WO2023284741A1 - Anticorps anti-pd-1 et son utilisation - Google Patents

Anticorps anti-pd-1 et son utilisation Download PDF

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WO2023284741A1
WO2023284741A1 PCT/CN2022/105210 CN2022105210W WO2023284741A1 WO 2023284741 A1 WO2023284741 A1 WO 2023284741A1 CN 2022105210 W CN2022105210 W CN 2022105210W WO 2023284741 A1 WO2023284741 A1 WO 2023284741A1
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seq
amino acid
binding protein
acid sequence
sequence shown
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康平
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南京吉盛澳玛生物医药有限公司
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Definitions

  • This application relates to the field of biomedicine, in particular to a PD-1 antibody and its use.
  • Human programmed cell death receptor-1 (PD-1) is one of the main known immune checkpoints, which is expressed on the surface of activated T lymphocytes, and it binds to the ligand PD-L1 (programmed death receptor- The combination of ligand 1, programmed cell death-Ligand 1) and PD-L2 (programmed cell death-ligand 2, programmed cell death-Ligand 2) can inhibit the activity of T lymphocytes and related cellular immune responses in vivo.
  • the PD-L1/PD-1 signaling pathway is a very important co-inhibitory signaling pathway in the immune response, which negatively regulates the immune response of T cells, inhibits the activity of T cells, and reduces the secretion of cytokines.
  • PD-L1 can be expressed in many tumor tissues, including gastric cancer, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, colon cancer, mast cell tumors, and malignant melanoma, as well as in bone marrow cells infiltrating the tumor microenvironment
  • gastric cancer lung cancer, breast cancer, pancreatic cancer, ovarian cancer, colon cancer, mast cell tumors, and malignant melanoma
  • ovarian cancer colon cancer
  • mast cell tumors and malignant melanoma
  • malignant melanoma as well as in bone marrow cells infiltrating the tumor microenvironment
  • anti-PD-1 antibodies still have the defects of poor selectivity and low affinity. Therefore, it is necessary to develop novel anti-PD-1 antibodies with high affinity and specificity for PD-1.
  • the application provides an isolated antigen-binding protein, which has one or more of the following properties: a) is specific to human PD -1 protein with a KD value of about 5E-07M or below in a Biacore assay and b) capable of blocking the binding of PD-1 to PD-L1.
  • the isolated antigen binding protein comprises HCDR3 comprising the amino acid sequence shown in SEQ ID NO:53.
  • the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10.
  • the isolated antigen binding protein comprises HCDR2 comprising the amino acid sequence shown in SEQ ID NO:6.
  • the isolated antigen binding protein comprises HCDR1 comprising the amino acid sequence shown in SEQ ID NO:3.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in SEQ ID NO:54.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3 of the heavy chain variable region VH shown in any one of SEQ ID NO:29 to SEQ ID NO:31.
  • the antigen binding protein of described isolation comprises heavy chain variable region VH
  • described VH comprises described HCDR1, HCDR2 and HCDR3, and described HCDR3 comprises the aminoacid sequence shown in SEQ ID NO:53
  • the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:6
  • the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR1, HCDR2 and HCDR3 of the isolated antigen binding protein comprise an amino acid sequence selected from any of the following groups:
  • HCDR1 SEQ ID NO:3
  • HCDR2 SEQ ID NO:6
  • HCDR3 SEQ ID NO:9
  • HCDR1 SEQ ID NO:3
  • HCDR2 SEQ ID NO:6
  • HCDR3 SEQ ID NO:10.
  • the isolated antigen binding protein comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the H-FR1 comprises SEQ ID NO : the amino acid sequence shown in 49.
  • the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2.
  • the isolated antigen binding protein comprises H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises SEQ ID NO:50 amino acid sequence.
  • the H-FR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5.
  • the antigen binding protein of described isolation comprises H-FR3, and described H-FR3 is positioned between described HCDR2 and described HCDR3, and described H-FR3 comprises SEQ ID NO:51 amino acid sequence.
  • the H-FR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8.
  • the isolated antigen binding protein comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and the H-FR4 comprises SEQ ID NO : the amino acid sequence shown in 52.
  • the H-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.
  • the antigen binding protein of described separation comprises H-FR1, H-FR2, H-FR3 and H-FR4, and described H-FR1 comprises the aminoacid sequence shown in SEQ ID NO:49; Said H-FR2 comprises the amino acid sequence shown in SEQ ID NO:50; the H-FR3 comprises the amino acid sequence shown in SEQ ID NO:51; and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:52.
  • said isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, said H-FR1 comprising any of SEQ ID NO:1 and SEQ ID NO:2
  • the H-FR2 includes the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5
  • the H-FR3 includes SEQ ID NO:7 and SEQ ID NO : the amino acid sequence shown in any one of 8
  • the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 11 and SEQ ID NO: 12.
  • the H-FR1, H-FR2, H-FR3 and H-FR4 in the isolated antigen binding protein comprise an amino acid sequence selected from any of the following groups:
  • H-FR1 SEQ ID NO:1
  • H-FR2 SEQ ID NO:4
  • H-FR3 SEQ ID NO:7
  • H-FR4 SEQ ID NO:11
  • H-FR1 SEQ ID NO:2
  • H-FR2 SEQ ID NO:5
  • H-FR3 SEQ ID NO:8
  • H-FR4 SEQ ID NO:12.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO:54.
  • the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.
  • the isolated antigen binding protein comprises LCDR3 comprising the amino acid sequence shown in SEQ ID NO:59.
  • the LCDR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.
  • the isolated antigen binding protein comprises LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding protein comprises LCDR1 comprising the amino acid sequence shown in SEQ ID NO:15.
  • the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in SEQ ID NO:60.
  • the isolated antigen binding protein comprises LCDR1, LCDR2 and LCDR3 of the light chain variable region VL shown in any one of SEQ ID NO:32 to SEQ ID NO:38.
  • the antigen binding protein of described isolation comprises light chain variable region VL, and described VL comprises described LCDR1, LCDR2 and LCDR3, and described LCDR3 comprises the aminoacid sequence shown in SEQ ID NO:59;
  • the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:18; and
  • the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:15.
  • said LCDR1, LCDR2 and LCDR3 in said isolated antigen binding protein comprise an amino acid sequence selected from any of the following groups:
  • LCDR1 SEQ ID NO:15
  • LCDR2 SEQ ID NO:18
  • LCDR3 SEQ ID NO:21;
  • LCDR1 SEQ ID NO:15
  • LCDR2 SEQ ID NO:18
  • LCDR3 SEQ ID NO:22;
  • LCDR1 SEQ ID NO:15
  • LCDR2 SEQ ID NO:18
  • LCDR3 SEQ ID NO:23;
  • LCDR1 SEQ ID NO:15
  • LCDR2 SEQ ID NO:18
  • LCDR3 SEQ ID NO:24;
  • LCDR1 SEQ ID NO: 15, LCDR2: SEQ ID NO: 18 and LCDR3: SEQ ID NO: 25;
  • LCDR1 SEQ ID NO:15
  • LCDR2 SEQ ID NO:18
  • LCDR3 SEQ ID NO:26.
  • the isolated antigen binding protein comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises SEQ ID NO : the amino acid sequence shown in 55.
  • the L-FR1 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:13 and SEQ ID NO:14.
  • said isolated antigen binding protein comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said L-FR2 comprises SEQ ID NO:56 amino acid sequence.
  • the L-FR2 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17.
  • said isolated antigen binding protein comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said L-FR3 comprises SEQ ID NO:57 amino acid sequence.
  • the L-FR3 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20.
  • the isolated antigen binding protein comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly linked to the C-terminus of the LCDR3, and the L-FR4 comprises SEQ ID NO : the amino acid sequence shown in 58.
  • the L-FR4 in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.
  • the antigen binding protein of described separation comprises L-FR1, L-FR2, L-FR3 and L-FR4, and described L-FR1 comprises the aminoacid sequence shown in SEQ ID NO:55; Said L-FR2 comprises the amino acid sequence shown in SEQ ID NO:56; said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:57; and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:58.
  • the antigen binding protein of described separation comprises L-FR1, L-FR2, L-FR3 and L-FR4, and described L-FR1 comprises SEQ ID NO:13 and SEQ ID NO:14 shown the amino acid sequence; the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17; the L-FR3 comprises any one of SEQ ID NO:19 and SEQ ID NO:20 an amino acid sequence shown in one; and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.
  • said L-FR1, L-FR2, L-FR3 and L-FR4 in said isolated antigen binding protein comprise an amino acid sequence selected from any of the following groups:
  • L-FR1 SEQ ID NO:13
  • L-FR2 SEQ ID NO:16
  • L-FR3 SEQ ID NO:19
  • L-FR4 SEQ ID NO:27;
  • L-FR1 SEQ ID NO:14
  • L-FR2 SEQ ID NO:17
  • L-FR3 SEQ ID NO:20
  • L-FR4 SEQ ID NO:28.
