WO2021000813A1 - 一种靶向pd-1的单克隆抗体及其应用 - Google Patents

一种靶向pd-1的单克隆抗体及其应用 Download PDF

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WO2021000813A1
WO2021000813A1 PCT/CN2020/098698 CN2020098698W WO2021000813A1 WO 2021000813 A1 WO2021000813 A1 WO 2021000813A1 CN 2020098698 W CN2020098698 W CN 2020098698W WO 2021000813 A1 WO2021000813 A1 WO 2021000813A1
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seq
antibody
amino acid
cancer
acid sequence
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French (fr)
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方树彬
林基祯
金荣
林金香
李修凤
翁焕娇
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方树彬
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of biopharmaceuticals, in particular to a monoclonal antibody against human PD-1 and its application in disease treatment.
  • PD-1 Programmed death receptor 1
  • CTLA-4 PD-1 (encoded by the gene Pdcd1) and CTLA-4 belong to the same CD28 family of receptors, and it has 23% amino acid sequence homology with CTLA-4 (Okazaki, T. & Honjo, T. Trends Immunol. 27, 195-201 (2006) )).
  • PD-1 protein is a 55kDa type I transmembrane protein, containing an extracellular IgV region, a transmembrane region and an intracellular region. The intracellular region contains an immune receptor tyrosine inhibitory motif (ITIM) close to the membrane and away from the membrane.
  • ITIM immune receptor tyrosine inhibitory motif
  • PD-1 tyrosine-based switching motif
  • ITMS tyrosine-based switching motif
  • PD-1 lacks the MYPPPY motif necessary to bind B7-1 and B7-2.
  • Two ligands for PD-1 have been identified, PD-L1 and PD-L2, which have been shown to down-regulate T cell activation after binding to PD-1.
  • Both PD-L1 and PD-L2 are B7 homologs, which bind to PD-1 but not to other CD28 family members.
  • PD-L1 is expressed on B cells, T cells, macrophages, and dendritic cells (DC), but also on peripheral cells (such as microvascular endothelial cells) and non-lymphoid organs (such as heart, lung, etc.).
  • PD-L2 is only present in activated macrophages and DCs.
  • PD-L1 and PD-L2 are also often expressed in a variety of tumor cells, and PD-1 (on tumor-infiltrating lymphocytes) and PD-1 (on tumor-infiltrating lymphocytes) have been found in many primary human tumors through immunohistochemical assessment and biopsy. / Or PD-L1 (on tumor cells).
  • Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors.
  • PCT International Application PCT/JP2006/309606 discloses isolated monoclonal antibodies that specifically bind to PD-1 with high affinity. The patent is believed Related to Opdivo of Bristol-Myers Squibb.
  • PCT International Application PCT/US2008/007463 discloses an antibody against the human programmed death receptor PD-1, which is believed to be related to Merck's Keytruda.
  • the PD-1/PD-L1 antibody has been officially approved by the US FDA for malignant melanoma, non-small cell lung cancer, liver cancer, gastric cancer, kidney cancer, bladder cancer, head and neck tumors, Hodgkin Lymphoma, cervical cancer, Merkel cell carcinoma and all solid tumors with highly unstable microsatellites (MSI-H).
  • MSI-H microsatellites
  • PD-1/PD-L1 drugs approved for marketing of which 6 PD-1 antibodies are Bristol-Myers Squibb’s Opdivo, Merck’s Keytruda, Sanofi (Sanofi) and Regeneron (Regeneron) Libtayo developed by Junshi Biologicals, Terelimumab developed by Junshi Bio, Sintilizumab developed by Xinda Biosciences, and Karelizumab developed by Jiangsu Hengrui.
  • the 3 PD-L1 antibodies are Roche’s Tecentriq, Imfinzi from AstraZeneca and Bavencio from Merck. The approved indications for these drugs are shown in the table above.
  • the present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, which specifically bind to PD-1 with high affinity. Also provided are nucleic acid molecules encoding the antibodies of the invention, expression vectors, host cells and methods for expressing the antibodies of the invention, and pharmaceutical compositions containing the antibodies of the invention.
  • Another aspect of the present invention relates to methods of using anti-PD-1 antibodies to modulate immune responses, providing their use for enhancing T cell function to up-regulate cell-mediated immune responses, and treating various diseases, including infections (such as acute And chronic) and tumor immunity methods.
  • the invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises:
  • the heavy chain variable region CDR1 which contains an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 13 consistent with the amino acid sequence shown;
  • the heavy chain variable region CDR2 which contains an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14 consistent with the amino acid sequence shown;
  • Heavy chain variable region CDR3 which includes an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15 consistent with the amino acid sequence shown;
  • the light chain variable region CDR1 which includes an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16 consistent with the amino acid sequence shown;
  • the light chain variable region CDR2 which includes an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17 consistent with the amino acid sequence shown;
  • the light chain variable region CDR3 which includes an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18;
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, the CDR sequence homology of the variable region of the heavy chain and the variable region of the light chain is at least 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the present invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises:
  • amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are consistent with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region CDR1
  • amino acid sequences of CDR2 and CDR3 are consistent with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; or
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises heavy chain and light chain variable region sequences:
  • the heavy chain variable region which includes an amino acid sequence selected from SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27;
  • the light chain variable region which comprises an amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 25, or SEQ ID NO: 29;
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody or an antigen-binding portion thereof, the heavy chain variable region of which is selected from SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 27
  • the sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology; its light chain variable region is selected from SEQ ID
  • the amino acid sequence of NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29 has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Sequence homology.
  • the invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises:
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the antibody is a full-length antibody comprising a human or murine constant region.
  • the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
  • the human constant region is IgG4.
  • the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from glycosylation.
  • the antibody or antibody fragment of the invention is a chimeric antibody or chimeric antibody fragment.
  • the antibody or antibody fragment of the invention is a human antibody or human antibody fragment.
  • the antibody or antibody fragment of the present invention is a humanized antibody or humanized antibody fragment.
  • the antibody fragment of the present invention is a Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2 antibody fragment.
  • the antibody fragments of the invention are diabodies.
  • the present invention also includes bispecific antibodies comprising any of the above-mentioned antibodies or antibody fragments that bind to human PD-1.
  • the invention provides an isolated monoclonal antibody comprising:
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody comprising:
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the invention provides isolated polynucleotides encoding the antibodies or antibody fragments.
  • the nucleotide sequence encoding the antibody heavy chain is such as SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35.
  • SEQ ID NO: 37 the nucleotide sequence encoding the antibody light chain is as SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 41 Or as shown in SEQ ID NO: 43.
  • the present invention provides an expression vector containing the isolated polynucleotide, and a host cell containing the expression vector.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antibody fragment and a pharmaceutically acceptable carrier.
  • the present invention provides a method for improving the activity of immune cells, which includes contacting immune cells with the antibody or antibody fragment.
  • the present invention provides a method of treating diseases, the method comprising administering a therapeutically effective amount of the antibody or antibody fragment of the present invention to a subject in need of treatment.
  • the present invention provides a method of modulating an immune response in a subject, which comprises administering to the subject the antibody of the present invention or an antigen binding portion thereof, so that the immune response in the subject is regulated.
  • the antibodies of the invention enhance, stimulate or increase the immune response in the subject.
  • the method includes the combined application of other drugs or therapies to the subject.
  • the present invention provides the use of the antibody or antibody fragment in the preparation of a medicine for treating diseases, wherein the disease is cancer or an infectious disease.
  • the present invention provides a method of inhibiting the growth of tumor cells in a subject, which comprises administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or antigen binding portion thereof.
  • the antibody of the present invention is preferably used in this method, although other anti-PD-1 antibodies can also be used instead (or in combination with the anti-PD-1 antibody of the present invention).
  • chimeric, humanized or fully human anti-PD-1 antibodies can be used in methods of inhibiting tumor growth.
  • the present invention provides a method of treating an infectious disease in a subject, which comprises administering to the subject a therapeutically effective amount of an anti-PD-1 antibody or antigen binding portion thereof.
  • the antibody of the present invention is preferably used in this method, although other anti-PD-1 antibodies can also be used instead (or in combination with the anti-PD-1 antibody of the present invention).
  • chimeric, humanized or fully human anti-PD-1 antibodies can be used in methods of treating infectious diseases.
  • the antibody of the present invention has higher affinity, better blocking effect, and higher cell killing activity when inhibiting the growth of tumor cells. Its affinity is about 10 times higher than that of existing therapeutic monoclonal antibodies, and it is very effective in blocking the interaction of PD-L1/PD-1 in vitro. Compared with Opdivo (Nivolumab), a marketed product of the American company (BMS), its blocking effect and in vitro killing activity are improved, and the monoclonal antibody of the present invention has more excellent effects in inhibiting the growth of tumor cells.
  • Figure 1 ELISA method to determine the serum titer of female Balb/c mice.
  • the horizontal axis represents the dilution factor
  • the vertical axis represents the measured OD value
  • UM1-1/UM1-2/UM1-3/UM1-4 represent different mice.
  • Figure 2 Flow cytometric determination of the blocking activity of mouse PD-1 monoclonal antibodies.
  • the horizontal axis represents the antibody concentration, and the vertical axis represents the blocking rate.
  • UM1-45/UM1-47/UM1-51/UM1-56/UM1-63/UM1-69 represent different murine PD-1 monoclonal antibodies.
  • Figure 3 ProteinA affinity separation and purification of humanized PD-1 monoclonal antibody.
  • the horizontal axis represents the mobile phase volume, and the vertical axis represents the UV absorbance value.
  • Figure 3-2 is a partial enlarged view of Figure 3-1.
  • Figure 4 SDS-PAGE to detect the molecular weight of antibodies.
  • Figure 4-1 SDS-PAGE (reduced)
  • Figure 4-2 SDS-PAGE (non-reduced)
  • FIG. 5 Ultra performance liquid molecular exclusion chromatography UPLC-SEC detects the purity of antibodies.
  • the horizontal axis represents time, and the vertical axis represents absorbance.
  • FIG. 6 Octet Red96 measures affinity.
  • the horizontal axis represents time, and the vertical axis represents bonding height.
  • Figure 7 Determination of blocking activity of humanized PD-1 monoclonal antibody (hUM1-30) by flow cytometry.
  • Figure 8 Detection of tumor elimination effect in SPC lung cancer model mice.
  • the horizontal axis is days, and the vertical axis is tumor volume.
  • Figure 9 Survival curve of SPC lung cancer model mice.
  • the horizontal axis is the number of days, and the vertical axis is the survival rate.
  • Figure 10 The treatment effect of each SPC lung cancer model mouse given the positive control antibody Opdivo.
  • the horizontal axis is days, and the vertical axis is tumor volume.
  • Figure 11 The therapeutic effect of humanized PD-1 monoclonal antibody (hUM1-30) administered to each SPC lung cancer model mouse.
  • the horizontal axis is days, and the vertical axis is tumor volume.
  • Figure 12 The treatment effect of each SPC lung cancer model mouse given the negative control antibody huIgG.
  • the horizontal axis is the number of days, and the vertical axis is the tumor volume.
  • the invention relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, which specifically bind to PD-1.
  • the antibody of the present invention exhibits one or more excellent functional properties, such as binding to human PD-1 with high affinity, inhibiting one or more PD-1 ligands (such as PD-L1 and / Or PD-L2) ability to bind, ability to stimulate antigen-specific memory response, ability to stimulate antibody response and/or ability to inhibit tumor cell growth in vivo.
  • PD-1 ligands such as PD-L1 and / Or PD-L2
  • the invention provides, for example, isolated antibodies, methods of preparing such antibodies, and pharmaceutical compositions containing the antibody molecules of the invention.
  • the present invention relates to a method of using anti-PD-1 antibodies to inhibit tumor cell growth in a subject.
  • anti-PD-1 antibodies can inhibit tumor cell growth in vivo.
  • the present invention also relates to methods of using the antibodies to modulate immune responses and treat diseases such as cancer or infectious diseases, or to stimulate a protective autoimmune response or stimulate an antigen-specific immune response (for example, by co-administering anti-PD-1 and a target antigen) .
  • programmed death 1 includes human PD-1 variants, isoforms, species homologs, and analogs that have at least one common epitope with PD-1.
  • the complete PD-1 sequence can be found according to GenBank number U64863.
  • Antibody refers to any form of antibody that exhibits a desired biological activity (for example, inhibiting the binding of a ligand to its receptor or inhibiting ligand-induced receptor signal transduction). Therefore, “antibody” is used in its broadest sense, and clearly includes but is not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (such as bispecific antibodies).
  • Antibody fragments and “antibody binding fragments” mean antigen-binding fragments of antibodies and antibody analogs, including but not limited to: Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies ); single-chain antibody molecules, such as scFv, single antibody; Nanobody; domain antibody (Nanobody); and multispecific antibody formed by antibody fragments.
  • the engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.
  • the "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • 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 containing the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.
  • the "Fab' fragment” contains a light chain and a portion of a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both the heavy and light chains, but lacks the constant region.
  • single chain Fv antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.
  • Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding.
  • “Diabodies” are small antibody fragments with two antigen binding sites.
  • the fragment comprises a heavy chain variable domain (VH) (VH-VL or VL-VH) connected to a light chain variable domain (VL) in the same polypeptide chain.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • Chimeric antibody is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal non-human immunoglobulin sequences.
  • the majority of humanized antibodies are human immunoglobulins (acceptor antibodies), in which the hypervariable region residues of the acceptor antibody are modified by non-human species (donor antibody) with the required specificity, affinity, and ability.
  • donor antibody non-human species
  • non-human species include mice, rats, rabbits, or non-human primates.
  • Fv framework region (FR) residues of human immunoglobulins are replaced with corresponding non-human residues.
  • humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance.
  • a humanized antibody contains almost all of at least one and usually two variable domains, wherein all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or almost all of the FR regions It is the FR region of human immunoglobulin sequence.
