WO2020143749A1 - 重组抗人pd-1抗体及其应用 - Google Patents

重组抗人pd-1抗体及其应用 Download PDF

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WO2020143749A1
WO2020143749A1 PCT/CN2020/071353 CN2020071353W WO2020143749A1 WO 2020143749 A1 WO2020143749 A1 WO 2020143749A1 CN 2020071353 W CN2020071353 W CN 2020071353W WO 2020143749 A1 WO2020143749 A1 WO 2020143749A1
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seq
antibody
fragment
amino acid
variable region
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PCT/CN2020/071353
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French (fr)
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张锦超
王荣娟
焦莎莎
王双
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迈威(上海)生物科技有限公司
北京科诺信诚科技有限公司
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Priority to EP20738344.9A priority Critical patent/EP3919514A4/en
Priority to JP2021540076A priority patent/JP2022517216A/ja
Priority to US17/422,123 priority patent/US20230104769A1/en
Priority to CA3126302A priority patent/CA3126302A1/en
Priority to KR1020217024728A priority patent/KR20210113635A/ko
Publication of WO2020143749A1 publication Critical patent/WO2020143749A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of antibody drugs, and in particular, the present invention relates to antibodies against human PD-1 and their use for the preparation of drugs.
  • PD-1 Programmed cell death protein 1
  • CD279 is a member of the CD28 family of T cell receptors and is expressed on the surface of various immune cells, such as T cells, B cells, monocytes, etc.
  • the key ligands of PD-1 include PD-L1 (B7-H1) and PD-L2 (B7-DC), which are expressed by immune cells and can also be induced on a variety of tissues when PD-1 on T cells When combined with PD-L1 or PD-L2, T cells receive inhibitory signals, thereby inhibiting T cell proliferation and cytokine production, which will effectively reduce the immune response that T cells participate in.
  • PD-L1 and PD-L2 are highly expressed on a variety of human tumor cells, so that tumor cells evade the supervision of T cells (Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12 (4), 252 –264(2012)).
  • Nivolumab a fully human antibody against human PD-1, has been shown to inhibit the binding of PD-1 to PD-L1 and PD-L2. It is clinically used to treat advanced melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, urothelial carcinoma, colorectal cancer, and hepatocellular carcinoma; the humanized antibody against human PD-1, pembrolizumab (Perbrolizumab), shows inhibition of PD-1 and PD- The combination of L1 and PD-L2 is clinically used to treat malignant melanoma, non-small cell lung cancer, classic Hodgkin lymphoma, head and neck squamous cell carcinoma, d-mmr mutation or MSI-H malignant tumor (Alsaab et al.
  • Libtayo monoclonal antibody (Cemiplimab) against human PD-1 can bind to PD-1 and block its interaction with PD-L1 and PD-L2, releasing the PD-1 pathway-mediated suppression of immune responses, including anti-tumor immunity Response, clinically used to treat patients with metastatic skin squamous cell carcinoma (CSCC) or patients with locally advanced CSCC (Markham, A. & Duggan, S. Drugs. 2018. doi: 10.1007/s40265-018-1012-5).
  • CSCC metastatic skin squamous cell carcinoma
  • the indications cover relapsed and refractory malignant lymphoma, Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, alveolar soft tissue sarcoma, Advanced or recurrent malignant tumor, esophageal cancer, gastric cancer or gastroesophageal junction cancer, nasopharyngeal cancer, head and neck squamous cell carcinoma, lung cancer, metastatic colorectal cancer, localized renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma , Melanoma, advanced triple negative breast cancer, pleural mesothelioma, advanced neuroendocrine tumors, unresectable or d-mmr mutations or MSI-H solid tumors.
  • anti-PD-1 antibodies there are three varieties of anti-PD-1 antibodies on the market, and many varieties are under clinical research.
  • the patents involved include: WO2004004771, WO2006121168, WO2008156712, WO2010029435, WO2012145493, WO2015085847, US20170044260, CN104250302, CN105330740, CN105531288 and so on.
  • anti-PD-1 antibodies can be obtained through hybridoma screening, humanization, or through phage display or yeast surface display technology, or through transgenic mouse technology.
  • the method of obtaining anti-human PD-1 antibody in WO2015085847 is: using hybridoma technology to obtain mouse antibodies, and after obtaining candidate mouse antibodies through antibody activity analysis (ELISA (binding, blocking), affinity kinetics);
  • ELISA binding, blocking
  • the sequence of the variable region of the chain gene was cloned upstream of the sequence encoding the constant region of the light and heavy chains of human antibodies, and expressed in mammalian cells to prepare chimeric antibodies, and then by antibody activity analysis, blocking PD-1 and its ligand binding experiments, and CD28 other Family member binding experiment, cell surface PD-1 binding experiment, and in vitro cytology experiment to determine the lead antibody; select the humanized template according to the Germline database, humanize the antibody sequence design, and obtain the humanized antibody again through antibody activity Analysis, blocking PD-1 and its ligand binding experiment, CD28 and other family members binding experiment, and cell surface PD-1 binding experiment, after in vitro cytology experiment verification; and then carry out humanized antibodies to inhibit tumor cell growth in
  • Antibody antigen recognition epitopes are one of the important characteristics of antibodies. Anti-PD-1 antibodies play a role in activating T cells by blocking the binding of PD-1 to its ligand PD-L1/PD-L2. Although the antibodies reported so far can block the interaction between PD-1 and ligands, the epitopes they target are not exactly the same (Tan S, Zhang H, Chai Y, et al. An unexpected N- terminal Loop in PD-1 dominates binding by nivolumab.Nat Commun. 2017; 8:14369). The difference in epitope may lead to unique characteristics of antibody in terms of drug efficacy and safety.
  • the technical problem to be solved by the present invention is to obtain high affinity antibodies that specifically bind to human PD-1 through hybridoma screening and humanization technology, which can not only effectively block PD-1/PD-L1, PD-1/ PD-L2 interacts and has a unique antigen-binding epitope, so it has unique properties in terms of drug efficacy and safety.
  • the object of the present invention is to provide an antibody or functional fragment thereof that specifically binds to human PD-1, and provide its use.
  • the invention provides an antibody or fragment thereof that binds to programmed cell death protein 1 (PD-1), the antibody or fragment thereof comprising a light chain variable region and/or a heavy chain variable region, wherein the The light chain variable region and/or the heavy chain variable region comprise the light chain CDR1 (LCDR1), CDR2 (LCDR2), CDR3 (LCDR3) and/or heavy chain CDR1 (HCDR1), CDR2 (HCDR2) shown in the following sequence, respectively , CDR3 (HCDR3):
  • the light chain variable region and/or the heavy chain variable region respectively include LCDR1, LCDR2, LCDR3 and/or HCDR1, HCDR2, HCDR3 shown in the following sequence:
  • the light chain variable region and/or the heavy chain variable region respectively include LCDR1, LCDR2, LCDR3 and/or HCDR1, HCDR2, HCDR3 shown in the following sequence:
  • LCDR1 selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11;
  • LCDR2 selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17;
  • SEQ ID NO: 18 From SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, LCDR3 of SEQ ID NO: 26 and SEQ ID NO: 27; and/or
  • HCDR1 selected from SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33;
  • SEQ ID NO: 34 From SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, HCDR2 of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46;
  • HCDR3 selected from SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51.
  • the light chain variable region comprises LCDR1, LCDR2, LCDR3 as shown in the following sequence combination:
  • the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 as shown in the following sequence combination:
  • the light chain variable region and the heavy chain variable region comprise LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3 as shown in the following sequence combination:
  • the antibody or fragment thereof provided by the present invention has one or more of the following activities:
  • the antibody or fragment thereof binds to the human PD-1N58 sugar chain; preferably, the heavy chain variable region of the antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO:47.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 52 or SEQ ID NO: 60 or an amino acid sequence having at least 75% identity with the amino acid sequence; and/or,
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 54 or SEQ ID NO: 62 or an amino acid sequence having at least 75% identity with the amino acid sequence;
  • the at least 75% identity is, for example, at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or even 99% identity, etc. ⁇ 75% identity of any percentage.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 52, and the heavy chain variable region includes SEQ ID NO: 54 The amino acid sequence shown; or the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 60, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 62.
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 60 or a variant of the amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 60
  • the variant has one or more, preferably 1-3 amino acid substitutions selected from the group consisting of K24R, E30S, V32A, V32Y, W50A, W50G, H55A, and SEQ ID NO: 60 H55Q, T56S, Y91A, Y91F, S92D, S92N, R93N, R93S, Y94F, W96G and W96Y;
  • the variant has one or more selected from SEQ ID NO: 60, preferably 1-3 Amino acid substitutions: K24R, V32A, V32Y, T56S, S92D, S92N, R93S and Y94F;
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 62 or a variant of the amino acid sequence, according to the residue numbering of the amino acid sequence shown in SEQ ID NO: 62, the variant is relative to SEQ ID NO : 62 has one or more, preferably 1-3 amino acid substitutions selected from S31D, Y32A, Y32N, D33S, D33Y, S52K, S52W, G53S, G53Y, G54D, G54S, G55S, S56G, Y57T, Y59A, Y59T, D100E, D100Y, S101A and Y106T; preferably, the variant has one or more, preferably 1-3 amino acid substitutions selected from the group consisting of S31D, Y32A, D33S, G53S, and SEQ ID NO: 62 G54S, G55S, Y59A and D100Y.
  • the present invention provides a variant antibody or fragment thereof.
  • the heavy chain variable region of the variant antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 62, while the light chain variable region is Compared with the residue number and composition of the amino acid sequence shown in SEQ ID NO: 60, with only the following amino acid substitutions: 1) K24R; 2) E30S; 3) V32A; 4) V32Y; 5) W50A; 6) W50G; 7) H55A ; 8) H55Q; 9) T56S; 10) Y91A; 11) Y91F; 12) S92D; 13) S92N; 14) R93N; 15) R93S; 16) Y94F; 17) W96G; 18) W96Y; 19) K24R and T56S ; 20) K24R and S92N; 21) K24R and Y94F; 22) T56S and S92N; or, 23) K24R, T56S and S92N
  • the present invention provides a variant antibody or fragment thereof.
  • the light chain variable region of the variant antibody or fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 60, while the heavy chain variable region
  • the residue number and composition of the amino acid sequence shown in SEQ ID NO: 62 have only the following amino acid substitutions: 1) S31D; 2) Y32A; 3) Y32N; 4) D33S; 5) D33Y; 6) S52K; 7) S52W 8) G53S; 9) G53Y; 10) G54D; 11) G54S; 12) G55S; 13) S56G; 14) Y57T; 15) Y59A; 16) Y59T; 18) D100E; 19) D100Y; 20) S101A; 21 ) Y106T; 22) D33S and G54S; 23) D33S and Y59A; or, 24) G54SY59A.
  • the present invention provides a variant antibody or fragment thereof, the residue chain number and composition of the light chain variable region of the variant antibody or fragment thereof compared to the amino acid sequence shown in SEQ ID NO: 60, Only the amino acid substitutions of K24R, T56S and S92N, and the heavy chain variable region is compared with the residue number and composition of the amino acid sequence shown in SEQ ID NO: 62, only the amino acid substitutions of D33S and G54S, or only G54S and Y59A Amino acid substitutions.
  • the antibody provided by the present invention may be in any form such as monoclonal antibody, single chain antibody, bifunctional antibody, single domain antibody, nanobody, fully or partially humanized antibody, or chimeric antibody; preferably, the antibody is IgA , IgD, IgE, IgG or IgM, more preferably IgG1 or IgG4;
  • the fragment provided by the present invention is a fragment that the antibody can specifically bind to PD-1 or any part thereof; preferably, the fragment is scFv, BsFv, dsFv, (dsFv) 2 of the antibody, Fab, Fab', F(ab') 2 or Fv fragment.
  • the heavy chain constant region of the antibody provided by the present invention is IgG1 or IgG4 subtype, and the light chain constant region is ⁇ type;
  • the light chain constant region of the antibody comprises the amino acid sequence shown in SEQ ID NO: 56 or SEQ ID NO: 64 or an amino acid sequence having at least 75% identity with the amino acid sequence; and/or
  • the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 58 or SEQ ID NO: 66 or an amino acid sequence having at least 75% identity with the amino acid sequence; wherein the at least 75% identity is, for example, at least 80 %, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity etc. ⁇ 75% Any percentage of identity.
  • the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 52, and the light chain constant region includes the amino acid sequence shown in SEQ ID NO: 56.
  • the chain variable region contains the amino acid sequence shown in SEQ ID NO:54, and the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO:58; or, the light chain variable region contains the amino acid sequence shown in SEQ ID NO:60,
  • the light chain constant region contains the amino acid sequence shown in SEQ ID NO: 62, the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 64, and the heavy chain constant region contains the amino acid sequence shown in SEQ ID NO: 66.
  • the present invention also provides a conjugate or fusion protein comprising the antibody or fragment of the present invention.
  • the conjugate or fusion protein may comprise other parts that are chemically or physically bound to the antibody or fragment thereof according to the invention, such as cell surface receptors, small molecule compounds such as amino acids and carbohydrates, small molecule polymers or the invention Any other part of the antibody that is modified, or even an active protein or polypeptide.
  • the conjugate or fusion protein may be a bispecific antibody comprising the antibody or fragment thereof of the invention.
  • the invention also provides a nucleic acid molecule that encodes the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain or light chain in any antibody or fragment thereof of the invention;
  • the nucleic acid molecule comprises SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65 or The nucleotide sequence shown in SEQ ID NO: 67.
  • the present invention provides a vector comprising the nucleic acid molecule of the present invention.
  • the vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
  • the vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preserving or expressing antibodies.
  • the present invention provides a host cell comprising the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected with the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant or animal cells.
  • the antibody or fragment thereof provided by the present invention and the corresponding conjugate or fusion protein, nucleic acid molecule, vector, and/or host cell can be obtained by using any conventional technical methods known in the art.
  • the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, carrier, and/or host cell may be included in a pharmaceutical composition, and more particularly in a pharmaceutical preparation, so as to be used in various purpose.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or fragment, conjugate or fusion protein, nucleic acid molecule, vector and/or host cell of the present invention, And optional pharmaceutically acceptable excipients.
  • the present invention also provides related applications of the above subject.
  • the present invention provides the antibody or fragment, conjugate or fusion protein, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the present invention are prepared for the prevention or treatment of tumor or Use in cancer medicine.
  • the tumor or cancer is selected from non-small cell lung cancer, classic Hodgkin's lymphoma, gastric cancer, (primary) liver cancer, melanoma, d-mmr mutation or MSI-H malignant tumor, cervical cancer, head and neck squamous cell carcinoma Cancer, urethral bladder cancer, primary mediastinal B-cell lymphoma, renal cell carcinoma, colorectal cancer, and urothelial carcinoma; more preferably, the tumor or cancer is non-small cell lung cancer, melanoma, colorectal cancer, Liver cancer, head and neck squamous cell carcinoma, classic Hodgkin's lymphoma, or urothelial carcinoma.
