in any order. The first agent and the additional agent (e.g., second, third agent) may be administered by the same route of administration or by different routes of administration. Typically, additional therapeutic agents used in combination are contemplated to be used at levels not exceeding those used alone. In some embodiments, the level used in the combination will be lower than the level used alone.

In some embodiments, the additional therapeutic agent is administered at or below the therapeutic dose. In certain embodiments, when the second therapeutic agent is administered in combination with the first therapeutic agent (e.g., an anti-tgfβ antibody molecule), the concentration of the second therapeutic agent required to achieve inhibition (e.g., growth inhibition) is lower than when the second therapeutic agent is administered alone (e.g., an anti-PD 1 antibody molecule). In certain embodiments, when the first therapeutic agent is administered in combination with the second therapeutic agent, the concentration of the first therapeutic agent required to achieve inhibition (e.g., growth inhibition) is lower than when the first therapeutic agent is administered alone. In certain embodiments, in combination therapy, the concentration of the second therapeutic agent required to achieve inhibition (e.g., growth inhibition) is lower than the therapeutic dose of the second therapeutic agent as monotherapy, e.g., 10% -20%, 20% -30%, 30% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80%, or 80% -90%. In certain embodiments, in combination therapy, the concentration of the first therapeutic agent required to achieve inhibition (e.g., growth inhibition) is lower than the therapeutic dose of the first therapeutic agent as monotherapy, e.g., by 10% -20%, 20% -30%, 30% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80%, or 80% -90%.

The term "inhibit", "inhibitor" or "antagonist" includes a decrease in certain parameters (e.g., activity) of a given molecule (e.g., an immune checkpoint inhibitor or tgfβ inhibitor). For example, the term includes inhibition of activity (e.g., tgfβ, PD-1 or PD-L1 activity) by at least 5%, 10%, 20%, 30%, 40% or more. Therefore, the inhibition need not be 100%.

The term "activation", "activator" or "agonist" includes an increase in certain parameters (e.g., activity) of a given molecule (e.g., a co-stimulatory molecule). For example, the term includes an increase in activity (e.g., co-stimulatory activity) of at least 5%, 10%, 25%, 50%, 75% or more.

The term "anti-cancer effect" refers to a biological effect that can be manifested by various means including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in proliferation of cancer cells, a decrease in survival of cancer cells, or an improvement in various physiological symptoms associated with cancer. "anticancer effect" can also be manifested by the ability of peptides, polynucleotides, cells and antibodies to first prevent the occurrence of cancer.

The term "anti-tumor effect" refers to a biological effect that can be manifested by various means including, but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in proliferation of tumor cells, or a decrease in the survival rate of tumor cells.

The term "cancer" refers to a disease characterized by rapid and uncontrolled growth of abnormal cells (e.g., proliferative disease). Cancer cells may spread to other parts of the body locally or through the blood stream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, solid tumors (e.g., lung, breast, prostate, ovarian, cervical, skin, pancreatic, colorectal, renal, liver, and brain cancers) and hematological malignancies (e.g., lymphomas and leukemias, etc.). The terms "tumor" and "cancer" are used interchangeably herein, e.g., both terms encompass solid and liquid tumors, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes premalignant as well as malignant cancers and tumors.

The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (e.g., B cell, dendritic cell, etc.), that displays a foreign antigen complexed with a Major Histocompatibility Complex (MHC) on its surface. T cells can recognize these complexes using their T Cell Receptor (TCR). APCs process antigens and present them to T cells.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activating molecules (SLAM proteins), activated NK cell receptors, BTLA, toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), 4-1BB (CD 137), B7-H3, CDS, ICAM-1, ICOS (CD 278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA4 VLA1, CD49a, ITGA4, IA4, CD49D, ITGA, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactive), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLASTAG (CD 162), SELPLG LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand binding specifically to CD 83.

As the term is used herein, "immune effector cell" or "effector cell" refers to a cell that is involved in an immune response (e.g., promotes an immune effector response). Examples of immune effector cells include T cells, such as α/β T cells and γ/δ T cells, B cells, natural Killer (NK) cells, natural Killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

As the term is used herein, "immune effector" or "effector" function "or" response "refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack by a target cell. For example, immune effector function or response refers to the characteristics of T or NK cells that promote killing or inhibit growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or helper activity (including secretion of cytokines).

As used herein, the terms "treat (treat, treatment and treating)" refer to reducing or ameliorating the progression, severity and/or duration of a disorder (e.g., a proliferative disorder) caused by administration of one or more therapies, or ameliorating one or more symptoms (preferably, one or more discernible symptoms) of the disorder. In particular embodiments, the terms "treatment" and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder (such as the growth of a tumor), which is not necessarily discernible to the patient. In other embodiments, the terms "treat (treat, treatment and treating)" refer to inhibiting the progression of a proliferative disorder, either physically, by, for example, stabilizing a discernible symptom, or physiologically, by, for example, stabilizing a physical parameter, or both. In other embodiments, the term "treating (treat, treatment and treating)" refers to reducing or stabilizing tumor size or cancer cell count.

The compositions, formulations and methods of the invention encompass polypeptides and nucleic acids having a specified sequence, or having a sequence that is substantially identical or similar to a specified sequence (e.g., a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or more to a specified sequence). In the context of amino acid sequences, the term "substantially identical" is used herein to refer to such first amino acid: it contains i) the same as the aligned amino acid residues in the second amino acid sequence, or ii) a sufficient or minimum number of amino acid residues that are conservative substitutions of aligned amino acid residues in the second amino acid sequence, such that the first amino acid sequence and the second amino acid sequence may have a common domain and/or a common functional activity. For example, an amino acid sequence that contains a common domain that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence (e.g., a sequence provided herein).

In the context of nucleotide sequences, the term "substantially identical" is used herein to refer to such first nucleic acid sequences: it contains a sufficient or minimal number of nucleotides that are identical to the aligned nucleotides in the second nucleic acid sequence such that the first nucleotide sequence and the second nucleotide sequence encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or have a common functional polypeptide activity. For example, a nucleotide sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a reference sequence (e.g., a sequence provided herein).

The term "functional variant" refers to a polypeptide having substantially the same amino acid sequence as a naturally occurring sequence, or a polypeptide encoded by substantially the same nucleotide sequence and capable of having one or more activities of the naturally occurring sequence.

The calculation of homology or sequence identity between sequences (these terms are used interchangeably herein) is performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps are introduced in one or both of the first amino acid and the second amino acid or the first nucleic acid sequence and the second nucleic acid sequence for optimal alignment, and non-homologous sequences are negligible for comparison purposes). In preferred embodiments, the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequences. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in a second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

Taking into account the number of gaps and the length of each gap, the percent identity between two sequences is a function of the number of identical positions shared by the sequences, which gaps need to be introduced for optimal alignment of the two sequences.

Sequence comparison and percent identity determination between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage identity between two amino acid sequences is determined using Needleman and Wunsch ((1970) j.mol.biol. [ journal of molecular biology ] 48:444-453) algorithms, which have been incorporated into the GAP program in the GCG software package (available from www.gcg.com), using the Blossum 62 matrix or PAM250 matrix and a GAP weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available from www.gcg.com), using the nwsgapdna.cmp matrix, and a GAP weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and parameters that should be used unless otherwise indicated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The percentage identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm of E.Meyers and W.Miller ((1989) computer application in CABIOS [ biosciences ]4:11-17, which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, the gap length penalty of 12 and the gap penalty of 4.

The nucleic acid sequences and protein sequences described herein may be used as "query sequences" to search public databases, for example, to identify other family members or related sequences. These searches can be performed using the NBLAST and XBLAST programs of Altschul et al (1990) J.mol.biol. [ journal of molecular biology ]215:403-10 (version 2.0). BLAST nucleotide searches can be performed using the NBLAST program (score=100, word length=12) to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain a gap alignment for comparison purposes, gap BLAST (Gapped BLAST) may be used as described in Altschul et al, (1997) Nucleic Acids Res [ nucleic acids Ind 25:3389-3402. When using BLAST and empty BLAST programs, default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology [ guidelines for molecular biology experiments ], john Wiley & Sons, N.Y [ John Weili father-son publishing company, new York ] (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in this reference and either may be used. Specific hybridization conditions referred to herein are as follows: 1) Low stringency hybridization conditions: hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45℃followed by washing twice in 0.2 XSSC, 0.1% SDS at least 50℃for low stringency conditions (the temperature of washing can be increased to 55 ℃); 2) Moderate stringency hybridization conditions: hybridization in 6 XSSC at about 45℃followed by one or more washes in 0.2 XSSC, 0.1% SDS at 60 ℃; 3) High stringency hybridization conditions: hybridization in 6 XSSC at about 45℃followed by one or more washes in 0.2 XSSC, 0.1% SDS at 65 ℃; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS,65℃followed by one or more washes in 0.2 XSSC, 1% SDS,65 ℃. Very high stringency condition (4) is a preferred condition and unless indicated otherwise, conditions should be used.

It will be appreciated that the molecules of the invention may have additional conservative or non-essential amino acid substitutions that have no substantial effect on the function of the molecule.

The term "amino acid" is intended to include all molecules, whether natural or synthetic, that include both amino and acid functionalities and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; their analogs, derivatives and congeners; amino acid analogs having variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D-optical isomers or L-optical isomers and peptide mimetics.

A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The terms "polypeptide", "peptide" and "protein" (if single-chain) are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation to a labeling component. The polypeptide may be isolated from a natural source, may be produced by recombinant techniques from a host eukaryotic or prokaryotic host, or may be the product of a synthetic procedure.

The terms "nucleic acid", "nucleic acid sequence", "nucleotide sequence" or "polynucleotide sequence" and "polynucleotide" are used interchangeably. They refer to nucleotides of any length in polymeric form, i.e., deoxyribonucleotides or ribonucleotides, or analogs thereof. The polynucleotide may be single-stranded or double-stranded, and if single-stranded, the polynucleotide may be the coding strand or the non-coding (antisense) strand. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The nucleic acid may be a recombinant polynucleotide, or a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin, which does not exist in nature or is linked to another polynucleotide in a non-natural arrangement.

As used herein, the term "isolated" refers to material removed from its original or natural environment (e.g., its naturally occurring natural environment). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide isolated from some or all of the coexisting materials in the natural system by human intervention is isolated. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition and still be isolated in that such vector or composition is not part of the environment in which it naturally occurs.

Various aspects of the invention are described in further detail below. Additional definitions are set forth throughout the application.

TGF beta inhibitors

Tgfβ belongs to a large family of structurally related cytokines including, for example, bone Morphogenic Proteins (BMP), growth and differentiation factors, activin, and inhibin. In some embodiments, a tgfβ inhibitor described herein may bind to and/or inhibit one or more isoforms of tgfβ (e.g., one, two, or all of tgfβ1, tgfβ2, or tgfβ3).

Transforming growth factor beta (also referred to as tgfβ, TGFb, or TGF- β, used interchangeably herein) inhibitors (e.g., anti-tgfβ antibody molecules) are described throughout and can be used in the methods described throughout.

In some embodiments, the TGF-beta inhibitor is NIS793, fresolimumab, PF-06952229, or AVID200.

In some embodiments, the tgfβ inhibitor comprises NIS973 or a compound disclosed in international application publication No. WO 2012/167143. NIS793 is also known as XOMA 089 or xpa.42.089.NIS793 is a fully human monoclonal antibody that specifically binds to and neutralizes TGF- β1 and 2 ligands.

The heavy chains CDR1, CDR2 and CDR3 of NIS793 have the following amino sequences, respectively: GGTFSSYAIS (SEQ ID NO: 1); GIIPIFGTANYAQKFQG (SEQ ID NO: 2); and GLWEVRALPSVY (SEQ ID NO: 3).

Light chain CDR1, CDR2 and CDR3 of NIS793 have the following amino sequences, respectively: GANDIGSKSVH (SEQ ID NO: 4); EDIRPS (SEQ ID NO: 5); QVWDRDSDQYV (SEQ ID NO: 6).

The heavy chain variable region of NIS793 has the following amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVY YCARGLWEVRALPSVYWGQGTLVTVSS (SEQ ID NO: 7) (disclosed as SEQ ID NO:6 in WO 2012/167143). The light chain variable region of NIS793 has the following amino acid sequence: SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQ KAGQAPVLVVSEDIIRPSGIPERISGSNSGNTATLTISRVEAGDEADYY CQVWDRDSDQYVFGTGTKVTVLG (SEQ ID NO: 8) (disclosed as SEQ ID NO:8 in WO 2012/167143).

The heavy chain of NIS793 has the following amino acid sequence: QVQLVQSGAEVKKPGSSV KVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGLWEVRALPSVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9). The light chain of NIS793 has the following amino acid sequence: SYELTQPPSVSVAPGQTARITCGANDIGSKSVHWYQQKAGQAPVLVVSEDIIRPSGIPERISGSNSGNTATLTISRVEAGDEADYYCQVWDRDSDQYVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 10).

NIS793 binds human tgfβ isoforms with high affinity. Typically, NIS793 binds tgfβ1 and tgfβ2 with high affinity and to a lesser extent tgfβ3. In the Biacore assay, the K of NIS793 on human TGF beta_D Is 14.6pM (for TGF-beta 1), 67.3pM (for TGF-beta 2), and 948pM (for TGF-beta 3). In view of the high affinity binding to all three tgfβ isoforms, in certain embodiments NIS793 is expected to bind tgfβ1, 2, and 3 at doses of NIS793 as described herein. NIS793 cross-reacts with rodent and cynomolgus tgfβ and shows functional activity in vitro and in vivo, making rodents and cynomolgus monkeys relevant species for toxicology studies.

In certain embodiments, the tgfβ inhibitor is used to treat cancer (e.g., pancreatic cancer (e.g., PDAC) or gastrointestinal cancer (e.g., colorectal cancer)). In some embodiments, the tgfβ inhibitor is used in combination with a checkpoint inhibitor (e.g., an inhibitor of PD1 described herein) for the treatment of cancer.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose of greater than 15 mg/kg. For example, the TGF-beta inhibitor is administered at a dose of between 15.1mg/kg and about 50 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose of between about 16mg/kg and about 50 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose between 16mg/kg and about 50 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose of between about 20mg/kg and about 40 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose of between about 25mg/kg and about 35 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose of about 20 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose of about 30 mg/kg. In some embodiments, the TGF-beta inhibitor is administered at a dose of about 45 mg/kg.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered in a fixed dose. For example, in some embodiments, the tgfβ inhibitor is administered at a dose of between about 1000mg to about 1600 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1100mg to about 1500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 1200 to about 1400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 1300mg to about 1400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1300mg to about 1500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1300mg to about 1600 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 1200mg to about 1500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 1200mg to about 1600 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 1400mg to about 1500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1400mg to about 1600 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1100mg to about 1600 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between 1100mg to about 1400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1100mg to about 1300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1100mg to about 1200 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between or about 1000mg to about 1500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between or about 1000mg to about 1400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between or about 1000mg to about 1300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between or about 1000mg to about 1200 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between or about 1000mg to about 1100 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1000 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1100 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1200 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 1600 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1200mg to about 2100 mg.

In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2000mg to about 2500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2000mg to about 2400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1900mg to about 2300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 1900mg to about 2200 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 2000mg to about 2100 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 2100mg to about 2500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 2100 to about 2400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2100 to about 2300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2100 to about 2200 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2200 to about 2500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2200 to about 2400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2200 to about 2300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2300mg to about 2500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose between about 2300mg to about 2400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of between about 2400mg to about 2500 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 2000 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 2100 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 2200 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 2300 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 2400 mg. In some embodiments, the tgfβ inhibitor is administered at a dose of about 2500 mg.

In some embodiments, the tgfβ inhibitor is administered weekly, biweekly, tricyclically, or weekly. In some embodiments, the tgfβ inhibitor is administered weekly. In some embodiments, the tgfβ inhibitor is administered once every two weeks. In some embodiments, the tgfβ inhibitor is administered once every three weeks. In some embodiments, the tgfβ inhibitor is administered once every three weeks.

In some embodiments, the tgfβ inhibitor is administered intravenously.

In some embodiments, the tgfβ inhibitor is administered over a period of about 20 minutes to about 40 minutes. For example, a tgfβ inhibitor is administered over a period of about 30 minutes. In some embodiments, the tgfβ inhibitor is administered over a period of about one hour. In some embodiments, the tgfβ inhibitor is administered over a period of about two hours. In some embodiments, the tgfβ inhibitor is administered over a period of about three hours. In some embodiments, the tgfβ inhibitor is administered over a period of about four hours. In some embodiments, the tgfβ inhibitor is administered over a period of about five hours. In some embodiments, the tgfβ inhibitor is administered over a period of about six hours.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously at a dose of between about 1300mg to about 1500mg (e.g., about 1400 mg) once every two weeks. In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously at a dose of between about 2000mg to about 2200mg (e.g., about 2100 mg) once every two weeks. In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously at a dose of between about 2000mg to about 2200mg (e.g., about 2100 mg) once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously every two weeks at a dose of between about 1300mg to about 1500mg (e.g., about 1400 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes). In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously every two weeks at a dose of between about 2000mg to about 2200mg (e.g., about 2100 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes). In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously every three weeks at a dose of between about 2000mg to about 2200mg (e.g., about 2100 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes).

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously at a dose of between about 40mg/kg to about 50mg/kg (e.g., about 45 mg/kg) once every three weeks. In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously at a dose of between about 25mg/kg to about 35mg/kg (e.g., about 30 mg/kg) once every three weeks. In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered intravenously at a dose of between about 15mg/kg to about 25mg/kg (e.g., about 20 mg/kg) once every three weeks.

In some embodiments, the methods described herein may further comprise one or more other therapeutic agents, procedures, or modes. In some embodiments, a tgfβ inhibitor (e.g., NIS 793) is administered in combination with a PD1 inhibitor (e.g., an anti-PD 1 antibody molecule) and/or a PD-L inhibitor (PD-L1 and/or PD-L2). In one embodiment, the methods described herein comprise administering an inhibitor of an inhibitory (or immune checkpoint) molecule PD-1, PD-L2, and/or tgfβ. In one embodiment, the inhibitor is an antibody or antibody fragment that binds to PD-1, PD-L2, and/or tgfβ.

Alternatively, or in combination with the foregoing methods, the methods described herein may be administered or used with one or more of the following: immunomodulators (e.g., activators of costimulatory molecules or inhibitors of inhibitory molecules, such as immune checkpoint molecules); vaccines, such as therapeutic cancer vaccines; or other forms of cellular immunotherapy.

In certain embodiments, the methods described herein are administered or used with a co-stimulatory molecule or a modulator of an inhibitory molecule (e.g., a co-inhibitory ligand or receptor).

Other exemplary TGF-beta inhibitors

In some embodiments, the TGF-beta inhibitor comprises freuzumab (CAS registry number 948564-73-6). The freuzumab is also known as GC1008. The fresolimumab is a human monoclonal antibody that binds to and inhibits TGF- β isoforms 1, 2 and 3.

The heavy chain of the fresolimumab has the following amino acid sequence:

the light chain of the fresolimumab has the following amino acid sequence:

fresolimumab is disclosed, for example, in International application publication No. WO 2006/086469, and U.S. Pat. Nos. 8,383,780 and 8,591,901.

In some embodiments, the TGF-beta inhibitor is PF-06952229.PF-06952229 is an inhibitor of TGF-beta R1, preventing signaling through receptors and TGF-beta R1 mediated immunosuppression, thereby enhancing anti-tumor immune responses. PF-06952229 is disclosed, for example, in Yano et al Immunology [ Immunology ]2019;157 (3) 232-47.

In some embodiments, the tgfβ inhibitor is AVID200.AVID200 is a tgfβ receptor extracellular domain-IgG Fc fusion protein that selectively targets and neutralizes tgfβ isoforms 1 and 3.AVID200 is disclosed, for example, in O' Connor-mccour, MD et al can.res. [ cancer research ]2018.78 (13).

PD-1 inhibitors

PD-1 is a CD28/CTLA-4 family member that is expressed on, for example, activated CD4+ and CD8+ T cells, tregs, and B cells. It down regulates effector T cell signaling and function. PD-1 is induced on tumor-infiltrating T cells and can lead to functional failure or dysfunction (Keir et al (2008) Annu. Rev. Immunol. [ immunology annual comment ]26:677-704; pardoll et al (2012) Nat Rev Cancer Nature comment ]12 (4): 252-64). PD-1 delivers a co-inhibitory signal when bound to one of its two ligands, programmed death-ligand 1 (PD-L1) or programmed death-ligand 2 (PD-L2). PD-L1 is expressed on many cell types, including T cells, natural Killer (NK) cells, macrophages, dendritic Cells (DCs), B cells, epithelial cells, vascular endothelial cells, and many types of tumors. High expression of PD-L1 on murine and human tumors is associated with poor clinical outcome in a variety of cancers (Keir et al (2008) Annu. Rev. Immunol [ immunology annual comment ]26:677-704; pardoll et al (2012) Nat Rev Cancer natural comment ]12 (4): 252-64). PD-L2 is expressed on dendritic cells, macrophages and some tumors. Blocking the PD-1 pathway has been clinically and clinically validated for cancer immunotherapy. Both preclinical and clinical studies demonstrate that anti-PD-1 blockade can restore effector T cell activity and produce a powerful anti-tumor response. For example, blocking the PD-1 pathway may restore depleted/dysfunctional effector T cell functions (e.g., proliferation, IFN-gamma secretion, or cytolytic functions) and/or inhibit Treg cell functions (Keir et al (2008) Annu. Rev. Immunol. [ immunology annual comment ]26:677-704; pardol et al (2012) Nat Rev Cancer Nature comment ]12 (4): 252-64). Blocking of the PD-1 pathway may be achieved with antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, or oligopeptides of PD-1, PD-L1 and/or PD-L2.

As used herein, the term "programmed death 1" or "PD-1" includes isoforms, mammalian (e.g., human PD-1), species homologs of human PD-1, and analogs that comprise at least one common epitope with PD-1. The amino acid sequence of PD-1 (e.g., human PD-1) is known in the art, e.g., shinohara T et al (1994) Genomics [ Genomics ]23 (3): 704-6; finger LR et al Gene [ Gene ] (1997) 197 (1-2): 177-87.

In certain embodiments, a tgfβ inhibitor as described herein is administered in combination with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is Stadalimumab (PDR 001, novartis, inc. (Novartis)), nawuzumab (Bristol-Myers Squibb), pembrolizumab (Merck & Co)), pituzumab (Curetech), MEDI0680 (England Mei Dimiao, inc. (Medimume)), REGN2810 (regenerator), TSR-042 (Tasa Luo Gongsi (Tesaro)), PF-06801591 (Pfizer), BGB-A317 (also known as Tiriluzumab) (Beigene), BGB-108 (BgIgIgIK), INCSHR1210 (INCyte)), or AMP-224 (An Puli (Amplmune)).

Exemplary PD-1 inhibitors

In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody molecule as described in US2015/0210769 published at 30/7/2015 entitled "antibody molecule of Antibody Molecules to PD-1and Uses Thereof[PD-1 and use thereof". In one embodiment, the anti-PD-1 inhibitor is swabber, also known as PDR001. In another embodiment, the anti-PD-1 inhibitor is tirelimumab, also known as BGB-A317.

In one embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising an amino acid sequence set forth in table 1 (e.g., a heavy and light chain variable region sequence from BAP 049-clone-E or BAP 049-clone-B disclosed in table 1), or an amino acid sequence encoded by a nucleotide sequence set forth in table 1. In some embodiments, CDRs are defined according to carbat (Kabat) (e.g., as set forth in table 1). In some embodiments, CDRs are defined according to Qiao Xiya (Chothia) (e.g., as listed in table 1). In some embodiments, the CDRs are defined according to the combined CDRs of both cabazite and Qiao Xiya (e.g., as listed in table 1). In one embodiment, the combination of the carboplatin and Qiao Xiya CDRs of VH CDR1 comprises the amino acid sequence GYTFTTYWMH (SEQ ID NO: 13). In one embodiment, one or more of the CDRs (or generally all CDRs) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 1, or the amino acid sequences encoded by the nucleotide sequences shown in table 1.

In one embodiment, the anti-PD-1 antibody molecule comprises: a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 14, the VH CDR2 amino acid sequence of SEQ ID NO. 15, and the VH CDR3 amino acid sequence of SEQ ID NO. 16; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO. 23, the VLCDR2 amino acid sequence of SEQ ID NO. 24, and the VLCDR3 amino acid sequence of SEQ ID NO. 25, each as disclosed in Table 1.

In one embodiment, the antibody molecule comprises: a VH comprising a VH CDR1 encoded by the nucleotide sequence of SEQ ID NO. 37, a VH CDR2 encoded by the nucleotide sequence of SEQ ID NO. 38, and a VH CDR3 encoded by the nucleotide sequence of SEQ ID NO. 39; and VL comprising VLCDR1 encoded by the nucleotide sequence of SEQ ID NO. 42, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO. 43, and VLCDR3 encoded by the nucleotide sequence of SEQ ID NO. 44, are each disclosed in Table 1.

In one embodiment, the anti-PD-1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 19, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 19. In one embodiment, the anti-PD-1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 33, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 33. In one embodiment, the anti-PD-1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 29, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 29. In one embodiment, the anti-PD-1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 19 and VL comprising the amino acid sequence of SEQ ID No. 33. In one embodiment, the anti-PD-1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 19 and VL comprising the amino acid sequence of SEQ ID No. 33.

In one embodiment, the antibody molecule comprises: VH encoded by the nucleotide sequence of SEQ ID No. 20, or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 20. In one embodiment, the antibody molecule comprises: VL encoded by the nucleotide sequence of SEQ ID No. 34 or 30, or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 34 or 30. In one embodiment, the antibody molecule comprises: a VH encoded by the nucleotide sequence of SEQ ID NO. 20 and a VL encoded by the nucleotide sequence of SEQ ID NO. 34 or 30.

In one embodiment, the anti-PD-1 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 21. In one embodiment, the anti-PD-1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID No. 35, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID No. 35. In one embodiment, the anti-PD-1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID No. 31, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID No. 31. In one embodiment, the anti-PD-1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 and a light chain comprising the amino acid sequence of SEQ ID NO. 35. In one embodiment, the anti-PD-1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 21 and a light chain comprising the amino acid sequence of SEQ ID NO. 31.

In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 22, or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID NO. 22. In one embodiment, the antibody molecule comprises: a light chain encoded by the nucleotide sequence of SEQ ID NO. 36 or 32, or a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID NO. 36 or 32. In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 22 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 36 or 32.

In another embodiment, the anti-PD-1 antibody molecule (e.g., tirelimumab) comprises the following heavy and/or light chains, VH, VL, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3:

in some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody comprising SEQ ID NO: 321. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody comprising SEQ ID NO. 322. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody comprising SEQ ID NOS 321 and 322. In some embodiments, the PD-1 inhibitor comprises HCDR and LCDR of tirelimumab as set forth in SEQ ID NOS: 325-330.

The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0210769.

In some embodiments, the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) is administered at a plateau dose of between about 100mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 100mg to about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 200mg to about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 300mg to about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 400mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 400mg to about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 500mg to about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 600mg to about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 700mg to about 800 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 800mg to about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of between about 900mg to about 1000 mg.

In some embodiments, the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) is administered at a plateau dose of about 100 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 200 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 300 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 400 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 500 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 600 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 700 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 800 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 900 mg. In some embodiments, the PD-1 inhibitor is administered at a dose of about 1000 mg.

In some embodiments, the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) is administered once every ten weeks. In some embodiments, the PD-1 inhibitor is administered once every nine weeks. In some embodiments, the PD-1 inhibitor is administered once every eight weeks. In some embodiments, the PD-1 inhibitor is administered once every seven weeks. In some embodiments, the PD-1 inhibitor is administered once every six weeks. In some embodiments, the PD-1 inhibitor is administered once every five weeks. In some embodiments, the PD-1 inhibitor is administered once every four weeks. In some embodiments, the PD-1 inhibitor is administered once every three weeks. In some embodiments, the PD-1 inhibitor is administered once every two weeks. In some embodiments, the PD-1 inhibitor is administered weekly.

In some embodiments, a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) is administered intravenously.

In some embodiments, the PD-1 inhibitor (e.g., swabs or tirelimumab) is administered over a period of about 20 minutes to 40 minutes (e.g., about 30 minutes). In some embodiments, the PD-1 inhibitor is administered over a period of about 30 minutes. In some embodiments, the PD-1 inhibitor is administered over a period of about one hour. In some embodiments, the PD-1 inhibitor is administered over a period of about two hours. In some embodiments, the PD-1 inhibitor is administered over a period of about three hours. In some embodiments, the PD-1 inhibitor is administered over a period of about four hours. In some embodiments, the PD-1 inhibitor is administered over a period of about five hours. In some embodiments, the PD-1 inhibitor is administered over a period of about six hours.

In some embodiments, the PD-1 inhibitor (e.g., swabs or tirelimumab) is administered intravenously at a dose of between about 300mg to about 500mg (e.g., about 400 mg) once every four weeks. In some embodiments, the PD-1 inhibitor is administered intravenously at a dose of between about 200mg to about 400mg (e.g., about 300 mg) once every three weeks. In some embodiments, the swabs are administered at a dose of 400mg once every four weeks. In some embodiments, the swabs are administered once every four weeks at a dose of 300 mg. In some embodiments, the swabs are administered once every three weeks at a dose of 300 mg. In some embodiments, the swabs are administered once every three weeks at a dose of 200 mg.

In some embodiments, the PD-1 inhibitor (e.g., swabs or tirelimumab) is administered intravenously at a dose of between about 300mg to about 500mg (e.g., about 400 mg) for a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes) once every two weeks. In some embodiments, the PD-1 inhibitor is administered intravenously at a dose of between about 200mg to about 400mg (e.g., about 300 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes) once every three weeks. In some embodiments, the PD-1 inhibitor is administered intravenously every four weeks at a dose of between about 200mg to about 400mg (e.g., about 300 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes). In some embodiments, the PD-1 inhibitor is administered intravenously at a dose of between about 100mg to about 300mg (e.g., about 200 mg) over a period of about 20 minutes to about 40 minutes (e.g., about 30 minutes) once every three weeks.

In some embodiments, the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) is administered weekly at a dose of about 100 mg. For example, if a patient is given a dose of 10 weeks, then a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 1000 mg. If administered at a dose of 9 weeks, then a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 900 mg. If administered at a dose of 8 weeks, then the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 800 mg. If administered at a dose of 7 weeks, then the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 700 mg. If administered at a dose of 6 weeks, then a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 600 mg. If administered at a dose of 5 weeks, then a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 500 mg. If administered at a dose of 4 weeks, the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 400 mg. If administered at a dose of 3 weeks, the PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 300 mg. If administered at a dose of 2 weeks, a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 200 mg. If administered at a dose of 1 week, a PD-1 inhibitor (e.g., swabs bead mab or tirelibead mab) can be administered at 100 mg.

For example, if an anti-PD-1 antibody (such as tirelizumab) is used, it can be administered as a 200mg dose by intravenous infusion once every three weeks. Alternatively, tirelimumab can be administered as a dose of 300mg by intravenous infusion once every four weeks. If an anti-PD-1 antibody (such as swabs bevacizumab or tirelimumab) is used, it can be administered as a 300mg dose by intravenous infusion once every three weeks. Alternatively, the swabs bead mab or tirelimumab can be administered at a dose of 400mg by intravenous infusion once every four weeks.

In some embodiments, a PD-1 inhibitor (e.g., swabber or tirelimumab) is administered in combination with a tgfβ inhibitor (e.g., NIS 793).

TABLE 1 amino acid and nucleotide sequences of exemplary anti-PD-1 antibody molecules

Tirelimumab is a humanized IgG monoclonal antibody against human programmed cell death receptor 1 (PD-1), approved by the chinese food and drug administration (China's Food and Drug Administration, CFDA) for different indications, including hodgkin's lymphoma, urothelial carcinoma, hepatocellular carcinoma and squamous, non-small cell lung carcinoma, and BLA against Esophageal Squamous Cell Carcinoma (ESCC) is accepted by the FDA.

In particular embodiments, tirelimumab may be administered in combination with a tgfβ antibody (e.g., NIS 793) and/or with any of the chemotherapeutic agents disclosed. The amino acid sequences of tirelimumab are disclosed above and include SEQ ID NOS 321 to 330.

In some embodiments, tirelimumab is administered to a patient (in need thereof) at a dose disclosed throughout. For example, tirelimumab can be administered in a smooth dose of about 200mg to 400 mg. More particularly, in these particular embodiments, tirelimumab can be administered to a patient at a dose of 300 mg.

When tirelimumab is used, the frequency of administration may be as described throughout. For example, a frequency of once every three weeks (Q3W) or once every four weeks (Q4W) may be used.

In one particular example, tirelimumab can be administered to a subject at a dose of 300mg once every four weeks. In another specific example, tirelimumab can be administered to a subject at a dose of 200mg once every three weeks.

Other exemplary PD-1 inhibitors

In one embodiment, the anti-PD-1 antibody molecule is Nawuzumab (BASEMERIUM Guibao), also known as MDX-1106, MDX-1106-04, ONO-4538, BMS-936558, orNivolumab (clone 5C 4) and other anti-PD-1 antibodies are disclosed in US 8,008,449 and WO 2006/121168. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of nivolumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 2.

In one embodiment, the anti-PD-1 antibody molecule is pembrolizumab (Merck, inc&Co)), also known as Lambrolizumab, MK-3475, MK03475, SCH-900475,Or (b)Pembrolizumab and other anti-PD-1 antibodies are disclosed in Hamid, o.et al (2013) New England Journal of Medicine [ journal of new england medicine ]]369 (2) 134-44, US 8,354,509, and WO 2009/114335. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of pembrolizumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 2.

In one embodiment, the anti-PD-1 antibody molecule is Pidazumab (Pidazuku Co., ltd.), also known as CT-011. Pittuzumab and other anti-PD-1 antibodies are disclosed in Rosenblatt, J.et al, (2011) J Immunotherapy [ J.Immunotherapy ]34 (5): 409-18; US 7,695,715; US 7,332,582; and US 8,686,119. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of pilzumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 2.

In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (England Mei Dimiao, inc.), also known as AMP-514.MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or overall all CDR sequences) of MEDI0680, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (regenerator corporation). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or overall all CDR sequences) of REGN2810, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (pyro). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of PF-06801591, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 or BGB-108 (BAIJISHENZHOUS Co.). In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of BGB-a317 or BGB-108, the heavy or light chain variable region sequences, or the heavy or light chain sequences.

In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Saint Co.), also known as INCSHR01210 or SHR-1210. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of incsshr 1210, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tassa Luo Gongsi), also known as ANB011. In one embodiment, the anti-PD-1 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of TSR-042, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

Other known anti-PD-1 antibodies include those described, for example, in the following: WO 2015/112800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209404, WO 2015/200119, US 8,735,553, US 7,488,802, US 8,927,697, US 8,993,731, and US 9,102,727.

In one embodiment, the anti-PD-1 antibody is an antibody that competes with one of the anti-PD-1 antibodies described herein for binding to the same epitope on PD-1 and/or binds to the same epitope on PD-1.

In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, e.g., as described in us 8,907,053. In one embodiment, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., fc region of an immunoglobulin sequence)). In one embodiment, the PD-1 inhibitor is AMP-224 (B7-DCIg (An Puli company), e.g., as disclosed in WO 2010/027827 and WO 2011/066342).

TABLE 2 amino acid sequences of other exemplary anti-PD-1 antibody molecules

PD-L1 inhibitors

In certain embodiments, the described methods further comprise administering a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is FAZ053 (North Co., ltd.), abtizumab (Atezolizumab) (Gentek/Roche), ablumab (Avelumab) (Merck Serono and pyroxene), devalumab (Durvaluab) (England Mei Dimiao Si Co., aspraZeneca), or BMS-936559 (BAIMEISHIGuinobody).

Exemplary PD-L1 inhibitors

In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule as disclosed in U.S. 2016/0108123, publication No. 21/4 of 2016 entitled "Antibody Molecules to PD-L1 and Users therapy of [ antibody molecules to PD-L1 and Uses Thereof ]".

In one embodiment, the anti-PD-L1 antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising an amino acid sequence set forth in table 3 (e.g., a heavy and light chain variable region sequence from BAP 058-clone O, or BAP 058-clone N, as disclosed in table 3), or an amino acid sequence encoded by a nucleotide sequence set forth in table 3. In some embodiments, the CDRs are according to the cabazite definition (e.g., as set forth in table 3). In some embodiments, CDRs are defined according to Qiao Xiya (e.g., as listed in table 3). In some embodiments, the CDRs are defined according to the combined CDRs of both cabazite and Qiao Xiya (e.g., as listed in table 3). In one embodiment, the combination of the carboplatin and Qiao Xiya CDRs of VH CDR1 comprises amino acid sequence GYTFTSYWMY (SEQ ID NO: 100). In one embodiment, one or more of the CDRs (or generally all CDRs) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 3, or the amino acid sequences encoded by the nucleotide sequences shown in table 3.

In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO:54, the VH CDR2 amino acid sequence of SEQ ID NO:55, and the VH CDR3 amino acid sequence of SEQ ID NO: 56; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:62, the VLCDR2 amino acid sequence of SEQ ID NO:63, and the VLCDR3 amino acid sequence of SEQ ID NO:64, each as disclosed in Table 3.

In one embodiment, the anti-PD-L1 antibody molecule comprises: a VH comprising a VH CDR1 encoded by the nucleotide sequence of SEQ ID NO. 81, a VH CDR2 encoded by the nucleotide sequence of SEQ ID NO. 82, and a VH CDR3 encoded by the nucleotide sequence of SEQ ID NO. 83; and VL comprising VLCDR1 encoded by the nucleotide sequence of SEQ ID NO:86, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO:87, and VLCDR3 encoded by the nucleotide sequence of SEQ ID NO:88, are each disclosed in Table 3.

In one embodiment, the anti-PD-L1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 59, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 59. In one embodiment, the anti-PD-L1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 69, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 69. In one embodiment, the anti-PD-L1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 73, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 73. In one embodiment, the anti-PD-L1 antibody molecule comprises: VL comprising the amino acid sequence of SEQ ID No. 77, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 77. In one embodiment, the anti-PD-L1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 59 and VL comprising the amino acid sequence of SEQ ID No. 69. In one embodiment, the anti-PD-L1 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 73 and VL comprising the amino acid sequence of SEQ ID No. 77.

In one embodiment, the antibody molecule comprises: VH encoded by the nucleotide sequence of SEQ ID No. 60, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 60. In one embodiment, the antibody molecule comprises: VL encoded by the nucleotide sequence of SEQ ID No. 70, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 70. In one embodiment, the antibody molecule comprises: VH encoded by the nucleotide sequence of SEQ ID No. 74, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 74. In one embodiment, the antibody molecule comprises: VL encoded by the nucleotide sequence of SEQ ID No. 78, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 78. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO. 60 and a VL encoded by the nucleotide sequence of SEQ ID NO. 70. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO:72 and a VL encoded by the nucleotide sequence of SEQ ID NO: 78.

In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID No. 61, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 61. In one embodiment, the anti-PD-L1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID No. 71, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 71. In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID No. 75, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 75. In one embodiment, the anti-PD-L1 antibody molecule comprises: a light chain comprising the amino acid sequence of SEQ ID No. 79, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 79. In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 61 and a light chain comprising the amino acid sequence of SEQ ID NO. 71. In one embodiment, the anti-PD-L1 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 75 and a light chain comprising the amino acid sequence of SEQ ID NO. 79.

In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 68, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID NO. 68. In one embodiment, the antibody molecule comprises: a light chain encoded by the nucleotide sequence of SEQ ID No. 72, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 72. In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 76, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID NO. 76. In one embodiment, the antibody molecule comprises: a light chain encoded by the nucleotide sequence of SEQ ID No. 80, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to SEQ ID No. 80. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 68 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 72. In one embodiment, the antibody molecule comprises: a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 76 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 80.

The antibody molecules described herein may be produced by vectors, host cells, and methods described in US 2016/0108123.

TABLE 3 amino acid and nucleotide sequences of exemplary anti-PD-L1 antibody molecules

Other exemplary PD-L1 inhibitors

In one embodiment, the anti-PD-L1 antibody molecule is alemtuzumab (GeneTek/Roche), also known as MPDL3280A, RG7446, RO5541267, YW243.55.S70, or TECENTRIQ^TM . Alemtuzumab and other anti-PD-L1 antibodies are disclosed in US 8,217,149. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of alemtuzumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 4.

In one embodiment, the anti-PD-L1 antibody molecule is avermectin (mercaptitude and febrile), also known as MSB0010718C. Avermectin and other anti-PD-L1 antibodies are disclosed in WO 2013/079174. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of avermectin, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 4.

In one embodiment, the anti-PD-L1 antibody molecule is dewaruzumab (imperial Mei Dimiao s/aliskiren), also known as MEDI4736. Dewaruzumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of dewaruzumab, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 4.

In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (BASEMERIUM CURRENT), also known as MDX-1105 or 12A4.BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081158. In one embodiment, the anti-PD-L1 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of BMS-936559, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 4.

Other known anti-PD-L1 antibodies include those described, for example, in the following: WO 2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, WO 2015/112805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927, and US 9,175,082.

In one embodiment, the anti-PD-L1 antibody is an antibody that competes with one of the anti-PD-L1 antibodies described herein for binding to the same epitope on PD-L1 and/or binding to the same epitope on PD-L1.

TABLE 4 amino acid sequences of other exemplary anti-PD-L1 antibody molecules

Antibodies and antibody-like molecules

As used herein, the term "antibody molecule" refers to a protein, such as an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes, for example, monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region). In embodiments, the antibody molecule comprises a full length antibody or a full length immunoglobulin chain. In embodiments, the antibody molecule comprises an antigen binding or functional fragment of a full length antibody or full length immunoglobulin chain. In embodiments, the antibody molecule is a multi-specific antibody molecule, e.g., comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope (e.g., a first target) and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope (e.g., a second target). In embodiments, the multispecific antibody molecule is a bispecific antibody molecule.

In embodiments, the antibody molecule is a monospecific antibody molecule and binds to a single epitope (e.g., a single target (e.g., tgfβ, like NIS 793)). For example, a monospecific antibody molecule may have multiple immunoglobulin variable domain sequences, each binding the same epitope.

In embodiments, the antibody molecule is a multi-specific antibody molecule, e.g., comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope (e.g., a first target) and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope (e.g., a second target). In embodiments, the first and second epitopes are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In embodiments, the first epitope and the second epitope overlap. In embodiments, the first epitope and the second epitope do not overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In embodiments, the first epitope and the second epitope overlap. In embodiments, the first epitope and the second epitope do not overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In an embodiment, the bispecific antibody molecule comprises a half-antibody having binding specificity for a first epitope and a half-antibody having binding specificity for a second epitope. In embodiments, the bispecific antibody molecule comprises a half-antibody, or fragment thereof, having binding specificity for a first epitope and a half-antibody, or fragment thereof, having binding specificity for a second epitope. In embodiments, the bispecific antibody molecule comprises an scFv, or fragment thereof, having binding specificity for a first epitope and an scFv, or fragment thereof, having binding specificity for a second epitope. In embodiments, the first epitope is located on tgfβ (1, 2, and/or 3) and the second epitope is located on PD-1 (or PD-L1, or PD-L2).

Protocols for generating multi-specific (e.g., bispecific or trispecific) or heterodimeric antibody molecules are known in the art; these schemes include, but are not limited to: the "knob in a hole" pathway, such as described in U.S. Pat. No. 5,731,168; electrostatically directed Fc pairing as described, for example, in WO 09/089004, WO 06/106905 and WO 2010/129304; chain exchange engineering domain (SEED) heterodimer formation as described, for example, in WO 07/110205; fab arm exchange as described for example in WO 08/119353, WO 2011/131746 and WO 2013/060867; diabody conjugates, for example using heterobifunctional reagents having amine-reactive groups and thiol-reactive groups, are cross-linked by antibodies to produce bispecific structures as described, for example, in US 4,433,059; bispecific antibody determinants produced by recombination of half antibodies (heavy-light chain pairs or Fab) from different antibodies by cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described for example in US 4,444,878; trifunctional antibodies, for example three Fab' fragments crosslinked by thiol-reactive groups, as described for example in US 5,273,743; biosynthesis of binding proteins, e.g. scFv pairs crosslinked by a C-terminal tail, preferably by disulfide or amine reactive chemical crosslinking, as described for example in US 5,534,254; bifunctional antibodies, e.g. Fab fragments with different binding specificities, dimerized by leucine zippers (e.g. c-fos and c-jun) that have replaced constant domains, as described for example in US 5,582,996; bispecific and oligospecific monovalent and oligovalent receptors, for example VH-CH1 regions of two antibodies (two Fab fragments), which VH-CH1 regions are linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody (typically with an associated light chain), as described for example in US 5,591,828; bispecific DNA-antibody conjugates, e.g. antibodies or Fab fragments, are cross-linked by double-stranded segments of DNA, as described, for example, in US 5,635,602; bispecific fusion proteins, for example expression constructs comprising two scFv (with a hydrophilic helical peptide linker between them) and one fully constant region, as described for example in US 5,637,481; multivalent and multispecific binding proteins, such as polypeptide dimers having a first domain of an Ig heavy chain variable region binding region and a second domain of an Ig light chain variable region binding region, are commonly referred to as diabodies (also disclosing higher order structures, resulting in bispecific, trispecific or tetraspecific molecules) as described, for example, in US 5,837,242; miniantibody constructs having linked VL and VH chains (which are further linked to an antibody hinge region and a CH3 region with a peptide spacer), which can dimerise to form bispecific/multivalent molecules, as described for example in US 5,837,821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or not linked at all at any orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described for example in US 5,844,094; a string of VH domains (or VL domains in family members) linked by peptide bonds to C-terminal crosslinkable groups which are further associated with the VL domains to form a series of FV (or scFv) as described, for example, in US 5,864,019; and single chain binding polypeptides having both VH and VL domains linked via peptide linkers are combined into multivalent structures by non-covalent or chemical cross-linking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures using scFv or diabody type formats, as described, for example, in US 5,869,620. Additional exemplary multispecific and bispecific molecules and methods of making the same are found, for example, US 5,910,573, US 5,932,448, US 5,959,083, US 5,989,830, US 6,005,079, US 6,239,259, US 6,294,353, US 6,333,396, US 6,476,198, US 6,511,663, US 6,670,453, US 6,743,896, US 6,809,185, US 6,833,441, US7,129,330, US7,183,076, US7,521,056, US7,527,787, US7,534,866, US7,612,181, US2002/004587A1, US2002/076406A1, US2002/103345A1, US 2003/207346A1, US2003/211078A1, US2004/219643A1, US 2004/220388A1, US 2004/2427A 1, US2005/003403A1, US 2005/352A 1, US2005/069552A1, US2005/079170A1, 2005/136049A1, US 2005/051A 1, US 2005/004587A 1, US 2006/2006A 1, US 2003/2110743A 1, US 2006/2006A 1, US 2006/2006A, US 0867A 1, US 2006/0881A 1, US 2006/0835A 1. US 2007/128150A1, US 2007/141049A1, US 2007/154901A1, US 2007/274985A1, US 2008/050370A1, US 2008/069820A1, US 2008/152645A1, US 2008/171855A1, US 2008/241884A1, US 2008/254512A1, US 2008/260738A1, US 2009/130106A1, US 2009/148905A1, US 2009/155275A1, US 2009/162359A1, US 2009/162360A1, US 2009/175851A1, US 2009/175867A1, US 2009/232811A1, US 2009/23410353 A1, US 2009/263392A1, US 2009/274649A1, EP 346087A2, WO 00/06605A2, WO 02/072635A2, WO 2008/051 A1, WO 06/020258A2, WO 2009/0487 A2, WO 2009/14838 A2, WO 2009/2007 A2, WO 2009/06353 A1, WO 2007/2007 A2, WO 2007/06353 A1 WO 91/03493A1, WO 93/23537A1, WO 94/09131A1, WO 94/12625A2, WO 95/09917A1, WO 96/37621A2, WO 99/64460A1.

"fusion protein" and "fusion polypeptide" refer to polypeptides having at least two moieties covalently linked together, wherein each moiety is a polypeptide having different properties. The property may be a biological property, such as in vitro or in vivo activity. The property may also be a simple chemical or physical property, such as binding to a target molecule, catalysis of a reaction, etc. The two moieties may be directly linked by a single peptide bond or by a peptide linker, but in frame with each other.

In embodiments, antibody molecules include diabodies, and single chain molecules, as well as antigen binding fragments of antibodies (e.g., fab, F (ab')₂ And Fv). For example, an antibody molecule may comprise a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In embodiments, the antibody molecule comprises or consists of heavy and light chains (referred to herein as half antibodies). In another example, an antibody molecule, such as Fab, fab ', F (ab'), comprises two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen binding sites₂ Fc, fd', fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by modification of an intact antibody or may be synthesized de novo using recombinant DNA techniques. These functional antibody fragments retain the ability to selectively bind to their respective antigens or receptors. Antibodies and antibody fragments may be from any class of antibodies, including but not limited to IgG, igA, igM, igD and IgE, as well as from any subclass of antibodies (e.g., igG1, igG2, igG3, and IgG 4). The preparation of antibody molecules may be monoclonal or polyclonal. The antibody molecule may also be a human, humanized, CDR-grafted or in vitro generated antibody. Antibodies may have heavy chain constant regions such as IgG1, igG2, igG3, or IgG 4. Antibodies may also have light chains such as kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody".

Examples of antigen binding fragments of antibody molecules include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, which is a bivalent fragment comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of an antibody, (v) diabody antibody (dAb) fragments consisting of VH domains; (vi) Camelidae (camelid) or camelized (camelized) variable domains; (vii) Single chain Fv (scFv) (see, e.g., bird et al (1988) Science [ Science ]242:423-426; and Huston et al (1988) Proc.Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ] 85:5879-5883); (viii) single domain antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for efficacy in the same manner as whole antibodies.

The term "antibody" includes intact molecules as well as functional fragments thereof. The constant region of an antibody can be altered (e.g., mutated) to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function).

The antibody molecule may also be a single domain antibody. Single domain antibodies may include antibodies whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies that naturally lack a light chain, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. The single domain antibody may be any of the antibodies described in the art, or any future single domain antibody. The single domain antibody may be derived from any species including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. According to another aspect of the invention, the single domain antibody is a naturally occurring single domain antibody, referred to as a heavy chain antibody lacking a light chain. Such single domain antibodies are disclosed, for example, in WO 94/04678. For clarity reasons, such variable domains derived from heavy chain antibodies that naturally lack light chains are referred to herein as VHHs or nanobodies to distinguish them from conventional VH's of four-chain immunoglobulins. Such VHH molecules may be derived from camelidae species, such as camels, llamas, dromedaries, alpacas and antibodies raised in dromedaries. Other species than camelidae can produce heavy chain antibodies that naturally lack light chains; such VHHs are within the scope of the invention.

VH and VL regions can be subdivided into regions of higher variability termed "complementarity determining regions" (CDRs) with more conserved regions termed "framework regions" (FR or FW) interposed therebetween.

The framework regions and CDR ranges have been precisely defined by a number of methods (see Kabat, E.A. et al (1991) Sequences of Proteins of Immunological Interest [ protein sequences of immunological interest ], 5 th edition, U.S. device of Health and Human Services [ U.S. health and public service ], NIH publication No. 91-3242; chothia, C. et al (1987) J.mol. Biol. [ journal of Molecular biology ]196:901-917; and AbM definitions used by Oxford Molecular AbM antibody modeling software (Oxford Molecular's AbM antibody modeling software). See, e.g., protein Sequence and Structure Analysis of Antibody Variable Domains [ protein sequence and structural analysis of antibody variable domains ] the literature is described in: antibody Engineering Lab Manual [ handbook of antibody engineering laboratories ] (editions: duebel, S. And Kontermann, R., springer-Verlag, sea Derburg).

