WO2021164692A1 - Protéine isolée se liant à l'antigène psma et son utilisation - Google Patents

Protéine isolée se liant à l'antigène psma et son utilisation Download PDF

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WO2021164692A1
WO2021164692A1 PCT/CN2021/076664 CN2021076664W WO2021164692A1 WO 2021164692 A1 WO2021164692 A1 WO 2021164692A1 CN 2021076664 W CN2021076664 W CN 2021076664W WO 2021164692 A1 WO2021164692 A1 WO 2021164692A1
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amino acid
seq
acid sequence
binding protein
antigen binding
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PCT/CN2021/076664
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Chinese (zh)
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黄冰
何云
戎一平
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和铂医药(上海)有限责任公司
和铂医药(苏州)有限公司
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Priority claimed from CN202010096322.6A external-priority patent/CN110922486B/zh
Priority claimed from CN202010143681.2A external-priority patent/CN111303288B/zh
Application filed by 和铂医药(上海)有限责任公司, 和铂医药(苏州)有限公司 filed Critical 和铂医药(上海)有限责任公司
Publication of WO2021164692A1 publication Critical patent/WO2021164692A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • This application relates to the field of biomedicine, in particular to an isolated protein that binds to the antigen PSMA and its use.
  • prostate cancer is the most commonly diagnosed cancer and the third leading cause of death. According to statistics, in 2017, 160,000 patients were diagnosed with this tumor, which caused 26,000 deaths (Siegel RL et al., (2017) CA Cancer J Clin. 67: 7-30). Patients with localized cancer usually receive surgical treatment or radiotherapy (Walsh PC et al., (2007) N Engl J Med. 357: 2696–705). However, 20-40% of patients undergoing radical prostatectomy and 30-50% of patients receiving radiotherapy will experience relapse (Paller CJ et al., (2013) Clin Adv Hematol Oncol. 11:14–23 ).
  • the standard treatment for metastatic cancer is usually androgen blockade, through bilateral orchiectomy or chemical castration (such as administration of luteinizing hormone receptor (LHR) agonists or antagonists) (Tannock IF et al., (2004) )N Engl J Med.351:1502-12).
  • LHR luteinizing hormone receptor
  • androgen blockade is very effective, but with obvious side effects, patients gradually suffer from castration refractory prostate cancer (CRPC) (Petrylak DP et al., (2004) N Engl J Med. 351: 1513-20).
  • CRPC castration refractory prostate cancer
  • mCRPC metastatic CRPC
  • PSMA prostate specific membrane antigen
  • PSMA is a homodimeric type II membrane glycoprotein, expressed in different tissues, such as prostate, kidney, small intestine, central nervous system and peripheral nervous system, but mainly expressed in the prostate.
  • PSMA is up-regulated in prostate cancer (Schulke N et al., (2003) Proc Natl Acad Sci USA. 100: 12590-5; Ross JS et al., (2003) Clin Cancer Res. 9: 6357-62), And it increases with the progression of the disease, and the expression is highest in metastatic, castration refractory prostate cancer (Su SL et al., (1995) Cancer Res. 55:1441-3).
  • PSMA is also expressed in large numbers in the neovascularization of most other solid tumors such as kidney cancer, breast cancer, and bowel cancer (Silver DA et al., (1997), Clin Cancer Res. 3: 81-5; Liu H et al. .,(1997),Cancer Res.57:3629-34;Chang SS et al.,(1999)Cancer Res.59:3192-8;Chang SS et al.,(1999)Clin Cancer Res 5:2674-81 ; Chang SS et al., (2001), Urology 57:801-5).
  • PSMA internalizes quickly and continuously, and delivers antibodies, antibody-drug conjugates, etc.
  • BAY2010112 a double antibody specific for the CD3 receptor of T cells and PSMA, is also under research in the treatment of prostate cancer.
  • a preclinical study of BAY2010112 conducted in a mouse model of prostate cancer found that the administration of this antibody can rapidly reduce tumor size and achieve complete remission (Friedrich M et al., (2012) Mol Cancer Ther. 11: 2664–73 ). The clinical trial of BAY2010112 is currently underway.
  • the conjugate of human IgG1PSMA antibody and the microtubule disrupting agent MMAE is being used in clinical trials for the treatment of taxane-refractory mCRPC.
  • phase 1 clinical trial about 50% of the treated patients had reduced PSA or decreased blood tumor cells (Petrylak DP et al., (2013) J Clin Oncol. 31:119).
  • phase 2 clinical trials 30% of patients had a PSA reduction of 30% or more, 14% of patients had a PSA reduction of 50% or more, 61% had stable disease, 13% had partial remission, and 26% had disease progression (Petrylak DP et al., (2015), J Clin Oncol. 33:144).
  • PSMA antibodies Although there have been advances in PSMA targets, PSMA antibodies have many deficiencies that cannot be ignored, such as the unknown epitope information that the antibody binds to, and the lack of cross-germline cross-reactivity data. Therefore, there is a need to develop more new PMSA antibodies with desired pharmaceutical properties.
  • the present application provides an isolated antigen binding protein comprising the amino acid sequence of SEQ ID NO: V H 15 shown in HCDR1, HCDR2 and the HCDR3; and which comprises the amino acid sequence of SEQ ID NO: V shown in FIG. 16 L of LCDR1, LCDR2 and LCDR3.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 1
  • the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 2
  • the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 3 Amino acid sequence.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 4
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 5
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 6 Amino acid sequence.
  • the V H includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 7; the H-FR2 Is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 8; the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR2 FR3 includes the amino acid sequence shown in SEQ ID NO: 9; the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 10.
  • the V L framework regions comprising L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 11; the L-FR2 Is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 12; the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR2 FR3 includes the amino acid sequence shown in SEQ ID NO: 13; the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 14.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  • the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 19.
  • 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 comprises the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein comprises an antibody heavy chain HC
  • the HC comprises the amino acid sequence shown in SEQ ID NO:21.
  • the isolated antigen binding protein comprises an antibody light chain LC
  • the LC comprises the amino acid sequence shown in SEQ ID NO:22.
  • the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , ScFv, di-scFv and/or dAb.
  • the isolated antigen binding protein has one or more of the following properties:
  • the present application provides an isolated antigen binding protein comprising the amino acid sequence of SEQ ID NO: V H 41 shown in HCDR1, HCDR2 and the HCDR3; and which comprises the amino acid sequence of SEQ ID NO: V 42 shown L of LCDR1, LCDR2 and LCDR3.
  • the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 28, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 29 Amino acid sequence.
  • the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 30
  • the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 31
  • the LCDR3 includes the amino acid sequence shown in SEQ ID NO: 32 Amino acid sequence.
  • the V H includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 includes the amino acid sequence shown in SEQ ID NO: 33; the H-FR2 Is located between the HCDR1 and the HCDR2, and the H-FR2 includes the amino acid sequence shown in SEQ ID NO: 34; the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR2 FR3 includes the amino acid sequence shown in SEQ ID NO: 35; the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 includes the amino acid sequence shown in SEQ ID NO: 36.
  • the V L framework regions comprising L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 includes the amino acid sequence shown in SEQ ID NO: 37; the L-FR2 Is located between the LCDR1 and the LCDR2, and the L-FR2 includes the amino acid sequence shown in SEQ ID NO: 38; the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR2 FR3 includes the amino acid sequence shown in SEQ ID NO: 39; the N-terminus of the L-FR4 is connected to the C terminal of the LCDR3, and the L-FR4 includes the amino acid sequence shown in SEQ ID NO: 40.
  • the isolated antigen binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from a human IgG heavy chain constant region.
  • the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 19.
  • 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 comprises the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein comprises an antibody heavy chain HC
  • the HC comprises the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein comprises an antibody light chain LC
  • the LC comprises the amino acid sequence shown in SEQ ID NO:44.
  • the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment includes Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , ScFv, di-scFv and/or dAb.
  • the isolated antigen binding protein has one or more of the following properties:
  • the present application provides a chimeric antigen receptor, which comprises the isolated antigen binding protein described in the present application.
  • this application provides an immunoconjugate comprising the isolated antigen binding protein described in this application.
  • the present application provides isolated one or more nucleic acid molecules, which encode the isolated antigen binding protein or the chimeric antigen receptor described in this application.
  • the present application provides a vector, which contains the nucleic acid molecule.
  • the present application provides a cell comprising the nucleic acid molecule or the vector.
  • the present application provides a pharmaceutical composition, which comprises the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the The carrier and/or the cell, and optionally a pharmaceutically acceptable adjuvant.
  • the present application provides a method for preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions that allow the expression of the isolated antigen binding protein.
  • the present application provides the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/ Or the use of the pharmaceutical composition in the preparation of medicaments, which are used to prevent, alleviate and/or treat tumors.
  • the tumor comprises prostate cancer.
  • the present application provides a method for detecting PSMA in a sample, the method comprising administering the isolated antigen binding protein.
  • the isolated antigen binding protein described in this application has at least one of the following beneficial effects:
  • the isolated antigen-binding protein described in this application can bind to the PSMA protein with a K D of 4 ⁇ 10 -10 M or lower, 1 ⁇ 10 -10 M or lower , wherein the K D value is determined by Octet determination.
  • the isolated antigen binding protein described in this application can specifically bind to the PSMA protein on the surface of HEK293T cells overexpressing human PSMA, CHO-K1 cells overexpressing monkey PSMA, or LNCAP cells.
  • the isolated antigen binding protein described in this application can be internalized into HEK293T cells or LNCAP cells overexpressing human PSMA.
  • the isolated antigen binding protein described in this application has ADCP activity on LNCAP cells.
  • the isolated antigen binding protein described in this application can be used to prepare drugs that can effectively prevent, alleviate and/or treat tumors.
