WO2021032174A1 - 一种抗cd47抗原结合蛋白及其应用 - Google Patents

一种抗cd47抗原结合蛋白及其应用 Download PDF

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WO2021032174A1
WO2021032174A1 PCT/CN2020/110342 CN2020110342W WO2021032174A1 WO 2021032174 A1 WO2021032174 A1 WO 2021032174A1 CN 2020110342 W CN2020110342 W CN 2020110342W WO 2021032174 A1 WO2021032174 A1 WO 2021032174A1
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amino acid
seq
acid sequence
sequence shown
antigen binding
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PCT/CN2020/110342
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French (fr)
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F• 格罗斯维尔德
D• 德拉贝克
M（R）• 范哈佩恩
R• 扬森
王路凡
刘斌
戎一平
何云
周伟
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和铂医药(上海)有限责任公司
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Priority to CN202080005950.7A priority Critical patent/CN112969715B/zh
Publication of WO2021032174A1 publication Critical patent/WO2021032174A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application relates to the field of biomedicine, in particular to an anti-CD47 antigen binding protein and its application.
  • CD47 is also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-associated antigen and MER6. It is a transmembrane protein widely expressed on the surface of cells and belongs to the immunoglobulin superfamily. CD47 can interact with integrin, thrombospondin (TSP-1), and signal regulatory protein alpha (SIRP ⁇ ). CD47 was initially identified as a tumor antigen on human ovarian cancer. Subsequent studies have confirmed that CD47 is also present in most human cancers (eg, lymphoma, leukemia, breast cancer, colon cancer, lung cancer, gastric cancer, glioblastoma, neuroglioma).
  • CD47 antibodies under clinical research include Forty Seven's Hu5F9-G4, Celgene's CC-90002, and Surface Oncology's SRF-231.
  • these antibodies also have the disadvantages of low affinity and cause hemagglutination reaction (see patent US9045541). Therefore, there is an urgent need to develop safer and more effective new therapies targeting CD47.
  • This application provides an anti-CD47 antigen binding protein, which has one or more of the following properties: 1) can bind CD47 derived from humans and monkeys; 2) can specifically bind to the surface of Jurkat cells and Raji cells CD47; 3) can inhibit the binding of CD47 and SIRP ⁇ ; 4) can block the binding of CD47 and SIRPa on the surface of Jurkat cells; 5) can bind to human red blood cells; 6) do not cause coagulation reaction; 7) can cause macrophages to tumor Phagocytosis of cells; 8) can inhibit tumor growth and/or tumor cell proliferation.
  • the application also provides the application of the antigen binding protein in the prevention and treatment of tumors.
  • the present application provides an isolated antigen binding protein comprising an antibody light chain variable region VL and an antibody heavy chain variable region VH, the VL comprising LCDR1, LCDR2 and LCDR3, and the VH comprising HCDR1 and HCDR2 And HCDR3, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 153, and the VH comprises the amino acid sequence shown in SEQ ID NO: 154.
  • the isolated antigen binding protein has one or more of the following properties:
  • the isolated antigen binding protein includes an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment in the isolated antigen-binding protein includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the antibody in the isolated antigen binding protein is a fully human antibody.
  • LCDR1 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 139.
  • the LCDR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 113, 114, and 115.
  • the LCDR2 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 140.
  • the LCDR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 31, 56, 57, 122, 126, and 127.
  • the LCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 141.
  • the HCDR1 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:142.
  • the HCDR1 in the isolated antigen binding protein comprises SEQ ID NO: 32, 34, 36, 38, 40, 42, 45, 47, 49, 51, and 53. The amino acid sequence shown.
  • the HCDR2 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:143.
  • the HCDR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 136-138.
  • the HCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:144.
  • the HCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 55 and 58-63.
  • the VL in the isolated antigen binding protein includes the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 in the isolated antigen binding protein is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the sequence shown in SEQ ID NO: 149 Amino acid sequence.
  • the L-FR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 156, 157, and 158.
  • the L-FR2 in the isolated antigen binding protein is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 150.
  • the L-FR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 191-194.
  • the L-FR3 in the isolated antigen binding protein is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid sequence shown in SEQ ID NO: 151.
  • the L-FR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 155, 162-166.
  • the N-terminus of the L-FR4 in the isolated antigen binding protein is directly or indirectly connected to the C-terminus of the LCDR3, and the L-FR4 includes the sequence shown in SEQ ID NO: 152 Amino acid sequence.
  • the L-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 159, 160, and 161.
  • the VL in the isolated antigen binding protein comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 The amino acid sequence shown in any one of and 29.
  • the isolated antigen binding protein includes an antibody light chain constant region, and the antibody light chain constant region includes a human Ig ⁇ constant region.
  • the antibody light chain constant region in the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO: 196.
  • the isolated antigen binding protein comprises an antibody light chain LC
  • the LC comprises the amino acid sequence shown in SEQ ID NO:197.
  • the isolated antigen binding protein comprises an antibody light chain LC
  • the LC comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 The amino acid sequence shown in any one of, 22, 24, 26, 28, and 30.
  • the VH in the isolated antigen binding protein includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 in the isolated antigen binding protein is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the sequence shown in SEQ ID NO: 145 Amino acid sequence.
  • the H-FR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 167-170.
  • the H-FR2 in the isolated antigen binding protein is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 146.
  • the H-FR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 184 and 187-190.
  • the H-FR3 in the isolated antigen binding protein is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid sequence shown in SEQ ID NO: 147.
  • the H-FR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID Nos: 171-182.
  • the N-terminus of the H-FR4 in the isolated antigen binding protein is directly or indirectly connected to the C-terminus of the HCDR3, and the H-FR4 includes the sequence shown in SEQ ID NO: 148 Amino acid sequence.
  • the H-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 183, 185 and 186.
  • the VH in the isolated antigen binding protein comprises SEQ ID NO: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 , 103, 105, 107, 109, and 111.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG constant region.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region includes a human IgG4 constant region.
  • the antibody heavy chain constant region in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO: 195.
  • the isolated antigen binding protein comprises an antibody heavy chain HC
  • the HC comprises the amino acid sequence shown in SEQ ID NO:198.
  • the isolated antigen binding protein comprises an antibody heavy chain HC
  • the HC comprises SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 , 98, 100, 102, 104, 106, 108, 110, and 112.
  • the present application provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein described in the present application.
  • the present application provides a vector, which contains the nucleic acid molecule described in the present application.
  • this application provides a cell comprising the nucleic acid molecule described in this application or the vector described in this application.
  • this application provides a method for preparing the isolated antigen binding protein described in this application, the method comprising culturing the isolated antigen binding protein described in this application under conditions Cell.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated antigen binding protein described in this application, the nucleic acid molecule described in this application, the vector described in this application, and/or the cell described in this application , And optionally a pharmaceutically acceptable carrier.
  • this application provides an isolated antigen binding protein described in this application, a nucleic acid molecule described in this application, a vector described in this application, a cell described in this application, and/or a cell described in this application.
  • the use, wherein the tumor includes solid tumors and/or hematological tumors.
  • this application provides a method for inhibiting the binding of CD47 to SIRP ⁇ , the method comprising administering the isolated antigen binding protein described in this application.
  • the present application provides a method for preventing, alleviating and/or treating tumors, the method comprising administering the isolated antigen binding protein described in this application to a subject in need.
  • FIGS 1A-1B show the binding activity of the antigen-binding protein described in this application to Jurkat cells (FACS detection);
  • Figure 1C shows the binding activity of the antigen binding proteins PR000806, PR000807 and PR000808 described in this application to Raji cells (FACS detection);
  • Figure 2 shows the binding activity of the antigen binding proteins PR000796, PR000802, PR000803, PR000804, PR000805, PR000806, PR000807, PR000808 and PR000811 with cynomolgus monkey PBMC cells (FACS detection) of the present application;
  • Figure 3A shows the binding activity of the antigen binding proteins PR000796, PR000802, PR000803, PR000804, PR000805, PR000806, PR000807, PR000808, and PR000811 with monkey CD47 protein (detected by ELISA) of the present application;
  • Figures 3B-3C show the binding activity of the antigen-binding proteins PR000806, PR000807 and PR000808 described in this application to human and monkey CD47 proteins (detected by ELISA);
  • Figures 4A-4B show the activity of the antigen binding proteins PR000796, PR000802, PR000803, PR000804, PR000805, PR000806, PR000807, PR000808 and PR000811 in inhibiting the binding of human CD47 to SIRP ⁇ (detected by ELISA);
  • Figures 5A-5B show the activity of the antigen binding proteins PR000796, PR000802, PR000803, PR000804, PR000805, PR000806, PR000807, PR000808 and PR000811 in blocking the binding of CD47 and SIRP ⁇ on the surface of Jurkat cells (FACS detection);
  • Figures 6A-6B show that the antigen binding protein described in this application does not cause agglutination of human erythrocytes
  • Figure 7 shows the binding ability of the antigen binding protein described in this application to red blood cells (FACS detection);
  • Figures 8A-8B show the ability of the antigen binding proteins PR000796, PR000802, PR000803, PR000804, PR000805, PR000806, PR000807, PR000808, and PR000811 described in this application to induce the phagocytosis of Jurkat cells by macrophages;
  • Figure 9 shows the ability of the antigen binding proteins PR000806, PR000807 and PR000808 of the present application to induce phagocytosis of OVCAR3 cells by macrophages;
  • Figure 10 shows the ability of the antigen binding proteins PR001265, PR001275, PR001276, PR000806, PR000808, and PR001281 of the present application to induce the phagocytosis of human red blood cells by macrophages;
  • Figures 11A-11B show the in vivo anti-tumor activity of the antigen-binding proteins PR000806 and PR000808 described in this application at different concentrations in the NCG-Raji mouse model, which are the tumor volume change (A) and the mouse body weight change (B), respectively ;
  • Figures 12A-12B show the in vivo anti-tumor activity of the antigen-binding proteins PR001265 and PR000806 described in this application at different concentrations in the B-hSIRPa/hCD47-MC38-hCD47 mouse model, which are tumor volume changes (A) and small Rat body weight change (B);
  • Figure 13 shows the trend of weight changes in mice before and after treatment with the antigen binding proteins PR001265, PR001275, PR001276, PR000806, PR000808, and PR001281 described in this application;
  • Figure 14 shows the routine blood test results of a mouse model 7 days before treatment with the antigen binding proteins PR001265, PR001275, PR001276, PR000806, PR000808 and PR001281 described in this application;
  • Figure 15 shows the routine blood test results of a mouse model on the first day after treatment with the antigen binding proteins PR001265, PR001275, PR001276, PR000806, PR000808, and PR001281 described in this application;
  • Figure 16 shows the routine blood test results of a mouse model on the 7th day after treatment with the antigen binding proteins PR001265, PR001275, PR001276, PR000806, PR000808 and PR001281 described in this application;
  • Figure 17 shows the use of the antigen binding proteins PR001265, PR001275, and PR001276 described in this application to highly specifically bind to CD47, but not to non-CD47 antigens.
  • the term "antigen binding protein” generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or framework portion that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen binding protein to the antigen. It may typically comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR-1, CDR1, FR-2, CDR2, FR-3, CDR3, and FR-4 .
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • Fab antigen-binding fragments
  • Fv fragments F(ab') 2
  • scFv fragments
  • di-scFv and/or dAb immunoconjugates
  • immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.
  • variable generally refers to the fact that certain parts of the sequence of the variable domain of an antibody change strongly, which forms the binding and specificity of various specific antibodies to their specific antigen.
  • variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light chain and heavy chain variable regions, which are called complementarity determining regions (CDR) or hypervariable regions (HVR).
  • CDR complementarity determining regions
  • HVR hypervariable regions
  • the more highly conserved parts of variable domains are called the framework (FR).
  • the variable domains of the natural heavy chain and light chain each contain four FR regions, most of which adopt a ⁇ -sheet configuration, are connected by three CDRs to form a loop connection, and in some cases form part of a ⁇ -sheet structure.
  • the CDRs in each chain are close together through the FR region, and together with the CDR from the other chain form the antigen binding site of the antibody.
  • the constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions. , Such as participating in antibody-dependent cytotoxicity.
  • the CDR of an antibody can be defined by various methods, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethe Star, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see, A1-Lazikani et al., JMol Biol 273:927-48, 1997).
  • the combined definition rule including Kabat definition and Chothia definition is also used to determine the amino acid residues in the variable domain sequence and the full-length antibody sequence.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rules) to position bb (Chothia coding rules);
  • Haa-Hbb can refer to the N-terminus of the antibody heavy chain , The amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule).
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and according to Chothia coding rules The amino acid sequence from position 26 to position 32.
  • isolated antigen binding protein generally refers to an antigen binding protein that has been identified, separated and/or recovered from components of its production environment (for example, natural or recombinant).
  • the pollutant components of the environment are usually substances that interfere with its research, diagnostic or therapeutic uses, and can include enzymes, hormones and other protein or non-protein solutes.
  • the isolated antigen binding protein or antibody will usually be prepared through at least one purification step.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a group of substantially homogeneous antibodies, that is, the individual antibodies in the group are the same, except for a small number of natural mutations that may exist.
  • Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • the modifier "monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population, and is not construed as requiring antibody production by any specific method.
  • the monoclonal antibody used according to the present invention can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • the term "fully human antibody” generally refers to an antibody expressed by the animal by transferring all the genes encoding the human antibody to a genetically engineered animal with a gene deletion of the antibody. All parts of the antibody (including the variable and constant regions of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, and the like.
  • the term "specific anti-CD47 antibody” generally refers to an antibody that can compete with the antibody of this application for binding to CD47.
  • the antigen binding protein of the present application may include those antibodies that have the same amino acid sequence as the specific anti-CD47 antibody.
  • the term "specific binding” generally means that the antibody binds to the epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, when an antibody binds to an epitope through its antigen-binding domain more easily than it will bind to a random, unrelated epitope, the antibody is said to “specifically bind” the antigen.
  • Epitope refers to a specific atomic group (for example, sugar side chain, phosphoryl, sulfonyl) or amino acid that binds to an antigen-binding protein (such as an antibody) on an antigen.
  • CD47 generally refers to the leukocyte surface antigen CD47, which can be a complete CD47 protein, or any form of CD47 and its variants that retain at least part of the activity of CD47.
  • the CD47 may include human CD47 and fragments thereof or monkey CD47 and fragments thereof.
  • human CD47 may contain the amino acid sequence shown in Accession No. Q08722 in the UniProtKB database.
  • the monkey may be a cynomolgus monkey.
  • cynomolgus CD47 may include the amino acid sequence of the UniProtKB database with the accession number No. A0A2K5X4I2.
  • human CD47 extracellular region and “hCD47.ECD” are used interchangeably, and generally refer to the domain of type V immunoglobulin at the extracellular N-terminal of CD47, which is also the CD47 ligand binding region.
  • the extracellular region of human CD47 may comprise the sequence from positions 19 to 141 of the amino acid sequence shown in UniProtKB with accession number No. Q08722.
  • SIRP ⁇ generally refers to signal regulatory protein ⁇ , also known as BIT, MFR, MYD1, PTPNS1, SHPS1 or SIRP, which is an immunoglobulin-like cell surface receptor for CD47, which can be a complete SIRP ⁇ protein It can also be any form of SIRP ⁇ and its variants that retain at least part of the activity of SIRP ⁇ .
  • the SIRP ⁇ may be human SIRP ⁇ , which may include the amino acid sequence shown in the UniProtKB database with the accession number P78324.
  • Jurkat cell generally refers to human leukemia T lymphocytes.
  • the Jurkat cells were derived from the peripheral blood of a 14-year-old boy.
  • the term "Raji cell” generally refers to human Burkitt's lymphoma cells.
  • the Raji cells were derived from Burkitt lymphoma of the left upper jaw of an 11-year-old black boy.
  • OVCAR 3 cell and “OVCAR-3 cell” are used interchangeably and generally refer to human ovarian adenocarcinoma cells.
  • the OVCAR 3 cells are derived from malignant ascites of patients with progressive ovarian adenocarcinoma.
  • the terms "coagulation reaction” and “agglutination” are used interchangeably, and generally refer to the homotypic interaction that occurs when red blood cells agglutinate or clump when incubated with different reagents.
  • the agglutination can refer to the phenomenon that the agglutination of red blood cells makes the edge of the blood circle blurred when the control antibody is present.
  • the existing CD47 antibody can cause RBC agglutination at a certain concentration, such as the existing antibody Tab1 (FortySeven, Hu5F9-G) in Example 7 of the present application. Therefore, blood coagulation can limit the therapeutic effect of CD47 antibodies.
  • the term “does not cause a coagulation reaction” generally means that no red blood cell aggregation or clumping occurs.
  • Tab1 usually refers to the anti-CD47 antibody Hu5F9-G from FortySeven.
  • Tab2 usually refers to the anti-CD47 antibody CC-90002 of Celgene.
  • Tab3 generally refers to the anti-CD47 antibody SRF-231 from Surface Oncology.
  • KD the terms “KD”, “Kd”, and “KD” are used interchangeably, generally refer to the equilibrium dissociation constant, and refer to the titration measurement, at equilibrium, or by dividing the dissociation rate constant ( koff) divided by the binding rate constant (kon).
  • the binding rate constant (kon), the dissociation rate constant (koff), and the equilibrium dissociation constant (KD) can be used to express the binding affinity of an antigen-binding protein (eg, an antibody) to an antigen.
  • an antigen-binding protein eg, an antibody
  • Methods of determining the association and dissociation rate constants are well known in the art.
  • fluorescence-based technology provides high sensitivity and the ability to check samples at equilibrium in physiological buffers.
  • Other experimental approaches and instruments such as BIAcore (Biomolecular Interaction Analysis) can be used.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having multiple functions described above.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the term "cell” generally refers to an individual cell, cell line, or cell line that can or already contains a plasmid or vector containing the nucleic acid molecule described in this application, or capable of expressing the antibody or antigen-binding fragment thereof described in this application.
  • the cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells and the original parent cells may not necessarily be completely the same in morphology or genome, but they can express the antibodies or antigen-binding fragments described in this application.
  • the cells can be obtained by transfecting cells in vitro using the vectors described in this application.
  • the cell may be a prokaryotic cell (such as Escherichia coli) or a eukaryotic cell (such as yeast cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells or Myeloma cells).
  • the cell is a mammalian cell.
  • the mammalian cell may be a CHO-K1 cell.
  • the term "recombinant cell” generally refers to a cell into which a recombinant expression vector is introduced.
  • the recombinant host cell includes not only certain specific cells, but also the progeny of these cells.
  • composition generally refers to a preparation that exists in a form that allows the biological activity of the active ingredient to be effective, and does not contain unacceptable toxicity to the subject to which the composition will be administered. Additional ingredients.
  • the composition is sterile.
  • “Sterile” compositions are sterile or free of all living microorganisms and their spores.
  • tumor generally refers to a neoplasm formed by the proliferation of local tissue cells in a mammalian body (for example, cells or their components) under the action of various tumorigenic factors.
  • tumors may include lymphoma, leukemia, and ovarian cancer.
  • tumors can include aggressive non-Hodgkin’s lymphoma, Burkitt’s lymphoma, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), ovarian cancer, etc.
  • the term "between” usually means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and that the N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment Indirect connection.
  • the N-terminus of the L-FR2 is directly or indirectly connected to the C-terminus of the LCDR1
  • the C-terminus of the L-FR2 is directly or indirectly connected to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly connected to the C-terminus of the LCDR2
  • the C-terminus of the L-FR3 is directly or indirectly connected to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly connected to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly connected to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly connected to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly connected to the N-terminus of the HCDR3.
  • first amino acid fragment" and "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides an antigen binding protein, which may include at least one CDR in the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 153.
  • the antigen binding protein may include LCDR1, and the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 139:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR1 may include at least substitutions at amino acid positions selected from: at X 1 , X 2 and/or X 3 The amino acid substitutions.
  • the LCDR1 may include at least the amino acid substitution at X 1 , X 2 or X 3 , wherein the amino acid at X 1 may be substituted Is V; the amino acid at X 2 can be substituted with Y or H; the amino acid at X 3 can be substituted with N.
  • the LCDR1 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 113-115.
  • the antigen binding protein may include LCDR2, and the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 140:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR2 may include at least substitutions at amino acid positions selected from: X 1 , X 2 , X 3 , X 4 , X 5 and/or X 6 amino acid substitutions.
  • the LCDR2 may include at least the amino acid substitutions at X 1 , X 2 , X 3 , X 4 , X 5 or X 6,
  • the amino acid at X 1 can be substituted with R, V or G; the amino acid at X 2 can be substituted with S; the amino acid at X 3 can be substituted with R, I or T; the amino acid at X 4 can be substituted Is R; the amino acid at X 5 can be substituted with D, Q or A; the amino acid at X 6 can be substituted with T.
  • the LCDR2 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 31, 56, 57, 122, 126, and 127.
  • the antigen binding protein may include LCDR3, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 141:
  • X 1 Q or R
  • X 2 Q, L or H
  • X 3 Y or -
  • X 4 N, I, Q or -
  • X 5 S, Y, T or N
  • X 6 Y, W, N or Q
  • X 7 S, T or -
  • X 8 T Or -
  • X 9 P or -
  • X 10 Y, R or L.
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the LCDR3 may include at least substitutions at amino acid positions selected from: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and/or X 10 amino acid substitutions.
  • the LCDR3 may be at least contained in X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8.
  • Amino acid substitution at X 9 and/or X 10 wherein the amino acid at X 1 can be substituted with Q; the amino acid at X 2 can be substituted with L or H; the amino acid at X 3 can be substituted with no amino acid ; The amino acid at X 4 can be substituted with I, Q or no amino acid; the amino acid at X 5 can be substituted with Y, T or N; the amino acid at X 6 can be substituted with Q, N or W; X 7
  • the amino acid at X can be replaced with T or no amino acid; the amino acid at X 8 can be substituted to increase T; the amino acid at X 9 can be replaced with no amino acid; the amino acid at X 10 can be replaced with R or L.
  • - means that compared with LCDR3 of the control antigen binding protein, this position can be without amino acid substitution.
  • the LCDR3 of the antigen-binding protein shown in SEQ ID NO: 74 has no amino acid substitution at X 9.
  • the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 67-74, 116-121.
  • the antigen binding protein described in the present application may include at least one CDR in the VH of the variable region of the antibody heavy chain, and the VH includes the amino acid sequence shown in SEQ ID NO: 154.
  • the antigen binding protein may include HCDR1, and the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 142:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the HCDR1 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , X 3 and/ Or X 4 amino acid substitution.
  • the HCDR1 may at least include amino acid substitutions at X 1 , X 2 , X 3 and/or X 4, wherein X
  • the amino acid at 1 can be replaced with D; the amino acid at X 2 can be replaced with L or V; the amino acid at X 3 can be replaced with S, D or Q; the amino acid at X 4 can be replaced with S or T .
  • the HCDR1 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 32, 34, 36, 38, 40, 42, 45, 47, 49, 51, and 53.
  • the antigen binding protein may include HCDR2, and the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 143:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • HCDR2 may comprise at least the following substituents selected from: amino acid substitution at X 1.
  • the HCDR2 may comprise at least the amino acid substitution at X 1, wherein X 2 at the amino acid may be substituted with S or T.
  • the HCDR2 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 136-138.
  • the antigen binding protein may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 144:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the HCDR3 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , X 3 , X 4, at the amino acid and X 5 / X 6 or substituted.
  • the HCDR3 may include at least amino acid substitutions at X 1 , X 2 , X 3 , X 4 , X 5 and/or X 6, wherein ,
  • the amino acid at X 1 can be replaced with G;
  • the amino acid at X 2 can be replaced with K;
  • the amino acid at X 3 can be replaced with A, T, L or W;
  • the amino acid at X 4 can be replaced with M , G, A or I;
  • the amino acid at X 5 can be substituted with Y, H, F or no amino acid;
  • the amino acid at X 6 can be substituted with S.
  • the HCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 55, 58-63.
  • the isolated antigen binding protein may comprise LCDR1-3.
  • the LCDR1 of the antigen-binding protein of the present application may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 113, 114 and 115; the LCDR2 may include any of the following The amino acid sequence shown in the item: SEQ ID NO: 31, 56, 57, 122, 126, and 127; the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 67-74, 116 -121.
  • the isolated antigen binding protein may comprise HCDR1-3.
  • the HCDR1 of the antigen binding protein of the present application may include the amino acid sequence shown in any one of the following: SEQ ID NO: 32, 34, 36, 38, 40, 42, 45, 47 , 49, 51 and 53;
  • the HCDR2 may include the amino acid sequence shown in any of the following: SEQ ID NO: 136-138; and the HCDR3 may include the amino acid sequence shown in any of the following: SEQ ID NO: 58-63 and 55.
