WO2018113258A1 - 抗pd-1抗体及其用途 - Google Patents

抗pd-1抗体及其用途 Download PDF

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WO2018113258A1
WO2018113258A1 PCT/CN2017/092026 CN2017092026W WO2018113258A1 WO 2018113258 A1 WO2018113258 A1 WO 2018113258A1 CN 2017092026 W CN2017092026 W CN 2017092026W WO 2018113258 A1 WO2018113258 A1 WO 2018113258A1
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antibody
seq
variable region
cdr
chain variable
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French (fr)
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李强
郑云程
杨璐
马心鲁
李媛丽
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Ampsource Biopharma Shanghai Inc
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Ampsource Biopharma Shanghai Inc
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Priority to EP17885356.0A priority Critical patent/EP3575318B1/en
Priority to MYPI2019003619A priority patent/MY197695A/en
Priority to KR1020197021501A priority patent/KR102340832B1/ko
Priority to US16/471,909 priority patent/US11161904B2/en
Priority to JP2019555527A priority patent/JP6908722B2/ja
Priority to RU2019120461A priority patent/RU2739610C1/ru
Publication of WO2018113258A1 publication Critical patent/WO2018113258A1/zh
Priority to PH12019501438A priority patent/PH12019501438A1/en
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • 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 invention is in the field of therapeutic monoclonal antibodies, and more particularly, the present invention relates to an antibody against a programmed death receptor (PD-1); and to the treatment of various diseases including cancer and infectious diseases Use in inflammatory diseases).
  • PD-1 programmed death receptor
  • PD-1 Programmed Death-1
  • TCR T cell receptor
  • BCR B cell receptor
  • the PD-1 molecule is composed of an extracellular region, a transmembrane region, and an intracellular region.
  • the extracellular domain contains an immunoglobulin variable region IgV domain
  • the intracellular region contains two tyrosine-based signal transduction motifs ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor) Tyrosine conversion phantom).
  • PD-1 aggregates tyrosine phospholipase SHP2 mainly through the ITSM motif, resulting in dephosphorylation of effector molecules including CD3 ⁇ , PKC ⁇ and ZAP70.
  • PD-1 ligands There are two PD-1 ligands: PD-L1 and PD-L2.
  • PD-L1 is also known as B7H1 or CD274, and PD-L2 is called B7DC or CD273.
  • B7H1 or CD274 B7DC or CD273.
  • PD-L1 and PD-L2 are expressed in different cell populations (Shimauchi, Kabashima et al, Int J Cancer, 2007; 121(12): 2585-2590).
  • PD-L2 expression is relatively limited, mainly in activated macrophages, dendritic cells and a few tumor cells.
  • PD-L1 is widely expressed on activated T cells, B cells, macrophages, dendritic cells and tumor cells, and is also in the body's immune shielding sites such as placenta, eyes and epithelium, muscle, liver and vascular endothelium. Organizational expression.
  • PD-1 is able to interact with ligands (Programmed Death-1 Ligands, PD-Ls) PD-L1 and PD-L2, and significantly inhibits CD3 and CD28-mediated T cell activation and cytokine production via intracellular signal transduction pathways. Therefore, it is an important immune checkpoint for regulating T cell responses. Under normal circumstances, The PD-1/PD-Ls signaling pathway can induce and maintain immune tolerance in peripheral tissues, and has a positive effect on preventing excessive inflammatory reactions in tissues and the development of autoimmune diseases (Latchman Y et al., Nat Immunol, 2001; 2:261). -268).
  • PD-1 interacts with ligands PD-L1 and PD-L2 to down-regulate the secretion of T-cell immunostimulatory cytokines such as IFN- ⁇ , IL-2 and TNF- ⁇ , and the expression of survivin.
  • T-cell immunostimulatory cytokines such as IFN- ⁇ , IL-2 and TNF- ⁇
  • the secretion of the immunosuppressive cytokine IL-10 is promoted, thereby suppressing the T cell immune response (Hamido et al, Expert Opin Biol Ther, 2013; 13(6): 847-861).
  • PD-1 synergistic inhibition signals are closely related to the occurrence of various diseases in humans and can be used as target molecules for disease treatment (Okazaki T et al, J Immunol. 2007; 19: 813-824).
  • PD-1/PD-L1 signaling pathway is closely related to tumor development.
  • high expression of PD-L1 can enhance tumor metastasis, leading to increased mortality and poor prognosis.
  • human lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer Highly expressed PD-L1 protein was detected in both.
  • tumor cells also highly expressed PD-L1 under the induction of various cytokines. This phenomenon is related to tumor immune escape.
  • invasive CD 8+ T cells in tumor sites are also affected by tumor microenvironment, PD-1 expression.
  • T cells in peripheral blood. It acts on the surface of tumor cells with PD-L1 and inhibits the activation and proliferation of T cells. Tumor cells can escape the killing effect of cytotoxic lymphocytes (CTL). , weaken the body's anti-tumor immune response. Blocking PD-1/PD-L1 signaling with blocking anti-PD-1 monoclonal antibody can up-regulate IFN- ⁇ , IL-2, IL-10 secretion and effectively reverse the proliferation of CD 4+ and CD 8+ T cells Inhibition, while significantly increasing the degree of activation and killing of T cells (Dong HD et al, Nat Med, 2002; 8: 793-800).
  • CTL cytotoxic lymphocytes
  • a variety of chronic and acute viral infections also evade human immune surveillance through PD-1/PD-Ls signaling.
  • Peripheral virus-specific CD 4+ and CD 8+ T cells overexpress PD-1, leading to dysfunction or inability to clean infected virus in a timely and effective manner (Narasimhan J et al., J Virol, 2008; 376: 140-153) .
  • Recently, a large number of studies have shown that blocking the PD-1/PD-Ls inhibition pathway by specific monoclonal antibodies can effectively activate the proliferation of virus-specific CD 4+ and CD 8+ T such as HIV, HBV and HCV, and produce IFN.
  • Killing factors such as ⁇ , TNF- ⁇ and granzyme B restore immune cell-specific antiviral function (Barber DL et al, Nature, 2006; 439: 682-687).
  • a specific anti-PD-1 monoclonal antibody can be prepared, which can block the action of the inhibitory pathway by specifically blocking the PD-1/PD-L1 signal, enhance the function of the CTL to kill tumor cells, and effectively inhibit the shape of the tumor. Growth and growth.
  • PD-1 inhibitor anti-tumor antibody drugs namely Pembrolizumab (trade name Keytruda, developed by Merck) and Nivolumab (trade name Opdivo, Ono Pharmaceutical / Bristol/Myers Squibb); developed by CureTech Pidilizumab is in Phase II clinical research; AMP-224 and AMP-514 developed by MedImmune are in Phase I clinical research.
  • the invention provides an isolated PD-1 binding monoclonal antibody comprising: a heavy chain variable region comprising a CDR-H1, CDR-H2 and CDR-H3 sequence; and a light chain variable region, It comprises CDR-L1, CDR-L2 and CDR-L3 sequences, wherein:
  • the heavy chain variable region comprises a CDR-H1 sequence selected from the group consisting of SEQ ID NOs: 1 and 2, and a CDR-H2 sequence selected from the group consisting of SEQ ID NOs: 3 and 4, and is selected from the group consisting of SEQ ID NOs : CDR-H3 sequences shown in 5 and 6;
  • a light chain variable region comprising a CDR-L1 sequence selected from the group consisting of SEQ ID NOs: 7 and 8, and a CDR-L2 sequence selected from the group consisting of SEQ ID NOs: 9 and 10, and selected from the group consisting of SEQ ID NOs : CDR-L3 sequences shown in 11 and 12.
  • the heavy chain variable region of the antibody comprises the CDR-H1 sequence set forth in SEQ ID NO: 1, and the CDR-H2 sequence set forth in SEQ ID NO: 3, and SEQ ID NO The CDR-H3 sequence shown in 5; and the light chain variable region thereof comprises the CDR-L1 sequence shown in SEQ ID NO: 7, and the CDR-L2 sequence shown in SEQ ID NO: 9, and SEQ ID NO: The CDR-L3 sequence shown in Figure 11.
  • the heavy chain variable region of the antibody comprises the CDR-H1 sequence set forth in SEQ ID NO: 2, and the CDR-H2 sequence set forth in SEQ ID NO: 4, and SEQ ID NO: 6 Shown CDR-H3 sequence; and the light chain variable region thereof comprises the CDR-L1 sequence set forth in SEQ ID NO:8, and the CDR-L2 sequence set forth in SEQ ID NO:10, and SEQ ID NO:12 CDR-L3 sequence.
  • the antibody comprising the above CDR sequences is murine, chimeric or humanized.
  • the antibody is murine or chimeric, the heavy chain variable region further comprising a heavy chain FR region of murine IgGl, IgG2, IgG3, IgG4 or variants thereof; and the light chain variable region thereof comprising the murine ⁇ The light chain FR region of the lambda chain or variant thereof.
  • the above murine or chimeric antibody comprises:
  • the murine antibody AB12N1 and the chimeric antibody AB12N2 comprise the amino acid sequence set forth in SEQ ID NO: 15; and the light chain variable region thereof comprises SEQ ID NO: The amino acid sequence shown.
  • the murine or chimeric antibody described above comprises:
  • the murine antibody AB12M1 and the chimeric antibody AB12M2 comprise the amino acid sequence set forth in SEQ ID NO: 13; and the light chain variable region thereof comprises SEQ ID NO: 14 The amino acid sequence shown.
  • the antibody is humanized.
  • Methods of making humanized antibodies are well known to those skilled in the art.
  • a humanized anti-PD-1 antibody of the invention can be prepared by transferring a CDR sequence of the invention into a variable region of a human antibody.
  • the humanized antibody does not produce an anti-antibody reaction (AAR) and a human anti-mouse antibody response (HAMA) and is not rapidly cleared by neutralization by anti-antibody.
  • AAR anti-antibody reaction
  • HAMA human anti-mouse antibody response
  • the murine antibody AB12M1 described above is humanized by CDR-grafting.
  • the humanized antibody thus produced, more preferably, the heavy chain variable region thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25, 27 and 29;
  • the chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, 26, 28 and 30.
  • the humanized antibodies AB12M3, AB12M4, AB12M5, AB12M6, AB12M7, AB12M8 and AB12M9 thus produced have heavy chain variable regions comprising SEQ ID NOs: 17, 19, 21, 23, 25, respectively. , amino acid sequences shown in 27 and 29; and its light chain
  • the variable regions comprise the amino acid sequences set forth in SEQ ID NOS: 18, 20, 22, 24, 26, 28 and 30, respectively.
  • the murine antibody AB12N1 described above is humanized by CDR-grafting.
  • the humanized antibody thus produced, more preferably, the heavy chain variable region thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 33 and 35; and the light chain variable region thereof comprises a SEQ ID NOs: amino acid sequences shown in 32, 34 and 36.
  • the humanized antibodies AB12N3, AB12N4 and AB12N5 thus produced have a heavy chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 31, 33 and 35, respectively; and a light chain variable thereof The regions comprise the amino acid sequences set forth in SEQ ID NOS: 32, 34 and 36, respectively.
  • sequences of the antibodies of the invention without substantial impact on antibody activity (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids to obtain variants of the antibody sequences. They are all considered to be included within the scope of the invention. Amino acids having similar properties are replaced as in the variable region.
  • the sequence of the variant of the invention may be at least 80% homologous to its source sequence; more preferably, the sequence of the variant of the invention may be at least 85%, 90%, 95%, 96%, 97% of its source sequence, 98% or 99% homologous.
  • the antibody of the present invention may be a full length antibody, for example, in some preferred embodiments, the anti-human PD-1 antibody of the present invention further comprises a heavy chain constant region of human IgG4 or IgG1 and a human kappa light chain constant region;
  • the antibody may comprise only an antigen binding fragment, such as a Fab or Fab' 2 fragment, or a single chain antibody ScFv.
  • the antibody of the invention is capable of binding PD-1 with a KD of about 1 nM or less; in a more preferred embodiment, the antibody or antigen binding thereof is capable of a KD of about 100 pM or less. Binding to PD-1; in a more preferred embodiment, the antibody is capable of binding PD-1 with a KD of about 10 pM or less; in a most preferred embodiment, the antibody is capable of a KD of about 1 pM or less Combine PD-1.
  • a DNA molecule encoding an antibody as described above is provided.
  • a DNA molecule encoding a heavy chain variable region of the preferred chimeric antibody AB12M2 of the invention is set forth in SEQ ID NO: 37, and the DNA molecule encoding its light chain variable region is set forth in SEQ ID NO:38.
  • a DNA molecule encoding a preferred humanized antibody AB12M3 heavy chain variable region of the invention is set forth in SEQ ID NO: 39, and the DNA molecule encoding its light chain variable region sequence is set forth in SEQ ID NO:40. .
  • a DNA molecule encoding another heavy human variable region of the humanized antibody AB12M4 of the present invention As shown in SEQ ID NO: 41, and the DNA molecule encoding its light chain variable region is set forth in SEQ ID NO:42.
  • an expression vector comprising a DNA molecule as described above is provided.
  • a host cell transformed with an expression vector as described above is provided.
  • the host cell is preferably a CHO cell.
  • an immunoconjugate comprising an antibody of the invention conjugated to a therapeutic agent.
  • the therapeutic agent is preferably a toxin, a radioisotope, a drug or a cytotoxic agent.
  • any bispecific molecule comprising an antibody of the invention.
  • the PD-1 antibody described above can be functionally linked to an antibody or antibody fragment having another antigen binding property to form a bispecific antibody.
  • the bispecific antibodies include, but are not limited to, antibodies against VEGF, EGFR, Her2/neu, VEGF receptor or other growth factor receptor, CD20, CD40, CTLA-4, OX-40, 4- IBB and ICOS.
  • composition comprising an antibody of the invention together with a pharmaceutically acceptable excipient, carrier or diluent.
  • a method of producing the antibody of the present invention which comprises: (a) cultivating the above-described host cell of the present invention under conditions permitting production of the antibody; (b) recovering, separating, and producing the same Said antibodies.
  • a further aspect of the present invention further relates to an antibody, or a pharmaceutical composition comprising the same, or a bispecific molecule comprising the same, or a bispecific molecule comprising the same, prepared according to the present invention, for use in the treatment Use in drugs for PD-1 mediated diseases or conditions.
  • the disease is preferably cancer; more preferably, a cancer that highly expresses PD-L1; and the cancer includes, but is not limited to, lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer. , glioma, renal cancer, gastric cancer, esophageal cancer, oral squamous cell carcinoma, head and neck cancer; preferably breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma; most preferably non-small cell lung cancer, Melanoma and kidney cancer.
  • the disease is preferably an infectious disease; for example, a chronic viral infection, a bacterial infection or a parasitic infection. More preferably, the infectious disease is HIV, HBV and HCV.
  • chimeric, humanized anti-PD-1 antibodies can be used in the preparation of a medicament for the treatment of cancer or an infectious disease; more preferably, humanized.
