WO2020156509A1 - 抗pd-1抗体、其抗原结合片段及医药用途 - Google Patents

抗pd-1抗体、其抗原结合片段及医药用途 Download PDF

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WO2020156509A1
WO2020156509A1 PCT/CN2020/074098 CN2020074098W WO2020156509A1 WO 2020156509 A1 WO2020156509 A1 WO 2020156509A1 CN 2020074098 W CN2020074098 W CN 2020074098W WO 2020156509 A1 WO2020156509 A1 WO 2020156509A1
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seq
antibody
variable region
chain variable
sequence
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PCT/CN2020/074098
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English (en)
French (fr)
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顾晓玲
叶鑫
葛虎
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to AU2020213579A priority Critical patent/AU2020213579A1/en
Priority to CA3128104A priority patent/CA3128104A1/en
Priority to BR112021015168-0A priority patent/BR112021015168A2/pt
Priority to CN202080005983.1A priority patent/CN112969716B/zh
Priority to KR1020217025236A priority patent/KR20210124993A/ko
Priority to MX2021009041A priority patent/MX2021009041A/es
Priority to US17/427,625 priority patent/US20220098304A1/en
Priority to EP20747692.0A priority patent/EP3922647A4/en
Priority to JP2021544436A priority patent/JP2022518588A/ja
Publication of WO2020156509A1 publication Critical patent/WO2020156509A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
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    • 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
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure belongs to the field of biotechnology. More specifically, the present disclosure relates to anti-PD-1 antibodies and applications thereof.
  • Tumor immunotherapy is a treatment method that fully utilizes and mobilizes the killer T cells in tumor patients to kill the tumor.
  • the escape of tumor cells is a huge obstacle to tumor immunotherapy.
  • Tumor cells use their own inhibitory effect on the immune system to promote the rapid growth of tumors.
  • tumor-specific killer T cells In the early stage of tumor immunotherapy, tumor-specific killer T cells have their biological activity, but as the tumor grows, they lose their killing function in the later stage.
  • T cells adopts two signal pathway systems.
  • a series of co-stimulatory molecules are also required to provide the second signal.
  • T cells produce a normal immune response.
  • This dual signal pathway system plays a vital role in the balance of the immune system in the body. It strictly regulates the body's different immune responses to self and non-self antigens. If the second signal provided by the costimulatory molecule is absent, it will lead to a non-response of T cells or a sustained specific immune response, thereby creating tolerance. Therefore, the second signaling pathway plays a very critical regulatory role in the entire process of the body's immune response.
  • PD-l Programmed death-l
  • CTL-4 cytotoxic T lymphocyte antigen 4
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (DC), and the expression on cells can be up-regulated after activation.
  • the expression of PD-L2 is relatively limited, and is mainly expressed on antigen-presenting cells, such as activated macrophages and dendritic cells.
  • PD-L1 inhibits the immune system by combining with PD-1 and B7-1.
  • Many tumor cells and immune cells in the tumor tissue microenvironment express PD-L1.
  • New research has found that breast cancer, lung cancer (for example, non-small cell lung cancer), gastric cancer, bowel cancer, kidney cancer, melanoma, colon cancer, bladder cancer, ovarian cancer, pancreatic cancer and liver cancer are highly detected in human tumor tissues.
  • the expression of PD-L1 protein, and the expression level of PD-L1 is closely related to the clinical and prognosis of patients.
  • the anti-PD-1 monoclonal antibody can block the binding between PD-L1/PD-1 and maximize the patient's own immune system response to tumors, thereby achieving the purpose of killing tumor cells.
  • the anti-PD-1 antibody Pembrolizumab also known as Merck keytruda, keytruda, Merck-Pemb, Merck-keytruda, Merck-PD-1, pembrolizumab
  • Novilumab also known as BMS Opdivo, Opdivo, BMS-Nivolumab, Na Wumab has been approved by the FDA for the treatment of melanoma, Hodgkin’s lymphoma patients, non-small cell lung cancer and other tumors.
  • patent documents WO200139722, WO2006121168, WO2010036959, WO2010089411, WO2011110604, WO2013173223, WO2013181634, US2014335093, US6803192B1, US8617546B2, WO2015085847, etc. also disclose a variety of anti-PD-1 monoclonal antibodies.
  • the present disclosure provides a new anti-PD-1 antibody, its antigen binding fragment and its medical use.
  • the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, which is an anti-PD-1 antibody or antigen-binding fragment thereof selected from any one of i) to iii) below Fragment:
  • an anti-PD-1 antibody or antigen-binding fragment thereof comprising the sequence shown in SEQ ID NO: 8 or HCDR1 with at most 3, 2 or 1 amino acid mutations, the sequence is as SEQ ID NO: 9 or HCDR2 with at most 3, 2 or 1 amino acid mutations, and sequence as shown in SEQ ID NO: 10 or at most 8, 3, 2 or 1 amino acids Mutated HCDR3;
  • the light chain variable region contains the sequence shown in SEQ ID NO: 11 or the LCDR1 with at most 4, 3, 2 or 1 amino acid mutations, the sequence shown in SEQ ID NO: 12 or the same LCDR2 with at most 3, 2 or 1 amino acid mutations, and the sequence shown in SEQ ID NO: 13 or LCDR3 with at most 3, 2 or 1 amino acid mutations;
  • an anti-PD-1 antibody or an antigen-binding fragment thereof the heavy chain variable region of which comprises the sequence shown in SEQ ID NO: 14 or the HCDR1 with at most 3, 2 or 1 amino acid mutations with the sequence shown in SEQ ID NO: 15 or HCDR2 with at most 3, 2 or 1 amino acid mutations, and sequence as shown in SEQ ID NO: 16, or at most 8, 3, 2 or 1 amino acids Mutated HCDR3;
  • the light chain variable region contains the sequence shown in SEQ ID NO: 17 or the LCDR1 with up to 4, 3, 2 or 1 amino acid mutations, the sequence shown in SEQ ID NO: 12 or the same LCDR2 with at most 3, 2 or 1 amino acid mutations, and the sequence shown in SEQ ID NO: 18 or LCDR3 with at most 3, 2 or 1 amino acid mutations;
  • An anti-PD-1 antibody or antigen-binding fragment thereof whose heavy chain variable region comprises the sequence shown in SEQ ID NO: 21 or HCDR1 with at most 3, 2 or 1 amino acid mutations with the sequence shown in SEQ ID NO: 22 or HCDR2 with at most 3, 2 or 1 amino acid mutations, and sequence as shown in SEQ ID NO: 23 or HCDR3 with at most 3, 2, or 1 amino acid mutations ;
  • the light chain variable region includes the sequence shown in SEQ ID NO: 24 or LCDR1 with at most 3, 2 or 1 amino acid mutations, and the sequence shown in SEQ ID NO: 25 or at most 3, 2 LCDR2 with one or one amino acid mutation, and the sequence shown in SEQ ID NO: 26 or with LCDR3 with at most 3, 2 or 1 amino acid mutation.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure binds to human PD-1 with a dissociation equilibrium constant equal to or less than 10 -7 M, and in some embodiments, it is equal to or less than 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 M dissociation equilibrium constant binds to human PD-1.
  • the present disclosure provides an anti-PD-1 antibody or antigen-binding fragment thereof, the heavy chain variable region of which comprises: HCDR1 with the sequence shown in SEQ ID NO: 65 and the sequence shown in SEQ ID NO : HCDR2 shown in 66, and HCDR3 shown in SEQ ID NO: 67; the light chain variable region includes: LCDR1 shown in SEQ ID NO: 68, and LCDR2 shown in SEQ ID NO: 12 , And the sequence of the LCDR3 region shown in SEQ ID NO: 69; the sequence is shown in Table 1 below:
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof comprises the HCDR1 sequence shown in SEQ ID NO: 8 and the sequence shown in SEQ ID NO: 9 HCDR2, and the sequence of HCDR3 shown in SEQ ID NO: 10;
  • the light chain variable region comprises the sequence of LCDR2 shown in SEQ ID NO: 12, the sequence of LCDR3 shown in SEQ ID NO: 13, and the sequence LCDR1 as shown in the general formula RSSQSX 13 VHSX 14 X 15 X 16 TYLE (SEQ ID NO: 68), wherein X 13 is selected from L, X 14 is selected from N, Q, L, T or D, and X 15 is selected from G , A or V, X 16 is selected from N.
  • the anti-PD-1 antibody or antigen-binding fragment thereof is selected from the anti-PD-1 antibody or the antigen-binding fragment thereof described in any one of (a) to (e) below:
  • An anti-PD-1 antibody or an antigen-binding fragment thereof which comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the sequence respectively LCDR2 and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13, and LCDR1 shown in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52;
  • An anti-PD-1 antibody or antigen-binding fragment thereof which comprises the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and the sequence respectively Such as the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18;
  • An anti-PD-1 antibody or antigen-binding fragment thereof which comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, and the sequence respectively Such as the LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26;
  • An anti-PD-1 antibody or an antigen-binding fragment thereof which comprises the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and the sequence respectively LCDR2 and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13, and LCDR1 shown in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52; and
  • the heavy chain variable region includes the sequence of HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the light chain variable region includes sequence The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 12, and SEQ ID NO: 18, respectively.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence as SEQ ID NO: 8 , SEQ ID NO: 9 and SEQ ID NO: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 10, and the light chain variable region comprising sequences are as shown in SEQ ID NO: 49, SEQ ID NO: 12 and SEQ ID NO: 13, respectively LCDR1, LCDR2 and LCDR3 shown.
  • the anti-PD-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a sequence as shown in SEQ ID NO: 21, respectively. , SEQ ID NO: 22 and SEQ ID NO: HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 23, and the light chain variable region comprising sequences are as shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively LCDR1, LCDR2 and LCDR3 shown.
  • the present disclosure provides an anti-PD-1 antibody or an antigen-binding fragment thereof, which is an anti-PD-1 antibody or an antigen-binding fragment thereof selected from any one of iv) to vi) below Fragment:
  • An anti-PD-1 antibody or antigen-binding fragment thereof whose heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 4, and light
  • the chain variable region includes LCDR1, LCDR2 and LCDR3 which have the same sequence as the light chain variable region shown in SEQ ID NO: 5;
  • an anti-PD-1 antibody or antigen-binding fragment thereof the heavy chain variable region of which comprises HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 6, and light
  • the chain variable region includes LCDR1, LCDR2, and LCDR3 that have the same sequence as the light chain variable region shown in the sequence of SEQ ID NO: 7;
  • An anti-PD-1 antibody or antigen-binding fragment thereof the heavy chain variable region of which comprises HCDR1, HCDR2 and HCDR3 having the same sequence as the heavy chain variable region shown in SEQ ID NO: 19, and light
  • the chain variable region includes LCDR1, LCDR2, and LCDR3 that have the same sequence as the light chain variable region shown in SEQ ID NO: 20.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof wherein said anti-PD-1 antibody or antigen-binding fragment thereof is a murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof , Fully human antibodies or antigen-binding fragments thereof, or humanized antibodies or antigen-binding fragments thereof.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof wherein the anti-PD-1 antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.
  • the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof.
  • the framework region variant is a back mutation having at most 11 amino acids on the basis of the light chain framework region and/or the heavy chain framework region of a human antibody.
  • the framework region variant comprises a mutation selected from any one of the following (f) to (h):
  • the light chain variable region contains a 2G amino acid back mutation, and/or the heavy chain variable region contains one or more amino acid back mutations selected from 27Y, 48I, 67T, 69L, 82F and 93T;
  • variable region of the light chain contains a 2V amino acid back mutation
  • variable region of the heavy chain contains a mutation selected from 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T One or more amino acid back mutations
  • the light chain variable region contains one or more amino acid back mutations selected from 42G, 44V and 71Y, and/or the heavy chain variable region contains 1K and/or 94S amino acid back mutations.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises an antibody variable region selected from the following:
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, and the heavy chain framework region includes 27Y, 48I, 67T, One or more amino acids in 69L, 82F and 93T are backmutated, and
  • the light chain variable region includes LCDR2 and LCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13, and LCDR1 shown in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52, respectively , And the light chain framework region contains a 2G amino acid back mutation;
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and the heavy chain framework region contains selected from 26D, 27F, One or more amino acid back mutations in 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F, and 93T; and
  • the light chain variable region includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, and the light chain framework region includes a 2V amino acid back mutation;
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, and the heavy chain framework region contains 1K and/or 94S amino acids Back mutation, and
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, and the light chain framework region includes one selected from 42G, 44V and 71Y Or more amino acid back mutations.
  • variable region of the heavy chain whose sequence is shown in SEQ ID NO: 4 or has at least 90% sequence identity with SEQ ID NO: 4, and/or
  • the light chain variable region has a sequence such as SEQ ID NO: 5 or has at least 90% sequence identity with SEQ ID NO: 5;
  • the light chain variable region whose sequence is as SEQ ID NO: 7 or has at least 90% sequence identity with SEQ ID NO: 7;
  • the light chain variable region whose sequence is shown in SEQ ID NO: 20 or has at least 90% sequence identity with SEQ ID NO: 20;
  • the heavy chain variable region whose sequence is as SEQ ID NO: 27, 30, 31 or 32 or has at least 90% sequence identity with SEQ ID NO: 27, 30, 31 or 32, and/or
  • the light chain variable region whose sequence is as SEQ ID NO: 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64, or with SEQ ID NO: 28, 29 , 34, 35, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 have at least 90% sequence identity;
  • the heavy chain variable region whose sequence is shown in SEQ ID NO: 33, 36, 37, 38, 39 or 40 or has at least 90% with SEQ ID NO: 33, 36, 37, 38, 39 or 40, respectively.
  • % Sequence identity, and/or light chain variable region whose sequence is as SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 Or 64 or SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 have at least 90% sequence identity;
  • the light chain variable region whose sequence is as SEQ ID NO: 42, 43 or 44 or has at least 90% sequence identity with SEQ ID NO: 42, 43 or 44 respectively;
  • the light chain variable region whose sequence is shown in 71 or has at least 90% sequence identity with SEQ ID NO: 71
  • the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 27 or has at least 90% identity with SEQ ID NO: 27, and the anti-PD-1 antibody
  • the light chain variable region sequence of the antibody is shown in SEQ ID NO: 55 or has at least 90% sequence identity with SEQ ID NO: 55.
  • the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 46 or has at least 90% identity with SEQ ID NO: 46
  • the anti-PD-1 antibody The light chain variable region sequence of the antibody is shown in SEQ ID NO: 43 or has at least 90% sequence identity with SEQ ID NO: 43.
  • the aforementioned "at least 90% identity” includes having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the heavy chain constant region of the antibody constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, the light chain constant region of the antibody constant region is selected from human antibody ⁇ and ⁇ chain constant regions and conventional variants thereof;
  • the The antibody constant region includes an IgG4 heavy chain constant region that introduces one or more mutations in S228P, F234A, and L235A, such as three amino acid mutations of S228P, F234A, and L235A; in other embodiments, the antibody includes sequences such as The heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 79 and the light chain constant region sequence shown in SEQ ID NO: 73.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof wherein the anti-PD-1 antibody comprises a light chain as shown in SEQ ID NO: 78 and a light chain as shown in SEQ ID NO: 77 or 82 The heavy chain; or
  • the anti-PD-1 antibody comprises:
  • the sequence of the heavy chain is shown in SEQ ID: 77 and the sequence of the light chain is shown in SEQ ID: 78.
  • an anti-PD-1 antibody or antigen-binding fragment thereof which competitively binds to human PD-1 or the same binding as any of the aforementioned anti-PD-1 antibodies or antigen-binding fragments thereof The human PD-1 epitope.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, wherein the antibody is a bispecific antibody or a multispecific antibody.
  • the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single-chain antibody (scFv), dimerized V region (diabody) and disulfide bond stabilized V region (dsFv).
  • an isolated monoclonal antibody or antigen-binding fragment thereof is also disclosed, which competitively binds to human PD-1 with the anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the foregoing.
  • the present disclosure also provides a pharmaceutical composition, which contains a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the foregoing, or a therapeutically effective amount of the foregoing isolated monoclonal Antibodies or antigen-binding fragments thereof, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg of the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof.
  • the present disclosure also provides a nucleic acid molecule that encodes the anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the foregoing, or encodes the foregoing isolated monoclonal antibody or antigen-binding fragment thereof.
  • the present disclosure also provides a host cell, which contains the aforementioned nucleic acid molecule.
  • the present disclosure also provides a method for immunodetection or determination of PD-1, the method comprising the step of using the anti-PD-1 antibody or antigen-binding fragment thereof according to any one of the foregoing, or The step of using the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof.
  • the present disclosure also provides a kit comprising the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof or the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof.
  • the use of the aforementioned anti-PD-1 antibody or its antigen-binding fragment or the aforementioned isolated monoclonal antibody or its antigen-binding fragment in the preparation of a diagnostic agent for PD-1 related diseases is also provided.
  • the present disclosure also provides a method of treating diseases, the method comprising administering to a subject a therapeutically effective amount of the anti-PD-1 antibody or antigen-binding fragment thereof described in any one of the foregoing, or the foregoing An isolated monoclonal antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition, or the aforementioned nucleic acid molecule.
