WO2017071625A1 - 一种抗pd-1单克隆抗体、其药物组合物及其用途 - Google Patents

一种抗pd-1单克隆抗体、其药物组合物及其用途 Download PDF

Info

Publication number
WO2017071625A1
WO2017071625A1 PCT/CN2016/103667 CN2016103667W WO2017071625A1 WO 2017071625 A1 WO2017071625 A1 WO 2017071625A1 CN 2016103667 W CN2016103667 W CN 2016103667W WO 2017071625 A1 WO2017071625 A1 WO 2017071625A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
monoclonal antibody
antigen
seq
binding fragment
Prior art date
Application number
PCT/CN2016/103667
Other languages
English (en)
French (fr)
Inventor
李百勇
夏瑜
***
张鹏
Original Assignee
中山康方生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中山康方生物医药有限公司 filed Critical 中山康方生物医药有限公司
Publication of WO2017071625A1 publication Critical patent/WO2017071625A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-PD-1 antibody, a pharmaceutical composition thereof and use thereof.
  • the invention relates to a monoclonal antibody against PD-1.
  • the transmembrane receptor PD-1 (programmed cell death 1, programmed cell death factor 1) is a member of the CD28 gene family and is expressed in activated T cells, B cells, and bone marrow cells.
  • PD-1 ligands PDL-1 and PDL-2 belong to the B7 superfamily, in which PDL-1 cells are expressed, including T cells, B cells, and endothelial cells and epithelial cells.
  • PDL-2 is expressed only in Antigen presenting cells such as dendritic cells and macrophages.
  • T cells play a very important role in clearing viral infections, but T cell antiviral responses are often associated with immunopathology.
  • PD-1 plays a very important role in the activation of negative regulatory T cells.
  • PD-1 mediated negative regulation of T cells can reduce tissue damage caused by the infection process, but block or inhibit PD-1. Negative regulation can lead to autoimmune diseases.
  • PD-1 knockout mice are more effective at clearing pancreatic virus infection, but they cause more severe liver damage (Isai et al., 2003, J. Exp. Med. 198: 39-50).
  • tumors that highly express PD-1 are accompanied by cancers that are difficult to detect (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. USA 104: 3360-5).
  • An effective method for the implementation is to regulate the expression of PD-1 by injecting antibodies in vivo.
  • Anti-PD-1 antibodies have become the hottest in the global pharmaceutical industry after the unprecedented clinical efficacy data released at the annual meeting of the American Cancer Society (AACR) in 2012 and 2013 and the annual meeting of the American Society of Clinical Oncology (ASCO). Research new drug targets.
  • the present inventors used a mammalian cell expression system to express recombinant PD-1 as an antigen to immunize mice, and fused the mouse spleen cells with myeloma cells to obtain hybridoma cells.
  • the inventors obtained the following hybridoma cell line by screening a large number of samples: hybridoma cell line LT004, which was deposited with the China Center for Type Culture Collection (CCTCC) on August 4, 2015 under the accession number CCTCC NO. :C2015132.
  • the hybridoma cell line LT004 is capable of secreting a specific monoclonal antibody (designated 6F5) which specifically binds to PD-1, and the monoclonal antibody is capable of blocking PD-1 and PDL very efficiently.
  • a specific monoclonal antibody designated 6F5
  • the monoclonal antibody is capable of blocking PD-1 and PDL very efficiently.
  • 6F5 designated 6F5 (Re)
  • 6F5H1L1, 6F5H2L2, respectively 6F5H1L1, 6F5H2L2, respectively.
  • the present inventors have also surprisingly found that the antibodies 6F5/6F5(Re), 6F5H1L1, 6F5H2L2 of the present invention can efficiently bind to human T cells, and activate T cells to induce secretion of IFN- ⁇ and IL-2 by human lymphocytes;
  • cancers such as lung cancer, melanoma, renal tumors, ovarian cancer, leukemia, and anemia.
  • One aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof, wherein
  • the heavy chain variable region ( VH ) of the monoclonal antibody comprises: a CDR having the amino acid sequence of SEQ ID NOs: 13-15;
  • the monoclonal antibody light chain variable region (V L) comprises: the amino acid sequence of SEQ ID NO: 16-18 of CDR.
  • amino acid sequence of the heavy chain variable region of the monoclonal antibody is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10;
  • amino acid sequence of the light chain variable region of the monoclonal antibody is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO: 12.
  • the monoclonal antibody or antigen-binding fragment thereof, wherein the single Cloning antibodies include:
  • VH as shown in SEQ ID NO: 2 and V L as shown in SEQ ID NO: 4;
  • variable regions of the light and heavy chains determine the binding of the antigen; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs) (the CDRs of the heavy chain (H) comprise HCDR1, HCDR2, HCDR3
  • CDRs of the light chain (L) comprise LCDR1, LCDR2, LCDR3; which is named by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), Vol. 1-3, NIH Publication 91-3242, Bethesda Md ).
  • the antibodies of the invention 6F5/6F5 (Re), 6F5H1L1, 6F5H2L2 have the same CDR;
  • amino acid sequences of the three CDR regions of the heavy chain variable region are as follows:
  • HCRD1 GFTFSSYG (SEQ ID NO: 13)
  • HCDR2 ISGGGSDT (SEQ ID NO: 14)
  • HCDR3 ARQLNYAWFAY (SEQ ID NO: 15);
  • amino acid sequences of the three CDR regions of the light chain variable region are as follows:
  • LCRD1 ESVDNYGISF (SEQ ID NO: 16)
  • LCDR2 TSS (SEQ ID NO: 17)
  • LCDR3 QQSKEVPWT (SEQ ID NO: 18).
  • the monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv, dAb, A complementarity determining region fragment, a single chain antibody (eg, scFv), a humanized antibody, a chimeric antibody, or a diabody.
  • the monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein said monoclonal antibody is less than about 100 nM, such as less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM EC 50 or less PD-1 binding protein.
  • the EC 50 measured by indirect ELISA.
  • the monoclonal antibody or antigen-binding fragment thereof according to any of the preceding claims, wherein said monoclonal antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 K D , 10 -9 M or 10 -10 M or less binds to the PD-1 protein.
  • the monoclonal antibody comprises a non-CDR region and the non-CDR region is from a species other than a murine, such as from a human antibody.
  • the monoclonal antibody or antigen-binding fragment thereof according to any one of the present invention, wherein the monoclonal antibody is a monoclonal antibody produced by a hybridoma cell line LT004, and the hybridoma cell line LT004 is preserved in a typical Chinese culture. Collection (CCTCC), the deposit number is CCTCC NO: C2015132.
  • CTCC Chinese culture. Collection
  • Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein
  • the heavy chain variable region of the antibody comprises: a CDR having the amino acid sequence of SEQ ID NOs: 13-15;
  • the heavy chain of the antibody has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 6 or SEQ ID NO: 10;
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO: 9.
  • a further aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence capable of encoding a variable region of an antibody light chain, wherein
  • the antibody light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NOs: 16-18;
  • the antibody light chain variable region has the amino acid sequence set forth in SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 12;
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:11.
  • a further aspect of the invention relates to a vector comprising the isolated nucleic acid molecule of any of the invention.
  • a further aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of any of the invention, or the vector of the invention.
  • a further aspect of the invention relates to a method of producing a monoclonal antibody or antigen-binding fragment thereof according to any of the invention, which comprises culturing a host cell of the invention under suitable conditions, and from cell culture The step of recovering the monoclonal antibody or antigen-binding fragment thereof.
  • a further aspect of the invention relates to a hybridoma cell line LT004 deposited with the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: C2015132.
  • a further aspect of the present invention relates to a conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof, and a conjugated portion, wherein the monoclonal antibody is the monoclonal antibody of any one of the present invention or An antigen-binding fragment, the coupled moiety being a detectable label; preferably, the coupled moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.
  • a further aspect of the invention relates to a kit comprising the monoclonal antibody or antigen-binding fragment thereof according to any of the invention, or comprising the conjugate of the invention;
  • the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance , luminescent substances, colored substances or enzymes.
  • a detectable label such as a radioisotope, a fluorescent substance , luminescent substances, colored substances or enzymes.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a kit for detecting PD-1 The presence or level of the sample.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate according to the invention; optionally further comprising a pharmacy Acceptable carriers and/or excipients.
  • a further aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a prophylactic and/or therapeutic and/or adjuvant treatment and/or diagnosis of a tumor or anemia
  • the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer, and leukemia.
  • a further aspect of the invention relates to the use of a monoclonal antibody or antigen-binding fragment thereof according to any of the invention or a conjugate of the invention for the preparation of a medicament as follows:
  • the ligand of PD-1 is PDL-1 or PDL-2, preferably PDL-1.
  • a further aspect of the invention relates to a method of in vivo or in vitro, comprising the step of applying a cell to an effective amount of a monoclonal antibody or antigen-binding fragment thereof according to any one of the invention or a conjugate of the invention,
  • the method is selected from the following:
  • the ligand of PD-1 is PDL-1 or PDL-2, preferably PDL-1.
  • the in vitro method is for non-therapeutic or diagnostic purposes.
  • a further aspect of the invention relates to a method of preventing and/or treating and/or adjunctively treating and/or diagnosing a tumor or anemia, comprising administering to a subject an effective amount of the monoclonal antibody of any of the invention Or a step of the antigen-binding fragment thereof or the monoclonal antibody conjugate of the present invention; preferably, the tumor is selected from the group consisting of melanoma, renal tumor, prostate cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer Non-small cell lung cancer, ovarian cancer and leukemia.
  • the dosage administered will depend on a number of factors, such as the severity of the condition being treated, the sex, age, weight and individual response of the patient or animal, as well as the condition and prior medical history of the patient to be treated. It is common practice in the art to start with a dose that is lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • the monoclonal antibody or antigen-binding fragment thereof for use in the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of a tumor or anemia; preferably, the tumor is selected from the group consisting of melanin Tumor, kidney tumor, front Adenocarcinoma, bladder cancer, colorectal cancer, gastrointestinal cancer, liver cancer, non-small cell lung cancer, ovarian cancer and leukemia.
  • a monoclonal antibody or antigen-binding fragment thereof for use in:
  • the ligand of PD-1 is PDL-1 or PDL-2, preferably PDL-1.
  • the monoclonal antibodies of the invention only block the binding of PD-1 to PDL-1.
  • PD-1 protein Programmed cell death protein 1, NCBI GenBank: NM_005018, it includes the full length of the PD-1 protein, or the extracellular fragment PD of PD-1.
  • -1 ECD dashed portion of SEQ ID NO: 19
  • a fragment comprising PD-1 ECD further comprising a fusion protein of PD-1 ECD, such as a fragment fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG (See the description in Preparation Example 1).
  • PD-1 protein shall include all such sequences, including the sequences shown in the wavy underlined portion of SEQ ID NO: 19, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the PD-1 protein, it includes not only the sequence fragment of the wavy underlined portion of SEQ ID NO: 19 but also the corresponding sequence fragment of its natural or artificial variant.
  • PDL-1 ECD wild-chain peptide-binding protein
  • mFc or hFc human IgG Fragment
  • FIG. 1 when referring to the amino acid sequence of the PDL-1 protein (Programmed death-ligand 1, NCBI Gene ID: 29126), it includes the full length of the PDL-1 protein, or an extracellular fragment of PDL-1.
  • PDL-1 ECD dashed portion of SEQ ID NO: 23
  • a fragment comprising PDL-1 ECD further comprising a fusion protein of PDL-1 ECD, for example, fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG Fragment (see preparation The description in Example 1).
  • the term "PDL-1 protein” shall include all such sequences, including the sequences shown in the wavy underlined portion of SEQ ID NO: 23, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of the PDL-1 protein, it includes not only the sequence fragment of the wavy underlined portion of SEQ ID NO: 23 but also the corresponding sequence fragment of its natural or artificial variant.
  • EC 50 refers to the term as used herein, half-maximal effective concentration (concentration for 50% of maximal effect ), the concentration refers to cause 50% of maximal effect.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of polypeptide chains (each pair having one "light” (L) chain and one "heavy” (H) chain. .
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full length antibody that retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody.
  • Specific binding to an antigen which is also referred to as an "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Or producing an antigen-binding fragment of an antibody by enzymatic or chemical cleavage of an intact antibody.
  • the antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDRs) Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • CDRs complementarity determining regions
  • Fd fragment means an antibody fragment consisting of V H and C H 1 domains
  • Fv fragment means a single arm of V H and V L domains of an antibody, antibody fragments
  • dAb fragment antibody fragment consisting of V H domains of a means (Ward et al., Nature 341: 544-546 (1989) );
  • Fab fragment is meant a V L, V H, C antibody fragments L and C H 1 domains;
  • F (ab ') 2 fragment means antibody fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • the antigen-binding fragment is a single chain antibody (e.g., the scFv), wherein V L and V H domains are paired to form so that it can be produced by a linker to a single polypeptide chain monovalent molecules (see, e.g., Bird Et al, Science 242: 423-426 (1988) and Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).
  • scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -V H - linker -V L -COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 may also be used variants (Holliger et al. (1993), Proc.Natl.Acad.Sci.USA 90: 6444-6448 ).
  • Other linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the antigen-binding fragment of an antibody is a diabody, ie, a bivalent antibody, wherein the VH and VL domains are expressed on a single polypeptide chain, but too short a linker is used such that two structures in the same chain are not allowed Pairing between domains forces the domain to pair with the complementary domain of another strand and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • a given antibody for example, the monoclonal antibody 6F5/6F5(Re), 6F5H1L1 or 6F5H2L2 provided by the present invention
  • can be used using conventional techniques known to those skilled in the art for example, recombinant DNA technology or enzymatic or chemical cleavage.
  • An antigen-binding fragment of an antibody for example, the above-described antibody fragment
  • specifically screening an antigen-binding fragment of the antibody in the same manner as used for the intact antibody.
  • antibody As used herein, unless the context clearly dictates otherwise, when referring to the term “antibody”, it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • mAb and “monoclonal antibody” refer to a fragment of an antibody or antibody from a population of highly homologous antibody molecules, ie, in addition to a natural mutation that may occur spontaneously, A group of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • Monoclonal antibodies are typically obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, U.S. Patent 4,816,567).
  • chimeric antibody refers to an antibody whose light chain or/and a portion of a heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or Subclass), and another portion of the light or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but in any case, it remains Binding activity to the antigen of interest (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • humanized antibody means that all or part of the CDR regions of a human immunoglobulin (receptor antibody) are replaced by a CDR region of a non-human antibody (donor antibody).
  • An antibody or antibody fragment, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody having the desired specificity, affinity or reactivity.
  • some of the amino acid residues of the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of the corresponding non-human antibody or by amino acid residues of other antibodies to further refine or optimize the performance of the antibody.
  • the terms "isolated” or “isolated” refer to artificially obtained from a natural state. If a certain "separated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or that the substance has been isolated from the natural environment, or both. For example, a certain living animal has a naturally isolated polynucleotide or polypeptide that is not isolated, and the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • separation of the term “separated” or “separated” It is not excluded to mix artificial or synthetic substances, nor to exclude other impure substances that do not affect the activity of the substance.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • the antibody e.g., the monoclonal antibody 6F5/6F5(Re), 6F5H1L1 or 6F5H2L2 of the invention
  • the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10
  • the dissociation equilibrium constant (K D ) of -9 M or 10 -10 M or less binds to an antigen (eg, L1 protein), for example, as measured using a surface plasmon resonance (SPR) in a BIACORE instrument.
  • an antigen eg, L1 protein
  • the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably; “Polypeptide” and “protein” have the same meaning and are used interchangeably.
  • the amino acid is usually a single word well known in the art. The mother and three-letter abbreviation are used to indicate.
  • alanine can be represented by A or Ala.
  • hybridomas and “hybridoma cell lines” are used interchangeably and, when referring to the terms “hybridomas” and “hybridoma cell lines”, they also include subclones of hybridomas. And progeny cells.