As used herein, the terms "complementarity determining regions" and "CDRs" refer to amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity. Typically, there are three CDRs (HCDR 1, HCDR2 and HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2 and LCDR 3) in each light chain variable region.

The exact amino acid sequence boundaries for a given CDR may be determined using any of a number of well-known schemes, including those described below: kabat et Al (1991), "Sequences of Proteins of Immunological Interest [ immunological protein sequence of interest ]", 5 th edition, public health agency (Public Health Service), national institutes of health (National Institutes of Health), bethesda, md. (Kabat numbering scheme), al-Lazikani et Al, (1997) JMB 273,927-948 (Qiao Xiya "numbering scheme). As used herein, CDRs defined according to the "Qiao Xiya" numbering scheme are sometimes also referred to as "hypervariable loops".

For example, according to cabazite, CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3). According to Qiao Xiya, the CDR amino acids in the VH are numbered 26-32 (HCDR 1), 52-56 (HCDR 2) and 95-102 (HCDR 3); and amino acid residues in VL are numbered 26-32 (LCDR 1), 50-52 (LCDR 2) and 91-96 (LCDR 3). By definition with CDRs binding to cabazite and Qiao Xiya, the CDRs consist of amino acid residues 26-35 (HCDR 1), 50-65 (HCDR 2) and 95-102 (HCDR 3) in human VH and amino acid residues 24-34 (LCDR 1), 50-56 (LCDR 2) and 89-97 (LCDR 3) in human VL.

Generally, unless specifically indicated, an antibody molecule may include any combination of one or more kappa CDRs and/or jotivia hypervariable loops. In one embodiment, the following definitions are used for the antibody molecules described in table 1: HCDR1, according to the combined CDR definition of both cabazite and Qiao Xiya, and HCCDR 2-3 and LCCDR 1-3, according to the CDR definition of cabazite. Each VH and VL typically comprises three CDRs and four FRs, according to all definitions, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

As used herein, "immunoglobulin variable domain sequence" refers to an amino acid sequence that can form the structure of an immunoglobulin variable domain. For example, the sequence may include all or part of the amino acid sequence of a naturally occurring variable domain. For example, the sequence may or may not include one, two or more N-or C-terminal amino acids, or may include other changes that are compatible with the formation of protein structures.

The term "antigen binding site" refers to a portion of an antibody molecule that comprises determinants that form an interface with a target (e.g., tgfβ) or epitope thereof. With respect to proteins (or protein mimics), an antigen binding site typically includes one or more loops (having at least four amino acids or amino acid mimics) that form an interface with a target polypeptide. Typically, the antigen binding site of an antibody molecule comprises at least one or two CDRs and/or hypervariable loops, or more typically at least three, four, five or six CDRs and/or hypervariable loops.

The terms "compete" or "cross-compete" are used interchangeably herein to refer to the ability of an antibody molecule to interfere with the binding of another antibody molecule (e.g., an anti-tgfβ antibody molecule provided herein) to a target (e.g., tgfβ1, 2, or 3). The interference with binding may be direct or indirect (e.g., through allosteric modulation of the antibody molecule or target). The extent to which an antibody molecule can interfere with the binding of another antibody molecule to a target and thus whether it can be referred to as competition can be determined using competition binding assays (e.g., FACS assays, ELISA or BIACORE assays). In some embodiments, the competitive binding assay is a quantitative competitive assay. In some embodiments, a first anti-tgfβ antibody molecule is said to compete with a second anti-tgfβ antibody molecule for binding to a target when binding of the first antibody molecule to the target is reduced by 10% or more, e.g., 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, in a competitive binding assay (e.g., a competitive assay described herein).

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecule composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Monoclonal antibodies can be prepared by hybridoma technology or methods that do not use hybridoma technology (e.g., recombinant methods).

An "effective human" protein is one that does not elicit a neutralizing antibody response, such as a human anti-mouse antibody (HAMA) response. In many cases, for example, HAMA can be problematic if antibody molecules are repeatedly administered, for example in the treatment of chronic or recurrent disease conditions. The HAMA response may render repeated antibody administration potentially ineffective due to increased antibody clearance in serum (see, e.g., saleh et al, cancer immunol. 32:180-190 (1990)), and also due to potential allergic reactions (see, e.g., loBuglio et al, hybridomas, 5:5117-5123 (1986)).

The antibody molecules described throughout may be polyclonal or monoclonal antibodies. In other embodiments, the antibodies may be recombinantly produced, such as by phage display or by combinatorial methods.

Phage display and combinatorial methods for producing antibodies are known in the art (as described in, for example: ladner et al, U.S. Pat. No. 5,223,409; kang et al, international publication No. WO 92/18619; dower et al, international publication No. WO 91/17271; winter et al, international publication No. WO 92/20791; markland et al, international publication No. WO 92/15679; breitling et al, international publication No. WO 93/01188; mcCafferty et al, international publication No. WO 92/01047; gargarard et al, international publication No. WO 92/09690; ladner et al, international publication No. WO 90/02 809; fuchs et al, (1991) Bio/Technology [ biological/Technology ]9:1370-1372; hay et al, (1992) Hum Antibody Hybridomas [ human antibody hybridoma ]3:81-85; huse et al, (1989) Science [ 246:5-1281 ]; grifmths et al, (1993) EMJ [ European molecular biology ] 12:12735:734:35:35:35:35:35:35:35; grfBull ] 6:35:35:35:35 (1991) nucleic Acid [ 1991 ] nucleic Acid journal 6:35:35:35:35:37 ] nucleic Acid (1991); barbas et al (1991) PNAS (Proc. Natl. Acad. Sci. USA) 88:7978-7982).

In one embodiment, the antibody is a fully human antibody (e.g., an antibody prepared in a mouse that has been genetically engineered to produce antibodies from human immunoglobulin sequences), or a non-human antibody, e.g., a rodent (mouse or rat), goat, primate (e.g., monkey), camel antibody. Preferably, the non-human antibody is a rodent (mouse or rat) antibody. Methods for producing rodent antibodies are known.

Transgenic mice carrying human immunoglobulin genes that are not mouse systems can be used to produce human monoclonal antibodies. Spleen cells from these transgenic mice immunized with the antigen of interest were used to generate hybridomas secreting human mAbs that have specific affinity for epitopes from human proteins (see, e.g., wood et al, international application WO 91/00906; kucherlapati et al, PCT publication WO 91/10741; lonberg et al, international application WO 92/03918; kay et al, international application 92/03917; lonberg, N. Et al, 1994Nature [ Nature ]368:856-859; green, L. Et al, 1994Nature Genet. [ Nature ]7:13-21; morrison, S.L. Et al, 1994Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. 81:6851-6855; bruggeman et al, 1993Year mu.Immunol [ Immunol ]7:33-40; tullon et al, 1994Nature [ Brug. Natl. Gen. Sci., 1993:3720; eur. Immunol, 1993:3724, J.1326:1326).

An antibody may be an antibody molecule whose variable regions or portions thereof (e.g., CDRs) are produced in a non-human organism (e.g., a rat or mouse). Chimeric antibodies, CDR-grafted antibodies, and humanized antibodies are within the scope of the invention. Antibodies produced in a non-human organism (e.g., a rat or mouse) and then modified in, for example, a variable framework or constant region to reduce antigenicity in humans are within the present invention.

Chimeric antibodies can be produced by recombinant DNA techniques known in the art (see: robinson et al, international patent publication PCT/US86/02269; akira et al, european patent application 184,187; taniguchi, M., european patent application 171,496; morrison et al, european patent application 173,494; neuberger et al, international application WO 86/01533; caplly et al, U.S. Pat. No. 4,816,567; capilli et al, european patent application 125,023; better et al (1988 Science [ Science ] 240:1041-1043); liu et al (1987) PNAS 84:3439-3443; liu et al, 1987, J. Immunol [ J.139:3521-3526; sun et al (1987) PNAS 84:214-218; niubera et al, 1987, canc. Res. 999 ] 47:314; WO-3880; nature, U.S. Pat. No. 4:446; nature, 1987:1559; nature, J.S. 4:3880).

At least one or two but typically all three acceptor CDRs (of the heavy and/or light immunoglobulin chains) of the humanized antibody or CDR-grafted antibody are replaced by donor CDRs. An antibody may be replaced with at least a portion of a non-human CDR, or only some CDRs may be replaced with non-human CDRs. Only the number of CDRs required for the humanized antibody to bind to its target (e.g., tgfβ) need be replaced. Preferably, the donor is a rodent antibody, such as a rat or mouse antibody, and the recipient will be a human framework or a human consensus framework. Typically, an immunoglobulin providing CDRs is referred to as a "donor" and an immunoglobulin providing framework is referred to as an "acceptor". In one embodiment, the donor immunoglobulin is non-human (e.g., rodent). The acceptor framework is a naturally occurring (e.g., human) framework or a consensus framework, or a sequence having about 85% or more, preferably 90%, 95%, 99% or more identity thereto.

As used herein, the term "consensus sequence" refers to a sequence formed by the most frequently occurring amino acids (or nucleotides) in the related sequence family (see, e.g., winnaker, from Genes to Clones [ from gene to clone ] (german Wei Yinhai m press (Verlagsgesellschaft, weinheim, germany) 1987)). In a family of proteins, each position in the consensus sequence is occupied by the amino acid most frequently present at that position in the family. If both amino acids occur equally frequently, either may be included in the consensus sequence. "consensus framework" refers to a framework region in a consensus immunoglobulin sequence.

Antibodies can be humanized by methods known in the art (see, e.g., morrison, S.L.,1985, science [ science ]229:1202-1207, oi et al, 1986, bioTechniques [ biotechnology ]4:214, and Queen et al, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761, and U.S. Pat. No. 5,693,762).

Humanized antibodies or CDR-grafted antibodies may be produced by CDR grafting or CDR substitution, wherein one, two or all of the CDRs of an immunoglobulin chain may be replaced. See, for example, U.S. Pat. nos. 5,225,539; jones et al 1986Nature 321:552-525; verhoeye et al 1988Science [ Science ]239:1534; beidler et al 1988J.Immunol. [ J.Immunol. ]141:4053-4060; winter US 5,225,539.Winter describes a CDR grafting method which can be used to prepare the humanized antibodies of the invention (uk patent application GB 2188638A;Winter US 5,225,539 filed on month 3, 26, 1987).

Humanized antibodies are also within the scope of the invention, wherein specific amino acids have been substituted, deleted or added. Criteria for selection of amino acids from donors are described in U.S. Pat. No. 5,585,089, e.g. in columns 12-16 of U.S. Pat. No. 5,585,089. Other techniques for humanizing antibodies are described in Padlan et al, EP519596A1, published at 12/23 1992.

The antibody molecule may be a single chain antibody. Single chain antibodies (scFv) can be engineered (see, e.g., colcher, D.et al (1999) Ann N Y Acad Sci [ New York academy of sciences annual. 880:263-80; and Reiter, Y. (1996) Clin Cancer Res [ clinical Cancer research ] 2:245-52). Single chain antibodies can be dimerized or multimerized to produce multivalent antibodies specific for different epitopes of the same target protein.

In yet other embodiments, the antibody molecule has a heavy chain constant region, such as the heavy chain constant region of IgG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE; in particular, the (human) heavy chain constant regions, e.g., igG1, igG2, igG3 and IgG 4. In another embodiment, the antibody molecule has a light chain constant region, such as a kappa or lambda (human) light chain constant region. The constant region can be altered (e.g., mutated) to modify the properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, and/or complement function). In one embodiment, the antibody has: effector function; and complement can be immobilized. In other embodiments, the antibody does not: recruiting effector cells; or fix complement. In another embodiment, the antibody has a reduced or no ability to bind to an Fc receptor. For example, it is an isoform or subtype, fragment or other mutant that does not support binding to Fc receptors, e.g., it has a mutagenized or deleted Fc receptor binding region.

Methods for altering the constant regions of antibodies are known in the art. Antibodies with altered function (e.g., altered affinity for effector ligands (e.g., fcrs on cells) or C1 components of complement) can be produced by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260). Similar types of changes can be described which, if applied to immunoglobulins of murine or other species, would reduce or eliminate these functions.

The antibody molecule may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody molecule is an antibody molecule that has been modified. Derivatization methods include, but are not limited to, addition of fluorescent moieties, radionucleotides, toxins, enzymes or affinity ligands such as biotin. Thus, the antibody molecules of the invention are intended to include derivatized forms and other modified forms of the antibodies described herein, including immunoadhesion molecules. For example, an antibody molecule may be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody antibody), a detectable agent, a cytotoxic agent, an agent, and/or a protein or peptide that may mediate the association of an antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody molecule is produced by cross-linking two or more antibodies (of the same type or different types, e.g., to produce bispecific antibodies). Suitable cross-linking agents include those that are heterobifunctional (having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from pierce chemical company (Pierce Chemical Company, rockford, ill) of rocford, il.

Useful detectable agents that can be derivatized (or labeled) with the antibody molecules of the invention include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, fluorescent emitting metal atoms (e.g., europium (Eu)) and other lanthanides (described below). Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. Antibodies may also be derivatized with detectable enzymes such as alkaline phosphatase, horseradish peroxidase, beta-galactosidase, acetylcholinesterase, glucose oxidase, and the like. Where the antibody is derived with a detectable enzyme, detection may be performed by adding other reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine results in a detectable colored reaction product. Antibody molecules may also be derivatized with prosthetic groups (e.g., streptavidin/biotin and avidin/biotin). For example, antibodies can be derivatized with biotin and detected by indirect measurement of avidin or streptavidin binding. Examples of suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of the light emitting material include luminol (luminol); and examples of bioluminescent materials include luciferase, luciferin and aequorin.

The labeled antibody molecules may be used in a variety of contexts, for example in diagnostics and/or experiments, including (i) isolation of a predetermined antigen by standard techniques such as affinity chromatography or immunoprecipitation; (ii) Detecting a predetermined antigen (e.g., in a cell lysate or cell supernatant) to assess the abundance and expression pattern of the protein; (iii) As part of a clinical test procedure, protein levels in tissue are monitored, for example, to determine the efficacy of a given treatment regimen.

The antibody molecule may be conjugated to another molecular entity (typically a label or therapeutic agent (e.g., a cytotoxic or cytostatic agent) or moiety). Radioisotopes may be used in diagnostic or therapeutic applications.

The present invention provides radiolabeled antibody molecules and methods of labeling the same. In one embodiment, a method of labeling an antibody molecule is disclosed. The method comprises contacting an antibody molecule with a chelator, thereby producing a conjugated antibody.

As described above, the antibody molecule may be conjugated to a therapeutic agent. Therapeutically active radioisotopes have been mentioned. Examples of other therapeutic agents include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthrax, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids such as maytansinol (see, e.g., U.S. Pat. No. 5,208,020), CC-1065 (see, e.g., U.S. Pat. No. 5,475,092, 5,585,499, 5,846,545), and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil amamide), alkylating agents (e.g., nitrogen mustard, chlorambucil, CC-1065, melphalan, carmustine (BSNU) and lomustine (CCNU)), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiammineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunorubicin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin and an Angustaine (AMC)), and antimitotics (e.g., vincristine, vinblastine, paclitaxel and maytansinoids).

In one aspect, the disclosure provides a method of providing a target binding molecule that specifically binds to a target throughout the disclosure. For example, the target binding molecule is an antibody molecule. The method comprises the following steps: providing a target protein comprising at least a portion of a non-human protein that is homologous (at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a corresponding portion of a human target protein, but differs by at least one amino acid (e.g., at least one, two, three, four, five, six, seven, eight, or nine amino acids); obtaining an antigen-binding antibody molecule; and evaluating the efficacy of the binding agent in modulating the activity of the target protein. The method may further comprise administering a binding agent (e.g., an antibody molecule) or derivative (e.g., a humanized antibody molecule) to the human subject.

The present disclosure provides isolated nucleic acid molecules (i.e., polynucleotides) encoding any of the antibody molecules described throughout. Vectors comprising the nucleic acid molecules and host cells thereof are also disclosed. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

Combination of two or more kinds of materials

The methods of treatment described herein may include the administration of two or more other therapeutic agents, procedures, or modes in combination.

In some embodiments, a tgfβ inhibitor (e.g., NIS 793) is administered in combination with a PD1 inhibitor (e.g., an anti-PD 1 antibody molecule). In some embodiments, the tgfβ inhibitor is administered on the same day as the PD1 inhibitor. In some embodiments, the tgfβ inhibitor is administered after administration of the PD1 inhibitor begins. In some embodiments, the tgfβ inhibitor is administered one hour after administration of the PD1 inhibitor is complete.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 1300mg and 1500mg (e.g., about 1400 mg), e.g., once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, e.g., swabber or tirelimumab) is administered at a dose between 300mg and 500mg (e.g., 400 mg), e.g., once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 1300mg and 1500mg (e.g., about 1400 mg), e.g., once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, e.g., swabber or tirelimumab) is administered at a dose between 200mg and 400mg (e.g., 300 mg), e.g., once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 1300mg and 1500mg (e.g., about 1400 mg), e.g., once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, e.g., swabber or tirelimumab) is administered at a dose between 200mg and 400mg (e.g., 300 mg), e.g., once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 1300mg and 1500mg (e.g., about 1400 mg), e.g., once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, e.g., swabber or tirelimumab) is administered at a dose between 100mg and 300mg (e.g., 200 mg), e.g., once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 1300mg and 1500mg (e.g., about 1400 mg), e.g., once every three weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, e.g., swabber or tirelimumab) is administered at a dose between 100mg and 300mg (e.g., 200 mg), e.g., once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose of between 1300mg and 1500mg (e.g., about 1400 mg), for example, once every three weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose of between 200mg and 400mg (e.g., 300 mg), for example, once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), such as once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose between 300mg and 500mg (e.g., 400 mg), such as once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), such as once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose between 200mg and 400mg (e.g., 300 mg), such as once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), e.g., once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose between 200mg and 400mg (e.g., 300 mg), e.g., once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), such as once every two weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose between 100mg and 300mg (e.g., 200 mg), such as once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), such as once every three weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bezumab or tirelimumab) is administered at a dose between 100mg and 300mg (e.g., 200 mg), such as once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose of between 2000mg and 2200mg (e.g., about 2100 mg), for example, once every three weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose of between 300mg to 500mg (e.g., 400 mg), for example, once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), for example, once every three weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose between 200mg and 400mg (e.g., 300 mg), for example, once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose between 2000mg and 2200mg (e.g., about 2100 mg), for example, once every three weeks, and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as swabs bead mab or tirelib bead mab) is administered at a dose between 200mg and 400mg (e.g., 300 mg), for example, once every three weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose of between 2000mg and 2200mg (e.g., about 2100 mg), for example, once every four weeks (e.g., on day 1 of a 28 day cycle), and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, such as tirelizumab) is administered at a dose of between 200mg and 400mg (e.g., 300 mg), for example, once every four weeks.

In some embodiments, the tgfβ inhibitor (e.g., NIS 793) is administered at a dose of between 2000mg and 2200mg (e.g., about 2100 mg), e.g., twice every four weeks (e.g., on days 1 and 15 of a 28 day cycle), and the PD1 inhibitor (e.g., an anti-PD 1 antibody molecule, e.g., tirelimumab) is administered at a dose of between 200mg and 400mg (e.g., 300 mg), e.g., once every four weeks.

In certain embodiments, the methods described herein can be administered in combination with one or more of other therapeutic agents (including antibody molecules, chemotherapeutic agents), other anti-cancer therapies (e.g., targeted anti-cancer therapies, gene therapies, viral therapies, RNA therapy bone marrow transplants, nanotherapies, or oncolytic agents), cytotoxic agents, immune-based therapies (e.g., cytokines or cell-based immunotherapy), surgical procedures (e.g., lumpectomy or lumpectomy), or radiological procedures, or a combination of any of the foregoing. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is an enzyme inhibitor (e.g., a small molecule enzyme inhibitor) or a metastasis inhibitor. Exemplary cytotoxic agents that may be administered in combination include antimicrotubular agents, topoisomerase inhibitors, antimetabolites, mitotic inhibitors, alkylating agents, anthracyclines, vinca alkaloids, intercalators, agents capable of interfering with the signal transduction pathway, agents that promote apoptosis, proteasome inhibitors, and radiation (e.g., localized or systemic radiation (e.g., gamma radiation)). In other embodiments, the additional therapy is surgery or radiation, or a combination thereof. In other embodiments, the additional therapy is a therapy targeting one or more of the PI3K/AKT/mTOR pathway, HSP90 inhibitor, or tubulin inhibitor.

Alternatively, or in combination with the foregoing, the methods described herein may be administered or used with one or more of the following: immunomodulators (e.g., activators of costimulatory molecules or inhibitors of inhibitory molecules, such as immune checkpoint molecules); vaccines, such as therapeutic cancer vaccines; or other forms of cellular immunotherapy.

In certain embodiments, a combination described herein is administered or used with a co-stimulatory molecule or a modulator of an inhibitory molecule (e.g., a co-inhibitory ligand or receptor).

In one embodiment, the combinations described herein are administered or used in combination with inhibitors of the inhibitory (or immune checkpoint) molecules PD-1, PD-L2 and/or tgfβ. In one embodiment, the inhibitor is an antibody or antibody fragment that binds to PD-1, PD-L2, or tgfβ.

For combination therapy, in some embodiments, the tgfβ inhibitor is administered on the same day as the checkpoint inhibitor. In other embodiments, the tgfβ inhibitor is administered prior to completion of administration of the checkpoint inhibitor. In further embodiments, the tgfβ inhibitor is administered after the administration of the checkpoint inhibitor is complete. In some embodiments, the tgfβ inhibitor is administered concurrently with the checkpoint inhibitor. In some embodiments, the tgfβ inhibitor is administered until (partially or completely) remission. In some embodiments, the checkpoint inhibitor is administered until (partially or fully) remission.

The compounds of the present disclosure may be administered in a therapeutically effective amount in combination therapy with one or more therapeutic agents (pharmaceutical combinations) or modes (e.g., non-pharmaceutical therapies). For example, other cancer agents may have synergistic effects. When the compounds of the application are administered in combination with other therapies, the dosage of the co-administered compounds will of course vary depending on the type of combination used, the particular drug used, the condition being treated, and the like.

The compounds may be administered simultaneously (as a single formulation or separate formulations) with other drug therapies or treatment modalities, sequentially, separately, or over a period of time. In general, combination therapies contemplate the administration of two or more drugs during a single cycle or course of treatment. A therapeutic agent is, for example, a chemical compound, peptide, antibody fragment, or nucleic acid, which has therapeutic activity or enhances therapeutic activity when administered to a patient in combination with a compound of the disclosure.

In one aspect, the tgfβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) may be combined with other therapeutic agents (e.g., other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetic agents), analgesics, cytoprotective agents, and combinations thereof).

In some embodiments, the tgfβ inhibitor is administered in combination with one or more second agents selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, cytokines, A2A antagonists, GITR agonists, TIM-3 inhibitors, STING agonists, and TLR7 agonists, to treat a disease (e.g., cancer).

In another embodiment, one or more chemotherapeutic agents are used in combination with a TGF-beta inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer), wherein the chemotherapeutic agents include, but are not limited to, anastrozoleBicalutamide->Bleomycin sulfate->Busulfan->Busulfan injection>Capecitabine->N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin +.>Carmustine>ChlorambucilCisplatin->Cladribine>CyclophosphamideOr->) Cytarabine, cytosine arabinoside +.>Cytarabine liposome injection>Dacarbazine->Dactinomycin (actinomycin D, cosmegan), daunorubicin hydrochloride +.>Daunorubicin citrate liposome injectionDexamethasone, docetaxel +.>Doxorubicin hydrochloride) Etoposide->Fludarabine phosphate->5-fluorouracil->Fluotamide->Tizalcitabine, gemcitabine (difluorodeoxycytidine), hydroxyurea >IdarubicinIfosfamide->Irinotecan->L-asparaginaseCalcium folinate, melphalan->6-mercaptopurine->Methotrexate>Mitoxantrone->Jituuzumab (mylotarg), paclitaxelPhoenix (Yttrium 90/MX-DTPA), implants of pravastatin, polifeprosan (polifeprosan) 20 and carmustine->Tamoxifen citrate->Teniposide6-thioguanine, thiotepa, tirapazamine>Topotecan hydrochloride for injectionVinblastine->Vincristine->VinorelbineEpirubicin->Oxaliplatin->ExemestaneLetrozole->And fulvestrant->For example, a tgfβ inhibitor (e.g., NIS 793) may be combined with gemcitabine. In another example, a tgfβ inhibitor (e.g., NIS 793) may be combined with albumin-bound paclitaxel. Tgfβ inhibitors (e.g., NIS 793) may also be combined with both gemcitabine and albumin-bound paclitaxel. In further examples, a tgfβ inhibitor (e.g., NIS 793) may be combined with cyclophosphamide. Tgfβ inhibitors (e.g., NIS 793) may also be combined with topotecan. In some cases, a tgfβ inhibitor (e.g., NIS 793) may be combined with both cyclophosphamide or topotecan. In some cases, a tgfβ inhibitor (e.g., NIS 793) may be combined with 5-fluorouracil, folinic acid (or levofolinic acid), irinotecan, bevacizumab, and optionally tirelimumab. In some cases, a tgfβ inhibitor (e.g., NIS 793) may be combined with 5-fluorouracil, folinic acid (or levofolinic acid), oxaliplatin, bevacizumab, and optionally tirelizumab. In some cases, tgfβ inhibitors (e.g., NIS 793) may be combined with oxaliplatin and capecitabine. In some cases, a tgfβ inhibitor (e.g., NIS 793) may be combined with oxaliplatin, folinic acid (or levofolinic acid), and 5-fluorouracil.

In other embodiments, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are conjugated to one or more other anti-HER 2 antibodies (e.g., trastuzumab, pertuzumab, mactuximab (margetuximab), or HT-19) as described above, or to other anti-HER 2 conjugates (e.g., ado-trastuzumab-maytansine) (also known asOr T-DM 1)) are used in combination.

In other embodiments, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more tyrosine kinase inhibitors (including, but not limited to, EGFR inhibitors, her3 inhibitors, IGFR inhibitors, and Met inhibitors) to treat diseases (e.g., cancer).

For example, tyrosine kinase inhibitors include, but are not limited to, erlotinib hydrochloride (erlotinib)Linifanib (N- [4- (3-amino-1H-indazol-4-yl) phenyl]-N' - (2-fluoro-5-methylphenyl) urea, also known as ABT 869, available from genetec company); sunitinib malate->Bai Shuti Nib (Bosutinib) (4- [ (2, 4-dichloro-5-methoxyphenyl) amino)]-6-methoxy-7- [3- (4-methylpiperazin-1-yl) propoxy]Quinoline-3-carbonitrile, also known as SKI-606, and described in U.S. Pat. No. 6,780,996); dasatinib- >Pazopanib->Sorafenib->Vandetanib (ZD 6474); and imatinib or imatinib mesylate (/ -for)>And->)。

Epidermal Growth Factor Receptor (EGFR) inhibitors include, but are not limited to, erlotinib hydrochloride (erlotinib)Gefitinib>N- [4- [ (3-chloro-4-fluorophenyl) amino group]-7- [ [ (3"S") -tetrahydro-3-furanyl group]Oxy group]-6-quinazolinyl]-4 (dimethylamino) -2-butenamide, -/->) The method comprises the steps of carrying out a first treatment on the surface of the Vandetanib (vanretanib)>Lapatinib->(3R, 4R) -4-amino-1- ((4- ((3-methoxyphenyl) amino) pyrrolo [2, 1-f)][1,2,4]Triazin-5-yl) methyl) piperidin-3-ol (BMS 690514); kanetinib dihydrochloride (CI-1033); 6- [4- [ (4-ethyl-1-piperazinyl) methyl]Phenyl group]-N- [ (1R) -1-phenylethyl]-7H-pyrrolo [2,3-d]Pyrimidine-4-amine (AEE 788, CAS 497839-62-0); xylolitinib (Mubritinib) (TAK 165); ceritinib (EKB 569); afatinib (Afatinib)>Lenatinib (Neratinib) (HKI-272); n- [4- [ [1- [ (3-fluorophenyl) methyl)]-1H-indazol-5-yl]Amino group]-5-methylpyrrolo [2,1-f][1,2,4]Triazin-6-yl]-carbamic acid, (3S) -3-morpholinomethyl ester (BMS 599626); n- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopenta [ c ]]Pyrrol-5-yl]Methoxy group]-4-quinazolinamine (XL 647, CAS 781613-23-8); and 4- [4- [ [ (1R) -1-phenylethyl ]Amino group]-7H-pyrrolo [2,3-d]Pyrimidin-6-yl]Phenol (PKI 166, CAS 187724-61-4).

EGFR antibodies include, but are not limited to, cetuximabPanitumumab->Matuzumab (EMD-72000); nituzumab (Nimotuzumab) (hR 3); zatuzumab (Zalutumumab); theraCIM h-R3; MDX0447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).

Other HER2 inhibitors include, but are not limited to, lenatinib (Neratinib) (HKI-272, (2E) -N- [4- [ [ 3-chloro-4- [ (pyridin-2-yl) methoxy)]Phenyl group]Amino group]-3-cyano-7-ethoxyquinolin-6-yl]-4- (dimethylamino) but-2-enamide and is described in PCT publication No. WO 05/028443); lapatinib or lapatinib xylene sulfonate(3R, 4R) -4-amino-1- ((4- ((3-methoxyphenyl) amino) pyrrolo [2, 1-f)][1,2,4]Triazin-5-yl) methyl) piperidin-3-ol (BMS 690514); (2E) -N- [4- [ (3-chloro-4-fluorophenyl) amino group]-7- [ [ (3S) -tetrahydro-3-furanyl]Oxy group]-6-quinazolinyl]-4- (dimethylamino) -2-butenamide (BIBW-2992, cas 850140-72-6); n- [4- [ [1- [ (3-fluorophenyl) methyl)]-1H-indazol-5-yl]Amino group]-5-methylpyrrolo [2,1-f][1,2,4]Triazin-6-yl]-carbamic acid, (3S) -3-morpholinomethyl ester (BMS 599626,CAS 714971-09-2); kanetinib dihydrochloride (PD 183805 or CI-1033); and N- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopenta [ c ] ]Pyrrol-5-yl]Methoxy group]-4-quinazolinamine (XL 647, CAS 781613-23-8).

HER3 inhibitors include, but are not limited to, LJM716, MM-121, AMG-888, RG7116, REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.

MET inhibitors include, but are not limited to, cabozantinib (Cabozantinib) (XL 184, CAS 849217-68-1); floritinib (Foretinib) (GSK 1363089, formerly XL880, CAS 849217-64-7); tivantinib (ARQ 197, CAS 1000873-98-2); 1- (2-hydroxy-2-methylpropyl) -N- (5- (7-methoxyquinolin-4-yloxy) pyridin-2-yl) -5-methyl-3-oxo-2-phenyl-2, 3-dihydro-1H-pyrazole-4-carboxamide (AMG 458); crizotinib @PF-0234766); (3Z) -5- (2, 3-dihydro-1H-indol-1-yl)Sulfonyl) -3- ({ 3, 5-dimethyl-4- [ (4-methylpiperazin-1-yl) carbonyl]-1H-pyrrol-2-yl } methylene) -1, 3-dihydro-2H-indol-2-one (SU 11271); (3Z) -N- (3-chlorophenyl) -3- ({ 3, 5-dimethyl-4- [ (4-methylpiperazin-1-yl) carbonyl]-1H-pyrrol-2-yl } methylene) -N-methyl-2-oxoindoline-5-sulfonamide (SU 11274); (3Z) -N- (3-chlorophenyl) -3- { [3, 5-dimethyl-4- (3-morpholin-4-ylpropyl) -1H-pyrrol-2-yl]Methylene } -N-methyl-2-oxoindoline-5-sulfonamide (SU 11606); 6- [ difluoro [6- (1-methyl-1H pyrazol-4-yl) -1,2, 4-triazolo [4,3-b ] ]Pyridazin-3-yl]Methyl group]Quinoline (JNJ 38877605, CAS 943540-75-8); 2- [4- [1- (quinolin-6-ylmethyl) -1H- [1,2,3]Triazolo [4,5-b ]]Pyrazin-6-yl]-1H-pyrazol-1-yl]Ethanol (PF 04217903, CAS 956905-27-4); n- ((2R) -1, 4-dioxan-2-ylmethyl) -N-methyl-N' - [3- (1-methyl-1H-pyrazol-4-yl) -5-oxo-5H-benzo [4,5]Cyclohepta [1,2-b ]]Pyridin-7-yl]Sulfonamide (MK 2461, CAS 917879-39-1); 6- [ [6- (1-methyl-1H-pyrazol-4-yl) -1,2, 4-triazolo [4,3-b]Pyridazin 3-yl]Thio-]Quinoline (SGX 523, CAS 1022150-57-7); and (3Z) -5- [ [ (2, 6-dichlorophenyl) methyl]Sulfonyl group]-3- [ [3, 5-dimethyl-4- [ [ (2R) -2- (1-pyrrolidinylmethyl) -1-pyrrolidinyl]Carbonyl group]-1H-pyrrol-2-yl]Methylene group]-1, 3-dihydro-2H-indol-2-one (PHA 665752, CAS 477575-56-7).

IGFR inhibitors include, but are not limited to BMS-754807, XL-228, OSI-906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. See, e.g., yee, JNCI [ J.State cancer institute ],104;975 Overview of (2012).

In another embodiment, a tgfβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) is used in combination with one or more inhibitors of the proliferation signaling pathway, including but not limited to MEK inhibitors, BRAF inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and also mTOR inhibitors and CDK inhibitors, to treat a disease (e.g., cancer).

For example, mitogen-activated protein kinase (MEK) inhibitors include, but are not limited to, XL-518 (also known as GDC-0973, CAS number 1029872-29-4, available from the ACC group (ACC Corp.)); 2- [ (2-chloro-4-iodophenyl) amino ] -N- (cyclopropylmethoxy) -3, 4-difluoro-benzamide (also known as CI-1040 or PD184352 and described in PCT publication No. WO 2000035436); n- [ (2R) -2, 3-dihydroxypropoxy ] -3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -benzamide (also known as PD0325901 and described in PCT publication No. WO 2002006213); 2, 3-bis [ amino [ (2-aminophenyl) thio ] methylene ] -succinonitrile (also known as U0126 and described in U.S. patent No. 2,779,780); n- [3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -6-methoxyphenyl ] -1- [ (2R) -2, 3-dihydroxypropyl ] -cyclopropanesulfonamide (also known as RDEA119 or BAY869766 and described in PCT publication No. WO 2007014011); (3 s,4r,5z,8s,9s, 11E) -14- (ethylamino) -8,9,16-trihydroxy-3, 4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxy-lene-1, 7 (8H) -dione ] (also known as E6201 and described in PCT publication No. WO 2003076424); 2 '-amino-3' -methoxyflavone (also known as PD98059, available from Biaffin GmbH & co., KG) of Biaffin, germany; (R) -3- (2, 3-dihydroxypropyl) -6-fluoro-5- (2-fluoro-4-iodophenylamino) -8-methylpyrido [2,3-d ] pyrimidine-4, 7 (3 h,8 h) -dione (TAK-733, cas 1035555-63-5); pimassertib (AS-703026, CAS 1204531-26-9); and dimethylsulfoxide trametinib (GSK-1120212, CAS 1204531-25-80).

BRAF inhibitors include, but are not limited to, vitamin Mo Feini (Vemurafenib) (orPLX-4032, CAS 918504-65-1), GDC-0879, PLX-4720 (available from Sabinas corporation (Symantis), darafenib (or GSK 2118436), LGX 818, CEP-32496, UI-152, RAF 265, regorafenib (BAY 73-4506), CCT239065, or sorafenib (or sorafenib tosylate or;)>)。

Phosphoinositide 3-kinase (PI 3K) inhibitors include, but are not limited to, 4- [2- (1H-indazol-4-yl) -6- [ [4- (methylsulfonyl) piperazin-1-yl ] methyl ] thieno [3,2-d ] pyrimidin-4-yl ] morpholine (also known as GDC0941, RG7321, GNE0941, pictrilixib, or pitilib; and are described in PCT publication Nos. WO 09/036082 and WO 09/055730); tozasertib (VX 680 or MK-0457, CAS 639089-54-6); (5Z) -5- [ [4- (4-pyridinyl) -6-quinolinyl ] methylene ] -2, 4-thiazolidinedione (GSK 1059615, CAS 958852-01-2); (1E, 4S,4aR,5R,6aS,9 aR) -5- [ (di-2-propenylamino) methylene ] -4,4a,5, 6a,8,9 a-octahydro-11-hydroxy-4- (methoxymethyl) -4a,6 a-dimethylcyclopenta [5,6] naphtho [1,2-c ] pyran-2,7,10 (1H) -trione (PX 866, CAS 502632-66-8); 8-phenyl-2- (morpholin-4-yl) -chromen-4-one (LY 294002, CAS 154447-36-6); (S) -N1- (4-methyl-5- (2- (1, 1-trifluoro-2-methylpropan-2-yl) pyridin-4-yl) thiazol-2-yl) pyrrolidine-1, 2-dicarboxamide (also known as BYL719 or apicalide); 2- (4- (2- (1-isopropyl-3-methyl-1H-1, 2, 4-triazol-5-yl) -5, 6-dihydrobenzo [ f ] imidazo [1,2-d ] [1,4] oxazepin-9-yl) -1H-pyrazol-1-yl) -2-methylpropanamide (also known as GDC0032, RG7604, or tasselisib).

mTOR inhibitors include, but are not limited to, temsirolimusGespholimus (ridaforolimus) (formally called deferolimus, (1R, 2R, 4S) -4- [ (2R) -2[ (1R, 9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S, 35R) -1, 18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentoxy-11, 36-dioxa-4-azatricyclo [30.3.1.04,9)]Thirty-sixteen-16,24,26,28-tetraen-12-yl]Propyl group]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669, and described in PCT publication No. WO 03/064383); everolimus (+)>Or RAD 001); rapamycin (AY 22989,) The method comprises the steps of carrying out a first treatment on the surface of the Plug Ma Mode (simapimod) (CAS 164301-51-3); (5- {2, 4-bis [ (3S) -3-methylmorpholin-4-yl)]Pyrido [2,3-d ]]Pyrimidin-7-yl } -2-methoxyphenyl) methanol (AZD 8055); 2-amino-8- [ trans-4- (2-hydroxyethoxy) cyclohexyl]-6- (6-methoxy-3-pyridinyl) -4-methyl-pyrido [2,3-d]Pyrimidin-7 (8H) -one (PF 04691502, CAS1013101-36-4); and N² - [1, 4-dioxo-4- [ [4- (4-oxo-8-phenyl-4H-1-benzopyran-2-yl) morpholinium-4-yl ]]Methoxy group]Butyl group]-L-arginyl glycyl-L-alpha-aspartyl L-serine-, inner salt (SF 1126, CAS 936487-67-1).

CDK inhibitors include, but are not limited to, pampers Bai Xili (also known as PD-0332991,6-acetyl-8-cyclopentyl-5-methyl-2- { [5- (1-piperazinyl) -2-pyridinyl]Amino } pyrido [2,3-d]Pyrimidin-7 (8H) -one).

In yet another embodiment, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more pro-apoptotics (including, but not limited to IAP inhibitors, BCL2 inhibitors, MCL1 inhibitors, TRAIL agents, CHK inhibitors) to treat a disease (e.g., cancer).

For example, IAP inhibitors include, but are not limited to, LCL161, GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include, but are not limited to, those disclosed in WO 04/005284, WO 04/007529, WO 05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/069063, WO 06/010118, WO 06/017295, and WO 08/134679.

BCL-2 inhibitors include, but are not limited to, 4- [4- [ [2- (4-chlorophenyl) -5, 5-dimethyl-1-cyclohexen-1-yl ]]Methyl group]-1-piperazinyl]-N- [ [4- [ [ (1R) -3- (4-morpholinyl) -1- [ (phenylthio) methyl]Propyl group]Amino group]-3- [ (trifluoromethyl) sulfonyl group]Phenyl group]Sulfonyl group]Benzamide (also known as ABT-263 and described in PCT publication No. WO 09/155386); four-step preparation of carcinomatous factor A; antimycin; gossypol ((-) BL-193); obatoclax (obatocrax); ethyl-2-amino-6-cyclopentyl-4- (1-cyano-2-ethoxy-2-oxoethyl) -4H chromone-3-carboxylate (HA 14-1); the method comprises the steps of (oimmerson) (G3139,) The method comprises the steps of carrying out a first treatment on the surface of the Bak BH3 peptide; (-) -gossypol acetic acid (AT-101); 4- [4- [ (4 '-chloro [1,1' -biphenyl)]-2-yl) methyl]-1-piperazinyl]-N- [ [4- [ [ (1R) -3- (dimethyl)Amino) -1- [ (phenylthio) methyl]Propyl group]Amino group]-3-nitrophenyl]Sulfonyl group]Benzamide (ABT-737, cas 852808-04-9); and Navitocline (ABT-263, CAS 923664-51-6).

Pro-apoptotic receptor agonists (PARA) include DR4 (TRAILR 1) and DR5 (TRAILR 2), including but not limited to Du Lale min (Dulanermin) (AMG-951, rhapo 2L/TRAIL); mapatumumab (HRS-ETR 1, CAS 658052-09-6); lexamumab (HGS-ETR 2, CAS 845816-02-6); apomabSitazidime beads (Conatumumab) (AMG 655, CAS 896731-82-1); and Tigatuzumab (CS 1008, CAS 946415-34-5, available from Daiichi Sankyo, inc.).

Checkpoint kinase (CHK) inhibitors include, but are not limited to, 7-hydroxy staurosporine (UCN-01); 6-bromo-3- (1-methyl-1H-pyrazol-4-yl) -5- (3R) -3-piperidylpyrazolo [1,5-a ] pyrimidin-7-amine (SCH 900776, CAS 891494-63-6); 5- (3-fluorophenyl) -3-ureidothiophene-2-carboxylic acid N- [ (S) -piperidin-3-yl ] amide (AZD 7762, CAS 860352-01-8); 4- [ ((3S) -1-azabicyclo [2.2.2] oct-3-yl) amino ] -3- (1H-benzoimidazol-2-yl) -6-chloroquinolin-2 (1H) -one (CHIR 124, cas 405168-58-3); 7-amino dactinomycin (7-AAD), isogranulatimide, debromohymenialdisine; n- [ 5-bromo-4-methyl-2- [ (2S) -2-morpholinylmethoxy ] -phenyl ] -N' - (5-methyl-2-pyrazinyl) urea (LY 2603618, CAS 911222-45-2); sulforaphane (CAS 4478-93-7, 4-methylsulfinylbutyl isothiocyanate); 9,10,11, 12-tetrahydro-9, 12-epoxy-1H-diindole [1,2,3-fg:3',2',1' -kl ] pyrrolo [3,4-i ] [1,6] benzodiazocine-1, 3 (2H) -dione (SB-218078, CAS 135897-06-2); and TAT-S216A (YGRKKRRQRRRLYRSPAMPENL (SEQ ID NO: 318)), and CBP501 ((d-Bpa) sws (d-Phe-F5) (d-Cha) rrrqrr).

In further embodiments, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more immunomodulatory agents (e.g., one or more of activators of co-stimulatory molecules or inhibitors of immune checkpoint molecules) to treat a disease (e.g., cancer).

In certain embodiments, the immunomodulator is an activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from the group consisting of an agonist (e.g., an agonistic antibody or antigen binding fragment thereof, or a soluble fusion) of OX40, CD2, CD27, CDs, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), 4-1BB (CD 137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, or CD83 ligand.

GITR agonists

In some embodiments, the GITR agonist is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the GITR agonist is GWN323 (norhua, inc.), BMS-986156, MK-4166, or MK-1248 (merck), TRX518 (Li Pu treatment company (Leap Therapeutics)), INCAGN1876 (indite)/Ai Jina s (aganus)), AMG 228 (meciano (amben)), or INBRX-110 (Yin Xi, inc. (Inhibrx)).

Exemplary GITR agonists

In one embodiment, the GITR agonist is an anti-GITR antibody molecule. In one embodiment, the GITR agonist is an anti-GITR antibody molecule, as described in WO 2016/057846, published under the heading "Compositions and Methods of Use for Augmented Immune Response and Cancer Therapy [ compositions and methods for enhancing immune responses and cancer treatment ]", month 4, 14.

In one embodiment, the anti-GITR antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising the amino acid sequences set forth in table 5 (e.g., the heavy and light chain variable region sequences from MAB7 disclosed in table 5), or the amino acid sequences encoded by the nucleotide sequences set forth in table 5. In some embodiments, these CDRs are defined according to cabazite. In some embodiments, these CDRs are defined according to Qiao Xiya. In one embodiment, one or more of the CDRs (or generally all of the CDRs) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequence, or the amino acid sequence encoded by the nucleotide sequence.

In one embodiment, the anti-GITR antibody molecule comprises: a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 109, the VH CDR2 amino acid sequence of SEQ ID NO. 111, and the VH CDR3 amino acid sequence of SEQ ID NO. 113; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:114, the VLCDR2 amino acid sequence of SEQ ID NO:116, and the VLCDR3 amino acid sequence of SEQ ID NO:118, each as disclosed in Table 5.

In one embodiment, the anti-GITR antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID No. 101, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 101. In one embodiment, the anti-GITR antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO. 102, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 102. In one embodiment, the anti-GITR antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 101 and VL comprising the amino acid sequence of SEQ ID No. 102.

In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID No. 105, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 105. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO. 106, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 106. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO. 105 and a VL encoded by the nucleotide sequence of SEQ ID NO. 106.

In one embodiment, the anti-GITR antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 103, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 103. In one embodiment, the anti-GITR antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID No. 104, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 104. In one embodiment, the anti-GITR antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 103 and a light chain comprising the amino acid sequence of SEQ ID NO. 104.

In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 107, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 107. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO. 108, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 108. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 107 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 108.

The antibody molecules described herein may be made by vectors, host cells, and methods described in WO 2016/057846.

Table 5: amino acid and nucleotide sequences of exemplary anti-GITR antibody molecules

Other exemplary GITR agonists

In one embodiment, the anti-GITR antibody molecule is BMS-986156 (bai meishi nobility corporation), also referred to as BMS986156 or BMS986156.BMS-986156 and other anti-GITR antibodies are disclosed, for example, in US 9,228,016 and WO 2016/196792. In one embodiment, the anti-GITR antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of BMS-986156, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 6.

In one embodiment, the anti-GITR antibody molecule is MK-4166 or MK-1248 (Merck). MK-4166, MK-1248, and other anti-GITR antibodies are disclosed, for example, in US 8,709,424, WO 2011/028683, WO 2015/026684, and Mahne et al, cancer Res 2017;77 (5):1108-1118. In one embodiment, the anti-GITR antibody molecule comprises one or more of the following: the CDR sequence (or generally all of the CDR sequence) of MK-4166 or MK-1248, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-GITR antibody molecule is TRX518 (Li Pu therapeutic company (Leap Therapeutics)). TRX518 and other anti-GITR antibodies are disclosed, for example, in US 7,812,135, US 8,388,967, US 9,028,823, WO 2006/105021, and Ponte J et al, (2010) Clinical Immunology [ clinical immunology ];135:S96. In one embodiment, the anti-GITR antibody molecule comprises one or more of the following: CDR sequences (or generally all CDR sequences) of TRX518, heavy or light chain variable region sequences, or heavy or light chain sequences.

In one embodiment, the anti-GITR antibody molecule is INCAGN1876 (as at siamet/Ai Jina s). INCAGN1876 and other anti-GITR antibodies are disclosed, for example, in US 2015/0368349 and WO 2015/184099. In one embodiment, the anti-GITR antibody molecule comprises one or more of the following: CDR sequences (or generally all CDR sequences) of INCAGN1876, heavy or light chain variable region sequences, or heavy or light chain sequences.

In one embodiment, the anti-GITR antibody molecule is AMG 228 (amagen). AMG 228 and other anti-GITR antibodies are disclosed, for example, in US 9,464,139 and WO 2015/031667. In one embodiment, the anti-GITR antibody molecule comprises one or more of the following: CDR sequences (or generally all CDR sequences) of AMG 228, heavy or light chain variable region sequences, or heavy or light chain sequences.

In one embodiment, the anti-GITR antibody molecule is INBRX-110 (Yin Xi, inc.). INBRX-110 and other anti-GITR antibodies are disclosed, for example, in US2017/0022284 and WO 2017/015623. In one embodiment, the GITR agonist comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of INBRX-110, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the GITR agonist (e.g., fusion protein) is MEDI1873 (intel Mei Dimiao s.c.), also known as MEDI1873.MEDI 1873 and other GITR agonists are disclosed, for example, in US 2017/0073086, WO 2017/025610, and Ross et al, cancer Res [ Cancer research ]2016;76 (14 journals) abstract nr 561. In one embodiment, the GITR agonist comprises one or more of an IgG Fc domain of MEDI1873, a functional multimerization domain, and a receptor binding domain of a glucocorticoid-induced TNF receptor ligand (GITRL).

Additional known GITR agonists (e.g., anti-GITR antibodies) include those described, for example, in WO 2016/054638.

In one embodiment, the anti-GITR antibody is an antibody that competes with one of the anti-GITR antibodies described herein for binding to and/or binding to the same epitope on GITR.

In one embodiment, the GITR agonist is a peptide that activates the GITR signaling pathway. In one embodiment, the GITR agonist is an immunoadhesin binding fragment (e.g., an immunoadhesin binding fragment comprising the extracellular portion of GITRL or the binding portion of GITR) fused to a constant region (e.g., the Fc region of an immunoglobulin sequence).

Table 6: amino acid sequences of other exemplary anti-GITR antibody molecules

In certain embodiments, the immunomodulator is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulatory agent is an inhibitor of PD-1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and/or TGFR beta. In one embodiment, the inhibitor of the immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3 or CTLA4, or any combination thereof.

Inhibition of the inhibitory molecule may be at the DNA, RNA or protein level. In some embodiments, an inhibitory nucleic acid (e.g., dsRNA, siRNA or shRNA) may be used to inhibit expression of an inhibitory molecule. In other embodiments, the inhibitor of the inhibitory signal is a polypeptide, e.g., a soluble ligand (e.g., PD-1-Ig or CTLA-4 Ig) or an antibody or antigen-binding fragment thereof that binds to an inhibitory molecule; for example, an antibody or fragment thereof (also referred to herein as an "antibody molecule") that binds to PD-1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and/or TGFR beta, or a combination thereof.

In one embodiment, the antibody molecule is a complete antibody or fragment thereof (e.g., fab, F (ab') 2, fv, or single chain Fv fragment (scFv)). In yet other embodiments, the antibody molecule has a heavy chain constant region (Fc) selected from the group consisting of, for example, the heavy chain constant regions of IgG1, igG2, igG3, igG4, igM, igA1, igA2, igD, and IgE; in particular, the heavy chain constant region is selected from, for example, igG1, igG2, igG3, and IgG4, more particularly, igG1 or IgG4 (e.g., human IgG1 or IgG 4). In one embodiment, the heavy chain constant region is human IgG1 or human IgG4. In one embodiment, the constant region is altered (e.g., mutated) to modify a property of the antibody molecule (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function).

In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody molecule has a first binding specificity for PD-1 or PD-L1 and a second binding specificity, for example, a second binding specificity for TGF-beta, TIM-3, LAG-3, or PD-L2. For one embodiment, the bispecific antibody molecule binds to PD-1 or PD-L1 and TIM-3. In another embodiment, the bispecific antibody molecule binds to PD-1 or PD-L1 and TGF-beta. In another embodiment, the bispecific antibody molecule binds to PD-1 and tgfβ. Any combination of the foregoing molecules may be prepared in a multispecific antibody molecule (e.g., a trispecific antibody comprising a first binding specificity for PD-1 or PD-1, and a second and third binding specificity for two or more of tgfβ, TIM-3, LAG-3, or PD-L2).

