WO2021244392A1 - 一种抗pd1×pdl1的双特异性抗体 - Google Patents

一种抗pd1×pdl1的双特异性抗体 Download PDF

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WO2021244392A1
WO2021244392A1 PCT/CN2021/096396 CN2021096396W WO2021244392A1 WO 2021244392 A1 WO2021244392 A1 WO 2021244392A1 CN 2021096396 W CN2021096396 W CN 2021096396W WO 2021244392 A1 WO2021244392 A1 WO 2021244392A1
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pdl1
seq
amino acid
cancer
acid sequence
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French (fr)
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朱祯平
黄浩旻
顾昌玲
祝海霞
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三生国健药业（上海）股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies, in particular to an anti-PD1 ⁇ PDL1 bispecific antibody, and a preparation method and application thereof.
  • PD-1 Human Programmed Cell Death Receptor-1
  • PD-1 is a type I membrane protein with 288 amino acids. It is one of the major known immune checkpoints (Blank et al, 2005, Cancer Immunotherapy) , 54: 307-314). PD-1 is expressed on activated T lymphocytes, and it interacts with the ligand PD-L1 (programmed cell death-Ligand 1) and PD-L2 (programmed cell death receptor- 1). Ligand 2, programmed cell death-Ligand 2) The combination can inhibit the activity of T lymphocytes and related cellular immune responses in the body.
  • PD-L2 is mainly expressed in macrophages and dendritic cells, while PD-L1 is widely expressed in B, T lymphocytes and peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells.
  • B T lymphocytes
  • peripheral cells such as microvascular epithelial cells, lung, liver, heart and other tissue cells.
  • PD-1 (encoded by the gene Pdcd1) is a member of the immunoglobulin superfamily related to CD28 and CTLA-4. Research results show that when PD-1 binds to its ligands (PD-L1 and/or PD-L2), it negatively regulates antigen receptor signal transduction.
  • PD-L1 and/or PD-L2 ligands
  • the structure of mouse PD-1 and the co-crystal structure of mouse PD-1 and human PD-L1 have been clarified (Zhang, X. et al. Immunity 20: 337-347 (2004); Lin et al., Proc. Natl. Acad. Sci. USA 105: 3011-6 (2008)).
  • PD-1 and similar family members are type I transmembrane glycoproteins, which contain an Ig variable (V-type) domain responsible for ligand binding and a cytoplasmic tail region responsible for binding signal transduction molecules.
  • the cytoplasmic tail of PD-1 contains two tyrosine-based signal transduction motifs, ITIM (Immunoreceptor Tyrosine Inhibition Motif) and ITSM (Immune Receptor Tyrosine Switch Motif).
  • PD-1 plays an important role in the immune evasion mechanism of tumors.
  • Tumor immunotherapy which uses the body’s own immune system to fight cancer, is a breakthrough tumor treatment method, but the tumor microenvironment can protect tumor cells from effective immune destruction. Therefore, how to break the tumor microenvironment has become an anti-tumor research Focus.
  • Existing research results have determined the role of PD-1 in the tumor microenvironment: PD-L1 is expressed in many mouse and human tumors (and can be induced by IFN- ⁇ in most PD-L1-negative tumor cell lines), It is presumed to be an important target for mediating tumor immune evasion (Iwai Y. et al., Proc. Natl. Acad. Sci.
  • PD-1 on tumor infiltrating lymphocytes
  • PD-L1 on tumor cells
  • Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell carcinoma, urothelial cell carcinoma and Pancreatic cancer and head and neck tumors. It can be seen that blocking the interaction of PD-1/PD-L1 can improve the immune activity of tumor-specific T cells and help the immune system to clear tumor cells. Therefore, PD-1 and PD-L1 have become the development of tumor immunotherapy drugs Popular target.
  • Bispecific antibodies refer to antibody molecules that can specifically bind to two antigens or two epitopes at the same time. According to symmetry, bispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent and multivalent molecules. Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancers and other diseases.
  • the present invention provides a bispecific antibody against PD1 ⁇ PDL1.
  • the first objective of the present invention is to provide a bispecific antibody against PD1 ⁇ PDL1.
  • the second object of the present invention is to provide an isolated nucleotide encoding the bispecific antibody.
  • the third object of the present invention is to provide an expression vector containing the nucleotide.
  • the fourth object of the present invention is to provide a host cell containing the expression vector.
  • the fifth object of the present invention is to provide a method for preparing the bispecific antibody.
  • the sixth object of the present invention is to provide a pharmaceutical composition containing the bispecific antibody.
  • the seventh object of the present invention is to provide the use of the bispecific antibody or the pharmaceutical composition in the preparation of drugs for the treatment of cancer.