  • an antineoplastic or immunogenic agent eg, a tumor antigen
  • an antigen presenting cell eg, a dendritic cell stimulated with a tumor-derived antigen or nucleic acid
  • an immunostimulatory cytokine eg, IL-2, IFNa2
  • GM-CSF tumor-derived antigen or nucleic acid
  • cells transfected with a gene encoding an immunostimulatory cytokine such as, but not limited to, GM-CSF
  • standard cancer treatment eg, chemotherapy, radiation, or surgery
  • antibodies including but not limited to Antibodies to the following molecules: VEGF, EGFR, Her2/neu, VEGF receptor or other growth factor receptor, CD20, CD40, CTLA-4, OX-40, 4-IBB and ICOS).
  • the anti-PD-1 humanized antibody prepared by the invention has higher binding affinity than the clinically used Keytruda and Opdivo, the affinity constant KD value is less than 1 pM, and has extremely strong specificity.
  • the humanized antibody provided by the present invention can significantly inhibit the growth of transgenic mouse xenografts, and even some of the mice have completely disappeared.
  • the antibody of the present invention is expressed by CHO cells, and has the advantages of high yield, high activity, simple purification process, and low production cost.
  • FR antibody framework region immunoglobulin variable region that excludes CDR regions
  • IC 50 produces a concentration of 50% inhibition
  • V region A sequence of variable IgG chain segments between different antibodies. It extends to the 109-position Kabat residue of the light chain and the 113th residue of the heavy chain.
  • antibody encompasses full length antibodies (eg, IgGl or IgG4 antibodies), various functional fragments thereof (eg, may comprise only antigen binding portions, such as Fab, F(ab')2 or scFv fragments) And modified antibodies (eg, humanization, glycosylation, etc.).
  • the invention also encompasses anti-PD-1 antibodies having glycosylation modifications. In some applications, modifications are made to remove undesired glycosylation sites, such as de-fucose modification on oligosaccharide chains to enhance antibody-dependent cellular cytotoxicity (ADCC) function; in other applications, galactose may be performed Modifications to alter complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cellular cytotoxicity
  • monoclonal antibody or mAb refers to an antibody obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic or phage clonal cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
  • Antibody fragments and “antigen-binding fragments” mean antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody.
  • the antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as ScFv, single antibodies (technology from Genmab); Antibodies (techniques from Domantis); domain antibodies (technology from Ablynx); and multispecific antibodies formed from antibody fragments.
  • Engineered antibody variants are reviewed in Holliger et al, 2005; Nat Biotechnol, 23: 1126-1136.
  • a "Fab fragment” consists of a light chain and a heavy chain CH1 and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. Two heavy chain pieces The segments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
  • a "Fab' fragment” contains a VH domain and a CH1 domain of a light chain and a heavy chain and a constant region portion between the CH1 and CH2 domains, thereby being between the two heavy chains of the two Fab' fragments
  • An interchain disulfide bond is formed to form a F(ab')2 molecule.
  • F(ab')2 fragment contains a VH domain and a CH1 domain of two light and two heavy chains and a constant region portion between the CH1 and CH2 domains, thereby forming a chain between the two heavy chains Inter-disulfide bond.
  • the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • a “single-chain Fv antibody” refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • scFv single-chain Fv antibody
  • an "antigen-binding fragment” is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain.
  • hypervariable region refers to an antibody amino acid residue responsible for antigen binding.
  • the hypervariable region comprises the following amino acid residues: amino acid residues from the "complementarity determining region" or "CDR" as defined by the sequence alignment, eg, 24-34 (L1), 50-56 of the light chain variable domain ( Residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of L2) and 89-97 (L3) residues and heavy chain variable domains, see Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md.; and/or from a "hyperbolic ring" (HVL) defined by structure Residues, for example, 26-32 (L1), 50-52 (L2) and 91-96 (L3) residues of the light chain variable domain and 26-32 (H1) of the heavy chain variable domain , 53-
  • CDR complementar
  • Chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody to a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is selected and then cloned from a mouse hybridoma cell.
  • the variable region gene is cloned according to the need to clone the constant region gene of the human antibody, and the mouse variable region gene and the human constant region gene are ligated into a chimeric gene and inserted into the vector, and finally in the eukaryotic expression system or the prokaryotic expression system. Expression of a chimeric antibody molecule.
  • the antibody light chain variable region of the PD-1 chimeric antibody further comprises a light chain FR region of a murine ⁇ , ⁇ chain or variant thereof.
  • the antibody heavy chain variable region of the PD-1 chimeric antibody further comprises a heavy chain FR region of murine IgGl, IgG2, IgG3 or IgG4 or variants thereof.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC after amino acid mutation (antibody dependent Cell-mediated cytotoxicity) toxic IgG1.
  • bispecific molecule means that an anti-PD-1 antibody or antigen-binding fragment thereof of the invention can be derivatized or linked to another functional molecule, such as another peptide or protein (eg, a tumor associated antigen, Cytokines and cell surface receptors) to generate bispecific molecules that bind to at least two different binding sites or target molecules.
  • another functional molecule such as another peptide or protein (eg, a tumor associated antigen, Cytokines and cell surface receptors) to generate bispecific molecules that bind to at least two different binding sites or target molecules.
  • an antibody of the invention can be functionally linked (eg, by chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other binding molecules, such as another An antibody, antibody fragment, peptide or binding mimetic that produces a bispecific molecule.
  • a "bispecific antibody” refers to a region comprising two variable domains or ScFv such that the resulting antibody recognizes two different antigens.
  • immunobinding and “immunobinding properties” refer to a non-covalent interaction that occurs between an immunoglobulin molecule and an antigen to which an immunoglobulin is specific for the antigen.
  • the intensity or affinity of the immunological binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction, wherein the smaller the K D value, the higher the affinity.
  • K D equilibrium dissociation constant
  • the immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One method involves measuring the rate of antigen binding site/antigen complex formation and dissociation.
  • Both the "binding rate constant” (Ka or Kon) and the “dissociation rate constant” (Kd or Koff) can be calculated from the concentration and the actual rate of association and dissociation. (See Malmqvist M, Nature, 1993, 361: 186-187). The ratio of Kd/Ka is equal to the dissociation constant K D (see generally Davies et al., Annual Rev Biochem, 1990; 59: 439-473). The K D , ka and kd values can be measured in any effective way. In a preferred embodiment, the dissociation constant is measured using bioluminescence interferometry (e.g., the ForteBio Octet method described in Example 3.4).
  • the dissociation constant can be measured using surface plasmon resonance techniques (e.g., Biacore) or Kinexa.
  • K D equilibrium binding constant
  • the antibody of the present invention is considered to specifically bind to the PD-1 epitope. .
  • the antibody of the invention comprises a heavy chain and a light chain variable region comprising an amino acid sequence homologous to the amino acid sequence of a preferred antibody described herein, and wherein the antibody retains an anti-PD-1 antibody of the invention Expected functional characteristics.
  • the invention provides a humanized PD-1 binding antibody or antigen binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises An amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25, 27, and 29 having at least 80% homology; more preferably, the heavy chain variable region comprises and selected from the group consisting of SEQ ID NOs: At least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous amino acid sequence of the amino acids of 17, 19, 21, 23, 25, 27 and 29; (b) said light chain The variable region comprises an amino acid sequence at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 20, 22, 24, 26, 28 and 30; more preferably, the light chain variable region comprises and is selected Amino acid sequences at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence of SEQ ID NOs: 18, 20, 22, 24, 26, 26,
  • conservative modification is intended to mean that the amino acid modification does not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include substitutions, additions and deletions of amino acids. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitutions mean that amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues having similar side chains has been described in detail in the art.
  • These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoleucine) , valine, phenylalanine, methionine), ⁇ -branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine) , tryptophan, histidine) amino acids.
  • basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine, Asparag
  • the antibodies of the invention comprise a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences and a light chain variable comprising CDR-L1, CDR-L2 and CDR-L3 sequences a region, wherein one or more of these CDR sequences comprises a particular amino acid sequence based on a preferred antibody (eg, AB12M1 or AB12N1) described herein, or a conservative modification thereof, and wherein the antibody retains the desired anti-PD-1 antibody of the invention Functional characteristics.
  • a preferred antibody eg, AB12M1 or AB12N1
  • the invention provides an isolated PD-1 binding antibody or antigen binding portion thereof comprising a heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 sequences and comprising CDR-L1, CDR-L2 And a light chain variable region of the CDR-L3 sequence, wherein: (a) the heavy chain variable region CDR-H1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 1 and 2, and conservative modifications thereof; And/or the heavy chain variable region CDR-H2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 3 and 4, and conservative modifications thereof; and/or the heavy chain variable region CDR-H3 sequence An amino acid sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 and 6, and conservative modifications thereof; and/or (b) the light chain variable region CDR-L1 sequence comprising selected from the group consisting of SEQ ID NOs: 7 and 8.
  • the light chain variable region CDR-L2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 9 and 10, and conservative modifications thereof;
  • said light chain variable region CDR-L3 sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 12 and conservatively modified amino groups thereof Acid sequence.
  • the antibodies of the invention which include bispecific, polyclonal, monoclonal, humanized antibodies, can be used as therapeutic agents. These agents are commonly used to treat or prevent cancer in a subject, increase vaccine efficacy or increase the innate immune response.
  • the antibody or fragment thereof of the present invention which specifically binds to the PD-1 protein can be administered in the form of a pharmaceutical composition for the treatment of cancer or chronic infection.
  • the therapeutically effective amount of an antibody of the invention generally relates to the amount required to achieve a therapeutic goal. As indicated above, this can be a binding interaction between the antibody and its target antigen. Furthermore, the amount required for administration will depend on the binding affinity of the antibody for its specific antigen and also on the pharmacokinetic properties of the antibody in the subject.
  • a typical range of therapeutically effective dosing of an antibody or antibody fragment of the invention may range from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Frequently administered frequencies can range, for example, from twice daily to once a week.
  • the minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
  • an antibody-based variable region sequence that retains the ability to bind to a target protein sequence can be chemically synthesized and/or prepared by recombinant DNA techniques. (See, for example, Marasco et al, Proc Natl Acad Sci USA, 1993, 90: 7889-7893).
  • the formulations may also contain more than one active compound, preferably those having complementary activities that do not adversely affect each other, as desired for the particular indication being treated.
  • the composition may comprise an agent that enhances its function, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • the antibodies or antigen-binding fragments of the invention can be used to treat cancer (i.e., inhibit the growth or survival of tumor cells).
  • Preferred cancers which can be inhibited from growth by the antibodies of the invention include those which are generally responsive to immunotherapy.
  • Non-limiting examples of preferred cancers for treatment include melanoma (e.g., malignant metastatic melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., prostate cancer that is difficult to control), pancreatic adenocarcinoma , breast cancer, colon cancer, lung cancer (eg non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymph Tumors and other malignant tumors.
  • melanoma e.g., malignant metastatic melanoma
  • renal cancer e.g., clear cell carcinoma
  • the antibodies or antibody fragments of the invention may also be used to prevent or treat infections and infectious diseases.
  • Antibodies or antibody fragments can be used alone or in combination with a vaccine to stimulate an immune response against pathogens, toxins, and autoantigens.
  • the antibody or antigen-binding fragment thereof can be used to stimulate an immune response against a human pathogenic virus, such as, but not limited to, some examples of pathogenic viruses including HIV, hepatitis (type A, B or C) viruses (hepatitis ( A, B, or C)), herpes virus (eg VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein Barr virus), adenovirus (adenovirus) , influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus, respiratory syncytial virus ), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV Virus, dengue virus
  • the antibodies or antibody fragments of the invention can be used in combination with other recombinant proteins and/or peptides (e.g., tumor antigens or cancer cells) to increase the immune response to these proteins (i.e., in a vaccination regimen).
  • other recombinant proteins and/or peptides e.g., tumor antigens or cancer cells
  • an anti-PD-1 antibody and antibody fragments thereof can be used to stimulate an antigen-specific immune response by co-administering an anti-PD-1 antibody with a target antigen (eg, a vaccine).
  • a target antigen eg, a vaccine
  • the invention provides, in another aspect, a method of enhancing an immune response of a subject to an antigen, the method comprising administering to the subject: (i) an antigen; and (ii) an anti-PD-1 antibody or antigen thereof of the invention
  • the binding moiety is to increase the immune response of the subject to the antigen.
  • the antigen can be a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen.
  • Non-limiting examples of such antigens include, but are not limited to, tumor antigens or antigens from viruses, bacteria or other pathogens.
  • Anti-PD-1 antibody commercials for non-therapeutic applications are already available, for example, J116 and J105 monoclonal anti-hPD-1 antibodies sold by eBioscience of San Diego, California, USA for flow cytometry analysis, immunohistochemistry Chemical and in vitro functional assays; Mab 1086 monoclonal anti-hPD-1 antibody sold by R&D Systems of Minneapolis, MN, USA for flow cytometry, Western blot and ELISA.
  • the antibodies of the invention can be used for any non-therapeutic purposes provided by current J116, J105 and/or Mab 1086.
  • the antibodies of the invention are useful as affinity purification reagents.
  • the antibodies can also be used in diagnostic assays, for example, to detect expression of PD-1 in a particular cell, tissue or serum.
  • antibodies are typically labeled (directly or indirectly) with a detectable moiety.
  • markers can be utilized, which are generally classified into the following classes: biotin, fluorescent dyes, radionucleotides, enzymes, iodine, and biosynthetic markers.
  • the antibodies of the invention can be used in any known assay, such as competitive binding assays, directly and Indirect sandwich assay and immunoprecipitation assay. Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).
  • Antibodies can also be used in in vivo diagnostic assays.
  • the antibody is typically labeled with a radionuclide (e.g., 111In, 99Tc, 4C, 31I, 125I, 3H, 32P, 35S, or 18F) such that the antigen or antibody expressing cells can be localized by immunoscintiography or positron mapping.
  • a radionuclide e.g., 111In, 99Tc, 4C, 31I, 125I, 3H, 32P, 35S, or 18F
  • the monoclonal antibodies (mAbs) of the invention can be prepared by a variety of techniques, including conventional monoclonal antibody methodology, such as the standard somatic cell hybridization techniques described in Kohler and Milstein, Nature, 1975; 256:495. Although somatic cell hybridization protocols are preferred, other methods of preparing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes, can also be used in principle.
  • a preferred animal system for preparing hybridomas is the murine system. Preparation of hybridomas in mice is a very well established procedure. Immune protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion protocols are also known.
  • DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (eg, PCR amplification or cDNA cloning of hybridomas expressing the antibody of interest), and DNA can be Insertion into an expression vector such that the gene of interest is operably linked to transcriptional and translational regulatory sequences, transfected into a host cell for expression, and the expression host is preferably a eukaryotic expression vector, more preferably a mammalian cell, such as CHO and its derived cell lines.
  • the antibody can be purified by well-known techniques such as affinity chromatography using Protein A or Protein G. Subsequently or alternatively, a specific antigen or epitope thereof can be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. Purification of immunoglobulins is for example discussed by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pages 25-28).