  • the humanized antibody optionally also comprises at least part of an immunoglobulin (usually a human immunoglobulin) constant region (Fc).
  • an “isolated” antibody is an antibody that has been identified and separated from a component of its natural environment (for example, an isolated antibody that specifically binds to PD-1 has no antibody that specifically binds to antigens other than PD-1).
  • the pollutant components of the natural environment are substances that interfere with the diagnostic or therapeutic application of the antibody, and may include enzymes, hormones and other protein solutes or non-protein solutes.
  • the antibody is purified to more than 95% purity, more preferably more than 99% purity, as determined by the Lowry method. Isolated antibodies are usually prepared by at least one purification step.
  • nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule (usually associated with it in the natural source of the antibody nucleic acid).
  • An isolated nucleic acid molecule is different from its naturally occurring form or environment.
  • monoclonal antibody or “monoclonal antibody composition” as used herein refers to a preparation of antibody molecules of a single molecular composition.
  • the monoclonal antibody composition exhibits a single binding specificity and affinity for a specific epitope
  • the monoclonal antibodies used in the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975, Nature 256:495), or can be prepared by recombinant DNA methods.
  • Monoclonal antibodies herein specifically include "chimeric" antibodies or Humanized.
  • Immune cell includes cells that have hematopoietic origin and play a role in immune responses.
  • Immune cells include: lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • a "variant" of a sequence refers to a sequence that differs from the shown sequence at one or more amino acid residues but retains the biological activity of the resulting molecule.
  • the term "about” as used herein means that the index value is within the acceptable error range of the specific value determined by a person of ordinary skill in the art, and the value depends in part on how it is measured or determined (ie, the limit of the measurement system). For example, “about” or “substantially comprising” can mean a range of up to 20%. Furthermore, especially for biological systems or processes, the term can mean at most an order of magnitude or at most 5 times the value. Unless otherwise stated, when a specific value appears in this application and claims, the meaning of "about” or “substantially comprising” should be assumed to be within the acceptable error range of the specific value.
  • administering and “treatment” are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means to combine exogenous drugs, therapeutic agents, diagnostic agents or compositions with animals, humans, and recipients. Contact with the treated person, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting the agent with the cell and contacting the agent with a fluid, where the fluid contacts the cell.
  • administering and “treatment” also mean treatment of cells in vitro and ex vivo, for example, by reagents, diagnostic agents, binding compositions, or by other cells.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of medical diseases.
  • An effective amount also means an amount sufficient to enable or facilitate diagnosis.
  • the effective amount for a specific subject may vary depending on various factors, such as the disease to be treated, the overall health of the patient, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • the antibodies of the present invention are characterized by specific functional characteristics or properties of the antibodies.
  • the antibody specifically binds to PD-1.
  • the antibody of the present invention binds to PD-1 with high affinity, for example, with a KD of 1 ⁇ 10 -9 M or less.
  • Standard assays to assess the ability of antibodies to bind PD-1 are well known in the art and include, for example, ELISA, Western blot, and RIA.
  • the binding kinetics (eg, binding affinity) of an antibody can also be assessed by standard assays known in the art, such as by Biacore or Octet analysis. The examples describe in detail assays suitable for evaluating any of the aforementioned characteristics.
  • the invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises:
  • the heavy chain variable region CDR1 which contains an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 7, or SEQ ID NO: 13 consistent with the amino acid sequence shown;
  • the heavy chain variable region CDR2 which contains an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 14 consistent with the amino acid sequence shown;
  • Heavy chain variable region CDR3 which includes an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 9, or SEQ ID NO: 15 consistent with the amino acid sequence shown;
  • the light chain variable region CDR1 which includes an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 10, or SEQ ID NO: 16 consistent with the amino acid sequence shown;
  • the light chain variable region CDR2 which includes an amino acid sequence selected from SEQ ID NO: 5, SEQ ID NO: 11, or SEQ ID NO: 17 consistent with the amino acid sequence shown;
  • the light chain variable region CDR3 which includes an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 12, or SEQ ID NO: 18;
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody or antigen-binding portion thereof, the CDR sequence homology of the variable region of the heavy chain and the variable region of the light chain is at least 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • mutations can be randomly or selectively introduced into the entire or part of the anti-PD-1 antibody coding sequence, and the resulting modified anti-PD-1 antibody can be as described herein The screening is performed for binding activity and/or other functional characteristics. Methods of mutation have been documented in the art. For example, Short’s PCT publication WO02/09278 describes methods for creating and screening antibody mutations through saturation mutagenesis, synthetic ligation assembly, or a combination thereof.
  • the invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises:
  • amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are consistent with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the light chain variable region CDR1
  • amino acid sequences of CDR2 and CDR3 are consistent with the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; or
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • spleen cells of immunized mice are fused with mouse myeloma cell line (SP2/0-Ag14) under the action of polyethylene glycol (PEG1450, Roche) fusion agent to obtain hybridomas
  • the cells are screened by a high-throughput transfection and screening system to find PD-1 antibodies that can bind to PD-1 positive cells.
  • the selected positive clones are subcloned and selected by the limiting dilution method more than 3 times to ensure that they are positive clones from a single source.
  • three particularly preferred positive clones UM1-51, UM1-56 and UM1-69 were sequenced, and the expressed antibody CDR sequence sequence is shown in the following table:
  • the present invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises heavy chain and light chain variable region sequences:
  • the heavy chain variable region which includes an amino acid sequence selected from SEQ ID NO: 19, SEQ ID NO: 23, or SEQ ID NO: 27;
  • the light chain variable region which comprises an amino acid sequence selected from SEQ ID NO: 21, SEQ ID NO: 25, or SEQ ID NO: 29;
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody or an antigen-binding portion thereof, the heavy chain variable region of which is selected from SEQ ID NO: 19, SEQ ID NO: 23 or SEQ ID NO: 27
  • the sequence has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology; its light chain variable region is selected from SEQ ID
  • the amino acid sequence of NO: 21, SEQ ID NO: 25 or SEQ ID NO: 29 has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Sequence homology.
  • Antibodies with high (ie 90% or higher) homology between the VH and VL regions of the heavy chain variable region (VH) and light chain variable region (VL) of the above sequence are obtained by conservative sequence modification, including amino acid substitutions , Additions and deletions, etc.
  • conservative sequence modification is intended to mean that the amino acid modification does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence.
  • the modification can be performed by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis of nucleic acid molecules encoding variable region sequences.
  • Conservative amino acid substitution refers to the replacement of amino acid residues with amino acid residues having similar side chains.
  • one or more amino acid residues outside the CDR region of the antibody of the present invention can be replaced with other amino acid residues from the same side chain family, and the modified antibody can be tested for the retained function using the functional assay described herein.
  • the preferred site of site-directed mutagenesis or PCR-mediated mutagenesis is located outside the CDR1-CDR3 of the heavy chain variable region and CDR1-CDR3 of the light chain variable region.
  • the invention provides an isolated monoclonal antibody or antigen binding portion thereof, which comprises:
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the antibody is a full-length antibody comprising a human or murine constant region.
  • the human constant region is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
  • the human constant region is IgG4.
  • the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from glycosylation.
  • the antibody or antibody fragment of the invention is a chimeric antibody or chimeric antibody fragment.
  • the antibody or antibody fragment of the invention is a human antibody or human antibody fragment.
  • the antibody or antibody fragment of the present invention is a humanized antibody or humanized antibody fragment.
  • the antibody fragment of the present invention is a Fab, Fab', Fab'-SH, Fv, scFv or F(ab')2 antibody fragment.
  • the antibody fragments of the invention are diabodies.
  • the present invention also includes bispecific antibodies comprising any of the above-mentioned antibodies or antibody fragments that bind to human PD-1.
  • the invention provides an isolated monoclonal antibody comprising:
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the present invention provides an isolated monoclonal antibody whose heavy chain (HC) and an amino acid sequence selected from SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38 have at least 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology; its light chain (LC) is selected from SEQ ID NO: 40, SEQ The amino acid sequence of ID NO: 42 or SEQ ID NO: 44 has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology .
  • Antibodies with high i.e.
  • homology to the heavy and light chains of the above sequence can be mutagenized (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) nucleic acid encoding heavy and light chain amino acids
  • the molecule is then tested for the retained function gain using the functional assay described herein on the encoded altered antibody.
  • the preferred site of site-directed mutagenesis or PCR-mediated mutagenesis is located outside the CDR1-CDR3 of the heavy chain variable region and CDR1-CDR3 of the light chain variable region.
  • the invention provides an isolated monoclonal antibody comprising:
  • the antibody or its antigen binding portion specifically binds to PD-1.
  • the antibody or antigen-binding portion thereof preferably specifically binds to human PD-1 or cynomolgus PD-1, and more preferably specifically binds to human PD-1.
  • the anti-PD-1 antibody of the present invention preferably exhibits one or more of the following characteristics:
  • the antibody binds to human PD-1 with a KD of 1 ⁇ 10 -10 M or less, and more preferably binds to human PD-1 with a KD of 1 ⁇ 10 -11 M or less.
  • the antibody (hUM1-30) at 3.34 ⁇ 10 -12 M binds to human PD-1, as a comparison, was measured in parallel antibody Opdivo BMS is listed at 2.58 ⁇ 10 - 11 M binds to human PD-1. It is shown that the affinity of the preferred antibody hUM1-30 to PD-1 is one order of magnitude higher than that of Opdivo. In the SPC lung cancer model mouse test, the antibody hUM1-30 can eliminate tumors better than the commercial Opdivo.
  • the antibodies of the present invention may exhibit any combination of the above-mentioned characteristics, such as two, three, four, or five of the above-mentioned characteristics.
  • nucleic acid molecules encoding the antibodies of the invention.
  • the nucleic acid of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences.
  • the nucleic acid is a cDNA molecule.
  • Standard molecular biology techniques can be used to obtain the nucleic acids of the invention.
  • standard PCR amplification or cDNA cloning techniques can be used to obtain cDNAs encoding the light and heavy chains of the antibody prepared by the hybridoma.
  • nucleic acid encoding the antibodies can be recovered from the library.
  • the preferred nucleic acid molecules of the present invention are those that encode the amino acid sequence of the CDR region, variable region, or full-length antibody of the anti-PD-1 antibody shown in the present invention.
  • these DNA fragments are further manipulated by standard recombinant DNA technology, such as converting variable region genes into full-length antibody chain genes, Fab Fragment gene or scFv gene.
  • a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operably linked” when used herein is intended to mean the connection of two DNA fragments so that the amino acid sequences encoded by the two DNA fragments remain in the same reading frame.
  • the isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3).
  • the sequence of the human heavy chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91- 3242), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is most preferably an IgG4 constant region.
  • the DNA encoding VH can be operably linked to another DNA molecule encoding only the constant region of the heavy chain CH1.
  • the isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operably linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL.
  • the sequence of the human light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991), Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91- 3242), DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but is most preferably a kappa constant region.
  • the DNA fragments encoding VH and VL are operably linked to a flexible linker, such as another fragment encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as adjacent single Chain protein in which the VL and VH regions are connected by a flexible linker (see, for example, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty (1990) Nature 348:552-554).
  • a flexible linker such as another fragment encoding the amino acid sequence (Gly4-Ser)3
  • the invention provides isolated polynucleotides encoding the antibodies or antibody fragments.
  • the nucleotide sequence encoding the antibody heavy chain is such as SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 35.
  • SEQ ID NO: 37 the nucleotide sequence encoding the antibody light chain is as SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 41 Or as shown in SEQ ID NO: 43.
  • the present invention provides an expression vector containing the isolated polynucleotide, and a host cell containing the expression vector.
  • a suitable vector will mainly depend on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector.
  • standard molecular biology techniques such as PCR amplification or use of cDNA clones of hybridomas expressing the target antibody
  • the DNA can be Insert into the expression vector, so that the gene is operably linked to transcription and translation control sequences.
  • the expression vector and expression control sequence suitable for the expression host cell used are selected.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into different vectors, or more commonly, the two genes can be inserted into the same expression vector.
  • the antibody gene is inserted into the expression vector by standard methods.
  • the light chain and heavy chain variable regions of the antibodies described herein can be used to create full-length antibody genes of any antibody isotype by inserting them into the heavy chain constant region and light chain constant region of the desired isotype.
  • the VH segment is operably connected to the CH segment in the vector
  • the VK segment is operably connected to the CL segment in the vector.
  • the recombinant expression vector may encode a signal peptide, which facilitates the secretion of antibody chains in host cells.
  • the antibody chain gene can be cloned into a vector so that the signal peptide and the amino terminus of the antibody chain gene are linked in the same reading frame.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).
  • the recombinant expression vector of the present invention also carries regulatory sequences, such as those derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (such as adenovirus major late promoter (AdMLP)) and polyoma
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus
  • the promoter and/or enhancer of the virus which regulate the expression of the antibody chain gene in the host cell.
  • the term "regulatory sequence” is intended to include promoters, enhancers and other expression control elements (such as polyadenylation signals) that regulate the transcription or translation of antibody chain genes.
  • regulatory sequences are described in, for example, Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques.
  • the various forms of the term "transfection” are intended to cover various techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.
  • it is theoretically possible to express the antibody of the present invention in either prokaryotic or eukaryotic host cells it is most preferred to express the antibody in eukaryotic cells (most preferably in mammalian host cells).
  • Preferred mammalian host cells for expressing the recombinant antibody of the present invention include Chinese hamster ovary cells (CHO cells), NSO myeloma cells, COS cells, SP2 cells, etc., and CHO cells are preferred.
  • the antibody When the recombinant expression vector encoding the antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for the expression of the antibody in the host cell, or more preferably, the antibody is secreted into the culture medium of the host cell . Standard protein purification methods can be used to recover antibodies from the culture broth.
