CN110551216B - Multivalent anti-OX40 antibody and use thereof - Google Patents
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Abstract
Translated fromChinese
Description
Translated fromChinese本发明涉及一种具有至少四个抗原结合位点的多价抗体，特别涉及新的特异性结合OX40的具有至少四个抗原结合位点的多价抗体。本发明还涉及含有所述多价抗体的组合物、编码所述抗体的核酸及包含所述核酸的宿主细胞。此外，本发明还涉及这些新型多价抗体、包含所述多价抗体的组合物、编码核酸以及包含所述核酸的宿主细胞的治疗和诊断用途。The present invention relates to a multivalent antibody with at least four antigen-combining sites, in particular to a novel multivalent antibody with at least four antigen-combining sites that specifically binds to OX40. The invention also relates to compositions comprising said multivalent antibodies, nucleic acids encoding said antibodies, and host cells comprising said nucleic acids. Furthermore, the present invention also relates to the therapeutic and diagnostic uses of these novel multivalent antibodies, compositions comprising said multivalent antibodies, encoding nucleic acids, and host cells comprising said nucleic acids.
背景技术Background technique
多价抗体multivalent antibody
大多数的天然抗体是二价抗体，其具有四个可变结构域，即两个VH结构域和两个VL结构域。天然存在的单克隆抗体可以作为多聚体结构如IgM存在，其增加对固定表面上的靶抗原的亲合性。因此，多价抗体在增加抗体抗原亲和力、增强抗体生物学功能方面具有一定的作用。Most natural antibodies are bivalent antibodies with four variable domains, two VH domains and two VL domains. Naturally occurring monoclonal antibodies may exist as multimeric structures such as IgM, which increase affinity for target antigens on immobilized surfaces. Therefore, multivalent antibodies play a certain role in increasing the affinity of antibody antigens and enhancing the biological functions of antibodies.
最近几年，已经开发了多种多样的重组多特异性抗体形式，例如通过融合IgG抗体形式和单链结构域的四价双特异性抗体(参见例如Coloma，M.J.等人，Nature Biotech.15(1997)159-163；WO 2001/077342；和Morrison，S.L.，Nature Biotech.25(2007)1233-1234。可以通过化学方式偶联两种不同的mAb来制造双特异性抗体(参见Staerz，U.D.等，Nature，1985.314(6012)：第628-31页)。其它方法使用了以化学方式偶联两种不同的单克隆抗体或较小的抗体片段(参见Brennan，M.等，Science，1985.229(4708)：第81-3页)。In recent years, a variety of recombinant multispecific antibody formats have been developed, for example tetravalent bispecific antibodies by fusing IgG antibody formats and single chain domains (see e.g. Coloma, M.J. et al., Nature Biotech. 15( 1997) 159-163; WO 2001/077342; and Morrison, S.L., Nature Biotech.25 (2007) 1233-1234. Bispecific antibodies can be produced by chemically coupling two different mAbs (see Staerz, U.D. et al. , Nature, 1985.314(6012): pages 628-31). Other methods have used chemically coupling two different monoclonal antibodies or smaller antibody fragments (see Brennan, M. et al., Science, 1985.229(4708 ): pp. 81-3).
在近期的研究中，报导了通过在转基因兔和牛的体内表达针对CD28和黑素瘤相关蛋白聚糖的串联scFv(参见Gracie，J.A.等，J Clin Invest，1999.104(10)：第1393-401页)。在这一构建体中，通过CH1连接子连接两个scFv分子，并且发现双特异性抗体的血清浓度高达100mg/L。现有少数研究报导了在细菌中使用极短的Ala3连接子或富含甘氨酸/丝氨酸的较长连接子的可溶性串联scFv分子的表达(参见Leung，B.P.等，J Immunol，2000.164(12)：第6495-502页；Ito，A.等，J Immunol，2003.170(9)：第4802-9页；Karni，A.等，JNeuroimmunol，2002.125(1-2)：第134-40页)。In a recent study, it was reported that tandem scFv targeting CD28 and melanoma-associated proteoglycans were expressed in transgenic rabbits and cattle (see Gracie, J.A. et al., J Clin Invest, 1999.104(10): pp. 1393-401 ). In this construct, two scFv molecules were linked via a CH1 linker, and serum concentrations of the bispecific antibody were found to be as high as 100 mg/L. The few existing studies report the expression of soluble tandem scFv molecules in bacteria using the very short Ala3 linker or the longer glycine/serine-rich linker (see Leung, B.P. et al., J Immunol, 2000.164(12): p. pp. 6495-502; Ito, A. et al., J Immunol, 2003.170(9): pp. 4802-9; Karni, A. et al., J Neuroimmunol, 2002.125(1-2): pp. 134-40).
双价抗体与Fc融合产生更类似Ig的分子，称为双双价抗体(参见Lu，D.等，J BiolChem，2004.279(4)：第2856-65页)。此外，也已描述了在IgG的重链中包含两个Fab重复并且能够结合四个抗原分子的多价抗体构建体(参见美国专利8,722,859B2，及Miller，K.等，JImmunol，2003.170(9)：第4854-61页)。Fusion of diabodies to Fc produces more Ig-like molecules called bidiabodies (see Lu, D. et al., J BiolChem, 2004. 279(4): pp. 2856-65). In addition, multivalent antibody constructs comprising two Fab repeats in the heavy chain of IgG and capable of binding four antigen molecules have also been described (see US Pat. : pp. 4854-61).
其他实例是四价IgG单链可变片段(scFv)融合物(Dong J等，2011MAbs 3：273-288；Coloma MJ，Morrison SL 1997Nat Biotechnol 15：159-163；Lu D等，2002J ImmunolMethods 267：213-226)与催化性抗体共价连接的药效团肽(DoppalapudiVR等，2010ProcNatl Acad Sci USA 107：22611-22616)、在一半IgG4分子之间使用动态交换产生双特异性抗体(van der Neut Kolfschoten M等，2007Science 317：1554-1557；Stubenrauch K等，2010Drug Metab Dispos 38：84-91)或通过在一半双特异性抗体的抗原结合片段(Fab)内交换重链结构域与轻链结构域(交叉Mab形式)(Schaefer W等，2011Proc Natl Acad Sci108：11187-92)。Other examples are tetravalent IgG single-chain variable fragment (scFv) fusions (Dong J et al., 2011 MAbs 3:273-288; Coloma MJ, Morrison SL 1997 Nat Biotechnol 15:159-163; Lu D et al., 2002 J Immunol Methods 267:213 -226) a pharmacophore peptide covalently linked to a catalytic antibody (DoppalapudiVR et al., 2010 ProcNatl Acad Sci USA 107:22611-22616), bispecific antibody generation using dynamic exchange between half IgG4 molecules (van der Neut Kolfschoten M et al., 2007 Science 317:1554-1557; Stubenrauch K et al., 2010 Drug Metab Dispos 38:84-91) or by exchanging the heavy and light chain domains (crossover) within the antigen-binding fragment (Fab) of half of the bispecific antibody Mab format) (Schaefer W et al., 2011 Proc Natl Acad Sci 108: 11187-92).
此外，美国专利8,722,859B2描述了在IgG的重链中包含两个Fab重复并且能够结合四个抗原分子的多价抗体构建体。Furthermore, US Patent 8,722,859 B2 describes a multivalent antibody construct comprising two Fab repeats in the heavy chain of IgG and capable of binding four antigen molecules.
CN106459182A提供了能够结合两个或更多个抗原，或者两个或更多个表位的多价和多特异性串联Fab免疫球蛋白(FIT-Ig)。CN106459182A provides multivalent and multispecific tandem Fab immunoglobulins (FIT-Ig) capable of binding two or more antigens, or two or more epitopes.
OX40OX40
OX40(也称为CD134、TNFRSF4和ACT35)最初被描述为大鼠CD4 T细胞上的T细胞激活标志物(Paterson DJ，Jefferies WA，Green JR，Brandon MR，Corthesy P，Puklavec M，Williams AF.Antigens of activated rat T lymphocytes including a molecule of50,000Mr detected only on CD4 positive T blasts.Mol Immunol.1987；24：1281-1290)并且随后显示为在TCR招募中被上调(Mallett S，Fossum S，BarclayAN.Characterization of the MRC OX40 antigen of activated CD4positive Tlymphocytes--a molecule related to nerve growth factor receptor.EMBO J.1990；9：1063-1068)。OX40信号传导能够促进对T细胞的共刺激信号，导致增强的细胞增殖、存活、效应子功能和迁移(Gramaglia I，WeinbergAD，Lemon M，Croft M.Ox-40ligand：a potentcostimulatory molecule fbr sustaining primary CD4 T cell responses.JImmunol.1998；161：6510-6517；Gramaglia I，Jember A，Pippig SD，Weinberg AD，KilleenN，Croft M.The OX40costimulatory receptor determines the development ofCD4memory by regulating primary clonal expansion.J Immunol.2000；165：3043-3050)。OX40 (also known as CD134, TNFRSF4 and ACT35) was originally described as a marker of T cell activation on rat CD4 T cells (Paterson DJ, Jefferies WA, Green JR, Brandon MR, Corthesy P, Puklavec M, Williams AF. Antigens of activated rat T lymphocytes including a molecule of 50,000Mr detected only on CD4 positive T blasts.Mol Immunol.1987;24:1281-1290) and subsequently shown to be upregulated in TCR recruitment (Mallett S, Fossum S, Barclay AN.Characterization of the MRC OX40 antigen of activated CD4positive Tlymphocytes--a molecule related to nerve growth factor receptor. EMBO J.1990; 9: 1063-1068). OX40 signaling can facilitate co-stimulatory signals to T cells, resulting in enhanced cell proliferation, survival, effector function and migration (Gramaglia I, Weinberg AD, Lemon M, Croft M. Ox-40ligand: a potent costimulatory molecule fbr sustaining primary CD4 T cell responses. J Immunol. 1998; 161: 6510-6517; Gramaglia I, Jember A, Pippig SD, Weinberg AD, Killeen N, Croft M. The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion. J Immunol. 2000: 165; 1 3043-3050).
OX40的配体，OX40L主要表达在抗原呈递细胞(APC)上并且其表达可以通过CD40和肥大细胞信号传导、toll样受体(TLR)以及炎性细胞因子被诱导。除了APC之外，非造血细胞例如平滑肌和血管内皮细胞也可以表达OX40L。在过量表达OX40L的转基因小鼠中，有增加的T-细胞激活，并且当被免疫时，这些小鼠产生增强的T细胞应答(Murata K，Nose M，Ndhlovu LC，Sato T，Sugamura K，Ishii N.Constitutive OX40/OX40ligand interactioninduces autoimmune-like diseases.J Immunol.2002；169：4628-4636和Sato T，IshiiN，Murata K，Kikuchi K，Nakagawa S，Ndhlovu LC，Sugamura K.Consequences of OX40-OX40ligand interactions in langerhans cell function：enhanced contacthypersensitivity responses in OX40L-transgenic mice.Eur J Immunol.2002；32：3326-3335)。这一数据表明OX40L表达是T细胞中OX40信号传导的限制性因子。The ligand of OX40, OX40L, is mainly expressed on antigen presenting cells (APCs) and its expression can be induced by CD40 and mast cell signaling, toll-like receptors (TLRs), and inflammatory cytokines. In addition to APCs, non-hematopoietic cells such as smooth muscle and vascular endothelial cells can also express OX40L. In transgenic mice overexpressing OX40L, there was increased T-cell activation, and these mice developed enhanced T-cell responses when immunized (Murata K, Nose M, Ndhlovu LC, Sato T, Sugamura K, Ishii N. Constitutive OX40/OX40ligand interaction induces autoimmune-like diseases. J Immunol. 2002;169:4628-4636 and Sato T, Ishii N, Murata K, Kikuchi K, Nakagawa S, Ndhlovu LC, Sugamura K. Consequences of OX40-OX40ligand langerhans cell function: enhanced contact hypersensitivity responses in OX40L-transgenic mice. Eur J Immunol. 2002; 32: 3326-3335). This data suggests that OX40L expression is a limiting factor for OX40 signaling in T cells.
在携带肿瘤的小鼠中，小鼠OX40的体内连接(通过可溶性小鼠OX40L-免疫球蛋白融合蛋白或小鼠OX40L模拟物，如抗小鼠CD134特异性抗体)增强抗肿瘤免疫，导致各种小鼠恶性肿瘤细胞系的小鼠模型例如淋巴瘤、黑素瘤、肉瘤、结肠癌、乳腺癌和神经胶质瘤中的无肿瘤存活(Sugamura et al.Nature Rev Imm2004；4：420-431)。In tumor-bearing mice, in vivo ligation of mouse OX40 (via soluble mouse OX40L-immunoglobulin fusion protein or mouse OX40L mimics, such as anti-mouse CD134-specific antibodies) enhances antitumor immunity, leading to various Tumor-free survival in mouse models of mouse malignancy cell lines such as lymphoma, melanoma, sarcoma, colon cancer, breast cancer and glioma (Sugamura et al. Nature Rev Imm 2004;4:420-431) .
在严重联合免疫缺陷(SCID)小鼠中，人OX40的体内连接(通过抗人OX40特异性抗体，其与人OX40上的OX40L结合结构域相互作用；US2009/0214560A1)增强抗肿瘤免疫，其导致各种人恶性肿瘤细胞系例如淋巴瘤、前列腺癌、结肠癌及乳腺癌的肿瘤生长抑制。In severe combined immunodeficiency (SCID) mice, in vivo linkage of human OX40 (by an anti-human OX40-specific antibody that interacts with the OX40L-binding domain on human OX40; US2009/0214560A1) enhances antitumor immunity, which leads to Tumor growth inhibition of various human malignancy cell lines such as lymphoma, prostate, colon and breast cancer.
因此，仍然需要开发新的具有至少四个抗原结合位点的多价抗体(例如抗OX40抗体)，其与相应的常规抗体(例如抗OX40抗体)相比，具有更好的激动剂活性，从而更好地用来治疗或延缓各种癌症、免疫相关疾病和T细胞功能障碍性疾病。Therefore, there is still a need to develop new multivalent antibodies (such as anti-OX40 antibodies) with at least four antigen-binding sites, which have better agonist activity compared with corresponding conventional antibodies (such as anti-OX40 antibodies), thereby It is better used to treat or delay various cancers, immune-related diseases and T-cell dysfunction diseases.
在免疫原性差的肿瘤中，单一的抗OX40治疗不能提供足够的抗肿瘤免疫原性。由于TNFR的激动作用需要受体聚集，本领域仍然需要关于TNF家族(尤其是OX40分子)的具有多价抗原结合功能的、具有改善的聚集和受体激动作用的单分子实体，以满足日益增加的健康和医疗需求。本发明满足了这些和其它需求。In poorly immunogenic tumors, a single anti-OX40 therapy does not provide sufficient antitumor immunogenicity. Since TNFR agonism requires receptor aggregation, there is still a need in the art for single-molecule entities with multivalent antigen-binding function, improved aggregation and receptor agonism for the TNF family (especially OX40 molecules) to meet the increasing health and medical needs. The present invention fulfills these and other needs.
发明概述Summary of the invention
申请人令人惊奇地发现，本发明提供的具有至少四个抗原结合位点的多价抗体能有效结合抗原，并且与相应的现有技术中已知的二价抗体相比，具有更好的激动剂活性。优选地，所述抗体或其片段能够活化T细胞，例如增强T效应细胞的免疫刺激/效应功能和/或使这些细胞增殖和/或下调T调节细胞的免疫抑制功能。因此，本发明的具有至少四个抗原结合位点的多价抗体能够用来治疗或延缓各种癌症、免疫相关疾病和T细胞功能障碍性疾病。在一个实施方案中，所述具有至少四个抗原结合位点的多价抗体是抗TNF的抗体。所述具有至少四个抗原结合位点的多价抗体是抗OX40的抗体。Applicants have surprisingly found that the multivalent antibodies provided by the present invention with at least four antigen-binding sites can efficiently bind antigens and have better Agonist activity. Preferably, the antibody or fragment thereof is capable of activating T cells, such as enhancing the immunostimulatory/effector function of T effector cells and/or proliferating these cells and/or down-regulating the immunosuppressive function of T regulatory cells. Therefore, the multivalent antibody with at least four antigen combining sites of the present invention can be used to treat or delay various cancers, immune-related diseases and T cell dysfunction diseases. In one embodiment, the multivalent antibody having at least four antigen binding sites is an anti-TNF antibody. The multivalent antibody having at least four antigen binding sites is an anti-OX40 antibody.
本发明提供了具有至少四个抗原结合位点的多价抗体。在一实施方案中，多价抗体含有二聚化结构域和至少四个抗原结合位点。优选的二聚化结构域含有Fc结构域和/或铰链区(或由其组成)。The invention provides multivalent antibodies having at least four antigen combining sites. In one embodiment, a multivalent antibody contains a dimerization domain and at least four antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc domain and/or a hinge region.
在一个实施方案中，本发明提供分离的含有二聚化结构域并在其氨基末端含有至少四个抗原结合位点的抗体。本发明进一步提供分离的含有Fc结构域和在Fc结构域氨基末端具有至少四个抗原结合位点的抗体。在一个实施方案中，所述至少四个抗原结合位点结合不同的抗原。在另一个实施方案中，所述至少四个抗原结合位点结合相同的抗原。在一个实施方案中，所述至少四个抗原结合位点为四个相同或者不同的Fv片段。在进一步优选的实施方案中，所述至少四个抗原结合位点为四个不同的Fab片段。在另一个实施方案中，所述Fab片段是相同的Fab片段。In one embodiment, the invention provides an isolated antibody comprising a dimerization domain and at least four antigen binding sites at its amino terminus. The invention further provides isolated antibodies comprising an Fc domain and having at least four antigen binding sites amino-terminal to the Fc domain. In one embodiment, said at least four antigen binding sites bind different antigens. In another embodiment, said at least four antigen binding sites bind the same antigen. In one embodiment, the at least four antigen binding sites are four identical or different Fv fragments. In a further preferred embodiment, said at least four antigen binding sites are four different Fab fragments. In another embodiment, said Fab fragments are the same Fab fragment.
在一个实施方案中，本发明提供分离的抗体，其包含Fc结构域和至少4个抗原结合片段，其中抗原结合片段两两串联，分别直接或者通过接头融合在Fc结构域的氨基末端。In one embodiment, the present invention provides an isolated antibody, which comprises an Fc domain and at least four antigen-binding fragments, wherein the antigen-binding fragments are connected in series two by two, respectively fused to the amino terminus of the Fc domain directly or through a linker.
在本发明提供分离的抗体的另一个实施方案中，Fc结构域和2个抗原结合片段组成2价的全长抗体，另外的2个抗原结合片段串联在所述全长抗体的重链的氨基末端，在另一个实施方案中，所述另外2个抗原结合片段通过接头融合在该全长抗体的重链氨基末端。In another embodiment of the invention that provides an isolated antibody, the Fc domain and two antigen-binding fragments constitute a bivalent full-length antibody, and the other two antigen-binding fragments are connected in tandem at the amino group of the heavy chain of the full-length antibody In another embodiment, the other two antigen-binding fragments are fused via a linker to the amino terminus of the heavy chain of the full-length antibody.
在一个优选的实施方案中，其中所述的另外的2个抗原结合片段是2个Fv片段或Fab片段，优选地，所述Fab片段分别通过其CH1端(该Fab片段称作远端Fab片段、或称作外Fab)直接或者通过接头(例如几个氨基酸的接头)融合到所述全长抗体的1条重链的氨基末端。在一个优选的实施方案中，所述Fab片段分别通过其CH1片段的氨基酸残基H113(Kabat编号)直接或借助于接头与所述全长抗体的1条重链的氨基末端融合，从而形成VH-CH1-重链(即，Fab-重链)的融合多肽。在一个实施方案中，形成的融合多肽的结构为VH-CH1-接头-重链(即，Fab-接头-重链)。在一个优选的实施方案中，所述接头为(GGGGS)n，其中n＝0-4，在进一步优选的实施方案中，所述接头是GGGGS(SEQ ID NO：57)。In a preferred embodiment, wherein the other two antigen-binding fragments are two Fv fragments or Fab fragments, preferably, the Fab fragments are respectively passed through their CH1 ends (the Fab fragments are called distal Fab fragments) , or called exo-Fab) is fused to the amino terminus of one heavy chain of the full-length antibody either directly or via a linker (eg, a linker of a few amino acids). In a preferred embodiment, the Fab fragments are respectively fused to the amino terminus of one heavy chain of the full-length antibody through amino acid residue H113 (Kabat numbering) of its CH1 fragment, directly or by means of a linker, thereby forming a VH - fusion polypeptide of CH1-heavy chain (ie Fab-heavy chain). In one embodiment, the structure of the resulting fusion polypeptide is VH-CH1-linker-heavy chain (ie, Fab-linker-heavy chain). In a preferred embodiment, the linker is (GGGGS)n , wherein n=0-4, in a further preferred embodiment, the linker is GGGGS (SEQ ID NO: 57).
在一个实施方案中，多价抗体包含2条重链融合多肽，其中每条重链融合多肽含有2个或更多个重链可变区。在一个优选实施方案中，多价抗体包含两条相同的重链融合多肽和四条相同的轻链，其中每条重链融合多肽含有2个可变区。In one embodiment, the multivalent antibody comprises two heavy chain fusion polypeptides, wherein each heavy chain fusion polypeptide comprises two or more heavy chain variable regions. In a preferred embodiment, the multivalent antibody comprises two identical heavy chain fusion polypeptides and four identical light chains, wherein each heavy chain fusion polypeptide contains two variable regions.
