WO2017148424A1 - 一种pdl-1抗体、其药物组合物及其用途 - Google Patents

一种pdl-1抗体、其药物组合物及其用途 Download PDF

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WO2017148424A1
WO2017148424A1 PCT/CN2017/075484 CN2017075484W WO2017148424A1 WO 2017148424 A1 WO2017148424 A1 WO 2017148424A1 CN 2017075484 W CN2017075484 W CN 2017075484W WO 2017148424 A1 WO2017148424 A1 WO 2017148424A1
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antibody
seq
antigen
monoclonal antibody
amino acid
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PCT/CN2017/075484
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English (en)
French (fr)
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李百勇
薛彤彤
夏瑜
***
肖亮
王利春
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to MX2017016851A priority Critical patent/MX2017016851A/es
Priority to US15/745,230 priority patent/US10465014B2/en
Priority to EP17759275.5A priority patent/EP3309177B1/en
Priority to JP2017565750A priority patent/JP6432121B2/ja
Application filed by 四川科伦博泰生物医药股份有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to RU2017145150A priority patent/RU2693661C2/ru
Priority to CN201780002182.8A priority patent/CN107922503B/zh
Priority to CA2987118A priority patent/CA2987118C/en
Priority to BR112017027877-4A priority patent/BR112017027877A2/pt
Priority to KR1020177036591A priority patent/KR102068600B1/ko
Priority to ES17759275T priority patent/ES2801873T3/es
Priority to AU2017226510A priority patent/AU2017226510C1/en
Publication of WO2017148424A1 publication Critical patent/WO2017148424A1/zh
Priority to HK18106097.9A priority patent/HK1247209A1/zh
Priority to HK18106688.4A priority patent/HK1247214B/zh
Priority to US16/516,991 priority patent/US11136413B2/en

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Definitions

  • the invention belongs to the field of tumor treatment and molecular immunology, and relates to an anti-PDL-1 antibody, a pharmaceutical composition thereof and use thereof.
  • the invention relates to a monoclonal antibody against PDL-1.
  • the PD-1/PDL-1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune escape.
  • the expression of PD-1 (programmed cell death 1, programmed cell death factor 1) is mainly in immune cells such as T cells, and the ligand PDL-1 of PD-1 is highly expressed in many human tumor tissues.
  • Application of immunohistochemical methods has been in breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, melanoma and other human tumors
  • the expression of PDL-1 protein was detected in the tissues, and the expression level of PDL-1 was closely related to the clinical and prognosis of the patients.
  • Blocking the PD-1/PDL-1 signaling pathway activates suppressed T cells and attacks cancer cells.
  • Blocking PD-1/PDL-1 signaling can promote the proliferation of tumor antigen-specific T cells and play a role in killing tumor cells, thereby inhibiting local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455) -2465); PDL-1 mAb up-regulates the secretion of IFN- ⁇ in infiltrating CD8+ T cells, suggesting that blockade of the PD-1/PDL-1 signaling pathway plays a role in tumor immune responses for the purpose of inducing immune responses ( Blank C et al., 2006, Int. J. Cancer. 119:317-327).
  • PDL-1 can also bind to B7-1 in vivo. Studies have shown that the PDL-1/B7-1 complex is also a negative signal for T cell activation, and the combination of the two can lead to decreased expression of T cell surface activation markers and inhibition of T cell proliferation.
  • IL-2 Interlukin 2, interleukin 2 is a lymphokine secreted by Th cells and has a wide range of immunological activities: 1 stimulates T cell proliferation and differentiation; 2 stimulates the production of killer T lymphocytes; 3 stimulates NK cell proliferation Differentiation and enhancement of NK activity; 5 stimulation of lymphokine-activated killer cells (LAK cells) cells, ie lymphocytes stimulated by IL-2, can be a strong killing effect on new tumor cells after 3-6 days of in vitro culture. The cells are called LAK cells.
  • LAK cells lymphokine-activated killer cells
  • IFN- ⁇ Interferon- ⁇ , interferon- ⁇
  • T cells T cells and has various anti-tumor effects such as directly inhibiting tumor cell proliferation, increasing surface MHC antigen and tumor necrosis factor expression, and anti-tumor angiogenesis.
  • IFN- ⁇ can regulate the expression of Fas/FasL in tumor cells and enhance tumor cells.
  • the sensitivity to Fas-mediated apoptosis pathways reduces the ability of tumor cells to evade attack by the immune system, thereby inhibiting the malignant proliferation of tumor cells.
  • the present inventors used a mammalian cell expression system to express recombinant PDL-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 strain by screening a large number of samples: hybridoma cell line LT005, which was deposited with the China Center for Type Culture Collection (CCTCC) on August 4, 2015 under the accession number CCTCC NO. :C2015133.
  • the hybridoma cell line LT005 is capable of secreting a specific monoclonal antibody (designated 5C10) which specifically binds to PDL-1, and the monoclonal antibody is capable of blocking PD-1 and PDL very efficiently.
  • a specific monoclonal antibody designated 5C10
  • the present inventors also found monoclonal antibodies 5F10 and 9F6 which block the binding of PD-1 to PDL-1.
  • the inventors have creatively produced humanized antibodies against PDL-1, which are named 5C10H1L1, 5C10H1L2, 5C10H2L1, and 5C10H2L2, respectively.
  • the inventors creatively mutated the constant region of 5C10H2L2 to obtain a 5C10H2L2-IgG1mt antibody, which effectively reduced ADCC (antibody-dependent cell-mediated cytotoxicity) and/or CDC (complement-dependent cytotoxicity).
  • the antibodies of the present invention can efficiently bind to human T cells and activate T cells to induce human lymphocyte secretion.
  • IFN- ⁇ and IL-2 have potential for the preparation of drugs for the prevention and treatment of 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 of the monoclonal antibody comprises a CDR having the amino acid sequence of SEQ ID NOs: 15-17, and/or the light chain variable region of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 18-20 CDR;
  • the heavy chain variable region of the monoclonal antibody comprises a CDR having the amino acid sequence of SEQ ID NOs: 29-31, and/or the light chain variable region of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 32-34 CDR;
  • the heavy chain variable region of the monoclonal antibody comprises a CDR having the amino acid sequence of SEQ ID NO: 35-37, and/or the light chain variable region of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 38-40 CDR.
  • amino acid sequences of the CDRs of the 5 monoclonal antibodies 5C10, 5C10H1L1, 5C10H1L2, 5C10H2L1 and 5C10H2L2 are identical:
  • amino acid sequences of the CDRs of 5F10 mAb are as follows:
  • amino acid sequences of the CDRs of 9F6 mAb are as follows:
  • the above CDRs can be obtained by technical means well known to those skilled in the art, for example, by analyzing the amino acid sequence of the heavy chain variable region or the amino acid sequence of the light chain variable region according to the IMGT definition by the VBASE2 database.
  • the monoclonal antibody or antigen-binding fragment thereof wherein
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10, and/or the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 4 SEQ ID NO: 8 and SEQ ID NO: 12;
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 21, and/or the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 23;
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 25 and/or the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 27.
  • the monoclonal antibody is selected from the group consisting of (1) to (7) below:
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 2
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 4 (5C10)
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8 (5C10H1L1);
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12 (5C10H2L2 or 5C10H2L2-IgG1mt);
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 12 (5C10H1L2);
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 10
  • amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 8 (5C10H2L1);
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 21, and the amino acid sequence of the light chain variable region is SEQ. ID NO: 23 (5F10);
  • amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 25, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO: 27 (9F6).
  • the monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody or antigen-binding fragment thereof is selected from the group consisting of Fab, Fab', F(ab') 2 , Fd, Fv a 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 wherein the 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, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM EC 50 or less PDL-1 binding protein; preferably, the EC 50 measured by indirect ELISA.
  • the monoclonal antibody or antigen-binding fragment thereof wherein
  • 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 constant region of the monoclonal antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4;
  • the constant region of the monoclonal antibody is selected from the mutated human IgG1 constant region, and more preferably, the mutated human IgG1 constant region is one of the following, 2 at positions 234, 235 and 237 according to the EU numbering system. Species or 3 mutations: L234A, L235A and G237A.
  • Typical Culture Collection (CCTCC) with accession number CCTCC NO: C2015133.
  • Another aspect of the invention relates to an isolated nucleic acid molecule A comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, wherein
  • the heavy chain variable region of the antibody comprises: the CDR of the amino acid sequence of SEQ ID NOs: 15-17;
  • 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;
  • the heavy chain variable region of the antibody comprises: the amino acid sequence is SEQ ID NO:29 -31 CDR,
  • the heavy chain of the antibody has the amino acid sequence set forth in SEQ ID NO:21.
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:22;
  • the heavy chain variable region of the antibody comprises: the CDRs of amino acid sequence SEQ ID NOs: 35-37,
  • the heavy chain of the antibody has the amino acid sequence set forth in SEQ ID NO:25,
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:26.
  • a further aspect of the invention relates to an isolated nucleic acid molecule B comprising a nucleic acid sequence capable of encoding an antibody light chain variable region, wherein
  • the antibody light chain variable region comprises a CDR having the amino acid sequence of SEQ ID NOs: 18-20;
  • 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;
  • the light chain variable region of the antibody comprises: the CDRs of amino acid sequence SEQ ID NOs: 32-34,
  • the light chain of the antibody has the amino acid sequence set forth in SEQ ID NO:23,
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:24;
  • the light chain variable region of the antibody comprises: the CDRs of amino acid sequence SEQ ID NOs: 38-40,
  • the light chain of the antibody has the amino acid sequence set forth in SEQ ID NO:27,
  • the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:28.
  • a further aspect of the invention relates to an isolated nucleic acid molecule C comprising the aforementioned nucleic acid molecule A, and the aforementioned nucleic acid molecule; optionally, the nucleic acid molecule C further comprises a ligation sequence for ligation of the nucleic acid molecule A and the nucleic acid Molecular B.
  • a further aspect of the invention relates to a vector comprising an isolated nucleic acid molecule A, an isolated nucleic acid molecule B or an isolated nucleic acid molecule C of the invention.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule A, an isolated nucleic acid molecule B or an isolated nucleic acid molecule C of the invention, or a vector comprising the invention.
  • isolated nucleic acid molecule A isolated nucleic acid molecule B
  • isolated nucleic acid molecule C isolated nucleic acid molecule C
  • a further aspect of the invention relates to a method of the monoclonal antibody or antigen-binding fragment thereof of any one of the invention, comprising culturing the host cell of the invention under suitable conditions, and recovering the cell culture The step of a monoclonal antibody or antigen-binding fragment thereof.
  • a further aspect of the invention relates to a hybridoma cell line LT005 deposited with the China Center for Type Culture Collection (CCTCC) under accession number CCTCC NO: C2015133.
  • a further aspect of the invention relates to a monoclonal antibody or antigen-binding fragment thereof which is capable of competing for an antigen binding epitope to which an antibody or fragment secreted by the hybridoma cell line LT005 is bound.
  • the antibody or antigen-binding fragment thereof has any of the following active effects:
  • a drug that increases the expression of IFN- ⁇ and/or IL-2 in T lymphocytes is provided.
  • 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 PDL-1 The presence of the sample or Level.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of the invention or a conjugate of the invention; optionally further comprising a pharmaceutically acceptable Accepted 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 Use in a diseased drug; preferably, the tumor is selected from the group consisting of breast cancer, lung cancer such as non-small cell lung cancer, liver cancer, stomach cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer , prostate cancer, bladder cancer, pancreatic cancer, glioma, melanoma 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:
  • a drug that increases the expression of IFN- ⁇ and/or IL-2 in T lymphocytes is provided.
  • 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:
  • a method of releasing PD-1 or PDL-1 against the body or
  • a method of increasing the expression of IFN- ⁇ and/or IL-2 in T lymphocytes is provided.
  • the method is for non-therapeutic purposes.
  • a further aspect of the invention relates to a method of treating and/or preventing a tumor or anemia comprising administering to a subject an effective amount of the monoclonal antibody or antigen-binding fragment thereof of any one of the invention or the invention a step of conjugate; preferably, the tumor is selected from the group consisting of breast cancer, lung cancer such as non-small cell lung cancer, liver cancer, gastric cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, renal cancer, Prostate cancer, bladder cancer, pancreatic cancer, glioma, melanoma and leukemia.
  • the amino acid sequence of the PDL-1 protein when referring to the amino acid sequence of the PDL-1 protein (Programmed death-ligand 1, NCBI GenBank ID: NP_054862.1), it includes the full length of the PDL-1 protein, or the cell of PDL-1.
  • the outer fragment PDL-1 ECD or a fragment comprising PDL-1 ECD also includes a fusion protein of PDL-1 ECD, such as a fragment fused to a Fc protein fragment (mFc or hFc) of mouse or human IgG.
  • Fc protein fragment mFc or hFc
  • the term "PDL-1 protein” shall include all such sequences, including the sequences shown, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of a PDL-1 protein, it includes not only sequence fragments but also corresponding sequence fragments in its natural or artificial variants.
  • 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 fragment
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al, Nature 341:544-546 (1989))
  • Fab fragment means by 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 can be used, but variants thereof can also be used (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 fragments are diabodies, i.e., bivalent antibodies in which V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the same chain in two 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)).
  • the antigen-binding fragment of an antibody is a "bispecific antibody” and refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) or antibody analog by a coupling arm, Ways of association include, but are not limited to, chemical reactions, gene fusion, and enzymatic.
