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WO2017197667A1 - 抗人pd-l1人源化单克隆抗体及其应用 - Google Patents

抗人pd-l1人源化单克隆抗体及其应用 Download PDF

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WO2017197667A1
WO2017197667A1 PCT/CN2016/084643 CN2016084643W WO2017197667A1 WO 2017197667 A1 WO2017197667 A1 WO 2017197667A1 CN 2016084643 W CN2016084643 W CN 2016084643W WO 2017197667 A1 WO2017197667 A1 WO 2017197667A1
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seq
sequence
sequences
human
antibody
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朱一翔
郭树华
张佳春
李戈
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Reyoung (suzhou) Biology Science & Technology Co Ltd
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Reyoung (suzhou) Biology Science & Technology Co Ltd
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Priority to US16/084,710 priority patent/US11155626B2/en
Priority to JP2018550775A priority patent/JP6753945B2/ja
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention relates to the field of biomedicine, in particular to an anti-human PD-L1 humanized monoclonal antibody and application thereof.
  • the adaptive response process of the human immune system mainly includes the activation, differentiation and proliferation of two types of immune cells, T cells and B cells.
  • T cell function is regulated by two types of signals.
  • One class is the antigen-specific signal provided by the T cell receptor (TCR) to recognize the MHC-antigen complex on antigen presenting cells (APC).
  • the other type is the costimulatory and inhibitory signal formed between the immunological checkpoint protein expressed on APC cells and T cells. This type of costimulatory or inhibitory signal often plays a crucial role in the proliferation, differentiation and activation of T cells.
  • immune checkpoints play an important role in maintaining the body's own tolerance (preventing autoimmunity) and protecting the body from external pathogen infection.
  • the PD-L1/PD1 signaling pathway is a very important co-suppression signaling pathway in the immune response.
  • Programmed Death Receptor-1 (PD-1, also known as CD279) has two cell surface glycoprotein ligands, PD-L1 (also known as B7-H1, CD274) and PD-L2 (also known as B7).
  • PD-L1 also known as B7-H1, CD274
  • PD-L2 also known as B7.
  • -DC CD273
  • the human PD-L1 gene encodes 290 amino acids (1-18 amino acids are signal peptides, 19-238 amino acids are extracellular segments, 239-259 amino acids are transmembrane segments, and 260-290 amino acids are intracellular segments) . It is a type I membrane protein that is typically expressed on T cells, B cells, dendritic cells, macrophages, and many non-hematopoietic cells. Studies have shown that when PD-L1 binds to PD-1, it will complement the protein tyrosine phosphatases SHP-1 and SHP-2 with the SH2 domain.
  • TAM immunoreceptor tyrosine activation motif
  • PD-L1 if the tumor tissue expresses PD-L1, it can weaken the killing effect of the immune system on the tumor tissue by binding to PD-1 of the immune cell. It has been found that PD-L1 is highly expressed in many tumor tissues (stomach cancer, breast cancer, pancreatic cancer, ovarian cancer, lung cancer, prostate cancer, malignant melanoma, etc.) and bone marrow cells infiltrating the tumor microenvironment. The expression of PD-L1 is also closely related to the poor prognosis of melanoma, breast cancer and ovarian cancer. If the ligation reaction between PD-L1 and PD-1 can be blocked, the effector function of T cells can be restored. Tumors such as melanoma can express PD-L1 at the beginning of their formation, and thus have an innate immune escape ability. The expression level of PD-L1 is also closely related to the prognosis of the disease.
  • the expression level of PD-L1 has become a very important biomarker when using immunotherapy against PD-1/PD-L1 signaling pathway, which can help researchers speculate which patients are more likely to have such immunotherapy. Produce a response.
  • Avel ⁇ mab Another PD-L1 monoclonal antibody, Avel ⁇ mab, jointly developed by Pfizer and Merck, is also being evaluated for efficacy and safety in patients with metastatic Merkel cell carcinoma.
  • PD-1 is found to be highly expressed on the surface of CD8+ T cells that specifically recognize HIV, and the virus specifically recognizes CD8+ T cells of HIV by activating the PD-L1/PD-1 signaling pathway.
  • the activity is inhibited, the cytokine secretion ability and the proliferation ability of the T cell itself are greatly weakened, and the acquired immune function defect is caused.
  • blocking the PD-L1/PD-1 signaling pathway has considerable application value in the treatment of this type of disease.
  • an object of the present invention is to provide an anti-human PD-L1 humanized monoclonal antibody having good specificity, high affinity and stability.
  • a first aspect of the invention relates to an anti-human PD-L1 humanized monoclonal antibody or antigen binding portion thereof comprising a CDR region selected from the group consisting of:
  • sequences of the heavy chain CDR1, CDR2, and CDR3 are set forth in SEQ ID NOs: 18-20, respectively, and the sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 34-36, respectively, or are combined with the above sequences. a sequence of the same antigenic epitope;
  • sequences of the heavy chain CDR1, CDR2, and CDR3 are set forth in SEQ ID NOs: 18-20, respectively, and the sequences of the light chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 52, 35, and 36, respectively, or A sequence that binds to a sequence of the same antigenic epitope.
  • the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof of the present invention further comprises a heavy chain variable region framework region selected from the group consisting of FR1, FR2, FR3, and FR4, respectively.
  • the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof of the present invention further comprises a light chain variable region framework region selected from the group consisting of FR1, FR2, FR3, and FR4, respectively.
  • the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof of the present invention comprises a heavy chain variable region selected from the group consisting of SEQ ID NO: 6, or A sequence that binds to a sequence of the same antigenic epitope.
  • the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof of the present invention comprises a light chain variable region selected from the group consisting of SEQ ID NO: 8, 45 or 51 , or The sequence identity with the above sequence is greater than 70%, 80%, 85%, 90%, 95%, 99%, respectively.
  • the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof of the present invention has a heavy chain sequence as shown in SEQ ID NO: 10.
  • the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof of the present invention has a light chain sequence as shown in SEQ ID NO: 26, 42 or 48.
  • a nucleic acid molecule according to any one of the second aspects of the invention comprising a nucleic acid sequence capable of encoding an antibody heavy chain variable region, the heavy chain variable region comprising an amino acid sequence selected from the group consisting of:
  • sequences that satisfies at least one of the following (1) sequences: a) binding to the same antigenic epitope; b) identity greater than 70%, 80%, 85%, 90% or 97%.
  • the heavy chain variable region comprises an amino acid sequence selected from the group consisting of:
  • SEQ ID NO: 6 or a sequence that satisfies at least one of the following three: a) binding to the same epitope, b) greater than 70%, 80%, 85%, 90% or 97% identity, c) a substitution with one to several nucleotides in the framework region of the aforementioned sequence.
  • the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 5.
  • nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 9.
  • a nucleic acid molecule according to any one of the third aspects of the invention comprising a nucleic acid sequence capable of encoding an antibody light chain variable region, the light chain variable region comprising an amino acid sequence selected from the group consisting of:
  • sequences satisfying at least one of the following (1) to (3) sequences: a) binding to the same antigenic epitope; b) identity greater than 70%, 80%, 85%, 90% or 97%.
  • the light chain variable region comprises an amino acid sequence selected from the group consisting of:
  • SEQ ID NO: 8, 45 or 51 or a sequence which satisfies at least one of the following three compared to the preceding sequence: a) binding to the same antigenic epitope, b) identity greater than 70%, 80%, 85%, 90% Or 97%, c) Substitution with one to several nucleotides in the framework region of the aforementioned sequence.
  • the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 7, 43, or 49.
  • nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NO: 25, 41 or 47.
  • a fourth aspect of the invention relates to a vector comprising the nucleic acid molecule of any of the second or third aspect of the invention.
  • the vector referred to in the present invention contains the nucleic acid molecule of any one of the second aspect of the invention and the nucleic acid molecule of any of the third aspect.
  • a fifth aspect of the invention relates to a host cell comprising the nucleic acid molecule of any one of the second or third aspects of the invention or the vector of any of the fourth aspect of the invention.
  • a sixth aspect of the invention relates to a conjugate comprising the anti-human PD-L1 humanized monoclonal antibody of any one of the first aspects of the invention, or an antigen-binding portion thereof, and other biologically active substance, said anti-human PD-
  • the L1 humanized monoclonal antibody or antigen binding portion thereof is conjugated to other biologically active substances either directly or through a linker.
  • the other biologically active substance is selected from the group consisting of chemicals and toxins which can directly or indirectly inhibit cell growth or kill cells, or inhibit the activation or killing of cells by activating the body's immune response, thereby treating tumors.
  • a seventh aspect of the invention relates to a composition (for example, a pharmaceutical composition) comprising the anti-human PD-L1 humanized monoclonal antibody of any one of the first aspects of the invention, or an antigen-binding portion thereof, the second aspect or the third aspect
  • a composition for example, a pharmaceutical composition
  • composition eg, a pharmaceutical composition
  • the other biologically active substance including but not limited to other antibodies, fusion proteins or drugs (eg anti-tumor drugs such as radiotherapy, chemotherapy) drug).