在一个实施方案中，多价抗体包含Fv1-接头1-Fv2-接头2-Fc的结构，其中Fv1是第一个Fv片段，Fv2是第二个Fv片段，其中Fv1与Fv2相同或者不同，在一个优选实施方案中，接头2是天然抗体的铰链区。In one embodiment, the multivalent antibody comprises the structure Fv1-linker1-Fv2-linker2-Fc, wherein Fv1 is the first Fv fragment, Fv2 is the second Fv fragment, wherein Fv1 and Fv2 are the same or different, in In a preferred embodiment,
在一个进一步优选的实施方案中，多价抗体具有Fab1-接头1-Fab2-接头2-Fc的结构，其中Fab1与Fab2相同或者不同。在更优选的实施方案中，接头1是(GGGGS)n，其中n＝0-4，接头2是天然抗体中连接CH1和CH2的铰链区。在另一个优选的实施方案中，多价抗体具有4个相同或者不同的Fab片段。In a further preferred embodiment, the multivalent antibody has the structure of Fab1-Linker1-Fab2-Linker2-Fc, wherein Fab1 and Fab2 are the same or different. In a more preferred embodiment,
在一个优选的实施方案中，多价抗体具有4个相同的Fab片段，其中一个Fab片段的重链H113(Kabat编号)通过接头与另一个Fab片段重链的氨基端相连。在更优选的实施方案中，所述接头是(GGGGS)n，其中n＝0-4。在一个实施方案中，所述Fab是抗TNF的Fab。在一个优选的实施方案中，所述Fab是抗OX40的Fab。In a preferred embodiment, the multivalent antibody has four identical Fab fragments, wherein the heavy chain H113 (Kabat numbering) of one Fab fragment is connected to the amino-terminal of the heavy chain of another Fab fragment through a linker. In a more preferred embodiment, the linker is (GGGGS)n , where n=0-4. In one embodiment, the Fab is an anti-TNF Fab. In a preferred embodiment, said Fab is an anti-OX40 Fab.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含表C所示任一抗体的VH区序列的一个、两个或三个CDR(优选三个CDR)、或其变体。在另一些实施方案中，本发明抗体包含表C所示任一抗体的VL区序列的一个、两个或三个CDR(优选三个CDR)、或其变体。在一些实施方案中，本发明抗体包含表A-E和I所示任一抗体的6个CDR区序列、或其变体。In one embodiment, the present invention provides a multivalent anti-OX40 antibody, wherein the multivalent anti-OX40 antibody comprises one, two or three CDRs (preferably three CDRs) of the VH region sequence of any antibody shown in Table C ), or variants thereof. In some other embodiments, the antibody of the present invention comprises one, two or three CDRs (preferably three CDRs) of the VL region sequence of any antibody shown in Table C, or a variant thereof. In some embodiments, the antibody of the present invention comprises the 6 CDR region sequences of any of the antibodies shown in Tables A-E and I, or variants thereof.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含3个重链互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含选自SEQ ID NO：1、2、3、59、66、67、68和69的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，HCDR2包含选自SEQ ID NO：4、5、6和60的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，且HCDR3包含选自SEQ ID NO：7、8、9、70、71和72的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列。在一个实施方案中，所述改变是氨基酸取代(例如保守性取代)、缺失或插入。In one embodiment, the present invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising three heavy chain complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 59, 66, 67, 68 and 69, or relative to an amino acid sequence comprising no more than 5 amino acid changes, HCDR2 comprises an amino acid sequence selected from From the amino acid sequence of SEQ ID NO: 4, 5, 6 and 60, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto, and HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 8, 9, 70, 71 and 72 amino acid sequence, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto. In one embodiment, the alteration is an amino acid substitution (eg, a conservative substitution), deletion or insertion.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含轻链可变区(VL)，其中所述LCVR包含互补决定区LCDR1、LCDR2和LCDR3，其中LCDR1包含选自SEQ ID NO：10和11所示的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，LCDR2包含选自SEQ ID NO：12和13所示的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，LCDR3包含选自SEQ IDNO：14、15和16所示的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列。在一个实施方案中，所述改变是氨基酸取代(例如保守性取代)、缺失或插入。In one embodiment, the invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising a light chain variable region (VL), wherein said LCVR comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises amino acid sequences selected from SEQ ID NO: 10 and 11, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto, and LCDR2 comprises amino acid sequences selected from SEQ ID NO: 10 and 11 The amino acid sequence shown in NO: 12 and 13, or relative to the amino acid sequence comprising no more than 5 amino acid changes, LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 14, 15 and 16, or relative to it comprising no more than 5 amino acid sequences Amino acid sequence with more than 5 amino acid changes. In one embodiment, the alteration is an amino acid substitution (eg, a conservative substitution), deletion or insertion.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中HCVR包含3个互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含选自SEQ ID NO：1、2、3、59、66、67、68和69的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，HCDR2包含选自SEQ ID NO：4、5、6和60的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，且HCDR3包含选自SEQ ID NO：7、8、9、70、71和72的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，其中LCVR包含LCDR1、LCDR2和LCDR3，其中LCDR1包含SEQ ID NO：10和11所示的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，LCDR2包含SEQ ID NO：12和13所示的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列，LCDR3包含SEQ ID NO：14、15和16所示的氨基酸序列，或相对于其包含不超过5个氨基酸改变的氨基酸序列。在一个实施方案中，所述改变是氨基酸取代(例如保守性取代)、缺失或插入。In one embodiment, the invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VH) Variable region (VL), wherein HCVR comprises 3 complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 59, 66, 67, 68 and 69 , or relative to an amino acid sequence comprising no more than 5 amino acid changes, HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 5, 6 and 60, or to an amino acid sequence comprising no more than 5 amino acid changes, And HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 7, 8, 9, 70, 71 and 72, or an amino acid sequence comprising no more than 5 amino acid changes relative to it, wherein LCVR comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 Comprising the amino acid sequence shown in SEQ ID NO: 10 and 11, or an amino acid sequence comprising no more than 5 amino acid changes relative to it, LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 12 and 13, or comprising no amino acid sequence relative to it For an amino acid sequence with more than 5 amino acid changes, LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 14, 15 and 16, or an amino acid sequence comprising no more than 5 amino acid changes relative thereto. In one embodiment, the alteration is an amino acid substitution (eg, a conservative substitution), deletion or insertion.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)，其中HCVR包含3个互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含选自SEQ ID NO：1、2、3、59、66、67、68和69的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含选自SEQ ID NO：4、5、6和60的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含选自SEQ ID NO：7、8、9、70、71和72的氨基酸序列或由所述氨基酸序列组成。In one embodiment, the invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH), wherein the HCVR comprises 3 complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 2, 3, 59, 66, 67, 68 and 69; HCDR2 comprises or consists of an amino acid sequence selected from SEQ ID NO: 4, 5, 6 and 60; HCDR3 comprises an amino acid sequence selected from SEQ ID NO: 7, 8, 9, 70, 71 and 72 or Consists of said amino acid sequence.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含轻链可变区(VL)，其中所述LCVR包含互补决定区LCDR1、LCDR2和LCDR3，其中LCDR1包含选自SEQ ID NO：10和11所示的氨基酸序列或由所述氨基酸序列组成；LCDR2包含选自SEQ ID NO：12和13所示的氨基酸序列或由所述氨基酸序列组成；LCDR3包含选自SEQ ID NO：14、15和16所示的氨基酸序列或由所述氨基酸序列组成。In one embodiment, the invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising a light chain variable region (VL), wherein said LCVR comprises complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of amino acid sequences shown in SEQ ID NO: 10 and 11; LCDR2 comprises amino acid sequences selected from SEQ ID NO: 12 and 13 The amino acid sequence or consists of the amino acid sequence; LCDR3 comprises the amino acid sequence selected from SEQ ID NO: 14, 15 and 16 or consists of the amino acid sequence.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中HCVR包含3个互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含选自SEQ ID NO：1、2、3、59、66、67、68和69的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含选自SEQ ID NO：4、5、6和60的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含选自SEQ ID NO：7、8、9、70、71和72的氨基酸序列或由所述氨基酸序列组成；其中LCVR包含LCDR1、LCDR2和LCDR3，其中LCDR1包含选自SEQ ID NO：10和11所示的氨基酸序列或由所述氨基酸序列组成；LCDR2包含选自SEQ ID NO：12和13所示的氨基酸序列或由所述氨基酸序列组成；LCDR3包含选自SEQID NO：14、15和16所示的氨基酸序列或由所述氨基酸序列组成。In one embodiment, the invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VH) Variable region (VL), wherein HCVR comprises 3 complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 59, 66, 67, 68 and 69 , or consist of the amino acid sequence; HCDR2 comprises the amino acid sequence selected from SEQ ID NO: 4, 5, 6 and 60, or consists of the amino acid sequence; HCDR3 comprises the amino acid sequence selected from SEQ ID NO: 7, 8, 9, The amino acid sequences of 70, 71 and 72 or consist of said amino acid sequences; wherein LCVR comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of amino acid sequences selected from SEQ ID NO: 10 and 11; LCDR2 comprises or consists of amino acid sequences selected from SEQ ID NO: 12 and 13; LCDR3 comprises or consists of amino acid sequences selected from SEQ ID NO: 14, 15 and 16.
在一个实施方案中，本发明提供了多价抗OX40抗体，其中所述多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中HCVR包含3个互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：1所示的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含SEQ ID NO：4所示的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：7所示的氨基酸序列或由所述氨基酸序列组成；其中LCDR1包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：12所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：14所示的氨基酸序列或由其组成。In one embodiment, the invention provides a multivalent anti-OX40 antibody, wherein said multivalent anti-OX40 antibody comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VH) Variable region (VL), wherein HCVR comprises 3 complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of said amino acid sequence; HCDR2 comprises SEQ ID NO : the amino acid sequence shown in 4, or consists of the amino acid sequence; HCDR3 contains the amino acid sequence shown in SEQ ID NO: 7 or consists of the amino acid sequence; wherein LCDR1 contains the amino acid sequence shown in SEQ ID NO: 10 or Composed of it, LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 12, and LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 14.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中HCVR包含3个互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：2所示的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含SEQ ID NO：5所示的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：8所示的氨基酸序列或由所述氨基酸序列组成；其中LCDR1包含SEQ ID NO：11所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：13所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：15所示的氨基酸序列或由其组成。In one embodiment, the multivalent anti-OX40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein HCVR comprises 3 A complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of said amino acid sequence; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, or consists of Said amino acid sequence composition; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8 or consists of said amino acid sequence; wherein LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 11, and LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 13 or consists of it, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 15 or consists of it.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中HCVR包含3个互补决定区(CDR)HCDR1、HCDR2和HCDR3，其中HCDR1包含SEQ ID NO：3所示的氨基酸序列，或由所述氨基酸序列组成；HCDR2包含SEQ ID NO：6所示的氨基酸序列，或由所述氨基酸序列组成；HCDR3包含SEQ ID NO：9所示的氨基酸序列或由所述氨基酸序列组成；其中LCDR1包含SEQ ID NO：10所示的氨基酸序列或由其组成，LCDR2包含SEQ ID NO：12所示的氨基酸序列或由其组成，LCDR3包含SEQ ID NO：16所示的氨基酸序列或由其组成。In one embodiment, the multivalent anti-OX40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein HCVR comprises 3 A complementarity determining region (CDR) HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 3, or consists of said amino acid sequence; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of Said amino acid sequence composition; HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of said amino acid sequence; wherein LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, and LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 12 or consists of it, LCDR3 contains the amino acid sequence shown in SEQ ID NO: 16 or consists of it.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区VH，其中重链可变区包含选自SEQ ID NO：36、37、38和61的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列。In one embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region VH, wherein the heavy chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 36, The amino acid sequence of 37, 38 and 61, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% therewith %, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical amino acid sequences, or amino acid sequences comprising no more than 10, no more than 5 amino acid changes relative thereto .
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含轻链可变区VL，其包含选自SEQ ID NO：39、40和41所示的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列。In one embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a light chain variable region VL comprising a sequence selected from the group consisting of SEQ ID NO: 39, 40 and 41 amino acid sequence, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, An amino acid sequence that is 94%, 95%, 96%, 97%, 98% or 99% or more identical, or an amino acid sequence comprising no more than 10, no more than 5 amino acid changes relative thereto.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区VH包含选自SEQ ID NO：36、37、38和61的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列；轻链可变区VL包含选自SEQ ID NO：39、40和41所示的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列。In one embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain can be The variable region VH comprises an amino acid sequence selected from SEQ ID NO: 36, 37, 38 and 61, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% thereof %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identical amino acid sequences, or contain no more than Amino acid sequence with 10, no more than 5 amino acid changes; the light chain variable region VL comprises an amino acid sequence selected from SEQ ID NO: 39, 40 and 41, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity amino acid sequence, or an amino acid sequence comprising no more than 10 and no more than 5 amino acid changes relative thereto.
在一个实施方案中，所述改变是氨基酸取代(例如保守性取代)、缺失或插入。In one embodiment, the alteration is an amino acid substitution (eg, a conservative substitution), deletion or insertion.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区VH，其包含选自SEQ ID NO：36、37、38和61的氨基酸序列或由其组成。In one embodiment, the multivalent anti-OX40 antibody of the invention comprises at least four identical Fab fragments comprising a heavy chain variable region VH comprising a sequence selected from SEQ ID NO: 36, 37, 38 and 61 The amino acid sequence of or consists of it.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含轻链可变区VL，其包含选自SEQ ID NO：39、40和41所示的氨基酸序列或由其组成。In one embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a light chain variable region VL comprising a sequence selected from the group consisting of SEQ ID NO: 39, 40 and 41 The amino acid sequence of or consists of it.
在一个实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区VH包含选自SEQ ID NO：36、37、38和61的氨基酸序列或由其组成；轻链可变区VL包含选自SEQ ID NO：39、40和41所示的氨基酸序列或由其组成。In one embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain can be The variable region VH comprises or consists of an amino acid sequence selected from SEQ ID NO: 36, 37, 38 and 61; the light chain variable region VL comprises an amino acid sequence selected from SEQ ID NO: 39, 40 and 41 or consists of its composition.
在优选的实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区VH包含SEQ ID NO：36所示的氨基酸序列或由其组成；轻链可变区VL包含SEQ ID NO：39所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain The variable region VH includes or consists of the amino acid sequence shown in SEQ ID NO:36; the light chain variable region VL includes or consists of the amino acid sequence shown in SEQ ID NO:39.
在优选的实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区VH包含SEQ ID NO：37所示的氨基酸序列或由其组成；轻链可变区VL包含SEQ ID NO：40所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain The variable region VH includes or consists of the amino acid sequence shown in SEQ ID NO: 37; the light chain variable region VL includes or consists of the amino acid sequence shown in SEQ ID NO: 40.
在优选的实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区VH包含SEQ ID NO：38所示的氨基酸序列或由其组成；轻链可变区VL包含SEQ ID NO：41所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain The variable region VH includes or consists of the amino acid sequence shown in SEQ ID NO:38; the light chain variable region VL includes or consists of the amino acid sequence shown in SEQ ID NO:41.
在优选的实施方案中，本发明的多价抗OX40抗体包含至少四个相同的Fab片段，所述Fab片段包含重链可变区(VH)和轻链可变区(VL)，其中重链可变区VH包含SEQ ID NO：61所示的氨基酸序列或由其组成；轻链可变区VL包含SEQ ID NO：40所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises at least four identical Fab fragments comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain The variable region VH includes or consists of the amino acid sequence shown in SEQ ID NO:61; the light chain variable region VL includes or consists of the amino acid sequence shown in SEQ ID NO:40.
在一个实施方案中，本发明的多价抗OX40抗体包含重链或重链融合多肽，其中重链或重链融合多肽包含选自SEQ ID NO：42、43、45、46、48、49、51、52、53、54、55、56、62、63、64和65的氨基酸序列，或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列。In one embodiment, the multivalent anti-OX40 antibody of the present invention comprises a heavy chain or a heavy chain fusion polypeptide, wherein the heavy chain or the heavy chain fusion polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO: 42, 43, 45, 46, 48, 49, The amino acid sequence of 51, 52, 53, 54, 55, 56, 62, 63, 64 and 65, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% therewith , an amino acid sequence with 98% or 99% or higher identity, or an amino acid sequence comprising no more than 10 and no more than 5 amino acid changes relative thereto.
在一些实施方案中，本发明的多价抗OX40抗体包含轻链，其中轻链包含选自SEQID NO：44、47、50所示的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列。In some embodiments, the multivalent anti-OX40 antibody of the invention comprises a light chain, wherein the light chain comprises an amino acid sequence selected from SEQ ID NO: 44, 47, 50, or at least 80%, 81%, 82% thereof , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 Amino acid sequences with % or higher identity, or amino acid sequences comprising no more than 10 and no more than 5 amino acid changes relative thereto.
在一些实施方案中，本发明的多价抗OX40抗体包含重链融合多肽和轻链，其中重链融合多肽包含选自SEQ ID NO：51、52、53、54、55、56、64、65的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列；轻链包含选自SEQ ID NO：44、47、50所示的氨基酸序列，或与其具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％或更高同一性的氨基酸序列，或相对于其包含不超过10个，不超过5个氨基酸改变的氨基酸序列。In some embodiments, the multivalent anti-OX40 antibody of the present invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises amino acid sequence, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Amino acid sequences of 94%, 95%, 96%, 97%, 98% or 99% or higher identity, or amino acid sequences comprising no more than 10, no more than 5 amino acid changes relative thereto; light chains comprising selected From the amino acid sequence shown in SEQ ID NO: 44, 47, 50, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical amino acid sequences, or contain no more than 10, no more than Amino acid sequence with 5 amino acid changes.
在优选的实施方案中，本发明的多价抗OX40抗体包含重链融合多肽和轻链，其中重链融合多肽包含选自SEQ ID NO：51、52所示的氨基酸序列或由其组成；轻链包含SEQ IDNO：44所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises or consists of amino acid sequences selected from SEQ ID NO: 51, 52; The chain comprises or consists of the amino acid sequence shown in SEQ ID NO:44.
在优选的实施方案中，本发明的多价抗OX40抗体包含重链融合多肽和轻链，其中重链融合多肽包含选自SEQ ID NO：53、54所示的氨基酸序列或由其组成；轻链包含SEQ IDNO：47所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises or consists of amino acid sequences selected from SEQ ID NOs: 53 and 54; The chain comprises or consists of the amino acid sequence shown in SEQ ID NO:47.
在优选的实施方案中，本发明的多价抗OX40抗体包含重链融合多肽和轻链，其中重链融合多肽包含选自SEQ ID NO：55、56所示的氨基酸序列或由其组成；轻链包含SEQ IDNO：50所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises or consists of amino acid sequences selected from SEQ ID NOs: 55 and 56; The chain comprises or consists of the amino acid sequence shown in SEQ ID NO:50.
在优选的实施方案中，本发明的多价抗OX40抗体包含重链融合多肽和轻链，其中重链融合多肽包含选自SEQ ID NO：64、65所示的氨基酸序列或由其组成；轻链包含SEQ IDNO：47所示的氨基酸序列或由其组成。In a preferred embodiment, the multivalent anti-OX40 antibody of the present invention comprises a heavy chain fusion polypeptide and a light chain, wherein the heavy chain fusion polypeptide comprises or consists of amino acid sequences selected from SEQ ID NOs: 64 and 65; The chain comprises or consists of the amino acid sequence shown in SEQ ID NO:47.
在一些实施方案中，本发明的抗体还涵盖抗OX40抗体的氨基酸序列的变体，以及与上文所述的任何抗体结合相同表位的抗体。In some embodiments, the antibodies of the invention also encompass variants of the amino acid sequence of the anti-OX40 antibodies, as well as antibodies that bind to the same epitope as any of the antibodies described above.
在一些实施方案中，本发明的多价抗OX40抗体是IgG1形式的抗体(在抗体命名中以g1表示)或IgG2形式的抗体(在抗体命名中以g2表示)。在一些实施方案中，抗OX40抗体是单克隆抗体。在一些实施方案中，抗OX40抗体是人源化的。在一些实施方案中，抗OX40抗体是人抗体。在一些实施方案中，至少部分的抗OX40抗体的框架序列是人共有框架序列。In some embodiments, a multivalent anti-OX40 antibody of the invention is an IgG1 antibody (represented as g1 in antibody nomenclature) or an IgG2 format antibody (represented as g2 in antibody nomenclature). In some embodiments, the anti-OX40 antibody is a monoclonal antibody. In some embodiments, anti-OX40 antibodies are humanized. In some embodiments, the anti-OX40 antibody is a human antibody. In some embodiments, at least a portion of the framework sequence of the anti-OX40 antibody is a human consensus framework sequence.
在一方面，本发明提供能够活化T细胞(例如CD4+T细胞)的具有激动剂活性的多价抗OX40抗体。In one aspect, the invention provides multivalent anti-OX40 antibodies having agonist activity capable of activating T cells (eg, CD4+ T cells).
在一些实施方案中，多价抗OX40抗体的激动剂活性由T细胞活化后释放的细胞因子的水平来评估。因此，本发明提供了与用IgG对照处理的CD4+T细胞的细胞因子生成相比，提高CD4+T细胞的细胞因子生成的多价抗OX40抗体。在一些实施方案中，该细胞因子是炎性细胞因子，例如IL-2或IFNg。In some embodiments, the agonist activity of the multivalent anti-OX40 antibody is assessed by the level of cytokines released upon T cell activation. Accordingly, the present invention provides multivalent anti-OX40 antibodies that increase cytokine production by CD4+ T cells as compared to cytokine production by CD4+ T cells treated with an IgG control. In some embodiments, the cytokine is an inflammatory cytokine, such as IL-2 or IFNg.
在一方面，本发明提供了编码以上任何抗OX40抗体或其片段的核酸。在一个实施方案中，提供包含所述核酸的载体。在一个实施方案中，载体是表达载体。在一个实施方案中，提供包含所述载体的宿主细胞。在一个实施方案中，宿主细胞是真核的。在另一个实施方案中，宿主细胞选自酵母细胞、哺乳动物细胞(例如CHO细胞或293细胞)或适用于制备抗体或其抗原结合片段的其它细胞。在另一个实施方案中，宿主细胞是原核的。In one aspect, the invention provides nucleic acids encoding any of the above anti-OX40 antibodies or fragments thereof. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising said vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (eg, CHO cells or 293 cells), or other cells suitable for the production of antibodies or antigen-binding fragments thereof. In another embodiment, the host cell is prokaryotic.
在一个实施方案中，本发明提供制备多价抗OX40抗体的方法，其中所述方法包含在将包含VH-CH1的构建体和包含VL-CL的构建体克隆到表达载体中，转染宿主细胞，并在适于表达编码所述多价抗体的核酸的条件下培养所述宿主细胞，以及任选地分离所述抗体。在某个实施方案中，所述方法还包括从宿主细胞回收抗多价抗OX40抗体。In one embodiment, the present invention provides a method for preparing a multivalent anti-OX40 antibody, wherein the method comprises cloning a construct comprising VH-CH1 and a construct comprising VL-CL into an expression vector, transfecting a host cell , and culturing said host cell under conditions suitable for expression of a nucleic acid encoding said multivalent antibody, and optionally isolating said antibody. In a certain embodiment, the method further comprises recovering the anti-multivalent anti-OX40 antibody from the host cell.
在一些实施方案中，本发明提供包含本文所述的任何多价抗OX40抗体的组合物，优选地组合物为药物组合物。在一个实施方案中，所述组合物还包含药用载体。在一些实施方案中，所述药物组合物用于治疗癌症，优选地肺癌(例如非小细胞肺癌)、肝癌、胃癌、结肠癌等。In some embodiments, the invention provides a composition, preferably a pharmaceutical composition, comprising any of the multivalent anti-OX40 antibodies described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is used to treat cancer, preferably lung cancer (eg, non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.
在一些实施方案中，本发明提供了免疫缀合物，其包含本文中提供的任何多价抗OX40抗体和细胞毒性剂。在一些实施方案中，所述免疫缀合物用于治疗癌症，优选地肺癌(例如非小细胞肺癌)、肝癌、胃癌、结肠癌等。In some embodiments, the invention provides an immunoconjugate comprising any of the multivalent anti-OX40 antibodies provided herein and a cytotoxic agent. In some embodiments, the immunoconjugate is used to treat cancer, preferably lung cancer (eg, non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.
在一方面中，本发明涉及在受试者中活化T细胞或诱导T细胞介导的抗肿瘤活性或增强机体的免疫应答的方法，所述方法包括向所述受试者施用有效量的本文所述的任何多价抗OX40抗体。本发明还涉及本文所述的任何多价抗OX40抗体制备用于在受试者中活化T细胞或诱导T细胞介导的抗肿瘤活性或增强机体的免疫应答的组合物或药物的用途。In one aspect, the present invention relates to a method for activating T cells or inducing T cell-mediated anti-tumor activity or enhancing the body's immune response in a subject, the method comprising administering to the subject an effective amount of Any of the multivalent anti-OX40 antibodies. The present invention also relates to the use of any multivalent anti-OX40 antibody described herein to prepare a composition or medicine for activating T cells in a subject or inducing T cell-mediated anti-tumor activity or enhancing the body's immune response.