  • the disease is a tumor.
  • the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system System cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, stomach Intestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, Leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myelody
  • the present disclosure also provides the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof, or the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition, or the aforementioned nucleic acid molecule. Use in medicine for the treatment or prevention of diseases.
  • the disease is a tumor.
  • the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system System cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, stomach Intestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, Leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myelody
  • the present disclosure also provides the anti-PD-1 antibody or antigen-binding fragment thereof described above, or the aforementioned isolated monoclonal antibody or antigen-binding fragment thereof, or the aforementioned nucleic acid molecule for use as a medicine , Or the aforementioned pharmaceutical composition.
  • the drug is used to treat or prevent diseases related to PD-1.
  • the disease is a tumor.
  • the disease is selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system System cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, stomach Intestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, Leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, myelody
  • Figure 1 Anti-PD-1 antibody blocking the binding test result of PD-1 and its ligand
  • Figure 2 The effect of anti-PD-1 antibody on the secretion of IFN ⁇ from PBMC cells
  • Figure 3 The curative effect of anti-PD-1 antibody on mouse colon cancer MC38 transplantation tumor
  • Figure 4 The effect of anti-PD-1 antibody on the tumor volume of mouse colon cancer MC38.
  • programmed death 1 means programmed death 1
  • protein PD-1 protein PD-1
  • PD-1 protein PD-1
  • PDCD1 protein PD-1
  • hPD-1 hPD-1
  • isotypes include human PD-1 Variants, isotypes, species homologs, and analogs that have at least one epitope in common with PD-1.
  • the complete PD-1 sequence can be found in GenBank accession number U64863.
  • P-L1 programmed death ligand-1
  • PD-L2 is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which down-regulates T when it binds to PD-1 Cell activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isotypes, and interspecies homologs of hPD-L1, and 5 species that have at least one of hPD-L1 Analogs of common epitopes. The complete hPD-L1 sequence can be found with GenBank accession number Q9NZQ7.
  • cytokine is a general term for proteins released by a cell population that act as intercellular mediators on other cells.
  • cytokines include lymphokines, monocytes, chemokines, and traditional polypeptide hormones.
  • exemplary cytokines include: human IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17 and IL-5.
  • the "antibody” mentioned in the present disclosure refers to an immunoglobulin generally.
  • a natural intact antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain, and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into ⁇ chain or ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig can have ⁇ chain or ⁇ chain.
  • the antibodies referred to in the present disclosure include antibodies or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof that have been modified on the basis of immunoglobulin while retaining the ability to bind antigen; including monospecific antibodies, bispecific antibodies or multispecific antibodies Sex antibodies; also include monovalent antibodies, bivalent antibodies or multivalent antibodies.
  • Antigen-binding fragments of antibodies may be antigen-binding fragments that contain at least one VH-CH1 and at least one VL-CL structure, where the VH and VL structures can approach based on the interaction between chains and retain the ability to bind antigen.
  • the antigen-binding fragment of the antibody is a monovalent Fab fragment (Fab1 fragment), a bivalent Fab fragment (F(ab)2), a trivalent Fab fragment (F(ab)3), a multivalent (two Or above) Fab fragments can also be other monospecific, bispecific or multispecific antigen binding fragments containing at least one Fab fragment.
  • Bispecific antibody refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single-chain antibodies) that can specifically bind to two different antigens or two different epitopes of the same antigen.
  • the prior art has disclosed bispecific antibodies with various structures. According to the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment bispecific antibodies. According to the number and configuration of the antigen binding region, they can be divided into Bivalent, trivalent, tetravalent or more bivalent antibodies can be divided into symmetrical structure bispecific antibodies and asymmetric structure bispecific antibodies according to whether the structure is symmetrical.
  • bispecific antibodies based on antibody fragments form bispecific antibodies by combining two or more Fab fragments in one molecule, which have low immunogenicity and Small molecular weight and high tumor tissue permeability.
  • Typical antibody structures of this type are bispecific antibodies such as F(ab)2, scFv-Fab, (scFv)2-Fab, etc.; IgG-like bispecific antibodies (such as With Fc fragment), this type of antibody has a relatively large molecular weight.
  • the Fc fragment helps the purification of the antibody and improves its solubility and stability.
  • the Fc part may also bind to the receptor FcRn and increase the serum half-life of the antibody.
  • Bispecific antibody structure models such as KiH, CrossMAb, Triomab quadroma, Fc ⁇ Adp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one/DAF , ScFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART- Bispecific antibodies such as Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585-608 (2019); Chen S1, etc., J Immunol Res. 2019
  • monovalent means that there is an antigen binding site in the antibody
  • monovalent polypeptide complex means that there is an antigen binding site in the polypeptide complex
  • bivalent antibody means that there are two antigen binding sites in the antibody
  • bivalent polypeptide complex means that there are two antigen binding sites in the polypeptide complex
  • trivalent antibody means that there are three antigen binding sites in the antibody
  • trivalent polypeptide complex means that there are in the polypeptide complex.
  • antibody fusion protein refers to a biologically active fusion protein formed by linking a target protein (polypeptide) with an immunoglobulin.
  • the fusion protein has the biological activity and immunoglobulin activity of the linked protein.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conservative sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies being preferred.
  • murine antibody in this disclosure is a monoclonal antibody against human PD-1 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with PD-1 antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • the murine anti-PD-1 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine ⁇ , ⁇ chain or a variant thereof, or further comprise murine origin The heavy chain constant region of IgG1, IgG2, IgG3, or variants thereof.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed, and replace the mouse variable region genes It is connected with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the PD-1 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or IgG1, IgG2, or IgG4 variants with amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations).
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the variable region framework of human antibodies, that is, different types of human germline antibodies The antibody produced in the framework sequence. It can overcome the heterogeneous reaction induced by the chimeric antibody due to the large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutations or back mutations to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further displayed by yeast to undergo affinity maturation mutations to the CDRs.
  • the grafting of the CDR may result in the reduced affinity of the produced antibody or its antigen-binding fragment to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of hypermutation of somatic cells. Therefore, it may still be necessary to transplant such donor framework amino acids to the framework of the humanized antibody.
  • the amino acid residues involved in antigen binding from non-human antibodies or antigen-binding fragments thereof can be identified by examining the sequence and structure of the variable region of animal monoclonal antibodies. Residues in the CDR donor framework that differ from the germline can be considered related.
  • the sequence can be compared with the consensus sequence of a subclass consensus sequence or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of hypermutation of somatic cells and thus play an important role in binding.
  • the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine ⁇ , ⁇ chain or a variant thereof, or further comprise human or murine IgG1 , IgG2, IgG3, IgG4 or variants thereof; preferably comprising the heavy chain constant region of human IgG1, IgG2 or IgG4, or using amino acid mutations (such as L234A and/or L235A mutations, and/or S228P mutations) IgG1, IgG2 or IgG4 variants.
  • the "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refer to the heavy chain constant region that has been disclosed in the prior art and does not change the structure and function of the antibody variable region. Or variants of the constant region of the light chain. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification of the heavy chain constant region and amino acid substitutions. The specific substitutions are YTE known in the art.
  • HumanMAb (HuMAb), “human antibody”, “fully human antibody” and “fully human antibody” can be used interchangeably, and can be antibodies derived from humans or antibodies obtained from a genetically modified organism.
  • the transgenic organism is "engineered” to produce specific human antibodies in response to antigen stimulation and can be produced by any method known in the art.
  • the elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and the endogenous heavy and light chain loci in these cell lines are targeted. Disruption of these cell lines contains the targeted endogenous heavy and light chain loci.
  • Transgenic organisms can synthesize human antibodies specific to human antigens, and the organisms can be used to produce human antibody-secreting hybridomas.
  • a human antibody can also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more personal DNA sources.
  • Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or constructed from B cells activated in vitro, all of which are known in the art.
  • full-length antibody “whole antibody”, “whole antibody” and “whole antibody” are used interchangeably herein and refer to an antibody in a substantially complete form, as distinguished from the antigen-binding fragments defined below.
  • the term specifically refers to an antibody whose heavy chain contains an Fc region.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments of the antibody that retain the ability to specifically bind to an antigen (eg, PD-1). It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of antibodies.
  • binding fragment contained in the term "antigen-binding fragment" of the antibody examples include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including A bivalent fragment of two Fab fragments connected by a disulfide bridge on the hinge region, (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody ; (V) Single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546), which is composed of VH domain; and (vi) isolated complementarity determining region (CDR) or (vii) can be either A combination of two or more isolated CDRs, optionally connected by a synthetic linker.
  • CDR complementarity determining region
  • single chain Fv single chain Fv
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of antibodies.
  • the antigen binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single-chain antibodies (scFv), dimerized V regions (diabodies), disulfide stabilized V regions (dsFv), including CDR peptides and so on.
  • Fab is an antibody fragment that has a molecular weight of about 50,000 and has antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (cleaving the amino acid residue at position 224 of the H chain), wherein the H chain is N-terminal About half of the side and the entire L chain are joined together by disulfide bonds.
  • the Fab of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain.
  • the Fab can be produced by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express the Fab.
  • F(ab')2 is obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of about 100,000 and has antigen binding activity and contains two Fab regions connected at the hinge position. Antibody fragments.
  • the F(ab')2 of the present disclosure can be produced by treating the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with pepsin.
  • the F(ab')2 can be produced by linking Fab' described below with a thioether bond or a disulfide bond.
  • Fab' is an antibody fragment obtained by cleaving the disulfide bond in the hinge region of F(ab')2 and having a molecular weight of about 50,000 and having antigen-binding activity.
  • the Fab' of the present disclosure can be produced by treating the F(ab')2 of the present disclosure that specifically recognizes PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab'.
  • single-chain antibody means to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker Molecule.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • a suitable prior art linker consists of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by the following steps: Obtain the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the scFv encoding The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryotic or eukaryotic organism to express scFv.
  • Diabodies are antibody fragments in which scFv is dimerized, and are antibody fragments with bivalent antigen-binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
  • the diabody of the present disclosure can be produced by the following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs
  • the DNA of scFv is such that the amino acid sequence of the peptide linker is 8 residues or less, the DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryote or true In nuclear organisms to express diabodies.
  • dsFv is obtained by connecting a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering, 7, 697 (1994)) based on the three-dimensional structure prediction of the antibody.
  • the dsFv of the present disclosure can be produced by the following steps: Obtain the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, and constructs the dsFv The DNA is inserted into a prokaryotic expression vector or a eukaryotic expression vector, and then the expression vector is introduced into a prokaryotic or eukaryotic organism to express dsFv.
  • the CDR-containing peptide is constructed by including one or more regions in the CDR of VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present disclosure can be produced by the following steps: constructing the CDR encoding of the VH and VL of the monoclonal antibody of the present disclosure that specifically recognizes human PD-1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure DNA, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then introducing the expression vector into a prokaryotic or eukaryotic organism to express the peptide.
  • the CDR-containing peptide can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • amino acid difference or “amino acid mutation” means that compared with the original protein or polypeptide, the variant protein or polypeptide has amino acid changes or mutations, including one, two, three or one mutation on the basis of the original protein or polypeptide. Insertion, deletion or substitution of more amino acids.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR amino acids in the heavy chain variable domain (VH) The residue numbers are 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residue numbers in the light chain variable domain (VL) are 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL
  • the base numbers are 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3).
  • the CDR is defined by the amino acid residues 26-35 (HCDR1) in human VH , 50-65 (HCDR2) and 95-102 (HCDR3) and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers of VL are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1), 50-52 ( CDR2) and 89-97 (CDR3).
  • the CDR region of an antibody can be determined using the program IMGT/DomainGap Align.
  • epitopes refers to a site on an antigen where an immunoglobulin or antibody specifically binds (for example, a specific site on a PD-L1 molecule).
  • Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10 -8 M, for example, about less than 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD or "Kd” refers to the dissociation equilibrium constant of a specific antibody-antigen interaction.
  • the antibodies of the present disclosure bind PD-1 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9 M, for example, as using surface plasmon resonance (SPR)
  • SPR surface plasmon resonance
  • the term “competition” in the context of antigen binding proteins that compete for the same epitope (e.g., neutralizing antigen binding protein or neutralizing antibody), it means competition between antigen binding proteins, which is determined by the following assay:
  • the antigen-binding protein to be detected for example, an antibody or immunological functional fragment thereof
  • prevents or inhibits for example, reduces
  • a reference antigen-binding protein for example, a ligand or a reference antibody
  • a common antigen for example, PD-1). Antigens or fragments thereof).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J.Immunol.137:3614-3619
  • solid Phase direct labeling assay solid-phase direct labeling sandwich assay (see for example Harlow and Lane, 1988, Antibodies, A Laboratory Manual (antibody, laboratory manual), Cold Spring Harbor Press); direct solid phase labeling with I-125 label RIA (see, for example, Morel et al., 1988, Molec.
  • the assay involves the use of purified antigen bound to a solid surface or cell carrying either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Competitive inhibition is measured by measuring the amount of label bound to a solid surface or cell in the presence of the antigen binding protein being tested. Usually the tested antigen binding protein is present in excess.
  • the antigen binding proteins identified by competition assays include: antigen binding proteins that bind to the same epitope as the reference antigen binding protein; and antigen binding that binds to adjacent epitopes that are sufficiently close to the binding epitope of the reference antigen binding protein Proteins, the two epitopes sterically hinder each other from binding. Additional details on the methods used to determine competitive binding are provided in the Examples herein.
  • the competing antigen binding protein is present in excess, it will inhibit (eg reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, and is preferably double-stranded DNA or single-stranded mRNA or modified mRNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • the vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., non-episomal mammalian vectors).
  • mice can be immunized with human PD-1 or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequence can be obtained from ImmunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT ImmunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easy to transform include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus (Streptococcus) and Haemophilus influenzae (Haemophilus influenzae).
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human PD-1. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer for purification. Wash away non-specifically bound components. Then the bound antibody was eluted by the pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects refers to treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition containing any one of the combination compounds of the present disclosure, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population, in order to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing method commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms can evaluate whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may not be effective in alleviating the symptoms of each target disease, according to any statistical test methods known in the art such as Student's t test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) replace amino acids in a protein so that they can be frequently Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)).
  • substitution of amino acids with similar structure or function is unlikely to disrupt biological activity. Exemplary conservative substitutions are set out in the table below "Exemplary Amino Acid Conservative Substitutions”.
  • Effective amount refers to the amount of the drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity, or delay of the onset of the disease, including the biochemistry, tissue, and tissue of the disease, its complications, and intermediate pathological phenotypes that appear during the development of the disease Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related disorders of the present disclosure or improving one or more symptoms of the disorder, and reducing the effectiveness of other agents required to treat the disorder Dosage, enhance the efficacy of another agent, and/or delay the progression of the patient’s target antigen-related disorder of the present disclosure.
  • Exogenous refers to substances produced outside organisms, cells, or humans according to circumstances.
  • Endogenous refers to substances produced in cells, organisms, or human bodies according to circumstances.
  • “Homology” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • BLAST algorithm BLAST ALGORITHMS: Altschul, SF et al., (1990) J. Mol. Biol.
  • PCR polymerase chain reaction
  • oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified.
  • the 5'terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences derived from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; Erlich edited, (1989) PCR Technology (Stockton Press, N.Y.).
  • PCR used herein is regarded as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, and the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Produce specific parts of nucleic acid.
  • isolated refers to a purified state, and in this case means that the designated molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth medium.
  • isolated is not intended to mean the complete absence of these materials or the absence of water, buffers or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compound as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in a formulation for the delivery of antibodies or antigen-binding fragments.
  • the carrier can be an anti-adhesive agent, binder, coating, disintegrant, filler or diluent, preservative (such as antioxidant, antibacterial or antifungal), sweetener, absorption delaying agent, wetting agent Agent, emulsifier, buffer, etc.
  • suitable pharmaceutically acceptable carriers include water, ethanol, polyol (e.g. glycerol, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oil (e.g. olive oil), saline, buffer, buffered saline and the like Penetrating agents such as sugars, polyols, sorbitol and sodium chloride.
  • the present disclosure includes agents for treating diseases related to target antigen (eg, PD-1) positive cells, the agents comprising the anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.
  • target antigen eg, PD-1
  • the agents comprising the anti-PD-1 antibody or antigen-binding fragment thereof of the present disclosure as an active ingredient.
  • the disease related to PD-1 in the present disclosure is not limited, as long as it is a disease related to PD-1.
  • the therapeutic response induced by the molecule of the present disclosure can be achieved by binding to human PD-1 and then inhibiting PD-1 from The binding of ligands PD-L1 and PD-L2 can kill tumor cells that overexpress PD-1. Therefore, when in preparations and formulations suitable for therapeutic applications, the molecules of the present disclosure are very useful for people who have tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric cancer, Colorectal cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.