  • HCDR1 in the antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 42;
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138;
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 59 The amino acid sequence.
  • the isolated antigen binding protein may comprise LCDR1-3 and HCDR1-3.
  • the LCDR1 of the antigen-binding protein of the present application may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 113, 114 and 115; the LCDR2 may include any of the following The amino acid sequence shown in the item: SEQ ID NO: 31, 56, 57, 122, 126, and 127; the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 67-74, 116 -121;
  • the HCDR1 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 32, 34, 36, 38, 40, 42, 45, 47, 49, 51 and 53; the HCDR2 may It includes the amino acid sequence shown in any one of the following: SEQ ID NO: 136-138; and the HCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 58-63 and 55.
  • LCDR1 in the antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 114; LCDR2 may include the amino acid sequence shown in SEQ ID NO: 126; LCDR3 may include the amino acid sequence shown in SEQ ID NO: 72 Amino acid sequence; its HCDR1 may include the amino acid sequence shown in SEQ ID NO: 38; HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138; and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 55.
  • LCDR1 in the antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 115; LCDR2 may include the amino acid sequence shown in SEQ ID NO: 31; LCDR3 may include the amino acid sequence shown in SEQ ID NO: 70 Amino acid sequence; its HCDR1 may include the amino acid sequence shown in SEQ ID NO: 36; HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138; and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 58.
  • LCDR1 in the antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 113; LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56; LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116 Amino acid sequence; its HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45; HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138; and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60.
  • LCDR1 in the antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 113; LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56; LCDR3 may include the amino acid sequence shown in SEQ ID NO: 74 Amino acid sequence; its HCDR1 may include the amino acid sequence shown in SEQ ID NO:53; HCDR2 may include the amino acid sequence shown in SEQ ID NO:137; and HCDR3 may include the amino acid sequence shown in SEQ ID NO:63.
  • the antigen binding protein described in the present application may also include the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
  • the antigen binding protein may include L-FR1, the C-terminus of L-FR1 is directly or indirectly connected to the N-terminus of LCDR1, and the L-FR1 includes SEQ ID NO: 149 Shown amino acid sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR1 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , Amino acid substitutions at X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and/or X 10.
  • the L-FR1 may be at least included in X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and/or X 10 amino acid substitutions, wherein the amino acid at X 1 may be substituted with E; the amino acid at X 2 may be substituted with V; the amino acid at X 3 may be substituted Replaced with A; the amino acid at X 4 can be replaced with S; the amino acid at X 5 can be replaced with V; the amino acid at X 6 can be replaced with P; the amino acid at X 7 can be replaced with E; X 8 The amino acid at X may be substituted with A; the amino acid at X 9 may be substituted with L; the amino acid at X 10 may be substituted with S.
  • the L-FR1 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 156-158.
  • the antigen-binding protein may include L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid shown in SEQ ID NO: 150 sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR2 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 and/or X 2 amino acid substitutions.
  • the L-FR2 may at least include amino acid substitutions at least included at X 1 and/or X 2, wherein, The amino acid at X 1 may be substituted with N or Q; the amino acid at X 2 may be substituted with R.
  • the L-FR2 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 191-194.
  • the antigen-binding protein may include L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 includes the amino acid shown in SEQ ID NO: 151 sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR3 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , Amino acid substitutions at X 3 , X 4 , X 5 , X 6 , X 7 , X 8 and/or X 9.
  • the L-FR3 may be at least contained in X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7.
  • Amino acid substitution at X 8 and/or X 9 wherein the amino acid at X 1 can be substituted with D; the amino acid at X 2 can be substituted with I; the amino acid at X 3 can be substituted with V; X The amino acid at 4 can be replaced by D; the amino acid at X 5 can be replaced by S; the amino acid at X 6 can be replaced by V; the amino acid at X 7 can be replaced by S; the amino acid at X 8 can be replaced by Replaced with E; the amino acid at X 9 can be substituted with I.
  • the L-FR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 155, 162-166.
  • the antigen binding protein may include L-FR4, the N-terminus of L-FR4 is connected to the C-terminus of LCDR3, and the L-FR4 includes SEQ ID NO: 152 Amino acid sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the L-FR4 may include at least substitutions at amino acid positions selected from the group consisting of: at X 1 and/or X 2 The amino acid substitutions.
  • the L-FR4 may include at least the amino acid substitution at least included at X 1 and/or X 2 , wherein the amino acid at X 1 It can be substituted with G; the amino acid at X2 can be substituted with V.
  • the L-FR4 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 159-161.
  • the antigen binding protein may include the light chain variable region VL, and the VL may include the amino acid sequence shown in SEQ ID NO: 153:
  • the VL may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 , X 39 , X 40 , X 41 and/or X 42 amino acid substitution
  • the VL may be at least included in X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 , X 36 , X 37 , X 38 , X 39 , X 40 , X 41 and/or Amino acid substitution at X 42 , wherein the amino acid at X 1 can be
  • the VL region may include the amino acid sequence shown in any one of the following: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29.
  • the antigen binding protein described in the present application may also include framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the antigen binding protein may comprise H-FR1, the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, and the H-FR1 comprises SEQ ID NO: 145 Shown amino acid sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the H-FR1 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 and the amino acid substitution and / or X 3.
  • the H-FR1 may include at least amino acid substitutions at X 1 , X 2 and/or X 3 , wherein the H-FR1 at X 1 Amino acids can be substituted with M; X 2 can be substituted with Q; X 3 can be substituted with S.
  • the H-FR1 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 167-170.
  • the antigen binding protein may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid shown in SEQ ID NO: 146 sequence:
  • sequence may be a sequence determined according to Chothia's definition rules.
  • the H-FR2 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , X 3 , Amino acid substitution at X 4 and/or X 5.
  • the H-FR2 may at least include amino acid substitutions at X 1 , X 2 , X 3 , X 4 and/or X 5, wherein the amino acids at X 1 may be substituted with T; amino acids at X 2 may be substituted is F; amino acid 3 of X may be substituted with a; amino acids at X 3 may be substituted with R < at X 3 is Amino acids can be substituted with N.
  • the H-FR2 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 184, 187-190.
  • the antigen binding protein may include H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 includes the amino acid shown in SEQ ID NO: 147 sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the H-FR3 may include at least substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , X 3 , X 4. Amino acid substitutions at X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 and/or X 15.
  • the H-FR3 may be at least contained in X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 and/or X 13 are substituted with amino acids, wherein the amino acid at X 1 can be substituted with D; the amino acid at X 2 can be Replaced with Q; the amino acid at X 3 can be replaced with P; the amino acid at X 4 can be replaced with T; the amino acid at X 5 can be replaced with S; the amino acid at X 6 can be replaced with M; X 7 The amino acid at X can be replaced with I; the amino acid at X 8 can be replaced with I; the amino acid at X 9 can be replaced with K; the amino acid at X 10 can be replaced with S or N; the amino acid
  • the H-FR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 171-182.
  • the antigen binding protein may include H-FR4, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 includes the sequence shown in SEQ ID NO: 148 Amino acid sequence:
  • the sequence may be a sequence determined according to Chothia's definition rules.
  • the H-FR4 may include at least a substitution at an amino acid position selected from: the amino acid at X 1 and/or X 2 replace.
  • the H-FR4 may include at least the amino acid substitution at X 1 and/or X 2 , wherein the amino acid at X 1 may be Replaced with S; the amino acid at X 2 can be substituted with I.
  • the H-FR4 may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 183, 185, and 186.
  • the antigen binding protein may include the heavy chain variable region VH, and the VH includes the amino acid sequence shown in SEQ ID NO: 154:
  • the VH may at least include substitutions at amino acid positions selected from the group consisting of: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 , X 27 , X 28 , X 29 , X 30 , X 31 , X 32 , X 33 , X 34 , X 35 and/or X 36
  • the amino acid substitutions are selected from the group consisting of: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8
  • the VH may include the amino acid sequence shown in any one of the following: SEQ ID NO: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 , 103, 105, 107, 109, and 111.
  • the antigen binding protein may comprise the variable region VL of the antibody light chain and the variable region VH of the heavy chain.
  • the VL of the antigen binding protein described in the present application may include the amino acid sequence SEQ ID NO shown in any one of the following: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29; the VH may include any of the following amino acid sequences SEQ ID NO: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, and 111.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 29, and the VH may include the amino acid sequence shown in SEQ ID NO: 111.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 29, and the VH may include the amino acid sequence shown in SEQ ID NO: 107.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 29, and the VH may include the amino acid sequence shown in SEQ ID NO: 109.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 1, and the VH may include the amino acid sequence shown in SEQ ID NO: 79.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include SEQ ID NO: 81, 83, 85, 89, 91, The amino acid sequence shown in any one of 93, 95, and 97.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 81.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 83.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 85.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 89.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 91.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 93.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in SEQ ID NO: 95.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 15, and the VH may include the amino acid sequence shown in any one of SEQ ID NO: 97 .
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 23, and the VH may include the amino acid sequence shown in SEQ ID NO: 99.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 21, and the VH may include the amino acid sequence shown in SEQ ID NO: 99.
  • the isolated antigen binding protein may include VL and VH, and its VL may include the amino acid sequence shown in SEQ ID NO: 25, and the VH may include the amino acid sequence shown in SEQ ID NO: 99.
  • the antigen binding protein includes a light chain constant region CL, and the antibody light chain constant region may include a human Ig ⁇ constant region.
  • the CL region may include the amino acid sequence shown below: SEQ ID NO: 196.
  • the antigen binding protein includes a light chain LC
  • the LC may include the amino acid sequence shown in SEQ ID NO:197.
  • the LC may include the amino acid sequence shown in any one of the following: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 And 30.
  • the antigen binding protein includes a heavy chain constant region CH, and the antibody heavy chain constant region may include a human IgG constant region.
  • the antibody heavy chain constant region described in this application may include a human IgG4 constant region.
  • the CH region may include the amino acid sequence shown below: SEQ ID NO: 195.
  • the antigen binding protein includes a light chain HC
  • the antibody HC may include the amino acid sequence shown in SEQ ID NO: 198.
  • the HC may include the amino acid sequence shown in any one of the following: SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102 , 104, 106, 108, 110, and 112.
  • the antigen binding protein described in the present application comprises an antibody light chain constant region CL and a heavy chain constant region CH.
  • the CL may include the amino acid sequence shown in SEQ ID NO: 196; and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein described in this application includes an antibody light chain LC and heavy chain HC.
  • the LC may include the amino acid sequence shown in SEQ ID NO: 197
  • the HC may include the amino acid sequence shown in SEQ ID NO: 198.
  • the LC may include the amino acid sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30.
  • the HC may include the amino acid sequence SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, and 112.
  • the antigen binding protein described in this application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 139
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 140
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 141
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 149
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 150
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 151
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 152.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 153, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 197.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 142
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 143
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 144.
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 145
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 146
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 147
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 148.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 154, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 198.
  • the antigen binding protein described in this application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in any one of SEQ ID NO: 113-115
  • the LCDR2 may include the amino acid sequence shown in any one of SEQ ID NO: 31, 56, 57, 122, 126 and 127
  • LCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 67-74, 116-121
  • the L-FR1 may include the amino acid sequence shown in any one of SEQ ID NO: 156-158
  • L -FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 191-194
  • L-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 155, 162-166
  • L-FR4 may Contains the amino acid sequence shown in any one of SEQ ID NO: 159-161.
  • the antigen binding protein may include VL and CL, and the VL may include SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29
  • the amino acid sequence shown in any one of the above, the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may comprise an antibody light chain, and the light chain may comprise SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 The amino acid sequence shown in any one of them.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in any one of SEQ ID NO: 32, 34, 36, 38, 40, 42, 45, 47, 49, 51 and 53
  • the HCDR2 may comprise SEQ ID NO: 136-
  • the amino acid sequence shown in any one of 138, HCDR3 may include the amino acid sequence shown in any one of SEQ ID NO: 55 and 58-63
  • the H-FR1 may include any one of SEQ ID NO: 167-170
  • the amino acid sequence shown in item, H-FR2 may include the amino acid sequence shown in any one of SEQ ID NO: 184, 187-190
  • H-FR3 may include the amino acid sequence shown in any one of SEQ ID NO: 171-182
  • the amino acid sequence, and H-FR4 may include the amino acid sequence shown in any one of SEQ ID NOs: 183, 185, and 186.
  • the antigen binding protein may include VH and CH, and the VH may include SEQ ID NO: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103
  • the CH may comprise the amino acid sequence shown in any one of SEQ ID NO: 195.
  • the antigen binding protein may comprise an antibody heavy chain, and the antibody heavy chain may comprise SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102 , 104, 106, 108, 110, and 112.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 74
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 162
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 5, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO:6.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 53
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 137
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 63
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 182
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 183.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 105, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 106.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001281.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 122
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 73
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 193
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 27, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 28.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 59
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 186
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 172
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 87, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 88.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR000806.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 114
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 126
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 72
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 156
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 163,
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 1, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 2.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 38
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 55
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 184
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 180
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 79, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:80.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR000808.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 114
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 127
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 71
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 156
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 192
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 164
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 161.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 3, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 4.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 61
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 167
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 189
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 171
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 75, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 76.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR000807.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 115
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 31
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 70
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 158
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 194
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 155
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 159.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 29, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may comprise an antibody light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 32
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 58
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 170
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 190
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 176
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 107, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 108.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001264.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 115
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 31
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 70
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 158
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 194
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 155
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 159.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 29, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 36
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 58
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 170
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 190
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 176
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 111, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 112.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001265.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 32
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60.
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 89, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:90.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001266.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 178
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 91, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 92.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001267.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 188
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 40
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 81, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 82.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001268.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 40
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 178
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 83, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 84.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001269.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 121
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 19, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may comprise an antibody light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 40
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 181
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 85, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 86.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR001270.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 120
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 17, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 18.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 40
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 181
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 85, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 86.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR001271.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 121
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 19, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may comprise an antibody light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 89, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO:90.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR001272.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 69
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 25, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 26.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 47
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 137
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 62
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 179
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 99, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001273.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 68
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 23, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 24.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 47
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 137
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 62
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 179
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 99, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR001274.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60.
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 174
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 97, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 98.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001275.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60.
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 175
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 93, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 94.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001276.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 119
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 165
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 11, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 12.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 168
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 77, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001277.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 120
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 165
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 13, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 14.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 168
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 77, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR001278.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 118
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 165
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 9, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 10.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 168
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 77, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR001279.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 74
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 162
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 5, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO:6.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 51
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 137
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 63.
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 182
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 183.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 103, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 104.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR001280.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 115
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 31
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 70
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 158
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 194
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 155
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 159.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 29, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 34
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 58
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 170
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 190
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 176
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 109, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 110.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR000796.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 40
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 136
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 181
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 186.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 85, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 86.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR000802.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 57
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 67
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 21, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may comprise an antibody light chain, and the light chain may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 47
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 137
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 62
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 179
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 99, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 100.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR000803.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 116
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 166
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 15, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 16.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 45
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60.
  • the H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 173
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 95, and the CH may include the amino acid sequence shown in SEQ ID NO: 195.
  • the antigen binding protein may comprise an antibody heavy chain, and the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 96.
  • the antigen binding protein contains the same antibody light chain and antibody heavy chain as PR000804.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 117
  • the L-FR1 may Contains the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 can include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 can include the amino acid sequence shown in SEQ ID NO: 165
  • L-FR4 can include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 7, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO: 8.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • HCDR1 may include the amino acid sequence shown in SEQ ID NO: 42
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 138
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 60
  • H-FR1 It may include the amino acid sequence shown in SEQ ID NO: 168
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 177
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 185.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 77, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 78.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR000805.
  • the antigen binding protein described in the present application may include LCDR1-3 and L-FR1-4.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO: 113
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 56
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 74
  • the L-FR1 may It includes the amino acid sequence shown in SEQ ID NO: 157
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 191
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 162
  • L-FR4 may include The amino acid sequence shown in SEQ ID NO: 160.
  • the antigen binding protein may include VL and CL, and the VL may include the amino acid sequence shown in SEQ ID NO: 5, and the CL may include the amino acid sequence shown in SEQ ID NO: 196.
  • the antigen binding protein may include an antibody light chain, and the light chain may include the amino acid sequence shown in SEQ ID NO:6.
  • the antigen binding protein may also include HCDR1-3 and H-FR1-4.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO: 49
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO: 137
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO: 63
  • the H-FR1 May include the amino acid sequence shown in SEQ ID NO: 169
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 188
  • H-FR3 may include the amino acid sequence shown in SEQ ID NO: 182
  • H-FR4 may Contains the amino acid sequence shown in SEQ ID NO: 183.
  • the antigen binding protein may include VH and CH, and the VH may include the amino acid sequence shown in SEQ ID NO: 101, and the CH may include the amino acid sequence shown in SEQ ID NO: 195. And the antigen binding protein may include an antibody heavy chain, and the antibody heavy chain may include the amino acid sequence shown in SEQ ID NO: 102.
  • the antigen binding protein includes the same antibody light chain and antibody heavy chain as PR000811.
  • the isolated antigen binding protein described in this application can compete with a specific anti-CD47 antibody for binding to the CD47, wherein the specific anti-CD47 antibody may comprise a light chain variable region and a heavy chain variable region, so
  • the light chain variable region of the specific anti-CD47 antibody may include LCDR1-3, and the LCDR1-3 may include the amino acid sequence shown in SEQ ID NO: 139-141; the heavy chain of the specific anti-CD47 antibody may
  • the variable region may include HCDR1-3, and the HCDR1-3 may include the amino acid sequence shown in SEQ ID NO: 142-144.
  • the amino acid sequence of the light chain variable region of the specific anti-CD47 antibody may be SEQ ID NO: 153, and the heavy chain variable region of the specific anti-CD47 antibody may be SEQ ID NO: 154.
  • the LCDR1 of the specific anti-CD47 antibody may include the amino acid sequence shown in any one of the following: SEQ ID NOs: 113, 114, and 115; the LCDR2 may include the amino acid sequence shown in any of the following The amino acid sequence of SEQ ID NO: 31, 56, 57, 122, 126, and 127; and the LCDR3 may include the amino acid sequence shown in any one of the following: SEQ ID NO: 67-74, 116-121.
  • the HCDR1 of the specific anti-CD73 antibody may include the amino acid sequence shown in any one of the following: SEQ ID NO: 32, 34, 36, 38, 40, 42, 45, 47, 49, 51 And 53;
  • the HCDR2 may include the amino acid sequence shown in any of the following: SEQ ID NO: 136-138;
  • the HCDR3 may include the amino acid sequence shown in any of the following: SEQ ID NO: 58-63 and 55.
  • the VL of the specific anti-CD47 antibody may comprise the amino acid sequence shown in any one of the following: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 , 21, 23, 25, 27, and 29.
  • the VH of the specific anti-CD47 antibody may comprise the amino acid sequence shown in any one of the following: SEQ ID NO: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93 , 95, 97, 99, 101, 103, 105, 107, 109, and 111.
  • the protein, polypeptide, and/or amino acid sequence involved in this application should also be understood to include at least the following range: a variant or homologue that has the same or similar functions as the protein or polypeptide.
  • a variant having the same or similar functions as the protein or polypeptide may be referred to as a functional variant.
  • the variant may be a substitution, deletion or addition of one or more in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the CD47 protein) Amino acid protein or peptide.
  • the variant e.g., functional variant
  • the variant may include at least 1, such as 1-30, 1-20, or 1-10, and another example, 1, 2, 3, 4 A protein or polypeptide with amino acid changes by one or five amino acid substitutions, deletions and/or insertions.
  • the variant e.g., functional variant
  • the variant e.g., functional variant
  • the variant can maintain at least 60%, 70%, 80%, 90% or 100% of the biological activity (e.g., antigen binding) of the protein or polypeptide before the change. ability).
  • the substitution may be a conservative substitution.
  • a part of the amino acid sequence of the antigen binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific category.
  • both the variable region and constant part of an antibody can be derived from the variable region and constant region of an antibody of an animal species (such as human).
  • the homologue may be at least about 85% (for example, having at least about 85% of the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to the CD47 protein) About 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence is the same Source protein or peptide.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology" can be calculated in the following way: the two sequences to be aligned are compared in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to produce the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu,
  • the alignment to determine the percent sequence homology can be achieved in a variety of ways known 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 the appropriate parameters for sequence alignment, including any algorithm needed to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST. For a description of the FASTA algorithm, see WRPearson and DJ Lipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • the isolated antigen binding protein can bind CD47 derived from human and monkey.
  • the monkey may refer to a cynomolgus monkey.
  • the binding affinity of an anti-CD47 antibody to CD47 can be determined by any method known in the art. In some cases, binding affinity can be determined by competitive ELISA, RIA, or surface plasmon resonance such as BIAcore. In some specific cases, the binding affinity is measured by surface plasmon resonance. For example, binding affinity and dissociation rate can be determined by BIAcore. In some cases, the KD that the isolated antigen binding protein binds to CD47 may be 3 ⁇ 10 -9 M or less.
  • the isolated antigen binding protein with a KD of binding CD47 may 3x10 -9 M, 2x10 is - 9 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M or less.
  • KD may be about 1 pM to about 500 pM. In other cases, KD may be about 500 pM to about 1 nM. In other cases, KD may be about 1 nM to about 10 nM.
  • the isolated antigen binding protein can specifically bind to CD47.
  • the CD47 described in this application may be CD47 on the surface of tumor cells.
  • the tumor cells may be Jurkat cells or Raji cells.
  • the EC 50 value of the antigen binding protein binding to CD47 on Jurkat cells may be about 10 pM to about 10 nM. In other cases, the EC 50 value may be about 100 pM to about 100 nM. In other cases, the EC 50 value may be about 1 pM to about 1 nM. In other cases, the EC 50 value may be about 0.1 nM to about 1 nM, for example, in FACS measurement.
  • the EC 50 value of the antigen binding protein binding to CD47 on Jurkat cells may be about 10 pM to about 10 nM, for example, about 10 pM to about 1 nM, to about 1 nM to about 10 nM.
  • the antigen binding protein does not bind or specifically binds to non-CD47 antigens, for example, does not bind or does not specifically bind SIPR ⁇ , CD147, PD-1, PD-L1, CTLA-4, CD80, CD86 , CD28H, B7H3, B7H4 and ICOS.
  • the antigen binding protein can also bind to CD47 on the surface of cynomolgus monkey cells, for example, it may be cynomolgus monkey peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the binding can be detected by FACS, and the resulting EC50 value can be about 0.02 nM to about 0.2 nM.
  • the EC50 measured by FACS may be about 0.01 nM to about 0.3 nM, about 0.01 nM to about 0.5 nM, or about 0.01 nM to about 1.0 nM.
  • the isolated antigen binding protein can inhibit the binding of CD47 to SIRP ⁇ .
  • the CD47 may be the extracellular region of human CD47.
  • the extracellular region of human CD47 described in the present application may include the sequence from position 19 to position 141 in the amino acid sequence shown in the accession number Q08722 in UniProt.
  • the IC 50 value of the isolated antigen binding protein inhibiting the binding of human CD47 to SIRP ⁇ may be about 0.5 nM to about 5.0 nM as measured by ELISA.
  • IC 50 values of the blocking binding may be about from about 1.0nM to 3.0nM. In other cases, it may be about 1.5 nM to about 2.5 nM.
  • the isolated antigen binding protein can inhibit the binding of human CD47 to SIRP ⁇ by greater than 90% (e.g., greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99% or higher).
  • the isolated antigen binding protein can block the binding of CD47 on the surface of tumor cells to SIRP ⁇ .
  • the tumor cell described in the present application may be Jurkat cell.
  • the CD47 antigen binding protein described in this application can block the binding of SIRP ⁇ to human CD47.
  • the isolated antigen binding protein can block the IC 50 value of CD47 on the surface of Jurkat cells from binding to SIRP ⁇ from about 0.01 nM to about 5.0 nM, from about 0.02 nM to about 3.0 nM, or from about 0.03 nM to about 2.0 nM.
  • the IC 50 value may be about 0.1 nM to about 1.0 nM, for example, as measured by FACS.
  • the isolated antigen binding protein may not cause a coagulation reaction.
  • the agglutination level in the presence of the CD47 antigen-binding protein of the present application is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99%, indicating that the CD47 antigen binding protein of the present application does not cause a coagulation reaction.
  • concentration is between about 0.65 nM to about 665 nM, the antigen binding protein does not cause a coagulation reaction.
