COMBINATION THERAPIES INVOLVING GREM1 ANTAGONISTS FOR TREATMENT OF CANCERTECHNICAL FIELDThe present disclosure generally relates to cancer therapy involving a gremlin1 (GREM1) antagonist in combination with an anti-angiogenesis therapy and/or a chemotherapy and/or an immunotherapy for treating a cancer, especially GREM1-expressing cancer.
BACKGROUND OF THE INVENTIONGREM1 is closely related to fibrotic lesions of kidney, lung, liver and retina as well as several tumor types including pancreatic, colon, lung, glioma, gastric and prostate cancers (Sneddon et al., PNAS 2006 October; 103 (40) : 14842-14847) . For example, aberrant gremlin1 upregulation endows colon cells outside of stem cell niche with tumorigenicity. It was also found that tumor stem cells highly express and secret gremlin1 to maintain their stemness in glioma (Yan, K., et al., Genes Dev 28, 1085-1100 (2014) ) . Accordingly, gremlin1 has been used as a therapeutic target in treating gremlin-related diseases.
While GREM1 antagonists have exhibited some therapeutic efficacy on several GREM1-expressing diseases, effective combination therapies involving GREM1 antagonists for treating GREM1-expressing cancers are still rare. Therefore, significant need exists for improved combination therapies for GREM1-expressing cancers to meet clinical needs.
SUMMARY OF THE INVENTIONThe present disclosure provides, among others, a method for treating a GREM1-expressing cancer in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a GREM1 antagonist in combination with: a) an anti-angiogenesis therapy, b) a chemotherapy, c) an immunotherapy, d) an anti-angiogenesis therapy and a chemotherapy, e) a chemotherapy and an immunotherapy; f) an anti-angiogenesis therapy and an immunotherapy; or g) an anti-angiogenesis therapy, a chemotherapy and an immunotherapy.
In certain embodiments, the anti-angiogenesis therapy comprises an antagonist of a VEGFA or VEGFR.
In certain embodiments, the antagonist of a VEGFA is an anti-VEFRA antibody, such as Bevacizumab
In certain embodiments, the antagonist of VEGFR is a small molecule VEGFR inhibitor or a large molecule VEGFR inhibitor.
In certain embodiments, the VEGFR is VEGFR-1, VEGFR-2 or VEGFR-3.
In certain embodiments, the antagonist of VEGFR is a large molecule VEGFR inhibitor, such as an anti-VEGFR-2 antibody. In certain embodiments, the anti-VEGFR-2 antibody is selected from the group consisting of: ramucirumab, olinvacimab, Gentuximab, alacizumab pegol, vulinacimab, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04 and HLX12.
In certain embodiments, the antagonist of VEGFR is a small molecule VEGFR inhibitor, such as Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, AxitinibCabozantinibFruquintiniband/or Regorafenib
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the anti-VEGFA antibody or the anti-VEGFR-2 antibody or the small molecule VEGFR inhibitor.
In certain embodiments, the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the cancer is resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or is determined to have low or no PD-L1 expression in a diseased tissue of the cancer.
In certain embodiments, the chemotherapy comprises a combination of chemotherapeutic agents.
In certain embodiments, the combination of chemotherapeutic agents comprises leucovorin calcium (folinic acid) , fluorouracil, and irinotecan hydrochloride (FOLFIRI) .
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the combination of chemotherapeutic agents and the anti-VEGFR-2 antibody, and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the cancer is resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or is determined to have low or no PD-L1 expression in a diseased tissue of the cancer.
In certain embodiments, the immunotherapy comprises a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the PD-1/PD-L1 axis inhibitor comprises a PD-1 inhibitor selected from the group consisting of antibody, small molecule, and combination thereof.
In certain embodiments, the PD-1 inhibitor comprises an anti-PD-1 antibody selected from the group consisting of: Nivolumab (OPDIVO; BMS-936558) , Dostarlimab (TSR-042) , Pembrolizumab (KEYTRUDA; MK-3475) , MEDI0680 (AMP-514) , MEDI4736, BI 754091, Pidilizumab (CT-011) , Cemiplimab (LIBTAYO, REGN2810) , Spartalizumab (PDR001) , Cetrelimab (JNJ 63723283) , Toripalimab (JS001) , PF-06801591, Tislelizumab (BGB-A317) , AMP-224 (GSK-2661380) , ABBV-181, Lambrolizumab, Camrelizuma (SHR-1210) , Sintilimab (Tyvyt, IBI308) , Penpulimab (AK105) , Zimberelimab, Retifanlimab, Serplulimab, Balstilimab, Geptanolimab, Prolgolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofazinlimab, Sindelizumab, MGA404, Sym021, BAT1306, and HX008.
In certain embodiments, the PD-1 inhibitor is Nivolumab (OPDIVO; BMS-936558) .
In certain embodiments, the PD-1/PD-L1 axis inhibitor comprises PD-L1 inhibitor selected from the group consisting of antibody, small molecule, and combination thereof.
In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody selected from the group consisting of: Atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446) , BMS-936559, Avelumab (bavencio) , lodapolimab (LY3300054) , Durvalumab (MEDI4736) , CX-072 (Proclaim-CX-072) , FAZ053, Envafolimab (KN035) , MDX-1105, STI-1040, CS1001, Adebrelimab (SHR-1316) , SHR-1701, TOB2450, Bintrafusp, LP002, STI-3031, Cosibelimab, Pacmilimab, NM01, LDP, AMP-224, Garivulimab (BGB-A333) , A167, SCD-135, Opucolimab, and GR1405.
In certain embodiments, the cancer is: a) resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor, and/or b) determined to have low or no PD-L1 expression in a diseased tissue of the GREM1-expressing cancer.
In certain embodiments, the method comprises administering the GREM1 antagonist to the subject, and after a period of time sufficient to increase expression level of PD-L1 in cancer cells of the subject, then administering the PD-1/PD-L1 axis inhibitor to the subject.
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the combination of chemotherapeutic agents (e.g. FOLFIRI) and the PD-1/PD-L1 axis inhibitor (e.g. PD-1 inhibitor, or Nivolumab) , and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the cancer is resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or is determined to have low or no PD-L1 expression in a diseased tissue of the cancer.
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the anti-VEGFR-2 antibody, the combination of chemotherapeutic agents, and the PD-1/PD-L1 axis inhibitor, and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the cancer is resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or is determined to have low or no PD-L1 expression in a diseased tissue of the cancer.
In certain embodiments, the subject is further determined to have low, medium or high GREM1 expression in a diseased tissue of the GREM1-expressing cancer. In certain embodiments, the subject is further determined to have 10-20%of the tumor cells positive in GREM1 as measured by IHC.
In another aspect, the present disclosure provides a method of improving responsiveness of a subject to treatment with PD-1/PD-L1 axis inhibitor, wherein the subject has been determined to have presence of GREM1 in a biological sample of a diseased tissue of the subject, or the subject has been determined to have expression level of GREM1 in the biological sample of the diseased tissue reaching a threshold level, the method comprising:
a) administering to the subject a therapeutically effective amount of a GREM1 antagonist such that expression of PD-L1 is increased in a diseased tissue, thereby improving responsiveness of the subject to PD-1/PD-L1 axis inhibitor.
In certain embodiments, the subject is a) determined to be resistant or refractory to PD-1/PD-L1 axis inhibitor and/or b) determined to have low or no PD-L1 expression in a diseased tissue.
In certain embodiments, the subject is determined to have medium or high PD-L1 expression in a diseased tissue.
In certain embodiments, the diseased tissue is a cancer tissue.
In certain embodiments, the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of PD-1/PD-L1 axis inhibitor after the expression of PD-L1 is increased in the diseased tissue of the subject.
In another aspect, the present disclosure provides a method of determining eligibility for or likelihood of responsiveness to treatment with a GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor in a subject, the method comprising:
a) determining presence or expression level of GREM1 in a biological sample of the diseased tissue of the subject,
wherein the presence or absence of or expression level of GREM1 is indicative of whether the subject is likely to be eligible for or responsive to treatment of the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the presence of GREM1 or the expression level of GREM1 above a threshold level determined in step a) indicates that the subject is likely to be eligible for or responsive to treatment of the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the absence of GREM1 or the expression level of GREM1 determined in step a) being not above a threshold level indicates that the subject is not eligible for or less likely to respond to treatment with the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the method provided herein further comprises a step i) before the step a) :
i) contacting the biological sample of the diseased tissue of the subject with a GREM1 diagnostic agent under conditions that allow detection of expression level of the GREM1 in the sample.
In certain embodiments, the subject is a) determined to be resistant or refractory to PD-1/PD-L1 axis inhibitor and/or b) determined to have low or no PD-L1 expression in a diseased tissue.
In certain embodiments, the subject is determined to have medium or high PD-L1 expression in a diseased tissue.
In certain embodiments, the method provided herein further comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist for a period of time sufficient to increase expression level of PD-L1 in cancer cells of the subject, and then administering the PD-1/PD-L1 axis inhibitor to the subject.
In certain embodiments, the GREM1 expression is detected by a GREM1 diagnostic reagent comprising an anti-GREM1 antibody or antigen-binding fragment thereof.
In certain embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or antigen-binding fragment thereof.
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 and/or light chain LCDR1, LCDR2 and LCDR3, wherein:
the HCDR1 comprises the amino acid sequence comprising TYGMA (SEQ ID NO: 1) , or a homologue sequence of at least 80%sequence identity thereof;
the HCDR2 comprises the amino acid sequence comprising
WINTLSGEPTYADDFKG (SEQ ID NO: 2) , or a homologue sequence of at least 80%sequence identity thereof;
the HCDR3 comprises the amino acid sequence comprising EPMDY (SEQ ID NO: 3) , or a homologue sequence of at least 80%sequence identity thereof;
the LCDR1 comprises the amino acid sequence comprising
KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologue sequence of at least 80%sequence identity thereof;
the LCDR2 comprises the amino acid sequence comprising LVSKLDS (SEQ ID NO: 5) or a homologue sequence of at least 80%sequence identity thereof; and
the LCDR3 comprises the amino acid sequence comprising s WQGAHFPLT (SEQ ID NO: 6) or a homologue sequence of at least 80%sequence identity thereof.
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7, and
the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
In certain embodiments, the method further comprises one or more amino acid residue substitutions or modifications yet retains specific binding specificity or affinity to hGREM1.
In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the non-CDR regions of the VH or VL sequences.
In certain embodiments, the method further comprises an immunoglobulin constant region, optionally a constant region of a human IgG.
In certain embodiments, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4, and optionally the constant region comprises a heavy chain constant region comprising a sequence of SEQ ID NO: 9 and/or a light chain constant region comprising a sequence of SEQ ID NO: 10.
In certain embodiments, the GREM1 antagonist or the anti-GREM1 diagnostic reagent is linked to one or more conjugate moieties.
In certain embodiments, the conjugate moiety comprises a clearance-modifying agent, therapeutic agent (e.g., a chemotherapeutic agent) , a toxin, a radioactive isotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, biotin/avidin, or an enzyme-substrate label) , a pharmacokinetic modifying moiety, a DNA-alkylator, a topoisomerase inhibitor, a tubulin-binders, other anticancer drugs such as androgen receptor inhibitor.
In certain embodiments, the subject is human.
In certain embodiments, the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
In certain embodiments, the administration of the GREM1 antagonist is prior to, simultaneously with, or after the administration of the anti-angiogenesis agent, and/or the chemotherapeutic agent, and/or the immunotherapy agent.