在另一方面中，本发明涉及治疗受试者癌症的方法，所述方法包括向所述受试者施用有效量的本文所述的任何多价抗OX40抗体。在一个实施方案中，癌症是肺癌(例如非小细胞肺癌)、肝癌、胃癌、结肠癌等。在另一方面，本发明还涉及本文所述的任何多价抗OX40抗体或其片段制备用于在受试者中治疗癌症的药物的用途。在一个实施方案中，癌症是肺癌(例如非小细胞肺癌)、肝癌、胃癌、结肠癌等。In another aspect, the invention relates to a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of any of the multivalent anti-OX40 antibodies described herein. In one embodiment, the cancer is lung cancer (eg, non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like. In another aspect, the present invention also relates to the use of any of the multivalent anti-OX40 antibodies or fragments thereof described herein for the manufacture of a medicament for treating cancer in a subject. In one embodiment, the cancer is lung cancer (eg, non-small cell lung cancer), liver cancer, gastric cancer, colon cancer, and the like.
在一些实施方案中，本文所述的方法还包括向所述受试者联合施用有效量的第二药物或活性剂，其中本文所述的多价抗OX40抗体是第一药物。在一些实施方案中，第二药物或活性剂选自化疗剂。在一些实施方案中，第二药物或活性剂选自PD-1轴结合拮抗剂(例如抗PD-1抗体或抗PD-L1抗体或抗PD-L2抗体)或者抗血管发生剂(例如贝伐珠单抗)。In some embodiments, the methods described herein further comprise co-administering to the subject an effective amount of a second drug or active agent, wherein the multivalent anti-OX40 antibody described herein is the first drug. In some embodiments, the second drug or agent is selected from a chemotherapeutic agent. In some embodiments, the second drug or agent is selected from a PD-1 axis binding antagonist (such as an anti-PD-1 antibody or an anti-PD-L1 antibody or an anti-PD-L2 antibody) or an anti-angiogenic agent (such as a bevacizumab Zhuzumab).
在一些实施方案中，受试者或个体是哺乳动物，优选是人。In some embodiments, the subject or individual is a mammal, preferably a human.
在一方面中，本发明涉及检测样品中OX40的方法，所述方法包括(a)将样品与本文所述的任何多价抗OX40抗体接触；和(b)检测多价抗OX40抗体和OX40间的复合物的形成。在一个实施方案中，多价抗OX40抗体是被可检测地标记的。In one aspect, the invention relates to a method of detecting OX40 in a sample comprising (a) contacting the sample with any of the multivalent anti-OX40 antibodies described herein; and (b) detecting the interaction between the multivalent anti-OX40 antibody and OX40 the formation of complexes. In one embodiment, the multivalent anti-OX40 antibody is detectably labeled.
在一些实施方案中，本发明涉及试剂盒或制品，其包含本文所述的任何多价抗OX40抗体。在一些实施方案中，所述试剂盒或制品包含本文所述的多价抗OX40抗体与任选的可药用载体。在一些实施方案中，该试剂盒或制品进一步包含关于施用药物来治疗癌症的说明书。In some embodiments, the invention relates to a kit or article of manufacture comprising any of the multivalent anti-OX40 antibodies described herein. In some embodiments, the kit or article of manufacture comprises a multivalent anti-OX40 antibody described herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the kit or article of manufacture further comprises instructions for administering the medicament to treat the cancer.
本发明还涵盖本文所述的任何实施方案的任意组合。本文所述的任何实施方案或其任何组合适用于本文所述的发明的任何和所有抗OX40抗体、方法和用途。The present invention also covers any combination of any of the embodiments described herein. Any embodiment described herein, or any combination thereof, applies to any and all anti-OX40 antibodies, methods and uses of the invention described herein.
附图说明Description of drawings
图1显示了在CHO细胞中产生的本发明的抗体阻断OX40L与CHO细胞上表达的OX40的结合。Figure 1 shows that antibodies of the invention raised in CHO cells block the binding of OX40L to OX40 expressed on CHO cells.
图2通过抗-CD3/抗-CD28刺激加重组OX40L与在CHO细胞中产生的IgG1或IgG2形式的本发明的抗体，在用人OX40和NFkB启动子-luc稳定转染的Jurkat中的荧光素酶报告基因检测。Fig. 2 increases recombinant OX40L by anti-CD3/anti-CD28 stimulation with the antibody of the present invention in the form of IgG1 or IgG2 produced in CHO cells, fluorescence in Jurkat stably transfected with human OX40 and NFkB promoter-luc Primer reporter gene assay.
图3评估了本发明的抗体对OX40信号的激动剂活性。用金黄色葡萄球菌肠毒素E(SEE)(1ng/ml)和在CHO细胞中产生的IgG1或IgG2形式的本发明的OX40抗体的DC共培养测定法进行测定。Figure 3 evaluates the agonist activity of antibodies of the invention on OX40 signaling. Assays were performed using a DC co-culture assay of S. aureus enterotoxin E (SEE) (1 ng/ml) and the OX40 antibody of the invention as IgG1 or IgG2 produced in CHO cells.
图4显示了4xFab mAb的结构，其含有通过外Fab的C末端上的CH1结构域和常规mAb结构中包含的内Fab的N末端与1×GS接头连接的4个相同Fab。Figure 4 shows the structure of a 4xFab mAb containing 4 identical Fabs linked by a CH1 domain at the C-terminus of the outer Fab and a 1xGS linker at the N-terminus of the inner Fab contained in the conventional mAb structure.
图5.显示了尺寸排阻色谱法对IgG2形式的人四价OX40 mAb：ADI-20057-g2-4xFab的测量Figure 5. Shows size exclusion chromatography measurements of human tetravalent OX40 mAb in IgG2 form: ADI-20057-g2-4xFab
图6.显示了尺寸排阻色谱法对IgG2形式的人四价OX40 mAb：ADI-23515-g2-4xFab的测量Figure 6. Shows size exclusion chromatography measurements of human tetravalent OX40 mAb in IgG2 form: ADI-23515-g2-4xFab
图7.显示了尺寸排阻色谱法对IgG1形式的人四价OX40 mAb：ADI-20112-g1-4xFab的测量Figure 7. Shows size exclusion chromatography measurements of human tetravalent OX40 mAb in IgG1 format: ADI-20112-g1-4xFab
图8显示各抗体的激动剂活性。通过抗-CD3/抗-CD28刺激与293细胞中产生的IgG1、IgG2和4xFab形式的本发明的抗体，用人OX40和NFkB启动子-luc稳定转染的Jurkat中的萤光素酶报告基因检测。Figure 8 shows the agonist activity of each antibody. Anti-CD3/anti-CD28 stimulation with antibodies of the invention produced in IgG1 , IgG2 and 4xFab formats in 293 cells, luciferase reporter in Jurkat stably transfected with human OX40 and NFkB promoter-luc detection.
图9显示各抗体的激动剂活性。通过抗-CD3/抗-CD28刺激与293细胞中产生的IgG1、IgG2和4xFab形式的本发明的抗体，混合Raii细胞，用人OX40和NFkB启动子-luc稳定转染的Jurkat中的荧光素酶报告基因检测。Figure 9 shows the agonist activity of each antibody. Anti-CD3/anti-CD28 stimulation with antibodies of the invention in the form of IgG1 , IgG2 and 4xFab produced in 293 cells, mixed Raii cells, fluorescein in Jurkat stably transfected with human OX40 and NFkB promoter-luc Enzyme reporter assay.
图10评估了本发明的抗-OX40抗体的阻断活性。通过抗-CD3/抗-CD28刺激与重组OX40L和293细胞中产生的IgG1、IgG2和4xFab形式的本发明的抗体，用人OX40和NFkB启动子-luc稳定转染的Jurkat中的荧光素酶报告基因检测。其中图中4xF是4xFab的缩写。Figure 10 evaluates the blocking activity of anti-OX40 antibodies of the invention. Anti-CD3/anti-CD28 stimulation with recombinant OX40L and antibodies of the invention in IgG1 , IgG2 and 4xFab formats produced in 293 cells, luciferase in Jurkat stably transfected with human OX40 and NFkB promoter-luc Reporter gene testing. 4xF in the figure is the abbreviation of 4xFab.
发明详述Detailed description of the invention
定义definition
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。应当理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。In order to explain this specification, the following definitions will be used, and whenever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated numerical value.
术语“和/或”应理解为意指可选项中的任一项或可选项的两项。The term "and/or" should be understood as meaning any one of the alternatives or both of the alternatives.
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。As used herein, the term "comprising" or "comprising" means including stated elements, integers or steps, but not excluding any other elements, integers or steps. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists of that particular sequence.
本文所用的术语“抗OX40抗体”、“抗OX40”、“OX40抗体”或“结合OX40的抗体”是指这样的抗体，所述抗体能够以足够的亲合力结合人或食蟹猴OX40蛋白或其片段以致所述抗体可以用作靶向人或食蟹猴OX40中的诊断剂和/或治疗剂。在一个实施方案中，抗OX40抗体与非人或非食蟹猴OX40蛋白结合的程度低于所述抗体与人或食蟹猴OX40结合的约10％，如例如通过放射性免疫测定(RIA)或生物光干涉测定法或MSD测定法测量的。在一些实施方案中，抗OX40的抗体的解离常数(Kd)≤100nM，≤10nM或≤1nM(例如10-7M以下，例如10-7M至10-10M，例如10-8M至10-9M)。As used herein, the term "anti-OX40 antibody", "anti-OX40", "OX40 antibody" or "OX40-binding antibody" refers to an antibody that binds with sufficient affinity to a human or cynomolgus OX40 protein or Fragments thereof such that the antibody can be used as a diagnostic and/or therapeutic agent targeting OX40 in humans or cynomolgus monkeys. In one embodiment, the anti-OX40 antibody binds to a non-human or non-cynomolgus OX40 protein to a degree that is less than about 10% of the binding of said antibody to human or cynomolgus OX40, as for example by radioimmunoassay (RIA) or Measured by biophotometric interferometry or MSD assay. In some embodiments, the dissociation constant (Kd) of the anti-OX40 antibody is ≤ 100 nM, ≤ 10 nM or ≤ 1 nM (e.g., less than 10−7 M, such as 10−7 M to 10−10 M, such as 10−8 M to10-9 M).
“亲和力”或“结合亲和力”指反映结合对的成员(例如抗体与抗原)之间相互作用的固有结合亲和力。分子X对其配偶体Y的亲和力通常可用平衡解离常数(KD)来表述。平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可通过本领域知道的常用方法来测量，包括现有技术已知以及本文中所描述的那些。"Affinity" or "binding affinity" refers to intrinsic binding affinity that reflects the interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be described in terms of an equilibrium dissociation constant (KD ). The equilibrium dissociation constant is the ratio of the dissociation rate constant and the association rate constant (kdis and kon , respectively). Affinity can be measured by common methods known in the art, including those known in the art and described herein.
如本文所用，“单克隆抗体”或“mAb”指来源于例如真核生物的、原核生物的或噬菌体克隆的单一拷贝或克隆的抗体，即，除了通常以很少量存在的可能变体抗体(例如，含有天然突变或在单克隆抗体制品的生产过程中产生的变体抗体)以外，构成所述群体的各个抗体是相同的和/或结合相同表位。修饰语“单克隆”表示抗体从基本上同质的抗体群获得的特征，并且不应解释为需要通过任何特定方法产生抗体。单克隆抗体可以例如通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术例如CDR嫁接、或此类或其它本领域已知的技术的组合来产生。As used herein, "monoclonal antibody" or "mAb" refers to a single copy or clone of an antibody derived, for example, from a eukaryotic, prokaryotic, or phage clone, i.e., the (eg, variant antibodies that contain natural mutations or arise during the production of monoclonal antibody preparations), the individual antibodies comprising the population are identical and/or bind the same epitope. The modifier "monoclonal" indicates the characteristics of an antibody acquired from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. Monoclonal antibodies can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or combinations of these or other techniques known in the art.
本领域技术人员明了，“全抗体”(在本文中可与“全长抗体”、“完全抗体”和“完整抗体”互换使用)包含至少两条重链(H)和两条轻链(L)。每条重链从N至C端由重链可变区(又称作可变重域或重链可变域，本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(又称作可变轻域或轻链可变域，本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。可变区是抗体的重链或轻链中参与抗体与其抗原的结合的结构域。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。在本文中，重链的融合多肽指全长抗体的重链与其他肽序列形成的融合多肽，在一个优选的实施方案中，其他肽序列通过其羧基端直接或者通过接头(例如几个氨基酸的接头)融合到所述全长抗体的1条重链的氨基末端。在一个优选的实施方案中，所述的其他肽序列是Fab片段，从而形成VH-CH1-重链(即，Fab-重链)的融合多肽。It will be apparent to those skilled in the art that a "whole antibody" (used herein interchangeably with "full length antibody", "complete antibody" and "intact antibody") comprises at least two heavy (H) chains and two light chains ( L). Each heavy chain consists, from N to C-terminus, of a heavy chain variable region (also referred to as a variable heavy domain or heavy chain variable domain, abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (also referred to as a variable light domain or light chain variable domain, abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs) interspersed with more conservative regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four The FR composition is arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region is the domain of the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions.Herein, the fusion polypeptide of the heavy chain refers to the fusion polypeptide formed by the heavy chain of the full-length antibody and other peptide sequences. In a preferred implementation In the scheme, other peptide sequences are fused to the amino terminus of one heavy chain of the full-length antibody directly or through a linker (such as a linker of several amino acids) through its carboxy terminus. In a preferred embodiment, the other The peptide sequence is a Fab fragment, forming a fusion polypeptide of VH-CH1-heavy chain (ie, Fab-heavy chain).
“天然抗体”指具有不同结构的天然存在的免疫球蛋白分子。“天然序列Fc结构域”包含与在自然界中找到的Fc结构域的氨基酸序列相同的氨基酸序列。天然序列人Fc结构域包括例如天然序列人IgG1Fc结构域(非A和A同种异型)；天然序列人IgG2Fc结构域；天然序列人IgG3Fc结构域；和天然序列人IgG4Fc结构域；及其天然存在变体。"Native antibody" refers to naturally occurring immunoglobulin molecules of varying structure. A "native sequence Fc domain" comprises an amino acid sequence identical to that of an Fc domain found in nature. Native sequence human Fc domains include, for example, native sequence humanIgG1 Fc domains (non-A and A allotypes); native sequence humanIgG2 Fc domains; native sequence human IgG3 Fc domains; and native sequence human IgG4 Fc domains; and naturally occurring variants thereof.
抗体的“结合位点”或“抗原结合位点”是抗体分子中与抗原实际结合的区域。在一个优选的实施方案中，抗原结合位点由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)组成的VH/VL对构成。The "binding site" or "antigen-binding site" of an antibody is the region of the antibody molecule that actually binds to the antigen. In a preferred embodiment, the antigen binding site consists of a VH/VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。抗原结合片段的例子包括但不限于Fv、Fab、Fab′、Fab′-SH、F(ab′)2、双链抗体(diabody)、单结构域抗体(sdAb)、单链抗体分子(例如scFv)、三链抗体(triabody)、四链抗体(tetrabody)、微抗体(minibody)、单结构域抗体(sdAb)。所述Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。可以借接头将Fab的轻链(L链)和重链(H链)融合构建成单一多肽链，即单链Fab(scFab)(参见例如US20070274985A1)。通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab′)2，其为Fab′的二聚体，是二价的抗体片段。F(ab′)2可以在中性条件下通过破坏铰链区中的二硫键而被还原，从F(ab′)2二聚体转化为Fab′单体。Fab′单体基本上是具有铰链区的Fab片段。Fab′-SH是这样的Fab′，其中恒定结构域的半胱氨酸残基带有游离巯基。抗体片段的其它化学偶联也是已知的。Fv片段由抗体单臂的VL和VH结构域组成。另外，可以使用重组方法，将独立编码Fv片段的两个结构域VL和VH的基因，通过编码连接肽(接头)的核酸序列连接在一起，重组表达形成单链Fv或“scFv”，在所述单条蛋白链中VL区和VH区配对以形成所需的结构。An "antigen-binding fragment" is a portion or segment of an intact or complete antibody having fewer amino acid residues than an intact or complete antibody, which is capable of binding antigen or competing for binding with the intact antibody (i.e., with the intact antibody from which the antigen-binding fragment is derived) antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2 , diabody, single domain antibody (sdAb), single chain antibody molecule (e.g. scFv ), triabody, tetrabody, minibody, single domain antibody (sdAb). The Fab fragment is a monovalent fragment consisting of VL, VH, CL and CH1 domains, for example, Fab fragments can be obtained by papain digestion of complete antibodies. The light chain (L chain) and heavy chain (H chain) of Fab can be fused to construct a single polypeptide chain, ie, single-chain Fab (scFab), by means of a linker (see, for example, US20070274985A1). Digestion of whole antibodies by pepsin below the disulfide bonds in the hinge region yields F(ab')2 , which is a dimer of Fab', a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, converting from F(ab')2 dimer to Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. Fab'-SH is a Fab' in which the cysteine residues of the constant domains bear a free sulfhydryl group. Other chemical couplings of antibody fragments are also known. The Fv fragment consists of the VL and VH domains of a single arm of an antibody. In addition, recombination methods can be used to link the genes independently encoding the two domains VL and VH of the Fv fragment through the nucleic acid sequence encoding the connecting peptide (linker), and recombinantly express to form a single-chain Fv or "scFv". The VL and VH regions in a single protein chain are paired to form the desired structure.
本领域已知根据重链恒定区氨基酸序列，可以将抗体划分为五个主要的类别：IgA，IgD，IgE，IgG和IgM，另外，这些抗体中的数个可以进一步被划分为亚类(同种型)，例如，IgG1，IgG2，IgG3，IgG4，IgA1和IgA2。对应于不同类别的免疫球蛋白的重链恒定结构域分别被称为α，δ，ε，γ和μ。根据轻链恒定区氨基酸序列，抗体轻链可归入两种类型中的一种，称作卡帕(κ)和拉姆达(入)。It is known in the art that antibodies can be divided into five major classes based on the amino acid sequence of the heavy chain constant region: IgA, IgD, IgE, IgG, and IgM, and that several of these antibodies can be further divided into subclasses (same as isotype), for example, IgG1 , IgG2 , IgG3 , IgG4 , IgA1 and IgA2 . The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Based on the amino acid sequence of the light chain constant region, antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (IN).
“IgG形式的抗体”是指抗体的重链恒定区是γ链。γ链有多种亚类，例如，IgG1、IgG2、IgG3、IgG4。在本文中对于IgG1类的抗体在抗体命名时加后缀-g1，在本文中对于IgG2类的抗体在抗体命名时加后缀-g2。"An antibody in IgG form" means that the heavy chain constant region of the antibody is a gamma chain. There are various subclasses of the γ chain, for example, IgG1 , IgG2 , IgG3 , IgG4 . In this paper, the suffix -g1 is added to the antibody name of IgG1 class antibody, and the suffix -g2 is added to the antibody name of IgG2 class antibody herein.
术语“双链抗体(diabody)”指具有两个抗原结合位点的抗体片段，该片段在同一多肽链中包含通过短接头连接的VL和VH。在双链抗体中，由于接头过短，同一链上的VH和VL两个结构域之间无法配对，而被迫与另一条链的互补结构域配对从而产生两个抗原结合位点。双链抗体可以是二价的或双特异性的。双链抗体更充分地描述于例如EP 404，097；WO1993/01161；Hudson等，Nat.Med.9：129-134(2003)；和Hollinger等，美国国家科学院学报(Proc.Natl.Acad.Sci.USA)90：6444-6448(1993)中。三链抗体和四链抗体同样描述于Hudson等，Nat.Med.9：129-134(2003)和邵荣光等人(编辑)，抗体药物研究与应用，人民卫生出版社(2013)。单结构域抗体(sdAb)通常指这样的抗体，其中单个可变结构域(例如，重链可变结构域(VH)或轻链可变结构域(VL)、衍生自骆驼科重链抗体的重链可变结构域、衍生自鱼类IgNAR的VH样单结构域(v-NAR))即可赋予抗原结合，而不需要与另一可变结构域相互作用以识别靶抗原。(关于抗体片段的更详细的描述，也可以参见：基础免疫学(Fundamental Immunology)，W.E.Paul编辑，Raven Press，N.Y.(1993))。The term "diabody" refers to an antibody fragment with two antigen-binding sites comprising a VL and a VH connected by a short linker in the same polypeptide chain. In diabodies, because the linker is too short, the VH and VL domains on the same chain cannot be paired, and are forced to pair with the complementary domains of another chain to generate two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP 404,097; WO1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA) 90:6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003) and Shao Rongguang et al. (eds.), Antibody Drug Research and Application, People's Health Publishing House (2013). A single domain antibody (sdAb) generally refers to an antibody in which a single variable domain (e.g., a heavy chain variable domain (VH) or a light chain variable domain (VL), derived from a camelid heavy chain antibody The heavy chain variable domain, a VH-like single domain derived from fish IgNAR (v-NAR), confers antigen binding without the need for interaction with another variable domain to recognize the target antigen. (For a more detailed description of antibody fragments, see also: Fundamental Immunology, edited by W.E. Paul, Raven Press, N.Y. (1993)).
术语“Fc结构域”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc结构域和变体Fc结构域。例如在天然抗体中，Fc结构域由两个相同的蛋白质片段组成，其源自IgG、IgA和IgD同种型中抗体的两条重链的第二和第三恒定结构域；IgM和IgE Fc结构域在每条多肽链中含有三个重链恒定结构域(CH结构域2-4)。在某些实施方案中，人IgG重链的Fc结构域从Cys226或Pro230延伸至重链的羰基端。然而，Fc结构域的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc结构域或恒定区中的氨基酸残基的编号是根据Kabat EU编号系统，其也被称为Kabat EU 索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.PublicHealth Service，National Institutes ofHealth，Bethesda，MD，1991中所述。The term "Fc domain" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region. The term includes native sequence Fc domains and variant Fc domains. For example in native antibodies, the Fc domain consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of antibodies in the IgG, IgA and IgD isotypes; IgM and IgE Fc Domains There are three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. In certain embodiments, the Fc domain of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc domain may or may not be present. Unless otherwise stated, the numbering of amino acid residues in the Fc domain or constant region is according to the Kabat EU numbering system, which is also known as the Kabat EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service , National Institutes of Health, Bethesda, MD, 1991.
“功能性Fc结构域”拥有天然序列Fc结构域的“效应器功能”。例示性的“效应器功能”包括C1q结合、CDC、Fc受体结合、ADCC、吞噬作用、细胞表面受体(例如B细胞受体、BCR)下调等。此类效应器功能一般要求Fc结构域与结合结构域(例如抗体可变域)联合，而且可以使用多种测定法来评估，例如本文定义中所公开的。A "functional Fc domain" possesses the "effector functions" of a native sequence Fc domain. Exemplary "effector functions" include Clq binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of cell surface receptors (eg, B cell receptor, BCR), and the like. Such effector functions generally require the association of an Fc domain with a binding domain (eg, an antibody variable domain), and can be assessed using a variety of assays, such as disclosed in the definitions herein.