  • hybridoma cell line LT004 when referring to the hybridoma cell line LT004, it also refers to the subcloned and progeny cells of the hybridoma cell line LT004.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancement. Agent.
  • pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.
  • adjuvant refers to a non-specific immunopotentiator that, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease eg, a tumor
  • an effective amount to prevent, prevent, or delay the onset of a disease eg, a tumor
  • treating an effective amount of the disease means sufficient to cure or at least partially arrest a patient already suffering from the disease.
  • the amount of disease and its complications Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • subject can refer to a patient or other animal that receives the composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, a cow, Horse and so on.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • the monoclonal antibodies of the present invention are capable of specifically binding to PD-1, and are capable of blocking PD-1 binding to PDL-1 very efficiently, specifically releasing PD-1 to immunosuppress the body, and activating T lymphocytes. cell.
  • Figure 1 Results of SDS-PAGE detection of the fusion protein PD-1 ECD-TEV-mFc.
  • the samples from the left to the right of the two lanes were: Marker; PD-1 ECD-TEV-mFc fusion protein.
  • Figure 2 Results of SDS-PAGE detection of the fusion protein PD-1 ECD-TEV-hFc.
  • the samples from the left to the right of the two lanes were: Marker; PD-1 ECD-TEV-hFc fusion protein.
  • FIG. 3 Results of SDS-PAGE detection of monoclonal humanized antibody 6F5H1L1.
  • the samples from the left to the right of the four lanes were: BSA; Marker; reduced protein electrophoresis loading buffer sample antibody; non-reduced protein electrophoresis loading buffer sample antibody.
  • Figure 4 Results of SDS-PAGE detection of monoclonal humanized antibody 6F5H2L2.
  • the samples from the left to the right of the four lanes were: non-reduced protein electrophoresis loading buffer sample antibody; reduced protein electrophoresis loading buffer sample antibody; Marker; BSA.
  • Figure 7 Binding of antibodies 6F5, 6F5 (Re) to PD-1 by indirect ELISA.
  • Figure 8 Binding of antibodies 6F5H1L1, 6F5H2L2 to PD-1 by indirect ELISA.
  • FIG. 10 Competition ELISA detection Antibodies 6F5H1L1, 6F5H2L2 compete with PDL-1 for binding to PD-1.
  • Figure 11 Binding activity of antibody 6F5H2L2 to cell surface antigen PD-1.
  • Figure 12 Binding activity of positive control antibody 5C4 to cell surface antigen PD-1.
  • Figure 13 Antibody 6F5H2L2 and positive control antibody 5C4 block the binding activity of the cell surface antigen PD-1 to PDL-1.
  • FIG. 14 Effect of antibody 6F5H2L2 on IFN- ⁇ secretion by mixed lymphocytes.
  • Figure 15 Effect of antibody 6F5H2L2 on IL-2 secretion by mixed lymphocytes.
  • the hybridoma cell line LT004 was deposited with the China Center for Type Culture Collection (CCTCC) on August 4, 2015 under the accession number CCTCC NO: C2015132.
  • the deposit address is Wuhan University, Wuhan, China, 430072.
  • the amino acid corresponding to the extracellular fragment PD-1ECD of the gene PD-1 (Programmed cell death protein 1, NCBI GenBank: NM_005018) and the Fc protein fragment (mFc) of TEV and mouse IgG, and the Fc protein of TEV and human IgG, respectively.
  • the fragment (hFc) was designed to enhance the expression efficiency of the target gene in the 293F cell expression system.
  • Jinsui Company was commissioned to optimize the nucleic acid sequence corresponding to the fusion protein sequence, and the optimization mainly considered the preference of the codon, GC. Content, secondary structure of mRNA, repeat sequence and other factors.
  • the sequence optimized for the PD-1ECD-TEV-mFc and PD-1ECD-TEV-hFc fusion protein genes was finally synthesized by Kingsray.
  • the recombinant PD-1ECD-TEV-mFc and PD-1ECD-TEV-hFc fusion genes were cloned into pUC57simple (provided by Kingsray) expression vector to obtain pUC57simple-PD-1ECD-TEV-mFc. And the pUC57simple-PD-1ECD-TEV-hFc plasmid.
  • the plasmid pUC57simple-PD-1ECD-TEV-mFc and pUC57simple-PD-1ECD-TEV-hFc were digested separately (Xba I and BamH I), and the fusion gene fragments PD-1ECD-TEV-mFc and PD- were recovered by electrophoresis.
  • 1ECD-TEV-hFc was ligated with pcDNA3.1 expression vector (purchased from Invitrogen) to obtain pcDNA3.1-PD-1ECD-TEV-mFc and pcDNA3.1-PD-1ECD-TEV-hFc, respectively, transfection feeling Large intestine Bacillus cell DH5a (purchased from TIANGEN) was transfected and cultured according to the instructions.
  • the positive pcDNA3.1-PD-1ECD-TEV-mFc and pcDNA3.1-PD-1ECD-TEV-hFc clone colonies were screened, and E.
  • coli was amplified according to the conventional method, and then the kit (purchased from Tiangen Biochemical Technology ( Beijing) Co., Ltd., DP103-03) and obtained the recombinant plasmid pcDNA3.1-PD-1ECD-TEV-mFc and pcDNA3.1-PD-1ECD-TEV-hFc according to the instructions of the kit.
  • Recombinant plasmids pcDNA3.1-PD-1ECD-TEV-mFc and pcDNA3.1-PD-1ECD-TEV-hFc were transfected into 293F cells according to the lipofectamin transfection kit (purchased from Invitrogen) (purchased from Invitrogen). the company).
  • the recombinant plasmid pcDNA3.1-PD-1ECD-TEV-mFc and pcDNA3.1-PD-1ECD-TEV-hFc were transfected into 293F cells for 7 days, respectively.
  • the culture medium was vacuum filtered by high-speed centrifugation and microfiltration membrane.
  • the Mabselect SuRe column was used to purify the PD-1ECD-TEV-mFc and PD-1ECD-TEV-hFc fusion proteins, and the purified sample was added to the reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis. 1, Figure 2 shows.
  • the synthesis method of the antigen PDL-1-hFc (PDL-1: Programmed death-ligand 1, NCBI Gene ID: 29126) is the same as the synthesis method of PD-1-hFc, and the obtained fusion protein is purified and subjected to SDS-PAGE electrophoresis. Detection.
  • sequence information involved in this preparation example is as follows:
  • PD-1 Programmed cell death protein 1, NCBI GenBank: NM_005018;
  • mFc Ig gamma-2A chain C region: ACCESSION: P01863, 99-330;
  • hFc Ig gamma-1 chain C region, ACCESSION: P01857, 106-330;
  • PDL-1 Programmed death-ligand 1, NCBI Gene ID: 29126.
  • the wavy underlined part is the PD-1ECD part
  • the lowercase letter part is the TEV cleavage site
  • the solid line underlined is the mFc part.
  • the wavy underlined part is the PD-1ECD part
  • the lowercase letter part is the TEV cleavage site
  • the solid line underlined is the mFc part.
  • the wavy underlined part is the PD-1ECD part
  • the lowercase letter part is the TEV cleavage site
  • the solid line underlined is the hFc part.
  • the wavy underlined part is the PD-1ECD part
  • the lowercase letter part is the TEV cleavage site
  • the solid line underlined is the hFc part.
  • the wavy underlined part is the PDL-1ECD part
  • the lowercase letter part is the TEV cleavage site
  • the solid line underlined is the hFc part.
  • the wavy underlined part is the PDL-1ECD part
  • the lowercase letter part is the TEV cleavage site
  • the solid line underlined is the hFc part.
  • Example 1 Obtainment of hybridoma cell line LT004 and preparation of monoclonal antibody 6F5
  • the mammalian cell expression system is used to express the recombinant PD-1ECD-TEV-mFc as an antigen to immunize the mouse, and the mouse spleen cells are fused with the myeloma cells to obtain hybridoma cells.
  • a hybridoma cell line LT004 was obtained, which was able to secrete monoclonal antibody 6F5 which specifically binds to PD-1.
  • the specific method is as follows:
  • PD-1ECD-TEV-mFc fusion protein ie PD-1-mFc, see Preparation 1
  • spleen cells and mouse myeloma of immunized BALB/C mice purchased from Guangdong Medical Laboratory Animal Center
  • Fusion of cells into hybridoma cells refer to currently established methods (eg, Stewart, SJ, "Monoclonal Antibody Production", in Basic Methods in antibody Production and Characterization, Eds. GC Howard and DR Bethell, Boca Raton: CRC Press, 2000 ).
  • hybridoma cell lines capable of secreting monoclonal antibodies that compete with ligand PDL-1 (see Preparation 1) for binding to PD-1 were screened by competition ELISA and stabilized by limiting dilution method.
  • the hybridoma cell line was obtained by a limiting dilution method to obtain a stable cell line of PD-1-6F5.
  • the hybridoma cell line is named LT004, and the secreted monoclonal antibody is named 6F5.
  • Hybridoma cell line LT004 accession number: CCTCC C2015132.
  • the LT004 cell line of the present invention was cultured in an IMDM medium containing 10% of low IgG fetal bovine serum, and after 7 days, the cell culture supernatant was collected and purified to prepare antibody 6F5.
  • the purified sample is separately added to a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and then boiled for detection.
  • the mRNA was extracted from the hybridoma cell line LT004 prepared in Example 1 according to the method of culturing the cell bacterial total RNA extraction kit (Tiangen, Cat. No. DP430).
  • cDNA was synthesized according to the TransScript First-Strand cDNA Synthesis SuperMix (Transgen, AT301) kit instructions and subjected to PCR amplification.
  • the PCR amplification product was directly subjected to TA cloning, and the specific procedure was carried out in accordance with the instructions of the pEASY-T1 Cloning Kit (Transgen CT101) kit.
  • Example 3 Design of light and heavy chain sequences of humanized antibodies 6F5H1L1, 6F5H2L2
  • the antibody obtained according to the three-dimensional crystal structure of the PD-1 protein (Shinohara T, et al., Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics 1995, 23(3): 704-6) and Example 2
  • the sequence of 6F5 was designed by computer simulation of the antibody model, and the variable region of the antibody 6F5H1L1, 6F5H2L2 was obtained according to the model design.
  • the heavy chain constant region was Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region was Ig kappa.
  • Chain C region, ACCESSION: P01834), the variable region sequence is as follows:
  • 6F5 heavy chain cDNA sequence (heavy chain variable region sequence as shown in SEQ ID NO: 1) (constant region sequence is immunoglobulin gamma 2b heavy chain precursor [Mus musculus] 140-475, ACCESSION: ACX70084.1) and light
  • the cDNA sequence of the strand (light chain variable region sequence as shown in SEQ ID NO: 3) (constant region is antibody kappa light chain, partial [Mus musculus], 106-213 GenBank: BAB 33404.1) was cloned into pUC57simple (Kings) In the vector provided by Rui Company, pUC57simple-6F5H and pUC57simple-6F5L plasmid.
  • the plasmids pUC57simple-6F5H and pUC57simple-6F5L were digested respectively (HindIII & EcoRI), and the heavy chain light chains recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmid was co-transfected into 293F cells. After 7 days of cell culture, the culture solution was vacuum filtered through a high-speed centrifugation, microfiltration membrane, and loaded onto a HiTrap MabSelectSuRe column, and the protein was eluted in one step with Elution Buffer, and the target sample was recovered and exchanged with HiTrap Desalting to PBS.
  • the heavy chain cDNA of 6F5H1L1, 6F5H2L2 (the heavy chain variable region sequences are SEQ ID NO: 5, 9 respectively) and the light chain cDNA (light chain variable region sequences are SEQ ID NO: 7, 11 respectively) were cloned into In the pUC57simple (provided by Kingsray) vector, the pUC57simple-6F5H1L1, pUC57simple-6F5H2L2 plasmid was obtained and subcloned into the pcDNA3.1 vector in the same manner as the above 6F5 (Re).
  • the recombinant plasmid was transfected into 293F cells, and the culture solution was purified and detected (the same method as 6F5 (Re) described above).
  • the target protein of the reduced protein sample was about 24.5 kD and 49 KD.
  • the non-reduced protein sample target protein is approximately 147 kD.
  • the kinetic parameters of binding of antibody 6F5H2L2 and positive control antibody 5C4 to antigen PD-1 were determined using a Fortebio molecular interaction instrument.
  • the PD-1-mFc protein was digested with TEV protease (see Preparation Example 1) and purified by column to obtain PD-1 antigen.
  • the antigen PD-1 (antigen concentration 1 ⁇ g/ml) was biotinylated and fixed on the surface of the SA sensor. After equilibration in PBST, it was bound to antibody 6F5H2L2 or 5C4, and the antibody was diluted three times from 200 nM with PBST. Dissociated in PBST.
  • the kinetic parameters of the antibodies 6F5H2L2 and 5C4 are shown in Table 1, and the results of the kinetic characteristic parameters are shown in Figures 5 and 6, respectively.
  • Example 6 Detection of binding activity of antibody to antigen PD-1 by indirect ELISA
  • hybridoma antibody 6F5, recombinant antibody 6F5 (Re) and humanized antibody 6F5H1L1, 6F5H2L2 and positive control antibody 5C4 was determined by indirect ELISA.
  • PD1-mFc or PD1-hFc was diluted to 0.5 ⁇ g/ml with CBS, and added to 96 wells of the plate, 50 ⁇ L per well, and incubated overnight at 4 °C. After washing one time, the cells were blocked with 1% BSA + PBS, 300 ⁇ L per well, and incubated at 37 ° C for 2 hours.
  • the antibodies were diluted with PBS to 1 ⁇ g/ml and initially diluted 1:3 with PBS as a blank control. 50 ⁇ l per well and incubate for 10 minutes at room temperature. After washing the plate three times, respectively, add HRP-labeled goat anti-human or goat anti-mouse secondary antibody, 50 ⁇ L per well, incubate at 37 ° C for 30 minutes, wash the plate four times, add TMB coloring solution, 50 ⁇ L per well, avoid at room temperature Light color for 5 minutes. The reaction was stopped by direct addition of stop solution at 50 ⁇ L per well. Immediately after termination of the reaction, the plate was placed in a microplate reader, and the OD value was measured at 450 nm, and the original data was saved. The raw data is entered into the software SoftMax Pro 6.2.1 for data processing.
  • antibodies 6F5, 6F5 (Re), 6F5H1L1, 6F5H2L2, positive control antibody 5C4 can effectively bind PD-1 protein, and the binding efficiency is dose-dependent.
  • the absorbance intensity of each dose is shown in Table 2, Table 3. .
  • the curve simulation binding efficiency EC 50 was 0.091 nM, 0.028 nM, 0.199 nM, respectively.
  • the recombinant antibody 6F5(Re) is different from the expression system for hybridoma antibody 6F5.
  • 6F5(Re) is expressed by human 293F cells
  • hybridoma antibody 6F5 is produced by mouse hybridoma cells
  • antibodies are produced. There are differences in the degree of expression of glycosylation during the process, and the affinity may vary.
  • binding efficiency simulated curve were EC 50 0.118nM, 0.100nM and 0.233nM. Visible, 6F5H1L1,6F5H2L2 EC 50 of about 1/2 of positive control antibody 5C4, binding efficiency is significantly better than the positive control antibody 5C4.
  • Example 7 Competitive ELISA method to detect the binding of antibodies to PDL-1 to bind antigen PD-1
  • PD-1-mFc or PD-1-hFc was diluted to 0.5 ⁇ g/ml with CBS, and added to 96 wells of the plate, 50 ⁇ L per well, and incubated overnight at 4 °C. After washing one time, the cells were blocked with 1% BSA + PBS, 300 ⁇ L per well, and incubated at 37 ° C for 2 hours.
  • the antibody was diluted with PBS to a concentration of 1.5 ⁇ g/ml, and the mixture was diluted 1:3, and PBS was used as a blank control. 50 ⁇ l per well and incubate for 10 minutes at room temperature. PDL-1-hFc or PDL-1-mFc was diluted to 2 ⁇ g/ml with PBS, 50 ⁇ l per well, mixed well with the antibody, and incubated at 37 ° C for 45 minutes. After washing the plate three times, HRP-labeled goat anti-human secondary antibody was added, respectively, 50 ⁇ L per well, and incubated at 37 ° C for 30 minutes.
  • Fig. 9 and Fig. 10 The results of the binding of the antibody 6F5 and the humanized antibody 6F5H1L1, 6F5H2L2 to PDL-1 for binding to the antigen PD-1 are shown in Fig. 9 and Fig. 10 .
  • both 6F5 and humanized antibodies 6F5H1L1 and 6F5H2L2 competed with PDL-1 for binding to PD-1 protein in a dose-dependent manner.
  • the curve simulation binding efficiency EC 50 was 0.198 nM, 0.979 nM, 1.175 nM and 1.461 nM, respectively.
  • Example 8 Flow cytometry method for detecting binding activity of antibody to cell surface antigen PD-1
  • Host cell 293T expressing the PD-1 antigen was first constructed, and then the host cell was labeled with the humanized antibody 6F5H2L2 (see Example 4) prepared in the present invention. Flow cytometry analysis was then used to verify the antigen-specific binding ability of antibody 6F5H2L2 to the native conformation of the cell surface.
  • the vector pLenti6.3-PD-1 (vector pLenti6.3 purchased from Invitrogen) containing PD-1 was transfected into 293T cells according to the lipofectamin transfection kit (purchased from Invitrogen), and the stably expressed PD- was obtained by screening. A clonal population of 1 293T-PD-1.
  • the PD-1 antigen-expressing host cell 293T-PD-1 obtained by the above steps of the conventional trypsin digestion method was used, and the number of cells in each collection tube was 2 ⁇ 10 5 , and the concentration was 50 nM in PBS (1% BSA).
  • the results of binding of the humanized antibody 6F5H2L2 to 293T-PD-1 cells are shown in Figs.
  • the 6F5H2L2 antibody can effectively bind to the target PD-1 protein on the surface of host cell 293T-PD-1, and its binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose is shown in Table 6.
  • Table 6 Fluorescence intensity analysis of 293T-PD-1 surface antigen in 6F5H2L2 binding to PD-1 host cells by flow cytometry
  • simulation 6F5H2L2 curves and positive control antibody 5C4 binding efficiency of EC 50 were 5.022 and 4.171nM.
  • Example 9 Flow cytometry method detection of antibody blocking cell surface antigen PD-1 binding activity to PDL-1
  • the host cell 293T-PD-1 expressing the PD-1 antigen obtained in Example 8 was subjected to a conventional trypsin digestion method, and the number of cells per collection tube was 2 ⁇ 10 5 , and the concentration was determined by using PBS (1% BSA). 100 nM, 50 nM, 20 nM, 10 nM, 3 nM, 1 nM PD-1 antibody 6F5H2L2 and positive control antibody 5C4 dilution, incubated with 293T cells expressing PD-1 for 1 hour on ice, adding appropriate amount of PDL to each cell dilution The -1-mFc protein was incubated at a final concentration of 20 nM and incubated on ice for 30 min.
  • Table 7 Fluorescence intensity analysis of 6F5H2L2 blocking human PDL-1 and PD-1 binding activity by flow cytometry
  • simulation curve 6F5H2L2 suppression efficiency of EC 50 and 5C4 are 8.088nM and 2.208nM.
  • Example 10 Mixed lymphocyte reaction: secretion of cytokine IFN- ⁇ , IL-2
  • PBMC peripheral blood mononuclear cells
  • Ficoll-Paque Plus GE Healthcare LOT No.: 171440-02
  • IL-4 Peprotech K2513, 1000 U/ml
  • GM-CSF Peprotech H1513, 1000 U/ml
  • DC cells were obtained by inducing TNF- ⁇ (Peprotech G1513, 200 U/ml) for 3 days.
  • T cells were isolated from PBMC, and the obtained DC cells were mixed with T cells in a ratio of 1:10, and different ratios of antibody 6F5H2L2 (hIgG as a negative control, Isotype control) were added for 5-6 days, and ELISA reagent was used.
  • the cassette was tested for secretion of IFN- ⁇ (purchased from Daktronics) and IL-2 (purchased from Daktronics).
  • 6F5H2L2 antibody can effectively induce mixed lymphocytes to secrete IFN- ⁇ and IL-2, and secrete them. Amount and antibody 6F5H2L2 was dose dependent.