In certain embodiments, the immunomodulatory agent is an inhibitor of PD-1 (e.g., human PD-1). In another embodiment, the immunomodulator is an inhibitor of PD-L1 (e.g., human PD-L1). In one embodiment, the inhibitor of PD-1 or PD-L1 is an antibody molecule of PD-1 or PD-L1. PD-1 or PD-L1 inhibitors may be administered alone or in combination with other immunomodulators, for example, with inhibitors of TGF beta, LAG-3, TIM-3 or CTLA 4. In exemplary embodiments, an inhibitor of PD-1 or PD-L1 (e.g., an anti-tgfβ, or an anti-PD-1 or PD-L1 antibody molecule) is administered in combination with a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody molecule). In another embodiment, an inhibitor of TGF-beta, PD-1, or PD-L1 (e.g., an anti-TGF-beta, or anti-PD-1 or PD-L1 antibody molecule) is administered in combination with a TIM-3 inhibitor (e.g., an anti-TIM-3 antibody molecule). In still other embodiments, an inhibitor of TGF-beta, PD-1, or PD-L1 (e.g., an anti-TGF-beta or anti-PD-1 antibody molecule) is administered in combination with a LAG-3 inhibitor (e.g., an anti-LAG-3 antibody molecule) and a TIM-3 inhibitor (e.g., an anti-TIM-3 antibody molecule).

Other combinations of immunomodulatory agents with PD-1 inhibitors (e.g., one or more of PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and/or TGFR) are also included within the disclosure. Any antibody molecule known in the art or disclosed herein may be used in combination with the checkpoint molecule inhibitors described above.

CTLA-4 inhibitors

In some embodiments, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with CTLA-4 inhibitors to treat a disease (e.g., cancer). In some embodiments, the PD-1 inhibitor is selected from Ipilimumab (Ipilimumab) (MDX-010, MDX-101, or Yervoy, BAIMEISHIBAO Co., ltd.), qu Meili mumab (tremeliumab) (ticalimumab, zilimumab/Abelikang Co., ltd.), AGEN1181 (Ai Jina S Co.), lycofulizumab (Zalifurlimab) (AGEN 1884, ai Jina S Co., ltd.), IBI310 (Xindabiological Co., innovent Biologics)),

LAG-3 inhibitors

In some embodiments, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with LAG-3 inhibitors to treat a disease (e.g., cancer). In some embodiments, the LAG-3 inhibitor is selected from LAG525 (North Co., ltd.), BMS-986016 (Bai Shi Guibao Co.), or TSR-033 (Tasa Luo Gongsi).

Exemplary LAG-3 inhibitors

In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule, as disclosed in US2015/0259420, publication No. 17, 9, 2015 entitled "antibody molecule of Antibody Molecules to LAG-3and Uses Thereof[LAG-3 and uses thereof".

In one embodiment, the anti-LAG-3 antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising an amino acid sequence set forth in table 7 (e.g., heavy and light chain variable region sequences from BAP 050-clone I or BAP 050-clone J disclosed in table 7), or encoded by a nucleotide sequence set forth in table 7. In some embodiments, these CDRs are according to the cabazite definition (e.g., as set forth in table 7). In some embodiments, these CDRs are defined according to Qiao Xiya (e.g., as set forth in table 7). In some embodiments, these CDRs are defined according to the combined CDRs of both cabazite and Qiao Xiya (e.g., as listed in table 7). In one embodiment, the combination of the carboplatin and Qiao Xiya CDRs of VH CDR1 comprises amino acid sequence GFTLTNYGMN (SEQ ID NO: 122). In one embodiment, one or more of the CDRs (or all of the CDRs in general) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 7, or encoded by the nucleotide sequences shown in table 7.

In one embodiment, the anti-LAG-3 antibody molecule comprises: a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO. 123, the VH CDR2 amino acid sequence of SEQ ID NO. 124, and the VH CDR3 amino acid sequence of SEQ ID NO. 125; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:132, the VLCDR2 amino acid sequence of SEQ ID NO:133, and the VLCDR3 amino acid sequence of SEQ ID NO:134, each as disclosed in Table 7.

In one embodiment, the anti-LAG-3 antibody molecule comprises: a VH comprising a VH CDR1 encoded by the nucleotide sequence of SEQ ID NO 158 or 159, a VH CDR2 encoded by the nucleotide sequence of SEQ ID NO 160 or 161, and a VH CDR3 encoded by the nucleotide sequence of SEQ ID NO 162 or 163; and VL comprising VLCDR1 encoded by the nucleotide sequence of SEQ ID NO:168 or 169, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO:170 or 171, and VLCDR3 encoded by the nucleotide sequence of SEQ ID NO:172 or 173, are each disclosed in Table 7. In one embodiment, the anti-LAG-3 antibody molecule comprises: a VH comprising a VH CDR1 encoded by the nucleotide sequence of SEQ ID NO. 180 or 159, a VH CDR2 encoded by the nucleotide sequence of SEQ ID NO. 181 or 161, and a VH CDR3 encoded by the nucleotide sequence of SEQ ID NO. 182 or 163; and VL comprising VLCDR1 encoded by the nucleotide sequence of SEQ ID NO:168 or 169, VLCDR2 encoded by the nucleotide sequence of SEQ ID NO:170 or 171, and VLCDR3 encoded by the nucleotide sequence of SEQ ID NO:172 or 173, are each disclosed in Table 7.

In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO. 128, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 128. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO. 140, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 140. In one embodiment, the anti-LAG-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO. 146, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 146. In one embodiment, the anti-LAG-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO. 152, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 152. In one embodiment, the anti-LAG-3 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 128 and VL comprising the amino acid sequence of SEQ ID No. 140. In one embodiment, the anti-LAG-3 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 146 and VL comprising the amino acid sequence of SEQ ID No. 152.

In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID No. 129 or 130, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 129 or 130. In one embodiment, the antibody molecule comprises a VL consisting of the nucleotide sequence of SEQ ID NO. 141 or 142, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 141 or 142. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID No. 147 or 148, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 147 or 148. In one embodiment, the antibody molecule comprises a VL consisting of the nucleotide sequence of SEQ ID NO 153 or 154, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO 153 or 154. In one embodiment, the antibody molecule comprises: a VH encoded by the nucleotide sequence of SEQ ID NO. 129 or 130 and a VL encoded by the nucleotide sequence of SEQ ID NO. 141 or 142. In one embodiment, the antibody molecule comprises: VH encoded by the nucleotide sequence of SEQ ID No. 147 or 148 and VL encoded by the nucleotide sequence of SEQ ID No. 153 or 154.

In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 131, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 131. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO. 143, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 143. In one embodiment, the anti-LAG-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 149 or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 149. In one embodiment, the anti-LAG-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO. 155, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 155. In one embodiment, the anti-LAG-3 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 131 and a light chain comprising the amino acid sequence of SEQ ID NO. 143. In one embodiment, the anti-LAG-3 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:149 and a light chain comprising the amino acid sequence of SEQ ID NO: 155.

In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 138 or 139, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 138 or 139. In one embodiment, the antibody molecule comprises a light chain consisting of the nucleotide sequence of SEQ ID NO. 144 or 145, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 144 or 145. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 150 or 151, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 150 or 151. In one embodiment, the antibody molecule comprises a light chain consisting of the nucleotide sequence of SEQ ID NO. 156 or 157, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 156 or 157. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 138 or 139 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 144 or 145. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 150 or 151 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 156 or 157.

The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0259420.

TABLE 7 amino acid and nucleotide sequences of exemplary anti-LAG-3 antibody molecules

Other exemplary LAG-3 inhibitors

In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986016 (BAIMEISHIGULAR), also known as BMS986016.BMS-986016 and other anti-LAG-3 antibodies are disclosed in WO2015/116539 and US 9,505,839. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of BMS-986016, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 8.

In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tassa Luo Gongsi). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of TSR-033, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima biomedical company (Prima BioMed)). IMP731 and other anti-LAG-3 antibodies are disclosed in WO 2008/132601 and US 9,244,059. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of IMP731, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 8. In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of GSK2831781, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

In one embodiment, the anti-LAG-3 antibody molecule is IMP761 (prima bio-pharmaceutical company). In one embodiment, the anti-LAG-3 antibody molecule comprises one or more of the following: the CDR sequence (or overall all CDR sequences) of IMP761, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

Other known anti-LAG-3 antibodies include, for example, those described in WO 2008/132601, WO 2010/019570, WO 2014/140180, WO 2015/116539, WO 2015/200119, WO 2016/028672, US 9,244,059, US 9,505,839.

In one embodiment, the anti-LAG-3 antibody is an antibody that competes with one of the anti-LAG-3 antibodies described herein for binding to the same epitope on LAG-3 and/or binds to the same epitope on LAG-3.

In one embodiment, the anti-LAG-3 inhibitor is a soluble LAG-3 protein, e.g., IMP321 (prima biomedical company), e.g., as disclosed in WO 2009/044273.

TABLE 8 amino acid sequences of other exemplary anti-LAG-3 antibody molecules

TIM-3 inhibitors

In certain embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of TIM-3. In some embodiments, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with TIM-3 inhibitors to treat a disease (e.g., cancer). In some embodiments, the TIM-3 inhibitor is MGB453 (North Co., ltd.), LY3321367 (Gift Lilly), sym023 (Symphogen), BGB-A425 (Beji China), INCAGN-2390 (Ai Jina Site Co., ltd.), MBS-986258 (BMS/Five columns (Five Prime), RO-7121661 (Roche Co., ltd.), LY-3415244 (Gift Lilly), or TSR-022 (Tasa Luo Gongsi).

Exemplary TIM-3 inhibitors

For one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule. In one embodiment, the TIM-3 inhibitor is an anti-TIM-3 antibody molecule, as disclosed in US2015/0218274 published 8/2015 entitled "antibody molecule of Antibody Molecules to TIM-3and Uses Thereof[TIM-3 and use thereof ]".

In one embodiment, the anti-TIM-3 antibody molecule comprises at least one, two, three, four, five, or six Complementarity Determining Regions (CDRs) (or generally all CDRs) from a heavy and light chain variable region comprising an amino acid sequence set forth in table 9 (e.g., heavy and light chain variable region sequences from ABTIM3-hum11 or ABTIM3-hum03 disclosed in table 9), or encoded by a nucleotide sequence set forth in table 9. In some embodiments, these CDRs are according to the cabazite definition (e.g., as set forth in table 9). In some embodiments, these CDRs are defined according to Qiao Xiya (e.g., as set forth in table 9). In one embodiment, one or more of the CDRs (or all of the CDRs in general) have one, two, three, four, five, six or more changes, such as amino acid substitutions (e.g., conservative amino acid substitutions) or deletions, relative to the amino acid sequences shown in table 9, or encoded by the nucleotide sequences shown in table 9.

For one embodiment, the anti-TIM-3 antibody molecule comprises: a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO:189, the VH CDR2 amino acid sequence of SEQ ID NO:190, and the VH CDR3 amino acid sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:198, the VLCDR2 amino acid sequence of SEQ ID NO:199, and the VLCDR3 amino acid sequence of SEQ ID NO:200, each as disclosed in Table 9. For one embodiment, the anti-TIM-3 antibody molecule comprises: a heavy chain variable region (VH) comprising the VH CDR1 amino acid sequence of SEQ ID NO:189, the VH CDR2 amino acid sequence of SEQ ID NO:208, and the VH CDR3 amino acid sequence of SEQ ID NO: 191; and a light chain variable region (VL) comprising the VLCDR1 amino acid sequence of SEQ ID NO:198, the VLCDR2 amino acid sequence of SEQ ID NO:199, and the VLCDR3 amino acid sequence of SEQ ID NO:200, each as disclosed in Table 9.

For one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO:194, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO: 194. For one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:204, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO: 204. For one embodiment, the anti-TIM-3 antibody molecule comprises a VH comprising the amino acid sequence of SEQ ID NO. 210, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 210. For one embodiment, the anti-TIM-3 antibody molecule comprises a VL comprising the amino acid sequence of SEQ ID NO:214, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO: 214. For one embodiment, the anti-TIM-3 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 194 and VL comprising the amino acid sequence of SEQ ID No. 204. For one embodiment, the anti-TIM-3 antibody molecule comprises: VH comprising the amino acid sequence of SEQ ID No. 210 and VL comprising the amino acid sequence of SEQ ID No. 214.

In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID No. 195, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 195. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO. 205, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 205. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID No. 211, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID No. 211. In one embodiment, the antibody molecule comprises a VL encoded by the nucleotide sequence of SEQ ID NO. 215, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 215. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO. 195 and a VL encoded by the nucleotide sequence of SEQ ID NO. 205. In one embodiment, the antibody molecule comprises a VH encoded by the nucleotide sequence of SEQ ID NO:211 and a VL encoded by the nucleotide sequence of SEQ ID NO: 215.

For one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 196, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 196. For one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO. 206, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 206. For one embodiment, the anti-TIM-3 antibody molecule comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 212, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 212. For one embodiment, the anti-TIM-3 antibody molecule comprises a light chain comprising the amino acid sequence of SEQ ID NO. 216, or an amino acid sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 216. For one embodiment, the anti-TIM-3 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 196 and a light chain comprising the amino acid sequence of SEQ ID NO. 206. For one embodiment, the anti-TIM-3 antibody molecule comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 212 and a light chain comprising the amino acid sequence of SEQ ID NO. 216.

In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 197, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 197. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO. 207, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 207. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO:213, or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO: 213. In one embodiment, the antibody molecule comprises a light chain encoded by the nucleotide sequence of SEQ ID NO. 217 or a nucleotide sequence having at least 85%, 90%, 95%, or 99% or more identity to SEQ ID NO. 217. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 197 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 207. In one embodiment, the antibody molecule comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO. 213 and a light chain encoded by the nucleotide sequence of SEQ ID NO. 217.

The antibody molecules described herein can be made by vectors, host cells, and methods described in US 2015/0218274.

TABLE 9 amino acid and nucleotide sequences of exemplary anti-TIM-3 antibody molecules

Other exemplary TIM-3 inhibitors

In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (An Naipu Tex Bio Inc. (AnaptysBio)/Tasa Luo Gongsi). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequence (or overall all CDR sequences) of TSR-022, the heavy or light chain variable region sequence, or the heavy or light chain sequence. For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of APE5137 or APE5121, the heavy or light chain variable region sequences, or the heavy or light chain sequences, e.g., as disclosed in table 10. APE5137, APE5121, and other anti-TIM-3 antibodies are disclosed in WO 2016/161270.

For one embodiment, the anti-TIM-3 antibody molecule is antibody clone F38-2E2. For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of F38-2E2, the heavy or light chain variable region sequence, or the heavy or light chain sequence.

For one embodiment, the anti-TIM-3 antibody molecule is LY3321367 (Gift). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of LY3321367, the heavy chain variable region sequence and/or the light chain variable region sequence, or the heavy chain sequence and/or the light chain sequence.

For one embodiment, the anti-TIM-3 antibody molecule is Sym023 (Symphogen). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of Sym023, the heavy chain variable region sequences and/or the light chain variable region sequences, or the heavy chain sequences and/or the light chain sequences.

For one embodiment, the anti-TIM-3 antibody molecule is BGB-A425 (Beigene). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences) of BGB-a425, the heavy chain variable region sequence and/or the light chain variable region sequence, or the heavy chain sequence and/or the light chain sequence.

For one embodiment, the anti-TIM-3 antibody molecule is INCAGN-2390 (Ai Jina Sitting Co., ltd./Legend./Incyte)). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of INCAGN-2390, the heavy chain variable region sequences and/or the light chain variable region sequences, or the heavy chain sequences and/or the light chain sequences.

For one embodiment, the anti-TIM-3 antibody molecule is BMS-986258 (BMS/five column Co.). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequences (or generally all CDR sequences) of BMS-986258, the heavy chain variable region sequences and/or the light chain variable region sequences, or the heavy chain sequences and/or the light chain sequences.

For one embodiment, the anti-TIM-3 antibody or inhibitor molecule is RO-7121661 (Roche). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences), heavy chain variable region sequence and/or light chain variable region sequence, or heavy chain sequence and/or light chain sequence of the TIM-3 binding arm of RO-7121661.

For one embodiment, the anti-TIM-3 antibody or inhibitor molecule is LY-3415244 (Gift). For one embodiment, the anti-TIM-3 antibody molecule comprises one or more of the following: the CDR sequence (or generally all CDR sequences), heavy and/or light chain variable region sequences, or heavy and/or light chain sequences of the TIM-3 binding arm of LY-3415244.

Other known anti-TIM-3 antibodies include, for example, those described in WO 2016/111947, WO 2016/071448, WO 2016/144803, US 8,552,156, US 8,841,418, and US 9,163,087.

For one embodiment, the anti-TIM-3 antibody is an antibody that competes with one of the anti-TIM-3 antibodies described herein for binding to the same epitope on TIM-3 and/or binding to the same epitope on TIM-3.

TABLE 10 amino acid sequences of other exemplary anti-TIM-3 antibody molecules

Cytokines and methods of use

In yet another embodiment, the TGF-beta inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more cytokines (including, but not limited to, interferon, IL-2, IL-15, IL-7, or IL 21). In certain embodiments, the TGF-beta inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) is administered in combination with the IL-15/IL-15Ra complex. In some embodiments, the IL-15/IL-15Ra complex is selected from NIZ985 (North Co., ltd.), ATL-803 (Altor) or CYP0150 (Cytune).

Exemplary IL-15/IL-15Ra complexes

In one embodiment, the cytokine is IL-15 complexed with a soluble form of IL-15 receptor alpha (IL-15 Ra). The IL-15/IL-15Ra complex can comprise a soluble form of IL-15 that is covalently or non-covalently bound to IL-15 Ra. In certain embodiments, human IL-15 is non-covalently bound to a soluble form of IL-15 Ra. In particular embodiments, the human IL-15 of the formulation comprises the amino acid sequence of SEQ ID NO:222 in Table 11 or an amino acid sequence having at least 85%, 90%, 95%, or 99%, or more identity to SEQ ID NO:222, and the human IL-15Ra of the soluble form comprises the amino acid sequence of SEQ ID NO:223 in Table 11 or an amino acid sequence having at least 85%, 90%, 95%, or 99%, or more identity to SEQ ID NO:223, as described in WO 2014/066527. The molecules described herein may be made by vectors, host cells, and methods described in WO 2007084342.

TABLE 11 amino acid and nucleotide sequences of exemplary IL-15/IL-15Ra complexes

Other exemplary IL-15/IL-15Ra complexes

In one embodiment, the IL-15/IL-15Ra complex is ALT-803, an IL-15/IL-15Ra Fc fusion protein (IL-15N 72D: IL-15RaSu/Fc soluble complex). ALT-803 is described in WO 2008/143794. In one embodiment, the IL-15/IL-15Ra Fc fusion protein comprises the sequences as disclosed in Table 12.

In one embodiment, the IL-15/IL-15Ra complex comprises IL-15 (CYP 0150, sambur. RTM.) fused to the sushi domain of IL-15 Ra. The sushi domain of IL-15Ra refers to a domain that starts at the first cysteine residue after the signal peptide of IL-15Ra and ends at the fourth cysteine residue after the signal peptide. Complexes of IL-15 fused to the sushi domain of IL-15Ra are described in WO 2007/04606 and WO 2012/175222. In one embodiment, the IL-15/IL-15Ra sushi domain fusion comprises a sequence as disclosed in Table 12.

TABLE 12 amino acid sequences of other exemplary IL-15/IL-15Ra complexes

In yet another embodiment, a tgfβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) of the present disclosure is used in combination with an agonist of one or more toll-like receptors (TLR, e.g., TLR7, TLR8, TLR 9) to treat a disease (e.g., cancer). In some embodiments, compounds of the present disclosure may be used in combination with a TLR7 agonist or a TLR7 agonist conjugate.

In some embodiments, the TLR7 agonist comprises a compound disclosed in international application publication No. WO 2011/049677. In some embodiments, the TLR7 agonist comprises 3- (5-amino-2- (4- (2- (3, 3-difluoro-3-phosphonopropoxy) ethoxy) -2-methylphenylethyl) benzo [ f ] [1,7] naphthyridin-8-yl) propionic acid. In some embodiments, the TLR7 agonist comprises a compound having the formula:

in another embodiment, a tgfβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) of the present disclosure is used in combination with one or more angiogenesis inhibitors to treat cancer, for example: bevacizumabAxitinib (a-xitinib)>Ala brinib (Brivanib alaninate) (BMS-582664, (S) - ((R) -1- (4- (4-fluoro-2-methyl-1H-indol-5-yloxy) -5-methylpyrrolo [2, 1-f)][1,2,4]Triazin-6-yloxy) propan-2-yl) 2-aminopropionic acid); sorafenib->Pazopanib->Sunitinib malate->Sildenib (Cediranib) (AZD 2171, CAS 288383-20-1); vegate (Vargatef) (BIBF 1120, CAS 928326-83-4); furitinib (fortinib) (GSK 1363089); tiratinib (Telatinib) (BAY 57-9352, cas 332012-40-5); apatinib (Apatinib) (YN 968D1, CAS 811803-05-1); imatinib (Imatinib)/(Imatinib) >Ponatinib (Ponatinib) (AP 24534, CAS 943319-70-8); tivozanib (AV 951, CAS 475108-18-0); regorafenib (BAY 73-4506, cas 755037-03-7); varanib dihydrochloride (Vatalanib dihydrochloride) (PTK 787, CAS 212141-51-0); brianib (BMS-540215,CAS 649735-46-6); vandetanib (>Or AZD 6474); motrasenib diphosphate (Motesanib diphosphate) (AMG 706, CAS 857876-30-3, N- (2, 3-dihydro-3, 3-dimethyl-1H-indol-6-yl) -2- [ (4-pyridylmethyl) amino)]-3-pyridinecarboxamide, described in PCT publication No. WO 02/066470); poly Wei Tini di-lactic acid (Dovitinib dilactic acid) (TKI 258, CAS 852433-84-2); lin Funi (Linfanib) (ABT 869, CAS 796967-16-3); cabotinib (XL 184, CAS 849217-68-1); litatinib (Lestaurtinib) (CAS 111358-88-4); n- [5- [ [ [5- (1, 1-dimethylethyl) -2-oxazolyl ]]Methyl group]Thio-]-2-thiazolyl]-4-piperidinecarboxamide (BMS 3803, CAS 345627-80-7); (3R, 4R) -4-amino-1- ((4- ((3-methoxyphenyl) amino) pyrrolo [2, 1-f)][1,2,4]Triazin-5-yl) methyl) piperidin-3-ol (BMS 690514); n- (3, 4-dichloro-2-fluorophenyl) -6-methoxy-7- [ [ (3 a alpha, 5 beta, 6a alpha) -octahydro-2-methylcyclopenta [ c ] ]Pyrrol-5-yl]Methoxy group]-4-quinazolinamine (XL 647, CAS 781613-23-8); 4-methyl-3- [ [ 1-methyl-6- (3-pyridinyl) -1H-pyrazolo [3,4-d ]]Pyrimidin-4-yl]Amino group]-N- [3- (trifluoromethyl) phenyl ]]Benzamide (BHG 712, CAS 940310-85-0); or Alepsieb->

Bevacizumab, when used in combination with other therapeutic agents (e.g., tgfβ inhibitors and/or PD-1 inhibitors), may be administered intravenously to a patient. For example, bevacizumab can be administered intravenously to a patient at a dose of 5 mg/kg. Bevacizumab may also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, bevacizumab is administered at a dose of 5mg/kg on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle).

In another embodiment, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more heat shock protein inhibitors to treat cancer, for example: tamsulosin (Tanespimycin) (17-allylamino-17-desmethoxygeldanamycin, also known as KOS-953 and 17-AAG, available from SIGMA (SIGMA) and described in U.S. Pat. No. 4,261,989); rithromycin (IPI 504), ganetespib (STA-9090); [ 6-chloro-9- (4-methoxy-3, 5-dimethylpyridin-2-ylmethyl) -9H-purin-2-yl ] amine (BIIB 021 or-CNF 2024, CAS 848695-25-0); trans-4- [ [2- (aminocarbonyl) -5- [4,5,6, 7-tetrahydro-6, 6-dimethyl-4-oxo-3- (trifluoromethyl) -1H-indazol-1-yl ] phenyl ] amino ] cyclohexylglycine ester (SNX 5422 or PF04929113, CAS 908115-27-5); 5- [2, 4-dihydroxy-5- (1-methylethyl) phenyl ] -N-ethyl-4- [4- (4-morpholinylmethyl) phenyl ] -3-isoxazolecarboxamide (AUY 922, CAS 747412-49-3); or 17-dimethylaminoethylamino-17-desmethoxygeldanamycin (17-DMAG).

In yet another embodiment, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more HDAC inhibitors or other epigenetic modifiers. Exemplary HDAC inhibitors include, but are not limited to vorinostat (Voninostat)Rockwell Mi Dixing (Romidepsin)>Trichostatin A (Treichostatin A) (TSA); oxamflatin; vorinostat (Vorinostat) (-in)>Suberoylanilide hydroxamic acid); pyroxamide (syberoyl-3-aminopyridine amide hydroxamic acid); trapoxin A (RF-1023A); trapoxin B (RF-10238); cyclo [ (alpha S, 2S) -alpha-amino-eta-oxo-2-oxirane octanoyl-O-methyl-D-tyrosyl-L-isoleucyl-L-prolyl](Cyl-1); cyclo [ (alpha S, 2S) -alpha-amino-eta-oxo-2-oxirane octanoyl-O-methyl-D-tyrosyl-L-isoleucyl- (2S) -2-piperidinecarbonyl](Cyl-2); cyclo [ L-alanyl-D-alanyl- (2S) -eta-oxo-L-alpha-amino oxiranyl octanoyl-D-prolyl](HC-toxin); cyclo [ (alpha S, 2S) -alpha-amino-eta-oxo-2-oxirane octanoyl-D-phenylalanyl-L-leucyl- (2S) -2-piperidinecarbonyl](WF-3161); chlorjiemycin (C)hlamydocin) ((S) -cyclo (2-methylalanyl-L-phenylalanyl-D-prolyl- η -oxo-L- α -amino oxiranyl); histone deacetylase inhibitor (Apicidin) (cyclo (8-oxo-L-2-aminodecanoyl-1-methoxy-L-tryptophanyl-L-isoleucyl-D-2-piperidinecarbonyl)), lol Mi Dixing ( >FR-901228); 4-phenylbutyrate; spiranthostatin a; mylprin (valproic acid); enronostat (Entinostat) (MS-275, N- (2-aminophenyl) -4- [ N- (pyridin-3-yl-methoxycarbonyl) -amino-methyl-)]-benzamide); depudecin (4, 5:8, 9-dianhydride-1,2,6,7,11-pentadeoxy-D-threo-D-ido-undec-1, 6-dienol); 4- (acetylamino) -N- (2-aminophenyl) -benzamide (also known as CI-994); n1- (2-aminophenyl) -N8-phenyl-octanediamide (also known as BML-210); 4- (dimethylamino) -N- (7- (hydroxyamino) -7-oxoheptyl) benzamide (also known as M344); (E) -3- (4- (((2- (1H-indol-3-yl) ethyl) (2-hydroxyethyl) amino) -methyl) phenyl) -N-hydroxyacrylamide; panobinostatMo Nuosi he (Mocetinostat) and belinostat (also known as PXD101,/-for example>Or (2E) -N-hydroxy-3- [3- (phenylsulfamoyl) phenyl]Prop-2-enamide) or cidamide (also known as CS055 or HBI-8000, (E) -N- (2-amino-5-fluorophenyl) -4- ((3- (pyridin-3-yl) acrylamido) methyl) benzamide. Other apparent modifiers include, but are not limited to, inhibitors of EZH2 (enhancers of zeste homolog 2), EED (embryonic ectodermal development) or LSD1 (lysine-specific histone demethylase 1A or KDM 1A).

In yet another embodiment, a tgfβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) of the present disclosure is used in combination with one or more inhibitors of indoleamine-pyrrole 2, 3-dioxygenase (IDO) (e.g., indoximod) (also known as NLG-8189), α -cyclohexyl-5H-imidazo [5,1-a ] isoindole-5-ethanol (also known as NLG 919), or (4E) -4- [ (3-chloro-4-fluoroanilino) -nitrosomethylene ] -1,2, 5-oxadiazol-3-amine (also known as INCB 024360)) to treat cancer.

Chimeric antigen receptor

The present disclosure provides tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) for use in combination with adoptive immunotherapy methods and agents, such as Chimeric Antigen Receptor (CAR) immune effector cells (e.g., T cells), or chimeric TCR transduced immune effector cells (e.g., T cells). This section describes CAR technology generally used in combination with tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors), and describes CAR reagents (e.g., cells and compositions) and methods.

In general, aspects of the disclosure relate to or include an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., an antibody or antibody fragment, a TCR or TCR fragment) that binds to a tumor antigen as described herein, a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain as described herein) (e.g., an intracellular signaling domain and/or a primary signaling domain (e.g., a primary signaling domain) comprising a costimulatory domain (e.g., a costimulatory domain as described herein)). In other aspects, the disclosure includes: a host cell comprising the above nucleic acid and an isolated protein encoded by such nucleic acid molecule. The CAR nucleic acid constructs, encoded proteins, vectors containing, host cells, pharmaceutical compositions, and methods of administration and treatment related to the present disclosure are disclosed in detail in international patent application publication No. WO 2015142675.

In one aspect, the disclosure relates to an isolated nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein), a transmembrane domain (e.g., a transmembrane domain as described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain as described herein) (e.g., an intracellular signaling domain and/or a primary signaling domain (e.g., a level signaling domain as described herein) comprising a co-stimulatory domain (e.g., a co-stimulatory domain as described herein)). In some embodiments, the antigen that supports a tumor is an antigen present on a stromal cell or Myeloid Derived Suppressor Cell (MDSC). In other aspects, the disclosure features polypeptides encoded by such nucleic acids, as well as host cells containing such nucleic acids and/or polypeptides.

Alternatively, aspects of the disclosure relate to isolated nucleic acids encoding chimeric T Cell Receptors (TCRs) (comprising tcra and/or tcrp variable domains, specific for the cancer antigens described herein). See, e.g., dembic et al, nature [ Nature ],320,232-238 (1986), schumacher, nat. Rev. Immunol. [ Nature summary of immunology ],2,512-519 (2002), kershaw et al, nat. Rev. Immunol. [ Immunol. Summary of immunology ],5,928-940 (2005), xue et al, clin. Exp. Immunol. [ clinical and experimental immunology ],139,167-172 (2005), rossig et al, mol. Ther. [ molecular therapy ],10,5-18 (2004), and Murphy et al, immunity [ immunology ],22,403-414 (2005); morgan et al J.Immunol. [ J.Immunol., 171,3287-3295 (2003), hughes et al, hum.Gene Ther. [ human gene therapy ],16,1-16 (2005), zhao et al J.Immunol. [ J.Immunol., 174,4415-4423 (2005), roszkowski et al, cancer Res. [ Cancer research ],65,1570-1576 (2005), and Engels et al, hum.Gene Ther. [ human gene therapy ],16,799-810 (2005); US2009/03046557. Such chimeric TCRs can recognize, for example, cancer antigens such as MART-1, gp-100, p53, and NY-ESO-1, MAGE A3/A6, MAGEA3, SSX2, HPV-16E6, or HPV-16E7. In other aspects, the disclosure features polypeptides encoded by such nucleic acids, as well as host cells containing such nucleic acids and/or polypeptides.

The sequences of non-limiting examples of different components that can be part of a CAR are listed in table 11a, where "aa" represents an amino acid and "na" represents a nucleic acid encoding the corresponding peptide.

Table 11a. Sequences of different components of CAR (aa-amino acid sequence, na-nucleic acid sequence).

Target(s)

The present disclosure provides cells, e.g., immune effector cells (e.g., T cells, NK cells) comprising or at any time comprising a gRNA molecule or CRISPR system as described herein, which are further engineered to contain one or more CARs that direct the immune effector cells to undesired cells (e.g., cancer cells). This is achieved by an antigen binding domain on the CAR that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs of the present disclosure: (1) a cancer-associated antigen expressed on the surface of a cancer cell; and (2) cancer-associated antigens themselves within cells, however, fragments of such antigens (peptides) are presented on the surface of cancer cells via MHC (major histocompatibility complex).

In some embodiments, the tumor antigen is selected from one or more of the following: CD19; CD123; CD22; CD30; CD171; CS-1 (also known as CD2 subgroup 1, CRACC, SLAMF7, CD319, and 19A 24); c-type lectin-like molecule-1 (CLL-1 or CLECL 1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD 2); ganglioside GD3 (aNeu 5Ac (2-8) aNeu5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer); TNF receptor family member B Cell Maturation (BCMA); tn antigen ((TnAg) or (GalNAcα -Ser/Thr)); prostate Specific Membrane Antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR 1); fms-like tyrosine kinase 3 (FLT 3); tumor-associated glycoprotein 72 (TAG 72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD 276); KIT (CD 117); interleukin-13 receptor subunit α -2 (IL-13 Ra2 or CD213A 2); mesothelin; interleukin 11 receptor alpha (IL-11 Ra); prostate Stem Cell Antigen (PSCA); protease serine 21 (testosterone or PRSS 21); vascular endothelial growth factor receptor 2 (VEGFR 2); lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her 2/neu); mucin 1, cell surface associated (MUC 1); epidermal Growth Factor Receptor (EGFR); neural Cell Adhesion Molecules (NCAM); a prostase enzyme; prostatectomy phosphatase (PAP); mutated elongation factor 2 (ELF 2M); liver accessory protein B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic Anhydrase IX (CAIX); proteasome (macropin) subunit, beta-form, 9 (LMP 2); glycoprotein 100 (gp 100); an oncogene fusion protein (BCR-Abl) consisting of a Breakpoint Cluster Region (BCR) and an Abelson murine leukemia virus oncogene homolog 1 (Abl); tyrosinase; ephrin-type a receptor 2 (EphA 2); fucosyl GM1; sialic acid Lewis adhesion molecules (sLe); ganglioside GM3 (aNeu 5Ac (2-3) bDGalp (1-4) bDGlcp (1-1) Cer); transglutaminase 5 (TGS 5); high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD 2 ganglioside (OAcGD 2); folate receptor beta; tumor endothelial marker 1 (TEM 1/CD 248); tumor endothelial marker 7-associated (TEM 7R); sealing protein 6 (CLDN 6); thyroid Stimulating Hormone Receptor (TSHR); g protein coupled receptor group C, member D (GPRC 5D); chromosome X open reading frame 61 (CXORF 61); CD97; CD179a; anaplastic Lymphoma Kinase (ALK); polysialic acid; placenta-specific 1 (PLAC 1); a hexose moiety of globoH glycosylceramide (globoH); breast differentiation antigen (NY-BR-1); urolysin 2 (UPK 2); hepatitis a virus cell receptor 1 (HAVCR 1); adrenergic receptor beta 3 (ADRB 3); ubiquitin 3 (PANX 3); g protein-coupled receptor 20 (GPR 20); lymphocyte antigen 6 complex, locus K9 (LY 6K); olfactory receptor 51E2 (OR 51E 2); tcrγ alternative reading frame protein (TARP); a wilms tumor protein (WT 1); cancer/testis antigen 1 (NY-ESO-1); cancer/testis antigen 2 (LAGE-1 a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation mutant gene 6, located on chromosome 12p (ETV 6-AML); sperm protein 17 (SPA 17); x antigen family, member 1A (XAGE 1); angiogenin binds to cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); fos-associated antigen 1; tumor protein p53 (p 53); a p53 mutant; prostate specific protein (prostein); survivin (survivin); telomerase; prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), T cell 1 recognized melanoma antigen (MelanA or MART 1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma cell apoptosis inhibitors (ML-IAPs); ERG (transmembrane protease, serine 2 (TMPRSS 2) ETS fusion gene); n-acetylglucosaminyl transferase V (NA 17); pairing box protein Pax-3 (Pax 3); androgen receptor; cyclin B1; v-myc avian myeloblastosis virus oncogene neuroblastoma source homolog (MYCN); ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP 1B 1); CCCTC-binding factor (zinc finger protein) -like (BORIS or imprinted site-regulatory factor-like protein (Brother of the Regulator of Imprinted Sites)), squamous cell carcinoma antigen (SART 3) recognized by T cell 3; pairing box protein Pax-5 (Pax 5); the preprotein binding protein sp32 (OY-TES 1); lymphocyte-specific protein tyrosine kinase (LCK); kinase ankyrin 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX 2); late glycosylation end product receptor (RAGE-1); renal ubiquitin 1 (RU 1); renal ubiquitin 2 (RU 2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; mutant heat shock protein 70-2 (mut hsp 70-2); CD79a; CD79b; CD72; leukocyte associated immunoglobulin-like receptor 1 (LAIR 1); an Fc fragment of IgA receptor (FCAR or CD 89); leukocyte immunoglobulin-like receptor subfamily a member 2 (LILRA 2); CD300 molecular-like family member f (CD 300 LF); c lectin domain family 12 member a (CLEC 12A); bone marrow stromal cell antigen 2 (BST 2); mucin-like hormone receptor-like 2 (EMR 2) containing EGF-like modules; lymphocyte antigen 75 (LY 75); glypican-3 (GPC 3); fc receptor like 5 (FCRL 5); immunoglobulin lambda-like polypeptide 1 (IGLL 1).

The CARs described herein can comprise an antigen binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein). In some embodiments, the antigen that supports a tumor is an antigen present on a stromal cell or Myeloid Derived Suppressor Cell (MDSC). Stromal cells may secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, the CAR-expressing cells destroy cells that support the tumor, thereby indirectly inhibiting tumor growth or survival.

In embodiments, the stromal cell antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST 2), fibroblast Activation Protein (FAP), and tenascin. In embodiments, the FAP-specific antibody is, competes for binding with, or has the same CDRs as, cetrimide. In embodiments, the MDSC antigen is selected from one or more of the following: CD33, CD11b, C14, CD15, and CD66b. Thus, in some embodiments, the tumor-supporting antigen is selected from one or more of the following: bone marrow stromal cell antigen 2 (BST 2), fibroblast Activation Protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b.

Antigen binding domain structure

In some embodiments, the antigen binding domain of the encoded CAR molecule comprises an antibody, an antibody fragment, scFv, fv, fab, (Fab') 2, a Single Domain Antibody (SDAB), a VH or VL domain, a camelidae VHH domain, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., lanzavecchia et al, eur.j. Immunol. [ journal of european immunology ]17,105 (1987)).

In some cases, scFv may be prepared according to methods known in the art (see, e.g., bird et al, (1988) Science [ Science ]242:423-426 and Huston et al, (1988) Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ] 85:5879-5883). ScFv molecules can be produced by joining VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly influence how the variable regions of the scFv fold and interact. Indeed, if a short polypeptide linker (e.g., between 5-10 amino acids) is employed, intra-strand folding may be prevented. Inter-strand folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientations and sizes, see, e.g., hollinger et al 1993Proc Natl Acad.Sci.U.S.A [ Proc. Natl. Acad. Sci. USA ]90:6444-6448, U.S. patent application publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. WO 2006/020258 and WO 2007/024715.

The scFv may comprise a linker having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequenceComprising a set of glycine and serine repeats, e.g. (Gly)₄ Ser) n, where n is a positive integer equal to or greater than 1 (SEQ ID NO: 232). In one embodiment, the linker may be (Gly)₄ Ser)₄ (SEQ ID NO: 230) or (Gly)₄ Ser)₃ (SEQ ID NO: 231). Variations in linker length can retain or enhance activity, resulting in superior efficacy in activity studies.

In another aspect, the antigen binding domain is a T cell receptor ("TCR") or a fragment thereof, such as a single chain TCR (scTCR). Methods for preparing such TCRs are known in the art. See, e.g., willemsen RA et al, gene Therapy [ Gene Therapy ]7:1369-1377 (2000); zhang T et al, cancer Gene Ther [ Cancer Gene therapy ]11:487-496 (2004); aggen et al, gene Ther [ Gene therapy ]19 (4): 365-74 (2012). For example, sctcrs can be engineered to contain the vα and vβ genes from T cell clones linked by a linker (e.g., a flexible peptide). This approach is very useful for targets associated with cancer that are themselves intracellular, however, fragments of this antigen (peptide) are presented on the surface of cancer cells via MHC.

In certain embodiments, the encoded antigen binding domain has 10^-4 M to 10^-8 Binding affinity KD of M.

In one embodiment, the encoded CAR molecule comprises an antigen binding domain having a binding affinity KD of 10 for a target antigen^-4 M to 10^-8 M, e.g. 10^-5 M to 10^-7 M, e.g. 10^-6 M or 10^-7 M. In one embodiment, the binding affinity of the antigen binding domain is at least 5-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or 1,000-fold lower than the binding affinity of a reference antibody (e.g., an antibody described herein). In one embodiment, the binding affinity of the encoded antigen binding domain is at least 5-fold lower than the binding affinity of a reference antibody (e.g., an antibody from which the antigen binding domain is derived). In one aspect, such antibody fragments are functional in that they provide a biological response, which may include, but is not limited to, activation of an immune response, signaling originating from their target antigenInhibition of the guide, inhibition of kinase activity, etc., as will be appreciated by the skilled artisan.

In one aspect, the antigen binding domain of the CAR is an scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

In one aspect, the antigen binding domain (e.g., scFv) of a CAR of the present disclosure is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell. In one aspect, the entire CAR construct of the disclosure is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in mammalian cells. Codon optimisation refers to the following findings: the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acid) in coding DNA varies among species. This codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods are known in the art and include, for example, the methods disclosed in at least U.S. Pat. nos. 5,786,464 and 6,114,148.

Antigen binding domains (and targeting antigens)

In one embodiment, the antigen binding domain to CD19 is an antigen binding portion (e.g., CDR) of a CAR, antibody, or antigen binding fragment thereof described, for example, in the following: PCT publication WO 2012/079000; PCT publication WO 2014/153270; kochenderfer, j.n. et al, j.immunother [ journal of immunotherapy ]32 (7), 689-702 (2009); kochenderfer, J.N., et al Blood [ Blood ],116 (20), 4099-4102 (2010); PCT publication WO 2014/031687; bejcek, cancer Research, 55,2346-2351,1995; or U.S. Pat. No. 7,446,190.

In one embodiment, the antigen binding domain to mesothelin is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described, for example, in PCT publication WO 2015/090230. In one embodiment, the antigen binding domain to mesothelin is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in, for example, PCT publication WO 1997/025068, WO 1999/028471, WO 2005/014652, WO 2006/099141, WO 2009/045957, WO 2009/068204, WO 2013/142034, WO 2013/040557, or WO 2013/063155. In one embodiment, the antigen binding domain to mesothelin is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in WO/2015/090230.

In one embodiment, the antigen binding domain to CD123 is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in, for example, PCT publication WO 2014/130635. In one embodiment, the antigen binding domain to CD123 is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in, for example, PCT publication WO 2014/138805, WO 2014/138819, WO 2013/173820, WO 2014/144622, WO 2001/66139, WO 2010/126066, WO 2014/144622, or US 2009/0252742. In one embodiment, the antigen binding domain to CD123 is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in WO/2016/028896.

In one embodiment, the antigen binding domain to egfrvlll is an antibody, antigen binding fragment or antigen binding portion (e.g. CDR) of a CAR described in, for example, WO/2014/130657.

In one embodiment, the antigen binding domain to CD22 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: haso et al Blood [ Blood ],121 (7): 1165-1174 (2013); wayne et al, clin Cancer Res [ clinical Cancer research ]16 (6): 1894-1903 (2010); kato et al, leuk Res [ leukemia study ]37 (1): 83-88 (2013); creative BioMart (creature biological Co., ltd.) MOM-18047-S (P).

In one embodiment, the antigen binding domain to CS-1 is an antigen binding portion (e.g., CDR) of erlotinib (BMS), see, e.g., tai et al, 2008, blood [ blood ]112 (4): 1329-37; tai et al, 2007, blood [ blood ]110 (5): 1656-63.

In one embodiment, the antigen binding domain for CLL-1 is an antigen binding portion (e.g., CDR) of an antibody obtainable from R & D company, electronic biosciences (ebiosciences), ai Bokang company (Abcam), such as PE-CLL1-hu catalog number 353604 (hundred-in biosciences); and PE-CLL1 (CLEC 12A) directory number 562566 (BD). In one embodiment, the antigen binding domain to CLL-1 is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in WO/2016/014535.

In one embodiment, the antigen binding domain to CD33 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: bross et al, clin Cancer Res [ clinical Cancer research ]7 (6): 1490-1496 (2001) (Gituzumab Ozomib, hP 67.6); caron et al, cancer Res [ Cancer Instructions ]52 (24): 6761-6767 (1992) (rituximab, huM 195); lapusan et al, investNew Drugs [ New drug Infinite (New drug Infinite) 30 (3): 1121-1131 (2012) (AVE 9633); aigner et al, leukemia [ Leukemia ]27 (5): 1107-1115 (2013) (AMG 330, CD33 BiTE); dutour et al, adv chemistry [ hematology progression ]2012:683065 (2012); and pizzila et al, leukemia [ Leukemia ] doi: 10.1038/Luc.2014.62 (2014). In one embodiment, the antigen binding domain to CD33 is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in WO/2016/014576.

In one embodiment, the antigen binding domain to GD2 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: mujoo et al, cancer Res [ Cancer Industry ]47 (4): 1098-1104 (1987); cheung et al, cancer Res [ Cancer Industry ]45 (6): 2642-2649 (1985); cheung et al, J Clin Oncol [ journal of clinical oncology ]5 (9): 1430-1440 (1987); cheung et al, J Clin Oncol 16 (9): 3053-3060 (1998); handgretinger et al Cancer Immunol Immunother [ cancer immunology and immunotherapy ]35 (3): 199-204 (1992). In some embodiments, the antigen binding domain to GD2 is an antigen binding portion of an antibody selected from the group consisting of: mabs 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see, e.g., WO 2012033885, WO 2013040371, WO 2013192294, WO 2013061273, WO 2013123061, WO 2013074916, and WO 201385552. In some embodiments, the antigen binding domain to GD2 is an antigen binding portion of an antibody described in U.S. publication No.: 20100150910 or PCT publication No.: WO 2011160119.

In one embodiment, the antigen binding domain to BCMA is the antigen binding portion (e.g., CDR) of an antibody described in, for example, WO 2012163805, WO 200112812, and WO 2003062401. In one embodiment, the antigen binding domain to BCMA is an antibody, antigen binding fragment, or antigen binding portion (e.g., CDR) of a CAR described in WO/2016/014565.

In one embodiment, the antigen binding domain to the Tn antigen is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US8,440,798; brooks et al, PNAS [ Proc. Natl. Acad. Sci. USA ]107 (22): 10056-10061 (2010); and Stone et al, oncomelanology [ tumor immunology ]1 (6): 863-873 (2012).

In one embodiment, the antigen binding domain to PSMA is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: parker et al, protein Expr Purif [ protein expression and purification ]89 (2): 136-145 (2013), US20110268656 (J591 ScFv); frigerio et al, european J Cancer [ J.European cancer ]49 (9): 2223-2232 (2013) (scFvD 2B); WO 2006125481 (mAbs 3/A12,3/E7 and 3/F11) and single chain antibody fragments (scFv A5 and D7).

In one embodiment, the antigen binding domain to ROR1 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: hudecek et al, clin Cancer Res [ clinical Cancer research ]19 (12): 3153-3164 (2013); WO 2011159847; and US 20130101607.

In one embodiment, the antigen binding domain directed against FLT3 is an antigen binding portion (e.g., CDR) of an antibody described in, for example, WO 2011076922, US 5777084, EP 0754230, US20090297529, as well as several commercial catalog antibodies (R & D company, electronic biosciences company, ai Bokang company).

In one embodiment, the antigen binding domain to TAG72 is an antigen binding portion (e.g., CDR) of the following antibody: antibodies described, for example, in Hombach et al, gastroenterology [ Gastroenterology ]113 (4): 1163-1170 (1997); and Abcam ab691.

In one embodiment, the antigen binding domain to FAP is an antigen binding portion (e.g., CDR) of an antibody that: antibodies described, for example, in Ostermann et al, clinical Cancer Research [ clinical cancer research ]14:4584-4592 (2008) (FAP 5), U.S. patent publication No. 2009/0304718; sibutrazol (see, e.g., hofheinz et al, oncology Research and Treatment [ oncology research and therapy ]26 (1), 2003); and Tran et al, J Exp Med [ journal of laboratory medicine ]210 (6): 1125-1135 (2013).

In one embodiment, the antigen binding domain to CD38 is an antigen binding portion (e.g., CDR) of the following antibody: daratumumab (see, e.g., groen et al, blood [ Blood ]116 (21): 1261-1262 (2010)); MOR202 (see, e.g., US 8,263,746); or antibodies described in US 8,362,211.

In one embodiment, the antigen binding domain to CD44v6 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Casucci et al, blood [ Blood ]122 (20): 3461-3472 (2013).

In one embodiment, the antigen binding domain for CEA is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Chuilewski et al, gastonterology [ gastroenterology ]143 (4): 1095-1107 (2012).

In one embodiment, the antigen binding domain to EPCAM is an antigen binding portion (e.g., CDR) of an antibody selected from the group consisting of: MT110, epCAM-CD3 bispecific Ab (see, e.g., clinicaltrias.gov/ct 2/show/NCT 00635596); ibrutinab; 3622W94; ING-1; and adalimumab (MT 201).

In one embodiment, the antigen binding domain to PRSS21 is an antigen binding portion (e.g., CDR) of an antibody described in: U.S. patent No.: 8,080,650.

In one embodiment, the antigen binding domain directed against B7H3 is the antigen binding portion (e.g., CDR) of antibody MGA271 (macrogenes).

In one embodiment, the antigen binding domain directed against KIT is an antigen binding portion (e.g., CDR) of an antibody described in, for example, US 7915391, US20120288506, and several commercial catalogue antibodies.

In one embodiment, the antigen binding domain to IL-13Ra2 is an antigen binding portion (e.g., CDR) of an antibody described in, for example, WO 2008/146911, WO 2004087758, several commercial catalogue antibodies, and antibodies in WO 2004087758.

In one embodiment, the antigen binding domain directed against CD30 is an antigen binding portion (e.g., CDR) of an antibody described in, for example, US 7090843B1 and EP 0805871.

In one embodiment, the antigen binding domain to GD3 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US 7253263; US 8,207,308; US 20120276046; EP 1013761; WO 2005035577; and US 6437098.

In one embodiment, the antigen binding domain to CD171 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Hong et al, J Immunothe J.Immunotherapy 37 (2): 93-104 (2014).

In one embodiment, the antigen binding domain for IL-11Ra is the antigen binding portion (e.g., CDR) of an antibody available from Ai Bokang (catalog No. ab 55262) or rofus biologies (Novus Biologicals) (catalog No. EPR 5446). In another embodiment, the antigen binding domain for IL-11Ra is a peptide, see, e.g., huang et al, cancer Res [ Cancer Industry ]72 (1): 271-281 (2012).

In one embodiment, the antigen binding domain to PSCA is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: morgenroth et al, prostate [ Prostate ]67 (10): 1121-1131 (2007) (scFv 7F 5); nejatollahi et al, J of Oncology journal of Oncology 2013 (2013), article ID 839831 (scFv C5-II); and U.S. patent publication No. 20090311181.

In one embodiment, the antigen binding domain to VEGFR2 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Chinnaamy et al, J Clin Invest [ journal of clinical research ]120 (11): 3953-3968 (2010).