  • the invention further relates to a diagnostic reagent or kit comprising the anti-human PD-L1 humanized monoclonal antibody of any of the first aspects of the invention, or an antigen binding portion thereof, for use in vitro ( For example, cells or tissues) or in vivo (eg, human or animal models) diagnose PD-L1-related diseases (eg, tumor or viral infections, such as viral infections with high expression of PD-L1 or tumors with high expression of PD-L1).
  • PD-L1-related diseases eg, tumor or viral infections, such as viral infections with high expression of PD-L1 or tumors with high expression of PD-L1.
  • the tumor includes, but is not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, Gliomas, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial cancer, osteosarcoma, thyroid cancer, prostate cancer;
  • the viral infections include, but are not limited to, acute, subacute or chronic HBV, HCV, HIV infection.
  • the present invention also relates to the anti-human PD-L1 humanized monoclonal antibody or antigen-binding portion thereof according to any one of the first aspects of the invention, the nucleic acid molecule according to any one of the second or third aspect, or the fourth aspect
  • the vector of any one of the fifth aspect, the conjugate of any one of the sixth aspect, or the composition of any of the seventh aspect for use in the preparation of a prophylactic or therapeutic disease associated with PD-L1 (eg, a tumor, a microorganism Use of a drug for viral infection, such as a tumor with high expression of PD-L1 or a virus with high expression of PD-L1.
  • a prophylactic or therapeutic disease associated with PD-L1 eg, a tumor, a microorganism
  • a drug for viral infection such as a tumor with high expression of PD-L1 or a virus with high expression of PD-L1.
  • the tumor includes, but is not limited to, lung cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematological malignancy, head and neck cancer, Gliomas, gastric cancer, nasopharyngeal cancer, laryngeal cancer, cervical cancer, endometrial cancer, osteosarcoma, thyroid cancer, prostate cancer;
  • the microbial infections include, but are not limited to, bacteria, fungi, protozoal infections; However, it is not limited to acute, subacute or chronic HBV, HCV, HIV infection.
  • the term “antibody” refers to an immunoglobulin molecule usually composed of two identical pairs of polypeptide chains each 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 portion" of an antibody refers to one or more portions of a full-length antibody that retain the ability to bind to the same antigen (eg, PD-L1) to which the antibody binds, competing with intact antibodies. Specific binding to antigen. 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 portion by enzymatic or chemical fragmentation of an intact antibody.
  • the antigen binding portion comprises a Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragment, 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.
  • the present invention has at least the following advantages: the present invention obtains an anti-human PD-L1 humanized monoclonal antibody having good specificity, high affinity and stability by screening, and the antibody can It specifically binds to human PD-L1 and does not bind to B7 family members, which can enhance the activation of T cells by binding to activated T cells, and has a significant inhibitory effect on tumor growth.
  • Figure 1 is a graph showing the results of ELISA-binding activity of a murine PD-L1 antibody
  • Figure 2 is a graph showing the results of ELISA inhibitory activity of a murine PD-L1 antibody
  • Figure 3 is a graph showing the results of cell binding activity of a murine PD-L1 antibody
  • Figure 4 is a graph showing the results of cytostatic activity of a murine PD-L1 antibody
  • Figure 5 is a graph showing the binding kinetics of the humanized PD-L1 antibody
  • Figure 6 is a graph showing the binding specificity of humanized PD-L1 antibody to other B7 family members and the binding results to different species of PD-L1 protein;
  • Figure 7 is a graph showing the results of binding specificity of a humanized PD-L1 antibody to CHO cells surface-expressing PD-L1;
  • Figure 8 is a graph showing the binding specificity of a humanized PD-L1 antibody and a recombinant human PD-L1 fusion protein
  • Figure 9 is a graph showing the results of blocking effect of PD-L1 binding to PD-1 by humanized PD-L1 antibody
  • Figure 10 is a graph showing the effect of humanized PD-L1 antibody on the secretion of cytokine IFN- ⁇ in a mixed lymphocyte reaction
  • Figure 11 is a graph showing the effect of humanized PD-L1 antibody on the secretion of cytokine IL-2 in a mixed lymphocyte reaction
  • Figure 12 is a graph showing the results of stability of humanized PD-L1 antibody in serum.
  • the BALB/c mice were immunized with the classic immunization schedule.
  • the immunogen was hPD-L1 (human PD-L1) protein (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) to make the animals produce anti-hPD-L1.
  • the specific protocol is shown in Table 1:
  • the harvested supernatant was first screened by direct ELISA binding method, and the screened positive clones were amplified and the supernatant was collected for screening.
  • the double screening was performed by cell-binding and cytostatic experiments.
  • the positive clones were subcloned by limiting dilution method and plated into 96-well plates at 5/well, 2/well and 1/well. After 7 days of culture, screening was performed by direct ELISA binding assay, and positive subclones were selected for amplification and conservation.
  • the coated hPD-L1-Fc was plated on the plate, and the antibody diluted with the gradient was added. After incubation and washing, goat anti-mouse-HRP was added, and the reaction was fitted to the reaction curve to calculate the EC50 value.
  • the hPD-L1-Fc overexpressing cells were plated one day in advance to the cell plate for the culture. After the next day of blocking, the gradient-diluted antibody was added, and then the anti-mouse-EU was added and the reading was performed.
  • the hPD-L1-Fc overexpressing cells were plated one day in advance to the cell plate for the culture. After the next day of blocking, the gradient-diluted antibody was added, and then PD1-Fc-Biotin was added, followed by Europium-labeled streptavidin, and the reading was performed.
  • Hybridoma cells with positive subclones were selected and inoculated into SFM medium for about 7 days.
  • the supernatant was collected, centrifuged, purified by Protein G purification column, and purified antibodies were ELISA-conjugated. Sex, ELISA inhibitory activity, cell binding activity, and cytostatic activity assay. After screening, one of the most active murine anti-PD-L1 monoclonal antibodies was obtained and named mouse anti-PD-L1.
  • the coated hPD-L1-Fc was coated on the plate, and the antibody diluted with the gradient was added. After incubation and washing, the goat anti-mouse-HRP was added and the color was developed. The reading was fitted to the reaction curve. The results are shown in Fig. 1. The EC50 value was calculated. Its EC50 with hPD-L1 binding activity was 1.67 ng/mL.
  • the gradient-diluted antibody and a certain concentration of hPD-L1-Fc-Biotin were simultaneously added to the plate coated with hPD-L1-Fc, and after washing and washing, SA-HRP was added to develop color, and the reading was fitted to the reaction curve.
  • the IC50 value was calculated and its inhibitory activity IC50 was 0.86 nM.
  • the hPD-L1-Fc overexpressing cells were plated one day in advance to the cell plate for detecting culture. After the next day of blocking, the gradient diluted antibody was added, and then the anti-mouse-EU was added, and the reaction curve was fitted, and the reaction curve was fitted. As shown in Fig. 3, the cell binding activity EC50 was calculated to be 30.29 ng/mL.
  • the hPD-L1-Fc overexpressing cells were plated one day in advance to the cell plate for the culture, and the diluted antibody was added after the next day of blocking, then PD1-Fc-Biotin was added, and then Europium-labeled streptavidin was added, and the reading was performed.
  • the reaction curve was fitted, and the results were as shown in Fig. 4.
  • the IC50 of the cell inhibitory activity was calculated to be 637.8 ng/mL.
  • Total RNA was prepared by reverse transcription of total mouse RNA anti-PD-L1 hybridoma.
  • the heavy chain and light chain variable regions of the antibody were amplified by Leader primer, and the reaction system and PCR conditions are shown in Table 2 and Table 3, respectively.
  • the PCR results were analyzed by electrophoresis, and 0.5 ⁇ l of LA Taq enzyme was added to a reaction tube having an amplification product, and reacted at 72 ° C for 10 min. After that, the enzyme was ligated, and the reaction system is shown in Table 4.
  • the transformation is carried out, and the clones are picked and preserved to obtain a mouse anti-human PD-L1 antibody.
  • the heavy chain variable region nucleic acid sequence and amino acid sequence are shown in SEQ ID NOS: 1 and 2, respectively, and the light chain variable region nucleic acid sequence and amino acid sequence are shown in SEQ ID NOS: 3 and 4, respectively.
  • the mouse antibody sequence obtained by screening is analyzed and compared with the human germline gene to determine KV1-9*01 is a light chain humanized framework sequence, and HV1-46*03 is a heavy chain humanized framework sequence.
  • the CDRs of the heavy and light chains are collocated into the framework sequences by CDR-grafting to construct a humanized antibody which synthesizes a fragment of the variable region of the humanized antibody.