  • the present invention provides a pharmaceutical combination comprising:
  • the antibody or fragment, conjugate or fusion protein, nucleic acid molecule, carrier, host cell and/or pharmaceutical composition of the present invention (2) other immune enhancing drugs or means, such as small molecule drugs, Targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiation therapy.
  • other immune enhancing drugs or means such as small molecule drugs, Targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiation therapy.
  • the present invention provides a method for preventing or treating a tumor or cancer, the method comprising administering the antibody or fragment, conjugate or fusion protein, nucleic acid molecule, vector, host cell to a subject in need thereof And/or pharmaceutical composition, and optionally other drugs or means.
  • the tumor or cancer is selected from non-small cell lung cancer, classic Hodgkin's lymphoma, gastric cancer, (primary) liver cancer, melanoma, d-mmr mutation or MSI-H malignant tumor, cervical cancer, head and neck squamous cell carcinoma Cancer, urethral bladder cancer, primary mediastinal B-cell lymphoma, renal cell carcinoma, colorectal cancer, and urothelial carcinoma; more preferably, the tumor or cancer is non-small cell lung cancer, melanoma, colorectal cancer, Liver cancer, head and neck squamous cell carcinoma, classic Hodgkin's lymphoma, or urothelial carcinoma.
  • the optional other drugs or means refer to other immune enhancing drugs or means that can be co-administered with the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the present invention, for example Recombinant protein drugs such as small molecule drugs, targeted drugs, antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiotherapy.
  • Recombinant protein drugs such as small molecule drugs, targeted drugs, antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutic drugs, and radiotherapy.
  • the joint administration of the two can take any form, for example, simultaneously, continuously or at certain intervals.
  • the subject is a mammal, preferably, the subject is a human.
  • the invention provides a kit comprising the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and/or pharmaceutical composition of the invention.
  • human PD-1 extracellular domain domain recombinant protein (sequence number: NP_005009.2, 21aa-167aa) is used to spleen cells after immunization of mice, and human PD-1 cells are screened using hybridoma technology
  • Recombinant proteins in the outer domain domain and human PD-1 on the cell surface have high affinity binding, and can specifically block antibody molecules that bind to PD-1/PD-L1, PD-1/PD-L2 ligand receptors (invention) It is named 317), and the humanized antibody with the same affinity and specificity (named h317 in the present invention) is further obtained through humanization technology, and its whole antibody IgG4 is bound, blocked, promotes T cell activation and
  • the antitumor efficacy in vivo was comprehensively evaluated, and the crystal complex of its Fab and PD-1-ECD was constructed, and its antigen-binding epitope was confirmed by crystal diffraction and further mutant verification.
  • the antibodies of the present invention can effectively block the binding of PD-1 and PD-L1/PD-L1, and at the same time have a competitive binding relationship with Nivolumab and Pembrolizumab (Keytruda); however, the crystal structure of the antigen-antibody complex
  • the antigen binding epitope of the antibody of the present invention is different from that of Nivolumab and Keytruda, and is the first antibody molecule that clearly defines the epitope associated with the N-glycosylation site of Glu at position 58 of PD-1.
  • the present invention has the following advantages:
  • the antibody of the present invention is an anti-PD-1 humanized antibody with high affinity.
  • the humanized anti-PD-1 antibody h317 of the present invention can specifically bind to human PD-1 protein, and its affinity (KD) is 6.16E-09M, while the control antibody Nivolumab affinity (KD) is 8.06E-09M, and is equivalent Under conditions, h317 dissociation (kd(1/s)9.50E-04) is better than Nivolumab dissociation (kd(1/s):1.69E-03), indicating that h317 has high affinity with human PD-1, which is h317 It provides a basis for the inhibition of PD-1/PD-L1 signaling pathway.
  • the antibodies of the present invention have good biological activity.
  • h317 of the present invention can effectively block the binding effect of recombinant human PD-1 and its ligands PD-L1 and PD-L2, both of which have IC50 of 1.4nM, and the control antibody Nivolumab (IC50: 1.3nM).
  • the activity is similar, and it is proved in vitro that h317 can promote the secretion of killer cytokines IL-2 and IFN- ⁇ by human mixed lymphocytes, which can induce Th1-type cell-mediated immune response.
  • the antibodies of the present invention have specific antigen recognition epitopes.
  • the antibody of the present invention specifically binds to human PD-1, and the antigen epitope to which it binds is unique.
  • PD-1 antigen extracellular region 1-167aa
  • the binding site of PD-1 and antibody is mainly in its loop area, including BC loop, C’D loop and FG loop area.
  • glycosylation at positions N49, N58, and N116 can be observed in the PD-1 antigen structure, and the glycosylation position at N74 is unclear, in which the sugar chain at position N58 participates in binding to h317-Fab.
  • other reported PD-1 antigen-antibody complex structures at this site did not participate in the interaction.
  • the four glycosylation sites on PD-1 were individually mutated to alanine (A) and transiently transferred to 293 cells, and the h317 and PD-1-mut molecules were detected by FACS. Binding situation, the results showed that the mutation of N58 of PD-1 to A lost glycosylation and h317 lost the binding ability to PD-1, so h317 is the first antibody molecule with a clear epitope associated with the N58 sugar chain.
  • the antibody of the present invention has a significant tumor suppressing effect in the PD-1 transgenic mouse xenograft model.
  • h317 can significantly inhibit the growth of PD-1 transgenic mouse subcutaneous xenografts at high doses (10 mg/kg) and medium doses (2 mg/kg).
  • the anti-tumor effect of the reference antibody Nivolumab is comparable.
  • Figure 1 shows the binding of the hybridoma supernatant detected by ELISA and the recombinant protein of the human PD-1 extracellular domain.
  • 1A, 1B, and 1C show the results of the first round, second round, and third round, respectively. .
  • Figure 2 shows the cross-reactivity between the hybridoma clone supernatant detected by ELISA and recombinant PD-1 of different species, where Nivo is nivolumab.
  • Figure 3 shows the antibody subtype identification of clone 317, which shows that the heavy chain is IgG1 and the light chain is Kappa chain.
  • Figure 4 shows the results of affinity analysis of 317 chimeric antibody (4A) and Nivolumab (4B) on recombinant protein of human PD-1 extracellular domain.
  • Figure 5 shows the results of affinity analysis of h317 antibody (5A) and Nivolumab (5B) on recombinant protein of human PD-1 extracellular domain.
  • Figure 6 shows the ELISA assay of h317 antibody and Nivolumab competitively inhibiting the binding of PD-1 to PD-L1 (6A) and PD-1 to PD-L2 (6B).
  • Figure 7 shows the analysis curve of h317 binding to different species of recombinant PD-1.
  • Figure 8 shows the binding analysis curve of h317 and CD28 family members.
  • Figure 9 shows the detection results of h317 stimulation of MLR to produce IL-2 (9A) and IFN- ⁇ (9B).
  • Figure 10 shows the results of h317-mediated ADCC effect of human PBMC on 293T/hPD-1 cells.
  • Figure 11 shows the tumor volume statistics of h317 inhibiting the growth of PD-1 transgenic mouse subcutaneously transplanted tumors.
  • Figure 12 shows PD-1 mediated endocytosis of h317.
  • Figure 13 shows the overall structure of the PD-1-h317-Fab complex.
  • Figure 14 shows the intermolecular interaction of PD-1-h317-Fab complex, where A is the binding site of PD-1 and h317-Fab, B is the interaction of BC loop and h317-Fab, and C is the C'D loop and h317-Fab interaction, D is FG loop interacts with h317-Fab.
  • Figure 15 shows the interaction of the sugar chain of PD-1N58 with h317-Fab.
  • Figure 16 shows the binding of h317 to human PD-1-mut recombinant protein, in which each antibody and MAPC of WT are used as the standard, the value is 1, the APC of other groups in this experiment is calculated, the three parallel experiments result.
  • Figure 17 shows that h317-Fab and PD-L1 competitively bind to PD-1, where A is the structure comparison of PD-1-h317-Fab complex and PD-1-PD-L1, gray is PD-1, and brick red is PD-L1, blue and green are the heavy and light chains of h317-Fab; B is the comparison of the action surface, gray is the PD-1 molecular surface, green is the 317-Fab action part, and brick red is the PD-L1 action part, Yellow is the part where both 317-Fab and PD-L1 act.
  • Example 1 Screening, identification and antibody sequence determination of anti-human PD-1 antibody hybridoma cell lines
  • mice were immunized with Freund's adjuvant and water-soluble adjuvant. Freund's adjuvant was given two intraperitoneal immunization injections on day 0 and day 14, and water-soluble adjuvant was given on day 0 and day 14. On the 21st day, 8-10 week old Balb/c mice were given two intramuscular injections of mice.
  • the immune antigen was: human PD-1/mFc recombinant protein (serial number: NP_005009.2, 21aa-167aa, Lot: 2016.3. 16) Recombinant expression in HEK293 cells by Beijing Kenuo Xincheng Technology Co., Ltd.
  • the dose for the first immunization was 50 ⁇ g, and the dose for the second immunization was 25 ⁇ g.
  • Freund's adjuvant was taken on the 28th day after the first immunization.
  • the water-soluble adjuvant was taken from the tail vein on the 35th day after the first immunization.
  • 96-well microplate was used to detect serum titers by ELISA; mice with serum titers reaching fusion requirements were boosted. 25 ⁇ g of antigen was diluted with D-PBS to 500 ⁇ l and injected intraperitoneally. On the 38th day after the initial immunization, the spleen cells of mice with high serum titers were taken for the next cell fusion.
  • Cell fusion and hybridoma preparation on day 38 after the initial immunization, select mice with the required titer, remove eyeballs for blood collection, and separate the serum as a positive control serum for antibody detection; and take the spleen of the mouse aseptically to prepare B lymph
  • the cell suspension was mixed with FO myeloma cells at a ratio of 5:1, and the two cells were fused under the action of PEG4000. After the fused cells were resuspended in HAT medium, the 96-well cell culture plates containing macrophages were aliquoted. Incubate at 37°C in a 5% CO 2 incubator. After two weeks, change to HT medium and continue culturing for 2 weeks, then change to R1640 complete medium.
  • Positive hybridoma screening 10-14 days after fusion, with human PD-1/His recombinant protein (sequence number: NP_005009.2, 21aa-167aa, Lot: 2016.2.22) (10 ⁇ g/ml, pH9.6, 0.1M NaHCO 3 ) coated microplate, 4°C, overnight; blocked with 4% skimmed milk powder-PBS, 37°C, 2hr; washed three times with PBST (0.05% Tween20-PBS), added hybridoma clone culture supernatant, 37 °C, 1hr.
  • human PD-1/His recombinant protein sequence number: NP_005009.2, 21aa-167aa, Lot: 2016.2.22
  • PBST 0.05% Tween20-PBS
  • Positive control mouse serum after immunization (diluted 1:1000 with PBS)
  • Negative control mouse serum before immunization (diluted 1:1000 with PBS)
  • blank control PBS. Wash three times with PBST (0.05% Tween20-PBS), add HRP-goat anti-mouse IgG (Fc ⁇ ), 1:20000 dilution, 37°C, 1hr; then wash five times with PBST (0.05% Tween20-PBS), add OPD color developing solution, avoid color development for 10-15min, add 2M H 2 SO 4 to stop the reaction; microplate reader read A490 value.
  • the test well A490 value is greater than the negative control well A490 value by more than 2.1 times and it is judged as positive.
  • Extract 317 hybridoma cells according to the instructions of the TRIzol kit (Cat: 15596026, Invitrogen); use M-MuLV reverse transcriptase (Cat: M0253S, NEB) to reverse transcribe the total RNA of the hybridoma cells into cDNA; Use degenerate primers (Table 1, Table 2) and Phusion kit (Cat: E0553L, NEB) to amplify the antibody light chain variable region IgVL ( ⁇ ) and heavy chain variable region V H sequences; use gel recovery kit ( Cat: AP-GX-250, Axygen) to purify the PCR amplification products; follow the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Bio) to connect the amplified PCR products to the T vector and transform E.
  • M-MuLV reverse transcriptase Cat: M0253S, NEB
  • Use degenerate primers Table 1, Table 2
  • Phusion kit Cat: E0553L, NEB
  • sequence of the variable region of the monoclonal antibody was shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region of the 317 antibody is deduced from the DNA sequence shown in SEQ ID NO: 52; the heavy chain of the 317 antibody can be The nucleotide sequence of the variable region DNA is shown in SEQ ID NO: 55, and the amino acid sequence of the variable region of the 317 antibody heavy chain is deduced from the DNA sequence shown in SEQ ID NO: 54.
  • the cloned monoclonal antibody light chain variable region and heavy chain variable region genes were introduced into the restriction sites by PCR using the following primers (Table 3), and cloned into human-kappa light chain constant region and human IgG4 heavy chain constant region, respectively.
  • human-mouse chimeric light chain (pKN019-317L) and human-mouse chimeric heavy chain (pKN034-317H) expression plasmids were obtained, transferred into E.
  • Plasmids containing human-mouse chimeric antibody light and heavy chains were mixed with 293fectin and co-transfected into HEK293 cells.
  • Cells were transfected for 5-6 days, the culture supernatant was taken, and the expression supernatant was purified using a ProA affinity chromatography column.
  • the obtained 317 chimeric antibody was captured with an anti-human antibody using Fortebio (BLITZ pro1.1.0.28) instrument. Method to determine antibody affinity.
  • the capture antibody (AHC) biological probe of the Fc segment of the anti-human antibody was soaked in PBS for 10 min; 4 ⁇ l of the diluted antibody sample (317 chimeric antibody, Nivolumab, 20 ⁇ g/mL) were loaded onto 2 AHC organisms The probe was then equilibrated in PBS for 30s, and the AHC probe was further diluted with human PD-1-His recombinant protein (sequence number: NP_005009.2, 21aa-167aa, Lot: 2016.2.22) (1 ⁇ M, 0.6 ⁇ M and 0nM) for the binding reaction, the binding time is 45s, and then the AHC probe is transferred to PBS for dissociation reaction, the time is 120s. After the experiment is completed, the blank control response value is deducted, and the software is used to fit the 1:1 Langmuir binding mode to calculate the kinetic constant of antigen-antibody binding.
  • Example 3 Humanization of anti-human PD-1 monoclonal antibody and construction of stable cell lines
  • a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework region supporting the antibody's conservative three-dimensional conformation. Then, based on the results of homology comparison, find the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and select VH3( 3-21) As a basic template, combined with the results of the full sequence blast, consider the frequency of rearranged antibodies at specific FR region sites (A49), perform CDR transplantation, consider the FR3 region (S98) close to the CDR3 region, and do not replace According to the CDR3 sequence (Pdssgvay), JH4 (wgqgtlvtvss) was selected as the sequence of the J region to achieve a highly humanized H1 in the framework region.
  • CDR3 sequence Pdssgva
  • the 317 antibody murine sequence was fully humanized and the sequence was synthesized.