In one embodiment, the antigen binding domain to lewis y is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: kelly et al Cancer Biother Radiopharm [ cancer biotherapy and radiopharmaceuticals ]23 (4): 411-423 (2008) (hu 3S193Ab (scFvs)); dolezal et al, protein Engineering [ protein engineering ]16 (1): 47-56 (2003) (NC 10 scFv).

In one embodiment, the antigen binding domain to CD24 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Maliar et al, gastroenterology 143 (5): 1375-1384 (2012).

In one embodiment, the antigen binding domain directed against PDGFR- β is the antigen binding portion (e.g., CDR) of antibody Abcam ab 32570.

In one embodiment, the antigen binding domain to SSEA-4 is the antigen binding portion (e.g., CDR) of antibody MC813 (Cell signaling), or other commercially available antibodies.

In one embodiment, the antigen binding domain directed against CD20 is the antigen binding portion (e.g., CDR) of the antibody rituximab, ofatuzumab, oreuzumab, veltuzumab, or GA 101.

In one embodiment, the antigen binding domain directed against folate receptor alpha is the antibody IMGN853 or the antigen binding portion (e.g., CDR) of the antibody described in: US 20120009181; US 4851332; LK26:US5952484.

In one embodiment, the antigen binding domain to ERBB2 (Her 2/neu) is the antigen binding portion (e.g., CDR) of the antibody trastuzumab, or pertuzumab.

In one embodiment, the antigen binding domain to MUC1 is an antigen binding portion (e.g., CDR) of antibody SAR 566658.

In one embodiment, the antigen binding domain directed against EGFR is the antibody cetuximab, panitumumab, zatuzumab, nituzumab, or an antigen-binding portion (e.g., CDR) of matuzumab.

In one embodiment, the antigen binding domain to NCAM is an antigen binding portion (e.g., CDR) of the following antibody: antibody clone 2-2B: MAB5324 (EMD Millipore).

In one embodiment, the antigen binding domain to ephrin B2 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in Abengozar et al, blood [ Blood ]119 (19): 4565-4576 (2012).

In one embodiment, the antigen binding domain to IGF-I receptor is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US 8344112 B2; EP 2322550A1; WO 2006/138315, or PCT/US2006/022995.

In one embodiment, the antigen binding domain to CAIX is an antigen binding portion (e.g., CDR) of antibody clone 303123 (R & D Systems ).

In one embodiment, the antigen binding domain to LMP2 is an antigen binding portion (e.g., CDR) of an antibody described in, for example, US 7,410,640, or US 20050129701.

In one embodiment, the antigen binding domain directed against gp100 is the antigen binding portion (e.g., CDR) of antibody HMB45, nkiβb, or an antibody described in WO 2013165940 or US 20130295007.

In one embodiment, the antigen binding domain to tyrosinase is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US 5843674; or US 19950504048.

In one embodiment, the antigen binding domain to EphA2 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Yu et al, mol Ther [ molecular therapy ]22 (1): 102-111 (2014).

In one embodiment, the antigen binding domain to GD3 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US 7253263; US 8,207,308; US 20120276046; EP1013761 A3;20120276046; WO 2005035577; or US 6437098.

In one embodiment, the antigen binding domain to fucosyl GM1 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US20100297138; or WO 2007/067992.

In one embodiment, the antigen binding domain to sLe is the antigen binding portion (e.g., CDR) of antibody G193 (for Lewis Y), see Scott AM et al, cancer Res [ Cancer Inquiry ]60:3254-61 (2000), also as described in Neeson et al, J Immunol [ J Immunol ]2013, 5 months 190 (conference abstract supplement) 177.10.

In one embodiment, the antigen binding domain directed against GM3 is the antigen binding portion (e.g., CDR) of antibody CA 2523449 (mAb 14F 7).

In one embodiment, the antigen binding domain to HMWMAA is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: kmiecik et al, oncoimiology [ tumor immunology ]3 (1) e27185 (2014) (PMID: 24575382) (mAb 9.2.27); US6528481; WO 2010033866; or US20140004124.

In one embodiment, the antigen binding domain to o-acetyl-GD 2 is an antigen binding portion (e.g. CDR) of antibody 8B 6.

In one embodiment, the antigen binding domain to TEM1/CD248 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: marty et al, cancer Lett [ Cancer flash ]235 (2): 298-308 (2006); zhao et al, J Immunol Methods J.Immunol.363 (2): 221-232 (2011).

In one embodiment, the antigen binding domain directed against CLDN6 is an antigen binding portion (e.g., CDR) of antibody IMAB027 (casimide pharmaceutical company (Ganymed Pharmaceuticals)), see, e.g., clinicaltrial.gov/show/NCT02054351.

In one embodiment, the antigen binding domain to the TSHR is an antigen binding portion (e.g. CDR) of an antibody described, for example, in the following: US 8,603,466; US 8,501,415; or US 8,309,693.

In one embodiment, the antigen binding domain to GPRC5D is the antigen binding portion (e.g., CDR) of the following antibody: antibody FAB6300A (R & D systems Co.); or LS-A4180 (Laishibang Biotechnology Co., ltd. (Lifespan Biosciences)).

In one embodiment, the antigen binding domain to CD97 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: US6,846,911; de Groot et al, JImmunol [ J.Immunol ]183 (6): 4127-4134 (2009); or antibodies from R & D MAB 3734.

In one embodiment, the antigen binding domain to ALK is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Mino-Kenudson et al, clin Cancer Res [ clinical Cancer research ]16 (5): 1561-1571 (2010).

In one embodiment, the antigen binding domain to polysialic acid is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Nagae et al, J Biol Chem [ journal of biochemistry ]288 (47): 33784-33796 (2013).

In one embodiment, the antigen binding domain to PLAC1 is an antigen binding portion (e.g., CDR) of an antibody described in: for example Ghods et al Biotechnol Appl Biochem [ Biochemical Biotechnology application ]2013doi:10.1002/bab.1177.

In one embodiment, the antigen binding domain to GloboH is the antigen binding portion of an antibody that: antibody VK9; or antibodies as described, for example, in Kudryashov V et al, glyconj J. [ journal of glycoconjugates ]15 (3): 243-9 (1998), lou et al, proc Natl Acad Sci USA [ Proc. Natl. Acad. Sci. USA ]111 (7): 2482-2487 (2014); MBr1: bremer E-G et al J Biol Chem [ journal of biochemistry ]259:14773-14777 (1984).

In one embodiment, the antigen binding domain to NY-BR-1 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Jager et al, appl Immunohistochem Mol Morphol [ application immunohistochemical molecular morphology ]15 (1): 77-83 (2007).

In one embodiment, the antigen binding domain to WT-1 is an antigen binding portion (e.g., CDR) of an antibody described in: for example, dao et al, sci Transl Med [ science conversion medical ]5 (176): 176ra33 (2013); or WO 2012/135854.

In one embodiment, the antigen binding domain for MAGE-A1 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Willemsen et al, J Immunol journal 174 (12): 7853-7858 (2005) (TCR-like scFv).

In one embodiment, the antigen binding domain to spimm protein 17 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in the following: song et al, target Oncol [ Target oncology ]2013, 8, 14 (PMID: 23943313); song et al, med Oncol [ medical oncology ]29 (4): 2923-2931 (2012).

In one embodiment, the antigen binding domain for Tie 2 is an antigen binding portion (e.g., CDR) of antibody AB33 (cell signaling technologies company (Cell Signalling Technology)).

In one embodiment, the antigen binding domain for MAD-CT-2 is an antigen binding portion (e.g., CDR) of an antibody described, for example, in PMID 2450952, US 7635753.

In one embodiment, the antigen binding domain for Fos-associated antigen 1 is an antigen binding portion (e.g., CDR) of antibody 12F9 (rofus biologies).

In one embodiment, the antigen binding domain to MelanA/MART1 is the antigen binding portion (e.g., CDR) of an antibody described in: EP 2514766 A2; or US 7,749,719.

In one embodiment, the antigen binding domain for a sarcoma translocation breakpoint is an antigen binding portion (e.g., CDR) of an antibody described in, for example, luo et al, EMBO mol. Med. [ EMBO molecular medicine ]4 (6): 453-461 (2012).

In one embodiment, the antigen binding domain directed against TRP-2 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Wang et al, J Exp Med [ journal of Experimental medicine ]184 (6): 2207-16 (1996).

In one embodiment, the antigen binding domain for CYP1B1 is the antigen binding portion (e.g., CDR) of an antibody described, for example, in Maecker et al, blood [ Blood ]102 (9): 3287-3294 (2003).

In one embodiment, the antigen binding domain directed against RAGE-1 is the antigen binding portion (e.g., CDR) of antibody MAB5328 (EMD Miibo).

In one embodiment, the antigen binding domain directed against human telomerase reverse transcriptase is the antigen binding portion (e.g., CDR) of the following antibody: antibody catalog number: LS-B95-100 (Laishibang biotechnology Co., ltd.)

In one embodiment, the antigen binding domain to the intestinal carboxylesterase is an antigen binding portion (e.g., CDR) of an antibody of the following: antibody 4F12: catalog number: LS-B6190-50 (Laishibang Biotech Co.).

In one embodiment, the antigen binding domain for mut hsp70-2 is an antigen binding portion (e.g., CDR) of the following antibody: antibodies (Laishibang Biotechnology Co., monoclonal: catalog number: LS-C133261-100 (Laishibang Biotechnology Co.).

In one embodiment, the antigen binding domain directed against CD79a is the antigen binding portion (e.g., CDR) of the following antibody: an antibody anti-CD79 a antibody [ HM47/A9] (ab 3121) available from Ai Bokang company; antibody CD79A antibody number 3351 available from cell signaling technologies company; or the antibody HPA 017748-anti-CD 79A antibody obtainable from Sigma Aldrich, which is produced from rabbit.

In one embodiment, the antigen binding domain to CD79b is the antigen binding portion (e.g., CDR) of the following antibody: antibody statin-perlattuzumab (polatuzumab vedotin) (anti-CD 79 b) (described in Dornan et al, "Therapeutic potential of an anti-CD79b anti-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma [ anti-CD79b antibody-drug conjugate anti-CD79b-vc-MMAE for the therapeutic potential for the treatment of non-Hodgkin lymphoma]"Blood]24 days of 2009 9 months; 114 (13) 2721-9.doi:10.1182/blood-2009-02-205500.Epub 2009 on 7-24), or bispecific antibody anti-CD79B/CD3 (described in "4507Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79B/CD3 As a Potential Therapy for B Cell Malignancies [4507T cell dependent Pre-clinical characterization of bispecific antibody anti-CD79B/CD 3) as potential therapy for B cell malignancy ]”Abstracts of 56^th ASH Annual Meeting and Exposition [ 56 th ASH annual meeting and exposition abstract ]]12 months, 6-9 days of 2014, san francisco, california).

In one embodiment, the antigen binding domain to CD72 is an antigen binding portion (e.g., CDR) of the following antibody: antibodies J3-109 (described in Myers and Uckun, "An anti-CD72 immunotoxin against therapy-return B-lineage acute lymphoblastic leukemia [ anti-CD72 immunotoxin anti-therapy refractory B-lineage acute lymphoblastic leukemia ]" Leuk Lymphoma [ leukemia Lymphoma ] month 6 1995; 18 (1-2): 119-22) or anti-CD72 (10D 6.8.1, mIgG 1) (described in Polson et al, "anti-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: target and Linker-Drug Selection [ Antibody-Drug conjugates for treating non-Hodgkin lymphomas: target and linker-Drug Selection ]" Cancer Res [ Cancer research ]2009 3 month 15 days 69; 2358).

In one embodiment, the antigen binding domain to LAIR1 is an antigen binding portion (e.g., CDR) of an antibody that: antibody ANT-301LAIR1 available from Prospec; or anti-human CD305 (LAIR 1) antibodies available from bai biotechnology company.

In one embodiment, the antigen binding domain to FCAR is the antigen binding portion (e.g., CDR) of antibody CD89/FCAR antibody (catalog No. 10414-H08H) available from Sino Biological Inc.

In one embodiment, the antigen binding domain directed against LILRA2 is the antigen binding portion (e.g., CDR) of the antibody LILRA2 monoclonal antibody (M17) (clone 3C 7) available from minodronate (Abnova), or the mouse anti-LILRA 2 antibody (monoclonal (2D 7)) available from leshibang biotechnology.

In one embodiment, the antigen binding domain to CD300LF is an antibody mouse anti-CMRF 35-like molecule 1 antibody available from bai biotechnology company (monoclonal [ UP-D2 ]); or an antigen binding portion (e.g., a CDR) of a rat anti-CMRF 35-like molecule 1 antibody (monoclonal [234903 ]) available from R & D systems company.

In one embodiment, the antigen binding domain to CLEC12A is an antigen binding portion (e.g., CDR) of the following antibody: antibody bispecific T cell engager (BiTE) scFv-antibodies and ADCs (described in Noordhuis et al, "Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1xCD3 BiTE Antibody [ by antibody-Drug-conjugates and ] Bispecific CLL-1xcd3 BiTE antibody targeting CLEC12A in acute myelogenous leukemia]”53^rd ASH Annual Meeting and Exposition [ 53 rd ASH annual meeting and exposition ]]10 to 13 days of 12 th 2011), and MCLA-117 (company Mei Lusi (Merus)).

In one embodiment, the antigen binding domain for BST2 (also known as CD 317) is an antigen binding portion (e.g., a CDR) of an antibody mouse anti-CD 317 antibody (monoclonal [3H4 ]) obtainable from Antibodies-Online or a mouse anti-CD 317 antibody (monoclonal [696739 ]) obtainable from R & D systems company.

In one embodiment, the antigen binding domain for EMR2 (also referred to as CD 312) is an antigen binding portion (e.g., a CDR) of an antibody mouse anti-CD 312 antibody (monoclonal [ LS-B8033 ]) available from Leisha Biotech, inc., or a mouse anti-CD 312 antibody (monoclonal [494025 ]) available from R & D systems, inc.

In one embodiment, the antigen binding domain to LY75 is an antigen binding portion (e.g., a CDR) of an antibody mouse anti-lymphocyte antigen 75 antibody available from EMD Milbot (monoclonal [ HD30 ]) or a mouse anti-lymphocyte antigen 75 antibody available from life technologies (Life Technologies) (monoclonal [ A15797 ]).

In one embodiment, the antigen binding domain to GPC3 is an antigen binding portion (e.g., CDR) of an antibody of: antibody hGC33 (described in Nakano K, ishiguro T, konishi H et al Generation of a humanized anti-Glypican 3antibody by CDR grafting and stability optimization [ production of humanized anti-Glypican 3antibody by CDR grafting and stability optimization ] anti-cancer Drugs [ anti-cancer drug ]2010 month 11; 21 (10): 907-916), or MDX-1414, HN3 or YP7 (all three antibodies described in Feng et al, "Glypican-3antibodies:a new therapeutic target for liver cancer) [ novel therapeutic target for Glypican-3 antibody: liver cancer ]" FEBS Lett. "European society of Biochemical Association" 2014, month 1; 588 (2): 377-82).

In one embodiment, the antigen binding domain to FCRL5 is the antigen binding portion (e.g., CDR) of an anti-FCRL 5 antibody described in the following: elkins et al, "FcRL5 as atarget of antibody-drug conjugates for the treatment of multiple myeloma [ FcRL5 as target for antibody-drug conjugate for treatment of multiple myeloma ]" Mol Cancer thers [ molecular Cancer therapeutics ]2012, month 10; 11 (10):2222-32. In one embodiment, the antigen binding domain to FCRL5 is the antigen binding portion (e.g., CDR) of an anti-FCRL 5 antibody described in the following: for example, WO 2001/038490, WO/2005/117986, WO 2006/039238, WO 2006/076691, WO 2010/114940, WO 2010/120561, or WO 2014/210064.

In one embodiment, the antigen binding domain to IGLL1 is an antigen binding portion (e.g., CDR) of the following antibody: antibody mouse anti-immunoglobulin lambda-like polypeptide 1 (monoclonal [ AT1G4 ]) available from Leisha Biotech company and mouse anti-immunoglobulin lambda-like polypeptide 1 antibody (monoclonal [ HSL11 ]) available from Baishibang Biotech company.

In one embodiment, the antigen binding domain comprises one, two, three (e.g., all three) heavy chain CDRs (HC CDR1, HC CDR2, and HC CDR 3) from the antibodies listed above, and/or one, two, three (e.g., all three) light chain CDRs (LC CDR1, LC CDR2, and LC CDR 3) from the antibodies listed above. In one embodiment, the antigen binding domain comprises the heavy chain variable region and/or variable light chain region of the antibodies listed above.

In another aspect, the antigen binding domain comprises a humanized antibody or antibody fragment. In some aspects, the non-human antibody is humanized, wherein specific sequences or regions of the antibody are modified to increase similarity to an antibody or fragment thereof naturally produced in a human. In one aspect, the antigen binding domain is humanized.

In embodiments, the antigen binding domain of a CAR (e.g., a CAR expressed by a cell of the disclosure) binds CD19. CD19 is found on B cells throughout lineage differentiation from the pre/progenitor B cell stage through the terminally differentiated plasma cell stage. In an embodiment, the antigen binding domain is a murine scFv domain that binds human CD19, such as the antigen binding domain of CTL019 (e.g., SEQ ID NO: 252). In embodiments, the antigen binding domain is a humanized antibody or antibody fragment derived from a murine CTL019 scFv (e.g., scFv domain). In embodiments, the antigen binding domain is a human antibody or antibody fragment that binds to human CD19. Exemplary scFv domains (and sequences thereof, e.g., CDR, VL, and VH sequences) that bind CD19 are provided in table 12 a. The scFv domain sequences provided in table 12a include a light chain variable region (VL) and a heavy chain variable region (VH). VL and VH are attached by a linker comprising the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 231), e.g., in the following orientations: VL-linker-VH.

TABLE 12a antigen binding domain that binds CD19

The sequences of CDR sequences of the scFv domains of the CD19 antigen binding domains provided in table 12a are shown in table 12b (for the heavy chain variable domain) and table 12c (for the light chain variable domain). "ID" represents the corresponding SEQ ID NO for each CDR.

TABLE 12b heavy chain variable domain CDRs

Description of the invention

FW

HCDR1

ID

HCDR2

ID

HCDR3

ID

mouse_CART 19

GVSLPDYGVS

319

VIWGSETTYYNSALKS

320

HYYYGGSYAMDY

246

Humanized_cart 19a

VH4

GVSLPDYGVS

319

VIWGSETTYYSSSLKS

296

HYYYGGSYAMDY

246

Humanized_cart 19b

VH4

GVSLPDYGVS

319

VIWGSETTYYQSSLKS

295

HYYYGGSYAMDY

246

Humanization_cart 19c

VH4

GVSLPDYGVS

319

VIWGSETTYYNSSLKS

284

HYYYGGSYAMDY

246

TABLE 12c light chain variable domain CDR

Description of the invention

FW

LCDR1

ID

LCDR2

ID

LCDR3

ID

mouse_CART19

RASQDISKYLN

251

HTSRLHS

250

QQGNTLPYT

247

Humanized_cart 19a

VK3

RASQDISKYLN

251

HTSRLHS

250

QQGNTLPYT

247

Humanized_cart 19b

VK3

RASQDISKYLN

251

HTSRLHS

250

QQGNTLPYT

247

Humanization_cart 19c

VK3

RASQDISKYLN

251

HTSRLHS

250

QQGNTLPYT

247

In embodiments, the antigen binding domain comprises an anti-CD 19 antibody or fragment thereof (e.g., scFv). For example, the antigen binding domain comprises the variable heavy and variable light chains listed in table 12d. The linker sequence connecting the variable heavy and variable light chains may be any of the linker sequences described herein, or alternatively may be GSTSGSGKPGSGEGSTKG (SEQ ID NO: 248). The light chain variable region and the heavy chain variable region of the scFv may be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

TABLE 12d additional anti-CD 19 antibody binding domains

In one embodiment, the CD19 binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR 1), light chain complementarity determining region 2 (LC CDR 2), and light chain complementarity determining region 3 (LC CDR 3) of a CD19 binding domain described herein (e.g., provided in table 12a or 15), and/or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR 1), heavy chain complementarity determining region 2 (HC CDR 2), and heavy chain complementarity determining region 3 (HC CDR 3) of a CD19 binding domain described herein (e.g., provided in table 12a or 16). In one embodiment, the CD19 binding domain comprises one, two or all of LC CDR1, LC CDR2 and LC CDR3 having any amino acid sequence as provided in table 12 c; and one, two or all of HC CDR1, HC CDR2 and HC CDR3 having any amino acid sequence as provided in table 12b.

The CAR may be constructed according to the present disclosure using any known CD19 CAR in the art, for example, any known CD19 antigen binding domain of a CD19 CAR. For example, LG-740; CD19 CAR is described in the following: U.S. patent No. 8,399,645; U.S. Pat. nos. 7,446,190; xu et al, leuk Lymphoma [ leukemia Lymphoma ]2013 54 (2): 255-260 (2012); cruz et al Blood 122 (17): 2965-2973 (2013); brentjens et al Blood 118 (18): 4817-4818 (2011); kochenderfer et al Blood 116 (20): 4099-102 (2010); kochenderfer et al Blood 122 (25): 4129-39 (2013); and 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) [ society for Gene and cell therapy (ASGCT) 16th annual meeting ] (5 months 15-18 days, salt lake City) 2013, abstract 10. In one embodiment, the antigen binding domain to CD19 is an antigen binding portion (e.g., CDR) of a CAR, antibody, or antigen binding fragment thereof described, for example, in the following: PCT publication WO 2012/079000; PCT publication WO 2014/153270; kochenderfer, j.n. et al, j.immunother [ journal of immunotherapy ]32 (7), 689-702 (2009); kochenderfer, J.N., et al Blood [ Blood ],116 (20), 4099-4102 (2010); PCT publication WO 2014/031687; bejcek, cancer Research, 55,2346-2351,1995; or U.S. Pat. No. 7,446,190.

In embodiments, the antigen binding domain of a CAR (e.g., a CAR expressed by a cell of the disclosure) binds BCMA. BCMA was found to be preferentially expressed in mature B lymphocytes. In an embodiment, the antigen binding domain is a murine scFv domain that binds to human BCMA. In embodiments, the antigen binding domain is a humanized antibody or antibody fragment (e.g., scFv domain) that binds human BCMA. In embodiments, the antigen binding domain is a human antibody or antibody fragment that binds to human BCMA. In an embodiment, an exemplary BCMACAR construct generated using VH and VL sequences from PCT publication WO 2012/0163805. In an example, additional exemplary BCMACAR constructs were generated using VH and VL sequences from PCT publication WO 2016/014565. In an embodiment, additional exemplary BCMACAR constructs are generated using VH and VL sequences from PCT publication WO 2014/122144. In embodiments, additional exemplary BCMACAR constructs are generated using the CAR molecule, and/or VH and VL sequences from PCT publication WO 2016/014789. In embodiments, additional exemplary BCMACAR constructs are generated using CAR molecules, and/or VH and VL sequences from PCT publication WO 2014/089335. In embodiments, additional exemplary BCMA CAR constructs are generated using CAR molecules from PCT publication WO 2014/140248, and/or VH and VL sequences.

Any BCMACAR known in the art, such as the BMCA antigen binding domain of any known BCMACAR, may be used according to the present disclosure. Such as those described herein.

Exemplary CAR molecules

In one aspect, a CAR (e.g., a CAR expressed by a cell of the disclosure) comprises a CAR molecule that contains an antigen binding domain that binds to a B cell antigen (e.g., CD19 or BCMA as described herein).

In one embodiment, the CAR comprises a CAR molecule comprising a CD19 antigen binding domain (e.g., a murine, human, or humanized antibody or antibody fragment that specifically binds CD 19), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain).

Exemplary CAR molecules described herein are provided in table 12e. The CAR molecule in table 12e comprises the amino acid sequence of a CD19 antigen binding domain, e.g., any of the CD19 antigen binding domains provided in table 12 a.

Table 12e exemplary CD19 CAR molecules

In one aspect, a CAR (e.g., a CAR expressed by a cell of the disclosure) comprises a CAR molecule comprising an antigen binding domain that binds to BCMA, e.g., a murine, human, or humanized antibody or antibody fragment that comprises a BCMA antigen binding domain (e.g., specifically binds BCMA (e.g., human BCMA)), a transmembrane domain, and an intracellular signaling domain (e.g., an intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain).

Exemplary CAR molecules for the CARs described herein are provided in table 1 of WO 2016/014565.

Transmembrane domain

Regarding the transmembrane domain, in various embodiments, the CAR can be designed to comprise a transmembrane domain attached to the extracellular domain of the CAR. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with an extracellular region of a transmembrane-derived protein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with an intracellular region of a transmembrane-protein-derived protein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is a domain associated with one of the other domains of the CAR, e.g., in one embodiment, the transmembrane domain can be from the same protein from which the signaling domain, co-stimulatory domain, or hinge domain is derived. In another aspect, the transmembrane domain is not derived from the same protein from which any other domain of the CAR is derived. In some cases, the transmembrane domains may be selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another CAR on the cell surface of the CAR-expressing cell. In various aspects, the amino acid sequence of the transmembrane domain can be modified or substituted so as to minimize interaction with the binding domain of a natural binding partner present in the same CAR-expressing cell.

The transmembrane domain may be derived from a natural source or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to one or more intracellular domains each time the CAR binds to a target. The transmembrane domains particularly used in the present disclosure may include at least one or more transmembrane regions such as the α, β or ζ chain of T cell receptors, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154. In some embodiments of the present invention, in some embodiments, the transmembrane domain may include at least one of, for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD 18), ICOS (CD 278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD160, CD19, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD C, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD 226), SLF 4 (CD 244, CD 2B 4), CD84, CD96 (Tactive) ACMA 1, CEBR 9, BLASTME 9, SLAMG 1, SLAMG 35, SLCD 35 (SLAMG 2), SLCD 35 (SLCD 35), SLAMG 2, SLCD 35 (SLAMG 2), SLCD 35 (SLASF 2), SLASGL 1, SLASGL 9, SLASGL 35 (SLASP 2) or SLASL 2 (SLASP 2).

In some cases, the transmembrane domain can be attached to the extracellular region of the CAR (e.g., the antigen binding domain of the CAR) by a hinge (e.g., a hinge from a human protein). For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker as described herein), a KIR2DS2 hinge, or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO. 265. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO 266.

In certain embodiments, the encoded transmembrane domain comprises an amino acid sequence of at least one, two or three modifications, but NO more than 20, 10 or 5 modified CD8 transmembrane domains having the amino acid sequence of SEQ ID NO 266, or a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO 266. In one embodiment, the encoded transmembrane domain comprises the sequence of SEQ ID NO. 266.

In other embodiments, the nucleic acid molecule encoding the CAR comprises a nucleotide sequence of the CD8 transmembrane domain, e.g., a sequence comprising SEQ ID No. 267 or SEQ ID No. 304, or a sequence thereof having at least 95% identity.

In certain embodiments, the encoded antigen binding domain is linked to the transmembrane domain by a hinge region. In one embodiment, the encoded hinge region comprises the amino acid sequence of a CD8 hinge, e.g., SEQ ID NO. 265; or the amino acid sequence of an IgG4 hinge, e.g., SEQ ID NO. 268, or a sequence having at least 95% identity to SEQ ID NO. 265 or SEQ ID NO. 268. In other embodiments, the nucleic acid sequence encoding the hinge region comprises the sequence of SEQ ID NO:269 or SEQ ID NO:270 corresponding to a CD8 hinge or an IgG4 hinge, respectively, or a sequence having at least 95% identity to SEQ ID NO:269 or 270.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of amino acid sequence ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 268). In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID NO: 270).

In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence of RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH (SEQ ID NO: 271). In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO: 272).

In one aspect, the transmembrane domain may be recombinant, in which case it will predominantly comprise hydrophobic residues such as leucine and valine. In one aspect, triplets of phenylalanine, tryptophan and valine can be found at each end of the recombinant transmembrane domain.

Optionally, a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length can form a linkage between the transmembrane domain and cytoplasmic region of the CAR. Glycine-serine doublets provide particularly suitable linkers. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 273). In some embodiments, the linker is encoded by the nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 274).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Signaling domains

In embodiments of the disclosure having intracellular signaling domains, such domains may contain, for example, one or more of a primary signaling domain and/or a co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain.

Intracellular signaling sequences within the cytoplasmic portion of the CARs of the disclosure can be linked to each other in random or specified order. Optionally, a short oligopeptide or polypeptide linker, for example, between 2 and 10 amino acids in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) may form a linkage between intracellular signaling sequences. In one embodiment, glycine-serine doublets may be used as suitable linkers. In one embodiment, a single amino acid (e.g., alanine, glycine) may be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed to comprise two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signaling domains. In embodiments, two or more (e.g., 2, 3, 4, 5, or more) co-stimulatory signaling domains are separated by a linker molecule (e.g., a linker molecule described herein). In one embodiment, the intracellular signaling domain comprises two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

Primary signaling domain

The primary signaling domain modulates primary activation of the TCR complex either in a stimulatory manner or in an inhibitory manner. The primary intracellular signaling domain acting in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

Examples of ITAMs containing primary intracellular signaling domains particularly useful in the present disclosure include those that: cd3ζ, common fcrγ (FCER 1G), fcγriia, fcrβ (fcεr1b), cd3γ, cd3δ, cd3ε, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3- ζ.

In one embodiment, the encoded primary signaling domain comprises a functional signaling domain of cd3ζ. The encoded CD3 zeta primary signaling domain may comprise at least one, two or three modified, but not more than 20, 10 or 5 modified amino acid sequences with the amino acid sequence of SEQ ID NO:275 or SEQ ID NO:276, or a sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:275 or SEQ ID NO: 276. In some embodiments, the encoded primary signaling domain comprises the sequence of SEQ ID NO:275 or SEQ ID NO: 276. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises the sequence of SEQ ID NO:277, SEQ ID NO:303, or SEQ ID NO:278, or a sequence thereof having at least 95% identity.

Costimulatory signaling domains

In some embodiments, the encoded intracellular signaling domain comprises a costimulatory signaling domain. For example, the intracellular signaling domain may comprise a primary signaling domain and a costimulatory signaling domain. In some embodiments, the encoded costimulatory signaling domain comprises a functional signaling domain of a protein selected from one or more of the following: CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B-H3, a ligand that specifically binds to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF 1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2 Rbeta, IL2 Rgamma, IL7 Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGATNM, CD11B, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, TRAA-1, GB7, RANFR 2, RANE/226, DNF 1, DNF 4, CD8 beta, IL2 Rbeta, IL2 Rgamma, IL7R gamma, IL7R alpha, IL7 alpha, ITGA4, CD49A 4, CD 35A 4, IA4, CD49A 6, CD49, SLA-9, SLA-35C 6, CD49, SLCD 35C 9, SLCD 35C 6, SLCD 35, SLASL, SLSLCD 35 (SLB 6) 6, SLSLSLSLSLSLSLB 62 9 (35) 6) 2, SLSLSLSLSLSLSLB 69 1, SLSLSLSLB 69 (35).

In certain embodiments, the encoded costimulatory signaling domain comprises at least one, two, or three modifications, but NO more than 20, 10, or 5 modified amino acid sequences having the amino acid sequence of SEQ ID NO:279 or SEQ ID NO:280, or a sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:279 or SEQ ID NO: 280. In one embodiment, the encoded costimulatory signaling domain comprises the sequence of SEQ ID NO. 279 or SEQ ID NO. 280. In other embodiments, the nucleic acid sequence encoding the costimulatory signaling domain comprises the sequence of SEQ ID NO:281, SEQ ID NO:305, or SEQ ID NO:282, or a sequence thereof having at least 95% identity.

In other embodiments, the encoded intracellular domain comprises the sequence of SEQ ID NO:279 or SEQ ID NO:280 and the sequence of SEQ ID NO:275 or SEQ ID NO:276, wherein the sequence comprising the intracellular signaling domain is expressed in the same frame and as a single polypeptide chain.

In certain embodiments, the nucleic acid sequence encoding the intracellular signaling domain comprises the sequence of SEQ ID NO:281, SEQ ID NO:305, or SEQ ID NO:282, and a sequence having at least 95% identity thereto, and the sequence of SEQ ID NO:277, SEQ ID NO:306, or SEQ ID NO:278, or a sequence having at least 95% identity thereto.

In some embodiments, the nucleic acid molecule further encodes a leader sequence. In one embodiment, the leader sequence comprises the sequence of SEQ ID NO: 283.

In one aspect, the intracellular signaling domain is designed to comprise a signaling domain of CD3- ζ and a signaling domain of CD 28. In one aspect, the intracellular signaling domain is designed to comprise a signaling domain of CD 3-zeta and a signaling domain of 4-1 BB. In one aspect, the signaling domain of 4-1BB is the signaling domain of SEQ ID NO. 279. In one aspect, the signaling domain of CD 3-zeta is the signaling domain of SEQ ID NO: 275.

In one aspect, the intracellular signaling domain is designed to comprise a signaling domain of CD3- ζ and a signaling domain of CD 27. In one aspect, the signaling domain of CD27 comprises the amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEP ACSP (SEQ ID NO: 280). In one aspect, the signaling domain of CD27 is encoded by the nucleic acid sequence of CAACGAAGGAAATATAGATCAAACAAAGGAGAAAGTCCTGTGGAGCCTGCAGAGCCTTGTCGTTACAGCTGCCCCAGGGAGGAGGAGGGCAGCACCATCCCCATCCAGGAGGATTACCGAAAACCGGAGCCTGCCTGCTCCCCC (SEQ ID NO: 282).

Carrier body

In another aspect, the disclosure relates to a vector comprising a nucleic acid sequence encoding a CAR described herein. In one embodiment, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In one embodiment, the vector is a lentiviral vector. These vectors, or portions thereof, can be used, among other things, to generate template nucleic acids, as described herein, for use with the CRISPR systems described herein. Alternatively, these vectors can be used to deliver nucleic acids directly to cells, such as immune effector cells, e.g., T cells, e.g., allogeneic T cells (independent of the CRISPR system).

The present disclosure also provides vectors into which the DNA of the present disclosure is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from tumor retroviruses such as murine leukemia virus in that they can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity. The retroviral vector may also be, for example, a gamma retroviral vector. The gamma retroviral vector may include, for example, a promoter, a packaging signal (ψ), a Primer Binding Site (PBS), one or more (e.g., two) Long Terminal Repeats (LTRs), and a transgene of interest (e.g., a gene encoding a CAR). The gamma retroviral vector may lack viral structural genes (e.g., gag, pol, and env). Exemplary gamma retrovirus vectors include Murine Leukemia Virus (MLV), spleen Focus Forming Virus (SFFV), and myeloproliferative sarcoma virus (MPSV), as well as vectors derived therefrom. Other gamma retroviral vectors are described, for example, in Tobias Maetzig et al, "Gammaretroviral Vectors:biology, technology and Application [ gamma retroviral vectors: biology/technology and application ] "Viruses @ [ virus ] month 6 of 2011; 3 (6):677-713.

In another embodiment, the vector comprising a nucleic acid encoding a desired CAR of the present disclosure is an adenovirus vector (A5/35). In another embodiment, expression of the nucleic acid encoding the CAR can be accomplished using transposons such as sleeping beauty systems, cresser, CAS9, and zinc finger nucleases. See June et al 2009Nature Reviews Immunology [ review of natural immunology ]9.10:704-716.

Nucleic acids can be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors and sequencing vectors.

Disclosed herein are methods for producing an in vitro transcribed RNA CAR. The disclosure also includes CAR-encoding RNA constructs that can be transfected directly into cells. Methods of generating mRNA for use in transfection may include In Vitro Transcription (IVT) of a template with specially designed primers followed by addition of polyA to generate a construct containing 3' and 5' untranslated sequences ("UTRs"), 5' caps and/or Internal Ribosome Entry Sites (IRES), nucleic acid to be expressed, and polyA tail, typically 50-2000 bases in length (SEQ ID NO: 310). The RNA thus produced can be used to efficiently transfect different cell types. In one aspect, the template includes the sequence of the CAR.

Non-viral delivery methods

In some aspects, nucleic acids encoding the CARs described herein can be delivered into a cell or tissue or subject using non-viral methods.

In some embodiments, the non-viral method includes the use of transposons (also referred to as transposable elements). In some embodiments, a transposon is a piece of DNA that can insert itself into one location in the genome, e.g., a piece of DNA that can self-replicate and insert its copy into the genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another location in the genome. For example, a transposon comprises a DNA sequence consisting of an inverted repeat sequence flanking a gene for transposition.

In some embodiments, cells, e.g., T cells or NK cells, expressing a CAR described herein are generated by using a combination of gene insertion (using SBTS) and gene editing (using a nuclease (e.g., zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas system, or engineered meganuclease re-engineered homing endonuclease)).

In some embodiments, cells of the disclosure, e.g., T cells or NK cells, e.g., allogeneic T cells (e.g., as described herein) (e.g., expressing a CAR as described herein) are produced by contacting these cells with (a) a composition comprising one or more gRNA molecules (e.g., as described herein) and one or more Cas molecules (e.g., cas9 molecules) (e.g., as described herein), and (b) a nucleic acid comprising a nucleic acid encoding a CAR sequence (e.g., as described herein) (e.g., a template nucleic acid molecule as described herein). Without being bound by theory, the composition of (a) above will induce a break at or near the genomic DNA targeted by the targeting domain of one or more gRNA molecules, and the nucleic acid of (b) will be incorporated (e.g., partially or fully) into the genome at or near the break, such that upon integration, the encoded CAR molecule is expressed. In embodiments, expression of the CAR will be controlled by a promoter endogenous to the genome or other regulatory element (e.g., a promoter that controls expression of a gene from the nucleic acid inserted into (b)). In other embodiments, the nucleic acid of (b) further comprises a promoter (e.g., as described herein) and/or other regulatory element (e.g., EF 1-a promoter) operably linked to the sequence encoding the CAR such that expression of the CAR upon integration is under the control of the promoter and/or other regulatory element. With respect to the use of a CRISPR/Cas9 system (e.g., as described herein) to direct the incorporation of a nucleic acid sequence encoding a CAR (e.g., as described herein), additional features of the disclosure are described elsewhere in the application, e.g., in the section regarding gene insertion and homologous recombination. In embodiments, the composition of a) above is a composition comprising RNP (containing one or more gRNA molecules). In embodiments, RNPs comprising grnas targeting unique target sequences are introduced into cells simultaneously, e.g., as a mixture of RNPs comprising one or more grnas. In an embodiment, RNPs comprising grnas targeting unique target sequences are sequentially introduced into cells.

In some embodiments, the use of non-viral delivery methods allows reprogramming of cells, such as T cells or NK cells, and infusion of these cells directly into a subject. Advantages of non-viral vectors include, but are not limited to, easy and relatively low cost production of sufficient amounts required to meet patient populations, stability during storage, and lack of immunogenicity.

Promoters

In one embodiment, the vector further comprises a promoter. In some embodiments, the promoter is selected from the group consisting of EF-1 promoter, CMV IE gene promoter, EF-1 alpha promoter, ubiquitin C promoter, or phosphoglycerate kinase (PGK) promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises the sequence of SEQ ID NO: 285.

Host cells for CAR expression

As described above, in some aspects, the disclosure relates to a cell, e.g., an immune effector cell (e.g., a population of cells, e.g., an immune effector cell population), comprising a nucleic acid molecule, CAR polypeptide molecule, or vector as described herein.

In certain aspects of the present disclosure, any number of techniques known to those skilled in the art (e.g., ficoll^TM Isolation) immune effector cells, such as T cells, are obtained from blood units collected from a subject. In a preferred aspect, cells from the circulating blood of the individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis can be washed to remove plasma fractions and optionally placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the invention, the cells are washed with Phosphate Buffered Saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many, if not all, divalent cations.

An initial activation step in the absence of calcium may result in amplified activation. As will be readily appreciated by one of ordinary skill in the art, the washing step may be accomplished by methods known to those of ordinary skill in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., cobe 2991 cell processor, baxter CytoMate, or Haemonetics Cell Saver 5) according to manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers, such as, for example, ca-free, mg-free PBS, crow's A, or other saline solutions with or without buffers. Alternatively, the unwanted components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

It will be appreciated that the methods of the application may utilize medium conditions comprising 5% or less (e.g., 2%) human AB serum, and use known medium conditions and compositions, such as those described below: smith et al, "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement [ ex vivo expansion of human T cells using novel Xeno CTS immune cell-free serum replacement for adoptive immunotherapy ]" Clinical & Translational Immunology [ Clinical and transplantation immunology ] (2015) 4, e31; doi 10.1038/cti.2014.31.

In one aspect, by, for example, PERCOL^TM Gradient centrifugation or panning by countercurrent centrifugation lyses erythrocytes and depletes monocytes, separating T cells from peripheral blood lymphocytes.

The methods described herein can include, for example, selecting a particular subpopulation of immune effector cells (e.g., T cells) that is a T regulatory cell depleted population, cd25+ depleted cells, using, for example, a negative selection technique (e.g., as described herein). Preferably, the cell population with T regulatory depletion contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% cd25+ cells.

In one embodiment, T regulatory cells (e.g., CD25+ T cells) are removed from the population using an anti-CD 25 antibody or fragment thereof, or CD25 binding ligand IL-2. In one embodiment, the anti-CD 25 antibody or fragment thereof, or CD25 binding ligand, is conjugated to, or otherwise coated on, a substrate (e.g., a bead). In one embodiment, an anti-CD 25 antibody or fragment thereof is conjugated to a substrate as described herein.

In one embodiment, a method from Miltenyi is used^TM The CD25 depleting agent of (a) removes T regulatory cells (e.g., cd25+ T cells) from the population. In one embodiment, the ratio of cells to CD25 depleting agent is 1e7 cells to 20uL, or 1e7 cells to 15uL, or 1e7 cells to 10uL, or 1e7 cells to 5uL, or 1e7 cells to 2.5uL, or 1e7 cells to 1.25uL. In one embodiment, for example, for T regulatory cells (e.g.Cd25+) depletion, greater than 5 hundred million cells/ml was used. In further aspects, a cell concentration of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to be depleted comprises about 6x 10⁹ Cd25+ T cells. In other aspects, the population of immune effector cells to be depleted comprises about 1x 10⁹ To 1x10¹⁰ Cd25+ T cells, and any integer value therebetween. In one embodiment, the resulting population of T regulatory depleted cells has a size of 2X 10⁹ T regulatory cells (e.g., cd25+ cells) or less (e.g., 1x 10)⁹ 5x 10⁸ 1x10⁸ 5x 10⁷ 1x10⁷ Cd25+ cells or less).

In one embodiment, T regulatory cells (e.g., cd25+ cells) are removed from the population using a clinic system with depletion tubing (e.g., like tubing 162-01). In one embodiment, the clinic mac system is run on a DEPLETION setting (such as, for example, delete 2.1).

Without wishing to be bound by a particular theory, reducing the level of negative regulator of immune cells in a subject (e.g., reducing unwanted immune cells (e.g., T_REG Cells) may reduce the risk of relapse in the subject. For example, deplete T_REG Methods for cells are known in the art. Reducing T_REG Methods of cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25 depletion, and combinations thereof.

In some embodiments, the method of manufacturing comprises reducing (e.g., depleting) T prior to manufacturing the CAR-expressing cells_REG Number of cells. For example, the method of manufacture comprises contacting a sample (e.g., an apheresis sample) with an anti-GITR antibody and/or an anti-CD 25 antibody (or fragment thereof, or CD25 binding ligand), e.g., to deplete T prior to manufacture of a CAR expressing cell (e.g., T cell, NK cell) product_REG And (3) cells.

In an embodiment, prior to collecting cells for CAR-expressing cell product manufacture,by one or more reduction of T_REG Cell therapy pre-treats the subject, thereby reducing the risk of relapse of the subject's treatment of the CAR-expressing cells. In an embodiment, T is reduced_REG Methods of cells include, but are not limited to, administering one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof to a subject. Administration of one or more of cyclophosphamide, anti-GITR antibodies, CD25 depletion, or a combination thereof can occur before, during, or after infusion of the CAR-expressing cell product.

In embodiments, the subject is pre-treated with cyclophosphamide prior to collecting cells for CAR-expressing cell product manufacture, thereby reducing the risk of relapse of the subject's treatment of CAR-expressing cells. In embodiments, the subject is pre-treated with an anti-GITR antibody prior to collecting cells for CAR-expressing cell product manufacture, thereby reducing the risk of relapse of the subject's treatment of CAR-expressing cells.

In one embodiment, the cell population to be removed is neither regulatory T cells, or tumor cells, nor cells that otherwise negatively affect the expansion and/or function of CART cells (e.g., cells that express CD14, CD11b, CD33, CD15, or other markers expressed by potentially immunosuppressive cells). In one embodiment, it is contemplated that such cells are removed in parallel with regulatory T cells and/or tumor cells, or after depletion, or in another order.

The methods described herein may include more than one selection step, such as more than one depletion step. Enrichment of the T cell population by negative selection may be accomplished, for example, with a combination of antibodies directed against surface markers specific for the cells of the negative selection. One approach is cell sorting and/or selection by negative magnetic immunoadsorption or flow cytometry using a mixture of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich for cd4+ cells by negative selection, a monoclonal antibody mixture may include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD 8.

The methods described herein can further comprise removing cells from a population that expresses a tumor antigen (e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11 b), thereby providing a population of T-regulatory depleted (e.g., cd25+ depleted) and tumor antigen depleted cells suitable for expressing a CAR (e.g., a CAR described herein). In one embodiment, cells expressing tumor antigens are removed simultaneously with T regulatory, e.g., cd25+ cells. For example, an anti-CD 25 antibody or fragment thereof, and an anti-tumor antigen antibody or fragment thereof may be attached to the same substrate (e.g., bead) that may be used to remove cells, or an anti-CD 25 antibody or fragment thereof, or an anti-tumor antigen antibody or fragment thereof, may be attached to separate beads (a mixture thereof may be used to remove cells). In other embodiments, the removal of T regulatory cells (e.g., cd25+ cells) and the removal of cells expressing tumor antigens are sequential and may occur, for example, in any order.

Also provided is a method comprising: cells (e.g., one or more of pd1+ cells, LAG3+ cells, and tim3+ cells) are removed from a population expressing a checkpoint inhibitor (e.g., a checkpoint inhibitor as described herein), thereby providing a population of T-regulatory depleted (e.g., cd25+ depleted) cells and checkpoint inhibitor depleted cells (e.g., pd1+, LAG3+ and/or tim3+ depleted cells). Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In one embodiment, cells expressing a checkpoint inhibitor are removed simultaneously with T-modulating, e.g., cd25+ cells. For example, the anti-CD 25 antibody or fragment thereof, and the anti-checkpoint inhibitor antibody or fragment thereof may be attached to the same bead that may be used to remove cells, or the anti-CD 25 antibody or fragment thereof, and the anti-checkpoint inhibitor antibody or fragment thereof, may be attached to separate beads (a mixture thereof may be used to remove cells). In other embodiments, the removal of T regulatory cells (e.g., cd25+ cells) and the removal of cells expressing the checkpoint inhibitor are continuous and may occur, for example, in any order.

The methods described herein mayTo include a positive selection step. For example, the anti-CD 3/anti-CD 28 (e.g., 3X 28) conjugate beads (e.g.)M-450CD3/CD 28T) for a period of time sufficient to positively select the desired T cells. In one embodiment, the period of time is about 30 minutes. In further embodiments, the period of time ranges from 30 minutes to 36 hours or more and all integer values therebetween. In further embodiments, the period of time is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the period of time is 10 to 24 hours, such as 24 hours. In any case where fewer T cells are present, such as in isolating Tumor Infiltrating Lymphocytes (TILs) from tumor tissue or immunocompromised individuals, longer incubation times may be used to isolate T cells as compared to other cell types. In addition, the use of longer incubation times may increase the efficiency of cd8+ T cell capture. Thus, by simply shortening or extending the time to bind T cells to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as further described herein), T cell subsets can be preferentially selected or targeted at the beginning of culture or at other points in time during the process. In addition, by increasing or decreasing the ratio of anti-CD 3 and/or anti-CD 28 antibodies on the bead or other surface, T cell subsets can be preferentially selected or targeted at the beginning of the culture or at other desired time points.

In one embodiment, a population of T cells may be selected that express one or more of the following: IFN-gamma, TNF alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules (e.g., other cytokines). Methods of screening for cell expression can be carried out, for example, by PCT publication No. WO 2013/126712.

To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles (e.g., beads)) can be varied. In certain aspects, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the concentration of cells) to ensure maximum contact of the cells and beads. For example, in one aspect, a concentration of 100 hundred million cells/ml, 90 hundred million cells/ml, 80 hundred million cells/ml, 70 hundred million cells/ml, 60 hundred million cells/ml, or 50 hundred million cells/ml is used. In one aspect, a concentration of 10 hundred million cells/ml is used. In yet another aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further aspects, a concentration of 125 or 150 million cells/ml may be used.

The use of high concentrations can lead to increased cell yield, cell activation, and cell expansion. In addition, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest (e.g., CD28 negative T cells), or cells from samples where many tumor cells are present (e.g., leukemia blood, tumor tissue, etc.). Such cell populations may be of therapeutic value and are desirable. For example, the use of high concentrations of cells allows for more efficient selection of cd8+ T cells that typically have weaker CD28 expression.

In related aspects, it may be desirable to use lower cell concentrations. Interactions between particles and cells are minimized by significantly diluting the mixture of T cells and surfaces (e.g., particles (e.g., beads)). This selects for cells that express a large number of desired antigens to be bound to the particle. For example, cd4+ T cells express higher levels of CD28 and are captured more efficiently than cd8+ T cells at diluted concentrations. In one aspect, the concentration of cells used is 5x 10⁶ /ml. In other aspects, the concentration used may be from about 1x 10⁵ Ml to 1x 10⁶ /ml, and any integer value therebetween.

In other aspects, the cells may be incubated on a rotator at different rates for different lengths of time at 2 ℃ to 10 ℃ or room temperature.

T cells used for stimulation may also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step to remove plasma and platelets, the cells may be suspended in a frozen solution. While many freezing solutions and parameters are known in the art and will be useful in this case, one approach involves using PBS containing 20% DMSO and 8% human serum albumin, or a medium containing 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or a medium containing 31.25% brio-a, 31.25% glucose 5%, 0.45% NaCl, 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing medium containing, for example, hespan and brio-a, and then freezing the cells to-80 ℃ at a rate of 1 ° per minute and storing in the gas phase of a liquid nitrogen storage tank. Other methods of controlling freezing may be used, with immediate uncontrolled freezing at-20 ℃ or in liquid nitrogen.

In certain aspects, the cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation using the methods of the present disclosure.

It is also contemplated in the context of the present disclosure to collect a blood sample or apheresis product from a subject for a period of time prior to the expansion of cells as described herein may be required. Thus, the source of cells to be expanded can be collected at any necessary point in time, and the desired cells (e.g., T cells) isolated and frozen for subsequent use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one aspect, the blood sample or apheresis is taken from a substantially healthy subject. In certain aspects, a blood sample or apheresis is taken from a substantially healthy subject at risk of developing a disease, but not yet suffering from a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, T cells may be expanded, frozen, and used at a later time. In certain aspects, a sample is collected from a patient after diagnosis of a particular disease as described herein, but shortly before any treatment. In a further aspect, cells are surgically isolated from a blood sample or apheresis of a subject prior to any number of relevant treatment modalities including, but not limited to, treatment with: agents (e.g., natalizumab), efalizumab, antiviral agents), chemotherapy, radiation, immune suppressors (e.g., cyclosporine, azathioprine, methotrexate, mycophenolic acid esters, and FK 506), antibodies or other immune scavengers (e.g., CAMPATH, anti-CD 3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR 901228), and radiation.