  • the antigen-binding fragment of the antibody may be a "multispecific antibody” including, for example, a trispecific antibody and a tetraspecific antibody, the former being an antibody having three different antigen binding specificities, and the latter having four different antigen binding specificities Antibodies.
  • a designer ankyrin repeat protein (DARPin), linked to an IgG antibody, an scFv-Fc antibody fragment, or a combination thereof, such as CN104341529A.
  • DARPin ankyrin repeat protein
  • the fynomer against IL-17a binds to an anti-IL-6R antibody, as described in WO2015141862A1.
  • the antigen-binding fragment of an antibody is a "bifunctional antibody conjugate", meaning that the first antibody (fragment) and or the second biologically functional fragment (non-antibody and its analog) are passed through a coupling arm.
  • the conjugate formed, including but not limited to chemical reaction, gene fusion and enzymatic, the second biological functional fragment comprises a binding active polypeptide, a protein, polyethylene glycol (PEG), a nuclides, a nucleic acid, Small molecule toxins, receptors or ligands, etc., which retain the activity of the respective antibodies, are bifunctional and bispecific.
  • Obtained from a given antibody e.g., monoclonal antibodies 5C10, 5C10H1L1, 5C10H1L2, 5C10H2L1, and 5C10H2L2 provided by the present invention
  • monoclonal antibodies 5C10, 5C10H1L1, 5C10H1L2, 5C10H2L1, and 5C10H2L2 provided by the present invention
  • conventional techniques known to those skilled in the art e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods.
  • 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 chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibodies Body or subclass), but in any case, retains 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 method of humanization is based on a combination of one or several of the currently used humanization methods. For example, the method described below is employed.
  • the method of humanization can adopt the method of CDR grafting.
  • the method first determines the CDR regions of the murine antibody, and then grafts the obtained CDRs of the murine heavy and light chains to a human template having a higher similarity to the murine FR region.
  • the selection of the human template can be an original germline sequence, such as a germline sequence derived from the IMGT database, or a sequence of a mature antibody, such as an antibody sequence derived from a Gene bank.
  • the CDR-grafted antibody can be subjected to a method of back mutation, that is, an individual human template amino acid is returned to the mouse amino acid to ensure antibody affinity.
  • the method of humanization can adopt the method of SDR grafting.
  • This method first requires the determination of the SDR region of the murine antibody.
  • the determination of the SDR region can be by alanine scanning.
  • the obtained murine SDR is then grafted onto a human template having a higher similarity to the murine source sequence.
  • the selection of the human template can be an original germline sequence, such as a germline sequence derived from the IMGT database, or a sequence of a mature antibody, such as an antibody sequence derived from a Gene bank.
  • the CDR-grafted antibody can be subjected to a method of back mutation, that is, an individual human template amino acid is returned to the mouse amino acid to ensure antibody affinity. Tamura, M., DEMilenic, M. Iwahashi, E.
  • the method of humanization can employ a method of surface resurfacing.
  • Mouse antibody A method of computer modeling or protein crystallization obtains a murine antibody model, and according to the model, amino acids of the accessible surface are determined, and then these amino acids are mutated to corresponding human amino acids.
  • the selection of human amino acids can be at the same site, with a higher proportion of amino acids in human antibodies.
  • Padlan, E.A. A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol. Immunol., 28, 489-98 (1991)
  • the method of humanization can adopt a method of superhumanization.
  • the method first determines the CDR region of the murine antibody, and then selects a human sequence having a higher similarity to the six CDRs of the mouse as a template, and grafts the six murine source CDRs onto the selected template.
  • the selection of the human template can be an original germline sequence, such as a germline sequence derived from the IMGT database, or a sequence of a mature antibody, such as an antibody sequence derived from a Gene bank.
  • the CDR-grafted antibody can be subjected to a method of back mutation, that is, an individual human template amino acid is returned to the mouse amino acid to ensure antibody affinity. Tan, P., DAMitchell, TNBuss, MAHolmes, C.
  • 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 the high purity of the same polynucleotide or polypeptide isolated from this natural state is called separation. of.
  • the term “isolated” or “isolated” does not exclude the inclusion of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
  • 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.
  • antibodies e.g., monoclonal antibodies 5C10, 5C10H1L1, 5C10H1L2, 5C10H2L1, and 5C10H2L2 of the invention
  • antibodies are 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 (for example, a PDL-1 protein), for example, as determined using a Fortebio molecular interaction meter.
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • 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 LT005 when referring to the hybridoma cell line LT005, it also refers to the subcloned and progeny cells of the hybridoma cell line LT005.
  • 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 adjusting agents, 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 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.
  • the monoclonal antibody 5C10 of the present invention can bind specifically to PDL-1, and can effectively block the binding of PD-1 to PDL-1, specifically deprive PDL-1 of immunosuppression, and activate T lymphocyte. cell.
  • Figure 1 Results of SDS-PAGE detection of PDL-1 ECD-mFc fusion protein.
  • the samples from the left to the right of the four lanes were loaded with M:marker 10 ⁇ L; 10 ⁇ L of the sample was loaded; 10 ⁇ L was passed through the column; and 10 ⁇ L was eluted from the column.
  • Figure 2 Results of SDS-PAGE detection of PD-1-hFc fusion protein.
  • the samples from the left to the right of the two lanes and their loadings were: 10 ⁇ L of the sample on the sample column; M: marker 10 ⁇ L.
  • Figure 3 Results of SDS-PAGE detection of B7-1-hFC fusion protein.
  • the samples from the left to the right of the two lanes and their loadings were: 10 ⁇ L of the sample on the sample column; M: marker 10 ⁇ L.
  • Figure 4 Results of SDS-PAGE detection of 5C10 antibody.
  • the samples from the left to the right of the four lanes were loaded with: M:marker 10 ⁇ L; reduced protein electrophoresis loading buffer sample antibody 1 ⁇ g; purified sample flowthrough; non-reduced protein electrophoresis 1 ⁇ g of buffer.
  • Figure 5 Results of SDS-PAGE detection of humanized antibody 5C10H1L1 of 5C10.
  • the samples from the left to the right of the four lanes were loaded with M:marker 10 ⁇ L; the column was eluted with 10 ⁇ L; the sample column was passed through 10 ⁇ L; and the sample column was loaded with 10 ⁇ L.
  • Figure 6 Results of SDS-PAGE detection of humanized antibody 5C10H1L2 of 5C10.
  • the samples from the left to the right of the four lanes were loaded with M:marker 10 ⁇ L; the column was eluted with 10 ⁇ L; the sample column was passed through 10 ⁇ L; and the sample column was loaded with 10 ⁇ L.
  • Figure 7 Results of SDS-PAGE detection of humanized antibody 5C10H2L1 of 5C10.
  • the samples from the left to the right of the four lanes were loaded with M:marker 10 ⁇ L; the column was eluted with 10 ⁇ L; the sample column was passed through 10 ⁇ L; and the sample column was loaded with 10 ⁇ L.
  • Figure 8 Results of SDS-PAGE detection of humanized antibody 5C10H2L2 of 5C10.
  • the samples from the left to the right of the four lanes were loaded with M:marker 10 ⁇ L; the column was eluted with 10 ⁇ L; the sample column was passed through 10 ⁇ L; and the sample column was loaded with 10 ⁇ L.
  • Figure 11 Results of the kinetic characteristic parameter detection of PCAB.
  • Figure 12 Fortebio assays 5C10, 5C10H2L2 and HpLp inhibit human PDL-1 binding to PD-1.
  • Figure 13 Results of binding of monoclonal antibody 5C10H1L1 to PDL-1 on the surface of 293T cells.
  • Figure 14 Results of binding of monoclonal antibody 5C10H1L2 to PDL-1 on the surface of 293T cells.
  • Figure 15 Results of binding of monoclonal antibody 5C10H2L1 to PDL-1 on the surface of 293T cells.
  • Figure 16 Results of binding of monoclonal antibody 5C10H2L2 to PDL-1 on the surface of 293T cells.
  • Figure 17 Results of binding of HpLp to PDL-1 on the surface of 293T cells.
  • Figure 18 Results of binding of PCAB to PDL-1 on the surface of 293T cells.
  • Figure 19 Binding of 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1 to human PDL-1 was determined by indirect ELISA.
  • Figure 20 Binding of 5C10H2L2, HpLp to monkey PDL-1 was determined by indirect ELISA.
  • Figure 21 ELISA detection of binding activity of 5C10H2L2 antibody to human PDL-1, human PD-L2 and murine PDL-1.
  • Figure 22 Competition ELISA results for 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1 and PD-1.
  • Figure 24 5C10H2L2 enhances IFN- ⁇ secretion by preventing binding of PDL-1 to PD-1.
  • FIG. 25 5C10H2L2 increases IL-2 secretion by preventing binding of PDL-1 to PD-1.
  • Figure 26A Dynamic affinity of 5C10H2L2-IgG1mt and FcyRIIIa Biacore assay.
  • FIG. 26B Dynamic affinity with FcyRIIIa was detected by Biacore.
  • Figure 27A Dynamic affinity of 5C10H2L2-IgG1mt and C1q for Biacore detection.
  • Figure 28 Effect of 5C10H2L2-IgG1mt on non-small cell lung cancer cells.
  • the hybridoma cell line LT005 was deposited with the China Center for Type Culture Collection (CCTCC) on August 4, 2015 under the accession number CCTCC NO: C2015133.
  • the deposit address is Wuhan University, Wuhan, China, 430072.
  • the BALB/C mice used were purchased from the Guangdong Medical Laboratory Animal Center.
  • the T cells used were from Zhongshan Kangfang Biomedical Co., Ltd.
  • PDL-1 ECD programmed cell death 1ligand 1, NCBI GenBank ID: NP_054862.1
  • PDL-1 and PD-L1 can be used interchangeably in the scientific literature.
  • the corresponding amino acid sequence was designed to be fused with the Fc protein fragment (mFc) of mouse IgG.
  • mFc Fc protein fragment
  • the corresponding nucleic acid sequence of the Jinseri company protein sequence was entrusted, and the corresponding fusion protein gene was synthesized by Kingsray.
  • the synthetic PDL-1 ECD-mFc fusion gene was cloned into the pUC57simple (provided by Kingsray) expression vector by Kingsray to obtain the pUC57simple-PDL-1 ECD-mFc plasmid.
  • the plasmid pUC57simple-PDL-1ECD-mFc was digested (Xba I and BamH I), and the fusion gene fragment PDL-1ECD-mFc obtained by electrophoresis was ligated with pcDNA3.1 expression vector (purchased from Invitrogen) to obtain pcDNA3. .1-PDL-1 ECD-mFc, transfected competent E. coli cell DH5a (purchased from TIANGEN), transfected and cultured according to the instructions.
  • the positive pcDNA3.1-PDL-1 ECD-mFc clone colony was screened, and E.
  • coli was amplified according to the conventional method, and then used in a kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., DP103-03) and according to the kit.
  • the pcDNA3.1-PDL-1 ECD-mFc recombinant plasmid was obtained by the instruction.
  • the recombinant plasmid pcDNA3.1-PDL-1 ECD-mFc was transfected into 293F cells (purchased from Invitrogen) according to the lipofectamin transfection kit (purchased from Invitrogen).
  • the culture medium was purified by high-speed centrifugation, microfiltration membrane filtration and HiTrap protein A HP column to purify PDL-1 ECD-mFc fusion protein.
  • the purified sample was added to a reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis. As shown in Figure 1, the target protein is approximately 53 kD.
  • the amino acid sequence corresponding to the extracellular fragment PD-1ECD (Programmed cell death protein 1, NCBI GenBank ID: NP_005009.2) of the gene PD-1 was fused with the Fc protein fragment (hFc) of human IgG.
  • hFc Fc protein fragment
  • the corresponding nucleic acid sequence of the Jinseri company protein sequence was entrusted, and the corresponding fusion protein gene was synthesized by Kingsray.
  • the synthetic and PD-1 ECD-TEV-hFc fusion gene was cloned into the pUC57simple (provided by Kingsray) expression vector by Kingsray to obtain the pUC57simple-PD-1ECD-TEV-hFc plasmid.
  • the plasmid pUC57simple-PD-1ECD-TEV-hFc was digested (Xba I and BamH I), and the fusion gene fragment PD-1ECD-TEV-hFc recovered by electrophoresis was ligated with the pcDNA3.1 expression vector (purchased from Invitrogen). Reaction, obtained pcDNA3.1-PD-1ECD-TEV-hFc, transfected competent E. coli cells DH5a (purchased From TIANGEN, transfection and culture were carried out according to the instructions. The positive pcDNA3.1-PD-1ECD-TEV-hFc clone colony was 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 according to the reagent
  • the pcDNA3.1-PD-1ECD-TEV-hFc recombinant plasmid was extracted from the instructions of the cassette.
  • the recombinant plasmid pcDNA3.1-PD-1ECD-TEV-hFc was transfected into 293F cells (purchased from Invitrogen) according to the lipofectamin transfection kit (purchased from Invitrogen).
  • the culture medium was purified by high-speed centrifugation, microfiltration membrane vacuum filtration and Mabselect SuRe column to purify PD-1ECD-TEV-
  • the hFc fusion protein was taken and the purified sample was added to the reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis.
  • the electrophoresis pattern of the reduced protein sample is shown in Fig. 2.
  • the synthetic and B7-1ECD-hFc fusion gene was cloned into the pUC57simple (provided by Kingsray) expression vector by Kingsray to obtain the pUC57simple-B7-1ECD-hFc plasmid.
