WO2019096136A1 - Anti-pd-1 antibody, preparation method therefor and use thereof - Google Patents

Anti-pd-1 antibody, preparation method therefor and use thereof Download PDF

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WO2019096136A1
WO2019096136A1 PCT/CN2018/115285 CN2018115285W WO2019096136A1 WO 2019096136 A1 WO2019096136 A1 WO 2019096136A1 CN 2018115285 W CN2018115285 W CN 2018115285W WO 2019096136 A1 WO2019096136 A1 WO 2019096136A1
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antibody
seq
cancer
functional fragment
amino acid
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PCT/CN2018/115285
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Chinese (zh)
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高荣凯
韩化敏
金瑾
徐义
魏晓莉
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拜西欧斯（北京）生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates to the field of antigen suppression and antibodies, and in particular to the modulation of immune responses regulated by programmed death 1 (PD-1) receptors and the treatment of tumors by PD-1 antigen conjugates.
  • PD-1 programmed death 1
  • the adaptive immune response involves activation, selection, and clonal proliferation of two major classes of lymphocytes called T cells and B cells. After encountering the antigen, the T cells proliferate and differentiate into antigen-specific effector cells, while the B cells proliferate and differentiate into antibody secreting cells.
  • T cell activation is a multi-step process that requires several signaling events between T cells and antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • both types of signals must be delivered to resting T cells.
  • the first type is mediated by the antigen-specific T cell receptor (TCR) and confers an immune response specificity; the second type of signal is co-stimulatory, and the amount of response is delivered and regulated by the helper receptor on the T cell. level.
  • the primary costimulatory signal is delivered by binding of the activated costimulatory molecule CD28 to its ligand B7-1 or B7-2.
  • CTLA-4 cytotoxic T lymphocyte-associated antigen 4
  • PD-1 is an inhibitory receptor CTLA-4 analog.
  • PD-1 is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line that undergoes activation-induced apoptosis.
  • PD-1 is a member of the immunoglobulin (Ig) superfamily, contains a single IgV-like domain in its extracellular region, has four important N-linked glycosylation sites and is highly glycosylated, possibly in combination with Play an important role in body binding.
  • Ig immunoglobulin
  • the cytoplasmic domain of PD-1 contains two tyrosines, of which tyrosine close to the membrane (VAYEEL in mouse PD-1) is located within the immunoreceptor tyrosine-based inhibition motif (ITIM).
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • the presence of ITIM on PD-1 indicates that the molecule acts by recruiting cytosolic phosphatase to attenuate the signaling of antigen receptors.
  • ITIM-like motifs around the ITIM and carboxy terminal tyrosines (TEYATI in humans and mice) in the cytoplasmic region are conserved between human and murine orthologues.
  • PD-1 is expressed on the surface of activated T cells, B cells, macrophages, and monocytes.
  • the ligand for PD-1 is the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).
  • B7-H1 B7 family members
  • B7-DC B7-DC
  • Experimental data in the literature in the field suggests the interaction of PD-1 with its ligand in down-regulating central and peripheral immune responses.
  • PD-1 deficient mice exhibit an autoimmune phenotype, leading to chronic progressive lupus-like glomerulonephritis and arthritis, and severe cardiomyopathy due to the presence of specific autoreactive antibodies in cardiac tissue.
  • PD-1 is also an important immunological checkpoint for modulating immune responses, and there is a need in the art to develop new modulators of PD-1 activity, thereby activating the immune system.
  • Such activity modulators can be used, for example, in the treatment of cancer immunotherapy and other conditions, such as chronic infections.
  • Therapeutic blockade of the PD-1 pathway will help overcome immune tolerance and aid in the treatment of cancer or infection and enhance immunity during vaccination (prophylactic or therapeutic).
  • the application provides a combination comprising a novel variable region, eg, a light chain variable region (eg, VL1, VL2, VL3, VL4, VL5, and VL6) and/or a heavy chain variable region (eg, VH1, VH2) Anti-PD-1 antibody molecules of VH3, VH4 and VH5).
  • a novel variable region eg, a light chain variable region (eg, VL1, VL2, VL3, VL4, VL5, and VL6) and/or a heavy chain variable region (eg, VH1, VH2) Anti-PD-1 antibody molecules of VH3, VH4 and VH5).
  • the present application also provides a nucleic acid molecule encoding an anti-PD-1 antibody molecule, an expression vector comprising the nucleic acid molecule, a host cell comprising the expression vector, and a method of producing a PD-1 antibody using a phage library. Immunoconjugates, dual or multi-specific antibody molecules, and pharmaceutical compositions comprising antibody molecules
  • the anti-PD-1 antibody molecules disclosed herein can treat, prevent, and/or diagnose diseases, such as cancer (eg, solid and soft tissue tumors), as well as infectious diseases, for example, chronic infectivity, either alone or in combination with other active agents or therapeutic forms. Disease or sepsis). Accordingly, disclosed herein are the use of anti-PD-1 antibody molecules or antigen-binding fragments, conjugates, conjugates, derivatives, compositions thereof, for the treatment of diseases, such as cancer diseases (eg, solid and soft tissue tumors), and infectious diseases A method (for example, a chronic infectious disease or sepsis). In addition, methods for detecting PD-1 using an anti-PD-1 antibody molecule and detection compositions are also provided.
  • the present application screens a plurality of specific light chains of anti-PD-1 antibodies by human phage antibody library screening method (L1, L2, L3, L4, L5 or L6, respectively, each comprising 3 LCDRs, wherein LCDR1 comprises SEQ NO.: any one of 1 to 5, LCDR2 comprising any one of SEQ NO.: 6 to 10, LCRD3 comprising any one of SEQ NO. 11 to 16) and specificity of a plurality of anti-PD-1 antibodies a strand (H1, H2, H3, H4 or H5, respectively, each comprising 3 HCDRs, wherein HCDR1 comprises any one of SEQ NO. 17-21, and HCDR2 comprises any one of SEQ NO. 22-26, HCRD3 comprises Any one of SEQ NO. 27 to 31), and further obtain various anti-PD-1 antibodies having high PD-1 binding activity.
  • human phage antibody library screening method L1, L2, L3, L4, L5 or L6, respectively, each comprising 3 LCDRs
  • LCDR1 comprises SEQ NO.: any one
  • the above antibodies are fully human or murine antibodies which (i) have a high affinity, for example, at least about 10 7 M -1 , usually about 10 8 M -1 , and more commonly, about 10 9 M -1 to 10 10 M -1 affinity constant, or stronger affinity binds PD-1; (ii) binds PD-1 with high specificity, while not substantially binding to CD28, CTLA-4, inducible T cell costimulator ( ICOS) or B cell and T cell attenuator (BTLA); (iii) inhibit or reduce binding of PD-1 to a PD-1 ligand (eg, PD-L1 or PD-L2, or both).
  • a PD-1 ligand eg, PD-L1 or PD-L2, or both.
  • the humanized high-affinity PD-1 antibody developed in the present application is of great significance for the clinical treatment of tumors, infectious diseases and immune-responsive diseases.
  • WHAT IS CLAIMED IS 1. An isolated antibody or functional fragment thereof, comprising a domain that specifically recognizes and binds to the immune cell surface antigen PD-1 and a constant region from an immunoglobulin constant region (Fc) that specifically recognizes and binds
  • the domain of the immunocyte surface antigen PD-1 includes a light chain variable region (anti-PD-1 VL) having three CDRs and a heavy chain variable region (anti-PD-1 VH) having three CDRs, wherein the light
  • the chain variable region (anti-PD-1 VL) comprises a light chain CDR (LCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 1-16; and the heavy chain variable region (anti-PD-1 VH) comprises a member selected from the group consisting of The heavy chain CDRs (HCDRs) of the amino acid sequences set forth in SEQ ID NOS: 17-31.
  • LCDR1 the amino acid sequence of which is set forth in any one of SEQ ID Nos: 1, 2, 3, 4 and 5,
  • LCDR2 the amino acid sequence of which is set forth in any one of SEQ ID Nos: 6, 7, 8, 9 and 10, and
  • LCDR3 the amino acid sequence of which is set forth in any one of SEQ ID Nos: 11, 12, 13, 14, 15 and 16;
  • the heavy chain variable region comprises:
  • HCDR1 the amino acid sequence of which is set forth in any one of SEQ ID NOs: 17, 18, 19, 20 and 21,
  • HCDR2 the amino acid sequence of which is set forth in any one of the SEQ ID NOs: 22, 23, 24, 25 and 26, and
  • HCDR3 the amino acid sequence of which is set forth in any one of SEQ ID NOs: 27, 28, 29, 30 and 31.
  • VL1 a light chain variable region VL1 comprising SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13;
  • VL2 a light chain variable region VL2 comprising SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 12;
  • VL3 a light chain variable region VL3 comprising SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 16;
  • VL4 a light chain variable region VL4 comprising SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11;
  • VL5 a light chain variable region VL5 comprising SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:13;
  • VL6 Light chain variable region VL6 comprising SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 14.
  • VH1 a heavy chain variable region VH1 comprising SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27;
  • VH2 a heavy chain variable region VH2 comprising SEQ ID NO: 18, SEQ ID NO: 23 and SEQ ID NO: 30;
  • VH3 a heavy chain variable region VH3 comprising SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 29;
  • VH4 a heavy chain variable region VH4 comprising SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 30;
  • Heavy chain variable region VH5 comprising SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 31.
  • the antibody or functional fragment thereof according to embodiment 1 or 2 comprising a light chain variable region selected from any one of VL1, VL2 and VL3, VL4, VL5 and VL6 and selected from the group consisting of VH1, VH2, VH3 a heavy chain variable region of any of VH4 and VH5.
  • the antibody or functional fragment thereof specifically binds to an epitope located within the extracellular domain of human PD-1.
  • An isolated antibody or a functional fragment thereof which is identical to an epitope that binds to the antibody or functional fragment thereof according to any one of embodiments 1-8, or The epitope to which the antibody or its functional fragment binds overlaps with an epitope that binds to the antibody or functional fragment thereof according to any of embodiments 1-8.
  • the constant region is a human constant region, such as a constant region of human IgA, IgD, IgE, IgG or IgM, preferably human IgG
  • the constant region is more preferably a constant region of human IgG1 or IgG4.
  • the antigen binding portion is selected from the group consisting of Fab, F(ab')2, Fv, scFv, Fd or dAb.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • nucleic acid sequence of embodiment 22 comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 54-58.
  • An expression vector comprising the nucleic acid of embodiment 22.
  • a host cell comprising the expression vector of embodiment 24, wherein the host cell is selected from the group consisting of E. coli, HEK293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and NSO cells.
  • An immunoconjugate or a derivative thereof comprising the antibody according to any one of embodiments 1 to 13 or a functional fragment thereof, which is a toxin, a radioisotope, Drug or cytotoxic agent.
  • a pharmaceutical composition comprising the antibody of any one of embodiments 1-21, or a functional fragment thereof, or the immunoconjugate of embodiment 25, or a derivative thereof, and a pharmaceutically acceptable excipient, Carrier or diluent.
  • the antibody of any one of embodiments 1 to 21, or a functional fragment thereof, or the immunoconjugate of embodiment 26, or a derivative thereof, or the pharmaceutical composition of embodiment 27, prepared for use in therapy or Use in a medicament for preventing a disorder selected from the group consisting of an autoimmune disorder, an immune response against a transplant, an allergy, and a cancer.
  • a method of treating a condition associated with PD-1 or a PD-1 mediated disease or condition in a subject comprising administering to the subject a therapeutically effective amount according to embodiment 1-21.
  • a conjugate comprising the antibody of any one of embodiments 1 to 21, or a functional fragment thereof, and a coupling moiety, wherein the coupling moiety is a detectable label; the coupling moiety is radioactive Isotopes, fluorescent substances, luminescent substances, colored substances or enzymes.
  • kits comprising the antibody of any one of embodiments 1 to 21, or a functional fragment thereof, or the conjugate of embodiment 32, further comprising a second antibody,
  • the second antibody specifically recognizes the antibody or a functional fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.
  • a method of enhancing T cell activation comprising contacting a T cell with an antibody or a functional fragment thereof according to any one of embodiments 1-21.
  • a method of reducing tumor growth in a subject or inhibiting tumor cell growth in a subject comprising administering to the subject a therapeutically effective amount of any of embodiments 1-21, according to any one of embodiments 1-21.
  • Antibody or functional fragment thereof comprising administering to the subject a therapeutically effective amount of any of embodiments 1-21, according to any one of embodiments 1-21.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibody or functional fragment thereof according to any one of embodiments 1-21 .
  • a method of treating a condition of a subject that would benefit from an up-regulated immune response comprising administering to the subject a therapeutically effective amount of the antibody of any of embodiments 1-21 or Its functional fragment.
  • the cancer is selected from the group consisting of gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, esophageal cancer, small intestine cancer, thyroid cancer, Parathyroid carcinoma, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer , rectal cancer, adrenal cancer, anal cancer, vulvar cancer, urethral cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non-Hodkin Lymphoma, endocrine system cancer, soft tissue sarcoma, central nervous system neoplasm, primary central nervous system lymphoma, tumor angiogenesis, spinal cord tumor, brains
  • infectious disease is selected from the group consisting of HIV, influenza, herpes, giardiasis, malaria, leishmaniasis, or an infection caused by a virus: hepatitis virus (eg hepatitis A, B or C), herpes virus (eg VZV, HSV-1, HAV-6, HSV-II, CMV or Epstein's virus), adenovirus, influenza virus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, round Virus measles virus, rubella virus, parvovirus, JC virus and arbovirus viral encephalitis virus, or infections caused by bacteria: pneumococcal, mycobacteria, staphylococcus, streptococcus, or an infection caused by a virus:
  • the disease is cancer, the cancer comprising lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma , kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer, intestinal cancer, melanoma, non-small cell lung cancer;
  • the infectious disease is a chronic viral infection, a bacterial infection or a parasitic infection,
  • the chronic virus is HIV, HBV or HCV.
  • a method of detecting the presence of PD-1 in a sample comprising the antibody of any one of embodiments 1-21, or a functional fragment thereof, or the immunoconjugate of claim 26. Or a derivative thereof or the conjugate of claim 32 is contacted with the sample.
  • composition for detecting the presence of PD-1 in a sample comprising the antibody of any of embodiments 1-21 or a functional fragment thereof.
  • Figure 1 is an electropherogram of the VH gene and VL gene of the amplified native human antibody.
  • Figure 2 is a DNA electropherogram of native human scFv.
  • Figure 3 is a diagram of the pCANTAB-5E vector after insertion of the native human scFv gene.
  • Figure 4 is an electropherogram of the VH and VL genes that amplify ⁇ PD-1 scFv.
  • Figure 5 is a DNA electrophoresis pattern of ⁇ PD-1 scFv.
  • Figure 6 is a ELISA result of phage library technology screening for monoclonal candidate phage comprising BXOS anti-PD-1 scFv.
  • Figure 7 shows the results of an experiment in which a monoclonal candidate phage comprising BXOS anti-PD-1 scFv blocked PD-1/PD-L1 binding.
  • Figure 8 is a map of the vector pBudCE4.1 for BXOS anti-PD-1 full antibody expression.
  • Figure 9 is a graph showing the results of affinity determination of BXOS anti-PD-1 whole antibody and PD-1 antigen.
  • 10A-10C are images of mature DC cell surface, DC cell purity detected by flow cytometry, and expression of PD-L1 on the surface of DC cells, respectively.
  • 11A-11C are images of PD-1 expression detected on the surface of T cells by flow cytometry.
  • 12A-12B are images of T cell apoptosis detected by flow cytometry.
  • Figure 13 shows the blocking effect of BXOS anti-PD-1 whole antibody on binding of PD-L1/293T-PD-1 cells.
  • Figure 14 shows the effect of BXOS anti-PD-1 whole antibody on PBMC killing HepG2-luc tumor cells.
  • Example 1 Establishment of a natural human phage antibody library
  • the primers used and their sequences are shown in Table 1, and the underlined portions are restriction endonuclease sites.
  • lymphocyte separation solution purchased from Tianjin Haoyang Biological Co., Ltd.
  • the lymphocytes were separated, centrifuged at 800 g for 15 minutes in a horizontal centrifuge, and the lymphocyte layer was pipetted into a new centrifuge tube with a pipette. After washing once with PBS buffer, the lymphocytes were resuspended in PBS, and the cell count was determined. The number of lymphocytes obtained per peripheral blood is approximately 5 ⁇ 10 7 .
  • Total RNA was extracted from the obtained lymphocytes using TRIzol Reagent (Invitrogen, 15596026).
  • VH human antibody heavy chain variable region
  • VL light chain variable region
  • V ⁇ Kappa
  • V ⁇ Lambda
  • the VH and VL genes are spliced by merging a portion of the DNA encoding the linker sequence (Gly 4 Ser) 3 as an overlapping complementary portion to form a single-chain variable region gene fragment (scFv).
  • the forward primer of VL introduces a linker sequence complementary to the heavy chain reverse primer, and the reverse primer introduces a Not I restriction site.
  • the PCR product was identified by 2.0% DNA agarose gel electrophoresis. The results showed that the amplified VH gene was about 390 bp in length and the VL gene was about 350 bp in length (Fig. 1).
  • the gene band of interest was recovered using QIAquick Glue Recovery Kit (QIAGEN, 28706).
  • VH and VL gene fragments obtained by the amplification of the respective subtypes of the human antibody germline gene were mixed, and ligated into the scFv gene library by overlap extension PCR using the added linker sequences in the VH and VL gene fragments (Fig. 2).
  • the scFv gene obtained as above was inserted into the phage vector pCANTAB-5E.
  • the scFv and pCANTAB-5E were digested with restriction endonuclease Sfi I (NEB, R0123S) and Not I (NEB, R3198L) respectively.
  • Sfi I restriction endonuclease
  • Not I NEB, R3198L
  • the scFv gene was digested with the digested pCANTAB-5E.
  • the vector was ligated using T4 DNA ligase (NEB, M0202L) (Fig. 3). The connection was carried out in batches.
  • the prepared phage was precipitated with PEG8000/NaCl to be concentrated and purified, and resuspended in a 1% initial volume of 15% glycerol solution to obtain a natural human scFv phage antibody library, and 1 ⁇ L was used for titer determination, and the remaining -80 °C save.
  • the natural human scFv phage antibody library was obtained by transformation of 10 batches of the ligation product with a library capacity of 1 ⁇ 10 9 and a titer of 1 ⁇ 10 12 .
  • the obtained phage antibody library was infected with the XL1-BLUE activating strain coated plate at 37 ° C overnight, and 20 colonies were randomly picked and cultured, and the plasmid was extracted to determine the gene sequence, and the sequencing results were subjected to Blast and comparative analysis. It was found that all 20 gene sequences belonged to the antibody sequence, but they were different, indicating that the phage antibody library diversity is very good.