In further aspects of the disclosure, T cells are obtained directly from the patient after the subject is taken out of treatment with functional T cells. In this regard, it has been observed that after certain cancer treatments (particularly treatments with drugs that disrupt the immune system), the quality of the T cells obtained may be optimal or improved due to their ability to expand ex vivo shortly after the patient will typically recover from the treatment period. As such, after ex vivo procedures using the methods described herein, these cells may be in a preferred state to enhance implantation and in vivo expansion. Thus, it is contemplated in the context of the present disclosure to collect blood cells during this recovery phase, including T cells, dendritic cells, or other cells of the hematopoietic lineage. Furthermore, in certain aspects, mobilization (e.g., mobilization with GM-CSF) and modulation schemes can be used to create conditions in a subject in which the re-proliferation, recycling, regeneration, and/or expansion of a particular cell type is beneficial, particularly in a time window determined after treatment. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

In one embodiment, immune effector cells expressing a CAR molecule (e.g., a CAR molecule described herein) are obtained from a subject who has received a low immunopotentiating dose of an mTOR inhibitor. In embodiments, a population of immune effector cells (e.g., T cells) engineered to express a CAR is harvested after a sufficient time (or after a sufficient dose of a low immunopotentiating dose of an mTOR inhibitor) such that the level of PD1 negative immune effector cells (e.g., T cells), or the ratio of PD1 negative immune effector cells (e.g., T cells)/PD 1 positive immune effector cells (e.g., T cells) in or harvested from the subject has been increased at least transiently.

In other embodiments, a population of immune effector cells (e.g., T cells) that have been, or are to be, engineered to express a CAR can be treated ex vivo by contacting with an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells (e.g., T cells), or increases the ratio of PD1 negative immune effector cells (e.g., T cells)/PD 1 positive immune effector cells (e.g., T cells).

In one embodiment, the T cell population is diacylglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA, or proteins, or have reduced, or inhibited DGK activity. DGK-deficient cells may be produced by genetic methods, such as administration of an RNA interfering agent (e.g., siRNA, shRNA, miRNA) to reduce or prevent DGK expression. Alternatively, DGK-deficient cells may be produced by treatment with a DGK inhibitor as described herein.

In one embodiment, the T cell population is Ikaros-deficient. Ikaros-defective cells include cells that do not express Ikaros RNA, or protein, or have reduced or inhibited Ikaros activity, and Ikaros-defective cells may be produced by genetic methods, such as the administration of an RNA interfering agent (e.g., siRNA, shRNA, miRNA) to reduce or prevent Ikaros expression. Alternatively, ikaros-deficient cells may be produced by treatment with Ikaros inhibitors (e.g., lenalidomide).

In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros deficient cells may be produced by any of the methods described herein.

In embodiments, NK cells are obtained from a subject. In another embodiment, the NK cell is an NK cell line such as the NK-92 cell line (Conkwest Co.).

In some aspects, the cells of the disclosure (e.g., immune effector cells of the disclosure, e.g., CAR-expressing cells of the disclosure) are induced pluripotent stem cells ("ipscs") or Embryonic Stem Cells (ESCs), or T cells produced from (e.g., distinct from) ipscs and/or ESCs. For example, ipscs may be produced from peripheral blood T lymphocytes (e.g., peripheral blood T lymphocytes isolated from healthy volunteers) by methods known in the art. Likewise, such cells may be differentiated into T cells by methods known in the art. See, e.g., thomeli m. Et al, nat. Biotechnol, [ natural biotechnology ], pages 31,928-933 (2013); doi 10.1038/nbt.2678; WO 2014/165707.

In another embodiment, the tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) of the present disclosure are used in combination with one or more of the therapeutic agents listed in table 13 or the patents and patent applications cited in table 13 to treat cancer. Each publication (including all structural formulas therein) is listed in table 13.

Table 13.

Estrogen receptor antagonists

In some embodiments, an Estrogen Receptor (ER) antagonist is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the estrogen receptor antagonist is a selective estrogen receptor degradation agent (SERD). SERD is an estrogen receptor antagonist that binds to a receptor and causes, for example, degradation or downregulation of the receptor (Boer K. Et al, (2017) Therapeutic Advances in Medical Oncology [ tumor medical treatment progression ]9 (7): 465-479). ER is a hormone-activated transcription factor that is important for, for example, the growth, development and physiology of the human reproductive system. ER is activated by, for example, the hormone estrogen (17β estradiol). ER expression and signaling are involved in cancers (e.g., breast cancer), such as ER positive (er+) breast cancer. In some embodiments, the SERD is selected from LSZ102, fulvestrant, cloth Li Siqun (brilanestant), or elanistrant (elacestrant).

Exemplary estrogen receptor antagonists

In some embodiments, the SERD comprises a compound disclosed in International application publication No. WO 2014/130310. In some embodiments, the SERD comprises LSZ102.LSZ102 has the chemical name: (E) -3- (4- ((2- (2- (1, 1-difluoroethyl) -4-fluorophenyl) -6-hydroxybenzo [ b ] thiophen-3-yl) oxy) phenyl) acrylic acid.

Other exemplary estrogen receptor antagonists

In some embodiments, the SERD comprises fulvestrant (CAS registry number 129453-61-8) or a compound disclosed in International application publication number WO 2001/051056. Fulvestrant is also known as ICI 182780, ZM 182780,Or (7α,17β) -7- {9- [ (4, 5-pentafluoropentyl) sulfinyl]Nonyl } estra-1, 3,5 (10) -triene-3, 17-diol. Fulvestrant is a high affinity estrogen receptor antagonist with an IC50 of 0.29 nM.

In some embodiments, the SERD comprises melarsoprol (CAS registry number 722533-56-4) or a compound disclosed in U.S. Pat. No. 7,612,114. Elastine is also known as RAD1901, ER-306323 or (6R) -6- {2- [ ethyl ({ 4- [2- (ethylamino) ethyl ] phenyl } methyl) amino ] -4-methoxyphenyl } -5,6,7, 8-tetrahydronaphthalen-2-ol. Melarsoprol is an orally bioavailable, non-steroidal-binding Selective Estrogen Receptor Modulator (SERM) and SERD. Elapril is also disclosed in, for example, garner F et al, (2015) Anticancer Drugs [ Anticancer drug ]26 (9): 948-56.

In some embodiments, the SERD is cloth Li Siqun (CAS registry number 1365888-06-7) or a compound disclosed in International application publication number WO 2015/136017. Cloth Li Siqun is also known as GDC-0810, ARN810, RG-6046, RO-7056118 or (2E) -3- {4- [ (1E) -2- (2-chloro-4-fluorophenyl) -1- (1H-indazol-5-yl) but-1-en-1-yl ] phenyl } prop-2-enoic acid. Cloth Li Siqun is a next generation oral bioavailable selective SERD with an IC50 of 0.7 nM. Cloth Li Siqun is also disclosed, for example, in Lai A. Et al (2015) Journal of Medicinal Chemistry [ J. Pharmaceutical chemistry ]58 (12): 4888-4904.

In some embodiments, the SERD is selected from RU 58686, GW7604, AZD9496, bazedoxifene, pirenzfen (pipendoxicene), arzoxifene, OP-1074, or acobifene, e.g., as disclosed in McDonell et al (2015) Journal of Medicinal Chemistry [ J.pharmaceutical chemistry ]58 (12) 4883-4887. Other exemplary estrogen receptor antagonists are disclosed, for example, in WO 2011/156518, WO 2011/159769, WO 2012/037410, WO 2012/037411, and US 2012/007465.

CDK4/6 inhibitors

In some embodiments, inhibitors of cyclin dependent kinase 4 or 6 (CDK 4/6) are used in combination with TGF-beta inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) to treat diseases (e.g., cancer). In some embodiments, the CDK4/6 inhibitor is selected from rebaudinib, abeyance (abemaciclib) (gili corporation) or pamphlet Bai Xili.

Exemplary CDK4/6 inhibitors

In some embodiments, the CDK4/6 inhibitor comprises Rabociclib (CAS registry number 1211441-98-3) or a compound disclosed in U.S. Pat. Nos. 8,415,355 and 8,685,980.

In some embodiments, the CDK4/6 inhibitor comprises a compound disclosed in International application publication No. WO 2010/020675, and U.S. Pat. Nos. 8,415,355 and 8,685,980.

In some embodiments, the CDK4/6 inhibitor comprises Rabociclib (CAS registry number 1211441-98-3). Rabociclib is also known as LEE011,Or 7-cyclopentyl-N, N-dimethyl-2- ((5- (piperazin-1-yl) pyridin-2-yl) amino) -7H-pyrrolo [2,3-d]Pyrimidine-6-carboxamide.

Other exemplary CDK4/6 inhibitors

In some embodiments, the CDK4/6 inhibitor comprises Abeli (CAS registry number 1231929-97-7). Abeli is also known as LY835219 or N- [5- [ (4-ethyl-1-piperazinyl) methyl ] -2-pyridinyl ] -5-fluoro-4- [ 4-fluoro-2-methyl-1- (1-methylethyl) -1H-benzimidazol-6-yl ] -2-pyrimidinamine. Abeli is a CDK inhibitor selective for CDK4 and CDK6 and is disclosed, for example, in Torres-Guzman R et al (2017) Oncostarget [ tumor target ] 10.18632/oncotargett.17778.

In some embodiments, the CDK4/6 inhibitor comprises Pa Bai Xili (CAS registry number 571190-30-2). Pa Bai Xili also referred to as PD-0332991,Or 6-acetyl-8-cyclopentyl-5-methyl-2- { [5- (1-piperazinyl) -2-pyridinyl]Amino } pyrido [2,3-d]Pyrimidin-7 (8H) -ones. Pampers Bai Xili inhibit CDK4 (with an IC50 of 11 nM) and inhibit CDK6 (with an IC50 of 16 nM), and are disclosed, for example, in Finn et al (2009) Breast Cancer Research [ breast cancer research ]11(5):R77。

CXCR2 inhibitors

In some embodiments, inhibitors of chemokine (C-X-C motif) receptor 2 (CXCR 2) are used in combination with tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) to treat a disease (e.g., cancer). In some embodiments, the CXCR2 inhibitor is selected from 6-chloro-3- ((3, 4-dioxo-2- (pentan-3-ylamino) cyclobut-1-en-1-yl) amino) -2-hydroxy-N-methoxy-N-methylbenzenesulfonamide, dani Li Xing (danirixin), repairixin, or novirixin (navalixin).

Exemplary CXCR2 inhibitors

In some embodiments, the CXCR2 inhibitor comprises compounds disclosed in U.S. patent nos. 7989497, 8288588, 8329754, 8722925, 9115087, U.S. application publication nos. US2010/0152205, US2011/0251205, and US2011/0251206, and international application publication nos. WO 2008/061740, WO 2008/061741, WO 2008/062026, WO 2009/106539, WO 2010/0631802, WO 2012/062713, WO 2013/16108, WO 2010/015613, and WO 2013/030803. In some embodiments, the CXCR2 inhibitor comprises 6-chloro-3- ((3, 4-dioxo-2- (pentan-3-ylamino) cyclobut-1-en-1-yl) amino) -2-hydroxy-N-methoxy-N-methylbenzenesulfonamide or a choline salt thereof. In some embodiments, the CXCR2 inhibitor comprises a 6-chloro-3- ((3, 4-dioxo-2- (pentan-3-ylamino) cyclobut-1-en-1-yl) amino) -2-hydroxy-N-methoxy-N-methylbenzenesulfonamide choline salt. In some embodiments, the CXCR2 inhibitor is 2-hydroxy-N, N-trimethylethyl-1-ammonium 3-chloro-6- ({ 3, 4-dioxo-2- [ (pentan-3-yl) amino ] cyclobut-1-en-1-yl } amino) -2- (N-methoxy-N-methylsulfinyl) phenol ester (i.e., 6-chloro-3- ((3, 4-dioxo-2- (pentan-3-ylamino) cyclobut-1-en-1-yl) amino) -2-hydroxy-N-methoxy-N-methylbenzenesulfonamide choline salt) and has the following chemical structure:

Other exemplary CXCR2 inhibitors

In some embodiments, the CXCR2 inhibitor comprises dani Li Xing (CAS registry number 954126-98-8). Danirisin is also known as GSK1325756 or 1- (4-chloro-2-hydroxy-3-piperidin-3-ylsulfonylphenyl) -3- (3-fluoro-2-methylphenyl) urea. Darnixin is disclosed, for example, in Miller et al Eur J Drug Metab Pharmacokinet [ J. European drug metabolism and pharmacokinetics ] (2014) 39:173-181; and Miller et al BMC Pharmacology and Toxicology [ BMC pharmacology and toxicology ] (2015), 16:18.

In some embodiments, the CXCR2 inhibitor comprises repairisin (CAS registry number 266359-83-5). Repairisin is also known as repitaxin or (2R) -2- [4- (2-methylpropyl) phenyl ] -N-methylsulfonylpropionamide. Repairisin is a non-competitive allosteric inhibitor of CXCR 1/2. Repairisin is disclosed, for example, in Zarbock et al, br J Pharmacol [ journal of pharmacology, uk ]2008;155 (3):357-64.

In some embodiments, the CXCR2 inhibitor comprises fluvalirudin. Navalidly also known as MK-7123, SCH 527123, PS291822, or 2-hydroxy-N, N-dimethyl-3- [ [2- [ [ (1R) -1- (5-methylfuran-2-yl) propyl ] amino ] -3, 4-dioxocyclobuten-1-yl ] amino ] benzamide. Novalacin is disclosed, for example, in Ning et al Mol Cancer therer [ molecular Cancer therapeutics ]2012;11 (6):1353-64.

CSF-1/1R binding agents

In some embodiments, a CSF-1/1R binding agent is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the CSF-1/1R binding agent is selected from inhibitors of macrophage colony-stimulating factor (M-CSF), e.g., monoclonal antibodies to M-CSF or Fab (e.g., MCS 110), CSF-1R tyrosine kinase inhibitors (e.g., 4- ((2- (((1R, 2R) -2-hydroxycyclohexyl) amino) benzo [ d ] thiazol-6-yl) oxy) -N-methylpyridine amide or BLZ 945), receptor tyrosine kinase inhibitors (RTKs) (e.g., peidatinib) or antibodies targeting CSF-1R (e.g., mi Tuozhu mab (emacuzumab) or FPA 008). In some embodiments, the CSF-1/1R inhibitor is BLZ945. In some embodiments, the CSF-1/1R binding agent is MCS110. In other embodiments, the CSF-1/1R binding agent is perlitinib.

Exemplary CSF-1 binding agents

In some embodiments, the CSF-1/1R binding agent comprises an inhibitor of macrophage colony-stimulating factor (M-CSF). M-CSF is sometimes also referred to as CSF-1. In certain embodiments, the CSF-1/1R binding agent is an antibody to CSF-1 (e.g., MCS 110). In other embodiments, the CSF-1/1R binding agent is an inhibitor of CSF-1R (e.g., BLZ 945).

In some embodiments, the CSF-1/1R binding agent comprises a monoclonal antibody to M-CSF or a Fab (e.g., MCS110/H-RX 1) or binding agent to CSF-1, as disclosed in International application publication Nos. WO 2004/045532 and WO 2005/068503 (including H-RX1 or 5H4, e.g., antibody molecules or Fab fragments directed against M-CSF) and US 9079956.

TABLE 13a amino acid and nucleotide sequences of exemplary anti-M-CSF antibody molecules (MCS 110)

In another embodiment, the CSF-1/1R binding agent comprises a CSF-1R tyrosine kinase inhibitor, 4- ((2- (((1R, 2R) -2-hydroxycyclohexyl) amino) benzo [ d ] thiazol-6-yl) oxy) -N-methylpyridine amide (BLZ 945), or a compound disclosed in international application publication No. WO 2007/121484 and U.S. patent nos. 7,553,854, 8,173,689 and 8,710,048.

Other exemplary CSF-1/1R binding agents

In some embodiments, the CSF-1/1R binding agent comprises certidine (CAS registry number 1029044-16-3). Pedaltinib is also known as PLX3397 or 5- ((5-chloro-1H-pyrrolo [2,3-b ] pyridin-3-yl) methyl) -N- ((6- (trifluoromethyl) pyridin-3-yl) methyl) pyridin-2-amine. Pedaltinib is a small molecule Receptor Tyrosine Kinase (RTK) inhibitor of KIT, CSF1R and FLT 3. FLT3, CSF1R and FLT3 are overexpressed or mutated in many cancer cell types and play a major role in tumor cell proliferation and metastasis. PLX3397 can bind to and inhibit phosphorylation of stem cytokine receptor (KIT), colony stimulating factor-1 receptor (CSF 1R) and FMS-like tyrosine kinase 3 (FLT 3), which may lead to inhibition of tumor cell proliferation and down-regulation of macrophages, osteoclasts and mast cells involved in osteolytic metastatic disease.

In some embodiments, the CSF-1/1R binding agent is emituzumab. The E Mi Tuozhu monoclonal antibody is also called RG7155 or RO5509554. The monoclonal antibody Mi Tuozhu was a humanized IgG1 mAb-targeted CSF1R. In some embodiments, the CSF-1/1R binding agent is FPA008.FPA008 is a humanized mAb that inhibits CSF1R.

A2aR antagonists

In some embodiments, an adenosine A2a receptor (A2 aR) antagonist (e.g., an inhibitor of the A2aR pathway, such as an adenosine inhibitor, e.g., an inhibitor of A2aR or CD-73) is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the A2aR antagonist is selected from the group consisting of PBF509/NIR178 (Palobiofma)/North Co., ltd.), CPI444/V81444 (Kawass (Corvus)/Gentek Co.), AZD4635/HTL-1071 (Aoslera/Haplotai (Heptares)), vipatadian (Vipatdan) (Lei Dasai/Cinnan (Redox/Juno)), GBV-2034 (Globavir), AB928 (Axobiosciences (Arcus Biosciences)), theophylline, itracine (Kyowa) and Kyowa (Kyowa Hakko Kogyo), tozadan/SYN-115 (Acorda), KW-6356 (Kyowa), ST-4206 (Likeshan bioscience (Leadiant Biosciences), and Primer/420814 (Schkrigy/Xie Lin).

Exemplary A2aR antagonists

In some embodiments, the A2aR antagonist comprises PBF509 (NIR 178) or a compound disclosed in U.S. patent No. 8,796,284 or international application publication No. WO 2017/025918. The PBF509 (NIR 178) is also known as NIR178.

Other exemplary A2aR antagonists

In certain embodiments, the A2aR antagonist comprises CPI444/V81444.CPI-444 and other A2aR antagonists are disclosed in International application publication No. WO 2009/156737. In certain embodiments, the A2aR antagonist is (S) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine. In certain embodiments, the A2aR antagonist is (R) -7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine, or a racemate thereof. In certain embodiments, the A2aR antagonist is 7- (5-methylfuran-2-yl) -3- ((6- (((tetrahydrofuran-3-yl) oxy) methyl) pyridin-2-yl) methyl) -3H- [1,2,3] triazolo [4,5-d ] pyrimidin-5-amine.

In certain embodiments, the A2aR antagonist is AZD 4635/HTL-1071. A2aR antagonists are disclosed in international application publication No. WO 2011/095625. In certain embodiments, the A2aR antagonist is 6- (2-chloro-6-methylpyridin-4-yl) -5- (4-fluorophenyl) -1,2, 4-triazin-3-amine.

In certain embodiments, the A2aR antagonist is ST-4206 (Lidean biosciences). In certain embodiments, the A2aR antagonist is an A2aR antagonist described in us patent No. 9,133,197.

In certain embodiments, the A2aR antagonist is an A2aR antagonist described in U.S. patent nos. 8,114,845 and 9,029,393, U.S. application publication nos. 2017/0015758 and 2016/0129108.

In some embodiments, the A2aR antagonist is itrafylline (CAS registry number 155270-99-8). Itratheophylline is also known as KW-6002 or 8- [ (E) -2- (3, 4-dimethoxyphenyl) vinyl ] -1, 3-diethyl-7-methyl-3, 7-dihydro-1H-purine-2, 6-dione. For example, itratheophylline is disclosed in Lewitt et al, (2008) Annals of Neurology [ neurological yearbook ]63 (3): 295-302).

In some embodiments, the A2aR antagonist is tolzadi nan (Biotie). Tolzadir is also known as SYN115 or 4-hydroxy-N- (4-methoxy-7-morpholin-4-yl-1, 3-benzothiazol-2-yl) -4-methylpiperidine-1-carboxamide. Tolzadi's blockade of endogenous adenosine at the A2a receptor results in enhanced dopamine effect at the D2 receptor and inhibition of glutamate at the mGluR5 receptor. In some embodiments, the A2aR antagonist is Prmidannan (CAS registry number 377727-87-2). Pralidoxime is also known as SCH 420814 or 2- (2-furyl) -7- [2- [4- [4- (2-methoxyethoxy) phenyl ] -1-piperazinyl ] ethyl ] 7H-pyrazolo [4,3-e ] [1,2,4] triazolo [1,5-c ] pyrimidin-5-amine. Pryidi's south was developed as a drug that acts as a potent and selective antagonist of the adenosine A2A receptor.

In some embodiments, the A2aR antagonist is verapamil. Wipaman is also known as BIIB014, V2006, or 3- [ (4-amino-3-methylphenyl) methyl ] -7- (furan-2-yl) triazolo [4,5-d ] pyrimidin-5-amine. Other exemplary A2aR antagonists include, for example, ATL-444, MSX-3, SCH-58261, SCH-412,348, SCH-442,416, VER-6623, VER-6947, VER-7835, CGS-15943, and ZM-241,385.

In some embodiments, the A2aR antagonist is an A2aR pathway antagonist (e.g., a CD-73 inhibitor, such as an anti-CD 73 antibody) is MEDI9447.MEDI9447 is a monoclonal antibody specific for CD 73. Extracellular production of adenosine by CD73 targeting can reduce the immunosuppressive effects of adenosine. MEDI9447 is reported to have a range of activities, for example, inhibition of CD73 exonuclease activity, alleviation of AMP-mediated lymphocyte suppression, and inhibition of syngeneic tumor growth. MEDI9447 can drive changes in both bone marrow and lymphoinfiltrating leukocyte populations in the tumor microenvironment. These changes include, for example, increases in CD8 effector cells and activated macrophages, and decreases in the proportion of myeloid-derived suppressor cells (MDSCs) and regulatory T lymphocytes.

IDO inhibitors

In some embodiments, inhibitors of indoleamine 2, 3-dioxygenase (IDO) and/or tryptophan 2, 3-dioxygenase (TDO) are used in combination with tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) to treat diseases (e.g., cancer). In some embodiments, the IDO inhibitor is selected from (4E) -4- [ (3-chloro-4-fluoroanilino) -nitrosomethylene ] -1,2, 5-oxadiazol-3-amine (also known as epocoadstat (epacoadstat) or INCB 24360), endomod (), (1-methyl-D-tryptophan), α -cyclohexyl-5H-imidazo [5,1-a ] isoindole-5-ethanol (also known as NLG 919), endomod, and BMS-986205 (previously known as F001287).

Exemplary IDO inhibitors

In some embodiments, the IDO/TDO inhibitor is endomod (neolin gene company (New Link Genetics)). The D isomer of endomod, 1-methyl-tryptophan, is an orally administered small molecule indoleamine 2, 3-dioxygenase (IDO) pathway inhibitor that disrupts the mechanism of tumor evasion immune-mediated destruction.

In some embodiments, the IDO/TDO inhibitor is NLG919 (neolin gene). NLG919 is a potent IDO (indoleamine- (2, 3) -dioxygenase) pathway inhibitor with a Ki/EC50 of 7nM/75nM in a cell-free assay.

In some embodiments, the IDO/TDO inhibitor is edostatin (CAS registry number 1204669-58-8). Epdostat is also known as INCB24360 or INCB024360 (available from Saint Co.). Epidorestat is a potent and selective indoleamine 2, 3-dioxygenase (IDO 1) inhibitor with an IC50 of 10nM and high selectivity for other related enzymes, such as IDO2 or tryptophan 2, 3-dioxygenase (TDO).

In some embodiments, the IDO/TDO inhibitor is F001287 (foster/BMS). F001287 is a small molecule inhibitor of indoleamine 2, 3-dioxygenase 1 (IDO 1).

STING agonists

In some embodiments, STING agonists are used in combination with tgfβ inhibitors (and/or PD1, PD-L1, or PD-L2 inhibitors) to treat a disease (e.g., cancer). In some embodiments, the STING agonist is a cyclic dinucleotide, e.g., a cyclic dinucleotide comprising a purine or pyrimidine nucleobase (e.g., an adenosine, guanine, uracil, thymine, or cytosine nucleobase). In some embodiments, the nucleobases of the cyclic dinucleotides comprise the same nucleobase or different nucleobases.

In some embodiments, the STING agonist comprises an adenosine or guanosine nucleobase. In some embodiments, the STING agonist comprises one adenosine nucleobase and one guanosine nucleobase. In some embodiments, the STING agonist comprises two adenosine nucleobases or two guanosine nucleobases.

In some embodiments, the STING agonist comprises a modified cyclic dinucleotide, e.g., comprises a modified nucleobase, a modified ribose, or a modified phosphoester linkage. In some embodiments, the modified cyclic dinucleotide comprises a modified phosphoester linkage, such as a phosphorothioate linkage.

In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) having a 2',5' or 3',5' phosphate linkage. In some embodiments, the STING agonist comprises a cyclic dinucleotide (e.g., a modified cyclic dinucleotide) having Rp or Sp stereochemistry around the phosphoester linkage.

In some embodiments, the STING agonist is MK-1454 (merck). MK-1454 is a cyclic dinucleotide stimulator of an interferon gene (STING) agonist that activates the STING pathway. Exemplary STING agonists are disclosed, for example, in PCT publication No. WO 2017/027645.

Galectin inhibitors

In some embodiments, a galectin (e.g., galectin-1 or galectin-3) inhibitor is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the combination comprises a galectin-1 inhibitor and a galectin-3 inhibitor. In some embodiments, the combination comprises a bispecific inhibitor (e.g., a bispecific antibody molecule) that targets both galectin-1 and galectin-3. In some embodiments, the galectin inhibitor is selected from an anti-galectin antibody molecule, GR-MD-02 (galectin therapeutics company (Galectin Therapeutics)), galectin-3C (Mandal Med), anginex, or OTX-008 (Weng Keai Stokes company (Oncoethix), merck company). Galectins are a family of proteins that bind to beta-galactosidase sugars.

The galectin family of proteins comprises at least galectin-1, galectin-2, galectin-3, galectin-4, galectin-7, and galectin-8. Galectins are also called S-type lectins and are soluble proteins with, for example, intracellular and extracellular functions.

Galectin-1 and galectin-3 are highly expressed in different tumor types. Galectin-1 and galectin-3 can promote angiogenesis and/or reprogram bone marrow cells to a pre-tumor phenotype, e.g., enhance immunosuppression of bone marrow cells. Soluble galectin-3 can also bind and/or inactivate infiltrating T cells.

Exemplary galectin inhibitors

In some embodiments, the galectin inhibitor is an antibody molecule. In embodiments, the antibody molecule is a monospecific antibody molecule and binds to a single epitope. For example, a monospecific antibody molecule having multiple immunoglobulin variable domain sequences, each binding to the same epitope. In embodiments, the galectin inhibitor is an anti-galectin (e.g., anti-galectin-1 or anti-galectin-3) antibody molecule. In some embodiments, the galectin inhibitor is an anti-galectin-1 antibody molecule. In some embodiments, the galectin inhibitor is an anti-galectin-3 antibody molecule.

In embodiments, the antibody molecule is a multi-specific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In embodiments, the first epitope and the second epitope overlap. In embodiments, the first epitope and the second epitope do not overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the multispecific antibody molecule comprises a third, fourth, or fifth immunoglobulin variable domain. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule, a trispecific antibody molecule, or a tetraspecific antibody molecule.

In embodiments, the galectin inhibitor is a multispecific antibody molecule. In embodiments, the multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies are specific for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In embodiments, the first and second epitopes are on the same antigen (e.g., the same protein (or subunit of a multimeric protein)). In embodiments, the first epitope and the second epitope overlap. In embodiments, the first epitope and the second epitope do not overlap. In embodiments, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In embodiments, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In an embodiment, the bispecific antibody molecule comprises a half-antibody having binding specificity for a first epitope and a half-antibody having binding specificity for a second epitope. In embodiments, the bispecific antibody molecule comprises a half-antibody, or fragment thereof, having binding specificity for a first epitope and a half-antibody, or fragment thereof, having binding specificity for a second epitope. In embodiments, the bispecific antibody molecule comprises an scFv, or fragment thereof, having binding specificity for a first epitope and an scFv, or fragment thereof, having binding specificity for a second epitope. In embodiments, the galectin inhibitor is a bispecific antibody molecule. In embodiments, the first epitope is located on galectin-1 and the second epitope is located on galectin-3.

Protocols for the production of bispecific or heterodimeric antibody molecules are known in the art; these schemes include, but are not limited to: the "knob in a hole" pathway, for example as described in US 5731168; electrostatically directed Fc pairing as described, for example, in WO 09/089004, WO 06/106905 and WO 2010/129304; chain exchange engineering domain (SEED) heterodimer formation as described, for example, in WO 07/110205; fab arm exchange as described for example in WO 08/119353, WO 2011/131746 and WO 2013/060867; diabody conjugates, for example using heterobifunctional reagents having amine-reactive groups and thiol-reactive groups, are cross-linked by antibodies to produce bispecific structures as described, for example, in US 4433059; bispecific antibody determinants produced by recombination of half antibodies (heavy-light chain pairs or Fab) from different antibodies by cycles of reduction and oxidation of disulfide bonds between the two heavy chains, as described for example in US 4444878; trifunctional antibodies, for example three Fab' fragments crosslinked by thiol-reactive groups, as described for example in US 5273743; biosynthesis of binding proteins, e.g. scFv pairs crosslinked by a C-terminal tail, preferably by disulfide or amine reactive chemical crosslinking, as described for example in US 5534254; bifunctional antibodies, e.g. Fab fragments with different binding specificities, dimerized by leucine zippers (e.g. c-fos and c-jun) that have replaced constant domains, as described for example in US 5582996; bispecific and oligospecific monovalent and oligovalent receptors, for example VH-CH1 regions of two antibodies (two Fab fragments), which VH-CH1 regions are linked by a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody (typically with an associated light chain), as described for example in US 5591828; bispecific DNA-antibody conjugates, e.g. antibodies or Fab fragments, are cross-linked by double-stranded segments of DNA, as described for example in US 5635602; bispecific fusion proteins, for example expression constructs comprising two scFv (with a hydrophilic helical peptide linker between them) and one fully constant region, as described for example in US 5637481; multivalent and multispecific binding proteins, such as polypeptide dimers having a first domain of an Ig heavy chain variable region binding region and a second domain of an Ig light chain variable region binding region, are commonly referred to as diabodies (also disclosing higher order structures, resulting in bispecific, trispecific or tetraspecific molecules) as described, for example, in US 5837242; miniantibody constructs having linked VL and VH chains (which are further linked to an antibody hinge region and a CH3 region with a peptide spacer), which can dimerise to form bispecific/multivalent molecules, as described for example in US 5837821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or not linked at all at any orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described for example in US 5844094; a string of VH domains (or VL domains in family members) linked by peptide bonds to C-terminal crosslinkable groups which are further associated with the VL domains to form a series of FV (or scFv) as described, for example, in US 5864019; and single chain binding polypeptides having both VH and VL domains linked via peptide linkers are combined into multivalent structures by non-covalent or chemical cross-linking to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures using scFV or diabody type formats, as described, for example, in US 5869620. Further exemplary multispecific and bispecific molecules and methods for their preparation are described, for example, in U.S. Pat. No. 3,814,393,956, U.S. Pat. No. 4,4639, 6239259, U.S. Pat. No. 5, 6294353, U.S. Pat. No. 4, 6333396, U.S. Pat. No. 3, 6511663, U.S. Pat. No. 2,372, 6743896, U.S. Pat. No. 5, 6833441, U.S. Pat. No. 5, 7129330, U.S. Pat. No. 5, 7521056, U.S. Pat. No. 7527787, U.S. Pat. No. 4, 7534866, U.S. Pat. No. 2,004587 A1, U.S. 2002/076406 A1, U.S. 2002/103345 A1, U.S. 2003/207346A1, U.S. Pat. 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In other embodiments, the anti-galectin (e.g., anti-galectin-1 or anti-galectin-3) antibody molecule (e.g., a monospecific, bispecific, or multispecific antibody molecule) is covalently linked (e.g., fused) to another partner (protein, e.g., as a fusion molecule, such as a fusion protein). In one embodiment, the bispecific antibody molecule has a first binding specificity for a first target (e.g., for galectin-1) and a second binding specificity for a second target (e.g., galectin-3).

The present invention provides isolated nucleic acid molecules encoding the above antibody molecules, vectors and host cells thereof. Nucleic acid molecules include, but are not limited to, RNA, genomic DNA, and cDNA.

In some embodiments, the galectin inhibitor is a peptide, such as a protein, that can bind to and inhibit galectin (e.g., galectin-1 or galectin-3) function. In some embodiments, the galectin inhibitor is a peptide that can bind to and inhibit galectin-3 function. In some embodiments, the galectin inhibitor is the peptide galectin-3C. In some embodiments, the galectin inhibitor is galectin-3 disclosed in U.S. patent 6,770,622.

galectin-3C is an N-terminally truncated protein of galectin-3 and functions, for example, as a competitive inhibitor of galectin-3. galectin-3C prevents endogenous galectin-3 from binding to glycoconjugates of, for example, cancer cells (e.g., laminin on the surface) and other beta-galactosidases on the extracellular matrix (ECM). galectin-3C and other exemplary galectin inhibiting peptides are disclosed in us patent 6,770,622.

In some embodiments, galectin-3C comprises or is substantially identical (e.g., 90%, 95% or 99% identical) to the amino acid sequence of SEQ ID NO: 294.

In some embodiments, the galectin inhibitor is a peptide that can bind to and inhibit galectin-1 function. In some embodiments, the galectin inhibitor is a peptide Anginex: anginex is an anti-angiogenic peptide that binds to galectin-1 (Salomonsson E, et al, (2011) Journal of Biological Chemistry [ J. Biochem., 286 (16): 13801-13804. Binding of Anginex to galectin-1 may interfere with e.g. the pro-angiogenic effect of galectin-1.

In some embodiments, the galectin (e.g., galectin-1 or galectin-3) inhibitor is a non-peptide topomimetic (topomimic) molecule. In some embodiments, the non-peptide topomimetic (topomimic) galectin inhibitor is OTX-008 (Oncoethix). In some embodiments, the topology simulation of the non-peptide is that disclosed in us patent 8,207,228. OTX-008 (also known as PTX-008 or Calixarene 0118) is a selective allosteric inhibitor of galectin-1. OTX-008 has the chemical name: n- [2- (dimethylamino) ethyl ] -2- { [26,27, 28-tris ({ [2- (dimethylamino) ethyl ] carbamoyl } methoxy) pentacyclo [19.3.1.1,7.1, ] twenty-eight-1 (25), 3 (28), 4,6,9 (27), 1012,15,17,19 (26), 21, 23-dodecen-25-yl ] oxy } acetamide.

In some embodiments, the galectin (e.g., galectin-1 or galectin-3) inhibitor is a carbohydrate-based compound. In some embodiments, the galectin inhibitor is GR-MD-02 (galectin therapeutics, inc (Galectin Therapeutics)).

In some embodiments, GR-MD-02 is a galectin-3 inhibitor. GR-MD-02 is a galactose-fork polysaccharide, also known as e.g. galactose arabino-rhamnogalacturonate. GR-MD-02 and other galactose-forked polysaccharide polymers (e.g., such as galactose arabino-rhamnogalacturonate) are disclosed in U.S. patent No.: 8,236,780 and us publication 2014/0086932.

MEK inhibitors

In some embodiments, a MEK inhibitor is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, semetinib, AS703026, BIX 02189, BIX 02188, CI-1040, PD0325901, PD98059, U0126, XL-518, G-38963, and G02443714. In some embodiments, the MEK inhibitor is trametinib.

Exemplary MEK inhibitors

In some embodiments, the MEK inhibitor is trametinib. Qu Meiti Ni is also known as JTP-74057, TMT212, N- (3- { 3-cyclopropyl-5- [ (2-fluoro-4-iodophenyl) amino ] -6, 8-dimethyl-2, 4, 7-trioxo-3, 4,6, 7-tetrahydropyrido [4,3-d ] pyrimidin-1 (2H) -yl } phenyl) acetamide, or Mekinist (CAS No. 871700-17-3).

Other exemplary MEK inhibitors

In some embodiments, the MEK inhibitor comprises semantenib, which has the chemical name: (5- [ (4-bromo-2-chlorophenyl) amino ] -4-fluoro-N- (2-hydroxyethoxy) -1-methyl-1H-benzimidazole e-6-carboxamide S. Semtinib is also known as AZD6244 or ARRY 142886, for example, as described in PCT publication No. WO 2003077914.

In some embodiments, the MEK inhibitor comprises AS703026, BIX 02189, or BIX 02188.

In some embodiments, the MEK inhibitor comprises 2- [ (2-chloro-4-iodophenyl) amino ] -N- (cyclopropylmethoxy) -3, 4-difluoro-benzamide (also known as CI-1040 or PD 184352), e.g., as described in PCT publication No. WO 2000035436.

In some embodiments, the MEK inhibitor comprises N- [ (2R) -2, 3-dihydroxypropoxy ] -3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -benzamide (also referred to as PD 0325901), for example, as described in PCT publication No. WO 2002006213.

In some embodiments, the MEK inhibitor comprises 2 '-amino-3' -methoxyflavone (also known as PD 98059), which is available from biafmin GmbH & co., KG, germany.

In some embodiments, the MEK inhibitor comprises 2, 3-bis [ amino [ (2-aminophenyl) thio ] methylene ] -succinonitrile (also known as U0126), e.g., as described in U.S. patent No. 2,779,780.

In some embodiments, the MEK inhibitor comprises XL-518 (also known as GDC-0973) having a CAS number 1029872-29-4 and is available from the ACC group (ACC Corp.).

In some embodiments, the MEK inhibitor comprises G-38963.

In some embodiments, the MEK inhibitor comprises G02443714 (also referred to AS 703206).

Further examples of MEK inhibitors are disclosed in WO 2013/019906, WO 03/077914, WO 2005/121142, WO 2007/04415, WO 2008/024325 and WO 2009/085983. Additional examples of MEK inhibitors include, but are not limited to, 2, 3-bis [ amino [ (2-aminophenyl) thio ] methylene ] -succinonitrile (also known as U0126 and described in U.S. patent No. 2,779,780); (3 s,4r,5z,8s,9s, 11E) -14- (ethylamino) -8,9,16-trihydroxy-3, 4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxy-lene-1, 7 (8H) -dione ] (also known as E6201, described in PCT publication No. WO 2003076424); vemurafenib (PLX-4032, CAS 918504-65-1); (R) -3- (2, 3-dihydroxypropyl) -6-fluoro-5- (2-fluoro-4-iodophenylamino) -8-methylpyrido [2,3-d ] pyrimidine-4, 7 (3 h,8 h) -dione (TAK-733, cas 1035555-63-5); pimasemide (pimasentib) (AS-703026, cas 1204531-26-9); 2- (2-fluoro-4-iodophenylamino) -N- (2-hydroxyethoxy) -1, 5-dimethyl-6-oxo-1, 6-dihydropyridine-3-carboxamide (AZD 8330); and 3, 4-difluoro-2- [ (2-fluoro-4-iodophenyl) amino ] -N- (2-hydroxyethoxy) -5- [ (3-oxo- [1,2] oxazinan-2-yl) methyl ] benzamide (CH 4987555 or Ro 4987555).

c-MET inhibitors

In some embodiments, the c-MET inhibitor is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). c-MET (receptor tyrosine kinase that is overexpressed or mutated in many tumor cell types) plays a key role in tumor cell proliferation, survival, invasion, metastasis and tumor angiogenesis. Inhibition of c-MET can induce cell death in tumor cells that overexpress the c-MET protein or express constitutively activated c-MET protein.

In some embodiments, the c-MET inhibitor is selected from the group consisting of carbamazepine (INC 280), JNJ-3887605, AMG 337, LY2801653, MSC2156119J, crizotinib (crizotinib), tivantinib, or govantinib (golvantinib).

Exemplary c-MET inhibitors

In some embodiments, the c-MET inhibitor comprises carbamazepine (INC 280) or a compound described in U.S. patent nos. 7,767,675 and 8,461,330.

Other exemplary c-MET inhibitors

In some embodiments, the c-MET inhibitor comprises JNJ-38877605.JNJ-38877605 is a small molecule inhibitor of orally available c-Met. JNJ-38877605 selectively binds c-MET, thereby inhibiting c-MET phosphorylation and disrupting the c-Met signaling pathway.

In some embodiments, the c-Met inhibitor is AMG 208.AMG 208 is a selective small molecule inhibitor of c-MET. AMG 208 inhibits ligand-dependent and ligand-independent activation of c-MET, inhibiting its tyrosine kinase activity, which may lead to inhibition of cell growth in tumors that overexpress c-MET.

In some embodiments, the c-Met inhibitor comprises AMG 337.AMG 337 is an orally bioavailable inhibitor of c-Met. AMG 337 selectively binds c-MET, disrupting the c-MET signaling pathway.

In some embodiments, the c-Met inhibitor comprises LY2801653.LY2801653 is a small molecule inhibitor of orally available c-Met. LY2801653 selectively binds to c-MET, thereby inhibiting c-MET phosphorylation and disrupting the c-Met signaling pathway.

In some embodiments, the c-Met inhibitor comprises MSC2156119J. MSC2156119J is an orally bioavailable c-Met inhibitor. MSC2156119J selectively binds to c-MET, inhibits c-MET phosphorylation and disrupts the c-Met-mediated signal transduction pathway.

In some embodiments, the c-MET inhibitor is camatinib (capmatinib). The carbamazepine is also known as INCB028060. The carbamazepine is an orally bioavailable c-MET inhibitor. The camatinib selectively binds to c-Met, thereby inhibiting c-Met phosphorylation and disrupting the c-Met signaling pathway.

In some embodiments, the c-MET inhibitor comprises crizotinib. Crizotinib is also known as PF-0234066. Crizotinib is an aminopyridine-based inhibitor of the orally available receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and c-Met/Hepatocyte Growth Factor Receptor (HGFR). Crizotinib binds to and inhibits ALK kinase and ALK fusion proteins in an ATP-competitive manner. In addition, crizotinib inhibits c-Met kinase and disrupts the c-Met signaling pathway. In summary, the agent inhibits tumor cell growth.

In some embodiments, the c-MET inhibitor comprises govastatin. Govastatin is an orally bioavailable dual kinase inhibitor of c-MET and VEGFR-2, which has potential anti-tumor activity. Gossypii binds and inhibits the activity of c-MET and VEGFR-2, which may inhibit tumor cell growth and survival of tumor cells overexpressing these receptor tyrosine kinases.

In some embodiments, the c-MET inhibitor is tivantinib (tivantinib). The alternative Mo Tini is also called ARQ 197. Instead Mo Tini is an orally bioavailable c-MET small molecule inhibitor. Tivantinib binds to the c-MET protein and disrupts the c-MET signaling pathway, inducing cell death in tumor cells that overexpress the c-MET protein or express constitutively activated c-MET protein.

IL-1 beta inhibitors

The interleukin-1 (IL-1) cytokine family is a group of secreted pleiotropic cytokines that play an important role in inflammation and immune responses. An increase in IL-1 was observed in a variety of clinical settings, including cancer (Apte et al (2006) Cancer Metastasis Rev [ cancer and metastasis reviews ] pages 387-408; dinarello (2010) Eur. J. Immunol [ European journal of immunology ] pages 599-606). The IL-1 family comprises, inter alia, IL-1β (IL-1 b) and IL-1α (IL-1 a). IL-1b is elevated in lung, breast and colorectal cancers (Voronov et al (2014) Front Physiol [ biological Front ] page 114) and is associated with poor prognosis (Apte et al (2000) adv. Exp. Med. Biol. [ experimental medicine and biological progression ] pages 277-88). Without wishing to be bound by theory, it is believed that in some embodiments, secreted IL-1b derived from the tumor microenvironment and through malignant cells promotes tumor cell proliferation, increases invasiveness, and inhibits anti-tumor immune responses (in part by recruiting inhibitory neutrophils) (Apte et al (2006) Cancer Metastasis Rev [ cancer and metastasis reviews ] pages 387-408; miller et al (2007) j.immunol [ journal of immunology ] pages 6933-42). Experimental data indicate that inhibition of IL-1b results in a reduction in tumor burden and metastasis (Voronov et al (2003) proc.Natl. Acad. Sci. [ Proc. Natl. Acad. Sci. U.S. 2645-50).

In some embodiments, interleukin-1 beta (IL-1 beta) is used in combination with a tgfβ inhibitor (and/or PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). In some embodiments, the IL-1β inhibitor is selected from the group consisting of canamab (canakinumab), lattice Wo Jizhu mab (gevokizumab), anakinra, or Li Naxi pride (Rilonacept). In some embodiments, the inhibitor of IL-1 β is cinacalcet.

Exemplary IL-1 beta inhibitors

In some embodiments, the inhibitor of IL-1 β is cinacalcet. Carneazumab is also known as ACZ885 orThe kanamab is a human monoclonal IgG 1/kappa antibody that neutralizes the biological activity of human IL-1β.

Canamazumab is disclosed in, for example, WO 2002/16436, US 7,446,175, and EP 1313769. The heavy chain variable region of the kanamab has the following amino acid sequence: MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSVYGMNWVRQAPGKGLEWVAIIWYDGDNQYYADSVKGRFTISRDNSKNTLYLQMNGLRAEDTAVYYCARDLRTGPFDYWGQGTLVTVSS (SEQ ID NO: 297) (disclosed as SEQ ID NO:1 in US 7,446,175). The light chain variable region of the kanamab has the following amino acid sequence: MLPSQLIGFLLLWVPASRGEIVLTQSPDFQSVTPKEKVTITCRASQSIG SSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLE AEDAAAYYCHQSSSLPFTFGPGTKVDIK (SEQ ID NO: 298) (disclosed in US 7,446,175 as SEQ ID NO: 2).

Carbamab has been used in the treatment of, for example, cryopyrin protein-associated periodic syndrome (CAPS) in adults and children, in the treatment of Systemic Juvenile Idiopathic Arthritis (SJIA), in symptomatic treatment of acute gouty arthritis episodes in adults, and in other IL-1β -driven inflammatory diseases. Without wishing to be bound by theory, it is believed that in some embodiments, an IL-1 β inhibitory agent (e.g., kanamab) may increase an anti-tumor immune response, such as by blocking one or more functions of IL-1b, including, for example, recruiting immunosuppressive neutrophils to the tumor microenvironment, stimulating tumor angiogenesis, and/or promoting metastasis (Dinarello (2010) eur.j. Immunol. [ journal of immunology ] pages 599-606).

In some embodiments, the combinations described herein include an inhibitor of IL-1 β, a kanamab, or a compound disclosed in WO 2002/16436, and an inhibitor of an immune checkpoint molecule (e.g., an inhibitor of PD-1 (e.g., an anti-PD-1 antibody molecule)). IL-1 is a secreted pleiotropic cytokine that plays an important role in inflammation and immune responses. An increase in IL-1 was observed in a variety of clinical settings, including cancer (Apte et al (2006) Cancer Metastasis Rev [ cancer and metastasis reviews ] pages 387-408; dinarello (2010) Eur. J. Immunol [ European journal of immunology ] pages 599-606). IL-1b is elevated in lung, breast and colorectal cancers (Voronov et al (2014) Front Physiol [ biological Front ] page 114) and is associated with poor prognosis (Apte et al (2000) adv. Exp. Med. Biol. [ experimental medicine and biological progression ] pages 277-88). Without wishing to be bound by theory, it is believed that in some embodiments, secreted IL-1b derived from the tumor microenvironment and through malignant cells promotes tumor cell proliferation, increases invasiveness, and inhibits anti-tumor immune responses (in part by recruiting inhibitory neutrophils) (Apte et al (2006) Cancer Metastasis Rev [ cancer and metastasis reviews ] pages 387-408; miller et al (2007) j.immunol [ journal of immunology ] pages 6933-42). Experimental data indicate that inhibition of IL-1b results in a reduction in tumor burden and metastasis (Voronov et al (2003) proc.Natl. Acad. Sci. [ Proc. Natl. Acad. Sci. U.S. 2645-50). The cinacalcet antibody can bind to IL-1b and inhibit IL-1 mediated signaling. Thus, in certain embodiments, an IL-1 β inhibitor (e.g., canamab) is enhanced or used to enhance the immune-mediated anti-tumor effect of a PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule).

In some embodiments, the IL-1 β inhibitor, the canamab, or the compounds disclosed in WO 2002/16436, and an inhibitor of an immune checkpoint molecule (e.g., an inhibitor of PD-1 (e.g., an anti-PD-1 antibody molecule)) are each administered in dosages and/or schedules, in combination, to achieve the desired anti-tumor activity.

MDM2 inhibitors

In some embodiments, the mouse double minute 2 homolog (MDM 2) is used in combination with a tgfβ inhibitor (and/or a PD1, PD-L1, or PD-L2 inhibitor) to treat a disease (e.g., cancer). A human homolog of MDM2 is also known as HDM2. In some embodiments, the MDM2 inhibitors described herein are also referred to as HDM2 inhibitors. In some embodiments, the MDM2 inhibitor is selected from HDM201 or CGM097.

In embodiments, the MDM2 inhibitor comprises (S) -1- (4-chlorophenyl) -7-isopropoxy-6-methoxy-2- (4- (methyl (((1 r, 4S) -4- (4-methyl-3-oxopiperazin-1-yl) cyclohexyl) methyl) amino) phenyl) -1, 2-dihydroisoquinolin-3 (4H) -one (also known as CGM 097) or a compound disclosed in PCT publication No. WO 2011/076786, to treat a disorder, such as the disorders described herein. In one embodiment, the therapeutic agents disclosed herein are used in combination with CGM097.

In embodiments, the MDM2 inhibitor comprises p53 and/or an inhibitor of the p53/MDM2 interaction. In embodiments, the MDM2 inhibitor comprises (S) -5- (5-chloro-1-methyl-2-oxo-1, 2-dihydropyridin-3-yl) -6- (4-chlorophenyl) -2- (2, 4-dimethoxypyrimidin-5-yl) -1-isopropyl-5, 6-dihydropyrrolo [3,4-d ] imidazol-4 (1H) -one (also known as HDM 201) or a compound disclosed in PCT publication No. WO 2013/111105, for treating a disorder, such as a disorder described herein. In one embodiment, the therapeutic agents disclosed herein are used in combination with HDM201. In some embodiments, HDM201 is administered orally.

In one embodiment, the combinations disclosed herein are useful for the treatment of cancer in vivo. For example, the combination may be used to inhibit the growth of cancerous tumors. The combination may also be used in combination with one or more of the following: standard of care treatment (standard of care treatment) (e.g., for cancer or infectious disorders), vaccines (e.g., therapeutic cancer vaccines), cell therapies, radiation therapies, surgery, or any other therapeutic agent or modality to treat the disorders herein. For example, to achieve antigen-specific enhancement of immunity, the combination may be administered with an antigen of interest.

Pharmaceutical compositions, formulations and kits

In another aspect, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising a combination as described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

The compositions described herein may take a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. These inhibitors (including antibody inhibitors) may be in the form of injectable or infusible solutions. The mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In embodiments, the antibody is administered by intravenous infusion or injection. In another embodiment, the antibody is administered by intramuscular or subcutaneous injection.

As used herein, the phrases "parenteral administration" and "parenteral administration" mean modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

The therapeutic composition should typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high antibody concentrations. Sterile injectable solutions can be prepared by: the active compound (e.g., antibody or antibody moiety) is incorporated in the desired amount, as desired, with one or a combination of the ingredients listed above, in an appropriate solvent, and then filter sterilized. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. For example, proper fluidity of the solution may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The absorption of the injectable composition may be prolonged by including agents (e.g., monostearates and gelatins) in the composition that delay absorption.