  • the present disclosure relates to methods for immunodetection or determination of target antigens (such as PD-1), reagents for immunodetection or determination of target antigens (such as PD-1), and reagents for immunodetection or determination of expression of target antigens (such as PD-1) cell method and a diagnostic agent for diagnosing a disease related to a target antigen (for example, PD-1) positive cell, which includes the specific recognition target antigen (for example, human PD-1) of the present disclosure and is combined with the cell
  • a target antigen for example, PD-1 positive cell
  • the antibody or antibody fragment combined with the amino acid sequence of the outer region or its three-dimensional structure is used as the active ingredient.
  • the method for detecting or measuring the amount of the target antigen can be any known method.
  • it includes immunoassay or assay methods.
  • the immunoassay or measurement method is a method that uses labeled antigen or antibody to detect or measure the amount of antibody or antigen.
  • immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescence immunoassay, western blotting, physicochemical method Wait.
  • the aforementioned diseases related to PD-1 positive cells can be diagnosed by detecting or measuring PD-1 expressing cells with the antibodies or antibody fragments of the present disclosure.
  • the living body sample used to detect or measure the target antigen for example, PD-1
  • the target antigen for example, PD-1
  • cells expressing the target antigen such as tissue cells , Blood, plasma, serum, pancreatic juice, urine, stool, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • the reagents used to perform the antigen-antibody reaction include buffers, salts, and the like.
  • the reagents used for detection include reagents commonly used in immunoassay or assay methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label.
  • the human PD-1-IgG1Fc fusion protein was designed and synthesized.
  • the N-terminus was 150 amino acids in the extracellular region of human PD-1, and the C-terminus was the Fc section of human IgG1 (hIgG1Fc).
  • Purified by Protein A affinity column, high-purity recombinant PD-1-Fc protein can be obtained for detecting the binding of anti-PD-1 antibody to antigen.
  • the underlined part is the signal peptide
  • the normal part is the extracellular region of human PD-1
  • the italicized part is hIgG1Fc (signal peptide + extracellular region + hIgG1Fc).
  • Transfected cell nucleic acid coded PD-1 antigen SEQ ID NO: 3:
  • Anti-human PD-1 antibodies can be produced by immunizing mice, and can also be obtained by anti-human PD-1 phage mouse immune library.
  • the method of preparing anti-human PD-1 antibody by immunizing mice is as follows:
  • mice Female, 6-8 weeks old and Balb/c white mice, female, 6-8 weeks old.
  • Feeding environment SPF level. After the mice are purchased, they are kept in a laboratory environment for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 °C; humidity 40-60%. The mice that have adapted to the environment were immunized according to different protocols, with 6-10 mice in each group.
  • the immune antigen can be the purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2), or PD-1 as the antigen (see SEQ ID NO: 3)
  • Transfected Jurkat/CHO-PD-1 cells can be cross-immunized with a single antigen combined with different immune adjuvants or different types of immunogens.
  • the immunization site can be the abdominal cavity or the back subcutaneous, or alternate immunization of the two positions.
  • Immune adjuvant Gold Adjuvant (hereinafter referred to as Titermax, purchased from Sigma, product number T2684) and Imject Alum Adjuvant (hereinafter referred to as Alum, purchased from Pierce, product number 77161) are cross-immunized.
  • Titermax 1:1
  • Alum 3:1
  • 25-50 ⁇ g/mouse first immunization
  • 50 ⁇ g/mouse booster immunization
  • 1 ⁇ 10 7 Jurkat/CHO-PD-1 cells/only On day 0, intraperitoneal injection of 25-50 ⁇ g/emulsified antigen, once a week or once every two weeks after the first immunization, Titermax and Alum are used alternately, 5-8 times in total.
  • the fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI), and aliquoted into 96-well cell culture plates (1 ⁇ 10 5 /150 ⁇ l) /Well), 37°C, 5% CO 2 incubate, about 10-30 seed plates.
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • Hybridoma cell screening 7-9 days after fusion, according to the cell growth density, carry out the ELISA method to detect the binding of antibody to PD-1, and carry out the PD-1/PDL1 binding blocking ELISA on the positive cells
  • the positive wells are exchanged, and the cells are expanded to 24-well plates in time according to the cell density.
  • the cell lines transferred into the 24-well plate were retested and then subcloned for the first time. Those who are positive in the first subcloning screening are preserved, and the second or third subcloning is performed until a single cell clone is obtained. Repeated fusions to obtain hybridoma cells that can block the combination of PD-1 and PDL1.
  • the method of obtaining anti-human PD-1 antibodies through the anti-human PD-1 phage mouse immune library is as follows:
  • the single-chain antibody fragment and the modified library vector pCantab5E were digested with Sfi1 (NEB Cat No.#R0123L), and used after electrophoresis Gel Extraction Kit (Omega Cat No. D2500-02) for purification and recovery. Then use T4DNA ligase (NEB Cat No.#M0202L) to ligate at 16°C for 16-18 hours, then use the above kit for purification and recovery, and finally elution with deionized water. Take 1 ⁇ g of the ligation product and mix it with one electrotransformation competent TG1 (Lucigen Cat No.
  • the phage mouse immune library uses biotinylated human PD-1-ECD-his antigen, after 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen), and finally obtains binding PD-1 and blocking PD-1
  • MACS screening streptomycin magnetic beads, Invitrogen
  • the monoclonal binding to PD-L1 was verified by sequencing, and the variable region sequence of the antibody was obtained.
  • ProteinG is the first choice for affinity chromatography.
  • the cultured hybridoma is centrifuged to take the supernatant, and 10-15% volume of 1M Tris-HCl (pH 8.0-8.5) is added according to the volume of the supernatant. Clear pH.
  • the ProteinG column uses 6M guanidine hydrochloride to wash 3-5 times the column volume, and then uses pure water to wash 3-5 times the column volume; use a buffer system such as 1 ⁇ PBS (pH7.4) as an equilibration buffer to equilibrate the column for 3-5 Times the column volume; the cell supernatant is combined with low flow rate and the flow rate is controlled so that the retention time is about 1 min or longer; the column is washed with 1 ⁇ PBS (pH7.4) 3-5 times the column volume until the UV absorption drops to Baseline: Use 0.1M acetic acid/sodium acetate (pH3.0) buffer for sample elution, collect elution peaks according to UV detection, and use 1M Tris-HCl (pH8.0) to quickly adjust the pH of the eluted product to 5-6 for temporary storage .
  • a buffer system such as 1 ⁇ PBS (pH7.4) as an equilibration buffer to equilibrate the column for 3-5
  • the eluted product can be replaced by a method well known to those skilled in the art, such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve sample purity.
  • a method well known to those skilled in the art such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve sample purity.
  • the supernatant of the cell culture expressing the antigen protein or antibody is centrifuged at a high speed to collect the supernatant.
  • the ProteinA affinity column is washed 3-5 times the column volume with 6M guanidine hydrochloride, and then washed 3-5 times the column volume with pure water.
  • Use a buffer system such as 1 ⁇ PBS (pH 7.4) as an equilibration buffer to equilibrate the chromatography column by 3-5 times the column volume.
  • the cell supernatant is loaded and bound at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer.
  • the column is washed with 1 ⁇ PBS (pH 7.4) 3-5 times the column volume until the UV absorption falls back to the baseline .
  • the sample was eluted with 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer, and the elution peaks were collected based on UV detection.
  • the eluted product was quickly adjusted to pH 5-6 with 1M Tris-HCl (pH8.0). Save.
  • the eluted product can be replaced by a method well known to those skilled in the art, such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve sample purity.
  • a method well known to those skilled in the art such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve sample purity.
  • the present disclosure is further described below in conjunction with embodiments, but these embodiments do not limit the scope of the present disclosure.
  • the experimental methods without specific conditions in the embodiments of the present disclosure usually follow conventional conditions, such as Cold Spring Harbor’s antibody technology experiment manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • Reagents without specific sources are conventional reagents purchased on the market.
  • Example 1 Obtaining anti-human PD-1 murine antibody
  • the anti-human PD-1 murine antibody obtained by the aforementioned method was subjected to antigen binding experiments, and multiple strains of antibodies with good activity were screened: including M23, M32 and M33.
  • the single cell clone was expanded and cultured, RNA was extracted, and mouse-Ig was used.
  • Reverse transcription amplification (RT-PCR) was performed on the degenerate primers to obtain the variable region sequence of the antibody.
  • the murine antibody variable region sequence is connected with the human antibody constant region sequence, the chimeric antibody of the murine monoclonal antibody is cloned and recombinantly expressed, and the in vitro activity experiment is performed to confirm that the obtained monoclonal antibody variable region sequence is correct.
  • variable region sequences of murine antibodies M23, M32 and M33 were determined as follows:
  • the heavy chain variable region of the murine antibody M23 (SEQ ID NO: 4):
  • the light chain variable region of the murine antibody M23 (SEQ ID NO: 5):
  • the heavy chain variable region of the murine antibody M32 (SEQ ID NO: 6):
  • the light chain variable region of the murine antibody M32 (SEQ ID NO: 7):
  • the heavy chain variable region of the murine antibody M33 (SEQ ID NO: 19)
  • the underline is the CDR sequence determined by the Kabat numbering system, followed by FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the antibody CDR sequences in the table are determined according to the Kabat numbering system.
  • the human germline heavy and light chain variable region germline genes with high identity with the M23, M32, and M33 light and heavy chain sequences were selected as Template, transplant the CDRs of the three murine antibodies into the corresponding human antibody templates to construct their corresponding humanized antibodies.
  • the humanized light chain templates of the murine antibody M23 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01.
  • the sequence of the variable region after humanization is as follows ( CDR sequence is underlined):
  • Hu23VH-CDR grafting (SEQ ID NO: 27)
  • Hu23VL-CDR grafting (SEQ ID NO: 28)
  • Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • I2G means that the 2nd I of the Kabat numbering system is mutated back to G.
  • the sequence of the light/heavy chain variable region of the humanized antibody of M23 is as follows:
  • Hu23-1 refers to an antibody whose light chain variable region is Hu23VL1 and heavy chain variable region is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (e.g. Hu23-1) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; unless otherwise specified in the present disclosure, a full-length antibody is formed.
  • the light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 are connected to form the antibody light chain, and the heavy chain variable region is connected to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO:
  • the IgG4-P heavy chain constant region shown in 79 is connected to form an antibody heavy chain, and the name in the table refers to the combination of antibody light/heavy chain variable region (such as Hu23-1) plus the suffix ".IgG4AA”
  • the full-length antibody formed by ligating the constant region of the heavy chain of IgG4-AA, plus the suffix ".IgG4P" indicates the full-length antibody formed by the constant region of the heavy chain of IgG4-P, for example, "Hu23-1.IgG4AA” indicates the heavy chain of Hu23VH1
  • the variable region and the heavy chain of the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 are connected to the Hu23VL1
  • Human23-1.IgG4P refers to the heavy chain formed by the Hu23VH1 heavy chain variable region and the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, which is combined with the Hu23VL1 light chain variable region and the ID NO: A full-length antibody formed by the light chain formed by connecting the constant regions of the Kappa chain shown in 73.
  • the humanized light chain templates of the murine antibody M32 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01.
  • the humanized variable region sequence is as follows (underlined CDR sequence):
  • Hu32VH-CDR grafting (SEQ ID NO: 33) IGHV1-69*02 and IGHJ6*01
  • Hu32VL-CDR grafting (SEQ ID NO: 34)
  • Grafted represents mouse antibody CDR implantation into human germline FR region sequence. Amino acid residues are determined and annotated by the Kabat numbering system. For example, I2V means that the second I of Kabat numbering is mutated back to V according to the Kabat numbering system.
  • variable region of the humanized antibody light and heavy chain of the murine antibody M32 is as follows:
  • the antibody and variable region combination obtained after humanization of murine antibody M32.
  • “Hu32-1” refers to the antibody light/heavy chain variable region combination in which the antibody light chain variable region is Hu32VL1 and the heavy chain variable region is Hu32VH1, and so on.
  • the antibody light/heavy chain variable region combinations (e.g. Hu32-1) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; unless otherwise specified in the present disclosure, a full-length antibody is formed.
  • the variable region of the light chain and the constant region of the Kappa chain shown in SEQ ID NO: 73 are connected to form an antibody light chain, and the variable region of the heavy chain is connected to the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4-P heavy chain constant region shown in ID NO: 79 is connected to form an antibody heavy chain, and the name in the table refers to the combination of antibody light/heavy chain variable regions (such as Hu32-1) plus the suffix ".IgG4AA”
  • the full-length antibody formed by the ligation of the constant region of the IgG4-AA heavy chain, plus the suffix ".IgG4P” indicates the full-length antibody formed by the ligation of the constant region of the IgG4-P heavy chain, for example, "Hu32-1.IgG4AA” indicates the heavy chain of Hu32VH1
  • the variable region and the heavy chain of the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 are connected to the Hu32VL1 light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 A full-length antibody formed by the light chain.
  • Human32-1.IgG4P refers to the heavy chain formed by the Hu32VH1 heavy chain variable region and the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, which is combined with the Hu32VL1 light chain variable region and the ID NO: 73 is a full-length antibody composed of light chains formed by linking constant regions of Kappa chains.
  • the humanized light chain templates of the murine antibody M33 are IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV3-7 and IGHJ6*01.
  • the humanized variable region sequences are as follows:
  • Hu33VL-CDR grafting (SEQ ID NO: 42):
  • Grafted represents mouse antibody CDR implantation into human germline FR region sequence. Amino acid residues are determined and annotated by the Kabat numbering system.
  • F71Y means that the F 71 of Kabat numbering is mutated back to Y according to the Kabat numbering system.
  • sequences of the light chain variable region and heavy chain variable region of the humanized antibody of the murine antibody M33 are as follows:
  • “Hu33-6” refers to the antibody light/heavy chain variable region combination in which the variable region of the antibody light chain is Hu33VL2 and the variable region of the heavy chain is Hu33VH3, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu33-6) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; if not explicitly stated in the present disclosure, a full-length antibody is formed.
  • the variable region of the light chain and the constant region of the Kappa chain shown in SEQ ID NO: 73 are connected to form an antibody light chain
  • the variable region of the heavy chain is connected to the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4-P heavy chain constant region shown in ID NO: 79 is connected to form an antibody heavy chain, and the name in the table refers to the combination of the antibody light/heavy chain variable region (such as Hu33-6) plus the suffix ".IgG4AA”
  • the full-length antibody formed by the ligation of the constant region of the heavy chain of IgG4-AA, plus the suffix ".IgG4P” means the full-length antibody formed by the constant region of the IgG4-P heavy chain, for example, "Hu33-6.IgG4AA", which means the heavy chain of Hu33VH3
  • the heavy chain formed by the chain variable region and the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 is connected with the Hu33VL2 light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 A full-length antibody formed by connected light chains.
  • Human33-6.IgG4P means the heavy chain formed by the Hu33VH3 heavy chain variable region and the IgG4-P heavy chain constant region as shown in SEQ ID NO: 79, and is combined with the Hu33VL2 light chain variable region and A full-length antibody formed by the light chain formed by connecting the constant regions of the Kappa chain shown in SEQ ID NO: 73.
  • Hu23LCDR1 (N28Q) represents the LCDR1 mutation sequence with the 28th N mutation to Q in the Kabat numbering rule of the Hu23 humanized antibody light chain variable region Hu23VL1 or Hu23VL2,
  • Hu23LCDR1 (G29A) represents the Hu23 humanized antibody light chain
  • the variable region Hu23VL1 or Hu23VL2 has a LCDR1 mutation sequence in which G is mutated to A at position 29 of the Kabat numbering rule (the CDR is determined by the Kabat numbering system).
  • the sequence of the light chain variable region of the Hu23 humanized antibody after LCDR1 mutation is as follows:
  • “Hu23-11” in the table refers to the antibody light/heavy chain variable region combination in which the light chain variable region of the antibody is Hu23VL1 (N28T) and the heavy chain variable region is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (e.g. Hu23-11) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; unless otherwise specified in the present disclosure, a full-length antibody is formed.
  • the light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 are connected to form the antibody light chain, and the heavy chain variable region is connected to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO:
  • the IgG4-P heavy chain constant region shown in 79 is connected to form an antibody heavy chain, and the name in the table refers to the combination of antibody light/heavy chain variable regions (such as Hu23-11) plus the suffix ".IgG4AA”
  • the full-length antibody formed by the ligation of the constant region of the heavy chain of IgG4-AA, plus the suffix ".IgG4P" means the full-length antibody formed by the constant region of the IgG4-P heavy chain, for example, "Hu23-11.IgG4AA", which means the heavy chain of Hu23VH1
  • the heavy chain formed by connecting the variable region of the chain and the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 72
  • “Hu23-11.IgG4P” means the heavy chain formed by the heavy chain variable region of Hu23VH1 and the constant region of the IgG4-P heavy chain shown in SEQ ID NO: 79, which is combined with the variable light chain of Hu23VL1 (N28T)
  • the sequence of the humanized antibody Hu23 derived from M23 and the humanized antibody Hu32 derived from M32 have high sequence identity.