  • the immunotube was coated with 1 ⁇ L of human PD-1 recombinant protein (Acro Biosystems, PD-1-H5221) at 10 ⁇ g/mL, the antigen coating amount was 10 ⁇ g/1 mL/tube, and coated at 4 ° C overnight; after washing with PBS for 1 time
  • the immune tube was blocked with 4 mL of 3% skim milk powder/PBS (MPBS) at room temperature for 3 h; 1 mL of the natural human phage antibody library prepared in Example 1 was added, and the input amount was 10 9 -10 12 /tube, and incubated for 3 h at room temperature.
  • MPBS skim milk powder/PBS
  • the cells were washed with PBST-PBS for 20 times, eluted with 0.1 M PH2.2 of Glycine-HCl, and the eluted phage solution was neutralized with 1.0 M PH8.8 Tris-HCl to pH 7.0 or so for infection of 1 mL XL1-BLUE.
  • the activated bacteria were allowed to stand in a 37 ° C incubator for 20 min, supplemented with 2YTA medium to 20 mL, and 10 ⁇ L of the bacterial solution was applied to 2YTA plates for counting the phage antibody production; the remaining bacterial solution was shake cultured at 37 ° C for 1 h, using 2YTA The culture solution was added to 100 mL, and after shaking to log phase, M13KO7 helper phage was added, and the mixture was incubated at 37 ° C, 220 rpm for 1 h. 50 ⁇ g/mL kanamycin was added and incubated overnight at 30 °C.
  • the VH and VL genes were chemically synthesized according to the gene sequence of the marketed anti-PD-1 mAb Nivolumab.
  • the VH/VL gene amplification primer of Example 1 was used for PCR amplification, and the product was identified by 2.0% DNA agarose gel electrophoresis, and the VH gene length was about 390 bp, and the VL gene length was about 350 bp (Fig. 4).
  • the gene band of interest was recovered using the QIAquick Glue Recovery Kit.
  • the linker overlapping complementary sequences of the VH and VL genes were ligated into an anti-PD-1 scFv positive control gene by PCR reaction, designated as ⁇ PD-1 scFv (Fig. 5).
  • the ⁇ PD-1scFv positive control gene was inserted into the phage vector pCANTAB-5E, and the ⁇ PD-1 scFv gene and the pCANTAB-5E vector were digested with restriction endonucleases SfiI and NotI, respectively. After purification, the ⁇ PD-1 scFv gene was digested. The product was ligated with the digested pCANTAB-5E vector using T4 DNA ligase. The cells were transformed into XL1-BLUE competent cells by electroporation, and appropriate transformants were uniformly coated on 2YTAG plates containing 100 ⁇ g/mL ampicillin, and cultured at 37 ° C overnight.
  • the helper phage M13KO7 was added, cultured at 37 ° C for 1 h, and then 50 ⁇ g/mL kanamycin was added, and cultured at 30 ° C overnight.
  • the supernatant was centrifuged the next day to obtain the ⁇ PD-1 scFv antibody positive control phage, and 1 ⁇ L of the supernatant was taken for titer measurement, and the titer was about 1 ⁇ 10 12 .
  • the phage obtained by the three-step panning in the step 1 was infected with the XL1-BLUE strain in the logarithmic growth phase at room temperature for 20 min, and the plate was incubated overnight at 37 ° C with a 2YTA plate. Single colony enrichment culture was picked, and M13KO7 helper phage was added after the logarithmic growth phase, and cultured overnight at 30 ° C with shaking, and the supernatant was centrifuged to obtain a phage antibody.
  • the BXOS anti-PD-1 phage candidate monoclonal 16 was screened by the biotin-labeled PD-1:PD-L1 inhibitor screening kit (Acro Biosystems, EP-101) for PD-1/. Blocking effect of PD-L1 binding. Referring to the product manual, briefly, the ELISA plate was coated with 100 ⁇ L/well 2 ⁇ g/mL of PD-L1 at 4 ° C overnight; and 200 ⁇ L/well of 3% MPBS was blocked at 37 ° C for 3 h.
  • Positive control ⁇ PD-1 monoclonal antibody Anti-PD1 mAb, Human (IgG4) Lot No. B52-63NS1-AS, ACRO Biosystems
  • VH and VL (V ⁇ ) genes of the monoclonal candidate phage 16 containing BXOS anti-PD-1 scFv were fused with the human antibody heavy chain constant region (C ⁇ 4) gene and the light chain constant region (C ⁇ 1) gene, respectively, to form a complete heavy chain.
  • light chain genes the nucleotide sequence of the BXOS anti-PD-1 full antibody encoding the HindIII and XbaI cleavage sites at both ends of the heavy chain gene and the NotI and XhoI cleavage sites at both ends of the light chain gene were obtained by PCR.
  • the coding nucleotide sequence of the whole antibody was inserted into the expression vector pBUdCE4.1 (Fig. 8).
  • the constructed expression vector was introduced into HEK293T/17 cells by electroporation, and whole antibody expression was carried out to obtain BXOS anti-PD-1 whole antibody of No. 16.
  • the light chain amino acid sequence of BXOS anti-PD-1 full antibody is shown in SEQ. ID. NO. 64, the nucleotide sequence is shown in SEQ. ID. NO. 66, and the amino acid sequence of LCDR1 is SEQ ID NO: 3, LCDR2 amino acid.
  • the sequence is as SEQ ID NO: 7, the LCDR3 amino acid sequence is SEQ ID NO: 13; the heavy chain amino acid sequence is shown in SEQ. ID. NO. 65, the nucleotide sequence is shown in SEQ ID NO. 67, and the HCDR1 amino acid sequence is as SEQ ID NO. 21, HCDR2 amino acid sequence is SEQ ID NO: 26, and HCDR3 amino acid sequence is SEQ ID NO: 31.
  • Example 5 Determination of the affinity of BXOS anti-PD-1 whole antibody of No. 16 obtained in Example 4 by plasma resonance method
  • Buffer PBST (0.05% Tween-20, 5% DMSO)
  • Regeneration fluid 10mM Gly PH2.0
  • PD-1-PE Commercially available PD-1 antigen
  • PD-1-PE Commercially available PD-1 antigen
  • PH4.5 10 mM sodium acetate commercially available PD-1 antigen
  • the positive control anti-PD-1 antibody (China/T&L, TL-106, Beijing Tongli Haiyuan Biotechnology Co., Ltd.) was diluted with 0.05% PBST buffer to prepare three concentrations of 100 nM, 30 nM and 10 nM, both at a flow rate of 25 ⁇ l/ The min injection was combined for 2 min and dissociated for 3 min. It was observed that all three concentrations reflected the value and the corresponding value of 100 nM was close to saturation, thus determining that the fixed amount of PD-1 antigen 50 RU could meet the test requirements.
  • Control antibody injection The control antibody was diluted with 0.05% PBST buffer and formulated into a concentration gradient of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, and injected at a flow rate of 25 ⁇ l/min for 2 min. Dissociate for 3 min.
  • the regeneration condition of the sensor chip was PH2.010 mM Gly solution, the flow rate was 30 ⁇ l/min, and the injection time was 30 s.
  • BXOS anti-PD-1 whole antibody HEK293T expression purified anti-PD-1 mAb (molecular weight about 150KD) injection: the same method.
  • PBMC Peripheral blood mononuclear cells
  • IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were added daily on days 1, 3, and 5 to maintain an IL-4 concentration of 10 ng/mL and a GM-CSF concentration of 20 ng/mL. .
  • the mature factor TNF- ⁇ (final concentration 10 ng/mL) was added on the 7th day.
  • LPS lipopolysaccharide
  • DC cells are the most important antigen presenting cells (APCs) in the body, and mature DC cells have many dendrites on the surface (see Figure 10A). Mature DC cells express CD80 and CD86 molecules, and the purity and maturity of DC cells can be calculated by flow cytometry to detect CD80 and CD86 molecules.
  • Figure 10B shows that the proportion of CD11c+CD86+ cells is 97.32%, indicating that the purity of cultured mature DC cells is 97.32%.
  • Figure 10C shows the expression of PD-L1 molecules on the surface of DC cells using PE anti-human CD274 (B7-H1, PD-L1) (KeMinbio, SZB12619), showing that all DC cells highly express PD-L1 molecules.
  • the mixed culture MNCs were divided into two groups, and one group was added with BXOS anti-PD-1 (5 ⁇ g/mL) obtained by the third step of Example 2, and one group was added with PBS.
  • the cultured T cells were resuspended in cold PBS, and the cell density was adjusted to 1 ⁇ 10 6 /mL.
  • Fig. 11A shows that the expression of PD-1 on the surface of T cells was 26.21% after 3 days of culture of MNC alone;
  • Fig. 11B shows that when MNC was mixed with DC, the expression of PD-1 was 32.65% (Fig. B) because DC cells The surface highly expressed PD-L1 and promoted the expression of PD-1 molecules on the surface of T cells;
  • Fig. 11C showed that MNC was mixed with DC and simultaneously added BXOS-anti-PD-1 whole antibody, and the expression of PD-1 was reduced to 0.25%.
  • FIGS 11A-11C illustrate that in the presence of the BXOS anti-PD-1 whole antibody of the present application, the commercial anti-PD-1 flow antibody PD-1-PE (biolegend, 329906) cannot be associated with T cell surface The PD-1 molecule binds, indicating that the BXOS anti-PD-1 whole antibody of the present application has bound to the PD-1 molecule on the T cell surface.
  • Example 7 BXOS anti-PD-1 antibody reduces PD-L1-induced T cell apoptosis on DC cell surface
  • CD3-APC article number: 300312, manufacturer: biolegend
  • Apoptosis Kit ANNEXIN V-FITC/PI Apoptosis Detection Kit (Solebao Biotechnology Co., Ltd., CA1020-50T)
  • the cells were resuspended in 1 mL of 1 ⁇ binding buffer to a cell density of 1 ⁇ 10 6 /mL.
  • the 293T-PD-1 cell line is a cell line constructed by the applicant to stably express the PD-1 antigen.
  • Example 9 The effect of 16X BXOS anti-PD-1 whole antibody on PBMC killing HepG2-luc tumor cells
  • lymphocyte separation solution Ficoll separation of human peripheral blood mononuclear cells PBMC
  • Cell count Count PBMC, adjust the concentration to 10 7 /mL; count HepG2-luc cells, adjust the concentration to 2 ⁇ 10 5 /mL;
  • HepG2-luc cells were added to a round-bottom 96-well plate at a concentration of 50 ⁇ L/well, and 50 ⁇ L of a 20 nM BXOS anti-PD1 whole antibody sample (final concentration 10 nM) was also added in groups according to the experiment, for a total of three replicate wells;
  • steps 3 and 4 were each allowed to stand in a 37 ° C incubator for 10 min;
  • the 4-step HepG2-luc cells were transferred to the 3-step PBMC, mixed uniformly, and the killing wells with a target ratio of 10:1 were prepared, and the final volume per well was 200 ⁇ L, and cultured in a 37 ° C incubator;
  • the anti-PD-1 antibody or derivative thereof provided by the embodiments of the present application is capable of effectively regulating an immune response in a subject, treating or preventing a PD-1-associated condition or a PD-1 mediated disease in a subject or
  • the disease is suitable for the development and industrial production of new drugs.

Abstract

An anti-PD-1 antibody molecule comprising a variable region combination, a nucleic acid molecule encoding the antibody molecule, a corresponding expression vector, a host cell, a method for screening the antibody using a phage library, and an immunoconjugate, a bis- or poly-specific antibody molecule and a pharmaceutical composition comprising the antibody molecule. The antibody molecule or a coupled compound, a conjugate, a derivative or a composition thereof is used for treating cancer diseases and infectious diseases. The antibody molecule is used to detect PD-1.

Description

抗PD-1抗体及其制备方法和应用Anti-PD-1 antibody and preparation method and application thereof 技术领域Technical field
本申请涉及抗原抑制和抗体领域，特别地涉及调节受程序性死亡1(PD-1)受体调控的免疫应答以及通过PD-1抗原结合物***。The present application relates to the field of antigen suppression and antibodies, and in particular to the modulation of immune responses regulated by programmed death 1 (PD-1) receptors and the treatment of tumors by PD-1 antigen conjugates.
背景background
适应性免疫应答涉及称为T细胞和B细胞的两大类淋巴细胞的激活、选择和克隆增殖。在遭遇抗原之后，T细胞增殖并分化为抗原特异性效应细胞，而B细胞增殖并分化为抗体分泌性细胞。The adaptive immune response involves activation, selection, and clonal proliferation of two major classes of lymphocytes called T cells and B cells. After encountering the antigen, the T cells proliferate and differentiate into antigen-specific effector cells, while the B cells proliferate and differentiate into antibody secreting cells.
T细胞激活是需要T细胞和抗原呈递细胞(APC)之间的数个信号传导事件的多步过程。为使T细胞激活，必须将两类信号递送给静息T细胞。第一类是由抗原特异性T细胞受体(TCR)介导的，并赋予免疫应答特异性；第二类信号是共刺激型的，通过T细胞上的辅助受体递送并调控应答的量级。初级共刺激信号是通过活化的共刺激分子CD28与其配体B7-1或B7-2的结合而递送的。相反，抑制性受体如细胞毒T淋巴细胞相关抗原4(CTLA-4)与相同配体B7-1或B7-2的结合却导致T细胞应答的削弱。可见，CTLA-4信号拮抗由CD28所介导的共刺激信号通路。在高抗原浓度下，CD28共刺激强于CTLA-4的抑制效应。CD28和CTLA-4表达的时序调节维持了激活和抑制信号之间的平衡，并确保了有效免疫应答的形成，同时预防了自身免疫性的发展。T cell activation is a multi-step process that requires several signaling events between T cells and antigen presenting cells (APCs). In order for T cells to activate, both types of signals must be delivered to resting T cells. The first type is mediated by the antigen-specific T cell receptor (TCR) and confers an immune response specificity; the second type of signal is co-stimulatory, and the amount of response is delivered and regulated by the helper receptor on the T cell. level. The primary costimulatory signal is delivered by binding of the activated costimulatory molecule CD28 to its ligand B7-1 or B7-2. Conversely, binding of an inhibitory receptor such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) to the same ligand, B7-1 or B7-2, results in a weakening of the T cell response. It can be seen that CTLA-4 signaling antagonizes the costimulatory signaling pathway mediated by CD28. At high antigen concentrations, CD28 co-stimulation is stronger than the inhibitory effect of CTLA-4. Timing regulation of CD28 and CTLA-4 expression maintains a balance between activation and suppression signals and ensures the formation of an effective immune response while preventing the development of autoimmunity.
本领域已鉴定了CD28和CTLA-4及其B-7样配体的分子同系物。PD-1为抑制性受体CTLA-4相似物。PD-1为50-55kDa的I型跨膜受体，其最初是在经历激活诱导的细胞凋亡的T细胞系中鉴定到的。PD-1是免疫球蛋白(Ig)超家族成员，在其胞外区含有单个IgV-样结构域，有4个重要的N连接糖基化位点并被高度糖基化，可能在与配体结合中发挥重要作用。PD-1的胞浆结构域含有两个酪氨酸，其中接近膜的酪氨酸(小鼠PD-1中的VAYEEL)位于基于免疫受体酪氨酸的抑制基序(ITIM)之内。PD-1上ITIM的存在预示 着该分子通过募集胞浆磷酸酶发挥作用以削弱抗原受体的信号传导。胞浆区中的ITIM以及羧基末端酪氨酸(人和小鼠中的TEYATI)周围的ITIM-样基序在人和鼠直系同源物(orthologue)之间是保守的。Molecular homologs of CD28 and CTLA-4 and their B-7-like ligands have been identified in the art. PD-1 is an inhibitory receptor CTLA-4 analog. PD-1 is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line that undergoes activation-induced apoptosis. PD-1 is a member of the immunoglobulin (Ig) superfamily, contains a single IgV-like domain in its extracellular region, has four important N-linked glycosylation sites and is highly glycosylated, possibly in combination with Play an important role in body binding. The cytoplasmic domain of PD-1 contains two tyrosines, of which tyrosine close to the membrane (VAYEEL in mouse PD-1) is located within the immunoreceptor tyrosine-based inhibition motif (ITIM). The presence of ITIM on PD-1 indicates that the molecule acts by recruiting cytosolic phosphatase to attenuate the signaling of antigen receptors. ITIM-like motifs around the ITIM and carboxy terminal tyrosines (TEYATI in humans and mice) in the cytoplasmic region are conserved between human and murine orthologues.
PD-1表达于活化的T细胞、B细胞、巨噬细胞和单核细胞表面。PD-1的配体为B7家族成员PD-L1(B7-H1)和PD-L2(B7-DC)。本领域文献中的实验数据暗示了PD-1与其配体在下调中枢和外周免疫应答中的相互作用。PD-1缺陷型小鼠表现自身免疫表型，导致慢性进行性狼疮样肾小球肾炎和关节炎，且由于心组织中特异性自身反应性抗体的存在而引起严重的心肌病。PD-1 is expressed on the surface of activated T cells, B cells, macrophages, and monocytes. The ligand for PD-1 is the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC). Experimental data in the literature in the field suggests the interaction of PD-1 with its ligand in down-regulating central and peripheral immune responses. PD-1 deficient mice exhibit an autoimmune phenotype, leading to chronic progressive lupus-like glomerulonephritis and arthritis, and severe cardiomyopathy due to the presence of specific autoreactive antibodies in cardiac tissue.
PD-1还是调节免疫应答的重要的免疫检查点，本领域需要研发新的PD-1活性的调节剂，由此激活免疫***。这样的活性调节剂可以用于例如癌症免疫治疗和其他病症的治疗，例如慢性感染。PD-1途径的治疗性阻断将有助于克服免疫耐受性且有助于癌症或感染的治疗并加强疫苗接种(预防性的或治疗性的)期间的免疫性。PD-1 is also an important immunological checkpoint for modulating immune responses, and there is a need in the art to develop new modulators of PD-1 activity, thereby activating the immune system. Such activity modulators can be used, for example, in the treatment of cancer immunotherapy and other conditions, such as chronic infections. Therapeutic blockade of the PD-1 pathway will help overcome immune tolerance and aid in the treatment of cancer or infection and enhance immunity during vaccination (prophylactic or therapeutic).
因此，本领域存在对用于治疗免疫紊乱的安全且有效的药物和方法的需要，所述免疫紊乱例如自身免疫病、炎性疾病、***反应、移植物排斥、癌症、免疫缺陷症以及其它免疫***相关紊乱。对这些紊乱中所涉及的免疫应答的调节可通过操纵PD-1通路而实现。Thus, there is a need in the art for safe and effective drugs and methods for treating immune disorders such as autoimmune diseases, inflammatory diseases, allergies, graft rejection, cancer, immunodeficiency, and other immunity. System related disorder. Modulation of the immune response involved in these disorders can be achieved by manipulating the PD-1 pathway.
发明概述Summary of invention
目前，尚需要开发新的具有更高的结合效率的抗PD-1抗体，以有效地阻断PD-1与PD-L1的结合。因此，研发新的抗PD-1抗体使其临床药效更佳以用于癌症的免疫治疗将给患者提供更多的药物选择。Currently, there is a need to develop new anti-PD-1 antibodies with higher binding efficiency to effectively block the binding of PD-1 to PD-L1. Therefore, the development of new anti-PD-1 antibodies to make them more clinically effective for the immunotherapy of cancer will provide patients with more drug choices.