  • the plasmid pUC57simple-B7-1ECD-hFc was digested (Xba I and BamH I), and the fusion gene fragment B7-1ECD-hFc obtained by electrophoresis was ligated with the pcDNA3.1 expression vector (purchased from Invitrogen) to obtain pcDNA3. .1-B7-1ECD-hFc, transfected competent E. coli cells DH5a (purchased from TIANGEN), transfection and culture were carried out according to the instructions. The positive pcDNA3.1-B7-1ECD-hFc clone colonies were screened, and E.
  • coli was amplified according to the conventional method, and then used in a kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., DP103-03) and according to the kit.
  • the pcDNA3.1-B7-1ECD-hFc recombinant plasmid was extracted by the instructions.
  • the recombinant plasmid pcDNA3.1-B7-1ECD-hFc was transfected into 293F cells for 7 days, and the B7-1ECD-hFc fusion protein was purified by high speed centrifugation, microfiltration membrane filtration and Mabselect SuRe column. The purified sample was added to the reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis. The electrophoresis pattern of the reduced protein sample is shown in Fig. 3.
  • Example 1 Obtainment of hybridoma cell line LT005 and preparation of monoclonal antibodies 5C10, 5F10, 9F6
  • the mammalian cell expression system is used to express the recombinant PDL-1 ECD-mFc fusion protein 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 LT005 was obtained, which was able to secrete the monoclonal antibody 5C10 which specifically binds to PDL-1.
  • Two other monoclonal antibodies, 5F10 and 9F6, were also made in the present invention.
  • the PDL-1 ECD-mFc fusion protein was prepared as the antigen by the preparation example 1 above, and the spleen cells of the immunized BALB/C mice (purchased from the Guangdong Medical Experimental Animal Center) were fused with the mouse myeloma cells into a hybridoma cell.
  • 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 cells secreting a novel antibody that specifically binds to PDL-1 ECD-mFc were obtained by indirect ELISA using PDL-1 ECD-mFc as an antigen-coated plate.
  • Hybridoma cells screened by indirect ELISA were screened by a competition ELISA for a hybridoma cell line capable of secreting a monoclonal antibody that competes with the ligand PD-1 (PD-1-hFc, preparation example 2) for binding to PDL-1.
  • a stable hybridoma cell line was obtained by limiting dilution method.
  • the hybridoma cell line was named LT005, and the secreted monoclonal antibody was named 5C10.
  • the hybridoma cell line LT005 was deposited with the China Center for Type Culture Collection (CCTCC) on August 4, 2015 under the accession number CCTCC NO: C2015133.
  • the deposit address is Wuhan University, Wuhan, China, 430072.
  • the inventors also constructed two additional hybridoma cell lines, the secreted antibodies were murine antibodies, and the antibodies were named 5F10 and 9F6, respectively.
  • the PDL-1-5C10 cell line of the present invention was cultured with 10% low IgG fetal bovine serum, and cells were collected 7 days later. The culture supernatant was purified to prepare antibody 5C10.
  • Antibodies 5F10 and 9F6 were prepared as described above.
  • the purified sample is separately added to a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and the purified sample flow is perforated and boiled for detection.
  • the results showed that the target protein of the reduced protein sample was approximately 50 kD and 25 kD, and the target protein of the non-reduced protein sample was approximately 150 kD (Fig. 4).
  • Example 9 For the ELISA affinity test method, see Example 9, ELISA competitive affinity test method. See Example 13 FACS cell affinity test method. See Example 8.
  • Table 1 Affinity of mouse antibody 5C10, 5F10, 9F6, competitive affinity and cell affinity for detection
  • the mRNA was extracted from the hybridoma cell line LT005 obtained 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 instructions of TransGen Biotech TransScript First-Strand cDNA Synthesis SuperMix kit 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.
  • the product of the TA clone was directly sequenced, and the sequencing results were as follows:
  • Example 3 Light and heavy chain sequences of humanized antibodies 5C10H1L1, 5C10H1L2, 5C10H2L1, 5C10H2L2 Column design
  • variable region sequences of the antibodies 5C10H1L1, 5C10H1L2, 5C10H2L1, 5C10H2L2 were obtained (the heavy chain constant region is Ig gamma-1 chain C region, ACCESSION: P01857, and the light chain constant region is Ig kappa chain C region, ACCESSION: P01834), the variable region sequence is as follows:
  • Nucleotide sequence encoding the heavy chain variable region of 5C10H1L2 SEQ ID NO: 5
  • Amino acid sequence of the 5C10H1L2 heavy chain variable region SEQ ID NO: 6.
  • Nucleotide sequence encoding the 5C10H1L2 light chain variable region SEQ ID NO: 11,
  • Amino acid sequence of the 5C10H1L2 light chain variable region SEQ ID NO: 12.
  • Amino acid sequence of the 5C10H2L1 heavy chain variable region SEQ ID NO: 10.
  • Nucleotide sequence encoding the 5C10H2L1 light chain variable region SEQ ID NO: 7,
  • Amino acid sequence of the 5C10H2L1 light chain variable region SEQ ID NO:8.
  • Example 4 Preparation and 5C10 humanized antibodies 5C10H1L1, 5C10H1L2, 5C10H2L1 and 5C10H2L2 SDS-PAGE electrophoresis
  • the heavy chain cDNAs of 5C10H1L1, 5C10H1L2, 5C10H2L1, and 5C10H2L2 (the heavy chain variable region sequences are shown in SEQ ID NO: 5, SEQ ID NO: 9, respectively; the heavy chain constant region sequence is hIgG1 sequence) and the light chain cDNA ( The light chain variable region sequences are shown in SEQ ID NO:7 and SEQ ID NO:11, respectively; the light chain constant region is human kappa sequence) cloned into pUC57simple (provided by Kingsray) vector to obtain pUC57simple-5C10H1.
  • pUC57simple5C10L1, pUC57simple-5C10H2 and pUC57simple-5C10L2 clone plasmids.
  • the recombinant plasmid was co-transfected into 293F cells. After 7 days of cell culture, the culture was purified by high speed centrifugation, vacuum filtration through a micropore filter, and HiTrap protein A HP column.
  • the purified sample was separately added to the reduced protein electrophoresis loading buffer and the non-reduced protein electrophoresis loading buffer, and then boiled and subjected to SDS-PAGE electrophoresis, and the results are shown in Fig. 5, Fig. 6, Fig. 7, and Fig. 8, respectively.
  • the reduced protein sample target protein is approximately 50 kD and 25 KD
  • the non-reduced protein sample target protein is approximately 150 kD.
  • Example 5 Determination of kinetic parameters of humanized antibody 5C10H2L2
  • the humanized antibody 5C10H2L2 was bound to the antigen PDL-1 (NCBI GenBank ID: NP_054862.1, encoding the nucleic acid sequence is SEQ ID NO: 13, and the encoded amino acid sequence is SEQ ID NO: 14) using a Fortebio molecular interaction instrument. Kinetic parameters.
  • a PDL-1-mFc protein was prepared by the method for producing PDL-1 ECD-mFc in Preparation Example 1, and the PDL-1-mFc protein was digested with TEV protease, and purified by column chromatography to obtain a PDL-1 antigen.
  • the present invention selects HpLp or PCAB as a positive control, wherein HpLp is a marketed Atezolizumab (trade name) ), PCAB is a clinically-derived PDL-1 antibody.
  • Atezolizumab (trade name ) purchased from Roche.
  • Preparation HpLp (also referred KF025HpLp) may refer to U.S. Patent Application US 2010 / 0203056A1 embodiment thereof e.g. Example 10, V H sequence of the sequence of the antibody, see patent 20, V L sequence see the sequence of the patent 21 .
  • Example 6 Fortebio assay 5C10, 5C10H2L2 and HpLp inhibit human PDL-1 binding to PD-1
  • Example 7 5C10H2L2 and HpLp block PD1/PDL-1 binding
  • the HTRF method was used to compare the binding of 5C10H2L2 to HpLp to block PD1/PDL-1.
  • a PD1/PDL-1 binding assay (CISBIO, Cat. No. 63ADK000CPLPEH) kit was used.
  • 5C10H2L2 and HpLp were diluted with dilution buffer, starting at 100 ⁇ g/mL, 3 fold dilution, and 10 concentration points. 2 ⁇ L of sample, 4 ⁇ L of PDL-1-EuK and 4 ⁇ L of Tag-PD1 were added, centrifuged, and incubated for 20 minutes at room temperature.
  • HpLp and 5C10H2L2 had the same ability to block PD1/PDL-1 binding, which were 67.29 ng/ml and 68.97 ng/ml, respectively. Both antibodies could effectively inhibit the binding of human PDL-1 to PD-1.
  • Example 8 Flow cytometry method for detecting the binding activity of humanized antibody to cell surface antigen PDL-1
  • Host cell 293T expressing the PDL-1 antigen was first constructed; then the host cells were labeled with the humanized antibodies 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1 and positive control antibodies (HpLp and PCAB) prepared in the present invention. Flow cytometry analysis was then used to verify the specific binding of antibodies 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1 and positive control antibodies (HpLp and PCAB) to antigens with a native conformation on the cell surface.
  • HpLp and PCAB positive control antibodies
  • the vector pLenti6.3-PDL-1 (vector pLenti6.3 purchased from Invitrogen) containing PDL-1 was transfected into 293F cells according to the lipofectamin transfection kit (purchased from Invitrogen), and the stably expressed PDL-1 was obtained by screening. Clonal population.
  • the host cell 293T expressing the PDL-1 antigen obtained by the above-described steps of the conventional trypsin digestion method was used, and the number of cells per collection tube was 2 ⁇ 10 5 .
  • Dilutions of each antibody were prepared in PBS (1% BSA) at 50 nM, 20 nM, 10 nM, 3 nM, 1 nM, 0.1 nM, 0.01 nM, 0 nM, and incubated with 293T cells expressing PDL-1 for 2 hours on ice, washed with PBS. 3 times.
  • FITC-Goat-Anti-Human IgG was diluted 1:100 with PBS, 100 ⁇ L was added to each tube, incubated on ice for 1 hour, and washed three times with PBS.
  • the fluorescent signal was detected on a flow cytometer using a FITC channel.
  • the tested antibodies can effectively bind to the target PDL-1 protein on the surface of the host cell 293T, and the binding efficiency is dose-dependent.
  • the fluorescence efficiency of the bound antibodies was analyzed by a curve, and the curve was used to simulate the binding efficiency EC 50 of each antibody, as shown in Table 4 below.
  • Table 4 Fluorescence intensity analysis of 5D10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1, HpLp, PCAB binding host cell 293T surface antigen PDL-1 by flow cytometry
  • Example 9 Detection of binding activity of humanized antibody to human PDL-1 by indirect ELISA
  • the binding activities of 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1 and positive control antibodies (HpLp and PCAB) to human PDL-1 were determined by indirect ELISA. Incubate the antigen in the plate, incubate at 4 ° C overnight, block with 1% BSA at 37 ° C for 2 h, add the antibody, incubate at 37 ° C for 30 min, add HRP-labeled goat anti-human IgG (H + L) secondary antibody ( Jackson, 109-035-088), a color reaction was carried out with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was measured in a microplate reader.
  • HRP-labeled goat anti-human IgG H + L
  • TMB Neogen, 308177
  • Fig. 19 The results of detecting the binding of the above antibody to human PDL-1 are shown in Fig. 19, respectively.
  • 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1, HpLp, PCAB can effectively bind human PDL-1 protein, and its binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose and the binding efficiency EC 50 of the curve simulation are shown in Table 5. .
  • the purpose of this experiment is to determine whether it can bind to the target antigen of monkeys. If it can be combined, monkeys can be used for pharmacokinetics and toxicology experiments.
  • the binding activity of 5C10H2L2 and the positive control antibody HpLp to monkey PDL-1 was determined by indirect ELISA. Incubate the antigen in the plate, incubate at 4 ° C overnight, block with 1% BSA at 37 ° C for 2 h, add the antibody, incubate at 37 ° C for 30 min, add HRP-labeled goat anti-human IgG (H + L) secondary antibody ( Jackson, 109-035-088), a color reaction was carried out with TMB (Neogen, 308177) for 5 min, and the absorbance at 450 nm was measured in a microplate reader.
  • Example 11 Indirect ELISA method for detection of 5C10H2L2 antibody with human PDL-1, human PD-L2 and murine PDL-1 Binding activity
  • the binding activity of 5C10H2L2 antibody to human PDL-1, human PD-L2 (purchased from Yishen Shenzhou, Cat. No. 10292-H08H) and murine PDL-1 (purchased from Yishen Shenzhou, Cat. No. 50010-M08H) was determined by indirect ELISA.
  • Human PDL-1, human PD-L2, and murine PDL-1 were incubated with 100 ⁇ L of 0.5 ⁇ g/ml antigen in the plate and overnight at 4 °C.
  • the antibody was diluted to a starting concentration of 1 ⁇ g/ml, diluted in a 3 fold ratio, and a total of 11 concentrations were diluted.
  • the antibodies were added separately and incubated at 37 ° C for 30 min. Diluted 1:20000, HRP-labeled goat anti-human IgG (H+L) secondary antibody (purchased from Jackson, Cat. No. 109-035-088), and color reaction with TMB (purchased from Neogen, Cat. No. 308177) for 5 min. The absorbance at 450 nm wavelength was measured in a microplate reader.
  • the results of binding of the 5C10H2L2 antibody to human PDL-1, human PD-L2, and murine PDL-1 are shown in FIG.
  • 5C10H2L2 can effectively bind human PDL-1 protein, and its binding efficiency is dose-dependent.