Abstract

本发明属于肿瘤治疗和分子免疫学领域，提供了一种抗PD-1的单克隆抗体、其抗原结合片段、其药物组合物及用途。所述单克隆抗体的重链可变区包含氨基酸序列如SEQ ID NO：13-15所示的CDR1-3，轻链可变区包含氨基酸序列如SEQ ID NO：16-18所示的CDR1-3。该单克隆抗体能够特异性与PD-1结合，解除PD-1对机体免疫抑制，激活T淋巴细胞。

Description

一种抗PD-1单克隆抗体、其药物组合物及其用途 技术领域
本发明属于肿瘤治疗和分子免疫学领域，涉及一种抗PD-1抗体、其药物组合物及其用途。优选地，本发明涉及一种抗PD-1的单克隆抗体。
背景技术
跨膜受体PD-1(programmed cell death 1，程序性细胞死亡因子1)是CD28基因家族成员之一，在活化的T细胞，B细胞以及骨髓系细胞都有表达。PD-1的配体PDL-1和PDL-2均属于B7超家族，其中PDL-1多种细胞都有表达，包括T细胞，B细胞以及内皮细胞和上皮细胞，PDL-2则仅表达于抗原呈递细胞如树突状细胞和巨噬细胞。
T细胞对清除病毒感染起着非常重要的作用，但T细胞抗病毒反应通常与免疫病理相关。PD-1在负调节T细胞的活化过程中起着非常重要的作用，PD-1介导的对T细胞负调节作用可减少促感染过程引起的组织损伤，但是阻断或抑制PD-1的负调节作用可导致自身免疫性疾病，例如，PD-1基因敲除小鼠能更有效的清除胰腺病毒感染，但是却导致了更严重的肝脏损伤(Isai et al.，2003，J.Exp.Med.198：39-50)。另外，高表达PD-1的肿瘤伴随着很难被检测到的癌症(Hamanishi et al.，2007，Proc.Natl.Acad.Sci.USA 104：3360-5)。一种实施有效的方法是通过体内注射抗体对PD-1的表达进行调控。
由于PD-1抗体的广谱抗肿瘤前景和惊人的药效，业界普遍认为针对PD-1通路的抗体将带来治疗多种肿瘤治疗的突破性的进展：用于治疗非小细胞性肺癌，肾细胞癌，卵巢癌，黑色素瘤(Homet M.B.，Parisi G.，et al.，Anti-PD-1 Therapy in Melanoma.Semin Oncol.2015Jun；42(3)：466-473)，白血病以及贫血病(Held SA，Heine A，et al.，Advances in immunotherapy of chronic myeloid leukemia CML.Curr Cancer Drug Targets.2013Sep；13(7)：768-74)。
在2012年和2013年的美国癌症协会(AACR)年会以及美国临床肿瘤协会(ASCO)年会上揭晓的前所未有的临床药效数据后，抗PD-1抗体已成为全球制药行业最炙手可热的在研新药靶点。
目前，尚需要开发新的具有更好的结合效率的抗PD-1抗体，以有效地阻断PD-1与 PDL-1的结合。
发明内容
本发明人经过深入的研究和创造性的劳动，利用哺乳动物细胞表达***表达出重组的PD-1作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过进行对大量样本的筛选，得到了如下的杂交瘤细胞株：杂交瘤细胞株LT004，其于2015年8月4日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015132。
本发明人惊奇地发现，杂交瘤细胞株LT004能够分泌产生与PD-1特异性结合的特异性单克隆抗体(命名为6F5)，并且该单克隆抗体能够十分有效地阻断PD-1与PDL-1的结合。
进一步地，本发明人通过人工表达制得了6F5(命名为6F5(Re))，并创造性地制得了抗PD-1的人源化抗体(分别命名为6F5H1L1、6F5H2L2)。
本发明人还惊奇地发现，本发明的抗体6F5/6F5(Re)、6F5H1L1、6F5H2L2能有效地结合人T细胞，并且激活T细胞，诱导人淋巴细胞分泌IFN-γ和IL-2；具有用于制备防治肺癌、黑色素瘤、肾肿瘤、卵巢癌、白血病等癌症以及贫血病的药物的潜力。
由此提供了下述发明：
本发明的一个方面涉及单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区(VH)包含：氨基酸序列为SEQ ID NO：13-15的CDR；
和/或
所述单克隆抗体的轻链可变区(VL)包含：氨基酸序列为SEQ ID NO：16-18的CDR。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，
所述单克隆抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6和SEQ ID NO：10；
和/或
所述单克隆抗体的轻链可变区的氨基酸序列选自SEQ ID NO：4、SEQ ID NO：8和SEQ ID NO：12。
在本发明的一个实施方案中，所述的单克隆抗体或其抗原结合片段，其中，所述单 克隆抗体包括：
(1)如SEQ ID NO：2所示的VH和如SEQ ID NO：4所示的VL；
(2)如SEQ ID NO：6所示的VH和如SEQ ID NO：8所示的VL；
(3)如SEQ ID NO：10所示的VH和如SEQ ID NO：12所示的VL。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)(重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3；其由Kabat等人命名，见Sequences of Proteins of Immunological Interest，Fifth Edition(1991)，第1-3卷，NIH Publication 91-3242，Bethesda Md)。
通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库分析上面的(1)-(3)项中的单克隆抗体序列的CDR区的氨基酸序列：
本发明的抗体6F5/6F5(Re)、6F5H1L1、6F5H2L2具有相同的CDR；
其重链可变区的3个CDR区的氨基酸序列如下：
HCRD1：GFTFSSYG(SEQ ID NO：13)
HCDR2：ISGGGSDT(SEQ ID NO：14)
HCDR3：ARQLNYAWFAY(SEQ ID NO：15)；
其轻链可变区的3个CDR区的氨基酸序列如下：
LCRD1：ESVDNYGISF(SEQ ID NO：16)
LCDR2：TSS(SEQ ID NO：17)
LCDR3：QQSKEVPWT(SEQ ID NO：18)。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段选自Fab、Fab’、F(ab’)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC50结合PD-1蛋白。优选地，所述EC50通过间接ELISA方法测得。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体 以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的KD结合PD-1蛋白。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中，
所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
根据本发明任一项所述的单克隆抗体或其抗原结合片段，其中所述单克隆抗体是有杂交瘤细胞株LT004产生的单克隆抗体，所述杂交瘤细胞株LT004保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015132。
本发明的另一方面涉及一种分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO：13-15的CDR；
优选地，所述抗体的重链具有SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的氨基酸序列；
更优选地，所述核酸分子具有SEQ ID NO：1、SEQ ID NO：5或SEQ ID NO：9所示的核苷酸序列。
本发明的再一方面涉及一种分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
所述抗体轻链可变区包含氨基酸序列为SEQ ID NO：16-18的CDR；
优选地，所述抗体轻链可变区具有SEQ ID NO：4、SEQ ID NO：8或SEQ ID NO：12所示的氨基酸序列；
更优选地，所述核酸分子具有SEQ ID NO：3、SEQ ID NO：7或SEQ ID NO：11所示的核苷酸序列。
本发明的再一方面涉及一种载体，其包含本发明中任一项所述的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明中任一项所述的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及一种制备本发明中任一项所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
本发明的再一方面涉及杂交瘤细胞株LT004，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015132。
本发明的再一方面涉及一种偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为本发明中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及一种试剂盒，其包括本发明中任一项所述的单克隆抗体或其抗原结合片段，或者包括本发明的偶联物；
优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PD-1在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病的药物中的用途；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、***癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物在制备如下药物中的用途：
阻断PD-1与PD-1的配体结合的药物，
调节(例如下调)PD-1活性或水平的药物，
解除PD-1对机体免疫抑制的药物，或者
提高T淋巴细胞中IFN-γ和/或IL-2表达的药物；
优选地，所述PD-1的配体为PDL-1或PDL-2，优选为PDL-1。
本发明的再一方面涉及一种在体内或体外方法，包括施加细胞以有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明的偶联物的步骤，所述方法选自如下：
阻断PD-1与PD-1的配体结合的方法，
调节(例如下调)PD-1活性或水平的方法，
解除PD-1对机体免疫抑制的方法，或者
提高T淋巴细胞中IFN-γ和/或IL-2表达的方法；
优选地，所述PD-1的配体为PDL-1或PDL-2，优选为PDL-1。
在本发明的一个具体的实施方案中，所述体外方法是非治疗或诊断目的的。
本发明的再一方面涉及一种预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病的方法，包括给予受试者有效量的本发明中任一项所述的单克隆抗体或其抗原结合片段或者本发明所述的单克隆抗体偶联物的步骤；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、***癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
给药剂量取决于许多因素，例如所治疗病况的严重程度，患者或动物的性别、年龄、体重及个体反应，以及待治疗患者的病况和既往病史来选定。本领域通常的做法是，剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。
根据本发明中任一项所述的单克隆抗体或其抗原结合片段，其用于预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、前 列腺癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
根据本发明中任一项所述的单克隆抗体或其抗原结合片段，其用于：
阻断PD-1与PD-1的配体结合，
调节(例如下调)PD-1活性或水平，
解除PD-1对机体免疫抑制，或者
提高T淋巴细胞中IFN-γ和/或IL-2表达；
优选地，所述PD-1的配体为PDL-1或PDL-2，优选为PDL-1。
在本发明的一个具体的实施方案中，本发明的单克隆抗体仅阻断PD-1与PDL-1的结合。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，当提及PD-1蛋白(Programmed cell death protein 1，NCBI GenBank：NM_005018)的氨基酸序列时，其包括PD-1蛋白的全长，或者PD-1的胞外片段PD-1ECD(SEQ ID NO：19中波浪下划线部分)或者包含PD-1ECD的片段；还包括PD-1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段(参见制备例1中的描述)。然而，本领域技术人员理解，在PD-1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PD-1蛋白”应包括所有此类序列，包括SEQ ID NO：19中波浪下划线部分所示的序列以及其天然或人工的变体。并且，当描述PD-1蛋白的序列片段时，其不仅包括SEQ ID NO：19中波浪下划线部分的序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，当提及PDL-1蛋白(Programmed death-ligand 1，NCBI Gene ID：29126)的氨基酸序列时，其包括PDL-1蛋白的全长，或者PDL-1的胞外片段PDL-1ECD(SEQ ID NO：23中波浪下划线部分)或者包含PDL-1ECD的片段；还包括PDL-1ECD的融合蛋白，例如与小鼠或人IgG的Fc蛋白片段(mFc或hFc)进行融合的片段(参见制备 例1中的描述)。然而，本领域技术人员理解，在PDL-1蛋白的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于置换，缺失和/或添加)，而不影响其生物学功能。因此，在本发明中，术语“PDL-1蛋白”应包括所有此类序列，包括SEQ ID NO：23中波浪下划线部分所示的序列以及其天然或人工的变体。并且，当描述PDL-1蛋白的序列片段时，其不仅包括SEQ ID NO：23中波浪下划线部分的序列片段，还包括其天然或人工变体中的相应序列片段。