本申请的发明人经过深入的研究和创造性的劳动，通过对大量样本的噬菌体抗体库进行筛选，获得了全人源化的具有高亲和力的抗PD-1抗体。该抗体既可以以高亲和力靶向结合人的PD-1蛋白而作为具有临床应用前景的抗肿瘤抗体将被进一步开发为抗肿瘤药物，同时又能够在ELISA、Western blot、免疫组化等应用中用作检测PD-1的抗体，大大节约科研成本和时间。The inventors of the present application obtained a fully humanized high-affinity anti-PD-1 antibody by screening a large sample of phage antibody libraries through intensive research and creative labor. The antibody can be targeted to bind human PD-1 protein with high affinity as an anti-tumor antibody with clinical application prospects, and will be further developed into an anti-tumor drug, and can be used in ELISA, Western blot, immunohistochemistry and the like. Used as an antibody to detect PD-1, saving scientific research costs and time.
本申请提供了包括新的可变区的组合，例如，轻链可变区(例如，VL1、VL2、VL3、VL4、VL5和VL6)和/或重链可变区(例如，VH1、VH2、VH3、 VH4和VH5)的抗PD-1抗体分子。本申请还提供了编码抗PD-1抗体分子的核酸分子、包含该核酸分子的表达载体、包含该表达载体的宿主细胞和使用噬菌体库制备PD-1抗体的方法。还提供了包括抗体分子的免疫缀合物、双或多-特异性抗体分子和药物组合物。本文公开的抗PD-1抗体分子可以单独地或以结合其他活性剂或治疗形式治疗、预防和/或诊断疾病，如癌症(例如，实体和软组织肿瘤)，以及传染病(例如，慢性感染性疾病或脓毒症)。相应地，本文公开了使用抗PD-1抗体分子或其抗原结合片段、偶联物、缀合物、衍生物、组合物治疗疾病，如癌症疾病(例如，实体和软组织肿瘤)，以及传染病(例如，慢性感染性疾病或脓毒症)的方法。此外，还提供了使用抗PD-1抗体分子检测PD-1的方法以及检测组合物。The application provides a combination comprising a novel variable region, eg, a light chain variable region (eg, VL1, VL2, VL3, VL4, VL5, and VL6) and/or a heavy chain variable region (eg, VH1, VH2) Anti-PD-1 antibody molecules of VH3, VH4 and VH5). The present application also provides a nucleic acid molecule encoding an anti-PD-1 antibody molecule, an expression vector comprising the nucleic acid molecule, a host cell comprising the expression vector, and a method of producing a PD-1 antibody using a phage library. Immunoconjugates, dual or multi-specific antibody molecules, and pharmaceutical compositions comprising antibody molecules are also provided. The anti-PD-1 antibody molecules disclosed herein can treat, prevent, and/or diagnose diseases, such as cancer (eg, solid and soft tissue tumors), as well as infectious diseases, for example, chronic infectivity, either alone or in combination with other active agents or therapeutic forms. Disease or sepsis). Accordingly, disclosed herein are the use of anti-PD-1 antibody molecules or antigen-binding fragments, conjugates, conjugates, derivatives, compositions thereof, for the treatment of diseases, such as cancer diseases (eg, solid and soft tissue tumors), and infectious diseases A method (for example, a chronic infectious disease or sepsis). In addition, methods for detecting PD-1 using an anti-PD-1 antibody molecule and detection compositions are also provided.
本申请通过人源噬菌体抗体库筛选法筛选出多个抗PD-1抗体的特异性轻链(分别为L1、L2、L3、L4、L5或L6，其各自包含3个LCDR，其中LCDR1包含SEQ NO.:1～5中的任一个，LCDR2包含SEQ NO.:6～10中的任一个，LCRD3包含SEQ NO.11～16中的任一个)和多个抗PD-1抗体的特异性重链(分别为H1，H2，H3，H4或H5，其各自包含3个HCDR，其中HCDR1包含SEQ NO.17～21中的任一个，HCDR2包含SEQ NO.22～26中的任一个，HCRD3包含SEQ NO.27～31中的任一个)，并进一步获得多种具备高的PD-1结合活性的抗PD-1抗体。The present application screens a plurality of specific light chains of anti-PD-1 antibodies by human phage antibody library screening method (L1, L2, L3, L4, L5 or L6, respectively, each comprising 3 LCDRs, wherein LCDR1 comprises SEQ NO.: any one of 1 to 5, LCDR2 comprising any one of SEQ NO.: 6 to 10, LCRD3 comprising any one of SEQ NO. 11 to 16) and specificity of a plurality of anti-PD-1 antibodies a strand (H1, H2, H3, H4 or H5, respectively, each comprising 3 HCDRs, wherein HCDR1 comprises any one of SEQ NO. 17-21, and HCDR2 comprises any one of SEQ NO. 22-26, HCRD3 comprises Any one of SEQ NO. 27 to 31), and further obtain various anti-PD-1 antibodies having high PD-1 binding activity.
以上抗体是全人源抗体或鼠源抗体，其(i)以高亲和力，例如，至少约10 7M -1，通常约10 8M -1，并且更常见地，约10 9M -1至10 10M -1的亲和力常数、或更强的亲和力结合PD-1；(ii)以高特异性结合PD-1，同时基本上不结合CD28、CTLA-4、诱导型T细胞共刺激物(ICOS)或B细胞和T细胞衰减器(BTLA)；(iii)抑制或降低PD-1与PD-1配体(例如，PD-L1或PD-L2，或两者)的结合。 The above antibodies are fully human or murine antibodies which (i) have a high affinity, for example, at least about 10 7 M -1 , usually about 10 8 M -1 , and more commonly, about 10 9 M -1 to 10 10 M -1 affinity constant, or stronger affinity binds PD-1; (ii) binds PD-1 with high specificity, while not substantially binding to CD28, CTLA-4, inducible T cell costimulator ( ICOS) or B cell and T cell attenuator (BTLA); (iii) inhibit or reduce binding of PD-1 to a PD-1 ligand (eg, PD-L1 or PD-L2, or both).
传统的抗体制备方法无法得到全人源抗体。本申请开发的人源化的高亲和力的PD-1抗体，对于肿瘤、传染病、免疫应答性疾病的临床治疗具有重要意义。Traditional antibody preparation methods fail to obtain fully human antibodies. The humanized high-affinity PD-1 antibody developed in the present application is of great significance for the clinical treatment of tumors, infectious diseases and immune-responsive diseases.
示例性地，本申请提供了以下实施方案：Illustratively, the present application provides the following embodiments:
1.一种分离的抗体或其功能性片段，包括特异性识别和结合免疫细胞表 面抗原PD-1的结构域和来自免疫球蛋白恒定区(Fc)的恒定区域，所述特异性识别和结合免疫细胞表面抗原PD-1的结构域包括具有3个CDR的轻链可变区(抗PD-1VL)和具有3个CDR的重链可变区(抗PD-1VH)，其中，所述轻链可变区(抗PD-1VL)包含选自SEQ ID NO：1-16所示的氨基酸序列的轻链CDR(LCDR)；并且所述重链可变区(抗PD-1VH)包含选自SEQ ID NO：17-31所示的氨基酸序列的重链CDR(HCDR)。WHAT IS CLAIMED IS: 1. An isolated antibody or functional fragment thereof, comprising a domain that specifically recognizes and binds to the immune cell surface antigen PD-1 and a constant region from an immunoglobulin constant region (Fc) that specifically recognizes and binds The domain of the immunocyte surface antigen PD-1 includes a light chain variable region (anti-PD-1 VL) having three CDRs and a heavy chain variable region (anti-PD-1 VH) having three CDRs, wherein the light The chain variable region (anti-PD-1 VL) comprises a light chain CDR (LCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 1-16; and the heavy chain variable region (anti-PD-1 VH) comprises a member selected from the group consisting of The heavy chain CDRs (HCDRs) of the amino acid sequences set forth in SEQ ID NOS: 17-31.
2.根据实施方案1所述的抗体或其功能性片段，其中所述轻链可变区包括：2. The antibody or functional fragment thereof of embodiment 1, wherein the light chain variable region comprises:
LCDR1，其氨基酸序列如SEQ ID NO：1、2、3、4和5中任一个所列，LCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 1, 2, 3, 4 and 5,
LCDR2，其氨基酸序列如SEQ ID NO：6、7、8、9和10中任一个所列，和LCDR2, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 6, 7, 8, 9 and 10, and
LCDR3，其氨基酸序列如SEQ ID NO：11、12、13、14、15和16中任一个所列；并且LCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 11, 12, 13, 14, 15 and 16;
所述重链可变区包括：The heavy chain variable region comprises:
HCDR1，其氨基酸序列如SEQ ID NO：17、18、19、20和21中任一个所列，HCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 17, 18, 19, 20 and 21,
HCDR2，其氨基酸序列如SEQ ID NO：22、23、24、25和26所列中任一个所列，和HCDR2, the amino acid sequence of which is set forth in any one of the SEQ ID NOs: 22, 23, 24, 25 and 26, and
HCDR3，其氨基酸序列如SEQ ID NO：27、28、29、30和31中任一个所列。HCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 27, 28, 29, 30 and 31.
3.根据实施方案1或2所述的抗体或其功能性片段，包括选自下组的轻链可变区：3. The antibody or functional fragment thereof according to embodiment 1 or 2, comprising a light chain variable region selected from the group consisting of:
a)轻链可变区VL1，其包括SEQ ID NO:1、SEQ ID NO:7和SEQ ID NO:13；a) a light chain variable region VL1 comprising SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13;
b)轻链可变区VL2，其包括SEQ ID NO:2、SEQ ID NO:6和SEQ ID NO:12；b) a light chain variable region VL2 comprising SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 12;
c)轻链可变区VL3，其包括SEQ ID NO:5、SEQ ID NO:10和SEQ ID NO:16；c) a light chain variable region VL3 comprising SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 16;
d)轻链可变区VL4，其包括SEQ ID NO:4、SEQ ID NO:8和SEQ ID NO:11；d) a light chain variable region VL4 comprising SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11;
e)轻链可变区VL5，其包括SEQ ID NO:3、SEQ ID NO:7和SEQ ID NO:13；以及e) a light chain variable region VL5 comprising SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:13;
f)轻链可变区VL6，其包括SEQ ID NO:4、SEQ ID NO:7和SEQ ID NO:14。f) Light chain variable region VL6 comprising SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 14.
4.根据实施方案1或2所述的抗体或其功能性片段，包括选自下组的重链可变区：4. The antibody or functional fragment thereof according to embodiment 1 or 2, comprising a heavy chain variable region selected from the group consisting of:
g)重链可变区VH1，其包括SEQ ID NO:17、SEQ ID NO:22和SEQ ID NO:27；g) a heavy chain variable region VH1 comprising SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27;
h)重链可变区VH2，其包括SEQ ID NO:18、SEQ ID NO:23和SEQ ID NO:30；h) a heavy chain variable region VH2 comprising SEQ ID NO: 18, SEQ ID NO: 23 and SEQ ID NO: 30;
i)重链可变区VH3，其包括SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:29；i) a heavy chain variable region VH3 comprising SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 29;
j)重链可变区VH4，其包括SEQ ID NO:20、SEQ ID NO:25和SEQ ID NO:30；j) a heavy chain variable region VH4 comprising SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 30;
k)重链可变区VH5，其包括SEQ ID NO:21、SEQ ID NO:26和SEQ ID NO:31。k) Heavy chain variable region VH5 comprising SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 31.
5.根据实施方案1或2所述的抗体或其功能性片段，包括选自VL1、VL2和VL3、VL4、VL5和VL6中的任一个的轻链可变区和选自VH1、VH2、VH3、VH4和VH5中的任一个的重链可变区。5. The antibody or functional fragment thereof according to embodiment 1 or 2, comprising a light chain variable region selected from any one of VL1, VL2 and VL3, VL4, VL5 and VL6 and selected from the group consisting of VH1, VH2, VH3 a heavy chain variable region of any of VH4 and VH5.
6.根据实施方案1所述的抗体或其功能性片段，其中所述抗体或其功能性片段包含6. The antibody or functional fragment thereof according to embodiment 1, wherein the antibody or functional fragment thereof comprises
分别为SEQ ID NO:1、7和13所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:17、22和27所示的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 1, 7, and 13, respectively, and the heavy chain CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 17, 22, and 27, respectively; or
分别为SEQ ID NO:2、6和12所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:18、23和28所示的氨基酸序列的重链 CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 2, 6 and 12, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 18, 23 and 28, respectively; or
分别为SEQ ID NO:5、10和16所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:19、24和29所示的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 5, 10 and 16, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 19, 24 and 29, respectively; or
分别为SEQ ID NO:2、6和12的氨基酸序列的轻链CDR1、CDR2和CDR3；以及分别为SEQ ID NO:21、26和31的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOs: 21, 26 and 31, respectively;
分别为SEQ ID NO:3、7和13所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:21、26和31所示的氨基酸序列的重链CDR1、CDR2和CDR3，并且The light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 3, 7 and 13, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 21, 26 and 31, respectively, and
其中所述抗体或其功能性片段特异性地结合位于人PD-1的胞外结构域内的表位。Wherein the antibody or functional fragment thereof specifically binds to an epitope located within the extracellular domain of human PD-1.
7.如实施方案3-6任一项所述的抗体或其功能性部分，其中，所述抗体包含一个或更多个氨基酸置换、添加和/或缺失，或在非CDR区域内的残基中具有一个或更多个保守氨基酸置换。7. The antibody or functional portion thereof of any one of embodiments 3-6, wherein the antibody comprises one or more amino acid substitutions, additions and/or deletions, or residues in non-CDR regions There are one or more conservative amino acid substitutions therein.
8.如实施方案7所述的抗体或其功能性部分，其中，所述抗体的氨基酸序列与其所来源的序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。8. The antibody or functional portion thereof according to embodiment 7, wherein the amino acid sequence of the antibody is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% of the sequence from which it is derived. Or 99% homologous.
9.一种分离的抗体或其功能性片段，所述抗体或其功能性片段与根据实施方案1-8中任一项所述的抗体或其功能性片段结合的表位相同，或所述抗体或其功能性片段结合的表位与根据实施方案1-8中任一项所述的抗体或其功能性片段结合的表位重叠。An isolated antibody or a functional fragment thereof, which is identical to an epitope that binds to the antibody or functional fragment thereof according to any one of embodiments 1-8, or The epitope to which the antibody or its functional fragment binds overlaps with an epitope that binds to the antibody or functional fragment thereof according to any of embodiments 1-8.
10.如实施方案1-9任一项所述的抗体或其功能性片段，其编码氨基酸序列包含SEQ ID NO:59-63任一所示序列。10. The antibody of any one of embodiments 1-9, or a functional fragment thereof, comprising an amino acid sequence comprising the sequence of any one of SEQ ID NOs: 59-63.
11.如实施方案10所述的抗体或其功能性片段，其编码氨基酸序列轻链氨基酸序列如SEQ ID NO:64所示序列，重链氨基酸序列如SEQ ID NO:65所示。The antibody or functional fragment thereof according to embodiment 10, which encodes the amino acid sequence light chain amino acid sequence as shown in SEQ ID NO: 64, and the heavy chain amino acid sequence is set forth in SEQ ID NO: 65.
12.如实施方案11所述的抗体或其功能性片段，其编码轻链核苷酸序列 如SEQ ID NO:66所示序列，编码重链氨基酸序列如SEQ ID NO:67所示。12. The antibody of claim 11, or a functional fragment thereof, which encodes a light chain nucleotide sequence, such as the sequence set forth in SEQ ID NO: 66, encoding a heavy chain amino acid sequence set forth in SEQ ID NO:67.
13.如实施方案1-12任一项所述的抗体或其功能性片段，其中所述抗体含有糖基化修饰。The antibody or functional fragment thereof of any one of embodiments 1 to 12, wherein the antibody comprises a glycosylation modification.
14.如实施方案1-13任一项所述的抗体或其功能性片段，其中所述抗体为天然人源的或人源化的。The antibody or functional fragment thereof of any of embodiments 1-13, wherein the antibody is naturally human or humanized.
15.实施方案1-14任一项所述的抗体或其功能性片段，其中所述重链由衍生自人VH种系的氨基酸序列构成；且所述轻链由衍生自人Vκ种系或Vλ种系的氨基酸序列构成。The antibody or functional fragment thereof of any one of embodiments 1 to 14, wherein the heavy chain is composed of an amino acid sequence derived from a human VH germline; and the light chain is derived from a human VK germline or The amino acid sequence of the Vλ germline is composed.
16.实施方案1-14任一项所述的抗体或其功能性片段，其中所述恒定区域是人恒定区域，例如人IgA、IgD、IgE、IgG或IgM的恒定区域，优选是人IgG的恒定区域，更优选是人IgG1或IgG4的恒定区域。The antibody or functional fragment thereof of any of embodiments 1-14, wherein the constant region is a human constant region, such as a constant region of human IgA, IgD, IgE, IgG or IgM, preferably human IgG The constant region is more preferably a constant region of human IgG1 or IgG4.
17.实施方案1-16任一项所述的抗体或其功能性片段，所述抗体是嵌合抗体、人源化抗体、人抗体、纳米抗体、域抗体或双价域抗体或其抗原结合部分，所述抗原结合部分选自Fab、F(ab′)2、Fv、scFv、Fd或dAb。17. The antibody or functional fragment thereof of any one of embodiments 1 to 16, which is a chimeric antibody, a humanized antibody, a human antibody, a Nanobody, a domain antibody or a bivalent domain antibody or antigen binding thereof In part, the antigen binding portion is selected from the group consisting of Fab, F(ab')2, Fv, scFv, Fd or dAb.
18.实施方案1-16任一项所述的抗体或其功能性片段，所述抗体是多特异性抗体，例如双特异性抗体或三特异性抗体。18. The antibody of any one of embodiments 1-16, or a functional fragment thereof, which is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.
19.实施方案1～18任一项所述的抗体或其功能性片段，其具备以下功能：The antibody or functional fragment thereof according to any one of embodiments 1 to 18, which has the following functions:
(a)结合PD-1，阻断PD-L1与PD-1结合；(a) binding PD-1 to block PD-L1 binding to PD-1;
(b)拮抗PD-1介导的信号转导；(b) antagonizing PD-1 mediated signal transduction;
(c)增加T细胞增殖；(c) increasing T cell proliferation;
(d)增强IFN-γ的产生；或(d) enhancing the production of IFN-γ; or
(e)以上功能的任意组合。(e) Any combination of the above functions.
20.实施方案19所述的抗体或其功能性片段，其中所述功能通过酶联免疫吸附测定(ELISA)、流式细胞术或表面等离子体共振(SPR)测定来测量，所述Kd值通过等离子共振结合法测定。20. The antibody of claim 19, or a functional fragment thereof, wherein said function is measured by an enzyme-linked immunosorbent assay (ELISA), flow cytometry or surface plasmon resonance (SPR) assay, said Kd value being passed Measured by plasma resonance binding method.