  • the isolated antigen binding protein described in this application can be used to detect the presence or content of PSMA in a sample.
  • Figure 1A and Figure 1B show the binding of antibodies to human PSMA protein on the surface of HEK293T cells overexpressing human PSMA protein
  • Figure 2A and Figure 2B show the binding of antibodies to CHO-K1 cells overexpressing monkey PSMA protein
  • Figure 3 shows the binding of antibodies to the PSMA protein expressed on the surface of LNCAP cells
  • Figure 4 shows the binding of antibodies to human PSMA protein on the surface of HEK293T cells overexpressing human PSMA protein
  • Figure 5 shows the binding of antibodies to CHO-K1 cells overexpressing monkey PSMA protein
  • Figure 6 shows the binding of antibodies to the PSMA protein expressed on the surface of LNCAP cells
  • Figure 7 shows the survival rate of HEK293T/human PSMA cells under co-treatment with antibody and NMAF
  • Figure 8 shows the survival rate of LNCAP cells under co-treatment with antibody and NMAF
  • Figure 9 shows the survival rate of HEK293 hPSMA cells co-treated with antibody and NMAF
  • Figure 10 shows the survival rate of LNCAP cells under co-treatment with antibody and NMAF
  • Figure 11 shows the phagocytosis of LNCAP mediated by antibodies in macrophages
  • Figure 12 shows the phagocytosis of LNCAP mediated by antibodies in macrophages
  • Figures 13A-13D show the endocytosis effect of antibodies on target cells.
  • isolated generally refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • isolated antigen binding protein generally refers to a protein with antigen binding ability obtained from a natural state by artificial means.
  • the "isolated antigen-binding protein” may comprise an antigen-binding portion and optionally, a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that facilitates the antigen-binding portion to bind to the antigen.
  • the antigen binding protein may comprise, for example, an antibody-derived protein scaffold or alternative protein scaffolds or artificial scaffolds with grafted CDRs or CDR derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Protéins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004).
  • peptide antibody mimics (“PAMs”) and scaffolds based on antibody mimics using fibronectin components can be used as scaffolds.
  • KD is the dissociation rate constant (kdis, also known as “dissociation rate constant”).
  • the binding rate constant (kon), the dissociation rate constant (kdis), and the equilibrium dissociation constant (KD) can be used to express the binding affinity of an antigen-binding protein (for example, an antibody) to an antigen.
  • Methods for determining the rate constants of association and dissociation are well known in the art, including but not limited to Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , Co-immunoprecipitation (Co-IP) and protein chip technology. If measured under different conditions (such as salt concentration, pH), the measured affinity of a specific protein-protein interaction can be different.
  • EC50 or also known as half maximal effect concentration “EC 50”,, generally it refers to an antibody concentration causing 50% maximal effect.
  • PSMA generally refers to prostate-specific membrane antigen, also known as glutamate carboxypeptidase type II (GCPII) or NAAG peptidase.
  • GCPII glutamate carboxypeptidase type II
  • NAAG peptidase NAAG peptidase.
  • an antibody specific for human PSMA can cross-react with the PSMA protein of another species, such as monkey, under certain circumstances.
  • antibodies specific for human PSMA protein may be completely specific for human PSMA protein without cross-reacting with other species or other types of proteins, or may cross-react with PSMA proteins of some other species but not all other species .
  • human PSMA generally refers to a PSMA protein having a human amino acid sequence, for example, a PSMA protein having an amino acid sequence of Genbank accession number NP_004467.
  • monkey PSMA refers to a PSMA protein having a monkey amino acid sequence, and the monkey may be a rhesus monkey or a cynomolgus monkey.
  • monkey PSMA can refer to the PSMA protein of cynomolgus monkeys with the amino acid sequence of Genbank Accession No. XP_005579379.
  • the term "specific binding” or “specific” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can be in a heterogeneous population of molecules (including biomolecules).
  • the existence of the situation can determine the existence of the target.
  • an antibody that specifically binds a target (which may be an epitope) is an antibody that binds to the target with greater affinity, affinity, easier, and/or longer duration than it binds to other targets.
  • the extent to which the antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA).
  • the isolated antigen binding protein can bind to the PSMA protein with a dissociation constant (KD) of ⁇ 4 ⁇ 10 -10 M or lower.
  • KD dissociation constant
  • the antibody specifically binds to an epitope on a protein that is conserved among proteins of different species.
  • specific binding may include but does not require exclusive binding.
  • the term "tumor” generally refers to neoplasms or solid lesions formed by abnormal cell growth.
  • the tumor may be a solid tumor or hematoma.
  • the tumor may be a PSMA-positive tumor, wherein the PSMA-positive tumor may include prostate cancer.
  • variable domain generally refers to the amino terminal domain of an antibody heavy or light chain.
  • the variable domain of the heavy chain and light chain may be referred to "V H” and “V L” (or referred to as “VH” and “VL”). These domains are usually the most varied parts of the antibody (relative to other antibodies of the same type) and contain antigen binding sites.
  • variable generally refers to the fact that certain segments of variable domains differ greatly in sequence between antibodies.
  • the V domain mediates antigen binding and determines the specificity of a specific antibody to its specific antigen.
  • CDRs or HVRs hypervariable regions
  • the more highly conserved parts of the variable domains are called the framework regions (FR).
  • the variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a ⁇ -sheet configuration, connected by three CDRs, which form a circular connection, and in some cases form part of a ⁇ -sheet structure .
  • the CDRs in each chain are held in close proximity by the FR region, and the CDRs from the other chain together promote the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fourth Edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, for example, antibodies are involved in antibody-dependent cytotoxicity.
  • antibody generally refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide that includes an antigen binding site, whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , Mutant and transplanted antibodies.
  • antibody also includes antibody fragments, such as Fab, F(ab') 2 , Fv, scFv, Fd, dAbs and other antibody fragments that retain the antigen binding function (e.g., specifically bind to PSMA). Generally, such fragments should include an antigen binding domain.
  • the basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgM antibody is composed of 5 basic heterotetrameric units and another polypeptide called J chain, and contains 10 antigen binding sites, while IgA antibody includes 2-5 that can be combined with J chain to form a multivalent The basic 4-chain unit of the combination.
  • the 4-chain unit is generally about 150,000 Daltons.
  • Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds depending on the isotype of the H chain.
  • Each H and L chain also has regularly spaced intra-chain disulfide bridges.
  • Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the ⁇ and ⁇ chains, and four CH domains for the ⁇ and ⁇ isotypes.
  • Each L chain has a variable domain (VL) at the N-terminus and a constant domain at the other end.
  • VL corresponds to VH
  • CL corresponds to the first constant domain (CH1) of the heavy chain.
  • Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
  • VH and VL pair together to form a single antigen binding site.
  • immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the gamma and alpha classes are further divided into subclasses.
  • humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.
  • CDR generally refers to a region of an antibody variable domain, the sequence of which is highly variable and/or forms a structural definition loop.
  • an antibody includes six CDRs; three in VH (HCDR1, HCDR2, HCDR3), and three in VL (LCDR1, LCDR2, LCDR3).
  • HCDR3 and LCDR3 show most of the diversity of the six CDRs, and in particular HCDR3 is considered to play a unique role in conferring fine specificity to antibodies.
  • variable domains of the natural heavy and light chains each contain four FR regions, namely, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL (L-FR1, L-FR2, L-FR3, and L-FR4).
  • VL of the isolated antigen binding protein described in this application may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the VH of the isolated antigen binding protein described in this application may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the term "antigen-binding fragment” generally refers to one or more fragments that have the ability to specifically bind to an antigen (for example, PSMA protein).
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the term "monoclonal antibody” or “monoclonal antibody” or “monoclonal antibody composition” generally refers to an antibody molecule product of a single molecular composition.
  • the monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope.
  • human antibody generally refers to an antibody whose variable region framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, it is also derived from human germline immunoglobulin sequences.
  • the human antibody of the present application may comprise amino acid residues not encoded by human germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or somatic mutations in vivo.
  • the term “human antibody” does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the human framework sequence.
  • murine antibody generally refers to an antibody whose variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains a constant region, it is also derived from mouse germline immunoglobulin sequences.
  • the murine antibody of the present application may comprise amino acid residues not encoded by the mouse germline immunoglobulin sequence, for example, mutations introduced by random or point mutations in vitro or somatic mutations in vivo. However, the term “murine antibody” does not include antibodies in which CDR sequences derived from other mammalian species are inserted into the mouse framework sequence.
  • chimeric antibody generally refers to an antibody obtained by combining non-human genetic material with human genetic material.
  • a chimeric antibody refers to an antibody that combines the genetic material of one species with the genetic material of another species.
  • humanized antibody generally refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to increase its similarity to human naturally-occurring antibodies.
  • antibody that recognizes an antigen and “antibody specific for an antigen” are used interchangeably with the term “antibody that specifically binds to an antigen” herein.
  • the term “directly connected” is opposite to the term “indirectly connected”, and the term “directly connected” generally refers to a direct connection.
  • the direct connection may be a case where the substances are directly connected without spacers.
  • the spacer may be a linker.
  • the linker may be a peptide linker.
  • the term “indirectly connected” usually refers to the situation where substances are not directly connected.
  • the indirect connection may be a case of connection through a spacer.
  • the C-terminus of L-FR1 and the N-terminus of LCDR1 may be directly or indirectly connected.
  • antibody-dependent cell-mediated phagocytosis or "ADCP” are used interchangeably, and generally refer to the binding of the antibody to the corresponding antigen on the target cell, the Fc segment of the antibody and the effector cell (such as The Fc receptor on phagocytes binds, thereby inducing the effect of effector cells to phagocytize target cells.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed.