如本文中所使用的，术语EC50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指，是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫***的各种细胞(例如，效应细胞)和经典补体***的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特 异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology，Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab’、F(ab’)2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由VH和CH1结构域组成的抗体片段；术语“Fv片段”意指由抗体的单臂的VL和VH结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人，Nature 341：544-546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中VL和VH结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423-426(1988)和Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883(1988))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)，和Poljak R.J.等人，Structure 2：1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体6F5/6F5(Re)、6F5H1L1或6F5H2L2)获得 抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片断，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly et al.；Morrison et al.，Proc.Natl.Acad.Sci.USA，81：6851 6855(1984))。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.，Nature，321：522 525(1986)；Reichmann et al.，Nature，332：323 329(1988)；Presta，Curr.Op.Struct.Biol.，2：593 596(1992)；和Clark，Immunol.Today 21：397 402(2000)。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的” 不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、***瘤病毒、***多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的单克隆抗体6F5/6F5(Re)、6F5H1L1或6F5H2L2)以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原(例如，L1蛋白)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用；术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字 母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“杂交瘤”和“杂交瘤细胞株”可互换使用，并且当提及术语“杂交瘤”和“杂交瘤细胞株”时，其还包括杂交瘤的亚克隆和后代细胞。例如，当提及杂交瘤细胞株LT004时，其还指杂交瘤细胞株LT004的亚克隆和后代细胞。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington’s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包括但不限于氯化钠。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
术语“受试者”可以指患者或者其它接受本发明组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。
发明的有益效果
本发明的单克隆抗体均能够很好地特异性与PD-1结合，并且能够十分有效地阻断PD-1与PDL-1的结合，特异地解除PD-1对机体免疫抑制，激活T淋巴细胞。
附图说明
图1：融合蛋白PD-1ECD-TEV-mFc的SDS-PAGE检测结果。从左至右的2个泳道的样品依次为：Marker；PD-1ECD-TEV-mFc融合蛋白。
图2：融合蛋白PD-1ECD-TEV-hFc的SDS-PAGE检测结果。从左至右的2个泳道的样品依次为：Marker；PD-1ECD-TEV-hFc融合蛋白。
图3：单克隆人源化抗体6F5H1L1的SDS-PAGE检测结果。从左至右的4个泳道的样品依次为：BSA；Marker；还原型蛋白电泳上样缓冲液样品抗体；非还原型蛋白电泳上样缓冲液样品抗体。
图4：单克隆人源化抗体6F5H2L2的SDS-PAGE检测结果。从左至右的4个泳道的样品依次为：非还原型蛋白电泳上样缓冲液样品抗体；还原型蛋白电泳上样缓冲液样品抗体；Marker；BSA。
图5：单抗6F5H2L2的动力学特征参数检测结果。
图6：阳性对照抗体5C4的动力学特征参数检测结果。
图7：间接ELISA检测抗体6F5、6F5(Re)与PD-1的结合。
图8：间接ELISA检测抗体6F5H1L1、6F5H2L2与PD-1的结合。
图9：竞争ELISA检测抗体6F5与PDL-1竞争结合PD-1。
图10：竞争ELISA检测抗体6F5H1L1、6F5H2L2与PDL-1竞争结合PD-1。
图11：抗体6F5H2L2与细胞表面抗原PD-1的结合活性。
图12：阳性对照抗体5C4与细胞表面抗原PD-1的结合活性。
图13：抗体6F5H2L2和阳性对照抗体5C4阻断细胞表面抗原PD-1与PDL-1的结合活性。
图14：抗体6F5H2L2对混合淋巴细胞的IFN-γ分泌的影响。
图15：抗体6F5H2L2对混合淋巴细胞的IL-2分泌的影响。
杂交瘤细胞株LT004，其于2015年8月4日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015132，保藏地址为中国.武汉.武汉大学，邮编：430072。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
制备例1：抗原PD-1-mFc、PD-1-hFc和PDL-1-hFc的合成
1.基因PD-1-mFc和PD-1-hFc的合成
对基因PD-1(Programmed cell death protein 1，NCBI GenBank：NM_005018)的胞外片段PD-1ECD所对应的氨基酸分别与TEV及小鼠IgG的Fc蛋白片段(mFc)和TEV及人IgG的Fc蛋白片段(hFc)进行融合设计，为提高目的基因在293F细胞表达***中的表达效率，委托金斯瑞公司分别对融合蛋白序列相对应的核酸序列进行优化，优化主要考虑密码子的偏好性、GC含量、mRNA的二级结构、重复序列等因素。最终将PD-1ECD-TEV-mFc和PD-1ECD-TEV-hFc融合蛋白基因优化后的序列，委托金斯瑞公司合成。
2.pUC57simple-PD-1ECD-TEV-mFc质粒的获得
分别将金斯瑞公司将合成的PD-1ECD-TEV-mFc和PD-1ECD-TEV-hFc融合基因克隆到pUC57simple(金斯瑞公司提供)表达载体中，获得pUC57simple-PD-1ECD-TEV-mFc和pUC57simple-PD-1ECD-TEV-hFc质粒。
3.pcDNA3.1-PD-1ECD-TEV-mFc和pcDNA3.1-PD-1ECD-TEV-hFc重组质粒的构建
分别将质粒pUC57simple-PD-1ECD-TEV-mFc和pUC57simple-PD-1ECD-TEV-hFc进行酶切(Xba I和BamH I)，电泳回收得到的融合基因片段PD-1ECD-TEV-mFc和PD-1ECD-TEV-hFc与pcDNA3.1表达载体(购自Invitrogen公司)进行连接反应，分别获得pcDNA3.1-PD-1ECD-TEV-mFc和pcDNA3.1-PD-1ECD-TEV-hFc，转染感受态大肠 杆菌细胞DH5a(购自TIANGEN公司)，转染和培养按照说明书进行。筛选得到阳性的pcDNA3.1-PD-1ECD-TEV-mFc和pcDNA3.1-PD-1ECD-TEV-hFc克隆菌落，按照常规方法扩增大肠杆菌，然后采用试剂盒(购自天根生化科技(北京)有限公司，DP103-03)并按照试剂盒的说明书提取得到pcDNA3.1-PD-1ECD-TEV-mFc和pcDNA3.1-PD-1ECD-TEV-hFc重组质粒。
4.按照lipofectamin转染试剂盒(购自Invitrogen公司)方法分别将重组质粒pcDNA3.1-PD-1ECD-TEV-mFc和pcDNA3.1-PD-1ECD-TEV-hFc转染293F细胞(购自Invitrogen公司)。
5.分别将重组质粒pcDNA3.1-PD-1ECD-TEV-mFc和pcDNA3.1-PD-1ECD-TEV-hFc转染293F细胞7天后，将培养液通过高速离心、微孔滤膜抽真空过滤以及Mabselect SuRe柱进行纯化PD-1ECD-TEV-mFc和PD-1ECD-TEV-hFc融合蛋白，并取纯化后样品加入还原型蛋白电泳上样缓冲液，进行SDS-PAGE电泳检测，检测结果如图1，图2所示。
6.抗原PDL-1-hFc的合成
抗原PDL-1-hFc(PDL-1：Programmed death-ligand 1，NCBI Gene ID：29126)的合成方法同PD-1-hFc的合成方法，将所获得的融合蛋白进行纯化并进行SDS-PAGE电泳检测。
本制备例中涉及的序列信息如下：
PD-1：Programmed cell death protein 1，NCBI GenBank：NM_005018；
mFc：Ig gamma-2A chain C region：ACCESSION：P01863，99-330；
hFc：Ig gamma-1 chain C region，ACCESSION：P01857，106-330；
PDL-1：Programmed death-ligand 1，NCBI Gene ID：29126。
PD-1-mFc融合蛋白序列：(389aa)
Figure PCTCN2016103667-appb-000001
Figure PCTCN2016103667-appb-000002
其中，带波浪下划线的为PD-1ECD部分，小写字母部分为TEV酶切位点，带实线下划线的为mFc部分。
PD-1-mFc融合蛋白相对应的基因编码序列：(1167bp)
Figure PCTCN2016103667-appb-000003
其中，带波浪下划线的为PD-1ECD部分，小写字母部分为TEV酶切位点，带实线下划线的为mFc部分。
PD-1-hFc融合蛋白序列：(382aa)
Figure PCTCN2016103667-appb-000004
Figure PCTCN2016103667-appb-000005
其中，带波浪下划线的为PD-1ECD部分，小写字母部分为TEV酶切位点，带实线下划线的为hFc部分。
PD-1-hFc融合蛋白相对应的基因编码序列：(1149bp)
Figure PCTCN2016103667-appb-000006
其中，带波浪下划线的为PD-1ECD部分，小写字母部分为TEV酶切位点，带实线下划线的为hFc部分。
PDL-1-hFc融合蛋白序列：(456aa)
Figure PCTCN2016103667-appb-000007
Figure PCTCN2016103667-appb-000008
其中，带波浪下划线的为PDL-1ECD部分，小写字母部分为TEV酶切位点，带实线下划线的为hFc部分。
PDL-1-hFc融合蛋白相对应的基因编码序列：(1371bp)
Figure PCTCN2016103667-appb-000010
其中，带波浪下划线的为PDL-1ECD部分，小写字母部分为TEV酶切位点，带实线下划线的为hFc部分。
实施例1：杂交瘤细胞株LT004的获得以及单克隆抗体6F5的制备
利用哺乳动物细胞表达***表达出重组的PD-1ECD-TEV-mFc作为抗原免疫小鼠，经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。通过大量的样本筛选，得到了一种杂交瘤细胞株LT004，该细胞株能够分泌产生与PD-1特异性结合的单克隆抗体6F5。具体方法如下：
1.杂交瘤细胞株LT004的建立
用PD-1ECD-TEV-mFc融合蛋白(即PD-1-mFc，见制备例1)作为抗原，取免疫BALB/C小鼠(购自广东医学实验动物中心)的脾细胞与小鼠骨髓瘤细胞融合成杂交瘤细胞，参照目前已确立的方法(e.g.，Stewart，S.J.，“Monoclonal Antibody Production”，in Basic Methods in antibody Production and Characterization，Eds.G.C.Howard and D.R.Bethell，Boca Raton：CRC Press，2000)。
用PD-1-hFc作为抗原包被酶标板，进行间接ELISA法筛选，得到分泌与PD-1特异性结合的新的抗体的杂交瘤细胞。对间接ELISA筛选得到的杂交瘤细胞，通过竞争ELISA筛选出能够分泌与配体PDL-1(见制备例1)竞争结合PD-1的单克隆抗体的杂交瘤细胞株，经过有限稀释法得到稳定的杂交瘤细胞株，并经过有限稀释法得到PD-1-6F5稳定细胞株。本发明中将该杂交瘤细胞株命名为LT004，其分泌的单克隆抗体命名为6F5。
杂交瘤细胞株LT004的保藏编号：CCTCC C2015132。
2.抗体6F5的制备：
用含10％的低IgG胎牛血清的IMDM培养基对本发明LT004细胞株进行培养，7天后收集细胞培养上清进行纯化制备抗体6F5。
3.抗体6F5的SDS-PAGE电泳检测：
将纯化后的样品分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液，煮沸后进行检测。
实施例2：单克隆抗体6F5的轻链和重链序列的获得
按照培养细胞细菌总RNA提取试剂盒(Tiangen，货号DP430)的方法，从实施例1制得的杂交瘤细胞株LT004中提取mRNA。
按照TransScript First-Strand cDNA Synthesis SuperMix(Transgen，AT301)试剂盒说明书合成cDNA，并进行PCR扩增。PCR扩增产物直接进行TA克隆，具体操作参照pEASY-T1Cloning Kit(Transgen CT101)试剂盒说明书进行。