  • the isolated antigen binding protein can cause phagocytosis of tumor cells by macrophages.
  • the tumor cells described in this application may include Jurkat and OVCAR 3 tumor cells.
  • Jurkat cells are labeled with CFSE and measured by flow cytometry.
  • the isolated antigen binding protein can cause the phagocytosis of Jurkat by macrophages.
  • the phagocytic index can be the percentage of CFSE + CD14 + cells to the total CD14 + cells And the phagocytic index may be at least greater than 30% (for example, at least greater than 40%, at least greater than 50%, at least greater than 60%, at least greater than 70%, at least greater than 80%, at least greater than 90% or more).
  • the isolated antigen binding protein can cause the EC 50 value of macrophage phagocytosis of OVCAR3 to be about 0.1 nM to about 5.0 nM (for example, about 0.2 nM to about 3.0 nM, about 0.2 nM to about 2.0 nM or about 0.5nM to about 1.5nM).
  • macrophages are labeled with CD14-APC and measured with a flow cytometer, which can be about 0.5 nM to about 1.5 nM.
  • the phagocytic index of the isolated antigen binding protein that can cause macrophage phagocytosis of human red blood cells can be lower than 35% (for example, it can be lower than 30%, lower than 25%, lower than 20%, less than 15%, less than 10%, less than 5% or less).
  • the isolated antigen binding protein described in this application causes the phagocytosis of macrophages to tumor cells to be stronger than the phagocytosis of red blood cells.
  • the antigen binding protein described in this application can inhibit tumor growth and/or tumor cell proliferation.
  • the antigen binding protein can slow down the growth of tumor volume, eliminate tumor growth, or can slow down the trend of weight loss. This effect can be tested by establishing a mouse tumor model, for example, the Raji mouse model or the MC38 mouse model.
  • the tumors include solid tumors and/or hematological tumors.
  • Solid tumors may include ovarian cancer, and hematomas may include leukemia and lymphoma.
  • Leukemia may include acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), etc.
  • Lymphomas may include Hodgkin's lymphoma, painless and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, etc.
  • the present application also provides isolated one or more nucleic acid molecules, which can encode the antibody or antigen-binding fragment thereof described in the present application.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the complete antibody, its antigen-binding fragment, or a part thereof (for example, HCDR1-3, LCDR1-3, VL, VH , One or more of light chain or heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody or its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides.
  • methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides.
  • overlapping extension PCR see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, for specific operations. New York NY, 1993.
  • the application provides one or more vectors, which comprise one or more nucleic acid molecules described in the application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a controllable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters may include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part can be fused to a certain part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), and it may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the application provides a host cell, which may comprise one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one nucleic acid molecule or vector described in this application.
  • each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in the present application can be introduced into the host cell, such as prokaryotic cells (for example, bacterial cells), CHO cells, NS/0 cells, HEK293T cells or HEK293A cells, or other eukaryotic cells, such as from plants Cells, fungi or yeast cells, etc.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the host cell can be COS, CHO, NSO, sf9, sf21, DH5a, BL21(DE3) or TG1.
  • this application provides methods for preparing the antibodies and antigen-binding fragments thereof described in this application.
  • the method may include culturing the host cell described in the present application under conditions that allow expression of the antibody or antigen-binding fragment thereof. For example, it is possible to use an appropriate medium, an appropriate temperature, a culture time, etc., and these methods are understood by those of ordinary skill in the art.
  • the method may also include a step of harvesting (for example, isolation and/or purification), the antibody or antigen-binding fragment thereof described in the present application.
  • a step of harvesting for example, isolation and/or purification
  • protein G-sepharose or protein A-sepharose can be used for affinity chromatography
  • gel electrophoresis and/or high performance liquid chromatography can also be used to purify and separate the antibodies and antigen-binding fragments of the present application. .
  • the application provides a pharmaceutical composition, which may comprise the antigen-binding fragment or variant described in the application, the nucleic acid molecule, the vector, the host cell, and optionally ⁇ pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier generally refers to a carrier that can be used to prepare a pharmaceutical composition or preparation, and is generally safe, non-toxic, and neither biologically nor otherwise undesirable.
  • the carrier used is usually a carrier suitable for administration to humans or other mammals.
  • the active ingredient is usually mixed with a carrier and diluted or enclosed by the carrier.
  • the carrier serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle or medium for the active ingredient of the antibody.
  • the pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or nonionic surface active ⁇ etc.
  • the pharmaceutical composition may include the antigen binding protein described in the present application, and may also include one or more active compounds in combination.
  • the active compound may be a compound that does not adversely affect the antigen binding protein, or may be an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitor.
  • the combined compounds may be combined in an amount effective for the desired purpose.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration Administration or via subcutaneous depot.
  • Solutions or suspensions for transdermal administration or via subcutaneous reservoirs may include the following components: sterile diluents such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial Agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate or phosphate; and conditioning Tonic substances such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical composition of the present application can inhibit or delay the development or progression of the disease, can reduce the tumor size (or even substantially eliminate the tumor), and/or can alleviate and/or stabilize the disease state.
  • it can be expressed by the degree of reduction in weight gain of tumor mice and the intensity of tumor cell fluorescence signals.
  • the pharmaceutical composition described in the present application may include a preventive and/or therapeutically effective amount of the antibody or antigen-binding fragment thereof.
  • the prophylactic and/or therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disease or disorder and/or any complications in a subject suffering from or at risk of development.
  • the application provides the use of the antibody and the antigen-binding fragment thereof in the preparation of drugs for preventing or treating diseases or disorders.
  • the antibodies and antigen-binding fragments provided in this application can be used to prevent or treat diseases or disorders.
  • the prevention or treatment of a disease or condition may refer to inhibiting or delaying the development or progression of the disease or condition.
  • it can be used to inhibit the development or progression of tumors.
  • it can inhibit tumor growth or tumor cell proliferation.
  • the present application provides the antibody or antigen-binding fragment thereof for preventing or treating tumors.
  • the present application provides a method for inhibiting the binding of CD47 to SIRP ⁇ , which comprises administering the antibody, the antigen-binding fragment thereof, the nucleic acid molecule, the vector, the host cell, and / Or said pharmaceutical composition.
  • the method can be an in vitro or ex vivo method.
  • the method can be ELISA or FACS.
  • the present application provides a method for preventing or treating tumors, which includes administering the antibody or antigen-binding fragment thereof, the molecular nucleic acid, the vector, the The host cell and/or the pharmaceutical composition.
  • the tumor includes solid tumor and/or hematological tumor.
  • the tumor includes lymphoma, leukemia, and ovarian cancer.
  • the antigen binding proteins, pharmaceutical compositions and methods of the application can be administered in combination with other therapeutic agents and/or forms.
  • the combined administration generally means that two (or more) different therapies are delivered to the subject in the process of administration to the subject, so that the therapeutic effect on the subject is within a certain range. Time points overlap. In some cases, when the delivery of the second therapy starts, the delivery of the first therapy is still taking place, so that there is overlap in administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery.” In other cases, the delivery of one therapy ends before the delivery of the other therapy starts. In some cases, the treatment is more effective due to the combined administration, and the effects of the two treatments can be partially superimposed, completely superimposed, or greater than superimposed.
  • the antigen binding protein or pharmaceutical composition can be combined with one or more additional therapies such as surgery, radiotherapy or the administration of another therapeutic agent.
  • the additional therapy may include chemotherapy, for example, a cytotoxic agent.
  • additional therapies may include targeted therapies, such as tyrosine kinase inhibitors, proteasome inhibitors, or protease inhibitors.
  • additional therapies may include: anti-inflammatory compounds, anti-angiogenic compounds, anti-fibrotic compounds, or anti-proliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNA , Antisense molecules, fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (such as methotrexate) or NSAIDs.
  • the additional therapies may include different types of combination therapies.
  • the antigen binding protein, pharmaceutical composition or formulation described in this application and another therapy can be administered simultaneously or sequentially.
  • An isolated antigen binding protein comprising an antibody light chain variable region VL and an antibody heavy chain variable region VH, the VL comprising any one of LCDR1, LCDR2 and LCDR3, and the VH comprising HCDR1, HCDR2 and HCDR3 Any one of, wherein the VL comprises the amino acid sequence shown in SEQ ID NO: 153, and the VH comprises the amino acid sequence shown in SEQ ID NO: 154.
  • LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 139
  • LCDR1 preferably comprises the amino acid sequence shown in any one of SEQ ID NO: 113, 114 and 115 The amino acid sequence shown.
  • LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 140, wherein LCDR2 preferably comprises SEQ ID NO: 31, 56, 57, 122, 126 and 127
  • LCDR2 preferably comprises SEQ ID NO: 31, 56, 57, 122, 126 and 127
  • LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 141, wherein LCDR3 preferably comprises any one of SEQ ID NO: 67-74, 116-121 The amino acid sequence shown in.
  • HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 142, wherein HCDR1 preferably comprises SEQ ID NO: 32, 34, 36, 38, 40, 42 The amino acid sequence shown in any one of, 45, 47, 49, 51 and 53.
  • HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 143, wherein HCDR2 preferably comprises the amino acid sequence shown in any one of SEQ ID NO: 136-138 Amino acid sequence.
  • HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 144, wherein HCDR3 preferably comprises the amino acid sequence shown in any one of SEQ ID NO: 55 and 58-63 The amino acid sequence shown.
  • VL comprises SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, The amino acid sequence shown in any of 23, 25, 27, and 29.
  • VH comprises SEQ ID NO: 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,
  • CD47 derived from human and monkey, preferably KD ⁇ 3x10-9M;
  • a vector comprising the nucleic acid molecule according to embodiment 13.
  • a cell comprising the nucleic acid molecule according to embodiment 13 or the vector according to embodiment 14.
  • a method for preparing the isolated antigen binding protein of any one of embodiments 1-12 comprising under conditions that allow expression of the isolated antigen binding protein of any one of embodiments 1-12 , Culturing the cells described in embodiment 15.
  • a pharmaceutical composition comprising the isolated antigen binding protein of any one of embodiments 1-12, the nucleic acid molecule of embodiment 13, the carrier of embodiment 14 and/or the embodiment of 15 The cells, and optionally a pharmaceutically acceptable carrier.
  • a method for inhibiting the binding of CD47 to SIRP ⁇ comprising administering the isolated antigen binding protein of any one of embodiments 1-12.
  • the CD47 antigen can be used to immunize experimental animals to obtain antibody molecules that specifically bind to CD47.
  • the experimental animals can be mice, rats, rabbits, sheep, camels, and the like.
  • the antibody molecules obtained are of non-human origin. After obtaining non-human antibodies, these molecules need to be humanized using antibody engineering technology to reduce immunogenicity and improve druggability.
  • the process of antibody humanization has its technical complexity, and the humanized molecules often reduce the affinity to the antigen.
  • transgenic technology have made it possible to breed genetically engineered mice that carry human immunoglobulin immune libraries and have their endogenous mouse immune libraries missing.
  • the antibody produced by this transgenic mouse has a fully human sequence, so there is no need for further humanization modification, which greatly improves the efficiency of therapeutic antibody development.
  • the Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune library.
  • the antibodies produced by it have complete human antibody variable domains and rat constant domains.
  • mice were immunized for multiple rounds with soluble recombinant human CD47-Fc fusion protein.
  • the antigen protein is mixed with an immune adjuvant to form an immunogenic reagent, and then injected subcutaneously through the inguinal or through the abdominal cavity.
  • the total injection dose received by each mouse is 100 microliters.
  • each mouse was immunized with an immunogen reagent prepared by mixing 50 ⁇ g antigen protein (human CD47-Fc) and complete Freund's adjuvant (Sigma#F5881) at a volume ratio of 1:1.
  • each mouse was immunized with immunogenic reagents mixed with 25 ⁇ g antigen protein and Sigma Adjuvant System adjuvant (Sigma#S6322).
  • the interval between each round of booster immunity is at least two weeks, usually no more than five rounds of booster immunity.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49, 77 days, the mouse serum antibody titers were detected.
  • the final booster immunization was performed at a dose of 25 ⁇ g antigen protein per mouse.
  • the spleen cells of the mouse are taken out and fused with the myeloma cell line to obtain hybridoma cells; the hybridoma cells are isolated after multiple rounds of screening and cloning Out of 21 hybridomas expressing anti-CD47 monoclonal antibody molecules.
  • the isolated hybridoma cells and the monoclonal antibodies expressed by them are represented by the corresponding clone numbers: 11B8, 15D1, 18C4, 32F1, 35A11, 39A8, 40D7, 73F1, 74B6, 75F6, 78B6, 80B6, 81E5, 83G1, 84G2 , 85G2, 86D7, 87E9, 87H6, 88G3, 89E3.
  • the isolated hybridoma expresses antibody molecules with intact human variable domains and rat constant domains of heavy and light chains.
  • the above monoclonal antibodies were further identified, and several hybridoma clones were selected for sequencing according to their binding ability to human CD47, cyno-CD47 binding ability, and inhibiting CD47 binding to SIRP ⁇ .
  • Conventional hybridoma sequencing methods are used to obtain the nucleotide sequence encoding the variable domain of the antibody molecule and the corresponding amino acid sequence.
  • the sequence of the variable domain of the anti-CD47 monoclonal antibody molecule obtained from the immunized Harbour H2L2 mouse is a human antibody sequence.
  • the CDR sequences of antibody variable domains can be analyzed by Kabat or Chothia or other CDR definition rules. In this embodiment, a combination of Kabat definition and Chothia definition is also used for analysis.
  • VH antibody heavy chain variable domain sequence
  • conventional recombinant DNA technology can be used to combine the light and heavy chain variable domain sequences and the corresponding human antibody light and heavy chain constant domain sequences. Fusion expression to obtain recombinant antibody molecules.
  • the antibody heavy chain variable domain sequence (VH) was synthesized by gene and cloned into mammalian cell expression plasmid vector encoding the human IgG4 antibody heavy chain constant domain sequence to encode the full length of the IgG4 antibody.
  • the antibody light chain variable domain sequence (VL) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig ⁇ light chain constant domain sequence to encode the full-length light chain of the antibody.
  • VL antibody light chain variable domain sequence
  • HEK293 cells were expanded in FreeStyle TM F17 Expression Medium medium (Thermo #A1383504). Before the start of transient transfection, adjust the cell concentration to 6-8 ⁇ 10 5 cells/ml, and incubate for 24 hours in a 37°C 8% CO2 shaker. The cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • Table 2 lists the amino acid sequences of the light and heavy chain variable domains of the CD47 antibody in this example, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG4) and the amino acid of the CDR defined according to Chothia's definition rules sequence.
  • the sequence of the variable domain of the heavy chain of an antibody is derived from events such as the gene rearrangement of the germline gene V, D, and J gene segments of the heavy chain gene group on the chromosome and somatic high frequency mutation;
  • the gene rearrangement of germline gene V and J gene fragments of light chain gene group and somatic high frequency mutation events are the main factors that increase antibody diversity.
  • Antibodies derived from the same germline V gene fragment may also produce different sequences, but the overall similarity is higher.
  • IMGT/DomainGapAlign http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi
  • NCBI/IgBLAST https://www.ncbi.nlm.nih.gov/igblast/
  • Protein or polypeptide amino acid chains sometimes introduce chemical modifications after translation and synthesis in cells, called post-translational modifications (PTM).
  • PTM post-translational modifications
  • some PTM sites are very conservative.
  • the conservative amino acid Asparagine Asn at position 297 (EU numbering) of the constant domain of human IgG1 antibodies usually undergoes glycosylation modification.
  • the sugar chain, and the structure of the sugar chain is critical to the structure of the antibody and related effector functions.
  • PTMs are present in the variable domains of antibodies, especially the antigen-binding regions (such as CDRs), the presence of these PTMs may have a greater impact on the binding of antigens, and may also affect the physicochemical properties of the antibody. To change.
  • glycosylation, deamidation, isomerization, oxidation, etc. may increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding some potential PTMs is very important for the development of therapeutic antibodies.
  • some PTMs are highly correlated with the composition of amino acid sequences, especially the "patterns" of adjacent amino acid composition, so that potential PTMs can be predicted from the primary amino acid sequence of the protein.
  • the sequence pattern of N-x-S/T (the first position is asparagine, the second position is any amino acid other than proline, and the third position is serine or threonine) sequence pattern predicts N-linked glycosylation sites.
  • the amino acid sequence pattern that causes PTM may be derived from the germline gene sequence.
  • human germline gene fragment IGHV3-33 naturally has a glycosylation pattern NST in the FR3 region; it may also be derived from somatic high-frequency mutations.
  • Table 3 lists the predicted PTMs of the variable domains VH and VL of the antibody of Example 1.
  • NSS or NLT may be glycosylation sites
  • NS or NT or NN may be deamidation sites.
  • Amino acid mutations can be used to disrupt the amino acid sequence pattern of PTM, thereby reducing or eliminating the formation of specific PTMs.
  • One method is to replace "hot spot" amino acids (such as N or S in the NS pattern) with amino acids with similar physical and chemical properties (such as mutating N to Q). If the PTM sequence pattern is derived from somatic high-frequency mutations and does not exist in the germline gene sequence, then another method can be to replace the sequence pattern with the corresponding germline gene sequence. In practice, multiple mutation design methods may be used for the same PTM sequence pattern.
  • Table 4 lists new antibody molecules (called PTM variants) obtained by performing amino acid mutations on the sequences of six antibodies with potential PTM sites from Example 1.
  • Table 5 lists the amino acid sequences of the light and heavy chain variable domains of these PTM variants in this example, the full-length amino acid sequence of the light chain, the full-length amino acid sequence of the heavy chain (human IgG4), and those defined according to Chothia's definition rules. The amino acid sequence of the CDR. All the designed PTM variants obtained purified recombinant antibodies according to the method described in Example 1.3, and were further verified in the subsequent functional experiments.
  • Example 3 Antigen binding protein can bind to CD47 on the surface of human and monkey cells
  • Jurkat cells ATCC#TIB-152 and Raji cells (ATCC#CCL-86) were expanded and cultured, and added to a 96-well plate (Corning#3799), 1 ⁇ 10 5 cells per well, and then 100 ⁇ l of different concentrations were added
  • the CD47 antibody obtained in Example 1 or 2 and the control antibody (Tab1, Tab2, Tab3), and the isotype hIgG4SP were used as the control. After mixing, incubate at 4°C for 1 hour. Wash 3 times with 1% BSA (Amresco#0332-100G), add 100 ⁇ l of 4 ⁇ g/mL goat anti-human IgG (H+L)-Alexa488, and incubate at 4°C for 1 hour.
  • Tab1, Tab2 and Tab3 are all derived from the prior art, which are Hu5F9-G4 of Forty Seven, CC-90002 of Celgene, and SRF-231 of Surface Oncology, respectively.
  • the results are shown in Figure 1 and Table 6-7 below.
  • the CD47 antibody obtained in Example 1 or 2 can bind to Jurkat cells, and the binding activity is not significantly different from that of Tab1 and Tab2 ( Figures 1A-1B), and has better binding capacity than Tab3.
  • PR000806, PR000807, PR000808 and Tab1 and Tab2 can all bind to CD47 on the surface of Jurkat (Figure 1B) and Raji ( Figure 1C) cells, with no significant difference in binding activity, and have stronger affinity than Tab3 to Jurkat and Raji.
  • Table 7 The binding of antigen binding proteins PR000806, PR000807, PR000808 to CD47 on the surface of Jurkat and Raji
  • the method 3.1 was used to detect the binding ability of CD47 antibody and cynomolgus monkey PBMC. The results are shown in Figure 2 and Table 8.
  • the CD47 antibody before PTM mutation obtained in Example 1 can all bind to CD47 on the surface of cynomolgus monkey PBMC cells, and the EC 50 value is less than Tab2.
  • Table 8 The binding of antigen binding protein to CD47 on the surface of cynomolgus monkey
  • Antigen binding protein can bind to human and cynomolgus CD47 protein
  • the CD47 antibody obtained in Example 1 or 2 and the three CD47 antibodies in the prior art Tab1( Forty Seven, Hu5F9-G4), Tab2 (Celgene, CC-90002), Tab3 (Surface Oncology, SRF-231) starting from the 66.7nM concentration, successively perform 5-fold concentration dilutions, a total of 10 concentration gradients, add 100 ⁇ l/well, Incubate at 37°C for 1 hour, and use the same type hIgG4SP (Crown Bio#C0045-4) as a control. After washing 3 times, add goat anti-human HRP secondary antibody (Invitrogen#A18805) diluted 4000 times and incubate at 37°C for 1 hour.
  • Figure 3A and Table 9 show that all the CD47 antibodies before PTM mutation obtained in Example 1 can bind to the CD47 protein derived from cynomolgus monkeys.
  • Figures 3B-3C and Table 10 show the activities of PR00806, PR000807, and PR000808 in binding to human ( Figure 3B) and monkey ( Figure 3C) CD47.
  • PR00806, PR000807, PR000808, Tab1, Tab2, Tab3 can all bind to human CD47 protein with no significant difference in binding activity, and the EC 50 of PR00806, PR000807, PR000808 is lower than 0.03nM, and the binding ability is better than Tab3.
  • PR00806, PR000807, PR000808, Tab1, Tab2 can all bind to monkey CD47 protein, and there is no significant difference in binding activity, but the binding ability of Tab3 to monkey CD47 protein is weak.
  • Antigen binding protein can inhibit the binding of human CD47 protein to SIRP ⁇
  • Human CD47-ECD-hFc (Sino Biological#12283-H02H) was diluted with PBS to 1 ⁇ g/ml and added to a 96-well plate (Corning#9018), 100 ⁇ l per well, coated overnight at 4°C. After washing 3 times, it was blocked with 100 ⁇ l of 2% BSA at room temperature for 1 hour.
  • the CD47 antibody and the control antibody (Tab1, Tab2, Tab3) obtained in Example 1 or 2 were diluted with PBST into different concentration gradients, and mixed with 1 ⁇ g/ml histidine-labeled SIRP ⁇ protein (Sino Biological#11612-H08H), Add the well plate with the same type hIgG4 as a control, and incubate for 1 hour at room temperature. After washing 3 times, add 100 ⁇ l of histidine-labeled HRP antibody (Biolegend#652504) to each well, and react for 1 hour at room temperature. After washing 3 times, add 100 ⁇ l TMB color developing solution, and darken the color for 5 minutes at room temperature.
  • the reaction was terminated by adding stop solution, and the absorbance (OD450) at 450 nm was measured with a microplate reader (PerkinElemer#Enspire).
  • the IC 50 value, the minimum OD value, and the maximum inhibition rate were used as the basis for evaluating the relative inhibitory activity.
  • Figure 4A and Table 11 show that the CD47 antibodies obtained in Example 1 can inhibit the binding of human CD47 to SIRP ⁇ .
  • Figure 4B and Table 12 show that PR00806, PR000807, and PR000808 can all inhibit the binding of human CD47 to SIRP ⁇ , the inhibition rate is all greater than 97%, and the inhibitory activity is not significantly different from Tab1, Tab2, and Tab3.
  • Antigen binding protein can block the binding of CD47 and SIRP ⁇ on Jurkat cells
  • Example 1 After expansion and culture of human Jurkat cells with high CD47 expression, 1 ⁇ 10 5 cells per well were added to a 96-well plate.
  • the CD47 antibody and control antibodies (Tab1, Tab2, Tab3) obtained in Example 1 or 2 were diluted with PBS into different concentration gradients, mixed with 1 ⁇ g/ml histidine-labeled SIRP ⁇ protein, and added to each well.
  • the isotype hIgG4SP was used as a control , Incubate for 1 hour at 4°C. Wash twice with 1% BSA, add 100 ⁇ l volume of the secondary antibody Biotin-conjugated anti-His Tag antibody (GenScript#A00613), and incubate for 1 hour at 4°C.
  • Figure 5A and Table 13 show that the CD47 antibodies obtained in Example 1 can block the binding of CD47 on the surface of Jurkat cells to SIRP ⁇ .
  • Figure 5B and Table 14 show that PR00806, PR000807, and PR000808 can all block the binding of CD47 on the surface of Jurkat cells to SIRP ⁇ , and their blocking activity is not significantly different from that of Tab1 and Tab2.
  • the IC 50 is less than 0.7nM, while the blocking ability of Tab3 is better. weak.
  • Example 7 Antigen binding protein does not cause agglutination of red blood cells
  • Example 1 It was analyzed whether the CD47 antibody obtained in Example 1 or 2 caused hemagglutination reaction. Dilute the human red blood cells to 4% with PBS and add them to a 96-well plate (Corning#3799), then add the test antibody and the control antibody (Tab1, Tab2, Tab3) starting from 200nM, and the isotype hIgG4SP as a control. Incubate for 1 hour. The effect of antibodies on human red blood cell agglutination is evaluated by comparing the sharpness of the edges of the red dots in the holes without antibodies.