  • the vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage And animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papilloma viruses such as papilloma viruses
  • papillary polyoma vacuoles Virus such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the carrier may
  • the term "cell” generally refers to a single cell, cell line, or cell culture that can be or has been a recipient of a subject's plasmid or vector, which includes the nucleic acid molecule of the present invention or the nucleic acid molecule of the present invention. a.
  • a cell can include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the offspring may not necessarily be exactly the same as the original parent cell (in the form of the total DNA complement or in the genome).
  • the cells may include cells transfected in vitro with the vectors described in this application.
  • the cell may be a bacterial cell (for example, Escherichia coli), a yeast cell or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells , NS0 cells.
  • the cell is a mammalian cell.
  • the mammalian cell is a HEK293 cell.
  • the term "pharmaceutical composition” generally refers to a composition suitable for administration to a patient, such as a human patient.
  • the pharmaceutical composition described in this application may comprise 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 adjuvant.
  • the pharmaceutical composition may also contain one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and/or preservatives. Preparations.
  • the acceptable ingredients of the composition are, for example, non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • pharmaceutically acceptable adjuvant generally refers to any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with the administration of drugs, and are generally safe and non-toxic. , And neither biologically nor otherwise undesirable.
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • 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 isolated antigen binding protein comprising the amino acid sequence as SEQ ID NO: V H 15 shown CDRs of at least one; and which comprises the amino acid sequence SEQ ID NO: 16 as shown in V At least one CDR in L.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V H 15 shown in HCDR1.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V H 15 shown in HCDR2.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V H 15 shown in HCDR3.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V L 16 shown in LCDR1.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V L 16 shown in LCDR2.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V L 16 shown in LCDR3.
  • the isolated antigen binding protein may have one or more of the following properties:
  • the isolated antigen binding protein can bind to the PSMA protein with a K D of 1 ⁇ 10 -10 M or lower , wherein the K D value is determined by Octet.
  • the K D value of the isolated antigen binding protein described in the present application for binding to the human-derived PSMA protein can be ⁇ 1 ⁇ 10 -10 M, ⁇ 5 ⁇ 10 -11 M, ⁇ 1 ⁇ 10 -11 M, ⁇ 1 ⁇ 10 -12 M, ⁇ 9 ⁇ 10 -13 M, ⁇ 8 ⁇ 10 - 13 M, ⁇ 7 ⁇ 10 -13 M, ⁇ 6 ⁇ 10 -13 M, ⁇ 5 ⁇ 10 -13 M, ⁇ 4 ⁇ 10 -13 M, ⁇ 3 ⁇ 10 -13 M, ⁇ 2 ⁇ 10 -13 M, or ⁇ 1 ⁇ 10 -13 M.
  • the isolated antigen binding protein described herein is derived from monkey PSMA-binding protein may ⁇ 1 ⁇ 10 - 10 M, ⁇ 5 ⁇ 10 -11 M, ⁇ 1 ⁇ 10 -11 M, ⁇ 1 ⁇ 10 -12 M, ⁇ 9 ⁇ 10 -13 M, ⁇ 8 ⁇ 10 -13 M, ⁇ 7 ⁇ 10 -13 M, ⁇ 6 ⁇ 10 -13 M, ⁇ 5 ⁇ 10 -13 M, ⁇ 4 ⁇ 10 -13 M, ⁇ 3 ⁇ 10 -13 M, ⁇ 2 ⁇ 10 -13 M, or ⁇ 1 ⁇ 10 -13 M.
  • the K D value can also be determined by ELISA, competitive ELISA, BIACORE or KINEXA.
  • the isolated antigen binding protein can specifically bind to the PSMA protein on the surface of HEK293T cells, CHO-K1 cells or LNCAP cells, and the specific binding can be determined by FACS.
  • the EC50 in the FACS assay can be used to reflect the specific binding of the antigen-binding protein described in this application to the PSMA protein on the surface of HEK293T cells, CHO-K1 cells or LNCAP cells. For example, the lower the EC50, the more specific binding. good.
  • the EC50 value of the isolated antigen binding protein that binds to the PSMA protein on the surface of HEK293T cells in the FACS measurement can be 0.01 ⁇ g/ml ⁇ 0.10 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.50 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.70 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.90 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 1.00 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 1.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 1.35 ⁇ g/ml or 0.01 ⁇ g/ml ⁇ 1.40 ⁇ g/ml.
  • the EC50 value of the isolated antigen binding protein that binds to the PSMA protein on the surface of CHO-K1 cells in the FACS measurement may be 0.01 ⁇ g/ml ⁇ 0.10 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.20 ⁇ g/ml, 0.01 ⁇ g /ml ⁇ 0.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.40 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.50 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.60 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.70 ⁇ g/ml, 0.01 ⁇ g /ml ⁇ 0.80 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.90 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.95 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 1.00 ⁇ g/ml or 0.01 ⁇ g/ml ⁇ 1.10 ⁇ g/ml.
  • the EC50 value of the isolated antigen binding protein bound to the PSMA protein on the LNCAP cell surface in the FACS measurement may be 0.01 ⁇ g/ml ⁇ 0.10 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.20 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.40 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.50 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.60 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.70 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.80 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.90 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.95 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 1.00 ⁇ g/ml or 0.01 ⁇ g/ml ⁇ 1.10 ⁇ g/ml.
  • the PSMA protein may comprise human PSMA protein or monkey PSMA protein.
  • the PSMA protein may comprise a PSMA protein having an amino acid sequence with Genbank accession number NP_004467.
  • the PSMA protein may comprise a PSMA protein having an amino acid sequence with Genbank accession number XP_014970879.
  • the PSMA protein may comprise a PSMA protein having an amino acid sequence with Genbank accession number XP_005579379.
  • the isolated antigen binding protein can be internalized into HEK293T cells or LNCAP cells overexpressing human PSMA.
  • the isolated antigen binding protein described in this application can mediate the internalization of the PSMA protein expressed on the cell surface by binding to the extracellular tail of PSMA.
  • the endocytosis rate of the isolated antigen binding protein described in this application in LNCAP cells may be ⁇ 2 hours, ⁇ 1.5 hours, ⁇ 1 hour, or ⁇ 0.5 hours.
  • the isolated antigen binding protein can show ADCP activity to PSMA-positive cells (also can be represented by "PSMA+ cells").
  • PSMA-positive cells may be LNCAP cells.
  • the isolated antigen binding protein can inhibit the growth of tumor cells by inducing antibody-dependent cell-mediated phagocytosis (ADCP).
  • the tumor cells may be PSMA-positive cells, for example, LNCAP cells.
  • the type of the isolated antigen binding protein is the type of the isolated antigen binding protein
  • the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.
  • the isolated antigen binding protein described in this application may include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, murine antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, bispecific antibodies, Antibodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab') 2 fragments and camelized single domain antibodies.
  • the antibody may be a humanized antibody.
  • the isolated antigen-binding protein described in the present application may be immunospecifically binding to a related antigen (such as human PSMA) and comprise a framework (FR) region that basically has the amino acid sequence of a human antibody and basically has The antibody or its variants, derivatives, analogs or fragments of the complementarity determining region (CDR) of the amino acid sequence of a non-human antibody.
  • CDR complementarity determining region
  • the humanized antibody may basically comprise all at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to non-human
  • the CDR regions of immunoglobulins (ie, antibodies) and all or substantially all of the framework regions are framework regions with consensus sequences of human immunoglobulins.
  • a humanized antibody also contains at least a portion of an immunoglobulin constant region (e.g., Fc), usually that of a human immunoglobulin.
  • a humanized antibody contains at least the variable domains of a light chain and a heavy chain.
  • the antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • humanized antibodies contain only humanized light chains. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or a humanized heavy chain.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the CDR of an antibody also known as the complementarity determining region, is a part of the variable region.
  • the amino acid residues in this region are in contact with the antigen or epitope.
  • Antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia and so on. These coding systems are known in the field. For details, please refer to http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in CDR regions.
  • the CDR of the isolated antigen binding protein described in this application can be determined using Kabat.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO:1.
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO:2.
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 1
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • HCDR3 may include the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence shown.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO:4.
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO:6.
  • LCDR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 4
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 6. The amino acid sequence shown.
  • the HCDR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 1
  • HCDR2 may include the amino acid sequence shown in SEQ ID NO: 2
  • HCDR3 may include SEQ ID NO: 3.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 6.
  • the VH of the isolated antigen binding protein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO:7.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO: 8.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include the amino acid sequence shown in SEQ ID NO:9.
  • the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 10.
  • H-FR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 7
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 8
  • H-FR3 may include The amino acid sequence shown in SEQ ID NO: 9
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 10.
  • the VL of the isolated antigen binding protein may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 11.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO: 12.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 13.
  • the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • L-FR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 11
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 12
  • L-FR3 may include The amino acid sequence shown in SEQ ID NO: 13
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • H-FR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 7
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 8
  • H-FR3 may Including the amino acid sequence shown in SEQ ID NO: 9
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 10
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 11
  • L-FR2 may include The amino acid sequence shown in SEQ ID NO: 12
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 13
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • Isolated antigen binding protein described herein may comprise an antibody light chain variable region and a V H antibody heavy chain variable regions V L.
  • the V H may comprise SEQ ID NO: 15 amino acid sequence shown
  • a V L may comprise SEQ ID NO: 16 amino acid sequence.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.
  • the antibody heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 19.
  • the isolated antigen binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Ig ⁇ constant region.
  • the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein may include an antibody heavy chain HC, and the HC may include the amino acid sequence shown in SEQ ID NO:21.
  • the isolated antigen binding protein may include an antibody light chain LC, and the LC may include the amino acid sequence shown in SEQ ID NO:22.