  • the chimeric or humanized antibody of the present invention can be produced according to the sequence of the murine monoclonal antibody prepared above.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from a target murine hybridoma and engineered using standard molecular biology techniques to include non-murine (eg, human) immunoglobulin sequences.
  • the murine variable region can be ligated to a human constant region using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.).
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgGl or IgG4 constant region.
  • the murine CDR regions can be inserted into a human framework sequence using methods known in the art (see U.S. Patent No. 5,225,539 to Winter, and U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).
  • Transgenic animals can also be utilized, for example, HuMAb mice (Medarex, Inc.) contain human immunoglobulin gene mini-locus (miniloci) encoding unrearranged human heavy chain ( ⁇ and ⁇ ) and kappa light chain immunoglobulin sequences.
  • Figure 1 ELISA assay for binding of AB12N1 and AB12M1 to human PD-1.
  • Figure 2-1 ELISA assay for the cross-reactivity of AB12N1 and AB12M1 with cynomolgus PD-1 and human ICOS.
  • FIG. 2-2 ELISA determines the cross-reactivity of AB12N1 and AB12M1 with human CTLA4.
  • Figure 2-3 ELISA assay for cross-reactivity of AB12N1 and AB12M1 with human CD28.
  • ELISA measures the titer and specificity of AB12M2, AB12M3 and AB12M4.
  • Figure 9 Binding of AB12M3 and AB12M4 to CHO cells overexpressing PD-1.
  • Figure 10 Binding of AB12M3 and AB12M4 to activated human T cells.
  • mice After the human PD-1 extracellular purified antigen 50 ⁇ g (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was fully emulsified with complete Freund's adjuvant, male Balb/C mice were immunized by multi-point immunization with an immune cycle of three. Once a week. On the 10th day after the third immunization, blood was taken through the eye socket, and plasma anti-human PD-1 antibody titer was measured by ELISA to monitor the degree of immune response in the mouse. The mice producing the highest anti-human PD-1 antibody titers were then boosted once 3 days before the fusion.
  • mice Three days later, the mice were sacrificed and the mouse spleens were removed and fused with mouse myeloma Sp2/0 cell line. 2 ⁇ 10 8 Sp2/0 cells were mixed with 2 ⁇ 10 8 splenocytes in a solution of 50% polyethylene glycol (molecular weight 1450) and 5% dimethyl sulfoxide (DMSO).
  • polyethylene glycol molecular weight 1450
  • DMSO dimethyl sulfoxide
  • spleen cells Adjust the number of spleen cells to 5 ⁇ 10 with Iscove medium (containing 10% fetal bovine serum, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, 0.1 mM hypoxanthine, 0.4 ⁇ M aminopterin and 16 ⁇ g thymidine) 5 / mL, added to the well of a 96-well culture plate at 0.3 ml, and placed in a 37 ° C, 5% CO 2 incubator. After 10 days of culture, the ability of the antibody in the supernatant to compete with biotinylated human PD-L1-Fc for binding to PD-1 was detected by ELISA in Example 3.2, and 8 competitively strong positive hybridomas were screened and identified. The cell lines were subcloned separately, and the purified mouse antibodies were screened and identified again to obtain two positive hybridoma monoclonal cell lines #22 and #32.
  • Iscove medium containing 10% fetal bovine serum, 100 units
  • Example 2 ELISA method for determination of anti-PD-1 murine antibody titer
  • the titer of the purified murine monoclonal antibody in the culture supernatant of the hybridoma cell line #22 (the secreted antibody was named AB12N1) and #32 (the secretory antibody was named AB12M1) was measured by ELISA.
  • PD-1 purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • PBS buffer was diluted to 0.1 ⁇ g/ml with PBS buffer, added to a 96-well plate at a volume of 100 ⁇ l/well, and placed at 4 ° C for 16-20 h.
  • the PBS buffer in the 96-well plate was aspirated, and the plate was washed once with PBST (pH 7.4, PBS containing 0.05% Tween 20) buffer, and then 200 ⁇ l/well of PBST/1% skim milk powder was added, and the mixture was incubated at room temperature for 1 hour.
  • the blocking solution was removed, and the plate was washed 3 times with PBST buffer, and then diluted with PBST/1% skim milk powder to a suitable concentration of the PD-1 mouse antibody to be tested, 100 ⁇ l/well, and incubated at room temperature for 1.5 h.
  • the reaction system was removed, and the plate was washed 3 times with PBST, and 50 ⁇ l/well was added to a HRP-labeled goat anti-mouse IgG secondary antibody (purchased from The Jackson Laboratory) diluted with PBST/1% skim milk powder (diluted 1:4000), room temperature. Incubate for 1 h. After washing the plate 3 times with PBST, 100 ⁇ l/well of TMB was added, and the color was incubated for 10-30 min at room temperature. The reaction was stopped by adding 50 ⁇ l/well of 0.2 M sulfuric acid. The microplate reader detects the absorbance (OD) at a dual wavelength of 450/620 nm and calculates the EC 50 value.
  • HRP-labeled goat anti-mouse IgG secondary antibody purchased from The Jackson Laboratory
  • PBST/1% skim milk powder diluted 1:4000
  • the murine monoclonal antibody AB12M1 expressed by the hybridoma clone #22 and the murine monoclonal antibody AB12N1 and #32 hybridoma clones were able to bind to PD-1.
  • AB12M1 antigen binding activity EC 50 value of about 0.002 ⁇ g / ml, whereas the EC 50 value of about AB12N1 0.1 ⁇ g / ml.
  • Human PD-1/His, human ICOS/Fc, human CTLA4/His, human CD28/Fc, cynomolgus PD-1/Fc and murine PD-1/His (both purchased from Beijing Yiqiao Shenzhou) in PBS buffer Biotechnology Co., Ltd.), diluted to 0.1 ⁇ g/ml, added to a 96-well plate in a volume of 100 ⁇ l/well, and placed at 4 ° C for 16-20 h.
  • the PBS buffer in the 96-well plate was aspirated, and the plate was washed once with PBST (pH 7.4, PBS containing 0.05% Tween 20) buffer.
  • AB12N1 and AB12M1 have no specific binding ability to the other three proteins of the PD-1 family.
  • AB12N1 and AB12M1 did not cross-react with the mouse PD-1, but AB12M1 specifically binds to PD-1 of cynomolgus monkey, while AB12N1 does not specifically bind to PD-1 of cynomolgus monkey.
  • Biotin-labeled human PD-L1 was used as a reagent.
  • PD-1 purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • PBS buffer a buffer for a 96-well plate
  • the coating solution was discarded, and 200 ⁇ l/well PBST/1% skim milk powder was added and incubated for 1 h at room temperature.
  • both murine antibodies AB12N1 and AB12M1 specifically blocked the binding of PD-1 to PD-L1.
  • AB12M1 blocked the binding ability of PD-L1 to PD-1 significantly better than AB12N1.
  • the purified murine monoclonal antibodies AB12N1 and AB12M1 were qualitatively and semi-quantitatively analyzed by SDS-PAGE electrophoresis and Western blotting (Western-Blot). A 12% strength PAGE gel was dispensed according to the gel configuration method, and 4 ⁇ g of the antibodies AB12N1, AB12M1, Keytruda and Opdivo were added to each lane. Electrophoresis until the dye reaches the bottom of the separation gel, disconnect the power supply, and observe the electrophoresis results in a gel imaging system.
  • the reductive SDS-PAGE electrophoresis results of the murine antibody AB12N1 and AB12M1 showed clear and uniform two bands, which were about 50KD heavy chain and about 25KD light chain (wherein each lane was loaded as follows: 1. Marker; 2. AB12N1; 3. AB12N1; 4. AB12M1; 5. AB12M1; 6. Keytruda; 7. Opdivo).
  • a 15% concentration of non-reducing PAGE gel was dispensed according to the glue configuration method.
  • the amount of human PD-1 was 5 ⁇ g, and the dye was transferred to the bottom of the separation gel to disconnect the power.
  • the gel was removed and placed on a uniform size NC membrane.
  • the power was applied at 1 mA/cm 2 according to the gel area, 100 mA, and electrotransferred for 2 to 4 hours.
  • HRP-goat anti-mouse IgG Fc secondary antibody diluted 1:5000 was used as a detection antibody, incubated for 1 h, and the membrane was washed 3 times with PBST buffer. In the DAB coloring solution, the color was developed in the dark for 15 min, and immediately after the band appeared, the water was washed to terminate the color reaction, and the photograph was taken for quantitative and qualitative analysis.
  • Biofilm interferometry was used to determine the characterization affinity and binding kinetics of purified murine monoclonal antibodies.
  • the assay was performed according to the standard operating method of the Octet Molecular Interaction Instrument (ForteBio Octet RED & QK System, PALL), and the control antibodies were Keytruda and Opidivo.
  • the multi-channel parallel quantitative analysis concentration gradient was set to 3.125, 6.25, 12.5, 25, 50, and 100 nM, and Human PD-1-His (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was coupled to the Ni-NTA sensor. Track antigen-antibody binding kinetics and dissociation kinetics.
  • the data obtained were analyzed, and the values of ka(kon), kd(koff) and KD measured by this method are shown in Table 1.
  • the equilibrium dissociation constant KD value of the murine monoclonal antibody AB12M1 and human PD-1 was ⁇ 1 ⁇ 10 -12 M, which was comparable to the binding affinities of the control antibodies Keytruda and Opidivo.
  • the KD value of AB12N1 was 3.508 ⁇ 10 -10 M, and the characterization affinity was lower than that of the control antibodies Keytruda and Opidivo.
  • Example 4 Subtype identification and variable region amplification of anti-PD-1 murine monoclonal antibody
  • Antibodies identified subtypes Take hybridoma cell culture supernatants, using an antibody subtype IsoStrip TM mouse monoclonal antibody subtype identification kit (Santa Cruz Biotechnology, NO sc-24958) identified.
  • the monoclonal antibody AB12N1 subtype was identified as IgG1 (Kappa) and the monoclonal antibody AB12M1 subtype was IgG2b (Kappa).
  • Antibody variable region amplification candidate hybridoma cells #22 and #32 were separately cultured to a total number of 10 7 cells, centrifuged at 1000 rpm for 10 minutes to collect cells, and total RNA was extracted with Trizol kit (Invitrogen) for reverse transcription.
  • the kit SMARTer RACE synthesizes the first strand cDNA, and uses the first strand cDNA as a subsequent template to amplify the antibody variable region DNA sequence corresponding to the hybridoma cells. According to the subtype identification result, the heavy chain and light chain constant region sequences of the antibody subtype are obtained, and a specific nested PCR primer is designed, and the primer sequence used in the amplification reaction and the first frame region of the antibody variable region are The constant regions are complementary.
  • the target gene is amplified by a conventional PCR method, and the amplified product is sequenced to obtain the heavy chain variable region sequence SEQ ID NO: 15 and the light chain variable region sequence SEQ ID NO: 16 of the hybridoma clone #22 secreting antibody AB12N1;
  • the amino acid sequences of the heavy chain CDRs (CDR-H1, CDR-H2 and CDR-H3) of the antibody are shown in SEQ ID NOs: 2, 4 and 6, respectively, and their light chain CDRs (CDR-L1, CDR-L2 and CDR)
  • the amino acid sequences of -L3) are shown in SEQ ID NOS: 8, 10 and 12, respectively.
  • Hybridoma clone #32 secretes the heavy chain variable region sequence of antibody AB12M1 SEQ ID NO: 13 and the light chain variable region sequence SEQ ID NO: 14; the heavy chain CDRs of the antibody (CDR-H1, CDR-H2 and CDR- The amino acid sequences of H3) are shown in SEQ ID NOS: 1, 3 and 5, respectively, and the amino acid sequences of the light chain CDRs (CDR-L1, CDR-L2 and CDR-L3) are SEQ ID NOS: 7, 9, and 11, respectively. Shown.
  • CDR-grafting is a common method of humanization of antibodies by replacing the FR of a human antibody with the FR of a mouse antibody to maintain activity and reduce immunogenicity.
  • the method for humanization of CDR grafting antibody in combination with Discovery Studio analysis tool mainly includes the following steps: (1) three-dimensional structure modeling of antibody; (2) analysis of key residues. Molecular docking was used to analyze the frame amino acid sequence of the variable region and its surrounding regions, and its spatial stereoscopic binding method was investigated to determine the key residues essential for maintaining the conformation of the CDR regions. There are mainly three types: 1.
  • VL and VH Residues Located at the interface between VL and VH Residues, which play a key role in the folding of the two domains; 2. Residues that are close to the CDR regions and are embedded inside the protein; 3. Residues that interact directly with the CDR regions, including: hydrophobic interactions / hydrogen bond / salt bridge; (3) human template selection.
  • the murine antibody AB12M1 has a human VH 3-23 heavy chain variable region and a human V K 3D-11 light chain variable region as a template sequence, and a total of 7 humanized antibodies are obtained, which are AB12M3, AB12M4, AB12M5, respectively.
  • a human-human chimeric antibody AB12M2 was constructed, which grafts the heavy chain variable region sequence of the murine antibody to the human IgG1 heavy chain constant region, and grafts the light chain variable region sequence of the mouse antibody to human Obtained from the Kappa light chain constant region.
  • the variable region amino acid sequence of the above humanized antibody is shown in Table 2.
  • the murine antibody AB12N1 was obtained by using human V H 3-33 heavy chain variable region and human V K 3-11 light chain variable region as template sequences, and three humanized antibodies were obtained, which were AB12N3, AB12N4 and AB12N5, respectively.
  • a human-human chimeric antibody AB12N2 was constructed by grafting the heavy chain variable region sequence of the murine antibody to the human IgG1 heavy chain constant region, and grafting the light chain variable region sequence of the murine antibody to human Obtained from the Kappa light chain constant region.
  • the variable region amino acid sequence of the above humanized antibody is shown in Table 2.
  • Another group of humanized antibodies AB12N3, AB12N4 and AB12N5 were more than 95% humanized compared with the murine antibody AB12N1 and the chimeric antibody AB12N2, and the affinity was not significantly decreased.
  • the KD values were all in the order of 10 -10 M.
  • Binding properties of humanized antibodies AB12M3 and AB12M4 and chimeric antibody AB12M2 to antigen PD-1 were determined by indirect ELISA. Keytruda and Opdivo were used as control antibodies and medium was used as a negative control. An HRP-labeled goat anti-human IgG antibody was used as a detection antibody (purchased from The Jackson Laboratory) in the same manner as in Example 2. The same method was used to detect whether humanized antibodies AB12M3 and AB12M4 and chimeric antibody AB12M2 cross-reacted with mouse PD-1 (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), also using Keytruda and Opdivo as controls. Resistance Body, using medium as a negative control.