In another aspect, the present disclosure provides use of a GREM1 antagonist in the manufacture of a medicament for treating a GREM1-expressing cancer in a subject in need thereof, wherein the treatment comprises administering to the subject the medicament in combination with: a) an anti-angiogenesis therapy, b) a chemotherapy, c) an immunotherapy, d) an anti-angiogenesis therapy and a chemotherapy, e) a chemotherapy and an immunotherapy; f) an anti-angiogenesis therapy and an immunotherapy; or g) an anti-angiogenesis therapy, a chemotherapy and an immunotherapy.
In another aspect, the present disclosure provides use of a GREM1 antagonist in the manufacture of a medicament for improving responsiveness of a subject to treatment with PD-1/PD-L1 axis inhibitor,
wherein the subject has been determined to have presence of GREM1 in a biological sample of a diseased tissue of the subject, or the subject has been determined to have expression level of GREM1 in the biological sample of the diseased tissue reaching a threshold level,
wherein the improvement comprises administering to the subject a therapeutically effective amount of a GREM1 antagonist such that expression of PD-L1 is increased in a diseased tissue, thereby improving responsiveness of the subject to PD-1/PD-L1 axis inhibitor.
In another aspect, the present disclosure provides use of an anti-GREM1 diagnostic reagent in the manufacture of a kit for determining the eligibility for or likelihood of responsiveness to treatment with a GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor in a subject,
wherein the anti-GREM1 diagnostic reagent is capable of determining presence or expression level of GREM1 in a biological sample of the diseased tissue of the subject,
wherein the presence or absence of or expression level of GREM1 is indicative of whether the subject is likely to be eligible for or responsive to treatment of the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the subject is a) determined to be resistant or refractory to PD-1/PD-L1 axis inhibitor and/or b) determined to have low or no PD-L1 expression in a diseased tissue
In certain embodiments, the subject is determined to have medium or high PD-L1 expression in a diseased tissue.
In another aspect, the present disclosure provides a kit useful in treating a GREM1-expressing cancer in a subject in need thereof, comprising a GREM1 antagonist and a package insert comprising instructions for using the GREM1 antagonist in combination with: a) an anti-angiogenesis therapy, b) a chemotherapy, c) an immunotherapy, d) an anti-angiogenesis therapy and a chemotherapy, e) a chemotherapy and an immunotherapy; f) an anti-angiogenesis therapy and an immunotherapy; or g) an anti-angiogenesis therapy, a chemotherapy and an immunotherapy.
In certain embodiments, the GREM1-expressing cancer is characterized in: a) being resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor, and/or b) having low or no expression of PD-L1 in the diseased tissue, and/or c) having GREM1 expression in a diseased tissue.
In another aspect, the present disclosure provides a method of improving tumor infiltrating lymphocytes in a subject having a solid tumor, comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist defined in any one of the preceding claims.
In certain embodiments, the solid tumor is a cold tumor.
In certain embodiments, the subject is resistant or refractory to anti-cancer therapies, such as immunotherapy, e.g., immune checkpoint inhibitors.
In another aspect, the present disclosure provides a method of promoting the transformation of a cold tumor into a hot tumor in a subject having a solid tumor, comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist defined in any one of the preceding claims.
In another aspect, the present disclosure provides a method of treating a cancer in a subject having a cold tumor, comprising: administering to the subject a therapeutically effective amount of a GREM1 antagonist defined in any one of the preceding claims.
In certain embodiments, the subject is resistant or refractory to anti-cancer therapies, such as immunotherapy, e.g., immune checkpoint inhibitors.
In certain embodiments, the method provided herein further comprises administering to the subject one or more therapies.
In certain embodiments, the one or more therapies are capable of promoting T cell proliferation, activation and/or tumor infiltration.
In certain embodiments, the T cell is CD3+ T cell or CD8+ T cell.
In certain embodiments, the one or more therapies are anti-angiogenesis therapy, immunotherapy and/or chemotherapy defined in any one of the preceding claims.
In certain embodiments, the subject is determined to have medium or high PD-L1 expression in a diseased tissue. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only, and are not restrictive of the invention. Further, the accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain principles of the invention.
BRIEF DESCRIPTION OF DRAWINGSThe drawings referenced herein form a part of the specification. Features shown in the drawing illustrate only some embodiments of the application, and not of all embodiments of the application, unless the detailed description explicitly indicates otherwise, and readers of the specification should not make implications to the contrary.
FIG. 1A shows that anti-GREM1 antibody in combination with DC101 inhibited CRC PDX tumor growth (mean ± S.E.M, n=8) .
FIG. 1B shows pathology information of the BZ-CRC-01 PDX tumor, where Gremlin-1 was detected in 10-20%of the tumor cells (A) by IHC method.
FIG. 2 shows that anti-GREM1 antibody in combination with FOLFIRI and DC101 inhibited CRC PDX tumor growth (mean ± S.E.M, n=8) .
FIG. 3 shows anti-GREM1 antibody in combination with FOLFIRI, Nivolumab and DC101 inhibited CRC PDX tumor growth (mean ± S.E.M, n=8) .
FIG. 4 shows representative IHC image of GREM1 expression on BZ-CRC-01 PDX tumor section.
FIG. 5 shows representative IHC image of PD-L1 expression before and after Hu14E3 HaLa treatment on BZ-CRC-01 PDX tumor sections.
FIG. 6 shows representative IHC image of tumor infiltrating lymphocytes (TILs) after Hu14E3 HaLa treatment on BZ-CRC-01 PDX tumor sections.
FIG. 7 shows safety profiles of Hu14E3 HaLa in NHP.
The same reference numbers will be used throughout the drawings to refer to the same or like parts.
DETAILED DESCRIPTION OF THE INVENTIONThe following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.
Definitions
As used herein, the term “a, ” “an, ” “the” and similar terms used in the context of the present invention (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context.
As used herein, the term “antagonist” with respect to GREM1 refers to any molecule that partially or completely inhibits, blocks, or neutralizes a biological activity of GREM1. Suitable GREM1 antagonists may include, without limitation, antibodies, antisense oligonucleotides, peptides, and small organic molecules. In certain embodiments, the GREM1 antagonist is an anti-GREM1 antibody.
The term “antibody” as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen, or any polypeptides that mimics an antibody in terms of being capable of binding to a specific antigen. A native intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each heavy chain consists of a variable region (VH) and a first, second, and third constant region (CH1, CH2, CH3, respectively) ; mammalian light chains are classified as λ or κ, while each light chain consists of a variable region (VL) and a constant region. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2, and LCDR3, heavy chain CDRs including HCDR1, HCDR2, HCDR3) . CDR boundaries for the antibodies and antigen-binding domains disclosed herein may be defined or identified by the conventions of Kabat, IMGT, AbM, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; N.R. Whitelegg et al, Protein Engineering, v13 (12) , 819-824 (2000) ; Chothia, C. et al., Nature. Dec 21-28; 342 (6252) : 877-83 (1989) ; Kabat E.A. et al., National Institutes of Health, Bethesda, Md. (1991) ; Marie-Paule Lefranc et al, Developmental and Comparative Immunology, 27: 55-77 (2003) ; Marie-Paule Lefranc et al, Immunome Research, 1 (3) , (2005) ; Marie-Paule Lefranc, Molecular Biology of B cells (second edition) , chapter 26, 481-514, (2015) ) . The three CDRs are interposed between flanking stretches known as framework regions (FRs) , which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen-binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (gamma1 heavy chain) , IgG2 (gamma2 heavy chain) , IgG3 (gamma3 heavy chain) , IgG4 (gamma4 heavy chain) , IgA1 (alpha1 heavy chain) , or IgA2 (alpha2 heavy chain) . In certain embodiments, the antibody provided herein encompasses any antigen-binding fragments thereof.
As used herein, the term “antigen-binding fragment” or “antigen-binding portion” refers to a fragment (e.g., antibody fragment) formed from a fragment of an antibody comprising one or more CDRs, or any other portion (e.g., antibody portion) that binds to an antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment/portion include, without limitation, a diabody, a Fab, a Fab', a F (ab') 2, a Fd, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single-chain antibody molecule (scFv) , an scFv dimer (bivalent diabody) , a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, and a bivalent domain antibody. An antigen-binding fragment/portion is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding fragment/portion may comprise one or more CDRs from a particular parent antibody.
“Fab” with regard to an antibody refers to a monovalent antigen-binding fragment of the antibody consisting of a single light chain (both variable and constant regions) bound to the variable region and first constant region of a single heavy chain by a disulfide bond. Fab can be obtained by papain digestion of an antibody at the residues proximal to the N-terminus of the disulfide bond between the heavy chains of the hinge region.
“Fab'” refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of an antibody at the residues proximal to the C-terminus of the disulfide bond between the heavy chains of the hinge region and thus is different from Fab in a small number of residues (including one or more cysteines) in the hinge region.
“F (ab') 2” refers to a dimer of Fab’ that comprises two light chains and part of two heavy chains.
“Fv” with regard to an antibody refers to the smallest fragment of the antibody to bear the complete antigen binding site. A Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. A “dsFv” refers to a disulfide-stabilized Fv fragment that the linkage between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond.
“Single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85: 5879 (1988) ) . A “scFv dimer” refers to a single chain comprising two heavy chain variable regions and two light chain variable regions with a linker. In certain embodiments, an “scFv dimer” is a bivalent diabody or bivalent ScFv (BsFv) comprising VH-VL (linked by a peptide linker) dimerized with another VH-VL moiety such that VH's of one moiety coordinate with the VL's of the other moiety and form two binding sites which can target the same antigens (or eptipoes) or different antigens (or eptipoes) . In other embodiments, a “scFv dimer” is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (also linked by a peptide linker) such that VH1 and VL1 coordinate and VH2 and VL2 coordinate and each coordinated pair has a different antigen specificity.
“Single-chain Fv-Fc antibody” or “scFv-Fc” refers to an engineered antibody consisting of a scFv connected to the Fc region of an antibody.
“Camelized single domain antibody, ” “heavy chain antibody, ” “nanobody” or “HCAb” refers to an antibody that contains two VH domains and no light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231 (1-2) : 25-38 (1999) ; Muyldermans S., J Biotechnol. Jun; 74 (4) : 277-302 (2001) ; WO94/04678; WO94/25591; U.S. Patent No. 6,005,079) . Heavy chain antibodies were originally obtained from Camelidae (camels, dromedaries, and llamas) . Although devoid of light chains, camelized antibodies have an authentic antigen-binding repertoire (Hamers-Casterman C. et al., Nature. Jun 3; 363 (6428) : 446-8 (1993) ; Nguyen VK. et al. “Heavy-chain antibodies in Camelidae; a case of evolutionary innovation, ” Immunogenetics. Apr; 54 (1) : 39-47 (2002) ; Nguyen VK. et al. Immunology. May; 109 (1) : 93-101 (2003) ) . The variable domain of a heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses (Koch-Nolte F. et al., FASEB J. Nov; 21 (13) : 3490-8. Epub 2007 Jun 15 (2007) ) . “Diabodies” include small antibody fragments with two antigen-binding sites, wherein the fragments comprise a VH domain connected to a VL domain in a single polypeptide chain (VH-VL or VL-VH) (see, e.g., Holliger P. et al., Proc Natl Acad Sci U S A. Jul 15; 90 (14) : 6444-8 (1993) ; EP404097; WO93/11161) . The two domains on the same chain cannot be paired, because the linker is too short, thus, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. The antigen–binding sites may target the same of different antigens (or epitopes) .
A “domain antibody” refers to an antibody fragment containing only the variable region of a heavy chain or the variable region of a light chain. In certain embodiments, two or more VH domains are covalently joined with a peptide linker to form a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.