  • the isolated antigen binding protein described in this application may comprise an antibody heavy chain and an antibody light chain.
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 21, and the light chain may include the amino acid sequence shown in SEQ ID NO: 22.
  • the heavy chain of the isolated antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 21, and the light chain may include the amino acid sequence shown in SEQ ID NO: 22.
  • the HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 1
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 2
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 3.
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 4
  • LCDR2 may include the amino acid sequence shown in SEQ ID NO: 5
  • LCDR3 may include the amino acid sequence shown in SEQ ID NO: 6.
  • H-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 7
  • H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 8
  • H-FR3 may comprise SEQ ID NO.
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 10
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 11
  • L-FR2 may include SEQ ID NO: In the amino acid sequence shown in 12
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 13
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 14.
  • the V H may comprise SEQ ID NO: 15 amino acid sequence, and may include the V L SEQ ID NO: 16 amino acid sequence.
  • the isolated antigen binding protein may be PR001104.
  • the isolated antigen binding protein described in the present application may include heavy chain and/or light chain sequences that have one or more conservative sequence modifications with the PR001104 antibody.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or change the binding properties of an antibody.
  • conservative modifications include amino acid substitutions, additions, and deletions.
  • Modifications can be introduced into the isolated antigen binding protein described in this application by standard techniques known in the art, such as point mutations and PCR-mediated mutations.
  • Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art.
  • amino acid residue groups include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and side chains without electrical polarity ( For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine) Acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains ( For example, tyrosine, phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • one or more amino acid residues in the CDR region of the isolated antigen binding protein described in this application can be replaced with other amino acid residues from the same side chain group. Those skilled in the art know that some conservative sequence modifications will not make the antigen binding properties disappear.
  • the application provides an isolated antigen binding protein, which comprises at least one CDR in the VH shown in the amino acid sequence of SEQ ID NO:41; and it comprises the VH in the amino acid sequence shown in SEQ ID NO:42. At least one CDR in L.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V H 41 shown in HCDR1.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V H 41 shown in HCDR2.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V H 41 shown in HCDR3.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V L 42 shown in LCDR1.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V L 42 shown in LCDR2.
  • the isolated antigen binding protein may comprise the amino acid sequence SEQ ID NO: V L 42 shown in LCDR3.
  • the isolated antigen binding protein may have one or more of the following properties:
  • the isolated antigen binding protein can bind to the PSMA protein with a K D of 4 ⁇ 10 -10 M or lower , wherein the K D value is determined by Octet.
  • the K D value of the isolated antigen binding protein described in the present application for binding to the human-derived PSMA protein may be ⁇ 4 ⁇ 10 -10 M, ⁇ 3.9 ⁇ 10 -10 M, ⁇ 3.8 ⁇ 10 -10 M, ⁇ 3.76 ⁇ 10 -10 M, ⁇ 3.7 ⁇ 10 -10 M, ⁇ 3.6 ⁇ 10 -10 M, ⁇ 3.5 ⁇ 10 -10 M, ⁇ 3.4 ⁇ 10 -10 M, ⁇ 3.3 ⁇ 10 -10 M, ⁇ 3.2 ⁇ 10 -10 M, ⁇ 3.1 ⁇ 10 -10 M, ⁇ 3 ⁇ 10 -10 M, ⁇ 2 ⁇ 10 -10 M, ⁇ 1 ⁇ 10 -10 M.
  • the K D value of the isolated antigen binding protein described in this application in binding to the mouse-derived PSMA protein may be ⁇ 4 ⁇ 10 -10 M, ⁇ 3.9 ⁇ 10 -10 M, ⁇ 3.8 ⁇ 10 -10 M, ⁇ 3.76 ⁇ 10 -10 M, ⁇ 3.7 ⁇ 10 -10 M, ⁇ 3.6 ⁇ 10 - 10 M, ⁇ 3.5 ⁇ 10 -10 M, ⁇ 3.4 ⁇ 10 -10 M, ⁇ 3.3 ⁇ 10 -10 M, ⁇ 3.2 ⁇ 10 -10 M, ⁇ 3.1 ⁇ 10 -10 M, ⁇ 3 ⁇ 10 -10 M, ⁇ 2 ⁇ 10 - 10 M, ⁇ 1 ⁇ 10 -10 M.
  • the K D value of the isolated antigen binding protein described in the present application for binding to the monkey-derived PSMA protein may be ⁇ 4 ⁇ 10 -10 M, ⁇ 3.9 ⁇ 10 -10 M, ⁇ 3.8 ⁇ 10 -10 M, ⁇ 3.76 ⁇ 10 -10 M, ⁇ 3.7 ⁇ 10 -10 M, ⁇ 3.6 ⁇ 10 -10 M, ⁇ 3.5 ⁇ 10 -10 M, ⁇ 3.4 ⁇ 10 -10 M, ⁇ 3.3 ⁇ 10 -10 M, ⁇ 3.2 ⁇ 10 -10 M, ⁇ 3.1 ⁇ 10 -10 M, ⁇ 3 ⁇ 10 -10 M, ⁇ 2 ⁇ 10 -10 M, ⁇ 1 ⁇ 10 -10 M.
  • the K D value can also be determined by ELISA, competitive ELISA, BIACORE or KINEXA.
  • the isolated antigen binding protein can specifically bind to the PSMA protein on the surface of HEK293 cells, CHO-K1 cells or LNCAP cells, and the specific binding can be determined by FACS.
  • the EC50 in the FACS measurement can be used to reflect the specific binding of the antigen-binding protein described in this application to the PSMA protein on the surface of HEK293 cells, CHO-K1 cells or LNCAP cells. For example, the lower the EC50, the more specific binding. good.
  • the EC50 value of the isolated antigen binding protein that binds to the PSMA protein on the surface of HEK293 cells in the FACS measurement can be 0.01 ⁇ g/ml ⁇ 0.10 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.15 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.20 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.25 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.35 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.40 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.45 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.50 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.55 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.60 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.65 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.67 ⁇ g/ml, 0.01 ⁇ g//
  • the EC50 value of the isolated antigen binding protein bound to the PSMA protein on the surface of the CHO-K1 cell in the FACS measurement may be 0.01 ⁇ g/ml ⁇ 0.10 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.15 ⁇ g/ml, 0.01 ⁇ g /ml ⁇ 0.16 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.20 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.25 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.35 ⁇ g/ml, 0.01 ⁇ g /ml ⁇ 0.40 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.45 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.50 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.55 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.60 ⁇ g/ml, 0.01 ⁇ g /ml ⁇ 0.65 ⁇ g/ml or 0.01 ⁇
  • the EC50 value of the isolated antigen binding protein bound to the PSMA protein on the surface of the LNCAP cell in the FACS assay may be 0.01 ⁇ g/ml ⁇ 0.09 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.10 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.11 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.12 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.15 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.20 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.25 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.30 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.35 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.40 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.45 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.50 ⁇ g/ml, 0.01 ⁇ g/ml ⁇ 0.55 ⁇ g/ml, 0.01 ⁇ g/ml
  • the PSMA protein may comprise human PSMA protein or monkey PSMA protein.
  • the PSMA protein may comprise a PSMA protein having an amino acid sequence with Genbank accession number NP_004467.1.
  • the PSMA protein may comprise a PSMA protein having an amino acid sequence with Genbank accession number XP_014970879.1.
  • the PSMA protein may include a PSMA protein having an amino acid sequence with Genbank accession number XP_005579379.1.
  • the isolated antigen binding protein can be internalized into HEK293 cells or LNCAP cells expressing human PSMA.
  • the isolated antigen binding protein described in this application can mediate the internalization of the PSMA protein expressed on the cell surface by binding to the extracellular tail of PSMA.
  • the isolated antigen binding protein can show ADCP activity to PSMA-positive cells (also can be represented by "PSMA+ cells").
  • PSMA-positive cells may be LNCAP cells.
  • the isolated antigen binding protein can inhibit the growth of tumor cells by inducing antibody-dependent cell-mediated phagocytosis (ADCP).
  • the tumor cells may be PSMA-positive cells, for example, LNCAP cells.
  • the type of the isolated antigen binding protein is the type of the isolated antigen binding protein
  • the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.
  • the isolated antigen binding protein described in this application may include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, murine antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, bispecific antibodies, Antibodies, triabodies, tetrabodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab') 2 fragments and camelized single domain antibodies.
  • the antibody may be a humanized antibody.
  • the isolated antigen-binding protein described in the present application may be immunospecifically binding to a related antigen (such as human PSMA) and comprise a framework (FR) region that basically has the amino acid sequence of a human antibody and basically has The antibody or its variants, derivatives, analogs or fragments of the complementarity determining region (CDR) of the amino acid sequence of a non-human antibody.
  • CDR complementarity determining region
  • the humanized antibody may basically comprise all at least one and usually two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to non-human
  • the CDR regions of immunoglobulins (ie, antibodies) and all or substantially all of the framework regions are framework regions with consensus sequences of human immunoglobulins.
  • a humanized antibody also contains at least a portion of an immunoglobulin constant region (e.g., Fc), usually that of a human immunoglobulin.
  • a humanized antibody contains at least the variable domains of a light chain and a heavy chain.
  • the antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • humanized antibodies contain only humanized light chains. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody only contains a humanized variable domain of a light chain and/or a humanized heavy chain.
  • the antigen-binding fragment may include Fab, Fab', F(ab) 2 , Fv fragment, F(ab') 2 , scFv, di-scFv and/or dAb.
  • the CDR of an antibody also known as the complementarity determining region, is a part of the variable region.
  • the amino acid residues in this region are in contact with the antigen or epitope.
  • Antibody CDR can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia and so on. These coding systems are known in the field. For details, please refer to http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR regions according to the sequence and structure of the antibody. Using different coding systems, there may be differences in CDR regions.