The combinations or compositions described herein can be formulated into a formulation (e.g., a dosage formulation or dosage form) suitable for administration (e.g., intravenous administration) to a subject described herein. The formulations described herein may be liquid formulations, lyophilized formulations, or reconstituted formulations.

In certain embodiments, the formulation is a liquid formulation. In some embodiments, the formulation (e.g., liquid formulation) comprises a tgfβ inhibitor (e.g., an anti-tgfβ antibody molecule as described herein) and a buffer. In some embodiments, the formulation (e.g., a liquid formulation) comprises a PD-1 inhibitor (e.g., an anti-PD-1 antibody molecule described herein) and a buffer. In some embodiments, the formulation (e.g., a liquid formulation) comprises a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody molecule described herein) and a buffer. In some embodiments, the formulation (e.g., a liquid formulation) comprises a PD-L2 inhibitor (e.g., an anti-PD-L2 antibody) and a buffer.

In some embodiments, the formulation (e.g., liquid formulation) comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule as disclosed herein, which is present at a concentration of about 25mg/mL to about 250 mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 50mg/mL to about 200mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 60mg/mL to about 180mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 70mg/mL to about 150mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 80mg/mL to about 120mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 90mg/mL to about 110mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 50mg/mL to about 150mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 50mg/mL to about 100mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 150mg/mL to about 200mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 100mg/mL to about 200mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 50mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 60mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 70mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 80mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 90mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 100mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 110mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 120mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 130mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 140mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 150mg/mL. In some embodiments, the formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/L2) antibody molecule at a concentration of about 80mg/mL to about 120mg/mL (e.g., about 100 mg/mL).

In some embodiments, the formulation (e.g., liquid formulation) comprises a buffer comprising histidine (e.g., histidine buffer). In certain embodiments, the buffer (e.g., histidine buffer) is present at the following concentrations: about 1mM to about 100mM, such as about 2mM to about 50mM, about 5mM to about 40mM, about 10mM to about 30mM, about 15 to about 25mM, about 5mM to about 40mM, about 5mM to about 30mM, about 5mM to about 20mM, about 5mM to about 10mM, about 40mM to about 50mM, about 30mM to about 50mM, about 20mM to about 50mM, about 10mM to about 50mM, or about 5mM to about 50mM, such as about 2mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM. In some embodiments, the buffer (e.g., histidine buffer) is present at a concentration of about 15mM to about 25mM (e.g., about 20 mM). In other embodiments, the buffer (e.g., histidine buffer) has a pH of about 4 to about 7 (e.g., about 5 to about 6 (e.g., about 5, about 5.5, or about 6)). In some embodiments, the buffer (e.g., histidine buffer) has a pH of about 5 to about 6 (e.g., about 5.5). In certain embodiments, the buffer comprises histidine buffer at a concentration of about 15mM to about 25mM (e.g., 20 mM) and has a pH of about 5 to about 6 (e.g., 5.5). In certain embodiments, the buffer comprises histidine and histidine-HCl.

In some embodiments, the formulation (e.g., liquid formulation) comprises: an antibody molecule as disclosed herein, present at a concentration of 80 to 120mg/mL (e.g., 100 mg/mL); and a buffer comprising histidine buffer at a concentration of 15mM to 25mM (e.g., 20 mM) and having a pH of 5 to 6 (e.g., 5.5).

In some embodiments, the formulation (e.g., liquid formulation) further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is present at the following concentrations: about 50mM to about 500mM, for example about 100mM to about 400mM, about 150mM to about 300mM, about 180mM to about 250mM, about 200mM to about 240mM, about 210mM to about 230mM, about 100mM to about 300mM, about 100mM to about 250mM, about 100mM to about 200mM, about 100mM to about 150mM, about 300mM to about 400mM, about 200mM to about 400mM, or about 100mM to about 400mM, for example about 100mM, about 150mM, about 180mM, about 200mM, about 220mM, about 250mM, about 300mM, about 350mM, or about 400mM. In some embodiments, the formulation comprises a carbohydrate or sucrose present at a concentration of about 200mM to about 250mM (e.g., about 220 mM).

In some embodiments, the formulation (e.g., liquid formulation) comprises: an antibody molecule as disclosed herein, present at a concentration of 80mg/mL to 120mg/mL (e.g., 100 mg/mL); a buffer comprising histidine buffer at a concentration of 15mM to 25mM (e.g., 20 mM) and having a pH of 5 to 6 (e.g., 5.5); and carbohydrate or sucrose present at a concentration of 200mM to 250mM (e.g., 220 mM).

In some embodiments, the formulation (e.g., liquid formulation) further comprises a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20 is present at the following concentrations: about 0.005% to about 0.1% (w/w), such as about 0.01% to about 0.08%, about 0.02% to about 0.06%, about 0.03% to about 0.05%, about 0.01% to about 0.06%, about 0.01% to about 0.05%, about 0.01% to about 0.03%, about 0.06% to about 0.08%, about 0.04% to about 0.08%, or about 0.02% to about 0.08% (w/w), such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% (w/w). In some embodiments, the formulation comprises a surfactant or polysorbate 20, which surfactant or polysorbate 20 is present at a concentration of about 0.03% to about 0.05% (e.g., about 0.04%) (w/w).

In some embodiments, the formulation (e.g., liquid formulation) comprises: an antibody molecule as disclosed herein, present at a concentration of about 80 to 120mg/mL (e.g., 100 mg/mL); a buffer comprising histidine buffer at a concentration of 15mM to 25mM (e.g., 20 mM) and a pH of 5 to 6 (e.g., 5.5); carbohydrates or sucrose present at a concentration of 200mM to 250mM (e.g., 220 mM); and surfactant or polysorbate 20 present at a concentration of 0.03% to 0.05% (e.g., 0.04%) (w/w).

In some embodiments, the formulation (e.g., liquid formulation) comprises: an antibody molecule as disclosed herein, present at a concentration of 100 mg/mL; a buffer comprising histidine buffer (e.g., histidine/histidine-HCL) at a concentration of 20mM and a pH of 5.5; carbohydrates or sucrose present at a concentration of 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.04% (w/w).

In some embodiments, the liquid formulation is prepared by diluting a formulation comprising an antibody molecule described herein. For example, the drug substance formulation may be diluted with a solution comprising one or more excipients (e.g., a concentrated excipient). In some embodiments, the solution comprises one, two, or all of histidine, sucrose, or polysorbate 20. In certain embodiments, the solution comprises one or more excipients that are the same as the pharmaceutical substance formulation. Exemplary excipients include, but are not limited to, amino acids (e.g., histidine), carbohydrates (e.g., sucrose), or surfactants (e.g., polysorbate 20). In certain embodiments, the liquid formulation is not a reconstituted lyophilized formulation. In other embodiments, the liquid formulation is a reconstituted lyophilized formulation. In some embodiments, the formulation is stored as a liquid. In other embodiments, the formulation is prepared as a liquid and then dried (e.g., by lyophilization or spray drying) prior to storage.

In certain embodiments, each container (e.g., vial) is filled with about 0.5mL to about 10mL (e.g., about 0.5mL to about 8mL, about 1mL to about 6mL, or about 2mL to about 5mL, such as about 1mL, about 1.2mL, about 1.5mL, about 2mL, about 3mL, about 4mL, about 4.5mL, about 5mL, about 5.5mL, about 6mL, about 6.5mL, about 7mL, about 7.5mL, about 8mL, about 8.5mL, about 9mL, about 9.5mL, or about 10 mL) of the liquid formulation. In other embodiments, the liquid formulation is filled into containers (e.g., vials) such that the extractable volume of the liquid formulation that each container (e.g., vial) can extract is at least 1mL (e.g., at least 1.2mL, at least 1.5mL, at least 2mL, at least 3mL, at least 4mL, or at least 5 mL). In certain embodiments, the liquid formulation is extracted from a container (e.g., a vial) without dilution at a clinical site. In certain embodiments, the liquid formulation is diluted from the drug substance formulation and extracted from a container (e.g., vial) at a clinical site. In certain embodiments, the formulation (e.g., liquid formulation) is injected into the infusion bag within 1 hour (e.g., within 45 minutes, 30 minutes, or 15 minutes) before infusion into the patient begins.

The formulations described herein may be stored in a container. Containers for any of the formulations described herein may include, for example, vials and optionally stoppers, caps, or both. In certain embodiments, the vial is a glass vial, e.g., a 6R white glass vial. In other embodiments, the stopper is a rubber stopper, for example, a gray rubber stopper. In other embodiments, the cover is a flip, for example, an aluminum flip. In some embodiments, the container comprises a 6R white glass vial, a gray rubber stopper, and an aluminum flip top. In some embodiments, the container (e.g., vial) is a container for single use. In certain embodiments, about 250mg to about 1500mg of an antibody molecule as described herein is present in the container. In some embodiments, the container comprises about 300mg to about 1250mg of antibody. In some embodiments, the container comprises about 350mg to about 1200mg of antibody. In some embodiments, the container comprises about 400mg to about 1100mg of antibody. In some embodiments, the container comprises about 450mg to about 1000mg of antibody. In some embodiments, the container comprises about 500mg to about 900mg of antibody. In some embodiments, the container comprises about 600mg to about 800mg of antibody. In some embodiments, the container comprises about 300mg of antibody. In some embodiments, the container contains about 400mg of antibody. In some embodiments, the container comprises about 500mg of antibody. In some embodiments, the container contains about 600mg of antibody. In some embodiments, the container comprises about 700mg of antibody. In some embodiments, the container comprises about 800mg of antibody. In some embodiments, the container contains about 900mg of antibody. In some embodiments, the container contains about 1000mg of antibody.

In some embodiments, the formulation is a lyophilized formulation. In certain embodiments, the lyophilized formulation is lyophilized or dried from a liquid formulation comprising the antibody molecules described herein. Each container (e.g., vial) may be filled with, for example, from about 1mL to about 10mL, such as from about 6mL to about 8mL, of the liquid formulation and lyophilized.

In some embodiments, the formulation is a reconstituted formulation. In certain embodiments, the reconstituted formulation is reconstituted from a lyophilized formulation comprising the antibody molecules described herein. For example, the reconstituted formulation is prepared by dissolving the lyophilized formulation in a diluent such that the protein is dispersed in the reconstituted formulation. In some embodiments, the lyophilized formulation is reconstituted with about 1mL to about 15mL, for example, about 5mL to about 9mL or about 7mL of water for injection or buffer. In certain embodiments, for example, at a clinical site, the lyophilized formulation is reconstituted with about 6mL to about 8mL of water for injection.

In some embodiments, the reconstituted formulation comprises an antibody molecule (e.g., an anti-tgfβ or anti-PD-1 (or anti-PD-L1/2) antibody molecule as disclosed herein) and a buffer.

In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 25mg/mL to about 250mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 50mg/mL to about 200mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 60mg/mL to about 180mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 70mg/mL to about 150mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 80mg/mL to about 120mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 90mg/mL to about 110mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 50mg/mL to about 150mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 50mg/mL to about 100mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 150mg/mL to about 200mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 100mg/mL to about 200mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 50mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 60mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 70mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 80mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 90mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 100mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 110mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 120mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 130mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 140mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 150mg/mL. In some embodiments, the reconstituted formulation comprises an anti-tgfβ or anti-PD 1 (or anti-PD-L1/2) antibody molecule at a concentration of about 80mg/mL to about 120mg/mL (e.g., about 100 mg/mL).

In some embodiments, the reconstituted formulation comprises a buffer comprising histidine (e.g., histidine buffer). In certain embodiments, the buffer (e.g., histidine buffer) is present at the following concentrations: about 1mM to about 100mM, such as about 2mM to about 50mM, about 5mM to about 40mM, about 10mM to about 30mM, about 15 to about 25mM, about 5mM to about 40mM, about 5mM to about 30mM, about 5mM to about 20mM, about 5mM to about 10mM, about 40mM to about 50mM, about 30mM to about 50mM, about 20mM to about 50mM, about 10mM to about 50mM, or about 5mM to about 50mM, such as about 2mM, about 5mM, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 35mM, about 40mM, about 45mM, or about 50mM. In some embodiments, the buffer (e.g., histidine buffer) is present at a concentration of about 15mM to about 25mM (e.g., about 20 mM). In other embodiments, the buffer (e.g., histidine buffer) has a pH of about 4 to about 7 (e.g., about 5 to about 6 (e.g., about 5, about 5.5, or about 6)). In some embodiments, the buffer (e.g., histidine buffer) has a pH of about 5 to about 6 (e.g., about 5.5). In certain embodiments, the buffer comprises histidine buffer at a concentration of about 15mM to about 25mM (e.g., 20 mM) and has a pH of about 5 to about 6 (e.g., 5.5). In certain embodiments, the buffer comprises histidine and histidine-HCl.

In some embodiments, the reconstituted formulation comprises: an antibody molecule as disclosed herein, present at a concentration of about 80 to about 120mg/mL (e.g., 100 mg/mL); and a buffer comprising histidine buffer at a concentration of about 15mM to about 25mM (e.g., 20 mM) and a pH of 5 to 6 (e.g., 5.5).

In some embodiments, the reconstituted formulation further comprises a carbohydrate. In certain embodiments, the carbohydrate is sucrose. In some embodiments, the carbohydrate (e.g., sucrose) is present at the following concentrations: from 50mM to about 500mM, for example from about 100mM to about 400mM, from about 150mM to about 300mM, from about 180mM to about 250mM, from about 200mM to about 240mM, from about 210mM to about 230mM, from about 100mM to about 300mM, from about 100mM to about 250mM, from about 100mM to about 200mM, from about 100mM to about 150mM, from about 300mM to about 400mM, from about 200mM to about 400mM, or from about 100mM to about 400mM, for example from about 100mM, about 150mM, about 180mM, about 200mM, about 220mM, about 250mM, about 300mM, about 350mM, or about 400mM. In some embodiments, the formulation comprises a carbohydrate or sucrose present at a concentration of about 200mM to about 250mM (e.g., about 220 mM).

In some embodiments, the reconstituted formulation comprises: the antibody molecules disclosed herein are present at a concentration of about 80 to about 120mg/mL (e.g., 100 mg/mL); a buffer comprising histidine buffer at a concentration of about 15mM to about 25mM (e.g., 20 mM) and a pH of about 5 to about 6 (e.g., 5.5); and carbohydrate or sucrose present at a concentration of about 200mM to about 250mM (e.g., 220 mM).

In some embodiments, the reconstituted formulation further comprises a surfactant. In certain embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant or polysorbate 20 is present at the following concentrations: about 0.005% to about 0.1% (w/w), such as about 0.01% to about 0.08%, about 0.02% to about 0.06%, about 0.03% to about 0.05%, about 0.01% to about 0.06%, about 0.01% to about 0.05%, about 0.01% to about 0.03%, about 0.06% to about 0.08%, about 0.04% to about 0.08%, or about 0.02% to about 0.08% (w/w), such as about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1% (w/w). In some embodiments, the formulation comprises a surfactant or polysorbate 20, which surfactant or polysorbate 20 is present at a concentration of about 0.03% to about 0.05% (e.g., about 0.04%) (w/w).

In some embodiments, the reconstituted formulation comprises: an antibody molecule as disclosed herein, present at a concentration of about 80 to about 120mg/mL (e.g., 100 mg/mL); a buffer comprising histidine buffer at a concentration of about 15mM to about 25mM (e.g., 20 mM) and a pH of about 5 to about 6 (e.g., 5.5); carbohydrates or sucrose present at a concentration of about 200mM to about 250mM (e.g., 220 mM); and surfactant or polysorbate 20 present at a concentration of about 0.03% to about 0.05% (e.g., 0.04%) (w/w).

In some embodiments, the reconstituted formulation comprises: an antibody molecule as disclosed herein, present at a concentration of 100 mg/mL; a buffer comprising histidine buffer (e.g., histidine/histidine-HCL) at a concentration of 20mM and a pH of 5.5; carbohydrates or sucrose present at a concentration of 220 mM; and a surfactant or polysorbate 20 present at a concentration of 0.04% (w/w).

In some embodiments, the formulation is reconstituted such that the extractable volume of the reconstituted formulation that can be withdrawn from the container (e.g., vial) containing the reconstituted formulation is at least 1mL (e.g., at least 1.2mL, 1.5mL, 2mL, 2.5mL, 3mL, 3.5mL, 4mL, 4.5mL, 5mL, 5.5mL, 6mL, 6.5mL, 7mL, 7.5mL, 8mL, 8.5mL, 9mL, 9.5mL, or 10 mL). In certain embodiments, the formulation is reconstituted and/or extracted from a container (e.g., vial) at a clinical site. In certain embodiments, the formulation (e.g., reconstituted formulation) is injected into the infusion bag within 1 hour (e.g., within 45 minutes, 30 minutes, or 15 minutes) before infusion into the patient begins.

In some embodiments, the reconstituted formulation has a fill volume of about 1mL to about 5 mL. In certain embodiments, the reconstituted formulation has a fill volume of about 2mL to about 4 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.2 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.4 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.6 mL. In some embodiments, the reconstituted formulation has a fill volume of about 3.8 mL.

Other exemplary buffers that may be used in the formulations described herein include, but are not limited to, arginine buffer, citrate buffer, or phosphate buffer. Other exemplary carbohydrates that may be used in the formulations described herein include, but are not limited to, trehalose, mannitol, sorbitol, or combinations thereof. The formulations described herein may also contain tonicity agents (e.g., sodium chloride) and/or stabilizers (e.g., amino acids (e.g., glycine, arginine, methionine, or combinations thereof)).

Antibody molecules can be administered by a variety of methods known in the art, but for many therapeutic applications, the preferred route/mode of administration is intravenous injection or infusion. For example, antibody molecules may be administered by intravenous infusion at a rate in excess of 20mg/min, such as 20-40mg/min, and typically greater than or equal to 40mg/min, to achieve about 35 to 440mg/m² Typically about 70mg/m² To about 310mg/m² And more typically about 110mg/m² To about 130mg/m² Is a dose of (a). In the case of an embodiment of the present invention,the antibody molecule may be administered by intravenous infusion at a rate of less than 10 mg/min; preferably less than or equal to 5mg/min to achieve about 1mg/m² To about 100mg/m² Preferably about 5mg/m² To about 50mg/m² About 7mg/m² To about 25mg/m² And more preferably about 10mg/m² Is a dose of (a). As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired result. In certain embodiments, the active compounds may be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Many methods for preparing such formulations are patented or generally known to those skilled in the art. See, e.g., sustained and Controlled Release Drug Delivery Systems [ sustained and controlled release drug delivery systems ]]Robinson editions, marcel Dekker, inc., new York, 1978.

In certain embodiments, the antibody molecule may be administered orally, e.g., with an inert diluent or an assimilable edible carrier. The compounds (and other ingredients, if desired) may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be mixed with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers (wafer), and the like. In order to administer the compounds of the present invention by means other than parenteral administration, it may be desirable to coat the compound with a material or co-administer the compound with a material to prevent its inactivation. The therapeutic composition may also be administered with medical devices known in the art.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the doses may be proportionally reduced or increased, as indicated by the emergency state of the treatment situation. Parenteral compositions may be particularly advantageously formulated in unit dosage form for ease of administration and to achieve dose uniformity. As used herein, a unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect and the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is specified by and directly depends on: (a) The unique characteristics of the active compounds and the particular therapeutic effect to be achieved, and (b) inherent limitations in the art of compounding such active compounds for the treatment of sensitivity in individuals.

Antibody molecules may be administered by intravenous infusion at a rate of greater than 20mg/min, such as 20-40mg/min, and typically greater than or equal to 40mg/min, to achieve about 35mg/m² To about 440mg/m² Typically about 70mg/m² To about 310mg/m² And more typically about 110mg/m² To about 130mg/m² Is a dose of (a). In an embodiment, about 110mg/m² To about 130mg/m² The infusion rate of (2) reaches a level of about 3 mg/kg. In other embodiments, the antibody molecule may be administered by intravenous infusion at a rate of less than 10mg/min, such as less than or equal to 5mg/min, to achieve about 1mg/m² To about 100mg/m² For example about 5mg/m² To about 50mg/m² About 7mg/m² To about 25mg/m² Or about 10mg/m² Is a dose of (a). In some embodiments, the antibody is infused over a period of about 30 minutes. It should be noted that the dosage value may vary with the type and severity of the condition to be alleviated. It will be further understood that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody portion of the invention. "therapeutically effective amount" means an amount effective to achieve the desired therapeutic result at the necessary dosage and for the necessary period of time. The therapeutically effective amount of the modified antibody or antibody fragment may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutic benefit exceeds any toxic or detrimental effect of the modified antibody or antibody fragment. The "therapeutically effective dose" preferably inhibits a measurable parameter, such as tumor growth rate inhibition of at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts the efficacy of a human tumor. Alternatively, such properties of the composition may be assessed by examining the ability of the compound to inhibit, such inhibition being performed in vitro by assays known to those of skill in the art.

"prophylactically effective amount" refers to an amount effective in dosimetry and for the period of time required to achieve the desired prophylactic result. Typically, such prophylactically effective amounts will be less than therapeutically effective amounts because the prophylactic dose is administered in the subject prior to or early in the disease.

Kits comprising the combinations, compositions or formulations described herein are also within the scope of the present disclosure. The kit may include one or more other elements, including: instructions for use (e.g., according to the dosage regimen described herein); other agents, such as labels, therapeutic agents, or agents for chelating or otherwise coupling antibodies to labels or therapeutic agents or radioprotective compositions; devices or other materials for preparing antibodies for administration; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

Chemotherapeutic agents

The compounds disclosed throughout (e.g., tgfβ inhibitors (e.g., NIS 793) or PD-1 inhibitors (e.g., tirelizumab)) can be used in combination with chemotherapeutic agents. The therapeutic agent may include, but is not limited to, anastrozoleBicalutamide->Bleomycin sulfate->Busulfan (Busulfan)Busulfan injection>Capecitabine->N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin +.>Carmustine>ChlorambucilCisplatin->Cladribine>CyclophosphamideOr->) Cytarabine, cytosine arabinoside +.>Cytarabine liposome injection>Dacarbazine->Dactinomycin (actinomycin D, cosmegan), daunorubicin hydrochloride->Daunorubicin citrate liposome injectionDexamethasone, docetaxel +.>Doxorubicin hydrochlorideEtoposide->Fludarabine phosphate5-fluorouracil->Fluotamide->tezacitibine, gemcitabine (difluoro deoxycytidine), hydroxyurea +.>IdarubicinIfosfamide->Irinotecan->L-asparaginaseCalcium folinate, melphalan->6-mercaptopurine->Methotrexate>Mitoxantrone->Mailuota, paclitaxel->Or albumin-bound paclitaxel, phoenix (Yttrium 90/MX-DTPA), penstatin, polifeprosan20 × with carmustine implant>Tamoxifen citrate->Teniposide6-thioguanine, thiotepa, tirapazamine +.>Hydrochloride topotecan for injectionVinblastine->Vincristine->VinorelbineEpirubicin->Oxaliplatin->ExemestaneLetrozole->And fulvestrant->

In some embodiments, gemcitabine may be used in combination with any of the therapeutic molecules disclosed throughout. For example, a tgfβ inhibitor (e.g., NIS 793) in combination with gemcitabine may be used to treat a patient. PD-1 inhibitors (e.g., PDR001, BGB-A317, or BGB-108) may be used in combination with gemcitabine or gemcitabine and a TGF-beta inhibitor.

In some embodiments, albumin-bound paclitaxel may be used in combination with any of the therapeutic molecules disclosed throughout. For example, a TGF-beta inhibitor (e.g., NIS 793) in combination with albumin-bound paclitaxel may be used to treat a patient. PD-1 inhibitors (e.g., PDR001, BGB-A317, or BGB-108) may be used in combination with albumin-bound paclitaxel or albumin-bound paclitaxel and a TGF-beta inhibitor (e.g., NIS 793). In some combinations, a tgfβ inhibitor (e.g., NIS 793) may be used in combination with gemcitabine and albumin-bound paclitaxel. In some combinations, a TGF-beta inhibitor (e.g., NIS 793) may be used in combination with a PD-1 inhibitor (e.g., PDR001, BGB-A317, or BGB-108), gemcitabine, and albumin-bound paclitaxel.

When gemcitabine is used in combination with other therapeutic agents, it may be administered intravenously to the patient. For example, gemcitabine may be present at 1000mg/m² Is administered intravenously to a patient. Gemcitabine may also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, at 1000mg/m on days 1, 8 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Gemcitabine is administered at a dose of (2). In some cases, gemcitabine may be present at 675mg/m² Is administered intravenously to a patient. For example, at 675mg/m on days 1 and 8 of each cycle (e.g., 21-day or 28-day cycle)² Gemcitabine is administered at a dose of (2).

Albumin-bound paclitaxel, when used in combination with other therapeutic agents, may be administered intravenously to the patient. For example, albumin-bound paclitaxel can be present at 125mg/m² Is administered intravenously to a patient. Albumin-bound paclitaxel may also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, at 125mg/m on days 1, 8 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Albumin-bound paclitaxel is administered at a dose of (a) an albumin-bound paclitaxel.

In some embodiments, 5-fluorouracil can be used in combination with any of the therapeutic molecules disclosed throughout. For example, a TGF-beta inhibitor (e.g., NIS 793) in combination with 5-fluorouracil may be used to treat a patient.

When 5-fluorouracil is used in combination with other therapeutic agents, it can be administered intravenously to a patient. For example, 5-fluorouracil can be present at 400mg/m² To 2400mg/m² Is administered intravenously (e.g., using intravenous bolus injection) to a patient. The 5-fluorouracil can also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, 5-fluoroUracil is present at 400mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Intravenous bolus injection followed by 2400mg/m² Administration was by continuous intravenous infusion over 46 hours.

In some embodiments, folinic acid may be used in combination with any of the therapeutic molecules disclosed throughout. For example, a combination of a tgfβ inhibitor (e.g., NIS 793) and folinic acid may be used to treat a patient.

When folinic acid is used in combination with other therapeutic agents, it may be administered intravenously to the patient. For example, folinic acid can be used in an amount of 400mg/m² Is administered intravenously to a patient. Folinic acid may also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, at 400mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Folinic acid is administered at a dose of (a) in the formulation.

In some embodiments, levofolinic acid may be used as a substitute for folinic acid. In those cases, L-folinic acid can be used in an amount of 200mg/mg² And (5) administration. Further, levofolinic acid may be administered on days 1 and 15 of a 28 day cycle.

In some embodiments, oxaliplatin can be used in combination with any of the therapeutic molecules disclosed throughout. For example, a tgfβ inhibitor (e.g., NIS 793) in combination with oxaliplatin may be used to treat a patient.

Oxaliplatin, when used in combination with other therapeutic agents, can be administered intravenously to a patient. For example, oxaliplatin can be present at 85mg/m² Is administered intravenously to a patient. Oxaliplatin can also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, at 85mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Oxaliplatin is administered at a dose of (a).

In some embodiments, irinotecan can be used in combination with any of the therapeutic molecules disclosed throughout. For example, a tgfβ inhibitor (e.g., NIS 793) in combination with irinotecan can be used to treat a patient.

Irinotecan can, when used in combination with other therapeutic agents, can be administered intravenously to a patient. For example, irinotecan can be present at 180mg/m² Is administered intravenously to a patient. Irinotecan can also be administered weekly, biweekly, tricyclically, or weekly for a given period of time. For example, at 180mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Irinotecan is administered at a dose of (a).

In some embodiments, a tgfβ inhibitor (e.g., NIS 793) may be combined with gemcitabine and albumin-bound paclitaxel. In some embodiments, a TGF-beta inhibitor (e.g., NIS 793) may be combined with a PD-1 inhibitor (e.g., PDR001, BGB-A317, or BGB-108), gemcitabine, and albumin-bound paclitaxel. In some embodiments, a tgfβ inhibitor (e.g., NIS 793) may be combined with bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin. In some embodiments, a TGF-beta inhibitor (e.g., NIS 793) may be combined with a PD-1 inhibitor (e.g., PDR001, BGB-A317, or BGB-108), bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin. In some embodiments, a tgfβ inhibitor (e.g., NIS 793) may be combined with bevacizumab, 5-fluorouracil, folinic acid, and irinotecan. In some embodiments, a TGF-beta inhibitor (e.g., NIS 793) may be combined with a PD-1 inhibitor (e.g., PDR001, BGB-A317, or BGB-108), bevacizumab, 5-fluorouracil, folinic acid, and irinotecan.

In some embodiments, cyclophosphamide may be used in combination with any of the therapeutic molecules disclosed throughout, such as a tgfβ inhibitor (e.g., NIS 793), to treat a patient. When cyclophosphamide is used in combination with other therapeutic agents, e.g., tgfβ inhibitors (e.g., NIS 793), it may be administered intravenously to a patient or as an oral drug. For example, cyclophosphamide can be used at 250mg/m² Is administered intravenously to a patient. Cyclophosphamide may also be administered daily for a certain amount of time. For example, cyclophosphamide may be administered for 5 days, e.g., five consecutive days. For example, 250mg/m for five consecutive days² Cyclophosphamide is administered at a dose of (a).

In some embodiments, topotecan can be used in combination with any of the therapeutic molecules disclosed throughout, such as tgfβ inhibitors (e.g., NIS 793), to treat a patient. Topotecan may be administered intravenously to a patient when used in combination with other therapeutic agents, such as tgfβ inhibitors (e.g., NIS 793). For example, topotecan can be used at 0.75mg/m² Is administered intravenously to a patient. Topotecan can also be administered over a certain amount of time course, e.g. 30 minutes. Topotecan can also be administered for 5 days, for example five consecutive days. For example, at 0.75mg/m for five consecutive days² Topotecan is administered at a dose of (a).

Additional exemplary combinations and dosage regimens

The compounds and/or therapeutic agents disclosed throughout can be used in any combination. Furthermore, each therapeutic agent can be used in a manner that is not overly toxic but is effective for its intended purpose. The following combinations and/or dosage regimens are for illustrative purposes only and do not fully encompass all of the contemplated combinations and/or dosage regimens. One of ordinary skill in the art can further contemplate various combinations and/or dosage regimens disclosed throughout the specification.

In combination, NIS793 is administered to a patient with gemcitabine and albumin-bound paclitaxel. The three component combination is administered intravenously to a patient. NIS793 is administered to the patient at 2100mg doses on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle) in the three-component combination. Gemcitabine in this three-component combination is 1000mg/m on days 1, 8, and 15 of each cycle (e.g., 21-day or 28-day cycle)² Is administered to a patient. Albumin bound paclitaxel in this three component combination was administered at 125mg/m on days 1, 8 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Is administered to a patient. In this combination, pancreatic cancer, e.g., metastatic pancreatic adenocarcinoma, is treated.

In combination, NIS793 is administered to a patient with bevacizumab, 5-fluorouracil, folinic acid and oxaliplatin. The five components are combined intravenously (in some cases, intravenous bolus injection or continuous intravenous infusionInjection) is administered to a patient. NIS793 is administered to the patient at 2100mg doses on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle) in the five component combination. Bevacizumab was administered to the patient at doses of 5mg/kg on days 1 and 15 of each cycle (e.g. 21 day or 28 day cycle) in this five component combination. For example, 5-fluorouracil in the five component combination is present at 400mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Intravenous bolus injection followed by 2400mg/m² The patients were administered as a continuous intravenous infusion over 46 hours. In this five-component combination folinic acid was present at 400mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Is administered to a patient. Oxaliplatin in this five-component combination was at 85mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Is administered to a patient. In this combination, colorectal cancer, e.g., metastatic colorectal cancer, is treated.

In combination, NIS793 is administered to a patient with bevacizumab, 5-fluorouracil, folinic acid and irinotecan. The five component combination is administered intravenously (in some cases, intravenous bolus or continuous intravenous infusion) to the patient. NIS793 is administered to the patient at 2100mg doses on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle) in the five component combination. Bevacizumab was administered to the patient at doses of 5mg/kg on days 1 and 15 of each cycle (e.g. 21 day or 28 day cycle) in this five component combination. For example, 5-fluorouracil in the five component combination is present at 400mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Intravenous bolus injection followed by 2400mg/m² The patients were administered as a continuous intravenous infusion over 46 hours. In this five-component combination folinic acid was present at 400mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Is administered to a patient. Irinotecan in this five-component combination was at 180mg/m on days 1 and 15 of each cycle (e.g., 21-day or 28-day cycle)² Is administered to a patient. In this combination, colorectal cancer, e.g., metastatic colorectal cancer, is treated.

In combination, NIS793 is administered to a patient with oxaliplatin and capecitabine. The three component combination is administered intravenously (in some cases, intravenous bolus or continuous intravenous infusion) to the patient. NIS793 was administered to the patient every three weeks (Q3W) at a dose of 2100mg in this three-component combination. Oxaliplatin in this three-component combination was at 130mg/m on day 1 of the Q3W cycle² Is administered intravenously to a patient. Capecitabine in this three-component combination was at 1000mg/m during the Q3W cycle² Is orally administered to the patient twice daily (days 1-14). Tirelimumab was optionally administered to the patient at a dose of 200mg once every three weeks (Q3W). In this combination, gastric cancer is treated.

In combination, NIS793 is administered to a patient with oxaliplatin, folinic acid (or levofolinic acid) and 5-fluorouracil. The combination is administered intravenously (in some cases, intravenous bolus or continuous intravenous infusion) to the patient. NIS793 was administered to the patient every three weeks (Q3W) at a dose of 2100mg in this four-component combination. Oxaliplatin at 85mg/m on day 1 of the Q2W cycle² Is administered to a patient. Folinic acid at 400mg/m on day 1 of the Q2W cycle² Is administered (or levofolinic acid is administered at 200 mg/m)² Is administered at a dose). 5-fluorouracil at 400mg/m on day 1 of the Q2W cycle² Then at 1200mg/m on days 1-2² Intravenous administration. Tirelimumab was optionally administered once every four weeks (Q4W) at a dose of 300 mg. In this combination, gastric cancer is treated.

In combination, NIS793 is administered to a patient with oxaliplatin and capecitabine. The combination is administered intravenously (in some cases, intravenous bolus or continuous intravenous infusion) to the patient. NIS793 was administered to the patient at a dose of 2100mg once every two weeks (Q2W) in this three-component combination. Oxaliplatin in this three-component combination was at 130mg/m on day 1 of the Q3W cycle² Is administered intravenously. Capecitabine in this three-component combination was at 1000mg/m during the Q3W cycle² Is administered orally twice daily (days 1-14). Tirelimumab was administered at a dose of 200mg once every three weeks (Q3W). In this combination, the treatmentIs gastric cancer.

In combination, NIS793 is administered to a patient with oxaliplatin, folinic acid (or levofolinic acid) and 5-fluorouracil. The combination is administered intravenously (in some cases, intravenous bolus or continuous intravenous infusion) to the patient. NIS793 was administered to the patient at a dose of 2100mg once every two weeks (Q2W) in this four-component combination. Oxaliplatin at 85mg/m on day 1 of the Q2W cycle² Is administered to a patient. Folinic acid at 400mg/m on day 1 of the Q2W cycle² Is administered (or levofolinic acid is administered at 200 mg/m)² Is administered at a dose). 5-fluorouracil at 400mg/m on day 1 of the Q2W cycle² Then at 1200mg/m on days 1-2² Intravenous administration. Tirelimumab was optionally administered once every four weeks (Q4W) at a dose of 300 mg. In this combination, gastric cancer is treated.

In combination, NIS793 is administered to a patient with cyclophosphamide or topotecan. The three component combination is administered intravenously to a patient. NIS793 is administered to the patient based on the patient's weight in the three component combination: (a) For patients weighing less than 20kg, administration is at a dose of 45 mg/kg; (b) For patients weighing between 20-40kg, at a dose of 30 mg/kg; and (c) for patients with a body weight greater than 40kg, at a dose of 20 mg/kg. Cyclophosphamide at 250mg/m in this three-component combination² Is administered intravenously to the patient for 5 consecutive days (on days 1-5). Topotecan at 0.75mg/m in this three-component combination² Is administered intravenously to the patient for 5 consecutive days (on days 1-5). In some cases, the three component combination can be used to treat neuroblastoma. In some cases, the patient population is a pediatric patient population (e.g., under 18 years of age).

In combination, NIS793 is administered to a patient with gemcitabine. The combination is administered intravenously to a patient. NIS793 was administered to the patient based on the patient's weight in this combination: (a) For patients weighing less than 20kg, administration is at a dose of 45 mg/kg; (b) For patients weighing between 20-40kg, at a dose of 30 mg/kg; and (c) for patients with a body weight greater than 40kg, at a dose of 20 mg/kg. In the combinationGemcitabine at 675mg/m² Is administered intravenously to the patient once a week (e.g., on days 1 and 8). In some cases, the combination can be used to treat osteosarcoma. In some cases, the patient population is a pediatric patient population (e.g., under 18 years of age).

Diagnostic subject and therapeutic subject

As used herein, the term "subject" is intended to include both human and non-human animals. In some embodiments, the subject is a human subject. The term "non-human animal" includes mammals and non-mammals, such as non-human primates. In some embodiments, the subject is a human. In some embodiments, the subject is a human patient in need of enhancing an immune response. The combinations described herein are useful for treating a human patient suffering from a disorder treatable by modulating (e.g., enhancing or inhibiting) an immune response. In certain embodiments, the patient has, or is at risk of, a disorder described herein, e.g., a cancer described herein.

In some cases, subjects treated using the methods disclosed herein are aware that they have a disease or disorder that, in some cases, would benefit from the methods described herein. For example, in some cases, the subject has been detected and/or diagnosed with a disease. The detection and/or diagnosis may be from a physician or other qualified medical personnel. In some cases, detection and/or diagnosis may be performed on its own based on one or more symptoms (e.g., raised masses, bumps, etc.). Thus, in some embodiments, a subject may need the methods described herein to treat a disease or disorder thereof. The term "in need thereof" is intended to mean that the subject (or a person treating the subject) is aware of the presence of a disorder or disease (e.g., a proliferative disease, such as cancer).

In certain embodiments, the subject has been identified as having tgfβ (1, 2, or 3) expression in one or more tumors (or tumor microenvironments) thereof. In certain embodiments, the subject has been identified as having PD-1 expression in one or more tumors (or tumor microenvironments) thereof. In certain embodiments, the subject has been identified as having PD-L1 expression in one or more tumors (or tumor microenvironments) thereof. In certain embodiments, the subject has been identified as having PD-L2 expression in one or more tumors (or tumor microenvironments) thereof. In some embodiments, the subject has been identified as having both tgfβ (1, 2, or 3) and PD-1 expression in one or more tumors (or tumor microenvironments) thereof. In some embodiments, the subject has been identified as having both tgfβ (1, 2, or 3) and PD-L1 expression in one or more tumors (or tumor microenvironments) thereof. In some embodiments, the subject has been identified as having both tgfβ (1, 2, or 3) and PD-L2 expression in one or more tumors (or tumor microenvironments) thereof. Once these biomarkers are found, treatment can be performed using the described methods.

In some embodiments, the subject is between about 5kg to about 500 kg. In some embodiments, the subject is between about 10kg to about 400 kg. In some embodiments, the subject is between about 15kg to about 300 kg. In some embodiments, the subject is between about 20kg to about 200 kg. In some embodiments, the subject is between about 25kg to about 150kg. In some embodiments, the subject is between about 40kg to about 125 kg. In some embodiments, the subject is between about 50kg to about 100kg. In some embodiments, the subject is between about 65kg to about 85kg. In some embodiments, the subject is about 40kg. In some embodiments, the subject is about 45kg. In some embodiments, the subject is about 50kg. In some embodiments, the subject is about 55kg. In some embodiments, the subject is about 60kg. In some embodiments, the subject is about 65kg. In some embodiments, the subject is about 70kg. In some embodiments, the subject is about 75kg. In some embodiments, the subject is about 80kg. In some embodiments, the subject is about 85kg. In some embodiments, the subject is about 90kg. In some embodiments, the subject is about 95kg. In some embodiments, the subject is about 100kg. In some embodiments, the subject is about 110kg. In some embodiments, the subject is about 120kg. In some embodiments, the subject is about 130kg. In some embodiments, the subject is about 140kg. In some embodiments, the subject is about 150kg.

Cancer of the human body

In some embodiments, these methods are used to treat cancer, such as myelofibrosis (e.g., primary Myelofibrosis (PMF), primary post-thrombocytosis myelofibrosis (PET-MF), post-polycythemia vera myelofibrosis (PPV-MF)), leukemia (e.g., acute Myelogenous Leukemia (AML), such as relapsed or refractory AML, or primary AML; or Chronic Lymphocytic Leukemia (CLL)), lymphoma (e.g., T-cell lymphoma, B-cell lymphoma, non-hodgkin's lymphoma, or Small Lymphocytic Lymphoma (SLL)), myeloma (e.g., multiple myeloma), lung cancer (e.g., non-small cell lung cancer (NSCLC) (e.g., squamous and/or non-squamous histological NSCLC, or NSCLC adenocarcinoma), or Small Cell Lung Cancer (SCLC)), skin cancer (e.g., merkel cell carcinoma or melanoma (e.g., advanced melanoma)), ovarian cancer, mesothelioma, bladder cancer, soft tissue sarcoma (e.g., vascular endothelial cell tumor (HPC)), bone cancer (osteosarcoma), kidney cancer (kidney cancer) (e.g., renal cell carcinoma)), liver cancer (e.g., hepatocellular carcinoma), cholangiocarcinoma, sarcoma, myelodysplastic syndrome (MDS) (e.g., low risk MDS), prostate cancer, breast cancer (e.g., without expressing one of the estrogen receptor, progesterone, or Her 2/neu) Two or all breast cancers, e.g., triple negative breast cancer), colorectal cancer, nasopharyngeal cancer, duodenal cancer, endometrial cancer, pancreatic cancer (e.g., pancreatic Ductal Adenocarcinoma (PDAC)), head and neck cancer (e.g., head and Neck Squamous Cell Carcinoma (HNSCC)), anal cancer, gastroesophageal cancer, thyroid cancer (e.g., thyroid undifferentiated cancer), cervical cancer, or neuroendocrine tumor (NET) (e.g., atypical lung carcinoid).

In certain embodiments, the patient is not suitable for standard treatment regimens with established benefits in patients with one or more cancers described herein. In some embodiments, the subject is not suitable for chemotherapy. In some embodiments, the chemotherapeutics are potentiated induction chemotherapeutics. For example, the methods described herein may be used to treat adult patients suffering from one or more cancers as described. In certain embodiments, the inhibitor (tgfβ and/or PD 1) is administered in an amount effective to treat cancer or a symptom thereof.

The compositions, formulations, or methods described herein can be used to inhibit the growth of cancerous tumors. Alternatively, the compositions, formulations, or methods described herein may be used in combination with one or more of the following: standard-of-care treatment for cancer, another antibody or antigen-binding fragment thereof, an immunomodulatory agent (e.g., an activator of a co-stimulatory molecule or an inhibitor of an inhibitory molecule); vaccines, such as therapeutic cancer vaccines; or other forms of cellular immunotherapy as described herein. In one embodiment, the method is suitable for the treatment of cancer in vivo.

In another aspect, a method of treating a subject, e.g., reducing or ameliorating a hyperproliferative condition or disorder (e.g., cancer), e.g., a solid tumor, hematological cancer, soft tissue tumor, or metastatic lesion in a subject, is provided. The method comprises performing a method as described herein, or a composition or formulation as described herein, according to a dosage regimen as described herein.

As used herein, the term "cancer" is meant to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of the histopathological type or stage of invasion. Examples of cancer disorders include, but are not limited to, hematological cancers, solid tumors, soft tissue tumors, and metastatic lesions.

Examples of solid tumors include, but are not limited to, malignant tumors (e.g., sarcomas) and carcinomas (including adenocarcinomas and squamous cell carcinomas) of various organ systems, such as those affecting the liver, lung, breast, lymph, gastrointestinal (e.g., colon), anal, genital and genitourinary tracts (e.g., kidney, urothelium, bladder), prostate, CNS (e.g., brain, nerve or glial cell), head and neck, skin, pancreas and pharynx. Adenocarcinomas include malignant tumors, such as most colon, rectum, kidney (e.g., renal cell carcinoma (e.g., clear cell or non-clear cell renal cell carcinoma)), liver cancer, lung cancer (e.g., non-small cell lung cancer (e.g., squamous or non-squamous non-small cell lung cancer)), small intestine, and esophageal cancer. Squamous cell carcinoma includes, for example, malignant tumors in the lung, esophagus, skin, head and neck area, mouth, anus, and cervix. In one embodiment, the cancer is a melanoma, such as advanced melanoma. The cancer may be in early, mid, late stages or may be metastatic cancer. The combinations described herein may also be used to treat or prevent metastatic lesions of the cancers described above.

In certain embodiments, the cancer is a solid tumor. In some embodiments, the cancer is ovarian cancer. In other embodiments, the cancer is lung cancer, such as Small Cell Lung Cancer (SCLC) or non-small cell lung cancer (NSCLC). In other embodiments, the cancer is mesothelioma. In other embodiments, the cancer is a skin cancer, such as merkel cell carcinoma or melanoma. In other embodiments, the cancer is a renal cancer, such as Renal Cell Carcinoma (RCC). In other embodiments, the cancer is bladder cancer. In other embodiments, the cancer is a soft tissue sarcoma, such as an angioepidermoid tumor (HPC). In other embodiments, the cancer is bone cancer, such as osteosarcoma. In other embodiments, the cancer is colorectal cancer. In other embodiments, the cancer is pancreatic cancer (e.g., PDAC). In other embodiments, the cancer is nasopharyngeal cancer. In other embodiments, the cancer is breast cancer. In other embodiments, the cancer is duodenal cancer. In other embodiments, the cancer is endometrial cancer. In other embodiments, the cancer is an adenocarcinoma, e.g., an unknown adenocarcinoma. In other embodiments, the cancer is liver cancer, e.g., hepatocellular carcinoma. In other embodiments, the cancer is cholangiocarcinoma. In other embodiments, the cancer is a sarcoma. In certain embodiments, the cancer is myelodysplastic syndrome (MDS) (e.g., high risk MDS or low risk MDS). In some embodiments, the cancer is a neuroblastoma. In certain embodiments, the cancer is osteosarcoma.

In another embodiment, the cancer is a cancer (e.g., advanced or metastatic cancer), melanoma, or lung cancer (e.g., non-small cell lung cancer). In one embodiment, the cancer is lung cancer, such as non-small cell lung cancer or small cell lung cancer. In some embodiments, the non-small cell lung cancer is stage I (e.g., stage Ia or stage Ib), stage II (e.g., stage IIa or stage IIb), stage III (e.g., stage IIIa or stage IIIb), or stage IV non-small cell lung cancer. In one embodiment, the cancer is a melanoma, such as advanced melanoma. In one embodiment, the cancer is advanced or unresectable melanoma that is unresponsive to other therapies. In other embodiments, the cancer is a melanoma with BRAF mutations (e.g., BRAF V600 mutations). In another embodiment, the cancer is liver cancer (e.g., advanced liver cancer) with or without a viral infection (e.g., chronic viral hepatitis). In another embodiment, the cancer is a prostate cancer, such as advanced prostate cancer. In yet another embodiment, the cancer is a myeloma, such as multiple myeloma. In yet another embodiment, the cancer is a renal cancer, such as Renal Cell Carcinoma (RCC) (e.g., metastatic RCC, non-clear cell renal cell carcinoma (ncrcc), or Clear Cell Renal Cell Carcinoma (CCRCC)).

In some embodiments, the cancer is a high MSI cancer. In some embodiments, the cancer is a metastatic cancer. In other embodiments, the cancer is advanced cancer. In other embodiments, the cancer is a recurrent or refractory cancer.

Exemplary cancers for which growth may be inhibited using the methods, compositions or formulations disclosed herein include cancers that are generally responsive to immunotherapy. In addition, refractory or recurrent malignancies can be treated using the combinations described herein.

Examples of other cancers that may be treated include, but are not limited to, basal cell carcinoma, biliary tract carcinoma; bladder cancer; bone cancer; brain and CNS cancers; primary CNS lymphoma; a Central Nervous System (CNS) tumor; breast cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; cancer of head and neck; stomach cancer; intraepithelial tumors; renal cancer; laryngeal carcinoma; leukemia (including acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic or acute leukemia); liver cancer; lung cancer (e.g., small cells and non-small cells); lymphomas (including hodgkin and non-hodgkin lymphomas); lymphocytic lymphoma; melanoma, for example, malignant melanoma of the skin or in the eye; a myeloma; neuroblastoma; oral cancers (e.g., lips, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancers of the respiratory system; sarcoma; skin cancer; stomach cancer; testicular cancer; thyroid cancer; uterine cancer; urinary system cancer, liver cancer, anal region cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, childhood solid tumors, spinal cord shaft tumors, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and other cancers and sarcomas, as well as combinations of these cancers.

The methods and therapeutic agents described herein can include compositions co-formulated and/or co-administered with one or more therapeutic agents (e.g., one or more anticancer agents, cytotoxic or cytostatic agents, hormonal therapy, vaccines, and/or other immunotherapies). In other embodiments, the antibody molecule is administered in combination with other therapeutic treatment modalities, including surgery, radiation, cryosurgery, and/or hyperthermia. Such combination therapies may advantageously use lower doses of the administered therapeutic agents, thereby avoiding the possible toxicity or complications associated with various monotherapy.

When administered in combination, the tgfβ inhibitor, PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor, one or more additional agents, or all may be administered in higher, lower, or the same amount or dose than each agent used alone (e.g., as monotherapy). In certain embodiments, the tgfβ inhibitor, PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor, one or more additional agents, or all are administered in an amount or dose that is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dose of each agent used alone (e.g., as monotherapy). In other embodiments, the amount or dose of the tgfβ inhibitor, PD-1 inhibitor, PD-L1 inhibitor, or PD-L2 inhibitor, one or more additional agents, or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) that results in the desired effect (e.g., treatment of cancer).

In other embodiments, the additional therapeutic agent is from the agents listed in table 6 of WO 2017/019897. In some embodiments, the additional therapeutic agent is one or more of the following: 1) Protein Kinase C (PKC) inhibitors; 2) Heat shock protein 90 (HSP 90) inhibitors; 3) Inhibitors of phosphoinositide 3-kinase (PI 3K) and/or rapamycin target protein (mTOR); 4) Inhibitors of cytochrome P450 (e.g., CYP17 inhibitors or 17α -hydroxylase/C17-20 lyase inhibitors); 5) An iron chelator; 6) An aromatase inhibitor; 7) Inhibitors of p53, e.g., inhibitors of the p53/Mdm2 interaction; 8) Apoptosis inducers; 9) An angiogenesis inhibitor; 10 Aldosterone synthase inhibitor; 11 A Smooth (SMO) receptor inhibitor; 12 A prolactin receptor (PRLR) inhibitor; 13 Wnt signaling inhibitors; 14 CDK4/6 inhibitors; 15 Fibroblast growth factor receptor 2 (FGFR 2)/fibroblast growth factor receptor 4 (FGFR 4) inhibitor; 16 Inhibitors of macrophage colony-stimulating factor (M-CSF); 17 Inhibitors of one or more of c-KIT, histamine release, flt3 (e.g., FLK2/STK 1), or PKC; 18 Inhibitors of one or more of VEGFR-2 (e.g., FLK-1/KDR), PDGFRβ, C-KIT or Raf kinase C; 19 A somatostatin agonist and/or a somatostatin release inhibitor; 20 Anaplastic Lymphoma Kinase (ALK) inhibitors; 21 Insulin-like growth factor 1 receptor (IGF-1R) inhibitors; 22 P-glycoprotein 1 inhibitors; 23 Vascular Endothelial Growth Factor Receptor (VEGFR) inhibitors; 24 BCR-ABL kinase inhibitors; 25 FGFR inhibitors; 26 Inhibitors of CYP11B 2; 27 An inhibitor of HDM2, such as an inhibitor of HDM2-p53 interaction; 28 Inhibitors of tyrosine kinase; 29 Inhibitors of c-MET; 30 Inhibitors of JAK; 31 DAC inhibitors; 32 11 beta-hydroxylase inhibitor; 33 An inhibitor of IAP; 34 Inhibitors of PIM kinase; 35 Inhibitors of bocavine; 36 Inhibitors of BRAF (e.g., BRAF V600E or wild-type BRAF); 37 An inhibitor of HER 3; 38 Inhibitors of MEK; or 39) inhibitors of lipid kinases (e.g. as described in table 6 of WO 2017/019897).

