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WO2017071625A1 - Anticorps monoclonal anti-pd-1, composition pharmaceutique et utilisation de celui-ci - Google Patents

Anticorps monoclonal anti-pd-1, composition pharmaceutique et utilisation de celui-ci Download PDF

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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
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antibody
monoclonal antibody
antigen
seq
binding fragment
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Chinese (zh)
Inventor
李百勇
夏瑜
王忠民
张鹏
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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    • 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.

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Abstract

L'invention concerne le domaine des traitements des tumeurs et de l'immunologie moléculaire, en particulier, un anticorps monoclonal anti-PD-1, et un fragment de liaison d'antigène, une composition pharmaceutique et une utilisation associée. L'anticorps monoclonal comprend une région variable de chaîne lourde comprenant une séquence d'acides aminés telle que CDR1-3 comme indiqué par SEQ ID NO. 13 à 15, et une région variable de chaîne légère comprenant une séquence d'acides aminés telle que CDR1-3 comme indiqué par SEQ ID NO. 16 à 18. L'anticorps monoclonal peut se lier spécifiquement à PD-1, supprimer l'immunosuppression de PD-1 par rapport à un organisme et activer un lymphocyte T.
PCT/CN2016/103667 2015-10-30 2016-10-28 Anticorps monoclonal anti-pd-1, composition pharmaceutique et utilisation de celui-ci Ceased WO2017071625A1 (fr)

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