术语“...价”抗体指抗体分子中存在的抗原结合位点的数目。“二价”、“三价”和“四价”抗体指抗体分子中分别存在2个抗原结合位点、3个结合位点和4个结合位点。在一个实施方案中，这些抗原结合位点可以结合不同的抗原表位，在另一个实施方案中，这些抗原结合位点结合相同的抗原表位。在一个实施方案中，本文中报道的抗体是“四价的”。The term "...valent" antibody refers to the number of antigen-binding sites present in the antibody molecule. "Bivalent", "trivalent" and "tetravalent" antibodies refer to the existence of 2 antigen-binding sites, 3 binding sites and 4 binding sites in the antibody molecule, respectively. In one embodiment, these antigen binding sites may bind different antigenic epitopes, in another embodiment, these antigen binding sites bind the same antigenic epitope. In one embodiment, the antibodies reported herein are "tetravalent".
“多价抗体”是指具有几个但至少四个可变结构域的抗体。有利的是多价抗体具有四个或八个可变结构域，即它是二价或四价的。此外，可变结构域可以彼此相同或不同，使得抗体构建体识别一种或几种抗原或抗原表位。多价抗体优选地识别一种或两种抗原，即它们分别是单特异性或双特异性的。"Multivalent antibody" refers to an antibody having several but at least four variable domains. Advantageously the multivalent antibody has four or eight variable domains, ie it is bivalent or tetravalent. Furthermore, the variable domains may be identical or different from each other such that the antibody construct recognizes one or several antigens or antigenic epitopes. Multivalent antibodies preferably recognize one or two antigens, ie they are monospecific or bispecific, respectively.
与抗体相关的术语“变体”在本文中指，包含已经通过至少1个，例如1-30，或1-20或1-10个，例如1或2或3或4或5个氨基酸取代、缺失和/或插入而具有氨基酸改变的目标抗体区域(例如重链可变区或轻链可变区或重链CDR区或轻链CDR区)的抗体，其中变体基本上保持改变之前的抗体分子的生物学特性。在一方面，本发明涵盖在本文中所述及的任何抗体的变体。在一个实施方案中，抗体变体保持改变前抗体的至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。在一些实施方案中，所述改变不导致抗体变体丧失对抗原的结合，但任选地可以赋予诸如提高的抗原亲和力和不同的效应子功能等性质。可以理解的，抗体的重链可变区或轻链可变区、或各CDR区可以单独改变或组合改变。在一些实施方案中，在一个或多个或全部三个重链CDR中的氨基酸改变不超过1个、2个、3个、4个、5个、6个、7个、8个、9个或10个。优选地，所述氨基酸改变为氨基酸取代，优选保守取代。The term "variant" in relation to an antibody refers herein to an antibody comprising at least 1, such as 1-30, or 1-20 or 1-10, such as 1 or 2 or 3 or 4 or 5 amino acid substitutions, deletions and/or insertions with amino acid changes in the antibody region of interest (e.g. heavy chain variable region or light chain variable region or heavy chain CDR region or light chain CDR region), wherein the variant substantially maintains the antibody molecule before the change biological characteristics. In one aspect, the invention encompasses variants of any of the antibodies described herein. In one embodiment, the antibody variant retains at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the pre-altered antibody. In some embodiments, the alterations do not result in the antibody variant losing binding to the antigen, but may optionally confer properties such as increased affinity for the antigen and different effector functions. It is understood that the antibody heavy chain variable region or light chain variable region, or each CDR region can be altered individually or in combination. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 amino acid changes in one or more or all three heavy chain CDRs or 10. Preferably, the amino acid changes are amino acid substitutions, preferably conservative substitutions.
在一些实施方案中，抗体变体与亲本抗体在目的抗体序列区域上具有至少80％、90％或95％或99％或更高的氨基酸同一性。在另一实施方案中，本发明抗体，与表A-E所列任一抗体相比，在重链可变区上具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％或更高的序列同一性。在再一实施方案中，本发明抗体与表A-E所列任一抗体相比，在轻链可变区上具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％或更高的序列同一性。在再一实施方案中，本发明抗体与表A-E所列任一抗体相比，在重链可变区和/或轻链可变区上具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％或更高的序列同一性。在再一实施方案中，本发明抗体与表A-E所列任一抗体相比，在重链(或重链融合多肽)和/或轻链上具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％或更高的序列同一性。In some embodiments, the antibody variant has at least 80%, 90% or 95% or 99% or more amino acid identity to the parent antibody over the antibody sequence region of interest. In another embodiment, the antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity. In yet another embodiment, the antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity. In yet another embodiment, the antibody of the invention has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more high sequence identity. In yet another embodiment, the antibody of the invention has at least 80%, 81%, 82%, 83% on the heavy chain (or heavy chain fusion polypeptide) and/or light chain compared to any antibody listed in Tables A-E. , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or Higher sequence identity.
表述“接头”是指适于将Fv片段，或者将Fab片段彼此连接的分子。在一个实施方案中，该分子可以是天然肽或者是人工合成的肽。肽接头可以含有任何氨基酸，优选氨基酸甘氨酸(G)，丝氨酸(S)和脯氨酸(P)。肽接头可以彼此相同或不同。肽接头的非限定性例子公开于Shen等人，Anal.Chem.80(6)：1910-1917(2008)、WO2012/138475和WO2014/087010，将其内容全文并入作为参考。此外，肽接头可以具有约0至10个氨基酸的长度。在前一种情况下，肽接头仅是来自可变结构域之一的COOH残基和另一个可变结构域的NH 2残基的肽键。肽接头优选包含氨基酸序列GG。肽接头也可以具有约3至10个氨基酸，特别是5个氨基酸的长度，最特别是氨基酸序列GGGGS，其用于实现Fv片段或者Fab片段的连接。在多价抗体的构建中，优选，接头将利于促使VH和VL配对，且不干扰VH和VL对形成功能有效的抗原结合位点。The expression "linker" refers to molecules suitable for linking Fv fragments, or Fab fragments, to each other. In one embodiment, the molecule may be a natural peptide or a synthetic peptide. The peptide linker may contain any amino acid, preferably the amino acids glycine (G), serine (S) and proline (P). The peptide linkers can be the same or different from each other. Non-limiting examples of peptide linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008), WO2012/138475 and WO2014/087010, the contents of which are incorporated by reference in their entirety. Additionally, the peptide linker can have a length of about 0 to 10 amino acids. In the former case, the peptide linker is simply a peptide bond from a COOH residue of one of the variable domains and an NH2 residue of the other variable domain. The peptide linker preferably comprises the amino acid sequence GG. The peptide linker may also have a length of about 3 to 10 amino acids, in particular 5 amino acids, most particularly the amino acid sequence GGGGS, which is used to effect linking of Fv fragments or Fab fragments. In the construction of multivalent antibodies, preferably, the linker will facilitate pairing of the VH and VL without interfering with the VH and VL pair forming a functionally efficient antigen binding site.
可以用于本发明的肽接头的实例包括：甘氨酸聚合物(G)n；甘氨酸-丝氨酸聚合物(G1-5S1-5)n，其中n是至少1、2、3、4或5的整数；甘氨酸-丙氨酸聚合物；丙氨酸-丝氨酸聚合物；以及本领域已知的其它柔性接头。本领域技术人员可以理解，在一些实施方案中，VH和VL之间的接头可以完全由柔性连接肽组成，或者接头可以由柔性连接肽部分以及赋予较小柔性结构的一个或多个部分组成。Examples of peptide linkers that can be used in the present invention include: glycine polymer (G)n; glycine-serine polymer (G1-5 S1-5 )n, wherein n is at least 1, 2, 3, 4 or 5 Glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Those skilled in the art will appreciate that in some embodiments, the linker between VH and VL may consist entirely of a flexible linker peptide, or the linker may consist of a flexible linker peptide portion and one or more portions that impart less flexible structure.
根据本发明的多价抗体可以通过常规方法产生。一种方法是有利的，其中编码肽接头的DNA与编码多价抗体Fv片段、Fab片段的DNA连接，使得肽接头将Fv片段、Fab片段彼此连接，并且所得的DNA分子在表达质粒中表达。Multivalent antibodies according to the present invention can be produced by conventional methods. A method is advantageous wherein DNA encoding a peptide linker is ligated to DNA encoding a multivalent antibody Fv fragment, Fab fragment such that the peptide linker joins the Fv fragment, Fab fragment to each other and the resulting DNA molecule is expressed in an expression plasmid.
编码根据本发明的多价抗体的DNA也是本发明的主题。The DNA encoding the multivalent antibody according to the invention is also a subject of the invention.
本发明的另一主题涉及试剂盒，其包含：Another subject of the invention relates to a kit comprising:
(a)根据本发明的多价抗体构建体，和/或(a) a multivalent antibody construct according to the invention, and/or
(b)根据本发明的表达质粒、含有质粒的载体或包含载体的宿主细胞，和(b) an expression plasmid, a vector containing the plasmid or a host cell containing the vector according to the invention, and
(c)常规助剂，例如缓冲剂，溶剂和对照。(c) Conventional auxiliaries such as buffers, solvents and controls.
根据本发明的多价抗体完全适于不仅用于诊断而且用于治疗目的。The multivalent antibodies according to the invention are well suited not only for diagnostic but also for therapeutic purposes.
与参照抗体“结合相同表位的抗体”是指这样的抗体，其在竞争测定中阻断50％以上的所述参照抗体与其抗原的结合，反之，参照抗体在竞争测定中阻断50％以上的该抗体与其抗原的结合。An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks more than 50% of the binding of said reference antibody to its antigen in a competition assay, whereas the reference antibody blocks more than 50% in a competition assay binding of the antibody to its antigen.
“抗体依赖性细胞介导的细胞毒性”或“ADCC”指其中结合到某些细胞毒性细胞(例如NK细胞，嗜中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)上的分泌型免疫球蛋白使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞的细胞毒性形式。介导ADCC的主要细胞，NK细胞，只表达FcγRIII，而单核细胞表达FcγRI，FcγRII和FcγRIII。Ravetch和Kinet，Annu.Rev.Immunol.9：457-92(1991)第464页表3总结了造血细胞上的FcR表达。为了评估目的分子的ADCC活性，可进行体外ADCC测定法，诸如美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。或者/另外，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes et al.，PNAS(USA)95：652-656(1998)中所披露的。本文实施例中提供了用于评估ADCC活性的一种例示性测定法。"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to cells that bind to Fc receptors (FcRs) present on certain cytotoxic cells such as NK cells, neutrophils, and macrophages. Secretory immunoglobulins are cytotoxic forms that allow these cytotoxic effector cells to specifically bind antigen-bearing target cells and subsequently kill target cells with cytotoxins. The primary cells that mediate ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta), can be performed. Useful effector cells for such assays include PBMCs and NK cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al., PNAS (USA) 95:652-656 (1998). An exemplary assay for assessing ADCC activity is provided in the Examples herein.
如本文中使用的，“抗体的激动剂活性”指抗体能活化它所结合的抗原的生物学活性。As used herein, "agonist activity of an antibody" refers to the ability of an antibody to activate the biological activity of the antigen to which it binds.
“免疫原性”指特定物质引发免疫应答的能力。肿瘤是免疫原性的，并且增强肿瘤免疫原性有助于通过免疫应答清除肿瘤细胞。"Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity facilitates clearance of tumor cells by immune responses.
“效应子功能”指那些可归于抗体Fc结构域且随抗体同种型而变化的生物学活性。抗体效应子功能的实例包括：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。"Effector functions" refer to those biological activities attributable to the Fc domain of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; body) downregulation; and B cell activation.
“人效应细胞”指表达一种或多种FcR并行使效应器功能的白细胞。在某些实施方案中，该细胞至少表达FcγRIII并行使ADCC效应器功能。介导ADCC的人白细胞的例子包括外周血单个核细胞(PBMC)，天然杀伤(NK)细胞，单核细胞，细胞毒性T细胞和嗜中性粒细胞。效应细胞可以从其天然来源分离，例如血液。"Human effector cells" refer to leukocytes that express one or more FcRs and perform effector functions. In certain embodiments, the cell expresses at least FcyRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils. Effector cells can be isolated from their natural source, such as blood.
适用于本发明的“抗体及其抗原结合片段”包括但不限于多克隆、单克隆、单价、双特异性、异缀合物、多特异性、重组、异源、异源杂合、嵌合、人源化(特别是嫁接有CDR的)、去免疫的、或人的抗体、Fab片段、Fab′片段、F(ab′)2片段、由Fab表达库产生的片段、Fd、Fv、二硫化物连接的Fv(dsFv)、单链抗体(例如scFv)、双链抗体或四抗体(Holliger P.等(1993)Proc.Natl.Acad.Sci.U.S.A.90(14)，6444-6448)、纳米抗体(nanobody)(也称为单域抗体)、抗独特型(抗Id)抗体(包括例如针对本发明抗体的抗Id抗体)和上述任一种的表位结合片段。"Antibodies and antigen-binding fragments thereof" suitable for use in the present invention include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, recombinant, heterologous, heterohybrid, chimeric , humanized (especially CDR-grafted), deimmunized, or human antibodies, Fab fragments, Fab' fragments, F(ab')2 fragments, fragments produced by Fab expression libraries, Fd, Fv, II Sulfide-linked Fv (dsFv), single chain antibody (eg scFv), diabody or tetrabody (Holliger P. et al. (1993) Proc. Natl. Acad. Sci. USA90 (14), 6444-6448), nano Nanobodies (also known as single domain antibodies), anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the foregoing.
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(CDR)。(参见，例如，Kindt等Kuby Immunology，6thed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。The term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, eg, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind a particular antigen can be used to isolate antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
“互补决定区”或“CDR区”或“CDR”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。A "complementarity determining region" or "CDR region" or "CDR" (used interchangeably herein with a hypervariable region "HVR") is the region of amino acids in an antibody variable region that is primarily responsible for binding to an antigenic epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus. The CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
本领域公知多种用于在一个给定的VH或VL氨基酸序列中确定其CDR序列的方案：Kabat互补决定区(CDR)是基于序列变异性确定的并且是最常用的(Kabat等人，Sequencesof Proteins of Immunological Interest，第5版，Public Health Service，NationalInstitutes of Health，Bethesda，Md.(1991))，而Chothia指的是结构环的位置(Chothia等人，(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：877-883)，AbMHVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用，“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。根据不同的CDR确定方案，这些HVR中的每一个HVR/CDR的残基如下所述。Several protocols are known in the art for determining the CDR sequences in a given VH or VL amino acid sequence: Kabat complementarity determining regions (CDRs) are determined based on sequence variability and are the most commonly used (Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)), while Chothia refers to the position of structural loops (Chothia et al., (1987) J.Mol.Biol.196 : 901-917; Chothia et al. (1989) Nature 342: 877-883), AbMHVR is a compromise between the Kabat HVR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software, "Contact" ( Contact) HVR is based on the analysis of available complex crystal structures. According to different CDR determination schemes, the residues of each of these HVRs/CDRs are described below.
在一个实施方案中，本发明抗体的HVR是根据Kabat编号系统位于如下位置的HVR序列：In one embodiment, the HVR of an antibody of the invention is an HVR sequence located according to the Kabat numbering system at the following positions:
VL中的位置24-34(LCDR1)、位置50-56(LCDR2)、和位置89-97位置(LCDR3)，以及VH中的位置27-35B或31-35B(HCDR1)、位置50-65(HCDR2)、和位置93-102或95-102(HCDR3)。Positions 24-34 (LCDR1), positions 50-56 (LCDR2), and positions 89-97 (LCDR3) in VL, and positions 27-35B or 31-35B (HCDR1), positions 50-65 ( HCDR2), and positions 93-102 or 95-102 (HCDR3).
在一个实施方案中，本发明抗体的HVR是根据Kabat编号系统位于如下位置的HVR序列：In one embodiment, the HVR of an antibody of the invention is an HVR sequence located according to the Kabat numbering system at the following positions:
VL中的位置24-34(LCDR1)、位置50-56(LCDR2)、和位置89-97(LCDR3)，以及VH中的位置27-35B(HCDR1)、位置50-65(HCDR2)、和位置93-102(HCDR3)。Positions 24-34 (LCDR1), positions 50-56 (LCDR2), and positions 89-97 (LCDR3) in VL, and positions 27-35B (HCDR1), positions 50-65 (HCDR2), and positions in VH 93-102 (HCDR3).
在一个实施方案中，本发明抗体的HVR是根据Kabat编号系统位于如下位置的HVR序列(Kabat定义的CDR)：In one embodiment, the HVRs of the antibodies of the invention are HVR sequences (CDRs defined by Kabat) located according to the Kabat numbering system at the following positions:
VL中的位置24-34(LCDR1)、位置50-56(LCDR2)、和位置89-97(LCDR3)，以及VH中的位置31-35B(HCDR1)、位置50-65(HCDR2)、和位置95-102(HCDR3)。Positions 24-34 (LCDR1), positions 50-56 (LCDR2), and positions 89-97 (LCDR3) in VL, and positions 31-35B (HCDR1), positions 50-65 (HCDR2), and positions in VH 95-102 (HCDR3).
HVR也可以基于与参考CDR序列(例如本发明示例性CDR之任一)具有相同的Kabat编号位置而确定。HVRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).
除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”或“HVR”或“HVR序列”涵盖以上述任一种方式确定的HVR或CDR序列。Unless otherwise stated, in the present invention, the terms "CDR" or "CDR sequence" or "HVR" or "HVR sequence" encompass HVR or CDR sequences determined in any of the above ways.
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences ofProteins of Immunological Interest，5th Ed.Public Health Service，NationalInstitutes of Health，Bethesda，Md.(1991))的编号位置。Unless otherwise stated, in the present invention, when referring to residue positions in antibody variable regions (including heavy chain variable region residues and light chain variable region residues), it is meant according to the Kabat numbering system ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
在一个实施方案中，本发明CDR序列如下表I所示。In one embodiment, the CDR sequences of the present invention are shown in Table I below.
表I：Table I:
在一个实施方案中，本申请要求保护的抗体的CDR来自表C中所列的VH和/或VL区的CDR。In one embodiment, the CDRs of the antibodies claimed herein are from the CDRs of the VH and/or VL regions listed in Table C.
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat，Chothia，AbM和Contact方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia定义的其余CDR残基可以被保守氨基酸残基替代。Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, the region of minimal overlap can be determined, thereby providing a "minimal binding unit" for antigen binding. A minimal binding unit may be a subsection of a CDR. As will be apparent to those skilled in the art, the residues of the remainder of the CDR sequences can be determined from the structure and protein folding of the antibody. Accordingly, the invention also contemplates variations of any of the CDRs presented herein. For example, in a variant of a CDR, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conserved amino acid residues.
在一些实施方案中，本发明的抗体包含与表A-E所列任一抗体的对应CDR相同的至少一个、两个、三个、四个、五个或六个CDR，或其变体。在一些实施方案中，本发明的抗体包含与表A-E所列任一抗体的对应重链CDR相同的至少一个、两个、或三个HCDR，或其变体。在一些实施方案中，本发明的抗体包含与表A-E所列任一抗体的对应轻链CDR相同的至少一个、两个、或三个HCDR，或其变体。在本文中，“对应CDR”是指在可变区氨基酸序列中位于基本相似位置上的CDR。在本文中，CDR变体是已经通过至少一个，例如1或2或3个氨基酸取代、缺失和/或插入而修饰的CDR，其中包含CDR变体的抗原结合分子基本上保持包含未修饰CDR的抗原结合分子的生物学特性，例如，保持至少60％，70％，80％，90％，或100％的生物学活性(例如抗原结合能力)。可以理解，各CDR可以单独修饰或组合修饰。优选地，氨基酸修饰为氨基酸取代，尤其是保守氨基酸取代，例如说明书中所列出的优选保守氨基酸置换。In some embodiments, an antibody of the invention comprises at least one, two, three, four, five, or six CDRs identical to the corresponding CDRs of any of the antibodies listed in Tables A-E, or a variant thereof. In some embodiments, an antibody of the invention comprises at least one, two, or three HCDRs identical to the corresponding heavy chain CDRs of any of the antibodies listed in Tables A-E, or variants thereof. In some embodiments, an antibody of the invention comprises at least one, two, or three HCDRs identical to the corresponding light chain CDRs of any of the antibodies listed in Tables A-E, or variants thereof. Herein, "corresponding CDRs" refer to CDRs located at substantially similar positions in the amino acid sequence of the variable region. Herein, a CDR variant is a CDR that has been modified by at least one, for example 1 or 2 or 3, amino acid substitutions, deletions and/or insertions, wherein the antigen binding molecule comprising the CDR variant substantially remains comprising the unmodified CDR. A biological property of an antigen binding molecule, eg, retaining at least 60%, 70%, 80%, 90%, or 100% of its biological activity (eg, antigen binding ability). It is understood that the individual CDRs may be modified individually or in combination. Preferably, the amino acid modification is an amino acid substitution, especially a conservative amino acid substitution, such as the preferred conservative amino acid substitutions listed in the specification.
“框架”或“F R”是指除高变区(HVR)(例如互补决定区)残基之外的可变结构域残基。可变结构域的FR通常由四个FR结构域组成：FR1，FR2，FR3和FR4。因此，HVR和FR序列通常出现在重链可变结构域(VH)(或轻链可变结构域(VL))的以下序列中：FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4。"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) (eg complementarity determining region) residues. The FR of a variable domain usually consists of four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences typically appear in the following sequence of the heavy chain variable domain (VH) (or the light chain variable domain (VL)): FR1-H1(L1)-FR2-H2(L2)-FR3 -H3(L3)-FR4.
除非另有说明，抗体各个结构域中的残基的编号根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5thEd.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。Unless otherwise stated, the numbering of residues in the individual domains of antibodies is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health , Bethesda, MD, 1991.
“Fv”是包含完整抗原结合位点的最小抗体片段。在一个实施方案中，双链Fv种类由一个重链可变结构域和一个轻链可变结构域以紧密的，非共价缔合的二聚体组成。在单链Fv(scFv)种类中，一个重链可变结构域和一个轻链可变结构域可以通过柔性肽接头共价连接从而使轻链和重链可以以类似于双链Fv种类的“二聚体”结构缔合。在这种构型中，每个可变结构域的三个HVRs相互作用从而限定在VH-VL二聚体的表面上的抗原结合位点。总而言之，六个HVRs将抗原结合特异性赋予抗体。然而，即使是单个可变结构域(或只包含对抗原特异的三个HVRs的Fv的一半)也具有识别和结合抗原的能力，尽管亲和性低于完整结合位点。关于scFv的综述参见例如Pluckthun于The Pharmacology of MonoclonalAntibodies，卷113，Rosenburg和Moore编辑，(Springer-Verlag，New York，1994)，pp.269-315中。"Fv" is the smallest antibody fragment that contains the complete antigen binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. In single-chain Fv (scFv) species, a heavy-chain variable domain and a light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can be formed in a "scFv" analogous to the two-chain Fv species. Dimer" structure association. In this configuration, the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Altogether, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only the three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the full binding site. For a review of scFv see eg Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York, 1994), pp. 269-315.