  • the humanized 317 antibody was named h317, and the humanized h317-VH1 was cloned into the eukaryotic expression vector pKN034 by enzyme digestion.
  • the humanized h317-VL2 was cloned into the eukaryotic expression vector pKN019 upstream of the gene encoding the human light chain C ⁇ by enzyme digestion to construct the humanized 317 light and heavy chain Expression vector, clone h317-H and L into pKN002 eukaryotic stable high expression vector by enzyme digestion method, after linearization by Pvu I, use Nucleofector 2b electroporator (300452, Lonza) to integrate h317 gene into CHO cell DNA In the meantime, MSX (Cat: M5379-500MG, Sigma) pressure screening and subcloning were used to obtain stable cell lines with high expression of h317.
  • Nucleofector 2b electroporator 300452, Lonza
  • SEQ ID NO: 61 The nucleotide sequence of the light chain variable region DNA after humanization is shown in SEQ ID NO: 61, the amino acid sequence is shown in SEQ ID NO: 60; the nucleotide sequence of the humanized heavy chain variable region DNA is shown in SEQ ID NO: 63. For the amino acid sequence, see SEQ ID NO: 62.
  • Example 4 Study on the binding kinetics of h317 and human PD-1 protein
  • Biotinylation of h317 and Nivolumab Place 200 ⁇ L of 5 mg/ml EZ-LinkNHS-LC-LC-Biotin (Cat: 21343, Thermo) at room temperature for 5 minutes, and 1 ml of 1 mg/mL of h317 (Lot: DP201805002, Jiangsu Taikang Biomedicine) Co., Ltd.) or Nivolumab (Lot: AAW4553, BMS) protein solution was mixed, and placed at room temperature until 30min. Concentrate by ultrafiltration to 0.5mL, determine the protein content with the absorption value of 280nm, aliquot, store at -80°C, freeze and thaw no more than once.
  • Binding kinetics determination using Octet QKe system instrument of Fortebio Company, Streptavidin capture Biotin labeled antibody method to determine antibody affinity.
  • Biotin-labeled antibody h317-biotin, Nivolumab-biotin
  • PBS buffer 5 ⁇ g/mL
  • Human PD-1 recombinant protein (sequence numbers: NP_005009.2, 21aa-167aa) was used as the mobile phase, with PBS for two-fold concentration gradient dilution, and its concentration range was 100nM to 12.5nM.
  • the combination time is 300s and the dissociation time is 300s.
  • the blank control response value is deducted, and the software is used to fit the 1:1 Langmuir binding mode to calculate the kinetic constant of antigen-antibody binding.
  • Example 5 ELISA detects the inhibitory effect of h317 on the binding of PD-1 and its ligands PD-L1, PD-L2
  • ELISA was used to detect the inhibitory effect of h317 on the binding of PD-1 to PD-L1: human PD-1-hFc (serial number: NP_005009.2, 21aa-167aa, Lot: 20180329) was diluted to 0.5 ⁇ g/mL at 4°C After being left overnight, it was sealed with a 5% BSA at 37°C incubator for 60 min.
  • the concentration of PD-1-hFc (serial number: NP_005009.2, 21aa-167aa, Lot: 20180329) was diluted to 0.5 ⁇ g/mL and coated at 4°C Overnight, closed with 5% BSA at 37°C incubator for 60 min.
  • h317 can effectively block the binding of recombinant human PD-1 to its ligands PD-L1 and PD-L2.
  • the competitive inhibitory effect of h317 and Nivolumab on the binding of PD-1 to its ligand PD-L1 was determined by ELISA, and its half effective inhibitory concentration (IC50) values were 1.4 nM and 1.3 nM, respectively ( Figure 6, 6A).
  • the competitive inhibitory effect of h317 and Nivolumab on the binding of PD-1 to its ligand PD-L2 was determined by ELISA, and its half effective inhibitory concentration (IC50) values were 1.2 nM and 1.0 nM, respectively (Fig. 6, 6A).
  • IC50 half effective inhibitory concentration
  • Example 6 Species-specific study on the detection of the binding of h317 to PD-1 by ELISA
  • PD-1 protein was coated at 4°C overnight with a coating concentration of 1 ⁇ g/mL; after washing the plate 3 times with PBS, 5% BSA was added and blocked at 37°C for 60 min.
  • Example 7 Specificity study of ELISA for detecting the binding of h317 to CD28 family members
  • Recombinant human PD-1 (serial number: NP_005009.2, 21aa-167aa, Lot: 20180423), CD28 (Cat: 11524-HCCH, Beijing Yiqiao Shenzhou), CTLA4 (Cat: 11159-H08H, Beijing Yiqiao Shenzhou) And ICOS (serial number: NP_036224.1, 1aa-199aa) protein coated overnight at 4 °C, coating concentration 1 ⁇ g/mL; after washing the plate 3 times with PBS, adding 5% BSA PBS, blocking 60 min at 37 °C, washing the plate with PBST 3 Times; add different dilution multiples of biotinylated h317 (same as Example 4) (the initial concentration is 1 ⁇ g/mL, 3 times the gradient is sequentially diluted 11 concentrations), incubate at 37°C for 60 min, wash the plate 4 times with PBST; add 1 : 100-diluted HRP-streptavidin (
  • Example 8 h317 in vitro stimulation of human T cell (PBMC) activity study
  • the volume of 5 ml was transferred to a T-25 cell culture flask and placed at 37°C, After culturing in a 5% CO 2 incubator for 3 days, centrifuge to change the medium, place it in a 5% CO 2 incubator at 37°C for 2 days, and add 50 ⁇ l of ImmunoCult TM Dendritic Cell Maturation Supplement (Cat: 10989, Lot: 17B77271, STEMCELL ), after mixing, put it in a 37°C, 5% CO 2 incubator for 2 days for MLR experiment.
  • ImmunoCult TM Dendritic Cell Maturation Supplement Cat: 10989, Lot: 17B77271, STEMCELL
  • test antibody (h317 (Lot: DP201805002, Jiangsu Taikang Biomedicine), Nivolumab (Lot: AAW4553, BMS), Human NC-IgG4 control (Lot: AB170090) solution with a complete medium at a concentration of 8 ⁇ g/mL, and then Perform a 10-fold gradient dilution to prepare a total of 6 concentrations. Then add 50 ⁇ L of antibody to the corresponding wells according to the design of the experimental plate. Adjust the concentration of CD4+ T cells to 1 ⁇ 10 6 /mL, add 100 ⁇ L (1 ⁇ 10 5 /well) cell suspension into the corresponding well.
  • IFN - ⁇ ELISA kit Cat: 430106, Lot: B247782, Biolegend
  • IL-2 ELISA kit Cat: DY202, Lot: P155804, R&D
  • Example 9 ADCC toxicity study of h317 on 293T/hPD-1 cells
  • ADCC medium phenol red-free RPMI1640 medium (Cat: 11835-030, Lot : 1860152, Gibco) + 2% FBS (Cat: SH30084.03, Lot: GAE0138, Hyclone)
  • the cell count of the obtained PBMC was performed, and NK cell isolation was performed according to the instructions of the NK cell isolation kit (Cat: 130-092-657, Lot: 5170911524, Miltenyi) for ADCC toxicity analysis.
  • the non-radioactive cytotoxicity test kit (Cat: G1781, Lot: 0000265175, Promega) indicates that 293T cells expressing human PD-1 protein in the logarithmic growth phase are taken, diluted with ADCC medium to adjust the concentration of the target cell suspension to 50 ⁇ 10 4 cells/mL, add 40 ⁇ L of cell suspension to each well in a 96-well round bottom plate, add 10 ⁇ L of drug solution to each well (three replicate wells per cell concentration), and incubate at room temperature for 30 minutes.
  • the tested antibodies (h317 (Lot: DP201805002, Jiangsu Taikang Biomedicine), h317-hIgG1 (Lot: 20180528), Human NC-IgG4 (Lot: 20180521)) final concentrations are 10000ng/mL, 1000ng/mL, 100ng/mL, 10ng/mL, 1ng/mL, 0.1ng/mL, 0.01ng/mL.
  • two E:T ratios of 10:1 and 5:1 were set.
  • the 96-well plate was centrifuged at 250 g for 4 min to allow the effector cells and target cells to fully contact, and the cells were cultured at 37° C., 5% CO 2 , and 95% air for 4 h.
  • 10 ⁇ L of lysis buffer (10 ⁇ L per 100 ⁇ L) was added to the maximum lysis control well (max lysis well).
  • centrifuge at 250g for 5min. Transfer 50 ⁇ L of supernatant to a clean 96-well plate. After adding 50 ⁇ L of reaction substrate and mixing for 30 s, incubate at room temperature for 30 min. After adding 50 ⁇ L of stop solution and mixing for 30 s, read the absorbance value (OD490nm).
  • the obtained value was used to calculate cytotoxicity using the following formula.
  • Example 10 In vivo antitumor efficacy evaluation of h317 in PD-1 transgenic mouse xenograft model
  • StudyDirector TM version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA
  • the number of animals in each group and the detailed administration route, dose and schedule are shown in Table 6.
  • Administration and tumor measurement All processes such as weighing and weighing are carried out in the biological safety cabinet or ultra-clean workbench.
  • the StudyDirector TM version number 3.1.399.19, supplier Studylog System, Inc.
  • the original data was directly imported into the software after measurement by balance and vernier calipers.
  • the mean tumor volume of the mice in the control group exceeded 2000 mm 3 or one week after the last administration, the experiment was terminated.
  • the administration volume is 10 ⁇ L/g; hIgG control (Lot: 20180521), h317 (Lot: DP201805002, Jiangsu Taikang Biomedicine), Nivolumab (Lot: AAW4553, BMS)
  • the HuGEMMPD-1 mouse model is a genetically engineered mouse.
  • the human PD-1 protein coding region is inserted into the ATG position of mouse PD-1 to express human PD-1 and replace the mouse.
  • MC38 is a murine intestinal cancer cell line induced by induction in C57BL/6 mice.
  • the mouse-derived PD-L1 of MC38 was knocked out, and the human-derived PD-L1 was knocked in to obtain the MC38 cell line expressing human-derived PD-L1, namely the MC38-hPD-L1 cell line.
  • h317 and its reference antibody Nivolumab The inhibitory effects of h317 and its reference antibody Nivolumab on tumor growth were evaluated and compared on a PD-1 transgenic mouse xenograft model. The results showed that (see Figure 11, Table 7), h317 can significantly inhibit the growth of PD-1 transgenic mouse subcutaneously transplanted tumors at high and medium doses, and at high and medium dose levels, h317 and its reference antibody Nivolumab have comparable tumor suppressive effects. .
  • the p-value was obtained by one-way ANOVA (one-way ANOVA) tumor volume, and the F-value was significantly different (p ⁇ 0.05). Dunnett’s T3 method was used for analysis.
  • Example 11 h317 binds to PD-1 receptor internalization
  • Transiently expressing human PD-1 293 cell line constructs HEK293 cells at 6 * 10 5 cells / ml in shake flasks access density, volume of 10ml, placed in 37 °C, 5% CO 2 incubator shaking of Shake at 130rpm for 24h.
  • the expression plasmid encoding full-length human PD-1 (NP_005009.2, 21aa-288aa) and 293fectin (Cat: 12347019, Gibco) were respectively in a ratio of 1:1.2 in 0.5mL Opti-MEM (Cat: 11058021, Gibco) medium
  • the two were mixed and allowed to stand at room temperature for 15 minutes, and then added to HEK293 cells. It was placed in a shaking incubator at 37°C, 5% CO 2 and shaking at 130 rpm for 48 h. 293/h PD-1 cells were used for FACS detection.
  • PD-1 mediated endocytosis of h317 Mix-n-Stain TM CF TM 488A (Cat: MX488AS100, Sigma) labeled h317 (Lot: DP201805002, Jiangsu Taikang Biomedical Co., Ltd.) and Nivolumab (Lot: AAW4553, BMS); Dilute the labeled antibody to 10 ⁇ g/mL with 5% BSA, wash 293/hPD-1 cells once with PBS, add the diluted h317-CF488A or Nivolumab-CF488A, and put them in a 37°C electric thermostat incubator, one The group was placed in a refrigerator at 4°C as a negative control; after 1h of incubation, the PBS was washed three times, and the culture was still kept at the original temperature of 37°C or 4°C; after 3h and 6h, it was observed with a fluorescence microscope and photographed.
  • PD-1-h317-Fab complex crystallization and crystal diffraction data collection and structure analysis using the sitting drop method, using a crystal screening robot to complete, the device consumes 0.2 ⁇ l protein sample per well, after the sample is added, the crystal plate is sealed and placed Constant temperature room cultivation.
  • the crystal growth temperature is 16°C and 4°C, and the protein concentration is 8mg/ml and 12mg/ml.
  • Screening kits include: HAMPTON: Crystalscreen (1-98), Index (1-96), PEGRX (1-96), SaltRx (1-96), Nartrix (1-96), PEG/ion (1- 96); QIAGEN: Protein Complex Suite (1-96); Emerald Biosystems: WIZARD I/II (1-96), WIZARD III/IV (1-96). Observe the crystal growth results within 15 days, and optimize the crystal growth conditions with better initial sieving crystal form. The crystals obtained after optimization were immersed in a crystallization solution supplemented with 10% ethylene glycol for several seconds before diffracting experiments, and then immersed in liquid nitrogen for rapid freezing. The crystal diffraction experiment was carried out at the Shanghai Synchrotron Radiation Light Source (SSRF) BL17U line station.
  • SSRF Synchrotron Radiation Light Source
  • the X-ray wavelength used was The crystal diffraction temperature is 100K, and the diffraction data is collected using ADSC Q315r detector.
  • the diffraction data processing uses HKL2000 software to determine the crystal space group as P2 and the crystal diffraction resolution as The structure was resolved by molecular replacement method using PHASER software.
  • the selected molecular replacement models were PD-1 individual protein structure (PDB ID 3RRQ) and PD-1Fab (PDB ID 5WT9) structure.
  • Use COOT software to rebuild the model, and use REFMAC5 and PHENIX software for structural refinement.
  • binding of deglycosylated human PD-1 recombinant protein to h317 In the structure of human PD-1 antigen (NP_005009.2), four glycosylation sites, N49, N58, N116, and N74, can be observed through site-directed mutation Method, the glycosylation site was mutated to alanine (A), loaded into an expression vector with EGFP to construct a total of 5 human PD-1 full-length expression plasmids (WT, N49A, N58A, N116A, N74A), The plasmids were mixed with 293fectin and co-transfected into HEK293 cells.
  • A alanine
  • the cells were transfected for 2 days, the cells were taken, the cells were collected in a 96-well plate, centrifuged at 1500rpm for 2min, and the supernatant was discarded.
  • the binding site of PD-1 and antibody is mainly in its loop area, including BC loop, C’D loop and FG loop area ( Figure 14).
  • the interaction modes include hydrogen bonds, salt bonds, van der Waals forces, and hydrophobic interactions, and the amino acids and forces involved in the interaction (Table 8).
  • glycosylation at positions N49, N58, and N116 can be observed.