  • humanized antibodies and chimeric antibodies AB12M3 and AB12M4 AB12M2 could specifically binds to human PD-1, and they were lower than the control antibody Keytruda Opdivo 50 and EC values antigen binding activity, about Between 0.001 and 0.01 ⁇ g/ml. This indicates that the binding ability of the anti-PD-1 humanized antibodies AB12M3 and AB12M4 and the chimeric antibody AB12M2 constructed by the present invention to PD-1 was not abolished by humanization, and the high affinity of the mouse parent antibody was retained. Moreover, they did not bind to mouse PD-1 and had strong species specificity (Fig. 6).
  • HRP Horseradish peroxidase
  • the blocking solution was removed, and the plate was washed 3 times with PBST buffer, and then 50 ⁇ L of growth medium (DMEM + 5% FBS) and 50 ⁇ L of HRP-labeled Keytruda or Opdivo antibody were added to each well, and unbound was washed away with PBS.
  • HRP-labeled Keytruda or Opdivo antibodies Antibodies AB12M2, AB12M3 and AB12M4 were then added separately; unlabeled Keytruda or Opdivo was used as a positive control. After sufficient incubation, unbound HRP-labeled Keytruda or Opdivo antibodies were washed away with PBS and the absorbance values were read at a dual wavelength of 450/620 nm using a microplate reader.
  • the His-tagged PD-1 protein extracellular domain fragment was coated with a 96-well microtiter plate, the plate was blocked, washed, and the PD-1 antibody to be tested was added, and biotin-labeled PD-L1-Fc was added to incubate the reaction. After washing the plate, the amount of biotin-labeled PD-L1/Fc binding was measured, and the IC 50 value of PD-1 antibody blocking of ligand PD-L1 binding was calculated.
  • PD1/His was diluted to 2 ⁇ g/ml with pH 7.2 PBS buffer, added to a 96-well microtiter plate in a volume of 100 ⁇ l per well, and incubated for 1 hour at room temperature with shaking.
  • the 96-well microtiter plate was aspirated in PBS buffer, and 200 ⁇ l per well of PBST (pH 7.2 PBS containing 0.05% Tween-20) / 1% skim milk powder was added, and the mixture was incubated at room temperature for 1 hour to block.
  • PBST pH 7.2 PBS containing 0.05% Tween-20
  • the reaction was stopped by adding 50 ⁇ l of 0.2 M sulfuric acid per well.
  • the absorbance value was read at 450 nm using a microplate reader, and the IC 50 value of PD-1 antibody blocking of ligand PD-L1 binding was calculated.
  • FACS Fluorescence Active Cell Sorter
  • AB12M3 or AB12M4 primary antibody
  • FITC-labeled goat anti-human IgG secondary antibody purchased from BD Biosciences
  • the effect of blocking the PD-1/PD-L1 pathway on lymphoid effector cells was demonstrated by a mixed lymphocyte reaction.
  • the effect of PD-1 antibody or isotype IgG control antibody on T cell proliferation and IFN- ⁇ secretion in mixed lymphocyte reaction was determined.
  • the freshly isolated human PBMC was adjusted to have a cell density of 2.0 ⁇ 10 6 /ml, and mononuclear cells were obtained by the adherence method. 100 ng/ml GM-CSF and 100 ng/ml IL-4 were added for 5 days, and 100 ng/ml TNF- ⁇ was added to induce DC cell maturation.
  • CD 4+ T cell positive selection kit (STEMCELL Corporation) CD 4+ T cells isolated from fresh human PBMC.
  • 96-well plate 250 ⁇ l per well of culture medium containing 10 5 T cells isolated 10 4 cells induce maturation of DC and a series of concentration gradients or AB12M3 AB12M4. A homotypic IgG control antibody was used as a negative control.
  • the mixed lymphocytes were cultured for 6 days in a 37 ° C, 5% CO 2 cell incubator, and then 100 ⁇ l of the culture supernatant was taken out from each well of a 96-well plate for IFN- ⁇ measurement.
  • IFN- ⁇ was measured using an OptEIA ELISA kit (BD Biosciences).
  • the remaining cells of the 96-well plate were assayed for cell proliferation using the Cell Titer Glo kit (Promega).
  • the results showed that AB12M3 and AB12M4 promoted T cell proliferation (Fig. 11) and IFN- ⁇ (Fig. 12) secretion in a concentration-dependent manner.
  • Freshly prepared human PBMC cells were resuspended in 10 6 /ml with RPMI 1640 medium (containing 10% inactivated FBS) containing 20 ⁇ g/ml AB12M3, AB12M4 or isotype IgG control antibody, seeded in 96-well plates, 100 ⁇ l/well .
  • the highest concentration of superantigen SEB was 2500 ng/ml, diluted 4 times in 10 folds, and added to a 96-well plate with three replicate wells. After culturing for 72 hours, the supernatant was taken and the IL-2 concentration was measured using an OptEIA ELISA kit (BD Biosciences). The results are shown in Figure 13, AB12M3 and AB12M4 can promote the secretion of IL-2 by T cells.
  • PD-L1 overexpressing human non-small cell lung cancer cell line HCC827 (Chinese Academy of Sciences Shanghai Cell Bank) was inoculated into a 96-well cell culture plate, and a series of concentrations of AB12M1, AB12M3, AB12M4 or huIgG were added, followed by a 10:1 target.
  • T cells activated with anti-CD3 antibody and IL-2 the cells were cultured for 48 hours, and most of the T cells were removed by washing with a medium, and the survival of HCC827 cells was measured by CCK-8 cell proliferation kit (Dojindo). Kill rate.
  • CCK-8 cell proliferation kit (Dojindo). Kill rate. The results are shown in Table 5, indicating that AB12M3, AB12M4 have the ability to enhance T cell killing of tumor cells.
  • mice constructed by Beijing Biotech Biotechnology Co., Ltd. were used to evaluate the in vivo efficacy of anti-human PD-1 antibody.
  • the mice were subjected to gene targeting technology using C57BL/6 background.
  • the second exon portion of the PD-1 gene, including the IgV domain portion, was humanized.
  • the successfully engineered mice carry human-chimeric chimeric PD-1, which is divided into hPD-1 and intracellularly divided into mPD-1. This chimeric PD-1 structure does not affect PD-1 normal signaling, and murine or human PD-L1 ligand binds to this PD-1 receptor to inhibit T cell activity.
  • MC38 murine colon cancer cells (purchased from Shanghai Biotechnology Co., Ltd.) were inoculated into the right anterior flank of female B-hPD-1 humanized mice at 5 ⁇ 10 5 /0.1 mL for tumor growth.
  • the tumor volume was randomly grouped into 8 groups of 3 groups, respectively: (1) solvent control group (PBS group), (2) AB12M4 treatment group, and (3) Keytruda control group (purchased from Merck, lot number: 5SNL80505), group (2) and group (3) were administered at a dose of 20 mg/kg and a dosing volume of 10 ml/kg. All groups were administered intraperitoneally, once every 3 days, and continuously for 6 times, and the experiment was completed on the 28th day after inoculation.
  • the median tumor volume of the solvent control group was 3405.2 mm 3 .
  • the mean tumor volume of the AB12M4 treated group was 277.4 mm 3 and the average tumor volume of the Keytruda administered group was 249 mm 3 . It indicates that AB12M4 has a significant anti-tumor effect, and its anti-tumor effect is comparable to that of Keytruda.
  • the animals were in good health and no animals died.