  • the invention provides a pharmaceutical composition comprising the anti-PD-1 antibody and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or a group of anti-PD-1 monoclonal antibodies of the present invention or antigen binding portions thereof, formulated together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and other physiologically compatible carriers.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the pharmaceutical composition must generally be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated into a solution, microemulsion, liposome, or lyophilized powder injection.
  • the preferred route of administration of the pharmaceutical composition of the present invention includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal/spinal or other parenteral administration routes, such as by injection or infusion.
  • the dosage of the antibody of the present invention ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg of host body weight.
  • the dosage can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
  • An exemplary treatment regimen requires administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the preferred dosage regimen includes intravenous administration of 1 mg/kg body weight or 3 mg/kg body weight, which uses one of the following dosage regimens to administer the antibody: (i) every four weeks Give six doses, then one dose every three months; (ii) dose every three weeks; (iii) give 3 mg/kg body weight once, then give 1 mg/kg body weight every three weeks.
  • the actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied, so as to obtain an amount of the active ingredient that is effective to achieve a desired therapeutic response for a specific patient, composition and mode of administration, and is non-toxic to the patient.
  • the "therapeutically effective amount" of the anti-PD-1 antibody of the present invention preferably leads to a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic period of the disease, or the prevention of damage or disability caused by the disease.
  • a "therapeutically effective amount” preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60% relative to untreated subjects.
  • a compound to inhibit tumor growth can be evaluated in an animal model system that can predict its efficacy in human tumors. Those of ordinary skill in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the symptoms of the subject, and the particular composition or route of administration selected.
  • the present invention provides a method for improving the activity of immune cells, which includes contacting immune cells with the antibody or antibody fragment.
  • the present invention provides a method of treating diseases, the method comprising administering a therapeutically effective amount of the antibody or antibody fragment of the present invention to a subject in need of treatment.
  • the present invention provides a method of modulating an immune response in a subject, which comprises administering to the subject the antibody of the present invention or an antigen binding portion thereof, so that the immune response in the subject is regulated.
  • the antibodies of the invention enhance, stimulate or increase the immune response in the subject.
  • subject includes humans and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. Mammals are preferred, such as non-human primates, sheep, dogs, cats, cows, and horses. More preferred subjects include human patients in need of enhanced immune response.
  • the antibodies, antibody compositions and methods of the present invention have many in vitro and in vivo applications, including, for example, detecting PD-1 or enhancing immune responses by blocking PD-1.
  • the antibody of the invention is a human antibody.
  • these molecules can be administered to cells cultured in vitro or ex vivo, or administered to human subjects, such as in vivo, to enhance immunity in a variety of situations.
  • the present invention provides a method for regulating the immune response in a subject, which comprises administering the antibody of the present invention or an antigen-binding portion thereof to the subject, so that the immune response in the subject is regulated.
  • the immune response is enhanced, stimulated or upregulated.
  • the present invention relates to the use of anti-PD-1 antibodies to treat a subject so that the growth of tumor cells in the subject is inhibited.
  • Anti-PD-1 antibodies can be used alone to inhibit the growth of cancerous tumors.
  • the anti-PD-1 antibody can be used in combination with the other immunogenic agents, standard cancer treatments, or other antibodies.
  • the combination of therapeutic antibodies can be administered sequentially.
  • the anti-CTLA-4 antibody and the anti-PD-1 antibody may be administered sequentially, such as anti-CTLA-4 first and then anti-PD-1, or anti-PD-1 first and then anti-CTLA-4.
  • Non-limiting examples of preferred cancers that can be treated with the antibodies of the present invention include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate gland Cancer), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer).
  • melanoma e.g., metastatic malignant melanoma
  • renal cancer e.g., clear cell carcinoma
  • prostate cancer e.g., hormone refractory prostate gland Cancer
  • breast cancer e.g., colon cancer
  • lung cancer e.g. non-small cell lung cancer
  • examples of other cancers that can be treated using the method of the present invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal area Cancer, gastrointestinal cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vagina cancer, vulva cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system Cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphoid Cellular leukemia), childhood solid tumors, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central nervous system (CN
  • the anti-PD-1 antibody of the present invention can also be combined with tinib products for cancer treatment.
  • tinib drugs have targets such as RAF/MEK, ALK, c-Met, JAK, etc.
  • Tinib drugs that can be used in combination with the anti-PD-1 antibody of the present invention for cancer treatment include Gefitinib (gefitinib), Osimertinib (Osimertinib), Icotinib (Icotinib), Afatinib (Afatinib), Erlotinib (erlotinib), Crizotinib (crizotinib), Alectinib (alatinib salt), Ceritinib (Seritinib), Trametinib (Trametinib), Cobimetinib (Cometinib), Axitinib (Axitinib), Lenvatinib (Luvatinib), Cabozantinib (Cabotinib), Sunitinib (Sunitinib) Nitinib), Bosutinib (Bosutinib), Nilotinib (Nilotinib), Radotinib
  • the present invention provides a method of treating an infectious disease in a subject, including administering an anti-PD-1 antibody or an antigen binding portion thereof to the subject, so that the infectious disease in the subject is treated.
  • Antibody-mediated PD-1 blockade can be used alone or as an adjuvant in combination with vaccines to stimulate immune responses against pathogens, toxins and autoantigens. Examples of pathogens for which this treatment method can be particularly useful include pathogens for which there is currently no effective vaccine, or pathogens for which conventional vaccines are not fully effective.
  • HIV hepatitis A
  • hepatitis B hepatitis C
  • influenza Influenza
  • herpes Herpes
  • giardia Gaardia
  • malaria Malaria
  • leishmaniasis Leishmania
  • Staphylococcus aureus Staphylococcus aureus
  • Pseudomonas aeruginosa Pseudomonas Aeruginosa
  • mice Eight-week-old female Balb/c mice were used to subcutaneously immunize with a mixture of hPD-1 extracellular segment fusion protein (purchased from abcam, catalog number ab221398) and Freund’s complete adjuvant (CFA, Sigma). , 80-100 ⁇ g protein per mouse, 3 weeks later with protein and incomplete adjuvant (IFA, Sigma) emulsion for booster immunization, a total of 3 immunizations. Each time the mice were immunized for about 2 weeks, the serum was taken to determine the antibody titer, and the serum titer was determined by the SpectraMax i3 microplate reader using ELISA method.
  • CFA complete adjuvant
  • Hybridoma cells are produced by fusion of spleen cells of immunized mice with mouse myeloma cell line (SP2/0-Ag14) under the action of polyethylene glycol (PEG1450, SIGMA) fusion agent, and the fused cells are suspended in a HAT containing HAT (Sigma Aldrich)
  • the culture medium was added to a 96-well culture plate and cultured for about 11 days, and the culture supernatant was identified by SpectraMax i3 using ELISA method.
  • the selected positive clones are subcloned and selected by the limiting dilution method more than 3 times to ensure that they are positive clones from a single source.
  • the culture supernatant was captured by HiTrap Protein G HP (purchased from GE Healthcare, catalog number 29048581) and eluted with 0.1M glycine (pH 3.0), and the elution peak was collected. Then use HiTrap Desalting (purchased from GE Healthcare, catalog number 29048684) column desalting, and the replacement buffer is 1 ⁇ PBS (pH 7.4). Store at -20 degrees for later use.
  • Figure 2 shows the determination of the blocking activity of mouse PD-1 antibodies by flow cytometry, where the horizontal axis represents the antibody concentration and the vertical axis represents the blocking rate, UM1-45/UM1-47/UM1-51/UM1-56/UM1 -63/UM1-69 represents different murine PD-1 monoclonal antibodies prepared by the inventors. As a result, it was found that UM1-51, UM1-56 and UM1-69 had better blocking effects (Figure 2).
  • RNA purification kits (RAeasy Mini Kit, Qiagen 74104, QIAshredder, Qiagen 79654) were used to extract and purify total RNA from hybridoma cells. Then use the reagent kit (SMARter RACE cDNA Amplification Kit, Clontech) and follow the company's reagent manual to reverse transcription of RNA into the first strand cDNA strand.
  • SMART first-strand cDNA was used as a template for PCR amplification. Then connect the PCR products to the T vector (Zero Blunt TOPO PCR Cloning Kit, Invitrogen K2875-J10).
  • the heavy chain variable region sequence of the UM1-51 antibody (UM1-51-mVH) is:
  • the DNA sequence encoding UM1-51-mVH is:
  • the light chain variable region sequence of the UM1-51 antibody (UM1-51-mVK) is:
  • the DNA sequence encoding UM1-51-mVK is:
  • the heavy chain variable region sequence (UM1-56-mVH) of the UM1-56 antibody is:
  • the DNA sequence encoding UM1-56-mVH is:
  • the light chain variable region sequence of the UM1-56 antibody (UM1-56-mVK) is:
  • the DNA sequence encoding UM1-56-mVK is:
  • the heavy chain variable region sequence of the UM1-69 antibody (UM1-59-mVH) is:
  • the DNA sequence encoding UM1-69-mVH is:
  • the light chain variable region sequence of the UM1-69 antibody (UM1-69-mVK) is:
  • the DNA sequence encoding UM1-69-mVK is:
  • humanized antibodies are prepared by analyzing the parent sequence and various conceptual humanized products with three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Almost all murine antibodies can be humanized by CDR grafting to maintain antigen binding.
  • Related technical literature can refer to Sims et al., 1987, J. Immunol., 151: 2296; Chothia et al., 1987, J. Mol.
  • the UM1-56 antibody it is preferable to humanize the UM1-56 antibody.
  • the sequences of the heavy chain variable region and the light chain variable region are humanized, and the key amino acids of the FR region are backmutated on the basis of CDR grafting.
  • the amino acids between FRs that cannot be determined to affect the binding of antigen and antibody were merged and synthesized to construct a series of IgG mutants.
  • the above light chain and heavy chain expression vectors are combined in pairs, a total of 9 light chain and heavy chain combinations, and 9 combinations of light and heavy chain expression vectors are co-transiently transfected into ExpiCHO-S (Invitrogen) cells to obtain 9 humanized antibodies.
  • the purified antibodies were screened by ELISA, and 4 humanized antibodies were selected, and hUM1-30 (heavy chain sequence Seq ID No: 34, light chain sequence Seq ID No. was further selected according to the method in Example 2 by flow cytometry: 40) is the best humanized antibody (Figure 7).
  • the optimized DNA sequence and amino acid sequence of the 3 heavy chain variants and 3 light chain variants are as follows:
  • Heavy chain 1 (UM1-56H-1)
  • Heavy chain 2 (UM1-56H-2)
  • Example 4 Prepared pcDNA3.1 plasmids containing the DNA sequences of the antibody light chain (Seq ID No: 39) and heavy chain (Seq ID No: 33) respectively, and transformed them into Escherichia coli DH5 ⁇ . After the correct sequencing was verified, the glycerol bacteria were stored in -80 degrees. Take the glycerol bacteria plate and streak it.
  • ExpiCHO-S cells were cultured with 30mL ExpiCHO expression medium (containing 0.5% Anti-clumping Agent) at 37°C, 135rpm, and 8% CO 2 at a cell density of 4-6X 10 6 cells/mL, at 5X10 5 cells Cells/mL seeding density for passage; after 2 to 3 consecutive passages, some of the passaged cells can be taken, counted by trypan blue staining, and transfected.
  • ExpiCHO-S cells were cultured with 30mL ExpiCHO expression medium (containing 0.5% Anti-clumping Agent) at 37°C, 135rpm, and 8% CO 2 at a cell density of 4-6X 10 6 cells/mL, at 5X10 5 cells Cells/mL seeding density for passage; after 2 to 3 consecutive passages, some of the passaged cells can be taken, counted by trypan blue staining, and transfected.
  • the purified protein hUM1-30 (about 2ml) into an ultrafiltration centrifuge tube with a molecular weight cutoff of 30kD, add about 15ml PBS buffer, centrifuge at 4000 ⁇ g for 5min*2 times, and centrifuge at 4000 ⁇ g for 10min*3 times. Aspirate the collected protein with a 2ml syringe, filter with 0.22 ⁇ m, aliquot, and store at -80C.
  • the light chain amino acid sequence of the purified antibody hUM1-30 is shown in Seq ID No: 40
  • the heavy chain amino acid sequence is shown in Seq ID No: 34.
  • Sample loading Add 5ul of processed sample to each hole. Run the concentrated gel for 60V electrophoresis, and run the separation gel at 120V to separate the samples. Coomassie brilliant blue staining for 40 min. Use a decolorizing solution to decolor until the band is clear, and take a photo with a gel imager for storage.
  • Figure 4-1 shows that the band under the reduction conditions of hUM1-30 is about 50KD for heavy chain and about 25KD for light chain, without other miscellaneous bands.
  • Figure 4-2 shows that the molecular weight of the main band is about 150kD under non-reducing conditions.
  • the affinity of hUM1-30 is 3.34*E-12M, the affinity of Opdivo is 2.58*E-11M, the dissociation coefficient of hUM1-30 is 5.43*E-06(1/s), and the dissociation coefficient of Opdivo is 4.02*E -05(1/s). It shows that hUM1-30 has a lower dissociation rate, and its affinity is one order of magnitude higher than Opdivo.
  • the human lung cancer cell line SPC-A1-PDL1 with high expression of PD-L1 was cultured in RPMI1640 medium containing 10% FBS, and passaged once to the appropriate number of cells in about 2-3 days, and healthy adults were extracted by density gradient centrifugation. PBMCs are washed and counted after extraction for use.
  • NCG mice purchased from Nanjing University-Nanjing Institute of Biomedicine, reared with IVC rack with independent water and gas supply system
  • the mice were inoculated at an inoculation density of 3x10 ⁇ 7cells/ml, and each mouse was inoculated with one place under the right wing.
  • tumors form within 3-5 days after inoculation, and tumors can reach 100-250mm3 in 7-9 days.
  • measure the body weight and tumor volume of mice measure the body weight and tumor volume of mice, and eliminate mice with tumors smaller than 80mm3 or larger than 250mm3 or other abnormal conditions. Then, the mice that meet the requirements will be randomly assigned to different experimental groups.