  • the glycosylation position at N74 is unclear, and the sugar chain at position N58 is involved in binding to h317-Fab ( Figure 15).
  • Other reported PD-1 antigen-antibody complex structure sugar chains at this site did not participate in the interaction between the two, so the interaction between h317-Fab and N58 sugar chain may improve the specificity of the recognition and binding of the two.
  • the glycosylation site on PD-1 was mutated and transiently transferred to 293 cells, and the binding of h317 to PD-1-mut molecule was detected by FACS; FACS results showed that the N58 sugar chain of human PD-1 was involved in h317 and PD -1 binding, N58 mutation to A loses glycosylation and makes h317 lose its binding to PD-1 expressing cells ( Figure 16), and the N58 sugar chain of PD-1 participates in the binding of h317 to PD-1.
  • the affinity comparison between the mutant and the parent antibody was performed using the Octet QKe system instrument from Fortebio.
  • the specific method is the same as that in Example 4, except that the affinity of all mutants is determined only by a single line to evaluate the relative affinity.
  • the antibody to be tested with the same target value was coated, and the relative affinity was determined using 100 nM human PD-1 recombinant protein (sequence number: NP_005009.2, 21aa-167aa) as the mobile phase.
  • the results of the affinity determination of the mutant antibody showed that the affinity of the antibody changed greatly after mutation at multiple sites.
  • the mutation site information is shown in Table 9, and part of the affinity changes are shown in Table 10.
  • the amino acid mutation at individual positions will have a greater impact on the affinity, and the affinity of some sites will be reduced by more than 10 times.
  • the affinity of the antibody has been improved after mutations at some sites.
  • the K at position 24 of the light chain is mutated to R
  • the T at position 56 is mutated to S
  • the S at position 92 is mutated to N.
  • S is mutated to S at position 54 and G is mutated to A at position 59
  • the affinity of the antibody can be improved to some extent, and these mutation sites can be further superimposed with mutations, and the affinity remains unchanged or further improved.
  • HCDR3 is essential for binding, and most of the affinity decreases after mutation.

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Abstract

本发明提供了一种结合程序性细胞死亡蛋白1(PD-1)的抗体或其片段，以及所述抗体或其片段用于预防或治疗肿瘤或癌症的用途。本发明的抗体或其片段能够有效阻断PD-1/PD-L1、PD-1/PD-L2相互作用，而且具有独特的抗原结合表位，在药效和安全性上具有独特的性质。

Description

重组抗人PD-1抗体及其应用
本专利申请要求于2019年1月10日提交的申请号为201910022548.9的中国专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明涉及抗体药物领域，具体而言，本发明涉及针对人PD-1的抗体及其用于制备药物的用途。
背景技术
程序性细胞死亡蛋白1(PD-1)，也被称为CD279，是T细胞受体CD28家族的成员，表达于多种免疫细胞，如T细胞，B细胞，单核细胞等的表面，是一种在肿瘤微环境中抑制CD4+和CD8+T细胞功能的重要的免疫检查点分子。PD-1的关键配体包括PD-L1(B7-H1)和PD-L2(B7-DC)，它们由免疫细胞表达，并且还可以在多种组织上诱导，当T细胞上的PD-1结合PD-L1或PD-L2时，T细胞接收抑制信号，从而抑制T细胞增殖和细胞因子产生，会有效降低T细胞参与的免疫应答。而PD-L1和PD-L2在多种人类肿瘤细胞上高表达，因此使得肿瘤细胞逃避T细胞的监督(Pardoll DM.The blockade of immune checkpoints in cancer immunotherapy.Nat.Rev.Cancer12(4),252–264(2012))。
目前在全球范围内已上市的抗人PD-1的单抗共有3种。抗人PD-1的全人源抗体纳武单抗(Nivolumab)显示抑制PD-1与PD-L1和PD-L2的结合，临床用于治疗晚期黑色素瘤，非小细胞肺癌，肾细胞癌，霍奇金淋巴瘤，头颈部鳞癌，尿路上皮癌，结直肠癌和肝细胞癌；抗人PD-1的人源化抗体派姆单抗(Perbrolizumab)显示抑制PD-1与PD-L1和PD-L2的结合，临床用于治疗恶性黑色素瘤，非小细胞肺癌，经典型霍奇金淋巴瘤，头颈部鳞癌，d-mmr突变或MSI-H恶性肿瘤(Alsaab et al.PD-1 and PD-L1Checkpoint Signaling Inhibition for Cancer Immunotherapy:Mechanism,Combinations,and Clinical Outcome.Front.Pharmacol.8:561)。抗人PD-1的Libtayo单抗(Cemiplimab)可与PD-1结合并阻断其与PD-L1和PD-L2的相互作用，释放PD-1通路介导的免疫应答抑制，包括抗肿瘤免疫应答,临床用于治疗转移性皮肤鳞状细胞癌(CSCC)患者或局部晚期CSCC患者 (Markham,A.&Duggan,S.Drugs.2018.doi:10.1007/s40265-018-1012-5)。国内处于临床阶段的抗人PD-1的单抗就有10多种，适应症覆盖复发难治恶性淋巴瘤，霍奇金淋巴瘤，B细胞非霍奇金淋巴瘤，腺泡状软组织肉瘤，晚期或复发性恶性肿瘤，食管癌、胃癌或胃食管结合部癌，鼻咽癌、头颈部鳞癌，肺癌，转移性结直肠癌，局限性肾细胞癌，尿路上皮癌，肝细胞癌，黑色素瘤，晚期三阴性乳腺癌，胸膜间皮瘤，晚期神经内分泌肿瘤，不可切除或d-mmr突变或MSI-H实体瘤。其中恒瑞的SHR-1210、百济神州的tislelizumab、信达的IBI-308已递交上市申请，君实的JS001已于12月17日有条件批准上市，名为特瑞普利单抗注射液(商品名：拓益)。
国际上，抗PD-1的抗体已经有三个上市品种，多个临床在研品种。涉及的专利包括：WO2004004771，WO2006121168，WO2008156712，WO2010029435，WO2012145493，WO2015085847，US20170044260，CN104250302，CN105330740，CN105531288等。通过分析这些专利发现，可以通过杂交瘤筛选、人源化途径，或通过噬菌体展示或者酵母表面展示技术，或通过转基因小鼠技术获得抗PD-1的抗体。其中WO2015085847中获得抗人PD-1的抗体的方法为：利用杂交瘤技术获得鼠抗体，通过抗体活性分析(ELISA(结合，阻断)，亲和力动力学)获得候选鼠抗体后；将鼠抗体轻重链基因可变区序列克隆至编码人抗体轻重链恒定区序列的上游，进行哺乳动物细胞表达，制备嵌合抗体，然后通过抗体活性分析，阻断PD-1与其配体结合实验，与CD28其他家族成员结合实验，与细胞表面PD-1结合实验，体外细胞学实验确定先导抗体后；根据Germline数据库选择人源化模板，进行抗体序列人源化设计，获得的人源化抗体再次通过抗体活性分析，阻断PD-1与其配体结合实验，与CD28其他家族成员结合实验，与细胞表面PD-1结合实验，体外细胞学实验验证后；再进行人源化抗体对体内外肿瘤细胞生长抑制实验进行最后的成药性评价，最终获得亲和力和特异性保持不变的PD-1人源化抗体序列。
抗体的抗原识别表位是抗体的重要特性之一。抗PD-1抗体，通过阻断PD-1与其配体PD-L1/PD-L2的结合发挥激活T细胞的作用。目前已报道的抗体虽然都能阻断PD-1与配体的相互作用，但其所针对的抗原表位却并不完全相同(Tan S,Zhang H,Chai Y,et al.An unexpected N-terminal loop in PD-1 dominates binding by nivolumab.Nat Commun.2017；8:14369)。而抗原表位的不同，可能导致抗体在药效和安全性上具有独特的特点。
发明内容
本发明要解决的技术问题是，通过杂交瘤筛选和人源化技术，获得特异性结合人PD-1的高亲和力抗体，其不但能够有效阻断PD-1/PD-L1、PD-1/PD-L2相互作用，而且具有独特的抗原结合表位，因此在药效和安全性上具有独特的性质。
针对上述技术问题，本发明的目的是提供一种特异性结合人PD-1的抗体或其功能片段，并提供其用途。
本发明的技术方案如下。
一方面，本发明提供一种结合程序性细胞死亡蛋白1(PD-1)的抗体或其片段，所述抗体或其片段包含轻链可变区和/或重链可变区，其中所述轻链可变区和/或重链可变区分别包含以下序列所示的轻链CDR1(LCDR1)、CDR2(LCDR2)、CDR3(LCDR3)和/或重链CDR1(HCDR1)、CDR2(HCDR2)、CDR3(HCDR3)：
Figure PCTCN2020071353-appb-000001
在本发明提供的抗体或其片段中，优选地，所述轻链可变区和/或重链可变区分别包含以下序列所示的LCDR1、LCDR2、LCDR3和/或HCDR1、HCDR2、HCDR3：
Figure PCTCN2020071353-appb-000002
在本发明提供的抗体或其片段中，优选地，所述轻链可变区和/或重链可变区分别包含以下序列所示的LCDR1、LCDR2、LCDR3和/或HCDR1、HCDR2、HCDR3：
选自SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11的LCDR1；
选自SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17的LCDR2；
选自SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27的LCDR3；和/或
选自SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33的HCDR1；
选自SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46的HCDR2；
选自SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、 SEQ ID NO:51的HCDR3。
在本发明提供的抗体或其片段中，优选地，所述轻链可变区包含以下序列组合所示的LCDR1、LCDR2、LCDR3：
组合 LCDR1 LCDR2 LCDR3
1 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18
2 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:18
3 SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:18
4 SEQ ID NO:10 SEQ ID NO:12 SEQ ID NO:18
5 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:18
6 SEQ ID NO:7 SEQ ID NO:13 SEQ ID NO:18
7 SEQ ID NO:7 SEQ ID NO:14 SEQ ID NO:18
8 SEQ ID NO:7 SEQ ID NO:15 SEQ ID NO:18
9 SEQ ID NO:7 SEQ ID NO:16 SEQ ID NO:18
10 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:18
11 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:19
12 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:20
13 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:21
14 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:22
15 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:23
16 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:24
17 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:25
18 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:26
19 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:27
20 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:18
21 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:22
22 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:25
23 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:22
24 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22
和/或，所述重链可变区包含以下序列组合所示的HCDR1、HCDR2、HCDR3：
组合 HCDR1 HCDR2 HCDR3
1 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
2 SEQ ID NO:29 SEQ ID NO:34 SEQ ID NO:47
3 SEQ ID NO:30 SEQ ID NO:34 SEQ ID NO:47
4 SEQ ID NO:31 SEQ ID NO:34 SEQ ID NO:47
5 SEQ ID NO:32 SEQ ID NO:34 SEQ ID NO:47
6 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:47
7 SEQ ID NO:28 SEQ ID NO:35 SEQ ID NO:47
8 SEQ ID NO:28 SEQ ID NO:36 SEQ ID NO:47
9 SEQ ID NO:28 SEQ ID NO:37 SEQ ID NO:47
10 SEQ ID NO:28 SEQ ID NO:38 SEQ ID NO:47
11 SEQ ID NO:28 SEQ ID NO:39 SEQ ID NO:47
12 SEQ ID NO:28 SEQ ID NO:40 SEQ ID NO:47
13 SEQ ID NO:28 SEQ ID NO:41 SEQ ID NO:47
14 SEQ ID NO:28 SEQ ID NO:42 SEQ ID NO:47
15 SEQ ID NO:28 SEQ ID NO:43 SEQ ID NO:47
16 SEQ ID NO:28 SEQ ID NO:44 SEQ ID NO:47
17 SEQ ID NO:28 SEQ ID NO:45 SEQ ID NO:47
18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:48
19 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:49
20 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:50
21 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:51
22 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47
23 SEQ ID NO:32 SEQ ID NO:44 SEQ ID NO:47
24 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47
25 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47
26 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47
优选地，所述轻链可变区和重链可变区包含以下序列组合所示的LCDR1、LCDR2、LCDR3和HCDR1、HCDR2、HCDR3：
组合 LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
1 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
2 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
3 SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
4 SEQ ID NO:10 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
5 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
6 SEQ ID NO:7 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
7 SEQ ID NO:7 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