  • the light chain variable region of Hu23 and the heavy chain variable region of Hu32 are combined into a new light and heavy chain. Chain variable region combination.
  • the experimental results showed that the humanized antibodies containing the new combination of light and heavy chain variable regions maintained their ability to bind to the PD-1 antigen (Table 16).
  • “Hu32a-85” refers to the antibody light chain variable region combination of Hu23VL1 (N28T) and the heavy chain variable region of Hu32VH6, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu32a-85) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; if not explicitly stated in the present disclosure, a full-length antibody is formed.
  • the light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 are connected to form the antibody light chain, and the heavy chain variable region is connected to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO:
  • the IgG4-P heavy chain constant region shown in 79 is connected to form an antibody heavy chain, and the name in the table refers to the combination of antibody light/heavy chain variable region (such as Hu32a-85) plus the suffix ".IgG4AA".
  • the full-length antibody formed by the ligation of the constant region of the heavy chain of IgG4-AA, plus the suffix ".IgG4P” means the full-length antibody formed by the constant region of the IgG4-P heavy chain, for example, "Hu32a-85.IgG4AA", which means that it is reproduced by Hu32VH6
  • the heavy chain formed by connecting the variable region of the chain and the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 72, and the variable region of the light chain of Hu23VL1 (N28T) and the Kappa chain shown in SEQ ID NO: 73 A full-length antibody formed by light chains connected by constant regions.
  • Human32a-85.IgG4P means the heavy chain formed by the heavy chain variable region of Hu32VH6 and the constant region of the IgG4-P heavy chain shown in SEQ ID NO: 79, which is combined with the variable light chain of Hu23VL1 (N28T) A full-length antibody formed by the light chain formed by linking the constant region of the Kappa chain as shown in SEQ ID NO: 73.
  • “Hu23a-57” in the table refers to the antibody light/heavy chain variable region combination in which the variable region of the antibody light chain is Hu32VL1 and the variable region of the heavy chain is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu23a-57) referred to in the above table can be respectively connected with the antibody light/heavy chain constant regions to form a full-length antibody; if not explicitly stated in the present disclosure, a full-length antibody is formed.
  • the light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 are connected to form the antibody light chain, and the heavy chain variable region is connected to the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO:
  • the IgG4-P heavy chain constant region shown in 79 is connected to form an antibody heavy chain, and the name in the table refers to the combination of antibody light/heavy chain variable region (such as Hu32a-85) plus the suffix ".IgG4AA".
  • the full-length antibody formed by the ligation of the constant region of the heavy chain of IgG4-AA, plus the suffix ".IgG4P" indicates the full-length antibody formed by the ligation of the constant region of the IgG4-P heavy chain, for example, "Hu23a-57.IgG4AA", which means that it is reproduced by Hu23VH1
  • the heavy chain formed by connecting the variable region of the chain and the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 72 is connected to the variable region of the light chain of Hu32VL1 and the constant region of the Kappa chain shown in SEQ ID NO: 73 A full-length antibody formed by connected light chains.
  • “Hu23a-57.IgG4P” means the heavy chain formed by the Hu23VH1 heavy chain variable region and the IgG4-P heavy chain constant region shown in SEQ ID NO: 79, and the heavy chain formed by the Hu32VL1 light chain variable region and SEQ ID NO: 73 is a full-length antibody formed by a light chain connected by the constant regions of the Kappa chain.
  • the affinity detection of different humanized antibodies was carried out by Biacore (see Test Example 3 for the method). The results are shown in Table 16. The results show that different humanized antibodies maintain their ability to bind to PD-1, and some humanized antibodies have even affinity It is basically close to its mouse antibody.
  • IgG4-P represents the mutation of S228P (corresponding to the 108th position of the sequence SEQ ID NO: 72 or SEQ ID NO: 79), and IgG4-AA represents the F234A (corresponding to the 114th position of the sequence SEQ ID NO: 72 or SEQ ID NO: 79) Position), L235A (corresponding to the 115th position of the sequence SEQ ID NO: 72 or SEQ ID NO: 79) and S228P (corresponding to the 108th position of the sequence SEQ ID NO: 72 or SEQ ID NO: 79) mutation, IgG4-AA And IgG4-P antibody format can be obtained by simple point mutation of IgG4 antibody format.
  • the sequence of the IgG4-AA heavy chain constant region is as follows (SEQ ID NO: 72):
  • the light chain (Kappa chain) constant region sequence of the antibody is as follows (SEQ ID NO: 73):
  • the constructed IgG4AA format full-length antibody sequence is exemplified as follows:
  • Hu23-11.IgG4AA antibody heavy chain (SEQ ID NO: 74):
  • Hu23-11.IgG4AA light chain (SEQ ID NO: 75):
  • Hu32a-85.IgG4AA heavy chain (SEQ ID NO: 76):
  • the light chain of Hu32a-85.IgG4AA (same as the light chain of Hu23-11.IgG4AA, SEQ ID NO: 75):
  • Hu33-6.IgG4AA heavy chain (SEQ ID NO: 77):
  • Hu33-6.IgG4AA light chain (SEQ ID NO: 78):
  • the heavy chain constant region sequence of IgG4-P is as follows (SEQ ID NO: 79):
  • the constructed IgG4-P format full-length antibody sequence is exemplified as follows:
  • IgG4P antibody heavy chain (SEQ ID NO: 80):
  • Hu23-11.IgG4P light chain (same as Hu23-11.IgG4AA light chain, SEQ ID NO: 75):
  • Hu32a-85.IgG4P heavy chain (SEQ ID NO: 81):
  • the light chain of Hu32a-85.IgG4P (same as the light chain of Hu23-11.IgG4AA, SEQ ID NO: 75):
  • Hu33-6.IgG4P heavy chain (SEQ ID NO: 82):
  • Hu33-6.IgG4P light chain (same as Hu33-6.IgG4AA light chain, SEQ ID NO: 78):
  • Test Example 1 Anti-PD-1 antibody in vitro PD-1 ligand binding and binding blocking ELISA experiment
  • PD-L1 on the surface of tumor cells binds to PD-1 on the surface of T cells, thereby inhibiting the proliferation of T cells.
  • the PD-1 antibody can block the PD-L1/PD-1 signaling pathway by binding to PD-1, thereby stimulating the proliferation of T cells.
  • the PD-1/PD-L1 binding blocking experiment is used to detect the blocking activity of anti-PD-1 antibodies on the signal pathway.
  • the anti-PD-1 antibodies to be tested including antibodies: Hu23-11.IgG4AA, Hu32a-85 .IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847), incubate; later, HRP-labeled goat anti-human IgG (H+L) antibody (Cat. #109-035-003, Jackson ImmunoResearch), incubate the reaction. After washing the plate, detect the amount of HRP-labeled goat anti-human IgG (H+L) binding, and calculate the binding of anti-PD-1 antibody to the ligand PD-1 EC 50 value.
  • the extracellular domain and Fc fused PD-1 protein (PD-1-Fc, sequence see SEQ ID NO:1) was coated in a 96-well plate, and the anti-PD-1 antibodies to be tested (including Antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (refer to H005-1 antibody in WO2015085847), carry out incubation reaction; add biotin-labeled PD later -L1/PD-L2, incubate the reaction. After washing the plate, detect the binding amount of biotin-labeled PD-L1/PD-L2, and calculate that the anti-PD-1 antibody blocks the binding of the ligand PD-L1/PD-L2 IC 50 value.
  • the anti-PD-1 antibodies to be tested including Antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody:
  • PBST pH 7.4 PBS containing 0.05% tween20
  • the exemplary anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA of the present disclosure can effectively block the binding of PD-1 and PD-L1/PD-L2, and their blocking activity is similar to The positive control antibody is similar.
  • Digest CHOK1/PD-L1 cells (Promega), add 100 ⁇ L/well to 96-well plates, and place them in a 37°C, 5% CO 2 incubator for 24 hours. Use PBS to dilute controls and samples to the desired concentration.
  • Count Jurkat/PD-1 cells (Jurkat cells that are stably transformed into PD-1), and plant CHOK1/PD-L1 cells in a certain proportion (90 ⁇ L/well) while adding 10 ⁇ L/well to the diluted antibody (antibody) :Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1), negative control IgG4 protein, antibody dilution concentrations are 0.3mg/ml, 3mg/ml, 30mg/ml ), placed in a 37°C, 5% CO 2 incubator for 5 hours. Take out the cell culture plate and place it at room temperature for 5 minutes, then add 50 ⁇ l Bio-Glo TM Re
  • Biacore T200 instrument detects PD-1 antibody and antigen PD-1 reaction signals in real time to obtain binding and dissociation curves. After the dissociation of each experimental cycle is completed, the biosensor chip is washed and regenerated with 10mM Glycine-HCl pH1.5 buffer. The experimental buffer system is 1 ⁇ HBS-EP buffer solution (Cat#BR-1001-88, GE). After the experiment, the GE Biacore T200 Evaluation version 3.0 software was used to fit the data with the (1:1) Langmuir model to obtain the affinity value. The results are shown in Table 18.
  • PBMC peripheral blood mononuclear cells
  • antibody samples including the antibodies of the present disclosure: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, the positive control antibody H005-1, and the negative control IgG4 protein.
  • the antibody concentration is 0.3mg/ml, 3mg/ml, 30mg/ml), diluted with PBS (B320, Shanghai Yuanpei Biotechnology Co., Ltd.), 10 ⁇ l per well.
  • the cell culture plate was placed in a 37°C, 5% CO 2 incubator and incubated for 3 days.
  • the test results are shown in Figure 2.
  • the results show that the anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA of the present disclosure can effectively activate the secretion of IFN- ⁇ .
  • Test Example 5 The effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38
  • mice Inoculate 5 ⁇ 10* 5 cells/mouse/100 ⁇ l of MC38 cells into 90 hPD-1TG mice (Biocytometer) subcutaneously in the right ribs. After 10 days, remove the animals with too large and small tumors. The average tumor volume is about 120mm ⁇ 3 mice were randomly divided into: blank control Vehicle (PBS), positive control H005-13mpk, Hu32a-85.IgG4AA1mpk, Hu32a-85.IgG4AA3mpk, Hu23-11.IgG4AA1mpk, Hu23-11.IgG4AA3mpk, Hu33-6 There are 7 groups of IgG4AA3mpk, 8 in each group.
  • PBS blank control Vehicle
  • H005-13mpk Hu32a-85.IgG4AA1mpk
  • Hu32a-85.IgG4AA3mpk Hu23-11.IgG4AA1mpk
  • Hu23-11.IgG4AA3mpk Hu33-6
  • the antibodies of each group were injected intraperitoneally three times a week. After the first week of administration, the tumor was found to be significantly inhibited. In the second and third weeks, the administration frequency was adjusted to once a week for a total of 5 administrations. The tumor volume and animal weight were monitored twice a week and the data was recorded. When the tumor volume exceeds 2000 mm 3 or most tumors appear ulcerated or lose weight by 20%, the tumor-bearing animals are euthanized as the experimental endpoint.
  • Tumor volume (TV) 1/2 ⁇ L long ⁇ L short 2
  • Tumor growth rate (T/C%) (T-T0)/(C-C0) ⁇ 100%
  • TGI%) 1-T/C%
  • T and T0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the antibody administration group, respectively
  • C and C0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the blank control group, respectively.
  • the test results are shown in Table 19 and attached Figure 3.
  • the test results show that, compared with the blank control, the antibodies of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 transplanted tumors.
  • Hu32a-85.IgG4AA has the highest tumor inhibition rate.
  • -3mpk group the tumor inhibition rate was 77.64% at the last measurement.
  • the results show that the tumor inhibition rate of the antibody of the present disclosure is significantly better than that of the positive control antibody H005-1 when tested on the seventh day; then the dosing frequency is reduced to once a week, After two administrations (Day 21), the efficacy of the antibodies of the present disclosure gradually widened, and showed a dose-dependent manner, among which Hu32a-85.IgG4AA was significantly better than the same dose of H005-1 (p ⁇ 0.05).
  • the tumor-bearing mice can tolerate the anti-PD-1 antibody well, and their weight rises steadily during the whole administration process, and no obvious drug-induced weight loss and other symptoms occur.
  • Test Example 6 The effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38
  • the transgenic PD-1 mice were derived from purchased transgenic PD-1 mice (ISIS INNOVATION LIMITED, University Offices, Wellington Square, Oxford OX1 2JD, England) and cultivated in Cephrim Biosciences, Inc. in the fifth generation of mice.
  • MC38 cells were inoculated into hPD-1 transgenic mice (male and female) at the rate of 5x10 5 cells/100 ⁇ l per subcutaneously on the posterior right side of the right rib.
  • the tumor-bearing mice are randomly divided into 5 groups (8 in each group) according to the tumor size: negative control hIgG control30mpk, H005-110mpk, H005-130mpk, Hu33-6.IgG4AA10mpk, Hu33-6.IgG4AA30mpk .
  • the group administration date is set to Day0.
  • each drug was administered intraperitoneally for a period of 22 days, once every two days, 11 times in total.
  • the tumor volume was measured twice a week, the weight was weighed, and the data was recorded.
  • the animal body weight and tumor volume of each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and graphpad Prism5 and Excel software were used for graphing, and student t test was used for statistical analysis.
  • Tumor growth rate T/C% (T-T0)/(C-C0) ⁇ 100%
  • T and T0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the antibody administration group, respectively
  • C and C0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the blank control group, respectively.
  • the test results are shown in Table 20 and Figure 4.
  • the test results show that compared with the control group, the antibodies of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 transplanted tumors, among which the highest tumor inhibition rate is Hu33-6.
  • the tumor inhibition rate was 80.4% when measured on the 20th day.
  • the efficacy of Hu33-6.IgG4AA-10mpk was better than the positive control H005-1-10mpk.
  • the unit of the average tumor volume of each group in the table is mm 3 .
  • Test Example 7 Pharmacokinetic test of anti-PD-1 antibody cynomolgus monkey
  • Feeding environment The room temperature is controlled at 18°C ⁇ 26°C, the relative humidity is between 40% ⁇ 70%, and the light is alternately light and dark for 12 hours. Except when fasting is required, unlimited feed and water are available.
  • the animals were weighed before administration, and the weight was between 2.81 ⁇ 3.52kg. Use a syringe pump to subcutaneously infuse the forelimbs or hindlimbs.
  • the dose of each group is 1mg/kg (1mpk).
  • the dose is given by a single intravenous injection, the administration rate is 0.1mL/kg/min, and the administration time is about 30 minutes. .