  • the body weight of each group increased. There was no significant difference between the animals in the AB12M4 treatment group and the solvent control group (p>0.05), indicating that the animals were well tolerated by AB12M4 and did not have obvious toxic effects on the experimental animals.

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Abstract

一种以高亲和力与PD-1特异性结合的抗体，还提供了编码所述抗体的核酸分子，用于表达所述抗体的表达载体和宿主细胞，以及所述抗体的生产方法。此外，还提供了包含所述抗体的免疫缀合物以及药物组合物，以及所述抗体在用于制备治疗癌症和感染性疾病、炎性疾病的药物中的用途。

Description

抗PD-1抗体及其用途 技术领域
本发明属于治疗性单克隆抗体领域，更具体地，本发明涉及一种针对程序性死亡受体(PD-1)的抗体；还涉及所述抗体在治疗多种疾病(包括癌症和感染性疾病、炎性疾病)中的用途。
背景技术
程序性死亡受体-1(Programmed Death-1，PD-1)是CD28家族成员，是在活化T细胞和B细胞表面表达的免疫抑制性受体(Yao Zhu等,Nat Rev Drug Discov,2013；12(2):130-146)，最初是在凋亡的T细胞杂交瘤中利用削减杂交技术得到的。PD-1主要表达于CD4+T细胞、CD8+T细胞、NK-T细胞、B细胞和活化的单核细胞表面，主要受T细胞受体(TCR)或B细胞受体(BCR)信号的诱导表达，TNFα可增强PD-1在这些细胞表面的表达(Francisco LM等,Immunol Rev,2010；236:219-242)。PD-1分子由胞外区、跨膜区和胞内区构成。胞外区含有一个免疫球蛋白可变区IgV结构域，胞内区含有两个基于酪氨酸的信号转导模体ITIM(免疫受体酪氨酸抑制作用模体)和ITSM(免疫受体酪氨酸转换作用模体)。T细胞被激活后，PD-1主要通过ITSM模体，将酪氨酸磷脂酶SHP2集合，导致包括CD3ζ、PKCθ和ZAP70等效应分子的去磷酸化。PD-1配体有两个：PD-L1和PD-L2。PD-L1又称B7H1或CD274，PD-L2称为B7DC或CD273。PD-L1和PD-L2表达于不同的细胞群体(Shimauchi,Kabashima等,Int J Cancer,2007；121(12):2585-2590)。其中，PD-L2表达比较局限，主要在活化的巨噬细胞、树突状细胞和少数肿瘤细胞上。PD-L1则在活化的T细胞、B细胞、巨噬细胞、树突状细胞和肿瘤细胞上广泛表达，同时在机体一些免疫屏蔽部位如胎盘、眼及其上皮、肌肉，肝和血管内皮等组织表达。
PD-1能够与配体(Programmed Death-1 Ligands,PD-Ls)PD-L1和PD-L2相互作用，通过细胞内信号转导通路显著抑制CD3和CD28介导的T细胞活化以及细胞因子产生，因此是调节T细胞反应的重要免疫哨卡。在正常情况下， PD-1/PD-Ls信号通路可以诱导和维持外周组织的免疫耐受，对防止组织的过度炎症反应以及自身免疫性疾病的发生具有积极作用(Latchman Y等,Nat Immunol,2001；2:261-268)。而在病理情况下，PD-1与配体PD-L1、PD-L2相互作用，下调T细胞免疫刺激性细胞因子如IFN-γ、IL-2和TNF-α的分泌以及存活蛋白的表达，促进免疫抑制性细胞因子IL-10分泌，从而抑制T细胞免疫反应(Hamido等,Expert Opin Biol Ther,2013；13(6):847-861)。研究表明，PD-1协同抑制信号与人类多种疾病发生密切相关，可作为疾病治疗的靶分子(Okazaki T等,J Immunol.2007；19:813-824)。
PD-1/PD-L1信号通路与肿瘤发展有着密切关系，在肿瘤患者体内，PD-L1高表达能增强肿瘤的转移能力，导致患者死亡率上升，并且与患者预后不良相关。研究发现在肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌及头颈癌等人类肿瘤组织中都检测到高表达的PD-L1蛋白。此外，肿瘤细胞在多种细胞因子诱导下也高表达PD-L1，这一现象与肿瘤免疫逃逸相关；同时，肿瘤部位浸润性CD8+T细胞也受到肿瘤微环境的影响，PD-1表达也比外周血中的T细胞要高，它与肿瘤细胞表面PD-L1作用，抑制T细胞的活化与增殖，肿瘤细胞可通过此途径逃避细胞毒性T淋巴细胞(cytotoxic lymphocyte，CTL)的杀伤作用，减弱机体抗肿瘤免疫应答。用阻断型抗PD-1单抗阻断PD-1/PD-L1信号可以通过上调IFN-γ、IL-2、IL-10分泌，并有效逆转CD4+和CD8+T细胞的增殖抑制，同时使T细胞的活化程度和杀伤能力显著增强(Dong HD等,Nat Med,2002；8:793-800)。
多种慢性和急性病毒感染也通过PD-1/PD-Ls信号逃避人体免疫监测。机体外周病毒特异性CD4+、CD8+T细胞高表达PD-1，导致功能失调或者无能，不能及时有效地清除感染的病毒(Narasimhan J等,J Virol,2008；376:140-153)。近来，已有大量研究表明，通过特异性单抗阻断PD-1/PD-Ls抑制途径能够有效地使HIV、HBV和HCV等病毒特异性CD4+、CD8+T活化增殖，产生IFN-γ、TNF-α以及颗粒酶B等杀伤因子，恢复免疫细胞特异性的抗病毒功能(Barber DL等,Nature,2006；439:682-687)。
因此，可制备特异性抗PD-1单抗，通过特异性阻断PD-1/PD-L1信号对该抑制途径的作用进行封闭，增强CTL杀伤肿瘤细胞的功能，有效抑制肿瘤的形 成和生长。目前已经有两种PD-1抑制剂抗肿瘤抗体药上市，分别是Pembrolizumab(商品名Keytruda，默克公司研发)和Nivolumab(商品名Opdivo，小野制药/百时美施贵宝研发)；CureTech公司研发的Pidilizumab处于II期临床研究阶段；MedImmune研发的AMP-224、AMP-514处于I期临床研究阶段。虽然已有多种抗PD-1、PD-L1和CTLA4等免疫哨卡的单克隆抗体应用于临床，但这些抗体作为单药使用的应答率还较低，平均只有15-20％。因此研发新型的抗PD-1单克隆抗体，具有更高的特异性、更低毒副作用、更佳临床药效，也将给癌症或感染性患者提供更多的用药选择。
发明内容
本发明的目的在于提供一种对PD-1分子具有高亲和力的抗PD-1单克隆抗体。
在一方面，本发明提供了分离的结合PD-1的单克隆抗体，其包含：重链可变区，其包含CDR-H1、CDR-H2和CDR-H3序列；和轻链可变区，其包含CDR-L1、CDR-L2和CDR-L3序列，其中：
(i)重链可变区包含选自SEQ ID NOs：1和2所示的CDR-H1序列，和选自SEQ ID NOs：3和4所示的CDR-H2序列，和选自SEQ ID NOs：5和6所示的CDR-H3序列；和
(ii)轻链可变区包含选自SEQ ID NOs：7和8所示的CDR-L1序列，和选自SEQ ID NOs：9和10所示的CDR-L2序列，和选自SEQ ID NOs：11和12所示的CDR-L3序列。
本发明的一优选实施例中，所述抗体的重链可变区包含SEQ ID NO：1所示的CDR-H1序列，和SEQ ID NO：3所示的CDR-H2序列，和SEQ ID NO：5所示的CDR-H3序列；且其轻链可变区包含SEQ ID NO：7所示的CDR-L1序列，和SEQ ID NO：9所示的CDR-L2序列，和SEQ ID NO：11所示的CDR-L3序列。
另一优选实施例中，所述抗体的重链可变区包含SEQ ID NO：2所示的CDR-H1序列，和SEQ ID NO：4所示的CDR-H2序列，和SEQ ID NO：6所示 的CDR-H3序列；且其轻链可变区包含SEQ ID NO：8所示的CDR-L1序列，和SEQ ID NO：10所示的CDR-L2序列，和SEQ ID NO：12所示的CDR-L3序列。
进一步地，包含上述CDR序列的抗体为鼠源的、嵌合的或人源化的。
例如，所述抗体为鼠源的或嵌合的，其重链可变区进一步包含鼠IgG1、IgG2、IgG3、IgG4或其变体的重链FR区；和其轻链可变区包含鼠κ、λ链或其变体的轻链FR区。
较优选地，上述鼠源或嵌合抗体包含：
(a)重链可变区，其包含SEQ ID NO：15所示的氨基酸序列；和
(b)轻链可变区，其包含SEQ ID NO：16所示的氨基酸序列。
例如，本发明的优选实施例中鼠源抗体AB12N1和嵌合抗体AB12N2，其重链可变区包含SEQ ID NO：15所示的氨基酸序列；和其轻链可变区包含SEQ ID NO：16所示的氨基酸序列。
更优选地，上述鼠源或嵌合抗体包含：
(a)重链可变区，其包含SEQ ID NO：13的氨基酸序列；和
(b)轻链可变区，其包含SEQ ID NO：14的氨基酸序列。
例如，本发明的优选实施例中鼠源抗体AB12M1和嵌合抗体AB12M2，其重链可变区包含SEQ ID NO：13所示的氨基酸序列；和其轻链可变区包含SEQ ID NO：14所示的氨基酸序列。
例如，所述抗体为人源化的。制备人源化抗体的方法是本领域技术人员公知的。例如，可以通过将本发明的CDR序列转移至人抗体可变区中来制备本发明的人源化抗PD-1抗体。所述人源化抗体不会产生抗抗体反应(AAR)和人抗鼠抗体反应(HAMA)，不会因被抗抗体中和而被快速清除。
本发明的一优选实施例中，将上述鼠源抗体AB12M1通过CDR移植(CDR-grafting)进行人源化改造。由此产生的所述人源化抗体，较优选地，其重链可变区包含选自SEQ ID NOs：17、19、21、23、25、27和29所示的氨基酸序列；和其轻链可变区包含选自SEQ ID NOs：18、20、22、24、26、28和30所示的氨基酸序列。更优选地，由此产生的所述人源化抗体AB12M3、AB12M4、AB12M5、AB12M6、AB12M7、AB12M8和AB12M9，其重链可变区分别包含如SEQ ID NO：17、19、21、23、25、27和29所示的氨基酸序列；和其轻链可 变区分别包含如SEQ ID NO：18、20、22、24、26、28和30所示的氨基酸序列。
本发明的另一优选实施例中，将上述鼠源抗体AB12N1通过CDR移植(CDR-grafting)进行人源化改造。由此产生的所述人源化抗体，较优选地，其重链可变区包含选自SEQ ID NOs：31、33和35所示的氨基酸序列；和其轻链可变区包含选自SEQ ID NOs：32、34和36所示的氨基酸序列。更优选地，由此产生的所述人源化抗体AB12N3、AB12N4和AB12N5，其重链可变区分别包含如SEQ ID NO：31、33和35所示的氨基酸序列；和其轻链可变区分别包含如SEQ ID NO：32、34和36所示的氨基酸序列。
在不实质性影响抗体活性的前提下，本领域技术人员可以对本发明抗体的序列进行替换、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体序列的变体。它们都被视为包括在本发明保护的范围内。如在可变区将具有类似性质的氨基酸进行替换。本发明所述变体的序列可以与其来源序列至少80％同源；更优地，本发明所述变体的序列可以与其来源序列至少85％、90％、95％、96％、97％、98％或99％同源。
本发明的抗体可以为全长抗体，例如，在一些优选的实施方案中，本发明的抗人PD-1抗体还包含人IgG4或IgG1的重链恒定区和人κ轻链恒定区；或者，所述抗体可以仅包含抗原结合片段，诸如Fab或Fab’2片段，或单链抗体ScFv。
在上述任何实施例中，本发明所述抗体能够以约1nM或更低的KD结合PD-1；在较优选的实施例中，所述抗体或其抗原结合能够以约100pM或更低的KD结合PD-1；在更优选的实施例中，所述抗体能够以约10pM或更低的KD结合PD-1；在最优选的实施例中，所述抗体能够以约1pM或更低的KD结合PD-1。
本发明另一方面，提供一种编码如上所述抗体的DNA分子。
例如，编码本发明优选嵌合抗体AB12M2重链可变区的DNA分子如SEQ ID NO：37所示，且编码其轻链可变区的DNA分子如SEQ ID NO：38所示。
又如，编码本发明的优选人源化抗体AB12M3重链可变区的DNA分子如SEQ ID NO：39所示，且编码其轻链可变区序列的DNA分子如SEQ ID NO：40所示。
再如，编码本发明另一优选人源化抗体AB12M4重链可变区的DNA分子 如SEQ ID NO：41所示，且编码其轻链可变区的DNA分子如SEQ ID NO：42所示。
本发明另一方面，提供一种包含如上所述DNA分子的表达载体。
本发明另一方面，提供一种用如上所述表达载体转化的宿主细胞。宿主细胞优选为CHO细胞。
本发明另一方面，提供一种包含与治疗剂缀合的本发明所述抗体的免疫缀合物。所述治疗剂优选为毒素、放射性同位素、药物或细胞毒剂。
本发明另一方面，还提供了包含本发明所述抗体的任何一种双特异性分子。例如，可以将上述PD-1抗体与具有另一种抗原结合特性的抗体或抗体片段功能性连接组成双特异性抗体。诸如，所述双特异性抗体包括但不限于针对以下分子的抗体：VEGF、EGFR、Her2/neu、VEGF受体或其它生长因子受体、CD20、CD40、CTLA-4、OX-40、4-IBB和ICOS。
本发明另一方面，还提供了一种药物组合物，其包含本发明所述抗体以及可药用赋形剂、载体或稀释剂。
本发明又一方面，还提供了制备本发明所述抗体的方法，其包括：(a)在允许产生所述抗体的条件下培养本发明的上述宿主细胞；(b)回收、分离产生的所述抗体。
本发明再一方面，还涉及根据本发明所述结合PD-1的抗体、或包含其的药物组合物、或包含其的免疫缀合物、或包含其的双特异性分子在制备用于治疗PD-1介导的疾病或病症的药物中的用途。
其中，所述的疾病优选为癌症；更优选为高表达PD-L1的癌症；所述的癌症包括但不限于肺癌、肝癌、卵巢癌、宫颈癌、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、头颈癌；优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌。
其中，所述疾病优选为感染性疾病；例如，慢性病毒感染、细菌感染或寄生虫感染疾病。所述感染性疾病更优选为HIV、HBV和HCV。
优选地，在制备治疗癌症或感染性疾病的药物中可以使用嵌合的、人源化的抗PD-1抗体；较优选地，使用人源化的。
其中，本发明所提供的抗体可单独使用或与其它治疗剂或治疗方法联合使 用：例如，抗肿瘤药或免疫原剂(例如，肿瘤抗原)、抗原呈递细胞(例如，用来源于肿瘤的抗原或核酸刺激的树突细胞)、免疫刺激细胞因子(例如IL-2、IFNa2、GM-CSF)和用编码免疫刺激细胞因子(例如，但不限于GM-CSF)的基因转染的细胞；标准癌症治疗(例如化疗、放疗或手术)；或其它抗体(包括但不限于针对以下分子的抗体：VEGF、EGFR、Her2/neu、VEGF受体或其它生长因子受体、CD20、CD40、CTLA-4、OX-40、4-IBB和ICOS)。
本发明制备的抗PD-1人源化抗体相对临床上使用的Keytruda和Opdivo具有更高的结合亲和力，亲和力常数KD值小于1pM，且具有极强的特异性。体内抗肿瘤研究数据显示，本发明所提供的人源化抗体能显著地抑制转基因小鼠移植瘤的生长，甚至部分小鼠的肿瘤完全消失。此外，本发明所述抗体采用CHO细胞表达，具有产量高、活性高、纯化工艺简单以及生产成本低的优势。
发明详述
缩写和定义
hPD-1  人PD-1蛋白
CDR    用Kabat编号系统界定的免疫球蛋白可变区中的互补决定区
EC50   产生50％功效或结合的浓度
ELISA  酶联免疫吸附测定
FR     抗体构架区：将CDR区排除在外的免疫球蛋白可变区
HRP    辣根过氧化物酶
IL-2   白细胞介素2
IFN    干扰素
IC50    产生50％抑制的浓度
IgG    免疫球蛋白G
Kabat  由Elvin A Kabat倡导的免疫球蛋白比对及编号系统
mAb    单克隆抗体
PCR    聚合酶链式反应
V区    在不同抗体之间序列可变的IgG链区段。其延伸到轻链的109位Kabat残基和重链的第113位残基。
VH    免疫球蛋白重链可变区
VK    免疫球蛋白κ轻链可变区
KD    平衡解离常数
ka    结合速率常数
Kd    解离速率常数
本发明所使用的术语“抗体”涵盖全长抗体(例如，IgG1或IgG4抗体)、其各种功能性片段(例如可仅包含抗原结合部分，如Fab、F(ab’)2或scFv片段)以及经过修饰的抗体(例如人源化、糖基化等)。本发明还包括具有糖基化修饰的抗PD-1抗体。在一些应用中，进行修饰以除去不期望的糖基化位点，例如在寡糖链上去岩藻糖修饰以增强抗体依赖性细胞毒性(ADCC)功能；在另一些应用中，可进行半乳糖基化修饰以改变补体依赖性细胞毒性(CDC)。