8 SEQ ID NO:7 SEQ ID NO:15 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
9 SEQ ID NO:7 SEQ ID NO:16 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
10 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
11 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:19 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
12 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:20 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
13 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:21 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
14 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
15 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:23 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
16 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:24 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
17 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:25 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
18 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:26 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
19 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
20 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
21 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
22 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:25 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
23 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
24 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
25 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:29 SEQ ID NO:34 SEQ ID NO:47
26 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:30 SEQ ID NO:34 SEQ ID NO:47
27 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:31 SEQ ID NO:34 SEQ ID NO:47
28 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:32 SEQ ID NO:34 SEQ ID NO:47
29 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:47
30 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:35 SEQ ID NO:47
31 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:36 SEQ ID NO:47
32 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:37 SEQ ID NO:47
33 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:38 SEQ ID NO:47
34 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:39 SEQ ID NO:47
35 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:40 SEQ ID NO:47
36 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:41 SEQ ID NO:47
37 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:42 SEQ ID NO:47
38 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:43 SEQ ID NO:47
39 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:44 SEQ ID NO:47
40 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:45 SEQ ID NO:47
41 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:48
42 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:49
43 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:50
44 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:51
45 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47
46 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:32 SEQ ID NO:44 SEQ ID NO:47
47 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47
48 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47
49 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47
本发明提供的抗体或其片段具有以下活性中的一个或多个：
(1)阻断PD-1与其配体PD-L1或PD-L2的结合；
(2)特异性结合灵长类动物PD-1，与非灵长类动物PD-1无交叉反应；
(3)不结合除PD-1之外的其他CD28家族成员；
(4)在CD4+T细胞中诱导IL-2和/或IFN-γ产生；
(5)无ADCC效应功能。
优选地，所述抗体或其片段结合人PD-1N58糖链；优选地，所述抗体或其片段的重链可变区包含SEQ ID NO:47所示氨基酸序列。
优选地，所述轻链可变区包含SEQ ID NO:52或SEQ ID NO:60所示氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，
所述重链可变区包含SEQ ID NO:54或SEQ ID NO:62所示的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；
优选地，所述至少75％同一性为例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性。
根据本发明的具体实施方式，在本发明提供的抗体或其片段中，所述轻链可变区包含SEQ ID NO:52所示氨基酸序列，所述重链可变区包含SEQ ID NO:54所示氨基酸序列；或者所述轻链可变区包含SEQ ID NO:60所示氨基酸序列，所述重链可变区包含SEQ ID NO:62所示氨基酸序列。
在本发明提供的抗体或其片段中，优选地，所述轻链可变区包含SEQ ID NO:60所示氨基酸序列或所述氨基酸序列的变体，根据SEQ ID NO:60所示氨基酸序列的残基编号和组成，所述变体相对于SEQ ID NO:60具有选自以下的一个或多个、优选1-3个氨基酸置换：K24R、E30S、V32A、V32Y、W50A、W50G、H55A、H55Q、T56S、Y91A、Y91F、S92D、S92N、R93N、R93S、Y94F、W96G和W96Y；优选地，所述变体相对于SEQ ID NO:60具有选自以下的一个或多个、优选1-3个氨基酸置换：K24R、V32A、V32Y、T56S、S92D、S92N、R93S和Y94F；
和/或
所述重链可变区包含SEQ ID NO:62所示氨基酸序列或所述氨基酸序列的变体，根据SEQ ID NO:62所示氨基酸序列的残基编号，所述变体相对于SEQ ID NO:62具有选自以下的一个或多个、优选1-3个氨基酸置换：S31D、Y32A、Y32N、D33S、D33Y、S52K、S52W、G53S、G53Y、G54D、G54S、G55S、S56G、Y57T、Y59A、Y59T、D100E、D100Y、S101A和Y106T；优选地，所述变体相对于SEQ ID NO:62具有选自以下的一个或多个、优选1-3个氨基酸置换：S31D、Y32A、D33S、G53S、G54S、G55S、Y59A和D100Y。
根据本发明的具体实施方式，本发明提供变体抗体或其片段，所述变体抗体或其片段的重链可变区包含SEQ ID NO:62所示氨基酸序列，而轻链可变区相比SEQ ID NO:60所示氨基酸序列的残基编号和组成，仅具有以下氨基酸置换：1)K24R；2)E30S；3)V32A；4)V32Y；5)W50A；6)W50G；7)H55A；8)H55Q；9)T56S；10)Y91A；11)Y91F；12)S92D；13)S92N；14)R93N；15)R93S；16)Y94F；17)W96G；18)W96Y；19)K24R和T56S；20)K24R和S92N；21)K24R和Y94F；22)T56S和S92N；或，23)K24R、T56S和S92N。
根据本发明的具体实施方式，本发明提供变体抗体或其片段，所述变体抗体或其片段的轻链可变区包含SEQ ID NO:60所示氨基酸序列，而重链可变区相比SEQ ID NO:62所示氨基酸序列的残基编号和组成，仅具有以下氨 基酸置换：1)S31D；2)Y32A；3)Y32N；4)D33S；5)D33Y；6)S52K；7)S52W；8)G53S；9)G53Y；10)G54D；11)G54S；12)G55S；13)S56G；14)Y57T；15)Y59A；16)Y59T；18)D100E；19)D100Y；20)S101A；21)Y106T；22)D33S和G54S；23)D33S和Y59A；或，24)G54SY59A。
根据本发明的具体实施方式，本发明提供变体抗体或其片段，所述变体抗体或其片段的轻链可变区相比SEQ ID NO:60所示氨基酸序列的残基编号和组成，仅具有K24R、T56S和S92N的氨基酸置换，并且重链可变区相比SEQ ID NO:62所示氨基酸序列的残基编号和组成，仅具有D33S和G54S的氨基酸置换，或仅具有G54S和Y59A的氨基酸置换。
本发明提供的抗体可以为单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式；优选地，所述抗体为IgA、IgD、IgE、IgG或IgM，更优选为IgG1或IgG4；
本发明提供的片段为所述抗体能够特异性结合PD-1或其任何部分的片段；优选地，所述片段为所述抗体的scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv片段。
优选地，本发明提供的抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型；
进一步优选地，所述抗体的轻链恒定区包含SEQ ID NO:56或SEQ ID NO:64所示氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述重链恒定区包含SEQ ID NO:58或SEQ ID NO:66所示的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；其中，所述至少75％同一性为例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性。
根据本发明的具体实施方式，在本发明提供的抗体中，所述轻链可变区包含SEQ ID NO:52所示氨基酸序列，轻链恒定区包含SEQ ID NO:56所示氨基酸序列，重链可变区包含SEQ ID NO:54所示氨基酸序列，重链恒定区包含SEQ ID NO:58所示氨基酸序列；或者，所述轻链可变区包含SEQ ID NO:60所示氨基酸序列，轻链恒定区包含SEQ ID NO:62所示氨基酸序列，重链可变区包含SEQ ID NO:64所示氨基酸序列，重链恒定区包含SEQ ID NO:66所示氨基酸序列。
基于本发明的抗体或其片段，本发明还提供包含本发明的抗体或其片段 的缀合物或融合蛋白。该缀合物或融合蛋白可包含通过化学或物理方法结合于本发明所述抗体或其片段的其他部分，例如细胞表面受体、小分子化合物如氨基酸和糖类、小分子聚合物或对本发明所述抗体进行修饰的任何其它部分，或者甚至是活性蛋白或多肽。例如，该缀合物或融合蛋白可以是包含本发明所述抗体或其片段的双特异性抗体。
另一方面，本发明还提供一种核酸分子，其编码本发明任意抗体或其片段中的重链CDR、轻链CDR、重链可变区、轻链可变区、重链或轻链；
优选地，所述核酸分子包含SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65或SEQ ID NO:67所示核苷酸序列。
还一方面，本发明提供一种载体，其包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。
本发明的载体或核酸分子可以用于转化或转染宿主细胞或以任何方式进入宿主细胞内，用于保存或表达抗体等目的。
因此，另一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
基于本发明的公开内容，本发明提供的抗体或其片段以及相应的缀合物或融合蛋白、核酸分子、载体和/或宿主细胞可以通过使用本领域已知的任何常规技术方法获得。所述抗体或其片段、缀合物或融合蛋白、核酸分子、载体和/或宿主细胞可以被包含在药物组合物中，更特别地被包含在药物制剂中，从而根据实际需要用于各种目的。
因此，在又一方面，本发明还提供一种药物组合物，所述药物组合物包含本发明所述的抗体或其片段、缀合物或融合蛋白、核酸分子、载体和/或宿主细胞，以及任选的药学上可接受的辅料。
作为特异性结合人PD-1或其任何部分的抗体或其片段，本发明还提供上述主题的相关应用。
具体而言，再一方面，本发明提供本发明所述的抗体或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物在制备用于预防或治疗肿瘤或癌症的药物中的用途。优选地，所述肿瘤或癌症选自非小细胞肺癌、经典霍德金淋巴瘤、胃癌、(原发性)肝癌、黑色素瘤、d-mmr突变或 MSI-H恶性肿瘤、宫颈癌、头颈部鳞癌、尿道膀胱癌、原发性纵隔B细胞淋巴瘤、肾细胞癌、结直肠癌和尿路上皮癌；更优选地，所述肿瘤或癌症为非小细胞肺癌、黑色素瘤、结直肠癌、肝癌、头颈部鳞癌、经典霍德金淋巴瘤或尿路上皮癌。
还一方面，本发明提供一种药物组合，其包括：
(1)本发明所述的抗体或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物；(2)其他免疫增强药物或手段，例如小分子化药、靶向药、抗体等重组蛋白药，疫苗、ADC、溶瘤病毒、基因和核酸治疗药物和放射疗法。
另外，本发明提供一种预防或治疗肿瘤或癌症的方法，所述方法包括给有此需要的受试者施用所述抗体或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物，以及任选的其他药物或手段。优选地，所述肿瘤或癌症选自非小细胞肺癌、经典霍德金淋巴瘤、胃癌、(原发性)肝癌、黑色素瘤、d-mmr突变或MSI-H恶性肿瘤、宫颈癌、头颈部鳞癌、尿道膀胱癌、原发性纵隔B细胞淋巴瘤、肾细胞癌、结直肠癌和尿路上皮癌；更优选地，所述肿瘤或癌症为非小细胞肺癌、黑色素瘤、结直肠癌、肝癌、头颈部鳞癌、经典霍德金淋巴瘤或尿路上皮癌。该任选的其他药物或手段是指可以与本发明抗体或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物联合施用的其他免疫增强药物或手段，例如小分子化药、靶向药、抗体等重组蛋白药，疫苗、ADC、溶瘤病毒、基因和核酸治疗药物和放射疗法。二者的联合施用可以采取任意形式进行，例如同时、连续或间隔一定时间进行。所述受试者为哺乳类动物，优选地，所述受试者为人。
再一方面，本发明提供一种试剂盒，所述试剂盒包括本发明的所述抗体或其片段、缀合物或融合蛋白、核酸分子、载体、宿主细胞和/或药物组合物。
在本发明中，利用人PD-1胞外区结构域重组蛋白(序列号：NP_005009.2，21aa-167aa)，免疫小鼠后取脾细胞，利用杂交瘤技术筛选获得与人PD-1胞外区结构域重组蛋白和细胞表面人PD-1均具有高亲和力结合，并且能特异性阻断PD-1/PD-L1、PD-1/PD-L2配受体结合的抗体分子(本发明中命名为317)，进一步通过人源化技术获得亲和力和特异性保持不变的人源化抗体(本发明中命名为h317)，对其全抗体IgG4的结合、阻断、促进T细胞活化及体内抗肿瘤药效进行了全面的评估，并且构建了其Fab和PD-1-ECD的晶体复合物，通过晶体衍射和进一步的突变体验证确认了其抗 原结合表位。
实验证明，本发明抗体能够有效阻断PD-1和PD-L1/PD-L1的结合，同时与Nivolumab和帕博利珠单抗(Keytruda)具有竞争结合关系；但是，通过抗原抗体复合物晶体结构分析发现，本发明抗体的抗原结合表位与Nivolumab和Keytruda均不同，是首个明确抗原表位与PD-1的第58位Glu的N糖基化位点相关的抗体分子。
具体而言，与现有技术相比，本发明具有以下有益之处：
第一，本发明的抗体为具有高亲和力的抗PD-1人源化抗体。
本发明的人源化抗PD-1抗体h317可以特异性结合人PD-1蛋白，其亲和力(KD)为6.16E-09M，而对照抗体Nivolumab亲和力(KD)为8.06E-09M，且在同等条件下，h317解离(kd(1/s)9.50E-04)优于Nivolumab解离(kd(1/s):1.69E-03)，表明h317与人PD-1具有高亲和力，为h317对PD-1/PD-L1信号通路的抑制作用提供了依据。
第二，本发明的抗体具有良好的生物学活性。
实验证明，本发明的h317可以有效阻断重组人PD-1与其配体PD-L1和PD-L2的结合作用，二者IC50均为1.4nM，与对照抗体Nivolumab(IC50：1.3nM)的阻断活性相近，并且在体外实验中证明h317可以促进人混合淋巴细胞分泌杀伤性细胞因子IL-2和IFN-γ，即可以诱导Th1型细胞介导的免疫反应。
第三，本发明的抗体具有特殊的抗原识别表位。
本发明抗体特异性结合人PD-1，其结合的抗原表位具有独特性。
PD-1-h317-Fab复合物整体结构分析显示，PD-1抗原(胞外区1-167aa)以单体形式存在，与PD-1抗体的h317-Fab片段形成一个1:1的复合物结构。PD-1与抗体的结合部位主要是在其loop区，包括BC loop，C’D loop和FG loop区域。此外，在PD-1抗原结构中可以观察到N49、N58、N116位点的糖基化，N74的糖基化位点不明确，其中N58位的糖链参与了与h317-Fab的结合。相比之下，其他已报道的PD-1抗原抗体复合物结构中该位点的糖链没有参与二者相互作用。并且，根据晶体复合物信息将PD-1上的4个糖基化位点分别定点突变为丙氨酸(A)，并瞬转至293细胞，通过FACS检测h317与PD-1-mut分子的结合情况，结果表明PD-1的N58位突变为A失去糖基化使得h317失去与PD-1的结合能力，因此h317是首个明确抗原表位与N58糖链相关的抗体分子。