  • the VL of the isolated antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:60.
  • the VL in the isolated antigen binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.
  • the VH and VL of the isolated antigen binding protein comprise amino acid sequences selected from any of the following groups:
  • VH SEQ ID NO: 29 and VL: SEQ ID NO: 32;
  • VH SEQ ID NO: 30 and VL: SEQ ID NO: 33;
  • VH SEQ ID NO: 30 and VL: SEQ ID NO: 34;
  • VH SEQ ID NO: 30 and VL: SEQ ID NO: 35;
  • VH SEQ ID NO: 30 and VL: SEQ ID NO: 36;
  • VH SEQ ID NO: 30 and VL: SEQ ID NO: 37;
  • VH SEQ ID NO: 30 and VL: SEQ ID NO: 38;
  • VH SEQ ID NO: 31 and VL: SEQ ID NO: 33;
  • VH SEQ ID NO: 31 and VL: SEQ ID NO: 36.
  • the isolated antigen binding protein has an increased half-life compared to a wild-type antibody.
  • the isolated antigen binding protein is capable of reducing antibody-dependent cellular cytotoxicity (ADCC) compared to a wild-type antibody.
  • ADCC antibody-dependent cellular cytotoxicity
  • the isolated antigen binding protein comprises a heavy chain constant region, and the heavy chain constant region comprises an IgG-derived constant region or an IgY-derived constant region.
  • said heavy chain constant region of said isolated antibody binding protein comprises an IgG-derived constant region.
  • the heavy chain constant region comprises a constant region derived from IgGl, IgG2, IgG3 or IgG4.
  • the heavy chain constant region comprises an IgG1 derived Fc region.
  • the N297 position of the heavy chain constant region is mutated, and the residues are numbered according to the Kabat system.
  • the M252 position of the heavy chain constant region is mutated, and the residues are numbered according to the Kabat system.
  • the S254 position of the heavy chain constant region is mutated, and the residues are numbered according to the Kabat system.
  • the T256 position of the heavy chain constant region is mutated, and the residues are numbered according to the Kabat system.
  • the Fc region in the heavy chain constant region comprises the N297A mutation.
  • the Fc region in the heavy chain constant region comprises the M252Y mutation.
  • the Fc region in the heavy chain constant region comprises the S254T mutation.
  • the Fc region in the heavy chain constant region comprises a T256E mutation.
  • the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:48.
  • the isolated antigen binding protein comprises a light chain constant region, and the light chain constant region comprises an Ig ⁇ -derived constant region or an Ig ⁇ -derived constant region.
  • the light chain constant region comprises a constant region derived from human Ig ⁇ .
  • the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:47.
  • the isolated antigen binding protein comprises a heavy chain HC comprising the amino acid sequence shown in any one of SEQ ID NO:39 and SEQ ID NO:40.
  • the isolated antigen binding protein comprises a light chain LC comprising the amino acid sequence shown in any one of SEQ ID NO:41 to SEQ ID NO:46.
  • the isolated antigen binding protein comprises an antibody or antigen binding fragment thereof.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the antibody is selected from the group consisting of monoclonal antibodies, single chain antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, multispecific antibodies, and fully human antibodies.
  • the present application provides one or more polypeptides comprising said isolated antigen-binding protein.
  • the present application provides one or more immunoconjugates comprising said isolated antigen-binding protein or said polypeptide.
  • the present application provides one or more isolated nucleic acid molecules encoding said isolated antigen binding protein, or said polypeptide.
  • the application provides one or more vectors comprising said isolated nucleic acid molecule.
  • the application provides one or more cells comprising said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule and/or said carrier.
  • the present application provides a method for preparing the isolated antigen-binding protein or the polypeptide, the method comprising culturing the cells.
  • the present application provides one or more pharmaceutical compositions comprising said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said carrier, the cells, and/or pharmaceutically acceptable adjuvants and/or excipients.
  • the present application provides a method for detecting or measuring PD-1, the method comprising using the isolated antigen-binding protein or the polypeptide.
  • the present application provides a PD-1 detection kit, which comprises the isolated antigen-binding protein or the polypeptide.
  • the present application provides the use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit for detecting the presence and/or content of PD-1.
  • the present application provides a method for inhibiting the interaction between PD-1 and PD-L1, which comprises administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the The immunoconjugate, the isolated nucleic acid molecule, the carrier, and/or the cell.
  • the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or Use of the pharmaceutical composition in the preparation of medicines for preventing and/or treating diseases or diseases.
  • the disease or condition comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • the tumor comprises melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer and/or breast cancer.
  • the present application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell and/or the A pharmaceutical composition for preventing, alleviating and/or treating diseases or conditions.
  • the disease or condition comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a non-solid tumor.
  • Such tumors include melanoma, lung cancer, squamous cell carcinoma of the head and neck, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal carcinoma and/or breast carcinoma.
  • the present application provides a method for preventing and/or treating a disease or condition, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the Immunoconjugate, said isolated nucleic acid molecule, said carrier, and/or said cell.
  • the disease or condition comprises a tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises a solid tumor.
  • the tumor comprises melanoma, lung cancer, head and neck squamous cell carcinoma, lymphoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer and/or breast cancer.
  • Figure 1 shows the ELISA detection results of the antigen binding protein described in this application inhibiting the binding of PD-L1 to PD-1.
  • FIGS 2a-2b show the results of MLR detection of the antigen binding proteins described in this application.
  • Figure 3 shows the ELISA detection results of the Fc engineered antigen-binding protein described in this application inhibiting the binding of PD-L1 to PD-1.
  • FIGS 4a-4b show the results of functional cell experiments of the Fc engineered antigen-binding protein described in this application.
  • isolated generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an 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 admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.
  • the term “antigen-binding protein” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • the term “antigen-binding protein” may include “antibody” or "antigen-binding fragment”.
  • the antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and may include any molecule comprising an antigen-binding portion thereof.
  • antibody may include monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single chain antibodies (e.g., scFv ), and antibody fragments (eg, Fab, Fab', VHH and (Fab)2 fragments) that bind to the antigen.
  • antibody may also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, aglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with an antigen (eg, human PD-1).
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the exact boundaries of the CDRs have been defined differently according to different systems.
  • the system described by Kabat Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a sequence that can be applied to any variable region of an antigen-binding fragment
  • a clear residue numbering system also provides the precise residue boundaries that define the CDRs. These CDRs can be referred to as Kabat CDRs.
  • the term "antigen-binding fragment” generally refers to one or more fragments of an antibody that specifically bind to an antigen.
  • the antigen-binding function of antibodies can be realized by full-length fragments of antibodies.
  • the antigen-binding function of an antibody can also be achieved by comprising a heavy chain of a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab'), or by comprising a Fv, scFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2.
  • Fab fragment usually a monovalent fragment consisting of VL, VH, CL and CH domains;
  • F(ab')2 fragment comprising two Fab fragments linked by a disulfide bond at the hinge region (3) Fd fragment composed of VH and CH domains; (4) Fv fragment composed of VL and VH domains of antibody single arm; (5) dAb fragment composed of VH domains (Ward et al., (1989) Nature 341:544-546); (6) an isolated complementarity determining region (CDR) and (7) a combination of two or more isolated CDRs optionally linked by a linker.
  • CDR complementarity determining region
  • the monovalent single-chain molecule Fv formed by the pairing of VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl.Acad.Sci.85 :5879-5883).
  • a type of antibody VHH that lacks the light chain of the antibody and only has the variable region of the heavy chain can also be included (for example, see Kang Xiaozhen et al., Acta Biological Engineering, 2018, 34(12): 1974-1984).
  • the "antigen binding portion” may also include an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • the term "monoclonal antibody” generally refers to a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that are present in minor amounts.
  • Monoclonal antibodies are highly specific, directed against a single antigenic site.
  • the monoclonal antibodies can be produced by hybridoma technology or produced in bacterial, eukaryotic or plant cells by using recombinant DNA methods.
  • Monoclonal antibodies can also be obtained from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991) conduct.
  • chimeric antibody generally refers to an antibody in which a part of each heavy or light chain amino acid sequence is homologous to the corresponding amino acid sequence in an antibody from a specific species, or belongs to a specific class, and The remainder of the chain is then homologous to the corresponding sequence in another species.
  • the variable regions of both the light and heavy chains are derived from the variable regions of antibodies from one animal species (e.g., mouse, rat, etc.), while the constant portions are homologous to antibody sequences from another species (e.g., human) .
  • B cells or hybridoma cells of non-human origin can be used to produce variable regions combined with constant regions of human origin.
  • variable region has the advantage of being easy to prepare and its specificity is not affected by the source of the constant region it is combined with.