In certain embodiments, a “ (dsFv) 2” comprises three peptide chains: two VH moieties linked by a peptide linker and bound by disulfide bridges to two VL moieties.
In certain embodiments, a “bispecific ds diabody” comprises VH1-VL2 (linked by a peptide linker) bound to VL1-VH2 (also linked by a peptide linker) via a disulfide bridge between VH1 and VL1.
In certain embodiments, a “bispecific dsFv” or “dsFv-dsFv'” comprises three peptide chains: a VH1-VH2 moiety wherein the heavy chains are bound by a peptide linker (e.g., a long flexible linker) and paired via disulfide bridges to VL1 and VL2 moieties, respectively. Each disulfide paired heavy and light chain has a different antigen specificity.
The term “humanized” as used herein means that the antibody or antigen-binding fragment comprises CDRs derived from non-human animals, FR regions derived from human, and when applicable, constant regions derived from human. In certain embodiments, the amino acid residues of the variable region framework of the humanized gremlin antibody are substituted for sequence optimization. In certain embodiments, the variable region framework sequences of the humanized gremlin antibody chain are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%or 100%identical to the corresponding human variable region framework sequences.
“Anti-GREM1 antibody” as used herein refers to an antibody that is capable of specific binding to GREM1 (e.g., human GREM1 or non-human GREM1) with a sufficient specificity and/or affinity, for example, to provide for diagnostic and/or therapeutic use.
The term “affinity” as used herein refers to the strength of non-covalent interaction between an immunoglobulin molecule (i.e. antibody) or fragment thereof and an antigen.
The term “specific binding” or “specifically binds” as used herein refers to a non-random binding reaction between two molecules, such as for example between an antibody and an antigen. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically bind to human and/or non-human gremlin1 with a binding affinity (KD) of ≤10-6 M (e.g., ≤5×10-7 M, ≤2×10-7 M, ≤10-7 M, ≤5×10-8 M, ≤2×10-8 M, ≤10-8 M, ≤5×10-9 M, ≤4×10-9M, ≤3×10-9M, ≤2×10-9 M, or ≤10-9 M. KD used herein refers to the ratio of the dissociation rate to the association rate (koff/kon) , which may be determined by using any conventional method known in the art, including but are not limited to surface plasmon resonance method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method. In certain embodiments, the KD value can be appropriately determined by using flow cytometry method. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow &Lane, Using Antibodies, A Laboratory Manual (1998) , for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity) . Typically a specific or selective binding reaction will produce a signal at least twice over the background signal and more typically at least 10 to 100 times over the background.
“Percent (%) sequence identity” with respect to amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum correspondence. Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al, J. Mol. Biol., 215: 403–410 (1990) ; Stephen F. et al, Nucleic Acids Res., 25: 3389–3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al, Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A. et al, Bioinformatics (Oxford, England) , 23 (21) : 2947-8 (2007) ) , and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by the tool, or may customize the parameters as appropriate for the alignment, such as for example, by selecting a suitable algorithm. In certain embodiments, the non-identical residue positions may differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity) . In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference.
As used herein, a “homologue sequence” refers to a polynucleotide sequence (or its complementary strand) or an amino acid sequence that has sequence identity of at least 80%(e.g. at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) to another sequence when optionally aligned.
The term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mouse, rat, cat, rabbit, sheep, dog, cow, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.
“Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
“Cancer” as used herein refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration, or metastasis, and includes both solid tumors and non-solid cancers (e.g., hematologic malignancies) such as leukemia. As used herein “solid tumor” refers to a solid mass of neoplastic and/or malignant cells.
The term “therapeutically effective amount” or “effective amount” means the amount of a pharmaceutical agent that produces some desired local or systemic therapeutic effect at a reasonable benefit/risk ratio applicable to any treatment alone or together with further doses. In the case of the treatment of a particular disease, the desired local or systemic therapeutic effect preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. When administered for preventing a disease, the amount is sufficient to avoid or delay onset of the disease. A therapeutically effective amount or an effective amount need not be curative or prevent a disease or condition from ever occurring. An effective amount of the pharmaceutical agent described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present) , the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical agent described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used. In certain embodiments, a therapeutically effective amount of a pharmaceutical agent will depend on its therapeutic index, solubility, and the like.
Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X. ” Numeric ranges are inclusive of the numbers defining the range. Generally speaking, the term “about” refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g. within the 95%confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. Where the term “about” is used within the context of a time period (years, months, weeks, days etc. ) , the term “about” means that period of time plus or minus one amount of the next subordinate time period (e.g. about 1 year means 11-13 months; about 6 months means 6 months plus or minus 1 week; about 1 week means 6-8 days; etc. ) , or within 10 percent of the indicated value, whichever is greater.
I. Methods of Improving Tumor Infiltrating Lymphocytes
The present disclosure provides a method of improving tumor infiltrating lymphocytes in a subject having a solid tumor, comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist. In certain embodiments, the solid tumor is a cold tumor. In certain embodiments, the subject is resistant or refractory to anti-cancer therapies, such as immunotherapy, e.g., immune checkpoint inhibitors (e.g., PD-1/PD-L1 axis inhibitors) .
The present disclosure also provides a method of promoting the transformation of a cold tumor into a hot tumor in a subject having a solid tumor, comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist. As used herein, the term “cold tumor” used interchangeably with the term “immune-desert tumor” or “immune-excluded tumor” , refers to a tumor lacking innate immunity or having ineffective innate antitumor immune features, which is characterized by 1) lack of T-cell infiltration, 2) low mutational load, 3) low major histocompatibility complex (MHC) class I expression, 4) low PD-L1 expression, and/or 5) presence of immunosuppressive cell populations (e.g., tumor-associated macrophages, T-regulatory cells, myeloid-derived suppressor cells) . As used herein, the term “hot tumor” refers to a tumor characterized by 1) high T-cell infiltration, 2) increased interferon-gamma signaling, 3) expression of PD-L1, and/or 4) high tumor mutational burden.
The present disclosure further provides a method of treating a cancer in a subject having a cold tumor, comprising administering to the subject a therapeutically effective amount of a GREM1 antagonist.
In certain embodiments, the subject is resistant or refractory to anti-cancer therapies, such as immunotherapy, e.g., immune checkpoint inhibitors.
In certain embodiments, the method further comprises administering to the subject one or more therapies. In certain embodiments, the one or more therapies are capable of promoting T cell proliferation, activation and/or tumor infiltration. In certain embodiments, the T cell is CD3+ T cell or CD8+ T cell. In certain embodiments, the one or more therapies are anti-angiogenesis therapy, immunotherapy and/or chemotherapy.
II. Combination Therapy
The present disclosure provides a method of treating a GREM1-expressing cancer in a subject in need thereof. In certain embodiments, the method comprises administering a therapeutically effective amount of a GREM1 antagonist in combination with a second therapy.
In another aspect, the present disclosure provides a method of treating a GREM1-expressing cancer in a subject in need thereof. In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of a GREM1 antagonist in combination with: a) an anti-angiogenesis therapy, b) a chemotherapy, c) an immunotherapy, d) an anti-angiogenesis therapy and a chemotherapy, e) a chemotherapy and an immunotherapy; f) an anti-angiogenesis therapy and an immunotherapy; or g) an anti-angiogenesis therapy, a chemotherapy and an immunotherapy.
In another aspect, the present disclosure provides use of a GREM1 antagonist in the manufacture of a medicament/pharmaceutical composition for treating a GREM1-expressing cancer in a subject in need thereof, wherein the treatment comprises administering to the subject the medicament/pharmaceutical composition in combination with: a) an anti-angiogenesis therapy, b) a chemotherapy, c) an immunotherapy, d) an anti-angiogenesis therapy and a chemotherapy, e) a chemotherapy and an immunotherapy; f) an anti-angiogenesis therapy and an immunotherapy; or g) an anti-angiogenesis therapy, a chemotherapy and an immunotherapy.
1. Immunotherapy
In certain embodiments, the immunotherapy used in the methods provided herein comprises a PD-1/PD-L1 axis inhibitor. The term “PD-1/PD-L1 axis inhibitor” is a molecule (e.g., small molecules, antibodies, etc. ) that inhibits the interaction between PD-1/PD-L1 axis binding partners, such as PD-1 and PD-L1, to reduce or eliminate inhibitory effect of T-cell function (e.g., proliferation, cytokine production, and target cell killing) resulting from signaling on the PD-1/PD-L1 signaling axis. The PD-1/PD-L1 axis inhibitor can include a PD-1 inhibitor or PD-L1 inhibitor.
In certain embodiments, the PD-1/PD-L1 axis inhibitor comprises a PD-1 inhibitor. The term “PD-1 inhibitor” , as used herein, refers to a molecule that decreases, abrogates, inhibits, blocks, or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1. In certain embodiments, PD-1 inhibitor is a molecule that blocks the binding of PD-1 to its binding partners, such as PD-L1. For example, a PD-1 inhibitor can be anti-PD-1 antibodies or antigen binding fragments thereof, fusion proteins, oligopeptides, immunoadhesins and other molecules that decrease, abrogate, inhibit, block, or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1.
In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from the group consisting of: Nivolumab (OPDIVO; BMS-936558) , Dostarlimab (TSR-042) , Pembrolizumab (KEYTRUDA; MK-3475) , MEDI0680 (AMP-514) , MEDI4736, BI 754091, Pidilizumab (CT-011) , Cemiplimab (LIBTAYO, REGN2810) , Spartalizumab (PDR001) , Cetrelimab (JNJ 63723283) , Toripalimab (JS001) , PF-06801591, Tislelizumab (BGB-A317) , AMP-224 (GSK-2661380) , ABBV-181, Lambrolizumab, Camrelizuma (SHR-1210) , Sintilimab (Tyvyt, IBI308) , Penpulimab (AK105) , Zimberelimab, Retifanlimab, Serplulimab, Balstilimab, Geptanolimab, Prolgolimab, Ezabenlimab, Sasanlimab, Pimivalimab, Budigalimab, Nofazinlimab, Sindelizumab, MGA404, Sym021, BAT1306, and HX008.
In certain embodiments, the PD-1 inhibitor is Nivolumab. Nivolumab (Bristol-Myers Squibb/Ono) is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and which is an anti-PD-1 antibody described in WO2006/121168.
In certain embodiment, the anti-PD-1 antibody used in the methods provided herein comprises a heavy chain and a light chain sequence, wherein the heavy chain comprises an amino acid sequence of SEQ ID NO: 19, and the light chain comprises an amino acid sequence of SEQ ID NO: 20.
In certain embodiments, the anti-PD-1 antibody used in the methods provided herein comprises six CDRs from SEQ ID NO: 17 and SEQ ID NO: 18 (e.g., the three heavy chain CDRs from SEQ ID NO: 17 and the three light chain CDRs from SEQ ID NO: 18) . In some embodiments, the anti-PD-1 antibody used in the methods provided herein comprises the heavy chain variable domain from SEQ ID NO: 19 and the light chain variable domain from SEQ ID NO: 20. In certain embodiments, the anti-PD-1 antibody used in the methods provided herein comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 18.
In certain embodiments, the anti-PD-1 antibody used in the methods provided herein comprises heavy chain HCDR1, HCDR2 and HCDR3 and/or light chain LCDR1, LCDR2 and LCDR3 sequences, wherein: the HCDR1 sequence comprises SEQ ID NO: 11, or a homologue sequence of at least 80%sequence identity thereof; the HCDR2 sequence comprises SEQ ID NO: 12, or a homologue sequence of at least 80%sequence identity thereof; the HCDR3 sequence comprises SEQ ID NO: 13, or a homologue sequence of at least 80%sequence identity thereof; the LCDR1 sequence comprises SEQ ID NO: 14 or a homologue sequence of at least 80%sequence identity thereof; the LCDR2 sequence comprises SEQ ID NO: 15 or a homologue sequence of at least 80%sequence identity thereof; the LCDR3 sequence comprises SEQ ID NO: 16 or a homologue sequence of at least 80%sequence identity thereof.