  • Example 12 Competitive ELISA method for detecting competitive binding activity of humanized antibodies to PDL-1 and PD-1
  • 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1 and positive control antibodies HpLp and PCAB
  • HpLp and PCAB positive control antibodies
  • HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (Jackson, 109-035-062) was added, and color reaction was carried out for 5 min with TMB (Neogen, 308177), and the absorbance at 450 nm was measured in a microplate reader.
  • Fig. 22 The results of detecting that the above antibody competes with PD-1 for binding to the antigen PDL-1 are shown in Fig. 22.
  • 5C10H1L1, 5C10H1L2, 5C10H2L2, 5C10H2L1, HpLp, PCAB can effectively compete with PD-1 for binding to antigen PDL-1, and its binding efficiency is dose-dependent.
  • the fluorescence intensity of each dose and the binding efficiency of curve simulation EC 50 is shown in Table 7.
  • Example 13 Competitive ELISA method for detecting competitive binding of 5C10H2L2 antibody to PDL-1 and B7-1
  • HRP-labeled goat anti-mouse IgG (H+L) secondary antibody (Jackson, 109-035-062) was added, and color reaction was carried out for 5 min with TMB (Neogen, 308177), and the absorbance at 450 nm was measured in a microplate reader.
  • Example 14 Analysis of cell biological activity of 5C10H2L2 and positive control antibodies (HpLp and PCAB)
  • T cells were isolated from PBMC, and the obtained DC cells were mixed with T cells at a ratio of 1:10, and 5C to 10 days after addition of different proportions of antibody 5C10H2L2 (hIgG as a control), IFN- was detected by ELISA kit.
  • the amount of ⁇ purchased from Daktronics for the company
  • IL-2 purchased from Daktronics for the company.
  • the secretion detection results of IFN- ⁇ and IL-2 after mixed culture of DC cells and T cells are shown in Fig. 24 and Fig. 25 respectively: 5C10H2L2, HpLp and PCAB can effectively induce the secretion of IFN- ⁇ and IL-2 by mixed lymphocytes. And its secretion amount is dose dependent on the antibody.
  • Example 15 Design and preparation of monoclonal antibody 5C10H2L2-IgG1mt modified human IgG1 constant region
  • the mutation in the heavy chain constant region is Ig gamma-1chain C region, ACCESSION: P01857, the light chain constant region is Ig kappa chain C region, ACCESSION: P01834 EU numbering system 234, 235, 237 is as follows: L234A, L235A, G237A, Named 5C10H2L2-IgG1mt.
  • 5C10H2L2-IgG1mt was prepared by the method of Example 4.
  • the purified Fc ⁇ RIIIa-Biotin was ligated to the SA chip (streptomycin-coated chip) by binding of biotin to streptomycin using standard methods and kits provided by ForteBio, and the conditions were: 1 ⁇ g/ml Fc ⁇ RIIIa-Biotin, 300s.
  • the antibody concentration was 4000 nM in combination with 120 S, and 180 S was dissociated in PBST (pH 7.4) buffer.
  • the binding and dissociation curve models were fitted using Octet software.
  • the purified antibody was ligated to the SA chip (streptomycin-coated chip) by binding of biotin to streptomycin using standard methods and kits provided by ForteBio, and the conditions were: 20 ug/ml Antibody-Biotin, 300 s.
  • the C1q concentration was 200 nM, a 2-fold concentration gradient, combined with 120S.
  • the binding and dissociation curve models were fitted using Octet software. In order to minimize the effects of affinity in the binding constant estimate, only the initial data segments corresponding to the binding and dissociation phases were fitted.
  • the measured K D , kon and koff values are shown in Table 9, and the specific maps are shown in Figures 27A and 27B.
  • Example 17 Determination of CDC cell activity of 5C10H2L2-IgG1mt
  • the first cultured tumor cell HCC1954 (purchased from ATCC Cat. No. CRL-2338) was a PDL-1 positive cell.
  • the culture was carried out using the corresponding medium (RPMI 1640 + 10% bovine serum).
  • HCC1954 assay medium (RPMI1640 + 10% human serum) was diluted with 5C10H2L2-IgG1mt drug 10000 ⁇ g/mL starting, 5 fold dilution, 10 concentration gradients.
  • the tumor cells were collected by conventional trypsin digestion method, and the number of tube cells was collected, and the corresponding detection medium (including RPMI1640+10%) was used.
  • the human serum was resuspended, and 10000 cells/well was added to the corresponding diluted antibody 96-well plate for 5 hours.
  • CCK8 reagent purchased from Dongren Chemical Technology Co., Ltd., product number CK04, Lot: JJ744.
  • the microplate reader 450 nm reading was used to detect mitochondria.
  • the activity of the dehydrogenase inside reflects the killing effect of the antibody on HCC1954 cells.
  • Example 18 In vivo efficacy against colon cancer
  • Human IgG is provided by Sichuan Kelun Pharmaceutical Research Institute Co., Ltd.; Purchased from Roche, human IgG was purchased from Chengdu Rongsheng Pharmaceutical Co., Ltd.
  • MC-38/H-11 cells are mouse colon cancer MC-38 (purchased from Cobioer, Cat. No. CBP60825) cells knocked out mouse endogenous PDL-1 by CRISPR/Cas9 technology, transfected and expressed human PDL-1 Monoclonal cells, therefore, MC-38/H-11 cells only express high levels of human PDL-1 protein.
  • mice C57BL/6 mice, 7-8 weeks old, purchased from Shanghai Slack Laboratory Animals Co., Ltd.
  • mice were subcutaneously inoculated with 1 ⁇ 10 5 MC-38/H-11 cells, and the next day after inoculation (D0), randomized and intraperitoneally injected (IP) drugs once every other day (Q2D); Volume of human IgG (15 mg/kg), 5C10H2L2- IgG1mt (1.5, 5 , 15 mg/kg), (15 mg/kg), injection volume 0.1 mL/10 g body weight. Each group was 10 mice.
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific indicators are T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter was measured twice a week with a vernier caliper.
  • the tumor volume (V) was calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width, respectively.
  • T/C% T/C ⁇ 100
  • C and T are the tumor volume or tumor weight of the solvent group and the treatment group, respectively.
  • Tumor inhibition rate (TGI) (%) (C-T) / C ⁇ 100, C, T are the tumor volume or tumor weight of the solvent group and the treatment group, respectively.
  • mice 5C10H2L2-IgG1mt (1.5, 5, 15 mg/kg), Efficacy of subcutaneous xenografts in mouse colon cancer MC-38/H-11 mice
  • the inhibitory rates of 5C10H2L2-IgG1mt (1.5, 5, 15 mg/kg) on subcutaneous xenografts of MC-38/H-11 mice were 63.9%, 75.8% and 68.6%, respectively (based on mean tumor volume); The individual differences of tumors are large. The median tumor volume is calculated to be reasonable, and the tumor inhibition rate is adjusted to 100%, 100% and 100%.
  • the inhibition rate of MC-38/H-11 was 93.8% (calculated according to the median tumor volume); the tumor inhibition rate was calculated by the median tumor weight, then 5C10H2L2-IgG1mt (1.5, 5, 15mg/ Kg) The inhibition rate of MC-38/H-11 is 100%, 100%, 100%, respectively.
  • the tumor inhibition rate was 93.7%; the median tumor volume was very consistent with the tumor weight calculated from the median tumor weight, indicating the reliability of the tumor volume measurement method.
  • 5C10H2L2-IgG1mt (1.5, 5, 15mg/kg) not only inhibited tumor growth, but also inhibited tumor formation.
  • 5C10H2L2-IgG1mt (1.5, 5, 15mg/kg) was 40%. 40% and 40%, The tumor formation rate of the group was 50%. The tumor-bearing mice were well tolerated by the above drugs, and no obvious weight loss and other symptoms occurred.
  • 5C10H2L2-IgG1mt (1.5, 5, 15 mg/kg) was compared to subcutaneous xenografts in mouse colon cancer MC-38/H-11 mice. Has a stronger anti-tumor effect.
  • Example 19 In vivo efficacy against lung cancer
  • Non-small cell lung cancer cells HCC827 (purchased from ATCC No. CRL-2868) were subcutaneously inoculated with NOG mice to construct a lung cancer-bearing mouse model, until the tumor grew to about 100 mm 3 , and the mice were intravenously activated before administration.
  • Human PBMCs mimic the human immune system and then administer.
  • Dosing schedule The dose was 10 mg/kg, intravenously, once every two days, for a total of four doses. Tumor volume was measured twice weekly after administration. Divided into control IgG, 5C10H2L2-IgG1mt, Three groups of six each.
  • the tumor growth curve is shown in Figure 28.

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Abstract