  • Figures 6A-6B show that, compared with the wells without antibody, except PR00796, other antigen-binding proteins do not cause agglutination, and the red dots have clear edges; while Tab1 causes agglutination, the red dots are fuzzy and diffuse.
  • the CD47 antibody obtained in Example 1 or 2 was tested for its binding activity to red blood cells. Dilute human red blood cells in PBS and add them to a 96-well plate (Corning#3799), 1 ⁇ 10 5 per well, and then add 100 ⁇ l of different concentrations of anti-CD47 antibodies and control antibodies (Tab1, Tab2, Tab3), with the same type hIgG4SP as a control After mixing, incubate for 1 hour at 4°C.
  • antigen binding protein The ability of antigen binding protein to bind to red blood cells is shown in Figure 7 and Tables 15-16 below.
  • Example 3 The ability of antigen binding protein to bind to Jurkat cells (Example 3) and red blood cell (RBC) binding was compared, and the ratio of EC 50 and maximum MFI of antigen binding protein to tumor cells and red blood cells was calculated.
  • Table 16 The results are shown in Table 16 below. It can be seen from the results that, compared with the control antibody, the antigen binding protein obtained in Example 1 or 2 has a greater ability to bind to Jurkat than to red blood cells.
  • Table 16 The ratio of the binding ability of antigen binding protein to tumor cells and red blood cells
  • Antigen binding protein can cause phagocytosis of tumor cells by macrophages
  • CD14+ monocytes (Miltenyi Biotec#130-050-201) were isolated from human peripheral blood mononuclear cells (PBMC) and cultured for 7 days by adding 100ng/ml M-CSF (R&D Systems#216-MC/CF) They were differentiated into macrophages, digested with trypsin, and seeded on a 96-well plate with 5 ⁇ 10 4 cells per well. At the same time, 0.25 ⁇ M carboxyfluorescein succinimidyl ester (CFSE) was added at a ratio of 1:1 ( Life-technology#C34570)-labeled Jurkat cells, then add diluted anti-CD47 antibody and control antibody (Tab1, Tab2, Tab3), mix and incubate at 37°C for 2 hours.
  • PBMC peripheral blood mononuclear cells
  • CFSE carboxyfluorescein succinimidyl ester
  • the Jurkat cells that were not phagocytosed were washed with 1% BSA, and the direct-labeled antibody CD14-APC (BD Pharmingen#561708) was added.
  • Flow cytometry (ACEA#Novocyte) is used for measurement, and the phagocytic index is used as the evaluation basis for phagocytosis.
  • the phagocytic index is the percentage of CFSE + CD14 + cells to the total CD14 + cells.
  • Figure 8A and Table 17 show that the CD47 antibody before PTM mutation obtained in Example 1 can all cause the phagocytosis of Jurkat cells by macrophages, and the ability to cause phagocytosis is better than Tab2.
  • Figure 8B and Table 18 show that PR000806, PR000807, and PR000808 can all cause the phagocytosis of Jurkat cells by macrophages, and the ability to cause phagocytosis is better than Tab2 and Tab3.
  • Table 19 shows that the CD47 antibody obtained in Example 1 or 2 caused the phagocytosis of Jurkat cells by macrophages at different concentrations.
  • Example 9.1 according to a method embodiment of detecting macrophage phagocytosis OVCAR 3 (ATCC # HTB-161 ) cells in flow cytometry and determination of EC 50 phagocytic index.
  • PR00806, PR000807, and PR000808 can all cause the phagocytosis of OVCAR3 cells by macrophages, and there is no significant difference; EC 50 value and maximum phagocytic index show that PR00806, PR000807, and PR000808 cause phagocytosis The ability to act is stronger than Tab3.
  • Example 9 Using the method of Example 1 or 2 for detecting the resultant antigen-binding protein induced phagocytosis embodiment macrophages human erythrocytes, flow cytometry EC 50 and phagocytic index.
  • Figure 10 and Tables 21-22 show the ability of the CD47 antibody after PTM removal to induce phagocytosis of human erythrocytes by macrophages.
  • Table 22 shows the ability of the CD47 antibody obtained in Example 1 or 2 to induce the phagocytosis of hRBC by macrophages at different concentrations.
  • the results show that the antigen binding protein (eg, CD47 antibody) described in this application can effectively cause macrophages to phagocytose hRBC, and the effect is similar to or higher than that of the control antibody.
  • the anti-tumor activity of the antigen binding protein was evaluated in the NCG-Raji mouse model. 1 ⁇ 10 7 cells/0.1 mL of Raji cells were inoculated into the tail vein of NCG mice. On the third day after cell inoculation, the mice were randomly divided into 10 groups, each with 8 mice, and the administration was started. Each test antibody was injected intraperitoneally at a dose of 0.3 mg/kg, 1 mg/kg and 3 mg/kg, 10 ⁇ l/g, once every 3 days for 6 consecutive administrations. The same type hIgG4 was used as a control. Record the mouse body weight and tumor volume.
  • Table 23 shows the tumor inhibition rate (TGI) of each antibody injected compared with the same type of IgG4.
  • TGI tumor inhibition rate
  • Figures 11A-11B show the trends of tumor volume and body weight in mice injected with PR000806, PR000808 and Tab1. The tumor volume of mice injected with PR000806 and PR000808 slowed down. The results show that PR000806 and PR000808 can inhibit tumor growth in vivo, and PR000808 is more effective than Tab1 at the doses of 3mg/kg and 1mg/kg.
  • the anti-tumor activity of the antigen binding protein was evaluated in the B-hSIRPa/hCD47-MC38-hCD47 mouse model.
  • 1 ⁇ 10 6 cells/0.1 mL of MC38 cells stably expressing human CD47 protein were inoculated subcutaneously on the right side of B-hSIRPA/hCD47 mice. After cell inoculation, when the average tumor volume reached about 80-120mm3, the mice were randomly divided into 7 groups, each with 8 mice, and the administration was started.
  • Each antibody to be tested was injected intraperitoneally at a dose of 5 mg/kg and 10 mg/kg, 10 ⁇ l/g, once every 3 days for 6 consecutive administrations, with the same type of hIgG4 as the control. Record the mouse body weight and tumor volume.
  • Figures 12A-12B show the trends of tumor volume and body weight in mice injected with PR000806, PR001265 and Tab1.
  • the tumor volume growth of mice injected with PR000806 and PR001265 slowed down.
  • the results showed that PR000806 and PR001265 can inhibit tumor growth in vivo, and PR000806 and PR001265 tumor suppressor effects are better than Tab1 at high doses.
  • B-hSIRPA/hCD47 homozygous mice were randomly grouped according to body weight, 4 mice in each group, a total of 8 groups, intraperitoneal injection of 10mpk was administered on the 8th day after grouping, and the first administration was the 0th day of treatment. A total of 1 administration (day 0). The body weight of the mice was measured once a day during the administration and observation period, and the measured value was recorded. In addition, blood was collected 7 days before administration (normal value), 1 day, and 7 days after treatment for routine blood analysis. The results of mouse body weight changes and blood routine analysis are shown in Figure 13-15.
  • Figure 13 shows the body weight changes of mice before and after treatment. Except for the homotype hIgG4, the weight of mice was reduced after treatment with other antigen binding proteins or antibodies. Among them, the weight loss of mice after Tab1, PR000806, PR000808 and PR001281 treatment was greater than that of the other three molecules, and the antibody molecules and antigens in vitro were on red blood cells. The results of the binding ability of the mice were consistent, but about 7 days after the treatment, the weight of the mice began to return to normal levels.
  • Figures 14-16 show the results of routine blood tests in the mouse model 7 days before treatment and on the 1st and 7th days after treatment. It can be seen from the image that on the first day after administration, some parameters in the blood routine of hIgG4SP in the sample group have changed from those in the control group, but after 7 days after administration, as the body's compensatory function exerts, All indicators returned to normal.
  • Example 13 Affinity test for antigen binding protein to bind to CD47 (Biacore)
  • each cycle includes antibody capture, analyte binding and chip regeneration.
  • the antibodies were all diluted to 1 ⁇ g/ml and injected into the 2, 3, and 4 channels at a flow rate of 10 ⁇ l/min for 40s, and each antibody was captured by the pre-coupled Protein A, and the capture amount was about 200RU.
  • glycine (10mM, pH1.5) was injected at the same flow rate for 30s to regenerate the chip.
  • Biacore T200 analysis software 2.0 was used to analyze the experimental results, 1 channel was used as the reference channel for subtraction, and the analysis model was a 1:1 kinetic fitting model.
  • Example 14 Antigen binding protein binds to CD47 with high specificity
  • the plate was washed 3 times with PBST, blocked with 2% BSA, and incubated for 1 hour at room temperature. The blocking solution was discarded, and the plate was washed 5 times with PBST buffer (pH7.4, containing 0.05% Tween-20), and the CD47 antibodies PR001265, PR001275, PR001276 and Tab1 (Forty Seven, Hu5F9- G4) Dilute to 10nM and 100nM, add 100 ⁇ l/well, and incubate at 37°C for 1 hour.
  • the isotype hIgG4SP (Crown Bio#C0045-4) is used as control.

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Abstract

一种分离的抗原结合蛋白，该抗原结合蛋白能够特异性结合肿瘤细胞表面的CD47，抑制CD47与SIRPα的结合，能够结合人红细胞，不引起凝血反应，能够引起巨噬细胞对肿瘤细胞的吞噬，抑制肿瘤生长和/或肿瘤细胞增殖。提供了该抗原结合蛋白在预防和***中的应用。

Description

一种抗CD47抗原结合蛋白及其应用
相关申请的交叉引用
本申请要求CN2019107757316优先权，将其通过引用整体并入全文。
技术领域
本申请涉及生物医药领域，具体的涉及一种抗CD47抗原结合蛋白及其应用。
背景技术
CD47也称为整联蛋白相关蛋白(IAP)、卵巢癌抗原OA3、Rh相关抗原和MER6，是一种广泛表达于细胞表面的跨膜蛋白，属于免疫球蛋白超家族。CD47可以与整联蛋白、血小板反应素(TSP-1)以及信号调节蛋白α(SIRPα)相互作用。CD47最初被鉴定为人卵巢癌上的肿瘤抗原，随后大量研究证实，CD47还在大部分人类癌症(例如，淋巴瘤、白血病、乳腺癌、结肠癌、肺癌、胃癌、成胶质细胞瘤、神经胶质瘤、卵巢癌、***、膀胱癌、***癌或黑色素瘤)的肿瘤细胞上高表达(参见Oldenborg PA.CD47:A Cell Surface Glycoprotein Which Regulates Multiple Functions of Hematopoietic Cells in Health and Disease.ISRN Hematol.2013；2013:614619.doi:10.1155/2013/614619)，与SIRPα结合后，能传递抑制性信号逃避巨噬细胞的吞噬。因此，阻断CD47-SIRPα相互作用的药剂，可以恢复CD47+靶细胞的吞噬摄取并降低巨噬细胞活化的阈值。
近年来，针对CD47与SIRPα信号通路介导的肿瘤逃逸机制的药物研发成为肿瘤免疫治疗的新的热门。目前，临床上正在研究的CD47抗体有Forty Seven的Hu5F9-G4、Celgene的CC-90002、Surface Oncology的SRF-231等。然而，这些抗体还存在亲和力较低、引起血细胞凝集反应等缺点(参见专利US9045541)。因此，急需研发更加安全有效的靶向CD47的新型疗法。
发明内容
本申请提供了一种抗CD47抗原结合蛋白，其具有下述性质中的一种或多种：1)能够结合源自人和猴的CD47；2)能够特异性结合Jurkat细胞和Raji细胞表面的CD47；3)能够抑制CD47与SIRPα的结合；4)能够阻断Jurkat细胞表面的CD47与SIRPa的结合；5)能够结合人红细胞；6)不引起凝血反应；7)能够引起巨噬细胞对肿瘤细胞的吞噬；8)能够抑制肿瘤生长和/或肿瘤细胞增殖。本申请还提供了所述抗原结合蛋白在预防和***中的应用。
一方面，本申请提供了一种分离的抗原结合蛋白，其包含抗体轻链可变区VL和抗体重链可变区VH，所述VL包含LCDR1，LCDR2和LCDR3，所述VH包含HCDR1，HCDR2和HCDR3，其中所述VL包含SEQ ID NO:153所示的氨基酸序列，所述VH包含SEQ ID NO:154所示的氨基酸序列。
在某些实施方式中，所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
1)能够以3x10 -9M或更高的KD值结合源自人和猴的CD47，其中所述KD值通过Biacore测定；
2)在FACS测定中，能够特异性结合Jurkat细胞和Raji细胞表面的CD47；
3)在ELISA测定中，能够抑制CD47与SIRPα的结合；
4)能够结合人血红细胞；
5)在红细胞凝血测定中，不引起凝血反应；
6)能够引起巨噬细胞对肿瘤细胞的吞噬；
7)能够抑制肿瘤生长和/或肿瘤细胞增殖。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述分离的抗原结合蛋白中所述抗原结合片段包括Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。
在某些实施方式中，所述分离的抗原结合蛋白中所述抗体为全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白中LCDR1包含SEQ ID NO:139所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述LCDR1包含SEQ ID NO:113、114和115中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述LCDR2包含SEQ ID NO:140所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述LCDR2包含SEQ ID NO:31、56、57、122、126和127中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述LCDR3包含SEQ ID NO:141所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述LCDR3包含SEQ ID NO:67-74、116-121中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR1包含SEQ ID NO:142所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR1包含SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR2包含SEQ ID NO:143所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR2包含SEQ ID NO:136-138中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR3包含SEQ ID NO:144所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述HCDR3包含SEQ ID NO:55和58-63中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:149所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR1包含SEQ ID NO:156、157和158中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:150所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR2包含SEQ ID NO:191-194中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:151所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR3包含SEQ ID NO:155、162-166中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO:152所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述L-FR4包含SEQ ID NO:159、160和161中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述VL包含SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27和29中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻 链恒定区包括人Igκ恒定区。
在某些实施方式中，所述分离的抗原结合蛋白中所述抗体轻链恒定区包含SEQ ID NO:196所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC包含SEQ ID NO:197所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC包含SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28和30中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:145所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR1包含SEQ ID NO:167-170中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:146所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR2包含SEQ ID NO:184、187-190中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:147所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR3包含SEQ ID NO:171-182中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:148所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述H-FR4包含SEQ ID NO:183、185和186中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白中所述VH包含SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
在某些实施方式中，所述分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG4恒定区。
在某些实施方式中，所述分离的抗原结合蛋白中所述抗体重链恒定区包含SEQ ID NO:195中任一项中所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链HC，且所述HC包含SEQ ID NO:198所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含抗体重链HC，且所述HC包含SEQ ID NO:76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110和112中任一项中所示的氨基酸序列。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码本申请所述的分离的抗原结合蛋白。
另一方面，本申请提供了一种载体，其包含本申请所述的核酸分子。
另一方面，本申请提供了一种细胞，其包含本申请所述的核酸分子或本申请所述的载体。
另一方面，本申请提供了一种制备本申请所述的分离的抗原结合蛋白的方法，所述方法包括在使得本申请所述的分离的抗原结合蛋白表达的条件下，培养本申请所述的细胞。
另一方面，本申请提供了一种药物组合物，其包含本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体和/或本申请所述的细胞，以及任选地药学上可接受的载体。
另一方面，本申请提供了一种本申请所述的分离的抗原结合蛋白、本申请所述的核酸分子、本申请所述的载体、本申请所述的细胞和/或本申请所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或***。
在某些实施方式中，所述的用途，其中所述肿瘤包括实体瘤和/或血液肿瘤。
另一方面，本申请提供了一种抑制CD47与SIRPα结合的方法，所述方法包括施用本申请所述的分离的抗原结合蛋白。
另一方面，本申请提供了一种预防、缓解和/或***的方法，所述方法包括向有需要的受试者施用本申请所述的分离的抗原结合蛋白。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1A-1B显示的是本申请所述抗原结合蛋白与Jurkat细胞的结合活性(FACS检测)；
图1C显示的是本申请所述抗原结合蛋白PR000806、PR000807和PR000808与Raji细胞的结合活性(FACS检测)；
图2显示的是本申请所述抗原结合蛋白PR000796、PR000802、PR000803、PR000804、PR000805、PR000806、PR000807、PR000808和PR000811与食蟹猴PBMC细胞的结合活性(FACS检测)；
图3A显示的是本申请所述抗原结合蛋白PR000796、PR000802、PR000803、PR000804、PR000805、PR000806、PR000807、PR000808和PR000811与猴CD47蛋白的结合活性(ELISA检测)；
图3B-3C显示的是本申请所述抗原结合蛋白PR000806、PR000807和PR000808分别与人和猴CD47蛋白的结合活性(ELISA检测)；
图4A-4B显示的是本申请所述抗原结合蛋白PR000796、PR000802、PR000803、PR000804、PR000805、PR000806、PR000807、PR000808和PR000811抑制人CD47与SIRPα结合的活性(ELISA检测)；
图5A-5B显示的是本申请所述抗原结合蛋白PR000796、PR000802、PR000803、PR000804、PR000805、PR000806、PR000807、PR000808和PR000811阻断Jurkat细胞表面的CD47与SIRPα结合的活性(FACS检测)；
图6A-6B显示的是本申请所述抗原结合蛋白不引起人红细胞凝集反应；
图7显示的是本申请所述抗原结合蛋白与红细胞的结合能力(FACS检测)；
图8A-8B显示的是本申请所述抗原结合蛋白PR000796、PR000802、PR000803、PR000804、PR000805、PR000806、PR000807、PR000808和PR000811引起巨噬细胞对Jurkat细胞吞噬作用的能力；
图9显示的是本申请所述抗原结合蛋白PR000806、PR000807和PR000808引起巨噬细胞对OVCAR3细胞吞噬作用的能力；
图10显示的是本申请所述抗原结合蛋白PR001265、PR001275、PR001276、PR000806、PR000808和PR001281引起巨噬细胞对人红细胞吞噬作用的能力；
图11A-11B显示的是不同浓度的本申请所述抗原结合蛋白PR000806和PR000808在NCG-Raji小鼠模型上的体内抗肿瘤活性，分别为肿瘤体积变化(A)和小鼠体重变化(B)；
图12A-12B显示的是不同浓度的本申请所述抗原结合蛋白PR001265和PR000806在B-hSIRPa/hCD47-MC38-hCD47小鼠模型上的体内抗肿瘤活性，分别为肿瘤体积变化(A)和小鼠体重变化(B)；
图13显示的是使用本申请所述抗原结合蛋白PR001265、PR001275、PR001276、PR000806、PR000808和PR001281治疗前后小鼠体重变化趋势；
图14显示的是使用本申请所述抗原结合蛋白PR001265、PR001275、PR001276、PR000806、PR000808和PR001281治疗前7天小鼠模型血常规检测结果；
图15显示的是使用本申请所述抗原结合蛋白PR001265、PR001275、PR001276、PR000806、PR000808和PR001281治疗后第1天小鼠模型血常规检测结果；
图16显示的是使用本申请所述抗原结合蛋白PR001265、PR001275、PR001276、PR000806、PR000808和PR001281治疗后第7天小鼠模型血常规检测结果；