  • the eighth object of the present invention is to provide a method of using the bispecific antibody or the pharmaceutical composition to treat cancer.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides an anti-PD1 ⁇ PDL1 bispecific antibody, the bispecific antibody comprising two polypeptide chains and two light chains:
  • Each of the polypeptide chains sequentially includes VH-PDL1—CH1—CH2—CH3—linker2—VL-PD1—linker1—VH-PD1 or VH-PDL1—CH1—CH2—CH3—linker2—VH from N-terminus to C-terminus -PD1—linker1—VL-PD1 or VL-PD1—linker1—VH-PD1—linker2—VH-PDL1—CH1—CH2—CH3 or VH-PD1—linker1—VL-PD1—linker2—VH-PDL1—CH1—CH2 —CH3, each of the light chains sequentially includes VL-PDL1-CL from N-terminus to C-terminus; or
  • Each of the polypeptide chains sequentially includes VH-PD1—CH1—CH2—CH3—linker2—VL-PDL1—linker1—VH-PDL1 or VH-PD1—CH1—CH2—CH3—linker2—VH from N-terminus to C-terminus -PDL1—linker1—VL-PDL1 or VL-PDL1—linker1—VH-PDL1—linker2—VH-PD1—CH1—CH2—CH3 or VH-PDL1—linker1—VL-PDL1—linker2—VH-PD1—CH1—CH2 —CH3, each of the light chains sequentially includes VL-PD1-CL from N-terminus to C-terminus,
  • the VH-PDL1 is the heavy chain variable region that binds PD-L1
  • the CH1-CH2-CH3 is the heavy chain constant region
  • the VL-PD1 is the light chain variable region that binds PD-1
  • the VH-PD1 is a heavy chain variable region that binds PD-1
  • the VL-PDL1 is a light chain variable region that binds PD-L1
  • the CL is a light chain constant region.
  • the VH-PDL1 and the VL-PDL1 form an antigen binding site that specifically binds to PD-L1, and the VL-PD1 and the VH-PD1 form specific Sexually binds to the antigen binding site of PD-1.
  • the anti-PD1 ⁇ PDL1 bispecific antibody further includes one or more of the following:
  • the VH-PDL1 includes the heavy chain CDR whose amino acid sequence is shown in SEQ ID NO: 1-3;
  • the VL-PDL1 includes a light chain CDR with an amino acid sequence as shown in SEQ ID NO: 4-6;
  • the VH-PD1 includes the heavy chain CDR whose amino acid sequence is shown in SEQ ID NO: 7-9;
  • the VL-PD1 includes a light chain CDR with an amino acid sequence as shown in SEQ ID NO: 10-12.
  • the VH-PDL1 includes an H-CDR1 whose amino acid sequence is shown in SEQ ID NO:1.
  • the VH-PDL1 includes an H-CDR2 whose amino acid sequence is shown in SEQ ID NO: 2.
  • the VH-PDL1 includes an H-CDR3 whose amino acid sequence is shown in SEQ ID NO: 3.
  • the VL-PDL1 includes L-CDR1 whose amino acid sequence is shown in SEQ ID NO:4.
  • the VL-PDL1 includes L-CDR2 whose amino acid sequence is shown in SEQ ID NO: 5.
  • the VL-PDL1 includes L-CDR3 whose amino acid sequence is shown in SEQ ID NO:6.
  • the VH-PD1 comprises H-CDR1 whose amino acid sequence is shown in SEQ ID NO:7.
  • the VH-PD1 includes an H-CDR2 whose amino acid sequence is shown in SEQ ID NO: 8.
  • the VH-PD1 includes an H-CDR3 whose amino acid sequence is shown in SEQ ID NO:9.
  • the VL-PD1 includes L-CDR1 whose amino acid sequence is shown in SEQ ID NO: 10.
  • the VL-PD1 includes L-CDR2 whose amino acid sequence is shown in SEQ ID NO: 11.
  • the VL-PD1 comprises L-CDR3 whose amino acid sequence is shown in SEQ ID NO: 12.
  • the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 13, and/or the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14, and/ Or, the VH-PD1 has an amino acid sequence as shown in SEQ ID NO: 15, and/or, the VL-PD1 has an amino acid sequence as shown in SEQ ID NO: 16.
  • the linker2 is 3 G 4 S, and the linker 1 is 4 G 4 S.
  • the heavy chain constant region includes an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region
  • the light chain constant region includes a kappa or lambda light chain constant region
  • the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 17 or SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 24, and/or said The light chain has an amino acid sequence as shown in SEQ ID NO: 18; or the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 25 or SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NO: 28 , And/or, the light chain has an amino acid sequence as shown in SEQ ID NO:21.
  • the second aspect of the invention provides an isolated nucleotide encoding the bispecific antibody.
  • the preparation method of the nucleotide of the present invention is a conventional preparation method in the field, and preferably includes the following preparation method: obtain the nucleotide encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or by artificial whole The method of sequence synthesis obtains the nucleotides encoding the above-mentioned monoclonal antibodies.