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Abstract

提供了抗PD-1抗体、其抗原结合片段及医药用途。具体地，提供了包含特定CDR区的人源化的抗PD-1抗体及其抗原结合片段，以及包含抗PD-1抗体及其抗原结合片段的药物组合物，及其作为药物的用途。特别地，提供了一种人源化的抗PD-1抗体在制备用于治疗PD-1相关的疾病或病症的药物中的用途。

Description

抗PD-1抗体、其抗原结合片段及医药用途 技术领域
本公开属于生物技术领域，更具体地，本公开涉及抗PD-1抗体及其应用。
背景技术
这里的陈述仅是提供与本发明有关的背景信息，而不必然地构成现有技术。
肿瘤免疫治疗是充分利用、调动肿瘤患者体内的杀伤性T细胞，对肿瘤进行杀伤作用的治疗方法。与此同时，肿瘤细胞逃逸是肿瘤免疫治疗面临的一个巨大障碍，肿瘤细胞利用其自身对免疫系统的抑制作用促进了肿瘤的快速生长。肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。在肿瘤免疫治疗早期肿瘤特异性的杀伤性T细胞是有其生物活性的，但随着肿瘤生长在后期失去了杀伤的功能。
人体内T细胞的活化采取了两条信号通路系统，除了需要通过抗原呈递细胞递呈MHC-抗原肽给T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，进而才能使T细胞产生正常的免疫应答。这个双信号通路系统对体内免疫系统的平衡起着至关重要的作用，它严格调控机体对自身和非自身抗原产生不同的免疫应答。如果缺少协同刺激分子提供的第二信号，将会导致T细胞的无应答或持续特异性免疫应答，从而产生耐受。因此，第二信号通路在机体免疫应答的整个过程中起着非常关键的调节作用。
程序性死亡分子1(programmed death-l，PD-l)是1992年发现的表达在T细胞表面的一个蛋白受体，参与到细胞的凋亡过程之中。PD-l属于CD28家族，与细胞毒性T淋巴细胞抗原4(cytotoxic T Iymphocyte antigen 4，CTLA-4)具有23％的氨基酸同源性，但其表达却与CTLA不同，主要表达在活化的T细胞、B细胞和髓系细胞上。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell，DC)上，在活化后细胞上的表达能够进行上调。而PD-L2的表达相对较局限，主要表达在抗原呈递细胞上，如活化的巨噬细胞和树突状细胞。
PD-L1通过和PD-1及B7-1的结合抑制免疫系统，很多肿瘤细胞及肿瘤组织微环境的免疫细胞表达PD-L1。新的研究发现乳腺癌、肺癌(例如，非小细胞肺癌)、胃癌、肠癌、肾癌、黑素瘤、结肠癌、膀胱癌、卵巢癌、胰腺癌及肝癌等人类肿瘤组织中检测到高PD-L1蛋白的表达，且PD-L1的表达水平和患者的临床及预后紧密相关。
抗PD-1单克隆抗体，它可通过阻断PD-L1/PD-1之间结合，最大限度的提高患者自身对肿瘤的免疫系统反应，从而达到对肿瘤细胞进行杀伤的目的。目前，抗PD-1抗体Pembrolizumab(也称Merck keytruda、keytruda、Merck-Pemb、 Merck-keytruda、Merck-PD-1、派姆单抗)和Novilumab(也称BMS Opdivo、Opdivo、BMS-Nivolumab、纳武单抗)已经获FDA批准用于治疗黑色素瘤、霍奇金淋巴瘤患者、非小细胞肺癌等肿瘤。另外专利文献WO200139722、WO2006121168、WO2010036959、WO2010089411、WO2011110604、WO2013173223、WO2013181634、US2014335093、US6803192B1、US8617546B2、WO2015085847等也公开了多种抗PD-1单克隆抗体。
发明内容
本公开提供了一种新的抗PD-1抗体、其抗原结合片段及其医药用途。
在一些可选的实施方案中，本公开提供一种抗PD-1抗体或其抗原结合片段，其为选自如下i)至iii)任一项所述的抗PD-1抗体或其抗原结合片段：
i)一种抗PD-1抗体或其抗原结合片段，其重链可变区包含序列如SEQ ID NO：8所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，序列如SEQ ID NO：9所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和序列如SEQ ID NO：10所示或与其具有至多8个、3个、2个或1个氨基酸突变的HCDR3；轻链可变区包含序列如SEQ ID NO：11所示或与其具有至多4个、3个、2个或1个氨基酸突变的LCDR1，序列如SEQ ID NO：12所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和序列如SEQ ID NO：13所示或与其具有至多3个、2个或1个氨基酸突变的LCDR3；
ii)一种抗PD-1抗体或其抗原结合片段，其重链可变区包含序列如SEQ ID NO：14所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，序列如SEQ ID NO：15所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和序列如SEQ ID NO：16所示或与其具有至多8个、3个、2个或1个氨基酸突变的HCDR3；轻链可变区包含序列如SEQ ID NO：17所示或与其具有至多4个、3个、2个或1个氨基酸突变的LCDR1，序列如SEQ ID NO：12所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和序列如SEQ ID NO：18所示或与其具有至多3个、2个或1个氨基酸突变的LCDR3；和
iii)一种抗PD-1抗体或其抗原结合片段，其重链可变区包含序列如SEQ ID NO：21所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，序列如SEQ ID NO：22所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和序列如SEQ ID NO：23所示或与其具有至多3个、2个或1个氨基酸突变的HCDR3；轻链可变区包含序列如SEQ ID NO：24所示或与其具有至多3个、2个或1个氨基酸突变的LCDR1，序列如SEQ ID NO：25所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和序列如SEQ ID NO：26所示或与其具有至多3个、2个或1个氨基酸突变的LCDR3。
在一些实施方式中，本公开的前述抗PD-1抗体或其抗原结合片段以等于或小 于10 -7M解离平衡常数与人PD-1结合，在一些实施方式中，以等于或小于10 -8M、10 -9M、10 -10M或10 -11M解离平衡常数与人PD-1结合。
在一些可选的实施方案中，本公开提供一种抗PD-1抗体或其抗原结合片段，其重链可变区包含：序列如SEQ ID NO：65所示的HCDR1，序列如SEQ ID NO：66所示的HCDR2，和序列如SEQ ID NO：67所示的HCDR3；轻链可变区包含：序列如SEQ ID NO：68所示的LCDR1，序列如SEQ ID NO：12所示的LCDR2，和序列如SEQ ID NO：69所示的LCDR3区；序列见下表1：
表1
Figure PCTCN2020074098-appb-000001
在一些可选的实施方案中，前述抗PD-1抗体或其抗原结合片段，所述重链可变区包含序列如SEQ ID NO：8所示的HCDR1，序列如SEQ ID NO：9所示的HCDR2，和序列如SEQ ID NO：10所示的HCDR3；所述轻链可变区包含序列如SEQ ID NO：12所示的LCDR2，序列如SEQ ID NO：13所示的LCDR3，和序列如通式RSSQSX 13VHSX 14X 15X 16TYLE(SEQ ID NO：68)所示的LCDR1，其中X 13选自L，X 14选自N、Q、L、T或D，X 15选自G、A或V，X 16选自N。
在一些可选的实施方案中，所述的抗PD-1抗体或其抗原结合片段是选自如下(a)至(e)任一项所述的抗PD-1抗体或其抗原结合片段：
(a)一种抗PD-1抗体或其抗原结合片段，其包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，和序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1；
(b)一种抗PD-1抗体或其抗原结合片段，其包含序列分别如SEQ ID NO： 14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3；
(c)一种抗PD-1抗体或其抗原结合片段，其包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；
(d)一种抗PD-1抗体或其抗原结合片段，其包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，以及序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1；和
(e)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述的抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述的重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：49、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，所述的抗PD-1抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述的重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3。
在一些可选的实施方案中，本公开提供一种抗PD-1抗体或其抗原结合片段，其为选自如下iv)至vi)任一项所述的抗PD-1抗体或其抗原结合片段：
iv)一种抗PD-1抗体或其抗原结合片段，其重链可变区包含与如SEQ ID NO：4序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，且轻链可变区包含与如SEQ ID NO：5序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；
v)一种抗PD-1抗体或其抗原结合片段，其重链可变区包含与如SEQ ID NO：6序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，且轻链可变区包含与如SEQ ID NO：7序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；和
vi)一种抗PD-1抗体或其抗原结合片段，其重链可变区包含与如SEQ ID NO： 19序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，且轻链可变区包含与如SEQ ID NO：20序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体或其抗原结合片段是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、全人抗体或其抗原结合片段，或人源化抗体或其抗原结合片段。
在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体或抗原结合片段为人源化抗体或其抗原结合片段。
在一些实施方案中，所述人源化抗体包含来源自人抗体的框架区或其框架区变体。
在一些实施方案中，所述框架区变体为在人抗体的轻链框架区和/或重链框架区基础上分别具有至多11个氨基酸的回复突变。
在一些实施方案中，所述框架区变体包含选自以下(f)至(h)任一所述的突变：
(f)轻链可变区中包含2G氨基酸回复突变，和/或重链可变区中包含选自27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变；
(g)轻链可变区中包含2V氨基酸回复突变，和/或重链可变区中包含选自26D、27F、30T、38K、43H、48I、66K、67A、69L、82F和93T中的一个或更多个氨基酸回复突变；和
(h)轻链可变区中包含选自42G、44V和71Y中的一个或更多个氨基酸回复突变，和/或重链可变区中包含1K和/或94S氨基酸回复突变。
在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体或其抗原结合片段包含选自如下所述的抗体可变区：
(a2)重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，且重链框架区包含27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变，和
轻链可变区包含分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，和序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1，且轻链框架区包含2G氨基酸回复突变；
(b2)重链可变区包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，且重链框架区包含选自26D、27F、30T、38K、43H、48I、66K、67A、69L、82F和93T中的一个或更多个氨基酸回复突变；和
轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3，且轻链框架区包含2V氨基酸回复突变；
(c2)重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID  NO：23所示的HCDR1、HCDR2和HCDR3，且重链框架区包含1K和/或94S氨基酸回复突变，和
轻链可变区包含分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3，且轻链框架区包含选自42G、44V和71Y中的一个或更多个氨基酸回复突变。在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体或其抗原结合片段包含选自如下(i)至(o)任一所述的抗体可变区；
(i)重链可变区，其序列如SEQ ID NO:4所示或与SEQ ID NO：4具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：5或与SEQ ID NO：5具有至少90％序列同一性；
(j)重链可变区，其序列如SEQ ID NO:6或与SEQ ID NO：6具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：7或与SEQ ID NO：7具有至少90％序列同一性；
(k)重链可变区，其序列如SEQ ID NO:19所示或与SEQ ID NO：19具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：20所示或与SEQ ID NO：20具有至少90％序列同一性；
(l)重链可变区，其序列如SEQ ID NO:27、30、31或32或分别与SEQ ID NO：27、30、31或32具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：28、29、53、54、55、56、57、58、59、60、61、62、63或64或分别与SEQ ID NO：28、29、34、35、53、54、55、56、57、58、59、60、61、62、63或64具有至少90％序列同一性；
(m)重链可变区，其序列如SEQ ID NO:33、36、37、38、39或40所示或分别与SEQ ID NO:33、36、37、38、39或40具有至少90％序列同一性，和/或轻链可变区，其序列如SEQ ID NO：34、35、28、29、53、54、55、56、57、58、59、60、61、62、63或64或分别与SEQ ID NO：34、35、28、29、53、54、55、56、57、58、59、60、61、62、63或64具有至少90％序列同一性；
(n)重链可变区，其序列如SEQ ID NO:41、45或46所示或分别与SEQ ID NO:41、45或46具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：42、43或44或分别与SEQ ID NO：42、43或44具有至少90％序列同一性；
(o)重链可变区，其序列如SEQ ID NO:70所示或与SEQ ID NO：70具有至少90％序列同一性，和/或
轻链可变区，其序列如71所示或与SEQ ID NO：71具有至少90％序列同一
Figure PCTCN2020074098-appb-000002
Figure PCTCN2020074098-appb-000003
在一些实施方案中，所述的抗PD-1抗体的重链可变区如SEQ ID NO：27所示或与SEQ ID NO：27具有至少90％的同一性，且所述抗PD-1抗体的轻链可变区序列如SEQ ID NO：55所示或与SEQ ID NO：55具有至少90％的序列同一性。
在一些实施方案中，所述的抗PD-1抗体的重链可变区如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％的同一性，且所述抗PD-1抗体的轻链可变区序列如SEQ ID NO：43所示或与SEQ ID NO：43具有至少90％的序列同一性。
前述的“至少90％同一性”包含具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
在另外一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗体进一步包含抗体恒定区；在另外一些实施方案中，所述抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；在另外一些实施方案中，所述抗体恒定区包含引入S228P、F234A和L235A中的一个或更多个突变的IgG4重链恒定区，例如含S228P、F234A和L235A三个氨基酸突变；在另外一些实施方案中，所述抗体包含序列如SEQ ID NO:72或如SEQ ID NO:79所示的重链恒定 区和序列如SEQ ID NO:73所示的轻链恒定区。
在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体包含如SEQ ID NO：78所示的轻链和如SEQ ID NO：77或82所示的重链；或
包含如SEQ ID NO：75所示的轻链和如SEQ ID NO：74、76、80或81所示的重链。
在一些实施方案中，所述的抗PD-1抗体包含：
序列如SEQ ID：74所示的重链和序列如SEQ ID：75所示的轻链；或
序列如SEQ ID：77所示的重链和序列如SEQ ID：78所示的轻链。
在一些实施方案中，还提供一种抗PD-1抗体或其抗原结合片段，所述抗体与前述的任一种抗PD-1抗体或其抗原结合片段竞争性结合人PD-1或结合相同的人PD-1表位。
在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗体是双特异性抗体或多特异抗体。
在一些实施方案中，前述的抗PD-1抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
在一些实施方案中，还公开一种分离的单克隆抗体或其抗原结合片段，所述抗体与前述任一项所述抗PD-1抗体或其抗原结合片段竞争性结合人PD-1。
在一些实施方案中，本公开还提供一种药物组合物，其含有治疗有效量的前述任一项所述的抗PD-1抗体或其抗原结合片段，或治疗有效量的前述分离的单克隆抗体或其抗原结合片段，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。在一些实施方案中，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的抗PD-1抗体或其抗原结合片段。
在一些实施方案中，本公开还提供一种核酸分子，其编码前述任一项所述的抗PD-1抗体或其抗原结合片段，或编码前述分离的单克隆抗体或其抗原结合片段。
在一些实施方案中，本公开还提供一种宿主细胞，其包含前述的核酸分子。
在一些实施方案中，本公开还提供一种用于免疫检测或测定PD-1的方法，所述方法包括使用前述任一项所述的抗PD-1抗体或其抗原结合片段的步骤，或使用前述分离的单克隆抗体或其抗原结合片段的步骤。
在一些实施方案中，本公开还提供一种试剂盒，其包含前述的抗PD-1抗体或其抗原结合片段或前述分离的单克隆抗体或其抗原结合片段。
在一些实施方案中，还提供前述抗PD-1抗体或其抗原结合片段或前述分离的单克隆抗体或其抗原结合片段在制备与PD-1相关的疾病的诊断剂中的应用。
在一些实施方案中，本公开还提供一种治疗疾病的方法，所述方法包括向受试者施用治疗有效量的前面任一项所述的抗PD-1抗体或其抗原结合片段，或前述 分离的单克隆抗体或其抗原结合片段，或前述的药物组合物，或前述的核酸分子。
在一些实施方案中，所述疾病为肿瘤。
在另一些实施方案中，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在其中一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；在另一些实施方案中，所述疾病选自：PD-L1阳性的黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌和结肠癌。
在一些实施方案中，本公开还提供前述的抗PD-1抗体或其抗原结合片段，或前述分离的单克隆抗体或其抗原结合片段，或前述的药物组合物，或前述的核酸分子在制备治疗或预防与疾病的药物中的用途。
在一些实施方案中，所述疾病为肿瘤。
在另一些实施方案中，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在其中一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；在另一些实施方案中，所述疾病选自： PD-L1阳性的黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌和结肠癌。
在一些实施方案中，本公开还提供用作药物的前面任一项所述的抗PD-1抗体或其抗原结合片段、或前述分离的单克隆抗体或其抗原结合片段，或前述的核酸分子、或前述的药物组合物。
在一些实施方案中，所述药物用于治疗或预防与PD-1相关的疾病。
在一些实施方案中，所述疾病为肿瘤。
在另一些实施方案中，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在其中一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；在另一些实施方案中，所述疾病选自：PD-L1阳性的黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌和结肠癌。
附图说明
图1：抗PD-1抗体阻断PD-1与其配体的结合测试结果；
图2：抗PD-1抗体对PBMC细胞分泌IFNγ的影响；
图3：抗PD-1抗体对小鼠结肠癌MC38移植瘤的疗效；
图4：抗PD-1抗体对小鼠结肠癌MC38肿瘤体积的影响。
具体实施方式
概述
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
术语“程序性死亡1”、“细胞程序性死亡1”、“蛋白PD-1”、“PD-1”、“PDCD1” 和“hPD-1”可互换使用，且包括人PD-1的变体、同种型、物种同源物、以及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以GenBank登录号U64863找到。
术语“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种为PD-L2)，它在与PD-1结合时下调T细胞活化和细胞因子分泌。如本文中使用的术语“PD-L1”包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型、和种间同源物，以及5种与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列可以用GenBank登录号Q9NZQ7查到。