21.实施方案1-18任一项所述的抗体或其功能性片段，其中所述抗体或 其功能性片段结合PD-1，其对PD-1具有10 -9mol/L的亲和力。 The antibody or functional fragment thereof of any one of embodiments 1 to 18, wherein the antibody or a functional fragment thereof binds to PD-1, which has an affinity for PD-1 of 10 -9 mol/L.
22.一种分离的核酸，其编码实施方案1所述的抗体或其功能性片段或实施方案3-6中任一项所述的重链可变区和轻链可变区或实施方案17所述的scFv。An isolated nucleic acid encoding the antibody of claim 1 or a functional fragment thereof, or the heavy chain variable region and light chain variable region of any of embodiments 3-6 or embodiment 17 Said scFv.
23.实施方案22所述的核酸序列包含SEQ ID NO:54-58任一所示核苷酸序列。23. The nucleic acid sequence of embodiment 22 comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 54-58.
24.一种包含实施方案22所述的核酸的表达载体。24. An expression vector comprising the nucleic acid of embodiment 22.
25.一种包含实施方案24所述的表达载体的宿主细胞，其中所述宿主细胞选自大肠杆菌、HEK293细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞和NSO细胞。25. A host cell comprising the expression vector of embodiment 24, wherein the host cell is selected from the group consisting of E. coli, HEK293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and NSO cells.
26.一种免疫缀合物或其衍生物，包含与治疗剂偶联的根据实施方案1至13中任一项所述的抗体或其功能性片段，所述治疗剂为毒素、放射性同位素、药物或细胞毒剂。An immunoconjugate or a derivative thereof, comprising the antibody according to any one of embodiments 1 to 13 or a functional fragment thereof, which is a toxin, a radioisotope, Drug or cytotoxic agent.
27.一种药物组合物，包含实施方案1-21任一项的抗体或其功能性片段或实施方案25所述的免疫缀合物或其衍生物，以及药学上可接受的赋形剂、载体或稀释剂。27. A pharmaceutical composition comprising the antibody of any one of embodiments 1-21, or a functional fragment thereof, or the immunoconjugate of embodiment 25, or a derivative thereof, and a pharmaceutically acceptable excipient, Carrier or diluent.
28.实施方案1-21任一项所述的抗体或其功能性片段或实施方案26所述的免疫缀合物或衍生物或实施方案27所述的药物组合物在制备用于调控受试者中的免疫应答、治疗或预防受试者中与PD-1相关的状况或PD-1介导的疾病或病症的药物中的用途。28. The antibody of any one of embodiments 1 to 21, or a functional fragment thereof, or the immunoconjugate or derivative of embodiment 26, or the pharmaceutical composition of embodiment 27, prepared for use in a regulatory test Use in an immune response, treatment or prevention of a condition associated with PD-1 or a PD-1 mediated disease or condition in a subject.
29.根据实施方案28所述的用途，所述药物包括阻断PD-1与PD-L1结合的药物，所述阻断PD-1与PD-L1结合的药物包括通过PD-1抗体结合PD-1抗原，解除PD-1对T细胞活性的弱化调控的药物、解除PD-1对机体免疫抑制的药物或者提高T淋巴细胞中IFN-γ表达的药物。29. The use according to embodiment 28, comprising a drug that blocks PD-1 binding to PD-L1, the drug that blocks PD-1 binding to PD-L1 comprising binding PD by PD-1 antibody -1 antigen, a drug that abolishes PD-1's weakening of T cell activity, a drug that relieves PD-1 against immunosuppression, or a drug that increases IFN-γ expression in T lymphocytes.
30.实施方案1～21任一项所述的抗体或其功能性片段或实施方案26所述的免疫缀合物或其衍生物或实施方案27所述的药物组合物在制备用于治疗或预防受试者中选自自身免疫紊乱、针对移植物的免疫应答、***反应和癌症的紊乱的药物中的用途。30. The antibody of any one of embodiments 1 to 21, or a functional fragment thereof, or the immunoconjugate of embodiment 26, or a derivative thereof, or the pharmaceutical composition of embodiment 27, prepared for use in therapy or Use in a medicament for preventing a disorder selected from the group consisting of an autoimmune disorder, an immune response against a transplant, an allergy, and a cancer.
31.一种治疗受试者中与PD-1相关的状况或PD-1介导的疾病或病症的方法，所述方法包括向所述受试者施用治疗有效量的根据实施方案1-21中任一项所述的分离的抗体或其功能性片段或如实施方案26所述的免疫缀合物或其衍生物或者如实施方案27所述的药物组合物。31. A method of treating a condition associated with PD-1 or a PD-1 mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount according to embodiment 1-21. The isolated antibody or functional fragment thereof of any one of the above, or the immunoconjugate or derivative thereof of embodiment 26 or the pharmaceutical composition of embodiment 27.
32.一种偶联物，包含实施方案1～21任一项所述的抗体或其功能性片段和偶联部分，其中所述偶联部分为可检测的标记；所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。32. A conjugate comprising the antibody of any one of embodiments 1 to 21, or a functional fragment thereof, and a coupling moiety, wherein the coupling moiety is a detectable label; the coupling moiety is radioactive Isotopes, fluorescent substances, luminescent substances, colored substances or enzymes.
33.一种试剂盒，包括实施方案1至21中任一项所述的抗体或其功能性片段，或实施方案32所述的偶联物，所述试剂盒还包括第二抗体，所述第二抗体特异性识别所述抗体或其功能性片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。33. A kit comprising the antibody of any one of embodiments 1 to 21, or a functional fragment thereof, or the conjugate of embodiment 32, further comprising a second antibody, The second antibody specifically recognizes the antibody or a functional fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.
34.一种增强T细胞活化的方法，所述方法包括使T细胞与根据实施方案1-21中任一项所述的抗体或其功能性片段接触。34. A method of enhancing T cell activation, the method comprising contacting a T cell with an antibody or a functional fragment thereof according to any one of embodiments 1-21.
35.一种减少受试者的肿瘤或抑制受试者的肿瘤细胞生长的方法，所述方法包括向所述受试者施用治疗有效量的根据实施方案1-21中任一项所述的抗体或其功能性片段。35. A method of reducing tumor growth in a subject or inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of any of embodiments 1-21, according to any one of embodiments 1-21. Antibody or functional fragment thereof.
36.一种治疗有需要的受试者的癌症的方法，所述方法包括向所述受试者施用治疗有效量的根据实施方案1-21中任一项所述的抗体或其功能性片段。36. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or functional fragment thereof according to any one of embodiments 1-21 .
37.根据实施方案28-30任一项所述的用途或实施方案34-36任一项所述的方法，其中所述受试者被鉴定为患有可能对PD-1拮抗剂响应的病症或状况，或者在来自所述个体的待测生物样品中呈PD-L1阳性或PD-L1水平上调。The method of any of embodiments 28-30, or the method of any one of embodiments 34-36, wherein the subject is identified as having a condition that is likely to respond to a PD-1 antagonist or The condition, or PD-L1 positive or PD-L1 level is up-regulated in the biological sample to be tested from the individual.
38.一种治疗会从上调的免疫响应中获益的受试者的状况的方法，包括对所述受试者施用治疗有效量的根据实施方案1-21中任意一项所述的抗体或其功能性片段。38. A method of treating a condition of a subject that would benefit from an up-regulated immune response, comprising administering to the subject a therapeutically effective amount of the antibody of any of embodiments 1-21 or Its functional fragment.
39.根据实施方案28-30任一项所述的用途或实施方案34-37任一项所述的方法，用于治疗选自癌症，感染性疾病和炎性疾病的状况。The method of any of embodiments 28-30, or the method of any one of embodiments 34-37, for treating a condition selected from the group consisting of cancer, an infectious disease, and an inflammatory disease.
40.根据实施方案39所述的用途或方法，其中所述癌症，感染性疾病和 炎性疾病与PD-1相关。The use or method of embodiment 39, wherein the cancer, infectious disease, and inflammatory disease are associated with PD-1.
41.根据实施方案39所述的用途或方法，其中所述癌症选自胃癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、***、***癌、食道癌、小肠癌、甲状腺癌、甲状旁腺癌、黑素瘤、肾癌、***癌、乳癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈部癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肾上腺癌、肛区癌、***癌、尿道癌、***癌、膀胱癌、肾或输尿管癌、肾盂癌、表皮样癌、鳞状细胞癌、何杰金氏病、非何杰金氏淋巴瘤、内分泌***的癌症、软组织肉瘤、中枢神经***的赘生物、原发性中枢神经***淋巴瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏肉瘤、T细胞淋巴瘤、慢性或急性白血病(其包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童期实体瘤和淋巴细胞性淋巴瘤中的一种或更多种。The use or method of embodiment 39, wherein the cancer is selected from the group consisting of gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, esophageal cancer, small intestine cancer, thyroid cancer, Parathyroid carcinoma, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer , rectal cancer, adrenal cancer, anal cancer, vulvar cancer, urethral cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non-Hodkin Lymphoma, endocrine system cancer, soft tissue sarcoma, central nervous system neoplasm, primary central nervous system lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi Sarcoma, T-cell lymphoma, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), childhood solid tumors One or more lymphocytic lymphoma.
42.根据实施方案39所述的用途或方法，其中所述感染性疾病选自HIV，流行性感冒，疱疹，贾第虫病，疟疾，利什曼病，或以下病毒引起的感染：肝炎病毒(例如甲型、乙型或丙型肝炎病毒)、疱疹病毒(例如VZV、HSV-1、HAV-6、HSV-II、CMV或埃巴二氏病毒)、腺病毒、流感病毒、牛痘病毒、HTLV病毒、登革热病毒、***瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、黄病毒、艾柯病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、腮腺炎病毒、轮状病毒麻疹病毒、风疹病毒、细小病毒、JC病毒和虫媒病毒性脑炎病毒，或以下细菌引起的感染：肺炎球菌、分枝杆菌、葡萄球菌、链球菌、脑膜炎球菌、***、沙雷氏菌、克雷伯氏菌、变形菌、假单胞菌、沙门氏菌、霍乱弧菌、白喉杆菌、肉毒杆菌、炭疽杆菌、破伤风梭菌、军团菌、鼠疫杆菌、钩端螺旋体病或莱姆氏病细菌，或以下真菌引起的感染：曲霉(例如烟曲霉、黑曲霉等)、假丝酵母(例如白色假丝酵母)、克鲁斯假丝酵母、光滑假丝酵母、热带假丝酵母、新型隐球酵母、皮炎芽酵母、毛霉目的属(例如毛霉属，犁头霉属或根霉属)、申克氏孢子丝菌、巴西副球孢子菌、粗球孢菌或加膜组织胞浆菌，或以下寄生虫引起的感染：痢疾内变形虫、结肠肠袋虫、福纳氏虫、棘变形虫、间日疟原虫、田鼠巴贝虫、吸吮贾第虫、隐孢子虫、卡氏肺囊虫、布鲁斯锥虫、克鲁兹锥虫、多氏利什曼虫、鼠弓浆 虫或巴西日圆线虫。The use or method of embodiment 39, wherein the infectious disease is selected from the group consisting of HIV, influenza, herpes, giardiasis, malaria, leishmaniasis, or an infection caused by a virus: hepatitis virus (eg hepatitis A, B or C), herpes virus (eg VZV, HSV-1, HAV-6, HSV-II, CMV or Epstein's virus), adenovirus, influenza virus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, round Virus measles virus, rubella virus, parvovirus, JC virus and arbovirus viral encephalitis virus, or infections caused by bacteria: pneumococcal, mycobacteria, staphylococcus, streptococcus, meningococcus, gonococcus, sand Ralstonia, Klebsiella, Proteobacteria, Pseudomonas, Salmonella, Vibrio cholerae, Diphtheria, Botox, Bacillus anthracis, Clostridium tetani, Legionella, Yersinia, Leptospira Diseases caused by disease or Lyme disease, or by fungi: Aspergillus (eg Aspergillus fumigatus, Aspergillus niger, etc.), Candida (eg Candida albicans), Candida krusei, Candida sphaeroides, tropical Candida, Cryptococcus neoformans, dermatitis bud, Mucor genus (such as Mucor, Absidia or Rhizopus), S. skrjab, Parastaminium, Coccidioides Or with tissue cytoplasmic bacteria, or infections caused by the following parasites: amoeba in the dysentery, colonic intestines, flucina, toxoplasmosis, Plasmodium vivax, squirrel Babe, sucking Giardia, Cryptosporidium, Pneumocystis carinii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania polygala, Rat toxoplasma gondii or Nematode.
43.根据实施方案39所述的用途或方法，其中所述疾病为癌症，所述癌症包括肺癌、肝癌、卵巢癌、***、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌或头颈癌、肠癌、黑素瘤、非小细胞肺癌、；所述感染性疾病为慢性病毒感染、细菌感染或寄生虫感染疾病，所述慢性病毒为HIV、HBV或HCV。The use or method of embodiment 39, wherein the disease is cancer, the cancer comprising lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma , kidney cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer, intestinal cancer, melanoma, non-small cell lung cancer; the infectious disease is a chronic viral infection, a bacterial infection or a parasitic infection, The chronic virus is HIV, HBV or HCV.
44.实施方案28～30任一项所述的用途或实施方案34-37任一项所述的方法，其中所述用途或方法是预防性的，并在癌症、自身免疫疾病、传染病或影响T细胞数目或影响健康的疾病的任何症状出现之前被提供。The method of any one of embodiments 28 to 30, wherein the use or method is prophylactic and is in cancer, autoimmune disease, infectious disease or Any symptoms that affect the number of T cells or diseases that affect health are provided before they appear.
45.实施方案1至21中任一项所述的抗体或其功能性片段或实施方案32所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PD-1在样品中的存在或其水平。The use of the antibody of any one of embodiments 1 to 21, or a functional fragment thereof, or the conjugate of embodiment 32, in the preparation of a kit for detecting PD-1 in a sample The existence or level of it.
46.一种检测样品中的PD-1的存在的方法，所述方法包括使实施方案1-21中任一项所述的抗体或其功能性片段或权利要求26所述的免疫缀合物或其衍生物或权利要求32所述的偶联物与所述样品接触。46. A method of detecting the presence of PD-1 in a sample, the method comprising the antibody of any one of embodiments 1-21, or a functional fragment thereof, or the immunoconjugate of claim 26. Or a derivative thereof or the conjugate of claim 32 is contacted with the sample.
47.一种用于检测样品中的PD-1的存在的组合物，所述组合物包含实施方案1-21中任一项所述的抗体或其功能性片段。47. A composition for detecting the presence of PD-1 in a sample, the composition comprising the antibody of any of embodiments 1-21 or a functional fragment thereof.
48.一种制备实施方案1-21中任一项所述的抗体的方法，所述方法是噬菌体展示法。48. A method of producing the antibody of any one of embodiments 1-21, which is a phage display method.
附图概述BRIEF abstract
图1是扩增的天然人源抗体的VH基因和VL基因的电泳图。Figure 1 is an electropherogram of the VH gene and VL gene of the amplified native human antibody.
图2是天然人源scFv的DNA电泳图。Figure 2 is a DNA electropherogram of native human scFv.
图3是***天然人源scFv基因后的pCANTAB-5E载体图。Figure 3 is a diagram of the pCANTAB-5E vector after insertion of the native human scFv gene.
图4是扩增αPD-1scFv的VH和VL基因的电泳图。Figure 4 is an electropherogram of the VH and VL genes that amplify αPD-1 scFv.
图5是αPD-1scFv的DNA电泳图。Figure 5 is a DNA electrophoresis pattern of αPD-1 scFv.
图6是噬菌体库技术筛选包含BXOS抗PD-1scFv的单克隆候选噬菌体的ELISA结果。Figure 6 is a ELISA result of phage library technology screening for monoclonal candidate phage comprising BXOS anti-PD-1 scFv.
图7示出了第16号包含BXOS抗PD-1scFv的单克隆候选噬菌体阻断PD-1/PD-L1结合的实验结果。Figure 7 shows the results of an experiment in which a monoclonal candidate phage comprising BXOS anti-PD-1 scFv blocked PD-1/PD-L1 binding.
图8是用于BXOS抗PD-1全抗体表达的载体pBudCE4.1的图谱。Figure 8 is a map of the vector pBudCE4.1 for BXOS anti-PD-1 full antibody expression.
图9是BXOS抗PD-1全抗体与PD-1抗原的亲和力测定的结果。Figure 9 is a graph showing the results of affinity determination of BXOS anti-PD-1 whole antibody and PD-1 antigen.
图10A-图10C分别是成熟的DC细胞表面、流式细胞术检测的DC细胞纯度及DC细胞表面上PD-L1的表达的图像。10A-10C are images of mature DC cell surface, DC cell purity detected by flow cytometry, and expression of PD-L1 on the surface of DC cells, respectively.
图11A-图11C是流式细胞术检测T细胞表面的PD-1表达的图像。11A-11C are images of PD-1 expression detected on the surface of T cells by flow cytometry.
图12A-图12B是流式细胞术检测T细胞凋亡的图像。12A-12B are images of T cell apoptosis detected by flow cytometry.
图13显示了BXOS抗PD-1全抗体对PD-L1/293T-PD-1细胞的结合的阻断作用。Figure 13 shows the blocking effect of BXOS anti-PD-1 whole antibody on binding of PD-L1/293T-PD-1 cells.
图14显示了BXOS抗PD-1全抗体促进PBMC杀伤HepG2-luc肿瘤细胞的作用。Figure 14 shows the effect of BXOS anti-PD-1 whole antibody on PBMC killing HepG2-luc tumor cells.
具体实施方式Detailed ways
下面通过实施例来描述本申请的实施方式，本领域的技术人员应当认识到，这些具体的实施例仅表明为了达到本申请的目的而选择的实施技术方案，并不是对技术方案的限制。根据本申请的教导，结合现有技术对本申请技术方案的改进是显然的，均属于本申请保护的范围。The embodiments of the present application are described below by way of examples, and those skilled in the art should understand that the specific embodiments are merely illustrative of the embodiments of the invention. Improvements to the technical solutions of the present application are apparent in light of the teachings of the present application, and are within the scope of the present disclosure.
以下实施例中所使用的药品和试剂，无特别说明，均为普通市售商品。The drugs and reagents used in the following examples are not generally described, and are all commercially available products.
实施例1：天然人源噬菌体抗体库的建立Example 1: Establishment of a natural human phage antibody library
所用的引物及其序列如表1，划线部分为限制性内切酶位点。The primers used and their sequences are shown in Table 1, and the underlined portions are restriction endonuclease sites.