  • the experimental antibody hUM1-30
  • positive control antibody Opdivo
  • negative control antibody human IgG4, denoted as hIgG4
  • the first dose of PD1 antibody is 250ug/bottle, and then 200ug/bottle/time, once every 3 days, until 6 times, ie q3dx6 times; measure the tumor volume before each administration, and measure 2-3 times a week Times, usually tested before administration.
  • the tumor volume growth in the hUM1-30 group was more significantly inhibited compared with the Opdivo group and the hIgG4 group. From 2 to 6 administrations, hUM1-30 is significantly better than Opdivo in inhibiting tumor proliferation. From the survival curve, as shown in Figure 9, the hUM1-30 group has a better survival rate. Specific to individuals, as shown in Figure 10, Figure 11, and Figure 12, the tumors of mice in the hUM1-30 group were significantly suppressed and even disappeared quickly. On Opdivo, only 1 mouse tumor was significantly inhibited, and 1 mouse tumor proliferated faster after administration, even faster than the negative control hIgG4 group, showing super-progression. Both hUM1-30 antibody and Opdivo antibody can prolong the survival period of model mice ( Figure 9), but the prolongation time is different, hUM1-30 is better than Opdivo.
  • hUM1-30 can eliminate tumors better than the commercial Opdivo on SPC lung cancer model mice.

Abstract

提供了以高亲和力与PD-1特异性结合的分离的单克隆抗体，特别是人单克隆抗体。还提供了编码抗体的核酸分子、用于表达抗体的表达载体、宿主细胞和方法，以及包含抗体的药用组合物。还涉及使用抗PD-1抗体调控免疫应答的方法，提供它们用于增强T细胞功能从而上调细胞介导的免疫应答的用途，以及治疗各种疾病，包括感染和肿瘤免疫的方法。

Description

一种靶向PD-1的单克隆抗体及其应用 技术领域
本发明涉及生物制药领域，具体而言，涉及针对人PD-1的单克隆抗体及其在疾病治疗中的应用。
背景技术
程序性死亡受体1(PD-1)是在T细胞、B细胞及骨髓细胞上表达的免疫抑制性受体。PD-1(由基因Pdcd1编码)与CTLA-4同属于CD28家族受体，其与CTLA-4有23％氨基酸序列同源性(Okazaki,T.&Honjo,T.Trends Immunol.27,195–201(2006))。PD-1蛋白是55kDa的I型跨膜蛋白，含有一个胞外IgV区、跨膜区及胞内区，胞内区包含接近膜的免疫受体酪氨酸抑制基序(ITIM)和远离膜的基于酪氨酸的转换基序(ITSM)(Thomas，M.L.(1995)J Exp Med 181：1953-6；Vivier，E和Daeron，M(1997)Immunol Today18：286-91)。虽然与CTLA-4结构类似，但PD-1缺少结合B7-1和B7-2所必需的MYPPPY基序。现已鉴定了PD-1的两种配体，PD-L1和PD-L2，其显示出在结合PD-1后下调T细胞活化。PD-L1和PD-L2都是B7同源物，其与PD-1结合，但不与其它CD28家族成员结合。PD-L1在B细胞、T细胞、巨噬细胞和树突状细胞(DC)上表达，但也在外周 细胞(例如微血管内皮细胞)和非淋巴器官(如心脏、肺等)中表达。PD-L2仅在活化的巨噬细胞和DC中存在。另外，PD-L1和PD-L2也常常在多种肿瘤细胞中表达，通过免疫组织化学评估活组织检查业已在人的很多原发性肿瘤中发现PD-1(在肿瘤浸润淋巴细胞上)和/或PD-L1(在肿瘤细胞上)。这样的组织包括肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、结肠癌、神经胶质瘤、膀胱癌、乳腺癌、肾癌、食道癌、胃癌、口腔鳞状细胞癌、尿道上皮细胞癌和胰腺癌以及头颈肿瘤。近年来研究表明阻断PD-1与其配体之间的相互作用可阻断T细胞内的负性调节信号，恢复肿瘤特异性CD8 +T细胞的杀伤活性，这表明肿瘤细胞可利用PD-1/PD-L1通路逃逸免疫监视，阻断该通路有助于免疫系统清除肿瘤细胞。Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway,Vassiliki A.Boussiotis,NEJM n engl j med 375；18 nejm.org November 3,2016中详细综述了PD-1及相关研究。
目前已经公开非常多的关于PD-1/PD-L1抗体相关专利，例如PCT国际申请PCT/JP2006/309606公开了以高亲和力与PD-1特异性结合的分离的单克隆抗体，该专利据信与百时美施贵宝的Opdivo相关。PCT国际申请PCT/US2008/007463披露了一种针对人程序性死亡受体PD-1的抗体，该专利据信与Merck公司的Keytruda相关。其他与PD-1/PD-L1抗体相关的专利可参考PCT/US2009/067104，PCT/CN2014/091090，PCT/CN2016/102238，PCT/CN2013/083467和CN201310258289.2等，在此全部引入作为参考。
目前，全球PD-1/PD-L1技术领域研究热点集中在四大领域：(1)联合免疫治疗，(2)联合小分子靶向药物治疗，(3)疗效预测生物标志物的检 测，(4)在治疗各种高发肿瘤中的应用。其中联合小分子靶向药物和联合其他免疫检查点治疗方案的文献最多，适应症主要集中在非小细胞肺癌、膀胱癌、头颈癌、前列腺癌、胰腺癌和恶性黑色素瘤等欧美人群高发肿瘤。除用于治疗肿瘤外，抗PD-1抗体还可用于慢性病毒性感染。最近，有研究显示PD-1在来自HIV感染的个体的T细胞中高度表达。
自2014年9月以来，PD-1/PD-L1抗体已经被美国FDA正式批准用于恶性黑色素瘤、非小细胞肺癌、肝癌、胃癌、肾癌、膀胱癌、头颈部肿瘤、霍奇金淋巴瘤、宫颈癌、Merkel细胞癌以及所有微卫星高度不稳定(MSI-H)的实体瘤。此外，在结直肠癌、鼻咽癌、卵巢癌、前列腺癌、子宫内膜癌、胶质瘤、神经内分泌肿瘤、恶性间皮瘤、非霍奇金淋巴瘤等其他多种实体瘤临床试验中，显示出了初步的、鼓舞人心的疗效。
已经批准上市的PD-1/PD-L1抗体药物
Figure PCTCN2020098698-appb-000001
目前已批准上市的PD-1/PD-L1的药物有9种，其中6款PD-1抗体为 百时美施贵宝的Opdivo、默沙东的Keytruda，赛诺菲(Sanofi)和再生元(Regeneron)联合开发的Libtayo、君实生物开发的特瑞普利单抗、信达生物开发的信迪利单抗和江苏恒瑞开发的卡瑞利珠单抗，3款PD-L1抗体为罗氏的Tecentriq、阿斯利康的Imfinzi以及默克的Bavencio。这几款药物批准的适应症如上表所示。
在中国，6款PD-1抗体除Libtayo外其余5款均获批准上市，药物之间的激烈竞争使得我国肿瘤患者在使用PD-1抗体药物的平均治疗费用低于欧美国家的费用。例如进口药物Opdivo在我国的治疗肺癌适应症一年费用大约48万元，而君实生物在国内国内首家批准用于治疗晚期黑色素瘤的特瑞普利单抗将治疗费用降到约18万元。
即使如此，动辄几十万的药物治疗费用对于全国居民人均可支配收入28228元(2018年国家统计局数字)的国内患者人群依然是非常大的负担。尽管目前已有多种PD-1抗体已上市或正在研究中，但是提供一种与人PD-1具有更高亲和力和特异性结合的PD-1抗体，具有更好的疗效和更低价格的抗PD-1抗体仍然是临床上所急需的。
发明概述
本发明提供了以高亲和力与PD-1特异性结合的分离的单克隆抗体，特别是人单克隆抗体。还提供了编码本发明抗体的核酸分子、用于表达本发明抗体的表达载体、宿主细胞和方法，以及包含本发明抗体的药用组合物。
本发明的另一方面涉及使用抗PD-1抗体调控免疫应答的方法，提供它 们用于增强T细胞功能从而上调细胞介导的免疫应答的用途，以及治疗各种疾病，包括感染(例如急性的和慢性的)和肿瘤免疫的方法。
在一方面，本发明提供了分离的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：1、SEQ ID NO：7、或SEQ ID NO：13所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：2、SEQ ID NO：8、或SEQ ID NO：14所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：3、SEQ ID NO：9、或SEQ ID NO：15所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：4、SEQ ID NO：10、或SEQ ID NO：16所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：5、SEQ ID NO：11、或SEQ ID NO：17所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：6、SEQ ID NO：12、或SEQ ID NO：18所示一致的氨基酸序列；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其重链可变区和轻链可变区的CDR序列同源性至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部 分，其包含：
(a)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的氨基酸序列一致；或
(b)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的氨基酸序列一致；或
(c)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在又一个实施方案中，本发明提供分离的单克隆抗体或其抗原结合部分，其包含重链和轻链可变区序列：
(a)重链可变区，其包含与选自SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27所示一致的氨基酸序列；
(b)轻链可变区，其包含与选自SEQ ID NO：21、SEQ ID NO：25或SEQ ID NO：29所示一致的氨基酸序列；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其重链可变区与选自SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链可变区与选自SEQ ID NO：21、SEQ ID NO：25或SEQ ID NO：29的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区，其氨基酸序列与SEQ ID NO：19所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：21所示的氨基酸序列一致；或
(b)重链可变区，其氨基酸序列与SEQ ID NO：23所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：25所示的氨基酸序列一致；或
(c)重链可变区，其氨基酸序列与SEQ ID NO：27所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：29所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在另一实施方案中，所述抗体为包含人或鼠恒定区的全长抗体。又一方面，所述人恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。在又一个具体方面中，所述人恒定区是IgG4。又一方面，所述鼠恒定区选自由IgGl、 IgG2A、IgG2B、IgG3组成的组。在又一个具体方面中，所述抗体具有降低的或最小的效应子功能。在又一个具体方面中，所述最小的效应子功能由去糖基化(glycosylation)产生。
在另一实施方案中，本发明抗体或抗体片段为嵌合抗体或嵌合抗体片段。
在另一实施方案中，本发明抗体或抗体片段为人抗体或人抗体片段。
在另一实施方案中，本发明抗体或抗体片段为人源化抗体或人源化抗体片段。
在另一实施方案中，本发明抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
在另一实施方案中，本发明抗体片段为双抗体。
本发明还包括包含上述结合人PD-1的抗体或抗体片段中的任一种的双特异性抗体。
在另一实施方案中，本发明提供了分离的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：34、SEQ ID NO：36或SEQ ID NO：38所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：40、SEQ ID NO：42或SEQ ID NO：44所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在本发明特别优选的实施方案中，本发明提供了分离的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：34所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：40所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
本发明提供了编码所述抗体或抗体片段的分离的多核苷酸。在一些实施方案中，编码抗体重链的核苷酸序列如SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:33、SEQ ID NO:35、或SEQ ID NO:37所示，编码抗体轻链的核苷酸序列如SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:39、SEQ ID NO:41或SEQ ID NO:43所示。