  • the heavy chain variable region nucleic acid sequence and amino acid sequence are shown in SEQ ID NOS: 5 and 6, respectively, and the light chain variable region nucleic acid sequence and amino acid sequence are shown in SEQ ID NOS: 7 and 8, respectively.
  • the DNA sequence of the murine antibody CDRs was analyzed to determine the mutation sites in the variable region CDRs. Design the primer sequence and design the position of the mutation site as NNS to encode any amino acid.
  • the scFv antibody library was amplified by PCR using the humanized antibody scFv as a template, and the antibody library of the scFv was ligated into the phage plasmid by the sfiI cleavage site to construct a secondary antibody library.
  • the phage plasmid containing the scFv antibody library was transformed into E. coli TG1 by electroporation, and after 37 ° C, 220 rpm, 1 h recovery, the helper phage was added to the remaining bacterial solution, and added. Ampicillin, 37 ° C, 220 rpm, 1 h. The supernatant was centrifuged at 2,500 rpm ⁇ 5 min, and the suspension was suspended in 2 ⁇ YT-AK medium, and cultured at 37 ° C, 220 rpm overnight;
  • coated antigen diluted hPD-L1-FC with coating buffer, mixed into the immune tube, coated at 4 ° C overnight;
  • Recombinant phage collection The above overnight culture solution was centrifuged at 2500 rpm ⁇ 5 min, 10 ml of the supernatant was collected, 2 ml of PEG/NaCl was added, mixed and placed on ice for 30-60 min, centrifuged at 10000 g ⁇ 20 min, and the supernatant was removed, and 2 ⁇ YT was used. The medium dissolves the phage library;
  • G, OUTPUT dilute the above bacterial liquid to 10 -1 , 10 -2 , respectively, take 100ul coated plate;
  • helper phage was added to 5 ml of the eluted bacterial solution, while adding 5 ⁇ l of ampicillin, 37 ° C, 220 rpm, 1 h. The supernatant was centrifuged at 2,500 rpm ⁇ 5 min, and the suspension was suspended with 10 ml of 2 ⁇ YT-AK, and cultured at 37 ° C, 220 rpm overnight.
  • anti-PD-L1-1 a total of three humanized antibodies were screened and named as anti-PD-L1-1, anti-PD-L1-2, and anti-PD-L1-3.
  • the heavy and light chain plasmid names of anti-PD-L1-1 are P3.1GS-anti-PD-L1-1–HC and P3.1GS-anti-PD-L1-1–LC, respectively.
  • the heavy and light chain plasmid names of PD-L1-2 are P3.1GS-anti-PD-L1-2–HC and P3.1GS-anti-PD-L1-2–LC, respectively, anti-PD-L1-3
  • the heavy and light chain plasmid names are P3.1GS-anti-PD-L1-3-HC and P3.1GS-anti-PD-L1-3-LC, respectively.
  • the sequence information is as follows:
  • the anti-PD-L1-1 heavy chain nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 9 and 10, respectively.
  • the heavy chain variable region nucleotide sequence is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 11-13, respectively; the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are respectively SEQ ID NO: 14- 17.
  • amino acid sequence of the heavy chain variable region is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 18-20, respectively; the uncrossed portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 21, respectively. twenty four.
  • the anti-PD-L1-1 light chain nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 25 and 26, respectively.
  • the light chain variable region nucleotide sequence is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 27-29, respectively; the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 30, respectively. 33.
  • amino acid sequence of the light chain variable region is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 34-36; the uncrossed portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 37-40, respectively.
  • the anti-PD-L1-2 heavy chain nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 9 and 10, respectively.
  • the heavy chain variable region nucleotide sequence is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 11-13, respectively; the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are respectively SEQ ID NO: 14- 17.
  • amino acid sequence of the heavy chain variable region is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 18-20, respectively; the uncrossed portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 21, respectively. twenty four.
  • the anti-PD-L1-2 light chain nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 41 and 42, respectively.
  • the light chain variable region nucleotide sequence is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 44, 28, and 29, respectively;
  • the uncrossed portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 30-33.
  • amino acid sequence of the light chain variable region is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 46, 35, and 36, respectively; the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 37-40.
  • the anti-PD-L1-3 heavy chain nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 9 and 10, respectively.
  • the heavy chain variable region nucleotide sequence is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 11-13, respectively; the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are respectively SEQ ID NO: 14- 17.
  • amino acid sequence of the heavy chain variable region is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 18-20, respectively; the uncrossed portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 21, respectively. twenty four.
  • the anti-PD-L1-3 light chain nucleotide sequence and amino acid sequence are shown in SEQ ID NOS: 47 and 48, respectively.
  • the light chain variable region nucleotide sequence is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 50, 28, and 29, respectively;
  • the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 30-33.
  • amino acid sequence of the light chain variable region is:
  • the horizontal line portions are CDR1, CDR2, and CDR3, respectively, and their sequence numbers are SEQ ID NO: 52, 35, and 36, respectively; the undrawn horizontal portions are FR1, FR2, FR3, and FR4, respectively, and their sequence numbers are SEQ ID NO: 37-40.
  • Reagent name Add volume Heavy chain / light chain template 1 ⁇ L 5 ⁇ Buffer 10 ⁇ L 2.5 ⁇ M dNTP Mix 4 ⁇ L Upstream and downstream primers (10 ⁇ M) 1 ⁇ L each Taq 0.5 ⁇ L ddH 2 O Up to 50 ⁇ L
  • the full length sequence of the light chain and heavy chain was recovered using a PCR product recovery kit.
  • the antibody fragment light chain, heavy chain and plasmid were separately digested, and after electrophoresis, the antibody was digested with the antibody and the plasmid was digested, and then the fragment was ligated.
  • the humanized antibody expression plasmid after enzyme ligation was named P3.1GS-PD-L1-1.
  • the reaction system is shown in Table 8 - Table 10.
  • the above enzyme product was added to 100 ⁇ L of XL1-10 competent state, iced for 30 minutes, then hot shocked at 42 ° C for 90 seconds, rapidly placed on ice for 2 minutes, then added to 500 ⁇ L of LB medium, and shaken at 37 ° C for 1 hour.
  • the bacterial solution was centrifuged at 4000 rpm for 5 minutes, 500 ⁇ L of the supernatant was discarded, the suspension was sprayed with a gun, and applied to an LB solid plate containing 50 ⁇ g/mL of AMP, and cultured overnight at 37 °C.
  • the colony was picked up into 5 mL LB liquid medium (50 ⁇ g/mL AMP), cultured at 37 ° C and 250 rpm for 6 hours.
  • the clones were verified by PCR, and the positive strains were preserved with 15% sterilized glycerol. Two clones per clone were taken and one frozen tube was taken. Sequencing was sent and the other was stored at -20
  • the humanized antibody expression plasmid P3.1GS-PD-L1-1 was linearized with PvuI before transfection; the linearized plasmid containing the humanized antibody light chain and heavy chain genes was electroporated. Transfected into CHO-KSM4 and transfected twice.
  • glutamylamine was removed for pressure screening, and the cells to be transfected were recovered for 2 days and then subjected to pressure plating. After about 30-40 days of culture, clones were observed to grow in 96-well plates, at which time yield identification was performed, and high-yielding clones were transferred and expanded. When the number of cells reached 2 ⁇ 10 6 cells/mL, the cells were inoculated for fed-batch culture, and after the completion of the culture, the supernatant was harvested for yield identification, and an alternate female clone was obtained.
  • High-yield clones were subcloned and screened: semi-solid plated, 3000-5000 cells per well in 6-well plate, 2.5 mL medium, plated, placed at 37 ° C, 5% CO2, cultured for 7-12 days. Monoclonal. Selected clones were subjected to yield identification to obtain alternative clones.
  • the shake flask feeding scheme is: inoculation with CDM4CHO as the basic medium, the inoculation density is 5 ⁇ 10 5 cells/mL, and after inoculation, it is cultured at 37° C., 5% CO 2 , 120 rpm, and the day of inoculation is recorded as the 0th day, culture By the third day, 70 g/L of cell Boost 5 was added, and 6% of the inoculation volume was added every day until the cells were harvested. After feeding, the highest yield of the cell line reached 1.97 g/L, and the expressed antibody was named anti-PD-L1-1.
  • the affinity and binding kinetics of the antibody expressed in the cell line of Example 4 were analyzed using Biacore.
  • Sheep anti-human IgG was covalently linked to the CM5 chip via a primary amine using standard amine coupling chemistry and a kit supplied by Biacore. Binding was measured by flowing the antibody in HBS EP buffer at a flow rate of 10 ⁇ L/min. The combination time is 300 seconds and the dissociation time is 1200 seconds.
  • the binding kinetic curves were determined as shown in Fig. 5.
  • the calculated values of ka, kd and KD are shown in Table 11.