In certain embodiments, the anti-PD-1 antibody used in the methods provided herein (e.g., Nivolumab) is administered at a dose of 120 mg, 160 mg, 200 mg, 240 mg, 280 mg, 320 mg, 360 mg, 400 mg, 480mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, or 1400mg intravenously; or 2mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12 mg/kg, 14 mg/kg, 16 mg/kg, 18 mg/kg, 20 mg/kg, 22 mg/kg, or 24 mg/kg intravenously. Nivolumab may be given intravenously according to institutional guidelines, published guidelines and the respective product prescribing information, and dosed according to this protocol.
In certain embodiments, the PD-1/PD-L1 axis inhibitor comprises PD-L1 inhibitor selected from the group consisting of antibody, small molecule, and combination thereof. In certain embodiments, the PD-L1 inhibitor comprises an anti-PD-L1 antibody selected from the group consisting of: Atezolizumab (TECENTRIQ; R05541267; MPDL3280A; RG7446) , BMS-936559, Avelumab (bavencio) , lodapolimab (LY3300054) , Durvalumab (MEDI4736) , CX-072 (Proclaim-CX-072) , FAZ053, Envafolimab (KN035) , MDX-1105, STI-1040, CS1001, Adebrelimab (SHR-1316) , SHR-1701, TOB2450, Bintrafusp, LP002, STI-3031, Cosibelimab, Pacmilimab, NM01, LDP, AMP-224, Garivulimab (BGB-A333) , A167, SCD-135, Opucolimab, and GR1405.
In certain embodiments, the anti-PD-L1 antibody used in the methods provided herein is administered at a dose of 120 mg, 160 mg, 200 mg, 240 mg, 280 mg, 320 mg, 360 mg, 400 mg, 480mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, or 1400mg intravenously; or 2mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 12 mg/kg, 14 mg/kg, 16 mg/kg, 18 mg/kg, 20 mg/kg, 22 mg/kg, or 24 mg/kg intravenously.
2. Chemotherapy
In certain embodiments, the chemotherapy used in the methods provided herein is a chemotherapeutic regimen (or a combination of chemotherapeutic agents) . In certain embodiments, the chemotherapy used in the methods provided herein comprises a combination of chemotherapeutic agents. The term “chemotherapeutic agent” is a biological (macromolecule) or chemical (small molecule) compound that can be used to treat cancer. The types of chemotherapeutic drugs include, but are not limited to, histone deacetylase inhibitor (HDACI) , alkylating agents, antimetabolites, alkaloids, cytotoxic/anti-cancer antibiotics, topoisomerase inhibitors, tubulin inhibitors, proteins, antibodies, kinase inhibitors, and the like. Examples of chemotherapeutic drugs include, erlotinib, afatinib, docetaxel, adriamycin, 5-FU (5-fluorouracil) , panobinostat, gemcitabine, cisplatin, pemetrexed, carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab, metformin, temozolomide, tamoxifen, oteracil, doxorubicin, rapamycin, lapatinib, hydroxycamptothecin, trametinib, tegafur, gimeracil, leucovorin calcium (folinic acid) (LV) , irinotecan hydrochloride (CPT-11) , platinum (e.g., cisplatin) , epirubicin, oxaliplatin, capecitabine. In certain embodiments, the chemotherapy comprises a combination of chemotherapeutic agents selected from the group consisting of: LV, 5-FU, CPT-11, epirubicin, oxaliplatin, capecitabine, platinum (e.g., cisplatin) , tegafur, gimeracil, oteracil, docetaxel, pemetrexed. In certain embodiments, the combination of chemotherapeutic agents comprises LV, 5-FU, and CPT-11. In certain embodiments, the combination of chemotherapeutic agents consists of LV, 5-FU, and CPT-11.
In certain embodiments, the chemotherapeutic regimen (or a combination of chemotherapeutic agents) may be selected from the group consisting of FOLFIRI chemotherapy, EOX chemotherapy, ECF chemotherapy, ECX chemotherapy, EOF chemotherapy, FLO chemotherapy, CAPOX chemotherapy, FOLFOX chemotherapy, DCF chemotherapy, SOX chemotherapy and FLOT chemotherapy. The drug combination used in FOLFIRI chemotherapy comprises or consists of: LV, 5-FU and CPT-11. The drug combination used in EOX chemotherapy comprises or consists of: epirubicin, oxaliplatin and capecitabine. The drug combination used in ECF chemotherapy comprises or consists of: epirubicin, cisplatin and 5-FU. The drug combination used in ECX chemotherapy comprises or consists of: epirubicin, cisplatin and capecitabine. The drug combination used in EOF chemotherapy comprises or consists of: epirubicin, oxaliplatin and 5-FU. The drug combination used in FLO chemotherapy comprises or consists of: 5-FU, LV and oxaliplatin. The drug combination used in SOX chemotherapy comprises or consists of: tegafur, gimeracil, oteracil and oxaliplatin.
In certain embodiments, the chemotherapy comprises CPT-11 of 20 mg/m2 to 400 mg/m2 (e.g., 40 mg/m2, 60 mg/m2, 80 mg/m2, 100 mg/m2, 120 mg/m2, 140 mg/m2, 160 mg/m2, 180 mg/m2, 200 mg/m2, 220 mg/m2, 240 mg/m2, 300 mg/m2, 340 mg/m2, 380 mg/m2 or 400 mg/m2) . In certain embodiments, the chemotherapy comprises LV of 20 mg/m2 to 400 mg/m2 (e.g., 40 mg/m2, 60 mg/m2, 80 mg/m2, 100 mg/m2, 120 mg/m2, 140 mg/m2, 160 mg/m2, 180 mg/m2, 200 mg/m2, 220 mg/m2, 240 mg/m2, 300 mg/m2, 340 mg/m2, 380 mg/m2 or 400 mg/m2) . In certain embodiments, the chemotherapy comprises a bolus dose of 5-FU of 100 mg/m2 to 800 mg/m2 (e.g., 100 mg/m2, 120 mg/m2, 140 mg/m2, 160 mg/m2, 180 mg/m2, 200 mg/m2, 220 mg/m2, 240 mg/m2, 300 mg/m2, 340 mg/m2, 380 mg/m2, 400 mg/m2, 500 mg/m2, 600 mg/m2, 700 mg/m2 or 800 mg/m2) .
In certain embodiments, the chemotherapy used in the methods provided herein is the FOLFIRI regimen. In certain embodiments, the FOLFIRI regimen consists of CPT-11 180 mg/m2 as a 90-min infusion on day 1 and LV 200 mg/m2 as a 2-h infusion during CPT-11, immediately followed by a bolus dose of 5-FU 400 mg/m2 and a 46-h continuous infusion of 2,400 mg/m2 every 2 weeks. In certain embodiments, the recommended dose schedule of FOLFIRI given every two weeks is as follows: Day 1: CTP-11 180 mg/m2 IV infusion and LV 400 mg/m2 IV infusion, followed by 5-FU 400 mg/m2 IV bolus, followed by 5-FU 2400 mg/m2 IV infusion as a 46-hour continuous infusion. There are several different FOLFIRI regimens that differ in the doses and ways in which the three drugs are given.
Other chemotherapeutic regimens mentioned above were described in PCT patent application PCT/JP2022/017017, which is herein incorporated by reference.
3. Anti-Angiogenesis Therapy
In certain embodiments, the anti-angiogenesis therapy used in the methods provided herein comprises an anti-angiogenesis agent that can block the growth of blood vessels that support tumor growth. Some of the anti-angiogenesis agent target vascular endothelial growth factor (VEGF) or its receptor VEGFR. In certain embodiments, the anti-angiogenesis therapy used in the methods provided herein comprises an antagonist of a vascular endothelial growth factor A (VEGFA) . In certain embodiments, the antagonist of a VEGFA is an anti-VEFRA antibody, such as Bevacizumab
In certain embodiments, the anti-angiogenesis therapy used in the methods provided herein comprises an antagonist of a VEGFR, such as VEGFR-1, VEGFR-2, and VEGFR-3. The term “antagonist of a VEGFR” , used interchangeably with the term “VEGFR inhibitor” , is a molecule (e.g., small molecules or large molecules (e.g., antibodies) ) that inhibits the interaction between VEGFR and VEGF ligands (e.g., those secreted by tumors, e.g., solid tumors) , thereby inhibiting angiogenesis and impeding tumor blood supply.
In certain embodiments, the antagonist of a VEGFR is an anti-VEGFR-2 antibody. In certain embodiments, the anti-VEGFR-2 antibody is selected from the group consisting of: Ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04 and HLX12, .
In certain embodiments, the antagonist of a VEGFR (or a VEGFR inhibitor) is a small molecule VEGFR inhibitor selected from the group consisting of: Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib and Regorafenib
In certain embodiments, the anti-angiogenesis therapy used in the methods provided herein (e.g., Bevacizumabramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib ) is administered at a dose of 120 mg, 160 mg, 200 mg, 240 mg, 280 mg, 320 mg, 360 mg, 400 mg, 500mg, 600mg, 700mg, 800mg, 900mg, 1000mg, 1100mg, 1200mg, 1300mg, or 1400mg intravenously; or 2 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg, 10 mg/kg, 12mg/kg, or 14 mg/kg intravenously . The anti-angiogenesis therapy used in the methods provided herein (e.g., Bevacizumabramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib ) may be given intravenously according to institutional guidelines, published guidelines and the respective product prescribing information, and dosed according to this protocol.
4. Specific Combination Therapies
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with a VEGFR inhibitor (e.g., Bevacizumab ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib In certain embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variant thereof (e.g., hu14E3, hu14E3 HaLa) .
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with a VEGFR inhibitor (e.g., Bevacizumab ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib ) , and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the combination of chemotherapeutic agents (or a chemotherapeutic regimen) (e.g., FOLFIRI) and a VEGFR inhibitor (e.g., Bevacizumab ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib ) . In certain embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variant thereof (e.g. hu14E3, hu14E3 HaLa) .
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the combination of chemotherapeutic agents (or a chemotherapeutic regimen) (e.g., FOLFIRI) and a VEGFR inhibitor (e.g., Bevacizumab ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib and/or Regorafenib and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the combination of chemotherapeutic agents (e.g. FOLFIRI) and the PD-1/PD-L1 axis inhibitor (e.g. Nivolumab) . In certain embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variant thereof (e.g. hu14E3, hu14E3 HaLa) .
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with the combination of chemotherapeutic agents (e.g. FOLFIRI) and the PD-1/PD-L1 axis inhibitor (e.g. Nivolumab) , and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with a VEGFR inhibitor (e.g., Bevacizumab ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, from Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib ) , the combination of chemotherapeutic agents (e.g., FOLFIRI) , and the PD-1/PD-L1 axis inhibitor (e.g. Nivolumab) . In certain embodiments, the GREM1 antagonist comprises an anti-GREM1 antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the GREM1 antagonist comprises anti-GREM1 antibody 14E3 or its humanized variant thereof (e.g. hu14E3, hu14E3 HaLa) .
