WO2021032174A1 - Protéine de liaison à l'antigène anti-cd47 et son utilisation - Google Patents

Protéine de liaison à l'antigène anti-cd47 et son utilisation Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
sequence shown
antigen binding
Prior art date
Application number
PCT/CN2020/110342
Other languages
English (en)
Chinese (zh)
Inventor
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
Original Assignee
和铂医药(上海)有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 和铂医药(上海)有限责任公司 filed Critical 和铂医药(上海)有限责任公司
Priority to CN202080005950.7A priority Critical patent/CN112969715B/zh
Publication of WO2021032174A1 publication Critical patent/WO2021032174A1/fr

Links

Images

Classifications

    • 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.

Abstract

L'invention concerne une protéine de liaison à un antigène séparée. La protéine de liaison à l'antigène peut être liée de manière spécifique à CD47 à la surface de cellules tumorales, peut inhiber la combinaison de CD47 et SIRPα, peut être liée à des érythrocytes humains, ne provoque pas de réponse de coagulation sanguine, peut provoquer la phagocytose de macrophages vers des cellules tumorales, et peut inhiber la croissance de tumeurs et/ou la prolifération des cellules tumorales. L'invention concerne une utilisation de la protéine de liaison à l'antigène dans la prévention et le traitement de tumeurs.
PCT/CN2020/110342 2019-08-21 2020-08-20 Protéine de liaison à l'antigène anti-cd47 et son utilisation WO2021032174A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202080005950.7A CN112969715B (zh) 2019-08-21 2020-08-20 一种抗cd47抗原结合蛋白及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910775731.6 2019-08-21
CN201910775731 2019-08-21

Publications (1)

Publication Number Publication Date
WO2021032174A1 true WO2021032174A1 (fr) 2021-02-25

Family

ID=74659805

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/110342 WO2021032174A1 (fr) 2019-08-21 2020-08-20 Protéine de liaison à l'antigène anti-cd47 et son utilisation

Country Status (3)

Country Link
CN (1) CN112969715B (fr)
TW (1) TW202108631A (fr)
WO (1) WO2021032174A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023025187A1 (fr) * 2021-08-24 2023-03-02 上海鑫湾生物科技有限公司 Anticorps se liant de manière spécifique à cd47, virus oncolytique recombinant de celui-ci et son utilisation

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018137705A1 (fr) * 2017-01-26 2018-08-02 Zai Lab (Shanghai) Co., Ltd. Unité de liaison à l'antigène cd47 et ses utilisations
CN109265547A (zh) * 2018-07-23 2019-01-25 中国科学院微生物研究所 一种抗cd47抗体及其应用
WO2019027903A1 (fr) * 2017-08-02 2019-02-07 Phanes Therapeutics, Inc. Anticorps anti-cd47 et leurs utilisations
WO2019086573A1 (fr) * 2017-11-01 2019-05-09 Hummingbird Bioscience Holdings Pte. Ltd. Molécules de liaison à l'antigène cd47
CN110066336A (zh) * 2019-05-12 2019-07-30 杭州科兴生物科技有限公司 抗cd47单克隆抗体、片段及其医药用途
WO2019144895A1 (fr) * 2018-01-24 2019-08-01 Nanjing Legend Biotech Co., Ltd. Anticorps anti-cd47 qui ne provoquent pas d'agglutination significative des globules rouges

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SI3515946T1 (sl) * 2016-09-23 2022-09-30 Regeneron Pharmaceuticals, Inc. Protitelesa proti-MUC16 (MUCIN 16)
CN109422811A (zh) * 2017-08-29 2019-03-05 信达生物制药(苏州)有限公司 抗cd47抗体及其用途
CN110305212A (zh) * 2018-03-27 2019-10-08 信达生物制药(苏州)有限公司 抗cd47抗体及其用途