术语“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的一般术语。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：人IL-2、IFN-γ、IL-6、TNFα、IL-17和IL-5。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本公开所述的“抗体”指一般是免疫球蛋白，一个天然完整抗体是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。本公开所称抗体包括抗体或其抗原结合片段，包括在免疫球蛋白基础上经过改造，同时保留结合抗原的能力的抗体或其抗原结合片段；包括单特异性抗体、双特异性抗体或多特异性抗体；还包括一价抗体、二价抗体或多价抗体。抗体的抗原结合片段，例如可以是包含至少一个VH-CH1和至少一个VL-CL结构，其中VH和VL结构能够基于链间相互作用而靠近，并保留结合抗原的能力的抗原结合片段，在一些实施方案中，所述抗体的抗原结合片段为一价Fab片段(Fab1片段)、二价Fab片段(F(ab)2)、三价Fab片段(F(ab)3)、多价(两个或以上)Fab片段，也可以是其它包含至少一个Fab片段的单特异性、双特异性或多特异性抗原结合片段。
“双特异性抗体”指能够对两个不同抗原或同一抗原的两个不同抗原表位特异性结合的抗体(包括抗体或其抗原结合片段，如单链抗体)。现有技术已公开了各种结构的双特异性抗体，根据IgG分子的完整性分可为IgG样双特异性抗体和抗体片段型双特异性抗体，根据抗原结合区域的数量构型可分为二价、三价、四价或更多价的双特异性抗体，根据结构左右是否对称性可分为对称结构双特异性抗体和不对称结构双特异性抗体。其中，基于抗体片段的双特异性抗体，例如缺 乏Fc片段的Fab片段，其通过将2个或多个Fab片段结合在一个分子中形成双特异性抗体，其具有较低的免疫原性，且分子量小，具有较高的肿瘤组织渗透性，该类型的典型的抗体结构如F(ab)2、scFv-Fab、(scFv)2-Fab等双特异性抗体；IgG样双特异性抗体(例如具有Fc片段)，这类抗体相对分子量较大，Fc片段有助于抗体后期的纯化，并提高其溶解性、稳定性，Fc部分还可能会与受体FcRn结合，增加抗体血清半衰期，典型的双特异性抗体结构模型如KiH、CrossMAb、Triomab quadroma、FcΔAdp、ART-Ig、BiMAb、Biclonics、BEAT、DuoBody、Azymetric、XmAb、2:1TCBs、1Fab-IgG TDB、FynomAb、two-in-one/DAF、scFv-Fab-IgG、DART-Fc、LP-DART、CODV-Fab-TL、HLE-BiTE、F(ab)2-CrossMAb、IgG-(scFv)2、Bs4Ab、DVD-Ig、Tetravalent-DART-Fc、(scFv)4-Fc、CODV-Ig、mAb2、F(ab)4-CrossMAb等等双特异性抗体(参见Aran F.Labrijn等，Nature Reviews Drug Discovery volume 18，pages585–608(2019)；Chen S1等，J Immunol Res.2019Feb11；2019:4516041)。
术语“一价”、“二价”、“三价”或“多价”是指抗体或多肽复合物中存在指定数量的抗原结合位点。例如“一价抗体”表示抗体中存在一个抗原结合位点，“一价多肽复合物”表示多肽复合物中存在一个抗原结合位点；“二价抗体”表示抗体中存在两个抗原结合位点，“二价多肽复合物”表示多肽复合物中存在两个抗原结合位点；“三价抗体”表示抗体中存在三个抗原结合位点，“三价多肽复合物”表示多肽复合物中存在三个抗原结合位点；“多价抗体”表示抗体中存在多个(三个或以上)抗原结合位点，“多价多肽复合物”表示多肽复合物中存在多个(两个或以上)抗原结合位点。
术语“抗体融合蛋白”是指将目的蛋白质(多肽)与免疫球蛋白连接形成的具有生物活性的融合蛋白，所述的融合蛋白具有所连接的蛋白质的生物学活性以及免疫球蛋白活性。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人抗体，优选人源化抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人PD-1的单克隆抗体。制备时用PD-1抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个优选的实施方案中，所述的鼠源抗PD-1抗体 或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的PD-L1嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的PD-1嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
由于抗原的接触残基，CDR的移植可由于与抗原接触的构架残基而导致产生的抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以可能是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查动物单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚类共有序列或具有高相似性百分数的动物抗体序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在本公开一个的实施方案中，所述的抗体或其抗原结合片段，可进一步包含人源或鼠源κ、λ链或其变体的轻链恒定区，或进一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区；优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变、和/或S228P突变)的IgG1、IgG2或IgG4变体。
本公开中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有 技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座的元素元件引入到源于胚胎干细胞系的生物体的细胞株中，这些细胞系中的内源性重链和轻链基因座被靶向破坏这些细胞系中包含靶向的内源性重链和轻链基因座破坏。转基因生物可以合成对人抗原特异的人抗体，并且该生物可以用于产生人抗体-分泌杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或更多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指重链包含Fc区的抗体。
术语抗体的“抗原结合片段”或“功能片段”是指抗体的保持特异性结合抗原(例如，PD-1)的能力的一个或更多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab′) 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人，(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2， IgD，IgE或IgM抗体。
本公开的抗原结合片段包括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)、包含CDR的肽等。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50，000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
本公开的Fab可以通过用木瓜蛋白酶处理本公开的特异性识别人PD-1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100，000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
本公开的F(ab′)2可以通过用胃蛋白酶处理本公开的特异性识别人PD-1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
Fab′是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50，000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的特异性识别PD-1并与胞外区的氨基酸序列或其三维结构结合的F(ab′)2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab′来生产所述Fab′。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得本公开的特异性识别人PD-1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
双抗体是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体 片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本公开的双抗体可以通过以下步骤来生产：获得本公开的特异性识别人PD-1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering，7，697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得本公开的特异性识别人PD-1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的编码cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
包含CDR的肽是通过包含VH或VL的CDR中的一个或更多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本公开的包含CDR的肽可以通过以下步骤来生产：构建本公开的特异性识别人PD-1并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体的VH和VL的CDR的编码DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典 格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，PD-L1分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合PD-1，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如PD-1抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记 的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人PD-1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码 重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PD-1特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
表3.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本公开靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那 么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4，683，195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
此外，本公开包括用于治疗与目标抗原(例如PD-1)阳性细胞相关的疾病的药剂，所述药剂包含本公开的抗PD-1抗体或其抗原结合片段作为活性成分。
本公开中与PD-1相关的疾病没有限制，只要它是与PD-1相关的疾病即可，例如利用本公开的分子诱导的治疗反应可通过结合人类PD-1，然后阻遏PD-1与其配体PD-L1、PD-L2的结合，或杀伤过表达PD-1的肿瘤细胞。因此，当处于适于治疗应用的制备物和制剂中时，本公开的分子对这样一些人是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌等。
此外，本公开涉及用于免疫检测或测定目标抗原(例如PD-1)的方法、用于免疫检测或测定目标抗原(例如PD-1)的试剂、用于免疫检测或测定表达目标抗原(例如PD-1)的细胞的方法和用于诊断与目标抗原(例如PD-1)阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别目标抗原(例如人PD-1)并与胞外区的氨基酸序列或其三维结构结合的抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定目标抗原(例如PD-1)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与PD-1阳性细胞相关的疾病可以通过用本公开的抗体或抗体片段检测或测定表达PD-1的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原(例如PD-1)的活体样品没有特别限制，只要它具有包含表达目标抗原(例如PD-1)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方 法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
抗原的制备
1.抗原构建：
设计并合成人PD-1-IgG1Fc融合蛋白，N端为人PD-1胞外区150个氨基酸，C端为人IgG1的Fc段(hIgG1Fc)。经Protein A的亲和柱纯化，可获得高纯度的重组PD-1-Fc蛋白，用于检测抗PD-1抗体与抗原的结合。
人PD-1-IgG1Fc(SEQ ID NO：1)：
Figure PCTCN2020074098-appb-000004
注释：下划线部分为信号肽，正体部分为人PD-1胞外区，斜体部分为hIgG1Fc(信号肽+胞外区+hIgG1Fc)。
人PD-1-his(SEQ ID NO：2)：
Figure PCTCN2020074098-appb-000005
转染细胞核酸编码的PD-1抗原(SEQ ID NO：3)：
Figure PCTCN2020074098-appb-000006
抗体的制备
抗人PD-1抗体可通过免疫小鼠产生，也可通过抗人PD-1噬菌体小鼠免疫文库获得。
通过免疫小鼠制备抗人PD-1抗体的方法如下：
1.免疫：实验用SJL白小鼠，雌性，6-8周龄和Balb/c白小鼠，雌性，6-8周龄。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按不同方案免疫，每组6-10只。免疫抗原可以是纯化的重组蛋白PD-1-IgG1Fc(见SEQ ID NO：1)、PD-1-his (见SEQ ID NO：2)、或PD-1作为抗原(见SEQ ID NO：3)转染的Jurkat/CHO-PD-1细胞，可以使用单独一种抗原配合不同的免疫佐剂或者不同类型免疫原交叉免疫。免疫部位可以是腹腔或者背部皮下，或者两种位置交替免疫。免疫佐剂
Figure PCTCN2020074098-appb-000007
Gold Adjuvant(以下简称Titermax，购自Sigma货号T2684)与Imject Alum Adjuvant(以下简称Alum，购自Pierce货号77161)交叉免疫。抗原与佐剂(Titermax)比例为1:1，抗原与佐剂(Alum)比例为3:1，25-50μg/只(首免)，50μg/只(加强免疫)，或是1×10 7个Jurkat/CHO-PD-1细胞/只。第0天腹膜内注射25-50μg/只的乳化后抗原，首免后每周一次或是每两周一次，Titermax和Alum交替使用，共5-8次。
2.细胞融合：选择血清中抗体滴度高的小鼠进行脾细胞融合，将冲刺免疫72小时后的小鼠眼球放血，拉颈处死，放入75％乙醇中消毒。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(中国科学院)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×10 5/150μl/孔)，37℃，5％CO 2孵育，种板10-30块左右。融合后的第5天加入HAT完全培养基，50μl/孔，37℃，5％CO 2孵育。融合后第7天至8天，根据细胞生长密度，全换液，200μl/孔，37℃，5％CO 2孵育。
3.杂交瘤细胞筛选：融合后第7-9天，根据细胞生长密度，进行抗体与PD-1结合的ELISA方法检测，并将检测的阳性孔细胞进行PD-1/PDL1结合的阻断ELISA检测，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选为阳性的进行保种，并进行第二次或第三次亚克隆，直至获得单细胞克隆。多次融合获得有阻断PD-1与PDL1结合效果的杂交瘤细胞。
通过抗人PD-1噬菌体小鼠免疫文库获得抗人PD-1抗体的方法如下：
1.构建抗人PD-1噬菌体小鼠免疫文库：选择血清中抗体滴度高的小鼠的脾脏，用Trizol(Invitrogen Cat No.15596-018)提取组织总RNA。使用PrimeScript TMII 1st Strand cDNA Synthesis Kit试剂盒(Takara Cat No.6210A)进行反转录获得cDNA。根据IMGT数据库设计并合成构建文库的引物。通过三轮PCR反应，获得单链抗体片段。将单链抗体片段和经过改造的建库载体pCantab5E(Amersham Biosciences/GE Cat No.27-9400-01)用Sfi1(NEB Cat No.#R0123L)进行酶切，电泳后用
Figure PCTCN2020074098-appb-000008
Gel Extraction Kit(Omega Cat No.D2500-02)进行纯化回收。然后用T4DNA连接酶(NEB Cat No.#M0202L)16℃连接16-18小时，再用上述试剂盒进行纯化回收，最后用去离子水洗脱。取1μg连接产物与1支电转化感受态TG1(Lucigen Cat No.60502-2)混合，电转化仪(Bio Rad Micropulser)参数设至2.5kV，200Ω，25uF，进行电转化。重复转化10次，涂平板，37℃倒置培养16-18小时。将所有菌落刮洗下来混和在一起，加入终浓度为15％的甘油，-80℃保存备 用。
2.抗人PD-1噬菌体小鼠免疫文库的筛选：将包装好的抗人PD-1噬菌体免疫文库(1×10 12-1×10 13)与100μl链菌素微珠(Mi1envi Biotec，Auburn，CA)加入1m1含2％脱脂牛奶-磷酸盐缓冲液(缩写MPBS)中于室温下孵育1小时，放置在磁力架上，取上清。上清加入10μg/ml生物素化的人PD-1-ECD-his蛋白(购自Sino Biological)中于室温下孵育1小时，再加入100μl链霉亲和素包被的磁珠(1ml MPBS预孵育)于室温下孵育1小时。并使其负载于磁力架系统上用于分选，吸去上清。加入1ml PBST(含0.1％Tween-20的磷酸盐缓冲液)，翻转多次，吸尽后再加入新鲜洗液，重复11次，以去除未结合的抗体片段，加入0.5ml洗脱液(50μl 10mg/ml trypsin stock solution(存储液)加入450μl PBS中)。室温下摇晃15min。放置在磁力架上，吸出上清至一新EP管中。TG1接入2YT培养基中扩增至培养细菌密度OD600＝0.4时。每管加入1.75ml TG1(OD600＝0.4)，并加入250μl洗脱后phage(噬菌体)，37℃水浴中静置孵育30min，梯度稀释涂板，用于测试滴度。其余TG1溶液离心，涂板，37℃过夜孵育。
噬菌体小鼠免疫文库利用生物素化的人PD-1-ECD-his抗原，经过2-3轮MACS筛选(链霉素磁珠，Invitrogen)，最终获得具有结合PD-1和阻断PD-1与PD-L1结合的单克隆，测序验证，得到抗体的可变区序列。
重组抗原蛋白/抗体的纯化
1.杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清纯化首选ProteinG进行亲和层析，将培养所得杂交瘤离心取上清，根据上清体积加入10-15％体积的1M Tris-HCl(pH8.0-8.5)调节上清pH。ProteinG柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M醋酸/醋酸钠(pH3.0)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1MTris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
2.Protein A亲和层析纯化蛋白或抗体：
首先将表达抗原蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M 醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗人PD-1鼠源抗体获得
将经前述方法获得的抗人PD-1鼠源抗体进行抗原结合实验，筛选得到多株活性良好的抗体：其中包括M23、M32和M33，将单细胞克隆扩培养，提取RNA，利用mouse-Ig的简并引物进行反转录扩增(RT-PCR)，得到抗体的可变区序列。将该鼠抗体可变区序列与人抗体恒定区序列连接，克隆并重组表达出该鼠单克隆抗体的嵌合抗体，进行体外活性实验，确认所得到的单克隆抗体可变区序列正确。
测得鼠源抗体M23、M32和M33的可变区序列如下：
鼠源抗体M23的重链可变区(SEQ ID NO：4)：
Figure PCTCN2020074098-appb-000009
鼠源抗体M23的轻链可变区(SEQ ID NO：5)：
Figure PCTCN2020074098-appb-000010
鼠源抗体M32的重链可变区(SEQ ID NO：6)：
Figure PCTCN2020074098-appb-000011
鼠源抗体M32的轻链可变区(SEQ ID NO：7)：
Figure PCTCN2020074098-appb-000012
鼠源抗体M33的重链可变区：(SEQ ID NO：19)
Figure PCTCN2020074098-appb-000013
鼠源抗体M33的轻链可变区：(SEQ ID NO：20)
Figure PCTCN2020074098-appb-000014
备注：上述抗体的重链可变区和轻链可变区序列中，下划线为Kabat编号系统确定的CDR序列，依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
表4.鼠源抗体M23、M32和M33重链及轻链CDR区序列
Figure PCTCN2020074098-appb-000015
备注：表中的抗体CDR序列是根据Kabat编号系统确定。
实施例2.抗人PD-1单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件分析，分别挑选与M23，M32，M33的轻重链序列同一性高的人种系重轻链可变区种系基因作为模板，将这3个鼠源抗体的CDR分别移植到相应的人源抗体模板中，分别构建其对应的人源化抗体。
1.鼠源抗体M23人源化
1.1鼠源抗体M23人源化构架选择
鼠源抗体M23的人源化轻链模板为IGKV2-40*01和IGKJ4*01，人源化重链 模板为IGHV1-69*02和IGHJ6*01，人源化改造后可变区序列如下(下划线为CDR序列)：
Hu23VH-CDR嫁接：(SEQ ID NO：27)
Figure PCTCN2020074098-appb-000016
Hu23VL-CDR嫁接：(SEQ ID NO：28)
Figure PCTCN2020074098-appb-000017
1.2鼠源抗体M23的人源化模板选择和回复突变设计
表5.鼠源抗体M23人源化抗体的回复突变
Figure PCTCN2020074098-appb-000018
注：嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列，氨基酸残基由Kabat编号系统确定并注释，如I2G表示依照Kabat编号系统，将Kabat编号的第2位I突变回G。