本发明提供了包含所述分离的多核苷酸的表达载体，以及包含所述表达载体的宿主细胞。
本发明提供了药物组合物，包含所述抗体或抗体片段以及可药用载体。
本发明提供了提高免疫细胞活性的方法，包括使免疫细胞与所述抗体或抗体片段接触。
本发明提供了治疗疾病的方法，所述方法包括将治疗有效量的本发明的抗体或抗体片段给予需要治疗的受试者。在又一方面，本发明提供了调控受试者中免疫应答的方法，其包括给受试者施用本发明的抗体或其抗原结合部分，使得受试者中的免疫应答得到调控。优选的是，本发明的抗体增强、刺激或增加受试者中的免疫应答。
在一些实施方案中，其中所述疾病为癌症或感染性疾病。在一些实施 方案中，所述方法包括对受试者联合应用其他药物或疗法。
本发明提供了所述抗体或抗体片段在制备用于治疗疾病的药物中的用途，其中所述疾病为癌症或感染性疾病。在一方面，本发明提供了抑制受试者中肿瘤细胞生长的方法，其包括给受试者施用治疗有效量的抗PD-1抗体或其抗原结合部分。在该方法中优选使用本发明的抗体，虽然其它抗PD-1抗体也能够替代(或与本发明的抗PD-1抗体联合)使用。例如，在抑制肿瘤生长的方法中可以使用嵌合的、人源化的或完全人的抗PD-1抗体。
在另一方面，本发明提供了治疗受试者中传染病的方法，其包括给受试者施用治疗有效量的抗PD-1抗体或其抗原结合部分。在该方法中优选使用本发明的抗体，虽然其它抗PD-1抗体也能够替代(或与本发明的抗PD-1抗体联合)使用。例如，在治疗传染病的方法中可以使用嵌合的、人源化的或完全人的抗PD-1抗体。
在一些实施方案中，利用本发明所述的抗体在抑制肿瘤细胞生长时其亲和力更高，阻断效果更好，细胞杀伤活力更高。其亲和力比现有治疗性单抗的亲和力高出大约10倍，体外阻断PD-L1/PD-1的相互作用非常有效。与美国公司(BMS)的已上市产品Opdivo(Nivolumab)相比，其阻断效果和体外杀伤活性均有所提高，在抑制肿瘤细胞生长方面本发明的单抗效果更加优异。
附图说明
图1：ELISA方法测定雌性Balb/c小鼠的血清效价。横轴表示稀释倍 数，纵轴表示测定的OD值，UM1-1/UM1-2/UM1-3/UM1-4代表不同的小鼠。
图2：流式细胞法测定鼠源PD-1单克隆抗体的阻断活性。横轴表示抗体浓度，纵轴表示阻断率，UM1-45/UM1-47/UM1-51/UM1-56/UM1-63/UM1-69代表不同的鼠源PD-1单克隆抗体。
图3：ProteinA亲和法分离纯化人源化PD-1单克隆抗体。横轴表示流动相体积，纵轴表示紫外吸光值。图3-2为图3-1的局部放大图。
图4：SDS‐PAGE检测抗体的分子量。图4-1SDS-PAGE(还原)，图4-2SDS-PAGE(非还原)
图5：超高效液相分子排阻色谱UPLC-SEC检测抗体的纯度。横轴表示时间，纵轴表示吸光度。
图6：Octet Red96测定亲和力。横轴表示时间，纵轴表示结合高度。
图7：流式细胞法测定人源化PD-1单克隆抗体(hUM1-30)阻断活性。
图8：SPC肺癌模型鼠检测消除肿瘤效果。横轴为天数，纵轴为肿瘤体积。
图9：SPC肺癌模型鼠生存曲线。横轴为天数，纵轴为生存百分率。
图10：每只SPC肺癌模型鼠给予阳性对照抗体Opdivo治疗效果。横轴为天数，纵轴为肿瘤体积。
图11：每只SPC肺癌模型鼠给予人源化PD-1单克隆抗体(hUM1-30)治疗效果。横轴为天数，纵轴为肿瘤体积。
图12：每只SPC肺癌模型鼠给予阴性对照抗体huIgG治疗效果。横轴 为天数，纵轴为肿瘤体积。
发明详述
在一方面，本发明涉及分离的单克隆抗体，特别是人单克隆抗体，其与PD-1特异性结合。在某些实施方式中，本发明的抗体展现出一种或多种优良的功能特性，诸如与人PD-1高亲和力结合，抑制一种或多种PD-1配体(例如PD-L1和/或PD-L2)结合的能力，刺激抗原特异性记忆应答的能力，刺激抗体应答的能力和/或抑制体内肿瘤细胞生长的能力。
本发明提供了例如分离的抗体、制备此类抗体的方法及含有本发明的抗体分子的药用组合物。
在另一方面，本发明涉及使用抗PD-1抗体抑制受试者中肿瘤细胞生长的方法。如本文所证明的，抗PD-1抗体能够抑制体内肿瘤细胞生长。本发明还涉及使用所述抗体调节免疫应答以及治疗诸如癌症或传染病的疾病，或者刺激保护性自身免疫应答或刺激抗原特异性免疫应答(例如通过共施用抗PD-1和目标抗原)的方法。
为使本发明更易于理解，首先定义某些术语。别的定义将在整个详述中阐明。
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内，这些技术在本领域的技术文献和通用教科书中有充分解释， 诸如Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)等。
术语“程序性死亡1”、“程序性细胞死亡1”、“蛋白PD-1”、“PD-1”、“PD1”、“PDCD1”、“hPD-1”和“hPD-1”可互换使用，包括人PD-1的变体、同种型(isoform)、物种同系物，及与PD-1具有至少一个共同表位的类似物。完整的PD-1序列可以根据GenBank编号U64863查到。
“抗体”是指表现出所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。因此，“抗体”以其最广泛的意义来使用，并明确包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体(例如双特异性抗体)。
“抗体片段”和“抗体结合片段”意即抗体的抗原结合片段及抗体类似物，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；纳米抗体；结构域抗体(Nanobody)；和由抗体片段形成的多特异性抗体等。工程改造的抗体变体综述于Holliger和Hudson(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”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，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包含CH1和CH2结构域之间的恒定区的部分的重链，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。一般而言，Fv多肽另外在VH和VL结构域之间包含多肽接头，该接头使得scFv能形成用于抗原结合的所需结构。
“双抗体”为具有两个抗原结合位点的小抗体片段。所述片段包含在相同的多肽链中与轻链可变结构域(VL)连接的重链可变结构域(VH)(VH-VL或VL-VH)。通过使用短至不能在同一链的两个结构域之间配对的接头，迫使所述结构域与另一条链的互补结构域配对并形成两个抗原结合位点。
“嵌合抗体”意图指可变区序列源自一种物种而恒定区序列源自另一种物种的抗体，诸如可变区序列源自小鼠抗体而恒定区序列源自人抗体的抗体。
非人类(例如鼠)抗体的“人源化”形式为含有最小限度的来源于非人类免疫球蛋白序列的嵌合抗体。人源化抗体的大部分为人免疫球蛋白(受体抗体)，其中受体抗体的高变区残基被具有所需特异性、亲和力和能力的非 人类物种(供体抗体)高变区的残基置换，非人类物种例如有小鼠、大鼠、兔或非人类灵长类。在某些情况下，人免疫球蛋白的Fv构架区(FR)残基被相应的非人类残基取代。此外，人源化抗体可包含不在受体抗体或供体抗体中存在的残基。进行这些修饰以进一步改进抗体性能。一般而言，人源化抗体包含至少一个且通常为两个可变结构域的几乎全部，其中全部或几乎全部超变环对应于非人类免疫球蛋白的超变环，全部或几乎全部FR区为人免疫球蛋白序列的FR区。人源化抗体还任选包含至少部分免疫球蛋白(通常为人免疫球蛋白)恒定区(Fc)。
“分离的”抗体为业已被鉴定并与其天然环境组分相分离的抗体(例如与PD-1特异性结合的分离的抗体基本没有与PD-1以外的其它抗原特异性结合的抗体)，其天然环境的污染组分是会干扰所述抗体的诊断性或治疗性应用的物质，可包括酶、激素和其它蛋白质溶质或非蛋白质溶质。在一些实施方案中，将所述抗体纯化到超过95％纯度，更优选超过99％纯度，其由Lowry法测定。分离的抗体通常由至少一个纯化步骤制备。
“分离的”核酸分子为被鉴定并与至少一种污染性核酸分子(通常在抗体核酸的天然来源中与其缔合)分离的核酸分子。分离的核酸分子不同于其天然存在的形式或环境。
术语“单克隆抗体””或“单克隆抗体组合物”在用于本文时指单一分子组合物的抗体分子的制备物。单克隆抗体组合物展现出对特定表位的单一结合特异性和亲和力。用于本发明的单克隆抗体可通过由Kohler等(1975，Nature 256：495)首先阐述的杂交瘤方法制备，或可通过重组DNA 方法制备。本文单克隆抗体明确包括“嵌合”抗体或人源化。
本文所用术语“免疫细胞”包括具有造血的起源并在免疫应答中起作用的细胞。免疫细胞包括：淋巴细胞，例如B细胞和T细胞；天然杀伤细胞；髓样细胞，例如单核细胞、巨噬细胞、嗜曙红细胞、肥大细胞、嗜碱细胞和粒细胞。
本文所用序列“变体”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
“有效量”包括足以改善或防止医学疾病的症状或病症的量。有效量还意指足以使得可以诊断或促进诊断的量。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。
本发明的各个方面将在下述分部中进一步详细描述。
抗PD-1抗体
本发明抗体的特征在于抗体的特定功能特征或特性。例如，所述抗体与PD-1特异性结合。优选的是，本发明的抗体以高亲和力，例如以1×10 -9M或更小的KD与PD-1结合。评估抗体对PD-1的结合能力的标准测定法是本领域公知的，包括例如ELISA、Western印迹和RIA。抗体的结合动力学(例如结合亲和力)也可以通过本领域已知的标准测定法来评估，诸如通过Biacore或Octet分析。实施例中详细描述了适于评估任何上述特征的测定法。
在一方面，本发明提供了分离的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1，其包含与选自SEQ ID NO：1、SEQ ID NO：7、或SEQ ID NO：13所示一致的氨基酸序列；
(b)重链可变区CDR2，其包含与选自SEQ ID NO：2、SEQ ID NO：8、或SEQ ID NO：14所示一致的氨基酸序列；
(c)重链可变区CDR3，其包含与选自SEQ ID NO：3、SEQ ID NO：9、 或SEQ ID NO：15所示一致的氨基酸序列；
(d)轻链可变区CDR1，其包含与选自SEQ ID NO：4、SEQ ID NO：10、或SEQ ID NO：16所示一致的氨基酸序列；
(e)轻链可变区CDR2，其包含与选自SEQ ID NO：5、SEQ ID NO：11、或SEQ ID NO：17所示一致的氨基酸序列；和
(f)轻链可变区CDR3，其包含与选自SEQ ID NO：6、SEQ ID NO：12、或SEQ ID NO：18所示一致的氨基酸序列；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其重链可变区和轻链可变区的CDR序列同源性至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。在本发明工程改造抗体的方法的某些实施方式中，可以向整个或部分抗PD-1抗体编码序列中随机或者有选择的引入突变，所产生的经修饰的抗PD-1抗体可如本文所述的进行结合活性和/或其它功能特性的筛选。本领域已经记载了突变的方法。例如，Short的PCT公开文本WO02/09278中描述了通过饱和诱变、合成的连接装配或其组合来创造和筛选抗体突变的方法。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：4、SEQ ID NO：5和SEQ  ID NO：6所示的氨基酸序列一致；或
(b)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的氨基酸序列一致；或
(c)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在本发明另一实施方案中，通过免疫小鼠的脾细胞与小鼠骨髓瘤细胞系(SP2/0-Ag14)在聚乙二醇(PEG1450，Roche)融合剂作用下进行融合，获得杂交瘤细胞，经高通量转染和筛选系统筛选到能与PD-1阳性表达细胞结合的PD-1抗体。挑选出的阳性克隆再经过3遍以上有限稀释法进行亚克隆选择，确保是单一来源的阳性克隆。其中，特别优选的3株阳性克隆UM1-51、UM1-56和UM1-69经测序，所表达的抗体CDR区序列如下表所示:
Figure PCTCN2020098698-appb-000002
在又一个实施方案中，本发明提供分离的单克隆抗体或其抗原结合部分，其包含重链和轻链可变区序列：
(a)重链可变区，其包含与选自SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27所示一致的氨基酸序列；
(b)轻链可变区，其包含与选自SEQ ID NO：21、SEQ ID NO：25或SEQ ID NO：29所示一致的氨基酸序列；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其重链可变区与选自SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链可变区与选自SEQ ID NO：21、SEQ ID NO：25或SEQ ID NO：29的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。重链可变区(VH)和轻链可变区(VL)与上述序列的VH和VL区具有高(即90％或更高)同源性的抗体通过保守序列修饰获得，包括氨基酸的取代、添加和缺失等。术语“保守序列修饰”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。修饰可以通过本领域已知的标准技术，例如定点诱变和PCR介导的诱变编码可变区序列的核酸分子。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区域外的一个或多个氨基酸残基，并使用本文所述的功能测定法对改变后的抗体测试保留的功能。优选的定点诱变或PCR介导的诱变位点位于重链可变区CDR1-CDR3和轻链可变区CDR1-CDR3之外的位点。
在另一实施方案中，本发明提供了分离的单克隆抗体或其抗原结合部分，其包含：
(a)重链可变区，其氨基酸序列与SEQ ID NO：19所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：21所示的氨基酸序列一致；或
(b)重链可变区，其氨基酸序列与SEQ ID NO：23所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：25所示的氨基酸序列一致；或
(c)重链可变区，其氨基酸序列与SEQ ID NO：27所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：29所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在另一实施方案中，所述抗体为包含人或鼠恒定区的全长抗体。又一 方面，所述人恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。在又一个具体方面中，所述人恒定区是IgG4。又一方面，所述鼠恒定区选自由IgGl、IgG2A、IgG2B、IgG3组成的组。在又一个具体方面中，所述抗体具有降低的或最小的效应子功能。在又一个具体方面中，所述最小的效应子功能由去糖基化(glycosylation)产生。