  • Example 6 ELISA assay binding specificity to other B7 family members and binding to different species of PD-L1 protein
  • Binding of the B7 family members B7-1, B7-2 and PD-L2 proteins and the murine, cynomolgus and human PD-L1 proteins to the humanized antibody anti-PD-L1-1 was tested. Different proteins were incubated overnight at 4 ° C in coating buffer at a concentration of 0.5 ⁇ g/mL. The solution in the well was discarded the next day and washed twice with PBST. Then 1% BSA was added, blocked at 37 ° C for 1 hour and then washed twice with PBST. 0.5 ⁇ g/mL antibody sample was added, incubated for 1 hour, and washed three times with PBST.
  • Sheep anti-human FAB-HRP diluted 1:10000, incubated for 1 hour at 37 ° C, washed three times with PBST. Color development was carried out by adding TMB for 15 min, quenching the reaction with 0.5 M H 2 SO 4 and reading the absorbance at 450 nm.
  • the humanized antibody anti-PD-L1-1 did not bind to other members of the B7 family.
  • the humanized antibody anti-PD-L1-1 binds human or cynomolgus PD-L1 protein with similar affinity.
  • Example 7 ELISA assay for binding specificity of antibodies to CHO cells expressing PD-L1 on the surface
  • a Chinese hamster ovary (CHO) cell line expressing recombinant human PD-L1 on the cell surface was constructed, and the binding specificity of the humanized antibody anti-PD-L1-1 was determined by ELISA.
  • the PD-L1 overexpressing cells were plated one day before the test, and the T75 flasks per well were 1/200 of the cells. Then 1% BSA was added and blocked at 37 ° C for 1 hour.
  • the antibody 5 ⁇ g/mL was sequentially subjected to a 3-fold dilution of a total 8-concentration gradient, 100 ⁇ L/well, and incubated at 25 ° C for 1 hour, and washed once with PBS.
  • the volume of 50 ng/mL anti-human-Eu was added to each well to be 100 ⁇ L, reacted at 25 ° C for 0.5 hour, and washed once with PBS.
  • a fluorescence enhancement solution was added and the excitation light was read at 337 nm/emitting light at 620 nm.
  • the humanized antibody anti-PD-L1-1 was able to efficiently bind to CHO cells transfected with PD-L1, and the EC50 reached 93.50 ng/mL.
  • Example 8 ELISA assay for binding specificity of antibodies to recombinant human PD-L1 fusion protein
  • 0.5 ⁇ g/mL of recombinant human PD-L1 fusion protein was overnight in coating buffer at 4 °C. The solution in the well was discarded the next day and washed twice with PBST. Then 1% BSA was added and blocked at 37 ° C for 1 hour. Wash twice with PBST. The antibody was initially subjected to a 3-fold dilution of a total concentration of 8 ⁇ g/mL, 100 ⁇ L/well, incubated at 25 ° C for 1 hour, and washed three times with PBS. Sheep anti-human FAB-HRP, diluted 1:10000, incubated for 1 hour at 37 ° C, washed three times with PBST. Color development was carried out by adding TMB for 15 min, quenching the reaction with 0.5 M H 2 SO 4 and reading the absorbance at 450 nm.
  • the humanized antibody anti-PD-L1-1 was able to efficiently interact with the recombinant human PD-L1 fusion protein with an EC50 of 19.47 ng/mL.
  • 0.5 ⁇ g/mL of recombinant human PD-L1 fusion protein was overnight in coating buffer at 4 °C. The solution in the well was discarded the next day and washed twice with PBST. Then 1% BSA was added, blocked at 37 ° C for 1 hour and then washed twice with PBST. 10 ⁇ g/mL of the antibody was sequentially subjected to a 2.5-fold dilution, and a total of 8 concentration gradients were mixed with 1 ⁇ g/mL of PD1-Fc-Biotin in an equal volume at 25 ° C for 1 hour, and then washed once with PBS.
  • the humanized antibody anti-PD-L1-1 blocked the binding of the ligand PD-L1 to PD-1 with an IC50 of 43.16 ng/mL.
  • the humanized antibody anti-PD-L1-1 was effective in promoting secretion of IFN- ⁇ and IL-2 by mixed lymphocytes.
  • the humanized antibody anti-PD-L1-1 was diluted with monkey serum at a concentration of 0.5 mg/mL. They were placed at 37 ° C for 0 days, 1 day, 4 days, and 7 days.
  • Recombinant human PD-L1 fusion protein was incubated overnight at 4 ° C in coating buffer at a concentration of 0.5 ⁇ g/mL. The solution in the well was discarded the next day and washed twice with PBST. Then 1% BSA was added, blocked at 37 ° C for 1 hour and then washed twice with PBST. The stable antibody samples were serially diluted 3 times at a concentration of 1 ⁇ g/mL for a total of 8 concentration gradients, incubated at 37 ° C for 1 hour, and washed three times with PBST. Sheep anti-human FAB-HRP, diluted 1:10000, incubated for 1 hour at 37 ° C, washed three times with PBST.

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Abstract