“人抗体”指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于这样抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。"Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody library or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
“人共有框架”是指这样的框架，即在选择人免疫球蛋白VL或VH框架序列中，其代表最常出现的氨基酸残基。一般而言，对人免疫球蛋白VL或VH序列的选择是从可变结构域序列的亚型中选择。一般而言，该序列的亚型是如Kabat等，Sequences of Proteins ofImmunological Interest，第五版，NIH Publication 91-3242，Bethesda MD(1991)，1-3卷中的亚型。在一个实施方案中，对于VL，该亚型是如Kabat等(见上文)中的亚型κI。在一个实施方案中，对于VH，该亚型是如Kabat等(见上文)中的亚型III。"Human consensus framework" refers to a framework that represents the most frequently occurring amino acid residues in selected human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences is from among subtypes of variable domain sequences. Generally, the subtypes of the sequences are as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subtype is subtype κI as in Kabat et al. (supra). In one embodiment, for VH, the subtype is subgroup III as in Kabat et al. (supra).
多价抗体根据不同的组成部分以及构建方式，可以分为许多种类。不同的多价抗体设计各有利弊，例如，虽然Blinatumomab可以通过重组中国仓鼠卵巢(CHO)细胞进行大规模培养生产，但是容易形成聚集物、在体内半衰期很短，实际使用的时候需要额外配备连续输液装置；Catumaxomab生产工艺复杂且鼠异源抗体比较容易在人体产生免疫原性问题。此外，四链免疫球蛋白(Ig)样多特异性抗体中非相关重链和轻链的不想要配对导致无活性抗原结合位点和/或其他无功能的不想要的副产物的形成，这在抗体的临床规模生产和治疗性应用中也是一个问题(Klein，C.等人，Progress in overcoming the chainassociation issue in bispecific heterodimeric IgG antibodies，mAbs，2012，4：653-663)。理论上，两条重链能够按四种不同的组合方式缔合，并且这些重链中每一者均可以按随机方式与轻链缔合，产生24(＝总计16)种可能的链组合。在这16种理论上可能的组合当中，实际上发现了10种组合，但其中仅一种组合对应于所需功能性的多特异性抗体。难以从复杂混合物分离出所需的一种多特异性抗体和理论上最大12.5％的固有不良产率致使在细胞表达体系中产生四链Ig样多特异性抗体是困难的。Multivalent antibodies can be divided into many types according to different components and construction methods. Different multivalent antibody designs have their own advantages and disadvantages. For example, although Blinatumomab can be cultured and produced on a large scale by recombinant Chinese hamster ovary (CHO) cells, it is easy to form aggregates and has a short half-life in vivo, so additional equipment is required for actual use. Infusion device; the production process of Catumaxomab is complex and mouse heterologous antibodies are more likely to cause immunogenicity problems in humans. Furthermore, unwanted pairing of unrelated heavy and light chains in four-chain immunoglobulin (Ig)-like multispecific antibodies leads to the formation of inactive antigen-binding sites and/or other non-functional unwanted by-products, which It is also a problem in the clinical-scale production and therapeutic application of antibodies (Klein, C. et al., Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies, mAbs, 2012, 4:653-663). Theoretically, two heavy chains can associate in four different combinations, and each of these heavy chains can associate with a light chain in a random fashion, resulting in24 (=16 total) possible chain combinations . Of these 16 theoretically possible combinations, 10 combinations were actually found, but only one of these combinations corresponded to the desired functional multispecific antibody. The difficulty of isolating the desired one multispecific antibody from a complex mixture and the inherently poor yield of a theoretical maximum of 12.5% make it difficult to produce four-chain Ig-like multispecific antibodies in cell expression systems.
本申请提供了一种新型的多价抗体，其具有4个Fab片段，该构建体在细胞中可以高的纯度和产量表达，优选纯度和/或产量至少为90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。The present application provides a novel multivalent antibody, which has 4 Fab fragments, and the construct can be expressed in cells with high purity and yield, preferably with a purity and/or yield of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
术语“功能障碍”在免疫功能障碍的背景中指降低的对抗原性刺激的免疫响应性的状态。如本文中使用的，术语“功能障碍”还包括对抗原识别的不感受或不响应，特别地，将抗原识别转化成下游T细胞效应器功能，诸如增殖，细胞因子生成(例如γ干扰素)和/或靶细胞杀伤的能力受损。The term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimuli. As used herein, the term "dysfunction" also includes insensitivity or unresponsiveness to antigen recognition, in particular, translation of antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (eg interferon gamma) and/or impaired ability to kill target cells.
“活化T细胞”意指诱导，引起或刺激效应或记忆T细胞具有更新，持续或放大的生物学功能。增强T细胞功能的例子包括：相对于干预前的此类水平，升高的来自CD8+效应T细胞的γ-干扰素(例如IFNg)或白细胞介素(例如IL-2)分泌，升高的来自CD4+记忆和/或效应T细胞的γ-干扰素(例如IFNg)或白细胞介素(例如IL-2)分泌，升高的CD4+效应和/或记忆T细胞增殖，升高的CD8+效应T细胞增殖，升高的抗原响应性(例如清除)。在一个实施方案中，增强的水平是至少50％，或者60％、70％、80％、90％、100％、120％、150％、2倍、3倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、20倍、30倍、40倍、50倍、60倍、70倍、80倍、90倍或100倍。测量此增强的方式是本领域普通技术人员已知的。"Activated T cells" means to induce, elicit or stimulate effector or memory T cells to have renewed, sustained or amplified biological functions. Examples of enhanced T cell function include: increased secretion of gamma-interferon (eg, IFNg) or interleukin (eg, IL-2) from CD8+ effector T cells relative to such levels before intervention, increased Gamma-interferon (eg, IFNg) or interleukin (eg, IL-2) secretion from CD4+ memory and/or effector T cells, elevated CD4+ effector and/or memory T cell proliferation, elevated CD8+ Proliferation of effector T cells, increased antigen responsiveness (eg clearance). In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 2-fold, 3-fold, 3-fold, 4-fold, 5-fold , 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or 100 times. Means of measuring this enhancement are known to those of ordinary skill in the art.
“肿瘤免疫”指肿瘤逃避免疫识别和清除的过程。如此，作为治疗概念，肿瘤免疫在此类逃避减弱时得到“治疗”，并且肿瘤被免疫系统识别并攻击。肿瘤识别的例子包括肿瘤结合，肿瘤收缩和肿瘤清除。"Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "cured" when such evasions are weakened, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
“抗血管发生剂”指阻断或在某种程度上干扰血管发育的化合物。抗血管发生剂可以是例如结合涉及促进血管发生的生长因子或生长因子受体的小分子或抗体。在一个实施方案中，抗血管发生剂是结合血管内皮生长因子(VEGF)的抗体，诸如贝伐单抗(AVASTIN)。"Anti-angiogenic agent" refers to a compound that blocks or interferes in some way with the development of blood vessels. Anti-angiogenic agents can be, for example, small molecules or antibodies that bind to growth factors or growth factor receptors involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds vascular endothelial growth factor (VEGF), such as bevacizumab (AVASTIN).
术语“PD-1轴结合拮抗剂”是指如下的分子，其抑制PD-1轴结合配偶与一种或多种它的结合配偶相互作用，从而去除源自PD-1信号传导轴上的信号传导的T细胞功能障碍，一项结果是恢复或增强T细胞功能(例如增殖，细胞因子生成，靶细胞杀伤)。如本文中使用的，PD-1轴结合拮抗剂包括PD-1结合拮抗剂(例如抗PD-1抗体，例如是WO2015/095423中公开的MDX-1106(nivolumab)、MK-3475(pembrolizumab)、CT-011(pidilizumab)或AMP-224)，PD-L1结合拮抗剂(例如抗PD-L1抗体，在一个具体方面，抗PD-L1抗体是WO2015/095423中公开的YW243.55.S70、MDX-1105、MPDL3280A或MEDI4736)和PD-L2结合拮抗剂(例如抗PD-L2抗体，免疫粘附素)。The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, thereby removing signals originating from the PD-1 signaling axis Transduced T cell dysfunction, one outcome is restoration or enhancement of T cell function (eg, proliferation, cytokine production, target cell killing). As used herein, PD-1 axis binding antagonists include PD-1 binding antagonists (such as anti-PD-1 antibodies, such as MDX-1106 (nivolumab), MK-3475 (pembrolizumab) disclosed in WO2015/095423, CT-011 (pidilizumab) or AMP-224), a PD-L1 binding antagonist (such as an anti-PD-L1 antibody, in a specific aspect, the anti-PD-L1 antibody is YW243.55.S70, MDX disclosed in WO2015/095423 -1105, MPDL3280A or MEDI4736) and PD-L2 binding antagonists (such as anti-PD-L2 antibodies, immunoadhesins).
如本文中使用的，术语“OX40”指来自任何脊椎动物来源，包括哺乳动物诸如灵长类(例如人、食蟹猴)和啮齿类(例如小鼠和大鼠)的任何天然OX40，除非另有说明。该术语涵盖“全长”，未加工的OX40以及因细胞中的加工所致的任何形式的OX40。该术语还涵盖OX40的天然发生变体，例如剪接变体或等位变体。As used herein, the term "OX40" refers to any native OX40 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), unless otherwise There are instructions. The term encompasses "full length", unprocessed OX40 as well as any form of OX40 that results from processing in the cell. The term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.
“OX40活化”指OX40的活化。通常，OX40活化导致信号转导。"OX40 activation" refers to the activation of OX40. Normally, OX40 activation leads to signal transduction.
术语“细胞毒性剂”用在本发明中指抑制细胞功能和/或引起细胞死亡或破坏的物质。细胞毒性剂例子参见WO2015/153513中所公开的那些，包括但不限于：放射性同位素；化学治疗剂或药物；生长抑制剂；酶及其片段，诸如溶核酶；抗生素；毒素，诸如小分子毒素或者细菌，真菌，植物或动物起源的酶活性毒素，包括其片段和/或变体；及各种抗肿瘤或抗癌剂。The term "cytotoxic agent" is used in the present invention to refer to a substance that inhibits cell function and/or causes cell death or destruction. Examples of cytotoxic agents are those disclosed in WO2015/153513, including but not limited to: radioisotopes; chemotherapeutic agents or drugs; growth inhibitors; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antineoplastic or anticancer agents.
“化疗剂”包括在治疗癌症中有用的化学化合物。化疗剂的例子参见WO2015/153513中所公开的那些。化疗剂还包括(i)起调节或抑制激素对肿瘤作用的作用的抗激素剂，诸如抗雌激素类和选择性雌激素受体调控物类(SERM)；(ii)抑制在肾上腺中调节雌激素生成的芳香酶的芳香酶抑制剂；(iii)抗雄激素类；(iv)蛋白质激酶抑制剂；(v)脂质激酶抑制剂；(vi)反义寡核苷酸，特别是抑制牵涉异常细胞增殖的信号传导途经中的基因表达的反义寡核苷酸，诸如例如PKC-α，Ralf和H-Ras；(vii)核酶，诸如VEGF表达抑制剂(例如
术语“细胞因子”是由一种细胞群释放，作为细胞间介质作用于另一细胞的蛋白质的通称。此类细胞因子的例子有淋巴因子，单核因子；白介素(IL)，诸如IL-1，IL-1α，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-11，IL-12，IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本文中使用的，术语细胞因子包括来自天然来源或来自重组细胞培养物的蛋白质及天然序列细胞因子的生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。The term "cytokine" is a general term for proteins released by one cell population to act as intercellular mediators on another cell. Examples of such cytokines are lymphokines, monokines; interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7. IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and kit ligand (KL) and gamma-interferon. As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of native sequence cytokines, including small molecular entities produced artificially, and their pharmaceutically acceptable derivatives and salts.
术语“有效量”指本发明的抗体或片段这样的量或剂量，其以单一或多次剂量施用患者后，在治疗的患者中产生预期效果。有效量可以由作为本领域技术人员的主治医师通过考虑以下多种因素来容易地确定：诸如哺乳动物的物种；它的大小、年龄和一般健康；涉及的具体疾病；疾病的程度或严重性；个体患者的应答；施用的具体抗体；施用模式；施用制剂的生物利用率特征；选择的给药方案；和任何伴随疗法的使用。The term "effective amount" refers to an amount or dose of an antibody or fragment of the present invention that produces the desired effect in a treated patient after administration to the patient in single or multiple doses. An effective amount can be readily determined by the attending physician, who is skilled in the art, by considering various factors such as the species of the mammal; its size, age and general health; the particular disease involved; the extent or severity of the disease; the individual patient's response; the particular antibody administered; the mode of administration; the bioavailability characteristics of the formulation administered; the chosen dosing regimen; and the use of any concomitant therapy.
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的子代。宿主细胞包括“转化体”和“转化的细胞”，其包括初级转化的细胞和来源于其的子代，而不考虑传代的数目。子代在核酸含量上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括与在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体子代。The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.
术语“癌症”和“癌性”指向或描述哺乳动物中特征通常为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃癌(包括胃肠癌和胃肠基质癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，尿道癌，肝瘤，乳腺癌，结肠癌，直肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌，前列腺癌，外阴癌，甲状腺癌，肝癌，肛门癌，阴茎癌，黑素瘤，浅表扩散性黑素瘤，恶性雀斑样痣黑素瘤，肢端黑素瘤，结节性黑素瘤，多发性骨髓瘤和B细胞淋巴瘤，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖，脑瘤和脑癌，以及头颈癌，及相关转移。在某些实施方案中，适合于通过本发明的抗体来治疗的癌症包括肺癌(例如非小细胞肺癌)、肝癌、胃癌或结肠癌，包括那些癌症的转移性形式。The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is often characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal carcinoma , hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, Rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome Symptoms associated with abnormal vascular proliferation, brain tumors and cancers, as well as head and neck cancers, and associated metastases. In certain embodiments, cancers suitable for treatment by antibodies of the invention include lung cancer (eg, non-small cell lung cancer), liver cancer, gastric cancer, or colon cancer, including metastatic forms of those cancers.
术语“细胞增殖性病症”和“增殖性病症”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，细胞增殖性病症指癌症。The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”，“癌性”，“细胞增殖性病症”，“增殖性病症”和“肿瘤”在本文中提到时并不互相排斥。The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive when referred to herein.
“免疫缀合物”是与一个或多个异源分子(包括但不限于细胞毒性剂)缀合的抗体。An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.
“个体”或“受试者”包括哺乳动物。哺乳动物包括但不限于，家养动物(例如，牛，羊，猫，狗和马)，灵长类动物(例如，人和非人灵长类动物如猴)，兔，以及啮齿类动物(例如，小鼠和大鼠)。在一些实施方案中，个体或受试者是人。"Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., , mice and rats). In some embodiments, the individual or subject is a human.
“分离的”抗体是这样的抗体，其已经与其天然环境的组分分离。在一些实施方案中，将抗体纯化至超过95％或99％纯度，如通过例如电泳(例如，SDS-PAGE，等电聚焦(IEF)，毛细管电泳)或层析(例如，离子交换或反相HPLC)确定的。对于用于评估抗体纯度的方法的综述，参见，例如，Flatman等，J.Chromatogr.B848：79-87(2007)。An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, antibodies are purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase determined by HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B848:79-87 (2007).
“分离的”核酸是指这样的核酸分子，其已经与其天然环境的组分分离。分离的核酸包括包含在通常包含该核酸分子的细胞中的核酸分子，但是该核酸分子存在于染色体外或在不同于其天然染色体位置的染色体位置处。An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
“分离的编码多价抗OX40抗体的核酸”是指一个或多个核酸分子，其编码抗体重和轻链，包括在单一载体或分开的载体中的这样的核酸分子，以及存在于宿主细胞中的一个或多个位置处的这样的核酸分子。"Isolated nucleic acid encoding a multivalent anti-OX40 antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains, including such nucleic acid molecules in a single vector or in separate vectors, and present in a host cell Such nucleic acid molecules at one or more positions of .
相对于参比多肽序列的“百分比(％)氨基酸序列同一性”定义为在将所述序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守置换视为序列同一性的部分之后，候选序列中的氨基酸残基与参比多肽序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the sequence after aligning the sequences (and introducing gaps, if necessary) for the greatest percent sequence identity, and not considering any conservative substitutions as Following the portion of sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence. Alignment of sequences to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
当在本申请中提到序列同一性的百分比时，若未另外特别指出，这些百分比相对于较长序列的全长计算。相对于较长序列的全长计算适用于核酸序列和多肽序列两者。Where percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. Full-length calculations relative to longer sequences apply to both nucleic acid and polypeptide sequences.
术语“药物组合物”指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical composition" refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered. .
术语“药用载体”指与治疗剂一起施用的稀释剂、佐剂(例如弗氏佐剂(完全和不完全的))、赋形剂或媒介物。The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient or vehicle with which the therapeutic agent is administered.
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。As used herein, "treating" means slowing, interrupting, arresting, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.
术语“载体”当在本文中使用时是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其可操作相连的核酸的表达。这样的载体在本文中被称为“表达载体”。The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."
“受试者/患者样品”指从癌症患者或癌症受试者得到的细胞或流体的集合。组织或细胞样品的来源可以是实体组织，像来自新鲜的、冷冻的和/或保存的器官或组织样品或活检样品或穿刺样品；血液或任何血液组分；体液，诸如脑脊液、羊膜液(羊水)、腹膜液(腹水)、或间隙液；来自受试者的妊娠或发育任何时间的细胞。组织样品可能包含在自然界中天然不与组织混杂的化合物，诸如防腐剂、抗凝剂、缓冲剂、固定剂、营养物、抗生素、等等。肿瘤样品的例子在本文中包括但不限于肿瘤活检、细针吸出物、支气管灌洗液、胸膜液(胸水)、痰液、尿液、手术标本、循环中的肿瘤细胞、血清、血浆、循环中的血浆蛋白质、腹水、衍生自肿瘤或展现出肿瘤样特性的原代细胞培养物或细胞系，以及保存的肿瘤样品，诸如福尔马林固定的、石蜡包埋的肿瘤样品或冷冻的肿瘤样品。A "subject/patient sample" refers to a collection of cells or fluid obtained from a cancer patient or subject with cancer. The source of the tissue or cell sample can be solid tissue like from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood components; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid ), peritoneal fluid (ascites), or interstitial fluid; cells from any time during pregnancy or development of a subject. Tissue samples may contain compounds that are not naturally intermingled with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsy, fine needle aspirate, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimen, circulating tumor cells, serum, plasma, circulating Plasma proteins in ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples such as formalin-fixed, paraffin-embedded tumor samples, or frozen tumors sample.
术语“包装插页”用于指治疗产品的商业包装中通常包含的用法说明书，其含有关于涉及此类治疗产品应用的适应症，用法，剂量，施用，联合疗法，禁忌症和/或警告的信息。The term "package insert" is used to refer to the instructions commonly included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapies, contraindications and/or warnings concerning the use of such therapeutic products .
实施例Example
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等人，Molecular Cloning：A LaboratoryManual(第3版，2001)；Sambrook等人，Molecular Cloning：A Laboratory Manual(第2版，1989)；Maniatis 等人，Molecular Cloning：A Laboratory Manual(1982)；Ausubel等人，Current Protocols in Molecular Biology(John Wiley和Sons，2008年7月更新)；ShortProtocols in Molecular Biology：A Compendium of Methods from Current Protocolsin Molecular Biology，Greene Pub.Associates和Wiley-Interscience；Glover，DNACloning：A Practical Approach，vol.I&II(IRL Press，Oxford，1985)；Anand，Techniquesfor the Analysis of Complex Genomes，(Academic Press，New York，1992)；Transcription and Translation(B.Hames&S.Higgins，Eds.，1984)；Perbal，A PracticalGuide to Molecular Cloning(1984)；Harlow和Lane，Antibodies，(Cold Spring HarborLaboratory Press，Cold Spring Harbor，N.Y.，1998)Current Protocols in ImmunologyQ.E.Coligan，A.M.Kruisbeek，D.H.Margulies，E.M.Shevach和W.Strober，eds.，1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。The practice of the present invention will employ, unless expressly indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology and cell biology, within the skill of the art. Descriptions of these methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed., 1989); Maniatis et al., Molecular Cloning : A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol.I&II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B.Hames&S Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and journal monographs such as Advances in Immunology.
实施例1.抗OX40单克隆抗体的制备Example 1. Preparation of anti-OX40 monoclonal antibody
抗体生产和纯化Antibody Production and Purification
本发明的示例抗体的CDR区、轻链可变区和重链可变区、轻链和重链的氨基酸序列，以及对应的核苷酸序列在本申请的表A-E部分及“序列表”部分列出。另外，上述本发明示例抗体的轻链可变区和重链可变区的序列编号如下表所示。The amino acid sequences of the CDR region, light chain variable region and heavy chain variable region, light chain and heavy chain of the exemplary antibody of the present invention, and the corresponding nucleotide sequences are listed in Tables A-E and "Sequence Listing" of this application listed. In addition, the sequence numbers of the light chain variable region and the heavy chain variable region of the above-mentioned exemplary antibodies of the present invention are shown in the table below.
表1Table 1
在酵母或CHO-S细胞或HEK293细胞中表达并且纯化本发明的如下抗OX40抗体Express and purify the following anti-OX40 antibodies of the present invention in yeast or CHO-S cells or HEK293 cells
对于酵母材料：For yeast material:
使表达相应抗体的酵母克隆在适当培养基上生长至饱和，然后在30℃下振荡培养并用IPTG诱导48小时。诱导后，将酵母细胞沉淀并收获上清液用于抗体的纯化。根据制造商的说明书，使用蛋白A柱纯化IgG并用乙酸(PH 2.0)洗脱。根据制造商的说明书，使用木瓜蛋白酶消化纯化的抗体以产生相应的Fab片段并通过KappaSelect(GE HealtheareLifeSciences)对该Fab进行纯化。Yeast clones expressing the corresponding antibodies were grown to saturation on appropriate medium, then cultured with shaking at 30°C and induced with IPTG for 48 hours. After induction, the yeast cells were pelleted and the supernatant harvested for antibody purification. IgG was purified using a protein A column and eluted with acetic acid (pH 2.0) according to the manufacturer's instructions. Purified antibodies were digested with papain to generate the corresponding Fab fragments and purified by KappaSelect (GE Healthare Life Sciences) according to the manufacturer's instructions.
对于CHO-S细胞的处理：For the treatment of CHO-S cells:
根据制造商的说明书使用
对于HEK293细胞的处理：For the treatment of HEK293 cells:
对于HEK293细胞中蛋白质的瞬时表达，使用载体PTT5，其中将编码抗体的重链和轻链的DNA通过酶切位点XhoI/NotI分别克隆到单独的载体中。使用本领域的常规标准步骤用PEI(Polysciences)进行对HEK293细胞的转染；之后培养7天，收集上清液，并在AKTA系统(GE)上纯化上清液中表达的相应抗体。For the transient expression of the protein in HEK293 cells, the vector PTT5 was used, in which the DNA encoding the heavy and light chains of the antibody were respectively cloned into separate vectors via restriction sites XhoI/NotI. HEK293 cells were transfected with PEI (Polysciences) using conventional standard procedures in the art; after culturing for 7 days, the supernatant was collected, and the corresponding antibodies expressed in the supernatant were purified on the AKTA system (GE).
获得上述四个示例抗体。Obtain the four example antibodies described above.