图17显示的是使用本申请所述抗原结合蛋白PR001265、PR001275和PR001276以高度特异性结合CD47，而不结合非CD47抗原。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为框架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR-1、CDR1、FR-2、CDR2、FR-3、CDR3和FR-4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“可变”通常是指这样的事实，即抗体的可变结构域的序列的某些部分变化强烈，它形成各种特定抗体对其特定抗原的结合和特异性。然而，变异性并非均匀地 分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)。可变域中更高度保守的部分被称为框架(FR)。天然重链和轻链的可变结构域各自包含四个FR区，大部分采用β-折叠构型，通过三个CDRs连接，形成环连接，并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点，恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)。在本申请中，还使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基。
表1 本申请抗体CDR定义方法(可参见http://bioinf.org.uk/abs/)
  Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。
在本申请中，术语“分离的抗原结合蛋白”通常是指已经从其产生环境(例如，天然的或重组的)的组分中识别，分离和/或回收的抗原结合蛋白。其产生环境的污染组分通常是干扰其研究、诊断或治疗用途的物质，可以包括酶、激素和其他蛋白质或非蛋白质溶质。分离的抗原结合蛋白或抗体通常将通过至少一个纯化步骤来制备。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即集群中的个别抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原 位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本发明使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“特定抗CD47抗体”通常是指可以与本申请的抗体竞争结合CD47的抗体。本申请的抗原结合蛋白可包括与特定抗CD47抗体具有相同氨基酸序列的那些抗体。
在本申请中，术语“特异性结合”通常是指抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“CD47”通常是指白细胞表面抗原CD47，可以为完整的CD47蛋白，也可以为保留CD47的至少部分活性的任何形式的CD47及其变体。所述CD47可包括人CD47及其片段或猴CD47及其片段。例如，人CD47可包含参见UniProtKB数据库中登录号No.Q08722所示的氨基酸序列。所述的猴可以为食蟹猴。例如，食蟹猴CD47可包含UniProtKB数据库中登录号为No.A0A2K5X4I2的氨基酸序列。在本申请中，术语“人CD47胞外区”和“hCD47.ECD”可互换地使用，通常是指CD47胞外N端的V型免疫球蛋白的结构域，也是CD47配体结合区域。例如，人CD47胞外区可包含UniProtKB中登录号为No.Q08722所示氨基酸序列的第19位至第141位序列。
在本申请中，术语“SIRPα”通常是指信号调节蛋白α，也称为BIT、MFR、MYD1、PTPNS1、SHPS1或SIRP，是CD47的免疫球蛋白样细胞表面受体，可以为完整的SIRPα蛋白，也可以为保留SIRPα的至少部分活性的任何形式的SIRPα及其变体。在某些情形中，所述SIRPα可以为人SIRPα，其可包含UniProtKB数据库中登录号为P78324所示的氨基酸序列。
在本申请中，术语“Jurkat细胞”通常是指人白血病T淋巴细胞。所述Jurkat细胞来源 于一个14岁的男孩的外周血。
在本申请中，术语“Raji细胞”通常是指人Burkitt's淋巴瘤细胞。所述Raji细胞源自一位11岁黑人男孩的左上颌骨的Burkitt淋巴瘤。
在本申请中，术语“OVCAR 3细胞”和“OVCAR-3细胞”可互换地使用，通常是指人卵巢腺癌细胞。所述OVCAR 3细胞源自患有进行性卵巢腺癌病人的恶性腹水。
在本申请中，术语“凝血反应”、“凝集作用”可互换地使用，通常是指当与不同试剂一起孵育时，血红细胞凝集或结块时发生的同型相互作用。所述凝集作用可以是指对照抗体存在时，红细胞的凝集使血液圆斑边缘模糊的现象。据报道，现有的CD47抗体在一定浓度下会引起RBC的凝集，如本申请实施例7中的现有抗体Tab1(FortySeven，Hu5F9-G)。因此，凝血作用会限制CD47抗体的治疗效果。术语“不引起凝血反应”通常是指不产生血红细胞凝集或结块。
在本申请中，术语“Tab1”通常是指FortySeven公司的抗CD47抗体Hu5F9-G。
在本申请中，术语“Tab2”通常是指Celgene公司的抗CD47抗体CC-90002。
在本申请中，术语“Tab3”通常是指Surface Oncology公司的抗CD47抗体SRF-231。
在本申请中，术语“KD”、“Kd”、“KD”可互换地使用，通常是指平衡解离常数，并指在滴定测量中，在平衡时、或者通过将解离速率常数(koff)除以结合速率常数(kon)所获得的值。可使用结合速率常数(kon)、解离速率常数(koff)和平衡解离常数(KD)表示抗原结合蛋白(例如抗体)对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知。使用基于荧光的技术可提供高灵敏度以及在生理缓冲液中在平衡时检查样品的能力。可以使用其他实验途径和仪器例如BIAcore(生物分子相互作用分析)测定。
在本申请中，术语“核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将***的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA***细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗体或其抗原结合片段的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞 与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在一些实施方案中，所述细胞是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。在本申请中，术语“重组细胞”通常是指在其中引入了重组表达载体的细胞。所述重组宿主细胞不仅包括某种特定的细胞，还包括这些细胞的后代。
在本申请中，术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。所述组合物是无菌的。“无菌”组合物是灭菌的，或不含所有活的微生物及它们的孢子。
在本申请中，术语“肿瘤”通常是指哺乳动物中的机体(例如，细胞或其组成部分)在各种致瘤因子作用下，局部组织细胞增生所形成的赘生物。在本申请中，肿瘤可以包括淋巴瘤、白血病和卵巢癌。例如，肿瘤可以包括侵袭性非霍奇金淋巴瘤、伯基特淋巴瘤、急性淋巴细胞性白血病(ALL)，急性骨髓性白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性骨髓性白血病(CML)，卵巢癌等。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
抗体、其抗原结合片段或变体
一方面，本申请提供一种抗原结合蛋白，其可包含轻链可变区VL中的至少一个CDR，所述VL可包含SEQ ID NO:153所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含LCDR1，且所述LCDR1可包含SEQ ID NO:139所示的氨基酸序列：
RASQSX 1SSX 2LX 3(SEQ ID NO:139)；其中，X 1＝I或V；X 2＝W，Y或H；X 3＝A或N。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:113所示抗原结合蛋白的LCDR1相比，所述LCDR1可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2和/或X 3处的氨基酸取代。
在本申请中，与SEQ ID NO:113所示抗原结合蛋白的LCDR1相比，所述LCDR1可至少包含在X 1、X 2或X 3处的氨基酸取代，其中X 1处的氨基酸可被取代为V；X 2处的氨基酸可被取代为Y或H；X 3处的氨基酸可被取代为N。
例如，所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:113-115。
在本申请中，所述抗原结合蛋白可包含LCDR2，且所述LCDR2可包含SEQ ID NO:140所示的氨基酸序列：
X 1X 2SX 3X 4X 5X 6(SEQ ID NO:140)；其中，X 1＝K，R，V或G；X 2＝A或S；X 3＝S，T，R或I；X 4＝L或R；X 5＝E，D，Q或A；X 6＝S或T。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:56所示抗原结合蛋白的LCDR2相比，所述LCDR2可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5和/或X 6处的氨基酸取代。
在本申请中，与SEQ ID NO:56所示抗原结合蛋白的LCDR2相比，所述LCDR2可至少包含在X 1、X 2、X 3、X 4、X 5或X 6处的氨基酸取代，其中X 1处的氨基酸可被取代为R、V或G；X 2处的氨基酸可被取代为S；X 3处的氨基酸可被取代为R、I或T；X 4处的氨基酸可被取代为R；X 5处的氨基酸可被取代为D、Q或A；X 6处的氨基酸可被取代为T。
例如，所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:31、56、57、122、126和127。
在本申请中，所述抗原结合蛋白可包含LCDR3，且所述LCDR3可包含SEQ ID NO:141所示的氨基酸序列：
X 1X 2X 3X 4X 5X 6X 7X 8X 9X 10T(SEQ ID NO:141)；其中，X 1＝Q或R；X 2＝Q，L或H；X 3＝Y或-；X 4＝N，I，Q或-；X 5＝S，Y，T或N；X 6＝Y，W，N或Q；X 7＝S，T或-；X 8＝T或-；X 9＝P或-；X 10＝Y，R或L。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:116所示抗原结合蛋白的LCDR3相比，所述LCDR3可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9和/或X 10处的氨基酸取代。
在本申请中，与SEQ ID NO:116所示抗原结合蛋白的LCDR3相比，所述LCDR3可至少包含在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9和/或X 10处的氨基酸取代，其中X 1处的氨基酸可被取代为Q；X 2处的氨基酸可被取代为L或H；X 3处的氨基酸可被取代为无氨基酸；X 4处的氨基酸可被取代为I、Q或无氨基酸；X 5处的氨基酸可被取代为Y、T或N；X 6处的氨基酸可被取代为Q、N或W；X 7处的氨基酸可被取代为T或无氨基酸；X 8处的无氨基酸可被取代增加为T；X 9处的氨基酸可被取代为无氨基酸；X 10处的氨基酸可被取代为R或L。
在本申请中，“-”表示与对照抗原结合蛋白的LCDR3相比，该位可以为无氨基酸取代。例如，与SEQ ID NO:116所示抗原结合蛋白的LCDR3(X 9处氨基酸为P)相比，SEQ ID NO:74所示的抗原结合蛋白的LCDR3在X 9处没有氨基酸取代。
例如，所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:67-74、116-121。
本申请所述的抗原结合蛋白可包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:154所示的氨基酸序列。
在本申请中，所述抗原结合蛋白可包含HCDR1，且所述HCDR1可包含SEQ ID NO:142所示的氨基酸序列：
GX 1SX 2X 3X 4Y(SEQ ID NO:142)；其中，X 1＝G或D；X 2＝I，L或V；X 3＝S，N，D或Q；X 4＝N，S或T。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:40所示的抗原结合蛋白的HCDR1相比，所述HCDR1可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3和/或X 4处的氨基酸取代。
在本申请中，与SEQ ID NO:40所示的抗原结合蛋白的HCDR1相比，所述HCDR1可至少包含在X 1、X 2、X 3和/或X 4处的氨基酸取代，其中，X 1处的氨基酸可被取代为D；X 2处的氨基酸可被取代为L或V；X 3处的氨基酸可被取代为S、D或Q；X 4处的氨基酸可被取代为S或T。
例如，所述HCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53。
在本申请中，所述抗原结合蛋白可包含HCDR2，且所述HCDR2可包含SEQ ID NO:143所示的氨基酸序列：
YYSGX 1(SEQ ID NO:143)；其中，X 1＝T，N或S。例如，该序列可以是根据Chothia 定义规则确定的序列。
在本申请中，与SEQ ID NO:136所示的抗原结合蛋白的HCDR2相比，所述HCDR2可至少包含在选自以下的氨基酸位置处的取代：在X 1处的氨基酸取代。
在本申请中，与SEQ ID NO:136所示的抗体的HCDR2相比，所述HCDR2可至少包含在X 1处的氨基酸取代，其中，X 2处的氨基酸可被取代为S或T。
例如，所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:136-138。
在本申请中，所述抗原结合蛋白可包含HCDR3，且所述HCDR3可包含SEQ ID NO:144所示的氨基酸序列：
X 1X 2X 3X 4X 5X 6Y(SEQ ID NO:144)；其中，X 1＝K或G；X 2＝R或K；X 3＝G，A，T，W或L；X 4＝V，M，G，I或A；X 5＝L，Y，H，F或-；X 6＝D或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:60所示的抗原结合蛋白的HCDR3相比，所述HCDR3可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5和/或X 6处的氨基酸取代。
在本申请中，与SEQ ID NO:60所示的HCDR3相比，所述HCDR3可至少包含在X 1、X 2、X 3、X 4、X 5和/或X 6处的氨基酸取代，其中，X 1处的氨基酸可被取代为G；X 2处的氨基酸可被取代为K；X 3处的氨基酸可被取代为A、T、L或W；X 4处的氨基酸可被取代为M、G、A或I；X 5处的氨基酸可被取代为Y、H、F或无氨基酸；X 6处的氨基酸可被取代为S。
例如，所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:55、58-63。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1-3。所述LCDR1可包含SEQ ID NO:139所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:140所示的氨基酸序列；所述LCDR3可包含SEQ ID NO:141所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:113、114和115；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:31、56、57、122、126和127；所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:67-74、116-121。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:113所示的氨基酸序列；LCDR2可包含SEQ ID NO:56所示的氨基酸序列；LCDR3可包含SEQ ID NO:116所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:113所示的氨基酸序列；LCDR2可包含SEQ ID NO:122所示的氨基酸序列；LCDR3可包含SEQ ID NO:73所示的氨 基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:114所示的氨基酸序列；LCDR2可包含SEQ ID NO:126所示的氨基酸序列；LCDR3可包含SEQ ID NO:72所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:113所示的氨基酸序列；LCDR2可包含SEQ ID NO:56所示的氨基酸序列；LCDR3可包含SEQ ID NO:116所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:115所示的氨基酸序列；LCDR2可包含SEQ ID NO:31所示的氨基酸序列；LCDR3可包含SEQ ID NO:70所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1-3。所述HCDR1可包含SEQ ID NO:142所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:143所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:144所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述HCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:136-138；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:58-63和55。
例如，本申请所述的抗原结合蛋白中HCDR1可包含SEQ ID NO:34所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:58所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中HCDR1可包含SEQ ID NO:42所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:59所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中HCDR1可包含SEQ ID NO:38所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:55所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中HCDR1可包含SEQ ID NO:36所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:58所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中HCDR1可包含SEQ ID NO:42所示的氨基酸序列； HCDR2可包含SEQ ID NO:136所示的氨基酸序列；且HCDR3可包含SEQ ID NO:60所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含LCDR1-3和HCDR1-3。所述LCDR1可包含SEQ ID NO:139所示的氨基酸序列；所述LCDR2可包含SEQ ID NO:140所示的氨基酸序列；所述LCDR3可包含SEQ ID NO:141所示的氨基酸序列；所述HCDR1可包含SEQ ID NO:142所示的氨基酸序列；所述HCDR2可包含SEQ ID NO:143所示的氨基酸序列；且所述HCDR3可包含SEQ ID NO:144所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白的所述LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:113、114和115；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:31、56、57、122、126和127；所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:67-74、116-121；所述HCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:136-138；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:58-63和55。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:113所示的氨基酸序列；LCDR2可包含SEQ ID NO:122所示的氨基酸序列；LCDR3可包含SEQ ID NO:73所示的氨基酸序列；其HCDR1可包含SEQ ID NO:42所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:59所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:114所示的氨基酸序列；LCDR2可包含SEQ ID NO:126所示的氨基酸序列；LCDR3可包含SEQ ID NO:72所示的氨基酸序列；其HCDR1可包含SEQ ID NO:38所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:55所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:115所示的氨基酸序列；LCDR2可包含SEQ ID NO:31所示的氨基酸序列；LCDR3可包含SEQ ID NO:70所示的氨基酸序列；其HCDR1可包含SEQ ID NO:36所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:58所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:113所示的氨基酸序列；LCDR2可包含SEQ ID NO:56所示的氨基酸序列；LCDR3可包含SEQ ID NO:116所示的氨基酸序列；其HCDR1可包含SEQ ID NO:45所示的氨基酸序列；HCDR2可包含SEQ ID NO:138所示的氨基酸序列；且HCDR3可包含SEQ ID NO:60所示的氨基酸序列。
例如，本申请所述的抗原结合蛋白中LCDR1可包含SEQ ID NO:113所示的氨基酸序列；LCDR2可包含SEQ ID NO:56所示的氨基酸序列；LCDR3可包含SEQ ID NO:74所示的氨基酸序列；其HCDR1可包含SEQ ID NO:53所示的氨基酸序列；HCDR2可包含SEQ ID NO:137所示的氨基酸序列；且HCDR3可包含SEQ ID NO:63所示的氨基酸序列。
本申请所述的抗原结合蛋白还可包含框架区L-FR1，L-FR2，L-FR3和L-FR4。
在本申请中，所述抗原结合蛋白可包含L-FR1，所述的L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述的L-FR1包含SEQ ID NO:149所示的氨基酸序列：
X 1IX 2MTQSPX 3X 4LSX 5SX 6GX 7RX 8TX 9X 10C(SEQ ID NO:149)；其中，X 1＝D或E；X 2＝Q或V；X 3＝S或A；X 4＝T或S；X 5＝A或V；X 6＝V或P；X 7＝D或E；X 8＝V或A；X 9＝I或L；X 10＝T或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:157所示的抗原结合蛋白的L-FR1相比，所述L-FR1可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9和/或X 10处的氨基酸取代。
在本申请中，与SEQ ID NO:157所示的抗原结合蛋白的L-FR1相比，所述L-FR1可至少包含在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9和/或X 10处的氨基酸取代，其中，X 1处的氨基酸可被取代为E；X 2处的氨基酸可被取代为V；X 3处的氨基酸可被取代为A；X 4处的氨基酸可被取代为S；X 5处的氨基酸可被取代为V；X 6处的氨基酸可被取代为P；X 7处的氨基酸可被取代为E；X 8处的氨基酸可被取代为A；X 9处的氨基酸可被取代为L；X 10处的氨基酸可被取代为S。
例如，所述L-FR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:156-158。
在本申请中，所述抗原结合蛋白可包含L-FR2，所述的L-FR2位于所述LCDR1与所述LCDR2之间，且所述的L-FR2包含SEQ ID NO:150所示的氨基酸序列：
WYQQKPGX 1APX 2LLIY(SEQ ID NO:150)；其中，X 1＝K，N或Q；X 2＝K或R。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:191所示的抗原结合蛋白的L-FR2相比，所述L-FR2可至少包含在选自以下的氨基酸位置处的取代：在X 1和/或X 2处的氨基酸取代。
在本申请中，与SEQ ID NO:191所示的抗原结合蛋白的L-FR2相比，所述L-FR2可至少包含在至少包含在X 1和/或X 2处的氨基酸取代，其中，X 1处的氨基酸可被取代为N或Q；X 2处的氨基酸可被取代为R。
例如，所述L-FR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:191-194。
在本申请中，所述抗原结合蛋白可包含L-FR3，所述的L-FR3位于所述LCDR2与所述LCDR3之间，且所述的L-FR3包含SEQ ID NO:151所示的氨基酸序列：
X 1X 2PX 3RFSGSGSGTX 4FX 5LTX 6SSLQX 7X 8DFAX 9YYC(SEQ ID NO:151)；其中，X 1＝G，D；X 2＝V或I；X 3＝S或V；X 4＝E或D；X 5＝T或S；X 6＝I或V；X 7＝P或S；X 8＝D或E；X 9＝T或I。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:166所示的抗原结合蛋白的L-FR3相比，所述L-FR3可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8和/或X 9处的氨基酸取代。
在本申请中，与SEQ ID NO:166所示的抗体的L-FR3相比，所述L-FR3可至少包含在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8和/或X 9处的氨基酸取代，其中，X 1处的氨基酸可被取代为D；X 2处的氨基酸可被取代为I；X 3处的氨基酸可被取代为V；X 4处的氨基酸可被取代为D；X 5处的氨基酸可被取代为S；X 6处的氨基酸可被取代为V；X 7处的氨基酸可被取代为S；X 8处的氨基酸可被取代为E；X 9处的氨基酸可被取代为I。
例如，所述L-FR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:155、162-166。
在本申请中，所述抗原结合蛋白可包含L-FR4，所述的L-FR4的N末端与所述LCDR3的C末端相连，且所述的L-FR4包含SEQ ID NO:152所示的氨基酸序列：
FGX 1GTKX 2EIK(SEQ ID NO:152)；其中，X 1＝Q或G；X 2＝L或V。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:160所示的抗体的L-FR4相比，所述L-FR4可至少包含在选自以下的氨基酸位置处的取代：在X 1和/或X 2处的氨基酸取代。
在本申请中，与SEQ ID NO:160所示的L-FR4相比，所述L-FR4可至少包含在至少包含在X 1和/或X 2处的氨基酸取代，其中，X1处的氨基酸可被取代为G；X2处的氨基酸可被取代为V。
例如，所述L-FR4可包含下述任一项中所示的氨基酸序列：SEQ ID NO:159-161。
在本申请中，所述抗原结合蛋白可包含轻链可变区VL，且所述的VL可包含SEQ ID NO:153所示的氨基酸序列：
X 1IX 2MTQSPX 3X 4LSX 5SX 6GX 7RX 8TX 9X 10CRASQSX 11SSX 12LX 13WYQQKPGX 14APX 15L LIYX 16X 17SX 18X 19X 20X 21X 22X 23PX 24RFSGSGSGTX 25FX 26LTX 27SSLQX 28X 29DFAX 30YYCX 31X 3 2X 33X 34X 35X 36X 37X 38X 39X 40TFGX 41GTKX 42EIK(SEQ ID NO:153)；其中，X 1＝D，E；X 2＝Q，V；X 3＝S或A；X 4＝T或S；X 5＝A或V；X 6＝V或P；X 7＝D或E；X 8＝V或A；X 9＝I或L； X 10＝T或S；X 11＝I或V；X 12＝W或Y或H；X 13＝A或N；X 14＝K，N或Q；X 15＝K或R；X 16＝K，R，V或G；X 17＝A或S；X 18＝S，T，R或I；X 19＝L或R；X 20＝E，D，Q或A；X 21＝S或T；X 22＝G或D；X 23＝V或I；X 24＝S或V；X 25＝E或D；X 26＝T或S；X 27＝I或V；X 28＝P或S；X 29＝D或E；X 30＝T或I；X 31＝Q或R；X 32＝Q，L或H；X 33＝Y或-；X 34＝N，I，Q或-；X 35＝S，T或N；X 36＝Y，W，N或Q；X 37＝S，T或-；X 38＝T或-；X 39＝P或-；X 40＝Y，R或L；X 41＝Q或G；X 42＝L或V。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:15所示的抗体的VL相比，所述VL可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9、X 10、X 11、X 12、X 13、X 14、X 15、X 16、X 17、X 18、X 19、X 20、X 21、X 22、X 23、X 24、X 25、X 26、X 27、X 28、X 29、X 30、X 31、X 32、X 33、X 34、X 35、X 36、X 37、X 38、X 39、X 40、X 41和/或X 42处的氨基酸取代。
在本申请中，与SEQ ID NO:15所示的VL相比，所述VL可至少包含在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9、X 10、X 11、X 12、X 13、X 14、X 15、X 16、X 17、X 18、X 19、X 20、X 21、X 22、X 23、X 24、X 25、X 26、X 27、X 28、X 29、X 30、X 31、X 32、X 33、X 34、X 35、X 36、X 37、X 38、X 39、X 40、X 41和/或X 42处的氨基酸取代，其中，X 1处的氨基酸可被取代为E；X 2处的氨基酸可被取代为V；X 3处的氨基酸可被取代为A；X 4处的氨基酸可被取代为S；X 5处的氨基酸可被取代为V；X 6处的氨基酸可被取代为P；X 7处的氨基酸可被取代为E；X 8处的氨基酸可被取代为A；X 9处的氨基酸可被取代为L；X 10处的氨基酸可被取代为S；X 11处的氨基酸可被取代为V；X 12处的氨基酸可被取代为H或Y；X 13处的氨基酸可被取代为N；X 14处的氨基酸可被取代为Q或N；X 15处的氨基酸可被取代为R；X 16处的氨基酸可被取代为G、R或V；X 17处的氨基酸可被取代为S；X 18处的氨基酸可被取代为I、R或T；X 19处的氨基酸可被取代为R；X 20处的氨基酸可被取代为A、Q或D；X 21处的氨基酸可被取代为T；X 22处的氨基酸可被取代为D；X 23处的氨基酸可被取代为I；X 24处的氨基酸可被取代为V；X 25处的氨基酸可被取代为D；X 26处的氨基酸可被取代为S；X 27处的氨基酸可被取代为V；X 28处的氨基酸可被取代为S；X 29处的氨基酸可被取代为E；X 30处的氨基酸可被取代为I；X 31处的氨基酸可被取代为Q；X 32处的氨基酸可被取代为L或H；X 33处的氨基酸可被取代为无氨基酸；X 34处的氨基酸可被取代为I或无氨基酸；X 35处的氨基酸可被取代为Y、T或N；X 36处的氨基酸可被取代为W或N；X 37处的氨基酸可被取代为无氨基酸；X 38处的氨基酸可被取代为T；X 39处的氨基酸可被取代为无氨基酸；X 40处的氨基酸可被取代为L或R；X 41处的氨基酸可被取代为G；X 42处的氨基酸可被取代为V。
例如，所述VL区可包含下述任一项中所示的氨基酸序列：SEQ ID NO:1、3、5、7、9、 11、13、15、17、19、21、23、25、27和29。
本申请所述的抗原结合蛋白还可包含框架区H-FR1，H-FR2，H-FR3和H-FR4。
在本申请中，所述抗原结合蛋白可包含H-FR1，所述的H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述的H-FR1包含SEQ ID NO:145所示的氨基酸序列：
QVQLQESGPGLX 1X 2PSETLSLTCX 3VS(SEQ ID NO:145)；其中，X 1＝V或M；X 2＝K或Q；X 3＝T或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:169所示的抗原结合蛋白的H-FR1相比，所述H-FR1可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2和/或X 3处的氨基酸取代。
在本申请中，与SEQ ID NO:169所示的H-FR1相比，所述H-FR1可至少包含在X 1、X 2和/或X 3处的氨基酸取代，其中，X 1处的氨基酸可被取代为M；X 2处的氨基酸可被取代为Q；X 3处的氨基酸可被取代为S。
例如，所述H-FR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:167-170。
在本申请中，所述抗原结合蛋白可包含H-FR2，所述的H-FR2位于所述HCDR1与所述HCDR2之间，且所述的H-FR2包含SEQ ID NO:146所示的氨基酸序列：
YWX 1WX 2RQX 3PGX 4GLEWIGX 5I(SEQ ID NO:146)；其中，X1＝S或T；X 2＝I或F；X 3＝P或A；X 4＝K或R；X 5＝Y或N。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:188所示的H-FR2相比，所述H-FR2可至少包含在选自以下的氨基酸位置处的取代：包含在X 1、X 2、X 3、X 4和/或X 5处的氨基酸取代。
在本申请中，与SEQ ID NO:188所示的H-FR2相比，所述H-FR2可至少包含在X 1、X 2、X 3、X 4和/或X 5处的氨基酸取代，其中，X 1处的氨基酸可被取代为T；X 2处的氨基酸可被取代为F；X 3处的氨基酸可被取代为A；X 3处的氨基酸可被取代为R；X 3处的氨基酸可被取代为N。
例如，所述H-FR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:184、187-190。
在本申请中，所述抗原结合蛋白可包含H-FR3，所述的H-FR3位于所述HCDR2与所述HCDR3之间，且所述的H-FR3包含SEQ ID NO:147所示的氨基酸序列：
RVX 1X 2SX 3DX 4SKNQFSLX 5LX 6SVX 7X 8X 9DTAX 10YYCX 11R(SEQ ID NO:147)；其中，X 1＝T或S；X 2＝I或M；X 3＝V或I；X 4＝T或I；X 5＝K或N；X 6＝S，T或N；X 7＝T或I；X 8＝A或S；X 9＝A或G；X 10＝V或M；X 11＝A或G。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:178所示的H-FR3相比，所述H-FR3可至少包含在选自以 下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9、X 10、X 11、X 12、X 13、X 14和/或X 15处的氨基酸取代。