提供了一种抗人PD-L1人源化单克隆抗体及其应用。该抗体能够特异性地与人PD-L1结合，其可以通过和活化的T细胞结合进而增强T细胞的活化作用，对肿瘤生长具有显著的抑制作用。

Description

抗人PD-L1人源化单克隆抗体及其应用
本申请要求了申请日为2016年5月20日，申请号为201610340678.3，发明名称为“抗人PD-L1人源化单克隆抗体及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物医药领域，尤其涉及一种抗人PD-L1人源化单克隆抗体及其应用。
背景技术
人体免疫系统的适应性应答过程主要包含了T细胞以及B细胞这两类免疫细胞的激活、分化和增殖。其中，T细胞功能的激活受到两类信号的调控。一类是由T细胞受体(TCR)识别抗原呈递细胞(APC)上的MHC-抗原复合物所提供的抗原特异性信号。另一类即为APC细胞上所表达的免疫检查点蛋白与T细胞间形成的共刺激和抑制信号。而这一类共刺激或抑制信号往往对T细胞的增殖、分化和激活起着至关重要的作用。正常情况下，免疫检查点对于维持机体自身耐受性(防止自身免疫)并保护机体免受外界病原体感染有重要作用。
PD-L1/PD1信号通路是免疫反应中非常重要的共抑制信号途径。程序性死亡受体-1(PD-1，亦称为CD279)具有两个细胞表面的糖蛋白配体，分别为PD-L1(亦称B7-H1、CD274)和PD-L2(亦称B7-DC、CD273)。
人PD-L1基因编码290个氨基酸(其中1-18位氨基酸为信号肽，19-238位氨基酸为胞外段，239-259位氨基酸为跨膜段，260-290位氨基酸为胞内段)。它是一个Ⅰ型膜蛋白，一般在T细胞、B细胞、树突状细胞、巨噬细胞及许多非造血细胞上表达。研究表明，当PD-L1与PD-1结合后，将会通过补充具有SH2结构域的蛋白酪氨酸磷酸酶SHP-1和SHP-2。这两种磷酸酶能够使CD3ζ链的免疫受体酪氨酸活化基序(ITAM)磷酸化程度降低，削弱ZAP-70活化，并抑制TCR下游信号传递，从而起到共抑制T细胞活化的作用，通过这种负向调节效应来防止效应T细胞过度激活而导致自身免疫损伤。
但是，如果肿瘤组织表达了PD-L1，则能够通过与免疫细胞的PD-1结合，削弱免疫系统对肿瘤组织的杀伤作用。目前发现PD-L1能在许多肿瘤组织(胃癌、乳腺癌、胰腺癌、卵巢癌、肺癌、前列腺癌以及恶性黑色素瘤等)以及浸润肿瘤微环境的骨髓细胞中高表达。而PD-L1的表达也与黑色素瘤、乳腺癌和卵巢癌的不良预后息息相关。若是能够阻断PD-L1与PD-1之间的连接反应，则能使T细胞的效应功能得到恢复。诸如黑色素瘤一类的肿瘤，在其形成之初便能表达PD-L1，从而具备了与生俱来的免疫逃逸能力，PD-L1的表达水平也往往与疾病的预后密切相关。
因此，在采用针对PD-1/PD-L1信号通路免疫疗法时，PD-L1的表达水平成为了非常重要的生物标志物，能帮助研究人员推测出哪些病人将更有可能对这类免疫疗法产生响应。
目前，以PD-L1为靶点的抗体药物，已经在临床上展示出了出色的应用前景。比如罗氏的全人IgG1单克隆抗体MPDL3280A，它能够阻断PD-L1与PD-1和CD80的结合，并且通过对其Fc片段的工程化改造削弱了抗体介导的细胞毒作用来提高安全性。在1期临床试验中，PD-L1表达阳性的转移性膀胱癌患者接受了12周MPDL3280A治疗后产生了52％的响应率，不良反应均为低级别的疲劳和恶心，没有证据显示其具有肾脏毒性。在黑色素瘤患者中，同样观察到了对药物的持续响应，因此MPDL3280A被FDA授予了突破疗法地位。其在晚期肾细胞癌和非小细胞肺癌患者中的临床研究也在同步进行。另一个PD-L1单克隆抗体，辉瑞与默克共同开发的Avelμmab，同样正在转移性默克尔细胞癌患者中进行有效性和安全性评价。
不仅如此，研究表明一些病毒感染也与PD-L1/PD-1信号通路息息相关。例如，在慢性HIV感染中，PD-1被发现在特异性识别HIV的CD8+T细胞表面高表达，病毒通过激活PD-L1/PD-1信号途径，使得特异性识别HIV的CD8+T细胞活性受到抑制，细胞因子的分泌能力及T细胞自身的增殖能力大大削弱，引起了获得性的免疫功能缺陷。由此可见，阻断PD-L1/PD-1信号通路，在这一类疾病的治疗中，同样具备相当的应用价值。
由此可见，研发具备阻断PD-L1/PD-1信号通路能力的药物，将为肿瘤、病毒感染及多种免疫系统相关疾病的治疗带来全新的方法，具备巨大的应用潜力 和市场价值。
发明内容
为解决上述技术问题，本发明的目的是提供一种具有良好特异性、较高的亲和性和稳定性的抗人PD-L1人源化单克隆抗体。
本发明第一方面涉及抗人PD-L1人源化单克隆抗体或其抗原结合部分，其包括选自于如下一组的CDR区：
(1)重链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：18-20所示，轻链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：34-36所示，或与上述序列结合相同抗原表位的序列；
(2)重链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：18-20所示，轻链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：46、35和36所示，或与上述序列结合相同抗原表位的序列；
(3)重链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：18-20所示，轻链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：52、35和36所示，或与上述序列结合相同抗原表位的序列。
进一步的，本发明中抗人PD-L1人源化单克隆抗体或其抗原结合部分，其还包括选自于如下的重链可变区框架区：FR1、FR2、FR3、FR4的序列分别如SEQ ID NO：21-24所示，或分别与上述序列的同一性大于70％、80％、85％、90％、95％、99％的序列。
进一步的，本发明中抗人PD-L1人源化单克隆抗体或其抗原结合部分，其还包括选自于如下的轻链可变区框架区：FR1、FR2、FR3、FR4的序列分别如SEQ ID NO：37-40所示，或分别与上述序列的同一性大于70％、80％、85％、90％、95％、99％的序列。
进一步的，本发明中抗人PD-L1人源化单克隆抗体或其抗原结合部分，其包括选自于如下的重链可变区：其序列如SEQ ID NO：6所示，或与上述序列结合相同抗原表位的序列。
进一步的，本发明中抗人PD-L1人源化单克隆抗体或其抗原结合部分，其包括选自于如下的轻链可变区：其序列如SEQ ID NO：8、45或51所示，或者 分别与上述序列的同一性大于70％、80％、85％、90％、95％、99％的序列。
具体的，本发明中抗人PD-L1人源化单克隆抗体或其抗原结合部分，其重链的序列如SEQ ID NO：10所示。
具体的，本发明中抗人PD-L1人源化单克隆抗体或其抗原结合部分，其轻链的序列如SEQ ID NO：26、42或48所示。
根据本发明第二方面任一项的核酸分子，其包含能够编码抗体重链可变区的核酸序列，所述重链可变区包含选自如下一组的氨基酸序列：
(1)SEQ ID NO：18-20；
(2)与前述(1)序列相比满足以下二者中至少一个的序列：a)结合相同抗原表位；b)同一性大于70％、80％、85％、90％或97％。
进一步的，所述重链可变区包含选自如下一组的氨基酸序列：
SEQ ID NO：6，或与前述序列相比满足以下三者中至少一个的序列：a)结合相同抗原表位、b)同一性大于70％、80％、85％、90％或97％、c)与前述序列框架区中含有一个到几个核苷酸的替换。
在本发明的实施方案中所述核酸分子包含选自如SEQ ID NO：5所示的序列。
进一步的，所述核酸分子包含选自如SEQ ID NO：9所示的序列。
根据本发明第三方面任一项的核酸分子，其包含能够编码抗体轻链可变区的核酸序列，所述轻链可变区包含选自如下一组的氨基酸序列：
(1)SEQ ID NO：34-36；
(2)SEQ ID NO：46、35和36；
(3)SEQ ID NO：52、35和36；
(4)与前述(1)-(3)序列相比满足以下二者中至少一个的序列：a)结合相同抗原表位；b)同一性大于70％、80％、85％、90％或97％。
进一步的，所述轻链可变区包含选自如下一组的氨基酸序列：
SEQ ID NO：8、45或51，或与前述序列相比满足以下三者中至少一个的序列：a)结合相同抗原表位、b)同一性大于70％、80％、85％、90％或97％、c) 与前述序列框架区中含有一个到几个核苷酸的替换。
在本发明的实施方案中，所述核酸分子包含选自如SEQ ID NO：7、43或49所示的序列。
进一步的，所述核酸分子包含选自如SEQ ID NO：25、41或47所示的序列。
本发明第四方面涉及载体，其含有本发明第二或第三方面任一项的核酸分子。
进一步的，本发明中所指的载体含有本发明第二方面任一项的核酸分子和第三方面任一项的核酸分子。
本发明第五方面涉及宿主细胞，其含有本发明第二或第三方面任一项的核酸分子或本发明第四方面任一项的载体。
本发明第六方面涉及偶联物，其含有本发明第一方面任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分，以及其它生物活性物质，所述抗人PD-L1人源化单克隆抗体或其抗原结合部分直接或通过连接片段与其它生物活性物质偶联。
在本发明的实施方案中，所述其它生物活性物质选自可直接或间接抑制细胞生长或杀灭细胞、或通过激活机体免疫反应从而抑制或杀灭细胞，从而达到治疗肿瘤的化学物质、毒素、多肽、酶、同位素、细胞因子或其他具有生物活性的单一物质或混合物质。
本发明第七方面涉及组合物(例如药物组合物)，其含有本发明第一方面任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分、第二方面或第三方面任一项的核酸分子、第四方面任一项的载体、第五方面任一项的宿主细胞、或者本发明第六方面任一项的偶联物，以及任选的药学上可接受的载体或赋形剂，以及任选的其它生物活性物质。
根据本发明第七方面任一项的组合物(例如药物组合物)，所述其它生物活性物质包括但不限于其它抗体、融合蛋白或药物(例如抗肿瘤药物，如放、化疗 药物)。
本发明还涉及诊断试剂或试剂盒，其含有本发明第一方面任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分，所述诊断试剂或试剂盒用于在体外(例如细胞或组织)或体内(例如人或动物模型)诊断与PD-L1相关的疾病(例如肿瘤或病毒感染，例如PD-L1高表达的病毒感染或PD-L1高表达的肿瘤)。