In certain embodiments, the method comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist in combination with a VEGFR inhibitor (e.g., Bevacizumab ramucirumab, Olinvacimab, Gentuximab, Alacizumab Pegol, Vulinacimab, Regorafenib, MSB0254, AK109, AT001, AC88, APX004, KDR-1121, VK-B8, mAb-04, HLX12, from Sitravatinib, Anlotinib, Apatinib, Telatinib, Altiratinib, Kanitinib, Lenvatinib mesylate, Pazopanib, Sorafenib, Sunitinib, Vandetanib, Axitinib Cabozantinib Fruquintinib or Regorafenib ) , the combination of chemotherapeutic agents (e.g., FOLFIRI) , and the PD-1/PD-L1 axis inhibitor (e.g. Nivolumab) , and wherein the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
5. GREM1-expressing cancer
“GREM1-expressing cancer” as used herein refers any cancer or tumor involving cancer cells that express or secret GREM1 (e.g., low expression, medium expression, or high expression) .
GREM1 expression can be determined using methods known in the art, including, without limitation, protein-based assays such as immunohistochemistry methods and ELISA, or nucleic acid based assays such as amplification assays, hybridization assays or sequencing assays. In certain embodiments, the GREM1 expression can be determined using methods or antibodies provided herein. In certain embodiments, the subject is further determined to have low, medium or high GREM1 expression in a diseased tissue of the GREM1-expressing cancer.
In certain embodiments, the subject is determined to have GREM1 expression (e.g., presence of GREM1 or the expression level of GREM1 above a threshold level) in a diseased tissue. As used herein, the “threshold level” refers to expression level of GREM1 at an intensity of 1+, 2+ or 3+ as measured by IHC.
In certain embodiments, GREM1 expression is determined from the diseased tissue (e.g. from the biological sample) .
The presence and/or expression level of GREM1 in the diseased tissue (e.g., cancerous tissue or tumor tissue) , can be determined by various methods known in the art. In certain embodiments, the biological sample may be further processed to, for example, isolate the analyte such as the nucleic acids or proteins. Presence and/or expression level of GREM1 can be determined by, for example, quantitative fluorescence cytometry, immunohistochemistry (IHC) , or nucleic acid-based methods. For example, the biological sample from the subject can be exposed to anti-GREM1 diagnostic reagent, which binds to and detects the expressed GREM1 protein.
In certain embodiments, the expression of GREM1 in the diseased tissue (e.g., cancerous tissue or tumor tissue) , is determined or measured by IHC. In certain embodiments, the expression level of human GREM1 protein on a cancerous tissue or tumor tissue from the subject can be determined in accordance to the methods described in Example 4 provided herein.
In certain embodiments, the subject is or has been determined to have high GREM1 expression in the diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from the subject. In certain embodiments, the diseased tissue is or has been determined to have GREM1 expression higher than or comparable to expression in healthy or noncancerous cells. The high GREM1 expression in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) refers to expression of GREM1 at an intensity of at least 2+ (e.g., 2+ or 3+ as measured by IHC.
In certain embodiments, the subject is or has been determined to have medium GREM1 expression in the diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from the subject. The medium GREM1 expression in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) refers to expression of GREM1 at an intensity of at least 1+ and below 2+ as measured by IHC.
In certain embodiments, the subject is or has been determined to have low GREM1 expression in the diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from the subject. In certain embodiments, the diseased tissue is or has been determined to have GREM1 expression comparable to expression in a healthy tissue and is detectable by an anti-GREM1 diagnostic reagent. The low GREM1 expression in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) refers to expression of GREM1 at an intensity of above 0 but below 1+ as measured by IHC.
6. GREM1-expressing cancer resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or having low or no PD-L1 expression
In certain embodiments, the GREM1-expressing cancer is resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or is determined to have low or no PD-L1 expression (i.e., low expression of PD-L1 or no expression of PD-L1) in a diseased tissue of the cancer.
The present disclosure surprisingly discovered that treating a tumor with a GREM1 antagonist resulted in upregulation of the programmed death ligand 1 (PD-L1) expression in the tumor. PD-L1 is a protein that interacts with programmed death protein 1 (PD-1) and is expressed on, for example, immune and tumor cells, in a tumor tissue. Studies have investigated the correlation between tumor PD-L1 expression and therapeutic efficacy of anti-PD-1 antibodies and have shown that PD-L1 overexpression is associated with significantly higher objective response rates (ORRs) (Gettinger et al., Overall survival and long-term safety of nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated advanced non-small-cell lung cancer. J Clin Oncol. 2015; 33: 2004–12.; Garon et al., Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015; 372: 2018–28. doi: 10.1056/NEJMoa1501824. ) . Publications and reports have also shown that patients having tumors with low PD-L1 expression can benefit less from treatment with PD-1/PD-L1 axis inhibitor.
Based, at least in part, on the discoveries as mentioned above, the methods provided herein have unexpected effects in treating a GREM1-expressing cancer which is resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor and/or is determined to have low or no PD-L1 expression (i.e., low expression of PD-L1 or no expression of PD-L1) in a diseased tissue of the cancer.
As used herein, “resistant” refers to non-responsiveness (which can be either naturally non-responsive or treatment-induced non-responsiveness) of a disease or condition to a treatment.
As used herein, “refractory” refers to the resistance or non-responsiveness of a disease or condition to a treatment (e.g., the number of neoplastic cells increases even though treatment is given) . Unless otherwise indicated, the term “refractory” refers a resistance or non-responsiveness to any previous treatment of PD-1/PD-L1 axis inhibitor.
PD-L1 expression can be determined using methods known in the art, including, without limitation, protein-based assays such as immunohistochemistry methods and ELISA, or nucleic acid based assays such as amplification assays, hybridization assays or sequencing assays. Methods as described above for detection of GREM1 expression can also be used in the detection of PD-L1 expression, except that detection reagent should be replaced with reagents for detection of PD-L1.
In certain embodiments, PD-L1 expression may be determined using the methods described in PCT/CN2022/131820, which is herein incorporated by reference. In certain embodiments, PD-L1 expression can be determined in accordance to the methods described in Example 4 provided herein. The reagents used to detect the PD-L1 expression throughout the specification can be an anti-PD-L1 diagnostic antibody, for example, 22C3, as described in US20170285037A1, disclosure of which has been incorporated by reference in its entirety, and a monoclonal rabbit anti-PD-L1, Clone 28-8, which is commercially available. In certain embodiments, the reagents used to detect the PD-L1 expression throughout the specification is a polypeptide comprising the antigen-binding portion of the 22C3, as described in US20170285037A1, disclosure of which has been incorporated by reference in its entirety, and a monoclonal rabbit anti-PD-L1, Clone 28-8, which is commercially available.
As used herein, the term “low expression of PD-L1” refers to the PD-L1 expression level that is lower than or no more than a reference level.
The term “reference level” with respect to the PD-L1 expression refer to the threshold (e.g., minimal) expression level of PD-L1 in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from a subject who is responsive to the treatment of PD-1/PD-L1 axis inhibitors.
In certain embodiments, the cancer having low PD-L1 expression has an intensity of no more than 1+, or no more than 2+ as measured by IHC using antibody 22C3 or antibody Clone 28-8, or has the PD-L1 positive percentage in tumor cell no more than 5%in CPS.
As used herein, the term “no expression of PD-L1” refers to the PD-L1 expression level that is lower than a baseline threshold level. In certain embodiments, a biological sample (e.g. a cancer cell) having no expression of PD-L1 is absent of any detectable PD-L1 signal as detected by an anti-PD-L1 diagnostic antibody using a proven technique, such as IHC.
The term “baseline threshold level” with respect to the PD-L1 expression refer to the threshold detectable expression level of PD-L1 in a biological sample.
Expression of PD-L1 can be measured by methods described in PCT/CN2022/131820, which is herein incorporated by reference. For different tumor types and/or using different PD-L1 detection assays, the threshold expression level of PD-L1 expression could be different, and can be determined using methods known in the art.
In some embodiments, the PD-L1 expression in the diseased tissue is low or non-detectable by an anti-PD-L1 diagnostic antibody.
In certain embodiments, the methods provided herein comprise administering to the subject a therapeutically effective amount of a GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor. In certain embodiments, the method comprises administering the GREM1 antagonist to the subject, and after a period of time sufficient to increase expression level of PD-L1 in cancer tissue/cancer cells of the subject, then administering the PD-1/PD-L1 axis inhibitor to the subject. In certain embodiments, the subject has been administered with the GREM1 antagonist for a period of time sufficient to increase expression level of PD-L1 in cancer tissue/cancer cells of the subject.
In certain embodiments, the GREM1-expressing cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the subject is human.
7. Pharmaceutical compositions and administration routes
The GREM1 antagonist, chemotherapeutic agent, immunotherapeutic agent, or anti-angiogenesis agent described above may each be administered in the form of any suitable pharmaceutical composition. The term "pharmaceutical composition" refers a formulation comprising a therapeutically effective agent (e.g., the GREM1 antagonist, chemotherapeutic agent, immunotherapeutic agent, anti-angiogenesis agents described above) , preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject.
Pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known in the art. A pharmaceutical composition may, for example, be in the liquid dosage form such as solution or suspension, or solid dosage forms such as tablets and capsules. The pharmaceutical compositions described herein are generally applied in a “therapeutically effective amount” and in a “pharmaceutically acceptable preparation” . The term “pharmaceutically acceptable” , as used herein, refers to the non-toxicity of a material that does not interact with the action of the active component of the pharmaceutical composition.
The pharmaceutical compositions described herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.
In certain embodiments, the administration is via oral, nasal, intravenous, subcutaneous, sublingual, or intramuscular administration.
In certain embodiments, the administration of the GREM1 antagonist is prior to, simultaneously with, or after the administration of the anti-angiogenesis agent, and/or the combination of chemotherapeutic agents, and/or the immunotherapy.
In certain embodiments, the method comprises administering the GREM1 antagonist to the subject, and after a period of time sufficient to increase expression level of PD-L1 in cancer tissues/cells of the subject, then administering the PD-1/PD-L1 axis inhibitor to the subject. In certain embodiments, the subject has been administered with the GREM1 antagonist for a period of time sufficient to increase expression level of PD-L1 in cancer tissue/cancer cells of the subject.
III. GREM1 as A Biomarker for Immunotherapy Eligibility
In another aspect, the present disclosure provides a method of determining eligibility for or likelihood of responsiveness to treatment with a GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor in a subject, the method comprising:
b) determining presence or expression level of GREM1 in a biological sample of the diseased tissue of the subject,
wherein the presence or absence of or expression level of GREM1 is indicative of whether the subject is likely to be eligible for or responsive to treatment of the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the presence of GREM1 or the expression level of GREM1 above a threshold level determined in step a) indicates that the subject is likely to be eligible for or responsive to treatment of the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the absence of GREM1 or the expression level of GREM1 determined in step a) being not above a threshold level indicates that the subject is not eligible for or less likely to respond to treatment with the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
In certain embodiments, the method further comprises a step i) before the step a) :
i) contacting the biological sample of the diseased tissue of the subject with a GREM1 diagnostic agent under conditions that allow detection of expression level of the GREM1 in the sample.
In certain embodiments, the subject has been a) determined to be resistant or refractory to PD-1/PD-L1 axis inhibitor and/or b) determined to have low or no PD-L1 expression in a diseased tissue. In certain embodiments, the subject has been determined to have medium or high PD-L1 expression in a diseased tissue.
In certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of the GREM1 antagonist for a period of time sufficient to increase expression level of PD-L1 in cancer tissues/cells of the subject, and then administering the PD-1/PD-L1 axis inhibitor to the subject.