将TA克隆的产物直接进行测序，测序结果如下：
重链可变区的DNA测序结果：(426bp)
Figure PCTCN2016103667-appb-000011
其编码的蛋白序列：(142aa)
Figure PCTCN2016103667-appb-000012
轻链可变区的DNA测序结果：(336bp)
Figure PCTCN2016103667-appb-000013
其编码的蛋白序列：(112aa)
Figure PCTCN2016103667-appb-000014
实施例3：人源化抗体6F5H1L1、6F5H2L2的轻链和重链序列的设计
根据PD-1蛋白的三维晶体结构(Shinohara T，et al.，Structure and chromosomal localization of the human PD-1gene(PDCD1).Genomics 1995，23(3)：704-6)以及实施例2获得的抗体6F5的序列，通过计算机模拟抗体模型，根据模型设计突变，得到抗体6F5H1L1、6F5H2L2的可变区序列(重链恒定区为Ig gamma-1 chain C region，ACCESSION：P01857，轻链恒定区为Ig kappa chain C region，ACCESSION：P01834)，可变区序列如下：
6F5H1L1重链可变区的DNA序列：(354bp)
Figure PCTCN2016103667-appb-000015
其编码的蛋白序列：(118aa)
Figure PCTCN2016103667-appb-000016
6F5H1L1轻链可变区的DNA序列：(333bp)
Figure PCTCN2016103667-appb-000017
其编码的蛋白序列：(111aa)
Figure PCTCN2016103667-appb-000018
6F5H2L2重链可变区的DNA序列：(354bp)
Figure PCTCN2016103667-appb-000019
其编码的蛋白序列：(118aa)
Figure PCTCN2016103667-appb-000020
6F5H2L2轻链可变区的DNA序列：(333bp)
Figure PCTCN2016103667-appb-000021
其编码的蛋白序列：(111aa)
Figure PCTCN2016103667-appb-000022
实施例4：6F5(Re)以及人源化抗体6F5H1L1、6F5H2L2的制备和SDS-PAGE电泳检测
1. 6F5重组抗体6F5(Re)的制备
将6F5的重链cDNA序列(重链可变区序列如SEQ ID NO：1所示)(恒定区序列为immunoglobulin gamma 2b heavy chain precursor[Mus musculus]140-475，ACCESSION：ACX70084.1)和轻链的cDNA序列(轻链可变区序列如SEQ ID NO：3所示)(恒定区为antibody kappa light chain，partial[Mus musculus]，106-213GenBank：BAB33404.1)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-6F5H和 pUC57simple-6F5L质粒。
分别将质粒pUC57simple-6F5H和pUC57simple-6F5L进行酶切(HindIII&EcoRI)，电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、微孔滤膜抽真空过滤后，上样至HiTrap MabSelectSuRe柱，用Elution Buffer一步洗脱蛋白，回收目标样品并用HiTrap Desalting换液至PBS。
2. 6F5人源化抗体6F5H1L1、6F5H2L2的制备和SDS-PAGE电泳检测
参照上面6F5(Re)的制备步骤进行。
将6F5H1L1、6F5H2L2的重链cDNA(重链可变区序列分别为SEQ ID NO：5、9)和轻链的cDNA(轻链可变区序列分别为SEQ ID NO：7、11)分别克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-6F5H1L1、pUC57simple-6F5H2L2质粒，并亚克隆到pcDNA3.1载体中，方法同前述6F5(Re)。
将重组质粒转染293F细胞，将培养液纯化后进行检测(同上所述6F5(Re)的方法)，结果如图3-4所示，还原型蛋白样品目标蛋白大约在24.5kD和49KD处，非还原型蛋白样品目标蛋白大约在147kD处。
实施例5：抗体的动力学参数测定
使用Fortebio分子相互作用仪测定抗体6F5H2L2和阳性对照抗体5C4与抗原PD-1结合的动力学参数。
1.用TEV蛋白酶酶切PD-1-mFc蛋白(见制备例1)，并过柱纯化获得PD-1抗原。
2.抗原PD-1(抗原浓度为1μg/ml)经生物素标记后固定于SA传感器表面，于PBST中平衡后，与抗体6F5H2L2或5C4结合，抗体用PBST从200nM往下三倍稀释，于PBST中解离。
抗体6F5H2L2和5C4的动力学参数见表1，动力学特征参数检测结果分别如图5、6所示。
表1：抗体6F5H2L2动力学参数
抗体名称 KD(M) kon(1/Ms) kon误差 kdis(1/s) kdis误差
6F5H2L2 2.34E-10 4.39E+05 7.05E+03 1.03E-04 7.41E-06
5C4 1.11E-10 9.27E+05 1.77E+04 1.03E-04 6.37E-06
KD为亲和力常数；kon为抗原抗体结合速率；kdis为抗原抗体解离速率；KD＝kdis/kon。
结果表明，6F5H2L2与抗原PD-1有良好的亲和力。
实施例6：间接ELISA方法检测抗体与抗原PD-1的结合活性
采用间接ELISA方法分别测定杂交瘤抗体6F5，重组抗体6F5(Re)和人源化抗体6F5H1L1、6F5H2L2及阳性对照抗体5C4的结合活性。将PD1-mFc或PD1-hFc用CBS稀释成0.5μg/ml，加至酶标板96孔中，每孔50μL，4℃下孵育过夜。洗板一次后用1％BSA+PBS封闭，每孔300μL，37℃下孵育2小时。洗板三次后，抗体分别用PBS稀释成1μg/ml起始作1∶3梯度稀释，以PBS做空白对照。每孔50μl，室温下孵育10分钟。洗板三次后，分别对应加入HRP标记的羊抗人或羊抗鼠二抗，每孔50μL，37℃下孵育30分钟洗板四次后，加TMB显色液，每孔50μL，室温下避光显色5分钟。直接加终止液终止反应，每孔50μL。终止反应后立即把酶标板放入酶标仪中，在450nm处检测OD值，保存原始数据。将原始数据输入到软件SoftMax Pro 6.2.1中进行数据处理。
检测杂交瘤抗体6F5，重组抗体6F5(Re)和人源化抗体6F5H1L1、6F5H2L2及阳性对照抗体5C4与抗原PD-1结合结果如图7、图8所示。
由图可见，抗体6F5、6F5(Re)、6F5H1L1、6F5H2L2、阳性对照抗体5C4均能有效地结合PD-1蛋白，并且其结合效率呈剂量依赖关系，各剂量的吸光度强度见表2，表3。
表2：抗体6F5、6F5(Re)与PD-1的结合(间接ELISA)
Figure PCTCN2016103667-appb-000023
Figure PCTCN2016103667-appb-000024
表3：抗体6F5H1L1、6F5H2L2以及阳性对照抗体5C4与PD-1的结合(间接ELISA)
Figure PCTCN2016103667-appb-000025
在对6F5、6F5(Re)以及阳性对照抗体5C4与人PD-1结合活性的平行对比实验中，曲线模拟结合效率EC50分别为0.091nM，0.028nM，0.199nM。不拘于理论的限制，重组抗体6F5(Re)跟杂交瘤抗体6F5用的表达***不一样，6F5(Re)由人293F细胞表达产生，杂交瘤抗体6F5用小鼠杂交瘤细胞产生，抗体在产生过程中表达糖基化程度有差异，亲和力也会有差别。
在对6F5H1L1、6F5H2L2以及阳性对照抗体5C4与人PD-1结合活性的平行对比实验 中，曲线模拟结合效率EC50分别为0.118nM，0.100nM和0.233nM。可见，6F5H1L1、6F5H2L2的EC50约为阳性对照抗体5C4的1/2，结合效率显著优于阳性对照抗体5C4。
实施例7：竞争ELISA方法检测抗体与PDL-1竞争结合抗原PD-1的活性
采用竞争ELISA方法分别测定杂交瘤细胞产生的单克隆抗体6F5及人源化抗体6F5H1L1、6F5H2L2与PDL-1竞争结合抗原PD-1的能力。将PD-1-mFc或PD-1-hFc用CBS稀释成0.5μg/ml，加至酶标板96孔中，每孔50μL，4℃下孵育过夜。洗板一次后用1％BSA+PBS封闭，每孔300μL，37℃下孵育2小时。洗板三次后，抗体分别用PBS稀释成浓度为1.5μg/ml起始作1∶3梯度稀释，以PBS做空白对照。每孔50μl，室温下孵育10分钟。将PDL-1-hFc或PDL-1-mFc用PBS稀释成2μg/ml，每孔50μl，与抗体充分混匀，37℃下孵育45分钟。洗板三次后，分别对应加入HRP标记的羊抗人二抗，每孔50μL，37℃下孵育30分钟。洗板四次后，加TMB显色液，每孔50μL，室温下避光显色5分钟。直接加终止液终止反应，每孔50μL。终止反应后立即把酶标板放入酶标仪中，在450nm处检测OD值，保存原始数据。将原始数据输入到软件SoftMax Pro 6.2.1中进行数据处理。
检测抗体6F5及人源化抗体6F5H1L1、6F5H2L2与PDL-1竞争结合抗原PD-1的结果如图9，图10所示。由图可见，6F5及人源化抗体6F5H1L1、6F5H2L2均能有效地与PDL-1竞争结合PD-1蛋白，并且其结合效率呈剂量依赖关系。通过对结合的抗体6F5及人源化抗体6F5H1L1、6F5H2L2和阳性对照抗体5C4进行吸光度检测分析，曲线模拟结合效率EC50分别为：0.198nM、0.979nM、1.175nM和1.461nM。
各剂量的吸光度强度见表4-5。
表4：抗体6F5与PDL-1竞争结合PD-1ELISA
Figure PCTCN2016103667-appb-000026
Figure PCTCN2016103667-appb-000027
表5：抗体6F5H1L1、6F5H2L2与PDL-1竞争结合PD-1ELISA
Figure PCTCN2016103667-appb-000028
实施例8：流式细胞仪方法检测抗体与细胞表面抗原PD-1的结合活性
首先构建表达PD-1抗原的宿主细胞293T，然后用本发明中制备的人源化抗体6F5H2L2(见实施例4)对改宿主细胞进行标记。然后采用流式细胞术分析验证抗体6F5H2L2对细胞表面天然构象的抗原特异性的结合能力。
具体步骤如下：
1.达PD-1抗原的宿主细胞293T的构建
表按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将包含PD-1的载体pLenti6.3-PD-1(载体pLenti6.3购自Invitrogen公司)转染293T细胞，经筛选获得稳定表达PD-1的克隆群体293T-PD-1。
2.抗体标记和流式细胞仪检测
采用常规胰酶消化方法上述步骤获得的表达PD-1抗原的宿主细胞293T-PD-1，并使每个收集管细胞数为2×105，用PBS(1％BSA)配制浓度分别为50nM，20nM，10nM，3nM，1nM，0.1nM，0.01nM的PD-1抗体6F5H2L2和阳性对照抗体5C4稀释液，冰上与表达PD-1的293T细胞孵育1小时，每管加入100μL FITC羊抗人IgG(1∶500)冰上孵育30min后，用PBS洗3次后加入300μL PBS重悬细胞，在流式细胞仪上用FITC通道检测荧光信号。
人源化抗体6F5H2L2与293T-PD-1细胞的结合结果如图11、12所示。由图可见，6F5H2L2抗体能有效地结合宿主细胞293T-PD-1表面的靶标PD-1蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度见表6。
表6：流式细胞仪检测6F5H2L2结合PD-1宿主细胞293T-PD-1表面抗原的荧光强度分析
Figure PCTCN2016103667-appb-000029
通过对结合的6F5H2L2和阳性对照抗体5C4进行荧光定量分析，曲线模拟6F5H2L2和阳性对照抗体5C4的结合效率EC50分别是5.022和4.171nM。
实施例9：流式细胞仪方法检测抗体阻断细胞表面抗原PD-1与PDL-1的结合活性
采用常规胰酶消化方法实施例8中获得的表达PD-1抗原的宿主细胞293T-PD-1，并 使每个收集管细胞数为2×105，用PBS(1％BSA)配制浓度分别为100nM，50nM，20nM，10nM，3nM，1nM的PD-1抗体6F5H2L2和阳性对照抗体5C4稀释液，冰上与表达PD-1的293T细胞孵育1小时，每管细胞稀释液中加入适量的PDL-1-mFc蛋白，使其终浓度均为20nM，冰上孵育30min。用PBS洗涤一次后，每管加入100μL FITC羊抗人IgG(1∶500)冰上孵育1小时用PBS洗3次后加入300μL PBS重悬细胞，在流式细胞仪上用FITC通道检测荧光信号。
人源化抗体6F5H2L2和阳性对照抗体5C4阻断人PDL-1与PD-1结合的结果如图13所示。由图可见，6F5H2L2和5C4能有效地抑制人PDL-1与PD-1的结合，并且其抑制效率呈剂量依赖关系，各剂量的荧光强度见表7。
表7：流式细胞仪检测6F5H2L2阻断人的PDL-1与PD-1结合活性的荧光强度分析
Figure PCTCN2016103667-appb-000030
通过对6F5H2L2和阳性对照抗体5C4抗体进行荧光定量分析，曲线模拟6F5H2L2和5C4的抑制效率EC50分别是8.088nM和2.208nM。
实施例10：混合淋巴细胞反应：细胞因子IFN-γ，IL-2的分泌
采用Ficoll-Paque Plus(GE Healthcare LOT No.：171440-02)分离PBMC，将分离出来的PBMC加入IL-4(Peprotech K2513，1000U/ml)和GM-CSF(Peprotech H1513，1000U/ml)诱导6天后，加入TNF-α(Peprotech G1513，200U/ml)诱导3天获得DC细胞。
PBMC中分离得到T细胞，将获得的DC细胞与T细胞按1∶10的比例混合培养，同时加入不同比例的抗体6F5H2L2(hIgG做为阴性对照，Isotype control)培养5-6天后，采用ELISA试剂盒检测IFN-γ(购自达科为公司)和IL-2(购自达科为公司)的分泌量。
DC细胞和T细胞混合培养后IFN-γ和IL-2的分泌检测结果分别如图14、图15所示：6F5H2L2抗体能有效地诱导混合淋巴细胞分泌IFN-γ和IL-2，并且其分泌量与抗体 6F5H2L2呈剂量依赖关系。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (22)