表1.引物及其序列Table 1. Primers and their sequences
Figure PCTCN2018115285-appb-000001
Figure PCTCN2018115285-appb-000001
Figure PCTCN2018115285-appb-000002
Figure PCTCN2018115285-appb-000002
Figure PCTCN2018115285-appb-000003
Figure PCTCN2018115285-appb-000003
人外周血淋巴细胞的分离及总RNA的提取Isolation of Human Peripheral Blood Lymphocytes and Extraction of Total RNA
分别采集50份年龄分布在20～50周岁之间的健康志愿者的新鲜外周血40ml，装入抗凝管，加入等体积的淋巴细胞分离液(购自天津灏洋生物公司)，按照厂家说明书进行淋巴细胞的分离，在水平离心机中于800g下离心15分钟，用移液枪吸取淋巴细胞层到新离心管中，加PBS缓冲液洗涤1次后用PBS重悬淋巴细胞，细胞计数确定每份外周血获得的淋巴细胞数量约为5×10 7。使用TRIzol Reagent(Invitrogen，15596026)从所获得的淋巴细胞中提取总RNA。 Collect 40 ml of fresh peripheral blood from healthy volunteers aged between 20 and 50 years old, place anticoagulation tubes, and add an equal volume of lymphocyte separation solution (purchased from Tianjin Haoyang Biological Co., Ltd.) according to the manufacturer's instructions. The lymphocytes were separated, centrifuged at 800 g for 15 minutes in a horizontal centrifuge, and the lymphocyte layer was pipetted into a new centrifuge tube with a pipette. After washing once with PBS buffer, the lymphocytes were resuspended in PBS, and the cell count was determined. The number of lymphocytes obtained per peripheral blood is approximately 5 × 10 7 . Total RNA was extracted from the obtained lymphocytes using TRIzol Reagent (Invitrogen, 15596026).
人源抗体重链和轻链可变区基因的扩增Amplification of human antibody heavy and light chain variable region genes
按照Superscript TM III First strand Synthesis system(Invitrogen，18080051)产品说明书以Oligo dT为反转录引物进行逆转录PCR反应，将淋巴细胞总RNA逆转录成cDNA。以该cDNA为模板，经PCR对人抗体重链可变区(VH)和轻链可变区(VL)基因进行扩增，其中VL又分为Kappa(Vκ)和Lambda(Vλ)两种类型，通过将编码接头序列(Gly 4Ser) 3的DNA的一部分作为重叠互补部分将VH与VL基因进行拼接，形成单链可变区基因片段(scFv)。采用一组能够覆盖全部VH或VL编码序列的特异性引物Library F(SEQ ID NO:68)和Library R(SEQ ID NO:69)(见以上表1)，使用Prime STAR
Figure PCTCN2018115285-appb-000004
DNA Polymerase(TAKARA，R405A)进行PCR基因扩增反应。扩增条件为：98℃5s；98℃5s，56℃5s，72℃5s，共30个循环；72℃90s。VH基因的正向引物引入Sfi I酶切位点，反向引物引入部分接头序列。VL的正向引物引入与重链反向引物互补的接头序列，反向引物引入Not I酶切位点。待反应结束后， 将PCR产物经2.0％的DNA琼脂糖凝胶电泳鉴定，结果显示扩增得到的VH基因长度为390bp左右，VL基因长度为350bp左右(图1)。使用QIAquick胶回收试剂盒(QIAGEN,28706)对目的基因条带进行回收。参照人抗体胚系基因各亚型出现比例分别混合扩增所得的VH和VL基因片段，并利用VH和VL基因片段中的添加的接头序列经重叠延伸PCR拼接为scFv基因库(图2)。 According Superscript TM III First strand Synthesis system ( Invitrogen, 18080051) the product specifications Oligo dT primer for reverse transcription PCR reverse transcription reaction, the total RNA was reverse transcribed into cDNA lymphocytes is. Using the cDNA as a template, the human antibody heavy chain variable region (VH) and light chain variable region (VL) genes were amplified by PCR, and VL was further divided into Kappa (Vκ) and Lambda (Vλ). The VH and VL genes are spliced by merging a portion of the DNA encoding the linker sequence (Gly 4 Ser) 3 as an overlapping complementary portion to form a single-chain variable region gene fragment (scFv). Using a set of specific primers, Library F (SEQ ID NO: 68) and Library R (SEQ ID NO: 69), which cover the entire VH or VL coding sequence (see Table 1 above), use Prime STAR
Figure PCTCN2018115285-appb-000004
DNA Polymerase (TAKARA, R405A) was subjected to PCR gene amplification reaction. The amplification conditions were: 98 ° C 5 s; 98 ° C 5 s, 56 ° C 5 s, 72 ° C 5 s, a total of 30 cycles; 72 ° C 90 s. The forward primer of the VH gene introduces a Sfi I restriction site, and the reverse primer introduces a partial linker sequence. The forward primer of VL introduces a linker sequence complementary to the heavy chain reverse primer, and the reverse primer introduces a Not I restriction site. After the reaction was completed, the PCR product was identified by 2.0% DNA agarose gel electrophoresis. The results showed that the amplified VH gene was about 390 bp in length and the VL gene was about 350 bp in length (Fig. 1). The gene band of interest was recovered using QIAquick Glue Recovery Kit (QIAGEN, 28706). The VH and VL gene fragments obtained by the amplification of the respective subtypes of the human antibody germline gene were mixed, and ligated into the scFv gene library by overlap extension PCR using the added linker sequences in the VH and VL gene fragments (Fig. 2).
天然人源scFv噬菌体抗体库的构建Construction of natural human scFv phage antibody library
将如上获得的scFv基因***噬菌体载体pCANTAB-5E中。分别用限制性内切酶Sfi I(NEB，R0123S)和Not I(NEB，R3198L)对scFv和pCANTAB-5E进行双酶切，纯化后将scFv基因的酶切产物与酶切处理的pCANTAB-5E载体使用T4DNA连接酶(NEB，M0202L)连接(图3)。连接分批进行，共进行10批，每批50个连接体系，经乙酸钠乙醇沉淀法浓缩后，溶于50μL无菌水中，经电击转化至5mL XL1-BLUE感受态细胞(购自Stratagen)后取适量转化菌均匀涂布于含100μg/mL氨苄青霉素的2YT平板上，以计算库容量，其余的转化菌培养至对数生长期，加入辅助噬菌体M13KO7，于37℃220rpm培养1h，加入50μg/mL卡那霉素，30℃培养过夜。次日，用PEG8000/NaCl将制备的噬菌体进行沉淀以浓缩纯化，用1％初始体积的15％甘油溶液重悬，即得到天然人源scFv噬菌体抗体库，取1μL进行滴度测定，其余-80℃保存。经10批连接产物的转化，获得天然人源scFv噬菌体抗体库，库容量为1x10 9，滴度为1x10 12。 The scFv gene obtained as above was inserted into the phage vector pCANTAB-5E. The scFv and pCANTAB-5E were digested with restriction endonuclease Sfi I (NEB, R0123S) and Not I (NEB, R3198L) respectively. After purification, the scFv gene was digested with the digested pCANTAB-5E. The vector was ligated using T4 DNA ligase (NEB, M0202L) (Fig. 3). The connection was carried out in batches. A total of 10 batches, 50 batches per batch system, were concentrated by sodium acetate ethanol precipitation, dissolved in 50 μL of sterile water, and electroporated to 5 mL of XL1-BLUE competent cells (purchased from Stratagen). Appropriate amount of transformant was uniformly coated on 2YT plate containing 100 μg/mL ampicillin to calculate the library capacity. The remaining transformants were cultured to logarithmic growth phase, and the helper phage M13KO7 was added and cultured at 37 ° C for 220 h at rpm for 50 h. mL kanamycin was incubated overnight at 30 °C. On the next day, the prepared phage was precipitated with PEG8000/NaCl to be concentrated and purified, and resuspended in a 1% initial volume of 15% glycerol solution to obtain a natural human scFv phage antibody library, and 1 μL was used for titer determination, and the remaining -80 °C save. The natural human scFv phage antibody library was obtained by transformation of 10 batches of the ligation product with a library capacity of 1×10 9 and a titer of 1×10 12 .
天然人源scFv噬菌体抗体库的多样性分析Diversity analysis of natural human scFv phage antibody library
以获得的噬菌体抗体库感染XL1-BLUE活化菌涂布平板37℃孵箱过夜，随机挑取20个菌落增菌培养，提取质粒测定基因序列，对测序结果进行Blast和比对分析。结果发现，20条基因序列都属于抗体序列，但各不相同，表明该噬菌体抗体库多样性很好。The obtained phage antibody library was infected with the XL1-BLUE activating strain coated plate at 37 ° C overnight, and 20 colonies were randomly picked and cultured, and the plasmid was extracted to determine the gene sequence, and the sequencing results were subjected to Blast and comparative analysis. It was found that all 20 gene sequences belonged to the antibody sequence, but they were different, indicating that the phage antibody library diversity is very good.
实施例2抗PD-1单克隆抗体的筛选Example 2 Screening of anti-PD-1 monoclonal antibodies
1.PD-1抗原对噬菌体抗体库淘选1. PD-1 antigen panning for phage antibody library
以10μg/mL的人PD-1重组蛋白(Acro Biosystems，PD-1-H5221)1mL包被免疫管，抗原包被量为10μg/1mL/管，4℃包被过夜；经PBS洗涤1遍后加4mL 3％脱脂奶粉/PBS(MPBS)封闭免疫管，室温3h；加1mL实施例1中 制备得到的天然人源噬菌体抗体库，投入量为10 9-10 12个/管，室温孵育3h。PBST-PBS洗涤20遍，0.1M PH2.2的Glycine-HCl洗脱，用1.0M PH8.8的Tris-HCl中和洗脱下来的噬菌体溶液至pH7.0左右，用于感染1mL XL1-BLUE活化菌，37℃培养箱中静置20min，补足2YTA培养基到20mL，吸取10μL菌液涂布于2YTA平板上，用于计数噬菌体抗体产出量；剩余菌液37℃振荡培养1h，用2YTA培养液补至100mL，摇至对数期后加入M13KO7辅助噬菌体，37℃，220rpm，孵育1h。加入50μg/mL卡那霉素，30℃培养过夜。次日，用PEG8000/NaCl进行沉淀以浓缩纯化，用1mL 15％甘油水溶液重悬。浓缩的噬菌体用于下一轮筛选。共进行三轮噬菌体库富集筛选，获得本发明的包含BXOS抗PD-1scFv的噬菌体库。 The immunotube was coated with 1 μL of human PD-1 recombinant protein (Acro Biosystems, PD-1-H5221) at 10 μg/mL, the antigen coating amount was 10 μg/1 mL/tube, and coated at 4 ° C overnight; after washing with PBS for 1 time The immune tube was blocked with 4 mL of 3% skim milk powder/PBS (MPBS) at room temperature for 3 h; 1 mL of the natural human phage antibody library prepared in Example 1 was added, and the input amount was 10 9 -10 12 /tube, and incubated for 3 h at room temperature. The cells were washed with PBST-PBS for 20 times, eluted with 0.1 M PH2.2 of Glycine-HCl, and the eluted phage solution was neutralized with 1.0 M PH8.8 Tris-HCl to pH 7.0 or so for infection of 1 mL XL1-BLUE. The activated bacteria were allowed to stand in a 37 ° C incubator for 20 min, supplemented with 2YTA medium to 20 mL, and 10 μL of the bacterial solution was applied to 2YTA plates for counting the phage antibody production; the remaining bacterial solution was shake cultured at 37 ° C for 1 h, using 2YTA The culture solution was added to 100 mL, and after shaking to log phase, M13KO7 helper phage was added, and the mixture was incubated at 37 ° C, 220 rpm for 1 h. 50 μg/mL kanamycin was added and incubated overnight at 30 °C. The next day, precipitation was carried out with PEG 8000/NaCl to concentrate and purify, and resuspended in 1 mL of 15% glycerin aqueous solution. The concentrated phage was used for the next round of screening. A total of three rounds of phage library enrichment screening were performed to obtain a phage library containing BXOS anti-PD-1 scFv of the present invention.
2.阳性对照αPD-1scFv噬菌体的制备2. Preparation of positive control αPD-1scFv phage
参照已上市的抗PD-1单抗Nivolumab的基因序列，分别化学合成其VH和VL基因。利用实施例1中的VH/VL基因扩增引物进行PCR扩增，产物经2.0％的DNA琼脂糖凝胶电泳鉴定，显示VH基因长度为390bp左右，VL基因长度为350bp左右(图4)。使用QIAquick胶回收试剂盒对目的基因条带进行回收。利用VH和VL基因的接头重叠互补序列经PCR反应拼接为抗PD-1scFv阳性对照基因，指定为αPD-1scFv(图5)。将αPD-1scFv阳性对照基因***噬菌体载体pCANTAB-5E中，分别用限制性内切酶SfiI和NotI对αPD-1scFv基因和pCANTAB-5E载体进行双酶切，纯化后将αPD-1scFv基因的酶切产物与酶切处理的pCANTAB-5E载体使用T4DNA连接酶进行连接。经电击转化转入XL1-BLUE感受态细胞，取适量转化菌均匀涂布于含100μg/mL氨苄青霉素的2YTAG平板上，37℃倒置过夜培养。次日挑取单克隆，震荡培养至对数生长期，加入辅助噬菌体M13KO7，37℃感染培养1h后加50μg/mL卡那霉素，30℃培养过夜。次日离心取上清液即得到αPD-1scFv抗体阳性对照噬菌体，取1μL上清液进行滴度测定，滴度约为1x10 12。 The VH and VL genes were chemically synthesized according to the gene sequence of the marketed anti-PD-1 mAb Nivolumab. The VH/VL gene amplification primer of Example 1 was used for PCR amplification, and the product was identified by 2.0% DNA agarose gel electrophoresis, and the VH gene length was about 390 bp, and the VL gene length was about 350 bp (Fig. 4). The gene band of interest was recovered using the QIAquick Glue Recovery Kit. The linker overlapping complementary sequences of the VH and VL genes were ligated into an anti-PD-1 scFv positive control gene by PCR reaction, designated as αPD-1 scFv (Fig. 5). The αPD-1scFv positive control gene was inserted into the phage vector pCANTAB-5E, and the αPD-1 scFv gene and the pCANTAB-5E vector were digested with restriction endonucleases SfiI and NotI, respectively. After purification, the αPD-1 scFv gene was digested. The product was ligated with the digested pCANTAB-5E vector using T4 DNA ligase. The cells were transformed into XL1-BLUE competent cells by electroporation, and appropriate transformants were uniformly coated on 2YTAG plates containing 100 μg/mL ampicillin, and cultured at 37 ° C overnight. The next day, the monoclonals were picked and shaken to the logarithmic growth phase. The helper phage M13KO7 was added, cultured at 37 ° C for 1 h, and then 50 μg/mL kanamycin was added, and cultured at 30 ° C overnight. The supernatant was centrifuged the next day to obtain the αPD-1 scFv antibody positive control phage, and 1 μL of the supernatant was taken for titer measurement, and the titer was about 1×10 12 .
3.包含BXOS抗PD-1scFv的单克隆候选噬菌体的ELISA筛选3. ELISA screening of monoclonal candidate phage containing BXOS anti-PD-1 scFv
(1)将步骤1经三轮淘筛获得的噬菌体感染对数生长期的XL1-BLUE菌，室温20min，涂2YTA板37℃培养过夜。挑取单菌落增菌培养，至对数生长期后加入M13KO7辅助噬菌体，30℃振荡培养过夜，离心取上清即为噬菌体 抗体。(1) The phage obtained by the three-step panning in the step 1 was infected with the XL1-BLUE strain in the logarithmic growth phase at room temperature for 20 min, and the plate was incubated overnight at 37 ° C with a 2YTA plate. Single colony enrichment culture was picked, and M13KO7 helper phage was added after the logarithmic growth phase, and cultured overnight at 30 ° C with shaking, and the supernatant was centrifuged to obtain a phage antibody.
(2)以50μL 2μg/mL的人PD-1重组蛋白包被ELISA板，4℃过夜。用200μL3％的MPBS封闭，37℃3h。将50μL(1)得到的噬菌体抗体加入ELISA板孔，37℃孵育1.5h；PBST洗板3遍，加入抗M13单克隆抗体/HRP(北京义翘神州科技有限公司,11973-MM05-50)进行孵育，37℃1h；TMB显色剂显色，2M硫酸终止后测A450值。同时用步骤2得到的αPD-1scFv阳性对照噬菌体作为阳性对照。筛选结果见表2和图6。(2) ELISA plates were coated with 50 μL of 2 μg/mL of human PD-1 recombinant protein at 4 ° C overnight. Block with 200 μL of 3% MPBS for 3 h at 37 °C. 50 μL of the phage antibody obtained in (1) was added to the well of the ELISA plate, and incubated at 37 ° C for 1.5 h; the plate was washed 3 times with PBST, and anti-M13 monoclonal antibody/HRP (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11973-MM05-50) was added. Incubate at 37 ° C for 1 h; TMB color developer was developed, and A450 value was measured after termination of 2 M sulfuric acid. At the same time, the αPD-1 scFv positive control phage obtained in the step 2 was used as a positive control. The screening results are shown in Table 2 and Figure 6.
表2抗PD-1scFv的单克隆候选噬菌体与PD-1的结合Table 2 Binding of monoclonal candidate phage against PD-1 scFv to PD-1
Figure PCTCN2018115285-appb-000005
Figure PCTCN2018115285-appb-000005
Figure PCTCN2018115285-appb-000006
Figure PCTCN2018115285-appb-000006
4.测序：4. Sequencing:
根据3的结果，确认高亲和力的scFv1、scFv2、scFv3、scFv4、scFv5，其核苷酸序列如SEQ.ID.NO.54、55、56、57和58所示，其氨基酸序列如SEQ.ID.NO.59、60、61、62和63所示。According to the results of 3, high affinity scFv1, scFv2, scFv3, scFv4, scFv5 were confirmed, and the nucleotide sequences thereof are shown in SEQ.ID.NO.54, 55, 56, 57 and 58 and the amino acid sequence thereof is SEQ.ID. .NO. 59, 60, 61, 62 and 63 are shown.