In certain embodiments, the GREM1 expression is detected by a GREM1 diagnostic reagent. In certain embodiments, the GREM1 diagnostic reagent/agent comprises a polypeptide comprising an antigen-binding portion of an anti-GREM1 antibody or antigen-binding fragment thereof. In certain embodiments, the GREM1 diagnostic reagent/agent comprises an anti-GREM1 antibody or antigen-binding fragment thereof, for example, an anti-GREM1 antibody or antigen-binding fragment thereof provided herein (such as 14E3 or a humanized variant thereof such as hu14E3 HaLa) . The detailed embodiments of detection methods are described in the below sections.
In another aspect, the present disclosure provides a method of improving responsiveness of a subject to treatment with PD-1/PD-L1 axis inhibitor, wherein the subject has been determined to have presence of GREM1 in a biological sample of a diseased tissue of the subject, or the subject has been determined to have expression level of GREM1 in the biological sample of the diseased tissue reaching a threshold level, the method comprising: a) administering to the subject a therapeutically effective amount of a GREM1 antagonist such that expression of PD-L1 is increased in a diseased tissue, thereby improving responsiveness of the subject to PD-1/PD-L1 axis inhibitor.
In certain embodiments, the subject has been a) determined to be resistant or refractory to PD-1/PD-L1 axis inhibitor and/or b) determined to have low or no PD-L1 expression in a diseased tissue. In certain embodiments, the subject has been determined to have medium or high PD-L1 expression in a diseased tissue. PD-L1 expression may be determined using the methods described in PCT/CN2022/131820, which is herein incorporated by reference. In certain embodiments, PD-L1 expression can be determined in accordance to the methods described in Example 4 provided herein. The reagents used to detect the PD-L1 expression throughout the specification can be an anti-PD-L1 diagnostic antibody, for example, 22C3, as described in US20170285037A1, disclosure of which has been incorporated by reference in its entirety, and a monoclonal rabbit anti-PD-L1, Clone 28-8, which is commercially available. In certain embodiments, the reagents used to detect the PD-L1 expression throughout the specification is a polypeptide comprising the antigen-binding portion of the 22C3, as described in US20170285037A1, disclosure of which has been incorporated by reference in its entirety, and a monoclonal rabbit anti-PD-L1, Clone 28-8, which is commercially available.
In certain embodiments, the diseased tissue is a cancer tissue. In certain embodiments, the cancer is colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of PD-1/PD-L1 axis inhibitor after the expression of PD-L1 is increased in the diseased tissue of the subject.
In another aspect, the present disclosure provides use of a GREM1 antagonist in the manufacture of a medicament for improving responsiveness of a subject to treatment with PD-1/PD-L1 axis inhibitor, wherein the subject has been determined to have presence of GREM1 in a biological sample of a diseased tissue of the subject, or the subject has been determined to have expression level of GREM1 in the biological sample of the diseased tissue reaching a threshold level, wherein the improvement comprises administering to the subject a therapeutically effective amount of a GREM1 antagonist such that expression of PD-L1 is increased in a diseased tissue, thereby improving responsiveness of the subject to PD-1/PD-L1 axis inhibitor.
In another aspect, the present disclosure provides use of an anti-GREM1 diagnostic reagent in the manufacture of a kit for determining the eligibility for or likelihood of responsiveness to treatment with a GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor in a subject, wherein the anti-GREM1 diagnostic reagent is capable of determining presence or expression level of GREM1 in a biological sample of the diseased tissue of the subject, wherein the presence or absence of or expression level of GREM1 is indicative of whether the subject is likely to be eligible for or responsive to treatment of the GREM1 antagonist in combination with a PD-1/PD-L1 axis inhibitor.
1. GREM1 Expression Determination
The methods or uses provided herein involves determination of expression of GREM1. In certain embodiments, the subject is determined to have GREM1 expression (e.g., presence of GREM1 or the expression level of GREM1 above a threshold level) in a diseased tissue. As used herein, the “threshold level” refers to expression level of GREM1 at an intensity of 1+, 2+ or 3+ as measured by IHC.
Suitable methods known in the art can be used and are described in detail below.
i. Sample preparation
In certain embodiments, the subject is human. In certain embodiments, the method provided herein further comprises providing a biological sample from the subject, wherein the biological sample comprises the diseased tissue (e.g., cancerous tissue or tumor tissue) .
Any biological sample suitable for conducting the methods provided herein can be obtained from the subject. As used herein, “biological sample” refers to a biological specimen taken by sampling from a subject, optionally with additional processing. The collection of a sample from a subject is performed in accordance with the standard protocol generally followed by hospital or clinics, such as during a biopsy.
In certain embodiments, the sample can be a biological sample comprising cancer cells, or non-cancer cells (e.g., stroma fibroblasts) . For example, non-cancer cells can be from the same tissue or organ as the cancer cells are also found. In certain embodiments, the biological sample containing or suspected of containing a cancer cell can be obtained from the subject. In some embodiments, the biological sample can be derived from a cancer cell or cancer tissue, or tumor infiltrating immune cells. In certain embodiment, a biological sample is a tumor tissue.
In some embodiments, the biological sample is a fresh or archived sample obtained from a tumor tissue, e.g., by a tumor biopsy or fine needle aspirate. In some embodiments, the sample can be any biological fluid containing cancer cells or non-cancer cells (e.g. peripheral blood mononuclear cells (PBMC) ) .
Examples of a biological sample include without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, pleural fluid, ascetic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, colon tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, colon tissue, renal tissue, endometrial tissue, cervical tissue, lymph node tissue, muscle tissue, or skin tissue) , a paraffin embedded tissue. In a further embodiment, a biological sample comprises cells, tissue, blood, plasma, serum, urine, mouthwash, stool, saliva, and any combination thereof.
In certain embodiments, the sample can be further processed by a desirable method for determining expression level of the at least one biomarker, such as GREM1.
ii. Determination of GREM1 expression
In certain embodiments, GREM1 expression is determined from the diseased tissue (e.g. from the biological sample) .
The presence and/or expression level of GREM1 in the diseased tissue (e.g., cancerous tissue or tumor tissue) , can be determined by various methods known in the art. In certain embodiments, the biological sample may be further processed to, for example, isolate the analyte such as the nucleic acids or proteins. Presence and/or expression level of GREM1 can be determined by, for example, quantitative fluorescence cytometry, immunohistochemistry (IHC) , or nucleic acid-based methods. For example, the biological sample from the subject can be exposed to anti-GREM1 diagnostic reagent, which binds to and detects the expressed GREM1 protein.
In certain embodiments, the expression of GREM1 in the diseased tissue (e.g., cancerous tissue or tumor tissue) , is determined or measured by IHC. In certain embodiments, the expression level of human GREM1 protein on a cancerous tissue or tumor tissue from the subject can be determined in accordance to the methods described in Example 4 provided herein.
In certain embodiments, the subject is or has been determined to have high GREM1 expression in the diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from the subject. In certain embodiments, the diseased tissue is or has been determined to have GREM1 expression higher than or comparable to expression in healthy or noncancerous cells. The high GREM1 expression in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) refers to expression of GREM1 at an intensity of at least 2+ (e.g., 2+ or 3+ as measured by IHC.
In certain embodiments, the subject is or has been determined to have medium GREM1 expression in the diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from the subject. The medium GREM1 expression in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) refers to expression of GREM1 at an intensity of at least 1+ and below 2+ as measured by IHC.
In certain embodiments, the subject is or has been determined to have low GREM1 expression in the diseased tissue (e.g., cancerous tissue or tumor tissue) , derived from the subject. In certain embodiments, the diseased tissue is or has been determined to have GREM1 expression comparable to expression in a healthy tissue and is detectable by an anti-GREM1 diagnostic reagent. The low GREM1 expression in a biological sample, such as a diseased tissue (e.g., cancerous tissue or tumor tissue) refers to expression of GREM1 at an intensity of above 0 but below 1+ as measured by IHC.
IV. The GREM1 Antagonist
The GREM1 antagonist used in the methods provided herein is capable of inducing the expression of PD-L1 in the diseased tissue of the subject. In certain embodiments, the GREM1 antagonist used in the methods provided herein comprises an anti-GREM1 antibody or antigen-binding fragment thereof, for example, any of those described in PCT patent application PCT/CN2022/072297, which is herein incorporated by reference. In certain embodiments, the GREM1 antagonist used in the methods provided herein is an anti-GREM1 antibody that has comparable properties as those of Hu14E3 HaLa described in PCT patent application PCT/CN2022/072297, which is herein incorporated by reference. The term “Hu14E3 HaLa” , as used herein, refers to a humanized anti-GREM1 antibody comprising a heavy chain variable region (Hu14E3-Ha VH) and a light chain variable region (Hu14E3-Ha VL) , wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
In certain embodiments, the GREM1 antagonist used in the methods provided herein comprises the antigen-binding portion of Hu14E3 HaLa. In certain embodiments, the GREM1 antagonist used in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are respectively the same as those of Hu14E3 HaLa. In certain embodiments, the GREM1 antagonist used in the methods provided herein comprises heavy chain HCDR1, HCDR2 and HCDR3 and/or light chain LCDR1, LCDR2 and LCDR3, wherein the heavy chain HCDR1, HCDR2 and HCDR3 and/or light chain LCDR1, LCDR2 and LCDR3 are respectively the same as those of Hu14E3 HaLa.
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3 and/or light chain LCDR1, LCDR2 and LCDR3, wherein: the HCDR1 comprises the amino acid sequence comprising TYGMA (SEQ ID NO: 1) , or a homologue sequence of at least 80%sequence identity thereof; the HCDR2 comprises the amino acid sequence comprising WINTLSGEPTYADDFKG (SEQ ID NO: 2) , or a homologue sequence of at least 80%sequence identity thereof; the HCDR3 comprises the amino acid sequence comprising EPMDY (SEQ ID NO: 3) , or a homologue sequence of at least 80%sequence identity thereof; the LCDR1 comprises the amino acid sequence comprising KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologue sequence of at least 80%sequence identity thereof; the LCDR2 comprises the amino acid sequence comprising LVSKLDS (SEQ ID NO: 5) or a homologue sequence of at least 80%sequence identity thereof; and the LCDR3 comprises the amino acid sequence comprising s WQGAHFPLT (SEQ ID NO: 6) or a homologue sequence of at least 80%sequence identity thereof.
CDRs are known to be responsible for antigen binding, however, it has been found that not all of the 6 CDRs are necessarily indispensable or unchangeable. In other words, it is possible to replace or change or modify 1, 2, or 3 CDRs in the anti-GREM1 antibody, yet substantially retain the specific binding affinity to GREM1.
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises a heavy chain CDR3 sequence of EPMDY (SEQ ID NO: 3) . Heavy chain CDR3 regions are located at the center of the antigen-binding site, and therefore are believed to make the most contact with antigen and provide the most free energy to the affinity of antibody to antigen. It is also believed that the heavy chain CDR3 is by far the most diverse CDR of the antigen-binding site in terms of length, amino acid composition and conformation by multiple diversification mechanisms (Tonegawa S. Nature. 302: 575-81) . The diversity in the heavy chain CDR3 is sufficient to produce most antibody specificities (Xu JL, Davis MM. Immunity. 13: 37-45) as well as desirable antigen-binding affinity (Schier R, etc. J Mol Biol. 263: 551-67) .
In some embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain. In one embodiment, the anti-GREM1 antibody is a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516) .
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof further comprises one or more amino acid residue substitutions or modifications yet retains specific binding specificity or affinity to GREM1 (e.g., human GREM1, i.e., hGREM1) .
In certain embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences, and/or in one or more of the non-CDR regions of the VH or VL sequences.