M23的人源化抗体轻/重链可变区序列如下：
>Hu23VL1(同Hu23VL-CDR grafted)：(SEQ ID NO：28)
Figure PCTCN2020074098-appb-000019
>Hu23VL2(SEQ ID NO：29)
Figure PCTCN2020074098-appb-000020
>Hu23VH1(同Hu23VH-CDR grafted)：(SEQ ID NO：27)
Figure PCTCN2020074098-appb-000021
>Hu23VH2(SEQ ID NO：30)
Figure PCTCN2020074098-appb-000022
Figure PCTCN2020074098-appb-000023
>Hu23VH3(SEQ ID NO：31)
Figure PCTCN2020074098-appb-000024
>Hu23VH4(SEQ ID NO：32)
Figure PCTCN2020074098-appb-000025
1.3鼠源抗体M23的人源化序列组合
鼠源抗体M23的人源化后获得的抗体及其可变区组合见下表。
表6.人源化Hu23抗体可变区组合
Figure PCTCN2020074098-appb-000026
备注：“Hu23-1”指代抗体轻链可变区为Hu23VL1且重链可变区为Hu23VH1的抗体，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23-1)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu23-1)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu23-1.IgG4AA”表示由Hu23VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu23VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu23-1.IgG4P”表示由Hu23VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu23VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
2.鼠源抗体M32人源化
2.1鼠源抗体M32人源化构架选择
鼠源抗体M32的人源化轻链模板为IGKV2-40*01和IGKJ4*01，人源化重链 模板为IGHV1-69*02和IGHJ6*01，人源化可变区序列如下(下划线为CDR序列)：
Hu32VH-CDR嫁接：(SEQ ID NO：33)IGHV1-69*02和IGHJ6*01
Figure PCTCN2020074098-appb-000027
Hu32VL-CDR嫁接：(SEQ ID NO：34)
Figure PCTCN2020074098-appb-000028
2.2鼠源抗体M32的人源化模板选择和回复突变设计
表7.鼠源抗体M32的人源化抗体的回复突变
Figure PCTCN2020074098-appb-000029
注：嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列。氨基酸残基由Kabat编号系统确定并注释，如I2V表示依照Kabat编号系统，将Kabat编号的第2位I突变回V。
鼠源抗体M32的人源化抗体轻重链可变区序列如下：
>Hu32VL1(同Hu32VL-CDR嫁接)：(SEQ ID NO：34)
Figure PCTCN2020074098-appb-000030
>Hu32VL2(SEQ ID NO：35)
Figure PCTCN2020074098-appb-000031
>Hu32VH1(同Hu32VH-CDR grafted)：(SEQ ID NO：33)
Figure PCTCN2020074098-appb-000032
Figure PCTCN2020074098-appb-000033
>Hu32VH2(SEQ ID NO：36)
Figure PCTCN2020074098-appb-000034
>Hu32VH3(SEQ ID NO：37)
Figure PCTCN2020074098-appb-000035
>Hu32VH4(SEQ ID NO：38)
Figure PCTCN2020074098-appb-000036
>Hu32VH5(SEQ ID NO：39)
Figure PCTCN2020074098-appb-000037
>Hu32VH6(SEQ ID NO：40)
Figure PCTCN2020074098-appb-000038
2.3鼠源抗体M32的人源化序列组合
鼠源抗体M32的人源化后获得的抗体及其可变区组合。
表8.人源化抗体Hu32轻/重链可变区组合
Figure PCTCN2020074098-appb-000039
备注：表中例如“Hu32-1”指代抗体轻链可变区为Hu32VL1且重链可变区为Hu32VH1的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu32-1)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P 重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu32-1)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu32-1.IgG4AA”表示由 Hu32VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由 Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu32-1.IgG4P”表示由 Hu32VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由 Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
3.鼠源抗体M33人源化
3.1鼠源抗体M33人源化构架选择
鼠源抗体M33的人源化轻链模板为IGKV1-39*01和IGKJ4*01，人源化重链模板为IGHV3-7和IGHJ6*01，人源化可变区序列如下：
Hu33VH-CDR嫁接(SEQ ID NO：41)：
Figure PCTCN2020074098-appb-000040
Hu33VL-CDR嫁接(SEQ ID NO：42)：
Figure PCTCN2020074098-appb-000041
3.2鼠源抗体M33的人源化模板选择和回复突变设计
表9.鼠源抗体M33人源化抗体的回复突变
Figure PCTCN2020074098-appb-000042
注：嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列。氨基酸残基由Kabat编号系统确定并注释，如F71Y表示依照Kabat编号系统，将Kabat编号的第71位F突变回Y。
鼠源抗体M33的人源化抗体轻链可变区和重链可变区序列如下：
>Hu33VL1(同Hu33VL-CDR grafted)：(SEQ ID NO：42)
Figure PCTCN2020074098-appb-000043
>Hu33VL2(SEQ ID NO：43)
Figure PCTCN2020074098-appb-000044
>Hu33VL3(SEQ ID NO：44)
Figure PCTCN2020074098-appb-000045
>Hu33VH1(同Hu33VH-CDR grafted)：(SEQ ID NO：41)
Figure PCTCN2020074098-appb-000046
>Hu33VH2(SEQ ID NO：45)
Figure PCTCN2020074098-appb-000047
>Hu33VH3(SEQ ID NO：46)
Figure PCTCN2020074098-appb-000048
3.3鼠源抗体M33的人源化序列组合
表10.人源化抗体轻重链可变区组合
Figure PCTCN2020074098-appb-000049
备注：表中例如“Hu33-6”指代抗体轻链可变区为Hu33VL2且重链可变区为Hu33VH3的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu33-6)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu33-6)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu33-6.IgG4AA”，表示由 Hu33VH3重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由 Hu33VL2轻链可变区和如SEQ ID NO： 73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu33-6.IgG4P”，表示由 Hu33VH3重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由 Hu33VL2轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
4.人源化抗体的突变体
4.1 Hu23人源化抗体的突变抗体
通过计算机模拟，对Hu23人源化抗体的轻链LCDR1(SEQ ID NO：11)的特定位点氨基酸进行定点突变，具体突变见表11：
表11.Hu23轻链LCDR1的突变序列:
Figure PCTCN2020074098-appb-000050
注：Hu23LCDR1(N28Q)表示对Hu23人源化抗体轻链可变区Hu23VL1或Hu23VL2的Kabat编号规则第28位N突变为Q的LCDR1突变序列，Hu23LCDR1(G29A)表示对Hu23人源化抗体轻链可变区Hu23VL1或Hu23VL2的Kabat编号规则第29位G突变为A的LCDR1突变序列(由Kabat编号系统确定CDR)。
LCDR1突变后的Hu23人源化抗体轻链可变区序列如下：
>Hu23VL1(N28Q)序列为：
Figure PCTCN2020074098-appb-000051
>Hu23VL1(N28L)序列为：
Figure PCTCN2020074098-appb-000052
>Hu23VL1(N28T)序列为：
Figure PCTCN2020074098-appb-000053
>Hu23VL1(N28D)序列为：
Figure PCTCN2020074098-appb-000054
>Hu23VL1(G29A)序列为：
Figure PCTCN2020074098-appb-000055
>Hu23VL1(G29V)序列为：
Figure PCTCN2020074098-appb-000056
>Hu23VL2(N28Q)序列为：
Figure PCTCN2020074098-appb-000057
>Hu23VL2(N28L)序列为：
Figure PCTCN2020074098-appb-000058
>Hu23VL2(N28T)序列为：
Figure PCTCN2020074098-appb-000059
>Hu23VL2(N28D)序列为：
Figure PCTCN2020074098-appb-000060
>Hu23VL2(G29A)序列为：
Figure PCTCN2020074098-appb-000061
>Hu23VL2(G29V)序列为：
Figure PCTCN2020074098-appb-000062
表12.Hu23人源化抗体轻/重链可变区组合
Figure PCTCN2020074098-appb-000063
Figure PCTCN2020074098-appb-000064
备注：表中例如“Hu23-11”指代抗体轻链可变区为Hu23VL1(N28T)且重链可变区为Hu23VH1的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23-11)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu23-11)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu23-11.IgG4AA”，表示由Hu23VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu23-11.IgG4P”，表示由Hu23VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
实验结果显示，Hu23LCDR1(N28Q)、Hu23LCDR1(N28L)、Hu23LCDR1(N28T)、Hu23LCDR1(N28D)、Hu23LCDR1(G29A)、Hu23LCDR1(G29V)位点突变后的人源化抗体均保持与PD-1的结合能力(表16)。
4.2Hu32人源化抗体的突变抗体
经序列分析，M23来源的系列人源化抗体Hu23和M32来源的系列人源化抗体Hu32的序列同一性较高，将Hu23轻链可变区和Hu32的重链可变区组合成新的轻重链可变区组合。实验结果显示，包含新组合轻重链可变区的人源化抗体均保持与PD-1抗原的结合能力(表16)。
表13.Hu32和Hu23抗体可变区共有序列通式
Figure PCTCN2020074098-appb-000065
Figure PCTCN2020074098-appb-000066
表14.Hu32重链可变区与Hu23轻链可变区的组合
Figure PCTCN2020074098-appb-000067
Figure PCTCN2020074098-appb-000068
备注：表中例如“Hu32a-85”指代抗体轻链可变区为Hu23VL1(N28T)且重链可变区为Hu32VH6的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu32a-85)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu32a-85)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu32a-85.IgG4AA”，表示由Hu32VH6重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu32a-85.IgG4P”，表示由Hu32VH6重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
表15.Hu23重链可变区与Hu32轻链可变区的组合
Figure PCTCN2020074098-appb-000069
备注：表中例如“Hu23a-57”指代抗体轻链可变区为Hu32VL1且重链可变区为Hu23VH1的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23a-57)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu32a-85)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu23a-57.IgG4AA”，表示由Hu23VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu23a-57.IgG4P”， 表示由Hu23VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
5.人源化抗体的筛选
通过Biacore进行不同人源化抗体的亲和力检测(方法参见测试例3)，结果见表16，结果显示不同的人源化抗体保持了对于PD-1的结合能力，部分人源化抗体的亲和力甚至和其鼠源抗体基本接近。
表16.Hu23人源化抗体与人PD-1的亲和力
抗体 ka(1/Ms) kd(1/s) KD(M)
M23 4.57E+04 1.32E-04 2.89E-09
Hu23-1.IgG4AA 3.43E+04 1.10E-04 3.20E-09
Hu23-5.IgG4AA 2.99E+04 1.14E-04 3.82E-09
Hu23-2.IgG4AA 2.74E+04 1.61E-04 5.86E-09
Hu23-6.IgG4AA 2.79E+04 1.55E-04 5.57E-09
Hu23-3.IgG4AA 2.93E+04 1.60E-04 5.46E-09
Hu23-7.IgG4AA 2.77E+04 1.66E-04 5.97E-09
Hu23-4.IgG4AA 4.24E+04 1.44E-04 3.39E-09
Hu23-8.IgG4AA 4.11E+04 1.47E-04 3.56E-09
Hu23-9.IgG4AA 6.16E+04 1.32E-04 2.15E-09
Hu23-10.IgG4AA 5.51E+04 1.53E-04 2.77E-09
Hu23-11.IgG4AA 4.22E+04 1.14E-04 2.71E-09
Hu23-12.IgG4AA 5.45E+04 1.10E-04 2.02E-09
Hu23-13.IgG4AA 4.24E+04 1.22E-04 2.88E-09
Hu23-14.IgG4AA 7.23E+04 1.61E-04 2.22E-09
M32 7.83E+04 4.15E-04 5.3E-09
Hu32a-15.IgG4AA 4.89E+04 2.52E-03 5.14E-08
Hu32a-29.IgG4AA 7.89E+04 6.20E-04 7.86E-09
Hu32a-43.IgG4AA 8.39E+04 6.85E-04 8.16E-09
Hu32a-57.IgG4AA 7.94E+04 6.24E-04 7.85E-09
Hu32a-71.IgG4AA 8.60E+04 3.96E-04 4.61E-09
Hu32a-85.IgG4AA 9.90E+04 3.15E-04 3.18E-09
M33 3.08E+05 2.27E-04 7.37E-10
Hu33-1.IgG4AA 6.61E+04 1.28E-03 1.93E-08
Hu33-4.IgG4AA 8.11E+04 2.55E-03 3.14E-08
Hu33-7.IgG4AA 7.69E+04 2.60E-03 3.38E-08
Hu33-2.IgG4AA 1.35E+05 1.43E-04 1.06E-09
Hu33-5.IgG4AA 1.46E+05 1.35E-04 9.26E-10
Hu33-8.IgG4AA 1.53E+05 1.62E-04 1.06E-09
Hu33-3.IgG4AA 1.25E+05 1.36E-04 1.09E-09
Hu33-6.IgG4AA 1.30E+05 1.40E-04 1.08E-09
Hu33-9.IgG4AA 1.40E+05 1.53E-04 1.09E-09
实施例3.构建和表达PD-1人源化抗体
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建抗体全长表达载体 VH-CH1-Fc-pHr/VK-CL-pHr。IgG4-P代表S228P(对应于序列SEQ ID NO：72或SEQ ID NO：79的第108位)突变，IgG4-AA代表F234A(对应于序列SEQ ID NO：72或SEQ ID NO：79的第114位)、L235A(对应于序列SEQ ID NO：72或SEQID NO：79的第115位)和S228P(对应于序列SEQ ID NO：72或SEQ ID NO：79的第108位)突变，IgG4-AA和IgG4-P抗体形式可以通过IgG4抗体形式简单点突变获得。
IgG4-AA重链恒定区序列如下(SEQ ID NO：72)：
Figure PCTCN2020074098-appb-000070
抗体的轻链(Kappa链)恒定区序列如下(SEQ ID NO：73)：
Figure PCTCN2020074098-appb-000071
构建的IgG4AA形式全长抗体序列示例性列举如下：
Hu23-11.IgG4AA抗体重链(SEQ ID NO：74)：
Figure PCTCN2020074098-appb-000072
Hu23-11.IgG4AA轻链(SEQ ID NO：75):
Figure PCTCN2020074098-appb-000073
Hu32a-85.IgG4AA重链(SEQ ID NO：76)：
Figure PCTCN2020074098-appb-000074
Figure PCTCN2020074098-appb-000075
Hu32a-85.IgG4AA的轻链(同Hu23-11.IgG4AA的轻链， SEQ ID NO：75)：
Figure PCTCN2020074098-appb-000076
Hu33-6.IgG4AA重链(SEQ ID NO：77)：
Figure PCTCN2020074098-appb-000077
Hu33-6.IgG4AA轻链(SEQ ID NO：78)：
Figure PCTCN2020074098-appb-000078
IgG4-P的重链恒定区序列如下(SEQ ID NO：79)：
Figure PCTCN2020074098-appb-000079
构建的IgG4-P形式全长抗体序列示例性列举如下：
Hu23-11. IgG4P抗体重链(SEQ ID NO：80)：
Figure PCTCN2020074098-appb-000080
Figure PCTCN2020074098-appb-000081
Hu23-11.IgG4P轻链(同Hu23-11.IgG4AA的轻链，SEQ ID NO：75):
Figure PCTCN2020074098-appb-000082
Hu32a-85.IgG4P重链(SEQ ID NO：81)：
Figure PCTCN2020074098-appb-000083
Hu32a-85.IgG4P的轻链(同Hu23-11.IgG4AA的轻链，SEQ ID NO：75)：
Figure PCTCN2020074098-appb-000084
Hu33-6.IgG4P重链(SEQ ID NO：82)：
Figure PCTCN2020074098-appb-000085
Hu33-6.IgG4P轻链(同Hu33-6.IgG4AA轻链，SEQ ID NO：78)：
Figure PCTCN2020074098-appb-000086
测试例
测试例1.抗PD-1抗体体外PD-1配体结合和结合阻断ELISA实验
肿瘤细胞表面的PD-L1通过和T细胞表面的PD-1的结合，从而对T细胞的增殖起到抑制的效果。PD-1的抗体能通过和PD-1的结合，而阻断PD-L1/PD-1的信号通路，进而刺激T细胞的增殖。PD-1/PD-L1的结合阻断实验用于检测抗PD-1抗体对于信号通路的阻断活性。
本实验中，将PD-1-His蛋白(Cat.#10377H08H，Sino Biological)包被96孔板后，分别加入待测的抗PD-1抗体(包括抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体：H005-1(参见WO2015085847中H005-1抗体)，进行孵育反应；稍后再HRP标记的goat anti-human IgG(H+L)抗体(Cat.#109-035-003，Jackson ImmunoResearch)，孵育反应。洗板后，检测HRP标记的goat anti-human IgG(H+L)结合量，计算得到抗PD-1抗体对配体PD-1结合的EC 50值。
本实验中，将胞外区与Fc融合的PD-1蛋白(PD-1-Fc，序列见SEQ ID NO：1)包被96孔板后，分别加入待测的抗PD-1抗体(包括抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体：H005-1(参见WO2015085847中H005-1抗体)，进行孵育反应；稍后再加入生物素标记的PD-L1/PD-L2，孵育反应。洗板后，检测生物素标记的PD-L1/PD-L2结合量，计算得到抗PD-1抗体对配体PD-L1/PD-L2结合阻断的IC 50值。