  • the CDR of the isolated antigen binding protein described in this application can be determined using Kabat.
  • the HCDR1 may include the amino acid sequence shown in SEQ ID NO:27.
  • the HCDR2 may include the amino acid sequence shown in SEQ ID NO:28.
  • the HCDR3 may include the amino acid sequence shown in SEQ ID NO:29.
  • the HCDR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 27, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 may include the amino acid sequence shown in SEQ ID NO: 29. The amino acid sequence shown.
  • the LCDR1 may include the amino acid sequence shown in SEQ ID NO:30.
  • the LCDR2 may include the amino acid sequence shown in SEQ ID NO:31.
  • the LCDR3 may include the amino acid sequence shown in SEQ ID NO:32.
  • LCDR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 30
  • 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: 32. The amino acid sequence shown.
  • HCDR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 27, HCDR2 may include the amino acid sequence shown in SEQ ID NO: 28, and HCDR3 may include SEQ ID NO: 29 LCDR1 may include the amino acid sequence shown in SEQ ID NO: 30, LCDR2 may include the amino acid sequence shown in SEQ ID NO: 31, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 32.
  • the VH of the isolated antigen binding protein may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.
  • the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the H-FR1 may include the amino acid sequence shown in SEQ ID NO: 33.
  • the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 may include the amino acid sequence shown in SEQ ID NO:34.
  • the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 may include the amino acid sequence shown in SEQ ID NO:35.
  • the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 may include the amino acid sequence shown in SEQ ID NO: 36.
  • the H-FR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 33
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 34
  • H-FR3 may include The amino acid sequence shown in SEQ ID NO: 35
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 36.
  • the VL of the isolated antigen binding protein may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.
  • the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may include the amino acid sequence shown in SEQ ID NO: 37.
  • the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may include the amino acid sequence shown in SEQ ID NO:38.
  • the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may include the amino acid sequence shown in SEQ ID NO: 39.
  • the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may include the amino acid sequence shown in SEQ ID NO:40.
  • L-FR1 of the isolated antigen binding protein described in this application may include the amino acid sequence shown in SEQ ID NO: 37
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO: 38
  • L-FR3 may include The amino acid sequence shown in SEQ ID NO: 39
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 40.
  • the H-FR1 of the isolated antigen binding protein described in the present application may include the amino acid sequence shown in SEQ ID NO: 33
  • H-FR2 may include the amino acid sequence shown in SEQ ID NO: 34
  • H-FR3 may Containing the amino acid sequence shown in SEQ ID NO: 35
  • H-FR4 can include the amino acid sequence shown in SEQ ID NO: 36
  • L-FR1 can include the amino acid sequence shown in SEQ ID NO: 37
  • L-FR2 can include The amino acid sequence shown in SEQ ID NO: 38
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 39
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 40.
  • Isolated antigen binding protein described herein may comprise an antibody light chain variable region and a V H antibody heavy chain variable regions V L.
  • the V H may comprise SEQ ID NO: 41 amino acid sequence shown
  • a V L may comprise SEQ ID NO: 42 amino acid sequence.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG heavy chain constant region.
  • the isolated antigen binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from a human IgG1 heavy chain constant region.
  • the antibody heavy chain constant region may include the amino acid sequence shown in SEQ ID NO: 19.
  • the isolated antigen binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Ig ⁇ constant region.
  • the antibody light chain constant region may include the amino acid sequence shown in SEQ ID NO:20.
  • the isolated antigen binding protein may include an antibody heavy chain HC, and the HC may include the amino acid sequence shown in SEQ ID NO: 43.
  • the isolated antigen binding protein may include an antibody light chain LC, and the LC may include the amino acid sequence shown in SEQ ID NO:44.
  • the isolated antigen binding protein described in this application may comprise an antibody heavy chain and an antibody light chain.
  • the heavy chain may include the amino acid sequence shown in SEQ ID NO: 43
  • the light chain may include the amino acid sequence shown in SEQ ID NO: 44.
  • the heavy chain of the isolated antigen binding protein may include the amino acid sequence shown in SEQ ID NO: 43, and the light chain may include the amino acid sequence shown in SEQ ID NO: 44.
  • HCDR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 27
  • HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 28
  • HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 29
  • LCDR1 may include the amino acid sequence shown in SEQ ID NO: 30
  • 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: 32.
  • H-FR1 of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 33
  • H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 34
  • H-FR3 may comprise SEQ ID NO
  • H-FR4 may include the amino acid sequence shown in SEQ ID NO: 36
  • L-FR1 may include the amino acid sequence shown in SEQ ID NO: 37
  • L-FR2 may include the amino acid sequence shown in SEQ ID NO:
  • L-FR3 may include the amino acid sequence shown in SEQ ID NO: 39
  • L-FR4 may include the amino acid sequence shown in SEQ ID NO: 40
  • the V H may comprise SEQ ID NO: 41 amino acid sequence, and may include the V L SEQ ID NO: 42 amino acid sequence.
  • the isolated antigen binding protein may be PR001331.
  • the isolated antigen binding protein described in the present application may include heavy chain and/or light chain sequences that have one or more conservative sequence modifications with the PR001331 antibody.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or change the binding properties of an antibody.
  • conservative modifications include amino acid substitutions, additions, and deletions.
  • Modifications can be introduced into the isolated antigen binding protein described in this application by standard techniques known in the art, such as point mutations and PCR-mediated mutations.
  • Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art.
  • amino acid residue groups include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and side chains without electrical polarity ( For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine) Acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains ( For example, tyrosine, phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • one or more amino acid residues in the CDR region of the isolated antigen binding protein described in this application can be replaced with other amino acid residues from the same side chain group. Those skilled in the art know that some conservative sequence modifications will not make the antigen binding properties disappear.
  • Chimeric antigen receptor, immunoconjugate, nucleic acid molecule, carrier, cell and pharmaceutical composition Chimeric antigen receptor, immunoconjugate, nucleic acid molecule, carrier, cell and pharmaceutical composition
  • the present application provides a chimeric antigen receptor, which may comprise the isolated antigen binding protein described in the present application.
  • the isolated antigen binding protein described in this application may be included in a PSMA-specific CAR in the form of scFv.
  • the CAR containing the isolated antigen binding protein described in this application may be contained in immune cells such as T cells and NK cells.
  • this application also provides an immunoconjugate, which may comprise the isolated antigen binding protein described in this application.
  • the isolated antigen binding protein described in this application can be cross-linked with a therapeutic agent to form the immunoconjugate.
  • a therapeutic agent for example, antibody-drug conjugates (ADC).
  • ADC antibody-drug conjugates
  • Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerases Inhibitors of I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents, such as SN-38.
  • the antibody and the therapeutic agent can be cross-linked by a linker, which can be cleaved, such as a peptide linker, a disulfide linker, or a hydrazone linker.
  • the linker may be a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val -Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu.
  • ADC can be like US Patent 7,087,600; 6,989,452; and 7,129,261; PCT Publication WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; US Patent Publication 20060024317; Prepare as described in 20060004081; and 20060247295.
  • the isolated antigen binding protein described in this application can also be fused with other functional molecules (for example, antibodies or receptor ligands) to form bispecific molecules.
  • the bispecific molecule can specifically bind to at least two different binding sites or targeting molecules.
  • the bispecific molecule can be prepared by genetic modification, somatic cell hybridization or chemical methods. For details, see, for example, Kufer et al, cited supra; Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644(1998); and van Spriel et al., Immunology Today, 21(8), 391-397( 2000).
  • this application also provides isolated one or more nucleic acid molecules, which can encode the isolated antigen binding protein described in this application or the chimeric antigen receptor described in this application.
  • the isolated one or more nucleic acid molecules described in this application can be nucleotides, deoxyribonucleotides or ribonucleotides in isolated form of any length, or analogs isolated from their natural environment or artificially synthesized , But can encode the isolated antigen binding protein described in this application or the chimeric antigen receptor described in this application.
  • the present application also provides a vector, which may contain the nucleic acid molecule described in the present application.
  • the vector can be transformed, transduced or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.
  • the vector may include: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage and Animal viruses and so on.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • the vector may contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication.
  • the carrier may also include components that assist it to enter cells, such as viral particles, liposomes or protein coats, but not only these substances.
  • this application also provides a cell, which may contain the nucleic acid molecule described in this application or the vector described in this application.
  • the cell may include the progeny of a single cell. Due to natural, accidental or deliberate mutations, the offspring may not necessarily be exactly the same as the original parent cell (in the form of the total DNA complement or in the genome).
  • the cells may also include cells transfected in vitro with the vectors of the present invention.
  • the cell may be a bacterial cell (e.g., E.
  • the cell may be a mammalian cell.
  • the mammalian cell may be a HEK293 cell.
  • this application also provides a pharmaceutical composition, which may comprise the isolated antigen binding protein described in this application, the chimeric antigen receptor described in this application, the immunoconjugate described in this application, and the immunoconjugate described in this application.
  • a pharmaceutical composition which may comprise the isolated antigen binding protein described in this application, the chimeric antigen receptor described in this application, the immunoconjugate described in this application, and the immunoconjugate described in this application.
  • the pharmaceutical composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/ Or a suitable formulation of preservative.
  • the acceptable ingredients of the composition are, for example, non-toxic to the recipient at the dosage and concentration used.
  • the pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with drug administration, and are generally safe and non-toxic. , And neither biologically nor otherwise undesirable.
  • the pharmaceutical composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal, and/or intranasal administration or direct injection into tissues.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015/036583.
  • the dosage regimen of the pharmaceutical composition may be the administration of a rapid infusion agent, multiple divided doses may be administered over time, or the dosage may be reduced or increased in proportion to the criticality of the treatment situation.