对照抗体是The control antibody is
如本文所用，pogalizumab是在HEK293细胞中瞬时表达的人IgG1 OX40抗体，其利用来自INN：列表114(参见http：//www.who.int/medicines/publications/druginformation/innlists/PL114.pdf)的重链和轻链序列。如本文所用，Hu106-222是在HEK293细胞中瞬时表达的人源化IgG1 OX40抗体，其利用来自US9006399的重链和轻链序列。如本文所用，11D4是在HEK293细胞中瞬时表达的人源化IgG1 OX40抗体，其利用来自US8236930的重链和轻链序列。如本文所用，tavolixizumab是在HEK293细胞中瞬时表达的人源化IgG1 OX40抗体，其利用来自INN：列表115(参见http：//www.who.int/medicines/publications/druginformation/innlists/PL115.pdf)的重链和轻链序列。As used herein, pogalizumab isa human IgG1 OX40 antibody transiently expressed in HEK293 cells using the drug from INN: List 114 (seehttp://www.who.int/medicines/publications/druginformation/innlists/PL114.pdf ) heavy and light chain sequences. As used herein, Hu106-222 is a humanized IgG1 OX40 antibody transiently expressed in HEK293 cells utilizing heavy and light chain sequences from US9006399. As used herein, 11D4 is a humanized IgG1 OX40 antibody transiently expressed in HEK293 cells utilizing heavy and light chain sequences from US8236930. As used herein, tavolixizumab is a humanized IgG1 OX40 antibody transiently expressed in HEK293 cells using the drug from INN: List 115 (seehttp://www.who.int/medicines/publications/druginformation/innlists/PL115. pdf ) of the heavy and light chain sequences.
本文中示例性的序列参见如下的表A-E：Exemplary sequences herein are found in Tables A-E below:
实施例2：抗OX40抗体的结合动力学和亲和力测定Example 2: Binding Kinetics and Affinity Determination of Anti-OX40 Antibodies
本申请采用生物光干涉测量(BLI，ForteBio)测定法测定本发明抗体结合人OX40的动力学和平衡解离常数(KD)。The present application adopts bio-light interferometry (BLI, ForteBio) assay to determine the kinetics and equilibrium dissociation constant (KD ) of the antibodies of the present invention binding to human OX40.
BLI KD测定(生物光干涉法)BLI KD determination (bio-light interferometry)
BLI亲和力测定按照现有的方法(Estep，P等人，High throughput solutionBased measurement of antibody-antigen affinity and epitope binning.MAbs，2013.5(2)：p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30分钟，然后线上检测60秒建立基线，在线加载如上所述获得的经纯化的抗体至AHQ传感器(ForteBio)上进行亲和测量。再将具有加载的IgG的传感器暴露于100nM的OX40抗原中作用5分钟，之后将传感器转移至分析缓冲液解离5分钟用于解离速率测量。使用1∶1结合模型进行动力学的分析。The BLI affinity measurement was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): p.270-8). Briefly, the sensor was equilibrated off-line in assay buffer for 30 minutes, followed by online detection for 60 seconds to establish a baseline, and the purified antibody obtained as described above was loaded online onto an AHQ sensor (ForteBio) for affinity measurements. Sensors with loaded IgG were then exposed to 100 nM OX40 antigen for 5 minutes, after which the sensors were transferred to assay buffer and dissociated for 5 minutes for off-rate measurement. Analysis of kinetics was performed using a 1:1 binding model.
在如该测定法所述进行的实验中，ADI-20057、ADI-20112和ADI-23515(在酵母中表达的IgG1形式的抗OX40抗体的Fab)在小于两位数nM范围内以单价KD结合人OX40_Fc(结合了抗体Fc部分的人OX40，购自R&D Systems)。当抗体在传感器尖端上时，ADI-20112、ADI-23515和ADI-20057(以IgG1形式且在酵母中表达)在个位数的纳摩尔至亚纳摩尔的范围以双价(avid)KD值结合人OX40_Fc，ADI-20112、ADI-23515和ADI-20057(以IgG1形式且在酵母中表达)以个位数的纳摩尔水平结合食蟹猴OX40_Fc(cyno OX40_Fc，购自AcroBiosystems)。当抗体在传感器尖端上时，ADI-23515(以IgG1形式且在酵母中表达)以8.98E-09nM结合小鼠OX40_Fc(购自Acro Biosystems)(表2)。作为阳性对照的11D4表现相似的结合亲和力。In experiments performed as described for this assay, ADI-20057, ADI-20112, and ADI-23515 (Fabs of the anti-OX40 antibody expressed in IgG1 format in yeast) exhibited a monovalent K in the less than double-digit nM range.D binds to human OX40_Fc (human OX40 bound to the Fc part of an antibody, purchased from R&D Systems). ADI-20112, ADI-23515, and ADI-20057 (as IgG1 and expressed in yeast) react with bivalent (avid) K in the single-digit nanomolar to subnanomolar range when the antibody is on the sensor tip.D values bind human OX40_Fc, ADI-20112, ADI-23515 and ADI-20057 (as IgG1 and expressed in yeast) bind cynomolgus OX40_Fc (cyno OX40_Fc, purchased from AcroBiosystems) at single digit nanomolar levels. ADI-23515 (as IgG1 expressed in yeast) bound mouse OX40_Fc (purchased from Acro Biosystems) at 8.98E-09nM when the antibody was on the sensor tip (Table 2). 11D4 as a positive control showed similar binding affinities.
表2：通过生物光干涉测量IgG1形式的本发明的抗体的结合Table 2: Binding of antibodies of the invention in IgG1 format measured by bio-optical interferometry
实施例3：本发明抗OX40抗体与人OX40的结合Example 3: Binding of anti-OX40 antibodies of the present invention to human OX40
结合CHO细胞上的人OX40Binds human OX40 on CHO cells
可以在基于流式细胞术的测定法中测量本发明的抗体与人OX40的结合。Binding of antibodies of the invention to human OX40 can be measured in flow cytometry-based assays.
通过多克隆酶切位点(AvrII/PacI)将编码人OX40的cDNA(Sinobiologics)克隆到pCHO1.0载体(Invitrogen)上，然后通过常规转染方法产生并筛选过表达人OX40的CHO细胞(CHO-hOX40细胞)。将CHO-hOX40细胞(细胞密度：0.2×106)与100nM的实验抗体在含0.1％BSA的PBS中在冰上孵育30分钟。然后将细胞洗涤至少两次，并与二抗(藻胆色素蛋白(Phycoerythrin，PE(SouthernBiotech))标记的，终浓度为5μg/ml)在含0.1％BSA的PBS中在冰上(避光)孵育30分钟。将细胞洗涤至少两次并通过流式细胞术进行分析。在Canto II系统(BD Biosciences)上进行流式细胞术，并相应地计算MFI。The cDNA (Sinobiologics) encoding human OX40 was cloned into the pCHO1.0 vector (Invitrogen) through multiple cloning restriction sites (AvrII/PacI), and then the CHO cells (CHO -hOX40 cells). CHO-hOX40 cells (cell density: 0.2×106 ) were incubated with 100 nM of the experimental antibody in PBS containing 0.1% BSA for 30 minutes on ice. Cells were then washed at least twice and incubated with secondary antibody (Phycoerythrin (Phycoerythrin, PE (Southern Biotech))-labeled, final concentration 5 μg/ml) in PBS containing 0.1% BSA on ice (protected from light) Incubate for 30 minutes. Cells were washed at least twice and analyzed by flow cytometry. Flow cytometry was performed on a Canto II system (BD Biosciences) and MFI was calculated accordingly.
实验结果表明：在本测定法中，ADI-20057、ADI-23515和ADI-20112对过表达人OX40的CHO细胞有高的结合强度，在MFI值方面与不表达OX40的CHO细胞相比，具有超过1500倍的差异(表3)。11D4作为阳性对照，也显示出＞1500倍的差异。The experimental results show that: in this assay, ADI-20057, ADI-23515 and ADI-20112 have high binding strength to CHO cells overexpressing human OX40, and have higher MFI values than CHO cells not expressing OX40. A difference of more than 1500-fold (Table 3). 11D4 served as a positive control and also showed a >1500-fold difference.
表3：通过流式细胞术的酵母中产生的本发明IgG1形式的抗体结合表达人OX40的CHO细胞Table 3: Binding of antibodies of the invention in IgG1 format produced in yeast by flow cytometry to CHO cells expressing human OX40
实施例4.在CHO细胞中产生的OX40抗体的药物动力学Example 4. Pharmacokinetics of OX40 Antibody Produced in CHO Cells
药物动力学测定法pharmacokinetic assay
将本发明抗体ADI-20078-IgG1、ADI-20078-IgG2、ADI-20112-IgG1、ADI-20112-IgG2i.v.以10mg/kg剂量给药给雌性Balb/C小鼠后，评估药物动力学数据。基于体重将相应的剂量体积注射到小鼠中。The pharmacokinetics of the antibodies ADI-20078-IgG1, ADI-20078-IgG2, ADI-20112-IgG1, ADI-20112-IgG2 of the present invention were administered i.v. to female Balb/C mice at a dose of 10 mg/kg data. Corresponding dose volumes were injected into mice based on body weight.
在开始给药时开始计时血液采集的时间。对于i.v.施用，时间点包括0.083小时(5分钟)直到第21天。在每个时间点从三只小鼠采集血液。将约100μl血液收集到微量离心管中。将血液样品在冰上在3000RPM的速度设置下储存至少10分钟，以获得约40-50ul的血清。The timing of the blood collection was started at the start of dosing. For i.v. administration, time points included 0.083 hours (5 minutes) until day 21. Blood was collected from three mice at each time point. Approximately 100 μl of blood was collected into microcentrifuge tubes. Blood samples were stored on ice for at least 10 minutes at a speed setting of 3000 RPM to obtain approximately 40-50ul of serum.
通过使用夹心ELISA方法的免疫测定法来测定给药小鼠的抗体的血清浓度。简言之，在0.2M碳酸盐缓冲溶液(CBS)(PH9.4)中在4℃下用lug/mL的OX40(ACRO，目录号#1044-5CIS1-AF)包被微量滴定板(Nunc，cat#442404)过夜。洗涤后，然后将板在环境温度下用5％脱脂乳封闭1.5小时，将小鼠血清样品施加到包被的板上，在室温下持续1.5小时。将抗-OX40抗体稀释于的正常小鼠血清中制备范围为0.315-80ng/mL的标准曲线。The serum concentration of the antibody administered to the mice was determined by immunoassay using the sandwich ELISA method. Briefly, microtiter plates (Nunc , cat#442404) overnight. After washing, the plates were then blocked with 5% skim milk for 1.5 hours at ambient temperature and mouse serum samples were applied to the coated plates for 1.5 hours at room temperature. A standard curve ranging from 0.315-80 ng/mL was prepared by diluting the anti-OX40 antibody in normal mouse serum.
使用在0.05％PBST(0.05％Tween的PBS溶液)、5％BSA中以1∶100,000稀释的辣根过氧化物酶标记的山羊抗人Fc抗体(Bethyl，cat#A80-104P-84)检测结合的抗-OX40抗体。在洗涤步骤后，将板用3，3′，5，5′-四甲基联苯胺盐酸盐溶液(TMB)在室温下显色10-15分钟，在5分钟后通过加入2N硫酸终止。使用Thermo ELISA板读数器(Multiskan FC)，在450nm下测量光密度，减去620nm参考波长。定量基于使用Skanit Software3.1(Thermo)制备的标准曲线的四参数逻辑(1/Y2)回归。Binding was detected using horseradish peroxidase-labeled goat anti-human Fc antibody (Bethyl, cat#A80-104P-84) diluted 1:100,000 in 0.05% PBST (0.05% Tween in PBS), 5% BSA anti-OX40 antibody. After the washing step, the plates were developed with 3,3',5,5'-tetramethylbenzidine hydrochloride solution (TMB) at room temperature for 10-15 minutes and stopped after 5 minutes by the addition of 2N sulfuric acid. Optical density was measured at 450 nm, subtracting the 620 nm reference wavelength, using a Thermo ELISA plate reader (Multiskan FC). Quantification was based on four parameter logistic (1/Y2) regression of a standard curve prepared using Skanit Software 3.1 (Thermo).
通过基于具有各组浓度平均值的非房室模型(non-compartmental model)的PKSolver软件分析PK参数(Cmax，AUC，t1/2，C1和Vss)。PK parameters (Cmax, AUC, t1/2, C1 and Vss) were analyzed by PKSolver software based on a non-compartmental model with mean values of concentrations for each group.
表4Table 4
实施例5.本发明抗体对人OX40L与OX40相互作用的阻断Example 5. Antibody of the present invention blocks interaction between human OX40L and OX40
1.本发明抗体对人OX40L与CHO细胞上的OX40相互作用的阻断1. The antibody of the present invention blocks the interaction between human OX40L and OX40 on CHO cells
可以通过流式细胞术测量本发明的抗体阻断人OX40与OX40L结合的能力。The ability of the antibodies of the invention to block the binding of human OX40 to OX40L can be measured by flow cytometry.
将CHO细胞(密度：0.2×106)与实验抗体(100nM)在含0.1％BSA的PBS中在冰上孵育30分钟。然后将细胞洗涤3次，并与融合至小鼠Fc(～10μg/ml)的OX40L(～10μg/ml)在含0.1％BSA的PBS中在冰上(避光)孵育30分钟。将细胞洗涤3次，随后在含0.1％BSA的PBS中在冰上与抗鼠FC-FITC二抗孵育30分钟。洗涤后通过流式细胞术分析细胞。在Accuri C6系统(BD Biosciences)上进行流式细胞术，并且在C6软件上计算MFI。CHO cells (density: 0.2×106 ) were incubated with experimental antibodies (100 nM) in PBS containing 0.1% BSA for 30 minutes on ice. Cells were then washed 3 times and incubated with OX40L (~10 μg/ml) fused to mouse Fc (~10 μg/ml) in PBS containing 0.1% BSA for 30 minutes on ice (protected from light). Cells were washed 3 times and then incubated with anti-mouse FC-FITC secondary antibody in PBS containing 0.1% BSA for 30 min on ice. Cells were analyzed by flow cytometry after washing. Flow cytometry was performed on an Accuri C6 system (BD Biosciences) and MFI was calculated on C6 software.
实验结果表明：在所述实验中，ADI-20057-g2、ADI-23515-g2和ADI-20112-g1(分别为IgG2(g2)和IgG1(g1)形式，并在CHO细胞中表达)阻断人OX40L(60μg/ml)与OX40(ACRO，目录号#1044-5CIS1-AF)的结合，在较高浓度下与Pogalizumab相比显示较低的阻断活性，但是与OX40L具有相似的水平(图1)。ADI-20057-g2显示非常低的阻断水平，类似于IgG对照(图1)。The experimental results showed that: in the experiment, ADI-20057-g2, ADI-23515-g2 and ADI-20112-g1 (IgG2 (g2) and IgG1 (g1) forms, respectively, and expressed in CHO cells) Blocks the binding of human OX40L (60 μg/ml) to OX40 (ACRO, Cat #1044-5CIS1-AF), showing lower blocking activity compared to Pogalizumab at higher concentrations, but at similar levels to OX40L (figure 1). ADI-20057-g2 showed a very low level of blocking, similar to the IgG control (Figure 1).
2.基于荧光素酶报告基因的OX40阻断测定法2. Luciferase Reporter-Based OX40 Blocking Assay
可以通过测量抗体阻断OX40L介导的OX40活化的性能来评估本发明的抗-OX40抗体的阻断活性。使用抗-CD3(2μg/ml，Biolegend)、抗-CD28(2μg/ml，Biolegend)和重组OX40L(60μg/ml；Acro Biosystems)，与增加浓度的抗-OX40抗体在溶液中持续18小时，活化过表达人OX40和NFkB-荧光素酶构建体的Jurkat细胞，然后在细胞裂解和加入底物后进行检测和吸光度测量。The blocking activity of the anti-OX40 antibodies of the invention can be evaluated by measuring the ability of the antibody to block OX40L-mediated activation of OX40. Using anti-CD3 (2 μg/ml, Biolegend), anti-CD28 (2 μg/ml, Biolegend) and recombinant OX40L (60 μg/ml; Acro Biosystems), with increasing concentrations of anti-OX40 antibodies in solution for 18 hours, activation Jurkat cells overexpressing human OX40 and NFkB-luciferase constructs were then subjected to detection and absorbance measurements following cell lysis and addition of substrate.
结果发现：Pogalizumab在大于约0.08nM或更高的浓度下容易阻断基于OX40L的活化，而在CHO细胞中产生的人IgG1型抗OX40抗体ADI-20112-g1和人IgG2型抗OX40抗体ADI-23515-g2，在20nM及以上浓度可以阻断基于OX40L的活化(图2)。此外发现，抗OX40抗体ADI-20057-g2增加了萤光素酶的水平，表明其具有非OX40L阻断属性并且对OX40聚集具有贡献(图1)。It was found that: Pogalizumab readily blocked OX40L-based activation at concentrations greater than about 0.08 nM or higher, while human IgG type1 anti-OX40 antibody ADI-20112-g1 and human IgG type2 anti-OX40 antibody produced in CHO cells ADI-23515-g2, at concentrations of 20 nM and above, can block OX40L-based activation (Figure 2). Furthermore, the anti-OX40 antibody ADI-20057-g2 was found to increase the level of luciferase, suggesting that it has non-OX40L blocking properties and contributes to OX40 aggregation (Figure 1).
实施例6.本发明的抗OX40抗体的激动剂活性Example 6. Agonist activity of anti-OX40 antibodies of the invention
可以通过测量T细胞活化后由T细胞释放的炎性细胞因子来评价本发明的抗-OX40抗体的激动剂活性。Agonist activity of anti-OX40 antibodies of the invention can be evaluated by measuring inflammatory cytokines released by T cells after T cell activation.
1.混合淋巴细胞反应/DC-T细胞共培养测定法1.Mixed lymphocyte reaction/DC-T cell co-culture assay
可以通过在混合淋巴细胞反应期间或DC-T细胞共培养测定法测量IL-2的释放评估本发明的抗体对OX40信号的激动剂活性。将2×106个PBMC铺板在6孔组织培养板的每个孔中或T25组织培养瓶中，在完全T细胞培养基中，37℃，5％CO2气氛中培养。将细胞孵育2-3小时，以允许单核细胞PBMC附着。如果附着不足，则使用无血清培养基。通过用3X新鲜培养基(X-VIVO 15，Lonza公司)轻轻地涡旋烧瓶除去未附着的细胞。The agonist activity of the antibodies of the invention on OX40 signaling can be assessed by measuring the release of IL-2 during a mixed lymphocyte reaction or in a DC-T cell co-culture assay.
通过在含有1％AB血清、10mM HEPES、50μMβ-Me、IL-4(1000U/ml)和GM-CSF(1000U/ml)，或各25-50ng/ml的X-VIVO 15培养基中培养来自PBMC的单核细胞(1×106个细胞/ml)产生的未成熟骨髓DC细胞。2天后，加入补充有IL-4和GM-CSF的新鲜培养基X-VIVO 15。在第5天，将细胞冷冻或通过加入含有rTNFa(1000U/ml)、IL-1b(5ng/ml)、IL-6(10ng/ml)和1μMPGE2的刺激混合物、以3×105个细胞/ml的细胞密度持续2天诱导成熟。根据在UntouchedCD4+T细胞分离试剂盒(Invitrogen)中制造商的说明书进行T细胞分离。装有1.5ml试管架的磁体用于去除不需要的磁珠(QIAGEN)。Cultured in X-VIVO 15 medium containing 1% AB serum, 10mM HEPES, 50μM β-Me, IL-4 (1000U/ml) and GM-CSF (1000U/ml), or 25-50ng/ml each Immature bone marrow DC cells derived from PBMC monocytes (1×106 cells/ml). After 2 days, fresh medium X-VIVO 15 supplemented with IL-4 and GM-CSF was added. On day 5, cells were either frozen or grown at 3 x105 cells by adding a stimulation mix containing rTNFa (1000 U/ml), IL-1b (5 ng/ml), IL-6 (10 ng/ml) and 1 μM PGE2 . /ml of cell density for 2 days to induce maturation. T cell isolation was performed according to the manufacturer's instructions in the Untouched CD4+ T cell isolation kit (Invitrogen). A magnet fitted with a 1.5ml tube rack was used to remove unwanted magnetic beads (QIAGEN).
将100,000-200,000个分离的T细胞与10,000-20,000个同种异体moDC以总体积200μl在96-圆底组织培养板中混合，在37℃下持续4-5天。加入金黄色葡萄球菌肠毒素E(staphylococcal entertoxin E，缩写为SEE)(1ng/ml)以增加T细胞活化。在混合淋巴细胞实验(MLR)的开始加入测试抗体，并在整个培养期间孵育。根据制造商(eBioscience)的说明书进行IL-2的检测。在Multiskan FC系统(Thermo)上测定OD测量值。100,000-200,000 isolated T cells were mixed with 10,000-20,000 allogeneic moDCs in a total volume of 200 μl in 96-round-bottom tissue culture plates at 37°C for 4–5 days. Staphylococcal enterotoxin E (abbreviated as SEE) (1 ng/ml) was added to increase T cell activation. Test antibodies were added at the beginning of the mixed lymphocyte assay (MLR) and incubated throughout the culture period. Detection of IL-2 was performed according to the manufacturer's (eBioscience) instructions. OD measurements were determined on a Multiskan FC system (Thermo).
结果表明，在本测定中，在CHO细胞中表达的IgG2形式的人抗体ADI-23515-g2和人IgG1形式抗体ADI-20112-g1和在CHO细胞中表达的人IgG2形式的抗体ADI-20057-g2，在～13.3μg/ml、～4.4μg/ml和～1.48μg/ml浓度下增加IL-2的分泌，其增加IL-2分泌的能力等同于或优于pogalizumab、11D4、Hu106-222和tavolixizumab(图3)。Pogalizumab在所有浓度下具有差的激动剂活性，40μg/ml的11D4、Hu106-222、tavolixizumab、ADI-23515-g1和ADI-20112-g1在40μg/ml的最高OX40抗体浓度下显示较低的激动剂活性(图3)。与较低浓度相比，只有40μg/ml的ADI-20057-g2具有相等或更好的IL-2分泌活化。The results show that, in this assay, human antibody ADI-23515-g2 in IgG2 form and antibody ADI-20112-g1 in human IgG1 form expressed in CHO cells and antibody ADI in human IgG2 form expressed in CHO cells -20057-g2, increases IL-2 secretion at ~13.3 μg/ml, ~4.4 μg/ml and ~1.48 μg/ml concentrations, its ability to increase IL-2 secretion is equal to or better than pogalizumab, 11D4, Hu106 -222 and tavolixizumab (Figure 3). Pogalizumab has poor agonist activity at all concentrations, 40 μg/ml of 11D4, Hu106-222, tavolixizumab, ADI-23515-g1 and ADI-20112-g1 showed lower agonism at the highest OX40 antibody concentration of 40 μg/ml agent activity (Figure 3). Only ADI-20057-g2 at 40 μg/ml had equal or better activation of IL-2 secretion compared to lower concentrations.