在本发明的实施方案中，所述肿瘤包括但不限于肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体癌、骨肉瘤、甲状腺癌、前列腺癌；所述病毒感染包括但不限于急性、亚急性或慢性HBV、HCV、HIV感染。
本发明还涉及本发明第一方面任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分、第二方面或第三方面任一项的核酸分子、第四方面任一项的载体、第五方面任一项的宿主细胞、第六方面任一项的偶联物或第七方面任一项组合物用于制备预防或治疗与PD-L1相关的疾病(例如肿瘤，微生物或病毒感染，例如PD-L1高表达的肿瘤或PD-L1高表达的病毒感染)的药物的用途。
在本发明的实施方案中，所述肿瘤包括但不限于肺癌、卵巢癌、结肠癌、直肠癌、黑色素瘤、肾癌、膀胱癌、乳腺癌、肝癌、淋巴瘤、恶性血液病、头颈癌、胶质瘤、胃癌、鼻咽癌、喉癌、宫颈癌、子宫体癌、骨肉瘤、甲状腺癌、前列腺癌；所述微生物感染包括但不限于细菌、真菌、原生动物感染；所述病毒感染包括但不限于急性、亚急性或慢性HBV、HCV、HIV感染。
以下对本发明做进一步描述：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，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。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。
在本发明中，术语“抗体”是指通常由两对相同的多肽链(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，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(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。
在本发明中，术语抗体的“抗原结合部分”是指全长抗体的一个或多个部分，所述部分保持结合抗体所结合的相同抗原(例如，PD-L1)的能力，与完整抗体竞争对抗原的特异性结合。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原结合部分。在一些情况下，抗原结合部分包括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。
借由上述方案，本发明至少具有以下优点：本发明通过筛选得到了具有良好特异性、较高的亲和性和稳定性的抗人PD-L1人源化单克隆抗体，该抗体能够 特异性地与人PD-L1结合，并且不与B7家族成员结合，其可以通过和活化的T细胞结合进而增强T细胞的活化作用，对肿瘤生长具有显著的抑制作用。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明如后。
附图说明
图1是鼠源PD-L1抗体的ELISA结合活性结果图；
图2是鼠源PD-L1抗体的ELISA抑制活性结果图；
图3是鼠源PD-L1抗体的细胞结合活性结果图；
图4是鼠源PD-L1抗体的细胞抑制活性结果图；
图5是人源化PD-L1抗体的结合动力学曲线图；
图6是人源化PD-L1抗体与其它B7家族成员的结合特异性以及与不同物种PD-L1蛋白的结合结果图；
图7是人源化PD-L1抗体与表面表达PD-L1的CHO细胞的结合特异性结果图；
图8是人源化PD-L1抗体与重组人PD-L1融合蛋白的结合特异性结果图；
图9是人源化PD-L1抗体对PD-L1结合PD-1的阻断作用结果图；
图10是人源化PD-L1抗体在混合淋巴细胞反应中对细胞因子IFN-γ分泌的影响结果图；
图11是人源化PD-L1抗体在混合淋巴细胞反应中对细胞因子IL-2分泌的影响结果图；
图12是人源化PD-L1抗体在血清中的稳定性结果图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例一鼠源抗体筛选
1.1动物免疫
采用经典的免疫时间表，对BALB/c小鼠免疫，免疫原为hPD-L1(人源PD-L1)蛋白(购自北京义翘神州生物技术有限公司)，以使动物产生抗hPD-L1的抗体，具体方案如表1所示：
表1 hPD-L1蛋白动物免疫方案
Figure PCTCN2016084643-appb-000001
Figure PCTCN2016084643-appb-000002
1.2细胞融合及杂交瘤细胞筛选
融合前调整小鼠骨髓瘤SP2/0的状态，保证其生长密度不超过于1.0×106个细胞，提前3天进行终免，终免采用尾静脉注射的方式，提前一天准备饲养细胞，铺板数量为2.0×104个细胞/孔。通过PEG融合，保证脾细胞与SP2/0细胞的数量比在10:1到5:1之间，每孔所铺脾细胞数量不超过1.0×105。融合7天以后收获上清液并更换培养基。
收获的上清液首先通过直接ELISA结合方法进行初筛，将筛得的阳性克隆扩增后收上清液进行复筛。
复筛采用细胞结合以及细胞抑制实验进行两轮筛选，筛选得到的阳性克隆采用有限稀释法进行亚克隆，铺96孔板，分别为5个/孔，2个/孔和1个/孔。培养7天后采用直接ELISA结合实验进行筛选，挑选阳性亚克隆进行扩增并保种。
其中，所涉及的各实验方法的具体步骤如下：
A、ELISA结合方法
包被hPD-L1-Fc在板上，加入梯度稀释的抗体，孵育洗涤后，再加入羊抗鼠-HRP，显色，读数拟合出反应曲线，计算出EC50值。
B、细胞结合实验
提前一天将hPD-L1-Fc过表达细胞铺至用于检测培养的细胞板，次日封闭后加入梯度稀释的抗体，再加入anti-mouse-EU，读数即可。
C、细胞抑制实验
提前一天将hPD-L1-Fc过表达细胞铺至用于检测培养的细胞板，次日封闭后加入梯度稀释的抗体，再加入PD1-Fc-Biotin，再加入Europium-labeled streptavidin，读数即可。
1.3鼠源抗体的制备及活性鉴定
将挑选阳性亚克隆的杂交瘤细胞接种至SFM培养基内，培养7天左右，收集上清，离心过滤后用Protein G纯化柱纯化，纯化抗体分别进行ELISA结合活 性、ELISA抑制活性、细胞结合活性、细胞抑制活性检测。经过筛选，获得活性最高的一株鼠源抗PD-L1单克隆抗体，命名为mouse anti-PD-L1。
其中，所涉及的各实验方法的具体步骤如下：
A、ELISA结合活性
包被hPD-L1-Fc在板上，加入梯度稀释的抗体，孵育洗涤后，再加入羊抗鼠-HRP，显色，读数拟合出反应曲线，结果如图1所示，计算出EC50值，其与hPD-L1结合活性EC50为1.67ng/mL。
B、ELISA抑制活性
梯度稀释的抗体和一定浓度的hPD-L1-Fc-Biotin同时加入到包被hPD-L1-Fc的板上，孵育洗涤后再加入SA-HRP，显色，读数拟合出反应曲线，结果如图2所示，计算IC50值，其抑制活性IC50为0.86nM。
C、细胞结合活性
提前一天将hPD-L1-Fc过表达细胞铺至用于检测培养的细胞板，次日封闭后加入梯度稀释的抗体，再加入anti-mouse-EU，读数即可，拟合出反应曲线，结果如图3所示，计算出其细胞结合活性EC50为30.29ng/mL。
D、细胞抑制活性
提前一天将hPD-L1-Fc过表达细胞铺至用于检测培养的细胞板，次日封闭后加入梯度稀释的抗体，再加入PD1-Fc-Biotin，再加入Europium-labeled streptavidin，读数即可，拟合出反应曲线，结果如图4所示，计算出其细胞抑制活性IC50为637.8ng/mL。
实施例二鼠源抗体人源化及亲和力成熟
2.1鼠源抗体基因获取
采用Purelink RNA Micro kit提取mouse anti-PD-L1杂交瘤总RNA，之后用PrimeScriptTMII 1st Strand cDNA Synthesis Kit逆转录总RNA制备cDNA。分别用Leader primer扩增抗体的重链及轻链可变区，反应体系及PCR条件分别如表2和表3所示。
表2鼠源抗体基因cDNA PCR反应体系
试剂名称 添加体积
10×Buffer 5μL
10μM dNTP Mix 1μL
50mM MgSO4 2μL
上下游引物 各1μL
cDNA模板 1μL
Taq 0.2μL
ddH2O up to 50μL
表3鼠源抗体基因cDNA PCR反应条件
Figure PCTCN2016084643-appb-000003
电泳分析PCR结果，在有扩增产物的反应管中加入0.5μl LA Taq酶，72℃反应10min。之后进行酶连，反应体系如表4所示。
表4酶连反应体系
Figure PCTCN2016084643-appb-000004
酶连完成后转化，挑克隆，保种，得到鼠源抗人PD-L1抗体。测序后，得到其重链可变区核酸序列和氨基酸序列分别如SEQ ID NO：1和2所示，轻链可变区核酸序列和氨基酸序列分别如SEQ ID NO：3和4所示。
2.2人源化设计
分析筛选得到的鼠源抗体序列，与人胚系(germline)基因比对，确定 KV1-9*01为轻链人源化框架序列，HV1-46*03为重链人源化框架序列。通过CDR-grafting，将重链和轻链的CDR并置到构架序列，构建人源化抗体，基因合成人源化抗体可变区的片段。得到其重链可变区核酸序列和氨基酸序列分别如SEQ ID NO：5和6所示，轻链可变区核酸序列和氨基酸序列分别如SEQ ID NO：7和8所示。
2.3抗体库构建
分析鼠源抗体CDR的DNA序列，确定可变区CDR中的突变位点。设计引物序列，将突变位点所在的位置设计为NNS，使之编码任意的氨基酸。以人源化抗体scFv为模板，PCR扩增scFv抗体库，将scFv的抗体库，通过sfiI酶切位点，构建到噬菌体质粒中，构建二级抗体库。
2.4抗体库筛选
然后通过噬菌体展示进行高亲和力抗体筛选，具体方法如下：
A、通过电转化，将含scFv的抗体库的噬菌体质粒转化到大肠杆菌TG1中，经过37℃，220rpm，1h的恢复后，将辅助噬菌体(helper phage)加入到剩余的菌液中，另加入氨苄西林，37℃，220rpm，1h。2500rpm×5min离心去上清，用2×YT-AK培养基吹悬菌泥，37℃，220rpm过夜培养；
B、包被抗原：用包被缓冲液稀释hPD-L1-FC，混匀加入到免疫管中，4℃包被过夜；
C、重组噬菌体收集：上述过夜培养菌液，2500rpm×5min离心，收集上清10ml，加入2ml PEG/NaCl，混匀放置冰上30-60min，10000g×20min离心，去上清，用2×YT培养基溶解噬菌体库；
D、封闭：免疫管用PBS洗两次，加入封闭液，室温1h。另外，取等体积封闭液与噬菌体库混合，室温封闭10-15min；
E、孵育噬菌体库：免疫管用PBS洗2次，加入封闭好的噬菌体库，37℃培养箱2-3h；