提供了一种针对程序性细胞死亡因子1配体（PDL-1）的单克隆抗体或者其抗原结合片段，该单克隆抗体能够特异性与PDL-1结合，解除PDL-1对机体免疫的抑制，从而激活T淋巴细胞。还提供了包含该单克隆抗体的药物组合物及用途。

Description

一种PDL-1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PDL-1抗体、其药物组合物及其用途。具体地，本发明涉及一种抗PDL-1的单克隆抗体。
背景技术
PD-1/PDL-1信号通路在调节免疫耐受、微生物感染及肿瘤免疫逃逸中发挥重要作用。PD-1(programmed cell death 1，程序性细胞死亡因子1)的表达主要在T细胞等免疫细胞，而PD-1的配体PDL-1主要在许多人类肿瘤组织呈高表达。应用免疫组织化学方法，已先后在乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤等多种人类肿瘤组织中检测到PDL-1蛋白的表达，且PDL-1的表达水平和患者的临床及预后紧密相关。
阻断PD-1/PDL-1信号通路可使被抑制的T细胞激活，进而攻击癌细胞。阻断PD-1/PDL-1信号可以促进肿瘤抗原特异性T细胞的增殖，发挥杀伤肿瘤细胞的作用，进而抑制局部肿瘤生长(Julie R et al.,2012，N Engl J Med.366:2455–2465)；PDL-1单抗可上调浸润CD8+T细胞IFN-γ的分泌，表明PD-1/PDL-1信号通路的阻断在以诱导免疫应答为目的的肿瘤免疫应答中发挥作用(Blank C et al.,2006，Int.J.Cancer.119:317–327)。
另外，PDL-1在体内还可以与B7-1结合。已有研究表明PDL-1/B7-1复合物也是T细胞活化的负信号，二者的结合可以导致T细胞表面活化标记物的表达下降，抑制T细胞增殖等。
IL-2(Interlukin 2，白细胞介素2)是由Th细胞分泌的一种淋巴因子，具有广泛的免疫活性：①刺激T细胞增殖分化；②刺激产生杀伤性T淋巴细胞；③刺激NK细胞增殖分化，增强NK活性；⑤刺激产生淋巴因子激活的杀伤细胞(LAK细胞)细胞，即淋巴细胞在IL-2刺激下，经3-6天体外培养可成为一种对新瘤细胞具有强烈杀伤作用的细胞，称为LAK细胞。IFN-γ(Interferon-γ，干扰素-γ)由T细胞产生，具有直接抑制肿瘤细胞增殖，增加表面MHC抗原和肿瘤坏死因子表达，抗肿瘤血管生成等多种抗肿瘤作用。近年来研究发现，IFN-γ可以通过调控肿瘤细胞的Fas/FasL表达以及增强肿瘤细胞 对Fas所介导凋亡途径的敏感性，使肿瘤细胞逃避免疫***攻击的能力降低，从而抑制肿瘤细胞的恶性增殖。
目前，业界普遍认为针对PDL-1通路的抗体将导致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-PD1Therapy 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)。
目前，尚需要开发新的具有更好的结合效率的抗PDL-1抗体，以有效地阻断PD-1与PDL-1的结合，并且激活T淋巴细胞。
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PDL-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LT005，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:C2015133。
本发明人惊奇地发现，杂交瘤细胞株LT005能够分泌产生与PDL-1特异性结合的特异性单克隆抗体(命名为5C10)，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。另外，本发明人还发现了阻断PD-1与PDL-1的结合的单克隆抗体5F10和9F6。
进一步地，本发明人创造性地制得了抗PDL-1的人源化抗体，分别命名为5C10H1L1、5C10H1L2、5C10H2L1和5C10H2L2。
更进一步地，本发明人创造性地对5C10H2L2的恒定区进行了突变，得到5C10H2L2-IgG1mt抗体，有效地降低了ADCC(antibody-dependent cell-mediated cytotoxicity)和/或CDC(complement-dependent cytotoxicity)。
本发明人还惊奇地发现，本发明的抗体特别是5C10、5C10H1L1、5C10H1L2、5C10H2L1、5C10H2L2、5F10、9F6和5C10H2L2-IgG1mt均能有效地结合人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一种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:15－17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:18－20的CDR；
或者
所述单克隆抗体的重链可变区包含氨基酸序列为SEQ ID NO:29－31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:32－34的CDR；
或者
所述单克隆抗体的重链可变区包含氨基酸序列为SEQ ID NO:35－37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:38－40的CDR。
5C10、5C10H1L1、5C10H1L2、5C10H2L1和5C10H2L2这5个单抗的CDR的氨基酸序列相同：
Figure PCTCN2017075484-appb-000001
5F10单抗的CDR的氨基酸序列如下：
Figure PCTCN2017075484-appb-000002
9F6单抗的CDR的氨基酸序列如下：
Figure PCTCN2017075484-appb-000003
Figure PCTCN2017075484-appb-000004
上述CDR可以通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库根据IMGT定义分析下面的重链可变区的氨基酸序列或者轻链可变区的氨基酸序列得到。
在本发明的一些实施方式中，所述的单克隆抗体或其抗原结合片段，其中，
所述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；
或者
所述重链可变区的氨基酸序列选自SEQ ID NO:21,和/或所述轻链可变区的氨基酸序列选自SEQ ID NO:23；
或者
所述重链可变区的氨基酸序列选自SEQ ID NO:25,和/或所述轻链可变区的氨基酸序列选自SEQ ID NO:27。
在本发明的一些实施方式中，所述的单克隆抗体，其选自如下的(1)至(7)：
(1)重链可变区的氨基酸序如SEQ ID NO:2所示，轻链可变区的氨基酸序列如SEQ ID NO:4所示(5C10)；
(2)重链可变区的氨基酸序如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示(5C10H1L1)；
(3)重链可变区的氨基酸序如SEQ ID NO:10所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示(5C10H2L2或5C10H2L2-IgG1mt)；
(4)重链可变区的氨基酸序如SEQ ID NO:6所示，轻链可变区的氨基酸序列如SEQ ID NO:12所示(5C10H1L2)；
(5)重链可变区的氨基酸序如SEQ ID NO:10所示，轻链可变区的氨基酸序列如SEQ ID NO:8所示(5C10H2L1)；
(6)重链可变区的氨基酸序如SEQ ID NO:21所示，轻链可变区的氨基酸序列如SEQ  ID NO:23所示(5F10)；
(7)重链可变区的氨基酸序如SEQ ID NO:25所示，轻链可变区的氨基酸序列如SEQ ID NO:27所示(9F6)。
在本发明的一些实施方式中，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。
在本发明的一些实施方式中，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PDL-1蛋白；优选地，所述EC50通过间接ELISA方法测得。
在本发明的一些实施方式中，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；
优选地，所述单克隆抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区；
优选地，所述单克隆抗体的恒定区选自突变的人IgG1恒定区，更优选地，突变的人IgG1恒定区为按照EU编号***在234、235和237位点进行如下的1种、2种或3种突变：L234A、L235A和G237A。
在本发明的一些实施方式中，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是有杂交瘤细胞株LT005产生的单克隆抗体，所述杂交瘤细胞株LT005保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015133。
本发明的另一方面涉及一种分离的核酸分子A，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:15－17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；
在另一个实施方案中，所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO:29 －31的CDR，
优选地，所述抗体的重链具有SEQ ID NO:21所示的氨基酸序列，
更优选地，所述核酸分子具有SEQ ID NO:22所示的核苷酸序列；
在另一个实施方案中，所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO:35－37的CDR，
优选地，所述抗体的重链具有SEQ ID NO:25所示的氨基酸序列，
更优选地，所述核酸分子具有SEQ ID NO:26所示的核苷酸序列。
本发明的再一方面涉及分离的核酸分子B，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:18－20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；
在另一个实施方案中，所述抗体的轻链可变区包含：氨基酸序列为SEQ ID NO:32－34的CDR，
优选地，所述抗体的轻链具有SEQ ID NO:23所示的氨基酸序列，
更优选地，所述核酸分子具有SEQ ID NO:24所示的核苷酸序列；
在另一个实施方案中，所述抗体的轻链可变区包含：氨基酸序列为SEQ ID NO:38－40的CDR，
优选地，所述抗体的轻链具有SEQ ID NO:27所示的氨基酸序列，
更优选地，所述核酸分子具有SEQ ID NO:28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C，其包含前述的核酸分子A，以及前述的核酸分子；可选地，核酸分子C还包括连接序列，其用于连接核酸分子A和核酸分子B。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子A、分离的核酸分子B或分离的核酸分子C。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子A、分离的核酸分子B或分离的核酸分子C，或者包含本发明的载体。
关于上述的“分离的核酸分子A”“分离的核酸分子B”或“分离的核酸分子C”， 其中字母A、B或C仅仅是为了描述清楚，或者用于区分，字母本身并不表示特别的含义。
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LT005，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015133。
本发明的再一方面涉及一种单克隆抗体或其抗原结合片段，其能够竞争结合杂交瘤细胞株LT005分泌的抗体或片段针对的抗原结合表位。优选的，所述抗体或其抗原结合片段具有以下任一活性效果：
阻断PDL-1与PD-1或B7-1结合的药物，
调节(例如下调)PDL-1活性或水平的药物，
解除PD-1或者PDL-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。
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PDL-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，所述肿瘤选自乳腺癌、肺癌例如非小细胞性肺癌、肝癌、胃癌、肠癌例如结肠癌或直肠癌、食管癌、卵巢癌、***、肾癌、***癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤和白血病。
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PDL-1与PD-1或B7-1结合的药物，
调节(例如下调)PDL-1活性或水平的药物，
解除PD-1或者PDL-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。
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PDL-1与PD-1或B7-1结合的方法，
调节(例如下调)PDL-1活性或水平的方法，
解除PD-1或者PDL-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。
在本发明的一个实施方案中，所述方法是非治疗目的的。
本发明的再一方面涉及一种治疗和/或预防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的偶联物的步骤；优选地，所述肿瘤选自乳腺癌、肺癌例如非小细胞性肺癌、肝癌、胃癌、肠癌例如结肠癌或直肠癌、食管癌、卵巢癌、***、肾癌、***癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤和白血病。
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PDL-1蛋白(Programmed death-ligand 1,NCBI GenBank ID:NP_054862.1)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。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))。
在另一些情况下，抗体的抗原结合片段是“双特异性抗体”，指由第一抗体(片段)和第二抗体(片段)或抗体类似物通过偶联臂所形成的偶联物，偶联的方式包括但不限于化学反应、基因融合和酶促。抗体的抗原结合片段可以是“多特异性抗体”包括例如：三特异性抗体和四特异性抗体，前者是具有三种不同抗原结合特异性的抗体，而后者是具有四种不同抗原结合特异性的抗体。例如，经设计的锚蛋白重复蛋白(DARPin)，与IgG抗体，scFv-Fc抗体片段相连或其组合，如CN104341529A。抗IL-17a的fynomer与抗IL-6R抗体结合，如WO2015141862A1。
在另一些情况下，抗体的抗原结合片段是“双功能抗体偶联物”，指由第一抗体(片段)和或第二生物学功能片段(非抗体及其类似物)通过偶联臂所形成的偶联物，偶联的方式包括但不限于化学反应、基因融合和酶促，第二生物学功能片段包括具有结合活性多肽、蛋白、聚乙二醇(PEG)，核素，核酸，小分子毒素，受体或配体等，该偶联物保留了各自抗体的活性，故具有双功能和双特异性。
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5C10、5C10H1L1、5C10H1L2、5C10H2L1和5C10H2L2)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)。
人源化的方法根据目前常用的人源化方法中的一种或几种的结合。例如采用下面所述的方法。
人源化的方法可以采用CDR移植(CDR grafting)的方法。本方法首先确定鼠源抗体的CDR区域，随后将获得的鼠源重链及轻链的6个CDR嫁接到与鼠源FR区具有较高相似度的人源模板上。人源模板的选择可以是原始种系序列(germline)，如来源于IMGT数据库中的germline序列，也可以是成熟抗体的序列，如来源于Gene bank中的抗体序列。CDR移植完成的抗体可以采用回复突变的方法，即将个别人源模板氨基酸，回复突变回鼠源氨基酸以保证抗体亲和力。
人源化的方法可以采用SDR移植(SDR grafting)的方法。本方法首先需要确定鼠源抗体的SDR区域。SDR区域的确定可以通过丙氨酸扫描(alanine scanning)的方法。随后将获得的鼠源SDR嫁接到与鼠源序列具有较高相似度的人源模板上。人源模板的选择可以是原始种系序列(germline)，如来源于IMGT数据库中的germline序列，也可以是成熟抗体的序列，如来源于Gene bank中的抗体序列。CDR移植完成的抗体可以采用回复突变的方法，即将个别人源模板氨基酸，回复突变回鼠源氨基酸以保证抗体亲和力。Tamura,M.,D.E.Milenic,M.Iwahashi,E.Padlan,J.Schlom&S.V.Kashmiri:Structural correlates of ananticarcinoma antibody:identification of specificitydeterminingresidues(SDRs)and development of aminimally immunogenic antibody variant by retention ofSDRs only.J.Immunol.,164,1432-41(2000)
人源化的方法可以采用表面重排(resurfacing)的方法。本方法首先通过鼠源抗体 计算机建模或者蛋白结晶的方法，获得鼠源抗体模型，根据模型，确定可及表面的氨基酸，随后将这些氨基酸突变为对应的人源氨基酸。人源氨基酸的选择可以是相同位点上，人源抗体中占有较高比例的氨基酸。Padlan,E.A.:A possible procedure for reducing theimmunogenicity of antibody variable domains whilepreserving their ligand-binding properties.Mol.Immunol.,28,489-98(1991)