  1. 单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区包含：氨基酸序列为SEQ ID NO：13-15的CDR；
    和/或
    所述单克隆抗体的轻链可变区包含：氨基酸序列为SEQ ID NO：16-18的CDR。
  2. 根据权利要求1所述的单克隆抗体或其抗原结合片段，其中，
    所述单克隆抗体的重链可变区的氨基酸序列选自SEQ ID NO：2、SEQ ID NO：6和SEQ ID NO：10；
    和/或
    所述单克隆抗体的轻链可变区的氨基酸序列选自SEQID NO：4、SEQ ID NO：8和SEQ ID NO：12。
  3. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述单克隆抗体或其抗原结合片段选自Fab、Fab’、F(ab’)2、Fd、Fv、dAb、互补决定区片段、单链抗体(例如，scFv)、人源化抗体、嵌合抗体或双抗体。
  4. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，所述的单克隆抗体以小于大约100nM，例如小于大约10nM、1nM、0.9nM、0.8nM、0.7nM、0.6nM、0.5nM、0.4nM、0.3nM、0.2nM、0.1nM或更小的EC50结合PD-1蛋白；优选地，所述EC50通过间接ELISA方法测得。
  5. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中，
    所述的单克隆抗体包括非-CDR区，且所述非-CDR区来自不是鼠类的物种，例如来自人抗体。
  6. 根据权利要求1至2中任一权利要求所述的单克隆抗体或其抗原结合片段，其中 所述单克隆抗体是有杂交瘤细胞株LT004产生的单克隆抗体，所述杂交瘤细胞株LT004保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015132。
  7. 分离的核酸分子，其包含能够编码抗体重链可变区的核酸序列，其中，
    所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO：13-15的CDR；
    优选地，所述抗体的重链具有SEQ ID NO：2、SEQ ID NO：6或SEQ ID NO：10所示的氨基酸序列；
    更优选地，所述核酸分子具有SEQ ID NO：1、SEQ ID NO：5或SEQ ID NO：9所示的核苷酸序列。
  8. 分离的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，其中，
    所述抗体轻链可变区包含氨基酸序列为SEQ ID NO：16-18的CDR；
    优选地，所述抗体轻链可变区具有SEQ ID NO：4、SEQ ID NO：8或SEQ ID NO：12所示的氨基酸序列；
    更优选地，所述核酸分子具有SEQ ID NO：3、SEQ ID NO：7或SEQ ID NO：11所示的核苷酸序列。
  9. 一种载体，其包含权利要求7和/或8所述的分离的核酸分子。
  10. 一种宿主细胞，其包含权利要求7和/或8所述的分离的核酸分子，或者权利要求9所述的载体。
  11. 制备权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段的方法，其包括在合适的条件下培养权利要求10的宿主细胞，以及从细胞培养物中回收所述单克隆抗体或其抗原结合片段的步骤。
  12. 杂交瘤细胞株LT004，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2015132。
  13. 偶联物，其包括单克隆抗体或其抗原结合片段以及偶联部分，其中，所述单克隆抗体为权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  14. 试剂盒，其包括权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，或者包括权利要求13所述的偶联物；
    优选地，所述试剂盒还包括第二抗体，其特异性识别所述单克隆抗体或其抗原结合片段；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  15. 权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物在制备试剂盒中的用途，所述试剂盒用于检测PD-1在样品中的存在或其水平。
  16. 一种药物组合物，其包含权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物；可选地，其还包括药学上可接受的载体和/或赋形剂。
  17. 权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物在制备治疗和/或预防和/或诊断肿瘤或者贫血病的药物中的用途；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、***癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  18. 权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物在制备如下药物中的用途：
    阻断PD-1与PD-1的配体结合的药物，
    调节(例如下调)PD-1活性或水平的药物，
    解除PD-1对机体免疫抑制的药物，或者
    提高T淋巴细胞中IFN-γ和/或IL-2表达的药物；
    优选地，所述PD-1的配体为PDL-1或PDL-2，更优选为PDL-1。
  19. 一种在体内或体外方法，包括施加细胞以有效量的权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物的步骤，所述方法选自如下：
    阻断PD-1与PD-1的配体结合的方法，
    调节(例如下调)PD-1活性或水平的方法，
    解除PD-1对机体免疫抑制的方法，或者
    提高T淋巴细胞中IFN-γ和/或IL-2表达的方法；
    优选体地，所述PD-1的配体为PDL-1或PDL-2，更优选为PDL-1。
  20. 一种治疗和/或预防和/或诊断肿瘤或者贫血病的方法，包括给予受试者有效量的权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段或者权利要求13所述的偶联物的步骤；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、***癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  21. 根据权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，其用于治疗和/或预防和/或诊断肿瘤或者贫血病；优选地，所述肿瘤选自黑色素瘤、肾肿瘤、***癌、膀胱癌、结肠直肠癌、胃肠道癌、肝癌、非小细胞性肺癌、卵巢癌和白血病。
  22. 根据权利要求1至6中任一权利要求所述的单克隆抗体或其抗原结合片段，其用于：
    阻断PD-1与PD-1的配体结合，
    调节(例如下调)PD-1活性或水平，
    解除PD-1对机体免疫抑制，或者
    提高T淋巴细胞中IFN-γ和/或IL-2表达；
    优选地，所述PD-1的配体为PDL-1或PDL-2，优选为PDL-1。
PCT/CN2016/103667 2015-10-30 2016-10-28 一种抗pd-1单克隆抗体、其药物组合物及其用途 WO2017071625A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510733389.5 2015-10-30
CN201510733389.5A CN106632674B (zh) 2015-10-30 2015-10-30 一种抗pd-1单克隆抗体、其药物组合物及其用途