术语“单克隆抗体或mAb”，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
“抗体片段”和“抗原结合片段”意即抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％5、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗体片段实例包括但不限于：Fab、Fab′、F(ab′)2和Fv片段；双抗体；线性抗体(linear antibody)；单链抗体分子，例如ScFv、单抗体(技术来自Genmab)；纳米抗体(技术来自Domantis)；结构域抗体(技术来自Ablynx)；和由抗体片段形成的多特异性抗体。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。
“Fc”区含有包含抗体的CH1和CH2结构域的两个重链片段。两个重链片 段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。
“Fab′片段”含有一条轻链和一条重链的VH结构域和CH1结构域以及CH1和CH2结构域之间的恒定区部分，由此可在两个Fab′片段的两条重链之间形成链间二硫键以形成F(ab′)2分子。
“F(ab′)2片段”含有两条轻链和两条重链的VH结构域和CH1结构域以及CH1和CH2结构域之间的恒定区部分，由此在两条重链间形成链间二硫键。因此，F(ab′)2片段由通过两条重链间的二硫键保持在一起的两个Fab′片段组成。
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。
“单链Fv抗体”(或“scFv抗体”)是指包含抗体的VH和VL结构域的抗体片段，其中这些结构域存在于单个多肽链中。对于scFv综述，可参见Pluckthun(1994)The Pharmacology of Monoclonal Antibodies(单克隆抗体药理学)，第113卷，Rosenburg和Moore主编，Springer-Verlag，New York，第269-315页。还参见国际专利申请公开号WO 88/01649和美国专利第4,946,778号和第5,260,203号。
“抗原结合片段”是仅含有重链可变区或轻链可变区链的具有免疫学功能的免疫球蛋白片段。
本文所用术语“超变区”是指负责抗原结合的抗体氨基酸残基。超变区包含以下氨基酸残基：来自序列比对所界定的“互补决定区”或“CDR”的氨基酸残基，例如，轻链可变结构域的24-34(L1)、50-56(L2)和89-97(L3)位残基和重链可变结构域的31-35(H1)、50-65(H2)和95-102(H3)位残基，参见Kabat等，1991，Sequences of Proteins of Immunological Interest(免疫目的物的蛋白质序列)，第5版，Public Health Service，National Institutes of Health，Bethesda，Md.；和/或来自根据结构来界定的“超变环”(HVL)的残基，例如，轻链可变结构域的26-32(L1)、50-52(L2)和91-96(L3)位残基和重链可变结构域的26-32(H1)、53-55(H2)和96-101(H3)位残基，参见Chothia和Leskl，1987，J.Mol.Biol.196：901-917。“构架”残基或“FR”残基为除本文定义的超变区残基之外的可变结构域残基。
术语“嵌合抗体(Chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克 隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入载体中，最后在真核表达系统或原核表达系统中表达嵌合抗体分子。在本发明的一优选实施方案中，所述的PD-1嵌合抗体的抗体轻链可变区进一步包含鼠源κ、λ链或其变体的轻链FR区。所述PD-1嵌合抗体的抗体重链可变区进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
术语“双特异性分子”，是指本发明的抗PD-1抗体或其抗原结合片段可以进行衍生化或连接至另一功能性分子上，例如另一种肽或蛋白质(例如肿瘤相关抗原、细胞因子和细胞表面受体)以生成与至少两种不同结合位点或靶分子结合的双特异性分子。为创建本发明的双特异性分子，可以将本发明的抗体在功能上连接(例如通过化学偶联、基因融合、非共价结合或其它方式)至一种或多种其它结合分子，诸如另一种抗体、抗体片段、肽或结合模仿物，从而产生双特异性分子。例如，“双特异性抗体”是指包含两个可变结构域或ScFv单位使得所得抗体识别两种不同抗原。
本文使用的术语“免疫结合”和“免疫结合性质”是指一种非共价相互作用，其发生在免疫球蛋白分子和抗原(对于该抗原而言免疫球蛋白为特异性的)之间。免疫结合相互作用的强度或亲和力可以相互作用的平衡解离常数(KD)表示，其中KD值越小，表示亲和力越高。所选多肽的免疫结合性质可使用本领域中公知的方法定量。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(Ka或Kon)和“解离速率常数”(Kd或Koff)两者都可通过浓度及缔合和解离的实际速率而计算得出。(参见Malmqvist M,Nature,1993,361:186-187)。Kd/Ka的比率等于解离常数KD(通常参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、ka和kd值。在优选的实施方案中，用生物发光干涉测量法(例如，实施例3.4中所述的ForteBio Octet法)来测量解离常数。在其它优选的实施方案中，可用表面等离子共振技术(例如Biacore)或Kinexa来测量解离常数。当平衡结合常数(KD)为≤10μM，优选为≤100nM，更优选为≤10nM，和最优选为≤100pM～约1pM时，本发明的抗体被认为 特异性地结合至PD-1表位。
同源抗体
在又一方面，本发明抗体包含的重链和轻链可变区所包含的氨基酸序列与本文所述的优选抗体的氨基酸序列同源，且其中所述抗体保留了本发明抗PD-1抗体的期望的功能特性。
例如，本发明提供了人源化的结合PD-1的抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：(a)所述重链可变区包含与选自SEQ ID NOs：17、19、21、23、25、27和29的氨基酸序列至少80％同源的氨基酸序列；更优地，所述重链可变区包含与选自SEQ ID NOs：17、19、21、23、25、27和29的氨基酸序列至少85％、90％、95％、96％、97％、98％或99％同源的氨基酸序列；(b)所述轻链可变区包含与选自SEQ ID NOs：18、20、22、24、26、28和30的氨基酸序列至少80％同源的氨基酸序列；更优选地，所述轻链可变区包含与选自SEQ ID NOs：18、20、22、24、26、28和30的氨基酸序列至少85％、90％、95％、96％、97％、98％或99％同源的氨基酸序列。
具有保守修饰的抗体
术语“保守修饰”意图指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。此类保守修饰包括氨基酸的取代、添加和缺失。修饰可以通过本领域已知的标准技术，例如定点诱变和PCR介导的优点引入到本发明的抗体中。保守氨基酸取代指氨基酸残基用具有类似侧链的氨基酸残基替换。本领域中对具有类似侧链的氨基酸残基家族已有详细说明。这些家族包括具有碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区中的一个或多个氨基酸残基。
在某些实施方式中，本发明的抗体包含含有CDR-H1、CDR-H2和CDR-H3序列的重链可变区和含有CDR-L1、CDR-L2和CDR-L3序列的轻链可变区，其中这些CDR序列中的一个或多个包含基于本文所述优选抗体(例如AB12M1或AB12N1)的特定氨基酸序列或其保守修饰，且其中所述抗体保留了本发明抗PD-1抗体的期望的功能特性。因此，本发明提供了分离的结合PD-1的抗体或其抗原结合部分，其包含含有CDR-H1、CDR-H2和CDR-H3序列的重链可变区和含有CDR-L1、CDR-L2和CDR-L3序列的轻链可变区，其中：(a)所述重链可变区CDR-H1序列包含选自SEQ ID NOs：1和2所示氨基酸序列及其保守修饰的氨基酸序列；和/或所述重链可变区CDR-H2序列包含选自SEQ ID NOs：3和4所示氨基酸序列及其保守修饰的氨基酸序列；和/或所述重链可变区CDR-H3序列包含选自SEQ ID NOs：5和6所示氨基酸序列及其保守修饰的氨基酸序列；和/或(b)所述轻链可变区CDR-L1序列包含选自SEQ ID NOs：7和8所示氨基酸序列及其保守修饰的氨基酸序列；和/或(b)所述轻链可变区CDR-L2序列包含选自SEQ ID NOs:9和10所示氨基酸序列及其保守修饰的氨基酸序列；和/或(b)所述轻链可变区CDR-L3序列包含选自SEQ ID NOs：11和12所示氨基酸序列及其保守修饰的氨基酸序列。
抗PD-L1的抗体的治疗用途
可将本发明的抗体(其包括双特异性、多克隆、单克隆、人源化抗体)用作治疗剂。这些药剂可通常用于在受试者中治疗或预防癌症，增加疫苗功效或提高天然免疫应答。
本发明的特异性结合PD-1蛋白的抗体或其片段可以药物组合物的形式给予，用于癌症或慢性感染的治疗。
本发明抗体的治疗上有效的量通常涉及实现治疗目标所需的量。如上指出，这可以是抗体和它的靶抗原之间的结合相互作用。此外，给予所需量取决于抗体对它的特异性抗原的结合亲和力，并也取决于抗体在受试者体内的药代动力学特性。本发明抗体或抗体片段的治疗上有效的剂量给药的常用范围(以非限制实例的方式)可为约0.1mg/kg体重—约50mg/kg体重。常用给药频率可在例如从每天两次至一周一次的范围内。
在使用抗体片段的情况下，特异性结合至靶蛋白的结合结构域的最小的抑制性片段是优选的。例如，基于抗体的可变区序列，其保持结合靶蛋白序列的能力。这类肽可化学合成和/或通过重组DNA技术制备。(参见，例如，Marasco等人,Proc Natl Acad Sci USA,1993,90:7889-7893)。制剂还可根据治疗的特定适应症的需要而含有超过一种活性化合物，优选相互间无不利影响的具有互补活性的那些。备选地或另外，组合物可包含增强它的功能的作用剂，例如，细胞毒性剂、细胞因子、化学治疗剂或生长抑制剂。
癌症
本发明抗体或抗原结合片段可用于治疗癌症(即抑制肿瘤细胞的生长或存活)。可用本发明抗体抑制其生长的优选的癌症包括通常对免疫疗法有反应的癌症。用于治疗的优选癌症的非限制性实例包括黑素瘤(例如恶性转移性黑素瘤)、肾癌(例如透明细胞癌)、前列腺癌(例如激素难控制的前列腺腺癌)、胰腺腺癌、乳腺癌、结肠癌、肺癌(例如非小细胞肺癌)、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、胶质母细胞瘤、神经胶质瘤、白血病、淋巴瘤和其它恶性肿瘤。
感染性疾病
本发明抗体或抗体片段还可用于防止或治疗感染和感染性疾病。抗体或抗体片段可单独使用，或与疫苗联合使用，以刺激针对病原体、毒素和自体抗原的免疫应答。抗体或其抗原结合片段可用于刺激对感染人的病原性病毒的免疫应答，这些病毒例如但不限于病原性病毒的一些实例包括HIV、肝炎(甲型、乙型或丙型)病毒(hepatitis(A,B,or C))、疱疹病毒(herpes virus)(例如VZV、HSV-1、HAV-6、HSV-II和CMV、埃巴二氏病毒(Epstein Barr virus))、腺病毒(adenovirus)、流感病毒(influenza virus)、黄病毒(flaviviruses)、艾柯病毒(echovirus)、鼻病毒(rhinovirus)、柯萨奇病毒(coxsackie virus)、冠状病毒(cornovirus)、呼吸道合胞病毒(respiratory syncytial virus)、腮腺炎病毒(mumps virus)、轮状病毒(rotavirus)、麻疹病毒(measles virus)、风疹病毒(rubella virus)、细小病毒(parvovirus)、牛痘病毒/痘苗病毒(vaccinia virus)、HTLV 病毒、登革热病毒(dengue virus)、乳头瘤病毒(papillomavirus)、软疣病毒(molluscum virus)、脊髓灰质炎病毒(poliovirus)、狂犬病病毒(rabies virus)、JC病毒和虫媒病毒性脑炎病毒(arboviral encephalitis virus)。抗体或其抗原结合片段还可用于刺激对细菌或真菌寄生虫及其它病原体引起的感染的免疫应答。
免疫佐剂
本发明的抗体或抗体片段可与其它重组蛋白和/或肽(例如肿瘤抗原或癌细胞)联合使用，以便提高对这些蛋白的免疫应答(即在接种方案中)。
例如，通过共同给予抗PD-1抗体与目标抗原(例如疫苗)，可将抗PD-1抗体及其抗体片段用于刺激抗原特异性免疫应答。因此，本发明在另一方面提供了增强受治疗者对抗原的免疫应答的方法，所述方法包括给予受治疗者：(i)抗原；和(ii)本发明抗PD-1抗体或其抗原结合部分，以便提高受治疗者对抗原的免疫应答。例如，抗原可为肿瘤抗原、病毒抗原、细菌抗原或来自病原体的抗原。所述抗原的非限制性实例包括但不限于肿瘤抗原或来自病毒、细菌或其它病原体的抗原。
本发明抗体及抗体片段的非治疗性应用
非治疗性应用的抗PD-1抗体商品已经存在，例如，由eBioscience of San Diego，California，USA所售的J116和J105单克隆抗hPD-1抗体，其用于流式细胞术分析、免疫组织化学和体外功能分析；由R&D Systems of Minneapolis，MN，USA所售的Mab1086单克隆抗hPD-1抗体，其用于流式细胞术、蛋白质印迹和ELISA。本发明抗体可用于当前J116、J105和/或Mab1086所提供的任何非治疗目的。
本发明抗体可用作亲和纯化试剂。
所述抗体还可用于诊断测定，例如用于检测PD-1在特定的细胞、组织或血清中的表达。为了诊断应用，通常用可检测的部分标记(直接或间接)抗体。可利用众多标记物，其通常分为以下类别：生物素、荧光染料、放射性核苷酸、酶、碘和生物合成标记物。
本发明抗体可用于任何已知的测定法中，例如竞争性结合测定法，直接和 间接夹心测定法和免疫沉淀测定法。Zola，Monoclonal Antibodies：A Manual of Techniques(单克隆抗体：技术手册)，第147-158页(CRC Press，Inc.1987)。
抗体还可用于体内诊断测定。通常用放射性核素(例如111In、99Tc、4C、31I、125I、3H、32P、35S或18F)标记抗体，以使可用免疫显像(immunoscintiography)或正电子成像术定位抗原或表达抗体的细胞。
单克隆抗体制备
本发明的单克隆抗体(mAb)可以通过多种技术进行制备，包括常规单克隆抗体方法学，例如Kohler和Milstein,Nature,1975；256:495中所述的标准体细胞杂交技术。虽然优选体细胞杂交规程，但是原则上也可以使用制备单克隆抗体的其它方法，例如B淋巴细胞的病毒或致癌转化。
用于制备杂交瘤的优选动物系统为鼠科动物系统。在小鼠中制备杂交瘤是非常完善的规程。分离用于融合的经免疫的脾细胞的免疫方案和技术是本领域已知的。融合配偶体(例如鼠骨髓瘤细胞)和融合规程也是已知的。
为表达抗体或其抗体片段，可以通过标准分子生物学技术(例如PCR扩增或使用表达目标抗体的杂交瘤的cDNA克隆)获得编码部分或全长轻链和重链的DNA，并且可以将DNA插入到表达载体中，从而使得目的基因与转录和翻译调控序列可操作的连接，转染宿主细胞进行表达，表达宿主优选真核表达载体，更优选哺乳动物细胞，例如CHO及其衍生细胞系。
抗体可通过公知的技术，例如使用蛋白A或蛋白G的亲和层析纯化。随后或备选地，可将特异性抗原或其表位固定在柱上以通过免疫亲和层析而纯化免疫特异性抗体。免疫球蛋白的纯化例如由D.Wilkinson论述(The Scientist,由The Scientist,Inc.,Philadelphia PA,Vol.14,No.8(2000年4月17日),25-28页公布)。