在本申请中，与SEQ ID NO:178所示的H-FR3相比，所述H-FR3可至少包含在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9、X 10、X 11、X 12、X 13、X 14和/或X 13处的氨基酸取代，其中，X 1处的氨基酸可被取代为D；X 2处的氨基酸可被取代为Q；X 3处的氨基酸可被取代为P；X 4处的氨基酸可被取代为T；X 5处的氨基酸可被取代为S；X 6处的氨基酸可被取代为M；X 7处的氨基酸可被取代为I；X 8处的氨基酸可被取代为I；X 9处的氨基酸可被取代为K；X 10处的氨基酸可被取代为S或N；X 11处的氨基酸可被取代为I；X 12处的氨基酸可被取代为S；X 13处的氨基酸可被取代为G；X 14处的氨基酸可被取代为M；X 15处的氨基酸可被取代为G。
例如，所述H-FR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:171-182。
在本申请中，所述抗原结合蛋白可包含H-FR4，所述的H-FR4的N末端与所述HCDR3的C末端相连，且所述的H-FR4包含SEQ ID NO:148所示的氨基酸序列：
X 1GQGX 2LVTVSS(SEQ ID NO:148)；其中，X 1＝W或S；X 2＝T或I。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:186所示的H-FR4相比，所述H-FR4可至少包含在选自以下的氨基酸位置处的取代：在X 1和/或X 2处的氨基酸取代。
在本申请中，与SEQ ID NO:186所示的H-FR4相比，所述H-FR4可至少包含在X 1和/或X 2处的氨基酸取代，其中，X 1处的氨基酸可被取代为S；X 2处的氨基酸可被取代为I。
例如，所述H-FR4可包含下述任一项中所示的氨基酸序列：SEQ ID NO:183、185和186。
在本申请中，所述抗原结合蛋白可包含重链可变区VH，且所述的VH包含SEQ ID NO:154所示的氨基酸序列：
QVQLQESGPGLX 1X 2PSETLSLTCX 3VSGX 4SX 5X 6X 7YYWX 8WX 9RQX 10PGX 11GLEWIGX 12IYYSGX 13TX 14YX 15X 16SLX 17SRVX 18X 19SX 20DX 21SKNQFSLX 22LX 23SVX 24X 25X 26DTAX 27YYCX 28RX 29X 30X 31X 32X 33X 34YX 35GQGX 36LVTVSS(SEQ ID NO:154)；其中，X 1＝V或M；X 2＝K或Q；X 3＝T或S；X 4＝G或D；X 5＝I，L或V；X 6＝S，N或D；X 7＝N，S或T；X 8＝S或T；X 9＝I或F；X 10＝P或A；X 11＝K或R；X 12＝Y或N；X 13＝T，S或N；X 14＝N或D；X 15＝N或Q；X 16＝P或S；X 17＝K或T；X 18＝T或S；X 19＝I或M；X 20＝V或I；X 21＝T或I；X 22＝K或N；X 23＝S，T或N；X 24＝T或I；X 25＝A或S；X 26＝A或G；X 27＝V或M；X 28＝A或G；X 29＝K或G；X 30＝R或K；X 31＝G，A，T，W或L；X 32＝V，M，G，I或A；X 33＝L，Y，H，F或-；X 34＝D或S；X 35＝W或S；X 36＝T或I。例如，该序列可以是根据Chothia定义规则确定的序列。
在本申请中，与SEQ ID NO:85所示的抗原结合蛋白的VH相比，所述VH可至少包含在选自以下的氨基酸位置处的取代：在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9、X 10、X 11、X 12、X 13、X 14、X 15、X 16、X 17、X 18、X 19、X 20、X 21、X 22、X 23、X 24、X 25、X 26、X 27、X 28、X 29、X 30、X 31、X 32、X 33、X 34、X 35和/或X 36处的氨基酸取代。
在本申请中，与SEQ ID NO:85所示的VH相比，所述VH可至少包含在X 1、X 2、X 3、X 4、X 5、X 6、X 7、X 8、X 9、X 10、X 11、X 12、X 13、X 14、X 15、X 16、X 17、X 18、X 19、X 20、X 21、X 22、X 23、X 24、X 25、X 26、X 27、X 28、X 29、X 30、X 31、X 32、X 33、X 34、X 35和/或X 36处的氨基酸取代，其中，X 1处的氨基酸可被取代为V；X 2处的氨基酸可被取代为Q；X 3处的氨基酸可被取代为S；X 4处的氨基酸可被取代为D；X 5处的氨基酸可被取代为L或V；X 6处的氨基酸可被取代为S、Q或D；X 7处的氨基酸可被取代为T；X 8处的氨基酸可被取代为T；X 9处的氨基酸可被取代为F；X 10处的氨基酸可被取代为A；X 11处的氨基酸可被取代为R；X 12处的氨基酸可被取代为N；X 13处的氨基酸可被取代为T或S；X 14处的氨基酸可被取代为D；X 15处的氨基酸可被取代为Q；X 16处的氨基酸可被取代为P；X 17处的氨基酸可被取代为T；X 18处的氨基酸可被取代为S；X 19处的氨基酸可被取代为M；X 20处的氨基酸可被取代为I；X 21处的氨基酸可被取代为I；X 22处的氨基酸可被取代为K；X 23处的氨基酸可被取代为N或T；X 24处的氨基酸可被取代为I；X 25处的氨基酸可被取代为S；X 26处的氨基酸可被取代为G；X 27处的氨基酸可被取代为M；X 28处的氨基酸可被取代为G；X 29处的氨基酸可被取代为G；X 30处的氨基酸可被取代为K；X 31处的氨基酸可被取代为T、A、W或L；X 32处的氨基酸可被取代为I、M、G或A；X 33处的氨基酸可被取代为H、F、Y或无氨基酸；X 34处的氨基酸可被取代为S；X 35处的氨基酸可被取代为S；X 36处的氨基酸可被取代为I。
例如，所述VH可包含下述任一项中所示的氨基酸序列：SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111。
在本申请中，所述抗原结合蛋白可包含抗体轻链可变区VL和重链可变区VH。所述VL可包含SEQ ID NO:153所示的氨基酸序列；所述VH可包含SEQ ID NO:154所示的氨基酸序列。
本申请所述抗原结合蛋白的所述VL可包含下述任一项中所示的氨基酸序列SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27和29；所述VH可包含下述任一项中所示的氨基酸序列SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:29所示的 氨基酸序列，且所述VH可包含SEQ ID NO:111所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:29所示的氨基酸序列，且所述VH可包含SEQ ID NO:107所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:29所示的氨基酸序列，且所述VH可包含SEQ ID NO:109所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:1所示的氨基酸序列，且所述VH可包含SEQ ID NO:79所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:81、83、85、89、91、93、95和97任一项中所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:81所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:83所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:85所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:89所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:91所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:93所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:95所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:15所示的氨基酸序列，且所述VH可包含SEQ ID NO:97任一项中所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:23所示的氨基酸序列，且所述VH可包含SEQ ID NO:99所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:21所示的氨基酸序列，且所述VH可包含SEQ ID NO:99所示的氨基酸序列。
例如，所述分离的抗原结合蛋白可包含VL和VH，其VL可包含SEQ ID NO:25所示的 氨基酸序列，且所述VH可包含SEQ ID NO:99所示的氨基酸序列。
在本申请中，所述抗原结合蛋白包含轻链恒定区CL，且所述抗体轻链恒定区可包括人Igκ恒定区。
例如，所述CL区可包含下述所示的氨基酸序列：SEQ ID NO:196。
在本申请中，所述抗原结合蛋白包含轻链LC，且所述LC可包含SEQ ID NO:197所示的氨基酸序列。
例如，所述LC可包含下述任一项中所示的氨基酸序列：SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28和30。
在本申请中，所述抗原结合蛋白包含重链恒定区CH，且所述抗体重链恒定区可包括人IgG恒定区。在某些情形中，本申请所述抗体重链恒定区可包括人IgG4恒定区。
例如，所述CH区可包含下述所示的氨基酸序列：SEQ ID NO:195。
在本申请中，所述抗原结合蛋白包含轻链HC，且所述抗体HC可包含SEQ ID NO:198所示的氨基酸序列。
例如，所述HC可包含下述任一项中所示的氨基酸序列：SEQ ID NO:76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110和112。
本申请所述抗原结合蛋白包含抗体轻链恒定区CL和重链恒定区CH。所述CL可包含SEQ ID NO:196所示的氨基酸序列；且所述CH可包含SEQ ID NO:195所示的氨基酸序列。
本申请所述抗原结合蛋白包含抗体轻链LC和重链HC。所述LC可包含SEQ ID NO:197所示的氨基酸序列，且所述HC可包含SEQ ID NO:198所示的氨基酸序列。
例如，所述LC可包含下述任一项中所示的氨基酸序列SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28和30，所述HC可包含下述任一项中所示的氨基酸序列SEQ ID NO:76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110和112。
本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:139所示的氨基酸序列，LCDR2可包含SEQ ID NO:140所示的氨基酸序列，LCDR3可包含SEQ ID NO:141所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:149所示的氨基酸序列，L-FR2可包含SEQ ID NO:150所示的氨基酸序列，L-FR3可包含SEQ ID NO:151所示的氨基酸序列，且L-FR4可包含SEQ ID NO:152所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:153所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID  NO:197所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:142所示的氨基酸序列，HCDR2可包含SEQ ID NO:143所示的氨基酸序列，HCDR3可包含SEQ ID NO:144所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:145所示的氨基酸序列，H-FR2可包含SEQ ID NO:146所示的氨基酸序列，H-FR3可包含SEQ ID NO:147所示的氨基酸序列，且H-FR4可包含SEQ ID NO:148所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:154所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:198所示的氨基酸序列。
在某些情形中，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含如SEQ ID NO:113-115中任一项所示的氨基酸序列，LCDR2可包含SEQ ID NO:31、56、57、122、126和127中任一项所示的氨基酸序列，LCDR3可包含SEQ ID NO:67-74、116-121中任一项所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:156-158中任一项所示的氨基酸序列，L-FR2可包含SEQ ID NO:191-194中任一项所示的氨基酸序列，L-FR3可包含SEQ ID NO:155、162-166中任一项所示的氨基酸序列，且L-FR4可包含SEQ ID NO:159-161中任一项所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27和29中任一项所示的氨基酸序列，所述CL可包含SEQ ID NO:196中所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28和30中任一项所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53中任一项所示的氨基酸序列，HCDR2可包含SEQ ID NO:136-138中任一项所示的氨基酸序列，HCDR3可包含SEQ ID NO:55和58-63中任一项所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:167-170中任一项所示的氨基酸序列，H-FR2可包含SEQ ID NO:184、187-190中任一项所示的氨基酸序列，H-FR3可包含SEQ ID NO:171-182中任一项所示的氨基酸序列，且H-FR4可包含SEQ ID NO:183、185和186中任一项所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111中任一项所示的氨基酸序列，所述CH可包含SEQ ID NO:195中任一项所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110和112中任一项所示的氨基酸序列。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ  ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:74所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:162所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:5所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:6所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:53所示的氨基酸序列，HCDR2可包含SEQ ID NO:137所示的氨基酸序列，HCDR3可包含SEQ ID NO:63所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:182所示的氨基酸序列，且H-FR4可包含SEQ ID NO:183所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:105所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:106所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001281相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:122所示的氨基酸序列，LCDR3可包含SEQ ID NO:73所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:193所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:27所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:28所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:59所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:186所示的氨基酸序列，H-FR3可包含SEQ ID NO:172所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:87所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:88所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000806相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:114所示的氨基酸序列，LCDR2可包含SEQ ID NO:126所示的氨基酸序列，LCDR3可包含SEQ ID NO:72所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:156所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:163所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:1所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:2所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:38所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:55所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:184所示的氨基酸序列，H-FR3可包含SEQ ID NO:180所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:79所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:80所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000808相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:114所示的氨基酸序列，LCDR2可包含SEQ ID NO:127所示的氨基酸序列，LCDR3可包含SEQ ID NO:71所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:156所示的氨基酸序列，L-FR2可包含SEQ ID NO:192所示的氨基酸序列，L-FR3可包含SEQ ID NO:164所示的氨基酸序列，且L-FR4可包含SEQ ID NO:161所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:3所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:4所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:61所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:167所示的氨基酸序列，H-FR2可包含SEQ ID NO:189所示的氨基酸序列，H-FR3可包含SEQ ID NO:171所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:75所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:76所示的氨基酸序列。例如，所述抗原结合蛋白包含与 PR000807相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:115所示的氨基酸序列，LCDR2可包含SEQ ID NO:31所示的氨基酸序列，LCDR3可包含SEQ ID NO:70所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:158所示的氨基酸序列，L-FR2可包含SEQ ID NO:194所示的氨基酸序列，L-FR3可包含SEQ ID NO:155所示的氨基酸序列，且L-FR4可包含SEQ ID NO:159所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:29所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:30所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:32所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:58所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:170所示的氨基酸序列，H-FR2可包含SEQ ID NO:190所示的氨基酸序列，H-FR3可包含SEQ ID NO:176所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:107所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:108所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001264相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:115所示的氨基酸序列，LCDR2可包含SEQ ID NO:31所示的氨基酸序列，LCDR3可包含SEQ ID NO:70所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:158所示的氨基酸序列，L-FR2可包含SEQ ID NO:194所示的氨基酸序列，L-FR3可包含SEQ ID NO:155所示的氨基酸序列，且L-FR4可包含SEQ ID NO:159所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:29所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:30所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:36所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:58所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:170所示的氨基酸序列，H-FR2可包含SEQ ID NO:190所示的氨基酸序列，H-FR3可包含SEQ ID NO:176所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:111所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链， 所述抗体重链可包含SEQ ID NO:112所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001265相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:32所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:89所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:90所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001266相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:178所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:91所示的氨基酸序列， 所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:92所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001267相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:188所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:40所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:81所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:82所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001268相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:40所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:178所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。 所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:83所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:84所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001269相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:121所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:19所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:20所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:40所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:181所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:85所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:86所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001270相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:120所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:17所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:18所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:40所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包 含SEQ ID NO:181所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:85所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:86所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001271相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:57所示的氨基酸序列，LCDR3可包含SEQ ID NO:121所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:19所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:20所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:89所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:90所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001272相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:57所示的氨基酸序列，LCDR3可包含SEQ ID NO:69所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:25所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:26所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:47所示的氨基酸序列，HCDR2可包含SEQ ID NO:137所示的氨基酸序列，HCDR3可包含SEQ ID NO:62所示的氨基酸序列，所述H-FR1可包含SEQ ID  NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:179所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:99所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:100所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001273相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:57所示的氨基酸序列，LCDR3可包含SEQ ID NO:68所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:23所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:24所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:47所示的氨基酸序列，HCDR2可包含SEQ ID NO:137所示的氨基酸序列，HCDR3可包含SEQ ID NO:62所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:179所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:99所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:100所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001274相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:45所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的 氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:174所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:97所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:98所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001275相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:45所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:175所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:93所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:94所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001276相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:119所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:165所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:11所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:12所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述 HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:168所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:77所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:78所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001277相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:120所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:165所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:13所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:14所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:168所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:77所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:78所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001278相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:118所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:165所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:9所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID  NO:10所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:168所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:77所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:78所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001279相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:74所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:162所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:5所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:6所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:51所示的氨基酸序列，HCDR2可包含SEQ ID NO:137所示的氨基酸序列，HCDR3可包含SEQ ID NO:63所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:182所示的氨基酸序列，且H-FR4可包含SEQ ID NO:183所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:103所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:104所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR001280相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:115所示的氨基酸序列，LCDR2可包含SEQ ID NO:31所示的氨基酸序列，LCDR3可包含SEQ ID NO:70所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:158所示的氨基酸序列，L-FR2可包含SEQ ID NO:194所示的氨基酸序列，L-FR3可包含SEQ ID NO:155所示的氨基酸序列，且L-FR4可包含SEQ ID NO:159所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:29所示的氨基酸序列，所述CL可包含SEQ  ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:30所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:34所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:58所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:170所示的氨基酸序列，H-FR2可包含SEQ ID NO:190所示的氨基酸序列，H-FR3可包含SEQ ID NO:176所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:109所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:110所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000796相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:40所示的氨基酸序列，HCDR2可包含SEQ ID NO:136所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:181所示的氨基酸序列，且H-FR4可包含SEQ ID NO:186所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:85所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:86所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000802相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:57所示的氨基酸序列，LCDR3可包含SEQ ID NO:67所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可 包含VL和CL，且所述VL可包含SEQ ID NO:21所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:22所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:47所示的氨基酸序列，HCDR2可包含SEQ ID NO:137所示的氨基酸序列，HCDR3可包含SEQ ID NO:62所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:179所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:99所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:100所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000803相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:116所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:166所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:15所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:16所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:45所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:173所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:95所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:96所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000804相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:117所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:165所 示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:7所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:8所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:42所示的氨基酸序列，HCDR2可包含SEQ ID NO:138所示的氨基酸序列，HCDR3可包含SEQ ID NO:60所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:168所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:177所示的氨基酸序列，且H-FR4可包含SEQ ID NO:185所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:77所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:78所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000805相同的抗体轻链和抗体重链。
例如，本申请所述抗原结合蛋白可包含LCDR1-3和L-FR1-4。所述LCDR1可包含SEQ ID NO:113所示的氨基酸序列，LCDR2可包含SEQ ID NO:56所示的氨基酸序列，LCDR3可包含SEQ ID NO:74所示的氨基酸序列，所述L-FR1可包含SEQ ID NO:157所示的氨基酸序列，L-FR2可包含SEQ ID NO:191所示的氨基酸序列，L-FR3可包含SEQ ID NO:162所示的氨基酸序列，且L-FR4可包含SEQ ID NO:160所示的氨基酸序列。所述抗原结合蛋白可包含VL和CL，且所述VL可包含SEQ ID NO:5所示的氨基酸序列，所述CL可包含SEQ ID NO:196所示的氨基酸序列。所述抗原结合蛋白可包含抗体轻链，所述轻链可包含SEQ ID NO:6所示的氨基酸序列。所述抗原结合蛋白还可包含HCDR1-3和H-FR1-4。其中所述HCDR1可包含SEQ ID NO:49所示的氨基酸序列，HCDR2可包含SEQ ID NO:137所示的氨基酸序列，HCDR3可包含SEQ ID NO:63所示的氨基酸序列，所述H-FR1可包含SEQ ID NO:169所示的氨基酸序列，H-FR2可包含SEQ ID NO:188所示的氨基酸序列，H-FR3可包含SEQ ID NO:182所示的氨基酸序列，且H-FR4可包含SEQ ID NO:183所示的氨基酸序列。所述抗原结合蛋白可包含VH和CH，且所述VH可包含SEQ ID NO:101所示的氨基酸序列，所述CH可包含SEQ ID NO:195所示的氨基酸序列。且所述抗原结合蛋白可包含抗体重链，所述抗体重链可包含SEQ ID NO:102所示的氨基酸序列。例如，所述抗原结合蛋白包含与PR000811相同的抗体轻链和抗体重链。
特定抗CD47抗体本申请所述的分离的抗原结合蛋白，其可与特定抗CD47抗体竞争结合所述CD47，其中所述特定抗CD47抗体可包含轻链可变区和重链可变区，所述特定抗CD47 抗体的轻链可变区可包含LCDR1-3，所述LCDR1-3的可包含依次为SEQ ID NO:139-141所示的氨基酸序列；所述特定抗CD47抗体的重链可变区可包含HCDR1-3，所述HCDR1-3可包含依次为SEQ ID NO:142-144所示的氨基酸序列。所述特定抗CD47抗体的轻链可变区氨基酸序列可为SEQ ID NO:153，且所述特定抗CD47抗体的重链可变区可为SEQ ID NO:154。
在本申请中，所述特定抗CD47抗体的LCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:113、114和115；所述LCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:31、56、57、122、126和127；且所述LCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:67-74、116-121。
在本申请中，所述特定抗CD73抗体的HCDR1可包含下述任一项中所示的氨基酸序列：SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53；所述HCDR2可包含下述任一项中所示的氨基酸序列：SEQ ID NO:136-138；且所述HCDR3可包含下述任一项中所示的氨基酸序列：SEQ ID NO:58-63和55。
在本申请中，所述特定抗CD47抗体的VL可包含下述任一项中所示的氨基酸序列：SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27和29。
在本申请中，所述特定抗CD47抗体的VH可包含下述任一项中所示的氨基酸序列：SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111。
在本申请中涉及的蛋白质、多肽和/或氨基酸序列，还应理解为至少包含以下的范围：与该所述蛋白质或多肽具备相同或类似功能的变体或同源物。与所述蛋白质或多肽具备相同或类似功能的变体可以称为功能性变体。
在本申请中，所述变体可以为，在所述蛋白质和/或所述多肽(例如，特异性结合CD47蛋白的抗体或其片段)的氨基酸序列中经过取代、缺失或添加一个或多个氨基酸的蛋白质或多肽。例如，所述变体(例如，功能性变体)可包含已经通过至少1个，例如1-30个、1-20个或1-10个，又例如1个、2个、3个、4个或5个氨基酸取代、缺失和/或***而具有氨基酸改变的蛋白质或多肽。所述变体(例如，功能性变体)可基本上保持改变(例如取代、缺失或添加)之前的所述蛋白质或所述多肽的生物学特性。例如，所述变体(例如，功能性变体)可保持改变之前的所述蛋白质或所述多肽的至少60％、70％、80％、90％或100％的生物学活性(例如抗原结合能力)。例如，所述取代可以为保守取代。
在本申请中，所述抗原结合蛋白的氨基酸序列的一部分可以与来自特定物种的抗体中相应的氨基酸序列同源，或者属于特定的类别。例如，抗体的可变区及恒定部分均可以来自一 个动物物种(如人)的抗体的可变区及恒定区。在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，特异性结合CD47蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
在本申请中，所述分离的抗原结合蛋白能结合源自人和猴的CD47。所述猴可以指食蟹猴。抗CD47抗体对CD47的结合亲和性可通过本领域已知的任何方法测定。在某些情形中，结合亲和性可通过竞争性ELISA、RIA或表面等离子共振如BIAcore来测定。在某些具体的情形中，结合亲和性通过表面等离子共振测定。例如，结合亲和性和解离速率可通过BIAcore测定。在某些情形中，所述分离的抗原结合蛋白与CD47结合的KD可以为3x10 -9M或以下。在一些具体的情形中，所述分离的抗原结合蛋白与CD47结合的KD可以为3x10 -9M、2x10 - 9M、1x10 -9M、1x10 -10M、1x10 -11M或以下。在某些情形中，KD可以为约1pM至约500pM。在另一些情形中，KD可以为约500pM至约1nM。在另一些情形中，KD可以为约1nM至约10nM。
在本申请中，所述分离的抗原结合蛋白可以特异性结合CD47。本申请所述CD47可以是肿瘤细胞表面的CD47。所述肿瘤细胞可以是Jurkat细胞或Raji细胞。在某些情形中，所述 抗原结合蛋白与Jurkat细胞上的CD47结合的EC 50值可以为约10pM至约10nM。在另一些情形中，所述EC 50值可以为约100pM至约100nM。在另一些情形中，EC 50值可以为约1pM至约1nM。在另一些情形中，所述EC 50值可以为约0.1nM至约1nM，例如，在FACS测量中。例如，在FACS测定中，所述抗原结合蛋白与Jurkat细胞上的CD47结合的EC 50值可以为约10pM至约10nM，如，约10pM至约1nM、至约1nM至约10nM。在本申请中，所述抗原结合蛋白不结合或不特异性结合非CD47的抗原，例如，不结合或不特异性结合SIPRα、CD147、PD-1、PD-L1、CTLA-4、CD80、CD86、CD28H、B7H3、B7H4和ICOS。
所述抗原结合蛋白还能与食蟹猴细胞表面的CD47结合，如，可以是食蟹猴外周血单个核細胞(PBMC)。在某些情形中，所述结合可通过FACS检测，得到的EC50值可以是约0.02nM至约0.2nM。在某些情形中FACS测得的EC50可以是约0.01nM至约0.3nM、约0.01nM至约0.5nM或约0.01nM至约1.0nM。
在本申请中，所述分离的抗原结合蛋白可以抑制CD47与SIRPα的结合。在本申请中，所述CD47可以为人CD47胞外区。本申请所述人CD47胞外区可包含UniProt中登录号为Q08722所示氨基酸序列中第19位至第141位的序列。例如，通过ELISA测量的，所述分离的抗原结合蛋白抑制人CD47与SIRPα结合的IC 50值可以是约0.5nM至约5.0nM。在某些情形中，所述阻断结合的IC 50值可以是约1.0nM至约3.0nM。在另一些情形中，可以是约1.5nM至约2.5nM。例如，通过ELISA测量的，所述分离的抗原结合蛋白抑制人CD47与SIRPα结合的抑制率可大于90％(例如，大于95％、大于96％、大于97％、大于98％、大于99％或更高)。
在本申请中，所述分离的抗原结合蛋白可以阻断肿瘤细胞表面的CD47与SIRPα结合。本申请所述的肿瘤细胞可以为Jurkat细胞。本申请所述CD47抗原结合蛋白能够通过阻断SIRPα与人CD47的结合。例如，所述分离的抗原结合蛋白阻断Jurkat细胞表面的CD47与SIRPα结合的IC 50值可以是约0.01nM至约5.0nM，约0.02nM至约3.0nM或约0.03nM至约2.0nM.在某些情形中，所述IC 50值可以是约0.1nM至约1.0nM，例如通过FACS测量的。
在本申请中，所述分离的抗原结合蛋白可以不引起凝血反应。在某些情形下，与现有CD47抗体存在时的凝集水平相比，本申请的CD47抗原结合蛋白存在时的凝集水平下降了至少5％、至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％或至少99％，说明本申请的CD47抗原结合蛋白不引起凝血反应。例如，在浓度介于约0.65nM至约665nM之间时，所述的抗原结合蛋白不引起凝血反应。
在本申请中，所述分离的抗原结合蛋白可以引起巨噬细胞对肿瘤细胞的吞噬。本申请所 述的肿瘤细胞可以包括Jurkat和OVCAR 3肿瘤细胞。例如，用CFSE标记Jurkat细胞，用流式细胞仪进行测量，所述分离的抗原结合蛋白可引起巨噬细胞对Jurkat的吞噬作用，吞噬指数可以为CFSE +CD14 +细胞占总CD14 +细胞的百分比，且所述吞噬指数可以至少大于30％(例如，至少大于40％、至少大于50％、至少大于60％、至少大于70％、至少大于80％、至少大于90％或以上)。所述分离的抗原结合蛋白可引起巨噬细胞对OVCAR3的吞噬作用的EC 50值可以是约0.1nM至约5.0nM(例如，约0.2nM至约3.0nM、约0.2nM至约2.0nM或约0.5nM至约1.5nM)。在某些情形中，例如，用CD14-APC标记巨噬细胞，用流式细胞仪进行测量，可以是约0.5nM至约1.5nM。
本申请检测了所述分离的抗原结合蛋白引起巨噬细胞对人红细胞的吞噬能力。例如，用FACS所测量的，所述分离的抗原结合蛋白可引起巨噬细胞对人红细胞的吞噬作用的吞噬指数可低于35％(例如，可低于30％、低于25％、低于20％、低于15％、低于10％、低于5％或更低)。本申请所述分离的抗原结合蛋白引起巨噬细胞对肿瘤细胞的吞噬作用强于对红细胞的吞噬作用。本申请所述的抗原结合蛋白能够抑制肿瘤生长和/或肿瘤细胞增殖。在某些情形中，所述抗原结合蛋白可以减缓肿瘤体积增长，消除肿瘤增长，或可以减缓体重减轻趋势。该效果可通过建立小鼠肿瘤模型检测，例如，Raji小鼠模型或MC38小鼠模型。其中所述肿瘤包括实体瘤和/或血液肿瘤。实体瘤可包括卵巢癌等，血液瘤可包括白血病、淋巴瘤。白血病可包括急性淋巴细胞性白血病(ALL)，急性骨髓性白血病(AML)，慢性淋巴细胞性白血病(CLL)，慢性骨髓性白血病(CML)等。淋巴瘤可包括霍奇金淋巴瘤，无痛性和侵袭性非霍奇金淋巴瘤，伯基特淋巴瘤等。
核酸、载体、细胞和制备方法
在另一个方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述的抗体、其抗原结合片段。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗体、其抗原结合片段，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体、其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR， 具体操作可参见Sambrook等人，Molecular Cloning，A Laboratory Manual，Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1989；和Ausube等人Current Protocols in Molecular Biology，Greene Publishing and Wiley-Interscience，New York N.Y.，1993。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调控的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。
在另一方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如原核细胞(例如，细菌细胞)、CHO细胞、NS/0细胞、HEK293T细胞或HEK293A细胞，或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。例如，所述宿主细胞可以为COS、CHO、NSO、sf9、sf21、DH5a、BL21(DE3)或TG1。
在另一个方面，本申请提供了制备本申请所述的抗体、其抗原结合片段的方法。所述方法可包括，在使得所述抗体、其抗原结合片段表达的条件下，培养本申请所述的宿主细胞。 例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
在某些情形中，所述方法还可包括收获(例如分离和/或纯化)，本申请所述的抗体、其抗原结合片段的步骤。例如，可以采用蛋白G-琼脂糖或蛋白A-琼脂糖进行亲和层析，还可通过凝胶电泳和/或高效液相色谱等来纯化和分离本申请所述的抗体、其抗原结合片段。
药物组合物、应用
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的抗原结合片段或变体，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载体。
所述药学上可接受的载体通常是指可用于制备药物组合物或制剂的载体，通常安全、无毒，且既不是生物学上也非其它方面不合需要的。所使用的载体通常是适合施用于人体或其他哺乳动物的载体。在制备组合物时，活性成分通常与载体混合，由载体稀释或封闭。当载体用作稀释剂时，其可以是固体，半固体或液体材料，其充当抗体活性成分的媒介物或介质。所述药学上可接受的载体可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。
所述药物组合物可包括本申请所述抗原结合蛋白，还可包括一种或多种联用的活性化合物。活性化合物可以为不与所述抗原结合蛋白产生不良影响的化合物，或者，可以为增强其功能的试剂，例如细胞毒剂、细胞因子、化疗制剂或生长抑制剂。所述联用的化合物可以以有效用于所需目的用量联合存在。
在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药，肌肉内给药，在肿瘤部位的原位给药，吸入，直肠给药，***给药，经皮给药或通过皮下储存库给药。用于经皮给药或通过皮下储存库的溶液或悬液可包括以下组分：无菌稀释剂如注射用水、盐水溶液、非挥发油、聚乙二醇、甘油、丙二醇或其它合成溶剂；抗菌剂如苯甲醇或对羟基苯甲酸甲酯；抗氧化剂如抗坏血酸或亚硫酸氢钠；螯合剂如乙二胺四乙酸(EDTA)；缓冲剂如乙酸盐、柠檬酸盐或磷酸盐；和调节张力的物质如氯化钠或右旋糖。pH可用酸或碱调节，例如盐酸或氢氧化钠。
所述药物组合物可以用于抑制肿瘤生长。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。例如，可通过肿瘤小鼠体重增长减缓程度和肿瘤细胞荧光信号强度表示。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述抗体、其抗原结合片段。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试 者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了所述抗体、其抗原结合片段在制备预防或治疗疾病或病症的药物中的用途。
另一方面，本申请提供的所述抗体、其抗原结合片段，可用于预防或治疗疾病或病症。所述预防或治疗疾病或病症可以指抑制或延缓疾病或病症的发展或进展。例如，可以用于抑制肿瘤的发展或进展。例如，可以抑制肿瘤增长或肿瘤细胞增殖。
另一方面，本申请提供了用于预防或***的所述的抗体或其抗原结合片段。
另一方面，本申请提供了一种抑制CD47与SIRPα结合的方法，其包括施用本申请所述的抗体、其抗原结合片段、所述的核酸分子、所述的载体、所述的宿主细胞和/或所述的药物组合物。例如，所述方法可以为体外或离体方法。例如，所述方法可以为ELISA或FACS。
另一方面，本申请提供了预防或***的方法，其包括向有需要的受试者施用本申请所述的抗体或其抗原结合片段、所述的分子核酸、所述的载体、所述的宿主细胞和/或所述的药物组合物。在本申请中，所述肿瘤包括实体瘤和/或血液肿瘤。例如，所述肿瘤包含淋巴瘤、白血病、卵巢癌。
联合用药
本申请的抗原结合蛋白、药物组合物和方法可以与其它治疗剂和/或形式组合施用。所述组合施用通常是指，在向受试者施用的过程中两种(或更多种)不同的疗法被递送至所述受试者，使得对所述受试者的治疗效果在某个时间点重叠。在某些情形中，当第二疗法的递送开始时，第一疗法的递送仍在发生，使得在施用方面存在重叠。这在本文中有时被称为“同时”或“并行递送”。在其它情形中，一个疗法的递送在其它疗法的递送开始之前结束。在某些情形中，由于组合施用，治疗更有效，两种处理的效果可以部分叠加、完全叠加或大于叠加。
在本申请中，所述的抗原结合蛋白或药物组合物可以与一种或多种另外的疗法如手术、放射治疗或另一治疗性制剂的施用组合。在一个实施方案中，另外的疗法可以包括化学疗法，例如，细胞毒性剂。在一个实施方案中，另外的疗法可以包括靶向治疗，例如酪氨酸激酶抑制剂、蛋白酶体抑制剂或蛋白酶抑制剂。在一个实施方案中，另外的疗法可以包括：抗炎化合物、抗血管生成化合物、抗纤维化化合物或抗增殖化合物，例如类固醇、生物免疫调节剂、单克隆抗体、抗体片段、适配子、siRNA、反义分子、融合蛋白、细胞因子、细胞因子受体、支气管扩张剂、他汀类药物、抗炎剂(例如甲氨蝶呤)或NSAID。在另一实施方案中，另外的疗法可以包括不同类别的组合疗法。可以同时或顺序地施用本申请所述的抗原结合蛋白、 药物组合物或制剂和另外的疗法。
另一方面，本申请还提供了如下实施方案：
1.分离的抗原结合蛋白，其包含抗体轻链可变区VL和抗体重链可变区VH，所述VL包含LCDR1，LCDR2和LCDR3中的任意一个，所述VH包含HCDR1，HCDR2和HCDR3中的任意一个，其中所述VL包含SEQ ID NO:153所示的氨基酸序列，所述VH包含SEQ ID NO:154所示的氨基酸序列。
2.如实施方案1所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:139所示的氨基酸序列，其中LCDR1优选包含SEQ ID NO:113、114和115中任一项中所示的氨基酸序列。
3.如实施方案1所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:140所示的氨基酸序列，其中LCDR2优选包含SEQ ID NO:31、56、57、122、126和127中任一项中所示的氨基酸序列。
4.如实施方案1所述的分离的抗原结合蛋白，其中所述LCDR3包含SEQ ID NO:141所示的氨基酸序列，其中LCDR3优选包含SEQ ID NO:67-74、116-121中任一项中所示的氨基酸序列。
5.如实施方案1所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:142所示的氨基酸序列，其中HCDR1优选包含SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53中任一项中所示的氨基酸序列。
6.如实施方案1所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:143所示的氨基酸序列，其中HCDR2优选包含SEQ ID NO:136-138中任一项中所示的氨基酸序列。
7.如实施方案1所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO:144所示的氨基酸序列，其中HCDR3优选包含SEQ ID NO:55和58-63中任一项中所示的氨基酸序列。
8.如实施方案1中任一项所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27和29中任一项中所示的氨基酸序列。
9.如实施方案1中任一项所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111中任一项中所示的氨基酸序列。
10.如实施方案1中所述的分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC包含 SEQ ID NO:197所示的氨基酸序列，其中所述LC优选包含SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28和30中任一项中所示的氨基酸序列。
11.如实施方案1中所述的分离的抗原结合蛋白，其包含抗体重链HC，且所述HC包含SEQ ID NO:198所示的氨基酸序列，其中所述HC优选包含SEQ ID NO:76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110和112中任一项中所示的氨基酸序列。
12.如实施方案1所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
1)能够特异性结合源自人和猴的CD47，优选KD≤3x10-9M；
2)能够抑制CD47与SIRPα的结合；
3)能够结合人血红细胞；
4)在红细胞凝血测定中，不引起凝血反应；
5)能够引起巨噬细胞对肿瘤细胞的吞噬；和
6)能够抑制肿瘤生长和/或肿瘤细胞增殖。
13.一种或多种分离的核酸分子，其编码实施方案1-12中任一项所述的分离的抗原结合蛋白。
14.载体，其包含如实施方案13所述的核酸分子。
15.细胞，其包含如实施方案13所述的核酸分子或如实施方案14所述的载体。
16.制备实施方案1-12中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得实施方案1-12中任一项所述的分离的抗原结合蛋白表达的条件下，培养如实施方案15所述的细胞。
17.药物组合物，其包含实施方案1-12中任一项所述的分离的抗原结合蛋白、实施方案13所述的核酸分子、实施方案14所述的载体和/或实施方案15所述的细胞，以及任选地药学上可接受的载体。
18.实施方案1-12中任一项所述的分离的抗原结合蛋白、实施方案13所述的核酸分子、实施方案14所述的载体、实施方案15所述的细胞和/或实施方案17所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或***。
19.抑制CD47与SIRPα结合的方法，所述方法包括施用实施方案1-12中任一项所述的分离的抗原结合蛋白。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
下面显示的实施例意在说明本发明的具体实施方案，并且不意在以任何方式限制本说明书或权利要求书的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因***到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook，J.，Fritsch，E.F.and Maniais，T.(1989)MolecuLar Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1 小鼠免疫和抗体分子的获得
可以利用CD47抗原对实验动物进行免疫以获得针对CD47特异性结合的抗体分子，该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常，其得到的抗体分子是非人源的。在获得非人源抗体后，需要对这些分子利用抗体工程技术进行人源化改造，以降低免疫原性并提高成药性。然而，抗体的人源化过程有其技术复杂性，经过人源化改造的分子往往会降低对抗原的亲和力。另一方面，转基因技术的进步使得可以培育出基因工程化小鼠，其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列，因而无需再进一步做人源化改造，大大提高了治疗性抗体开发的效率。Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。
1.1用CD47抗原免疫小鼠
用可溶的重组人CD47-Fc融合蛋白对Harbour H2L2小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100微升。在首轮免疫中，每只小鼠接受用50μg抗原蛋白(人CD47-Fc)与完全弗氏佐剂(Sigma#F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25μg抗原蛋白与Sigma Adjuvant System佐剂(Sigma#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行细胞融合前3天，以每只小鼠25μg抗原蛋白的剂量进行最后一次增强免疫。
1.2获得杂交瘤单克隆和抗体序列
当检测小鼠血清中CD47特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出并与骨髓瘤细胞系融合得到杂交瘤细胞；对杂交瘤细胞经过多轮筛选和克隆之后，分离出21个表达抗-CD47单克隆抗体分子的杂交瘤。分离的杂交瘤细胞及其表达的单克隆抗体都使用对应的克隆号来表示：11B8，15D1，18C4，32F1，35A11，39A8，40D7，73F1，74B6，75F6，78B6，80B6，81E5，83G1，84G2，85G2，86D7，87E9，87H6，88G3，89E3。分离的杂交瘤表达具有完整的人可变结构域和大鼠恒定结构域的重链和轻链的抗体分子。对上述单克隆抗体进行进一步的鉴定，根据其对人CD47的结合能力、食蟹猴CD47的结合能力、抑制CD47与SIRPα结合能力等参数，选出若干个杂交瘤克隆进行测序。利用常规的杂交瘤测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中，从免疫的Harbour H2L2小鼠得到的抗CD47单克隆抗体分子可变结构域的序列是人源抗体序列。抗体可变结构域的CDR序列可以通过Kabat或者Chothia或者其他CDR定义规则进行分析。在本实施例中，还使用了一种结合了Kabat定义和Chothia定义的称为Combined定义规则进行分析。
1.3制备全人重组抗体
在得到编码抗体分子的轻、重链可变结构域序列以后，可以采用常规的重组DNA技术，将轻、重链可变结构域序列和相应的人的抗体轻、重链恒定结构域序列进行融合表达，得到重组抗体分子。在本实施例中，抗体重链可变结构域序列(VH)通过基因合成并克隆到编码人IgG4抗体重链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生IgG4抗体的全长重链，并且在该IgG4重链恒定区引入S228P突变(根据EU编号，第228位丝氨酸取代成脯氨酸)以增加IgG4抗体的稳定性。抗体轻链可变结构域序列(VL)通过基因合成并克隆到编码人抗体Igκ轻链恒定结构域序列的哺乳动物细胞表达质粒载体中，以编码产生抗体的全长轻链。在本实施例中，由于从免疫的Harbour H2L2小鼠得到的抗CD47单克隆抗体分子可变结构域的序列是人源抗体序列，因而本实施例也得到全人源的抗CD47重组IgG4抗体。
将编码抗体重链的质粒和编码抗体轻链的质粒同时转染哺乳动物宿主细胞(如人胚肾细胞HEK293)，利用常规的重组蛋白表达和纯化技术，可以得到具有轻重链正确配对组装的纯化的CD47重组抗体。具体说来，将HEK293细胞在FreeStyle TM F17 Expression Medium培养基(Thermo #A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6～8x10 5细胞/ml，于37℃ 8％CO2摇床中培养24小时，细胞浓度在1.2X10 6细胞/ml。准备30ml培养的细胞。将上述编码抗体重链的质粒和编码抗体轻链的质粒以2:3的比例混合共计30μg质粒溶 解于1.5ml Opti-MEM减血清培养基(Thermo,31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,Inc#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃ 8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelect TM(GE Healthcare Life Science#71-5020-91AE)的重力柱(Bio-Rad#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,UFC901024)浓缩换液至PBS缓冲液，得到纯化的CD47抗体溶液。最后用NanoDrop(Thermo Scientific TM NanoDrop TM One)测定浓度，分装、存储备用。