用pH 9.6 CB缓冲液(1.59g Na 2CO 3和2.93g NaHCO 3溶于1L蒸馏水)将PD-1-Fc稀释至1μg/ml，以100μl/孔的体积加于96孔板中，于4℃放置16h-20h。将96孔板中PBS缓冲液吸掉，用PBST(pH7.4 PBS含0.05％tween20)缓冲液洗板1次后，加入120μl/孔PBST/1％milk，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板1次后，加入90μl用样品稀释液(pH7.4 PBS含5％BSA，0.05％Tween20)稀释至合适浓度的待测抗PD-1抗体，置4℃预孵育1h。以10μl/孔的体积加入10×浓度的生物素标记PD-L1/PD-L2(北京义翘神州生物技术有限公司)(10μg/ml)，在振荡器上振荡、混匀后，置37℃孵育1h。移去反应体系，用PBST洗板6次后，加入100μl/孔用PBST缓冲液1:400稀释的Streptavidin–Peroxidase Polymer(链霉亲和素-过氧化物酶聚合物)，室温振荡孵育50分钟。用PBST洗板6次后，加入100μl/孔TMB，于室温孵育5-10min。加入100μl/孔1M H 2SO 4终止反应。用酶标仪在450nm处读取吸收值，计算抗PD-1抗体对配体PD-L1/PD-L2 结合阻断的IC 50值。数据详见下表17。
表17.本公开的抗PD-1抗体和PD-1结合及
对配体PD-L1/PD-L2结合阻断ELISA
Figure PCTCN2020074098-appb-000087
本公开示例性抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA都能够有效阻断PD-1与PD-L1/PD-L2的结合，其阻断活性与阳性对照抗体相似。
测试例2.示例性抗体的配体阻断试验
研究抗体对PD-1与PD-L1结合的阻断作用。实验过程简单描述如下：
消化CHOK1/PD-L1细胞(Promega)，按照100μL/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养24小时。使用PBS稀释对照品和样品至所需浓度。计数Jurkat/PD-1细胞(稳转PD-1的Jurkat细胞)，按一定比例种CHOK1/PD-L1细胞的细胞培养板中(90μL/孔)同时加入10μL/孔加入稀释后的抗体(抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体：H005-1)，阴性对照IgG4蛋白，抗体梯度稀释浓度为0.3mg/ml、3mg/ml、30mg/ml)，置于37℃，5％CO 2培养箱培养5小时。取出细胞培养板，置于室温放置5分钟，然后每孔加入50μl Bio-Glo TMReagent，室温孵育5分钟，读板。实验结果见附图1。
结果表明，本公开中示例性的抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA能够有效阻断PD-1与PD-L1的结合。
测试例3.示例性抗体的BIAcore抗体亲和力实验
用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获IgG，human PD-1抗原(Cat.#10377H08H，Sino Biological)、Cyno PD-1抗原(购自Sino Biological)流过芯片表面，Biacore T200仪器实时检测PD-1抗体和抗原PD-1反应信号获得结合和解离曲线。在每个实验循环解离完成后，用10mM Glycine-HCl pH1.5的缓冲液将生物传感芯片洗净再生。实验缓冲体系为1×HBS-EP缓冲溶液(Cat#BR-1001-88，GE)。实验结束后用GE Biacore T200Evaluation version3.0软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，结果见表18。
表18.抗PD-1抗体与人PD-1和猴PD-1的亲和力
Figure PCTCN2020074098-appb-000088
结果显示，本公开示例性的抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA均能够与人PD-1和猴PD-1结合。
测试例4.抗体在PBMC-T淋巴细胞激活实验中对细胞IFNγ的分泌作用
为了研究抗PD-1抗体对人原代T淋巴细胞功能的影响，收集和纯化人外周血单核细胞(PBMC)，采用结核菌素(TB)体外刺激5天后，检测细胞因子IFNγ分泌水平。实验过程简单描述如下：
新鲜血液利用Ficoll-Hypaque(17-5442-02，GE)，密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI1640(SH30809.01，GE)培养基中培养，该培养基中添加10％(v/v)FBS(10099-141，Gibco)，37℃，5％CO 2条件下培养。
新鲜分离纯化的PBMC以RPMI1640培养基调整密度为2×10 6个/ml，20mL细胞悬液中加入40μl结核菌素(97-8800，Synbiotics)，37℃，5％CO 2培养箱培养5天。第5天，收集上述培养的细胞离心，重悬至新鲜的RPMI1640培养基中，调整密度为1.1×10 6个/ml，接种至96孔细胞培养板，每孔90μl。同时加入梯度稀释的抗体样品(包括本公开的抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体H005-1，和阴性对照IgG4蛋白，抗体梯度稀释浓度为0.3mg/ml、3mg/ml、30mg/ml)，用PBS(B320，上海源培生物科技股份有限公司)稀释，每孔10μl。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)收集细胞培养上清，采用ELISA的方法(人IFN-γ检测试剂盒(EHC102g.96，欣博盛)，检测IFN-γ的水平。具体操作参考试剂说明书。
试验结果见图2，结果表明本公开的抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA、和Hu33-6.IgG4AA均能有效激活IFN-γ的分泌。
测试例5.抗PD-1抗体在转基因PD-1小鼠结肠癌模型MC38中的作用
将MC38细胞5×10* 5细胞/小鼠/100μl接种于90只hPD-1TG小鼠(百奥赛图)右肋部皮下，10天后去除肿瘤体积过大过小的动物，按平均肿瘤体积约120mm^3将小鼠随机分为：空白对照Vehicle(PBS)、阳性对照H005-13mpk、Hu32a-85.IgG4AA1mpk、Hu32a-85.IgG4AA3mpk、Hu23-11.IgG4AA1mpk、Hu23-11.IgG4AA3mpk、Hu33-6.IgG4AA3mpk共7组，每组8只。Day0(第0天)起每周三次腹腔注射各组抗体，第一周给药结束后发现肿瘤被明显抑制，第二、 三周调整给药频率为每周一次，共给药5次。每周2次监测肿瘤体积、动物重量并记录数据。当肿瘤体积超过2000mm 3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率(T/C％)＝(T-T0)/(C-C0)×100％
抑瘤率(TGI％)＝1-T/C％
其中，T、T0分别表示抗体给药组试验结束和试验开始时的肿瘤体积，C、C0分别表示空白对照组试验结束和试验开始时的肿瘤体积。
试验结果见表19和附图3，试验结果表明，与空白对照相比，本公开的抗体均能显著抑制小鼠结肠癌MC38移植瘤的生长，其中抑瘤率最高的是Hu32a-85.IgG4AA-3mpk组，末次测量时抑瘤率为77.64％。当给药频率为一周三次给药3次，在第七天检测时，结果显示本公开的抗体的抑瘤率均明显优于阳性对照抗体H005-1；其后给药频率降为一周一次，给药2次后(Day21)，本公开的抗体间药效逐渐拉开差距，且表现出剂量依赖性，其中Hu32a-85.IgG4AA明显优于同等剂量的H005-1(p＜0.05)。而且荷瘤小鼠对抗PD-1抗体均能很好耐受，在整个给药过程中体重平稳上升，无明显药物致体重减轻等症状发生。
表19.抗PD-1抗体对小鼠结肠癌MC38的抑瘤率影响(mm 3)
Figure PCTCN2020074098-appb-000089
测试例6.抗PD-1抗体在转基因PD-1小鼠结肠癌模型MC38中的作用
转基因PD-1小鼠来源于购买的转基因PD-1小鼠(ISIS INNOVATION LIMITED，University Offices，Wellington Square，Oxford OX1 2JD，England)在Cephrim Biosciences，Inc.培育的第五代小鼠。将MC38细胞以5x10 5个/100μl/只接种到hPD-1转基因小鼠(雌雄各半)右肋后部皮下，待小鼠平均肿瘤体积达到80-100mm3之间时，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为5组(每组8只)：阴性对照hIgG control30mpk、H005-110mpk、H005-130mpk、Hu33-6.IgG4AA10mpk、Hu33-6.IgG4AA30mpk。分组给药日期设定为Day0。分组后腹腔给予各药物，给药周期22天，每两天给药一次，共11次。每周测2次瘤体积，称体重，记录数据。各组动物体重、肿瘤体积均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism5和Excel软件作图，使用student t test统计分析。
肿瘤体积(TV)＝0.5236×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
抑瘤率％TGI＝1-T/C％
其中，T、T0分别表示抗体给药组试验结束和试验开始时的肿瘤体积，C、C0分别表示空白对照组试验结束和试验开始时的肿瘤体积。
试验结果见表20和附图4所示，试验结果表明，与对照组相比，本公开的抗体能显著抑制小鼠结肠癌MC38移植瘤的生长，其中抑瘤率最高的是Hu33-6.IgG4AA30mpk组，第20天测量时抑瘤率为80.4％。在低剂量组(10mpk)，Hu33-6.IgG4AA-10mpk的药效好于阳性对照H005-1-10mpk。
表20.抗PD-1抗体对小鼠结肠癌MC38肿瘤体积影响
Figure PCTCN2020074098-appb-000090
备注：表中各组肿瘤平均体积的单位为：mm 3。
测试例7.抗PD-1抗体食蟹猴的药代动力学试验
实验用食蟹猕猴，雄性，6只，2-5岁，2-5公斤。购于于广东前沿生物科技有限公司，许可证号：SCXK(粤)2015-0037，动物合格证号：44613900000219。
饲养环境：室温控制在18℃～26℃，相对湿度在40％～70％，光照12小时明暗交替。除了需要禁食的情况外，无限量获取饲料和水。
动物给药前称重，体重介于2.81～3.52kg之间。使用注射泵于前肢或后肢皮下静脉输注给药，各组给药剂量均为1mg/kg(1mpk)，单次静脉注射给药，给药速度0.1mL/kg/min，给药时间约30min。动物于给药前，静脉输注开始后5min、0.25h、0.5h(给药结束即刻)，1h、2h、4h、8h，1d、2d、3d、4d、5d、7d、10d、13d、14d、21d和28d，于后肢静脉采集全血，分离血清。其中给药前，静脉输注开始后14d，21d，28d采集全血约2mL，其余采血点采集全血约1mL。用ELISA检测血清中的血药浓度，进行PK分析，结果见表21。
表21.人源化抗PD1抗体食蟹猕猴药代动力学
  Hu23-11.IgG4AA Hu33-6.IgG4AA
t1/2(day) 5.5±0.7 4.6±1.3
Cmax(μg/mL) 23.75±2.29 21.47±2.13
AUC(h*μg/ml) 2775±241 2319±518
CL(ml/day/kg) 8.7±0.7 10.7±2.3
Vz(mL/kg) 69±3.6 67.9±3.4
结果显示，Hu23-11.IgG4AA和Hu33-6.IgG4AA的药代动力学活性良好。
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本公开的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (17)

  1. 一种抗PD-1抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    所述重链可变区包含：序列如SEQ ID NO：65所示的HCDR1，序列如SEQ ID NO：66所示的HCDR2，和序列如SEQ ID NO：67所示的HCDR3；且
    所述轻链可变区包含：序列如SEQ ID NO：68所示的LCDR1，序列如SEQ ID NO：12所示的LCDR2，和序列如SEQ ID NO：69所示的LCDR3；
    优选地，
    所述重链可变区包含序列如SEQ ID NO：8所示的HCDR1，序列如SEQ ID NO：9所示的HCDR2，和序列如SEQ ID NO：10所示的HCDR3；且
    所述轻链可变区包含序列如SEQ ID NO：12所示的LCDR2，序列如SEQ ID NO：13所示的LCDR3，和序列如通式RSSQSX 13VHSX 14X 15X 16TYLE(SEQ ID NO：68)所示的LCDR1，其中X 13选自L，X 14选自N、Q、L、T和D，X 15选自G、A和V，X 16选自N。
  2. 一种抗PD-1抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述的重链可变区和轻链可变区组合选自如下(a)至(e)任一项：
    (a)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，和序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1；
    (b)所述重链可变区包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3；
    (c)所述重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；
    (d)所述重链可变区包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，以及序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1；和
    (e)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3；优选地，所述的抗PD-1抗体包含如下所示的重链可变区和轻链可变区组合：
    (a1)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：49、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3；或
    (a2)所述重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，且
    所述轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3。
  3. 一种抗PD-1抗体或其抗原结合片段，其包含选自如下iv)至vi)任一项所述的重链可变区和轻链可变区组合：
    iv)所述重链可变区包含与如SEQ ID NO：4序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含与如SEQ ID NO：5序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；
    v)所述重链可变区包含与如SEQ ID NO：6序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含与如SEQ ID NO：7序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3；和
    vi)所述重链可变区包含与如SEQ ID NO：19序列所示的重链可变区具有相同序列的HCDR1、HCDR2和HCDR3，且所述轻链可变区包含与如SEQ ID NO：20序列所示的轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3。
  4. 根据权利要求1至3任一项中所述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体或其抗原结合片段是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、全人抗体或其抗原结合片段，或人源化抗体或其抗原结合片段。
  5. 根据权利要求4所述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体为人源化抗体，所述人源化抗体包含源自人抗体的框架区或其框架区变体，其中：
    所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多11个氨基酸的回复突变；
    优选地，所述框架区变体包含选自以下(f)至(h)中任一所述的突变：
    (f)轻链可变区中包含2G氨基酸回复突变，和/或
    重链可变区中包含选自27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变；
    (g)轻链可变区中包含2V氨基酸回复突变，和/或
    重链可变区中包含选自26D、27F、30T、38K、43H、48I、66K、67A、69L、82F和93T中的一个或更多个氨基酸回复突变；和
    (h)轻链可变区中包含选自42G、44V和71Y中的一个或更多个氨基酸回复突变，和/或
    重链可变区中包含1K和/或94S氨基酸回复突变；其中所述的氨基酸位点依照kabat规则编号确定。
  6. 根据权利要求4所述的抗PD-1抗体或其抗原结合片段，所述的抗体重链可变区和轻链可变区组合选自如下(i)至(o)任一项所述：
    (i)重链可变区，其序列如SEQ ID NO：4所示或与SEQ ID NO：4具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：5所示或与SEQ ID NO：5具有至少90％序列同一性；
    (j)重链可变区，其序列如SEQ ID NO：6所示或与SEQ ID NO：6具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：7所示或与SEQ ID NO：7具有至少90％序列同一性；
    (k)重链可变区，其序列如SEQ ID NO：19所示或与SEQ ID NO：19具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：20所示或与SEQ ID NO：20具有至少90％序列同一性；
    (l)重链可变区，其序列如SEQ ID NO：27、30、31或32所示或与SEQ ID NO：27、30、31或32具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：28、29、34、35、53、54、55、56、57、58、59、60、61、62、63或64所示或与SEQ ID NO：28、29、34、35、53、54、55、56、57、58、59、60、61、62、63或64具有至少90％序列同一性；
    (m)重链可变区，其序列如SEQ ID NO：33、36、37、38、39或40所示或与SEQ ID NO：33、36、37、38、39或40具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：34、35、28、29、53、54、55、56、57、58、59、60、61、62、63或64所示或与SEQ ID NO：34、35、28、29、53、54、55、56、57、58、59、60、61、62、63或64具有至少90％序列同一性；
    (n)重链可变区，其序列如SEQ ID NO：41、45或46所示或与SEQ ID NO:41、 45或46具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：42、43或44所示或与SEQ ID NO：42、43或44具有至少90％序列同一性；和
    (o)重链可变区，其序列如SEQ ID NO：70所示或与SEQ ID NO：70具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：71所示或与SEQ ID NO：71具有至少90％序列同一性；优选地，所述的抗体重链可变区和轻链可变区组合选自：
    (p)重链可变区，其序列如SEQ ID NO：27所示或与SEQ ID NO：27具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：55所示或与SEQ ID NO：55具有至少90％序列同一性；或
    (q)重链可变区，其序列如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％序列同一性，和/或
    轻链可变区，其序列如SEQ ID NO：43所示或与SEQ ID NO：43具有至少90％序列同一性。
  7. 根据权利要求1至6任一项所述的抗PD-1抗体或其抗原结合片段，其中所述抗体进一步包含抗体恒定区；优选地，所述抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗体包含序列如SEQ ID NO：72或79所示的重链恒定区和序列如SEQ ID NO：73所示的轻链恒定区。
  8. 根据权利要求1至7任一项所述的抗PD-1抗体或其抗原结合片段，其中所述抗PD-1抗体包含：如SEQ ID NO：78所示的轻链和如SEQ ID NO：77或82所示的重链；或所述抗PD-1抗体包含如SEQ ID NO：75所示的轻链和如SEQ ID NO：74、76、80或81所示的重链；优选地，所述的抗PD-1抗体包含：
    序列如SEQ ID：74所示的重链和序列如SEQ ID：75所示的轻链；或
    序列如SEQ ID：77所示的重链和序列如SEQ ID：78所示的轻链。
  9. 根据权利要求1至8任一项所述的抗PD-1抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
  10. 根据权利要求1至9任一项所述的抗PD-1抗体或其抗原结合片段，其中所述抗体是双特异性抗体、多特异抗体或抗体融合蛋白。
  11. 一种分离的单克隆抗体或其抗原结合片段，所述抗体与权利要求1至10 任一项所述的抗PD-1抗体或其抗原结合片段竞争性结合人PD-1。
  12. 一种药物组合物，其含有：
    治疗有效量的权利要求1至10任一项所述的抗PD-1抗体或其抗原结合片段或治疗有效量的权利要求11所述分离的单克隆抗体或其抗原结合片段，以及一种或更多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  13. 一种核酸分子，其编码权利要求1至10任一项所述的抗PD-1抗体或其抗原结合片段，或编码权利要求11所述分离的单克隆抗体或其抗原结合片段。
  14. 一种宿主细胞，其包含如权利要求13所述的核酸分子。
  15. 一种用于免疫检测或测定PD-1的方法，所述方法包括使用权利要求1至10任一项的抗PD-1抗体或其抗原结合片段的步骤，或
    使用权利要求11所述分离的单克隆抗体或其抗原结合片段的步骤。
  16. 一种试剂盒，其包含根据权利要求1至10任一项所述的抗PD-1抗体或其抗原结合片段或权利要求11所述分离的单克隆抗体或其抗原结合片段。
  17. 一种治疗与PD-1相关的疾病的方法，所述方法包括向受试者施用治疗有效量的权利要求1至10任一项所述的抗PD-1抗体或其抗原结合片段，或权利要求11所述的分离的单克隆抗体或其抗原结合片段，或权利要求12所述的药物组合物，或权利要求13所述的核酸分子；其中所述疾病优选为肿瘤；更优选所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；最优选的，所述疾病选自：PD-L1阳性的黑色素瘤、肺癌、非小细胞肺 癌、乳腺癌、胃癌、肾癌、膀胱癌和肠癌和结肠癌。
PCT/CN2020/074098 2019-02-03 2020-01-31 抗pd-1抗体、其抗原结合片段及医药用途 WO2020156509A1 (zh)

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KR1020217025236A KR20210124993A (ko) 2019-02-03 2020-01-31 항-pd-1 항체, 이의 항원-결합 단편 및 이의 약학적 용도
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