本发明的嵌合或人源化抗体可以根据上述制备的鼠单克隆抗体的序列进行制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。例如，为创造嵌合抗体，可使用本领域已知的方法将鼠可变区连接至人恒定区(参见例如Cabilly等人的美国专利No.4,816,567)。通过将编码VH的DNA可操作 的连接至编码重链恒定区(CH1、CH2和CH3)的另一DNA分子可以将编码VH区的分离的DNA转变为全长重链基因。人重链恒定区基因的序列是本领域已知的(参见例如Kabat,E.A.等人(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)，包含这些区的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区，但是最优选为IgG1或IgG4恒定区。
为创造人源化抗体，可以使用本领域已知的方法将鼠CDR区插入人源框架序列(参见Winter的美国专利No.5,225,539及Queen等人的美国专利Nos.5,530,101；5,585,089；5,693,762和6,180,370)。还可以利用转基因动物，例如，HuMAb小鼠(Medarex,Inc.)含有编码未重排的人重链(μ和γ)和κ轻链免疫球蛋白序列的人免疫球蛋白基因微型基因座(miniloci)，加之使内源μ和κ链基因座失活的靶向突变(参见例如Lonberg等人(1994)Nature 368(6474):856-859)；或携带人重链转基因和人轻链转染色体的“KM小鼠TM”(参见专利WO02/43478)进行抗体人源化改造。其他抗体人源化改造的方法包括噬菌体展示技术。
通过下列实施例进一步说明本发明，所述实施例不应解释为进一步限制。在此将整篇申请中引用的所有附图和所有参考文献、专利和已公开专利申请的内容明确收入本文作为参考。
附图说明
图1、ELISA测定AB12N1和AB12M1与人PD-1的结合。
图2-1、ELISA测定AB12N1和AB12M1与食蟹猴PD-1和人ICOS的交叉反应。
图2-2、ELISA测定AB12N1和AB12M1与人CTLA4的交叉反应。
图2-3、ELISA测定AB12N1和AB12M1与人CD28的交叉反应。
图3、竞争ELISA测定AB12N1和AB12M1阻断人PD-1和PD-L1结合的能力。
图4、SDS-PAGE还原电泳定性分析AB12N1和AB12M1。
图5、ELISA测定AB12M2、AB12M3和AB12M4效价及特异性。
图6、ELISA测定AB12M2、AB12M3和AB12M4与鼠PD-1交叉反应。
图7、竞争ELISA测定AB12M2、AB12M3和AB12M4与Keytruda的相对亲和力。
图8、竞争ELISA测定AB12M2、AB12M3和AB12M4与Opdivo的相对亲和力。
图9、AB12M3和AB12M4与过表达PD-1的CHO细胞的结合。
图10、AB12M3和AB12M4与活化人T细胞的结合。
图11、AB12M3和AB12M4以浓度依赖方式促进T细胞增殖。
图12、AB12M3和AB12M4以浓度依赖方式促进IFN-γ分泌。
图13、AB12M3和AB12M4促进T细胞分泌IL-2。
图14、AB12M4对小鼠肿瘤体积增长的抑制作用。
具体实施方式
实施例1：抗PD-1的鼠源单克隆抗体的制备
将人PD-1胞外段纯化抗原50μg(购自北京义翘神州生物技术有限公司)以完全弗氏佐剂充分乳化后，采用多点免疫方式免疫雄性Balb/C小鼠，免疫周期为三周一次。在第3次免疫后第10天，通过眼窝取血，ELISA测试血浆抗人PD-1抗体滴度以监测小鼠免疫应答程度。然后在融合前3天，对产生抗人PD-1抗体滴度最高的小鼠加强免疫一次。3天后，处死小鼠并取出该小鼠脾脏与小鼠骨髓瘤Sp2/0细胞株融合。混合2×108Sp2/0细胞与2×108脾细胞在50％聚乙二醇(分子量为1450)和5％二甲基亚砜(DMSO)溶液中融合。用Iscove培养基(含有10％胎牛血清，100单位/mL青霉素，100μg/mL链霉素，0.1mM次黄嘌呤，0.4μM氨基蝶呤和16μg胸苷)来调整脾脏细胞数至5×105/mL，以0.3ml加入96孔培养板孔内，并置于37℃，5％CO2培养箱内。培养10天后，采用实施例3.2中ELISA法分别检测上清中抗体与生物素标记的人PD-L1-Fc竞争结合PD-1的能力，筛选并鉴定了8个竞争性较强的阳性杂交瘤细胞株，分别进行亚克隆，再次对上清纯化鼠源抗体进行筛选及鉴定，获得2个阳性杂交瘤单克隆细胞株#22和#32。
实施例2、ELISA法测定抗PD-1鼠源抗体效价
采用ELISA对杂交瘤细胞株#22(所分泌抗体被命名为AB12N1)和#32(该 分泌抗体被命名为AB12M1)培养上清中纯化的鼠源单克隆抗体的效价进行测定。用PBS缓冲液将PD-1(购自北京义翘神州生物技术有限公司)稀释至0.1μg/ml，以100μl/孔的体积加于96孔板中，4℃放置16-20h。将96孔板中PBS缓冲液吸掉，用PBST(pH 7.4，PBS含0.05％吐温20)缓冲液洗板1次后，加入200μl/孔PBST/1％脱脂奶粉，室温孵育1h封闭。移去封闭液，用PBST缓冲液洗板3次后，加入用PBST/1％脱脂奶粉稀释至适合浓度的待测PD-1鼠源抗体，100μl/孔，室温孵育1.5h。移去反应体系，用PBST洗板3次后，50μl/孔加入用PBST/1％脱脂奶粉稀释(稀释比例1：4000)HRP标记的羊抗鼠IgG二抗(购自The Jackson Laboratory)，室温孵育1h。PBST洗板3次后，加入100μl/孔TMB，室温孵育显色10-30min。加入50μl/孔0.2M硫酸终止反应。酶标仪在双波长450/620nm处检测吸光值(O.D.)，计算EC50值。
从图1可知，杂交瘤克隆#22表达的鼠单抗AB12N1和#32杂交瘤克隆表达的鼠单抗AB12M1均能与PD-1结合。AB12M1与抗原结合活性的EC50值约为0.002μg/ml，而AB12N1的EC50值约为0.1μg/ml。
实施例3、抗PD-1鼠源单克隆抗体的筛选与鉴定
3.1、鼠源抗体的结合特异性测定
为检测PD-1抗体对于PD-1同一家族其他蛋白的特异结合活性，人CTLA4、人CD28及人ICOS被用于进行结合检测。同时，为了检测PD-1抗体对于人以外的不同种属的差异性，对小鼠和食蟹猴的PD-1也进行了结合检测。
用PBS缓冲液将人PD-1/His、人ICOS/Fc，人CTLA4/His、人CD28/Fc、食蟹猴PD-1/Fc及鼠PD-1/His(均购自北京义翘神州生物技术有限公司)，稀释至0.1μg/ml，以100μl/孔的体积加于96孔板中，4℃放置16～20h。将96孔板中PBS缓冲液吸掉，PBST(pH 7.4，PBS含0.05％吐温20)缓冲液洗板1次后，加入200μl/孔PBST/1％脱脂奶粉，室温孵育1h封闭。移去封闭液，PBST缓冲液洗板3次后，加入待测PD-1抗体，100μl/孔，室温孵育1.5h。移去反应体系，PBST洗板3次后，50μl/孔加入1：4000稀释的HRP标记的羊抗鼠IgG二抗(购自The Jackson Laboratory)，室温孵育1小时。PBST洗板3次后，加入100μl/孔TMB，室温孵育5-10min。加入50μl/孔0.2M硫酸终止反应。酶标仪在双波长450/620 nm处读取吸收值。
如图2-1至图2-3所示，AB12N1和AB12M1对PD-1家族的其他三种蛋白都没有特异的结合能力。同时，AB12N1和AB12M1与鼠源PD-1也没有种属交叉反应，但AB12M1能特异性结合食蟹猴的PD-1，而AB12N1与食蟹猴的PD-1无特异性结合。
3.2、鼠源抗体阻断PD-1结合PD-L1实验
以生物素标记的人PD-L1作为试剂。用PBS缓冲液将PD-1(购自北京义翘神州生物技术有限公司)稀释至2.0μg/ml，以100μl/孔的体积加于96孔板中，室温过夜。弃去包被溶液，加入200μl/孔PBST/1％脱脂奶粉，室温孵育1h封闭。移去封闭液，PBST缓冲液洗板3次后，然后每孔加入50μL稀释好的鼠源单抗AB12N1和AB12M1与50μl生物素标记的人PD-L1混合液，充分孵育后以PBST洗去未结合的抗体及生物素标记的PD-L1，然后加入100μl每孔的HRP标记的亲和素，充分孵育后以PBST洗去未结合的HRP标记的亲和素，加入100μl每孔的TMB显色液，显色30分钟。用0.2M的硫酸终止反应，以酶标仪在双波长450/620nm处读取吸光度值。如图3中所示，鼠源抗体AB12N1和AB12M1都能特异地阻断PD-1与PD-L1的结合。AB12M1阻断PD-L1与PD-1结合能力明显优于AB12N1。
3.3、纯化鼠源抗体SDS-PAGE分析和Western-Blot鉴定
采用SDS-PAGE电泳和免疫印迹法(Western-Blot)对纯化的鼠单克隆抗体AB12N1和AB12M1进行定性、半定量分析。按胶配置方法配置12％浓度的PAGE胶，各泳道分别加入4μg的抗体AB12N1、AB12M1、Keytruda和Opdivo。电泳至染料抵达分离胶底部，断开电源，以凝胶成像系统观察电泳结果。如图4所示，鼠源抗体AB12N1和AB12M1还原性SDS-PAGE电泳结果均呈现清晰、均一的两条带，分别是约50KD重链和约25KD轻链(其中，各泳道上样如下：1.Marker；2.AB12N1；3.AB12N1；4.AB12M1；5.AB12M1；6.Keytruda；7.Opdivo)。
按胶配置方法配置15％浓度的非还原PAGE胶，人PD-1上样量为5μg，电 泳至染料抵达分离胶底部，断开电源。取下凝胶，平放于大小一致的NC膜上，根据凝胶面积按1mA/cm2接通电源，100mA，电转移2～4小时。浸在封闭液中，4℃封闭过夜，用PBST缓冲液洗膜3次，每次10min。再分别加入过量AB12N1和AB12M1抗体孵育1h，PBST缓冲液洗膜3次，每次10min。以1：5000稀释的HRP-羊抗鼠IgG Fc二抗作为检测抗体，孵育1h，PBST缓冲液洗膜3次。在DAB显色液中避光显色15min，出现条带后立即以水冲洗以终止显色反应，拍照后进行定量和定性分析。
Western-Blot结果显现约34KD大小的PD-1目的条带，说明AB12N1和AB12M1均能特异性结合人PD-1。
3.4、抗PD-1鼠源抗体的亲和力测定以及动力学分析
我们采用生物薄膜干涉技术(BLI)对纯化的鼠单克隆抗体的表征亲和力及结合动力学进行测定。按照Octet分子相互作用仪(ForteBio Octet RED&QK系统，PALL公司)标准操作方法进行测定，对照抗体为Keytruda和Opidivo。多通道平行定量分析浓度梯度设定为：3.125、6.25、12.5、25、50和100nM，将Human PD-1-His(北京义翘神州生物技术有限公司)偶联Ni-NTA传感器。追踪抗原—抗体结合动力学及解离动力学。分析所得数据，以此法测定的ka(kon)、kd(koff)和KD值显示于表1。鼠单克隆抗体AB12M1与人PD-1的平衡解离常数KD值<1×10-12M，与对照抗体Keytruda和Opidivo的结合亲和力相当。而AB12N1的KD值为3.508×10-10M，表征亲和力低于对照抗体Keytruda和Opidivo。
表1、鼠单抗AB12M1和AB12N1的亲和力测定结果
Figure PCTCN2017092026-appb-000001
实施例4、抗PD-1鼠源单抗的亚型鉴定及可变区扩增
抗体亚型鉴定：取杂交瘤细胞培养上清液，采用IsoStripTM小鼠单克隆抗体 亚型鉴定试剂盒(Santa Cruz Biotechnology，货号sc-24958)鉴定抗体亚型。单抗AB12N1亚型经鉴定为IgG1(Kappa)，单抗AB12M1亚型为IgG2b(Kappa)。
抗体可变区扩增：将候选杂交瘤细胞#22和#32分别培养至总数量107个细胞，1000rpm离心10分钟收集细胞，并以Trizol试剂盒(Invitrogen)提取总RNA，用反转录试剂盒SMARTer RACE合成第一链cDNA，以第一链cDNA为后续模板扩增杂交瘤细胞所对应的抗体可变区DNA序列。根据亚型鉴定结果，获取该抗体亚型的重链和轻链恒定区序列，设计特异性的巢式PCR引物，该扩增反应中所使用的引物序列与抗体可变区第一框架区和恒定区互补。采用常规PCR方法扩增目的基因，将扩增产物测序后，得到杂交瘤克隆#22分泌抗体AB12N1的重链可变区序列SEQ ID NO：15和轻链可变区序列SEQ ID NO：16；该抗体的重链CDR(CDR-H1、CDR-H2和CDR-H3)的氨基酸序列分别如SEQ ID NO：2、4和6所示，其轻链CDR(CDR-L1、CDR-L2和CDR-L3)的氨基酸序列分别如SEQ ID NO：8、10和12所示。杂交瘤克隆#32分泌抗体AB12M1的重链可变区序列SEQ ID NO：13和轻链可变区序列SEQ ID NO：14；该抗体的重链CDR(CDR-H1、CDR-H2和CDR-H3)的氨基酸序列分别如SEQ ID NO：1、3和5所示，其轻链CDR(CDR-L1、CDR-L2和CDR-L3)的氨基酸序列分别如SEQ ID NO：7、9和11所示。
实施例5、抗PD-1鼠源抗体的人源化
根据上述获得的AB12N1和AB12M1抗体可变区序列，利用计算机辅助抗体三维建模以及结构分析进行抗体人源化改造。CDR移植(CDR-Grafting)是一种常见的抗体人源化方法，是通过将人源抗体的FR替换鼠源抗体的FR来达到保持活性、降低免疫原性的目的。结合Discovery Studio分析工具进行CDR移植抗体人源化改造的方法主要包括以下步骤：(1)抗体三维结构建模；(2)关键残基分析。利用分子对接分析可变区及其周边的框架氨基酸序列，考察其空间立体结合方式以确定对于保持CDR区构象至关重要的关键残基，主要有三类：1、位于VL和VH结合界面上的残基，对于两个结构域的折叠起到关键作用；2、靠近CDR区并且包埋于蛋白内部的残基；3、与CDR区有直接相互作用的残基，相互作用包括：疏水相互作用/氢键/盐桥；(3)人源模板选择。须同时满足下面 两个条件：首先，把各杂交瘤细胞分泌的抗体氨基酸序列与人胚胎系抗体氨基酸序列进行比对，找出同源性高的序列；其次，选择其中与MHC II(HLA-DR)亲和力低的人胚胎系(germline)抗体框架序列，以降低其免疫原性；(4)基于关键残基分析的反向嫁接，获得人源化抗体序列。
其中，鼠源抗体AB12M1以人VH3-23重链可变区和人VK3D-11轻链可变区为模板序列，共获得7个人源化抗体，分别是AB12M3、AB12M4、AB12M5、AB12M6、AB12M7、AB12M8和AB12M9。同时，构建了它的一株人鼠嵌合抗体AB12M2，是将鼠源抗体的重链可变区序列嫁接至人IgG1重链恒定区，将鼠源抗体的轻链可变区序列嫁接至人Kappa轻链恒定区而获得。上述人源化抗体的可变区氨基酸序列如表2所示。
鼠源抗体AB12N1以人VH3-33重链可变区和人VK3-11轻链可变区为模板序列，共获得3个人源化抗体，分别是AB12N3、AB12N4和AB12N5。同时，构建了它的一株人鼠嵌合抗体AB12N2，是将鼠源抗体的重链可变区序列嫁接至人IgG1重链恒定区，将鼠源抗体的轻链可变区序列嫁接至人Kappa轻链恒定区而获得。上述人源化抗体的可变区氨基酸序列如表2所示。
从表3中各人源化抗体的亲和力常数及动力学参数可知，AB12M3、AB12M4、AB12M5、AB12M6、AB12M7、AB12M8和AB12M9与鼠源抗体AB12M1以及嵌合抗体AB12M2相比人源化程度超过95％，但亲和力没有明显损失，KD值均小于1×10-12M，保留了亲本鼠单克隆抗体的亲和力和特异性，大大降低了其免疫原性。
另一组人源化抗体AB12N3、AB12N4和AB12N5与鼠源抗体AB12N1以及嵌合抗体AB12N2相比人源化程度也超过95％，亲和力也未出现明显下降，KD值均在10-10M数量级。
表2、人源化抗体可变区氨基酸序列
Figure PCTCN2017092026-appb-000002
Figure PCTCN2017092026-appb-000003
表3、人源化抗体亲和力比较
Figure PCTCN2017092026-appb-000004
实施例6、抗PD-1人源化抗体功能鉴定
6.1、间接法ELISA测定人源化抗体的效价及结合特异性
采用间接法ELISA测定人源化抗体AB12M3和AB12M4及嵌合抗体AB12M2与抗原PD-1的结合特性。以Keytruda和Opdivo作为对照抗体，以培养基作为阴性对照。使用HRP标记的羊抗人IgG抗体作为检测抗体(购自The Jackson Laboratory)，具体方法同实施例2所述。同样的方法，被用于检测人源化抗体AB12M3和AB12M4及嵌合抗体AB12M2对小鼠PD-1(购自北京义翘神州生物技术有限公司)有无交叉反应，同样以Keytruda和Opdivo作为对照抗 体，以培养基作为阴性对照。