人源化的方法可以采用过人源化(superhumanization)的方法。本方法首先确定鼠源抗体的CDR区域，随后选择与鼠源6个CDR有较高相似度的人源序列作为模板，将6个鼠源CDR嫁接到选择的模板上。人源模板的选择可以是原始种系序列(germline)，如来源于IMGT数据库中的germline序列，也可以是成熟抗体的序列，如来源于Gene bank中的抗体序列。CDR移植完成的抗体可以采用回复突变的方法，即将个别人源模板氨基酸，回复突变回鼠源氨基酸以保证抗体亲和力。Tan,P.,D.A.Mitchell,T.N.Buss,M.A.Holmes,C.Anasetti&J.Foote:"Superhumanized"antibodies:reduction of immunogenic potential by complementaritydeterminingregion grafting with human germlinesequences:application to an anti-CD28.J.Immunol.,169,1119-25(2002)
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5C10、5C10H1L1、5C10H1L2、5C10H2L1和5C10H2L2)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，PDL-1蛋白)，例如，如使用Fortebio分子相互作用仪测定的。
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LT005时，其还指杂交瘤细胞株LT005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。
发明的有益效果
本发明的单克隆抗体5C10能够很好地特异性与PDL-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PDL-1对机体免疫抑制，激活T淋巴细胞。
附图说明
图1：PDL-1ECD-mFc融合蛋白的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M：marker 10μL；上样层析柱样品10μL；层析柱穿透10μL；层析柱洗脱10μL。
图2：PD-1-hFc融合蛋白的SDS-PAGE检测结果。从左至右的2个泳道的样品及其上样量依次为：上样层析柱样品10μL；M：marker 10μL。
图3：B7-1-hFC融合蛋白的SDS-PAGE检测结果。从左至右的2个泳道的样品及其上样量依次为：上样层析柱样品10μL；M：marker 10μL。
图4：5C10抗体的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M:marker 10μL；还原型蛋白电泳上样缓冲液样品抗体1μg；纯化的上样流穿液；非还原型蛋白电泳上样缓冲液1μg。
图5：5C10的人源化抗体5C10H1L1的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M:marker 10μL；层析柱洗脱10μL；上样层析柱穿透10μL；上样层析柱样品10μL。
图6：5C10的人源化抗体5C10H1L2的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M:marker 10μL；层析柱洗脱10μL；上样层析柱穿透10μL；上样层析柱样品10μL。
图7：5C10的人源化抗体5C10H2L1的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M:marker 10μL；层析柱洗脱10μL；上样层析柱穿透10μL；上样层析柱样品10μL。
图8：5C10的人源化抗体5C10H2L2的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为：M:marker 10μL；层析柱洗脱10μL；上样层析柱穿透10μL；上样层析柱样品10μL。
图9：单抗5C10H2L2的动力学特征参数检测结果。
图10：HpLp的动力学特征参数检测结果。
图11：PCAB的动力学特征参数检测结果。
图12：Fortebio测定5C10、5C10H2L2和HpLp抑制人PDL-1与PD-1结合。
图13：单抗5C10H1L1与293T细胞表面PDL-1的结合结果。
图14：单抗5C10H1L2与293T细胞表面PDL-1的结合结果。
图15：单抗5C10H2L1与293T细胞表面PDL-1的结合结果。
图16：单抗5C10H2L2与293T细胞表面PDL-1的结合结果。
图17：HpLp与293T细胞表面PDL-1的结合结果。
图18：PCAB与293T细胞表面PDL-1的结合结果。
图19：采用间接ELISA方法分别测定5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1与人PDL-1的结合。
图20：采用间接ELISA方法分别测定5C10H2L2、HpLp与猴PDL-1的结合。
图21：5C10H2L2抗体与人PDL-1、人PD-L2以及鼠PDL-1的结合活性ELISA检测。
图22：5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1与PD-1竞争ELISA结果。
图23：5C10H2L2与B7-1竞争ELISA结果。
图24：5C10H2L2通过阻止PDL-1与PD-1的结合提高IFN-γ的分泌。
图25：5C10H2L2通过阻止PDL-1与PD-1的结合提高IL-2的分泌。
图26A：5C10H2L2-IgG1mt与FcγRIIIa的动态亲和力Biacore检测。
图26B：
Figure PCTCN2017075484-appb-000005
与FcγRIIIa的动态亲和力Biacore检测。
图27A：5C10H2L2-IgG1mt与C1q的动态亲和力Biacore检测。
图27B：
Figure PCTCN2017075484-appb-000006
与C1q的动态亲和力Biacore检测。
图28：5C10H2L2-IgG1mt对非小细胞肺癌细胞疗效。
关于生物材料保藏的说明：
杂交瘤细胞株LT005，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:C2015133，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，均为可以通过市场购买获得的常规产品。
在本发明的下述实施例中，使用的BALB/C小鼠购自广东省医学实验动物中心。使用的T细胞来自中山康方生物医药有限公司。
制备例1：融合蛋白PDL-1ECD-mFc的制备
1.基因PDL-1ECD-mFc的合成
对基因PDL-1的胞外片段PDL-1ECD(programmed cell death 1ligand 1,NCBI GenBank ID:NP_054862.1)，在科技文献中PDL-1和PD-L1可以互换使用，本文统一使用PDL-1，所对应的氨基酸序列与小鼠IgG的Fc蛋白片段(mFc)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，委托金斯瑞公司蛋白序列相对应的核酸序列进行优化，并委托金斯瑞公司合成相对应的融合蛋白基因。
2.pUC57simple-PDL-1ECD-mFc质粒的获得：
由金斯瑞公司将合成的PDL-1ECD-mFc融合基因克隆到pUC57simple(金斯瑞公司提供)表达载体中，获得pUC57simple-PDL-1ECD-mFc质粒。
3.pcDNA3.1-PDL-1ECD-mFc重组质粒的构建：
将质粒pUC57simple-PDL-1ECD-mFc进行酶切(Xba I和BamH I)，电泳回收得到的融合基因片段PDL-1ECD-mFc与pcDNA3.1表达载体(购自Invitrogen公司)进行连接反应，获得pcDNA3.1-PDL-1ECD-mFc，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-PDL-1ECD-mFc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-PDL-1ECD-mFc重组质粒。
4.按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将重组质粒pcDNA3.1-PDL-1ECD-mFc转染293F细胞(购自Invitrogen公司)。
5.将重组质粒pcDNA3.1-PDL-1ECD-mFc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HiTrap protein A HP柱进行纯化PDL-1ECD-mFc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。如图1所示，目标蛋白大约在53kD处。
制备例2：融合蛋白PD-1-hFc的制备
1.基因PD-1-hFc的合成
对基因PD-1的胞外片段PD-1ECD(Programmed cell death protein 1,NCBI GenBank ID:NP_005009.2)所对应的氨基酸序列与人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，委托金斯瑞公司蛋白序列相对应的核酸序列进行优化，并委托金斯瑞公司合成相对应的融合蛋白基因。
2.pUC57simple-PD-1ECD-TEV-hFc质粒的获得
将金斯瑞公司将合成的和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-hFc质粒。
3.pcDNA3.1-PD-1ECD-TEV-hFc重组质粒的构建
将质粒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-hFc与pcDNA3.1表达载体(购自Invitrogen公司)进行连接反应，获得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-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-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-hFc转染293F细胞(购自Invitrogen公司)。
5.分别将重组质粒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-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，还原型蛋白样品电泳图如图2所示。
制备例3：B7-1-hFc的制备
1.基因B7-1-hFc的合成
对基因B7-1(Cluster of Differentiation 80(also CD80and B7-1),NCBI GenBank ID:NP_005182.1)的胞外片段B7-1ECD所对应的氨基酸与人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，委托金斯瑞公司蛋白序列相对应的核酸序列进行优化，并委托金斯瑞公司合成相对应的融合蛋白基因。
2.pUC57simple-B7-1ECD-hFc质粒的获得
将金斯瑞公司将合成的和B7-1ECD-hFc融合基因克隆到pUC57simple(金斯瑞公司提供)表达载体中，获得pUC57simple-B7-1ECD-hFc质粒。
3.pcDNA3.1-B7-1ECD-hFc重组质粒的构建
将质粒pUC57simple-B7-1ECD-hFc进行酶切(Xba I和BamH I)，电泳回收得到的融合基因片段B7-1ECD-hFc与pcDNA3.1表达载体(购自Invitrogen公司)进行连接反应，获得pcDNA3.1-B7-1ECD-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-B7-1ECD-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-B7-1ECD-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-B7-1ECD-hFc转染293F细胞(购自Invitrogen公司)。
5.分别将重组质粒pcDNA3.1-B7-1ECD-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B7-1ECD-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，还原型蛋白样品电泳图如图3所示。
实施例1：杂交瘤细胞株LT005的获得以及单克隆抗体5C10、5F10、9F6的制备
利用哺乳动物细胞表达***表达出重组的PDL-1ECD-mFc融合蛋白作为抗原免疫小鼠，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LT005，该细胞株能够分泌产生与PDL-1特异性结合的单克隆抗体5C10。本发明还制得了另外两种单克隆抗体5F10和9F6。
具体方法如下：
1.杂交瘤细胞株的建立
用前面制备例1制得PDL-1ECD-mFc融合蛋白作为抗原，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PDL-1ECD-mFc作为抗原包被酶标板，进行间接ELISA法筛选，得到分泌与PDL-1ECD-mFc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PD-1(PD-1-hFc，制备例2值得)竞争结合PDL-1的单克隆抗体的杂交瘤细胞株，并经过有限稀释法得到稳定的杂交瘤细胞株。将该杂交瘤细胞株命名LT005，其分泌的单克隆抗体命名为5C10。
杂交瘤细胞株LT005，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:C2015133，保藏地址为中国.武汉.武汉大学，邮编：430072。
类似地，本发明人还构建了另外2株杂交瘤细胞株，其分泌的抗体为鼠源抗体，抗体分别命名为5F10和9F6。
2.抗体5C10、5F10和9F6的制备
用含10％的低IgG胎牛血清对本发明PDL-1-5C10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5C10。
抗体5F10、9F6按照上述方法制备。
3.抗体5C10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，纯化的上样流穿液，煮沸后进行检测。检测结果显示，还原型蛋白样品目标蛋白大约在50kD和25kD处，非还原型蛋白样品目标蛋白大约在150kD处(图4)。
4.鼠源抗体5C10、5F10和9F6的亲和力、竞争亲和力以及细胞亲和力的检测：
ELISA亲和力测试方法请参见实施例9，ELISA竞争亲和力测试方法请参见实施例13FACS细胞亲和力测试方法请参见实施例8。
结果如下面的表1所示。
表1:鼠源抗体5C10、5F10、9F6亲和力，竞争亲和力以及细胞亲和力以检测
Figure PCTCN2017075484-appb-000007
结果表明，三个抗体鼠源抗体在亲和力和竞争亲和力方面不逊于对照抗体PCAB(制备见实施例5)，其中，5C10在细胞亲和力及阳性率表现最好，5F10细胞亲和力优于PCAB，9F6阳性率优于对照抗体PCAB。
实施例2：单克隆抗体5C10、5F10和9F6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获得的杂交瘤细胞株LT005中提取mRNA。
按照TransGen Biotech TransScript First-Strand cDNA Synthesis SuperMix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-T1 Cloning 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：
编码单抗5C10重链可变区的核苷酸：(360bp)
Figure PCTCN2017075484-appb-000008
单抗5C10重链可变区的氨基酸序列：(120aa)
Figure PCTCN2017075484-appb-000009
编码单抗5C10轻链可变区的核苷酸序列：(318bp)
Figure PCTCN2017075484-appb-000010
单抗5C10轻链可变区的氨基酸序列：(106aa)
Figure PCTCN2017075484-appb-000011
类似地，可获得单克隆抗体9F6和5F10的轻链和重链序列。
单抗5F10重链可变区的氨基酸序列：(117aa)
Figure PCTCN2017075484-appb-000012
编码单抗5F10重链可变区的核苷酸序列：(351bp)
Figure PCTCN2017075484-appb-000013
单抗5F10轻链可变区的氨基酸序列：(106aa)
Figure PCTCN2017075484-appb-000014
编码单抗5F10轻链可变区的核苷酸序列：(318bp)
Figure PCTCN2017075484-appb-000015
单抗9F6重链可变区的氨基酸序列：(124aa)
Figure PCTCN2017075484-appb-000016
编码单抗9F6重链可变区的核苷酸序列：(372bp)
Figure PCTCN2017075484-appb-000017
单抗9F6轻链可变区的氨基酸序列：(107aa)
Figure PCTCN2017075484-appb-000018
编码单抗9F6轻链可变区的核苷酸序列：(321bp)
Figure PCTCN2017075484-appb-000019
Figure PCTCN2017075484-appb-000020
实施例3：人源化抗体5C10H1L1、5C10H1L2、5C10H2L1、5C10H2L2的轻链和重链序 列的设计
根据PDL-1蛋白的三维晶体结构(PDB Code 3BIK，Lin,DY et.al.,PNAS USA105(8):3011-6(2008)以及实施例2获得的抗体5C10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5C10H1L1、5C10H1L2、5C10H2L1、5C10H2L2的可变区序列(重链恒定区为Ig gamma-1chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834)，可变区序列如下：
1. 5C10人源化单克隆抗体5C10H1L1的轻链和重链序列
编码5C10H1L1重链可变区的核苷酸序列：(360bp)
Figure PCTCN2017075484-appb-000021
5C10H1L1重链可变区的氨基酸序列：(120aa)
Figure PCTCN2017075484-appb-000022
编码5C10H1L1轻链可变区的核苷酸序列：(321bp)
Figure PCTCN2017075484-appb-000023