  • the constant region of the chimeric antibody can be derived from humans, the possibility of the chimeric antibody triggering an immune response when injected is lower than that of an antibody whose constant region is of non-human origin.
  • humanized antibody generally refers to a chimeric antibody that contains less sequence from a non-human immunoglobulin, thereby reducing the immunogenicity of a heterologous antibody when introduced into a human, while simultaneously Preserves the full antigen-binding affinity and specificity of the antibody.
  • CDR grafting (Jones et al., Nature 321:522 (1986)) and variants thereof; including “reshaping", (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann , et al., 1988 Nature 332:323-337; Tempest, et al., Bio/Technol 1991 9:266-271), "high addition” (hyperchimerization), (Queen, et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co, et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co, et al., 1992 J Immunol 148:1149-1154) and "veneering", (Mark, et al., "Derivation of therapeutically active humanized and veneered anti-CD18 antibodies.” In: Metcalf B W, Dalton B J, eds.
  • 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 comprises a constant region, these also are derived from mouse germline immunoglobulin sequences.
  • the murine antibody of the present application may contain amino acid residues not encoded by the mouse germline immunoglobulin sequence, for example, may include mutations introduced by random mutation or point mutation in vitro or by somatic mutation in vivo.
  • PD-1 protein protein
  • PD-1 PD-1 antigen
  • PD-1 may be human PD-1, whose accession number in UniProt/Swiss-Prot is Q15116.
  • PD-1 can be a functionally active fragment of human PD-1.
  • such "functionally active fragments” may include fragments that retain at least one endogenous function of a naturally occurring protein (eg, binding to an antigen binding protein described herein).
  • the "functionally active fragment” may include a domain that binds to the antigen-binding protein of the present application.
  • the present application may also include functionally active fragments, derivatives, analogs, homologues and fragments thereof.
  • a functionally active fragment refers to a polypeptide having substantially the same amino acid sequence or encoded by a substantially identical nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence.
  • a functionally active fragment of any given sequence is one in which a specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least A sequence of endogenous function.
  • Sequences encoding functionally active fragments can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in naturally occurring proteins and/or polynucleotides , as long as the original functional activity is maintained.
  • derivative generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and and/or added, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.
  • analogue generally refers to polypeptides or polynucleotides, including any mimetic of polypeptides or polynucleotides, that is, having at least one endogenous function of the polypeptide or polynucleotide simulated by the mimetic of chemical compounds.
  • amino acid substitutions e.g., at least 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made so long as the modified sequence remains substantially as desired. activity or ability. Amino acid substitutions may involve the use of non-naturally occurring analogs.
  • homologue generally refers to an amino acid sequence or nucleotide sequence having a certain homology to a naturally occurring sequence.
  • the term “homology” may be equated with sequence "identity”.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will comprise the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity.
  • sequence having a percentage identity of any one of the SEQ ID NOs of the mentioned amino acid sequence or nucleotide sequence means having said percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.
  • sequence alignment can be performed by various means known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment across the full-length sequences being compared.
  • proteins or polypeptides used in this application may also have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar headgroups with similar hydrophilicity values include aspartic acid.
  • Paragine, Glutamine, Serine, Threonine and Tyrosine are examples of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • the term "tumor” generally refers to a neoplasm formed by the proliferation of local tissue cells under the action of various tumorigenic factors.
  • the tumor can include a solid tumor.
  • the tumor can include a non-solid tumor.
  • the tumor can include a tumor associated with expression of PD-L1.
  • the term "tumor associated with the expression of PD-L1” generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance.
  • the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance.
  • the tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor.
  • the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • solid tumor generally refers to a tangible mass that can be detected clinically (eg, X-ray film, CT scan, B-ultrasound or palpation).
  • said solid tumor may be selected from the group consisting of melanoma, lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma, colorectal cancer and breast cancer.
  • non-solid tumor generally refers to a tumor that cannot be seen or touched by X-ray films, CT scans, B-ultrasound and palpation.
  • the non-solid tumor can include leukemia.
  • the non-solid tumor can include lymphoma.
  • the non-solid tumor can include multiple myeloma.
  • the term “immunoconjugate” generally refers to a conjugate formed by conjugating (for example, covalently linking through a linker molecule) the other therapeutic agent to the isolated antigen-binding protein, the conjugate
  • the other therapeutic agent can be delivered to a target cell (eg, a tumor cell) by specific binding of the isolated antigen binding protein to an antigen on the target cell.
  • the antigen may also be secreted by the target cell and located in the space outside the target cell.
  • 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.
  • nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length isolated from their natural environment or artificially synthesized.
  • the term "vector” generally refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vectors can transfer inserted nucleic acid molecules into and/or between cells.
  • the vectors may include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and vectors mainly used for expression of transcription and/or translation of DNA or RNA.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell.
  • the vector produces the desired expression product by culturing appropriate cells containing the vector.
  • the vector may comprise a lentiviral vector.
  • the term "cell” generally refers to an individual cell that can or has contained a plasmid or vector comprising a nucleic acid molecule described herein, or is capable of expressing a polypeptide described herein or an antigen binding protein described herein , cell line or cell culture.
  • the cells may include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cells, but it is sufficient to be able to express the polypeptide or antigen-binding protein described in this application.
  • the cells can be obtained by transfecting cells in vitro with the vectors described in this application.
  • the cells can be prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells or myeloma cells).
  • the cells can be immune cells.
  • the immune cells may be selected from the group consisting of T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.
  • treating generally refers to: (i) preventing a disease, disorder, and/or condition from occurring in a patient who may be predisposed to it but has not been diagnosed with it; (ii) inhibiting the disease , disorder or condition, i.e. arresting its development; and (iii) relieving the disease, disorder or condition, i.e. making the disease, disorder and/or condition and/or symptoms associated with the disease, disorder and/or condition subside.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to a polymer of amino acids of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component).
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably and generally refer to nucleosides of any length
  • a polymeric form of an acid such as deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, multiple loci (one locus) defined by junctional analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Isolated DNA of any sequence, isolated RNA, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA Transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.
  • K D (likewise, “K D " or “K D ”) generally refers to "affinity constant” or "equilibrium dissociation constant” and refers to a titration measurement at equilibrium, or Value obtained by dividing the dissociation rate constant (kd) by the association rate constant (ka).
  • the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) are used to express the binding protein (such as the isolated antigen binding protein described in this application) to the antigen (such as PD -1 protein) binding affinity.
  • Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers.
  • the KD value can be determined by Biacore (Biomolecular Interaction Analysis) (for example, an instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden), and other experimental approaches and instruments such as Octet can also be used.
  • the KD value can also be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho), or using a surface plasmon resonance (SPR) instrument.
  • the KD value can also be determined by an amine coupling kit.
  • 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. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen binding protein, which can be measured in a Biacore assay with a KD value of about 5E- 07M or below (for example, the KD is not higher than about 5E- 07M , not higher than About 4.9E-07M, not higher than about 4.8E-07M, not higher than about 4.7E-07M, not higher than about 4.6E-07M, not higher than about 4.5E-07M, not higher than about 4.4E-07M , not higher than about 4.3E-07M, not higher than about 4.0E-07M, not higher than about 3.5E-07M, not higher than about 3.0E-07M, not higher than about 2.5E-07M, not higher than about 2.0E-07M, not higher than about 1.5E-07M, not higher than about 1.0E-07M, not higher than about 9E-08M, or not higher than about 8.5E-08M or less) specific for human PD-1 protein combined.
  • the KD is not higher than about 5E-
  • the application provides an isolated antigen-binding protein, which may comprise at least one CDR in the variable region VH of an antibody heavy chain, and the VH may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the VH may comprise the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.
  • the HCDR of the isolated antigen-binding protein can be divided in any form, as long as the VH is identical to the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31, it can be divided in any form to obtain All HCDRs can fall within the protection scope of the present application.
  • the CDR of an antibody also known as the complementarity determining region, is part of the variable region.
  • the amino acid residues in this region may make contacts with the antigen or antigenic epitope.
  • Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, comprehensive consideration of Kabat/Chothia, etc. These numbering systems are known in the art, see, for example, http://www.bioinf.org.uk/abs/index.html#kabatnum. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems.
  • the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids .
  • amino acids For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.
  • the isolated antigen binding proteins described herein are defined by the Kabat coding system.
  • the antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the HCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:53.
  • the HCDR3 sequence of the antigen binding protein can be defined according to the Kabat coding system.
  • the HCDR3 of the antigen-binding protein has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the following group: X 1 .
  • X 1 HYGTSPFVY (SEQ ID NO: 53), wherein X 1 can be D or E.
  • the HCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10.
  • the HCDR3 sequence of the antigen binding protein can be defined according to the Kabat coding system.