F、洗脱：取100μl TG1菌液(前一天接种)到10ml 2×YT中，37℃，220rpm培养到A600值0.4-0.5。用PBST洗涤免疫管8次，再用PBS洗2次，加入5ml对数期生长的菌液，37℃，220rpm，1h；
G、OUTPUT：稀释上述菌液至10-1、10-2，分别取100ul涂平板；
H、下一轮筛选：取200μl helper phage加入到5ml洗脱后的菌液中，同时加入5μl氨苄西林，37℃，220rpm，1h。2500rpm×5min离心去上清，用10ml 2×YT-AK吹悬菌泥，37℃，220rpm过夜培养。
重复步骤B-H。
经过三轮筛选，挑选单克隆，制备重组噬菌体，通过Phage ELISA方法，检测重组噬菌体活性，具体如下：
A、包被hPD-L1-FC，4℃过夜；
B、PBST洗两次，加入phage上清，25℃，1h；
C、PBST洗三次，加入稀释的anti-M13-biotinAb，25℃，1h；
D、PBST洗三次，加入稀释的HRP-streptavidin，25℃，1h；
E、PBST洗三次，加入预热的TMB，25℃，10min，加入1M H2SO4中止反应，OD450检测吸光值。挑选阳性克隆，送测序，通过PCR，重链可变区或轻链可变区拼接至其对应的人源抗体的恒定区序列，扩增的抗体重链和轻链全长片段(包含信号肽)分别克隆入pcDNA3.1GS。
通过以上实验，共筛选得到3株人源化抗体，分别命名为anti-PD-L1-1，anti-PD-L1-2，anti-PD-L1-3。相应的，anti-PD-L1-1的重链和轻链质粒名称分别为P3.1GS-anti-PD-L1-1–HC和P3.1GS-anti-PD-L1-1–LC，anti-PD-L1-2的重链和轻链质粒名称分别为P3.1GS-anti-PD-L1-2–HC和P3.1GS-anti-PD-L1-2–LC，anti-PD-L1-3的重链和轻链质粒名称分别为P3.1GS-anti-PD-L1-3–HC和P3.1GS-anti-PD-L1-3–LC。序列信息具体如下：
1)anti-PD-L1-1重链核苷酸序列和氨基酸序列分别如SEQ ID NO：9和10所示。其中，重链可变区核苷酸序列为：
Figure PCTCN2016084643-appb-000005
Figure PCTCN2016084643-appb-000006
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：11-13；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：14-17。
对应的，重链可变区氨基酸序列为：
Figure PCTCN2016084643-appb-000007
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：18-20；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：21-24。
anti-PD-L1-1轻链核苷酸序列和氨基酸序列分别如SEQ ID NO：25和26所示。其中，轻链可变区核苷酸序列为：
Figure PCTCN2016084643-appb-000008
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：27-29；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：30-33。
对应的，轻链可变区氨基酸序列为：
Figure PCTCN2016084643-appb-000009
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO： 34-36；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：37-40。
2)anti-PD-L1-2重链核苷酸序列和氨基酸序列分别如SEQ ID NO：9和10所示。其中，重链可变区核苷酸序列为：
Figure PCTCN2016084643-appb-000010
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：11-13；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：14-17。
对应的，重链可变区氨基酸序列为：
Figure PCTCN2016084643-appb-000011
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：18-20；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：21-24。
anti-PD-L1-2轻链核苷酸序列和氨基酸序列分别如SEQ ID NO：41和42所示。其中，轻链可变区核苷酸序列为：
Figure PCTCN2016084643-appb-000012
Figure PCTCN2016084643-appb-000013
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：44、28、29；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：30-33。
对应的，轻链可变区氨基酸序列为：
Figure PCTCN2016084643-appb-000014
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：46、35、36；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：37-40。
3)anti-PD-L1-3重链核苷酸序列和氨基酸序列分别如SEQ ID NO：9和10所示。其中，重链可变区核苷酸序列为：
Figure PCTCN2016084643-appb-000015
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：11-13；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：14-17。
对应的，重链可变区氨基酸序列为：
Figure PCTCN2016084643-appb-000016
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：18-20；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：21-24。
anti-PD-L1-3轻链核苷酸序列和氨基酸序列分别如SEQ ID NO：47和48所示。其中，轻链可变区核苷酸序列为：
Figure PCTCN2016084643-appb-000017
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：50、28、29；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：30-33。
对应的，轻链可变区氨基酸序列为：
Figure PCTCN2016084643-appb-000018
横线部分分别为CDR1、CDR2、CDR3，其序列编号分别为SEQ ID NO：52、35、36；未划横线部分分别为FR1、FR2、FR3、FR4，其序列编号分别为SEQ ID NO：37-40。
实施例三人源化抗体表达质粒构建
由于三株抗体均表达出较好的特异性，本实施例仅以P3.1GS-PD-L1-1-HC和P3.1GS-PD-L1-1-LC为模板作进一步说明。通过PCR扩增全长抗体重链片段和轻链片段，构建人源化抗体表达质粒。
轻链和重链上下游引物、反应体系及PCR条件如表5、表6和表7所示。
表5人源化抗体轻链和重链PCR反应的上下游引物
Figure PCTCN2016084643-appb-000019
表6人源化抗体轻链和重链PCR反应体系
试剂名称 添加体积
重链/轻链模板 1μL
5×Buffer 10μL
2.5μM dNTP Mix 4μL
上下游引物(10μM) 各1μL
Taq 0.5μL
ddH2O up to 50μL
表7人源化抗体轻链和重链PCR反应条件
Figure PCTCN2016084643-appb-000020
用PCR产物回收试剂盒回收轻链、重链的全长序列。对抗体片段轻链、重链以及质粒分别进行双酶切，电泳后胶回收抗体酶切和质粒酶切片段，之后对片段进行酶连。酶连之后的人源化抗体表达质粒命名为P3.1GS-PD-L1-1。反应体系如表8-表10所示。
表8人源化抗体轻链和重链双酶切反应体系
Figure PCTCN2016084643-appb-000021
表9表达质粒双酶切反应体系
Figure PCTCN2016084643-appb-000022
表10抗体片段与表达质粒片段酶连反应体系
Figure PCTCN2016084643-appb-000023
将上述酶连产物加入到100μL XL1-10感受态中，冰上30分钟，然后，42℃热击90秒，迅速放置冰上2分钟，接着加入500μL LB培养基，37℃摇床培养1小时，将菌液于4000rpm离心5分钟，弃500μL上清，用枪吹悬菌泥，涂布于含50μg/mL AMP的LB固体平板上，37℃过夜培养。挑单菌落到5mL LB液体培养基(50μg/mL AMP)，37℃ 250rpm培养6小时，PCR验证克隆，用15％灭菌甘油保藏阳性菌种，每个克隆2根，取一份冻存管送测序，另一份保存-20℃。
实施例四稳定表达细胞株开发
人源化抗体表达质粒P3.1GS-PD-L1-1，转染前采用PvuⅠ对其进行线性化；采用电转染的方法，将含有人源化抗体轻链、重链基因的线性化质粒转染至CHO-KSM4，转染2次。
转染后撤谷氨酰氨进行加压筛选，待转染细胞恢复2天后进行加压铺板。培养约30-40天后，96孔板可观察到克隆长出，此时进行产量鉴定，将高产克隆转移并扩增培养。待细胞数目达到2×106cells/mL左右接种进行补料分批培养，培养结束后收获上清进行产量鉴定，获得备选母克隆。将高产的克隆开展亚克隆筛选：半固体铺板，6孔板每孔3000-5000个细胞，培养基2.5mL，铺板后置于37℃、5％CO2静置培养，培养7-12天可挑选单克隆。将挑选的单克隆进行产量鉴定，获得备选克隆。