In certain embodiments, the anti-GREM1 antibody or antigen-binding fragment thereof further comprises an immunoglobulin constant region, optionally a constant region of a human IgG. In certain embodiments, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4, and optionally the constant region comprises a heavy chain constant region comprising a sequence of SEQ ID NO: 9 and/or a light chain constant region comprising a sequence of SEQ ID NO: 10.
In certain embodiments, the GREM1 antagonist is linked to one or more conjugate moieties. In certain embodiments, the conjugate moiety comprises a clearance-modifying agent, therapeutic agent (e.g., a chemotherapeutic agent) , a toxin, a radioactive isotope, a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, or an enzyme-substrate label) , a pharmacokinetic modifying moiety, a DNA-alkylator, a topoisomerase inhibitor, a tubulin-binders, other anticancer drugs such as androgen receptor inhibitor, as those described in PCT patent application PCT/CN2022/072297, which is herein incorporated by reference.
V. The GREM1 Diagnostic Reagent/Agent
The GREM1 diagnostic reagent/agent used in the methods provided herein is capable of specifically detecting low, medium, or high expression of GREM1 in vivo (e.g., inside the body of the subject) or in vitro (e.g., in a biological sample of a diseased tissue of the subject) . In certain embodiments, the subject is further determined to have 5-50% (e.g., 10-40%, 10-30%, 10-20%or 15%) of the tumor cells positive in GREM1 as measured by IHC. In certain embodiments, the GREM1 diagnostic reagent/agent used in the methods provided herein is a polypeptide comprising the antigen-binding portion of any of the anti-GREM1 antibodies or antigen-binding fragments thereof described in the PCT patent application PCT/CN2022/072297, which is herein incorporated by reference. In certain embodiments, the GREM1 diagnostic reagent/agent used in the methods provided herein comprises any of the anti-GREM1 antibodies or antigen-binding fragments thereof described in the PCT patent application PCT/CN2022/072297, which is herein incorporated by reference.
In certain embodiments, the GREM1 diagnostic reagent/agent is a polypeptide comprising the antigen-binding fragment of Hu14E3 HaLa, as described in PCT/CN2022/072297, which is herein incorporated by reference. In certain embodiments, the antigen-binding fragment comprises heavy chain HCDR1, HCDR2 and HCDR3 and/or light chain LCDR1, LCDR2 and LCDR3, wherein: the HCDR1 comprises the amino acid sequence comprising TYGMA (SEQ ID NO: 1) , or a homologue sequence of at least 80%sequence identity thereof; the HCDR2 comprises the amino acid sequence comprising WINTLSGEPTYADDFKG (SEQ ID NO: 2) , or a homologue sequence of at least 80%sequence identity thereof; the HCDR3 comprises the amino acid sequence comprising EPMDY (SEQ ID NO: 3) , or a homologue sequence of at least 80%sequence identity thereof; the LCDR1 comprises the amino acid sequence comprising KSSQSLLDSDGKTYLS (SEQ ID NO: 4) or a homologue sequence of at least 80%sequence identity thereof; the LCDR2 comprises the amino acid sequence comprising LVSKLDS (SEQ ID NO: 5) or a homologue sequence of at least 80%sequence identity thereof; and the LCDR3 comprises the amino acid sequence comprising s WQGAHFPLT (SEQ ID NO: 6) or a homologue sequence of at least 80%sequence identity thereof. In certain embodiments, the antigen-binding fragment comprises a heavy chain CDR3 sequence of EPMDY (SEQ ID NO: 3) .
In certain embodiments, the antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence of SEQ ID NO: 7, and the light chain variable region comprises an amino acid sequence of SEQ ID NO: 8.
In certain embodiments, the GREM1 diagnostic reagent/agent further comprises an immunoglobulin constant region, optionally a constant region of a human IgG. In certain embodiments, the constant region comprises a constant region of human IgG1, IgG2, IgG3, or IgG4, and optionally the constant region comprises a heavy chain constant region comprising a sequence of SEQ ID NO: 9 and/or a light chain constant region comprising a sequence of SEQ ID NO: 10.
In some embodiments, the GREM1 diagnostic reagent/agent comprises all or a portion of the heavy chain variable domain and/or all or a portion of the light chain variable domain. In one embodiment, the GREM1 diagnostic reagent/agent is a single domain antibody which consists of all or a portion of the heavy chain variable domain provided herein. More information of such a single domain antibody is available in the art (see, e.g., U.S. Pat. No. 6,248,516) .
In certain embodiments, the GREM1 diagnostic reagent/agent comprises Hu14E3 HaLa or antigen-binding fragment thereof.
In certain embodiments, the GREM1 diagnostic reagent/agent further comprises one or more conjugate moieties linked to the polypeptide or the Hu14E3 HaLa or antigen-binding fragment thereof described above. In certain embodiments, the conjugate moiety comprises a detectable label (e.g., a lanthanide, a luminescent label, a fluorescent label, biotin/avidin, or an enzyme-substrate label) .
VI. Kit
In another aspect, the present disclosure provides kits useful in treating a GREM1-expressing cancer in a subject in need thereof, comprising a GREM1 antagonist and a package insert comprising instructions for using the GREM1 antagonist in combination with: a) an anti-angiogenesis therapy, b) a chemotherapy, c) an immunotherapy, d) an anti-angiogenesis therapy and a chemotherapy, e) a chemotherapy and an immunotherapy; f) an anti-angiogenesis therapy and an immunotherapy; or g) an anti-angiogenesis therapy, a chemotherapy and an immunotherapy.
In certain embodiments, the GREM1-expressing cancer is characterized in: a) being resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor, and/or b) having low or no expression of PD-L1 in the diseased tissue, and/or c) having GREM1 expression in a diseased tissue. In certain embodiments, the subject is determined to have medium or high PD-L1 expression in a diseased tissue.
As used herein, the term “package insert” refers to instructions included in a commercial package of medicines that contain information about, for example, indications, dosage, usage, administration, contraindications, other medicines to be combined with the packaged product, and/or warnings concerning the use of such medicines. In certain embodiments, the instructions comprise selecting a subpopulation a) being resistant or refractory to the treatment with a PD-1/PD-L1 axis inhibitor, and/or b) having low or no expression of PD-L1 in the diseased tissue, and/or c) having GREM1 expression in a diseased tissue and/or d) having been administered with the GREM1 antagonist for a period of time sufficient to increase expression level of PD-L1 in cancer tissue/cancer cells of the subject. In certain embodiments, the instructions comprise administering a therapeutically effective amount of the GREM1 antagonist for a period of time sufficient to increase expression level of PD-L1 in cancer tissues/cells of the subject, and then administering the PD-1/PD-L1 axis inhibitor to the subject
The kit may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
VII. Cancer
In certain embodiments, the cancer of the methods provided herein is GREM1-expressing cancer. In certain embodiments, the cancer is selected from the group consisting of:solid tumors or hematological tumors. In certain embodiments, the solid tumor is adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, colorectal cancer, emphysema, endometrial cancer, esophageal cancer, Ewing's sarcoma, retinoblastoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas) , Kaposi sarcoma, kidney cancer (renal cell cancer) , laryngeal cancer, liver cancer, lung cancer, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer) , retinoblastoma, Ewing family of tumors, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, or vaginal cancer.
In certain embodiments, the cancer is selected from the group consisting of: colorectal cancer, gastric cancer, lung cancer, endometrical cancer, esophageal cancer, bladder cancer, prostate cancer, breast cancer, or pancreatic cancer.
In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is a subtype of colorectal cancer, such as CMS1, CMS2, CMS3, CMS4. Detailed description of the subtypes of colorectal cancer can be found in, for example, PCT patent application PCT/US2022/076717, which is herein incorporated by reference.
EXAMPLES
While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments) , it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.
Example 1: Efficacy of Anti-Gremlin1 Antibody in Combination with DC101 on BZ-CRC-0001 PDX Tumor Model on NOG Mice
This example shows that the combination use of anti-Gremlin 1 antibody (i.e., anti-GREM1 antibody) and anti-angiogenesis therapy achieved synergistic therapeutic effect in treating colorectal cancer (CRC) . The anti-GREM1 antibody used in this example is Hu14E3 HaLa described herein. The safety profile of Hu14E3 HaLa is shown in Figure 7. The anti-angiogenesis therapy comprises an anti-VEGFR-2 antibody DC101, which is a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g. under catalog #BE0060 from BioXell) .
BZ-CRC-0001 colorectal cancer PDX was obtained from Beijing Cancer Hospital passage in NODSCID mice and established PDX bank. The pathology information of BZ-CRC-0001 colorectal cancer is shown in Figure 1B. Each NOG mouse was subcutaneously inoculated with a small tumor tissue block approximately 3 mm in diameter which sheared from a tumor decollement from a tumor bearing moue. 19 days after inoculation animals with tumor size at about 50mm^3 were selected and randomly divided into 4 groups each group consisting of 8 mice. Animals were intravenously inoculated with 5*10^6 human PBMC in 0.1mL. Animal were selected and dosed a week later after human PBMC infusion. Animals from groups 1 to groups 4 were administered with 10 mg/kg hIgG1 control, 10 mg/kg anti-Gremlin1 antibody, 5 mg/kg DC101 and combination of 10 mg/kg anti-Gremlin1 antibody and 5 mg/kg DC101.
hIgG1 control, Hu14E3 HaLa and DC101 were administrated by intraperitoneal injection twice weekly for 4 weeks. Animals were sacrificed at the end of the study with CO2 inhalation. Tumor size was measured twice or triple times a week in two dimensions using a caliper (INSIZE) and the volume was expressed in mm3 using the formula: V=0.5 a*b^2 where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean=S.E.M. Comparisons between two groups were made by T-test. and the difference is considered significant if p is *<0.05 and **<0.01. As shown in the Figure 1A, tumor growth inhibition rates of anti-Gremlin1 antibody single, DC101, combination of anti-Gremlin1 antibody and DC101 were 46.80%, 54.12%and 66.53%, respectively, on the sacrifice day. Anti-Gremlin1 antibody combination with DC101 had a better anti-tumor activity than anti-Gremlin1 antibody and improved antitumor activity than DC101 single.
Example 2: Efficacy of Anti-gremlin1 antibody in Combination with FOLFIRI and DC101 on BZ-CRC-0001 PDX Tumor Model on NOG Mice
This example shows that the combination use of anti-Gremlin 1 antibody (i.e., anti-GREM1 antibody) , chemotherapy and anti-angiogenesis therapy achieved synergistic therapeutic effect in treating CRC. The anti-GREM1 antibody used in this example is Hu14E3 HaLa described herein. The chemotherapy is FOLFIRI. The anti-angiogenesis therapy comprises an anti-VEGFR-2 antibody DC101, which is a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g. under catalog #BE0060 from BioXell) .
BZ-CRC-0001 colorectal cancer PDX was obtained from Beijing Cancer Hospital passage in NODSCID mice and established PDX bank. Each NOG mouse was subcutaneously inoculated with a small tumor tissue block approximately 3 mm in diameter which sheared from a tumor decollement from a tumor bearing moue. 18 days after inoculation animals with tumor size at about 50mm^3 were selected and randomly divided into 5 groups each group consisting of 8 mice. Tumor bearing mice were intravenously inoculated with 5*10^6 human PBMC in 0.1mL. Animals were selected and dosed a week later after human PBMC infusion. Animals from groups 1 to groups 5 were administered with 35mg/kg isotype control and vehicle, 30 mg/kg anti-gremlin1 antibody, combination of 30 mg/kg anti-gremlin1 antibody and 5 mg/kg DC101 , combination of FOLFIRI (5-fluororacil (5-FU) : 7.5 mg/kg, Leucovorin (LV) : 22.5 mg/kg, Irinotecan (CPT-11) : 4 mg/kg) and 5 mg/kg DC101, combination of 30 mg/kg anti-gremlin1 antibody, FOLFIRI (5-FU: 7.5 mg/kg, LV: 22.5 mg/kg, CPT-11: 4 mg/kg) and 5 mg/kg DC101.