  • the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the HCDR2 sequence of the antigen binding protein can be defined according to the Kabat coding system.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR1 sequence of the antigen binding protein can be defined according to the Kabat coding system.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the amino acid sequence shown may comprise antibodies 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10 or have the same HCDR3 (e.g., have the same HCDR1-3 as the antigen-binding fragment).
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10
  • the amino acid sequence shown may comprise antibody 19D4-25-3C11, 19D4-25-1C2-3C11 or an antigen binding fragment thereof having the same HCDR3 (eg, having the same HCDR1-3).
  • the VH of the antigen binding protein may comprise framework regions H-FR1, H-FR2, H-FR3 and H-FR4.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:49.
  • H-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 5 , X 13 , X 16 , X 17 and X 20 .
  • X 1 VQLX 5 ESGPGLVX 13 PSX 16 X 17 LSX 20 TCTVSGFSLT (SEQ ID NO: 49), wherein, X 1 can be E or Q, X 5 can be K or Q, X 13 can be A or K, X 16 can be is E or Q, X 17 can be S or T and X 20 can be I or L.
  • the H-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:50.
  • H-FR2 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 2 and X13 .
  • WX 2 RQPPGKGLEWX 13 G (SEQ ID NO: 50), wherein X 2 can be I or V, and X 13 can be I or L.
  • the H-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:4 and SEQ ID NO:5.
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:51.
  • H-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 2 , X 3 , X 8 , X 11 , X 14 , X 17 , X 18 , X 20 , X 21 , X 22 , X 23 and X 27 .
  • X 2 can be L or V
  • X 3 can be S or T
  • X 8 can be N or T
  • X 11 can be N or S
  • X 14 can be F or S
  • X 17 can be L or M
  • X 18 can be N or S
  • X 20 can be L or V
  • X 21 can be Q
  • the H-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8.
  • the H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:52.
  • H-FR4 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 11 .
  • WGQGTLVTVSX 11 (SEQ ID NO: 52), wherein X 11 can be A or S.
  • the H-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:49; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:50; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:51; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:52.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:2; the H-FR2 may comprise SEQ ID NO:4 and The amino acid sequence shown in any one of SEQ ID NO:5; the H-FR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:7 and SEQ ID NO:8; and the H-FR4 can be Comprising the amino acid sequence shown in any one of SEQ ID NO:11 and SEQ ID NO:12.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:1; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:4; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:7; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:11.
  • the antigen binding protein may comprise antibody 19D4F1 or an antigen binding fragment thereof having the same H-FR1-4.
  • the H-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:8; and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:12.
  • the antigen binding protein can include antibodies hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, 19D4-25-3C11, 19D4-25-1C2-3C11 or an antigen-binding fragment thereof with the same H-FR1-4.
  • the antigen binding protein may comprise a heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the antigen binding protein comprises a VH having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of the sequence shown in SEQ ID NO:54 compared to the VH: X1 , X5 , X13 , X16 , X17 , X20 , X37 , X48 , X67 , X68 , X73 , X76 , X79 , X82 , X83 , X85 , X86 , X 87 , X 88 , X 92 , X 98 and X 118 .
  • the heavy chain variable region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.
  • the antigen-binding protein may comprise a heavy chain constant region, which may include an IgG-derived constant region or an IgY-derived constant region.
  • the heavy chain constant region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:48.
  • the antigen-binding protein may comprise at least one CDR in the VL of the antibody light chain variable region, and the VL may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the VL may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.
  • the LCDR of the isolated antigen-binding protein can be divided in any form, as long as the VL is identical to the amino acid sequence shown in any one of SEQ ID NO: 32 to SEQ ID NO: 38, it can be divided in any form to obtain All LCDRs can fall within the protection scope of the present application.
  • the antigen binding protein may comprise a light chain variable region VL, and the VL may comprise at least one, at least two or at least three of LCDR1, LCDR2 and LCDR3.
  • the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:59.
  • the LCDR3 of the antigen binding protein can be defined according to the Kabat numbering system.
  • the LCDR3 of the antigen-binding protein has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 4 , X5 , X7 and X9 .
  • X 1 QSX 4 X 5 VX 7 WX 9 (SEQ ID NO: 59), wherein, X 1 can be Q or S, X 4 can be K, L or S, X 5 can be E, H, K or R, X 7 can be N or P, and X 9 can be S or T.
  • the LCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.
  • the LCDR3 of the antigen binding protein can be defined according to the Kabat numbering system.
  • the LCDR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the LCDR2 of the antigen binding protein can be defined according to the Kabat numbering system.
  • the LCDR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:15.
  • the LCDR1 of the antigen binding protein can be defined according to the Kabat numbering system.
  • the LCDR1 of the antigen binding protein described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 21.
  • the antigen binding protein may comprise antibody 19D4F1, hu19D4-25, 19D4-25-3C11 or an antigen binding fragment thereof having the same LCDR3 (eg, having the same LCDR1-3).
  • the antigen binding protein can comprise antibody 19D4-25-1A3 or an antigen binding fragment having the same LCDR3 therewith (eg, having the same LCDR1-3 as it).
  • the antigen binding protein may comprise antibody 19D4-25-1B3 or an antigen binding fragment having the same LCDR3 therewith (eg, having the same LCDR1-3 as it).
  • the antigen binding protein may comprise antibody 19D4-25-1C2, 19D4-25-1C2-3C11, or an antigen binding fragment thereof having the same LCDR3 (eg, having the same LCDR1-3).
  • the antigen binding protein may comprise antibody 19D4-25-1E4 or an antigen binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.
  • the antigen binding protein may comprise antibody 19D4-25-2E10 or an antigen binding fragment having the same LCDR3 (eg, having the same LCDR1-3) therewith.
  • the VL of the antigen binding protein may comprise the framework regions L-FR1, L-FR2, L-FR3 and L-FR4.
  • the L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:55.
  • L-FR1 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 7 , X 9 , X 12 , X 14 , X 15 , X 18 and X 20 .
  • DIVLTQX 7 PX 9 SLX 12 VX 14 X 15 GQX 18 AX 20 ISC SEQ ID NO:55
  • X 7 can be S or T
  • X 9 can be A or L
  • X 12 can be A or S
  • X 14 can be S or T
  • X 15 can be L or P
  • X 18 can be P or R
  • X 20 can be S or T.
  • the L-FR1 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:13 and SEQ ID NO:14.
  • the L-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:56.
  • L-FR2 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 3 and X11 .
  • WFX 3 QKPGQPPX 11 LLIY (SEQ ID NO: 56), wherein X 3 can be L or Q, and X 11 can be K or Q.
  • the L-FR2 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:16 and SEQ ID NO:17.
  • the L-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:57.
  • the L-FR3 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 4 , X 16 , X 18 , X 20 , X 21 , X 22 , X 24 , X 25 , X 27 , X 28 and X 29 .
  • GVPX 4 RFSGSGSGTDFX 16 LX 18 IX 20 X 21 X 22 EX 24 X 25 DX 27 X 28 X 29 YFC (SEQ ID NO: 57), wherein, X 4 can be A or D, X 16 can be S or T, X 18 can be K or N, X 20 can be H or S, X 21 can be P or R, X 22 can be M or V, X 24 can be A or E, X 25 can be D or E, X 27 can is T or V, X 28 can be A or G, and X 29 can be M or V.
  • the L-FR3 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20.
  • the L-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:58.
  • L-FR4 of the antigen binding protein has amino acid substitutions (for example, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 7 .
  • FGGGTKX7EIK (SEQ ID NO: 58), wherein X7 can be L or V.
  • the L-FR4 of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.
  • the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:55; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:56; the L-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:57; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:58.
  • the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 13 and SEQ ID NO: 14; the L-FR2 may comprise SEQ ID NO: 16 and The amino acid sequence shown in any one of SEQ ID NO:17;
  • the L-FR3 can comprise the amino acid sequence shown in any one of SEQ ID NO:19 and SEQ ID NO:20;
  • the L-FR4 can be Comprising the amino acid sequence shown in any one of SEQ ID NO:27 and SEQ ID NO:28.
  • the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 13; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 16; the L-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:19; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:27.
  • the antigen binding protein may comprise antibody 19D4F1 or an antibody having the same L-FR1-4 therewith.
  • the L-FR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO: 14; the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 17; the L-FR3 It may comprise the amino acid sequence shown in SEQ ID NO:20; and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:28.
  • the antigen binding protein can include antibodies hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10, 19D4-25-3C11, 19D4-25-1C2-3C11 or an antibody with the same H-FR1-4 as it.
  • the antigen-binding protein may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the VL of the antigen binding protein has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 7 , X 9 , X 12 , X 14 , X 15 , X 18 , X 20 , X 41 , X 49 , X 64 , X 76 , X 78 , X 80 , X 81 , X 82 , X 84 , X 85 , X 87 , X 88 , X 89 , X 93 , X 96 , X 97 , X 99 , X 101 and X 108 .