实施例7：四价(4xFab)mAb的构建、表达、纯化和分析Example 7: Construction, expression, purification and analysis of tetravalent (4xFab) mAb
1.四价(4xFab)mAb的构建和表达1. Construction and expression of tetravalent (4xFab) mAb
天然存在的单克隆抗体可以作为多聚体结构如IgM存在，其增加对固定表面上的其靶抗原的亲合性。因为TNFR的激动作用需要受体聚集，所以四价或六价单克隆抗体可以提供增加的聚集和受体激动作用。本发明是一种mAb，其包含四个相同的Fab区域，具有将远端Fab的C末端CH1区域通过接头或者没有接头而连接到常规mAb上的Fab的N末端上，所述接头例如是GGGGS接头。简言之，为了产生四价mAb，将2个构建体(构建体#1和构建体#2)分别通过常规方法采用XhoI和NotI的酶切位点克隆到HEK293细胞的表达载体pTT5中。具体而言，构建体#1包含编码测试抗体VH-CH1的基因序列，其中测试抗体的VH-CH1(从H1到H113，Kabat编号)直接或通过接头(GGGGS)n(其中n可以为0-4的整数，在本实施例中采用了n＝1)融合到相同抗体重链的N末端(图4)。构建体#2是抗体的正常VL-CL(从L1到L110，Kabat编号)链。将2种构建体在HEK293细胞中共转染，筛选表达和分泌的4xFab mAb分子，并选择正确的分子用于进一步的实验。Naturally occurring monoclonal antibodies may exist as multimeric structures such as IgM, which increase the affinity for their target antigens on immobilized surfaces. Because agonism of TNFR requires receptor aggregation, tetravalent or hexavalent monoclonal antibodies can provide increased aggregation and receptor agonism. The present invention is a mAb comprising four identical Fab regions with the C-terminal CH1 region of the distal Fab joined to the N-terminus of the Fab on a conventional mAb with or without a linker such as GGGGS connector. Briefly, in order to produce tetravalent mAbs, two constructs (
蛋白表达过程如下：The protein expression process is as follows:
1)根据所需转染体积传代HEK293细胞(Invitrogen)，转染前一天将细胞密度调整至1×106/ml。转染当天细胞密度约为2×106/ml；1) Passage HEK293 cells (Invitrogen) according to the required transfection volume, and adjust the cell density to 1×106 /ml one day before transfection. On the day of transfection, the cell density is about 2×106 /ml;
2)取终体积1/10的F17(Gibco，A13835-01)培养基作为转染缓冲液，加入适当的质粒，混匀；2) Take F17 (Gibco, A13835-01) medium of 1/10 of the final volume as the transfection buffer, add appropriate plasmids, and mix well;
3)加合适的PEI(Polysciences，23966)到质粒中，混匀后室温孵10分钟。将混合物轻柔倒入细胞，36.5℃，8％CO2培养；3) Add appropriate PEI (Polysciences, 23966) to the plasmid, mix well and incubate at room temperature for 10 minutes. Gently pour the mixture into the cells and incubate at 36.5°C, 8%CO2 ;
4)过夜后各补加转染体积1/50的200g/L的FEED(Sigma，H6784-100G)和200g/L的葡萄糖母溶液，20小时后加VPA(Gibco，11140-050)至2mM/L；4) Add
5)连续培养至第7天或者细胞活力≤60％时，收集细胞上清用以纯化。5) After continuous culture until day 7 or when the cell viability is ≤60%, the cell supernatant is collected for purification.
2.四价(4xFab)mAb的纯化和分析2. Purification and Analysis of Tetravalent (4xFab) mAb
A.纯化和尺寸排阻色谱法(SEC)A. Purification and Size Exclusion Chromatography (SEC)
上述获得的4xFab mAb分子在HiTrapTMMabSelect SuReTM5ml柱(GE 11-0034-93)上根据制造商的说明进行纯化，其中使用20mMTris+150mM氯化钠(pH7.2)作为结合缓冲液。采用洗脱缓冲液(0.1M柠檬酸钠，pH 3.5)洗脱抗体，并用1M Tris-Hcl将洗脱的抗体溶液的pH调节至6。使用Mono S 5/50柱(GE 17-5168-01)根据制造商的说明书，通过离子交换法(CEX)进一步纯化各个4xFab mAb分子，例如ADI-20112-g1-4xFab。The 4xFab mAb molecules obtained above were purified on a HiTrap™ MabSelect SuRe™ 5 ml column (GE 11-0034-93) according to the manufacturer's instructions using 20 mM Tris + 150 mM NaCl (pH 7.2) as binding buffer. The antibody was eluted with an elution buffer (0.1M sodium citrate, pH 3.5), and the pH of the eluted antibody solution was adjusted to 6 with 1M Tris-Hcl. Each 4xFab mAb molecule, eg ADI-20112-g1-4xFab, was further purified by ion exchange (CEX) using a Mono S 5/50 column (GE 17-5168-01) according to the manufacturer's instructions.
SEC分析方法可用于测试纯化后4xFab抗体的聚集倾向。使用TSKgel SuperSW3000柱(18675)以0.35mL/min的速率进行mAb的快速SEC分析，循环时间为15分钟/运行。将20mM磷酸钠和150mM氯化钠的溶液调节至pH 6.8，用作流动相。注射体积为20μl，在280nm检测UV信号。The SEC analysis method can be used to test the aggregation propensity of purified 4xFab antibodies. Fast SEC analysis of mAb was performed using a TSKgel SuperSW3000 column (18675) at a rate of 0.35 mL/min with a cycle time of 15 min/run. A solution of 20 mM sodium phosphate and 150 mM sodium chloride adjusted to pH 6.8 was used as the mobile phase. The injection volume was 20 [mu]l and the UV signal was detected at 280 nm.
结果表明，作为四价分子的IgG2形式的人OX40抗体ADI-20057-g2-4xFab、ADI-23515-g2-4xFab(图4)、作为四价分子的IgG1形式的人OX40抗体ADI-20112-g1-4xFab(图4)，可以在单一蛋白A纯化步骤之后以高纯度表达和纯化，ADI-20057-g2-4xFab和ADI-23515-g2-4xFab的纯度分别为98.72％(图5)和98.51％(图6)。以亲和层析+离子交换+分子筛法的三步法将ADI-20112-g1-4xFab纯化至99.22％纯度(图7)。The results showed that the human OX40 antibody ADI-20057-g2-4xFab, ADI-23515-g2-4xFab (Figure 4) as a tetravalent molecule in the IgG2 format, the human OX40 antibody ADI-20112 in the IgG1 format as a tetravalent molecule -g1-4xFab (Figure 4), which can be expressed and purified with high purity after a single protein A purification step, the purity of ADI-20057-g2-4xFab and ADI-23515-g2-4xFab were 98.72% (Figure 5) and 98.51% (Figure 6). ADI-20112-g1-4xFab was purified to a purity of 99.22% by a three-step method of affinity chromatography + ion exchange + molecular sieve ( FIG. 7 ).
实施例8.本发明的抗OX40抗体的激动剂活性Example 8. Agonist activity of anti-OX40 antibodies of the invention
荧光素酶报告基因T细胞活化测定法Luciferase Reporter T Cell Activation Assay
可以通过在荧光素酶报告基因测定法中测量NFkB介导的转录活化的促进来评估本发明的抗-OX40抗体的激动剂活性。通过常规方法构建过表达人OX40(Sinobiologics)和NFkB-荧光素酶(Promega)构建体的Jurkat细胞，然后用抗-CD3(2μg/ml；Biolegend)与抗-CD28(2μg/ml；Biolegend)抗体活化过表达人OX40和NFkB-荧光素酶构建体的Jurkat细胞，37℃持续18小时，然后根据本领域技术人员已知的常规方法裂解细胞，向细胞裂解液中添加底物(购自Promega公司)并引发生物发光，之后在检测装置(Molecular Devices)上测试，其指示诱导表达的荧光素酶的相对值。Agonist activity of anti-OX40 antibodies of the invention can be assessed by measuring the promotion of NFkB-mediated transcriptional activation in a luciferase reporter gene assay. Construct Jurkat cells overexpressing human OX40 (Sinobiologics) and NFkB-luciferase (Promega) constructs by conventional methods, and then use anti-CD3 (2 μg/ml; Biolegend) and anti-CD28 (2 μg/ml; Biolegend) antibodies Activation of Jurkat cells overexpressing human OX40 and NFkB-luciferase constructs was continued at 37°C for 18 hours, and then the cells were lysed according to conventional methods known to those skilled in the art, and substrates (purchased from Promega Corporation) were added to the cell lysate. ) and elicit bioluminescence before testing on a detection device (Molecular Devices), which indicates the relative value of luciferase induced expression.
在如上所述测定法进行的实验中，添加Raji细胞以提供共刺激信号和用于IgG交联的表达FcgRIIb的细胞表面，并使用本发明的以下抗体：在CHO细胞中表达的IgG2形式的人抗-OX40抗体ADI-23515-g2、IgG1形式的人抗-OX40抗体ADI-20112-g1和IgG2形式的人抗OX40抗体ADI-20057-g2。In experiments performed with the assay described above, Raji cells were added to provide co-stimulatory signals and FcgRIIb-expressing cell surfaces for IgG crosslinking, and the following antibodies of the invention were used:IgG2 form expressed in CHO cells Human anti-OX40 antibody ADI-23515-g2, human anti-OX40 antibody ADI-20112-g1 in IgG1 format and human anti-OX40 antibody ADI-20057-g2 in IgG2 format.
结果表明，使用抗CD3/抗CD28抗体作为T细胞活化剂，本发明的抗OX40抗体均具有良好的激动剂活性。例如，人抗-OX40抗体ADI-23515-g2、ADI-20112-g1和ADI-20057-g2具有良好的激动剂活性。The results show that the anti-OX40 antibodies of the present invention all have good agonist activity using anti-CD3/anti-CD28 antibodies as T cell activators. For example, human anti-OX40 antibodies ADI-23515-g2, ADI-20112-g1 and ADI-20057-g2 have good agonist activity.
根据上述公开的方法和步骤，在HEK293细胞中瞬时表达本发明的4xFab分子人抗-OX40抗体ADI-23515-g2-4xFab(IgG2形式)、人抗-OX40-4xFab抗体ADI-20112-g1-4xFab(IgG1形式)和人抗-OX40抗体ADI-20057-g2-4xFab(IgG2形式)。然后评估本发明的4xFab分子人抗-OX40抗体的激动剂活性。According to the methods and steps disclosed above, the 4xFab molecules of the present invention, human anti-OX40 antibody ADI-23515-g2-4xFab (IgG2 format), human anti-OX40-4xFab antibody ADI-20112-g1- 4xFab (IgG1 format) and human anti-OX40 antibody ADI-20057-g2-4xFab (IgG2 format). The 4xFab molecule human anti-OX40 antibodies of the invention were then assessed for agonist activity.
实验结果表明，在较低浓度，与各个4xFab分子相应的常规IgG对应物相比，所有4xFab抗-OX40抗体均具有更好的激动剂活性(图8)。此外，相比较于在本测定中显示很少或没有激动剂活性的tavolixumab和pogalizumab基准，上述4xFab分子的激动剂活性很高。Experimental results showed that at lower concentrations, all 4xFab anti-OX40 antibodies had better agonist activity than the respective conventional IgG counterparts of the respective 4xFab molecules (Figure 8). Furthermore, the agonist activity of the 4xFab molecules described above was high compared to the benchmarks tavolixumab and pogalizumab which showed little or no agonist activity in this assay.
与没有Raji细胞相比，当将Raji细胞引入实验中时，Tavolixizumab、pogalizumab和ADI-20112-g1在较高浓度下都具有增加的激动剂活性(图9)。Tavolixizumab, pogalizumab and ADI-20112-g1 all had increased agonist activity at higher concentrations when Raji cells were introduced into the experiment compared to no Raji cells (Figure 9).
实施例9.基于荧光素酶报告基因的OX40阻断测定法Example 9. Luciferase Reporter Based OX40 Blocking Assay
可以通过测量抗体阻断OX40L介导的OX40活化的性能来评估本发明的抗-OX40抗体的阻断活性。使用抗-CD3(2μg/ml；Biolegend)、抗-CD28(2μg/ml(Biolegend)和重组OX40L(60μg/ml；Acro Biosystems)，与增加浓度的抗-OX40抗体在溶液中持续18小时，活化过表达人OX40和NFkB-荧光素酶构建体的Jurkat细胞，然后在细胞裂解和加入底物后进行检测和吸光度测量。The blocking activity of the anti-OX40 antibodies of the invention can be evaluated by measuring the ability of the antibody to block OX40L-mediated activation of OX40. Using anti-CD3 (2 μg/ml; Biolegend), anti-CD28 (2 μg/ml (Biolegend) and recombinant OX40L (60 μg/ml; Acro Biosystems), with increasing concentrations of anti-OX40 antibodies in solution for 18 hours, activation Jurkat cells overexpressing human OX40 and NFkB-luciferase constructs were then subjected to detection and absorbance measurements following cell lysis and addition of substrate.
在基本上如上述测定法中进行实验，其中在CHO细胞中表达人抗-OX40抗体ADI-23515-g2、人抗-OX40抗体ADI-20112-g1、人抗-OX40抗体ADI-20057-g2。在HEK293细胞中瞬时表达人抗-OX40抗体ADI-23515-g2-4xFab、人抗-OX40IgG1-4xFab抗体ADI-20112-g1-4xFab，和人抗-OX40抗体ADI-20057-g2-4xFab。Experiments were performed in assays essentially as described above, wherein human anti-OX40 antibody ADI-23515-g2, human anti-OX40 antibody ADI-20112-g1, human anti-OX40 antibody ADI-20057-g2 were expressed in CHO cells. Human anti-OX40 antibody ADI-23515-g2-4xFab, human anti-OX40 IgG1-4xFab antibody ADI-20112-g1-4xFab, and human anti-OX40 antibody ADI-20057-g2-4xFab were transiently expressed in HEK293 cells.
结果表明，ADI-20112-g1和ADI-20112-g1-4xFab具有低的OX40L阻断功能。ADI-23515-g2在高于～0.5ug/ml的浓度下显示OX40L阻断，而ADI-23515-g2-4xFab在～0.18ug/ml或以上显示增加的信号(图10)。所有浓度的ADI-20057-g2和ADI-20057-g2-4xFab显示OX40的活化增加(图10)。由于ADI-20057-g2是非配体阻断剂(图2)，这解释了将ADI-20057-g2和ADI-20057-g2-4xFab加入实验中如何可以加强其受体的基于OX40L的活化。这也在DC-T细胞共培养实验中观察到(图3)。The results showed that ADI-20112-g1 and ADI-20112-g1-4xFab have low OX40L blocking function. ADI-23515-g2 showed OX40L blocking at concentrations above ~0.5ug/ml, while ADI-23515-g2-4xFab showed increased signal at ~0.18ug/ml or above (Figure 10). All concentrations of ADI-20057-g2 and ADI-20057-g2-4xFab showed increased activation of OX40 (Figure 10). Since ADI-20057-g2 is a non-ligand blocker (Figure 2), this explains how the addition of ADI-20057-g2 and ADI-20057-g2-4xFab to the experiment can enhance OX40L-based activation of their receptors. This was also observed in DC-T cell co-culture experiments (Fig. 3).
序列表 sequence listing
<110> 信达生物制药（苏州）有限公司<110> Innovent Biopharmaceutical (Suzhou) Co., Ltd.
<120> 多价抗OX40抗体及其用途<120> Multivalent anti-OX40 antibody and use thereof
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<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 10<400> 10
Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu AsnGln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
1 5 101 5 10
<210> 11<210> 11
<211> 12<211> 12
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 11<400> 11
Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu AlaArg Ala Ser Gln Ser Val Ser Ser Ser Ser Tyr Leu Ala
1 5 101 5 10
<210> 12<210> 12
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 12<400> 12
Asp Ala Ser Asn Leu Glu ThrAsp Ala Ser Asn Leu Glu Thr
1 51 5
<210> 13<210> 13
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 13<400> 13
Gly Ala Ser Ser Arg Ala ThrGly Ala Ser Ser Arg Ala Thr
1 51 5
<210> 14<210> 14
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 14<400> 14
Gln Gln Ser Ala Asn Tyr Pro Tyr ThrGln Gln Ser Ala Asn Tyr Pro Tyr Thr
1 51 5
<210> 15<210> 15
<211> 9<211> 9
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 15<400> 15
Gln Gln Ser Glu Arg Ser Pro Phe ThrGln Gln Ser Glu Arg Ser Pro Phe Thr
1 51 5
<210> 16<210> 16
<211> 8<211> 8
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 16<400> 16
Gln Gln Ser Asp His Tyr Pro ThrGln Gln Ser Asp His Tyr Pro Thr
1 51 5
<210> 17<210> 17
<211> 26<211> 26
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 17<400> 17
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser GlySer Val Lys Val Ser Cys Lys Ala Ser Gly
20 25 20 25
<210> 18<210> 18
<211> 26<211> 26
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 18<400> 18
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly
20 25 20 25
<210> 19<210> 19
<211> 26<211> 26
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 19<400> 19
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly ArgGln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser GlySer Leu Arg Leu Ser Cys Ala Ala Ser Gly
20 25 20 25
<210> 20<210> 20
<211> 14<211> 14
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 20<400> 20
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met GlyTrp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
1 5 101 5 10
<210> 21<210> 21
<211> 14<211> 14
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 21<400> 21
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile GlyTrp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly
1 5 101 5 10
<210> 22<210> 22
<211> 14<211> 14
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 22<400> 22
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val AlaTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala
1 5 101 5 10
<210> 23<210> 23
<211> 30<211> 30
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 23<400> 23
Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met GluArg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu
1 5 10 151 5 10 15
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysLeu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
20 25 30 20 25 30
<210> 24<210> 24
<211> 30<211> 30
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 24<400> 24
Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu LysArg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys
1 5 10 151 5 10 15
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr CysLeu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
20 25 30 20 25 30
<210> 25<210> 25
<211> 30<211> 30
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 25<400> 25
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu GlnArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln
1 5 10 151 5 10 15
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
20 25 30 20 25 30
<210> 26<210> 26
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 26<400> 26
Trp Gly Arg Gly Thr Leu Val Thr Val Ser SerTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
1 5 101 5 10
<210> 27<210> 27
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 27<400> 27
Trp Gly Gln Gly Thr Thr Val Thr Val Ser SerTrp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
1 5 101 5 10
<210> 28<210> 28
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 28<400> 28
Trp Gly Gln Gly Thr Met Val Thr Val Ser SerTrp Gly Gln Gly Thr Met Val Thr Val Ser Ser
1 5 101 5 10
<210> 29<210> 29
<211> 23<211> 23
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 29<400> 29
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr CysAsp Arg Val Thr Ile Thr Cys
20 20
<210> 30<210> 30
<211> 23<211> 23
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 30<400> 30
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro GlyGlu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 151 5 10 15
Glu Arg Ala Thr Leu Ser CysGlu Arg Ala Thr Leu Ser Cys
20 20
<210> 31<210> 31
<211> 15<211> 15
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 31<400> 31
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile TyrTrp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr
1 5 10 151 5 10 15
<210> 32<210> 32
<211> 15<211> 15
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 32<400> 32
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile TyrTrp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
1 5 10 151 5 10 15
<210> 33<210> 33
<211> 32<211> 32
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 33<400> 33
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrGly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 151 5 10 15
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr CysPhe Thr Ile Ser Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys
20 25 30 20 25 30
<210> 34<210> 34
<211> 32<211> 32
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 34<400> 34
Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrGly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 151 5 10 15
Leu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr CysLeu Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
20 25 30 20 25 30
<210> 35<210> 35
<211> 10<211> 10
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 35<400> 35
Phe Gly Gly Gly Thr Lys Val Glu Ile LysPhe Gly Gly Gly Thr Lys Val Glu Ile Lys
1 5 101 5 10
<210> 36<210> 36
<211> 123<211> 123
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 36<400> 36
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp MetTyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys PheGly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val TyrGln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp LeuAla Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu
100 105 110 100 105 110
Trp Gly Arg Gly Thr Leu Val Thr Val Ser SerTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser
115 120 115 120
<210> 37<210> 37
<211> 122<211> 122
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 37<400> 37
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly
20 25 30 20 25 30
Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluAla Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser SerGly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 115 120
<210> 38<210> 38
<211> 124<211> 124
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 38<400> 38
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly ArgGln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe AspAla Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp
100 105 110 100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser SerIle Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 115 120
<210> 39<210> 39
<211> 107<211> 107
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 39<400> 39
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn TyrAsp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro TyrGlu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr
85 90 95 85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile LysThr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 100 105
<210> 40<210> 40
<211> 108<211> 108
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 40<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro GlyGlu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 151 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser SerGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuTyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe SerIle Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu GluGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 8065 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Glu Arg Ser ProPro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Glu Arg Ser Pro
85 90 95 85 90 95
Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile LysPhe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 100 105
<210> 41<210> 41
<211> 106<211> 106
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 41<400> 41
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn TyrAsp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp His Tyr Pro ThrGlu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp His Tyr Pro Thr
85 90 95 85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile LysPhe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 100 105
<210> 42<210> 42
<211> 453<211> 453
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 42<400> 42
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp MetTyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys PheGly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val TyrGln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp LeuAla Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu
100 105 110 100 105 110
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys GlyTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125 115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly GlyPro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140 130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro ValThr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr PheThr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175 165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValPro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val
180 185 190 180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn ValThr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205 195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro LysAsn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys
210 215 220 210 215 220
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu LeuSer Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240225 230 235 240
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp ThrLeu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
245 250 255 245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp ValLeu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
260 265 270 260 265 270
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly ValSer His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
275 280 285 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn SerGlu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300 290 295 300
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp LeuThr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
305 310 315 320305 310 315 320
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro AlaAsn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
325 330 335 325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu ProPro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
340 345 350 340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn GlnGln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
355 360 365 355 360 365
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile AlaVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
370 375 380 370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr ThrVal Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
385 390 395 400385 390 395 400
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys LeuPro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
405 410 415 405 410 415
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys SerThr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
435 440 445 435 440 445
Leu Ser Pro Gly LysLeu Ser Pro Gly Lys
450 450
<210> 43<210> 43
<211> 448<211> 448
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 43<400> 43
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp MetTyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys PheGly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val TyrGln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp LeuAla Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu
100 105 110 100 105 110
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys GlyTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125 115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu SerPro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
130 135 140 130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro ValThr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr PheThr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175 165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValPro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val
180 185 190 180 185 190
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn ValThr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val
195 200 205 195 200 205
Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg LysAsp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys
210 215 220 210 215 220
Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly ProCys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
225 230 235 240225 230 235 240
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu AspArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
260 265 270 260 265 270
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnPro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
275 280 285 275 280 285
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg ValAla Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val
290 295 300 290 295 300
Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys GluVal Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320305 310 315 320
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu LysTyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys
325 330 335 325 330 335
Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrThr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350 340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu ThrLeu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
355 360 365 355 360 365
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp GluCys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met LeuSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu
385 390 395 400385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp LysAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
405 410 415 405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His GluSer Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyAla Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445 435 440 445
<210> 44<210> 44
<211> 214<211> 214
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 44<400> 44
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn TyrAsp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro TyrGlu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Ala Asn Tyr Pro Tyr
85 90 95 85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala AlaThr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser GlyPro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu AlaThr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser GlnLys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu SerGlu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val TyrSer Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys SerAla Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 195 200 205
Phe Asn Arg Gly Glu CysPhe Asn Arg Gly Glu Cys
210 210
<210> 45<210> 45
<211> 452<211> 452
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 45<400> 45
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly
20 25 30 20 25 30
Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluAla Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser
210 215 220 210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu LeuCys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255 245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270 260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val GluHis Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285 275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser ThrVal His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnTyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala ProGly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335 325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro GlnIle Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350 340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln ValVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365 355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380 370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415 405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430 420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 435 440 445
Ser Pro Gly LysSer Pro Gly Lys
450 450
<210> 46<210> 46
<211> 447<211> 447
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 46<400> 46
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly
20 25 30 20 25 30
Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluAla Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser ThrSer Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val AspVal Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys CysHis Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
210 215 220 210 215 220
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro SerCys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
225 230 235 240225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255 245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp ProThr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270 260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaGlu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285 275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val ValLys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
290 295 300 290 295 300
Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu TyrSer Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys ThrLys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335 325 330 335
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuIle Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr CysPro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365 355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380 370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu AspAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
385 390 395 400385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys SerSer Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaArg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445 435 440 445
<210> 47<210> 47
<211> 215<211> 215
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 47<400> 47
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro GlyGlu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 151 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser SerGlu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser
20 25 30 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuTyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 35 40 45
Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe SerIle Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60 50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu GluGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 8065 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Glu Arg Ser ProPro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Glu Arg Ser Pro
85 90 95 85 90 95
Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val AlaPhe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110 100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerAla Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125 115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg GluGly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140 130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerAla Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuGln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175 165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys ValSer Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190 180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr LysTyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 195 200 205
Ser Phe Asn Arg Gly Glu CysSer Phe Asn Arg Gly Glu Cys
210 215 210 215
<210> 48<210> 48
<211> 454<211> 454
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 48<400> 48
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly ArgGln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe AspAla Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp
100 105 110 100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr LysIle Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140 130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProGly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His ThrVal Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175 165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValPhe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val
180 185 190 180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys AsnVal Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu ProVal Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro
210 215 220 210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro GluLys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspLeu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255 245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val AspThr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270 260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp GlyVal Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285 275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr AsnVal Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300 290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp TrpSer Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu ProLeu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335 325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg GluAla Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350 340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys AsnPro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
355 360 365 355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp IleGln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380 370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrAla Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser LysThr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415 405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser CysLeu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430 420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser LeuSer Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445 435 440 445
Ser Leu Ser Pro Gly LysSer Leu Ser Pro Gly Lys
450 450
<210> 49<210> 49
<211> 449<211> 449
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 49<400> 49
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly ArgGln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe AspAla Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp
100 105 110 100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr LysIle Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser GluGly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
130 135 140 130 135 140
Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProSer Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His ThrVal Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175 165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValPhe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val
180 185 190 180 185 190
Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys AsnVal Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn
195 200 205 195 200 205
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu ArgVal Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg
210 215 220 210 215 220
Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala GlyLys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly
225 230 235 240225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met IlePro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255 245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluSer Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270 260 265 270
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285 275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe ArgAsn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
290 295 300 290 295 300
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly LysVal Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile GluGlu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu
325 330 335 325 330 335
Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val TyrLys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350 340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser LeuThr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365 355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu TrpThr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380 370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro MetGlu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met
385 390 395 400385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLeu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415 405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met HisLys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser ProGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445 435 440 445
GlyGly
<210> 50<210> 50
<211> 213<211> 213
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 50<400> 50
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val GlyAsp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 151 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn TyrAsp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser GlyTyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln ProSer Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 8065 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp His Tyr Pro ThrGlu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Asp His Tyr Pro Thr
85 90 95 85 90 95
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala ProPhe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110 100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly ThrSer Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125 115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala LysAla Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140 130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln GluVal Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser SerSer Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175 165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr AlaThr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190 180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser PheCys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205 195 200 205
Asn Arg Gly Glu CysAsn Arg Gly Glu Cys
210 210
<210> 51<210> 51
<211> 688<211> 688
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 51<400> 51
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp MetTyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys PheGly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val TyrGln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp LeuAla Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu
100 105 110 100 105 110
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys GlyTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125 115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly GlyPro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140 130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro ValThr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr PheThr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175 165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValPro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val
180 185 190 180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn ValThr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205 195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro LysAsn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220 210 215 220
Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln LeuSer Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu
225 230 235 240225 230 235 240
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys ValVal Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
245 250 255 245 250 255
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His TrpSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser Tyr Tyr Met His Trp
260 265 270 260 265 270
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile AsnVal Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn
275 280 285 275 280 285
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg ValPro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val
290 295 300 290 295 300
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu SerThr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser
305 310 315 320305 310 315 320
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp HisSer Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp His
325 330 335 325 330 335
Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu Trp Gly Arg GlyAla Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu Trp Gly Arg Gly
340 345 350 340 345 350
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val PheThr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
355 360 365 355 360 365
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala LeuPro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
370 375 380 370 375 380
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
385 390 395 400385 390 395 400
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 405 410 415
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro SerGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
420 425 430 420 425 430
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys ProSer Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
435 440 445 435 440 445
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp LysSer Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
450 455 460 450 455 460
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly ProThr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
465 470 475 480465 470 475 480
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
485 490 495 485 490 495
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu AspArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
500 505 510 500 505 510
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnPro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
515 520 525 515 520 525
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValAla Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
530 535 540 530 535 540
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys GluVal Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
545 550 555 560545 550 555 560
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu LysTyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
565 570 575 565 570 575
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr ThrThr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
580 585 590 580 585 590
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu ThrLeu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
595 600 605 595 600 605
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp GluCys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