5C10H1L1轻链可变区的氨基酸序列：(107aa)
Figure PCTCN2017075484-appb-000024
2. 5C10人源化单克隆抗体5C10H2L2的轻链和重链序列
编码5C10H2L2重链可变区的核苷酸序列：(360bp)
Figure PCTCN2017075484-appb-000025
5C10H2L2重链可变区的氨基酸序列：(120aa)
Figure PCTCN2017075484-appb-000026
编码5C10H2L2轻链可变区的核苷酸序列：(321bp)
Figure PCTCN2017075484-appb-000027
5C10H2L2轻链可变区的氨基酸序列：(107aa)
Figure PCTCN2017075484-appb-000028
3. 5C10人源化单克隆抗体5C10H1L2的轻链和重链序列
编码5C10H1L2重链可变区的核苷酸序列：SEQ ID NO:5，
5C10H1L2重链可变区的氨基酸序列：SEQ ID NO:6。
编码5C10H1L2轻链可变区的核苷酸序列：SEQ ID NO:11，
5C10H1L2轻链可变区的氨基酸序列：SEQ ID NO:12。
4. 5C10人源化单克隆抗体5C10H2L1的轻链和重链序列
编码5C10H2L1重链可变区的核苷酸序列：SEQ ID NO:9，
5C10H2L1重链可变区的氨基酸序列：SEQ ID NO:10。
编码5C10H2L1轻链可变区的核苷酸序列：SEQ ID NO:7，
5C10H2L1轻链可变区的氨基酸序列：SEQ ID NO:8。
实施例4：5C10人源化抗体5C10H1L1、5C10H1L2、5C10H2L1和5C10H2L2的制备和 SDS-PAGE电泳检测
将5C10H1L1、5C10H1L2、5C10H2L1和5C10H2L2的重链cDNA(重链可变区序列分别如SEQ ID NO:5、SEQ ID NO:9所示；重链恒定区序列为hIgG1序列)和轻链的cDNA(轻链可变区序列分别如SEQ ID NO:7、SEQ ID NO:11所示；轻链恒定区为human kappa序列)分别克隆到pUC57simple(金斯瑞公司提供)载体中，获得pUC57simple-5C10H1、pUC57simple5C10L1、pUC57simple-5C10H2和pUC57simple-5C10L2克隆质粒。
然后参照制备例1中的方法，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以及HiTrap protein A HP柱进行纯化。
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SDS-PAGE电泳检测，结果分别如图5、图6、图7和图8所示，还原型蛋白样品目标蛋白大约在50kD和25KD处，非还原型蛋白样品目标蛋白大约在150kD处。
实施例5：人源化抗体5C10H2L2的动力学参数测定
使用Fortebio分子相互作用仪测定人源化抗体5C10H2L2与抗原PDL-1(NCBI GenBank ID:NP_054862.1，编码核酸序列为SEQ ID NO:13，所编码的氨基酸序列为SEQ ID NO:14)结合的动力学参数。
1.前期的实验样品准备
(1)参照制备例1中的PDL-1ECD-mFc的制备方法制备PDL-1-mFc蛋白，并用TEV蛋白酶酶切PDL-1-mFc蛋白，过柱纯化获得PDL-1抗原。
PDL-1的基因序列:(870bp)
Figure PCTCN2017075484-appb-000029
Figure PCTCN2017075484-appb-000030
PDL-1蛋白的氨基酸序列:(290aa)
Figure PCTCN2017075484-appb-000031
(2)阳性对照抗体HpLp和PCAB的获得
本发明选择HpLp或PCAB作为阳性对照，其中，HpLp是已上市的Atezolizumab(商品名
Figure PCTCN2017075484-appb-000032
)，PCAB是进入临床的PDL-1抗体。
Atezolizumab(商品名
Figure PCTCN2017075484-appb-000033
)购自Roche公司。HpLp(亦称为KF025HpLp)的制备可参照美国专利申请US 2010/0203056A1例如其实施例10，该抗体的VH序列请参见该专利中的序列20，VL序列请参见该专利中的序列21。
PCAB的制备可参照美国专利US 7,943,743 B2例如其实施例1，该抗体的VH序列请参见该专利中的序列1，VL序列请参见该专利中的序列11。
2.实验方法
为检测5C10H2L2、HpLp和PCAB与抗原PDL-1的亲和力，将5μg/ml的抗原PDL-1采用氨基偶联的方式固定于AR2G传感器表面，经乙醇胺封闭，于PBST中平衡后，将传感器固定的抗原与抗体结合。抗体浓度从200nM三倍稀释，缓冲液为10Mm PBST。
采用Fortebio Data Analysis 7.0软件，分析5C10H2L2、HpLp和PCAB与抗原PDL-1的亲和力。
3.实验结果
抗体5C10H2L2、HpLp和PCAB与PDL-1结合的动力学参数见表2，动力学特征参数检测结果分别如图9-11所示。
表2：抗体5C10H2L2、HpLp、PCAB动力学参数
抗体名称 KD(M) kon(1/Ms) kon误差 kdis(1/s) kdis误差
5C10H2L2 8.08E-11 5.58E+06 2.06E+05 4.51E-04 1.66E-05
HpLp 3.68E-11 4.07E+06 1.02E+05 1.50E-04 9.99E-06
PCAB 1.28E-10 6.55E+06 3.88E+05 8.37E-04 2.25E-05
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，3种抗体均与抗原有较好的亲和力，5C10H2L2和HpLp与抗原的亲和力强于PCAB。
实施例6：Fortebio测定5C10、5C10H2L2和HpLp抑制人PDL-1与PD-1结合
为检测5C10、5C10H2L2和HpLp抑制人PDL-1与PD-1的结合，将5μg/ml的抗原PDL-1采用氨基偶联的方式固定于AR2G传感器表面，经乙醇胺封闭，于PBST中平衡。然后将传感器固定的抗原与抗体结合，抗体浓度从33.33nM三倍稀释，缓冲液为10Mm PBST。然后把传感器于10μg/ml配体PD-1中浸泡420s。
5C10、5C10H2L2和HpLp抑制人PDL-1与PD-1结合结果如图12所示。由图可见，各抗体均能够有效地抑制人PDL-1与PD-1的结合，并且其结合效率呈剂量依赖关系，各剂量的荧光强度及曲线模拟的结合效率EC50见表3。
表3：抗体5C10、5C10H2L2和HpLp抑制人PDL-1与PD-1结合
抗体(nM) 5C10 5C10H2L2 HpLp
33.33 0.0058 -0.0109 0.0127
11.11 0.0038 -0.0078 0.0149
3.704 0.0088 -0.0007 0.0073
1.235 0.0289 0.0103 0.0268
0.4115 0.0599 0.0425 0.0697
0.1372 0.0739 0.0732 0.0867
0.04572 0.0773 0.0601 0.0947
EC50(nM) 0.817 0.654 0.625
结果表明，3种抗体均能够有效地抑制人PDL-1与PD-1的结合，并且其结合效率呈剂量依赖关系。
实施例7：5C10H2L2与HpLp阻断PD1/PDL-1结合
采用HTRF的方法比较5C10H2L2与HpLp阻断PD1/PDL-1结合。使用PD1/PDL-1binding assay(CISBIO，货号:63ADK000CPLPEH)试剂盒。用稀释缓冲液稀释5C10H2L2与HpLp，100μg/mL起始，3倍稀释，10个浓度点。加入2μL样品、4μL PDL-1-EuK和4μL Tag-PD1，离心，室温孵育20分钟。加入10μL anti-Tag-XL665，瞬时离心，室温孵育2小时。使用PHERA star Fs(BMG)进行读数，将数据导入Graph Prism进行计算。
结果表明，HpLp和5C10H2L2对PD1/PDL-1结合阻断的能力一致，分别为67.29ng/ml和68.97ng/ml，2种抗体均能够有效地抑制人PDL-1与PD-1的结合。
实施例8：流式细胞仪方法检测人源化抗体与细胞表面抗原PDL-1的结合活性
首先构建表达PDL-1抗原的宿主细胞293T；然后用本发明中制备的人源化抗体5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1及阳性对照抗体(HpLp和PCAB)对宿主细胞进行标记。然后采用流式细胞术分析验证抗体5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1及阳性对照抗体(HpLp和PCAB)对细胞表面具有天然构象的抗原的特异性结合。
具体步骤如下：
1.表达PDL-1抗原的宿主细胞293T的构建
按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将包含PDL-1的载体pLenti6.3-PDL-1(载体pLenti6.3购自Invitrogen公司)转染293F细胞，经筛选获得稳定表达PDL-1的克隆群体。
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PDL-1抗原的宿主细胞293T，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，0nM的各抗体稀释液，冰上与表达PDL-1的293T细胞孵育2小时，PBS清洗3次。用PBS按1:100稀释FITC-Goat-Anti-Human IgG,每管加入100μL,冰上孵育1小时，PBS清洗3次。
并加入300μL PBS重悬后，在流式细胞仪上用FITC通道检测荧光信号。
3.实验结果
检测5C10H1L1、5C10H1L2、5C10H2L1、5C10H2L2及阳性对照抗体(HpLp和PCAB)与293T细胞表面PDL-1的结合结果分别如图13-18所示。
由图可见，被检测抗体均能有效地结合宿主细胞293T表面的靶标PDL-1蛋白，并且其结合效率呈剂量依赖关系。通过对结合的被检测抗体进行荧光定量分析，曲线模拟各抗体的结合效率EC50，如下的表4所示。
表4：流式细胞仪检测5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1、HpLp、PCAB结合宿主细胞293T表面抗原PDL-1的荧光强度分析
Figure PCTCN2017075484-appb-000034
结果表明，被检测抗体均能有效地结合宿主细胞293T表面的靶标PDL-1蛋白，并且其结合效率呈剂量依赖关系。
实施例9：间接ELISA方法检测人源化抗体与人PDL-1的结合活性
采用间接ELISA方法分别测定5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1及阳性对照抗体(HpLp和PCAB)与人PDL-1的结合活性。酶标板中加入抗原孵育，4℃度过夜，用1％的BSA 37℃度封闭2h后，分别加入抗体，37℃度孵育30min，加入HRP标记羊抗人IgG(H+L)二抗(Jackson，109-035-088)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测上述抗体与人PDL-1结合结果分别如图19所示。由图可见，5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1、HpLp、PCAB能有效地结合人PDL-1蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强度及曲线模拟的结合效率EC50见表5。
表5：5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1、HpLp、PCAB与人PDL-1的结合(间接ELISA)
Figure PCTCN2017075484-appb-000035
结果表明，本发明的抗体能有效地结合人PDL-1蛋白，并且其结合效率呈剂量依赖关系。
实施例10：间接ELISA方法检测5C10H2L2抗体与猴PDL-1的结合活性
考虑到药代动力学实验和毒理学实验需要用到实验动物，本实验目的在于判定是不是和猴子的靶抗原可以结合，如果可以结合就可以采用猴子做药代动力学和毒理实验。
采用间接ELISA方法分别测定5C10H2L2和阳性对照抗体HpLp与猴PDL-1的结合活性。酶标板中加入抗原孵育，4℃度过夜，用1％的BSA 37℃度封闭2h后，分别加入抗体，37℃度孵育30min，加入HRP标记羊抗人IgG(H+L)二抗(Jackson，109-035-088)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测5C10H2L2和HpLp与猴PDL-1结合结果分别如图20所示。由图可见，5C10H2L2和HpLp能有效地结合猴PDL-1蛋白，并且其结合效率呈剂量依赖关系，各剂量的荧光强 度及曲线模拟的结合效率EC50见表6。
表6：5C10H2L2和HpLp与猴PDL-1的结合(间接ELISA)
Figure PCTCN2017075484-appb-000036
结果表明，5C10H2L2和HpLp能有效地结合猴PDL-1蛋白，且5C10H2L2与猴PDL-1蛋白的结合能力强于HpLp,并且其结合效率呈剂量依赖关系。
实施例11：间接ELISA方法检测5C10H2L2抗体与人PDL-1、人PD-L2以及鼠PDL-1 的结合活性
采用间接ELISA方法测定5C10H2L2抗体与人PDL-1、人PD-L2(购自义翘神州，货号10292-H08H)以及鼠PDL-1(购自义翘神州，货号50010-M08H)的结合活性。人PDL-1、人PD-L2以及鼠PDL-1，酶标板中加入0.5μg/ml抗原100μL孵育，4℃度过夜。抗体稀释至起始浓度为1μg/ml，按3倍比例梯度稀释，共稀释11个浓度。用1％的BSA 37℃度封闭2h后，分别加入抗体，37℃度孵育30min。按1:20000稀释，加入HRP标记羊抗人IgG(H+L)二抗(购自Jackson，货号109-035-088)，用TMB(购自Neogen，货号308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
5C10H2L2抗体与人PDL-1、人PD-L2以及鼠PDL-1结合结果如图21所示。由图可见，5C10H2L2能有效地结合人PDL-1蛋白，并且其结合效率呈剂量依赖关系，各剂量的吸光强度及曲线模拟的结合效率EC50＝9.16ng/ml；而5C10H2L2与人PD-L2和鼠PDL-1不存在结合。
结果表明，5C10H2L2抗体与人PDL-1特异性结合，而Atezolizumab与鼠PDL-1结合 (参见
Figure PCTCN2017075484-appb-000037
在FDA的公开审评资料PHARMACOLOGY REVIEW，Application number761034Orig1s000),本发明具有优良的特异性。
实施例12：竞争ELISA方法检测人源化抗体与PDL-1、PD-1的竞争结合活性
采用竞争ELISA方法分别测定5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1及阳性对照抗体(HpLp和PCAB)与PD-1竞争结合抗原PDL-1的能力。酶标板中加入受体孵育，4℃度过夜，用1％的BSA 37℃度封闭2h，抗体与抗原在室温混合孵育15分钟后，混合液转移到酶标板中，37℃度孵育30min，加入HRP标记羊抗鼠IgG(H+L)二抗(Jackson，109-035-062)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测上述抗体与PD-1竞争结合抗原PDL-1的结果如图22所示。由图可见，5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1、HpLp、PCAB均能有效地与PD-1竞争结合抗原PDL-1，并且其结合效率呈剂量依赖关系，各剂量的荧光强度及曲线模拟的结合效率EC50见表7。
表7：5C10H1L1、5C10H1L2、5C10H2L2、5C10H2L1、HpLp、PCAB与PD-1竞争结合PDL-1ELISA
Figure PCTCN2017075484-appb-000038
Figure PCTCN2017075484-appb-000039
结果表明，被检测抗体均能有效地与PD-1竞争结合抗原PDL-1，并且其结合效率呈剂量依赖关系。
实施例13：竞争ELISA方法检测5C10H2L2抗体与PDL-1、B7-1的竞争结合
采用竞争ELISA方法分别测定5C10H2L2及阳性对照抗体(HpLp和PCAB)与B7-1(B7-1-hFc，制备例3制得)竞争结合抗原PDL-1的能力。酶标板中加入受体孵育，4℃度过夜，用1％的BSA 37℃度封闭2h，抗体与抗原在室温混合孵育15分钟后，混合液转移到酶标板中，37℃度孵育30min，加入HRP标记羊抗鼠IgG(H+L)二抗(Jackson，109-035-062)，用TMB(Neogen，308177)进行显色反应5min，并在酶标仪中检测450nm波长吸光度。
检测上述抗体与B7-1竞争结合抗原PDL-1的结果如图23所示。由图可见，5C10H2L2、HpLp、PCAB均能有效地与B7-1竞争结合抗原PDL-1，各剂量的荧光强度及曲线模拟的结合效率EC50见表8。
表8：5C10H2L2、HpLp、PCAB与B7-1竞争结合PDL-1ELISA
Figure PCTCN2017075484-appb-000040
*抑制不彻底，应该理解为竞争实验中的数据窗口与另外两个抗体相比较小；另如表7所示，HpLp在1:27开始再增加浓度时，如1:9，1:3，3μg/ml时，OD读数随浓度增加降低不明显，不能达到5C10H2L2和PCAB 3ug/ml时的数值。
结果表明，被检测抗体均能与B7-1竞争结合抗原PDL-1，其中5C10H2L2的竞争结合活性较强，EC50是PCAB的1/2倍；HpLp随着浓度增高，但是抑制程度并未呈现明显增加趋势。
实施例14：5C10H2L2及阳性对照抗体(HpLp和PCAB)的细胞生物学活性分析
为检测单克隆抗体5C10H2L2及阳性对照抗体(HpLp和PCAB)对外周血单核细胞(Peripheral blood mononuclear cell:PBMC)IL-2和IFN-γ分泌的影响，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5C10H2L2(hIgG做为对照)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24、图25所示：5C10H2L2、HpLp和PCAB均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呈剂量依赖关系。
实施例15：经修饰人类IgG1恒定区的单抗5C10H2L2-IgG1mt的设计和制备