  • DIVLTQX 7 PX 9 SLX 12 VX 14 X 15 GQX 18 AX 20 ISCRASESVDNYGISFMNWFX 41 QKPGQPPX 49 LLIYAASNQGSGVPX 64 RFSGSGSGTDFX 76 LX 78 IX 80 X 81 X 82 EX 84 X 85 DX 87 X 88 X 89 YFCX 93 QSX 96 X 97 VX 99 WX 101 FGGGTKX 108 EIK (SEQ ID NO:60), wherein, X 7 can be S or T, X 9 can be A or L, X 12 can be A or S, X 14 can be S or T, X 15 can be L Or P, X 18 can be P or R, X 20 can be S or T, X 41 can be L or Q, X 49 can be K or Q, X 64 can be A or D, X 76 can be S or T , X 78 can be K or N,
  • the light chain variable region of the antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.
  • the antigen-binding protein may comprise a light chain constant region, which may include a constant region derived from Ig ⁇ or a constant region derived from Ig ⁇ .
  • the light chain constant region may comprise a constant region derived from Ig ⁇ .
  • the light chain constant region of the antigen binding protein comprises the amino acid sequence shown in SEQ ID NO:47.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the antigen binding protein may comprise antibody 19D4F1, hu19D4-25, or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4F1, hu19D4-25.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; the LCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the antigen binding protein can comprise antibody 19D4-25-1A3 or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1A3.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the antigen binding protein can comprise antibody 19D4-25-1B3 or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1B3.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; The LCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the antigen binding protein can comprise antibody 19D4-25-1C2 or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1C2.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the antigen binding protein can comprise antibody 19D4-25-1E4 or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1E4.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:26.
  • the antigen binding protein may comprise antibody 19D4-25-2E10 or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-2E10.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:10; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; the LCDR3 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the antigen binding protein can comprise antibody 19D4-25-3C11 or an antigen binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-3C11.
  • the antigen binding protein may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the antigen binding protein may comprise Comprising the amino acid sequence shown in SEQ ID NO:10; the LCDR1 of the antigen-binding protein may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the antigen-binding protein may include the amino acid shown in SEQ ID NO:18 Sequence; The LCDR3 of the antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the antigen binding protein may comprise antibody 19D4-25-1C2-3C11 or an antigen binding fragment thereof that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:21.
  • the L-FR3 can include the amino acid sequence shown in SEQ ID NO: 19;
  • the L-FR4 can include the amino acid sequence shown in SEQ ID NO: 27.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:29.
  • the antigen binding protein may comprise antibody 19D4F1 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:32.
  • the antigen binding protein may comprise antibody 19D4F1 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:21.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may comprise antibody hu19D4-25 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the antigen binding protein may comprise antibody hu19D4-25 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:22.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may include antibody 19D4-25-1A3 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:34.
  • the antigen binding protein may comprise antibody 19D4-25-1A3 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:23.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen-binding protein may include the antigen-binding fragment 19D4-25-1B3 or an antigen-binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:35.
  • the antigen binding protein may comprise antibody 19D4-25-1B3 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:24.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may comprise antibody 19D4-25-1C2 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the antigen binding protein may comprise antibody 19D4-25-1C2 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region of the antigen binding protein may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region of the antigen binding protein may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:25.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may comprise antibody 19D4-25-1E4 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:37.
  • the antigen binding protein may comprise antibody 19D4-25-1E4 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3;
  • the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6;
  • the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15;
  • the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18;
  • the LCDR3 can include the amino acid sequence shown in SEQ ID NO:26.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:30.
  • the antigen binding protein may comprise antibody 19D4-25-2E10 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:38.
  • the antigen binding protein may comprise antibody 19D4-25-2E10 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18; the LCDR3 can include the amino acid sequence shown in SEQ ID NO:21.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:31.
  • the antigen binding protein may comprise antibody 19D4-25-3C11 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:33.
  • the antigen binding protein may comprise antibody 19D4-25-3C11 or an antigen binding protein having the same light chain variable region as it.
  • the antigen binding protein may comprise a heavy chain variable region and a light chain variable region, and the heavy chain variable region may comprise HCDR1-3 and H-FR1-4.
  • the light chain variable region may comprise LCDR1-3 and L-FR1-4.
  • the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10;
  • the LCDR1 can include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 can include the amino acid sequence shown in SEQ ID NO:18; the LCDR3 can include the amino acid sequence shown in SEQ ID NO:24.
  • the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:2; the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO:5; the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO:8 The amino acid sequence shown; the H-FR4 can comprise the amino acid sequence shown in SEQ ID NO:12; the L-FR1 can comprise the amino acid sequence of SEQ ID NO:14; the L-FR2 can comprise the amino acid sequence of SEQ ID NO The amino acid sequence shown in: 17; The L-FR3 can comprise the amino acid sequence shown in SEQ ID NO: 20; The L-FR4 can comprise the amino acid sequence shown in SEQ ID NO: 28.
  • the heavy chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:31.
  • the antigen binding protein may comprise antibody 19D4-25-1C2-3C11 or an antigen binding protein having the same heavy chain variable region as it.
  • the light chain variable region of the antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:36.
  • the antigen binding protein may comprise antibody 19D4-25-1C2-3C11 or an antigen binding protein having the same light chain variable region as it.
  • the isolated antigen-binding protein can also compete with a reference antibody for binding to the human PD-1 protein, and the reference antibody can comprise a heavy chain variable region VH, and the VH of the reference antibody can be Contains at least one, two or three of HCDR1, HCDR2 and HCDR3.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:53.
  • the HCDR3 sequence of the reference antibody can be defined according to the Kabat coding system.
  • the HCDR3 of the reference antibody has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the following group: X 1 .
  • X 1 HYGTSPFVY (SEQ ID NO: 53), wherein X 1 can be D or E.
  • the HCDR3 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:9 and SEQ ID NO:10.
  • the HCDR3 sequence of the reference antibody can be defined according to the Kabat coding system.
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the HCDR2 sequence of the reference antibody can be defined according to the Kabat coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3.
  • the HCDR1 sequence of the reference antibody can be defined according to the Kabat coding system.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:9
  • the amino acid sequence shown may comprise antibody 19D4F1, hu19D4-25, 19D4-25-1A3, 19D4-25-1B3, 19D4-25-1C2, 19D4-25-1E4, 19D4-25-2E10 or have the same HCDR3 (e.g., have the same HCDR1-3 as the antigen-binding fragment).
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:6; and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:10 The amino acid sequence shown.
  • the reference antibody may comprise antibody 19D4-25-3C11, 19D4-25-1C2-3C11, or an antigen-binding fragment having the same HCDR3 (eg, having the same HCDR1-3) therewith.
  • the reference antibody may comprise a heavy chain variable region, which may comprise the amino acid sequence shown in SEQ ID NO:54.
  • the reference antibody comprises a VH having amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group consisting of: X1 , X5 , X13 , X16 , X17 , X20 , X37 , X48 , X67 , X68 , X73 , X76 , X79 , X82 , X83 , X85 , X86 , X 87 , X 88 , X 92 , X 98 and X 118 .
  • the heavy chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:29 to SEQ ID NO:31.
  • the reference antibody may comprise a heavy chain constant region, which may comprise an IgG-derived constant region or an IgY-derived constant region.
  • the heavy chain constant region of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO:48.
  • the reference antibody may comprise at least one CDR in the variable region VL of the light chain of the antibody, and the VL may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the VL of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.
  • the LCDR of the isolated reference antibody can be divided in any form, as long as the VL is identical to the amino acid sequence shown in any one of SEQ ID NO: 32 to SEQ ID NO: 38, it can be divided in any form to obtain All LCDRs can fall within the protection scope of the present application.
  • the reference antibody may comprise a light chain variable region VL, which may comprise at least one, at least two or at least three of LCDR1, LCDR2 and LCDR3.
  • the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:59.
  • the LCDR3 of the reference antibody can be defined according to the Kabat numbering system.
  • the LCDR3 of the reference antibody has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 1 , X 4 , X5 , X7 and X9 .
  • X 1 QSX 4 X 5 VX 7 WX 9 (SEQ ID NO: 59), wherein, X 1 can be Q or S, X 4 can be K, L or S, X 5 can be E, H, K or R, X 7 can be N or P, and X 9 can be S or T.
  • the LCDR3 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:21 to SEQ ID NO:26.
  • the LCDR3 of the reference antibody can be defined according to the Kabat numbering system.
  • the LCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:18.
  • the LCDR2 of the reference antibody can be defined according to the Kabat numbering system.
  • the LCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:15.
  • the LCDR1 of the reference antibody can be defined according to the Kabat numbering system.
  • the LCDR1 of the reference antibody described in the present application may comprise the amino acid sequence shown in SEQ ID NO: 15; the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 18; and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 21.