  • the treatment regimen may be once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the dosing regimen includes intravenous administration, 1 mg/kg body weight or 3 mg/kg body weight, and the antibody is administered in one of the following dosing schedules: (i) six times every four weeks, then Once every three months; (ii) once every three weeks; (iii) 3 mg/kg body weight once, and then 1 mg/kg body weight once every three weeks.
  • the dosage is adjusted to achieve a blood concentration of about 1-1000 ⁇ g/ml, for example, it may be about 25-300 ⁇ g/ml.
  • this application also provides a method for preparing the isolated antigen binding protein described in this application, and the method may include culturing the isolated antigen binding protein described in this application under conditions Cell.
  • the application also provides the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell and /Or the use of the pharmaceutical composition in the preparation of a medicament for the prevention, alleviation and/or treatment of tumors.
  • the present application also provides a method for preventing, alleviating or treating tumors.
  • the method may include administering the isolated antigen binding protein, the chimeric antigen receptor, and the antigen binding protein of the present application to a subject in need.
  • the administration can be carried out in different ways, such as intravenous, intratumor, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the isolated antigen binding protein, the chimeric antigen receptor, the immunoconjugate, the nucleic acid molecule, the vector, the cell and/or the cell can be used to prevent, alleviate or treat tumors.
  • the tumor may be a solid tumor or hematoma.
  • the tumor may include a PSMA-positive tumor
  • the PSMA-positive tumor may include prostate cancer
  • the subject may include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the isolated antigen binding protein can be administered with one or more other antibodies in order to effectively inhibit tumor growth in the subject.
  • the isolated antigen binding protein and one or more other antibodies such as LAG-3 antibodies, PD-1 antibodies, and/or CTLA-4 antibodies, can be administered to the subject.
  • the isolated antigen binding protein can be administered together with a chemotherapeutic agent, which can be a cytotoxic agent, for example, SN-38, epirubicin, oxaliplatin, and/or 5- FU.
  • a chemotherapeutic agent which can be a cytotoxic agent, for example, SN-38, epirubicin, oxaliplatin, and/or 5- FU.
  • this application also provides a method for detecting PSMA in a sample, the method comprising administering the isolated antigen binding protein described in this application.
  • the administration can be carried out in different ways, such as intravenous, intratumor, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • Cells after 48 hours of infection are passaged at a ratio of 1:10 with a screening antibody containing 8 ⁇ g/ml F2K medium with 10% (w/v) fetal bovine serum for about 1 week until the uninfected control group CHO-K1 was killed by screening resistance, while the infected group’s CHO-K1 cells still survived.
  • the lentivirus After digestion of CHO-K1 cells in the infection group, limited dilution was carried out in a 96-well plate to 0.5 cells per well, and the medium containing screening resistance was added to culture for about 5 days. Then, observe whether they are monoclonal and mark them. Continue to 37 degrees carbon dioxide Culture in the incubator for about 1 week to 50% full, and the single clones are expanded to 6 wells.
  • Anti-PSMA Tab antibody (recombinant human IgG1 antibody prepared by the VH and VL sequences of Pasotuxizumab) is used to detect its expression by flow cytometry.
  • the clone CHO-K1/cyno PSMA B6 with high growth rate and high fluorescence intensity detected by FACS will continue to expand and be cultured and stored in liquid nitrogen.
  • H2L2 transgenic mice (WO2010/070263Al) can produce immune responses and antibody titers comparable to wild-type mice (such as BALB/C).
  • the immunogen was recombinant PSMA ECD-his (Sino-Biological, cat#15877-H07H) to immunize 6-8 week-old Harbour H2L2 transgenic mice and bred them under specific pathogen-free conditions (SPF).
  • PSMA ECD-his Session-Betadium-Ag
  • SPF pathogen-free conditions
  • each mouse was injected with 50 ⁇ g immunogen protein and 0.22ml complete Freund's adjuvant (CFA, Sigma, cat#F5881) in the abdominal cavity and axillary lymph nodes and inguinal lymph nodes respectively.
  • CFA complete Freund's adjuvant
  • the blood of the mice was collected, and the blood was diluted 10-fold to obtain 6 concentrations (1:100, 1:1000, 1:10000, 1:100,000, 1 :1000000), the ELISA plate coated with human PSMA ECD protein (Sino-Biological, cat#15877-H07H) was tested by enzyme-linked immunosorbent assay (ELISA) to determine the titer of anti-human PMSA in the blood of mice.
  • ELISA enzyme-linked immunosorbent assay
  • Flow cytometry was used to detect the specific reactivity of three concentrations of mouse blood (1:100, 1:1000, 1:10000) to LNCAP cells (Cobioer, Nanjing, China) that highly express human PSMA.
  • the blank control group (PB) is the serum of the mice before immunization.
  • mice with a specific immune response against human PSMA for fusion inject 100 ⁇ g of purified PSMA ECD-his into the abdominal cavity for booster immunization. Three days later, the mice were sacrificed, and their spleen cells and lymph node cells were collected. NH 4 OH was added to the spleen cells and lymph node samples at a final concentration of 1% (w/w) to dissolve the red blood cells in the samples. The samples were centrifuged at a speed of 1000 rpm and washed three times with DMEM medium to determine the survival rate and number of cells.
  • Mouse myeloma cells sp2/0 (ATCC, cat#CRL-1581) were washed twice with serum-free DMEM to determine the survival rate and number of cells. Then live spleen cells and mouse myeloma cells sp2/0 (ATCC, cat#CRL-1581) were fused with a high-efficiency electrofusion method at a ratio of 5:1.
  • FBS ultra-low IgG, Fetal Bovine Serum, cat#16250086, Life Technologies
  • FBS ultra-low IgG, Fetal Bovine Serum, cat#16250086, Life Technologies
  • 1X hypoxanthine, aminopterin, and thymidine 50X HAT supplement , cat # 21060017, life technologies
  • Hybridoma-SFM cat # 12045084 , Life Technologies
  • the selected positive clones (OD450>2) are then screened by flow cytometry for clones that specifically bind to HEK293T/human PSMA cells (KYinno, Beijing, China) and CHO-K1/cyno PSMA B6 cells. After scoring the fluorescence intensity, the 25 parent hybridoma clones with the strongest fluorescence intensity were selected and subcloned by the limiting dilution method. PSMA on the cell binds the strongest sub-monoclonal. Ready-SET-GO by mouse Ig typing! ELISA (Life technologies, cat#88-50640-88) confirmed that the sub-monoclonal IgG subtype will be sequenced and analyzed.
  • Example 2 The monoclonal PSMA antibody screened in Example 1 was sequenced, and the amino acid sequence is shown in Table 1 or Table 2.
  • the heavy chain variable region sequence of the monoclonal PSMA antibody of Table 1 was subcloned into the pTT5 expression vector containing the signal peptide and the human heavy chain IgG1 constant region (SEQ ID NO: 19).
  • the light chain variable region sequence of the monoclonal PSMA antibody was subcloned into an expression vector containing the signal peptide and the human antibody light chain kappa constant region (SEQ ID NO: 20).
  • Recombinant plasmids were confirmed by sequencing and used a large extraction kit (Macherey-Nagel, Xtra Midi) extract plasmids to improve the purity and quality of recombinant plasmids.
  • the plasmids are filtered through a 0.22 ⁇ m filter (millpore). The purified plasmid is used for transfection.
  • HEK293 human embryonic kidney cells
  • PSMA recombinant antibody Specifically, HEK293F cells (invitrogen, cat#A14527) were expanded in FreeStyle TM F17 Expression Medium (Thermo #A1383504).
  • the cell concentration is 6-8 ⁇ 10 5 cells/ml, and incubate for 24 hours in a 37°C 8% CO 2 shaker, and the cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • the above-mentioned plasmid encoding the heavy chain of the antibody and the plasmid encoding the light chain of the antibody were mixed at a ratio of 2:3, a total of 30 ⁇ g plasmid was dissolved in 1.5ml Opti-MEM reduced serum medium (Thermo, 31985088), and filtered with a 0.22 ⁇ m filter to sterilize .
  • NanoDrop Thermo Scientific TM NanoDrop TM One
  • high performance liquid chromatography-mass spectrometry was used to determine the purity of the antibody
  • endotoxin detection kit (lonza) was used to determine the endotoxin content, packaged and stored for later use.
  • the monoclonal PSMA antibody PR001104 (ie, the isolated antigen binding protein described in this application) was obtained, which is of the IgG1 subtype, and the amino acid sequence of the variable region is shown in Table 1 above.
  • the heavy chain variable region sequence of the monoclonal PSMA antibody of Table 2 was subcloned into the pTT5 expression vector containing the signal peptide and the human heavy chain IgG1 constant region (SEQ ID NO: 19).
  • the light chain variable region sequence of the monoclonal PSMA antibody was subcloned into an expression vector containing the signal peptide and the human antibody light chain kappa constant region (SEQ ID NO: 20).
  • Recombinant plasmids were confirmed by sequencing and used a large extraction kit (Macherey-Nagel, Xtra Midi) extract plasmids to improve the purity and quality of recombinant plasmids.
  • the plasmids are filtered through a 0.22 ⁇ m filter (millpore). The purified plasmid is used for transfection.
  • Epi 293F cells (invitro gen, cat#A14527) were cultured in FreeStyle 293 medium (invitrogen, cat#12338026) at 37°C, 130 rpm, 8% carbon dioxide (v/v). Adjust HEK293F cells to 1-1.5x10 6 /ml cell density before transfection. HEK293F cells were co-transfected with heavy chain plasmid and light chain plasmid by PEI (sigma) for one week. Approximately on days 5-7, the antibody titer is determined. Around days 6-7, the HEK293E culture was centrifuged (30 minutes, 3500 rpm), the supernatant was collected, and filtered through a 0.22 ⁇ m filter for purification.