610 615 620 610 615 620
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuSer Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
625 630 635 640625 630 635 640
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp LysAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
645 650 655 645 650 655
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His GluSer Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
660 665 670 660 665 670
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyAla Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685 675 680 685
<210> 52<210> 52
<211> 684<211> 684
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 52<400> 52
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly AlaGln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 151 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser TyrSer Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 20 25 30
Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp MetTyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45 35 40 45
Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys PheGly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
50 55 60 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val TyrGln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr
65 70 75 8065 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr CysMet Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp LeuAla Arg Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu
100 105 110 100 105 110
Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys GlyTrp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
115 120 125 115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly GlyPro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140 130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro ValThr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr PheThr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175 165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValPro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val Val
180 185 190 180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn ValThr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205 195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro LysAsn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
210 215 220 210 215 220
Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln LeuSer Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu
225 230 235 240225 230 235 240
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys ValVal Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val
245 250 255 245 250 255
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His TrpSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser Tyr Tyr Met His Trp
260 265 270 260 265 270
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile AsnVal Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn
275 280 285 275 280 285
Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg ValPro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val
290 295 300 290 295 300
Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu SerThr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser
305 310 315 320305 310 315 320
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp HisSer Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp His
325 330 335 325 330 335
Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu Trp Gly Arg GlyAla Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu Trp Gly Arg Gly
340 345 350 340 345 350
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val PheThr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
355 360 365 355 360 365
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala LeuPro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
370 375 380 370 375 380
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
385 390 395 400385 390 395 400
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuAsn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
405 410 415 405 410 415
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro SerGln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
420 425 430 420 425 430
Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys ProSer Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro
435 440 445 435 440 445
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val GluSer Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu
450 455 460 450 455 460
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe LeuCys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
465 470 475 480465 470 475 480
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro GluPhe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
485 490 495 485 490 495
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val GlnVal Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
500 505 510 500 505 510
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr LysPhe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
515 520 525 515 520 525
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val LeuPro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
530 535 540 530 535 540
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys LysThr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
545 550 555 560545 550 555 560
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser LysVal Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
565 570 575 565 570 575
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro SerThr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
580 585 590 580 585 590
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val LysArg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
595 600 605 595 600 605
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly GlnGly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
610 615 620 610 615 620
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp GlyPro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
625 630 635 640625 630 635 640
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp GlnSer Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
645 650 655 645 650 655
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His AsnGln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
660 665 670 660 665 670
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 675 680
<210> 53<210> 53
<211> 687<211> 687
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 53<400> 53
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly
20 25 30 20 25 30
Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluAla Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220 210 215 220
Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu GlnCys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
225 230 235 240225 230 235 240
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu ThrGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
245 250 255 245 250 255
Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly Ala Tyr Tyr Trp SerCys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly Ala Tyr Tyr Trp Ser
260 265 270 260 265 270
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr IleTrp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile
275 280 285 275 280 285
Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg ValTyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val
290 295 300 290 295 300
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu SerThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
305 310 315 320305 310 315 320
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp ValSer Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val
325 330 335 325 330 335
Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr ThrGly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
340 345 350 340 345 350
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
355 360 365 355 360 365
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly CysAla Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
370 375 380 370 375 380
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SerLeu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
385 390 395 400385 390 395 400
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
405 410 415 405 410 415
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser SerSer Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
420 425 430 420 425 430
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser AsnLeu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
435 440 445 435 440 445
Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr HisThr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His
450 455 460 450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser ValThr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPhe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495 485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro GluPro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510 500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala LysVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525 515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val SerThr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540 530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr LysVal Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr IleCys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575 565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu ProSer Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
580 585 590 580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys LeuPro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
595 600 605 595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620 610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp SerGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640625 630 635 640
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgAsp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655 645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala LeuTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670 660 665 670
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685 675 680 685
<210> 54<210> 54
<211> 682<211> 682
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 54<400> 54
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly
20 25 30 20 25 30
Ala Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluAla Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
210 215 220 210 215 220
Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu GlnCys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
225 230 235 240225 230 235 240
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu ThrGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
245 250 255 245 250 255
Cys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly Ala Tyr Tyr Trp SerCys Thr Val Ser Gly Gly Ser Ile Arg Ser Gly Ala Tyr Tyr Trp Ser
260 265 270 260 265 270
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr IleTrp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile
275 280 285 275 280 285
Tyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg ValTyr Tyr Asp Gly Gln Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val
290 295 300 290 295 300
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu SerThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
305 310 315 320305 310 315 320
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp ValSer Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val
325 330 335 325 330 335
Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr ThrGly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
340 345 350 340 345 350
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
355 360 365 355 360 365
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly CysAla Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
370 375 380 370 375 380
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SerLeu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
385 390 395 400385 390 395 400
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
405 410 415 405 410 415
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser AsnSer Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Asn
420 425 430 420 425 430
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser AsnPhe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
435 440 445 435 440 445
Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys ProThr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
450 455 460 450 455 460
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe ProPro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
465 470 475 480465 470 475 480
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val ThrPro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
485 490 495 485 490 495
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe AsnCys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
500 505 510 500 505 510
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro ArgTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
515 520 525 515 520 525
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr ValGlu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
530 535 540 530 535 540
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val SerVal His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
545 550 555 560545 550 555 560
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr LysAsn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
565 570 575 565 570 575
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg GluGly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
580 585 590 580 585 590
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheGlu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
595 600 605 595 600 605
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluTyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
610 615 620 610 615 620
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser PheAsn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
625 630 635 640625 630 635 640
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyPhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
645 650 655 645 650 655
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His TyrAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
660 665 670 660 665 670
Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyThr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 675 680
<210> 55<210> 55
<211> 691<211> 691
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 55<400> 55
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly ArgGln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe AspAla Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp
100 105 110 100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr LysIle Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140 130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProGly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His ThrVal Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175 165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValPhe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val
180 185 190 180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys AsnVal Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu ProVal Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro
210 215 220 210 215 220
Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val GlnLys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln
225 230 235 240225 230 235 240
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu ArgLeu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg
245 250 255 245 250 255
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met HisLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His
260 265 270 260 265 270
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val IleTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile
275 280 285 275 280 285
Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly ArgSer Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg
290 295 300 290 295 300
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln MetPhe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met
305 310 315 320305 310 315 320
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg GlyAsn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
325 330 335 325 330 335
Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile Trp GlyArg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile Trp Gly
340 345 350 340 345 350
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerGln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
355 360 365 355 360 365
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr AlaVal Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
370 375 380 370 375 380
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
385 390 395 400385 390 395 400
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
405 410 415 405 410 415
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
420 425 430 420 425 430
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisPro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
435 440 445 435 440 445
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser CysLys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys
450 455 460 450 455 460
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyAsp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
465 470 475 480465 470 475 480
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu MetGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
485 490 495 485 490 495
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
500 505 510 500 505 510
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValGlu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
515 520 525 515 520 525
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrHis Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
530 535 540 530 535 540
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyArg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
545 550 555 560545 550 555 560
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleLys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
565 570 575 565 570 575
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValGlu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
580 585 590 580 585 590
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val SerTyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
595 600 605 595 600 605
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluLeu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
610 615 620 610 615 620
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
625 630 635 640625 630 635 640
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValVal Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
645 650 655 645 650 655
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetAsp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
660 665 670 660 665 670
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerHis Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
675 680 685 675 680 685
Pro Gly LysPro Gly Lys
690 690
<210> 56<210> 56
<211> 686<211> 686
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 56<400> 56
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly ArgGln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 151 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser TyrSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ValGly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser ValAla Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu TyrLys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 8065 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr CysLeu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Ala Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe AspAla Arg Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp
100 105 110 100 105 110
Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr LysIle Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125 115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140 130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu ProGly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His ThrVal Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175 165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValPhe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Ser Val
180 185 190 180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys AsnVal Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205 195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu ProVal Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro
210 215 220 210 215 220
Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val GlnLys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln
225 230 235 240225 230 235 240
Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu ArgLeu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg
245 250 255 245 250 255
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met HisLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met His
260 265 270 260 265 270
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val IleTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Val Ile
275 280 285 275 280 285
Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly ArgSer Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg
290 295 300 290 295 300
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln MetPhe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met
305 310 315 320305 310 315 320
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg GlyAsn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly
325 330 335 325 330 335
Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile Trp GlyArg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile Trp Gly
340 345 350 340 345 350
Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerGln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
355 360 365 355 360 365
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr AlaVal Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
370 375 380 370 375 380
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr ValAla Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
385 390 395 400385 390 395 400
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaSer Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
405 410 415 405 410 415
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValVal Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
420 425 430 420 425 430
Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp HisPro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His
435 440 445 435 440 445
Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys CysLys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys
450 455 460 450 455 460
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser ValVal Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
465 470 475 480465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPhe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495 485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro GluPro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510 500 505 510
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala LysVal Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525 515 520 525
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val SerThr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
530 535 540 530 535 540
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr LysVal Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560545 550 555 560
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr IleCys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575 565 570 575
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu ProSer Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
580 585 590 580 585 590
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys LeuPro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
595 600 605 595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620 610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp SerGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
625 630 635 640625 630 635 640
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgAsp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655 645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala LeuTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670 660 665 670
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 685 675 680 685
<210> 57<210> 57
<211> 5<211> 5
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 57<400> 57
Gly Gly Gly Gly SerGly Gly Gly Gly Ser
1 51 5
<210> 58<210> 58
<211> 20<211> 20
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 58<400> 58
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val ProMet Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 151 5 10 15
Gly Ser Thr GlyGly Ser Thr Gly
20 20
<210> 59<210> 59
<211> 11<211> 11
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 59<400> 59
Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp SerGly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp Ser
1 5 101 5 10
<210> 60<210> 60
<211> 16<211> 16
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 60<400> 60
Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys SerTyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser
1 5 10 151 5 10 15
<210> 61<210> 61
<211> 122<211> 122
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 61<400> 61
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Gly
20 25 30 20 25 30
Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluSer Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser SerGly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 115 120
<210> 62<210> 62
<211> 452<211> 452
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 62<400> 62
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Gly
20 25 30 20 25 30
Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluSer Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser
210 215 220 210 215 220
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu LeuCys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
225 230 235 240225 230 235 240
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
245 250 255 245 250 255
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerMet Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
260 265 270 260 265 270
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val GluHis Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285 275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser ThrVal His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
290 295 300 290 295 300
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnTyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
305 310 315 320305 310 315 320
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala ProGly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
325 330 335 325 330 335
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro GlnIle Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
340 345 350 340 345 350
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln ValVal Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
355 360 365 355 360 365
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValSer Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
370 375 380 370 375 380
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProGlu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
385 390 395 400385 390 395 400
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrPro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
405 410 415 405 410 415
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValVal Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
420 425 430 420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuMet His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
435 440 445 435 440 445
Ser Pro Gly LysSer Pro Gly Lys
450 450
<210> 63<210> 63
<211> 447<211> 447
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 63<400> 63
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Gly
20 25 30 20 25 30
Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluSer Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser ThrSer Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val AspVal Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys CysHis Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys
210 215 220 210 215 220
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro SerCys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
225 230 235 240225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser ArgVal Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255 245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp ProThr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270 260 265 270
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaGlu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285 275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val ValLys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
290 295 300 290 295 300
Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu TyrSer Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320305 310 315 320
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys ThrLys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335 325 330 335
Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuIle Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr CysPro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365 355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380 370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu AspAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
385 390 395 400385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys SerSer Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415 405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu AlaArg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyLeu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445 435 440 445
<210> 64<210> 64
<211> 687<211> 687
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 64<400> 64
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Gly
20 25 30 20 25 30
Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluSer Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser
210 215 220 210 215 220
Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu GlnCys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
225 230 235 240225 230 235 240
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu ThrGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
245 250 255 245 250 255
Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp SerCys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp Ser
260 265 270 260 265 270
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr IleTrp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile
275 280 285 275 280 285
Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg ValTyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val
290 295 300 290 295 300
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu SerThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
305 310 315 320305 310 315 320
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp ValSer Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val
325 330 335 325 330 335
Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr ThrGly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
340 345 350 340 345 350
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
355 360 365 355 360 365
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly CysAla Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
370 375 380 370 375 380
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SerLeu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
385 390 395 400385 390 395 400
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
405 410 415 405 410 415
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser SerSer Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
420 425 430 420 425 430
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser AsnLeu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
435 440 445 435 440 445
Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr HisThr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys Asp Lys Thr His
450 455 460 450 455 460
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser ValThr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
465 470 475 480465 470 475 480
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg ThrPhe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
485 490 495 485 490 495
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro GluPro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
500 505 510 500 505 510
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala LysVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
515 520 525 515 520 525
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val SerThr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
530 535 540 530 535 540
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr LysVal Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
545 550 555 560545 550 555 560
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr IleCys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
565 570 575 565 570 575
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu ProSer Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
580 585 590 580 585 590
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys LeuPro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
595 600 605 595 600 605
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
610 615 620 610 615 620
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp SerGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
625 630 635 640625 630 635 640
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgAsp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
645 650 655 645 650 655
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala LeuTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
660 665 670 660 665 670
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly LysHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
675 680 685 675 680 685
<210> 65<210> 65
<211> 682<211> 682
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 65<400> 65
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser GlnGln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 151 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser GlyThr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser Gly
20 25 30 20 25 30
Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu GluSer Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 35 40 45
Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro SerTrp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln PheLeu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
65 70 75 8065 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr TyrSer Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95 85 90 95
Cys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val TrpCys Ala Arg Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp
100 105 110 100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly ProGly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
115 120 125 115 120 125
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrSer Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
130 135 140 130 135 140
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrAla Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
145 150 155 160145 150 155 160
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProVal Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175 165 170 175
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val ThrAla Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
180 185 190 180 185 190
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnVal Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys SerHis Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser
210 215 220 210 215 220
Cys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu GlnCys Asp Lys Thr His Thr Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
225 230 235 240225 230 235 240
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu ThrGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
245 250 255 245 250 255
Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp SerCys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Ser Tyr Tyr Trp Ser
260 265 270 260 265 270
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr IleTrp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile
275 280 285 275 280 285
Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg ValTyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val
290 295 300 290 295 300
Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu SerThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
305 310 315 320305 310 315 320
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp ValSer Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Val
325 330 335 325 330 335
Gly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr ThrGly Tyr Pro His Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr
340 345 350 340 345 350
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuVal Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
355 360 365 355 360 365
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly CysAla Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys
370 375 380 370 375 380
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn SerLeu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser
385 390 395 400385 390 395 400
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln SerGly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
405 410 415 405 410 415
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser AsnSer Gly Leu Tyr Ser Leu Ser Ser Ser Val Val Thr Val Pro Ser Ser Asn
420 425 430 420 425 430
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser AsnPhe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
435 440 445 435 440 445
Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys ProThr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro
450 455 460 450 455 460
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe ProPro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro
465 470 475 480465 470 475 480
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val ThrPro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
485 490 495 485 490 495
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe AsnCys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn
500 505 510 500 505 510
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro ArgTrp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
515 520 525 515 520 525
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr ValGlu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
530 535 540 530 535 540
Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val SerVal His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
545 550 555 560545 550 555 560
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr LysAsn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
565 570 575 565 570 575
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg GluGly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
580 585 590 580 585 590
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly PheGlu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
595 600 605 595 600 605
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro GluTyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
610 615 620 610 615 620
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser PheAsn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
625 630 635 640625 630 635 640
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln GlyPhe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
645 650 655 645 650 655
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His TyrAsn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
660 665 670 660 665 670
Thr Gln Lys Ser Leu Ser Leu Ser Pro GlyThr Gln Lys Ser Leu Ser Leu Ser Pro Gly
675 680 675 680
<210> 66<210> 66
<211> 5<211> 5
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 66<400> 66
Ser Tyr Tyr Met HisSer Tyr Tyr Met His
1 51 5
<210> 67<210> 67
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 67<400> 67
Ser Gly Ala Tyr Tyr Trp SerSer Gly Ala Tyr Tyr Trp Ser
1 51 5
<210> 68<210> 68
<211> 5<211> 5
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 68<400> 68
Ser Tyr Gly Met HisSer Tyr Gly Met His
1 51 5
<210> 69<210> 69
<211> 7<211> 7
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 69<400> 69
Ser Gly Ser Tyr Tyr Trp SerSer Gly Ser Tyr Tyr Trp Ser
1 51 5
<210> 70<210> 70
<211> 14<211> 14
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 70<400> 70
Asp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp LeuAsp His Ala Ser Ser Ser Trp Tyr Thr Thr His Leu Asp Leu
1 5 101 5 10
<210> 71<210> 71
<211> 12<211> 12
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 71<400> 71
Asp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp ValAsp Val Gly Tyr Pro His Tyr Tyr Gly Met Asp Val
1 5 101 5 10
<210> 72<210> 72
<211> 15<211> 15
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 抗体/抗体片段序列<223> Antibody/antibody fragment sequence
<400> 72<400> 72
Gly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp IleGly Arg Pro Trp Tyr Ser Glu Thr Gly Thr Ser Ala Phe Asp Ile
1 5 10 151 5 10 15
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Applications Claiming Priority (2)
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| CN113045654A (en)* | 2019-12-27 | 2021-06-29 | 南开大学 | anti-OX 40 antibodies and uses thereof |
| CN111704671B (en)* | 2020-08-19 | 2020-11-24 | 广东赛尔生物科技有限公司 | OX40 antibody and its application in the treatment of cancer |
| CN117229396A (en)* | 2022-06-06 | 2023-12-15 | 普米斯生物技术(珠海)有限公司 | anti-CD 40 antibodies and uses thereof |
| CN114966061B (en)* | 2022-07-28 | 2022-10-21 | 中国食品药品检定研究院 | A kind of biological activity detection method of anti-OX40 antibody |
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| EP2857516B1 (en)* | 2000-04-11 | 2017-06-14 | Genentech, Inc. | Multivalent antibodies and uses therefor |
| CA2756244A1 (en)* | 2009-04-02 | 2010-10-07 | Roche Glycart Ag | Multispecific antibodies comprising full length antibodies and single chain fab fragments |
| GB201005063D0 (en)* | 2010-03-25 | 2010-05-12 | Ucb Pharma Sa | Biological products |
| GB0920127D0 (en)* | 2009-11-17 | 2009-12-30 | Ucb Pharma Sa | Antibodies |
| WO2014100490A1 (en)* | 2012-12-19 | 2014-06-26 | Adimab, Llc | Multivalent antibody analogs, and methods of their preparation and use |
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