第四，本发明抗体在PD-1转基因鼠移植瘤模型中有明显的抑瘤效果。
在PD-1转基因鼠移植瘤模型中，h317在高剂量(10mg/kg)和中剂量(2mg/kg)下可显著抑制PD-1转基因鼠皮下移植瘤的生长，在高中剂量水平下h317与其参比抗体Nivolumab的抑瘤效果相当。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了ELISA检测阳性杂交瘤上清与人PD-1胞外区结构域重组蛋白的结合情况，其中1A、1B、1C分别示出了第一轮、第二轮、第三轮的结果。
图2显示了ELISA检测阳性杂交瘤克隆上清与不同种属重组PD-1之间的交叉反应情况，其中Nivo为纳武单抗。
图3显示了克隆317的抗体亚型鉴定，其中显示重链为IgG1，轻链为Kappa链。
图4显示了317嵌合抗体(4A)和Nivolumab(4B)对人PD-1胞外区结构域重组蛋白的亲和力分析结果。
图5显示了h317抗体(5A)和Nivolumab(5B)对人PD-1胞外区结构域重组蛋白的亲和力分析结果。
图6显示了ELISA测定h317抗体和Nivolumab竞争抑制PD-1与PD-L1(6A)、PD-1与PD-L2(6B)的结合情况。
图7显示了h317与不同种属重组PD-1的结合分析曲线。
图8显示了h317与CD28家族成员的结合分析曲线。
图9显示了h317刺激MLR生成IL-2(9A)和IFN-γ(9B)的检测结果。
图10显示了h317介导人PBMC对293T/hPD-1细胞ADCC效应的结果。
图11显示了h317抑制PD-1转基因鼠皮下移植瘤增长的肿瘤体积统计结果。
图12显示了PD-1介导的h317内吞情况。
图13显示了PD-1-h317-Fab复合物整体结构。
图14显示了PD-1-h317-Fab复合物分子间相互作用，其中A为PD-1与h317-Fab的结合部位，B为BC loop与h317-Fab相互作用，C为C’D  loop与h317-Fab相互作用，D为FG loop与h317-Fab相互作用。
图15显示了PD-1N58的糖链与h317-Fab的相互作用。
图16显示了h317与人PD-1-mut重组蛋白结合情况，其中每次实验各抗体与WT的MAPC做为标准，取值为1，计算本次实验其他各组的APC，三次平行实验的结果。
图17显示了h317-Fab与PD-L1竞争性结合PD-1，其中A为PD-1-h317-Fab复合物与PD-1-PD-L1结构比较，灰色为PD-1，砖红色为PD-L1，蓝色和绿色为h317-Fab的重链和轻链；B为作用表面比较，灰色为PD-1分子表面，绿色为317-Fab作用部分，砖红色为PD-L1作用部分，黄色为317-Fab和PD-L1都作用的部分。
实施发明的最佳方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1：抗人PD-1抗体杂交瘤细胞株的筛选、鉴定及抗体序列测定
免疫：小鼠免疫采用弗氏佐剂和水溶佐剂两种免疫方法，弗氏佐剂在第0天和第14天给小鼠进行两次腹腔免疫注射，水溶佐剂在第0天和第21天给8-10周龄Balb/c小鼠进行两次肌肉免疫注射小鼠，免疫抗原为：人PD-1/mFc重组蛋白(序列号：NP_005009.2，21aa-167aa，Lot：2016.3.16)，由北京科诺信诚科技有限公司在HEK293细胞中重组表达。第一次免疫剂量为50μg，第二次免疫剂量为25μg。免疫前取小鼠血清作为检测时的阴性对照，弗氏佐剂在初次免疫后第28天，水溶佐剂在初次免疫后第35天尾静脉取血，用包被重组人PD-1蛋白的96孔酶标板以ELISA法检测血清滴度；血清滴度达到融合要求的小鼠加强免疫，将25μg抗原用D-PBS稀释成500μl，腹腔注射。初次免疫后第38天取血清滴度高的小鼠的脾细胞用于下一步的细胞融合。
细胞融合及杂交瘤制备：初次免疫后第38天，选取滴度达到要求的小鼠，摘除眼球采血，并分离血清作为抗体检测时的阳性对照血清；并无菌取小鼠脾脏，制备B淋巴细胞悬液，与FO骨髓瘤细胞以5:1的比例混合，在 PEG4000作用下使两种细胞融合。融合后的细胞用HAT培养基重悬后，分装含有巨噬细胞的96孔细胞培养板。置37℃，5％CO 2培养箱内培养。两周后换HT培养基继续培养2周，然后换R1640完全培养基。
阳性杂交瘤筛选：融合后10-14天，以人PD-1/His重组蛋白(序列号：NP_005009.2，21aa-167aa，Lot：2016.2.22)(10μg/ml，pH9.6，0.1M NaHCO 3)包被酶标板，4℃，过夜；用4％脱脂奶粉-PBS封闭，37℃，2hr；用PBST(0.05％Tween20-PBS)洗三遍，加入杂交瘤克隆培养上清，37℃，1hr。设以下对照：(1)阳性对照：免疫后小鼠血清(用PBS 1:1000稀释)(2)阴性对照：免疫前小鼠血清(用PBS 1:1000稀释)(3)空白对照：PBS。经PBST(0.05％Tween20-PBS)洗三遍，加入HRP-羊抗小鼠IgG(Fcγ)，1:20000稀释，37℃，1hr；再经PBST(0.05％Tween20-PBS)洗五遍，加入OPD显色液，避光显色10-15min，加入2M H 2SO 4终止反应；酶标仪读A490值。检测孔A490值大于阴性对照孔A490值2.1倍以上判断为阳性。为确定阳性克隆的可靠性，在一次筛选换液后隔一天再进行第二轮筛选，共进行三轮筛选。经检测鉴定，共获得多个抗体分泌阳性细胞株(图1)：6，317，500，763，795，904号。
将重组人PD-1/His(序列号：NP_005009.2，21aa-167aa，Lot：2016.2.22)、食蟹猴(Cat：90311-C08H，北京义翘神州)、大鼠(Cat：80448-R08H，北京义翘神州)及小鼠(Cat：50124-M08H，北京义翘神州)PD-1蛋白4℃包被过夜，包被浓度1μg/mL；PBS洗板3次后，加入5％BSA PBS，37℃封闭60min，PBST洗板3次；加入稀释100倍的杂交瘤上清，37℃孵育60min，PBST洗板4次；加入1:5000稀释的HRP-羊抗小鼠IgG(Fcr)(Cat：115-035-071，Jackson Immuno Research)，37℃孵育30min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。共选用五个(317，500，763，795，904号)杂交瘤上清进行不同种属重组PD-1之间的交叉反应检测，ELISA结果显示五个抗体均可以特异性地与重组人、食蟹猴PD-1结合，但与重组大鼠、小鼠PD-1则无结合活性，不产生交叉反应(图2)。选择其中317号克隆用于下一步的序列鉴定。
将分泌抗人PD-1抗体的杂交瘤细胞317号克隆扩大培养后，用Mouse Monoclonal Antibody IgG Subclass Test Card(Cat：A12403)及Mouse Monoclonal Antibody Light/Heavy Chain Test Card(Cat:A12401)按照试剂操 作规程进行亚型检测，亚型鉴定为：重链为IgG1，轻链为Kappa链(图3)，为317抗体基因的克隆提供依据。
将317杂交瘤细胞按照TRIzol试剂盒(Cat：15596026，Invitrogen)说明书步骤提取细胞总RNA；利用M-MuLV反转录酶(Cat：M0253S，NEB)将杂交瘤细胞总RNA反转录成cDNA；使用简并引物(表1，表2)和Phusion试剂盒(Cat：E0553L，NEB)扩增抗体轻链可变区IgVL(κ)和重链可变区V H序列；利用胶回收试剂盒(Cat：AP-GX-250，Axygen)纯化PCR扩增产物；按照T载体克隆试剂盒(Cat：ZC205，庄盟生物)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞，菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。测序结果显示317抗体轻链可变区DNA的核苷酸序列见SEQ ID NO:53，由该DNA序列推测得到317抗体轻链可变区氨基酸序列见SEQ ID NO:52；317抗体重链可变区DNA的核苷酸序列见SEQ ID NO:55，由该DNA序列推测得到317抗体重链可变区氨基酸序列见SEQ ID NO:54。
表1.轻链PCR扩增引物
Figure PCTCN2020071353-appb-000003
表2.重链PCR扩增引物
Figure PCTCN2020071353-appb-000004
Figure PCTCN2020071353-appb-000005
实施例2：抗人PD-1嵌合抗体的制备
将克隆获得的单克隆抗体轻链可变区和重链可变区基因通过如下引物(表3)PCR引入酶切位点，分别克隆至装有人-kappa轻链恒定区和人IgG4重链恒定区编码基因上游的真核表达载体中，获得人-鼠嵌合轻链(pKN019-317L)和人-鼠嵌合重链(pKN034-317H)表达质粒，转入大肠杆菌扩增，分离获得大量含人-鼠嵌合抗体轻链和重链的质粒，将二者与293fectin混合后共转染入HEK293细胞中。细胞转染5-6天，取培养上清，利用ProA亲和层析柱对表达上清进行纯化，获得的317嵌合抗体利用Fortebio(BLITZ pro1.1.0.28)仪器，采用抗人抗体捕获法测定抗体亲和力。测定时将抗人抗体Fc段的捕获抗体(AHC)生物探针浸泡于PBS，10min；将4μl稀释后的抗体样品(317嵌合抗体，Nivolumab，20μg/mL)分别上样到2根AHC生物探针上，然后在PBS中平衡30s，进一步将AHC探针与不同稀释浓度的人PD-1-His重组蛋白(序列号：NP_005009.2，21aa-167aa，Lot：2016.2.22)(1μM，0.6μM及0nM)进行结合反应，结合时间45s，之后将AHC探针转移至PBS中，进行解离反应，时间为120s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
317嵌合抗体(图4，4A)及对照抗体Nivolumab(图4，4B)与人PD-1重组蛋白的反应曲线如图4所示，拟合曲线并计算亲和力，结果317嵌合抗 体亲和力(KD)为1.02E-09M，Nivolumab亲和力(KD)为1.01E-09M。详细动力学参数如表4所示。结果表明317嵌合抗体与人PD-1具有高亲和力，且在同等条件下，解离值与对照抗体Nivolumab相近。该结果为317抗体的人源化及对PD-1/PD-L1信号通路的抑制作用提供了理论依据。
表3. 317抗体轻链及重链可变区克隆引物
Figure PCTCN2020071353-appb-000006
表4. 317嵌合抗体与人PD-1胞外区结构域重组蛋白的亲和力测定结果
  KD值(M) kon(1/Ms) kd(1/s)
317 2.64E-09 2.15E+05 5.66E-04
Nivolumab 2.018E-09 3E+05 6.05E-04
实施例3：抗人PD-1单克隆抗体的人源化及稳定细胞株的构建
首先对鼠源抗体重链序列进行综合分析，确定抗体与抗原结合的抗原互补决定簇(CDR)区域及支撑抗体保守三维构象的框架区(framework)。随后根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似人源抗体模板，选择VH3(3-21)为基础模板，结合全序列blast结果，考虑重排(rearranged)抗体在特定FR区位点氨基酸出现频率(A49)，进行CDR移植，考虑FR3区(S98)靠近CDR3区，不进行更换，根据CDR3序列(Pdssgvay)情况，选择JH4(wgqgtlvtvss)为J区序列，实现了H1在框架区的高度人源化。根据同源性比对结果，在人抗体germline库(http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX)寻找最相似 人源抗体模板，选择VK II(A19)为基础模板，结合全序列blast结果，考虑重排抗体在特定FR区位点氨基酸出现频率(R45)，进行CDR移植，根据CDR3序列(Qqysrypw)情况，选择JK1(fgqgtkveik)为JK区序列，实现了轻链框架区的全人源化。
将317抗体鼠源序列进行全人源化设计并全合成该序列，并将人源化后的317抗体命名为h317，将人源化的h317-VH1通过酶切克隆入真核表达载体pKN034的人IgG4的重链恒定区编码基因的上游，将人源化的h317-VL2通过酶切克隆入真核表达载体pKN019的人轻链Cκ的编码基因的上游，构建人源化317轻、重链表达载体，将h317-H和L通过酶切的方法克隆至pKN002真核稳定高表达载体，通过Pvu I线性化后，利用Nucleofector 2b电转仪(300452，Lonza)，将h317基因整合到CHO细胞DNA中，经过MSX(Cat：M5379-500MG，Sigma)加压筛选及亚克隆得到最终高表达h317的稳定细胞株。317人源化后轻链可变区DNA的核苷酸序列见SEQ ID NO:61，氨基酸序列见SEQ ID NO:60；人源化后重链可变区DNA的核苷酸序列见SEQ ID NO:63，氨基酸序列见SEQ ID NO:62。
实施例4：h317与人PD-1蛋白的结合动力学研究
h317及Nivolumab的生物素化：将200μL的5mg/ml EZ-LinkNHS-LC-LC-Biotin(Cat：21343，Thermo)室温放置5分钟，与1ml1mg/mL的h317(Lot：DP201805002，江苏泰康生物医药有限公司)或Nivolumab(Lot：AAW4553，BMS)蛋白溶液混匀，室温放至30min。超滤浓缩至0.5mL，以280nm的吸收值测定蛋白含量，分装，-80℃保存，冻融不超过1次。
结合动力学测定：利用Fortebio公司的Octet QKe system仪器，采用Streptavidin捕获Biotin标记抗体法测定抗体亲和力。测定时将Biotin标记的抗体(h317-biotin，Nivolumab-biotin)用PBS缓冲液稀释至5μg/mL，流经预包被了Streptavidin的探针(Cat：1612101，PALL)表面，时间为120s。人PD-1重组蛋白(序列号：NP_005009.2，21aa-167aa)作为流动相，用PBS作两倍浓度梯度稀释，其浓度范围为100nM～12.5nM。结合时间为300s，解离时间为300s。实验完毕，扣除空白对照响应值，用软件进行1:1Langmuir结合模式拟合，计算抗原抗体结合的动力学常数。
结果显示h317(图5，5A)及对照抗体Nivolumab(图5，5B)与人PD-1 重组蛋白的反应曲线如图5所示，拟合曲线并计算亲和力，h317亲和力(KD)为6.16E-09M，Nivolumab亲和力(KD)为8.06E-09M。详细动力学参数如表5所示。结果表明h317与人PD-1具有高亲和力，且在同等条件下，解离值优于对照抗体Nivolumab。该结果为h317对PD-1/PD-L1信号通路的抑制作用提供了理论依据。
表5. h317与人PD-1胞外区结构域重组蛋白的亲和力测定结果
  KD值(M) kon(1/Ms) kd(1/s)
h317 6.16E-09 1.54E+05 9.50E-04
Nivolumab 8.06E-09 2.09E+05 1.69E-03
实施例5：ELISA检测h317对PD-1与其配体PD-L1、PD-L2结合的抑制作用
ELISA检测h317对PD-1与PD-L1结合的抑制作用：将人PD-1-hFc(序列号：NP_005009.2，21aa-167aa，Lot：20180329)浓度稀释至0.5μg/mL，4℃包被过夜，用5％BSA于37℃恒温培养箱封闭60min。加入h317(Lot：DP201805002，江苏泰康生物医药有限公司)或Nivolumab(Lot：AAW4553，BMS)(起始浓度为3μg/mL，1.5倍连续稀释)，37℃恒温培养箱反应120min，加入1μg/mL的PD-L1-mFc(序列号：NP_054862.1，19aa-238aa，Lot：20180412)与抗体共孵育，37℃恒温培养箱反应60min。然后加入1:5000稀释的HRP-anti-mouse Fc(Cat：115-035-071，Jackson Immuno Research)，反应45min，加入TMB(Cat：ME142，北京泰天河生物)底物显色15min，2M HCl终止后读板。以630nm为参比波长，读取并记录波长450nm下孔板的吸光度值A450nm-630nm。
ELISA检测h317对PD-1与PD-L2结合的抑制作用：将PD-1-hFc(序列号：NP_005009.2，21aa-167aa，Lot：20180329)浓度稀释至0.5μg/mL，4℃包被过夜，用5％BSA于37℃恒温培养箱封闭60min。加入h317(Lot：DP201805002，江苏泰康生物医药)或Nivolumab(Lot：AAW4553，BMS)(起始浓度为3μg/mL，1.5倍连续稀释)，37℃恒温培养箱反应120min，加入2μg/mL的PD-L2-hFc-Biotin(序列号：NP_079515.2，20aa-220aa，Lot：2016.12.05)，与抗体共孵育，37℃恒温培养箱反应60min。然后加入1:2000 稀释的HRP-streptavidin(Cat：S2438，Sigma)反应30min，加入TMB底物显色15min，2M HCl终止后读板。以630nm为参比波长，读取并记录波长450nm下孔板的吸光度值A450nm-630nm。
结果显示h317可以有效阻断重组人PD-1与其配体PD-L1和PD-L2的结合作用。通过ELISA测定了h317和Nivolumab对PD-1与其配体PD-L1结合的竞争抑制效应，其半数有效抑制浓度(IC50)值分别1.4nM和1.3nM(图6，6A)。通过ELISA测定了h317和Nivolumab对PD-1与其配体PD-L2结合的竞争抑制效应，其半数有效抑制浓度(IC50)值分别1.2nM和1.0nM(图6，6A)。与Nivolumab相比，h317阻断活性相近。
实施例6：ELISA检测h317与PD-1结合的种属特异性研究
将重组人(序列号：NP_005009.2，21aa-167aa，Lot：20180423)、食蟹猴(Cat：90311-C08H，北京义翘神州)、大鼠(Cat：80448-R08H，北京义翘神州)及小鼠(Cat：50124-M08H，北京义翘神州)PD-1蛋白4℃包被过夜，包被浓度1μg/mL；PBS洗板3次后，加入5％BSA PBS，37℃封闭60min，PBST洗板3次；加入不同稀释倍数的生物素化的h317(同实施例4)(起始浓度为1μg/mL，3倍梯度依次稀释11个浓度)，37℃孵育60min，PBST洗板4次；加入1:100稀释的HRP-streptavidin(Cat：019-035-098，Jackson Immuno Research)，37℃孵育30min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。
实验结果表明，h317可以特异性地与重组人、食蟹猴PD-1结合(图7)，其半数有效结合浓度(EC50)值分别为0.004703nM及0.01823nM，但与重组大鼠、小鼠PD-1则无结合活性，不产生交叉反应(图7)，为h317进行体内外药效学实验提供依据。
实施例7：ELISA检测h317与CD28家族成员结合的专一性研究
将重组人PD-1(序列号：NP_005009.2，21aa-167aa，Lot:20180423)、CD28(Cat：11524-HCCH，北京义翘神州)、CTLA4(Cat：11159-H08H，北京义翘神州)及ICOS(序列号：NP_036224.1，1aa-199aa)蛋白4℃包被过夜，包被浓度1μg/mL；PBS洗板3次后，加入5％BSA PBS，37℃封闭60min，PBST洗板3次；加入不同稀释倍数的生物素化的h317(同实施例 4)(起始浓度为1μg/mL，3倍梯度依次稀释11个浓度)，37℃孵育60min，PBST洗板4次；加入1:100稀释的HRP-streptavidin(Cat：019-035-098，Jackson Immuno Research)，37℃孵育30min，PBST洗板4次；加入TMB底物显色，37℃孵育10min后，加入2M HCl终止反应；以630nm为参比波长，读取并记录波长450nm下孔板的吸光度A450nm-630nm。
实验结果表明，h317可以特异性地与重组人PD-1结合，与其他CD28家族成员无结合活性，不产生交叉反应(图8)，为h317进行体内外药效学实验提供依据。
实施例8：h317体外刺激人T细胞(PBMC)活性研究