将获得9株高产细胞株进行摇瓶补料实验。摇瓶补料方案为：采用CDM4CHO为基础培养基进行接种，接种密度为5×105cells/mL，接种后置于37℃、5％CO2、120rpm培养，接种当天记为第0天，培养至第3天开始补加70g/L的cell Boost 5，每天补加接种体积的6％直至细胞收获。经过补料，细胞株的最高产量达到1.97g/L，表达的抗体命名为anti-PD-L1-1。
实施例五抗体的结合特异性和结合动力学比较
采用Biacore分析实施例四中的细胞株表达抗体的亲和力及结合动力学。利用标准胺偶联化学和由Biacore提供的试剂盒，经伯胺将羊抗人IgG共价连接至CM5芯片。将抗体以10μL/min的流速在HBS EP缓冲液中流动而测量结合。结合时间300秒，解离时间1200秒。测定结合动力学曲线如图5所示，计算得到的ka、kd和KD值如表11所示。
表11人源化抗体anti-PD-L1-1的结合动力学结果
样品 ka(1/Ms) kd(1/s) KD(M)
anti-PD-L1-1 1.76×105 1.72×10-4 4.06×10-10
实施例六ELISA测定与其它B7家族成员的结合特异性及与不同物种的PD-L1蛋白的结合
测试B7家族成员B7-1、B7-2和PD-L2蛋白及鼠、食蟹猴和人的PD-L1蛋白与人源化抗体anti-PD-L1-1的结合。不同蛋白以0.5μg/mL的浓度在包被缓冲 液中4℃过夜。次日弃去孔内溶液用PBST洗两次。然后加入1％BSA，37℃封闭1小时然后用PBST洗两次。加入0.5μg/mL抗体样品，孵育1小时，PBST洗三次。用羊抗人FAB-HRP，1:10000稀释，37℃孵育1小时，PBST洗三次。加入TMB显色15min，以0.5M的H2SO4中止反应并在450nm读出吸光度。
结果如图6所示，人源化抗体anti-PD-L1-1不结合B7家族其它成员。人源化抗体anti-PD-L1-1以类似的亲和力结合人或食蟹猴PD-L1蛋白。
实施例七ELISA测定抗体与表面表达PD-L1的CHO细胞的结合特异性
构建在细胞表面表达重组人PD-L1的中国仓鼠卵巢(CHO)细胞系，并通过ELISA测定人源化抗体anti-PD-L1-1的结合特异性。检测前一天PD-L1过表达细胞铺板，每孔铺细胞T75瓶长满的1/200。然后加入1％BSA，37℃封闭1小时。抗体5μg/mL起始依次进行3倍稀释共8浓度梯度，100μL/孔，25℃孵育1小时，PBS洗一次。每孔加入50ng/mL anti-human-Eu的体积是100μL，25℃反应0.5小时，PBS洗一次。加入荧光增强液，激发光337nm/发射光620nm读数。
结果如图7所示，人源化抗体anti-PD-L1-1能有效结合经PD-L1转染的CHO细胞，EC50达到93.50ng/mL。
实施例八ELISA测定抗体与重组人PD-L1融合蛋白的结合特异性
0.5μg/mL的重组人PD-L1融合蛋白在包被缓冲液中4℃过夜。次日弃去孔内溶液用PBST洗两次。然后加入1％BSA，37℃封闭1小时。用PBST洗两次。抗体1μg/mL起始依次进行3倍稀释共8浓度梯度，100μL/孔，25℃孵育1小时，PBS洗三次。用羊抗人FAB-HRP，1:10000稀释，37℃孵育1小时，PBST洗三次。加入TMB显色15min，以0.5M的H2SO4中止反应并在450nm读出吸光度。
结果如图8所示，人源化抗体anti-PD-L1-1能有效的与重组人PD-L1融合蛋白相互作用，EC50为19.47ng/mL。
实施例九抗体对PD-L1结合PD-1的阻断作用
0.5μg/mL的重组人PD-L1融合蛋白在包被缓冲液中4℃过夜。次日弃去孔内溶液用PBST洗两次。然后加入1％BSA，37℃封闭1小时然后用PBST洗两次。抗体10μg/mL起始依次进行2.5倍稀释共8个浓度梯度与1μg/mL的 PD1-Fc-Biotin两者等体积混合25℃孵育1小时后，用PBS洗一次。用链亲和素-HRP，1:10000，37℃孵育1小时，PBST洗三次。加入TMB显色15min，以0.5M的H2SO4中止反应并在450nm读出吸光度。
结果如图9所示，人源化抗体anti-PD-L1-1能阻断配体PD-L1与PD-1的结合，IC50为43.16ng/mL。
实施例十抗体在混合淋巴细胞反应中对细胞因子分泌的影响
先用PBS缓冲液1:1稀释血液，移取3mL的LSM至离心管内，加入稀释过的血液4mL，注意加入的时候，确保使稀释后的血液至于LSM的上层，不可混匀。400g，RT离心30-40min。最后吸出分离出的上层的PBMC，100g离心10min。使用BD公司CD4+细胞分离磁珠分离CD4+T细胞，使用BD公司DC细胞分离磁珠分离DC细胞。96孔板每孔CD4+T细胞数量1×105，DC数量1×104，体积共计100μL共培养。加入梯度稀释的抗体，培养5天后检测IFN-γ，IL-2的浓度。
结果如图10和11所示，人源化抗体anti-PD-L1-1能有效的促进混合淋巴细胞分泌IFN-γ和IL-2。
实施例十一抗体在血清中的稳定性
用猴血清稀释人源化抗体anti-PD-L1-1，浓度为0.5mg/mL。37℃分别放置0天、1天、4天、7天。
重组人PD-L1融合蛋白以0.5μg/mL的浓度在包被缓冲液中4℃过夜。次日弃去孔内溶液用PBST洗两次。然后加入1％BSA，37℃封闭1小时然后用PBST洗两次。稳定性抗体样品以1μg/mL起始依次进行3倍稀释共8个浓度梯度，37℃孵育1小时，PBST洗三次。用羊抗人FAB-HRP，1:10000稀释，37℃孵育1小时，PBST洗三次。加入TMB显色15min，以0.5M的H2SO4中止反应并在450nm读出吸光度。结果见图12所示，人源化抗体anti-PD-L1-1显示出了良好的血清稳定性，7天之内均未显示明显的活性衰减。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (14)

  1. 一种抗人PD-L1人源化单克隆抗体或其抗原结合部分，其特征在于：包括选自于如下一组的CDR区：
    (1)重链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：18-20所示，轻链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：34-36所示，或与上述序列结合相同抗原表位的序列；
    (2)重链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：18-20所示，轻链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：46、35和36所示，或与上述序列结合相同抗原表位的序列；
    (3)重链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：18-20所示，轻链CDR1、CDR2、CDR3的序列分别如SEQ ID NO：52、35和36所示，或与上述序列结合相同抗原表位的序列。
  2. 根据权利要求1所述的抗人PD-L1人源化单克隆抗体或其抗原结合部分，其特征在于：还包括选自于如下的重链可变区框架区：FR1、FR2、FR3、FR4的序列分别如SEQ ID NO：21-24所示，或分别与上述序列的同一性大于70％、80％、85％、90％、95％、99％的序列。
  3. 根据权利要求2所述的抗人PD-L1人源化单克隆抗体或其抗原结合部分，其特征在于：其重链的序列如SEQ ID NO：10所示。
  4. 根据权利要求1所述的抗人PD-L1人源化单克隆抗体或其抗原结合部分，其特征在于：还包括选自于如下的轻链可变区框架区：FR1、FR2、FR3、FR4的序列分别如SEQ ID NO：37-40所示，或分别与上述序列的同一性大于70％、80％、85％、90％、95％、99％的序列。
  5. 根据权利要求4所述的抗人PD-L1人源化单克隆抗体或其抗原结合部分，其特征在于：其轻链的序列如SEQ ID NO：26、42或48所示。
  6. 一种核酸分子，其特征在于：其包含能够编码抗体重链可变区的核酸序列，所述重链可变区包含选自如下一组的氨基酸序列：
    (1)SEQ ID NO：18-20；
    (2)与前述(1)序列相比满足以下二者中至少一个的序列：a)结合相同抗原表位；b)同一性大于70％、80％、85％、90％或97％。
  7. 根据权利要求6所述的核酸分子，其特征在于：所述重链可变区包含选 自如下一组的氨基酸序列：
    SEQ ID NO：6，或与前述序列相比满足以下三者中至少一个的序列：a)结合相同抗原表位、b)同一性大于70％、80％、85％、90％或97％、c)与前述序列框架区中含有一个到几个核苷酸的替换。
  8. 一种核酸分子，其特征在于：其包含能够编码抗体轻链可变区的核酸序列，所述轻链可变区包含选自如下一组的氨基酸序列：
    (2)SEQ ID NO：34-36；
    (2)SEQ ID NO：46、35和36；
    (3)SEQ ID NO：52、35和36；
    (4)与前述(1)-(3)序列相比满足以下二者中至少一个的序列：a)结合相同抗原表位；b)同一性大于70％、80％、85％、90％或97％。
  9. 根据权利要求8所述的核酸分子，其特征在于：所述轻链可变区包含选自如下一组的氨基酸序列：
    SEQ ID NO：8、45或51，或与前述序列相比满足以下三者中至少一个的序列：a)结合相同抗原表位、b)同一性大于70％、80％、85％、90％或97％、c)与前述序列框架区中含有一个到几个核苷酸的替换。
  10. 一种载体，其特征在于：其含有权利要求6-9任一项的核酸分子。
  11. 一种宿主细胞，其特征在于：其含有权利要求6-9任一项的核酸分子或权利要求10的载体。
  12. 一种偶联物，其特征在于：其含有权利要求1-5任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分，以及其它生物活性物质，所述抗人PD-L1人源化单克隆抗体或其抗原结合部分直接或通过连接片段与其它生物活性物质偶联。
  13. 一种组合物，其特征在于：其含有权利要求1-5任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分、权利要求6-9的核酸分子、权利要求10的载体、权利要求11的宿主细胞、或者权利要求12的偶联物，以及任选的药学上可接受的载体或赋形剂，以及任选的其它生物活性物质。
  14. 权利要求1-5任一项的抗人PD-L1人源化单克隆抗体或其抗原结合部分、权利要求6-9的核酸分子、权利要求10的载体、权利要求11的宿主细胞、 权利要求12的偶联物、或者权利要求13的组合物用于制备预防或治疗肿瘤、免疫系统相关疾病(T细胞功能障碍)、微生物或病毒引起的感染的用途。
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