Publications (1)

Publication Number Publication Date
WO2017071625A1 true WO2017071625A1 (zh) 2017-05-04

Family

ID=58631322

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/103667 WO2017071625A1 (zh) 2015-10-30 2016-10-28 一种抗pd-1单克隆抗体、其药物组合物及其用途

Country Status (2)

Country Link
CN (1) CN106632674B (zh)
WO (1) WO2017071625A1 (zh)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017220990A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 antibodies
WO2018217227A1 (en) * 2017-05-24 2018-11-29 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1
WO2019206281A1 (zh) * 2018-04-28 2019-10-31 齐鲁制药有限公司 一种抗人pd-1的单克隆抗体制剂、联合用药物及其用途
CN110423277A (zh) * 2019-03-21 2019-11-08 南京东极医药科技有限公司 Pd-1的纳米抗体及其临床应用
WO2019217457A1 (en) 2018-05-07 2019-11-14 Genmab A/S Methods of treating cancer with a combination of an anti-pd-1 antibody and an anti-tissue factor antibody-drug conjugate
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
US10669338B2 (en) 2016-06-17 2020-06-02 Immunomedics, Inc. Anti-PD-1 checkpoint inhibitor antibodies that block binding of PD-L1 to PD-1
WO2020239558A1 (en) 2019-05-24 2020-12-03 Pfizer Inc. Combination therapies using cdk inhibitors
US10894830B2 (en) 2015-11-03 2021-01-19 Janssen Biotech, Inc. Antibodies specifically binding PD-1, TIM-3 or PD-1 and TIM-3 and their uses
CN112300283A (zh) * 2019-07-29 2021-02-02 深圳华大生命科学研究院 一种抗人胰腺癌单克隆抗体及其制备方法和应用
WO2022108931A2 (en) 2020-11-17 2022-05-27 Seagen Inc. Methods of treating cancer with a combination of tucatinib and an anti-pd-1/anti-pd-l1 antibody
WO2022118197A1 (en) 2020-12-02 2022-06-09 Pfizer Inc. Time to resolution of axitinib-related adverse events
WO2022254227A1 (en) 2021-06-04 2022-12-08 Kymab Limited Treatment of pd-l1 negative or low expressing cancer with anti-icos antibodies
WO2023285552A1 (en) 2021-07-13 2023-01-19 BioNTech SE Multispecific binding agents against cd40 and cd137 in combination therapy for cancer
CN115785269A (zh) * 2022-11-01 2023-03-14 四川大学 抗pd-l1的抗体及其应用
WO2023057534A1 (en) 2021-10-06 2023-04-13 Genmab A/S Multispecific binding agents against pd-l1 and cd137 in combination
WO2023057882A1 (en) 2021-10-05 2023-04-13 Pfizer Inc. Combinations of azalactam compounds with a pd-1 axis binding antagonist for the treatment of cancer
WO2023079428A1 (en) 2021-11-03 2023-05-11 Pfizer Inc. Combination therapies using tlr7/8 agonist
WO2023083439A1 (en) 2021-11-09 2023-05-19 BioNTech SE Tlr7 agonist and combinations for cancer treatment
WO2023218046A1 (en) 2022-05-12 2023-11-16 Genmab A/S Binding agents capable of binding to cd27 in combination therapy