A.分离CD14+单核细胞和DC细胞的分化
根据Ficoll-Paque PLUS(Cat：17-1440-03，Lot：10246684，GE Healthcare)试剂提取说明提取PBMC后，按照CD14磁珠(Cat：130-050-201，Lot：5170814438，Miltenyi)说明，阳选CD14+单核细胞。随后将细胞用ImmunoCult TM DC Differentiation Medium(Cat：10988，Lot：17G83035，STEMCELL)重悬至5×10 5细胞/ml，5ml体积接入到一个T-25细胞培养瓶中，置于37℃、5％CO 2培养箱中培养3天后，离心换液，置于37℃、5％CO 2培养箱中培养2天后，加入50μl的ImmunoCult TM Dendritic Cell Maturation Supplement(Cat：10989，Lot：17B77271，STEMCELL)，混匀后，置于37℃、5％CO 2培养箱中培养2天，用于MLR实验。
B.CD4+T细胞的分离提取
根据Ficoll-Paque PLUS(Cat：17-1440-03，Lot：10246684，GE Healthcare)试剂提取说明提取PBMC后，按照CD4+T细胞分离试剂盒(Cat：130-096-533，Lot：5171016623，Miltenyi)说明，从PBMC中分离CD4+T细胞，用于MLR实验。
C.MLR实验
先用完全培养基配置浓度为8μg/mL的待测抗体(h317(Lot：DP201805002，江苏泰康生物医药)，Nivolumab(Lot：AAW4553，BMS)，Human NC-IgG4对照(Lot：AB170090)溶液，再进行10倍梯度稀释，共配制6个浓度。随后根据实验板的设计加50μL的抗体到相应的孔中。将CD4+T细胞的浓度调整到1×10 6/mL，加100μL(1×10 5/孔)的细胞悬液到相应的孔中。用2mM的EDTA消化DC细胞，1500rpm离心5min，调整DC细胞 的浓度为2×10 5/mL，加50μL(1×10 4/孔)DC细胞悬液到相应孔中，总体积为200μL，CD4+T细胞:DC细胞＝10:1。将96孔细胞培养板置37℃细胞培养箱中培养5天后，收集细胞上清液，用IFN-γELISA试剂盒(Cat：430106，Lot：B247782,Biolegend)，IL-2ELISA试剂盒(Cat：DY202，Lot：P155804，R&D)检测IFN-γ和IL-2的浓度。
检测结果表明，h317体外刺激人混合淋巴细胞，可促进其分泌杀伤性细胞因子IL-2(图9，9A)和IFN-γ(图9，9B)，提示h317可以诱导Th1型细胞介导的免疫反应。
实施例9：h317对293T/hPD-1细胞的ADCC毒性作用研究
根据Ficoll-Paque PLUS(Cat：17-1440-03，Lot：10246684，GE Healthcare)试剂提取说明提取PBMC后，细胞重悬在ADCC培养基(无酚红RPMI1640培养基(Cat：11835-030，Lot：1860152，Gibco)+2％FBS(Cat：SH30084.03，Lot：GAE0138，Hyclone))中。对得到的PBMC进行细胞计数，按照NK细胞分离试剂盒(Cat：130–092–657，Lot：5170911524，Miltenyi)说明，进行NK细胞分离，用于ADCC毒性分析。按照CytoTox
Figure PCTCN2020071353-appb-000007
非放射性细胞毒性检测试剂盒(Cat：G1781，Lot：0000265175，Promega)说明，取在对数生长期的表达人PD-1蛋白的293T细胞，用ADCC培养基稀释调整目的细胞悬液浓度为50×10 4个/mL，在96孔圆底板中每孔加入40μL细胞悬液，每孔加入10μL药物溶液(每个细胞浓度设置三个复孔)，并在室温孵育30分钟。被测抗体(h317(Lot：DP201805002，江苏泰康生物医药)，h317-hIgG1(Lot：20180528)，Human NC-IgG4(Lot：20180521))终浓度为10000ng/mL，1000ng/mL，100ng/mL，10ng/mL，1ng/mL，0.1ng/mL，0.01ng/mL。通过加入调整至不同浓度的50μL NK细胞细胞悬液，设置两个E:T比例10:1、5:1。将96孔板于250g离心4min，让效应细胞和目的细胞的充分接触，在37℃、5％CO 2、95％空气条件下进行细胞培养4h。转移上清前45min时，在最大裂解对照孔(max lysis孔)中加入10μL裂解液(每100μL加入10μL)。孵育4h后，250g离心5min。将50μL上清转移到干净的96孔平板中。加入50μL反应底物并混合30s后，在室温孵育30min。加入50μL终止液并混合30s后，读取吸光度值(OD490nm)。
将实验组孔数值，目的细胞对照孔数值，NK细胞+目的细胞孔数值减去培养基对照孔数值。将得到的数值用下列公式计算细胞毒性。
Figure PCTCN2020071353-appb-000008
实验结果表明，h317与293T细胞表面的hPD-1结合后并不启动人PBMC(外周血单个核细胞)中NK细胞对靶细胞的杀伤作用(图10)。与不加抗hPD-1抗体的对照组相比，h317对高表达hPD-1的293T细胞没有ADCC活性。结论是h317没有ADCC效应功能。
实施例10：h317在PD-1转基因鼠移植瘤模型中的药效体内抗肿瘤药效评价
试验共选用65只HuGEMMPD-1小鼠(6-8周，上海南方模式生物)，在受试小鼠右侧皮下接种MC38-hPD-L1肿瘤细胞(1×10 6/只，1×10 6细胞重悬于100μL PBS)。当荷瘤鼠平均肿瘤体积到达约60-100mm 3时，测定动物体重，并用游标卡尺测量肿瘤体积，开始给药，给药前使用StudyDirector TM(版本号3.1.399.19，供应商Studylog System,Inc.，S.San Francisco，CA，USA)进行随机分组，共分为6组，进行给药(D0)，每组的动物数及详细的给药途径、剂量和方案见表6，给药、肿瘤测量及体重称量等全部过程都在生物安全柜或超净工作台中进行。实验中使用StudyDirector TM(版本号3.1.399.19，供应商Studylog System,Inc.)软件收集数据，包括肿瘤的长短径的测量和动物体重的称量，原始数据由天平和游标卡尺测量后直接导入软件，当对照组小鼠瘤体积均值超过2000mm 3时或最后一次给药后一星期终止实验。并收集肿瘤块，测量肿瘤重量，拍摄肿瘤照片，结束实验当天取瘤做FACS，每组4只(与采集血液动物编号对应)，Marker：CD3，CD4，CD8，CD45，Live/Dead。结实验当天取全血做FACS，每组4只(与采集肿瘤动物编号对应)，Marker：CD3，CD4，CD8，CD45。
表6.药效学实验设计
Figure PCTCN2020071353-appb-000009
Figure PCTCN2020071353-appb-000010
注：给药体积为10μL/g；hIgG对照(Lot：20180521)，h317(Lot：DP201805002，江苏泰康生物医药)，Nivolumab(Lot：AAW4553，BMS)
HuGEMMPD-1小鼠模型是基因工程鼠，是在C57BL/6J遗传背景下，将人源PD-1蛋白编码区插入到小鼠PD-1的ATG位置，表达人源PD-1，取代小鼠PD-1的表达。MC38是C57BL/6小鼠中诱导获得的鼠源肠癌细胞系。通过基因工程的方法，敲除MC38的鼠源PD-L1，并敲入人源PD-L1，获得了表达人源PD-L1的MC38细胞系，即MC38-hPD-L1细胞系。在PD-1转基因鼠移植瘤模型上评价并比较了h317及其参比抗体Nivolumab对肿瘤生长的抑制效果。结果表明(见图11，表7)，h317在高剂量和中剂量下可显著抑制PD-1转基因鼠皮下移植瘤的生长，并且在高中剂量水平下h317与其参比抗体Nivolumab的抑瘤效果相当。
表7.各组PD-1转基因鼠皮下移植瘤的瘤重数据及统计分析
Figure PCTCN2020071353-appb-000011
Figure PCTCN2020071353-appb-000012
注释：a.数据以“平均值±标准误差”表示；n＝8
b.p值运用单因素方差分析(one way ANOVA)肿瘤体积所得，F值有显著性差异(p<0.05)，应用Dunnett’s T3法进行分析。
实施例11：h317结合PD-1受体内化
瞬时表达人PD-1的293细胞株的构建：将HEK293细胞按6*10 5个/ml的密度接入摇瓶中，体积10ml，置于37℃，5％CO 2的振摇培养箱中130rpm振摇培养24h。将编码全长人PD-1(NP_005009.2，21aa-288aa)表达质粒与293fectin(Cat：12347019，Gibco)按1：1.2的比例分别在0.5mL Opti-MEM(Cat：11058021，Gibco)培养基中静置5min后，二者混匀室温静置15min，然后加入到HEK293细胞中。置于37℃，5％CO 2的振摇培养箱中130rpm振摇培养48h，293/hPD-1细胞用于FACS检测。
PD-1介导的h317内吞：Mix-n-Stain TM CF TM 488A(Cat：MX488AS100，Sigma)标记h317(Lot：DP201805002，江苏泰康生物医药有限公司)和Nivolumab(Lot：AAW4553，BMS)；用5％的BSA将标记好的抗体稀释至10μg/mL，PBS洗涤293/hPD-1细胞1次，加入稀释好的h317-CF488A或Nivolumab-CF488A，一组放37℃电热恒温培养箱，一组放4℃冰箱做为阴性对照；孵育1h后PBS洗涤3次，仍继续放置于原来温度37℃或4℃培养；放置3h和6h后各用荧光显微镜观察一次并拍照。
通过荧光显微镜观察PD-1介导的h317内吞，实验结果(见图12)表明h317和Nivolumab在37℃条件下都能够被PD-1介导内吞。
实施例12：317识别表位研究
PD-1-h317-Fab复合物的制备：人PD-1胞外区结构域(序列号：NP_005009.2，21aa-167aa，Lot：20170626)重组蛋白分子量为16.4Kd，h317-Fab(Lot：20170626)分子量约为46.5KD，在20mM Tris 150mM NaCl溶液，根据二者的摩尔比计算，选择PD1:h317-Fab＝1:1.5的比例混合，冰上放置30min，复合物形成以后，将复合物超滤浓缩至浓度为12mg/ml，用于结晶分析。
PD-1-h317-Fab复合物结晶及晶体衍射数据的收集和结构解析：采用坐滴法，利用结晶筛选机器人完成，该装置每孔消耗0.2μl蛋白样品，加样结 束后密封晶体板，放置恒温室培养。晶体生长温度为16℃和4℃，蛋白的浓度为8mg/ml、12mg/ml。筛选的试剂盒包括：HAMPTON公司：Crystalscreen(1-98)、Index(1-96)、PEGRX(1-96)、SaltRx(1-96)、Nartrix(1-96)、PEG/ion(1-96)；QIAGEN公司：Protein Complex Suite(1-96)；Emerald Biosystems公司：WIZARD I/II(1-96)、WIZARD III/IV(1-96)。15天内观察晶体生长结果，对初筛晶型较好的晶体生长条件进行优化。优化后获得的晶体，衍射实验前将晶体在添加10％乙二醇的结晶溶液中浸泡数秒，然后浸入液氮快速冷冻。晶体衍射实验在上海同步辐射光源(SSRF)BL17U线站，使用的X射线波长为
Figure PCTCN2020071353-appb-000013
晶体衍射温度为100K，衍射数据使用ADSC公司Q315r探测器收集。衍射数据处理使用HKL2000软件，确定晶体空间群为P2，晶体衍射分辨率为
Figure PCTCN2020071353-appb-000014
使用PHASER软件通过分子置换法解析结构，所选用的分子置换模型为PD-1单独蛋白结构(PDB ID 3RRQ)和PD-1Fab(PDB ID 5WT9)结构。使用COOT软件重新搭建模型，并用REFMAC5和PHENIX软件进行结构精修。
去糖基化人PD-1重组蛋白与h317的结合：在人PD-1抗原(NP_005009.2)结构中可以观察到N49、N58、N116、N74位四个糖基化位点，通过定点突变的方法，将糖基化位点突变为丙氨酸(A)，装入带有EGFP的表达载体共构建5种人PD-1全长表达质粒(WT，N49A、N58A、N116A、N74A)，将质粒分别与293fectin混合后共转染入HEK293细胞中。细胞转染2天，取细胞，收集细胞于96孔板中，1500rpm，离心2min，弃上清。加入200μl PBS重悬，1500rpm，离心2min，弃上清。加入200μl用5％BSA稀释的一抗(h317(Lot：2017.05.24)，Nivolumab(Lot：2017.04.26))，工作浓度为2μg/ml，阴性对照不加一抗，室温孵育1h。1500rpm，离心2min，弃上清。加入200μl PBS重悬，1500rpm，离心2min，弃上清。加入200μl用5％BSA稀释的羊抗人IgG(Fc)-Cy5(Cat：12136，Lot：017M4800V，Sigma)，4℃孵育1h。1500rpm，离心2min，弃上清。加入200μl PBS重悬，1500rpm，离心2min，弃上清。加入200μl PBS重悬，流式细胞仪(型号B49007AD，SNAW31211)检测。
PD-1-h317-Fab复合物整体结构分析显示，PD-1抗原(胞外区1-167aa)以单体形式存在，与PD-1抗体的h317-Fab片段形成一个1:1的复合物结构(图13)。PD-1与抗体的结合部位主要是在其loop区，包括BC loop，C’D loop和FG loop区域(图14)。相互作用方式包括氢键、盐键、范德华力以及疏 水相互作用，参与相互作用的氨基酸及作用力(表8)。
表8.PD-1与Fab317相互作用的氨基酸
Figure PCTCN2020071353-appb-000015
Figure PCTCN2020071353-appb-000016
此外，在PD-1抗原结构中可以观察到N49、N58、N116位点的糖基化，N74的糖基化位点不明确，其中N58位的糖链参与了与h317-Fab的结合(图15)。其他已报道的PD-1抗原抗体复合物结构中该位点的糖链没有参与二者相互作用，因此h317-Fab与N58糖链的相互作用有可能提高了二者识别和结合的特异性。将PD-1上的糖基化位点突变，并瞬转至293细胞，通过FACS检测h317与PD-1-mut分子的结合情况；FACS结果显示人PD-1的N58糖链参与h317与PD-1的结合，N58突变为A失去糖基化使h317失去与PD-1表达细胞结合(图16)，PD-1的N58糖链参与h317与PD-1的结合。通过比较PD-1-h317-Fab复合物结构与PD-1/PD-L1的复合物结构发现，h317-Fab和PD-L1在与PD-1相互作用界面上存在很大面积的重叠(图17A)，大部 分重叠部位在PD-1的FGloop(图17B)，表明h317-Fab结合PD-1与PD-L1具有竞争性，能够阻断PD-1与PD-L1之间的相互作用。
实施例13 h317轻、重链CDR区定向突变
1.根据PD-1-h317Fab晶体复合物结构解析，构建h317轻、重链CDR区突变体，表达载体的构建采用质粒定点突变的方法进行，具体可参照文献[王荣浩，陈瑞川，刘润忠。快速点突变的优化方法。厦门大学学报(自然科学版)，2008,Vol 47,sup 2,282-285]。突变体抗体表达和纯化方法同上。
2.突变体相对亲和力比较
突变体与亲本抗体亲和力比较采用Fortebio公司的Octet QKe system仪器完成。具体方法同实施例4，不同之处在于所有突变体亲和力仅测定单线以评估相对亲和力。首先包被相同目标值的待测抗体，并以100nM的人PD-1重组蛋白(序列号：NP_005009.2，21aa-167aa)为流动相进行相对亲和力测定。
对突变体抗体亲和力测定结果显示，多个位点突变后抗体的亲和力发生较大改变，突变位点信息如表9，部分亲和力变化如表10所示。
表9. h317突变体CDR区的氨基酸序列
Figure PCTCN2020071353-appb-000017
Figure PCTCN2020071353-appb-000018
表10. h317 CDR区突变体亲和力变化情况统计表(除所示突变位点外，可变区其余氨基酸与h317相同)
Figure PCTCN2020071353-appb-000019
Figure PCTCN2020071353-appb-000020
从表中数据看出，个别位置的氨基酸突变后，对亲和力会产生较大影响，有些位点亲和力降低10倍以上。同时，有些位点突变后抗体的亲和力有所提升，比如轻链第24位K突变为R、第56位T突变成S、第92位S突变为N后，重链第33位D突变为S、第54位G突变成S、第59位Y突变为A后，均可使抗体亲和力得到一定提高，并且这些突变位点可以进一步叠加突变，其亲和力保持不变或进一步提高。HCDR3对结合至关重要，突变后大多亲和力下降。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (17)

  1. 一种结合程序性细胞死亡蛋白1(PD-1)的抗体或其片段，所述抗体或其片段包含轻链可变区和/或重链可变区，其中所述轻链可变区和/或重链可变区分别包含以下序列所示的轻链CDR1(LCDR1)、CDR2(LCDR2)、CDR3(LCDR3)和/或重链CDR1(HCDR1)、CDR2(HCDR2)、CDR3(HCDR3)：
    Figure PCTCN2020071353-appb-100001
  2. 根据权利要求1所述的抗体或其片段，其特征在于，所述轻链可变区和/或重链可变区分别包含以下序列所示的LCDR1、LCDR2、LCDR3和/或HCDR1、HCDR2、HCDR3：
    Figure PCTCN2020071353-appb-100002
    Figure PCTCN2020071353-appb-100003
  3. 根据权利要求1或2所述的抗体或其片段，其特征在于，所述轻链可变区和/或重链可变区分别包含以下序列所示的LCDR1、LCDR2、LCDR3和/或HCDR1、HCDR2、HCDR3：
    选自SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11的LCDR1；
    选自SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17的LCDR2；
    选自SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27的LCDR3；和/或
    选自SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32和SEQ ID NO:33的HCDR1；
    选自SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45和SEQ ID NO:46的HCDR2；
    选自SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51的HCDR3。
  4. 根据权利要求1至3中任一项所述的抗体或其片段，其特征在于，所述轻链可变区包含以下序列组合所示的LCDR1、LCDR2、LCDR3：
    组合 LCDR1 LCDR2 LCDR3 1 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 2 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:18 3 SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:18