Isotype control, anti-gremlin1 antibody and DC101 were administrated by intraperitoneal injection twice weekly for 4 weeks. LV was administrated by intraperitoneal injection once weekly for 4 weeks; 5-Fu and CPT-11 were administrated by intravenous injection once weekly for 4 weeks. Animals were sacrificed at the end of the study with CO2 inhalation. Tumor size was measured twice or triple times a week in two dimensions using a caliper (INSIZE) and the volume was expressed in mm3 using the formula: V=0.5 a*b^2 where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean=S.E.M. Comparisons between two groups were made by T-test. and the difference is considered significant if p is *<0.05 and **<0.01. As shown in the Figure 2, tumor growth inhibition rates of anti-gremlin1 antibody at 30mg/kg, combination of anti-gremlin1 antibody and DC101, combination of FOLFIRI and DC101, combination of anti-gremlin1 antibody, FOLFIRI and DC101 were 30.10%, 47.47%, 58.75%and 68.91%, respectively, on the sacrifice day. Anti-gremlin1 antibody in combination with DC101 had a better anti-tumor activity than anti-gremlin1 antibody alone, and anti-gremlin1 antibody in combination with FOLFIRI and DC101 had a better anti-tumor activity than anti-gremlin1 antibody alone and improve anti-tumor activity than FOLFIRI combination with DC101.
Example 3: Efficacy of Anti-gremlin1 antibody in Combination with FOLFIRI and DC101 and Nivolumab on BZ-CRC-0001 PDX Tumor Model on NOG Mice
This example shows that the combination use of anti-Gremlin 1 antibody (i.e., anti-GREM1 antibody) , chemotherapy, anti-angiogenesis therapy and immunotherapy achieved synergistic therapeutic effect in treating CRC. The anti-GREM1 antibody used in this example is Hu14E3 HaLa described herein. The chemotherapy is FOLFIRI. The anti-angiogenesis therapy comprises an anti-VEGFR-2 antibody DC101, which is a monoclonal antibody that reacts with mouse VEGFR-2 and is commercially available (e.g. under catalog #BE0060 from BioXell) . The immunotherapy comprises an anti-PD-1 antibody Nivolumab.
BZ-CRC-0001 colorectal cancer PDX was obtained from Beijing Cancer Hospital passage in NODSCID mice and established PDX bank. Each NOG mouse was subcutaneously inoculated with a small tumor tissue block approximately 3 mm in diameter which sheared from a tumor decollement from a tumor bearing moue. 18 days after inoculation animals with tumor size at about 50mm^3 were selected and randomly divided into 4 groups each group consisting of 8 mice. Animals were intravenously inoculated with 5*10^6 human PBMC in 0.1mL. Animal were selected and dosed a week later after human PBMC infusion. Animals from groups 1 to groups 4 were administered with 43 mg/kg hIgG1 control and vehicle, 30 mg/kg anti-gremlin1 antibody, 10 mg/kg Nivolumab, combination of FOLFIRI (5-FU: 5 mg/kg, LV: 20 mg/kg, CPT-11: 3 mg/kg) , 3 mg/kg DC101, 10 mg/kg Nivolumab (acommercially available anti-PD-1 antibody) and 30 mg/kg anti-gremlin1 antibody.
hIgG1 control, anti-gremlin1 antibody, Nivolumab and DC101 were administrated by intraperitoneal injection twice weekly for 4 weeks; LV was administrated by intraperitoneal injection twice on the 1st and 3rd week; 5-Fu and CPT11 were administrated by intravenous injection twice on the 1st and 3rd week. Animals were sacrificed at the end of the study with CO2 inhalation. Tumor size was measured twice or triple times a week in two dimensions using a caliper (INSIZE) and the volume was expressed in mm3 using the formula: V=0.5 a*b^2 where a and b are the long and short diameters of the tumor, respectively. Results were analyzed using Prism GraphPad and expressed as mean=S.E.M. Comparisons between two groups were made by T-test. and the difference is considered significant if p is *<0.05 and **<0.01. As shown in the Figure 3, tumor growth inhibition rates of anti-gremlin1 antibody single, Nivolumab, combination of FOLFIRI, DC101, Nivolumab and anti-gremlin1 antibody were 56.03%, 26.86%, and 80.72%, respectively, on the sacrifice day, the combination group had better antitumor effect than anti-gremlin1 antibody single and Nivolumab single.
Example 4: Evaluation of Tumor infiltrating lymphocytes (TILs) , Gremlin and PD-L1 Expression in Sections BZ-CRC-0001 PDX Tumor Model using IHC Assay
This example showed that anti-GREM1 antibody Hu14E3 HaLa could be used as a diagnostic reagent to detect GREM1 expression on a disease tissue. The example also showed that the CRC tumor tissue has medium to high GREM1 expression and low or no PD-L1 expression. The example further showed that treatment of anti-GREM1 antibody Hu14E3 HaLa could upregulate the expression level of PD-L1 in a dose-dependent manner.
1. Gremlin1 Expression Status in BZ-CRC-0001 PDX Tumor Model
To investigate the expression level and staining pattern of Gremlin 1 in BZ-CRC-01 PDX tumor samples, biotinylated 14E3 (Hu14E3 HaLa Biotin) was prepared and used for detection. Briefly, EZ-LinkTM Sulfo-NHS-LC-Biotin (ThermoFisher, A39257) was dissolved in ultrapure water to prepare a 10mM solution of the biotin reagent. 27μL biotin solution was added for each 2mg 14E3 antibody to be labelled and mix gently for 0.5hr at room temperature. Reaction products of low molecular weight were removed by desalting the product on ZebaTM Spin Desalting Columns (ThermoFisher, 89890) according to the manufacturer’s instruction.
Immunohistochemistry (IHC) was performed on slides of 4%neutral buffered formalin fixed paraffin embedded PDX samples. After deparaffinization and rehydration, all slides were proceeded to antigen retrieval by boiling in EnVisionTM FLEX Target Retrieval Solution (Dako, K8002) for 25minutes at 97-99 ℃, subsequently quenched, blocked with IHC Biotin Block Kit (MaiXin, BLK-0001) following instruction and incubated with 20 ug/mL in-house biotinylated monoclonal mouse anti-Gremlin1 (Hu14E3 HaLa Biotin) for 30 min at 37℃. Antibody binding was visualized with horseradish peroxidase labeled streptavidin (MaiXin, SP KIT-D1) and EnVisionTM FLEX Substrate Working Solution (Dako, K8002) . Sections were finally counterstained with Hematoxylin and mounted with permanent mounting medium.
All viable stroma fibroblasts/tumor cells on the entire slide were evaluated and included in the scoring method under light field by microscope. In general, at least 100 viable stromal fibroblasts and/or tumor cells were suggested for a percent score. Positivity for gremlin1 expression was defined as viable stromal fibroblast/tumor cells showing partial or complete cytoplasmic staining in stromal fibroblasts/tumor cells. The percentage of stromal fibroblasts/tumor cells at four different staining intensities were estimated: 0 (no staining) , 1+ (weak) , 2+ (moderate) , and 3+ (strong) . The sum of all 4 percentages should equal 100%. The total percent positivity was determined for each specimen, which was defined as the percentage of viable tumor cells showing ≥1+ staining intensity. The gremlin1 positivity was observed in BZ-CRC-01 PDX model with moderate to strong intensity showing punctate cytoplasmic staining (see Figure 4) .
2. PD-L1 Expression Regulation post Anti-gremlin1 antibody (Hu14E3 HaLa) Treatment in BZ-CRC-0001 PDX Tumor Model
PD-L1 expression status was also assessed to better understand the immune modulation pre-and post anti-gremlin1 antibody treatment (refer to example 1 and 2) in BZ-CRC-01 PDX tumor samples. Immunohistochemistry (IHC) was performed on these 4%neutral buffered formalin fixed paraffin-embedded (FFPE) tumor sections using commercially available Rabbit anti-human PD-L1 (SP263) monoclonal antibodies. After deparaffinization and rehydration, all slides were proceeded to antigen retrieval in BOND Epitope Retrieval Solution 2 (Leica, AR9640) for 30minutes at 97-99 ℃. Subsequently quenched, blocked with peroxidase inhibitor and incubated with appropriately diluted SP263 (0.2 ug/mL) antibodies for 30minutes at room temperature (RT) . Antibody binding was visualized with BOND Polymer Detection (Leica, DS9800) for 8minutes on auto-staining Leica BOND III. Sections were finally counterstained with Hematoxylin and mounted with permanent mounting medium.
All samples were scored by combined positive score (CPS) of total stained immune and tumor cell relative to all visible tumor cell for PD-L1 with membrane staining of different intensity (neg (0) , weak (1+) , moderate (2+) , strong (3+) ) as depicted in Table 1. Interestingly, the staining results displayed the treatment of anti-gremlin1 antibody could upregulate the expression level of PD-L1 as compared with that of isotype control and the staining proportion was augmented as the dosage increase from 10 mg/kg to 30 mg/kg in the BZ-CRC-01 PDX model as depicted in Figure 5 and Table 2.
Table 1. PD-L1 IHC result interpretation
Table 2. PD-L1 IHC result scoring on BZ-CRC-01 PDX tumor sections across Hu14E3 HaLa dose levels
3. Penetration of CD3+ and CD8+ TILs post Anti-gremlin1 antibody (Hu14E3 HaLa) Treatment in BZ-CRC-0001 PDX Tumor Model
On BZ-CRC-01 PDX tumor sections, immune cell was stained with CD3 and CD8 biomarkers to see infiltration of T lymphocytes pre-and post anti-gremlin1 antibody treatment (refer to 1 and 2) in tumor microenvironment. Immunohistochemistry (IHC) was performed on these 4%neutral buffered formalin fixed paraffin-embedded (FFPE) tumor sections using commercially available CD8α (D8A8Y) Rabbit mAb (CST, 85336S) and CD3ε (D7A6ETx) Rabbit mAb (CST, 85061S) , respectively. After deparaffinization and rehydration, all slides were proceeded to antigen retrieval in BOND Epitope Retrieval Solution 2 (Leica, AR9640) for 30minutes at 97-99 ℃. Subsequently quenched, blocked with peroxidase inhibitor and incubated with appropriately diluted CD8α (1: 200) and CD3ε (1: 200) antibodies for 30minutes at room temperature (RT) , respectively. Antibody binding was visualized with BOND Polymer Detection (Leica, DS9800) for 8minutes on auto-staining Leica BOND III. Sections were finally counterstained with Hematoxylin and mounted with permanent mounting medium.
All samples were analyzed by totally stained immune cell at any intensity. Remarkably, the results indicated the treatment of anti-gremlin1 antibody could facilitate the infiltration of CD3 and CD8 positive T cells relative to that of isotype control as depicted in Figure 6.
Various embodiments have been described herein with reference to the accompanying drawings. It will, however, be evident that various modifications and changes may be made thereto, and additional embodiments may be implemented, without departing from the broader scope of the invention as set forth in the claims that follow. Further, other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of one or more embodiments of the invention disclosed herein. It is intended, therefore, that this application and the examples herein be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following listing of exemplary claims.
Table 3. Sequences mentioned or used in the present application