  • the reference antibody can comprise antibody 19D4F1, hu19D4-25, 19D4-25-3C11, or an antigen-binding fragment that has the same LCDR3 (eg, has the same LCDR1-3) therewith.
  • the reference antibody can comprise antibody 19D4-25-1A3 or an antigen-binding fragment that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
  • the reference antibody can comprise antibody 19D4-25-1B3 or an antigen-binding fragment that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
  • the reference antibody can include antibody 19D4-25-1C2, 19D4-25-1C2-3C11, or an antigen-binding fragment that has the same LCDR3 (eg, has the same LCDR1-3) therewith.
  • the reference antibody can comprise antibody 19D4-25-1E4 or an antigen-binding fragment that has the same LCDR3 as it (e.g., has the same LCDR1-3 as it).
  • the reference antibody can comprise antibody 19D4-25-2E10 or an antigen-binding fragment that has the same LCDR3 as it (eg, has the same LCDR1-3 as it).
  • the reference antibody may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:60.
  • the VL of the reference antibody has amino acid substitutions (eg, conservative amino acid substitutions, etc.) at one or more amino acids selected from the group: X 7 , X 9 , X 12 , X 14 , X 15 , X 18 , X 20 , X 41 , X 49 , X 64 , X 76 , X 78 , X 80 , X 81 , X 82 , X 84 , X 85 , X 87 , X 88 , X 89 , X 93 , X 96 , X 97 , X 99 , X 101 and X 108 .
  • DIVLTQX 7 PX 9 SLX 12 VX 14 X 15 GQX 18 AX 20 ISCRASESVDNYGISFMNWFX 41 QKPGQPPX 49 LLIYAASNQGSGVPX 64 RFSGSGSGTDFX 76 LX 78 IX 80 X 81 X 82 EX 84 X 85 DX 87 X 88 X 89 YFCX 93 QSX 96 X 97 VX 99 WX 101 FGGGTKX 108 EIK (SEQ ID NO:60), wherein, X 7 can be S or T, X 9 can be A or L, X 12 can be A or S, X 14 can be S or T, X 15 can be L Or P, X 18 can be P or R, X 20 can be S or T, X 41 can be L or Q, X 49 can be K or Q, X 64 can be A or D, X 76 can be S or T , X 78 can be K or N,
  • the light chain variable region of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NO:32 to SEQ ID NO:38.
  • the reference antibody may comprise a light chain constant region, which may comprise a constant region derived from Ig ⁇ or a constant region derived from Ig ⁇ .
  • the light chain constant region may comprise a constant region derived from Ig ⁇ .
  • the light chain constant region of the reference antibody comprises the amino acid sequence shown in SEQ ID NO:47.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the reference antibody can include antibody 19D4F1, hu19D4-25, or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as it.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the reference antibody can comprise antibody 19D4-25-1A3 or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1A3.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the reference antibody can comprise antibody 19D4-25-1B3 or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1B3.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the reference antibody can comprise antibody 19D4-25-1C2 or an antigen-binding fragment having the same HCDR3 (eg, the same HCDR1-3) and LCDR3 (eg, the same LCDR1-3) as antibody 19D4-25-1C2.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:25.
  • the reference antibody can comprise antibody 19D4-25-1E4 or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as it.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:9; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:26.
  • the reference antibody can include antibody 19D4-25-2E10 or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-2E10.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:10; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:21.
  • the reference antibody can comprise antibody 19D4-25-3C11 or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-3C11.
  • the reference antibody may comprise HCDR1-3 and LCDR1-3.
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:3; the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:6; the HCDR3 of the reference antibody may comprise Comprising the amino acid sequence shown in SEQ ID NO:10; the LCDR1 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:15; the LCDR2 of the reference antibody may include the amino acid sequence shown in SEQ ID NO:18 Sequence; the LCDR3 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the reference antibody can include antibody 19D4-25-1C2-3C11 or an antigen-binding fragment that has the same HCDR3 (eg, has the same HCDR1-3) and LCDR3 (eg, has the same LCDR1-3) as antibody 19D4-25-1C2-3C11.
  • the application provides one or more polypeptides, which may comprise an isolated antigen binding protein of the application.
  • the polypeptide can include a fusion protein.
  • the polypeptides can include multispecific antibodies (eg, bispecific antibodies).
  • the application provides one or more immunoconjugates, which may comprise an isolated antigen binding protein of the application.
  • the immunoconjugate may further comprise a pharmaceutically acceptable therapeutic agent, label and/or detection agent.
  • the present application also provides one or more isolated nucleic acid molecules that encode the isolated antigen-binding protein described herein.
  • each of the one or more nucleic acid molecules may encode the entirety of the antigen binding protein, or may encode a portion thereof (e.g., HCDR1-3, one of the heavy chain variable regions, or variety).
  • the products encoded by the nucleic acid molecules together can form a functional (for example, capable of binding to PD-1) isolated antigen-binding protein of the present application.
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.
  • the isolated nucleic acid can be a nucleic acid molecule prepared by recombinant DNA techniques.
  • nucleic acid encoding the isolated antigen-binding protein can be prepared by various methods known in the art, including but not limited to, using reverse transcription PCR and PCR to obtain the isolated antigen-binding protein described in the application. protein nucleic acid molecule.
  • the present application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • Each vector may contain one or more such nucleic acid molecules.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host.
  • control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like.
  • the expression control sequences are regulatable elements.
  • the specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may comprise a promoter region which may comprise a promoter sequence for transcriptional control of the functionally linked nucleic acid.
  • the expression control sequences may also include enhancer sequences or upstream activator sequences.
  • suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, human U6 RNA promoters, CMV promoters, and artificial hybrid promoters thereof (such as CMV), wherein the promoter's Portions may be fused to portions of gene promoters of other cellular proteins (eg, human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described herein can be operably linked to the expression control element.
  • Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering.
  • the vector may be an expression vector.
  • the vector can be a viral vector.
  • Viral vectors may be administered directly to the patient (in vivo) or may be indirect, for example, in vitro by treating cells with virus and then administering the treated cells to the patient (ex vivo).
  • Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers.
  • Lentiviral vectors may contain long terminal repeat 5'LTR and truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and/or post-translational regulatory element (WPRE).
  • the vectors described herein can be introduced into cells.
  • the present application provides a cell.
  • the cells may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, one or more nucleic acid molecules and/or one or more carriers described herein .
  • each or each cell may comprise one or more of the nucleic acid molecules or vectors described herein.
  • each or each cell may contain multiple (eg, 2 or more) or multiple (eg, 2 or more) nucleic acid molecules or vectors described herein.
  • the vectors described herein can be introduced into said host cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS/0 cells, HEK293T cells, 293F cells or HEK293A cells, or other eukaryotic cells, Such as cells from plants, fungal or yeast cells, etc.
  • the vectors described in this application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection and the like.
  • the cells can include yeast cells.
  • the cells may include E. coli cells.
  • the cells can include mammalian cells.
  • the cells can include immune cells.
  • the cells may include immune cells.
  • the cells may include immune cells.
  • the cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells cell.
  • NK natural killer
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may comprise the isolated antigen binding protein described herein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, the cell, and/or Or pharmaceutically acceptable adjuvants and/or excipients.
  • the pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and /or nonionic surfactants. Unless incompatible with the cells described herein, any conventional media or reagents are contemplated for use in the pharmaceutical compositions of the present application.
  • the pharmaceutically acceptable excipients may include additives other than the main drug in the pharmaceutical preparation, and may also be referred to as auxiliary materials.
  • the excipients may include binders, fillers, disintegrants, lubricants in tablets.
  • the excipients may include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills.
  • the excipient may comprise the base part of a semi-solid formulation ointment, cream.
  • the excipients may include preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations.
  • the present application provides a method for detecting or measuring PD-1, which may include using the isolated antigen-binding protein or the polypeptide.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the method may include a method for detecting the presence and/or amount of PD-1 for non-diagnostic purposes, which may include the steps of:
  • the present application provides a PD-1 kit, which may include the use of the isolated antigen-binding protein or the polypeptide.
  • the kit may further include instructions for use, which describe methods for detecting the presence and/or content of PD-1.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the present application provides a use of the isolated antigen-binding protein or the polypeptide in the preparation of a kit, and the kit can be used in a method for detecting the presence and/or content of PD-1.
  • the methods may include in vitro methods, ex vivo methods, methods for non-diagnostic or non-therapeutic purposes.
  • the present application provides a method for modulating an immune response, comprising administering to a subject in need an effective amount of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, Said isolated nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition, and/or a pharmaceutically acceptable therapeutic agent.
  • the method for modulating immune response may include in vitro method, ex vivo method, non-diagnostic or non-therapeutic method.