在另一实施方案中，本发明抗体或抗体片段为嵌合抗体或嵌合抗体片段。
在另一实施方案中，本发明抗体或抗体片段为人抗体或人抗体片段。
在另一实施方案中，本发明抗体或抗体片段为人源化抗体或人源化抗体片段。
在另一实施方案中，本发明抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
在另一实施方案中，本发明抗体片段为双抗体。
本发明还包括包含上述结合人PD-1的抗体或抗体片段中的任一种的双特异性抗体。
在另一实施方案中，本发明提供了分离的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：34、SEQ ID NO：36或SEQ ID NO：38所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：40、SEQ ID NO：42或SEQ ID NO： 44所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
在优选的实施方案中，本发明提供了分离的单克隆抗体，其重链(HC)与选自SEQ ID NO：34、SEQ ID NO：36或SEQ ID NO：38的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链(LC)与选自SEQ ID NO：40、SEQ ID NO：42或SEQ ID NO：44的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。与上述序列的重链和轻链具有高(即90％或更高)同源性的抗体可以通过诱变(例如定点诱变或PCR介导的诱变)编码重链和轻链氨基酸的核酸分子，然后使用本文所述的功能测定法对所编码的改变后的抗体测试所保留的功能获得。优选的定点诱变或PCR介导的诱变位点位于重链可变区CDR1-CDR3和轻链可变区CDR1-CDR3之外的位点。
在特别优选的实施方案中，本发明提供了分离的单克隆抗体，其包含：
(a)重链，其序列与SEQ ID NO：34所示的氨基酸序列一致；以及
(b)轻链，其序列与SEQ ID NO：40所示的氨基酸序列一致；
其中所述抗体或其抗原结合部分与PD-1特异性结合。
优选的，其中所述抗体或其抗原结合部分优选与人PD-1或猕猴PD-1特异性结合，更优选的与人PD-1特异性结合。本发明的抗PD-1抗体优选展现出一种或多种下列特征：
(a)以1×10 -9M或更小的KD与人PD-1结合；
(b)抑制PD-L1和/或PD-L2与PD-1的结合；
(c)刺激抗原特异性记忆应答；
(d)刺激抗体应答；
(e)抑制体内肿瘤细胞生长。
优选的是，所述抗体以1×10 -10M或更小的KD与人PD-1结合，更优选以1×10 -11M或更小的KD与人PD-1结合。在本发明另一优选实施方案中，所述抗体(hUM1-30)以3.34×10 -12M与人PD-1结合，作为比较，在平行测定中BMS公司已上市抗体Opdivo以2.58×10 -11M与人PD-1结合。显示优选抗体hUM1-30与PD-1的亲和力比Opdivo的高1个数量级。在SPC肺癌模型鼠试验中，抗体hUM1-30对比商品化的Opdivo能更好地消除肿瘤。
本发明的抗体可展现出上述特征的任意组合，诸如两个、三个、四个、或五个上述特征。
编码本发明抗体的核酸分子
本发明的另一方面涉及编码本发明抗体的核酸分子。本发明的核酸可以是例如DNA或RNA，而且可以含有或不含内含子序列。在一优选的实施方式中，核酸是cDNA分子。
可以使用标准分子生物学技术来获得本发明的核酸。对于由杂交瘤表达的抗体，可以通过标准PCR扩增或cDNA克隆技术获得编码杂交瘤所制备的抗体轻链和重链的cDNAs。对于从免疫球蛋白基因库(例如使用噬菌体展示技术)中获得的抗体，可以从文库中回收编码抗体的核酸。
本发明优选的核酸分子是那些编码本发明中所示抗PD-1抗体CDR区、 可变区或者全长抗体的氨基酸序列的核酸分子。在获得编码本发明所述的抗PD-1抗体的VH和VL区段的DNA片段后，进一步通过标准重组DNA技术操作这些DNA片段，例如将可变区基因转变为全长抗体链基因、Fab片段基因或scFv基因。在这些操作中，将编码VL或VH的DNA片段可操作的连接至编码另一蛋白质，诸如抗体恒定区或柔性接头的另一DNA片段。术语“可操作的连接”在用于本文时意图表示连接两DNA片段从而使得这两个DNA片段所编码的氨基酸序列保持在同一读码框中。
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IgG4恒定区。为获得Fab片段重链基因，可以将编码VH的DNA可操作的连接至仅编码重链CH1恒定区的另一DNA分子。
通过将编码VL的DNA可操作的连接至编码轻链恒定区CL另一DNA分子可以将编码VL区的分离的DNA转变为全长轻链基因(以及Fab轻链基因)。人轻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。轻链恒定区可以是κ或λ恒定区，但最优选为κ恒定区。
为创造scFv基因，将编码VH和VL的DNA片段可操作的连接至编码柔性接头，例如编码氨基酸序列(Gly4-Ser)3的另一片段，从而使得VH和VL序列可以表达成相邻的单链蛋白质，其中VL和VH区通过柔性接头连接(参见例如Bird等人(1988)Science 242：423-426；Huston等人(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883；McCafferty等人(1990)Nature 348：552-554)。
本发明提供了编码所述抗体或抗体片段的分离的多核苷酸。在一些实施方案中，编码抗体重链的核苷酸序列如SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:33、SEQ ID NO:35、或SEQ ID NO:37所示，编码抗体轻链的核苷酸序列如SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:39、SEQ ID NO:41或SEQ ID NO:43所示。
表达载体及宿主细胞
本发明提供了包含所述分离的多核苷酸的表达载体，以及包含所述表达载体的宿主细胞。
适宜载体的选择将主要取决于将要插入载体的核酸的大小和将要用载体转化的具体宿主细胞。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 使得基因与转录和翻译调控序列可操作的连接。
选择适合所用表达宿主细胞的表达载体和表达调控序列。可将抗体轻链基因和抗体重链基因插入到不同的载体中，或者更通常的，将两个基因插入到同一表达载体中。通过标准方法将抗体基因插入到表达载体中。可以使用本文所述抗体的轻链和重链可变区来创造任何抗体同种型的全长抗体基因，其通过将它们插入已编码期望同种型的重链恒定区和轻链恒定区的表达载体中，从而使得VH区段与载体中的CH区段可操作的连接，VK区段与载体中的CL区段可操作的连接。或者，重组表达载体可以编码信号肽，其利于宿主细胞中抗体链的分泌。可将抗体链基因克隆到载体中以使信号肽与抗体链基因的氨基末端连接于同一读码框中。信号肽可以是免疫球蛋白信号肽或异源信号肽(即来自非免疫球蛋白的信号肽)。
除了抗体链基因，本发明的重组表达载体还携带调控序列，诸如源自巨细胞病毒(CMV)、猿病毒40(SV40)、腺病毒(例如腺病毒主要晚期启动子(AdMLP))和多瘤病毒的启动子和/或增强子，其调控抗体链基因在宿主细胞中的表达。术语“调控序列”意图包括启动子、增强子和其它表达调控元件(例如聚腺苷酸化信号)，其调控抗体链基因的转录或翻译。此类调控序列记载于例如Goeddel，Gene Expression Technology.Methodsin Enzymology 185，Academic Press，San Diego，CA(1990)中。
为表达轻链和重链，通过标准技术将编码重链和轻链的表达载体转染到宿主细胞中。各种形式的术语“转染”意图涵盖多种通常用于将外源DNA导入原核或真核宿主细胞的技术，例如电穿孔、磷酸钙沉淀、DEAE-右旋糖 苷转染等。虽然理论上在原核或真核宿主细胞中都有可能表达本发明的抗体，但是最优选在真核细胞中(最优选在哺乳动物宿主细胞中)表达抗体。用于表达本发明重组抗体的优选哺乳动物宿主细胞包括中国仓鼠卵巢细胞(CHO细胞)、NSO骨髓瘤细胞、COS细胞和SP2细胞等，优选CHO细胞。
当将编码抗体基因的重组表达载体导入哺乳动物宿主细胞时，通过培养宿主细胞一段足以使宿主细胞中抗体表达的时间生产抗体，或者更优选的是，将抗体分泌到培养宿主细胞的培养基中。可以使用标准蛋白质纯化方法从培养物的培养液中回收抗体。
药物组合物
本发明提供了药物组合物，包含所述的抗PD-1抗体和药学可接受的载体。
一方面，本发明提供了一种药用组合物，其包含一种或一组本发明的抗PD-1单克隆抗体或其抗原结合部分，与药学可接受载体配制在一起。药剂学可接受载体包括任何和所有溶剂、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂等生理学相容的载体。优选的是，载体适于静脉内、肌肉内、皮下、胃肠外、脊髓或表皮施用(例如通过注射或输注)。
药物组合物在生产和贮存条件下通常必须是无菌的和稳定的。可以将组合物配制成溶液、微乳液、脂质体或冻干粉针等剂型。本发明药物组合物的优选给药途径包括静脉内、肌肉内、皮内、腹膜内、皮下、脊髓/脊柱或其它胃肠外施用路径，例如通过注射或输注。
本发明的抗体的给药剂量的范围为大约0.0001-100mg/kg，更加通常的 为0.01-5mg/kg宿主体重。例如，剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重或10mg/kg体重或在1-10mg/kg的范围内。例示性的治疗方案要求每周施用一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次或每3-6个月一次。对于本发明的抗PD-1抗体而言，优选的剂量方案包括经静脉内施用1mg/kg体重或3mg/kg体重，其利用下列剂量给药方案之一来给予抗体：(i)每四周一剂达六个剂量，然后每三个月一剂；(ii)每三周一剂；(iii)给予一次3mg/kg体重，随后每三周给予1mg/kg体重。
本发明药用组合物中的活性成分的实际剂量水平可以变化，从而获得对于特定患者、组合物和施用模式有效实现期望治疗性应答而对患者是无毒的活性成分量。“治疗有效量”的本发明抗PD-1抗体优选导致疾病症状严重性降低、无疾病症状期的频率和持续时间提高、或对患病所造成的损害或残疾的预防。例如，对于肿瘤的治疗而言，“治疗有效量”优选相对于未治疗受试者抑制细胞生长或肿瘤生长达至少大约20％、更优选达至少大约40％、甚至更优选达至少大约60％、且仍更优选达至少大约80％。可以在动物模型系统中评估化合物抑制肿瘤生长的能力，所述动物模型系统可以预测在人肿瘤中的功效。本领域普通技术人员将会能够基于诸如受试者体型大小、受试者症状的严重性、及所选择的特定组合物或施用路径的因素来确定此类量。
本发明的用途和方法
本发明提供了提高免疫细胞活性的方法，包括使免疫细胞与所述抗体 或抗体片段接触。
本发明提供了治疗疾病的方法，所述方法包括将治疗有效量的本发明的抗体或抗体片段给予需要治疗的受试者。在又一方面，本发明提供了调控受试者中免疫应答的方法，其包括给受试者施用本发明的抗体或其抗原结合部分，使得受试者中的免疫应答得到调控。优选的是，本发明的抗体增强、刺激或增加受试者中的免疫应答。术语“受试者”包括人和非人动物。非人动物包括所有的脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类、绵羊、犬、猫、牛、马、鸡、两栖类和爬行类。优选哺乳动物，诸如非人灵长类、绵羊、犬、猫、牛和马。更优选的受试者包括需要增强免疫应答的人类患者。
本发明的抗体、抗体组合物和方法具有众多体外和体内用途，包括例如检测PD-1或通过阻断PD-1来增强免疫应答。在一个优选的实施方案中，本发明的抗体是人抗体。例如，可以将这些分子施用于体外或离体培养的细胞，或者施用于人受试者，例如在体内，用以在多种情况中增强免疫力。
因此，一方面，本发明提供了调控受试者体内的免疫应答的方法，包括给受试者施用本发明的抗体或其抗原结合部分，使得受试者体内的的免疫应答得到调控。优选的是，免疫应答得到增强、刺激或上调。
一方面，本发明涉及使用抗PD-1抗体治疗受试者，使得受试者中肿瘤细胞的生长得到抑制。可以单独使用抗PD-1抗体来抑制癌性肿瘤的生长。或者，可以联合所述其它免疫原性剂、标准癌症治疗、或其它抗体来使用抗PD-1抗体。在一个实施方案中，可以序贯施用治疗性抗体的组合。例如， 可以序贯施用抗CTLA-4抗体和抗PD-1抗体，诸如先施用抗CTLA-4后施用抗PD-1，或者先施用抗PD-1后施用抗CTLA-4。
可以使用本发明的抗体来进行治疗的优选癌症的非限制性例子包括黑素瘤(例如转移性恶性黑素瘤)、肾癌(例如透明细胞癌)、前列腺癌(例如激素不应性前列腺腺癌)、乳癌、结肠癌和肺癌(例如非小细胞肺癌)。此外，可以使用本发明的方法来治疗的其它癌症的例子包括骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃肠、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、宫颈癌、阴道癌、阴户癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统的癌症、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病(包括急性髓细胞样白血病、慢性髓细胞样白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病)、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)的赘生物/肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊髓轴(spinal axis)肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏(Kaposi)肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症(包括由石棉诱发的那些癌症)、及所述癌症的组合。本发明还可用于治疗转移性癌症，尤其是表达PD-L1的转移性癌症(Iwai等人，Int.Immunol.17：133-144)。
优选的，为提高临床疗效，本发明的抗PD-1抗体还可以联合替尼类产品用于癌症的治疗。目前替尼类药物有RAF/MEK、ALK、c-Met、JAK等靶点， 可以与本发明的抗PD-1抗体联合用于癌症治疗的替尼类药物包括Gefitinib(吉非替尼)，Osimertinib(奥希替尼)，Icotinib(埃克替尼)，Afatinib(阿法替尼)，Erlotinib(厄洛替尼)，Crizotinib(克唑替尼)，Alectinib(阿来替尼盐)，Ceritinib(色瑞替尼)，Trametinib(曲莫替尼)，Cobimetinib(可美替尼)，Axitinib(阿西替尼)，Lenvatinib(乐伐替尼)，Cabozantinib(卡博替尼)，Sunitinib(舒尼替尼)，Bosutinib(伯舒替尼)，Nilotinib(尼洛替尼)，Radotinib(拉多替尼)，Dasatinib(达沙替尼)，Apatinib(阿帕替尼)，Ponatinib(泊那替尼)，Ruxolitinib(芦可替尼)，Neratinib(来那替尼)，Lapatinib(拉帕替尼)，Ibrutinib(依鲁替尼)，Tofacitinib(托法替尼)，Baricitinib(巴瑞克替尼)和Olmutinib(奥莫替尼)等或其组合。优选的替尼类药物有依鲁替尼、厄洛替尼、阿西替尼、卡博替尼和曲美替尼。