Pediatric patient

In some cases, the patient population may be an adult population or a pediatric population. For pediatric patients, NIS793 may be used to treat children with recurrent or refractory solid tumors. The single agent and/or combination therapy may be used to treat pediatric patients with recurrent or refractory solid tumors.

In some embodiments, NIS793 (or other tgfβ inhibitors), cyclophosphamide and topotecan may be used to treat patients with neuroblastomas (e.g., recurrent or refractory neuroblastomas), such as pediatric patients.

In some embodiments, NIS793 (or other tgfβ inhibitor) and gemcitabine may be used to treat patients with osteosarcoma (e.g., a recurrent measurable osteosarcoma), such as pediatric patients.

In some embodiments, the patient population is older than 12 months and younger than 21 years. For example, in the treatment of neuroblastoma, a patient population may be older than 12 months and younger than 21 years. In some embodiments, the patient population may be greater than or equal to 12 months of age and the patient population encompasses adult patients having an age less than 39 years (between 12 months and 39 years of age).

Patients receiving the various therapies (and combinations) described throughout must be histologically validated for malignancy at the time of initial diagnosis or recurrence. Patients with recurrent or refractory solid tumors can be treated with various therapies (and combinations) as described throughout. In some embodiments, patients with recurrent or refractory neuroblastoma can be treated with various therapies (and combinations) described throughout. In some embodiments, patients with recurrent or refractory osteosarcoma may be treated with various therapies (and combinations) described throughout.

In some embodiments, for those patients weighing less than 20kg, they may be administered a dose of 45mg/kg of a tgfβ inhibitor (e.g., NIS 793). In some embodiments, for those patients weighing between 20kg and 40kg, they may be administered a tgfβ inhibitor (e.g., NIS 793) at a dose of 30 mg/kg. In some embodiments, for those patients having a body weight greater than 40kg, they may be administered a dose of 20mg/kg of a tgfβ inhibitor (e.g., NIS 793). The administration of each group may be once every 3 weeks, which may be considered a single cycle. In some cases, treatment of a patient may last for up to 35 cycles.

For some patients (e.g., those suffering from neuroblastoma and thus "in need thereof"), cyclophosphamide and/or topotecan may be administered in combination with a tgfβ inhibitor (e.g., NIS 793). For some patients (e.g., those suffering from osteosarcoma and thus "in need thereof"), gemcitabine may be administered in combination with a tgfβ inhibitor (e.g., NIS 793).

In some embodiments, a tgfβ inhibitor (e.g., NIS 793) may be used in combination with an additional therapeutic agent. In some cases, the additional therapeutic agent comprises cyclophosphamide or topotecan. When cyclophosphamide is used, it can be used at 250mg/m² And (5) administration. In some embodiments, the cyclophosphamide is administered for 5 days, e.g., five consecutive days. In some embodiments, the additional therapeutic agent comprises at 0.75mg/m² Topotecan is administered. When topotecan is used, its administration can last for 5 days, for example five consecutive days. In some embodiments, the proliferative disease is neuroblastoma.

In some embodiments, the additional therapeutic agent comprises gemcitabine. When gemcitabine is used, it may be used at 675mg/m² And (5) administration. In some embodiments, the administration of gemcitabine is continued for 2 days, for example, on days 1 and 8. In some embodiments, the proliferative disease is osteosarcoma.

In some cases, each period is 21 days long. Treatment of the patient may last for about 2 years.

Examples

Example 1: pharmaceutical product

Tgfβ inhibitors called NIS793 are powdered and can be used as infusion solutions. The powder is provided in glass vials with rubber stoppers, which are sealed with flip-flops. Each vial contained 100mg of NIS793 lyophilizate. Pharmaceutical products are produced using standard aseptic processes. In addition to NIS793, the pharmaceutical product contains the following pharmaceutical excipients: L-histidine/L-histidine hydrochloride monohydrate, polysorbate 20 and sucrose. The vial had 20% overfilling to allow the entire dose to be withdrawn.

The drug product was designed to be reconstituted with 1mL of sterile water for injection prior to administration to give 100mg/mL of NIS793 solution.

NIS793 concentrate for infusion solutions is provided in glass vials with rubber stoppers that are sealed with flip-caps. Each vial contained 700mg of NIS793 in 7mL of solution. The drug product solution contained the same quantitative and qualitative excipients as was used for the NIS793 powder (for infusion solution after reconstitution in sterile water). Similarly, 7% overfilling is provided to allow the entire dose to be withdrawn.

Example 2: human study

The pharmaceutical product containing NIS793 as described in example 1 was used in clinical trials. Table 5 below provides an overview of the ongoing human trials.

TABLE 0 ongoing human study

One study has begun and is ongoing: for the first human study, CNIS793X2101, "stage I/Ib, open label, multicenter dose escalation study in adult patients with advanced malignancy in combination with PDR 001". Total 120 patients were treated with NIS793 as a single agent or in combination with PDR001TABLE 5)。

Pharmacokinetics, metabolism and pharmacodynamics in humans

PK data from NIS793 from the CNIS793X2101 study (75 patients, 5 months 4 days of the expiration date 2020) were characterized. In the ascending cohort, a summary of PK parameter estimates derived for NIS793 as a single agent (NIS 793:0.3-1mg/kg Q3W) and in combination with PDR001 (NIS 793/PDR001:0.3mg/kg/100mg Q3W, 0.3-30mg/kg/300mg Q3W and 20-30mg/kg Q2W/400mg Q4W) is presented in Table 6 (cycle 1) and Table 7 (cycle 3). In addition, in an extended group of MSS-CRC and NSCLC, an overview of PK parameter estimates derived for NIS793 in combination with PDR001 (NIS 793/PDR001:2100mg/300mg Q3W) is presented in Table 8. The mean concentration versus time curves for each dose group of NIS793 are plotted in fig. 1 (cycle 1) and fig. 2 (cycle 3).

After administration of NIS793 via 30 minutes of intravenous infusion, an approximate dose ratio increase from 0.3mg/kg to 30mg/kg was observed in terms of NIS793 exposure (i.e., cycle 1Cmax and AUClast). Moderate accumulation (approximately up to 2.0 times) of NIS793 was observed based on the ratio of aucast and Cmax at cycle 3 compared to cycle 1. As shown by inter-subject variability (CV%), PK variability was low to moderate (e.g., cmax was 12.1% to 73.3%).

PDR001 was administered in combination with NIS793 (100 or 300mg q3w and 400mg q4 w) at three doses and two dosing schedules. The PK of PDR001 in combination with NIS793 was similar to single agent data from PDR001 clinical trial studies.

Table 6 overview of PK parameters of NIS793 by cycle 1 treatment in single agent and combinatorial study (incremental)

N: number of patients with primary PK; n: the number of patients with available PK parameter values; "-": inapplicable; CV%: coefficient of variation (%) =sd/average value 100.

Table 7 summary of PK parameters of NIS793 by cycle 3 treatment in single agent and combinatorial study (boost)

N: number of patients with primary PK; n: the number of patients with available PK parameter values; "-": inapplicable; CV%: coefficient of variation (%) =sd/average value 100.

Table 8 overview of PK parameters at NIS793 2100mg q3w of cycles 1 and 3 in combinatorial studies (extensions)

300mg Q3W，NSCLC(N＝20)

N: number of patients with primary PK; n: the number of patients with available PK parameter values; "-": inapplicable; CV%: coefficient of variation (%) =sd/average value 100.

Population PK analysis performed using concentration data from the up-dosing phase of study CNIS793X2101 described pharmacokinetic profiles of NIS793, including body weight as a covariate effect on clearance and distribution volume. Analysis showed that the pharmacokinetics of NIS793 can be well described using a two-compartment model with primary elimination from the central compartment. This is consistent with the observations of dose proportionality and time dependence of NIS793 PK based on non-compartmental analysis.

While Body Weight (BW) is a covariate of clearance in the population PK model, the estimated index of the power model is 0.55 (CV% = 40%), the predicted exposure between the weight-based and fixed dosing regimens and the trough concentration at steady state are comparable across different BW categories. The analysis supports the use of fixed or smooth dosing on a mg basis, regardless of patient weight, as weight-based dosing does not reduce inter-individual variability. Model-based simulations indicate that a dose of 2100mg will match the exposure observed at 30 mg/kg. In addition, a dose of 1400mg will match the exposure observed at 20 mg/kg.

Example 3: pediatric study design

Biomarker assays (including genomics) required for correlation studies and PK/PD studies

* PK/PD: pharmacokinetics/pharmacodynamics

* If the assay results are to be reported to the patient/patient family or patient physician at any time (whether at the time of the study or at the time of the study discontinuation), the assay must be performed in a CLIA-approved laboratory.

Equivalents (Eq.)

While specific embodiments of the invention have been discussed, the above description is illustrative and not restrictive. Many modifications of the invention will be apparent to those skilled in the art after review of this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification.

Sequence listing

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<120> use of anti-TGFb antibodies and other therapeutic agents for the treatment of proliferative diseases

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Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe Gln

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Gly

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Gly Leu Trp Glu Val Arg Ala Leu Pro Ser Val Tyr

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Gly Ala Asn Asp Ile Gly Ser Lys Ser Val His

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Glu Asp Ile Ile Arg Pro Ser

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Gln Val Trp Asp Arg Asp Ser Asp Gln Tyr Val

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

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Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Leu Trp Glu Val Arg Ala Leu Pro Ser Val Tyr Trp Gly

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Gln Gly Thr Leu Val Thr Val Ser Ser

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Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

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Thr Ala Arg Ile Thr Cys Gly Ala Asn Asp Ile Gly Ser Lys Ser Val

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His Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Val Ser

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Glu Asp Ile Ile Arg Pro Ser Gly Ile Pro Glu Arg Ile Ser Gly Ser

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Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

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Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg Asp Ser Asp Gln

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Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

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Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

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Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Leu Trp Glu Val Arg Ala Leu Pro Ser Val Tyr Trp Gly

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Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

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Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

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Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

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Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

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Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

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Thr Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His

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Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys

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Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu Val His Asn Ala Lys

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Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser

290 295 300

Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

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His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Ala Asn Asp Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Ala Gly Gln Ala Pro Val Leu Val Val Ser

35 40 45

Glu Asp Ile Ile Arg Pro Ser Gly Ile Pro Glu Arg Ile Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Arg Asp Ser Asp Gln

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Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys

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Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

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Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

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Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly

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Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

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Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

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Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Asn

20 25 30

Val Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Val Ile Pro Ile Val Asp Ile Ala Asn Tyr Ala Gln Arg Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Thr Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Leu Gly Leu Val Leu Asp Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

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Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

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Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

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Glu Thr Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Leu Gly Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Pro Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ala Asp Ser Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

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Gly Tyr Thr Phe Thr Thr Tyr Trp Met His

1 5 10

<210> 14

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Thr Tyr Trp Met His

1 5

<210> 15

<211> 17

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Asn Ile Tyr Pro Gly Thr Gly Gly Ser Asn Phe Asp Glu Lys Phe Lys

1 5 10 15

Asn

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Trp Thr Thr Gly Thr Gly Ala Tyr

1 5

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Gly Tyr Thr Phe Thr Thr Tyr

1 5

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Tyr Pro Gly Thr Gly Gly

1 5

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Thr Gly Gly Ser Asn Phe Asp Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Thr Arg Trp Thr Thr Gly Thr Gly Ala Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

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gaggtgcagc tggtgcagtc aggcgccgaa gtgaagaagc ccggcgagtc actgagaatt 60

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accggcacag gcgcctactg gggtcaaggc actaccgtga ccgtgtctag c 351

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asn Ile Tyr Pro Gly Thr Gly Gly Ser Asn Phe Asp Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Trp Thr Thr Gly Thr Gly Ala Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 22

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gaggtgcagc tggtgcagtc aggcgccgaa gtgaagaagc ccggcgagtc actgagaatt 60

agctgtaaag gttcaggcta caccttcact acctactgga tgcactgggt ccgccaggct 120

accggtcaag gcctcgagtg gatgggtaat atctaccccg gcaccggcgg ctctaacttc 180

gacgagaagt ttaagaatag agtgactatc accgccgata agtctactag caccgcctat 240

atggaactgt ctagcctgag atcagaggac accgccgtct actactgcac taggtggact 300

accggcacag gcgcctactg gggtcaaggc actaccgtga ccgtgtctag cgctagcact 360

aagggcccgt ccgtgttccc cctggcacct tgtagccgga gcactagcga atccaccgct 420

gccctcggct gcctggtcaa ggattacttc ccggagcccg tgaccgtgtc ctggaacagc 480

ggagccctga cctccggagt gcacaccttc cccgctgtgc tgcagagctc cgggctgtac 540

tcgctgtcgt cggtggtcac ggtgccttca tctagcctgg gtaccaagac ctacacttgc 600

aacgtggacc acaagccttc caacactaag gtggacaagc gcgtcgaatc gaagtacggc 660

ccaccgtgcc cgccttgtcc cgcgccggag ttcctcggcg gtccctcggt ctttctgttc 720

ccaccgaagc ccaaggacac tttgatgatt tcccgcaccc ctgaagtgac atgcgtggtc 780

gtggacgtgt cacaggaaga tccggaggtg cagttcaatt ggtacgtgga tggcgtcgag 840

gtgcacaacg ccaaaaccaa gccgagggag gagcagttca actccactta ccgcgtcgtg 900

tccgtgctga cggtgctgca tcaggactgg ctgaacggga aggagtacaa gtgcaaagtg 960

tccaacaagg gacttcctag ctcaatcgaa aagaccatct cgaaagccaa gggacagccc 1020

cgggaacccc aagtgtatac cctgccaccg agccaggaag aaatgactaa gaaccaagtc 1080

tcattgactt gccttgtgaa gggcttctac ccatcggata tcgccgtgga atgggagtcc 1140

aacggccagc cggaaaacaa ctacaagacc acccctccgg tgctggactc agacggatcc 1200

ttcttcctct actcgcggct gaccgtggat aagagcagat ggcaggaggg aaatgtgttc 1260

agctgttctg tgatgcatga agccctgcac aaccactaca ctcagaagtc cctgtccctc 1320

tccctggga 1329

<210> 23

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Lys Ser Ser Gln Ser Leu Leu Asp Ser Gly Asn Gln Lys Asn Phe Leu

1 5 10 15

Thr

<210> 24

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Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 25

<211> 9

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Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr

1 5

<210> 26

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Ser Gln Ser Leu Leu Asp Ser Gly Asn Gln Lys Asn Phe

1 5 10

<210> 27

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Trp Ala Ser

1

<210> 28

<211> 6

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Asp Tyr Ser Tyr Pro Tyr

1 5

<210> 29

<211> 113

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<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 30

<211> 339

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gagatcgtcc tgactcagtc acccgctacc ctgagcctga gccctggcga gcgggctaca 60

ctgagctgta aatctagtca gtcactgctg gatagcggta atcagaagaa cttcctgacc 120

tggtatcagc agaagcccgg taaagcccct aagctgctga tctactgggc ctctactaga 180

gaatcaggcg tgccctctag gtttagcggt agcggtagtg gcaccgactt caccttcact 240

atctctagcc tgcagcccga ggatatcgct acctactact gtcagaacga ctatagctac 300

ccctacacct tcggtcaagg cactaaggtc gagattaag 339

<210> 31

<211> 220

<212> PRT

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<400> 31

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 32

<211> 660

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 32

gagatcgtcc tgactcagtc acccgctacc ctgagcctga gccctggcga gcgggctaca 60

ctgagctgta aatctagtca gtcactgctg gatagcggta atcagaagaa cttcctgacc 120

tggtatcagc agaagcccgg taaagcccct aagctgctga tctactgggc ctctactaga 180

gaatcaggcg tgccctctag gtttagcggt agcggtagtg gcaccgactt caccttcact 240

atctctagcc tgcagcccga ggatatcgct acctactact gtcagaacga ctatagctac 300

ccctacacct tcggtcaagg cactaaggtc gagattaagc gtacggtggc cgctcccagc 360

gtgttcatct tcccccccag cgacgagcag ctgaagagcg gcaccgccag cgtggtgtgc 420

ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg 480

cagagcggca acagccagga gagcgtcacc gagcaggaca gcaaggactc cacctacagc 540

ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcataaggt gtacgcctgc 600

gaggtgaccc accagggcct gtccagcccc gtgaccaaga gcttcaacag gggcgagtgc 660

<210> 33

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr

65 70 75 80

Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 34

<211> 339

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 34

gagatcgtcc tgactcagtc acccgctacc ctgagcctga gccctggcga gcgggctaca 60

ctgagctgta aatctagtca gtcactgctg gatagcggta atcagaagaa cttcctgacc 120

tggtatcagc agaagcccgg tcaagcccct agactgctga tctactgggc ctctactaga 180

gaatcaggcg tgccctctag gtttagcggt agcggtagtg gcaccgactt caccttcact 240

atctctagcc tggaagccga ggacgccgct acctactact gtcagaacga ctatagctac 300

ccctacacct tcggtcaagg cactaaggtc gagattaag 339

<210> 35

<211> 220

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 35

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Gly Asn Gln Lys Asn Phe Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ala Pro Arg Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr

65 70 75 80

Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 36

<211> 660

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 36

gagatcgtcc tgactcagtc acccgctacc ctgagcctga gccctggcga gcgggctaca 60

ctgagctgta aatctagtca gtcactgctg gatagcggta atcagaagaa cttcctgacc 120

tggtatcagc agaagcccgg tcaagcccct agactgctga tctactgggc ctctactaga 180

gaatcaggcg tgccctctag gtttagcggt agcggtagtg gcaccgactt caccttcact 240

atctctagcc tggaagccga ggacgccgct acctactact gtcagaacga ctatagctac 300

ccctacacct tcggtcaagg cactaaggtc gagattaagc gtacggtggc cgctcccagc 360

gtgttcatct tcccccccag cgacgagcag ctgaagagcg gcaccgccag cgtggtgtgc 420

ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg 480

cagagcggca acagccagga gagcgtcacc gagcaggaca gcaaggactc cacctacagc 540

ctgagcagca ccctgaccct gagcaaggcc gactacgaga agcataaggt gtacgcctgc 600

gaggtgaccc accagggcct gtccagcccc gtgaccaaga gcttcaacag gggcgagtgc 660

<210> 37

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 37

acctactgga tgcac 15

<210> 38

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 38

aatatctacc ccggcaccgg cggctctaac ttcgacgaga agtttaagaa t 51

<210> 39

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 39

tggactaccg gcacaggcgc ctac 24

<210> 40

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 40

ggctacacct tcactaccta c 21

<210> 41

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 41

taccccggca ccggcggc 18

<210> 42

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 42

aaatctagtc agtcactgct ggatagcggt aatcagaaga acttcctgac c 51

<210> 43

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 43

tgggcctcta ctagagaatc a 21

<210> 44

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 44

cagaacgact atagctaccc ctacacc 27

<210> 45

<211> 39

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 45

agtcagtcac tgctggatag cggtaatcag aagaacttc 39

<210> 46

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 46

tgggcctct 9

<210> 47

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 47

gactatagct acccctac 18

<210> 48

<211> 440

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 48

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 49

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 49

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 50

<211> 447

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 50

Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 51

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 51

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 52

<211> 447

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 52

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Ser Gly Glu Ser Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Thr Ser Leu Thr Ala Glu Asp Thr Gly Met Tyr Phe Cys

85 90 95

Val Arg Val Gly Tyr Asp Ala Leu Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 53

<211> 213

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 53

Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Arg Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Trp Ile Tyr

35 40 45

Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Cys Leu Thr Ile Asn Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Phe Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 54

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 54

Ser Tyr Trp Met Tyr

1 5

<210> 55

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 55

Arg Ile Asp Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 56

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 56

Asp Tyr Arg Lys Gly Leu Tyr Ala Met Asp Tyr

1 5 10

<210> 57

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 57

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210> 58

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 58

Asp Pro Asn Ser Gly Ser

1 5

<210> 59

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 59

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Arg Lys Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 60

<211> 360

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 60

gaagtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctac cgtgaagatt 60

agctgtaaag tctcaggcta caccttcact agctactgga tgtactgggt ccgacaggct 120

agagggcaaa gactggagtg gatcggtaga atcgacccta atagcggctc tactaagtat 180

aacgagaagt ttaagaatag gttcactatt agtagggata actctaagaa caccctgtac 240

ctgcagatga atagcctgag agccgaggac accgccgtct actactgcgc tagagactat 300

agaaagggcc tgtacgctat ggactactgg ggtcaaggca ctaccgtgac cgtgtcttca 360

<210> 61

<211> 446

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 61

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Arg Lys Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 62

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 62

Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala

1 5 10

<210> 63

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 63

Trp Ala Ser Thr Arg His Thr

1 5

<210> 64

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 64

Gln Gln Tyr Asn Ser Tyr Pro Leu Thr

1 5

<210> 65

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 65

Ser Gln Asp Val Gly Thr Ala

1 5

<210> 66

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 66

Trp Ala Ser

1

<210> 67

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 67

Tyr Asn Ser Tyr Pro Leu

1 5

<210> 68

<211> 1338

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 68

gaagtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctac cgtgaagatt 60

agctgtaaag tctcaggcta caccttcact agctactgga tgtactgggt ccgacaggct 120

agagggcaaa gactggagtg gatcggtaga atcgacccta atagcggctc tactaagtat 180

aacgagaagt ttaagaatag gttcactatt agtagggata actctaagaa caccctgtac 240

ctgcagatga atagcctgag agccgaggac accgccgtct actactgcgc tagagactat 300

agaaagggcc tgtacgctat ggactactgg ggtcaaggca ctaccgtgac cgtgtcttca 360

gctagcacta agggcccgtc cgtgttcccc ctggcacctt gtagccggag cactagcgaa 420

tccaccgctg ccctcggctg cctggtcaag gattacttcc cggagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggagtg cacaccttcc ccgctgtgct gcagagctcc 540

gggctgtact cgctgtcgtc ggtggtcacg gtgccttcat ctagcctggg taccaagacc 600

tacacttgca acgtggacca caagccttcc aacactaagg tggacaagcg cgtcgaatcg 660

aagtacggcc caccgtgccc gccttgtccc gcgccggagt tcctcggcgg tccctcggtc 720

tttctgttcc caccgaagcc caaggacact ttgatgattt cccgcacccc tgaagtgaca 780

tgcgtggtcg tggacgtgtc acaggaagat ccggaggtgc agttcaattg gtacgtggat 840

ggcgtcgagg tgcacaacgc caaaaccaag ccgagggagg agcagttcaa ctccacttac 900

cgcgtcgtgt ccgtgctgac ggtgctgcat caggactggc tgaacgggaa ggagtacaag 960

tgcaaagtgt ccaacaaggg acttcctagc tcaatcgaaa agaccatctc gaaagccaag 1020

ggacagcccc gggaacccca agtgtatacc ctgccaccga gccaggaaga aatgactaag 1080

aaccaagtct cattgacttg ccttgtgaag ggcttctacc catcggatat cgccgtggaa 1140

tgggagtcca acggccagcc ggaaaacaac tacaagacca cccctccggt gctggactca 1200

gacggatcct tcttcctcta ctcgcggctg accgtggata agagcagatg gcaggaggga 1260

aatgtgttca gctgttctgt gatgcatgaa gccctgcaca accactacac tcagaagtcc 1320

ctgtccctct ccctggga 1338

<210> 69

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 69

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 70

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 70

gctattcagc tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta aagcctctca ggacgtgggc accgccgtgg cctggtatct gcagaagcct 120

ggtcaatcac ctcagctgct gatctactgg gcctctacta gacacaccgg cgtgccctct 180

aggtttagcg gtagcggtag tggcaccgac ttcaccttca ctatctcttc actggaagcc 240

gaggacgccg ctacctacta ctgtcagcag tataatagct accccctgac cttcggtcaa 300

ggcactaagg tcgagattaa g 321

<210> 71

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 71

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 72

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 72

gctattcagc tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta aagcctctca ggacgtgggc accgccgtgg cctggtatct gcagaagcct 120

ggtcaatcac ctcagctgct gatctactgg gcctctacta gacacaccgg cgtgccctct 180

aggtttagcg gtagcggtag tggcaccgac ttcaccttca ctatctcttc actggaagcc 240

gaggacgccg ctacctacta ctgtcagcag tataatagct accccctgac cttcggtcaa 300

ggcactaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 73

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 73

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Arg Lys Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 74

<211> 360

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 74

gaagtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctac cgtgaagatt 60

agctgtaaag tctcaggcta caccttcact agctactgga tgtactgggt ccgacaggct 120

accggtcaag gcctggagtg gatgggtaga atcgacccta atagcggctc tactaagtat 180

aacgagaagt ttaagaatag agtgactatc accgccgata agtctactag caccgcctat 240

atggaactgt ctagcctgag atcagaggac accgccgtct actactgcgc tagagactat 300

agaaagggcc tgtacgctat ggactactgg ggtcaaggca ctaccgtgac cgtgtcttca 360

<210> 75

<211> 446

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 75

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Asn Ser Gly Ser Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Arg Lys Gly Leu Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440 445

<210> 76

<211> 1338

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 76

gaagtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctac cgtgaagatt 60

agctgtaaag tctcaggcta caccttcact agctactgga tgtactgggt ccgacaggct 120

accggtcaag gcctggagtg gatgggtaga atcgacccta atagcggctc tactaagtat 180

aacgagaagt ttaagaatag agtgactatc accgccgata agtctactag caccgcctat 240

atggaactgt ctagcctgag atcagaggac accgccgtct actactgcgc tagagactat 300

agaaagggcc tgtacgctat ggactactgg ggtcaaggca ctaccgtgac cgtgtcttca 360

gctagcacta agggcccgtc cgtgttcccc ctggcacctt gtagccggag cactagcgaa 420

tccaccgctg ccctcggctg cctggtcaag gattacttcc cggagcccgt gaccgtgtcc 480

tggaacagcg gagccctgac ctccggagtg cacaccttcc ccgctgtgct gcagagctcc 540

gggctgtact cgctgtcgtc ggtggtcacg gtgccttcat ctagcctggg taccaagacc 600

tacacttgca acgtggacca caagccttcc aacactaagg tggacaagcg cgtcgaatcg 660

aagtacggcc caccgtgccc gccttgtccc gcgccggagt tcctcggcgg tccctcggtc 720

tttctgttcc caccgaagcc caaggacact ttgatgattt cccgcacccc tgaagtgaca 780

tgcgtggtcg tggacgtgtc acaggaagat ccggaggtgc agttcaattg gtacgtggat 840

ggcgtcgagg tgcacaacgc caaaaccaag ccgagggagg agcagttcaa ctccacttac 900

cgcgtcgtgt ccgtgctgac ggtgctgcat caggactggc tgaacgggaa ggagtacaag 960

tgcaaagtgt ccaacaaggg acttcctagc tcaatcgaaa agaccatctc gaaagccaag 1020

ggacagcccc gggaacccca agtgtatacc ctgccaccga gccaggaaga aatgactaag 1080

aaccaagtct cattgacttg ccttgtgaag ggcttctacc catcggatat cgccgtggaa 1140

tgggagtcca acggccagcc ggaaaacaac tacaagacca cccctccggt gctggactca 1200

gacggatcct tcttcctcta ctcgcggctg accgtggata agagcagatg gcaggaggga 1260

aatgtgttca gctgttctgt gatgcatgaa gccctgcaca accactacac tcagaagtcc 1320

ctgtccctct ccctggga 1338

<210> 77

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 77

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 78

gacgtcgtga tgactcagtc acccctgagc ctgcccgtga ccctggggca gcccgcctct 60

attagctgta aagcctctca ggacgtgggc accgccgtgg cctggtatca gcagaagcca 120

gggcaagccc ctagactgct gatctactgg gcctctacta gacacaccgg cgtgccctct 180

aggtttagcg gtagcggtag tggcaccgag ttcaccctga ctatctcttc actgcagccc 240

gacgacttcg ctacctacta ctgtcagcag tataatagct accccctgac cttcggtcaa 300

ggcactaagg tcgagattaa g 321

<210> 79

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 79

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Asp Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 80

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 80

gacgtcgtga tgactcagtc acccctgagc ctgcccgtga ccctggggca gcccgcctct 60

attagctgta aagcctctca ggacgtgggc accgccgtgg cctggtatca gcagaagcca 120

gggcaagccc ctagactgct gatctactgg gcctctacta gacacaccgg cgtgccctct 180

aggtttagcg gtagcggtag tggcaccgag ttcaccctga ctatctcttc actgcagccc 240

gacgacttcg ctacctacta ctgtcagcag tataatagct accccctgac cttcggtcaa 300

ggcactaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 81

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 81

agctactgga tgtac 15

<210> 82

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 82

agaatcgacc ctaatagcgg ctctactaag tataacgaga agtttaagaa t 51

<210> 83

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 83

gactatagaa agggcctgta cgctatggac tac 33

<210> 84

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 84

ggctacacct tcactagcta c 21

<210> 85

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 85

gaccctaata gcggctct 18

<210> 86

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 86

aaagcctctc aggacgtggg caccgccgtg gcc 33

<210> 87

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 87

tgggcctcta ctagacacac c 21

<210> 88

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 88

cagcagtata atagctaccc cctgacc 27

<210> 89

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 89

tctcaggacg tgggcaccgc c 21

<210> 90

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 90

tgggcctct 9

<210> 91

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 91

tataatagct accccctg 18

<210> 92

<211> 448

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 92

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 93

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 93

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 94

<211> 450

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 94

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ile Lys Leu Gly Thr Val Thr Thr Val Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 95

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 95

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 96

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 96

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 97

<211> 215

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 97

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 98

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 98

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 99

<211> 106

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 99

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 100

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 100

Gly Tyr Thr Phe Thr Ser Tyr Trp Met Tyr

1 5 10

<210> 101

<211> 121

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 101

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Val Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr Ala Ser Ser Leu Met

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg His Ala Tyr Gly His Asp Gly Gly Phe Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 102

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 102

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 103

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 103

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Val Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr Ala Ser Ser Leu Met

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg His Ala Tyr Gly His Asp Gly Gly Phe Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 104

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 104

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Ser Asn

20 25 30

Val Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 105

<211> 363

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 105

gaggtgcagc tggtggaatc tggcggcgga ctggtgcagt ccggcggctc tctgagactg 60

tcttgcgctg cctccggctt ctccctgtcc tcttacggcg tggactgggt gcgacaggcc 120

cctggcaagg gcctggaatg ggtgggagtg atctggggcg gaggcggcac ctactacgcc 180

tcttccctga tgggccggtt caccatctcc cgggacaact ccaagaacac cctgtacctg 240

cagatgaact ccctgcgggc cgaggacacc gccgtgtact actgcgccag acacgcctac 300

ggccacgacg gcggcttcgc catggattat tggggccagg gcaccctggt gacagtgtcc 360

tcc 363

<210> 106

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 106

gagatcgtga tgacccagtc ccccgccacc ctgtctgtgt ctcccggcga gagagccacc 60

ctgagctgca gagcctccga gtccgtgtcc tccaacgtgg cctggtatca gcagagacct 120

ggtcaggccc ctcggctgct gatctacggc gcctctaacc gggccaccgg catccctgcc 180

agattctccg gctccggcag cggcaccgac ttcaccctga ccatctcccg gctggaaccc 240

gaggacttcg ccgtgtacta ctgcggccag tcctactcat accccttcac cttcggccag 300

ggcaccaagc tggaaatcaa g 321

<210> 107

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 107

gaggtgcagc tggtggaatc tggcggcgga ctggtgcagt ccggcggctc tctgagactg 60

tcttgcgctg cctccggctt ctccctgtcc tcttacggcg tggactgggt gcgacaggcc 120

cctggcaagg gcctggaatg ggtgggagtg atctggggcg gaggcggcac ctactacgcc 180

tcttccctga tgggccggtt caccatctcc cgggacaact ccaagaacac cctgtacctg 240

cagatgaact ccctgcgggc cgaggacacc gccgtgtact actgcgccag acacgcctac 300

ggccacgacg gcggcttcgc catggattat tggggccagg gcaccctggt gacagtgtcc 360

tccgctagca ccaagggccc aagtgtgttt cccctggccc ccagcagcaa gtctacttcc 420

ggcggaactg ctgccctggg ttgcctggtg aaggactact tccccgagcc cgtgacagtg 480

tcctggaact ctggggctct gacttccggc gtgcacacct tccccgccgt gctgcagagc 540

agcggcctgt acagcctgag cagcgtggtg acagtgccct ccagctctct gggaacccag 600

acctatatct gcaacgtgaa ccacaagccc agcaacacca aggtggacaa gagagtggag 660

cccaagagct gcgacaagac ccacacctgc cccccctgcc cagctccaga actgctggga 720

gggccttccg tgttcctgtt cccccccaag cccaaggaca ccctgatgat cagcaggacc 780

cccgaggtga cctgcgtggt ggtggacgtg tcccacgagg acccagaggt gaagttcaac 840

tggtacgtgg acggcgtgga ggtgcacaac gccaagacca agcccagaga ggagcagtac 900

aacagcacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960

aaagaataca agtgcaaagt ctccaacaag gccctgccag ccccaatcga aaagacaatc 1020

agcaaggcca agggccagcc acgggagccc caggtgtaca ccctgccccc cagccgggag 1080

gagatgacca agaaccaggt gtccctgacc tgtctggtga agggcttcta ccccagcgat 1140

atcgccgtgg agtgggagag caacggccag cccgagaaca actacaagac caccccccca 1200

gtgctggaca gcgacggcag cttcttcctg tacagcaagc tgaccgtgga caagtccagg 1260

tggcagcagg gcaacgtgtt cagctgcagc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgagcct gagccccggc aag 1353

<210> 108

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 108

gagatcgtga tgacccagtc ccccgccacc ctgtctgtgt ctcccggcga gagagccacc 60

ctgagctgca gagcctccga gtccgtgtcc tccaacgtgg cctggtatca gcagagacct 120

ggtcaggccc ctcggctgct gatctacggc gcctctaacc gggccaccgg catccctgcc 180

agattctccg gctccggcag cggcaccgac ttcaccctga ccatctcccg gctggaaccc 240

gaggacttcg ccgtgtacta ctgcggccag tcctactcat accccttcac cttcggccag 300

ggcaccaagc tggaaatcaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 109

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 109

Ser Tyr Gly Val Asp

1 5

<210> 110

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 110

Gly Phe Ser Leu Ser Ser Tyr

1 5

<210> 111

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 111

Val Ile Trp Gly Gly Gly Gly Thr Tyr Tyr Ala Ser Ser Leu Met Gly

1 5 10 15

<210> 112

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 112

Trp Gly Gly Gly Gly

1 5

<210> 113

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 113

His Ala Tyr Gly His Asp Gly Gly Phe Ala Met Asp Tyr

1 5 10

<210> 114

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 114

Arg Ala Ser Glu Ser Val Ser Ser Asn Val Ala

1 5 10

<210> 115

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 115

Ser Glu Ser Val Ser Ser Asn

1 5

<210> 116

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 116

Gly Ala Ser Asn Arg Ala Thr

1 5

<210> 117

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 117

Gly Ala Ser

1

<210> 118

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 118

Gly Gln Ser Tyr Ser Tyr Pro Phe Thr

1 5

<210> 119

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 119

Ser Tyr Ser Tyr Pro Phe

1 5

<210> 120

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 120

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Glu Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Met Val Arg Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 121

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 121

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 122

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 122

Gly Phe Thr Leu Thr Asn Tyr Gly Met Asn

1 5 10

<210> 123

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 123

Asn Tyr Gly Met Asn

1 5

<210> 124

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 124

Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys

1 5 10 15

Gly

<210> 125

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 125

Asn Pro Pro Tyr Tyr Tyr Gly Thr Asn Asn Ala Glu Ala Met Asp Tyr

1 5 10 15

<210> 126

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 126

Gly Phe Thr Leu Thr Asn Tyr

1 5

<210> 127

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 127

Asn Thr Asp Thr Gly Glu

1 5

<210> 128

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 128

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Leu Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Pro Pro Tyr Tyr Tyr Gly Thr Asn Asn Ala Glu Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 129

<211> 375

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 129

caagtgcagc tggtgcagtc gggagccgaa gtgaagaagc ctggagcctc ggtgaaggtg 60

tcgtgcaagg catccggatt caccctcacc aattacggga tgaactgggt cagacaggcc 120

cggggtcaac ggctggagtg gatcggatgg attaacaccg acaccgggga gcctacctac 180

gcggacgatt tcaagggacg gttcgtgttc tccctcgaca cctccgtgtc caccgcctac 240

ctccaaatct cctcactgaa agcggaggac accgccgtgt actattgcgc gaggaacccg 300

ccctactact acggaaccaa caacgccgaa gccatggact actggggcca gggcaccact 360

gtgactgtgt ccagc 375

<210> 130

<211> 375

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 130

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg cctctggctt caccctgacc aactacggca tgaactgggt gcgacaggcc 120

aggggccagc ggctggaatg gatcggctgg atcaacaccg acaccggcga gcctacctac 180

gccgacgact tcaagggcag attcgtgttc tccctggaca cctccgtgtc caccgcctac 240

ctgcagatct ccagcctgaa ggccgaggat accgccgtgt actactgcgc ccggaacccc 300

ccttactact acggcaccaa caacgccgag gccatggact attggggcca gggcaccacc 360

gtgaccgtgt cctct 375

<210> 131

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 131

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Leu Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Arg Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Pro Pro Tyr Tyr Tyr Gly Thr Asn Asn Ala Glu Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly

450

<210> 132

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 132

Ser Ser Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 133

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 133

Tyr Thr Ser Thr Leu His Leu

1 5

<210> 134

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 134

Gln Gln Tyr Tyr Asn Leu Pro Trp Thr

1 5

<210> 135

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 135

Ser Gln Asp Ile Ser Asn Tyr

1 5

<210> 136

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 136

Tyr Thr Ser

1

<210> 137

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 137

Tyr Tyr Asn Leu Pro Trp

1 5

<210> 138

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 138

caagtgcagc tggtgcagtc gggagccgaa gtgaagaagc ctggagcctc ggtgaaggtg 60

tcgtgcaagg catccggatt caccctcacc aattacggga tgaactgggt cagacaggcc 120

cggggtcaac ggctggagtg gatcggatgg attaacaccg acaccgggga gcctacctac 180

gcggacgatt tcaagggacg gttcgtgttc tccctcgaca cctccgtgtc caccgcctac 240

ctccaaatct cctcactgaa agcggaggac accgccgtgt actattgcgc gaggaacccg 300

ccctactact acggaaccaa caacgccgaa gccatggact actggggcca gggcaccact 360

gtgactgtgt ccagcgcgtc cactaagggc ccgtccgtgt tccccctggc accttgtagc 420

cggagcacta gcgaatccac cgctgccctc ggctgcctgg tcaaggatta cttcccggag 480

cccgtgaccg tgtcctggaa cagcggagcc ctgacctccg gagtgcacac cttccccgct 540

gtgctgcaga gctccgggct gtactcgctg tcgtcggtgg tcacggtgcc ttcatctagc 600

ctgggtacca agacctacac ttgcaacgtg gaccacaagc cttccaacac taaggtggac 660

aagcgcgtcg aatcgaagta cggcccaccg tgcccgcctt gtcccgcgcc ggagttcctc 720

ggcggtccct cggtctttct gttcccaccg aagcccaagg acactttgat gatttcccgc 780

acccctgaag tgacatgcgt ggtcgtggac gtgtcacagg aagatccgga ggtgcagttc 840

aattggtacg tggatggcgt cgaggtgcac aacgccaaaa ccaagccgag ggaggagcag 900

ttcaactcca cttaccgcgt cgtgtccgtg ctgacggtgc tgcatcagga ctggctgaac 960

gggaaggagt acaagtgcaa agtgtccaac aagggacttc ctagctcaat cgaaaagacc 1020

atctcgaaag ccaagggaca gccccgggaa ccccaagtgt ataccctgcc accgagccag 1080

gaagaaatga ctaagaacca agtctcattg acttgccttg tgaagggctt ctacccatcg 1140

gatatcgccg tggaatggga gtccaacggc cagccggaaa acaactacaa gaccacccct 1200

ccggtgctgg actcagacgg atccttcttc ctctactcgc ggctgaccgt ggataagagc 1260

agatggcagg agggaaatgt gttcagctgt tctgtgatgc atgaagccct gcacaaccac 1320

tacactcaga agtccctgtc cctctccctg gga 1353

<210> 139

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 139

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg cctctggctt caccctgacc aactacggca tgaactgggt gcgacaggcc 120

aggggccagc ggctggaatg gatcggctgg atcaacaccg acaccggcga gcctacctac 180

gccgacgact tcaagggcag attcgtgttc tccctggaca cctccgtgtc caccgcctac 240

ctgcagatct ccagcctgaa ggccgaggat accgccgtgt actactgcgc ccggaacccc 300

ccttactact acggcaccaa caacgccgag gccatggact attggggcca gggcaccacc 360

gtgaccgtgt cctctgcttc taccaagggg cccagcgtgt tccccctggc cccctgctcc 420

agaagcacca gcgagagcac agccgccctg ggctgcctgg tgaaggacta cttccccgag 480

cccgtgaccg tgtcctggaa cagcggagcc ctgaccagcg gcgtgcacac cttccccgcc 540

gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc 600

ctgggcacca agacctacac ctgtaacgtg gaccacaagc ccagcaacac caaggtggac 660

aagagggtgg agagcaagta cggcccaccc tgccccccct gcccagcccc cgagttcctg 720

ggcggaccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 780

acccccgagg tgacctgtgt ggtggtggac gtgtcccagg aggaccccga ggtccagttc 840

aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 900

tttaacagca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 960

ggcaaagagt acaagtgtaa ggtctccaac aagggcctgc caagcagcat cgaaaagacc 1020

atcagcaagg ccaagggcca gcctagagag ccccaggtct acaccctgcc acccagccaa 1080

gaggagatga ccaagaacca ggtgtccctg acctgtctgg tgaagggctt ctacccaagc 1140

gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200

ccagtgctgg acagcgacgg cagcttcttc ctgtacagca ggctgaccgt ggacaagtcc 1260

agatggcagg agggcaacgt ctttagctgc tccgtgatgc acgaggccct gcacaaccac 1320

tacacccaga agagcctgag cctgtccctg ggc 1353

<210> 140

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 140

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ser Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 141

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 141

gatattcaga tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta gctctagtca ggatatctct aactacctga actggtatct gcagaagccc 120

ggtcaatcac ctcagctgct gatctactac actagcaccc tgcacctggg cgtgccctct 180

aggtttagcg gtagcggtag tggcaccgag ttcaccctga ctatctctag cctgcagccc 240

gacgacttcg ctacctacta ctgtcagcag tactataacc tgccctggac cttcggtcaa 300

ggcactaagg tcgagattaa g 321

<210> 142

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 142

gacatccaga tgacccagtc cccctccagc ctgtctgctt ccgtgggcga cagagtgacc 60

atcacctgtt cctccagcca ggacatctcc aactacctga actggtatct gcagaagccc 120

ggccagtccc ctcagctgct gatctactac acctccaccc tgcacctggg cgtgccctcc 180

agattttccg gctctggctc tggcaccgag tttaccctga ccatcagctc cctgcagccc 240

gacgacttcg ccacctacta ctgccagcag tactacaacc tgccctggac cttcggccag 300

ggcaccaagg tggaaatcaa g 321

<210> 143

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 143

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ser Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Leu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Asn Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 144

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 144

gatattcaga tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta gctctagtca ggatatctct aactacctga actggtatct gcagaagccc 120

ggtcaatcac ctcagctgct gatctactac actagcaccc tgcacctggg cgtgccctct 180

aggtttagcg gtagcggtag tggcaccgag ttcaccctga ctatctctag cctgcagccc 240

gacgacttcg ctacctacta ctgtcagcag tactataacc tgccctggac cttcggtcaa 300

ggcactaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 145

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 145

gacatccaga tgacccagtc cccctccagc ctgtctgctt ccgtgggcga cagagtgacc 60

atcacctgtt cctccagcca ggacatctcc aactacctga actggtatct gcagaagccc 120

ggccagtccc ctcagctgct gatctactac acctccaccc tgcacctggg cgtgccctcc 180

agattttccg gctctggctc tggcaccgag tttaccctga ccatcagctc cctgcagccc 240

gacgacttcg ccacctacta ctgccagcag tactacaacc tgccctggac cttcggccag 300

ggcaccaagg tggaaatcaa gcgtacggtg gccgctccca gcgtgttcat cttcccccca 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gtctgctgaa caacttctac 420

cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gtgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 146

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 146

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Leu Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Pro Pro Tyr Tyr Tyr Gly Thr Asn Asn Ala Glu Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 147

<211> 375

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 147

caggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctag tgtgaaagtc 60

agctgtaaag ctagtggctt caccctgact aactacggga tgaactgggt ccgccaggcc 120

ccaggtcaag gcctcgagtg gatgggctgg attaacaccg acaccggcga gcctacctac 180

gccgacgact ttaagggcag attcgtgttt agcctggaca ctagtgtgtc taccgcctac 240

ctgcagatct ctagcctgaa ggccgaggac accgccgtct actactgcgc tagaaacccc 300

ccctactact acggcactaa caacgccgag gctatggact actggggtca aggcactacc 360

gtgaccgtgt ctagc 375

<210> 148

<211> 375

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 148

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg cctctggctt caccctgacc aactacggca tgaactgggt gcgacaggcc 120

cctggacagg gcctggaatg gatgggctgg atcaacaccg acaccggcga gcctacctac 180

gccgacgact tcaagggcag attcgtgttc tccctggaca cctccgtgtc caccgcctac 240

ctgcagatct ccagcctgaa ggccgaggat accgccgtgt actactgcgc ccggaacccc 300

ccttactact acggcaccaa caacgccgag gccatggact attggggcca gggcaccacc 360

gtgaccgtgt cctct 375

<210> 149

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 149

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Thr Leu Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Pro Pro Tyr Tyr Tyr Gly Thr Asn Asn Ala Glu Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly

450

<210> 150

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 150

caggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctag tgtgaaagtc 60

agctgtaaag ctagtggctt caccctgact aactacggga tgaactgggt ccgccaggcc 120

ccaggtcaag gcctcgagtg gatgggctgg attaacaccg acaccggcga gcctacctac 180

gccgacgact ttaagggcag attcgtgttt agcctggaca ctagtgtgtc taccgcctac 240

ctgcagatct ctagcctgaa ggccgaggac accgccgtct actactgcgc tagaaacccc 300

ccctactact acggcactaa caacgccgag gctatggact actggggtca aggcactacc 360

gtgaccgtgt ctagcgctag cactaagggc ccgtccgtgt tccccctggc accttgtagc 420

cggagcacta gcgaatccac cgctgccctc ggctgcctgg tcaaggatta cttcccggag 480

cccgtgaccg tgtcctggaa cagcggagcc ctgacctccg gagtgcacac cttccccgct 540

gtgctgcaga gctccgggct gtactcgctg tcgtcggtgg tcacggtgcc ttcatctagc 600

ctgggtacca agacctacac ttgcaacgtg gaccacaagc cttccaacac taaggtggac 660

aagcgcgtcg aatcgaagta cggcccaccg tgcccgcctt gtcccgcgcc ggagttcctc 720

ggcggtccct cggtctttct gttcccaccg aagcccaagg acactttgat gatttcccgc 780

acccctgaag tgacatgcgt ggtcgtggac gtgtcacagg aagatccgga ggtgcagttc 840

aattggtacg tggatggcgt cgaggtgcac aacgccaaaa ccaagccgag ggaggagcag 900

ttcaactcca cttaccgcgt cgtgtccgtg ctgacggtgc tgcatcagga ctggctgaac 960

gggaaggagt acaagtgcaa agtgtccaac aagggacttc ctagctcaat cgaaaagacc 1020

atctcgaaag ccaagggaca gccccgggaa ccccaagtgt ataccctgcc accgagccag 1080

gaagaaatga ctaagaacca agtctcattg acttgccttg tgaagggctt ctacccatcg 1140

gatatcgccg tggaatggga gtccaacggc cagccggaaa acaactacaa gaccacccct 1200

ccggtgctgg actcagacgg atccttcttc ctctactcgc ggctgaccgt ggataagagc 1260

agatggcagg agggaaatgt gttcagctgt tctgtgatgc atgaagccct gcacaaccac 1320

tacactcaga agtccctgtc cctctccctg gga 1353

<210> 151

<211> 1353

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 151

caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgcctc cgtgaaggtg 60

tcctgcaagg cctctggctt caccctgacc aactacggca tgaactgggt gcgacaggcc 120

cctggacagg gcctggaatg gatgggctgg atcaacaccg acaccggcga gcctacctac 180

gccgacgact tcaagggcag attcgtgttc tccctggaca cctccgtgtc caccgcctac 240

ctgcagatct ccagcctgaa ggccgaggat accgccgtgt actactgcgc ccggaacccc 300

ccttactact acggcaccaa caacgccgag gccatggact attggggcca gggcaccacc 360

gtgaccgtgt cctctgcttc taccaagggg cccagcgtgt tccccctggc cccctgctcc 420

agaagcacca gcgagagcac agccgccctg ggctgcctgg tgaaggacta cttccccgag 480

cccgtgaccg tgtcctggaa cagcggagcc ctgaccagcg gcgtgcacac cttccccgcc 540

gtgctgcaga gcagcggcct gtacagcctg agcagcgtgg tgaccgtgcc cagcagcagc 600

ctgggcacca agacctacac ctgtaacgtg gaccacaagc ccagcaacac caaggtggac 660

aagagggtgg agagcaagta cggcccaccc tgccccccct gcccagcccc cgagttcctg 720

ggcggaccca gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 780

acccccgagg tgacctgtgt ggtggtggac gtgtcccagg aggaccccga ggtccagttc 840

aactggtacg tggacggcgt ggaggtgcac aacgccaaga ccaagcccag agaggagcag 900

tttaacagca cctaccgggt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 960

ggcaaagagt acaagtgtaa ggtctccaac aagggcctgc caagcagcat cgaaaagacc 1020

atcagcaagg ccaagggcca gcctagagag ccccaggtct acaccctgcc acccagccaa 1080

gaggagatga ccaagaacca ggtgtccctg acctgtctgg tgaagggctt ctacccaagc 1140

gacatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200

ccagtgctgg acagcgacgg cagcttcttc ctgtacagca ggctgaccgt ggacaagtcc 1260

agatggcagg agggcaacgt ctttagctgc tccgtgatgc acgaggccct gcacaaccac 1320

tacacccaga agagcctgag cctgtccctg ggc 1353

<210> 152

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 152

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ser Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Leu Gly Ile Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu Ser

65 70 75 80

Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln Tyr Tyr Asn Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 153

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 153

gatattcaga tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta gctctagtca ggatatctct aactacctga actggtatca gcagaagccc 120

ggtaaagccc ctaagctgct gatctactac actagcaccc tgcacctggg aatcccccct 180

aggtttagcg gtagcggcta cggcaccgac ttcaccctga ctattaacaa tatcgagtca 240

gaggacgccg cctactactt ctgtcagcag tactataacc tgccctggac cttcggtcaa 300

ggcactaagg tcgagattaa g 321

<210> 154

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 154

gacatccaga tgacccagtc cccctccagc ctgtctgctt ccgtgggcga cagagtgacc 60

atcacctgtt cctccagcca ggacatctcc aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctactac acctccaccc tgcacctggg catcccccct 180

agattctccg gctctggcta cggcaccgac ttcaccctga ccatcaacaa catcgagtcc 240

gaggacgccg cctactactt ctgccagcag tactacaacc tgccctggac cttcggccag 300

ggcaccaagg tggaaatcaa g 321

<210> 155

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 155

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ser Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Thr Leu His Leu Gly Ile Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Ile Glu Ser