如图5所示，人源化抗体AB12M3和AB12M4及嵌合抗体AB12M2均能特异性地结合人PD-1，且它们与抗原结合活性的EC50值均低于对照抗体Keytruda和Opdivo，约在0.001～0.01μg/ml之间。这表明本发明所构建的抗PD-1人源化抗体AB12M3和AB12M4及嵌合抗体AB12M2与PD-1的结合能力未因人源化改造而消减，仍保留了鼠源亲本抗体的高亲和力。并且，它们与小鼠PD-1均不结合，具有较强的种属特异性(图6)。
6.2、PD-1人源化抗体相对亲和力测定
以辣根过氧化物酶(HRP)标记的Keytruda和Opdivo作为试剂。用PBS缓冲液将PD-1(购自北京义翘神州生物技术有限公司)稀释至0.1μg/ml，以100μl/孔的体积加于96孔板中，室温过夜。弃去包被溶液，加入200μl/孔PBST/1％脱脂奶粉，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板3次后，然后每孔加入50μL生长培养基(DMEM+5％FBS)与50μL HRP标记的Keytruda或Opdivo抗体的混合液，以PBS洗去未结合的HRP标记的Keytruda或Opdivo抗体。然后分别加入抗体AB12M2、AB12M3和AB12M4；以未标记的Keytruda或Opdivo作为阳性对照。充分孵育后以PBS洗去未结合的HRP标记的Keytruda或Opdivo抗体，然后以酶标仪在双波长450/620nm处读取吸光度值。
结果如图7及图8所示，抗体AB12M2和AB12M3及AB12M4都能显著地竞争性阻断Keytruda或Opdivo与PD-1的结合，且它们与Keytruda-HRP或Opdivo-HRP竞争结合PD-1的EC50值均低于Keytruda及Opdivo，但都在0.1～1μg/ml之间。因此，可以判断抗体AB12M2、AB12M3及AB12M4与Keytruda和Opdivo的亲和力大小相当。
6.3、PD-1人源化抗体体外阻断PD-1与PD-L1结合实验
将带His标签的PD-1蛋白胞外区片段包被96孔酶标板后，封闭、洗板，加入待测PD-1抗体，同时加入生物素标记的PD-L1-Fc，孵育反应。洗板后，检测生物素标记的PD-L1/Fc结合量，计算PD-1抗体对配体PD-L1结合阻断的IC50值。
用pH 7.2PBS缓冲液将PD1/His稀释至2μg/ml，以100μl每孔的体积加入96孔酶标板中，室温振荡孵育1小时。将96孔酶标板中PBS缓冲液吸掉，加入200μl每孔PBST(pH 7.2PBS含0.05％吐温-20)/1％脱脂奶粉，室温孵育1小时封闭。PBST洗板3次，加入50μl每孔用封闭液稀释至合适浓度的待测PD-1抗体，同时加入50μl每孔用封闭液稀释至200ng/ml的生物素标记的PD-L1/Fc，室温孵育1小时，PBST洗板3次，加入100μl每孔用封闭液1：250稀释的SA-Avidin-HRP(HRP标记的链霉亲和素)，室温孵育1小时。PBST洗板3次，加入100μl每孔TMB，室温孵育5-10分钟。加入50μl每孔0.2M硫酸终止反应。用酶标仪在450nm处读取吸收值，计算PD-1抗体对配体PD-L1结合阻断的IC50值。
如表4中实验结果所示，AB12M3和AB12M4抗体都能有效阻断PD-L1与PD-1的结合，并且优于Keytruda，与Opdivo结果相似。
表4、AB12M3和AB12M4体外阻断PD-1与PD-L1结合的IC50值
Figure PCTCN2017092026-appb-000005
6.4、抗PD-1人源化抗体体外细胞结合实验
FACS(荧光活性细胞分选仪)是一个用于检测蛋白和细胞结合的试验方法。本测试用于检测本发明的PD-1人源化抗体与细胞表面表达的天然PD-1的结合活性。本测试所用的细胞为PD-1过表达的CHO细胞。3×105个CHO细胞与一系列梯度浓度待测的AB12M3或AB12M4(一抗)孵育30分钟，经洗涤后加入FITC标记的羊抗人IgG二抗(购自BD Biosciences公司)结合30分钟，流式细胞仪检测FITC信号。结果显示在图9中，AB12M3和AB12M4可以与过表达于CHO细胞表面的PD-1特异性结合。
6.5、抗PD-1人源化抗体与活化人T细胞特异性结合试验
采用密度梯度离心法(LymphoprepTM，人淋巴细胞分离液，STEMCELL公司)从人外周血获得新鲜单个核细胞，使用T细胞分选试剂(STEMCELL公司)获得高纯度T淋巴细胞。经过5μg/ml抗CD3抗体刺激48h，加入250IU/ml的 人IL-2培养7天获得大量活化T淋巴细胞。3×105个T淋巴细胞与一系列浓度梯度的AB12M3或AB12M4(一抗)孵育30分钟，经洗涤后加入FITC标记的羊抗人IgG二抗(BD Biosciences公司)，流式细胞仪检测FITC信号。结果显示在图10中，AB12M3和AB12M4可以与表达于活化人T细胞表面的PD-1受体特异性结合。
实施例7、抗人PD-1人源化抗体的生物学活性测定
7.1、抗PD-1人源化抗体在混合淋巴细胞反应中对细胞增殖和细胞因子分泌的影响
利用混合淋巴细胞反应证明阻断PD-1/PD-L1途径对淋巴效应细胞的影响。测定PD-1抗体或者同型IgG对照抗体对混合淋巴细胞反应中T细胞增殖以及IFN-γ分泌的影响。
新鲜分离的人PBMC调整细胞密度为2.0×106个/ml，采用贴壁法获得单核细胞。添加100ng/ml GM-CSF和100ng/ml IL-4培养5天，补加100ng/ml TNF-ɑ诱导DC细胞成熟。使用CD4+T细胞正选试剂盒(STEMCELL公司)从人新鲜PBMC中分离CD4+T细胞。96孔板中，每孔250μl培养液含有105个分离的T细胞，104个诱导成熟的DC细胞以及一系列浓度梯度的AB12M3或AB12M4。使用同型IgG对照抗体作为阴性对照。混合淋巴细胞在37℃，5％CO2细胞培养箱中培养6天，然后从96孔板中每孔取出100μl培养上清进行IFN-γ测定。IFN-γ采用OptEIA ELISA试剂盒(BD Biosciences公司)测定。96孔板剩余细胞用Cell Titer Glo试剂盒(Promega公司)测定细胞增殖情况。结果显示，AB12M3和AB12M4以浓度依赖方式促进T细胞增殖(图11)和IFN-γ(图12)分泌。
7.2、抗PD-1人源化抗体对超抗原刺激诱导人PBMC细胞分泌细胞因子的影响
新鲜制备的人PBMC细胞，用含20μg/ml AB12M3、AB12M4或同型IgG对照抗体的RPMI 1640培养基(含10％灭活FBS)重悬为106/ml，接种于96孔板，100μl/孔。超抗原SEB的最高浓度为2500ng/ml，10倍稀释4个梯度，加入到96孔板中，设三复孔。培养72小时，取上清使用OptEIA ELISA试剂盒(BD  Biosciences公司)测定IL-2浓度。结果显示在图13中，AB12M3和AB12M4可以促进T细胞分泌IL-2。
7.3、体外测定抗PD-1人源化抗体刺激T细胞杀伤肿瘤细胞效应
将PD-L1过表达的人非小细胞肺癌细胞株HCC827(中科院上海细胞库)接种于96孔细胞培养板，加入一系列浓度的AB12M1、AB12M3、AB12M4或huIgG，然后按照10：1的效靶比加入用抗CD3抗体和IL-2活化的T细胞，培养48小时，使用培养基洗板，去除大部分T细胞，用CCK-8细胞增殖试剂盒(Dojindo公司)测定HCC827细胞存活情况，计算杀伤率。结果显示在表5中，表明AB12M3、AB12M4具备增强T细胞杀伤肿瘤细胞的能力。
表5、人源化抗体AB12M3和AB12M4对肿瘤细胞的杀伤率(％)
Figure PCTCN2017092026-appb-000006
7.4、抗PD-1人源化抗体在人源化PD-1小鼠体内皮下移植结肠癌MC38细胞模型中的药效学研究
采用北京百奥赛图生物技术有限公司构建的B-hPD-1人源化小鼠进行抗人PD-1抗体的体内药效评价，该小鼠采用C57BL/6背景，采用基因打靶技术将小鼠PD-1基因的包括IgV结构域部分在内的第二个外显子部分进行人源化改造。改造成功的小鼠带有人鼠嵌合的PD-1，即胞外部分为hPD-1而胞内部分为mPD-1。这类嵌合PD-1结构不影响PD-1正常信号传递，鼠源或人源PD-L1配体与此PD-1受体结合后可抑制T细胞活性。
将MC38鼠结肠癌细胞(购自舜冉上海生物科技有限公司)以5×105个/0.1mL接种于雌性B-hPD-1人源化小鼠右侧前胁肋部皮下，待肿瘤生长到约150mm3时按肿瘤体积随机分组，每组8只，共3组，分别为：(1)溶剂对照组(PBS组)、(2)AB12M4处理组和(3)Keytruda对照组(购自Merck公司，批号：5SNL80505)，组(2)和组(3)的给药剂量为20mg/kg，给药体积10ml/kg。所有组给药途径均为腹腔注射，每3天给药1次，连续给药6次，当实验进行到接种后第28天结 束。
使用游标卡尺测量肿瘤长径(L)和短径(W)，通过公式V＝1/2(L×W2)计算肿瘤体积(V)，每周测量3次，同时测量小鼠体重。
如图14所示，实验结束时，溶剂对照组平均肿瘤体积为3405.2mm3。AB12M4处理组的平均肿瘤体积为277.4mm3，Keytruda给药组的平均肿瘤体积为249mm3。表明AB12M4具有显著抑瘤作用，其抑瘤效果与Keytruda相当。另外，整个实验过程中，动物健康状态良好，无动物死亡。实验结束时，各组动物体重均出现增长，AB12M4处理组动物与溶剂对照组动物体重比较无显著性差异(p>0.05)，表明动物对AB12M4耐受良好，未对实验动物产生明显毒性作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (27)

  1. 分离的结合PD-1的单克隆抗体，其包含：
    重链可变区，其包含CDR-H1、CDR-H2和CDR-H3序列；和
    轻链可变区，其包含CDR-L1、CDR-L2和CDR-L3序列，
    其中：
    (i)重链可变区包含选自SEQ ID NOs：1和2所示的CDR-H1序列，和选自SEQ ID NOs：3和4所示的CDR-H2序列，和选自SEQ ID NOs：5和6所示的CDR-H3序列；和
    (ii)轻链可变区包含选自SEQ ID NOs：7和8所示的CDR-L1序列，和选自SEQ ID NOs：9和10所示的CDR-L2序列，和选自SEQ ID NOs：11和12所示的CDR-L3序列。
  2. 如权利要求1所述抗体，其特征在于，其重链可变区包含SEQ ID NO：1所示的CDR-H1序列，和SEQ ID NO：3所示的CDR-H2序列，和SEQ ID NO：5所示的CDR-H3序列；和其轻链可变区包含SEQ ID NO：7所示的CDR-L1序列，和SEQ ID NO：9所示的CDR-L2序列，和SEQ ID NO：11所示的CDR-L3序列。
  3. 如权利要求2所述抗体，其特征在于，所述抗体为鼠源的或嵌合的，其重链可变区包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区；和其轻链可变区包含鼠源κ、λ链或其变体的轻链FR区。
  4. 如权利要求3所述抗体，其特征在于，其重链可变区包含SEQ ID NO：13的氨基酸序列；和其轻链可变区包含SEQ ID NO：14的氨基酸序列。
  5. 如权利要求2所述抗体，其特征在于，所述抗体为人源化的。
  6. 如权利要求5所述抗体，其特征在于，其重链可变区包含选自SEQ ID NOs：17、19、21、23、25、27和29所示的氨基酸序列；和其轻链可变区包含选自SEQ ID NOs：18、20、22、24、26、28和30所示的氨基酸序列。
  7. 如权利要求6所述抗体，其特征在于，其重链可变区分别包含SEQ ID NO：17、19、21、23、25、27和29所示的氨基酸序列；和其轻链可变区分别包含SEQ ID NO：18、20、22、24、26、28和30所示的氨基酸序列。
  8. 如权利要求1所述抗体，其特征在于，其重链可变区包含SEQ ID NO：2所示的CDR-H1序列；和SEQ ID NO：4所示的CDR-H2序列；和SEQ ID NO： 6所示的CDR-H3序列；和其轻链可变区包含SEQ ID NO：8所示的CDR-L1序列；和SEQ ID NO：10所示的CDR-L2序列；和SEQ ID NO：12所示的CDR-L3序列。
  9. 如权利要求8所述抗体，其特征在于，所述抗体为鼠源的或嵌合的，其重链可变区包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区；和其轻链可变区包含鼠源κ、λ链或其变体的轻链FR区。
  10. 如权利要求9所述抗体，其特征在于，其重链可变区包含SEQ ID NO：15所示的氨基酸序列；和其轻链可变区包含SEQ ID NO：16所示的氨基酸序列。
  11. 如权利要求8所述抗体，其特征在于，所述抗体为人源化的。
  12. 如权利要求11所述抗体，其特征在于，其重链可变区包含选自SEQ ID NOs：31、33和35所示的氨基酸序列；和其轻链可变区包含选自SEQ ID NOs：32、34和36所示的氨基酸序列。
  13. 如权利要求12所述抗体，其特征在于，其重链可变区分别包含SEQ ID NO：31、33和35所示的氨基酸序列；和其轻链可变区分别包含SEQ ID NO：32、34和36所示的氨基酸序列。
  14. 如权利要求1-13任一项权利要求所述抗体，其特征在于，所述抗体包含一个或更多个氨基酸的替换、添加和/或缺失。
  15. 如权利要求14所述抗体，其特征在于，包含一个或更多个氨基酸的替换、添加和/或缺失的抗体与其来源序列至少80％、85％、90％、95％、96％、97％、98％或99％同源。
  16. 如权利要求1-15任一项权利要求所述抗体，其特征在于，所述抗体为包含人IgG4或IgG1的重链恒定区和人κ轻链恒定区的全长抗体；或仅包含Fab或Fab’2或ScFv的抗原结合片段。
  17. 如权利要求1-15任一项权利要求所述抗体，其特征在于，所述抗体含有糖基化修饰。
  18. 如权利要求1-15任一项权利要求所述抗体，其特征在于，所述抗体以1nM或更低的KD结合PD-1，较优选地，以100pM或更低的KD结合PD-1；更优选地，以10pM或更低的KD结合PD-1；最优选地，以1pM或更低的KD结合PD-1。
  19. 编码如权利要求1-15任一项所述抗体的DNA分子。
  20. 如权利要求19所述的DNA分子，其特征在于，编码所述抗体重链可变区的DNA分子选自SEQ ID NO：37、39和41，和编码所述抗体轻链可变区的DNA分子选自SEQ ID NO：38、40和42。
  21. 包含如权利要求19所述DNA分子的表达载体。
  22. 用如权利要求21所述的表达载体转化的宿主细胞，所述宿主细胞优选为CHO细胞。
  23. 包含如权利要求1-15任一项所述抗体的双特异性分子，优选地，所述双特异性分子还包括但不限于针对以下分子的抗体：VEGF、EGFR、Her2/neu、VEGF受体或其它生长因子受体、CD20、CD40、CTLA-4、OX-40、4-1-BB和ICOS。
  24. 包含如权利要求1-15任一项所述抗体的免疫缀合物，所述免疫缀合物还包含治疗剂，其中，治疗剂优选为毒素、放射性同位素、药物或细胞毒剂。
  25. 药物组合物，其包含如权利要求1-15任一项所述抗体以及可药用赋形剂、载体或稀释剂。
  26. 制备如权利要求1-15任一项所述抗体的方法，其包括：在允许产生所述抗体的条件下培养包含如权利要求22所述的宿主细胞，以及回收、分离产生的所述抗体。
  27. 如权利要求1至15任一项所述抗体或如权利要求23所述双特异性分子或如权利要求24所述免疫缀合物或如权利要求25所述的药物组合物在制备用于治疗PD-1介导的疾病或病症的药物中的用途，其中所述的疾病或病症优选为癌症；更优选为高表达PD-L1的癌症；所述的癌症包括但不限于肺癌、肝癌、卵巢癌、宫颈癌、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、头颈癌；优选为乳腺癌、肺癌、胃癌、肠癌、肾癌、黑素瘤；最优选为非小细胞肺癌、黑素瘤和肾癌；其中，所述疾病优选为感染性疾病；所述感染性疾病包括但不限于慢性病毒感染、细菌感染或寄生虫感染疾病，所述感染性疾病更优选为HIV、HBV和HCV。
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