在一些实施方案中，本发明提供了治疗受试者中的传染病的方法，包括给受试者施用抗PD-1抗体或其抗原结合部分，使得受试者中的传染病得到治疗。抗体介导的PD-1阻断可以单独使用，或作为佐剂，与疫苗联合，以刺激针对病原体、毒素和自身抗原的免疫应答。这种治疗方法可以对它特别有用的病原体的例子包括目前没有有效疫苗的病原体，或常规疫苗并非完全有效的病原体。这些包括但不限于HIV、甲型肝炎、乙型肝炎、丙型肝炎、流行性感冒(Influenza)、疱疹(Herpes)、贾第虫病(Giardia)、疟疾(Malaria)、利什曼虫病(Leishmania)、金黄色葡萄球菌 (Staphylococcus aureus)引起的感染和铜绿假单胞菌(Pseudomonas Aeruginosa)引起的感染等。
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。
实施例
一、抗PD-1单抗(鼠源)的产生
1.1免疫小鼠
取8周龄大小的雌性Balb/c小鼠，用hPD-1胞外段融合蛋白(购自abcam，货号为ab221398)与福氏完全佐剂(CFA,Sigma)混合乳化液皮下多点接种免疫，每只小鼠80-100μg蛋白，3周后用蛋白与不完全佐剂(IFA,Sigma)乳化液进行加强免疫，共免疫3次。每次在小鼠免疫2周左右取血清测定抗体效价，血清效价用ELISA方法通过酶标仪SpectraMax i3测定。当血清效价达到足够高时(图1)，用100μg hPD胞外段融合蛋白溶于PBS中，腹腔注射加强免疫一次，3天后取出脾脏进行融合。
1.2制备和筛选杂交瘤
杂交瘤细胞的产生通过免疫小鼠的脾细胞与小鼠骨髓瘤细胞系(SP2/0-Ag14)在聚乙二醇(PEG1450，SIGMA)融合剂作用下进行融合，融合的细胞悬于含有HAT(Sigma Aldrich)选择培养液加入96孔培养板培养11 天左右，利用ELISA方法通过SpectraMax i3鉴定培养上清。挑选出的阳性克隆再经过3遍以上有限稀释法进行亚克隆选择，确保是单一来源的阳性克隆。
1.3抗PD-1单抗(鼠源)的纯化
1.2所述亚克隆经常规杂交瘤细胞培养后，培养上清经HiTrap Protein G HP(购自GE Healthcare，货号为29048581)捕获后，以0.1M甘氨酸(pH 3.0)洗脱，收集洗脱峰，再以HiTrap Desalting(购自GE Healthcare，货号为29048684)柱脱盐，置换缓冲液为1×PBS(pH7.4)。于-20度保存待用。
二、抗PD-1单抗(鼠源)的特性(阻断活性)
采用高表达PD-L1的人肺癌SPC-A1-PDL1细胞株、经过Alexa Fluor 488 Microscale Protein Labeling Kit(购自Thermo，货号为A30006)标记的hPD1/Fc of human IgG1和按上述1.3纯化的抗PD-1单抗(鼠源)充分混合，4度孵育20～30分钟，洗涤后，在FACSVerse上机检测，结果如图2所示。图2为通过流式细胞法测定鼠PD-1抗体的阻断活性，其中横轴表示抗体浓度，纵轴表示阻断率，UM1-45/UM1-47/UM1-51/UM1-56/UM1-63/UM1-69代表发明人制备的不同的鼠源PD-1单克隆抗体，结果发现UM1-51、UM1-56和UM1-69阻断效果较好(图2)。
三、抗PD-1抗体(鼠源)的序列测定
根据杂交瘤制备技术(Kohler和Milstein，1975)及《分子克隆实验指南》(第3版)【Molecular Cloning，J.萨姆布鲁克，D.W.拉塞尔著，黄培堂等译】中cDNA文库制备及其基因鉴定和DNA测序等章节操作。对UM1-51、UM1-56和UM1-69杂交瘤细胞提取总RNA，合成cDNA后进行测序。
首先利用RNA纯化试剂盒(RAeasy Mini Kit，Qiagen 74104，QIAshredder，Qiagen 79654)从杂交瘤细胞提取和纯化总RNA。然后用试剂盒(SMARter RACE cDNA Amplification Kit,Clontech)并按公司试剂操作手册将RNA反转录合成第一链cDNA链。利用5’RACE技术，用试剂盒提供的通用引物UPM(universal primer)作为上游引物，和根据小鼠IgG1重链可变区和轻链κ链基因序列设计的基因特异引物GSP(gene specific primer)作为下游引物，以SMART第一链cDNA为模板进行PCR扩增。然后将PCR产物分别连接到T载体(Zero Blunt TOPO PCR Cloning Kit，Invitrogen K2875-J10)。挑选克隆进行序列测定和分析，分别得到抗体的轻链和重链可变区序列，再根据重链可变区序列和小鼠IgG1序列设计引物，通过PCR获得重链全长序列，利用IGBLAST分析工具(KABAT)对序列进行分析，确定轻、重链CDR区域，如下表所示。
Figure PCTCN2020098698-appb-000003
相对应的，UM1-51抗体的重链可变区序列(UM1-51-mVH)为：
Figure PCTCN2020098698-appb-000004
编码UM1-51-mVH的DNA序列为：
Figure PCTCN2020098698-appb-000005
UM1-51抗体的轻链可变区序列(UM1-51-mVK)为：
Figure PCTCN2020098698-appb-000006
编码UM1-51-mVK的DNA序列为：
Figure PCTCN2020098698-appb-000007
UM1-56抗体的重链可变区序列(UM1-56-mVH)为：
Figure PCTCN2020098698-appb-000008
编码UM1-56-mVH的DNA序列为：
Figure PCTCN2020098698-appb-000009
UM1-56抗体的轻链可变区序列(UM1-56-mVK)为：
Figure PCTCN2020098698-appb-000010
编码UM1-56-mVK的DNA序列为：
Figure PCTCN2020098698-appb-000011
UM1-69抗体的重链可变区序列(UM1-59-mVH)为：
Figure PCTCN2020098698-appb-000012
编码UM1-69-mVH的DNA序列为：
Figure PCTCN2020098698-appb-000013
UM1-69抗体的轻链可变区序列(UM1-69-mVK)为：
Figure PCTCN2020098698-appb-000014
编码UM1-69-mVK的DNA序列为：
Figure PCTCN2020098698-appb-000015
四、抗PD-1抗体(鼠源)的人源化
为了使抗体能够更好的应用于临床病人治疗，需要对抗体序列进行人源化改造，使其保留对抗原的高亲和力和其它有利的生物学性质(如降低其在人体中的免疫原性)。为了达到这一目的，根据优选的方法，通过用母体和人源化序列的三维模型分析母体序列及各种概念上的人源化产物的方法来制备人源化抗体。三维免疫球蛋白模型一般是可利用的，并为本领域技术人员所熟悉。几乎所有鼠抗体都可通过CDR嫁接来人源化，从而保持抗原结合性。相关的技术文献可以参考Sims等，1987，J.Immunol.，151：2296；Chothia等，1987，J.Mol.Biol.，196：901；Presta等，1993，J.Immnol.，151：2623；Carter等，1992，Proc.Natl.Acad.Sci.USA，89：4285；Lo Benny  K.C.等，载于Antibody Engineering：Methods and Protocols(抗体工程：方法和方案)，第248卷，Humana Press，New Jersey，2004。
在前述工作基础上，优选对UM1-56抗体进行人源化改造。首先将抗体重链恒定区换为人IgG4恒定区序列，同时把抗体轻链恒定区换成人的κ链恒定区，得到人鼠嵌合的母本抗体(Chimeric Ab)。在确认母本抗体的功能活性后对重链可变区和轻链可变区序列进行人源化改造，在CDR移植的基础上进行FR区关键氨基酸的回复突变。对于不能确定是否影响抗原和抗体结合的FR间氨基酸进行兼并合成，构建一系列IgG突变体。经过基于抗体亲和力和表达水平的筛选后，共得到3个重链变异体(重链UM1-56-H1，UM1-56-H2和UM1-56-H3)和3个轻链变异体序列(轻链UM1-56K-2，UM1-56K-6和UM1-56K-8)。将1个母本和6个变异体序列分别采用合适酶切位点使用常规分子克隆技术克隆到pcDNA3.1表达载体中，轻链或重链序列5’端携带Kozak序列及人IL-2信号肽：MYRMQLLSCIALSLALVTNS(Seq ID No：31)，其相应的核苷酸序列为：atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaattcg(Seq ID No：32)。上述轻链及重链表达载体经两两组合，共9种轻链重链组合，9种组合的轻重链表达载体分别共瞬时转染ExpiCHO-S(Invitrogen)细胞可得到9个人源化抗体，纯化后的抗体经ELISA筛选后优选出4个人源化抗体，经流式细胞法按实施例二中方法进一步优选出hUM1-30(重链序列Seq ID No：34，轻链序列Seq ID No：40)为最优人源化抗体(图7)。优化后的3 个重链变异体和3个轻链变异体的DNA序列和氨基酸序列如下所示：
1.重链1(UM1-56H-1)
(1)重链UM1-56H-1DNA序列：
Figure PCTCN2020098698-appb-000016
(2)重链UM1-56H-1蛋白质序列：
Figure PCTCN2020098698-appb-000017
2.重链2(UM1-56H-2)
(3)重链UM1-56H-2DNA序列：
Figure PCTCN2020098698-appb-000018
(4)重链UM1-56H-2蛋白质序列：
Figure PCTCN2020098698-appb-000019
3.重链3(UM1-56-H3)
(5)重链UM1-56-H3DNA序列：
Figure PCTCN2020098698-appb-000020
Figure PCTCN2020098698-appb-000021
(6)重链UM1-56-H3蛋白质序列：
Figure PCTCN2020098698-appb-000022
4.轻链1(UM1-56K-2)
(1)轻链UM1-56K-2 DNA序列：
Figure PCTCN2020098698-appb-000023
Figure PCTCN2020098698-appb-000024
(2)轻链UM1-56K-2蛋白质序列：
Figure PCTCN2020098698-appb-000025
5.轻链2(UM1-56K-6)
(3)轻链UM1-56K-6 DNA序列：
Figure PCTCN2020098698-appb-000026
(4)轻链UM1-56K-6蛋白质序列：
Figure PCTCN2020098698-appb-000027
6.轻链3(UM1-56K-8)
(5)轻链UM1-56K-8 DNA序列
Figure PCTCN2020098698-appb-000028
Figure PCTCN2020098698-appb-000029
(6)轻链UM1-56K-8蛋白质序列：
Figure PCTCN2020098698-appb-000030
五、人源化抗体的特性和功能
5.1人源化抗体的表达和纯化
实施例四制备得到分别含有抗体轻链(Seq ID No：39)和重链(Seq ID No：33)DNA序列的pcDNA3.1质粒，将其转化大肠杆菌DH5α，测序验证正确后保存甘油菌于-80度。取甘油菌平板划线，过夜培养后挑单菌落接种至3mL含100μg/mL氨苄青霉素的LB培养基中培养过夜，再次接种至150mL LB液体培养基中(添加终浓度100μg/mL氨苄青霉素)，于37℃过夜培养；使用天根无内毒素质粒大提试剂盒，分别提取抗体轻重链质粒(具体操作过程详见说明书)，测定质粒浓度后，于-20℃保存，待转染。
ExpiCHO-S细胞用30mL ExpiCHO expression medium(含0.5％Anti-clumping Agent)于37℃，135rpm，8％CO 2条件下培养，细胞密度为4-6X 10 6个细胞/mL时，以5X10 5个细胞/mL接种密度传代；连续传代2至3次后，可取部分传代细胞，台盼蓝染色法计数，进行转染。用ExpiFectamine CHO Transfection Kit瞬时转染试剂盒；取1.5X 10 8个细胞离心，弃上清，加入25.5mL新鲜的ExpiCHO expression medium重悬 细胞；取20μg抗体质粒(轻链质粒10μg，重链质粒10μg)，溶于1mL预冷的OptiPRO SFM无血清培养基中，轻轻混匀；取80μL转染试剂，加入920μL预冷的OptiPRO SFM无血清培养基，轻轻混匀；将转染试剂溶液与质粒溶液混合，室温静置1-5min后加入ExpiCHO-S细胞培养液中；采用Standard培养模式，转染后18-22h，添加150μL Enhancer和6mL Feed；待细胞活率降至75％时，收获细胞；将离心后获得的细胞上清0.22μm滤器过滤后在AKTAavant上将细胞培养上清上样，过ProA预装柱(GE Hitrap ProteinA HP 1ml)。分离条件：上样结束后用PBS缓冲液洗柱，用0.1M甘氨酸(pH3.5)洗脱，0.1M甘氨酸(pH2.0)再生(图3)。
将纯化后蛋白hUM1-30(2ml左右)吸入截留分子量为30kD的超滤离心管，加入约15ml PBS缓冲液，4000×g离心5min*2次，4000×g离心10min*3次。将收集的蛋白用2ml注射器吸出，0.22μm过滤，分装，‐80C保存。根据DNA测序和蛋白N端测序结果，纯化获得的抗体hUM1-30的轻链氨基酸序列如Seq ID No：40所示，重链氨基酸序列如Seq ID No：34所示。
5.2 SDS‐PAGE检测抗体的分子量
1、配制6％或12％分离胶、5％浓缩胶；
2、样品处理：取样品6ul，分别加入1.5ul 5*还原loading buffer和1.5ul 5*非还原loading buffer，95℃金属浴煮3min；
3、上样：每孔加入5ul处理后的样品。60V电泳跑浓缩胶，120V跑分 离胶至样品分离。考马斯亮蓝染色40min。用脱色液脱色至条带清晰，用凝胶成像仪拍照保存。
图4-1为hUM1‐30还原条件下条带为重链50KD左右，轻链25KD左右，没有其他杂带。图4-2为非还原条件下显示主条带分子量在150kD左右。
5.3 UPLC-SEC检测抗体的纯度
配置100mM磷酸钠缓冲溶液调节pH至6.80，0.1mL/min平衡色谱柱(Waters,BEH SEC 200 4.6*150mm)至压力波动<10psi,然后将流速调至0.4mL/min。样品经0.22um滤膜过滤，过滤后加到相应的样品盘位置，每个样至少20uL。基线平稳后，选定相应仪器磷酸钠100mM，，进样体积2uL,时间7min，点击运行。进样结束后，低流速(小于0.2mL/min)冲洗色谱柱至少10个柱体积，保存于20％甲醇溶液中。
hUM1‐30抗体纯化如图5所示,主峰占96.70％。
5.4 Octet测定亲和力