  • the protein A column (GE) was washed with 0.1M NaOH for 30 minutes or with 5 column volumes of 0.5M NaOH to remove endotoxin.
  • the protein A column that has not been used for a long time is first soaked in 1mNaOH for at least 1 hour, then washed with endotoxin-free water until the pH is neutral, and finally washed with 10 times the column volume of 1% Triton X100. Then equilibrate the protein column with 5 column volumes of PBS (PBS phosphate buffered saline, pH 7.4). Load the supernatant collected above on the column, and collect the flowing liquid if necessary.
  • PBS PBS phosphate buffered saline, pH 7.4
  • the column was washed with 5 column volumes of PBS, and then eluted with 5 column volumes of 0.1M glycine-HCl (pH 3.0).
  • the eluate containing the monoclonal PSMA antibody was neutralized with 0.5 column volume of 1M Tris-HCl (Nacl 1.5M) pH 8.5.
  • the anti-human PSMA antibody ie, the aforementioned monoclonal PSMA antibody was dialyzed in 1X PBS for 4 hours to avoid endotoxin contamination.
  • the concentration of anti-PSMA antibody was determined by spectrophotometry or kit, antibody purity was determined by high performance liquid chromatography-mass spectrometry, and endotoxin content was determined by endotoxin detection kit (lonza).
  • monoclonal PSMA antibody PR001331 (ie, the isolated antigen binding protein described in this application) was obtained, which is of IgG1 subtype, and the amino acid sequence of its variable region is shown in Table 2 above.
  • the constant region sequence of the comparative example 1 antibody PR001086 is the same as PR001104, and the variable region sequence is different from PR001104; the constant region sequence of the comparative example 1 antibody PR001086 is the same as PR001331, and the variable region sequence is different from PR001331, specifically, its
  • the amino acid sequence of V H is shown in SEQ ID NO: 17, the amino acid sequence of VL is shown in SEQ ID NO: 18, the amino acid sequence of heavy chain is shown in SEQ ID NO: 23, and the amino acid sequence of light chain is shown in SEQ ID NO. :24 shown.
  • Antibody Tab is a self-made comparative antibody with human IgG1 subtype based on the sequence of PSMA x CD3Pasotuxizumab from Amgen. The amino acid sequences of its heavy chain and light chain are shown in SEQ ID NOs: 25 and 26, respectively. .
  • Example 3 The binding ability of monoclonal PSMA antibodies to PSMA on the cell surface
  • HEK293T cells overexpressing human PSMA (HEK293T/human PSMA, Kangyuan Bochuang, cat#KC-1005) or CHO-K1 cells overexpressing cynomolgus monkey PSMA (CHO-K1/cyno PSMA) or highly expressing human PSMA Tumor cells (LNCAP) were cultured and expanded in T-75 culture flasks, and after reaching 90% confluence, the culture medium was aspirated, and the cells were washed twice with PBS. The cells are treated with trypsin (Invitrogen, cat#15050065) for about 1 minute, and then the trypsin is neutralized with culture medium.
  • trypsin Invitrogen, cat#15050065
  • the cells were washed twice with PBS, the cell count was measured, and the cells were resuspended in PBS to 2 ⁇ 10 6 cells/ml.
  • the cells were washed twice with PBS and incubated with goat anti-human (H+L)-Alexa Fluor 647 (Life technology, cat#A21445) at 4°C for 30-45 minutes. After washing again with PBS, the median fluorescence intensity (MFI) of the cells was analyzed on a FACS CatonII flow cytometer.
  • the control group was human IgG1.
  • both hIgG1k or hIgG1 in the figure represent the control group (ie, human IgG1).
  • the PSMA antibody PR001104 of the present application can specifically bind to human PSMA and detect The obtained antibody binding capacity increases in a positive correlation with the antibody concentration; on the contrary, the binding of the antibody PR001086 of Comparative Example 1 to human PSMA is weak.
  • the EC50 value of the PSMA antibody PR001104 of the present application is equivalent to that of Tab, but at the same concentration, the PSMA antibody PR001104 of the present application shows a higher Emax, indicating that the antibody can be used in HEK293T/human PSMA cells bind more human PSMA protein.
  • Antibody PR001086 Tab EC50( ⁇ g/ml) 10.14 1.137 Maximum MFI 2565 19488
  • Antibody PR001104 Tab EC50( ⁇ g/ml) 1.357 0.9895 Maximum MFI 30199 16,794
  • hIgG1, k or hIgG1 all represent the control group (ie, human IgG1). It can be seen that the PSMA antibody PR001104 of the present application can specifically bind to CHO-K1 /cyno Cynomolgus monkey PSMA expressed on the surface of PSMA cells. And compared with the reference antibody Tab, the binding EC50 value of this antibody is much lower than that of Comparative Example 2, indicating that this antibody can more sensitively bind monkey PSMA at a lower concentration.
  • the PSMA antibody PR001104 of the present application showed a higher Emax, indicating that the antibody can bind more cyno PSMA protein (ie, monkey PSMA protein) on CHO-K1/monkey PSMA cells.
  • PR001086 binds weakly to monkey PSMA.
  • Antibody PR001086 Tab EC50( ⁇ g/ml) 24.38 2.659 Maximum MFI 177.9 2172
  • Antibody PR001104 Tab EC50( ⁇ g/ml) 0.9995 2.659 Maximum MFI 2750 2172
  • hIgG1 in the figure represents the control group (ie, human IgG1).
  • the PSMA antibody PR001104 of the present application can specifically bind to the PSMA expressed on the surface of LNCAP cells, and is compared with the comparative example 2 antibody Tab
  • the EC50 value of the PSMA antibody PR001104 of the present application is equivalent to that of Tab, but at the same concentration, the PSMA antibody PR001104 of the present application shows a higher Emax, indicating that the antibody can bind more human PSMA on LNCAP cells. protein.
  • Antibody PR001104 Tab EC50( ⁇ g/ml) 0.9303 0.8662 Maximum MFI 1657 819.1
  • IgG1, k or hIgG1k in the figure all represent the control group (ie, human IgG1).
  • the PSMA antibody PR001331 of the present application can specifically bind to human PSMA, and the detected antibody binding The ability increases in a positive correlation with the antibody concentration; on the contrary, the binding of the antibody PR001086 of Comparative Example 1 to human PSMA is weak.
  • the PSMA antibody PR001331 of the present application showed a higher Emax, indicating that the antibody can bind more hPSMA protein (ie, human PSMA) on HEK293 hPSMA cells. protein).
  • the EC50 value of the PSMA antibody PR001331 of the present application is lower than that of Tab, indicating that the antibody can more sensitively bind to human PSMA at a lower concentration.
  • Antibody PR001086 Tab hIgG1k EC50( ⁇ g/ml) 10.14 1.137 0.05266
  • Antibody PR001331 Tab IgG1,k EC50( ⁇ g/ml) 0.6738 1.501 0.007228
  • IgG1,k or hIgG1,k in the figure represents the control group (ie human IgG1).
  • the PSMA antibody PR001331 of the present application can specifically bind to CHO -K1/cyno Monkey PSMA expressed on the surface of PSMA cells.
  • the binding EC50 value of this antibody is much lower than that of the comparative example 2, indicating that the antibody can more sensitively bind monkey PSMA at a lower concentration.
  • PR001086 binds weakly to monkey PSMA.
  • Antibody PR001086 Tab hIgG1,k EC50( ⁇ g/ml) 24.38 2.659 ⁇ 0.01842
  • Antibody PR001331 Tab IgG1,k EC50( ⁇ g/ml) 0.1537 1.591 ⁇ 0.02004
  • IgG1, k in the figure represents the control group (ie, human IgG1).
  • the PSMA antibody PR001331 of the present application can specifically bind to the PSMA expressed on the surface of LNCAP cells, and compares with Comparative Example 2.
  • the EC50 value of the antibody PR001331 is much lower, indicating that the antibody can more sensitively bind to PSMA expressed by tumor cell lines at a lower concentration.
  • Antibody PR001331 Tab IgG1,k EC50( ⁇ g/ml) 0.1056 0.6958 ⁇ 11.12
  • the PSMA antibody can mediate the internalization of the PSMA protein expressed on the cell surface by binding to the extracellular tail of PSMA.
  • the degree of internalization of the PSMA antibody of the present application and the susceptibility of PSMA+ cells to PSMA antibody killing were tested.
  • HEK293T/human PSMA or LNCAP cells were cultured and expanded in T-75 culture flasks. After reaching 90% confluence, the culture medium was aspirated, and the cells were washed twice with PBS. The cells are treated with trypsin (Invitrogen, cat#15050065) for about 1 minute, and then the trypsin is neutralized with culture medium. Transfer the cells to a 15ml sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature to pellet the cells. Aspirate the medium and resuspend the cells in their respective medium. Gently pipette the cells to obtain a single cell suspension.
  • trypsin Invitrogen, cat#15050065
  • Luminescent cell viability reagent Promega, USA, cat#G7570
  • the 96-well plate was incubated at room temperature for 10 minutes to stabilize the light signal.
  • PE Enspire microplate reader Perkin Elmer, EnSpire
  • Figure 7 shows the survival rate of HEK293T/human PSMA cells under co-treatment with antibody PR001104 and MMAF.
  • hIgG1 represents the control group (ie, human IgG1). It can be seen that under the treatment of PSMA antibody PR001104 and MMAF of the present application, the cell survival rate is equivalent to that of Comparative Example 2 antibody Tab, and its EC50 value is equivalent to that of Comparative Example 2 antibody (see Table 13).