表3table 3
Figure PCTCN2018115285-appb-000007
Figure PCTCN2018115285-appb-000007
实施例3包含BXOS抗PD-1scFv的单克隆候选噬菌体16号对PD-1/PD-L1结合的阻断作用Example 3 Blocking of PD-1/PD-L1 Binding by a Monoclonal Candidate Phage 16 Containing BXOS Anti-PD-1 scFv
利用生物素标记的PD-1:PD-L1抑制剂筛选试剂盒(Acro Biosystems，EP-101)检测实施例2步骤3筛选所得的BXOS抗PD-1噬菌体候选单克隆16号对PD-1/PD-L1结合的阻断作用。参照产品说明书进行，简要来说，以100μL/孔2μg/mL的PD-L1包被ELISA板4℃过夜；加200μL/孔3％MPBS37℃封闭3h。在离心管中加入0.6μL生物素化PD-1(100μg/mL)和不同稀释度待测样品溶液100μL，37℃孵育1.5h，同时设置实施例2步骤2制备的噬菌体为阳性对照，将M13KO7噬菌体设置为阴性对照。将测试样品和对照样品加入ELISA板，37℃孵育1.5h；PBST洗涤4遍，加100μL/孔HRP标记的链霉亲和素(0.4μg/mL)进行孵育，37℃1h。PBST洗涤4遍后显色测A450值(图7)。The BXOS anti-PD-1 phage candidate monoclonal 16 was screened by the biotin-labeled PD-1:PD-L1 inhibitor screening kit (Acro Biosystems, EP-101) for PD-1/. Blocking effect of PD-L1 binding. Referring to the product manual, briefly, the ELISA plate was coated with 100 μL/well 2 μg/mL of PD-L1 at 4 ° C overnight; and 200 μL/well of 3% MPBS was blocked at 37 ° C for 3 h. Add 0.6 μL of biotinylated PD-1 (100 μg/mL) and 100 μL of different dilutions of the sample solution to the test tube, incubate at 37 ° C for 1.5 h, and set the phage prepared in step 2 of Example 2 as a positive control, M13KO7 The phage was set as a negative control. Test and control samples were added to the ELISA plate, incubated at 37 ° C for 1.5 h; washed 4 times with PBST, and incubated with 100 μL/well of HRP-labeled streptavidin (0.4 μg/mL) for 1 h at 37 °C. The A450 value was measured after washing 4 times with PBST (Fig. 7).
阻断结果见下表4。The blocking results are shown in Table 4 below.
表4抗PD-1scFv的单克隆候选噬菌体对PD-1/PD-L1的阻断率Table 4 Blocking rate of PD-1/PD-L1 by monoclonal candidate phage against PD-1 scFv
  阻断率 Blocking rate
噬菌体16号原液Phage 16 stock solution 46.1％46.1%
噬菌体16号稀释2倍 Phage 16 diluted 2 times 32.3％32.3%
噬菌体16号稀释5倍Phage 16 diluted 5 times 13.8％13.8%
噬菌体16号稀释10倍Phage 16 diluted 10 times 16.9％16.9%
阳性对照αPD-1scFv噬菌体Positive control αPD-1scFv phage 47.7％47.7%
阳性对照αPD-1单克隆抗体Positive control αPD-1 monoclonal antibody 70.7％70.7%
阳性对照αPD-1单克隆抗体：Anti-PD1mAb，Human(IgG4)Lot No.B52-63NS1-AS,ACRO BiosystemsPositive control αPD-1 monoclonal antibody: Anti-PD1 mAb, Human (IgG4) Lot No. B52-63NS1-AS, ACRO Biosystems
结果表明：16号噬菌体原液可以明显阻断PD1/PDL1的结合，阻断率为46.1％，与阳性对照αPD-1scFv噬菌体(阻断率为47.7％)相当，且存在剂量依赖关系，浓度越大，阻断率越高。The results showed that the 16th phage stock solution could significantly block the binding of PD1/PDL1, the blocking rate was 46.1%, which was equivalent to the positive control αPD-1scFv phage (blocking rate 47.7%), and there was a dose-dependent relationship. The higher the blocking rate.
实施例4 16号BXOS抗PD-1全抗体的制备Example 4 Preparation of 16-BXOS Anti-PD-1 Whole Antibody
将包含BXOS抗PD-1scFv的单克隆候选噬菌体16号的VH和VL(Vκ)基因分别与人类抗体重链恒定区(Cγ4)基因和轻链恒定区(Cκ1)基因融合，形成完整的重链和轻链基因，通过PCR获得在重链基因两端引入HindⅢ和XbaI酶切位点，轻链基因两端引入NotI和XhoI酶切位点的BXOS抗PD-1全抗体的编码核苷酸序列，将该全抗体的编码核苷酸序列***表达载体pBUdCE4.1(图8)。The VH and VL (Vκ) genes of the monoclonal candidate phage 16 containing BXOS anti-PD-1 scFv were fused with the human antibody heavy chain constant region (Cγ4) gene and the light chain constant region (Cκ1) gene, respectively, to form a complete heavy chain. And light chain genes, the nucleotide sequence of the BXOS anti-PD-1 full antibody encoding the HindIII and XbaI cleavage sites at both ends of the heavy chain gene and the NotI and XhoI cleavage sites at both ends of the light chain gene were obtained by PCR. The coding nucleotide sequence of the whole antibody was inserted into the expression vector pBUdCE4.1 (Fig. 8).
将构建好的表达载体经电穿孔导入HEK293T/17细胞，进行全抗体表达，得到16号BXOS抗PD-1全抗体。The constructed expression vector was introduced into HEK293T/17 cells by electroporation, and whole antibody expression was carried out to obtain BXOS anti-PD-1 whole antibody of No. 16.
BXOS抗PD-1全抗体的轻链氨基酸序列如SEQ.ID.NO.64所示，核苷酸序列如SEQ.ID.NO.66所示，LCDR1氨基酸序列如SEQ ID NO:3，LCDR2氨基酸序列如SEQ ID NO:7，LCDR3氨基酸序列如SEQ ID NO:13；重链氨基酸序列如SEQ.ID.NO.65所示，核苷酸序列如SEQ ID NO.67所示，HCDR1氨基酸序列如SEQ ID NO.21，HCDR2氨基酸序列如SEQ ID NO:26，HCDR3氨基酸序列如SEQ ID NO:31。The light chain amino acid sequence of BXOS anti-PD-1 full antibody is shown in SEQ. ID. NO. 64, the nucleotide sequence is shown in SEQ. ID. NO. 66, and the amino acid sequence of LCDR1 is SEQ ID NO: 3, LCDR2 amino acid. The sequence is as SEQ ID NO: 7, the LCDR3 amino acid sequence is SEQ ID NO: 13; the heavy chain amino acid sequence is shown in SEQ. ID. NO. 65, the nucleotide sequence is shown in SEQ ID NO. 67, and the HCDR1 amino acid sequence is as SEQ ID NO. 21, HCDR2 amino acid sequence is SEQ ID NO: 26, and HCDR3 amino acid sequence is SEQ ID NO: 31.
实施例5：等离子共振法测定实施例4所得的16号BXOS抗PD-1全抗体的亲和力Example 5: Determination of the affinity of BXOS anti-PD-1 whole antibody of No. 16 obtained in Example 4 by plasma resonance method
仪器和试剂：Instruments and reagents:
仪器：Reichert 4SPR(表面等离子体共振仪)Instrument: Reichert 4SPR (surface plasmon resonance)
芯片:Planar Mixed SAM,13206061Chip: Planar Mixed SAM, 13206061
反应温度:25℃Reaction temperature: 25 ° C
缓冲液:PBST(0.05％Tween-20,5％DMSO)Buffer: PBST (0.05% Tween-20, 5% DMSO)
再生液：10mM Gly PH2.0Regeneration fluid: 10mM Gly PH2.0
1.抗原固定：1. Antigen fixation:
1)取商品化PD-1抗原(PD-1-PE，货号：329906，厂家：biolegend)，使用PH4.5 10mM乙酸钠将其稀释成1μg/mL的浓度，以25μL/min流速进样40s将其负载到表面等离子体共振仪(Reichert4SPR，赫航科学仪器(上海)有限公司)，使最终芯片抗原结合量达到50RU。1) Commercially available PD-1 antigen (PD-1-PE, Cat. No. 329906, manufacturer: biolegend), diluted to a concentration of 1 μg/mL using PH4.5 10 mM sodium acetate, and injected at a flow rate of 25 μL/min for 40 s. It was loaded onto a surface plasmon resonance instrument (Reichert 4SPR, Herae Scientific Instruments (Shanghai) Co., Ltd.) to achieve a final chip antigen binding amount of 50 RU.
2)使用0.05％PBST缓冲液稀释阳性对照抗PD-1抗体(China/T&L，TL-106，北京同立海源生物科技有限公司)配制成100nM、30nM、10nM三个浓度，均以流速25μl/min进样结合2min，解离3min，观察到这3个浓度均有反映值并且100nM对应的反映值接近饱和，由此确定PD-1抗原的固定量50RU能满足试验需求。2) The positive control anti-PD-1 antibody (China/T&L, TL-106, Beijing Tongli Haiyuan Biotechnology Co., Ltd.) was diluted with 0.05% PBST buffer to prepare three concentrations of 100 nM, 30 nM and 10 nM, both at a flow rate of 25 μl/ The min injection was combined for 2 min and dissociated for 3 min. It was observed that all three concentrations reflected the value and the corresponding value of 100 nM was close to saturation, thus determining that the fixed amount of PD-1 antigen 50 RU could meet the test requirements.
2.对照抗体进样：使用0.05％PBST缓冲液稀释对照抗体，将其配制成3.125nM、6.25nM、12.5nM、25nM、50nM和100nM的浓度梯度，以流速25μl/min进样，结合2min，解离3min。传感器芯片的再生条件为PH2.010mM Gly溶液，流速30μl/min，进样时间30s。2. Control antibody injection: The control antibody was diluted with 0.05% PBST buffer and formulated into a concentration gradient of 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM, and injected at a flow rate of 25 μl/min for 2 min. Dissociate for 3 min. The regeneration condition of the sensor chip was PH2.010 mM Gly solution, the flow rate was 30 μl/min, and the injection time was 30 s.
3.BXOS抗PD-1全抗体(HEK293T表达纯化的抗PD-1单抗(分子量约为150KD))进样：方法同2。3. BXOS anti-PD-1 whole antibody (HEK293T expression purified anti-PD-1 mAb (molecular weight about 150KD)) injection: the same method.
实验结果参见图9，其中本申请的BXOS抗-PD-1全抗体的Ka(1/(M*s))为1.24E+05，Kd(1/s)为6.57E-04，KD(M)为5.32E-9。结果显示，本申请的抗PD-1抗体与抗原的亲和力可满足治疗需求。The experimental results are shown in Figure 9, in which the BXOS anti-PD-1 whole antibody of the present application has a Ka (1/(M*s)) of 1.24E+05 and a Kd (1/s) of 6.57E-04, KD (M). ) is 5.32E-9. The results show that the affinity of the anti-PD-1 antibody of the present application to the antigen can meet the therapeutic needs.
实施例6 16号BXOS抗PD-1抗体对流式细胞抗PD-1抗体与T细胞表面PD-1结合的阻断作用Example 6 Blocking effect of 16X BXOS anti-PD-1 antibody on binding of PD-1 antibody to T cell surface by anti-PD-1 antibody
实验方案：Experimental program:
1、成熟DC培养1. Mature DC culture
(1)抽取50mL志愿者A捐献的外周血。(1) Extract 50 mL of peripheral blood donated by volunteer A.
(2)使用淋巴分离液(天津市灏洋生物制品科技有限责任公司，LTS1077)按照厂家说明分离外周血单核细胞(PBMC)。(2) Peripheral blood mononuclear cells (PBMC) were isolated using a lymphatic separation solution (Tianjin Yuyang Biological Products Co., Ltd., LTS1077) according to the manufacturer's instructions.
(3)使用1640无血清培养基重悬PBMC，调整细胞密度2×10 6个/mL，加入到细胞培养瓶，于37℃，5％CO 2培养箱孵育1.5-2h。弃去悬浮细胞和培养基，使用培养基洗涤2遍，然后加入完全培养基(含10％FBS、10ng/mL IL-4、20ng/mL GM-CSF)。 (3) Resuspend PBMC in 1640 serum-free medium, adjust the cell density to 2×10 6 /mL, add to the cell culture flask, and incubate at 37 ° C, 5% CO 2 incubator for 1.5-2 h. The suspension cells and medium were discarded, washed twice with medium, and then complete medium (containing 10% FBS, 10 ng/mL IL-4, 20 ng/mL GM-CSF) was added.
(4)在第1、3、5天每天加入IL-4和粒细胞-巨噬细胞集落刺激因子(GM-CSF)以保持IL-4浓度为10ng/mL，GM-CSF浓度为20ng/mL。(4) IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were added daily on days 1, 3, and 5 to maintain an IL-4 concentration of 10 ng/mL and a GM-CSF concentration of 20 ng/mL. .
(5)在第7天加入成熟因子TNF-α(终浓度10ng/mL)。(5) The mature factor TNF-α (final concentration 10 ng/mL) was added on the 7th day.
(6)继续培养至第9天，加入终浓度10μg/mL的脂多糖(LPS)以促进树突状细胞(Dendritic Cells,DC)表达PD-L1分子。(6) The culture was continued until day 9, and lipopolysaccharide (LPS) at a final concentration of 10 μg/mL was added to promote the expression of PD-L1 molecules by dendritic cells (DCs).
(7)加入LPS后24h，流式细胞术分析DC表面B7-H1(PD-L1)的表达。(7) Flow cytometry was used to analyze the expression of B7-H1 (PD-L1) on the DC surface 24 h after the addition of LPS.
结果参见图10。DC细胞为体内最重要的抗原呈递细胞(APC)，成熟的DC细胞表面有许多树突状突起(参见图10A)。成熟的DC细胞高表达CD80和CD86分子，通过流式细胞术检测CD80和CD86等分子，可以计算出DC细胞纯度及成熟程度。图10B显示CD11c+CD86+细胞占比例为97.32％，说明培养的成熟DC细胞纯度达到97.32％。图10C使用PE抗-human CD274(B7-H1,PD-L1)(KeMinbio，SZB12619)，检测DC细胞表面PD-L1分子表达，显示所有的DC细胞都高表达PD-L1分子。See Figure 10 for the results. DC cells are the most important antigen presenting cells (APCs) in the body, and mature DC cells have many dendrites on the surface (see Figure 10A). Mature DC cells express CD80 and CD86 molecules, and the purity and maturity of DC cells can be calculated by flow cytometry to detect CD80 and CD86 molecules. Figure 10B shows that the proportion of CD11c+CD86+ cells is 97.32%, indicating that the purity of cultured mature DC cells is 97.32%. Figure 10C shows the expression of PD-L1 molecules on the surface of DC cells using PE anti-human CD274 (B7-H1, PD-L1) (KeMinbio, SZB12619), showing that all DC cells highly express PD-L1 molecules.
2、混合淋巴细胞培养2, mixed lymphocyte culture
(1)抽取志愿者B的外周血，提取单核细胞(MNC)，将所得MNC与成熟的DC按照10:1的比例加入到96孔板(U型底)，每孔MNC 0.1×10 6个，每孔终体积200μl。 (1) Extract the peripheral blood of volunteer B, extract monocytes (MNC), and add the obtained MNC and mature DC to a 96-well plate (U-bottom) in a ratio of 10:1, MNC 0.1×10 6 per well. The final volume of each well is 200 μl.
(2)将混合培养的MNC分为2组，一组加入实施例2第3步筛选得到的BXOS抗-PD-1(5μg/mL)，一组加入PBS。(2) The mixed culture MNCs were divided into two groups, and one group was added with BXOS anti-PD-1 (5 μg/mL) obtained by the third step of Example 2, and one group was added with PBS.
(3)混合培养第3天流式细胞术检测MNC中T细胞的PD-1表达。(3) On the third day of mixed culture, flow cytometry was used to detect PD-1 expression of T cells in MNC.
3、16号BXOS抗PD-1抗体对流式细胞抗-PD-1抗体与T细胞表面PD-1结合的阻断作用：Blocking effect of BXOS anti-PD-1 antibody on the binding of flow-cell anti-PD-1 antibody to PD-1 on the surface of T cells:
(1)收集混合培养的T细胞，使用冷的PBS洗涤2次。(1) Mixed cultured T cells were collected and washed twice with cold PBS.
(2)用冷的PBS重悬混合培养的T细胞，调整细胞密度1×10 6个/mL。 (2) The cultured T cells were resuspended in cold PBS, and the cell density was adjusted to 1 × 10 6 /mL.
(3)每管取100μL(1×10 5个)细胞悬浮液。 (3) Take 100 μL (1 × 10 5 ) cell suspension per tube.
(4)加入CD3-APC(1mg/ml)和PD-1-PE(1mg/ml)各1μL。(4) 1 μL each of CD3-APC (1 mg/ml) and PD-1-PE (1 mg/ml) was added.
(5)冰上避光孵育30min。(5) Incubate on ice for 30 min in the dark.
(6)PBS洗涤2次，100μL PBS重悬浮细胞。(6) Wash twice with PBS and resuspend the cells in 100 μL of PBS.
(7)流式细胞术检测T细胞表面PD-1表达。(7) Flow cytometry was used to detect PD-1 expression on T cell surface.
结果参见图11A-11C。图11A显示，单独培养MNC 3天后，T细胞表面PD-1表达量为26.21％；图11B显示，当MNC与DC混合培养后，PD-1表达量为32.65％(图B)，因为DC细胞表面高表达PD-L1，促进T细胞表面PD-1分子的表达；图11C显示，MNC与DC混合培养并同时添加BXOS-抗-PD-1全抗体，PD-1表达量减少为0.25％。图11A-11C说明，在本申请的BXOS抗-PD-1全抗体存在的情况下，商品化的抗PD-1的流式抗体PD-1-PE(biolegend，329906)不能与T细胞表面的PD-1分子结合，这表明本申请的BXOS抗PD-1全抗体已经与T细胞表面PD-1分子的结合。The results are shown in Figures 11A-11C. Fig. 11A shows that the expression of PD-1 on the surface of T cells was 26.21% after 3 days of culture of MNC alone; Fig. 11B shows that when MNC was mixed with DC, the expression of PD-1 was 32.65% (Fig. B) because DC cells The surface highly expressed PD-L1 and promoted the expression of PD-1 molecules on the surface of T cells; Fig. 11C showed that MNC was mixed with DC and simultaneously added BXOS-anti-PD-1 whole antibody, and the expression of PD-1 was reduced to 0.25%. Figures 11A-11C illustrate that in the presence of the BXOS anti-PD-1 whole antibody of the present application, the commercial anti-PD-1 flow antibody PD-1-PE (biolegend, 329906) cannot be associated with T cell surface The PD-1 molecule binds, indicating that the BXOS anti-PD-1 whole antibody of the present application has bound to the PD-1 molecule on the T cell surface.
实施例7 16号BXOS抗PD-1抗体减少了DC细胞表面PD-L1诱导的T细胞凋亡Example 7 16 BXOS anti-PD-1 antibody reduces PD-L1-induced T cell apoptosis on DC cell surface
流式抗体：Flow antibody:
CD3-APC，货号：300312，厂家：biolegendCD3-APC, article number: 300312, manufacturer: biolegend
PD-1-PE，货号：329906，厂家：biolegendPD-1-PE, article number: 329906, manufacturer: biolegend
凋亡试剂盒：ANNEXIN V-FITC/PI凋亡检测试剂盒(索莱宝生物科技有限公司，CA1020-50T)Apoptosis Kit: ANNEXIN V-FITC/PI Apoptosis Detection Kit (Solebao Biotechnology Co., Ltd., CA1020-50T)
操作步骤：Steps:
(1)使用27mL的去离子水稀释3ml结合缓冲液(10×)至30mL，每次用 3mL。(1) Dilute 3 ml of binding buffer (10x) to 30 mL using 27 mL of deionized water, 3 mL each time.
(2)收集细胞(1×10 6个/次)，然后使用冷的PBS洗涤。 (2) Cells were collected (1 × 10 6 /time) and then washed with cold PBS.