65 70 75 80

Glu Asp Ala Ala Tyr Tyr Phe Cys Gln Gln Tyr Tyr Asn Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 156

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 156

gatattcaga tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta gctctagtca ggatatctct aactacctga actggtatca gcagaagccc 120

ggtaaagccc ctaagctgct gatctactac actagcaccc tgcacctggg aatcccccct 180

aggtttagcg gtagcggcta cggcaccgac ttcaccctga ctattaacaa tatcgagtca 240

gaggacgccg cctactactt ctgtcagcag tactataacc tgccctggac cttcggtcaa 300

ggcactaagg tcgagattaa gcgtacggtg gccgctccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcataag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 157

<211> 642

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 157

gacatccaga tgacccagtc cccctccagc ctgtctgctt ccgtgggcga cagagtgacc 60

atcacctgtt cctccagcca ggacatctcc aactacctga actggtatca gcagaagccc 120

ggcaaggccc ccaagctgct gatctactac acctccaccc tgcacctggg catcccccct 180

agattctccg gctctggcta cggcaccgac ttcaccctga ccatcaacaa catcgagtcc 240

gaggacgccg cctactactt ctgccagcag tactacaacc tgccctggac cttcggccag 300

ggcaccaagg tggaaatcaa gcgtacggtg gccgctccca gcgtgttcat cttcccccca 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gtctgctgaa caacttctac 420

cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtca ccgagcagga cagcaaggac tccacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gtgaggtgac ccaccagggc 600

ctgtccagcc ccgtgaccaa gagcttcaac aggggcgagt gc 642

<210> 158

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 158

aattacggga tgaac 15

<210> 159

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 159

aactacggca tgaac 15

<210> 160

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 160

tggattaaca ccgacaccgg ggagcctacc tacgcggacg atttcaaggg a 51

<210> 161

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 161

tggatcaaca ccgacaccgg cgagcctacc tacgccgacg acttcaaggg c 51

<210> 162

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 162

aacccgccct actactacgg aaccaacaac gccgaagcca tggactac 48

<210> 163

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 163

aacccccctt actactacgg caccaacaac gccgaggcca tggactat 48

<210> 164

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 164

ggattcaccc tcaccaatta c 21

<210> 165

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 165

ggcttcaccc tgaccaacta c 21

<210> 166

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 166

aacaccgaca ccggggag 18

<210> 167

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 167

aacaccgaca ccggcgag 18

<210> 168

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 168

agctctagtc aggatatctc taactacctg aac 33

<210> 169

<211> 33

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 169

tcctccagcc aggacatctc caactacctg aac 33

<210> 170

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 170

tacactagca ccctgcacct g 21

<210> 171

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 171

tacacctcca ccctgcacct g 21

<210> 172

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 172

cagcagtact ataacctgcc ctggacc 27

<210> 173

<211> 27

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 173

cagcagtact acaacctgcc ctggacc 27

<210> 174

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 174

agtcaggata tctctaacta c 21

<210> 175

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 175

agccaggaca tctccaacta c 21

<210> 176

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 176

tacactagc 9

<210> 177

<211> 9

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 177

tacacctcc 9

<210> 178

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 178

tactataacc tgccctgg 18

<210> 179

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 179

tactacaacc tgccctgg 18

<210> 180

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 180

aactacggga tgaac 15

<210> 181

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 181

tggattaaca ccgacaccgg cgagcctacc tacgccgacg actttaaggg c 51

<210> 182

<211> 48

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 182

aaccccccct actactacgg cactaacaac gccgaggcta tggactac 48

<210> 183

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 183

ggcttcaccc tgactaacta c 21

<210> 184

<211> 447

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 184

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Tyr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Arg Gly Ser Thr Asn Ser Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Leu Ser Leu Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Phe Gly Tyr Ser Asp Tyr Glu Tyr Asn Trp Phe Asp Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 185

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 185

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Asn Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 186

<211> 446

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 186

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ala Tyr

20 25 30

Gly Val Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Asp Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Glu Gly Asp Val Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 187

<211> 220

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 187

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Gly

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln

85 90 95

His Phe Gly Thr Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 188

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 188

Gly Phe Thr Leu Thr Asn Tyr Gly Met Asn

1 5 10

<210> 189

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 189

Ser Tyr Asn Met His

1 5

<210> 190

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 190

Asp Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 191

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 191

Val Gly Gly Ala Phe Pro Met Asp Tyr

1 5

<210> 192

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 192

Gly Tyr Thr Phe Thr Ser Tyr

1 5

<210> 193

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 193

Tyr Pro Gly Asn Gly Asp

1 5

<210> 194

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 194

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 195

<211> 354

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 195

caggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggctctag cgtgaaagtt 60

tcttgtaaag ctagtggcta caccttcact agctataata tgcactgggt tcgccaggcc 120

ccagggcaag gcctcgagtg gatgggcgat atctaccccg ggaacggcga cactagttat 180

aatcagaagt ttaagggtag agtcactatc accgccgata agtctactag caccgtctat 240

atggaactga gttccctgag gtctgaggac accgccgtct actactgcgc tagagtgggc 300

ggagccttcc ctatggacta ctggggtcaa ggcactaccg tgaccgtgtc tagc 354

<210> 196

<211> 444

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 196

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Asp Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 197

<211> 1332

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 197

caggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggctctag cgtgaaagtt 60

tcttgtaaag ctagtggcta caccttcact agctataata tgcactgggt tcgccaggcc 120

ccagggcaag gcctcgagtg gatgggcgat atctaccccg ggaacggcga cactagttat 180

aatcagaagt ttaagggtag agtcactatc accgccgata agtctactag caccgtctat 240

atggaactga gttccctgag gtctgaggac accgccgtct actactgcgc tagagtgggc 300

ggagccttcc ctatggacta ctggggtcaa ggcactaccg tgaccgtgtc tagcgctagc 360

actaagggcc cgtccgtgtt ccccctggca ccttgtagcc ggagcactag cgaatccacc 420

gctgccctcg gctgcctggt caaggattac ttcccggagc ccgtgaccgt gtcctggaac 480

agcggagccc tgacctccgg agtgcacacc ttccccgctg tgctgcagag ctccgggctg 540

tactcgctgt cgtcggtggt cacggtgcct tcatctagcc tgggtaccaa gacctacact 600

tgcaacgtgg accacaagcc ttccaacact aaggtggaca agcgcgtcga atcgaagtac 660

ggcccaccgt gcccgccttg tcccgcgccg gagttcctcg gcggtccctc ggtctttctg 720

ttcccaccga agcccaagga cactttgatg atttcccgca cccctgaagt gacatgcgtg 780

gtcgtggacg tgtcacagga agatccggag gtgcagttca attggtacgt ggatggcgtc 840

gaggtgcaca acgccaaaac caagccgagg gaggagcagt tcaactccac ttaccgcgtc 900

gtgtccgtgc tgacggtgct gcatcaggac tggctgaacg ggaaggagta caagtgcaaa 960

gtgtccaaca agggacttcc tagctcaatc gaaaagacca tctcgaaagc caagggacag 1020

ccccgggaac cccaagtgta taccctgcca ccgagccagg aagaaatgac taagaaccaa 1080

gtctcattga cttgccttgt gaagggcttc tacccatcgg atatcgccgt ggaatgggag 1140

tccaacggcc agccggaaaa caactacaag accacccctc cggtgctgga ctcagacgga 1200

tccttcttcc tctactcgcg gctgaccgtg gataagagca gatggcagga gggaaatgtg 1260

ttcagctgtt ctgtgatgca tgaagccctg cacaaccact acactcagaa gtccctgtcc 1320

ctctccctgg ga 1332

<210> 198

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 198

Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln

1 5 10 15

<210> 199

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 199

Ala Ala Ser Asn Val Glu Ser

1 5

<210> 200

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 200

Gln Gln Ser Arg Lys Asp Pro Ser Thr

1 5

<210> 201

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 201

Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu

1 5 10

<210> 202

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 202

Ala Ala Ser

1

<210> 203

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 203

Ser Arg Lys Asp Pro Ser

1 5

<210> 204

<211> 111

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 204

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Asp Pro Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 205

<211> 333

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 205

gctattcagc tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta gagctagtga atcagtcgag tactacggca ctagcctgat gcagtggtat 120

cagcagaagc ccgggaaagc ccctaagctg ctgatctacg ccgcctctaa cgtggaatca 180

ggcgtgccct ctaggtttag cggtagcggt agtggcaccg acttcaccct gactatctct 240

agcctgcagc ccgaggactt cgctacctac ttctgtcagc agtctaggaa ggaccctagc 300

accttcggcg gaggcactaa ggtcgagatt aag 333

<210> 206

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 206

Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Arg

85 90 95

Lys Asp Pro Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 207

<211> 654

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 207

gctattcagc tgactcagtc acctagtagc ctgagcgcta gtgtgggcga tagagtgact 60

atcacctgta gagctagtga atcagtcgag tactacggca ctagcctgat gcagtggtat 120

cagcagaagc ccgggaaagc ccctaagctg ctgatctacg ccgcctctaa cgtggaatca 180

ggcgtgccct ctaggtttag cggtagcggt agtggcaccg acttcaccct gactatctct 240

agcctgcagc ccgaggactt cgctacctac ttctgtcagc agtctaggaa ggaccctagc 300

accttcggcg gaggcactaa ggtcgagatt aagcgtacgg tggccgctcc cagcgtgttc 360

atcttccccc ccagcgacga gcagctgaag agcggcaccg ccagcgtggt gtgcctgctg 420

aacaacttct acccccggga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc 480

ggcaacagcc aggagagcgt caccgagcag gacagcaagg actccaccta cagcctgagc 540

agcaccctga ccctgagcaa ggccgactac gagaagcata aggtgtacgc ctgcgaggtg 600

acccaccagg gcctgtccag ccccgtgacc aagagcttca acaggggcga gtgc 654

<210> 208

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 208

Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 209

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 209

Tyr Pro Gly Gln Gly Asp

1 5

<210> 210

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 210

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 211

<211> 354

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 211

caggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctag tgtgaaagtt 60

agctgtaaag ctagtggcta tactttcact tcttataata tgcactgggt ccgccaggcc 120

ccaggtcaag gcctcgagtg gatcggcgat atctaccccg gtcaaggcga cacttcctat 180

aatcagaagt ttaagggtag agctactatg accgccgata agtctacttc taccgtctat 240

atggaactga gttccctgag gtctgaggac accgccgtct actactgcgc tagagtgggc 300

ggagccttcc caatggacta ctggggtcaa ggcaccctgg tcaccgtgtc tagc 354

<210> 212

<211> 444

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 212

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Pro Gly Gln Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Gly Gly Ala Phe Pro Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 213

<211> 1332

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 213

caggtgcagc tggtgcagtc aggcgccgaa gtgaagaaac ccggcgctag tgtgaaagtt 60

agctgtaaag ctagtggcta tactttcact tcttataata tgcactgggt ccgccaggcc 120

ccaggtcaag gcctcgagtg gatcggcgat atctaccccg gtcaaggcga cacttcctat 180

aatcagaagt ttaagggtag agctactatg accgccgata agtctacttc taccgtctat 240

atggaactga gttccctgag gtctgaggac accgccgtct actactgcgc tagagtgggc 300

ggagccttcc caatggacta ctggggtcaa ggcaccctgg tcaccgtgtc tagcgctagc 360

actaagggcc cgtccgtgtt ccccctggca ccttgtagcc ggagcactag cgaatccacc 420

gctgccctcg gctgcctggt caaggattac ttcccggagc ccgtgaccgt gtcctggaac 480

agcggagccc tgacctccgg agtgcacacc ttccccgctg tgctgcagag ctccgggctg 540

tactcgctgt cgtcggtggt cacggtgcct tcatctagcc tgggtaccaa gacctacact 600

tgcaacgtgg accacaagcc ttccaacact aaggtggaca agcgcgtcga atcgaagtac 660

ggcccaccgt gcccgccttg tcccgcgccg gagttcctcg gcggtccctc ggtctttctg 720

ttcccaccga agcccaagga cactttgatg atttcccgca cccctgaagt gacatgcgtg 780

gtcgtggacg tgtcacagga agatccggag gtgcagttca attggtacgt ggatggcgtc 840

gaggtgcaca acgccaaaac caagccgagg gaggagcagt tcaactccac ttaccgcgtc 900

gtgtccgtgc tgacggtgct gcatcaggac tggctgaacg ggaaggagta caagtgcaaa 960

gtgtccaaca agggacttcc tagctcaatc gaaaagacca tctcgaaagc caagggacag 1020

ccccgggaac cccaagtgta taccctgcca ccgagccagg aagaaatgac taagaaccaa 1080

gtctcattga cttgccttgt gaagggcttc tacccatcgg atatcgccgt ggaatgggag 1140

tccaacggcc agccggaaaa caactacaag accacccctc cggtgctgga ctcagacgga 1200

tccttcttcc tctactcgcg gctgaccgtg gataagagca gatggcagga gggaaatgtg 1260

ttcagctgtt ctgtgatgca tgaagccctg cacaaccact acactcagaa gtccctgtcc 1320

ctctccctgg ga 1332

<210> 214

<211> 111

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 214

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Asp Pro Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 215

<211> 333

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 215

gatatcgtcc tgactcagtc acccgatagc ctggccgtca gcctgggcga gcgggctact 60

attaactgta gagctagtga atcagtcgag tactacggca ctagcctgat gcagtggtat 120

cagcagaagc ccggtcaacc ccctaagctg ctgatctacg ccgcctctaa cgtggaatca 180

ggcgtgcccg ataggtttag cggtagcggt agtggcaccg acttcaccct gactattagt 240

agcctgcagg ccgaggacgt ggccgtctac tactgtcagc agtctaggaa ggaccctagc 300

accttcggcg gaggcactaa ggtcgagatt aag 333

<210> 216

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 216

Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Arg

85 90 95

Lys Asp Pro Ser Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 217

<211> 654

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 217

gatatcgtcc tgactcagtc acccgatagc ctggccgtca gcctgggcga gcgggctact 60

attaactgta gagctagtga atcagtcgag tactacggca ctagcctgat gcagtggtat 120

cagcagaagc ccggtcaacc ccctaagctg ctgatctacg ccgcctctaa cgtggaatca 180

ggcgtgcccg ataggtttag cggtagcggt agtggcaccg acttcaccct gactattagt 240

agcctgcagg ccgaggacgt ggccgtctac tactgtcagc agtctaggaa ggaccctagc 300

accttcggcg gaggcactaa ggtcgagatt aagcgtacgg tggccgctcc cagcgtgttc 360

atcttccccc ccagcgacga gcagctgaag agcggcaccg ccagcgtggt gtgcctgctg 420

aacaacttct acccccggga ggccaaggtg cagtggaagg tggacaacgc cctgcagagc 480

ggcaacagcc aggagagcgt caccgagcag gacagcaagg actccaccta cagcctgagc 540

agcaccctga ccctgagcaa ggccgactac gagaagcata aggtgtacgc ctgcgaggtg 600

acccaccagg gcctgtccag ccccgtgacc aagagcttca acaggggcga gtgc 654

<210> 218

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 218

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ala Ser Gly Phe Thr Phe Ser Ser

20 25 30

Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp

35 40 45

Val Ser Thr Ile Ser Gly Gly Gly Thr Tyr Thr Tyr Tyr Gln Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Ser Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

100 105 110

Ser Ala

<210> 219

<211> 108

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 219

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Arg Tyr

20 25 30

Leu Asn Trp Tyr His Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser His Ser Ala Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 220

<211> 120

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 220

Glu Val Gln Val Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Tyr Cys Val Ala Ser Gly Phe Thr Phe Ser Gly Ser

20 25 30

Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Lys Lys Tyr Tyr Val Gly Pro Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly

115 120

<210> 221

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 221

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Ile Glu Val

100 105 110

Lys

<210> 222

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 222

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 223

<211> 170

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 223

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val

65 70 75 80

Thr Thr Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly

85 90 95

Lys Glu Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr

100 105 110

Thr Ala Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro

115 120 125

Ser Thr Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr

130 135 140

Pro Ser Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser

145 150 155 160

His Gln Pro Pro Gly Val Tyr Pro Gln Gly

165 170

<210> 224

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 224

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 225

<211> 297

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 225

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

65 70 75 80

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

85 90 95

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

100 105 110

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

115 120 125

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

130 135 140

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

145 150 155 160

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

165 170 175

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

180 185 190

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

195 200 205

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

210 215 220

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

225 230 235 240

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

245 250 255

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

260 265 270

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

275 280 285

Lys Ser Leu Ser Leu Ser Pro Gly Lys

290 295

<210> 226

<211> 114

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 226

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 227

<211> 77

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 227

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro

65 70 75

<210> 228

<211> 248

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 228

Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Ser Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro

115 120 125

Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Leu Val Leu Thr Gln Ser

130 135 140

Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys

145 150 155 160

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn

180 185 190

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe

210 215 220

Tyr Phe Cys Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr

225 230 235 240

Lys Leu Glu Ile Lys Arg Arg Ser

245

<210> 229

<211> 111

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 229

Glu Leu Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile

35 40 45

Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser

65 70 75 80

Lys Asp Leu Ala Asp Tyr Phe Tyr Phe Cys Gln Tyr Asn Arg Tyr Pro

85 90 95

Tyr Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Arg Ser

100 105 110

<210> 230

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 230

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 231

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 231

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 232

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 232

Gly Gly Gly Ser

1

<210> 233

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 233

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

180 185 190

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

195 200 205

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

225 230 235 240

Ser Ser

<210> 234

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 234

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 235

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 235

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 236

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 236

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 237

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 237

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 238

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 238

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 239

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 239

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 240

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 240

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 241

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 241

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 242

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 242

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

115 120 125

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

130 135 140

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

145 150 155 160

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

165 170 175

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

180 185 190

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

195 200 205

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

210 215 220

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 243

<211> 247

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 243

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

130 135 140

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

145 150 155 160

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

180 185 190

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys

245

<210> 244

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 244

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu

115 120 125

Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys

130 135 140

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser

165 170 175

Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser

180 185 190

Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr

195 200 205

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

210 215 220

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser

<210> 245

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 245

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala

130 135 140

Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly

165 170 175

Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly

180 185 190

Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu

195 200 205

Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys

<210> 246

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 246

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

1 5 10

<210> 247

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 247

Gln Gln Gly Asn Thr Leu Pro Tyr Thr

1 5

<210> 248

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 248

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

1 5 10 15

Lys Gly

<210> 249

<211> 119

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 249

Gln Val Gln Leu Leu Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ser

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Ser Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr

115

<210> 250

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 250

His Thr Ser Arg Leu His Ser

1 5

<210> 251

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 251

Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn

1 5 10

<210> 252

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 252

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

195 200 205

Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys

210 215 220

Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 253

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 253

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 254

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 254

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 255

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 255

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 256

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 256

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met

145 150 155 160

Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr

165 170 175

Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln

245 250 255

Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 257

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 257

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 258

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 258

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 259

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 259

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Ser Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 260

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 260

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Gln Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 261

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 261

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 262

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 262

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val

145 150 155 160

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser

165 170 175

Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly

180 185 190

Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn

195 200 205

Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn

210 215 220

Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

225 230 235 240

Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp

245 250 255

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 263

<211> 491

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 263

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys

50 55 60

Gly Leu Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ser Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys

85 90 95

Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala

100 105 110

Val Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro

165 170 175

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

180 185 190

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

195 200 205

Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro Ala Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln

225 230 235 240

Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

245 250 255

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro

260 265 270

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

275 280 285

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

305 310 315 320

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

325 330 335

Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

340 345 350

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

355 360 365

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

370 375 380

Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr

385 390 395 400

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

405 410 415

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn

420 425 430

Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu

435 440 445

Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly

450 455 460

His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr

465 470 475 480

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 264

<211> 486

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 264

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu

20 25 30

Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala

50 55 60

Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile

85 90 95

Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

130 135 140

Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr

145 150 155 160

Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly

165 170 175

Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly

180 185 190

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

195 200 205

Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys

210 215 220

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys

225 230 235 240

His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

245 250 255

Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

275 280 285

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

305 310 315 320

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

325 330 335

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

340 345 350

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

355 360 365

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

370 375 380

Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

385 390 395 400

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

405 410 415

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

420 425 430

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

<210> 265

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 265

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 266

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 266

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 267

<211> 72

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 267

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 268

<211> 230

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 268

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys Met

225 230

<210> 269

<211> 135

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 269

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 270

<211> 690

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 270

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaacc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc cggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgtccct gggcaagatg 690

<210> 271

<211> 282

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 271

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala

1 5 10 15

Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala

20 25 30

Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys

35 40 45

Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro

50 55 60

Ser His Thr Gln Pro Leu Gly Val Tyr Leu Leu Thr Pro Ala Val Gln

65 70 75 80

Asp Leu Trp Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val Val Gly

85 90 95

Ser Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly Lys Val

100 105 110

Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser Asn Gly

115 120 125

Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser Leu Trp Asn

130 135 140

Ala Gly Thr Ser Val Thr Cys Thr Leu Asn His Pro Ser Leu Pro Pro

145 150 155 160

Gln Arg Leu Met Ala Leu Arg Glu Pro Ala Ala Gln Ala Pro Val Lys

165 170 175

Leu Ser Leu Asn Leu Leu Ala Ser Ser Asp Pro Pro Glu Ala Ala Ser

180 185 190

Trp Leu Leu Cys Glu Val Ser Gly Phe Ser Pro Pro Asn Ile Leu Leu

195 200 205

Met Trp Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe Ala Pro

210 215 220

Ala Arg Pro Pro Pro Gln Pro Gly Ser Thr Thr Phe Trp Ala Trp Ser

225 230 235 240

Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr Tyr Thr

245 250 255

Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu Asn Ala Ser Arg

260 265 270

Ser Leu Glu Val Ser Tyr Val Thr Asp His

275 280

<210> 272

<211> 847

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 272

aggtggcccg aaagtcccaa ggcccaggca tctagtgttc ctactgcaca gccccaggca 60

gaaggcagcc tagccaaagc tactactgca cctgccacta cgcgcaatac tggccgtggc 120

ggggaggaga agaaaaagga gaaagagaaa gaagaacagg aagagaggga gaccaagacc 180

cctgaatgtc catcccatac ccagccgctg ggcgtctatc tcttgactcc cgcagtacag 240

gacttgtggc ttagagataa ggccaccttt acatgtttcg tcgtgggctc tgacctgaag 300

gatgcccatt tgacttggga ggttgccgga aaggtaccca cagggggggt tgaggaaggg 360

ttgctggagc gccattccaa tggctctcag agccagcact caagactcac ccttccgaga 420

tccctgtgga acgccgggac ctctgtcaca tgtactctaa atcatcctag cctgccccca 480

cagcgtctga tggcccttag agagccagcc gcccaggcac cagttaagct tagcctgaat 540

ctgctcgcca gtagtgatcc cccagaggcc gccagctggc tcttatgcga agtgtccggc 600

tttagcccgc ccaacatctt gctcatgtgg ctggaggacc agcgagaagt gaacaccagc 660

ggcttcgctc cagcccggcc cccaccccag ccgggttcta ccacattctg ggcctggagt 720

gtcttaaggg tcccagcacc acctagcccc cagccagcca catacacctg tgttgtgtcc 780

catgaagata gcaggaccct gctaaatgct tctaggagtc tggaggtttc ctacgtgact 840

gaccatt 847

<210> 273

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 273

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 274

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 274

ggtggcggag gttctggagg tggaggttcc 30

<210> 275

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 275

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 276

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 276

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 277

<211> 336

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 277

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 278

<211> 336

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 278

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 279

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 279

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 280

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 280

Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

1 5 10 15

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

20 25 30

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

35 40 45

<210> 281

<211> 126

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 281

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 282

<211> 144

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 282

caacgaagga aatatagatc aaacaaagga gaaagtcctg tggagcctgc agagccttgt 60

cgttacagct gccccaggga ggaggagggc agcaccatcc ccatccagga ggattaccga 120

aaaccggagc ctgcctgctc cccc 144

<210> 283

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 283

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 284

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 284

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ser Leu Lys Ser

1 5 10 15

<210> 285

<211> 1184

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 285

cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60

tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120

aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360

ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420

cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480

ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540

tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600

gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660

tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720

tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780

caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840

ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900

ttccgtcctc agccgtcgct tcatgtgact ccacggagta ccgggcgccg tccaggcacc 960

tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020

cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080

tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140

agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184

<210> 286

<211> 448

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 286

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Asp Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Phe Asp Tyr Ala His Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 287

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 287

Asp Ile Val Leu Thr Gln Ser Pro Ala Phe Leu Ser Val Thr Pro Gly

1 5 10 15

Glu Lys Val Thr Phe Thr Cys Gln Ala Ser Gln Ser Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Lys Thr Asp Gln Ala Pro Lys Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Glu Ala

65 70 75 80

Glu Asp Ala Ala Asp Tyr Tyr Cys Gln Gln Ile Asn Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 288

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 288

Ser Asp Tyr Ala Trp Asn

1 5

<210> 289

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 289

Tyr Ile Ser Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 290

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 290

Phe Asp Tyr Ala His Ala Met Asp Tyr

1 5

<210> 291

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 291

Gln Ala Ser Gln Ser Ile Gly Thr Ser Ile His

1 5 10

<210> 292

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 292

Tyr Ala Ser Glu Ser Ile Ser

1 5

<210> 293

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 293

Gln Gln Ile Asn Ser Trp Pro Thr Thr

1 5

<210> 294

<211> 143

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 294

Gly Ala Pro Ala Gly Pro Leu Ile Val Pro Tyr Asn Leu Pro Leu Pro

1 5 10 15

Gly Gly Val Val Pro Arg Met Leu Ile Thr Ile Leu Gly Thr Val Lys

20 25 30

Pro Asn Ala Asn Arg Ile Ala Leu Asp Phe Gln Arg Gly Asn Asp Val

35 40 45

Ala Phe His Phe Asn Pro Arg Phe Asn Glu Asn Asn Arg Arg Val Ile

50 55 60

Val Cys Asn Thr Lys Leu Asp Asn Asn Trp Gly Arg Glu Glu Arg Gln

65 70 75 80

Ser Val Phe Pro Phe Glu Ser Gly Lys Pro Phe Lys Ile Gln Val Leu

85 90 95

Val Glu Pro Asp His Phe Lys Val Ala Val Asn Asp Ala His Leu Leu

100 105 110

Gln Tyr Asn His Arg Val Lys Lys Leu Asn Glu Ile Ser Lys Leu Gly

115 120 125

Ile Ser Gly Asp Ile Asp Ile Thr Ser Ala Ser Tyr Thr Met Ile

130 135 140

<210> 295

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 295

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Gln Ser Ser Leu Lys Ser

1 5 10 15

<210> 296

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 296

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser

1 5 10 15

<210> 297

<211> 137

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 297

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Val Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ile Ile Trp Tyr Asp Gly Asp Asn Gln Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Leu Arg Thr Gly Pro Phe Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Leu Val Thr Val Ser Ser

130 135

<210> 298

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 298

Met Leu Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala

1 5 10 15

Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val

20 25 30

Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile

35 40 45

Gly Ser Ser Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys

50 55 60

Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser

85 90 95

Leu Glu Ala Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser

100 105 110

Leu Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

115 120 125

<210> 299

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 299

atggccctgc ctgtgacagc cctgctgctg cctctggctc tgctgctgca tgccgctaga 60

ccc 63

<210> 300

<211> 63

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 300

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 301

<211> 72

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 301

atctacattt gggcccctct ggctggtact tgcggggtcc tgctgctttc actcgtgatc 60

actctttact gt 72

<210> 302

<211> 126

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 302

aagcgcggtc ggaagaagct gctgtacatc tttaagcaac ccttcatgag gcctgtgcag 60

actactcaag aggaggacgg ctgttcatgc cggttcccag aggaggagga aggcggctgc 120

gaactg 126

<210> 303

<211> 336

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 303

cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

<210> 304

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 304

Gly Gly Gly Gly Ser

1 5

<210> 305

<211> 150

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 305

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val

145 150

<210> 306

<211> 450

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 306

cccggatggt ttctggactc tccggatcgc ccgtggaatc ccccaacctt ctcaccggca 60

ctcttggttg tgactgaggg cgataatgcg accttcacgt gctcgttctc caacacctcc 120

gaatcattcg tgctgaactg gtaccgcatg agcccgtcaa accagaccga caagctcgcc 180

gcgtttccgg aagatcggtc gcaaccggga caggattgtc ggttccgcgt gactcaactg 240

ccgaatggca gagacttcca catgagcgtg gtccgcgcta ggcgaaacga ctccgggacc 300

tacctgtgcg gagccatctc gctggcgcct aaggcccaaa tcaaagagag cttgagggcc 360

gaactgagag tgaccgagcg cagagctgag gtgccaactg cacatccatc cccatcgcct 420

cggcctgcgg ggcagtttca gaccctggtc 450

<210> 307

<211> 394

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 307

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

20 25 30

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

35 40 45

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

50 55 60

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

65 70 75 80

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

85 90 95

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

100 105 110

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

115 120 125

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

130 135 140

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

145 150 155 160

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

165 170 175

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

180 185 190

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

195 200 205

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

210 215 220

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

225 230 235 240

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

245 250 255

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

260 265 270

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

275 280 285

Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn

290 295 300

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

305 310 315 320

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

325 330 335

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

340 345 350

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

355 360 365

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

370 375 380

Ala Leu His Met Gln Ala Leu Pro Pro Arg

385 390

<210> 308

<211> 1182

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 308

atggccctcc ctgtcactgc cctgcttctc cccctcgcac tcctgctcca cgccgctaga 60

ccacccggat ggtttctgga ctctccggat cgcccgtgga atcccccaac cttctcaccg 120

gcactcttgg ttgtgactga gggcgataat gcgaccttca cgtgctcgtt ctccaacacc 180

tccgaatcat tcgtgctgaa ctggtaccgc atgagcccgt caaaccagac cgacaagctc 240

gccgcgtttc cggaagatcg gtcgcaaccg ggacaggatt gtcggttccg cgtgactcaa 300

ctgccgaatg gcagagactt ccacatgagc gtggtccgcg ctaggcgaaa cgactccggg 360

acctacctgt gcggagccat ctcgctggcg cctaaggccc aaatcaaaga gagcttgagg 420

gccgaactga gagtgaccga gcgcagagct gaggtgccaa ctgcacatcc atccccatcg 480

cctcggcctg cggggcagtt tcagaccctg gtcacgacca ctccggcgcc gcgcccaccg 540

actccggccc caactatcgc gagccagccc ctgtcgctga ggccggaagc atgccgccct 600

gccgccggag gtgctgtgca tacccgggga ttggacttcg catgcgacat ctacatttgg 660

gctcctctcg ccggaacttg tggcgtgctc cttctgtccc tggtcatcac cctgtactgc 720

aagcggggtc ggaaaaagct tctgtacatt ttcaagcagc ccttcatgag gcccgtgcaa 780

accacccagg aggaggacgg ttgctcctgc cggttccccg aagaggaaga aggaggttgc 840

gagctgcgcg tgaagttctc ccggagcgcc gacgcccccg cctataagca gggccagaac 900

cagctgtaca acgaactgaa cctgggacgg cgggaagagt acgatgtgct ggacaagcgg 960

cgcggccggg accccgaaat gggcgggaag cctagaagaa agaaccctca ggaaggcctg 1020

tataacgagc tgcagaagga caagatggcc gaggcctact ccgaaattgg gatgaaggga 1080

gagcggcgga ggggaaaggg gcacgacggc ctgtaccaag gactgtccac cgccaccaag 1140

gacacatacg atgccctgca catgcaggcc cttccccctc gc 1182

<210> 309

<211> 4

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 309

Gly Gly Gly Ser

1

<210> 310

<211> 50

<212> RNA

<213> artificial sequence

<220>

<223> biology

<400> 310

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 50

<210> 311

<211> 373

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 311

Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr

1 5 10 15

Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe

20 25 30

Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr

35 40 45

Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu

50 55 60

Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu

65 70 75 80

Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn

85 90 95

Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala

100 105 110

Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg

115 120 125

Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly

130 135 140

Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

145 150 155 160

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

165 170 175

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

180 185 190

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

195 200 205

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

210 215 220

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

225 230 235 240

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

245 250 255

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

260 265 270

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

275 280 285

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

290 295 300

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

305 310 315 320

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

325 330 335

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

340 345 350

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

355 360 365

Ala Leu Pro Pro Arg

370

<210> 312

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 312

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

1 5 10 15

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

20 25 30

Val Thr Leu

35

<210> 313

<211> 105

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 313

acaaaaaaga agtattcatc cagtgtgcac gaccctaacg gtgaatacat gttcatgaga 60

gcagtgaaca cagccaaaaa atccagactc acagatgtga cccta 105

<210> 314

<211> 69

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 314

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu

35 40 45

Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile

50 55 60

Leu Ile Cys Trp Leu

65

<210> 315

<211> 207

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 315

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatttctg gttacccata ggatgtgcag cctttgttgt agtctgcatt 180

ttgggatgca tacttatttg ttggctt 207

<210> 316

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 316

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 317

<211> 123

<212> DNA

<213> artificial sequence

<220>

<223> biology

<400> 317

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 318

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 318

Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Tyr Arg Ser Pro

1 5 10 15

Ala Met Pro Glu Asn Leu

20

<210> 319

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 319

Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

1 5 10

<210> 320

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> biology

<400> 320

Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser

1 5 10 15

<210> 321

<211> 445

<212> PRT

<213> artificial sequence

<220>

<223> heavy chain

<400> 321

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Ala Asp Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Tyr Gly Asn Tyr Trp Tyr Ile Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Ala Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 322

<211> 214

<212> PRT

<213> artificial sequence

<220>

<223> light chain

<400> 322

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Asn Tyr Ala Phe His Arg Phe Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys His Gln Ala Tyr Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 323

<211> 118

<212> PRT

<213> artificial sequence

<220>

<223> VHC

<400> 323

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Tyr Ala Asp Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ala Tyr Gly Asn Tyr Trp Tyr Ile Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 324

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> tirelib monoclonal antibody LHC

<400> 324

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Glu Ser Val Ser Asn Asp

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile

35 40 45

Asn Tyr Ala Phe His Arg Phe Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala

65 70 75 80

Glu Asp Val Ala Val Tyr Tyr Cys His Gln Ala Tyr Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 325

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> HCDR1

<400> 325

Gly Phe Ser Leu Thr Ser Tyr Gly Val His

1 5 10

<210> 326

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> HCDR2

<400> 326

Val Ile Tyr Ala Asp Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 327

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> HCDR3

<400> 327

Ala Arg Ala Tyr Gly Asn Tyr Trp Tyr Ile Asp Val

1 5 10

<210> 328

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> LCDR1

<400> 328

Lys Ser Ser Glu Ser Val Ser Asn Asp Val Ala

1 5 10

<210> 329

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> LCDR2

<400> 329

Tyr Ala Phe His Arg Phe Thr

1 5

<210> 330

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> LCDR3

<400> 330

His Gln Ala Tyr Ser Ser Pro Tyr Thr

1 5

Claims (101)

1. A method of treating a proliferative disease in a subject in need thereof, said method comprising administering to said subject a tgfβ antibody and at least one additional therapeutic agent, wherein said tgfβ antibody is administered once every two or three weeks from about 1400mg to about 2100mg or from about 20mg/kg to about 45 mg/kg.

2. The method of claim 1, wherein the tgfβ antibody is administered at about 1400mg once every two weeks.

3. The method of claim 1, wherein the tgfβ antibody is administered about 30mg/kg once every two weeks.

4. The method of claim 1, wherein the tgfβ antibody is administered about 2100mg once every two, three or four weeks.

5. The method of claim 1, wherein the tgfβ antibody is administered about 20mg/kg once every three weeks.

6. The method of claim 1, wherein the tgfβ antibody is administered about 45mg/kg once every three weeks.

7. The method of any one of claims 1 to 6, wherein the tgfβ antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs 4, 5 and 6, respectively.

8. A method according to any one of claims 1 to 7, wherein the tgfβ antibody comprises a heavy chain variable region and a light chain variable region as set out in amino acid sequences SEQ ID NOs 7 and 8 respectively.

9. A method according to any one of claims 1 to 8, wherein the tgfβ antibody comprises heavy and light chains as set out in amino acid sequences SEQ ID NOs 9 and 10 respectively.

10. The method of any one of claims 1 to 9, wherein the tgfβ antibody is a monoclonal antibody.

11. The method of any one of claims 1 to 10, wherein the tgfβ antibody is a fully human antibody.

12. The method of any one of claims 1 to 11, wherein the tgfβ inhibitor is administered over a period of about 30 minutes.

13. The method of any one of claims 1 to 12, wherein the additional therapeutic agent is one or more of the following: PD-1 inhibitors, gemcitabine, albumin-bound paclitaxel, leucovorin (calcium folinate), levofolinic acid, fluorouracil (5-FU), oxaliplatin (eloxatin), bevacizumab, irinotecan, cyclophosphamide, capecitabine or topotecan.

14. The method of any one of claims 1-13, wherein the additional therapeutic agent comprises a PD-1 inhibitor.

15. The method of claim 14, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

16. The method of claim 15, wherein the anti-PD-1 antibody is PDR001 (swabber), BGB-a317 (tirelizumab), or BGB-108.

17. The method of claim 16, wherein the anti-PD-1 antibody is tirelimumab.

18. The method of any one of claims 15-17, wherein the anti-PD-1 antibody is administered at 300mg every 28 day cycle or 200mg every 21 day cycle.

19. The method of any one of claims 1-18, wherein the additional therapeutic agent comprises gemcitabine.

20. The method of claim 19, wherein gemcitabine is at about 1000mg/m² And (5) administration.

21. The method of claim 20, wherein gemcitabine is administered on days 1, 8, and 15 of a 28 day cycle.

22. The method of claim 19, wherein gemcitabine is at 675mg/m² And (5) administration.

23. The method of any one of claims 19-22, wherein gemcitabine is administered on days 1 and 8 of a 21 day cycle.

24. The method of any one of claims 1-23, wherein the additional therapeutic agent comprises albumin-bound paclitaxel.

25. The method of claim 24, wherein albumin-bound paclitaxel is at about 125mg/m² And (5) administration.

26. The method of claim 24 or 25, wherein albumin-bound paclitaxel is administered on days 1, 8, and 15 of a 28-day cycle.

27. The method of any one of claims 1-26, wherein the additional therapeutic agent comprises gemcitabine and albumin-bound paclitaxel.

28. The method of any one of claims 1-27, wherein the additional therapeutic agent comprises a PD-1 inhibitor, gemcitabine, and albumin-bound paclitaxel.

29. The method of any one of claims 1-28, wherein the additional therapeutic agent comprises bevacizumab.

30. The method of claim 29, wherein bevacizumab is administered at about 5 mg/kg.

31. The method of claim 29 or 30, wherein bevacizumab is administered on days 1 and 15 of a 28 day cycle.

32. The method of any one of claims 1-31, wherein the additional therapeutic agent comprises 5-fluorouracil.

33. The method of claim 32, wherein 5-fluorouracil is present at about 400mg/m² And (5) administration.

34. The method of claim 32, wherein 5-fluorouracil is present at about 2400mg/m² And (5) administration.

35. The method of any one of claims 32-34, wherein 5-fluorouracil is administered on days 1 and 15 of a 28 day cycle.

36. The method of any one of claims 32-35, wherein 5-fluorouracil is administered as an intravenous bolus.

37. The method of any one of claims 32-36, wherein 5-fluorouracil is present at 400mg/m on days 1 and 15 of each 28 day cycle² Intravenous bolus injection followed by 2400mg/m² Administration was by continuous intravenous infusion over 46 hours.

38. The method of any one of claims 1-37, wherein the additional therapeutic agent comprises folinic acid or levofolinic acid.

39. The method of claim 38, wherein the folinic acid or levofolinic acid, respectively, is about 400mg/m² Or 200mg/m² And (5) administration.

40. The method of claim 38 or 39, wherein the folinic acid or levofolinic acid is administered on days 1 and 15 of a 28 day cycle.

41. The method of any one of claims 1-40, wherein the additional therapeutic agent comprises oxaliplatin.

42. The method of claim 41, wherein oxaliplatin is about 85mg/m² And (5) administration.

43. The method of claim 41 or 42, wherein oxaliplatin is administered on days 1 and 15 of a 28 day cycle.

44. The method of any one of claims 1-43, wherein the additional therapeutic agent comprises irinotecan.

45. The method of claim 44, wherein irinotecan is at about 180mg/m² And (5) administration.

46. The method of claim 44 or 45, wherein irinotecan is to be administered on days 1 and 15 of the 28 day cycle.

47. The method of any one of claims 1-46, wherein the additional therapeutic agent comprises bevacizumab, 5-fluorouracil, folinic acid (or levofolinic acid), and oxaliplatin.

48. The method of any one of claims 1-47, wherein the additional therapeutic agent comprises a PD-1 inhibitor, bevacizumab, 5-fluorouracil, folinic acid (or levofolinic acid), and oxaliplatin.

49. The method of any one of claims 1-46, wherein the additional therapeutic agent comprises bevacizumab, 5-fluorouracil, folinic acid, and irinotecan.

50. The method of any one of claims 1-46 and 49, wherein the additional therapeutic agent comprises a PD-1 inhibitor, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan.

51. The method of any one of claims 1-50, wherein the tgfβ antibody and the one or more additional therapeutic agents are administered to the subject over 2 or more cycles.

52. The method of any one of claims 1-51, wherein the proliferative disease is pancreatic cancer or colorectal cancer.

53. The method of any one of claims 1 to 52, wherein the additional therapeutic agent comprises cyclophosphamide and topotecan.

54. The method of claim 53, wherein cyclophosphamide is present at 250mg/m² And (5) administration.

55. The method of claim 53 or 54, wherein cyclophosphamide is administered for five (5) days.

56. The method of claim 55, wherein the five (5) days are consecutive days.

57. The method of any one of claims 53-56, wherein topotecan is at 0.75mg/m² And (5) administration.

58. The method of any one of claims 53-57, wherein topotecan is administered for five (5) days.

59. The method of claim 58, wherein the five (5) days are consecutive days.

60. The method of any one of claims 1-59, wherein the proliferative disease is neuroblastoma or osteosarcoma.

61. The method of any one of claims 1-60, wherein the subject is a pediatric patient.

62. A method of treating pancreatic adenocarcinoma in a subject in need thereof, the method comprising administering 2100mg tgfβ antibody, 1000mg/m to the subject² Gemcitabine, and 125mg/m² Wherein the TGF-beta antibody is administered on days 1 and 15 of a 28 day cycle and gemcitabine and albumin-bound paclitaxel are administered on days 1, 8 and 15 of the cycle, and wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO:1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO:4, 5 and 6, respectively.

63. The method of claim 62, further comprising administering a PD-1 inhibitor to the patient.

64. The method of claim 63, wherein the PD-1 inhibitor is swabs bevacizumab or tirelimumab.

65. The method of claim 64, wherein the PD-1 inhibitor is swastimulizumab and is administered at 400mg once every four (4) weeks.

66. The method of claim 64, wherein the PD-inhibitor is tirelimumab and is administered weekly at 100 mg.

67. The method of claim 66, wherein tirelimumab is administered at 400mg once every four (4) weeks.

68. The method of any one of claims 62-67, wherein the tgfβ antibody, gemcitabine, and albumin-bound paclitaxel (and optionally a PD-1 inhibitor) are administered to the subject over 2 or more cycles.

69. The method of any one of claims 62-68, wherein the tgfβ antibody, gemcitabine, and albumin-bound paclitaxel (and optionally a PD-1 inhibitor) are administered intravenously to the subject.

70. A method of treating colorectal cancer in a subject in need thereof, the method comprising administering to the subject 2100mg of tgfβ antibody, 5mg/kg bevacizumab, 400 to 2400mg/m² 5-fluorouracil, 400mg/m² And 85mg/m² Wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin are administered on days 1 and 15 of a 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs 4, 5, and 6, respectively.

71. The method of claim 70, wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin are administered to the subject in 2 or more cycles.

72. The method of claim 70 or 71, wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and oxaliplatin are administered intravenously to the subject.

73. The method of any one of claims 70-72, wherein 5-fluorouracil is administered as a 400mg/m2 intravenous bolus followed by a 2400mg/m2 continuous intravenous infusion over 46 hours.

74. A method of treating colorectal cancer in a subject in need thereof, the method comprising administering 2100mg of TGF to the subjectBeta antibody, bevacizumab of 5mg/kg, 400 to 2400mg/m² 5-fluorouracil, 400mg/m² And 180mg/m² Wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan are administered on days 1 and 15 of a 28 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs 4, 5, and 6, respectively.

75. The method of claim 74, wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan are administered to the subject in 2 or more cycles.

76. The method of claim 74 or 75, wherein the tgfβ antibody, bevacizumab, 5-fluorouracil, folinic acid, and irinotecan Kang Jingmai are administered to the subject internally.

77. The method of any one of claims 74-76, wherein 5-fluorouracil is at 400mg/m² Intravenous bolus administration was then performed as a 46 hour continuous intravenous infusion at 2400mg/m 2.

78. A method of treating neuroblastoma in a subject in need thereof, the method comprising administering 20, 30, or 45mg/kg of a tgfp antibody, 250mg/m, to the subject² Cyclophosphamide of (C), and 0.75mg/m² Wherein the tgfβ antibody is administered every 3 weeks and cyclophosphamide and topotecan are administered on days 1 through 5 of a 21 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs 4, 5 and 6, respectively.

79. A method of treating osteosarcoma in a subject in need thereof, the method comprising administering 20, 30, or 45mg/kg of a tgfβ antibody and 675mg/m to the subject² Gemcitabine of (C)A shore, wherein the tgfβ antibody is administered every 3 weeks and gemcitabine is administered on days 1 and 8 of the 21 day cycle, and wherein the tgfβ antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NOs 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NOs 4, 5 and 6, respectively.

80. The method of claim 78 or 79, wherein the subject has a body weight of less than 20kg and receives administration of 45mg/kg of the tgfβ antibody.

81. The method of claim 78 or 79, wherein the subject has a body weight of between 20kg and 40kg and receives administration of 30mg/kg of the tgfβ antibody.

82. The method of claim 78 or 79, wherein the subject has a body weight of greater than 40kg and receives administration of 20mg/kg of the tgfβ antibody.

83. The method of claim 78 or 79, wherein the subject is under the age of eighteen (18) years.

84. The method of any one of claims 1-83, wherein the tgfβ inhibitor is administered concurrently with the one or more additional therapeutic agents.

85. The method of any one of claims 1-83, wherein the tgfβ inhibitor is administered prior to the one or more additional therapeutic agents.

86. The method of any one of claims 1-83, wherein the tgfβ inhibitor is administered after the one or more additional therapeutic agents.

87. The method of any one of claims 1 to 83, wherein the tgfβ inhibitor and/or the one or more additional therapeutic agents are administered until remission.

88. A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising administering to the subject:

(a) 2100mg of TGF-beta antibody,

(b) At 500mg/m² Intravenous bolus injection followed by 2400mg/m² 5-fluorouracil infused continuously over 46 hours,

(c) At 400mg/m² Folinic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of levofolinic acid,

(d) At 180mg/m² Irinotecan for intravenous administration, and

(e) 5mg/kg of bevacizumab,

wherein the tgfβ antibody is administered on day 1 of a 28 day cycle (and optionally day 15), and wherein the 5-fluorouracil, folinic acid (or levofolinic acid), irinotecan, and bevacizumab are administered on days 1 and 15 of the 28 day cycle, and

wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively.

89. A method of treating pancreatic adenocarcinoma or colorectal cancer in a subject in need thereof, the method comprising administering to the subject:

(a) 2100mg of TGF-beta antibody,

(b) At 500mg/m² Intravenous bolus injection followed by 2400mg/m² 5-fluorouracil infused continuously over 46 hours,

(c) At 400mg/m² Folinic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of levofolinic acid,

(d) At 85mg/m² Oxaliplatin for intravenous administration, and

(e) 5mg/kg of bevacizumab,

wherein the tgfβ antibody is administered on day 1 of a 28 day cycle (and optionally day 15), and wherein the 5-fluorouracil, folinic acid (or levofolinic acid), oxaliplatin, and bevacizumab are administered on days 1 and 15 of the 28 day cycle, and

wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively.

90. The method of claim 88 or 89, further comprising administering to the patient a PD-1 inhibitor.

91. The method of claim 90, wherein the PD-1 inhibitor is tirelimumab with a heavy chain of SEQ ID No. 321 and a light chain of SEQ ID No. 322.

92. The method of claim 91, wherein tirelimumab is administered intravenously at 300mg on day 1 of each 28 day cycle.

93. The method of any one of claims 88-92, wherein the tgfβ antibody, 5-fluorouracil, folinic acid (or levofolinic acid), oxaliplatin, irinotecan, bevacizumab, or PD-1 inhibitor is administered to the subject in 2 or more cycles.

94. A method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject:

(a) 2100mg of TGF-beta antibody,

(b) At 130mg/m² Oxaliplatin for intravenous administration, and

(c) At 1000mg/m² Capecitabine is administered orally twice daily,

wherein the TGF-beta antibody is administered once every three weeks, and wherein oxaliplatin is administered on day 1 of the Q3W cycle and capecitabine is administered on days 1-14 of the cycle, and

wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively.

95. A method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject:

(a) 2100mg of TGF-beta antibody,

(b) At 130mg/m² Oxaliplatin for intravenous administration, and

(c) At 1000mg/m² Capecitabine is administered orally twice daily,

wherein the TGF-beta antibody is administered once every two weeks, and wherein oxaliplatin is administered on day 1 of the Q3W cycle and capecitabine is administered on days 1-14 of the cycle, and

Wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively.

96. A method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject:

(a) 2100mg of TGF-beta antibody,

(b) At 85mg/m² Oxaliplatin for intravenous administration,

(c) At 400mg/m² Folinic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of levofolinic acid, and

(d) At 400mg/m² Intravenous bolus injection followed by 1200mg/m² 5-fluorouracil administered intravenously daily,

wherein the TGF-beta antibody is administered once every three weeks, and wherein the oxaliplatin, folinic acid (or levofolinic acid), 5-fluorouracil are administered on day 1 of the Q2W cycle, and the 1200mg are administered intravenously on days 1-2 of the cycle, and

wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively.

97. A method of treating gastric cancer in a subject in need thereof, the method comprising administering to the subject:

(a) 2100mg of TGF-beta antibody,

(b) At 85mg/m² Oxaliplatin for intravenous administration,

(c) At 400mg/m² Folinic acid (leucovorin) administered intravenously or at 200mg/m² Intravenous administration of levofolinic acid, and

(d) At 400mg/m² Intravenous bolus injection followed by 1200mg/m² 5-fluorouracil administered intravenously daily,

wherein the TGF-beta antibody is administered once every two weeks, and wherein the oxaliplatin, folinic acid (or levofolinic acid), 5-fluorouracil are administered on day 1 of the Q2W cycle, and the 1200mg are administered intravenously on days 1-2 of the cycle, and

wherein the TGF-beta antibody comprises heavy chain CDR1, CDR2 and CDR3 of SEQ ID NO 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 of SEQ ID NO 4, 5 and 6, respectively.

98. The method of claim 94 or 97, further comprising administering a PD-1 inhibitor to the patient.

99. The method of claim 98, wherein the PD-1 inhibitor is tirelimumab with a heavy chain of SEQ ID No. 321 and a light chain of SEQ ID No. 322.

100. The method of claim 99, wherein tirelimumab is administered intravenously at 300mg on day 1 of each 28 day cycle.

101. The method of claim 99, wherein tirelimumab is administered intravenously at 200mg every three weeks.