1.4抗CD47全人重组抗体的序列
表2列出了本实施例中CD47抗体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG4)全长氨基酸序列和根据Chothia定义规则定义的CDR的氨基酸序列。
实施例2 抗体的序列分析和表达纯化
抗体的重链可变结构域序列来源于染色体上重链基因群的胚系基因V、D、J基因片段的基因重排和体细胞高频突变等事件；轻链可变结构域序列来源于轻链基因群的胚系基因V、J基因片段的基因重排和体细胞高频突变等事件。基因重排和体细胞高频突变是增加抗体多样性的主要因素。来源于相同胚系V基因片段的抗体也可能产生不同的序列，但总体上相似性较高。利用一些算法，例如IMGT/DomainGapAlign(http://imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi)或者NCBI/IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)可以从抗体的可变结构域序列推测出其发生基因重排时可能的胚系基因片段。将实施例1和表2中的抗体序列进行分析，其重链可变结构域(VH)和轻链可变结构域(VL)的胚系基因V基因片段列于表3。
蛋白质或多肽氨基酸链在细胞中翻译合成后有时会引入化学修饰，称为翻译后修饰(PTM)。对于抗体而言，一些PTM的位点是非常保守的，例如，在人的IgG1抗体的恒定结构域的第297位(EU编号)的保守的氨基酸天冬酰胺Asn通常会发生糖基化修饰形成糖链，而该糖链结构对于抗体结构和相关的效应子功能是至关重要的。但是，如果在抗体的可变结构域尤其是抗原结合区域(如CDR)中存在PTM，那么这些PTM的存在有可能会对抗原的结合有较大的影响，也可能对抗体的物理化学性质带来变化。例如，糖基化、脱酰胺、 异构化、氧化等都可能增加抗体分子的不稳定性或异质性，从而增加抗体开发的难度和风险。因而避免一些潜在的PTM对于治疗性抗体的开发是非常重要的。随着经验的积累，人们发现一些PTM是和氨基酸序列的组成尤其是相邻氨基酸组成的“模式”是高度相关的，这样使得可以从蛋白质的一级氨基酸序列预测出潜在的PTM。例如，N-x-S/T(第一位是天冬酰胺，第二位是非脯氨酸以外的任意氨基酸，第三位是丝氨酸或者苏氨酸)的序列模式预测出N-连接糖基化位点。引起PTM的氨基酸序列模式有可能来源于胚系基因序列，例如人胚系基因片段IGHV3-33天然地在FR3区域存在糖基化模式NST；也可能来源于体细胞高频突变。表3列出了实施例1的抗体的可变结构域VH和VL的预测的PTM。具体说来，NSS或NLT可能是糖基化位点，NS或NT或NN可能是脱酰胺位点。
可以通过氨基酸突变来破坏PTM的氨基酸序列模式，从而降低或者去除特定PTM的形成。根据抗体序列和PTM序列模式的不同，有不同的突变设计方法。一种方法是将“热点”氨基酸(如NS模式中的N或S)替换成物理化学性质相似的氨基酸(如把N突变为Q)。如果PTM序列模式来源于体细胞高频突变，而并不存在于胚系基因序列中，那么另一种方法可以是把该序列模式替换成对应的胚系基因序列。实际操作中，对同一个PTM序列模式可能采用多种突变设计方法。
表4列出了对六个来自实施例1的具有潜在PTM位点的抗体的序列进行氨基酸突变得到的新的抗体分子(称为PTM变体)。表5列出了本实施例中这些PTM变体的轻、重链可变结构域氨基酸序列，轻链全长氨基酸序列，重链(人IgG4)全长氨基酸序列，和根据Chothia定义规则定义的CDR的氨基酸序列。所有设计出来的PTM变体按照实施例1.3中描述的方法得到纯化的重组抗体，并在后续的功能实验中进一步验证。
表2 筛选得到的抗CD47杂交瘤单克隆及其重组抗体
Figure PCTCN2020110342-appb-000001
Figure PCTCN2020110342-appb-000002
表3 CD47抗体序列的胚系基因分析和翻译后修饰位点(PTM)分析
Figure PCTCN2020110342-appb-000003
表4 CD47抗体序列的突变位点设计
Figure PCTCN2020110342-appb-000004
Figure PCTCN2020110342-appb-000005
表5 突变PTM后得到的抗体
Figure PCTCN2020110342-appb-000006
Figure PCTCN2020110342-appb-000007
Figure PCTCN2020110342-appb-000008
实施例3 抗原结合蛋白能够与人和猴细胞表面的CD47结合
3.1能够与Jurkat以及Raji靶细胞表面的CD47结合
分别将Jurkat细胞(ATCC#TIB-152)和Raji细胞(ATCC#CCL-86)扩增培养，加入96孔板(Corning#3799)，每孔1×10 5个，再加入100μl不同浓度的实施例1或2得到的CD47抗体和对照抗体(Tab1、Tab2、Tab3)，同型hIgG4SP作为对照。混匀后4℃下孵育1小时。1％BSA(Amresco#0332-100G)洗涤3次，加入100μl的4μg/mL山羊抗人IgG(H+L)-Alexa488，4℃下孵育1小时。洗涤3次后进行流式细胞上机测试(BD Biosciences#Canto II cytometer)。根据测定结果计算半数最大结合效应浓度(EC 50)，并以EC 50值和最大MFI值作为相对结合活性评价依据。在本申请中，Tab1、Tab2和Tab3均来自现有技术，分别是Forty Seven的Hu5F9-G4、Celgene的CC-90002、Surface Oncology的SRF-231。
结果如图1及下表6-7所示。实施例1或2得到的CD47抗体均能结合Jurkat细胞，结合活性与Tab1、Tab2没有显著差别(图1A-1B)，且比Tab3的结合能力好。
PR000806、PR000807、PR000808和Tab1、Tab2均能与Jurkat(图1B)和Raji(图1C)细胞表面的CD47结合，结合活性没有显著性区别，且比Tab3与Jurkat和Raji的亲和能力强。
表6 抗原结合蛋白与Jurkat细胞表面CD47的结合
Figure PCTCN2020110342-appb-000009
Figure PCTCN2020110342-appb-000010
表7 抗原结合蛋白PR000806、PR000807、PR000808与Jurkat和Raji表面CD47的结合
Figure PCTCN2020110342-appb-000011
3.2能够与食蟹猴PBMC细胞表面的CD47结合
采用3.1的方法检测CD47抗体与食蟹猴PBMC的结合能力，结果如图2及表8所示。实施例1得到的PTM突变前的CD47抗体均能与食蟹猴PBMC细胞表面的CD47结合，且EC 50值小于Tab2。
表8 抗原结合蛋白与食蟹猴表面CD47的结合
Figure PCTCN2020110342-appb-000012
实施例4 抗原结合蛋白能够与人和食蟹猴CD47蛋白结合
分别将人CD47(Sino Biological#12283-HCCH)和猴CD47(Acro Biosystems#CD7-C52H1)用PBS稀释为0.25μg/ml，加至96孔板(Corning#9018)中，每孔100μl，4℃下孵育过夜。弃去液体后用PBST洗板3次，加入2％BSA封闭，室温条件下孵育1小时。弃去封闭液，并用PBST缓冲液(pH7.4，含0.05％吐温-20)洗板5次，将实施例1或2得到的CD47抗体和三种现有技术中的CD47抗体：Tab1(Forty Seven，Hu5F9-G4)、Tab2(Celgene，CC-90002)、Tab3(Surface Oncology，SRF-231)自66.7nM浓度起，依次进行5倍浓度稀释，共10个浓度梯度，加入100μl/孔，37℃孵育1小时，同型hIgG4SP(Crown Bio#C0045-4)为对照。清洗3次后，加入稀释4000倍的羊抗人HRP二抗(Invitrogen#A18805)，37℃孵育1小时。清洗后添加100μl/孔TMB(Beyotime#P0209)，室温避光放置5分钟；每孔加入100μl/孔终止液(BBI life sciences#E661006-0200)终止反应，置酶标仪(PerkinElemer#Enspire)中测定450nm吸光度(OD450)值。根据测定结果计算半数最大结合效应浓度(EC 50)，并以EC 50值和最大OD值作为相对结合活性评价依据。
结果如图3及下表9-10所示。图3A和表9显示，实施例1得到的PTM突变前的CD47抗体均能与源自食蟹猴的CD47蛋白结合。图3B-3C和表10显示PR00806、PR000807、PR000808与人(图3B)和猴(图3C)CD47结合的活性。PR00806、PR000807、PR000808、Tab1、Tab2、Tab3均能与人CD47蛋白结合，结合活性没有显著差别，且PR00806、PR000807、PR000808的EC 50低于0.03nM，结合能力优于Tab3。PR00806、PR000807、PR000808、Tab1、Tab2均能与猴CD47蛋白结合，结合活性没有显著差别，但Tab3与猴CD47蛋白的结合能 力较弱。
表9 抗原结合蛋白与猴CD47蛋白的结合
Figure PCTCN2020110342-appb-000013
表10 抗原结合蛋白与人或猴CD47蛋白的结合
Figure PCTCN2020110342-appb-000014
实施例5 抗原结合蛋白能够抑制人CD47蛋白与SIRPα结合
将人CD47-ECD-hFc(Sino Biological#12283-H02H)用PBS稀释成1μg/ml加入到96孔 板(Corning#9018)中，每孔100μl，4℃包被过夜。洗涤3次后用100μl的2％BSA室温下封闭1小时。将实施例1或2得到的CD47抗体和对照抗体(Tab1、Tab2、Tab3)用PBST稀释成不同浓度梯度，与1μg/ml组氨酸标记的SIRPα蛋白(Sino Biological#11612-H08H)混合后，加入孔板，同型hIgG4作为对照，室温下孵育1小时。洗涤3次后每孔加入100μl组氨酸标记的HRP抗体(Biolegend#652504)，室温下反应1小时。洗涤3次后加入100μl TMB显色液，室温下避光显色5分钟。加入终止液终止反应，用酶标仪(PerkinElemer#Enspire)测定450nm吸光度(OD450)值。以IC 50值、最小OD值、最大抑制率作为相对抑制活性评价依据。
结果图4及下表11-12所示。图4A和表11显示，实施例1得到的CD47抗体均能抑制人CD47与SIRPα结合。图4B和表12显示PR00806、PR000807、PR000808均能抑制人CD47与SIRPα结合，抑制率均大于97％，且抑制活性与Tab1、Tab2、Tab3没有显著差别。
表11 抗原结合蛋白抑制人CD47与SIRPα结合
Figure PCTCN2020110342-appb-000015
表12 抗原结合蛋白抑制人CD47与SIRPα结合
Figure PCTCN2020110342-appb-000016
Figure PCTCN2020110342-appb-000017
实施例6 抗原结合蛋白能够阻断Jurkat细胞上的CD47与SIRPα结合
将CD47高表达的人Jurkat细胞扩增培养后，以每孔1×10 5个加入96孔板。将实施例1或2得到的CD47抗体和对照抗体(Tab1、Tab2、Tab3)用PBS稀释成不同浓度梯度，与1μg/ml组氨酸标记的SIRPα蛋白混合后，加入每孔，同型hIgG4SP作为对照，4℃下孵育1小时。1％BSA洗涤2次，加入二抗Biotin-conjugated anti-His Tag antibody(GenScript#A00613)100μl体积，4℃下孵育1小时。离心之后1％BSA洗涤2次，加入三抗PE-Conjugated streptavidin(共轭链霉抗生物素蛋白)(Affymetrix#DC)100μl体积，洗涤3次后进行流式细胞上机测试。以IC 50值、最小MFI值、最大抑制率作为相对阻断活性评价依据。
结果如图5及下表13-14所示。图5A和表13显示实施例1得到的CD47抗体均能阻断Jurkat细胞表面的CD47与SIRPα结合。图5B和表14显示PR00806、PR000807、PR000808均能阻断Jurkat细胞表面的CD47与SIRPα结合，且其阻断活性与Tab1、Tab2没有显著差别，IC 50均小于0.7nM，而Tab3阻断能力较弱。
表13 抗原结合蛋白阻断Jurkat细胞上的CD47与SIRPα结合
Figure PCTCN2020110342-appb-000018
Figure PCTCN2020110342-appb-000019
表14 抗原结合蛋白阻断Jurkat细胞上的CD47与SIRPα结合
Figure PCTCN2020110342-appb-000020
实施例7 抗原结合蛋白不引起红细胞的凝集反应
分析实施例1或2得到的CD47抗体是否引起红细胞凝集反应。将人红细胞用PBS稀释至4％后加入96孔板(Corning#3799)，再加入从200nM开始2倍稀释待测抗体和对照抗体(Tab1、Tab2、Tab3)，同型hIgG4SP作为对照，在37℃下孵育1小时。通过与无抗体的孔内红点边缘的清晰程度对比来评价抗体对人红细胞凝集的作用效果。
图6A-6B显示，与不加抗体的孔相比，除PR00796，其他抗原结合蛋白不引起凝集反应，红点边缘清晰；而Tab1引起凝集反应，红点模糊，呈弥散状。
实施例8 抗原结合蛋白与红细胞结合能力检测
检测实施例1或2得到的CD47抗体与红细胞结合的活性。将人红细胞在PBS中稀释，加入96孔板(Corning#3799)，每孔1×10 5个，再加入100μl不同浓度的抗CD47抗体和对照抗体(Tab1、Tab2、Tab3)，同型hIgG4SP作为对照，混匀后4℃下孵育1小时。1％BSA(Amresco#0332-100G)洗涤3次，加入100μl的4μg/mL山羊抗人IgG(H+L)-Alexa 488，4℃下孵育1小时，洗涤3次后进行流式细胞上机测试(BD Biosciences#Canto II cytometer)。以EC 50值、最大MFI值作为相对结合活性评价依据。
抗原结合蛋白与红细胞结合的能力如图7和下表15-16显示。
表15 抗原结合蛋白与红细胞结合
Figure PCTCN2020110342-appb-000021
比较抗原结合蛋白与Jurkat细胞结合(实施例3)和红细胞(RBC)结合的能力，计算抗原结合蛋白与肿瘤细胞和红细胞结合的EC 50和最大MFI的比值，结果见下表16。从结果看出，与对照抗体相比，实施例1或2所得抗原结合蛋白与Jurkat结合的能力大于与红细胞结合的能力。
表16 抗原结合蛋白与肿瘤细胞和红细胞结合能力比值
Figure PCTCN2020110342-appb-000022
实施例9 抗原结合蛋白能够引起巨噬细胞对肿瘤细胞的吞噬作用
9.1引起巨噬细胞对Jurkat细胞的吞噬作用
从人外周血单核细胞(PBMC)中分离得到CD14 +的单核细胞(Miltenyi Biotec#130-050-201)，加入100ng/ml M-CSF(R&D Systems#216-MC/CF)培养7天使其分化为巨噬细胞，用胰酶消化，接种于96孔板上，每孔5×10 4个，同时以1：1的比例加入用0.25μM羧基荧光素琥珀酰亚胺酯(CFSE)(Life-technology#C34570)-标记的Jurkat细胞，再加入稀释好的 抗CD47的抗体和对照抗体(Tab1、Tab2、Tab3)，混匀后37℃下孵育2小时。用1％BSA洗去未被吞噬的Jurkat细胞，加入直标抗体CD14-APC(BD Pharmingen#561708)。流式细胞仪(ACEA#Novocyte)进行测量，以吞噬指数作为吞噬作用的评价依据，吞噬指数为CFSE +CD14 +细胞占总CD14 +细胞的百分比。
结果如图8和下表17-19所示。图8A和表17显示的是实施例1得到的PTM突变前的CD47抗体均能引起巨噬细胞对Jurkat细胞的吞噬作用，且引起吞噬作用的能力优于Tab2。图8B和表18显示的是PR000806、PR000807、PR000808均能引起巨噬细胞对Jurkat细胞的吞噬作用，且引起吞噬作用的能力优于Tab2和Tab3。表19显示的是实施例1或2得到的CD47抗体在不同浓度引起巨噬细胞对Jurkat细胞的吞噬作用。
表17 抗原结合蛋白引起巨噬细胞对Jurkat细胞的吞噬作用
Figure PCTCN2020110342-appb-000023
表18 抗原结合蛋白引起巨噬细胞对Jurkat细胞的吞噬作用
Figure PCTCN2020110342-appb-000024
表19 抗原结合蛋白引起巨噬细胞对Jurkat细胞的吞噬作用
Figure PCTCN2020110342-appb-000025
9.2引起巨噬细胞对OVCAR 3细胞的吞噬作用
按实施例9.1的方法检测巨噬细胞对OVCAR 3(ATCC#HTB-161)细胞的吞噬作用，流式细胞仪测定EC 50和吞噬指数。
结果如图9和下表20所示，PR00806、PR000807、PR000808均能引起巨噬细胞对OVCAR3细胞的吞噬作用，且无显著差别；EC 50值和最大吞噬指数显示，PR00806、PR000807、PR000808引起吞噬作用的能力比Tab3更强。
表20 抗原结合蛋白引起巨噬细胞对OVCAR3细胞的吞噬作用
Figure PCTCN2020110342-appb-000026
实施例10 抗原结合蛋白引起巨噬细胞对人红细胞吞噬作用的能力检测
采用实施例9的方法检测实施例1或2所得的抗原结合蛋白引起巨噬细胞对人红细胞的吞噬作用，流式细胞仪测定EC 50和吞噬指数。
结果如图10和下表21-22所示。图10和表21显示的是PTM移除后的CD47抗体引起巨噬细胞对人红细胞吞噬作用的能力。表22显示的是实施例1或2得到的CD47抗体在不同浓度引起巨噬细胞对hRBC吞噬作用的能力检测。结果显示，本申请所述的抗原结合蛋白(如，CD47抗体)能够有效引起巨噬细胞对hRBC吞噬，且效果与对照抗体相近或高于对照抗体。
表21 抗原结合蛋白引起巨噬细胞对人红细胞的吞噬作用
Figure PCTCN2020110342-appb-000027
表22 抗原结合蛋白引起巨噬细胞对人红细胞的吞噬作用
Figure PCTCN2020110342-appb-000028
实施例11 抗原结合蛋白的体内抗肿瘤活性
11.1抗原结合蛋白在NCG-Raji小鼠模型上的体内抗肿瘤活性
在NCG-Raji小鼠模型中评价抗原结合蛋白的抗肿瘤活性。将Raji细胞1×10 7个/0.1mL接种于NCG小鼠尾静脉。细胞接种后第三天，将小鼠随机分成10组，每组8只小鼠，开始给药。分别以0.3mg/kg、1mg/kg和3mg/kg剂量经腹腔注射各待测抗体，10μl/g，每3天给药1次，连续给药6次，同型hIgG4为对照。记录小鼠体重和肿瘤体积。
表23显示的是与同型IgG4相比，注射各抗体的肿瘤抑制率(TGI)，肿瘤抑制率与剂量有关。图11A-11B显示的是注射PR000806、PR000808和Tab1后小鼠体内肿瘤体积和体重变化趋势。注射PR000806、PR000808的组小鼠肿瘤体积增长减缓。结果显示，PR000806和PR000808具能在体内抑制肿瘤生长，PR000808在3mg/kg和1mg/kg剂量下比Tab1效果更好。
表23 抗原结合蛋白抑制肿瘤生长
Figure PCTCN2020110342-appb-000029
11.2抗原结合蛋白在B-hSIRPa/hCD47-MC38-hCD47小鼠模型上的体内抗肿瘤活性
在B-hSIRPa/hCD47-MC38-hCD47小鼠模型中评价抗原结合蛋白的抗肿瘤活性。将稳定表达人CD47蛋白的MC38细胞1×10 6个/0.1mL接种于B-hSIRPA/hCD47小鼠的右侧皮下。细胞接种后，当平均肿瘤体积达到大约80-120mm3时，将小鼠随机分成7组，每组8只小鼠，开始给药。分别以5mg/kg和10mg/kg剂量经腹腔注射各待测抗体，10μl/g，每3天给药1次，连续给药6次，同型hIgG4为对照。记录小鼠体重和肿瘤体积。
图12A-12B显示的是注射PR000806、PR001265和Tab1后小鼠体内肿瘤体积和体重变化趋势。注射PR000806、PR001265的组小鼠肿瘤体积增长减缓。结果显示，PR000806和PR001265能够在体内抑制肿瘤生长，高剂量时PR000806和PR001265肿瘤抑制效果比Tab1更好。
实施例12 抗原结合蛋白的毒性检测
将B-hSIRPA/hCD47纯合小鼠按体重随机分组，每组4只，共8组，分组后第8天以 10mpk的剂量腹腔注射给药一次，第一次给药为治疗第0天，共给药1次(第0天)。给药和观察期间每天测量1次小鼠体重，并记录测量值。另外，分别于给药前7天(正常值)、治疗后1天、7天取血，进行血常规分析。小鼠体重变化和血常规分析结果如图13-15所示。
给药11天后结束实验，实验结束时，动物安乐死，小鼠拍照。
图13显示的是治疗前后小鼠的体重变化。除同型hIgG4，其他抗原结合蛋白或抗体治疗后小鼠体重均减轻，其中，Tab1、PR000806、PR000808和PR001281治疗后小鼠体重减轻程度较其它三个分子大，与体外抗体分子与抗原在红细胞上的结合能力结果一致，但是在治疗后大约7天，小鼠体重开始恢复至正常水平。
图14-16显示的是治疗前7天和治疗后小鼠模型第1天、第7天血常规检测结果。从图像中可以看出，给药后第一天，样品组较对照组hIgG4SP血常规中的一些参数发生一定的变化，但是在给药后7天之后，随着机体代偿功能的作用发挥，指标均恢复正常。
最后，从体重和血常规的数据来看，靶向CD47的抗体分子PR000806、PR000808和PR001281、PR001265、PR001275和PR001276均未显示出不可恢复的血液毒性。
实施例13 抗原结合蛋白能够与CD47结合的亲和力测试(Biacore)
13.1 Protein A的偶联
以1x HBS-EP+(GE Healthcare#BR-1006-69)为运行缓冲液，流速10ul/min,在CM5的4个通道上偶联Protein A(Thermo Fisher#21181)：1)设置注入时间800s，将50mM NHS和200mM EDC以1：1体积比混合后注入4个通道；2)用pH4.5的醋酸钠将Protein A稀释至20μg/ml，注入800s；3)注入1M乙醇胺800s，以封闭芯片表面剩余的活性羧基。封闭后继续用1x HBS-EP+缓冲液平衡仪器两小时，Protein A最终偶联量约为2000RU。
13.2动力学测试
设置多循环动力学模式，每个循环包括抗体的捕获、分析物的结合以及芯片的再生。将抗体均稀释至1μg/ml，以10μl/min的流速注入2、3、4通道40s，由预先偶联的Protein A捕获各抗体，捕获量约为200RU。分别将人CD47(Sino biological#12283-HCCH)和食蟹猴CD47(Acro Biosystems#CD7-C52H1)依次按照0nM、1.25nM、2.5nM、5nM、10nM、20nM、40nM的浓度梯度注入四个通道，流速为30ul/min，设置注入时间180s，解离时间900s。最后以同样流速注入甘氨酸(10mM，pH1.5)30s，以再生芯片。
13.3数据分析
用Biacore T200分析软件2.0对实验结果进行分析，1通道作为参比通道扣除，分析模型选用1：1动力学拟合模型。
结果如下表24所示。实验结果表明，5个抗体均与人CD47和食蟹猴CD47有良好的结合。
表24 抗原结合蛋白与食蟹猴和人CD47结合的结合亲和力
Figure PCTCN2020110342-appb-000030
实施例14 抗原结合蛋白以高度特异性结合CD47
分别将组氨酸标记的人CD47(Sino Biological#12283-HCCH)、SIPRα(Sino Biological#11612-H08H)、CD147(Sino Biological#10186-H08H)、PD-1(Novoprotein#CD91)、PD-L1(Novoprotein#C315)、CTLA-4(Sino Biological#11159-H08H)、CD80(Sino Biological#10698-H08H)、CD86(Sino Biological#10699-H08H)、CD28H(AcroBiosystem#CDH-H52H3)、B7H3(Sino Biological#11188-H08H)、B7H4(Sino Biological#10738-H08H)和ICOS(AcroBiosystem#ICS-H52H6)用PBS稀释为1μg/ml，加至96孔板(Corning#9018)中，每孔100μl，4℃下孵育过夜。弃去液体后用PBST洗板3次，加入2％BSA封闭，室温条件下孵育1小时。弃去封闭液，并用PBST缓冲液(pH7.4，含0.05％吐温-20)洗板5次，将实施例1或2得到的CD47抗体PR001265、PR001275、PR001276以及Tab1(Forty Seven，Hu5F9-G4)稀释至10nM和100nM，加入100μl/孔，37℃孵育1小时，同型hIgG4SP(Crown Bio#C0045-4)为对照。清洗3次后，加入稀释4000倍的羊抗人HRP二抗(Invitrogen#A18805)，37℃孵育1小时。清洗后添加100μl/孔TMB(Beyotime#P0209)，室温避光放置5分钟；每孔加入100μl/孔终止液(BBI life sciences#E661006-0200)终止反应，置酶标仪(PerkinElemer#Enspire)中测定450nm吸光度值和570nm吸光度值。计算OD450/570的比值。
结果显示(图17)，本申请的抗原结合蛋白以高度特异性结合CD47，不会结合非CD47的抗原SIPRα、CD147、PD-1、PD-L1、CTLA-4、CD80、CD86、CD28H、B7H3、B7H4和ICOS。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (51)

  1. 分离的抗原结合蛋白，其包含抗体重链可变区VH中的至少一个CDR，所述VH包含SEQ ID NO:154所示的氨基酸序列。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其包含抗体轻链可变区中的至少一个CDR，其中所述VL包含SEQ ID NO:153所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  4. 根据权利要求3所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。
  5. 根据权利要求3-4中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  6. 根据权利要求1-5中任一项所述的分离的抗原结合蛋白，其中所述VH包含HCDR1，HCDR2和HCDR3，其中所述HCDR3包含SEQ ID NO:144所示的氨基酸序列，其中所述HCDR3优选包含SEQ ID NO:55和58-63中任一项中所示的氨基酸序列。
  7. 根据权利要求6所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO:143所示的氨基酸序列，其中所述HCDR2优选包含SEQ ID NO:136-138中任一项中所示的氨基酸序列。
  8. 根据权利要求6-7中任一项所述的分离的抗原结合蛋白，其中所述HCDR1包含SEQ ID NO:142所示的氨基酸序列，其中所述HCDR1优选包含SEQ ID NO:32、34、36、38、40、42、45、47、49、51和53中任一项中所示的氨基酸序列。
  9. 根据权利要求2-8中任一项所述的分离的抗原结合蛋白，其中所述VL包含LCDR1，LCDR2和LCDR3，其中所述LCDR3包含SEQ ID NO:141所示的氨基酸序列，其中所述LCDR3优选包含SEQ ID NO:67-74、116-121中任一项中所示的氨基酸序列。
  10. 根据权利要求9所述的分离的抗原结合蛋白，其中所述LCDR2包含SEQ ID NO:140所示的氨基酸序列，其中所述LCDR2优选包含SEQ ID NO:31、56、57、122、126和127中任一项中所示的氨基酸序列。
  11. 根据权利要求2-10中任一项所述的分离的抗原结合蛋白，其中所述LCDR1包含SEQ ID NO:139所示的氨基酸序列，其中所述LCDR1优选包含SEQ ID NO:113、114和115中任一项中所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR1，H-FR2，H-FR3，和H-FR4。
  13. 根据权利要求12所述的分离的抗原结合蛋白，其中所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:145所示的氨基酸序列。
  14. 根据权利要求12-13中任一项所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:167-170中任一项中所示的氨基酸序列。
  15. 根据权利要求12-14中任一项所述的分离的抗原结合蛋白，其中所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:146所示的氨基酸序列。
  16. 根据权利要求12-15中任一项所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:184、187-190中任一项中所示的氨基酸序列。
  17. 根据权利要求12-16中任一项所述的分离的抗原结合蛋白，其中所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:147所示的氨基酸序列。
  18. 根据权利要求12-17中任一项所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:171-182中任一项中所示的氨基酸序列。
  19. 根据权利要求12-18中任一项所述的分离的抗原结合蛋白，其中所述H-FR4的N末端与所述HCDR3的C末端直接或间接相连，且所述H-FR4包含SEQ ID NO:148所示的氨基酸序列。
  20. 根据权利要求12-19中任一项所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:183、185和186中任一项中所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含VH，其中所述VH包含SEQ ID NO:154所示的氨基酸序列。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:75、77、79、81、83、85、87、89、91、93、95、97、99、101、103、105、107、109和111中任一项中所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG恒定区。
  24. 根据权利要求1-23中任一项所述的分离的抗原结合蛋白，其包括抗体重链恒定区，且所述抗体重链恒定区包括人IgG4恒定区。
  25. 根据权利要求23-24中任一项所述的分离的抗原结合蛋白，其中所述抗体重链恒定区 包含SEQ ID NO:195中所示的氨基酸序列。
  26. 根据权利要求1-25中任一项所述的分离的抗原结合蛋白，其包含抗体重链HC，且所述HC包含SEQ ID NO:198所示的氨基酸序列。
  27. 根据权利要求26所述的分离的抗原结合蛋白，其中所述HC包含SEQ ID NO:76、78、80、82、84、86、88、90、92、94、96、98、100、102、104、106、108、110和112中任一项中所示的氨基酸序列。
  28. 根据权利要求2-27中任一项所述的分离的抗原结合蛋白，其中所述VL包括框架区L-FR1，L-FR2，L-FR3，和L-FR4。
  29. 根据权利要求28所述的分离的抗原结合蛋白，其中所述L-FR1的C末端与所述LCDR1的N末端直接或间接相连，且所述L-FR1包含SEQ ID NO:149所示的氨基酸序列。
  30. 根据权利要求28-29中任一项所述的分离的抗原结合蛋白，其中所述L-FR1包含SEQ ID NO:156、157和158中任一项中所示的氨基酸序列。
  31. 根据权利要求28-30中任一项所述的分离的抗原结合蛋白，其中所述L-FR2位于所述LCDR1与所述LCDR2之间，且所述L-FR2包含SEQ ID NO:150所示的氨基酸序列。
  32. 根据权利要求28-31中任一项所述的分离的抗原结合蛋白，其中所述L-FR2包含SEQ ID NO:191-194中任一项中所示的氨基酸序列。
  33. 根据权利要求28-32中任一项所述的分离的抗原结合蛋白，其中所述L-FR3位于所述LCDR2与所述LCDR3之间，且所述L-FR3包含SEQ ID NO:151所示的氨基酸序列。
  34. 根据权利要求28-33中任一项所述的分离的抗原结合蛋白，其中所述L-FR3包含SEQ ID NO:155、162-166中任一项中所示的氨基酸序列。
  35. 根据权利要求28-34中任一项所述的分离的抗原结合蛋白，其中所述L-FR4的N末端与所述LCDR3的C末端直接或间接相连，且所述L-FR4包含SEQ ID NO:152所示的氨基酸序列。
  36. 根据权利要求28-35中任一项所述的分离的抗原结合蛋白，其中所述L-FR4包含SEQ ID NO:159、160和161中任一项中所示的氨基酸序列。
  37. 根据权利要求1-36中任一项所述的分离的抗原结合蛋白，其包含VL，其中所述VL包含SEQ ID NO:153所示的氨基酸序列。
  38. 根据权利要求2-37中任一项所述的分离的抗原结合蛋白，其中所述VL包含SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27和29中任一项中所示的氨基酸序列。
  39. 根据权利要求1-38中任一项所述的分离的抗原结合蛋白，其包括抗体轻链恒定区，且所述抗体轻链恒定区包括人Igκ恒定区。
  40. 根据权利要求39所述的分离的抗原结合蛋白，其中所述抗体轻链恒定区包含SEQ ID NO:196中所示的氨基酸序列。
  41. 根据权利要求1-40中任一项所述的分离的抗原结合蛋白，其包含抗体轻链LC，且所述LC包含SEQ ID NO:197所示的氨基酸序列。
  42. 根据权利要求41中所述的分离的抗原结合蛋白，其中所述LC包含SEQ ID NO:197所示的氨基酸序列，其中所述LC优选包含SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28和30中任一项中所示的氨基酸序列。
  43. 根据权利要求1所述的分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    1)能够特异性结合源自人和猴的CD47，优选KD≤3x10 -9M；
    2)能够抑制CD47与SIRPα的结合；
    3)能够结合人血红细胞；
    4)在红细胞凝血测定中，不引起凝血反应；
    5)能够引起巨噬细胞对肿瘤细胞的吞噬；和
    6)能够抑制肿瘤生长和/或肿瘤细胞增殖。
  44. 一种或多种分离的核酸分子，其编码权利要求1-43中任一项所述的分离的抗原结合蛋白。
  45. 载体，其包含根据权利要求44所述的核酸分子。
  46. 细胞，其包含根据权利要求44所述的核酸分子或根据权利要求45所述的载体。
  47. 制备权利要求1-43中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-43中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求46所述的细胞。
  48. 药物组合物，其包含权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的核酸分子、权利要求45所述的载体和/或权利要求46所述的细胞，以及任选地药学上可接受的载体。
  49. 权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的核酸分子、权利要求45所述的载体、权利要求46所述的细胞和/或权利要求48所述的药物组合物在制备药物中的用途，所述药物用于预防、缓解和/或***。
  50. 抑制CD47与SIRPα结合的方法，所述方法包括施用权利要求1-43中任一项所述的 分离的抗原结合蛋白。
  51. 预防、缓解和/或***的方法，所述方法包括向有需要的受试者施用权利要求1-43中任一项所述的分离的抗原结合蛋白、权利要求44所述的核酸分子、权利要求45所述的载体、权利要求46所述的细胞和/或权利要求48所述的药物组合物。
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