(3)使用1mL 1×的结合缓冲液悬浮细胞，300g离心10min，弃上清。(3) The cells were suspended using 1 mL of 1× binding buffer, centrifuged at 300 g for 10 min, and the supernatant was discarded.
(4)使用1mL 1×的结合缓冲液重悬细胞，使细胞密度达到1×10 6个/mL。 (4) The cells were resuspended in 1 mL of 1× binding buffer to a cell density of 1 × 10 6 /mL.
(5)每管加入100μL细胞(1X10 5个)和CD3-APC(1mg/mL)1μL，避光冰浴30min。 (5) 100 μL of cells ( 1 ×10 5 ) and CD3-APC (1 mg/mL) 1 μL were added to each tube, and the ice bath was protected from light for 30 min.
(6)冷PBS洗涤2次，100μl PBS重悬细胞，加入5μL Annexin V-FITC，室温，避光10min。(6) Wash twice with cold PBS, resuspend the cells in 100 μl PBS, add 5 μL of Annexin V-FITC, and keep at room temperature for 10 min.
(7)加入5μL PI，室温，避光孵育5min。(7) Add 5 μL of PI, incubate at room temperature for 5 min in the dark.
(8)加PBS至体积为500μL，混匀。(8) Add PBS to a volume of 500 μL and mix.
(9)在1h内在流式细胞仪上完成检测。(9) The test was completed on a flow cytometer within 1 h.
结果参见图12。从图12A我们可以看出，AnnexinV-FITC+PI+双阳性细胞占11.72％(晚期凋亡T细胞)，而在图12B中AnnexinV-FITC+和PI+双阳性细胞占5.41％，说明本申请的BXOS抗PD-1全抗体通过与T细胞表面PD-1分子结合，阻止了DC细胞表面PD-L1分子与PD-1分子的结合，减少了T细胞的凋亡。See Figure 12 for the results. From Fig. 12A, we can see that AnnexinV-FITC+PI+ double positive cells accounted for 11.72% (late apoptotic T cells), while in Figure 12B, AnnexinV-FITC+ and PI+ double positive cells accounted for 5.41%, indicating BXOS resistance of the present application. The PD-1 whole antibody prevents the binding of PD-L1 molecules on the surface of DC cells to PD-1 molecules by binding to PD-1 molecules on the surface of T cells, and reduces the apoptosis of T cells.
实施例8 16号BXOS抗PD-1全抗体对PD-L1/293T-PD-1细胞的结合的阻断作用Example 8 Blocking effect of BXOS anti-PD-1 whole antibody on binding of PD-L1/293T-PD-1 cells
通过流式细胞术(FACS)检测BXOS抗PD-1全抗体与293T-PD-1细胞的结合。293T-PD-1细胞系是由申请人自行构建的可以稳定表达PD-1抗原的细胞系。Binding of BXOS anti-PD-1 whole antibodies to 293T-PD-1 cells was detected by flow cytometry (FACS). The 293T-PD-1 cell line is a cell line constructed by the applicant to stably express the PD-1 antigen.
方法如下：Methods as below:
1)取293T-PD-1细胞，1.5E+5细胞/50μL/管，每管加入2μL PD-L1(ACRO Biosystems,PD1-H5229)(0.4μg/μL)，分别加入BXOS抗PD1全抗体(0.95μg/μL)0、0.3、0.5、1、2、4、8μl，于4℃孵育60min；1) Take 293T-PD-1 cells, 1.5E+5 cells/50μL/tube, add 2μL PD-L1 (ACRO Biosystems, PD1-H5229) (0.4μg/μL) to each tube, and add BXOS anti-PD1 whole antibody ( 0.95 μg/μL) 0, 0.3, 0.5, 1, 2, 4, 8 μl, incubated at 4 ° C for 60 min;
2)PBS洗涤两遍；2) Wash PBS twice;
3)每管加入1.5μL Anti-PD-L1antibody APC流式抗体 (Biolegend,329707)，混匀，4℃孵育35min3) Add 1.5 μL of Anti-PD-L1antibody APC flow antibody (Biolegend, 329707) to each tube, mix and incubate for 35 min at 4 °C.
4)PBS洗涤2次，并在流式细胞仪上测定结果。4) Wash twice with PBS and measure the results on a flow cytometer.
结果如图13所示。随着BXOS抗PD-1全抗体加入量越高，PD-L1与293-PD-1细胞的结合能力越弱，且呈剂量依赖关系，说明BXOS抗PD-1全抗体对PD-1/PD-L1的结合有明显的阻断作用。The result is shown in FIG. The higher the amount of anti-PD-1 total antibody added to BXOS, the weaker the binding ability of PD-L1 to 293-PD-1 cells in a dose-dependent manner, indicating that BXOS anti-PD-1 whole antibody to PD-1/PD The binding of -L1 has a significant blocking effect.
实施例9 16号BXOS抗PD-1全抗体促进PBMC杀伤HepG2-luc肿瘤细胞的作用Example 9 The effect of 16X BXOS anti-PD-1 whole antibody on PBMC killing HepG2-luc tumor cells
测定了16号BXOS抗PD-1全抗体对PBMC杀伤epG2-luc肿瘤细胞的作用。方法如下：The effect of BXOS anti-PD-1 whole antibody on the killing of epG2-luc tumor cells by PBMC was determined. Methods as below:
1)PBMC的分离：淋巴细胞分离液Ficoll分离人外周血单个核细胞PBMC；1) separation of PBMC: lymphocyte separation solution Ficoll separation of human peripheral blood mononuclear cells PBMC;
2)细胞计数：计数PBMC，调整浓度至10 7/mL；计数HepG2-luc细胞，调整浓度至2×10 5/mL； 2) Cell count: Count PBMC, adjust the concentration to 10 7 /mL; count HepG2-luc cells, adjust the concentration to 2 × 10 5 /mL;
3)将PBMC以50μL/孔的浓度加入圆底96孔板中，向50μL PBMC细胞中加入20nM BXOS抗PD1全抗体样品50μL(终浓度10nM)，共三个复孔；阴性对照加入等体积的PBS溶液；3) Add PBMC to a round-bottom 96-well plate at a concentration of 50 μL/well, and add 50 μL of 20 μM BXOS anti-PD1 whole antibody sample (final concentration 10 nM) to 50 μL of PBMC cells for a total of three replicate wells; PBS solution;
4)将HepG2-luc细胞以50μL/孔的浓度加入圆底96孔板中，同样按照实验分组加入20nM BXOS抗PD1全抗体样品50μL(终浓度10nM)，共三个复孔；4) HepG2-luc cells were added to a round-bottom 96-well plate at a concentration of 50 μL/well, and 50 μL of a 20 nM BXOS anti-PD1 whole antibody sample (final concentration 10 nM) was also added in groups according to the experiment, for a total of three replicate wells;
5)将3和4步骤的细胞分别于37℃培养箱静置10min；5) The cells of steps 3 and 4 were each allowed to stand in a 37 ° C incubator for 10 min;
6)把4步骤的HepG2-luc细胞移至3步骤的PBMC中，混合均匀，制备成效靶比为10:1的杀伤孔，每孔终体积为200μL，置于37℃培养箱培养；6) The 4-step HepG2-luc cells were transferred to the 3-step PBMC, mixed uniformly, and the killing wells with a target ratio of 10:1 were prepared, and the final volume per well was 200 μL, and cultured in a 37 ° C incubator;
7)16h后取出96孔板，每孔加入100μL D-luc底物，用小动物成像仪检测生物发光的荧光强度。7) After 16 h, 96-well plates were taken, 100 μL of D-luc substrate was added to each well, and the fluorescence intensity of bioluminescence was measured with a small animal imager.
结果如下表5和图14所示。The results are shown in Table 5 and Figure 14 below.
表5table 5
  H+PH+P BXOS抗PD1全抗体10nMBXOS anti-PD1 full antibody 10nM
杀伤率％Kill rate% 43.2543.25 62.6662.66
  40.6640.66 51.5251.52
  43.6243.62 63.0063.00
平均值average value 42.5142.51 59.0659.06
表5和图14的数据表明，BXOS抗PD-1全抗体在10nM浓度下将PBMC对HepG2的杀伤率由42.51％提高到了59.06％，显著促进了PBMC细胞对肿瘤细胞HepG2-luc的杀伤。The data in Table 5 and Figure 14 show that the BXOS anti-PD-1 whole antibody increased the killing rate of PBMC against HepG2 from 42.51% to 59.06% at 10 nM, which significantly promoted the killing of tumor cells HepG2-luc by PBMC cells.
虽然本申请所披露的实施方式如上，但所述的内容仅为便于理解本申请而采用的实施方式，并非用以限定本申请。任何本申请所属领域内的技术人员，在不脱离本申请所揭露的精神和范围的前提下，可以在实施的形式及细节上进行任何的修改与变化，但本申请的专利保护范围，仍须以所附的权利要求书所界定的范围为准。The embodiments disclosed in the present application are as described above, but the description is only for the purpose of understanding the present application, and is not intended to limit the present application. Any modifications and changes in the form and details of the embodiments may be made by those skilled in the art without departing from the spirit and scope of the disclosure. The scope defined by the appended claims shall prevail.
工业实用性Industrial applicability
本申请实施方案提供的抗PD-1抗体或其衍生物能够有效地调控受试者中的免疫应答、治疗或预防受试者中与PD-1相关的状况或PD-1介导的疾病或病症，适合新药的开发和工业化生产。The anti-PD-1 antibody or derivative thereof provided by the embodiments of the present application is capable of effectively regulating an immune response in a subject, treating or preventing a PD-1-associated condition or a PD-1 mediated disease in a subject or The disease is suitable for the development and industrial production of new drugs.

Claims (48)

  1. 一种分离的抗体或其功能性片段，包括特异性识别和结合免疫细胞表面抗原PD-1的结构域和来自免疫球蛋白恒定区(Fc)的恒定区域，所述特异性识别和结合免疫细胞表面抗原PD-1的结构域包括具有3个CDR的轻链可变区(抗PD-1 VL)和具有3个CDR的重链可变区(抗PD-1 VH)，其中，所述轻链可变区(抗PD-1 VL)包含选自SEQ ID NO：1-16所示的氨基酸序列的轻链CDR(LCDR)；并且所述重链可变区(抗PD-1 VH)包含选自SEQ ID NO：17-31所示的氨基酸序列的重链CDR(HCDR)。An isolated antibody or functional fragment thereof, comprising a domain that specifically recognizes and binds to the immune cell surface antigen PD-1 and a constant region from an immunoglobulin constant region (Fc) that specifically recognizes and binds to immune cells The domain of the surface antigen PD-1 includes a light chain variable region (anti-PD-1 VL) having three CDRs and a heavy chain variable region (anti-PD-1 VH) having three CDRs, wherein the light The chain variable region (anti-PD-1 VL) comprises a light chain CDR (LCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 1-16; and the heavy chain variable region (anti-PD-1 VH) comprises A heavy chain CDR (HCDR) selected from the amino acid sequences set forth in SEQ ID NOS: 17-31.
  2. 根据权利要求1所述的抗体或其功能性片段，其中所述轻链可变区包括：The antibody or functional fragment thereof according to claim 1, wherein the light chain variable region comprises:
    LCDR1，其氨基酸序列如SEQ ID NO：1、2、3、4和5中任一个所列，LCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 1, 2, 3, 4 and 5,
    LCDR2，其氨基酸序列如SEQ ID NO：6、7、8、9和10中任一个所列，和LCDR2, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 6, 7, 8, 9 and 10, and
    LCDR3，其氨基酸序列如SEQ ID NO：11、12、13、14、15和16中任一个所列；并且LCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 11, 12, 13, 14, 15 and 16;
    所述重链可变区包括：The heavy chain variable region comprises:
    HCDR1，其氨基酸序列如SEQ ID NO：17、18、19、20和21中任一个所列，HCDR1, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 17, 18, 19, 20 and 21,
    HCDR2，其氨基酸序列如SEQ ID NO：22、23、24、25和26所列中任一个所列，和HCDR2, the amino acid sequence of which is set forth in any one of the SEQ ID NOs: 22, 23, 24, 25 and 26, and
    HCDR3，其氨基酸序列如SEQ ID NO：27、28、29、30和31中任一个所列。HCDR3, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 27, 28, 29, 30 and 31.
  3. 根据权利要求1或2所述的抗体或其功能性片段，包括选自下组的轻链可变区：The antibody or functional fragment thereof according to claim 1 or 2, comprising a light chain variable region selected from the group consisting of:
    a)轻链可变区VL1，其包括SEQ ID NO:1、SEQ ID NO:7和SEQ ID NO:13；a) a light chain variable region VL1 comprising SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 13;
    b)轻链可变区VL2，其包括SEQ ID NO:2、SEQ ID NO:6和SEQ ID NO:12；b) a light chain variable region VL2 comprising SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 12;
    c)轻链可变区VL3，其包括SEQ ID NO:5、SEQ ID NO:10和SEQ ID NO:16；c) a light chain variable region VL3 comprising SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 16;
    d)轻链可变区VL4，其包括SEQ ID NO:4、SEQ ID NO:8和SEQ ID NO:11；d) a light chain variable region VL4 comprising SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 11;
    e)轻链可变区VL5，其包括SEQ ID NO:3、SEQ ID NO:7和SEQ ID NO:13；以及e) a light chain variable region VL5 comprising SEQ ID NO:3, SEQ ID NO:7 and SEQ ID NO:13;
    f)轻链可变区VL6，其包括SEQ ID NO:4、SEQ ID NO:7和SEQ ID NO:14。f) Light chain variable region VL6 comprising SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 14.
  4. 根据权利要求1或2所述的抗体或其功能性片段，包括选自下组的重链可变区：The antibody or functional fragment thereof according to claim 1 or 2, comprising a heavy chain variable region selected from the group consisting of:
    g)重链可变区VH1，其包括SEQ ID NO:17、SEQ ID NO:22和SEQ ID NO:27；g) a heavy chain variable region VH1 comprising SEQ ID NO: 17, SEQ ID NO: 22 and SEQ ID NO: 27;
    h)重链可变区VH2，其包括SEQ ID NO:18、SEQ ID NO:23和SEQ ID NO:30；h) a heavy chain variable region VH2 comprising SEQ ID NO: 18, SEQ ID NO: 23 and SEQ ID NO: 30;
    i)重链可变区VH3，其包括SEQ ID NO:19、SEQ ID NO:24和SEQ ID NO:29；i) a heavy chain variable region VH3 comprising SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 29;
    j)重链可变区VH4，其包括SEQ ID NO:20、SEQ ID NO:25和SEQ ID NO:30；j) a heavy chain variable region VH4 comprising SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO: 30;
    k)重链可变区VH5，其包括SEQ ID NO:21、SEQ ID NO:26和SEQ ID NO:31。k) Heavy chain variable region VH5 comprising SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 31.
  5. 根据权利要求1或2所述的抗体或其功能性片段，包括选自VL1、VL2和VL3、VL4、VL5和VL6中的任一个的轻链可变区和选自VH1、VH2、VH3、VH4和VH5中的任一个的重链可变区。The antibody or functional fragment thereof according to claim 1 or 2, comprising a light chain variable region selected from any one of VL1, VL2 and VL3, VL4, VL5 and VL6 and selected from the group consisting of VH1, VH2, VH3, VH4 And a heavy chain variable region of any of VH5.
  6. 根据权利要求1所述的抗体或其功能性片段，其中所述抗体或其功能性片段包含The antibody or functional fragment thereof according to claim 1, wherein the antibody or functional fragment thereof comprises
    分别为SEQ ID NO:1、7和13所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:17、22和27所示的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 1, 7, and 13, respectively, and the heavy chain CDR1, CDR2, and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 17, 22, and 27, respectively; or
    分别为SEQ ID NO:2、6和12所示的氨基酸序列的轻链CDR1、CDR2 和CDR3以及分别为SEQ ID NO:18、23和28所示的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 2, 6 and 12, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 18, 23 and 28, respectively; or
    分别为SEQ ID NO:5、10和16所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:19、24和29所示的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences set forth in SEQ ID NOS: 5, 10 and 16, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NOs: 19, 24 and 29, respectively; or
    分别为SEQ ID NO:2、6和12的氨基酸序列的轻链CDR1、CDR2和CDR3；以及分别为SEQ ID NO:21、26和31的氨基酸序列的重链CDR1、CDR2和CDR3；或The light chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOS: 2, 6 and 12, respectively; and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences of SEQ ID NOs: 21, 26 and 31, respectively;
    分别为SEQ ID NO:3、7和13所示的氨基酸序列的轻链CDR1、CDR2和CDR3以及分别为SEQ ID NO:21、26和31所示的氨基酸序列的重链CDR1、CDR2和CDR3，并且The light chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 3, 7 and 13, respectively, and the heavy chain CDR1, CDR2 and CDR3 of the amino acid sequences shown by SEQ ID NOS: 21, 26 and 31, respectively, and
    其中所述抗体或其功能性片段特异性地结合位于人PD-1的胞外结构域内的表位。Wherein the antibody or functional fragment thereof specifically binds to an epitope located within the extracellular domain of human PD-1.
  7. 如权利要求3-6任一项所述的抗体或其功能性部分，其中，所述抗体包含一个或更多个氨基酸置换、添加和/或缺失，或在非CDR区域内的残基中具有一个或更多个保守氨基酸置换。The antibody or functional portion thereof according to any of claims 3-6, wherein the antibody comprises one or more amino acid substitutions, additions and/or deletions, or has residues in non-CDR regions One or more conservative amino acid substitutions.
  8. 如权利要求7所述的抗体或其功能性部分，其中，所述抗体的氨基酸序列与其所来源的序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。The antibody or functional portion thereof according to claim 7, wherein the amino acid sequence of the antibody is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 of the sequence from which it is derived. % homologous.
  9. 一种分离的抗体或其功能性片段，所述抗体或其功能性片段与根据权利要求1-8中任一项所述的抗体或其功能性片段结合的表位相同，或所述抗体或其功能性片段结合的表位与根据权利要求1-8中任一项所述的抗体或其功能性片段结合的表位重叠。An isolated antibody or a functional fragment thereof, the antibody or a functional fragment thereof having the same epitope as the antibody or functional fragment thereof according to any one of claims 1-8, or the antibody or The epitope to which the functional fragment binds overlaps with an epitope that binds to the antibody or functional fragment thereof according to any one of claims 1-8.
  10. 如权利要求1-9任一项所述的抗体或其功能性片段，其编码氨基酸序列包含SEQ ID NO:59-63任一所示序列。The antibody or functional fragment thereof according to any one of claims 1 to 9, which encodes an amino acid sequence comprising the sequence of any of SEQ ID NOS: 59-63.
  11. 如权利要求10所述的抗体或其功能性片段，其编码氨基酸序列轻链氨基酸序列如SEQ ID NO:64所示序列，重链氨基酸序列如SEQ ID NO:65所示。The antibody or functional fragment thereof according to claim 10, which encodes the amino acid sequence light chain amino acid sequence as set forth in SEQ ID NO: 64, and the heavy chain amino acid sequence set forth in SEQ ID NO: 65.