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109696461B (zh) * 2017-10-24 2023-05-26 上海药明生物技术有限公司 一种治疗性抗体的放行检测方法
CA3106055A1 (en) * 2018-07-18 2020-01-23 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Therapeutic combination of quinoline derivative and antibody
CN109053895B (zh) 2018-08-30 2020-06-09 中山康方生物医药有限公司 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途
CN110878122B (zh) * 2018-09-06 2023-07-28 上海张江生物技术有限公司 重组抗pd-l1单克隆抗体
CN111423510B (zh) * 2019-01-10 2024-02-06 迈威(上海)生物科技股份有限公司 重组抗人pd-1抗体及其应用
CN110095612B (zh) * 2019-04-12 2022-05-10 河北仁博科技有限公司 一种基于spr快速筛选单克隆抗体的方法
CN110903391B (zh) * 2019-10-25 2021-05-28 东大生物技术(苏州)有限公司 一组pd-l1单克隆抗体及其医药用途
EP4079763A4 (en) * 2019-12-20 2023-10-11 Guangdong Feipeng Pharmaceutical Co., Ltd MONOCLONAL ANTIBODY AGAINST HUMAN PROGRAMMED DEATH-1 (PD-1)
CN113372445B (zh) * 2020-03-10 2023-09-12 鲁南制药集团股份有限公司 一种抗pd-1单克隆抗体
CN112724208A (zh) * 2020-12-25 2021-04-30 中山大学 一种SADS-CoV重组S蛋白胞外段及其制备方法与应用
CN115521379B (zh) * 2021-07-16 2023-12-22 南京吉盛澳玛生物医药有限公司 Pd-1抗体及其用途
CA3233205A1 (en) 2021-09-29 2023-04-06 Yu Xia Anti-lag3 antibody, pharmaceutical composition and use
CN114958774B (zh) * 2022-05-08 2023-10-27 中国医学科学院医学生物学研究所 抗狂犬病病毒单克隆抗体及分泌该抗体的杂交瘤细胞株与应用
CN115873117B (zh) * 2022-11-11 2023-07-18 广州国家实验室 一种pd-l1单克隆抗体或其抗原结合片段及检测试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056875A1 (en) * 2002-12-23 2004-07-08 Wyeth Antibodies against pd-1 and uses therefor
WO2006121168A1 (en) * 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2011110621A1 (en) * 2010-03-11 2011-09-15 Ucb Pharma, S.A. Biological products: humanised agonistic anti-pd-1 antibodies

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112457403B (zh) * 2013-09-13 2022-11-29 广州百济神州生物制药有限公司 抗pd1抗体及其作为治疗剂与诊断剂的用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004056875A1 (en) * 2002-12-23 2004-07-08 Wyeth Antibodies against pd-1 and uses therefor
WO2006121168A1 (en) * 2005-05-09 2006-11-16 Ono Pharmaceutical Co., Ltd. Human monoclonal antibodies to programmed death 1(pd-1) and methods for treating cancer using anti-pd-1 antibodies alone or in combination with other immunotherapeutics
WO2011110621A1 (en) * 2010-03-11 2011-09-15 Ucb Pharma, S.A. Biological products: humanised agonistic anti-pd-1 antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CAO JB ET AL.: "Application of PD-1 antibody in therapy of tumors", CHINESE JOURNAL OF BILOGICALS, vol. 27, no. 6, 30 June 2014 (2014-06-30), pages 857 - 859 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10513558B2 (en) 2015-07-13 2019-12-24 Cytomx Therapeutics, Inc. Anti-PD1 antibodies, activatable anti-PD1 antibodies, and methods of use thereof
US10894830B2 (en) 2015-11-03 2021-01-19 Janssen Biotech, Inc. Antibodies specifically binding PD-1, TIM-3 or PD-1 and TIM-3 and their uses
US10669338B2 (en) 2016-06-17 2020-06-02 Immunomedics, Inc. Anti-PD-1 checkpoint inhibitor antibodies that block binding of PD-L1 to PD-1
WO2017220989A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 and il-2 cytokines
WO2017220988A1 (en) 2016-06-20 2017-12-28 Kymab Limited Multispecific antibodies for immuno-oncology
WO2017220990A1 (en) 2016-06-20 2017-12-28 Kymab Limited Anti-pd-l1 antibodies
WO2018217227A1 (en) * 2017-05-24 2018-11-29 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1
WO2019206281A1 (zh) * 2018-04-28 2019-10-31 齐鲁制药有限公司 一种抗人pd-1的单克隆抗体制剂、联合用药物及其用途
CN111971062A (zh) * 2018-04-28 2020-11-20 齐鲁制药有限公司 一种抗人pd-1的单克隆抗体制剂、联合用药物及其用途
CN111971062B (zh) * 2018-04-28 2022-07-26 上海齐鲁制药研究中心有限公司 一种抗人pd-1的单克隆抗体制剂、联合用药物及其用途
WO2019217457A1 (en) 2018-05-07 2019-11-14 Genmab A/S Methods of treating cancer with a combination of an anti-pd-1 antibody and an anti-tissue factor antibody-drug conjugate
CN110423277A (zh) * 2019-03-21 2019-11-08 南京东极医药科技有限公司 Pd-1的纳米抗体及其临床应用
CN110423277B (zh) * 2019-03-21 2022-09-06 南京东极医药科技有限公司 Pd-1的纳米抗体及其临床应用
WO2020239558A1 (en) 2019-05-24 2020-12-03 Pfizer Inc. Combination therapies using cdk inhibitors
CN112300283A (zh) * 2019-07-29 2021-02-02 深圳华大生命科学研究院 一种抗人胰腺癌单克隆抗体及其制备方法和应用
CN112300283B (zh) * 2019-07-29 2024-01-02 深圳华大生命科学研究院 一种抗人胰腺癌单克隆抗体及其制备方法和应用
WO2022108931A2 (en) 2020-11-17 2022-05-27 Seagen Inc. Methods of treating cancer with a combination of tucatinib and an anti-pd-1/anti-pd-l1 antibody
WO2022118197A1 (en) 2020-12-02 2022-06-09 Pfizer Inc. Time to resolution of axitinib-related adverse events
WO2022254227A1 (en) 2021-06-04 2022-12-08 Kymab Limited Treatment of pd-l1 negative or low expressing cancer with anti-icos antibodies
WO2023285552A1 (en) 2021-07-13 2023-01-19 BioNTech SE Multispecific binding agents against cd40 and cd137 in combination therapy for cancer
WO2023057882A1 (en) 2021-10-05 2023-04-13 Pfizer Inc. Combinations of azalactam compounds with a pd-1 axis binding antagonist for the treatment of cancer
WO2023057534A1 (en) 2021-10-06 2023-04-13 Genmab A/S Multispecific binding agents against pd-l1 and cd137 in combination
WO2023079428A1 (en) 2021-11-03 2023-05-11 Pfizer Inc. Combination therapies using tlr7/8 agonist
WO2023083439A1 (en) 2021-11-09 2023-05-19 BioNTech SE Tlr7 agonist and combinations for cancer treatment
WO2023083868A1 (en) 2021-11-09 2023-05-19 BioNTech SE Tlr7 agonist and combinations for cancer treatment
WO2023218046A1 (en) 2022-05-12 2023-11-16 Genmab A/S Binding agents capable of binding to cd27 in combination therapy
CN115785269A (zh) * 2022-11-01 2023-03-14 四川大学 抗pd-l1的抗体及其应用
CN115785269B (zh) * 2022-11-01 2023-09-22 四川大学 抗pd-l1的抗体及其应用

Also Published As

Publication number Publication date
CN106632674B (zh) 2018-11-16
CN106632674A (zh) 2017-05-10

Similar Documents

Publication Publication Date Title
WO2017071625A1 (zh) 一种抗pd-1单克隆抗体、其药物组合物及其用途
JP6783886B2 (ja) 抗ｃｔｌａ４モノクローナル抗体またはその抗原結合断片、医薬組成物および使用
WO2018036472A1 (zh) 一种抗pd1单克隆抗体、其药物组合物及其用途
JP7425604B2 (ja) 抗ｃｔｌａ４－抗ｐｄ－１二機能性抗体、その医薬組成物および使用
CN107151269B (zh) 一种pdl-1抗体、其药物组合物及其用途
WO2020043184A1 (zh) 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途
KR20210018336A (ko) 항인터류킨-17a 항체, 그의 약학 조성물 및 용도
WO2021104302A1 (zh) 抗pd-1-抗vegfa的双特异性抗体、其药物组合物及其用途
CN110407941B (zh) Cd39的高亲和力抗体及其用途
WO2018219327A1 (zh) 抗cd40抗体、其抗原结合片段及其医药用途
WO2019242619A1 (zh) 全人源的抗lag-3抗体及其应用
WO2021213475A1 (zh) 抗cd73-抗pd-1双特异性抗体及其用途
WO2016127912A1 (zh) Pcsk9抗体、其药物组合物及其用途
KR20210142638A (ko) Cd3 항원 결합 단편 및 이의 응용
BR112020020637A2 (pt) anticorpo anti-pd-l1, fragmento de ligação ao antígeno ou variante do mesmo, anticorpo biespecífico, composição farmacêutica e uso dos mesmos para aliviar ou tratar tumores
WO2023151693A1 (zh) 包含抗tigit抗体和抗pd-1-抗vegfa双特异性抗体的药物组合物及用途
WO2022174813A1 (zh) 抗GPRC5DxBCMAxCD3三特异性抗体及其用途
WO2022089392A1 (zh) 抗tigit抗体、其药物组合物及用途
CN115521379B (zh) Pd-1抗体及其用途
WO2023241656A1 (zh) 包含抗cldn18.2抗体的双特异性抗体、药物组合物及用途
CN115947855B (zh) 抗cd24抗体的制备及其用途
WO2023001303A1 (zh) 药物组合物及用途
WO2024051223A1 (zh) 药物组合及用途
CN113164601B (zh) 一种分离的抗原结合蛋白及其用途
WO2024102962A1 (en) Cytotoxic bispecific antibodies binding to dr5 and muc16 and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16859060

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16859060

Country of ref document: EP

Kind code of ref document: A1