将待测抗体配成50nM倍半稀释的6个浓度，同时设置一个Buffer空白孔和50nM抗原(Human PD1/PDCD1 Protein Biotinylated,Sinobiological，Cat.No.10377-H03H-B)空白孔；将5ug/ml抗原固定在SA传感器上，固化高度约0.6nm；按照设定好的程序；(Baseline:120s；Loading:60s；Baseline:120s；Association:300s；Dissoc iation:1800s)在Octet Red96仪器(Fortebio公司)上测定待测抗体的亲 和力。用Octet Red96配套软件拟合计算亲和力数据(图6)。
抗体 KD(M) Kon(1/Ms) Kdis(1/s) Full R 2
Opdivo 2.58E-11 1.56E+06 4.02E-05 0.9965
hUM1-30 3.34E-12 1.63E+06 5.43E-06 0.9974
hUM1-30的亲和力为3.34*E-12M，Opdivo的亲和力为2.58*E-11M，hUM1-30的解离系数为5.43*E-06(1/s)，Opdivo的解离系数为4.02*E-05(1/s)。显示hUM1-30具有更低的解离速率，亲和力比Opdivo高1个数量级。
5.5流式检测测定阻断活性
贴壁培养表达PD-L1的人肺癌细胞株SPC-A1-PDL1至合适细胞密度，吸弃培养基，用PBS清洗两次，加入0.2‐0.5ml 0.25％胰酶‐EDTA放置细胞培养箱中消化1‐2min，不可消化时间过长，用RPMI‐1640(10％FBS)终止消化，细胞计数，每管1～5*10^5个cells。离心机2000rpm离心，3分钟，吸弃上层培养基，加入适量1*PBS重悬细胞，再次2000rpm离心，3分钟，重复2次。加不同浓度的待测抗体和荧光标记的PD1(Dimeric)混合物；并设置同型、阳性对照每管加100ng的hPD‐1/hFc‐FITC和不同浓度的待检抗PD‐1抗体(0ng、50ng、100ng、200ng、400ng、800ng、1600ng 和3200ng)，混匀，一管同型对照，4度放置20～30Min。1*PBS洗涤1～3次，加适量1*PBS调整细胞浓度，上机检测(结果如图7所示)。数据显示随着抗体浓度的增加，PD-1与PD-L1的结合被阻断的更多，加入hUM1-30至1.6ug可以阻断PD-1与PDL1的结合。
5.6肺癌模型鼠检测消除肿瘤效果
将高表达PD-L1的人肺癌细胞株SPC-A1-PDL1在含有10％FBS的RPMI1640培养基中进行培养，约2-3天传代1次至合适细胞数量，另密度梯度离心法提取健康成人PBMC，提取后进行洗涤、计数，备用。采用4-5周龄雌性免疫缺陷NCG小鼠(购自南京大学-南京生物医药研究院，IVC架子独立供水和供气系统饲养)，将SPC-A1-PDL1细胞消化后与PBMC按合适比例混合后按3x10^7cells/ml的接种密度接种在小鼠上，每只小鼠接种1处在右侧翼下。一般接种后3-5天成瘤，7-9天瘤体可达100-250mm3，入组时测量小鼠体重和瘤体体积，剔除掉瘤体小于80mm3或大于250mm3的小鼠或其它异常状态小鼠，然后将符合要求的小鼠按随机分配方法分配至不同的实验组别中。
入组后，根据组别给予实验抗体(hUM1-30)、阳性对照抗体(Opdivo)及阴性对照抗体(human IgG4，记为hIgG4)，抗体浓度为1mg/ml，腹腔注射，首剂量加大，PD1抗体首剂量为250ug/只，之后按200ug/只/次，每3天给药1次，直至给6次，即q3dx6次；每次给药前测量瘤体体积，每周测2-3次，一般在给药前测试。
经过6次的抗体治疗，如图8所示，hUM1-30组与Opdivo组，hIgG4组相比，肿瘤体积增长得到更显著的抑制。从给药2次到6次，hUM1-30抑制瘤体增殖的能力明显优于Opdivo。从生存曲线看，如图9所示，hUM1-30组具有更好的生存率。具体到个体上，如图10，图11，图12所示，hUM1-30组小鼠瘤体明显被抑制，甚至快消失。而Opdivo只有1只小鼠瘤体被明显抑制，有1只小鼠给药后瘤体反而增殖更快，甚至比阴性对照组hIgG4组更快，出现超进展现象。hUM1-30抗体和Opdivo抗体均能延长模型鼠的生存期(图9)，但延长时间不同，hUM1-30优于Opdivo。
比较而言，hUM1-30对SPC肺癌模型鼠，比商品化的Opdivo能更好地消除肿瘤。
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Claims (32)

  1. 一种分离的单克隆抗体或其抗原结合部分，其包含：
    (a)重链可变区CDR1，其包含与选自SEQ ID NO：1、SEQ ID NO：7、或SEQ ID NO：13所示一致的氨基酸序列；
    (b)重链可变区CDR2，其包含与选自SEQ ID NO：2、SEQ ID NO：8、或SEQ ID NO：14所示一致的氨基酸序列；
    (c)重链可变区CDR3，其包含与选自SEQ ID NO：3、SEQ ID NO：9、或SEQ ID NO：15所示一致的氨基酸序列；
    (d)轻链可变区CDR1，其包含与选自SEQ ID NO：4、SEQ ID NO：10、或SEQ ID NO：16所示一致的氨基酸序列；
    (e)轻链可变区CDR2，其包含与选自SEQ ID NO：5、SEQ ID NO：11、或SEQ ID NO：17所示一致的氨基酸序列；和
    (f)轻链可变区CDR3，其包含与选自SEQ ID NO：6、SEQ ID NO：12、或SEQ ID NO：18所示一致的氨基酸序列；
    其中所述抗体或其抗原结合部分与PD-1特异性结合。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其特征在于，其重链可变区和轻链可变区的CDR序列同源性至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％。
  3. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其包含：
    (a)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO： 4、SEQ ID NO：5和SEQ ID NO：6所示的氨基酸序列一致；或
    (b)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的氨基酸序列一致；或
    (c)重链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的氨基酸序列一致，以及轻链可变区CDR1、CDR2和CDR3的氨基酸序列分别与SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的氨基酸序列一致；
    其中所述抗体或其抗原结合部分与PD-1特异性结合。
  4. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其包含重链和轻链可变区序列：
    (a)重链可变区，其包含与选自SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27所示一致的氨基酸序列；
    (b)轻链可变区，其包含与选自SEQ ID NO：21、SEQ ID NO：25或SEQ ID NO：29所示一致的氨基酸序列；
    其中所述抗体或其抗原结合部分与PD-1特异性结合。
  5. 根据权利要求4所述的单克隆抗体或其抗原结合部分，其特征在于：其重链可变区与选自SEQ ID NO：19、SEQ ID NO：23或SEQ ID NO：27的氨基酸序列具有至少为91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性；其轻链可变区与选自SEQ ID NO：21、SEQ ID NO：25或SEQ ID NO：29的氨基酸序列具有至少为91％， 92％，93％，94％，95％，96％，97％，98％，99％或100％的序列同源性。
  6. 根据权利要求4所述的单克隆抗体或其抗原结合部分，其包含：
    (a)重链可变区，其氨基酸序列与SEQ ID NO：19所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：21所示的氨基酸序列一致；或
    (b)重链可变区，其氨基酸序列与SEQ ID NO：23所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：25所示的氨基酸序列一致；或
    (c)重链可变区，其氨基酸序列与SEQ ID NO：27所示的氨基酸序列一致；以及轻链可变区，其氨基酸序列与SEQ ID NO：29所示的氨基酸序列一致；
    其中所述抗体或其抗原结合部分与PD-1特异性结合。
  7. 根据权利要求1所述的单克隆抗体或其抗原结合部分，其为包含人或鼠恒定区的全长抗体。
  8. 根据权利要求7所述的单克隆抗体或其抗原结合部分，其特征在于：所述人恒定区选自由IgGl、IgG2、IgG3、IgG4组成的组。
  9. 根据权利要求8所述的单克隆抗体或其抗原结合部分，其特征在于：所述人恒定区是IgG4。
  10. 根据权利要求7所述的单克隆抗体或其抗原结合部分，其特征在于：所述鼠恒定区选自由IgGl、IgG2A、IgG2B、IgG3组成的组。
  11. 根据权利要求7所述的单克隆抗体或其抗原结合部分，其特征在于：所述抗体具有降低的或最小的效应子功能。
  12. 根据权利要求11所述的单克隆抗体或其抗原结合部分，其特征在于：所述最小的效应子功能由去糖基化(glycosylation)产生。
  13. 根据权利要求7所述的单克隆抗体或其抗原结合部分，其特征在于：所述抗体或抗体片段为嵌合抗体或嵌合抗体片段。
  14. 根据权利要求13所述的单克隆抗体或其抗原结合部分，其特征在于：所述抗体或抗体片段为人抗体或人抗体片段。
  15. 根据权利要求13所述的单克隆抗体或其抗原结合部分，其特征在于：所述抗体或抗体片段为人源化抗体或人源化抗体片段。
  16. 根据权利要求7所述的单克隆抗体或其抗原结合部分，其特征在于：所述抗体片段为Fab、Fab’、Fab’-SH、Fv、scFv或F(ab’)2抗体片段。
  17. 根据权利要求7所述的单克隆抗体或其抗原结合部分，其特征在于：所述抗体片段为双抗体。
  18. 一种分离的单克隆抗体，其包含：
    (a)重链，其序列与SEQ ID NO：34、SEQ ID NO：36或SEQ ID NO：38所示的氨基酸序列一致；以及
    (b)轻链，其序列与SEQ ID NO：40、SEQ ID NO：42或SEQ ID NO：44所示的氨基酸序列一致；
    其中所述抗体或其抗原结合部分与PD-1特异性结合。
  19. 根据权利要求18所述的单克隆抗体，其包含：
    (a)重链，其序列与SEQ ID NO：34所示的氨基酸序列一致；以 及
    (b)轻链，其序列与SEQ ID NO：40所示的氨基酸序列一致；
    其中所述抗体或其抗原结合部分与PD-1特异性结合。
  20. 编码权利要求1-19任一项所述的单克隆抗体或抗原结合部分的分离的多核苷酸。
  21. 根据权利要求20所述的多核苷酸，其特征在于：编码抗体重链的核苷酸序列如SEQ ID NO:20、SEQ ID NO:24、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:33、SEQ ID NO:35、或SEQ ID NO:37所示，编码抗体轻链的核苷酸序列如SEQ ID NO:21、SEQ ID NO:25、SEQ ID NO:29、SEQ ID NO:39、SEQ ID NO:41或SEQ ID NO:43所示。
  22. 包含权利要求20所述多核苷酸的表达载体
  23. 包含权利要求22所述表达载体的宿主细胞。
  24. 药物组合物，其包含权利要求1-19任一项所述的单克隆抗体或抗原结合部分以及可药用载体。
  25. 权利要求1-19任一项所述的单克隆抗体或抗原结合部分用于调控受试者中免疫应答的用途。
  26. 根据权利要求25所述的用途，其特征在于：所述用途包括将治疗有效量的单克隆抗体或抗原结合部分给予需要治疗的受试者。
  27. 权利要求1-19任一项所述的单克隆抗体或抗原结合部分用于治疗癌症疾病的用途。
  28. 根据权利要求27所述的用途，其特征在于：可以单独使用抗PD-1抗体来抑制癌性肿瘤的生长，或者联合所述其它免疫原性剂、标准癌 症治疗、或其它抗体来使用抗PD-1抗体。
  29. 根据权利要求27所述的用途，其特征在于：所述的癌症包括黑素瘤、肾癌、前列腺癌、乳癌、结肠癌、肺癌、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内恶性黑素瘤、子宫癌、卵巢癌、直肠癌、肛区癌、胃肠、睾丸癌、子宫癌、输卵管癌、子宫内膜癌、宫颈癌、阴道癌、阴户癌、何杰金氏病、非何杰金氏淋巴瘤、食道癌、小肠癌、内分泌系统的癌症、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织肉瘤、尿道癌、阴茎癌、慢性或急性白血病、儿童期实体瘤、淋巴细胞性淋巴瘤、膀胱癌、肾或输尿管癌、肾盂癌、中枢神经系统(CNS)的赘生物/肿瘤、原发性CNS淋巴瘤、肿瘤血管发生、脊髓轴(spinal axis)肿瘤、脑干胶质瘤、垂体腺瘤、卡波西氏(Kaposi)肉瘤、表皮样癌、鳞状细胞癌、T细胞淋巴瘤、环境诱发的癌症及所述癌症的组合。
  30. 根据权利要求27所述的用途，其特征在于：所述的抗PD-1抗体可以联合替尼类产品用于癌症的治疗，替尼类药物包括Gefitinib(吉非替尼)，Osimertinib(奥希替尼)，Icotinib(埃克替尼)，Afatinib(阿法替尼)，Erlotinib(厄洛替尼)，Crizotinib(克唑替尼)，Alectinib(阿来替尼盐)，Ceritinib(色瑞替尼)，Trametinib(曲莫替尼)，Cobimetinib(可美替尼)，Axitinib(阿西替尼)，Lenvatinib(乐伐替尼)，Cabozantinib(卡博替尼)，Sunitinib(舒尼替尼)，Bosutinib(伯舒替尼)，Nilotinib(尼洛替尼)，Radotinib(拉多替尼)，Dasatinib(达沙替尼)，Apatinib(阿帕替尼)，Ponatinib(泊那替尼)，Ruxolitinib(芦 可替尼)，Neratinib(来那替尼)，Lapatinib(拉帕替尼)，Ibrutinib(依鲁替尼)，Tofacitinib(托法替尼)，Baricitinib(巴瑞克替尼)和Olmutinib(奥莫替尼)等或其组合。
  31. 权利要求1-19任一项所述的单克隆抗体或抗原结合部分用于治疗感染性疾病的用途。
  32. 根据权利要求31所述的用途，其特征在于：所述的感染性疾病包括HIV、甲型肝炎、乙型肝炎、丙型肝炎、流行性感冒(Influenza)、疱疹(Herpes)、贾第虫病(Giardia)、疟疾(Malaria)、利什曼虫病(Leishmania)、金黄色葡萄球菌(Staphylococcus aureus)或铜绿假单胞菌(Pseudomonas Aeruginosa)。
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CN113425856B (zh) * 2021-07-07 2022-01-04 浙江康佰裕生物科技有限公司 一种含有基因修饰的溶瘤病毒的药物组合物及其在治疗癌症的用途

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