本发明对重链恒定区为Ig gamma-1chain C region，ACCESSION:P01857，轻链恒定区为Ig kappa chain C region，ACCESSION:P01834的EU编号***234,235，237进行突变如下：L234A，L235A，G237A，命名为5C10H2L2-IgG1mt。
参照实施例4的方法制备5C10H2L2-IgG1mt。
实施例16：ForteBio测定5C10H2L2-IgG1mt与FcγRIIIa、C1q的动态亲和力
1.通过ForteBio(购自Pall货号Octet,Qke)分析表征了5C10H2L2-IgG1mt、
Figure PCTCN2017075484-appb-000041
与FcγRIIIa的亲和力和结合动力学。
具体步骤如下：
使用ForteBio提供的标准方法和试剂盒，通过生物素与链霉素的结合将纯化的FcγRIIIa-Biotin连接到SA芯片(链霉素包被的芯片)上，固定条件：1μg/ml FcγRIIIa-Biotin,300s。抗体浓度为4000nM结合120S，的，在PBST(pH7.4)缓冲液解离180S。用Octet软件将结合和解离曲线模型拟合。
实验结果如图26A和26B所示。
结果表明：5C10H2L2-IgG1mt、
Figure PCTCN2017075484-appb-000042
均不与FcγRIIIa结合，间接表明其都不具备ADCC活性。
2.通过ForteBio(购自Pall货号Octet,Qke)分析表征了5C10H2L2-IgG1mt、
Figure PCTCN2017075484-appb-000043
与C1q(购自fitzgerald，货号32R-AC049)的亲和力和结合动力学。
具体步骤如下：
使用ForteBio提供的标准方法和试剂盒，通过生物素与链霉素的结合将纯化抗体连接到SA芯片(链霉素包被的芯片)上，固定条件：20ug/ml Antibody-Biotin,300s。C1q浓度为200nM,2倍浓度梯度，结合120S。在PBST(pH7.4)缓冲液解离180S。用Octet软件将结合和解离曲线模型拟合。为了使结合常数估计中的亲合力的影响降至最小，只用对应于结合和解离阶段的最初的数据段进行拟合。测定的KD、kon和koff值显示在表9中，具体图谱如图27A和图27B。
表9：5C10H2L2-IgG1mt、
Figure PCTCN2017075484-appb-000044
与C1q动态亲和力
Figure PCTCN2017075484-appb-000045
结果表明，5C10H2L2-IgG1mt比
Figure PCTCN2017075484-appb-000046
与C1q动态亲和力更低。
实施例17：测定5C10H2L2-IgG1mt的CDC细胞活性
首先培养肿瘤细胞HCC1954(购自ATCC货号CRL-2338)是PDL-1阳性的细胞。用对应培养基(RPMI1640+10％牛血清)进行培养。HCC1954检测培养基(RPMI1640+10％人血清)稀释5C10H2L2-IgG1mt药物10000μg/mL起始，5倍稀释，10个浓度梯度。采用常规胰酶消化方法上述的肿瘤细胞，收集管细胞数，用对应检测培养基(含RPMI1640+10％ 人血清)重悬，加入10000细胞/孔至对应的稀释好抗体96孔板中，共培养5小时。然后每孔加入CCK8试剂20μL(购自东仁化学科技有限公司，货号CK04,Lot:JJ744)，反应至3小时，酶标仪450nm读数(厂家：Molecular Devices,型号：SpectraMax M2)，通过检测线粒体内的脱氢酶的活性，反映出抗体对HCC1954细胞的杀伤作用。
结果显示，5C10H2L2-IgG1mt未对HCC1954细胞产生CDC杀伤作用。
实施例18：对结肠癌的体内疗效
1.受试药物
5C10H2L2-IgG1mt，
Figure PCTCN2017075484-appb-000047
人IgG均由四川科伦药物研究院有限公司提供；其中
Figure PCTCN2017075484-appb-000048
购自Roche公司，人IgG购自成都蓉生药业有限责任公司。
配制方法：三药均用含0.1％BSA生理盐水稀释成所需浓度。
1.试验细胞和动物
MC-38/H-11细胞是小鼠结肠癌MC-38(购自Cobioer,货号CBP60825)细胞通过CRISPR/Cas9技术敲除小鼠内源性PDL-1，转染并表达人PDL-1的单克隆细胞,因此，MC-38/H-11细胞只高表达人PDL-1蛋白。
C57BL/6小鼠，7-8周，♀，购自上海斯莱克实验动物有限责任公司。
2.实验步骤
每只小鼠皮下接种1×105MC-38/H-11细胞，接种后第二天(D0)随机分组并腹腔注射(IP)药物，隔1天1次(Q2D)；溶剂组注射相同体积的人IgG(15mg/kg)，5C10H2L2-IgG1mt(1.5、5、15mg/kg)、
Figure PCTCN2017075484-appb-000049
(15mg/kg)，注射体积0.1mL/10g体重。各组均为10只小鼠。
3.实验指标
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为：
V＝1/2×a×b2其中a、b分别表示长、宽。
T/C％＝T/C×100,C、T分别为溶剂组和治疗组的肿瘤体积或肿瘤重量。
抑瘤率(TGI)(％)＝(C-T)/C×100,C、T分别为溶剂组和治疗组的肿瘤体积或肿瘤重量。
4.实验结果
如下面的表10所示。
表10：5C10H2L2-IgG1mt(1.5、5、15mg/kg)、
Figure PCTCN2017075484-appb-000050
对小鼠结肠癌MC-38/H-11小鼠皮下移植瘤的疗效
Figure PCTCN2017075484-appb-000051
注：随机分组，第一次给药时间为D0；D27为给药后第27天。
5C10H2L2-IgG1mt(1.5、5、15mg/kg)对MC-38/H-11小鼠皮下移植瘤的抑瘤率分别为63.9％、75.8％和68.6％(根据平均肿瘤体积计算)；鉴于每组肿瘤的个体差异较大，采用中位肿瘤体积计算抑瘤率比较合理，则抑瘤率调整为100％、100％和100％；参比药物
Figure PCTCN2017075484-appb-000052
(15mg/kg)对MC-38/H-11的抑瘤率为93.8％(根据中位肿瘤体积计算)；采用中位肿瘤重量计算抑瘤率，则5C10H2L2-IgG1mt(1.5、5、15mg/kg)对MC-38/H-11的抑瘤率分别为100％、100％、100％，
Figure PCTCN2017075484-appb-000053
的抑瘤率为93.7％；中位肿瘤体积与中位肿瘤重量计算所得抑瘤率非常一致，说明肿瘤体积测量方法的可靠性。5C10H2L2-IgG1mt(1.5、5、15mg/kg)不但抑制肿瘤生长，还抑制肿瘤成瘤，实验结束时(D27)，5C10H2L2-IgG1mt(1.5、5、15mg/kg)剂量组成瘤率分别为40％、40％和40％，
Figure PCTCN2017075484-appb-000054
组的成瘤率为50％。荷瘤小鼠对以上药物均能很好耐受，没有明显体重减轻等症状发生。相比较，5C10H2L2-IgG1mt(1.5、5、15mg/kg)对小鼠结肠癌MC-38/H-11小鼠皮下移植瘤较
Figure PCTCN2017075484-appb-000055
具有更强的抑瘤作用。
实施例19：对肺癌的体内疗效
建模方法：将非小细胞肺癌细胞HCC827(购自ATCC货号CRL-2868)皮下接种NOG鼠，构建肺癌荷瘤鼠模型，待肿瘤长到约100mm3,给药前小鼠静脉注射激活后的人PBMCs 模拟人免疫***，然后给药。
给药方案：给药剂量为10mg/kg，静脉注射、每两天一次，共计给药四次。给药后每周两次测定肿瘤体积。分为对照IgG，5C10H2L2-IgG1mt，
Figure PCTCN2017075484-appb-000056
三组，每组6只。
肿瘤生长曲线见图28。
结果显示，在第4天开始，5C10H2L2-IgG1mt组的肿瘤体积明显小于
Figure PCTCN2017075484-appb-000057
组和IgG对照组，5C10H2L2-IgG1mt的肿瘤生长几乎被完全抑制，
Figure PCTCN2017075484-appb-000058
组和IgG对照组肿瘤在不断生长，证明了本发明抗体较
Figure PCTCN2017075484-appb-000059
具有更强的体内抑瘤作用。
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 (27)

  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:15－17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:18－20的CDR；
    或者
    所述单克隆抗体的重链可变区包含氨基酸序列为SEQ ID NO:29－31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:32－34的CDR；
    或者
    所述单克隆抗体的重链可变区包含氨基酸序列为SEQ ID NO:35－37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:38－40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；
    或者
    所述重链可变区的氨基酸序列选自SEQ ID NO:21,和/或所述轻链可变区的氨基酸序列选自SEQ ID NO:23；
    或者
    所述重链可变区的氨基酸序列选自SEQ ID NO:25,和/或所述轻链可变区的氨基酸序列选自SEQ ID NO:27。
  3. 根据权利要求1至2中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。
  4. 根据权利要求1至2中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PDL-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；
    优选地，所述单克隆抗体的恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区；
    优选地，所述单克隆抗体的恒定区选自突变的人IgG1恒定区。
  6. 根据权利要求5所述的单克隆抗体或其抗原结合片段，其中所述突变的人IgG1恒定区中，按照EU编号***重链恒定区包括N297A突变。
  7. 根据权利要求5或6所述的单克隆抗体或其抗原结合片段，所述突变的人IgG1恒定区中，按照EU编号***重链恒定区在234、235、237位点至少一个发生突变，突变后抗体或其抗原结合片段与FcγRIIIa和/或C1q的动态亲和力下降。
  8. 根据权利要求5或6所述的单克隆抗体或其抗原结合片段，所述突变的人IgG1恒定区中，按照EU编号***重链恒定区在234、235和237位点进行如下的1种、2种或3种突变：L234A、L235A或G237A。
  9. 根据权利要求5-8任一所述的单克隆抗体或其抗原结合片段，所述突变使抗体或抗原结合片段具有降低的ADCC和/或CDC活性。
  10. 根据权利要求1至2中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LT005产生的单克隆抗体，所述杂交瘤细胞株LT005保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015133。
  11. 分离的核酸分子A，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:15－17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；
    在另一个实施方案中，所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO:29－31的CDR，
    优选地，所述抗体的重链具有SEQ ID NO:21所示的氨基酸序列，
    更优选地，所述核酸分子具有SEQ ID NO:22所示的核苷酸序列；
    在另一个实施方案中，所述抗体的重链可变区包含：氨基酸序列为SEQ ID NO:35－37的CDR，
    优选地，所述抗体的重链具有SEQ ID NO:25所示的氨基酸序列，
    更优选地，所述核酸分子具有SEQ ID NO:26所示的核苷酸序列。
  12. 分离的核酸分子B，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:18－20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；
    在另一个实施方案中，所述抗体的轻链可变区包含：氨基酸序列为SEQ ID NO:32－34的CDR，
    优选地，所述抗体的轻链具有SEQ ID NO:23所示的氨基酸序列，
    更优选地，所述核酸分子具有SEQ ID NO:24所示的核苷酸序列；
    在另一个实施方案中，所述抗体的轻链可变区包含：氨基酸序列为SEQ ID NO:38－40的CDR，
    优选地，所述抗体的轻链具有SEQ ID NO:27所示的氨基酸序列，
    更优选地，所述核酸分子具有SEQ ID NO:28所示的核苷酸序列。
  13. 分离的核酸分子C，其包含权利要求11所述的核酸分子A，以及权利要求12所述的核酸分子；可选地，核酸分子C还包括连接序列，其用于连接核酸分子A和核酸分子B。
  14. 一种载体，其包含权利要求11所述的核酸分子A、权利要求12所述的核酸分子B或权利要求13所述的核酸分子C。
  15. 一种宿主细胞，其包含权利要求11所述的核酸分子A、权利要求12所述的核酸分子B或权利要求13所述的核酸分子C，或者包含权利要求14所述的载体。
  16. 制备权利要求1至9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权利要求15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。
  17. 杂交瘤细胞株LT005，其保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:C2015133。
  18. 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9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。
  19. 双功能抗体偶联物，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9中任一项所述的单克隆抗体或其抗原结合片段，所述偶联部分为第二生物学功能片段；优选的，所述第二生物学功能片段为具有结合活性多肽、蛋白、聚乙二醇(PEG)，核素，核酸，小分子毒素，受体或配体。
  20. 多特异性抗体，所述抗体由第一抗体或其片段与其他抗体或其片段或抗体类似物通过偶联形成，各抗体或其片段或抗体类似物保持原结合特异性，所述第一抗体或其片段为权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段；优选的，所述多特异性抗体为双特异性抗体或三特异性抗体或四特异性抗体。
  21. 试剂盒，其包括权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段，或者包括权利要求18或19所述的偶联物，或者包括权利要求20所述的多特异性抗体；
    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。
  22. 权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段或者权利要求18或19所述的偶联物，或者权利要求20所述的多特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测PDL-1在样品中的存在或其水平。
  23. 一种药物组合物，其包含权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段或者权利要求18或19所述的偶联物，或者权利要求20所述的多特异性抗体；可选地，其还包括药学上可接受的载体和/或赋形剂。
  24. 权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段或者权利要求18或19所述的偶联物，或者权利要求20所述的多特异性抗体在制备预防和/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，所述肿瘤选自乳腺癌、肺癌例如非小细胞性肺癌、肝癌、胃癌、肠癌例如结肠癌或直肠癌、食管癌、卵巢癌、***、肾癌、***癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤和白血病。
  25. 权利要求1至9中任一项所述的单克隆抗体或其抗原结合片段或者权利要求18或19所述的偶联物，或者权利要求20所述的多特异性抗体在制备如下药物中的用途：
    阻断PDL-1与PD-1或B7-1结合的药物，
    调节(例如下调)PDL-1活性或水平的药物，
    解除PD-1或者PDL-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。
  26. 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9中任一项所述的单克隆抗体或其抗原结合片段或者权利要求18或19所述的偶联物，或者权利要求20所述的多特异性抗体的步骤，所述方法选自如下：
    阻断PDL-1与PD-1或B7-1结合的方法，
    调节(例如下调)PDL-1活性或水平的方法，
    解除PD-1或者PDL-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。
  27. 一种治疗和/或预防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9中任一项所述的单克隆抗体或其抗原结合片段或者权利要求18或19所述的偶联物或者权利要求20所述的多特异性抗体的步骤；优选地，所述肿瘤选自乳腺癌、肺癌例如非小细胞性肺癌、肝癌、胃癌、肠癌例如结肠癌或直肠癌、食管癌、卵巢癌、***、肾癌、***癌、膀胱癌、胰腺癌、神经胶质瘤、黑素瘤和白血病。
PCT/CN2017/075484 2016-03-04 2017-03-02 一种pdl-1抗体、其药物组合物及其用途 WO2017148424A1 (zh)

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