  • the present application provides a method for regulating immune response, which comprises administering an effective amount of the drug combination, and/or a pharmaceutically acceptable therapeutic agent to a subject in need.
  • the method for modulating immune response may include in vitro method, ex vivo method, non-diagnostic or non-therapeutic method.
  • the application provides the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the carrier, and the pharmaceutical composition for preventing, alleviating and/or treat a disease or condition.
  • the disease or condition may include tumors.
  • the tumor can include a non-solid tumor.
  • the tumor can include a tumor associated with expression of PD-L1.
  • the term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance.
  • the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance.
  • the tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor.
  • the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • the application provides a kind of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said carrier, said cell and /or the use of the pharmaceutical composition in the preparation of medicaments for preventing, alleviating and/or treating diseases or conditions.
  • the disease or condition may include tumors.
  • the tumor can include a solid tumor.
  • the tumor can include a non-solid tumor.
  • the tumor can include a tumor associated with expression of PD-L1.
  • the term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance.
  • the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance.
  • the tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor.
  • the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • the present application provides a method for preventing and/or treating a disease or disorder, comprising administering the isolated antigen-binding protein, the isolated nucleic acid molecule, the The carrier, the cell, the pharmaceutical composition.
  • the disease or condition may include tumors.
  • the tumor can include a non-solid tumor.
  • the tumor can include a tumor associated with expression of PD-L1.
  • the term "tumor associated with the expression of PD-L1" generally refers to a tumor formed due to changes in the expression of PD-L1 leading to disease progression or escape from immune surveillance.
  • the "tumor associated with the expression of PD-L1" may be a tumor formed by the upregulation of PD-L1 expression leading to disease progression or escape from immune surveillance.
  • the tumor associated with the protein expression of PD-L1 may be a PD-L1 positive tumor.
  • the protein expression of PD-L1 on the surface of tumor cells or in the tumor microenvironment is about 1%, 5%, 10%, 15%, 20%, 25% higher , 30%, 35%, 40%, 50%, 60%, 70%, 80% or higher.
  • the pharmaceutical compositions and methods described herein can be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, and the like.
  • the pharmaceutical compositions and methods described in this application can be used in other disease conditions that depend on immune responses, such as inflammation, immune diseases and infectious diseases.
  • the subject may include humans or non-human animals.
  • the non-human animal can be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice and rats.
  • non-human animals may also include any animal species other than humans, such as livestock animals, or rodents, or primates, or domestic animals, or poultry animals.
  • the human can be Caucasian, African, Asian, Semitic, or other ethnicity, or a hybrid of various ethnicities.
  • the human can be elderly, adult, adolescent, child or infant.
  • the effective amount in humans can be inferred from the effective amount in experimental animals.
  • Freireich et al. describe the correlation of doses in animals and humans (based on milligrams per square meter of body surface) (Freiheim et al., Cancer Chemother. Rep. 50, 219 (1966)).
  • Body surface area can be approximately determined from the patient's height and weight. See, eg, Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 537 (1970).
  • the anti-PD-1 antibody was produced by immunizing mice by intramuscular injection of a plasmid encoding human PD1 with a gene gun, and the feeding environment: SPF grade. After the mice were purchased, they were bred in a laboratory environment for one week, and the mice that had adapted to the environment were immunized with intramuscular injection of a plasmid encoding human PD1 and cardiotoxin adjuvant with a gene gun. Multiple immunizations were carried out, during which the serum antibody level was monitored, and mice with high antibody titers in the serum were selected for booster immunization. After that, the mice were sacrificed, spleen cells were collected, and fused with myeloma cells. Fusion of spleen lymphocytes and myeloma cells Sp2/0 cells to obtain hybridoma cells. The preparation method of hybridoma can be carried out according to the methods published in many documents in this field.
  • Antigen binding protein inhibits the binding of PD-L1 and PD-1 ELISA experiment
  • PD1 protein Label PD1 protein with biotin (Sulfo-NHS-Biotin, Pierce), dilute with PBS buffer; coat PD-L1 protein in 96-well plate (1ug/mL, 100uL/well, overnight at 4°C); add 200uL/ The wells were blocked with blocking solution, and the plate was washed 3 times with PBST; anti-PD-1 antibody (starting from 5ug/mL, diluted 2 times to 0.078ug/mL, 7 dilution points) and PD-1 [B ] (0.5ug/mL, within the linear range of PD1-PDL1 binding), incubate for 60 minutes, wash the plate 3 times with PBST; add HRP*-streptavidin, and detect the OD value by ELISA.
  • biotin Sulfo-NHS-Biotin, Pierce
  • the results are shown in Figure 1.
  • the antigen-binding protein of the present application can effectively inhibit the binding of PD-L1 and PD-1, and the inhibitory effect is equivalent to that of the positive control antibody (Pembrolizumab).
  • the IC50 value is shown in Table 1.
  • Antigen binding protein 19D4F1 (0.5 mg) was labeled with biotin. Dilute with PBS buffer; coat PD-1 protein in a 96-well plate (0.1ug/mL, 100uL/well, overnight at 4°C); add blocking solution to block, 200uL/well, wash the plate 3 times with PBST; each well Add unlabeled antigen-binding protein 19D4F1 (2ug/mL, 40x detection antibody) and incubate for 30 minutes; add detection antibody (biotin-labeled antigen-binding protein 19D4F1, 50ng/mL) to the corresponding wells, incubate for 60 minutes, and use Wash the plate 3 times with PBST; add HRP*-streptavidin, and detect the OD value by ELISA.
  • Table 2-Table 3 19D4F1 can compete with Pembrolizumab (trade name: Keytruda) for binding to PD-1.
  • VH/VL CDR residues were identified and annotated with the Kabat numbering system.
  • the typical structures of VH/VL CDRs were determined from the published literature. Based on the canonical structure of VH/VL CDRs, human germline framework acceptors with the same canonical structure were selected.
  • the Q1E mutation is to eliminate the formation of N-terminal pyroglutamate. Mutations may also include mutations identified from CDR binding regions based on three-dimensional structural homology modeling.
  • the JH region of the humanized antibody was selected based on optimal sequence homology.
  • VH/VLs Synthetically designed VH/VLs, recombinant DNA constructs are used to express humanized antibodies on a human IgG4/k wild-type backbone.
  • Various humanized LC/HC constructs will be co-expressed in parallel, which will generate a panel of antibodies expressed by 293 cells. These antibodies will be formulated in PBS pH 7.4 (default). Titers and yields will be recorded.
  • Each purified human antibody will be analyzed for physicochemical properties.
  • Immobilized anti-human Fc With HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA and 0.05% P20, pH 7.4) as the running buffer, the target protein immobilization level was set to 8000RU. Prepare enough 50 mM NaOH, 50 mM N-hydroxysuccinimide (NHS), 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 20 ⁇ g/mL Anti-human Fc (diluted in 10mmol/L NaAc pH 4.5) and 1M ethanolamine, and immobilized anti-human Fc to flow cells 1 and 2 using the wizard method. To maintain equilibrium after fixation, the buffer flow rate was maintained at 10 [mu]L/min.
  • each kinetic cycle included capture of the tested antibody, injection of serially diluted PD-1, and surface regeneration.
  • Antibodies to be tested are captured onto flow cell 2 and flow cell 1 is used as a reference.
  • the diluted PD-1 fraction was then injected into flow cells 1 and 2 (30 ⁇ L/min) with a correlation time of 180 s. Buffer flow was maintained for 900 seconds for dissociation.
  • regeneration was performed using a 30 s injection of 10 mM glycine-hydrochloric acid (pH 1.5).
  • the isolated human PBMCs were diluted with RPMI-1640 medium and distributed to cell culture dishes. Incubate at 37°C for 3 hours; (2) Absorb and discard the supernatant. Add fresh medium containing IL-4 and GM-CSF. Incubate at 37°C for 1 day; (3) Absorb and discard the supernatant. Add fresh medium containing IL-4 and GM-CSF. Incubate at 37 °C for 1 day. Incubate at 37°C in an incubator; (4) replace one-third of the cell culture medium with fresh IL-4 and GM-CSF on days 3 and 5; (5) mature DC cells with LPS, Wash twice with PBS.
  • CD4+ T cells were purified from PBMCs from another donor according to the manufacturer's guidelines (EasySep TM Human CD4+ T Cell Isolation Kit) procedure.
  • the experimental results are shown in Fig. 2a-2b.
  • the antigen binding protein of the present application can promote the release of IL-2 and IFN- ⁇ from lymphocytes.
  • the oligonucleotides used for codon-based mutagenesis were synthesized by Shanghai Baisheng Biotechnology Co., Ltd. CDRH3 and CDRL3 were selected for mutation. These two CDRs of the VH and VL chains are targets for mutagenesis because they occur in the center of the antibody binding interface and thus for affinity maturation.
  • the VH and VL fragments of hu19D4-25 were amplified using codon-based oligonucleotides.