  12. 如权利要求11所述的抗体或其功能性片段，其编码轻链核苷酸序列如SEQ ID NO:66所示序列，编码重链氨基酸序列如SEQ ID NO:67所示。The antibody or functional fragment thereof according to claim 11, which encodes a light chain nucleotide sequence as set forth in SEQ ID NO: 66 and a heavy chain amino acid sequence as set forth in SEQ ID NO:67.
  13. 如权利要求1-12任一项所述的抗体或其功能性片段，其中所述抗体含有糖基化修饰。The antibody or functional fragment thereof of any of claims 1 to 12, wherein the antibody comprises a glycosylation modification.
  14. 如权利要求1-13任一项所述的抗体或其功能性片段，其中所述抗体为天然人源的或人源化的。The antibody or functional fragment thereof of any of claims 1 to 13, wherein the antibody is naturally human or humanized.
  15. 权利要求1-14任一项所述的抗体或其功能性片段，其中所述重链由衍生自人VH种系的氨基酸序列构成；且所述轻链由衍生自人Vκ种系或Vλ种系的氨基酸序列构成。The antibody or functional fragment thereof of any one of claims 1 to 14, wherein the heavy chain is composed of an amino acid sequence derived from a human VH germline; and the light chain is derived from a human VK germline or Vλ species The amino acid sequence of the system is composed.
  16. 权利要求1-14任一项所述的抗体或其功能性片段，其中所述恒定区域是人恒定区域，例如人IgA、IgD、IgE、IgG或IgM的恒定区域，优选是人IgG的恒定区域，更优选是人IgG1或IgG4的恒定区域。The antibody or functional fragment thereof of any one of claims 1 to 14, wherein the constant region is a human constant region, such as a constant region of human IgA, IgD, IgE, IgG or IgM, preferably a constant region of human IgG More preferably, it is a constant region of human IgG1 or IgG4.
  17. 权利要求1-16任一项所述的抗体或其功能性片段，所述抗体是嵌合抗体、人源化抗体、人抗体、纳米抗体、域抗体或双价域抗体或其抗原结合部分，所述抗原结合部分选自Fab、F(ab′)2、Fv、scFv、Fd或dAb。The antibody or functional fragment thereof according to any one of claims 1 to 16, which is a chimeric antibody, a humanized antibody, a human antibody, a Nanobody, a domain antibody or a bivalent domain antibody or an antigen binding portion thereof, The antigen binding portion is selected from the group consisting of Fab, F(ab')2, Fv, scFv, Fd or dAb.
  18. 权利要求1-16任一项所述的抗体或其功能性片段，所述抗体是多特异性抗体，例如双特异性抗体或三特异性抗体。The antibody or functional fragment thereof according to any one of claims 1 to 16, which is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.
  19. 权利要求1～18任一项所述的抗体或其功能性片段，其具备以下功能：The antibody or functional fragment thereof according to any one of claims 1 to 18, which has the following functions:
    (a)结合PD-1，阻断PD-L1与PD-1结合；(a) binding PD-1 to block PD-L1 binding to PD-1;
    (b)拮抗PD-1介导的信号转导；(b) antagonizing PD-1 mediated signal transduction;
    (c)增加T细胞增殖；(c) increasing T cell proliferation;
    (d)增强IFN-γ的产生；或(d) enhancing the production of IFN-γ; or
    (e)以上功能的任意组合。(e) Any combination of the above functions.
  20. 权利要求19所述的抗体或其功能性片段，其中所述功能通过酶联免疫吸附测定(ELISA)、流式细胞术或表面等离子体共振(SPR)测定来测量，所述Kd值通过等离子共振结合法测定。The antibody or functional fragment thereof of claim 19, wherein said function is measured by an enzyme-linked immunosorbent assay (ELISA), flow cytometry or surface plasmon resonance (SPR) assay, said Kd value by plasmon resonance Determine by binding method.
  21. 权利要求1-18任一项所述的抗体或其功能性片段，其中所述抗体或 其功能性片段结合PD-1，其对PD-1具有10 -9mol/L的亲和力。 The antibody or functional fragment thereof of any one of claims 1 to 18, wherein the antibody or a functional fragment thereof binds to PD-1, which has an affinity for PD-1 of 10 -9 mol/L.
  22. 一种分离的核酸，其编码权利要求1所述的抗体或其功能性片段或权利要求3-6中任一项所述的重链可变区和轻链可变区或权利要求17所述的scFv。An isolated nucleic acid encoding the antibody of claim 1 or a functional fragment thereof, or the heavy chain variable region and the light chain variable region of any one of claims 3-6 or claim 17 The scFv.
  23. 权利要求22所述的核酸序列包含SEQ ID NO:54-58任一所示核苷酸序列。The nucleic acid sequence of claim 22 comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 54-58.
  24. 一种包含权利要求22所述的核酸的表达载体。An expression vector comprising the nucleic acid of claim 22.
  25. 一种包含权利要求24所述的表达载体的宿主细胞，其中所述宿主细胞选自大肠杆菌、HEK293细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞和NSO细胞。A host cell comprising the expression vector of claim 24, wherein the host cell is selected from the group consisting of Escherichia coli, HEK293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and NSO cells.
  26. 一种免疫缀合物或其衍生物，包含与治疗剂偶联的根据权利要求1至13中任一项所述的抗体或其功能性片段，所述治疗剂为毒素、放射性同位素、药物或细胞毒剂。An immunoconjugate or a derivative thereof, comprising an antibody according to any one of claims 1 to 13 or a functional fragment thereof, which is a toxin, a radioisotope, a drug or Cytotoxic agent.
  27. 一种药物组合物，包含权利要求1-21任一项的抗体或其功能性片段或权利要求25所述的免疫缀合物或其衍生物，以及药学上可接受的赋形剂、载体或稀释剂。A pharmaceutical composition comprising the antibody of any one of claims 1 to 21, or a functional fragment thereof, or the immunoconjugate of claim 25 or a derivative thereof, and a pharmaceutically acceptable excipient, carrier or Thinner.
  28. 权利要求1-21任一项所述的抗体或其功能性片段或权利要求26所述的免疫缀合物或衍生物或权利要求27所述的药物组合物在制备用于调控受试者中的免疫应答、治疗或预防受试者中与PD-1相关的状况或PD-1介导的疾病或病症的药物中的用途。The antibody or functional fragment thereof according to any one of claims 1 to 21, or the immunoconjugate or derivative of claim 26 or the pharmaceutical composition according to claim 27, for use in the preparation of a subject for modulation Use of an immune response, treatment or prevention of a PD1-related condition or a PD-1 mediated disease or condition in a subject.
  29. 根据权利要求28所述的用途，所述药物包括阻断PD-1与PD-L1结合的药物，所述阻断PD-1与PD-L1结合的药物包括通过PD-1抗体结合PD-1抗原，解除PD-1对T细胞活性的弱化调控的药物、解除PD-1对机体免疫抑制的药物或者提高T淋巴细胞中IFN-γ表达的药物。The use according to claim 28, wherein the drug comprises a drug that blocks PD-1 binding to PD-L1, and the drug that blocks PD-1 binding to PD-L1 comprises binding PD-1 by PD-1 antibody An antigen, a drug that abolishes the weakening regulation of T cell activity by PD-1, a drug that relieves PD-1 against immunosuppression of the body, or a drug that increases the expression of IFN-γ in T lymphocytes.
  30. 权利要求1～21任一项所述的抗体或其功能性片段或权利要求26所述的免疫缀合物或其衍生物或权利要求27所述的药物组合物在制备用于治疗或预防受试者中选自自身免疫紊乱、针对移植物的免疫应答、***反应和癌症的紊乱的药物中的用途。The antibody or functional fragment thereof according to any one of claims 1 to 21, or the immunoconjugate of claim 26 or a derivative thereof, or the pharmaceutical composition according to claim 27, for use in the preparation of a therapeutic or prophylactic Among the subjects, the use of a drug selected from the group consisting of an autoimmune disorder, an immune response against a graft, an allergy, and a disorder of cancer.
  31. 一种治疗受试者中与PD-1相关的状况或PD-1介导的疾病或病症的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求1-21中任一项所述的分离的抗体或其功能性片段或如权利要求26所述的免疫缀合物或其衍生物或者如权利要求27所述的药物组合物。A method of treating a condition associated with PD-1 or a PD-1 mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of any of claims 1-21. An isolated antibody or functional fragment thereof, or an immunoconjugate or derivative thereof according to claim 26 or a pharmaceutical composition according to claim 27.
  32. 一种偶联物，包含权利要求1～21任一项所述的抗体或其功能性片段和偶联部分，其中所述偶联部分为可检测的标记；所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。A conjugate comprising the antibody of any one of claims 1 to 21, or a functional fragment thereof, and a coupling moiety, wherein the coupling moiety is a detectable label; the coupling moiety is a radioisotope, A fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  33. 一种试剂盒，包括权利要求1至21中任一项所述的抗体或其功能性片段，或权利要求32所述的偶联物，所述试剂盒还包括第二抗体，所述第二抗体特异性识别所述抗体或其功能性片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。A kit comprising the antibody of any one of claims 1 to 21, or a functional fragment thereof, or the conjugate of claim 32, further comprising a second antibody, said second The antibody specifically recognizes the antibody or a functional fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.
  34. 一种增强T细胞活化的方法，所述方法包括使T细胞与根据权利要求1-21中任一项所述的抗体或其功能性片段接触。A method of enhancing T cell activation, the method comprising contacting a T cell with an antibody or a functional fragment thereof according to any one of claims 1 to 21.
  35. 一种减少受试者的肿瘤或抑制受试者的肿瘤细胞生长的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求1-21中任一项所述的抗体或其功能性片段。A method of reducing tumor growth in a subject or inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of claims 1-21 or Its functional fragment.
  36. 一种治疗有需要的受试者的癌症的方法，所述方法包括向所述受试者施用治疗有效量的根据权利要求1-21中任一项所述的抗体或其功能性片段。A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or functional fragment thereof according to any one of claims 1-21.
  37. 根据权利要求28-30任一项所述的用途或权利要求34-36任一项所述的方法，其中所述受试者被鉴定为患有可能对PD-1拮抗剂响应的病症或状况，或者在来自所述个体的待测生物样品中呈PD-L1阳性或PD-L1水平上调。The use of any one of claims 28-30, or the method of any one of claims 34-36, wherein the subject is identified as having a condition or condition that is likely to respond to a PD-1 antagonist, Or it is up-regulated in PD-L1 positive or PD-L1 levels in the biological sample to be tested from the individual.
  38. 一种治疗会从上调的免疫响应中获益的受试者的状况的方法，包括对所述受试者施用治疗有效量的根据权利要求1-21中任意一项所述的抗体或其功能性片段。A method of treating a condition of a subject who would benefit from an up-regulated immune response, comprising administering to the subject a therapeutically effective amount of the antibody of any of claims 1-21 or a function thereof Sexual fragment.
  39. 根据权利要求28-30任一项所述的用途或权利要求34-37任一项所述的方法，用于治疗选自癌症，感染性疾病和炎性疾病的状况。Use according to any one of claims 28 to 30 or a method according to any one of claims 34 to 37 for the treatment of a condition selected from the group consisting of cancer, infectious diseases and inflammatory diseases.
  40. 根据权利要求39所述的用途或方法，其中所述癌症，感染性疾病和 炎性疾病与PD-1相关。The use or method of claim 39, wherein the cancer, infectious disease, and inflammatory disease are associated with PD-1.
  41. 根据权利要求39所述的用途或方法，其中所述癌症选自胃癌、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、***、***癌、食道癌、小肠癌、甲状腺癌、甲状旁腺癌、黑素瘤、肾癌、***癌、乳癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头颈部癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肾上腺癌、肛区癌、***癌、尿道癌、***癌、膀胱癌、肾或输尿管癌、肾盂癌、表皮样癌、鳞状细胞癌、何杰金氏病、非何杰金氏淋巴瘤、内分泌***的癌症、软组织肉瘤、中枢神经***的赘生物、原发性中枢神经***淋巴瘤、肿瘤血管发生、脊髓轴肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏肉瘤、T细胞淋巴瘤、慢性或急性白血病(其包括急性髓细胞样白血病、慢性髓细胞样白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童期实体瘤和淋巴细胞性淋巴瘤中的一种或更多种。The use or method according to claim 39, wherein the cancer is selected from the group consisting of gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, esophageal cancer, small intestine cancer, thyroid cancer, and thyroid Adenocarcinoma, melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectum Cancer, adrenal cancer, anal cancer, vulvar cancer, urinary tract cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, epidermoid carcinoma, squamous cell carcinoma, Hodgkin's disease, non-Hodgkin's lymph Tumor, endocrine system cancer, soft tissue sarcoma, central nervous system neoplasm, primary central nervous system lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, T-cell lymphoma, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia), childhood solid tumors and One or more bar-cell lymphoma.
  42. 根据权利要求39所述的用途或方法，其中所述感染性疾病选自HIV，流行性感冒，疱疹，贾第虫病，疟疾，利什曼病，或以下病毒引起的感染：肝炎病毒(例如甲型、乙型或丙型肝炎病毒)、疱疹病毒(例如VZV、HSV-1、HAV-6、HSV-II、CMV或埃巴二氏病毒)、腺病毒、流感病毒、牛痘病毒、HTLV病毒、登革热病毒、***瘤病毒、软疣病毒、脊髓灰质炎病毒、狂犬病病毒、黄病毒、艾柯病毒、鼻病毒、柯萨奇病毒、冠状病毒、呼吸道合胞病毒、腮腺炎病毒、轮状病毒麻疹病毒、风疹病毒、细小病毒、JC病毒和虫媒病毒性脑炎病毒，或以下细菌引起的感染：肺炎球菌、分枝杆菌、葡萄球菌、链球菌、脑膜炎球菌、***、沙雷氏菌、克雷伯氏菌、变形菌、假单胞菌、沙门氏菌、霍乱弧菌、白喉杆菌、肉毒杆菌、炭疽杆菌、破伤风梭菌、军团菌、鼠疫杆菌、钩端螺旋体病或莱姆氏病细菌，或以下真菌引起的感染：曲霉(例如烟曲霉、黑曲霉等)、假丝酵母(例如白色假丝酵母)、克鲁斯假丝酵母、光滑假丝酵母、热带假丝酵母、新型隐球酵母、皮炎芽酵母、毛霉目的属(例如毛霉属，犁头霉属或根霉属)、申克氏孢子丝菌、巴西副球孢子菌、粗球孢菌或加膜组织胞浆菌，或以下寄生虫引起的感染：痢疾内变形虫、结肠肠袋虫、福纳氏虫、棘变形虫、间日疟原虫、田鼠巴贝虫、吸吮贾第虫、隐孢子虫、卡氏肺囊虫、布鲁斯锥虫、克鲁兹锥虫、多氏利什曼虫、鼠弓浆 虫或巴西日圆线虫。The use or method according to claim 39, wherein the infectious disease is selected from the group consisting of HIV, influenza, herpes, giardiasis, malaria, leishmaniasis, or an infection caused by a virus: Hepatitis A, B or C virus, herpes virus (eg VZV, HSV-1, HAV-6, HSV-II, CMV or Epstein's virus), adenovirus, influenza virus, vaccinia virus, HTLV virus , dengue virus, papillomavirus, soft prion, poliovirus, rabies virus, flavivirus, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus Measles virus, rubella virus, parvovirus, JC virus and arbovirus viral encephalitis virus, or infections caused by bacteria: pneumococcal, mycobacteria, staphylococcus, streptococcus, meningococcus, gonococcus, serrano Bacteria, Klebsiella, Proteobacteria, Pseudomonas, Salmonella, Vibrio cholerae, Diphtheria, Botox, Bacillus anthracis, Clostridium tetani, Legionella, Yersinia, Leptospirosis Or Lyme disease bacteria, or infections caused by the following fungi: Aspergillus (eg Aspergillus fumigatus, Aspergillus niger, etc.), Candida (eg Candida albicans), Candida krusei, Candida glabrata, tropical leave Saccharomyces cerevisiae, Cryptococcus neoformans, dermatitis bud, Mucor genus (such as Mucor, Absidia or Rhizopus), S. skrjab, Parastaminium, Coccidioides or Addition of tissue cytoplasmic bacteria, or infections caused by the following parasites: amoeba in the dysentery, colonic intestines, flucler, anatomium, vivax malaria, squirrel babe, sucking giardia, hidden Sporozoans, Pneumocystis carinii, Trypanosoma brucei, Trypanosoma cruzi, Leishmania polygala, Rat toxoplasma gondii or Nematode.
  43. 根据权利要求39所述的用途或方法，其中所述疾病为癌症，所述癌症包括肺癌、肝癌、卵巢癌、***、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌或头颈癌、肠癌、黑素瘤、非小细胞肺癌、；所述感染性疾病为慢性病毒感染、细菌感染或寄生虫感染疾病，所述慢性病毒为HIV、HBV或HCV。The use or method according to claim 39, wherein the disease is cancer, and the cancer includes lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney. Cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma or head and neck cancer, intestinal cancer, melanoma, non-small cell lung cancer; the infectious disease is a chronic viral infection, a bacterial infection or a parasitic infection disease, the chronic The virus is HIV, HBV or HCV.
  44. 权利要求28～30任一项所述的用途或权利要求34-37任一项所述的方法，其中所述用途或方法是预防性的，并在癌症、自身免疫疾病、传染病或影响T细胞数目或影响健康的疾病的任何症状出现之前被提供。The use according to any one of claims 28 to 30, or the method of any one of claims 34 to 37, wherein the use or method is prophylactic and is in cancer, autoimmune disease, infectious disease or affecting T The number of cells or any symptoms of a disease affecting health is provided before the onset of any symptoms.
  45. 权利要求1至21中任一项所述的抗体或其功能性片段或权利要求32所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PD-1在样品中的存在或其水平。The use of the antibody of claim 1 or a functional fragment thereof, or the conjugate of claim 32, in a kit for detecting PD-1 in a sample Exist or its level.
  46. 一种检测样品中的PD-1的存在的方法，所述方法包括使权利要求1-21中任一项所述的抗体或其功能性片段或权利要求26所述的免疫缀合物或其衍生物或权利要求32所述的偶联物与所述样品接触。A method of detecting the presence of PD-1 in a sample, the method comprising the antibody of any one of claims 1 to 21, or a functional fragment thereof, or the immunoconjugate of claim 26, or The derivative or the conjugate of claim 32 is contacted with the sample.
  47. 一种用于检测样品中的PD-1的存在的组合物，所述组合物包含权利要求1-21中任一项所述的抗体或其功能性片段。A composition for detecting the presence of PD-1 in a sample, the composition comprising the antibody of any one of claims 1 to 21 or a functional fragment thereof.
  48. 一种制备权利要求1-21中任一项所述的抗体的方法，所述方法是噬菌体展示法。A method of producing the antibody of any one of claims 1 to 21, which is a phage display method.
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