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018137705A1 (fr) * 2017-01-26 2018-08-02 Zai Lab (Shanghai) Co., Ltd. Unité de liaison à l'antigène cd47 et ses utilisations
WO2019027903A1 (fr) * 2017-08-02 2019-02-07 Phanes Therapeutics, Inc. Anticorps anti-cd47 et leurs utilisations
WO2019086573A1 (fr) * 2017-11-01 2019-05-09 Hummingbird Bioscience Holdings Pte. Ltd. Molécules de liaison à l'antigène cd47
WO2019144895A1 (fr) * 2018-01-24 2019-08-01 Nanjing Legend Biotech Co., Ltd. Anticorps anti-cd47 qui ne provoquent pas d'agglutination significative des globules rouges
CN109265547A (zh) * 2018-07-23 2019-01-25 中国科学院微生物研究所 一种抗cd47抗体及其应用
CN110066336A (zh) * 2019-05-12 2019-07-30 杭州科兴生物科技有限公司 抗cd47单克隆抗体、片段及其医药用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIE LIU, WANG LIJUAN, ZHAO FEIFEI, TSENG SERENA, NARAYANAN CYNDHAVI, SHURA LEI, WILLINGHAM STEPHEN, HOWARD MAUREEN, PROHASKA SUSAN: "Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, vol. 10, no. 9, 21 September 2015 (2015-09-21), pages 1 - 23, XP055223677, DOI: 10.1371/journal.pone.0137345 *
LIANG DONGWEI, LIU JIHONG: "Preparement and Identification of Monoclonal Antibody Against Xxtracellular Region of Human CD47 Protein", INTERNATIONAL JOURNAL OF LABORATORY MEDICINE, vol. 36, no. 21, 1 November 2015 (2015-11-01), pages 3125 - 3127, XP055782564 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023025187A1 (fr) * 2021-08-24 2023-03-02 上海鑫湾生物科技有限公司 Anticorps se liant de manière spécifique à cd47, virus oncolytique recombinant de celui-ci et son utilisation

Also Published As

Publication number Publication date
TW202108631A (zh) 2021-03-01
CN112969715B (zh) 2022-03-18
CN112969715A (zh) 2021-06-15

Similar Documents

Publication Publication Date Title
US11254746B2 (en) Anti-PD-1 monoclonal antibody, and preparation method therefor and application thereof
JP7196076B2 (ja) がんの処置のための抗ｔｎｆ関連アポトーシス誘発リガンド受容体２及び抗カドヘリン１７結合性二重特異的分子
JP2023134490A (ja) リンパ球における阻害経路の中和
KR20180004277A (ko) Pdl-1 항체, 그 약학적 조성물 및 그 용도
CA2959841A1 (fr) Traitement des cancers au moyen d'agents anti-nkg2a
CA2977621C (fr) Nouvel anticorps se liant a la tfpi et composition le comprenant
CN105829345B (zh) 结合egfr和met的多功能抗体
KR20190035863A (ko) 암 치료를 위한 항Siglec-7 항체
US20220041744A1 (en) Cd73 blocking antibodies
AU2015280436A1 (en) Antibodies that bind LGR4
WO2023036281A1 (fr) Anticorps anti-cd47 et son utilisation
KR20220042258A (ko) 항 tigit 항체 및 그의 응용
JP2024026170A (ja) 癌を処置するための組成物及び方法
CN114181310B (zh) 抗tigit抗体、其药物组合物及用途
WO2021032174A1 (fr) Protéine de liaison à l'antigène anti-cd47 et son utilisation
TW202334228A (zh) B7h6抗體及其應用
EP4292611A1 (fr) Anticorps anti-cd112r et son utilisation
AU2021223063A1 (en) Anti-IL-2 antibody, and antigen-binding fragment thereof and medical use thereof
TWI834867B (zh) Cd73阻斷抗體
CN113166264B (zh) 一种分离的抗原结合蛋白及其用途
CN113164601B (zh) 一种分离的抗原结合蛋白及其用途
WO2021013061A1 (fr) Anticorps anti-vegfr2 humanisé et son utilisation
US20240052065A1 (en) Binding molecules for the treatment of cancer
WO2022247826A1 (fr) Protéine de liaison spécifique ciblant pd-l1 et cd73
WO2024078558A1 (fr) Anticorps anti-cd100 et son utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20854099

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20854099

Country of ref document: EP

Kind code of ref document: A1