  • the VH and VL fragments were combined into scFv by overlap PCR.
  • the scFv fragment was digested with Sfi I and cloned into pCANTab5E vector digested with the same restriction enzymes.
  • the ligation product was purified using the QIAquick purification kit from Qiagen, and electroporated into TG1 cells at a voltage of 2.5KV. Transformed cells were resuspended in 2YT medium and incubated at 37°C and 250 rpm for 1 hour. On 2YT-GA agar plates, have 10 ⁇ l of cell suspension for each dilution to determine transformation efficiency. The rest of the cells in suspension were plated on 25 x 25 cm plates and incubated overnight at 37 °C. Colonies were collected from the plates and stored at -80 °C with the addition of glycerol. The library was validated by sequencing the clones on the plates used to determine transformation efficiency.
  • the suspension was centrifuged at 3200 g for 10 minutes, and the supernatant was collected.
  • One-fifth volume of PEG solution (20% PEG6000, 2.5M NaCl) was added and incubated on ice for 1 hour. Centrifuge the phage at 3200xg for 30 minutes, discard the supernatant, and resuspend the phage with 1ml PBS. Centrifuge again at 16,000 g to remove debris and residual cells, and transfer the supernatant to a new tube. Phage preparations were titrated and used for panning.
  • the beads carrying the phage in solution were separated in a magnetic particle concentrator, and the supernatant was discarded. Beads were washed with fresh wash buffer, 10 times with PBST and 10 times with PBS (pH 7.4). Add 1 ml of 10 ⁇ g/ml trypsin (Sigma, USA) diluted in PBS, and incubate at 37° C. for 30 minutes to elute the phage. Titrate the resulting phage and prepare for the next round of panning, decreasing the antigen concentration round by round.
  • Clones were selected, inoculated into each well of a 96-well U-bottom plate containing 2YT-AG medium, and cultured overnight in a microplate shaker at 37°C and 1000rpm.
  • To prepare stock plates dispense 50 ⁇ l of cell culture into fresh plates, mix with 50 ⁇ l of 50% glycerol and store at -80 °C.
  • Another 96-well U-bottom plate containing 2YT-A medium (0.05% glucose) was inoculated with the overnight culture and incubated at 37° C. and 1000 rpm on a microplate shaker for 2 hours. 1M IPTG was added to the medium to induce scFv expression at a final concentration of 1 mM.
  • each well was coated with 200 ng of Neutravidin (Pierce, USA) diluted in PBS, overnight at 4°C. Plates were washed three times with PBST and blocked with blocking buffer (PBST+1% BSA) for 1 hour at room temperature. Plates were washed three times with PBST, and 100 ng of biotinylated antigen was added for 1 hour at room temperature. After washing 3 times with PBST, the supernatant was added and incubated at room temperature for 1 hour.
  • anti-myc-tag HRP-conjugated antibody (Abcam, USA) diluted 1:10000 was added to blocking buffer and incubated at room temperature for 1 hour. Wash the plate three times with PBST, then add 100 ⁇ l of freshly prepared TMB solution and incubate for 15 min. Add 100 ⁇ l 2M H 2 SO 4 to stop color development, and measure OD450 by ELISA. ELISA positive clones were identified and sent for sequencing.
  • the clones were inoculated with 4 ml of 2YT-GA medium and cultured overnight at 37 °C and 250 rpm. Another 4ml of 2YT-A medium (0.05% glucose) was used to inoculate 40 ⁇ l of overnight culture, and cultivated at 37°C and 250rpm until OD600 ⁇ 0.5. IPTG (1M) was added to the medium at a final concentration of 1 mM to induce scFv expression. Incubate overnight at 30 °C and 250 rpm, centrifuge at 3200 g for 15 minutes, and discard the supernatant.
  • the cell pellet was resuspended in 200 ⁇ l of ice-cold 1XTES buffer (0.2M Tris-HCl, 0.5mM EDTA, 0.5M sucrose, pH 8.0). Then add 300 ⁇ l 1/5XTES buffer and vortex to resuspend. Cell pellets were incubated on ice for 30 minutes and centrifuged at 12000 g for 10 minutes. Transfer the supernatant containing soluble antibody fragments to a clean tube for off-rate check.
  • 1XTES buffer 0.2M Tris-HCl, 0.5mM EDTA, 0.5M sucrose, pH 8.0.
  • N297 was mutated to Alanin to remove the glycosylation site.
  • W52F refers to the 52nd amino acid changed from W to F.
  • test sample antigen binding protein
  • each kinetic cycle included capture of FcRn, injection of serially diluted test antibodies, and surface regeneration.
  • 0.4 ⁇ g/mL of FcRn was captured to flow cell 2 of each channel, and flow cell 1 of the same channel was used as reference.
  • Serially diluted samples were then injected (30 ⁇ l/min) in two flow cells per channel and dissociated.
  • interface regeneration was performed using 30 s injections of 10 mM glycine-HCl (pH 1.5).
  • 1C2-A refers to the antigen-binding protein obtained by fusing 19D4-25-1C2 with YTE mutant Fc
  • 1C2-B refers to the antigen-binding protein obtained by fusing 19D4-25-1C2 with unmodified Fc
  • 1C2-3C11-A refers to the antigen binding protein obtained after fusion of 19D4-25-1C2-3C11 and YTE mutant Fc
  • 3C11-A refers to the fusion of 19D4-25-3C11 and YTE mutant Fc antigen binding protein.
  • 1C2-A refers to the antigen-binding protein obtained by fusing 19D4-25-1C2 with YTE mutant Fc
  • 1C2-B refers to the antigen-binding protein obtained by fusing 19D4-25-1C2 with unmodified Fc Protein
  • 1C2-3C11-A refers to the antigen binding protein obtained after fusion of 19D4-25-1C2-3C11 and YTE mutant Fc
  • 3C11-A refers to the fusion of 19D4-25-3C11 and YTE mutant Fc
  • WT-A refers to the antigen-binding protein obtained after the fusion of hu19D4-25 and YTE mutant Fc.
  • 1C2-Agly-YTE represents the antigen-binding protein obtained by fusing 19D4-25-1C2 with Fc with YTE mutation and N297 mutation
  • 1C2-Agly represents the fusion of 19D4-25-1C2 with Fc with N297 mutation
  • 1C2-3C11-Agly-YTE refers to the antigen-binding protein obtained by fusing 19D4-25-1C2-3C11 with YTE mutation and N297 mutation Fc
  • 3C11-Agly-YTE refers to 19D4- 25-3C11 is the antigen-binding protein obtained by fusing Fc with YTE mutation and N297 mutation
  • WT-Agly-YTE refers to the antigen-binding protein obtained by fusing hu19D4-25 with Fc with YTE
  • affinity increase factor KD value of antibody/KD value of WT-AGY-YTE. As shown in Table 10, the affinity of 1C2-3C11-A for FcRn is 10 times that of pembrolizumab.
  • PD1 protein Label PD1 protein with biotin (Sulfo-NHS-Biotin, Pierce), dilute with PBS buffer; coat PD-L1 protein in 96-well plate (1ug/mL, 100uL/well, overnight at 4°C); add 200uL/ The wells were blocked with blocking solution, and the plate was washed 3 times with PBST; anti-PD-1 antibody (starting from 5ug/mL, diluted 2 times to 0.078ug/mL, 7 dilution points) and PD-1 [B ] (0.5ug/mL, within the linear range of PD1-PDL1 binding), incubate for 60 minutes, wash the plate 3 times with PBST; add HRP*-streptavidin, and detect the OD value by ELISA.
  • biotin Sulfo-NHS-Biotin, Pierce
  • Antibody IC50(ug/ml) Pembrolizumab 0.2445 1C2-3C11-W52F-Agly-YTE 0.4953 1C2-Agly-YTE 0.4916
  • WT-Ag-YTE the antigen-binding protein obtained after fusion of hu19D4-25 without affinity maturation and Fc of YTE mutation and N297 mutation
  • WT-Ag-YTE has the ability to stimulate IL-2 secretion, all Affinity-matured antibodies all significantly stimulated IL-2 secretion.

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Abstract

L'invention concerne un anticorps anti-PD-1 et son utilisation. L'anticorps anti-PD-1 se lie spécifiquement à une protéine PD-1 humaine ayant une valeur KD inférieure ou égale à environ 5E-07 M, et peut bloquer la liaison de PD-1 à PD-L1. La présente invention concerne en outre la modification de la région Fc dans l'anticorps et un immunoconjugué contenant l'anticorps anti-PD-1, un procédé de préparation de l'anticorps anti-PD-1, ainsi que l'utilisation de l'anticorps anti-PD-1.
PCT/CN2022/105210 2021-07-16 2022-07-12 Anticorps anti-pd-1 et son utilisation WO2023284741A1 (fr)

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