  • Figure 8 shows the survival rate of LNCAP cells under the co-treatment of antibody PR001104 and MMAF.
  • hIgG1 represents the control group (ie, human IgG1). It can be seen that under the treatment of the PSMA antibody PR001104 and MMAF of the present application, compared with the comparative example 2 antibody Tab, the LNCAP cell survival rate is lower, and the EC50 value is lower than that of the comparative example 2 antibody Tab (see Table 14), It shows that it can achieve the maximum antibody internalization effect at a lower concentration.
  • Figure 9 shows the survival rate of HEK293T/hPSMA cells under the co-treatment of antibody PR001331 and MMAF.
  • hIgG1, k represents the control group (ie, human IgG1).
  • the cell survival rate is lower, and its EC50 value is much lower than that of the comparative example 2 antibody (see Table 15), indicating that it can achieve the maximum antibody internalization effect at a lower concentration.
  • Figure 10 shows the survival rate of LNCAP cells under co-treatment with antibody PR001331 and MMAF.
  • hIgG1, k represent the control group (ie, human IgG1).
  • the LNCAP cell survival rate is lower, and the EC50 value is lower than that of the comparative example 2 antibody (see Table 16 ), indicating that it can achieve the maximum antibody internalization effect at a lower concentration.
  • Antibody PR001331 Tab hIgG1,k EC50( ⁇ g/ml) 0.1181 9.879 /
  • Antibody PR001331 Tab hIgG1,k
  • CD14+ monocytes from PBMC (Allcells, cat#PB005-C, Lot#LP191125) with human CD14 sorting magnetic beads (Meltenyi, 130-050-201), and resuspend them at a density of 1*10 6 /mL RPMI1640 medium containing 10% FBS, adding 100ng/ml GM-CSF (PeproTech, cat#300-03-A). Take 2*10 6 monocytes per well in a 6-well plate and culture in a 37°C CO2 incubator for 9 days to differentiate into macrophages. Change the solution every 3-4 days (containing 100ng/ml GM-CSF).
  • the macrophages were digested with trypsin, and the trypsin reaction was terminated with RPMI1640 containing 10% FBS.
  • the cells were collected, washed once with PBS, and resuspended in PBS to a density of 1*10 6 /ml.
  • Collect LNCAP cells in the same way and resuspend them in PBS to a density of 1*10 6 /ml.
  • Macrophages were stained with 0.1 ⁇ M Far-red (lifetechnologies, cat#C34572), and LNCAP cells were stained with 0.5 ⁇ M CFSE (lifetechnologies, cat#C34544) at 4 degrees for 10 minutes.
  • the stained cells were centrifuged and washed once with >20ml of RPMI1640+10% FBS medium. Resuspend the washed cells in 1% BSA-RPMI1640 medium, and adjust the cell density to 1.6*10 6 /ml. In a 96-well V-shaped plate (Corning, cat#3894), 25 ⁇ l LNCAP cells (cell number per well 4*10 4 ) and 25 ⁇ l macrophages (cell number per well 4*10 4 ) were added to each well. The antibody was diluted with 1% BSA-RPMI1640 to an intermediate concentration of 20 nM, and then diluted 5 times into 7 gradients.
  • FIG 11 shows the phagocytosis of LNCAP mediated by the PSMA antibody PR001104 of the present application in macrophages.
  • hIgG1 represents the control group (ie, human IgG1)
  • LNCAP+Macrophage represents the absence of antibodies.
  • the PR001104 antibody showed ADCP effect on LNCAP cells.
  • the highest killing% of antibody PR001104 is slightly higher than that of Comparative Example 2 antibody Tab, and its EC50 value is equivalent to that of Tab.
  • the maximum lethality equal to or higher than that of Comparative Example 2 antibody can be achieved at a lower concentration.
  • Figure 12 shows the phagocytosis of LNCAP mediated by the PSMA antibody PR001331 of the present application in macrophages.
  • IgG1, k represent the control group (ie human IgG1), and no Ab represents blank (ie no antibody).
  • Binding Table 20 It can be seen that the PR001331 antibody showed ADCP effect on LNCAP cells. Among them, in the case of using donor 4051# PBMC, from the mean value and standard error of specific killing rate, the highest killing% of antibody PR001331 is closest to that of Comparative Example 2 antibody Tab, and its EC50 value is less than Tab, indicating that it can At a lower concentration, the maximum lethality equal to or higher than that of Comparative Example 2 antibody was achieved.
  • the Octet Red96e instrument (Fortebio) was used to conduct epitope competition experiments on the antigen binding proteins Tab and PR001104.
  • Use 1xkinetics buffer (Fortebio; article number 18-1105) as the buffer, dilute Tab to 200nM, and dilute the remaining antibodies to 100nM.
  • HIS1K sensor Formebio; article number 18-5120
  • His-tagged human PSMA protein Session Biological; article number 15877-H07H
  • the inhibition rate is greater than 80%, it means that the two antibodies have very similar epitopes; if the inhibition rate is between 40-80%, it means that the two antibodies have relatively close but not completely overlapping epitopes; if The inhibition rate is less than 40%, which means that the two antibodies have non-overlapping epitopes.
  • the PSMA antibody can mediate the internalization of the PSMA protein expressed on the cell surface by binding to the outside of the PSMA cell.
  • the internalization of the PSMA antibody at different time points in a short time (3 hours) was tested.
  • LNCAP cells were cultured and expanded in RPMI 1640 medium (Life technologies, cat#: 61870-036) containing 10% serum (BI, fetal bovine serum, cat#: 04-002-1A) in T-75 culture flasks After reaching 90% confluence, aspirate the medium and wash the cells twice with PBS. The cells are treated with trypsin (Invitrogen, cat#15050065) for about 1 minute, and then the trypsin is neutralized with culture medium. Transfer the cells to a 15ml sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature to pellet the cells. Aspirate the medium and resuspend the cells in their respective medium. Gently pipette the cells to obtain a single cell suspension.
  • trypsin Invitrogen, cat#15050065
  • Count with a cell counting plate then resuspend LNCAP cells into 2*10 6 cells/ml in ice-cold FACS buffer (PBS+2% FBS), and transfer 100 ⁇ l of cell suspension per well to 96-well V-plate (Corning ,3894), centrifuge at 400g for 5 minutes. After the supernatant was discarded, 100 ⁇ l of FACS buffer containing PR001104 or Comparative Example 2 antibody Tab or hIgG1 (ie, human IgG1) at a final concentration of 100 nM was added, and incubated at 4° C. for one hour. Wash twice with ice-cold FACS buffer to remove unbound antibody. Add 100ul of ice-cold FACS buffer to each well.

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Abstract

La présente invention concerne une protéine isolée de liaison à un antigène, comprenant une HCDR1, HCDR2 et HCDR3 dans des VH représentées par des séquences d'acides aminés telles que SEQ ID NO : 15, et comprenant une LCDR1, LCDR2 et LCDR3 dans VL représentées par des séquences d'acides aminés telles que SEQ ID NO : 16, ou comprenant une HCDR1, HCDR2 et HCDR3 dans VH représentées par des séquences d'acides aminés telles que SEQ ID NO : 41, et comprenant une LCDR1, LCDR2 et LCDR3 dans VL représentées par des séquences d'acides aminés telles que SEQ ID NO : 42. La présente invention concerne également un récepteur antigénique chimérique et un immunoconjugué qui comprennent la protéine isolée de liaison à l'antigène, un acide nucléique qui code la protéine isolée de liaison à l'antigène, un support qui comprend la protéine de liaison à l'antigène isolée, une cellule qui comprend l'acide nucléique ou le support, un procédé de préparation de la protéine de liaison à l'antigène isolée, et une utilisation de la protéine isolée de liaison à l'antigène.
PCT/CN2021/076664 2020-02-18 2021-02-17 Protéine isolée se liant à l'antigène psma et son utilisation WO2021164692A1 (fr)

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CN202010096322.6A CN110922486B (zh) 2020-02-18 2020-02-18 一种分离的结合抗原psma的蛋白及其用途
CN202010143681.2A CN111303288B (zh) 2020-03-04 2020-03-04 一种分离的结合抗原psma的蛋白及其用途
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1652821A (zh) * 2002-01-28 2005-08-10 米德列斯公司 抗前列腺特异性膜抗原(psma)的人单克隆抗体
EP2363486A1 (fr) * 2005-05-27 2011-09-07 Universitätsklinikum Freiburg Anticorps monoclonaux et fragments d'anticorps monocaténaires contre l'antigène spécifique de surface membranaire de la prostate
CN109641037A (zh) * 2016-04-13 2019-04-16 奥里马布斯有限公司 抗psma抗体及其用途
CN110922486A (zh) * 2020-02-18 2020-03-27 和铂医药(上海)有限责任公司 一种分离的结合抗原psma的蛋白及其用途
CN111303288A (zh) * 2020-03-04 2020-06-19 和铂医药(苏州)有限公司 一种分离的结合抗原psma的蛋白及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1652821A (zh) * 2002-01-28 2005-08-10 米德列斯公司 抗前列腺特异性膜抗原(psma)的人单克隆抗体
EP2363486A1 (fr) * 2005-05-27 2011-09-07 Universitätsklinikum Freiburg Anticorps monoclonaux et fragments d'anticorps monocaténaires contre l'antigène spécifique de surface membranaire de la prostate
CN109641037A (zh) * 2016-04-13 2019-04-16 奥里马布斯有限公司 抗psma抗体及其用途
CN110922486A (zh) * 2020-02-18 2020-03-27 和铂医药(上海)有限责任公司 一种分离的结合抗原psma的蛋白及其用途
CN111303288A (zh) * 2020-03-04 2020-06-19 和铂医药(苏州)有限公司 一种分离的结合抗原psma的蛋白及其用途

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