    4 SEQ ID NO:10 SEQ ID NO:12 SEQ ID NO:18 5 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:18 6 SEQ ID NO:7 SEQ ID NO:13 SEQ ID NO:18 7 SEQ ID NO:7 SEQ ID NO:14 SEQ ID NO:18 8 SEQ ID NO:7 SEQ ID NO:15 SEQ ID NO:18 9 SEQ ID NO:7 SEQ ID NO:16 SEQ ID NO:18 10 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:18 11 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:19 12 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:20 13 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:21 14 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:22 15 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:23 16 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:24 17 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:25 18 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:26 19 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:27 20 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:18 21 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:22 22 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:25 23 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:22 24 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22
    和/或，所述重链可变区包含以下序列组合所示的HCDR1、HCDR2、HCDR3：
    组合 HCDR1 HCDR2 HCDR3 1 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 2 SEQ ID NO:29 SEQ ID NO:34 SEQ ID NO:47 3 SEQ ID NO:30 SEQ ID NO:34 SEQ ID NO:47 4 SEQ ID NO:31 SEQ ID NO:34 SEQ ID NO:47 5 SEQ ID NO:32 SEQ ID NO:34 SEQ ID NO:47 6 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:47 7 SEQ ID NO:28 SEQ ID NO:35 SEQ ID NO:47
    8 SEQ ID NO:28 SEQ ID NO:36 SEQ ID NO:47 9 SEQ ID NO:28 SEQ ID NO:37 SEQ ID NO:47 10 SEQ ID NO:28 SEQ ID NO:38 SEQ ID NO:47 11 SEQ ID NO:28 SEQ ID NO:39 SEQ ID NO:47 12 SEQ ID NO:28 SEQ ID NO:40 SEQ ID NO:47 13 SEQ ID NO:28 SEQ ID NO:41 SEQ ID NO:47 14 SEQ ID NO:28 SEQ ID NO:42 SEQ ID NO:47 15 SEQ ID NO:28 SEQ ID NO:43 SEQ ID NO:47 16 SEQ ID NO:28 SEQ ID NO:44 SEQ ID NO:47 17 SEQ ID NO:28 SEQ ID NO:45 SEQ ID NO:47 18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:48 19 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:49 20 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:50 21 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:51 22 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47 23 SEQ ID NO:32 SEQ ID NO:44 SEQ ID NO:47 24 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47 25 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47 26 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47
    优选地，所述轻链可变区和重链可变区包含以下序列组合所示的LCDR1、LCDR2、LCDR3和HCDR1、HCDR2、HCDR3：
    组合 LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 1 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 2 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 3 SEQ ID NO:9 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 4 SEQ ID NO:10 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 5 SEQ ID NO:11 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 6 SEQ ID NO:7 SEQ ID NO:13 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 7 SEQ ID NO:7 SEQ ID NO:14 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 8 SEQ ID NO:7 SEQ ID NO:15 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 9 SEQ ID NO:7 SEQ ID NO:16 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47
    10 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 11 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:19 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 12 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:20 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 13 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:21 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 14 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 15 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:23 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 16 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:24 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 17 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:25 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 18 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:26 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 19 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 20 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 21 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 22 SEQ ID NO:8 SEQ ID NO:12 SEQ ID NO:25 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 23 SEQ ID NO:7 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 24 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:47 25 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:29 SEQ ID NO:34 SEQ ID NO:47 26 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:30 SEQ ID NO:34 SEQ ID NO:47 27 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:31 SEQ ID NO:34 SEQ ID NO:47 28 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:32 SEQ ID NO:34 SEQ ID NO:47 29 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:33 SEQ ID NO:34 SEQ ID NO:47 30 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:35 SEQ ID NO:47 31 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:36 SEQ ID NO:47 32 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:37 SEQ ID NO:47 33 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:38 SEQ ID NO:47 34 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:39 SEQ ID NO:47 35 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:40 SEQ ID NO:47 36 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:41 SEQ ID NO:47 37 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:42 SEQ ID NO:47 38 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:43 SEQ ID NO:47 39 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:44 SEQ ID NO:47 40 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:45 SEQ ID NO:47
    41 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:48 42 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:49 43 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:50 44 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:34 SEQ ID NO:51 45 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47 46 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:32 SEQ ID NO:44 SEQ ID NO:47 47 SEQ ID NO:7 SEQ ID NO:12 SEQ ID NO:18 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47 48 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:32 SEQ ID NO:40 SEQ ID NO:47 49 SEQ ID NO:8 SEQ ID NO:17 SEQ ID NO:22 SEQ ID NO:28 SEQ ID NO:46 SEQ ID NO:47
  5. 根据权利要求1至4中任一项所述的抗体或其片段，其特征在于，所述抗体或其片段具有以下活性中的一个或多个：
    (1)阻断PD-1与其配体PD-L1或PD-L2的结合；
    (2)特异性结合灵长类动物PD-1，与非灵长类动物PD-1无交叉反应；
    (3)不结合除PD-1之外的其他CD28家族成员；
    (4)在CD4+T细胞中诱导IL-2和/或IFN-γ产生；
    (5)无ADCC效应功能；
    优选地，所述抗体或其片段结合人PD-1N58糖链；优选地，所述抗体或其片段的重链可变区包含SEQ ID NO:47所示氨基酸序列；
    优选地，所述轻链可变区包含SEQ ID NO:52或SEQ ID NO:60所示氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述重链可变区包含SEQ ID NO:54或SEQ ID NO:62所示的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；
    优选地，所述至少75％同一性为例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性。
  6. 根据权利要求1至5中任一项所述的抗体或其片段，其特征在于，所述轻链可变区包含SEQ ID NO:60所示氨基酸序列或所述氨基酸序列的变体，根据SEQ ID NO:60所示氨基酸序列的残基编号，所述变体相对于SEQ ID NO:60具有选自以下的一个或多个、优选1-3个氨基酸置换：K24R、E30S、V32A、V32Y、W50A、W50G、H55A、H55Q、T56S、Y91A、Y91F、S92D、S92N、R93N、R93S、Y94F、W96G和W96Y；
    优选地，所述变体相对于SEQ ID NO:60具有选自以下的一个或多个、优选1-3个氨基酸置换：K24R、V32A、V32Y、T56S、S92D、S92N、R93S和Y94F。
  7. 根据权利要求1至6中任一项所述的抗体或其片段，其特征在于，所述重链可变区包含SEQ ID NO:62所示氨基酸序列或所述氨基酸序列的变体，根据SEQ ID NO:62所示氨基酸序列的残基编号，所述变体相对于SEQ ID NO:62具有选自以下的一个或多个、优选1-3个氨基酸置换：S31D、Y32A、Y32N、D33S、D33Y、S52K、S52W、G53S、G53Y、G54D、G54S、G55S、S56G、Y57T、Y59A、Y59T、D100E、D100Y、S101A和Y106T；
    优选地，所述变体相对于SEQ ID NO:62具有选自以下的一个或多个、优选1-3个氨基酸置换：S31D、Y32A、D33S、G53S、G54S、G55S、Y59A和D100Y。
  8. 根据权利要求1至7中任一项所述的抗体或其片段，其特征在于，所述抗体为单克隆抗体、单链抗体、双功能抗体、单域抗体、纳米抗体、完全或部分人源化的抗体或者嵌合抗体等任意形式；优选地，所述抗体为IgA、IgD、IgE、IgG或IgM，更优选为IgG1或IgG4；
    优选地，所述片段为所述抗体能够特异性结合PD-1或其任何部分的片段；更优选地，所述片段为所述抗体的scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv片段；
    更优选地，所述抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型；
    进一步优选地，所述抗体的轻链恒定区包含SEQ ID NO:56或SEQ ID NO:64所示氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，
    所述重链恒定区包含SEQ ID NO:58或SEQ ID NO:66所示的氨基酸序列或者与所述氨基酸序列具有至少75％同一性的氨基酸序列；
    优选地，所述至少75％同一性为例如至少80％、优选至少85％、更优选至少90％、进一步优选至少91％、92％、93％、94％、95％、96％、97％、98％或甚至99％同一性等≥75％的任何百分比的同一性。
  9. 一种缀合物或融合蛋白，所述缀合物或融合蛋白包含权利要求1至8中任一项所述的抗体或其片段。
  10. 一种核酸分子，其编码权利要求1至8中任一项限定的所述抗体或 其片段中的重链CDR、轻链CDR、重链可变区、轻链可变区、重链或轻链；
    优选地，所述核酸分子包含SEQ ID NO:53、SEQ ID NO:55、SEQ ID NO:57、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:63、SEQ ID NO:65或SEQ ID NO:67所示核苷酸序列。
  11. 一种载体，其包含权利要求10所述的核酸分子。
  12. 一种宿主细胞，所述宿主细胞包含权利要求10所述的核酸分子和/或权利要求11所述的载体，或者所述宿主细胞被权利要求10所述的核酸分子和/或权利要求11所述的载体转化或转染。
  13. 一种药物组合物，所述药物组合物包含权利要求1至8中任一项所述的抗体或其片段、权利要求9所述的缀合物或融合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体和/或权利要求12所述的宿主细胞，以及任选的药学上可接受的辅料。
  14. 权利要求1至8中任一项所述的抗体或其片段、权利要求9所述的缀合物或融合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞和/或权利要求13所述的药物组合物在制备用于预防或治疗肿瘤或癌症的药物中的用途。
  15. 一种药物组合，其包括：
    (1)权利要求1至8中任一项所述的抗体或其片段、权利要求9所述的缀合物或融合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞和/或权利要求13所述的药物组合物；
    (2)其他免疫增强药物或手段；
    所述其他免疫增强药物或手段例如小分子化药、靶向药、抗体等重组蛋白药，疫苗、ADC、溶瘤病毒、基因和核酸治疗药物和放射疗法。
  16. 一种试剂盒，所述试剂盒包括权利要求1至8中任一项所述的抗体或其片段、权利要求9所述的缀合物或融合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞和/或权利要求13所述的药物组合物。
  17. 一种预防或治疗肿瘤或癌症的方法，所述方法包括给有此需要的受试者施用权利要求1至8中任一项所述的抗体或其片段、权利要求9所述的缀合物或融合蛋白、权利要求10所述的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞和/或权利要求13所述的药物组合物，以及任选的其他药物或手段。
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