WO2019096136A1 - Anticorps anti-pd-1, son procédé de préparation et son utilisation - Google Patents

Anticorps anti-pd-1, son procédé de préparation et son utilisation 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

L'invention concerne une molécule d'anticorps anti-PD-1 comprenant une combinaison de régions variables, une molécule d'acide nucléique codant pour la molécule d'anticorps, un vecteur d'expression correspondant, une cellule hôte, un procédé de criblage de l'anticorps à l'aide d'une bibliothèque de phages, et un immunoconjugué, une molécule d'anticorps bis-spécifique ou poly-spécifique et une composition pharmaceutique comprenant la molécule d'anticorps. La molécule d'anticorps ou un composé couplé, un conjugué, un dérivé ou une composition de celui-ci sont utilisés pour traiter des maladies cancéreuses et des maladies infectieuses. La molécule d'anticorps est utilisée pour détecter PD-1.
PCT/CN2018/115285 2017-11-14 2018-11-13 Anticorps anti-pd-1, son procédé de préparation et son utilisation WO2019096136A1 (fr)

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TW202204418A (zh) * 2020-07-16 2022-02-01 大陸商和鉑醫藥（上海）有限責任公司 Ｐｄ－１抗原結合蛋白及其應用

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