  • nucleotide sequence encoding the amino acid sequence of the bispecific antibody can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homolog.
  • the polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the bispecific antibody within the scope of maintaining the activity of the antibody.
  • the third aspect of the present invention provides an expression vector containing the above-mentioned nucleotides.
  • the expression vector is a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate regulatory sequences.
  • the expression vector may be a virus or a plasmid, such as an appropriate phage or phagemid.
  • a virus or a plasmid such as an appropriate phage or phagemid.
  • the expression vector of the present invention is preferably pTT5, pDR1, pcDNA3.1, pcDNA3.4, pDHFF, pGM-CSF or pCHO1.0, more preferably pcDNA3.4, pTT5.
  • the fourth aspect of the present invention provides a host cell, which contains the expression vector as described above.
  • the host cell of the present invention is a variety of conventional host cells in the field, as long as it can make the above-mentioned recombinant expression vector stably replicate itself and the nucleotides carried can be effectively expressed.
  • the host cells include prokaryotic expression cells and eukaryotic expression cells.
  • the host cell is, for example, COS, CHO (Chinese Hamster Ovary), NS0, sf9, sf21, DH5 ⁇ , BL21 (DE3), E. coli TG1, BL21 (DE3) cells, or CHO-K1 Cells, 293E cells or TG1.
  • the host cell is more preferably a CHO cell and/or 293E cell.
  • the fifth aspect of the present invention provides a method for preparing the bispecific antibody, the method comprising the following steps:
  • the host cell culture method of the present invention, the separation and purification method of the anti-PD1 ⁇ PDL1 bispecific antibody are conventional methods in the field, and the specific operation method please refer to the corresponding cell culture technical manual and the separation of bispecific antibody Purification technology manual.
  • the preparation method of the anti-PD1 ⁇ PDL1 bispecific antibody disclosed in the present invention includes: culturing the above-mentioned host cell under expression conditions to express the anti-PD1 ⁇ PDL1 bispecific antibody; and isolating and purifying the anti-PD1 ⁇ PDL1 bispecific antibody. Using the above method, the recombinant protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.
  • the anti-PD1 ⁇ PDL1 bispecific antibody disclosed in the present invention can be separated and purified by affinity chromatography. According to the characteristics of the affinity column used, conventional methods such as high-salt buffer, pH change, etc. can be used. The bispecific antibody bound to the affinity column is eluted.
  • the inventors of the present invention conducted detection experiments on the obtained bispecific antibody against PD1 ⁇ PDL1, and the experimental results showed that the bispecific antibody can bind to PD-1 and PDL-1 well and has a higher affinity.
  • the sixth aspect of the present invention provides a pharmaceutical composition containing the bispecific antibody as described above and a pharmaceutically acceptable carrier.
  • the anti-PD1 ⁇ PDL1 bispecific antibody provided by the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition, thereby exerting a more stable therapeutic effect.
  • the pharmaceutical composition has a certain preparation form that can ensure the conformational integrity of the amino acid core sequence of the anti-PD1 ⁇ PDL1 bispecific antibody disclosed in the present invention, and at the same time protects the protein’s multifunctional groups from degradation (including but not limited to aggregation and degrading). Ammonia or oxidation).
  • the seventh aspect of the present invention provides the use of the above-mentioned bispecific antibody or the above-mentioned pharmaceutical composition in the preparation of a medicament for the treatment of cancer.
  • the cancer is selected from one or more of melanoma, kidney cancer, lung cancer, liver cancer, gastric cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases. kind.
  • the anti-PD1 ⁇ PDL1 bispecific antibody of the present invention can be used alone or in combination with other anti-tumor drugs.
  • the cancer treatment drugs referred to in the present invention refer to drugs that inhibit and/or treat tumors, which may include delays in the development of tumor-related symptoms and/or reduction in the severity of these symptoms, and further include symptoms associated with existing tumors. Reduce and prevent the appearance of other symptoms, including reducing or preventing tumor metastasis.
  • the dosage depends on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Different, you can refer to the results of animal experiments and various situations, and the total dose cannot exceed a certain range.
  • the eighth aspect of the present invention provides a method of treating cancer, comprising administering the bispecific antibody as described above or the pharmaceutical composition as described above to a subject in need.
  • the cancer is selected from one or more of melanoma, kidney cancer, lung cancer, liver cancer, gastric cancer, lymphoma, breast cancer, colorectal cancer, leukemia, prostate cancer, bone marrow cancer and other neoplastic malignant diseases. kind.
  • the administered dose When the anti-PD1 ⁇ PDL1 bispecific antibody and the pharmaceutical composition thereof are administered to a subject, the administered dose must be a therapeutically effective amount.
  • the therapeutically effective amount refers to an amount effective in treating cancer.
  • the dosage of administration varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. You can refer to the results of animal experiments and various situations, and the total dose cannot exceed a certain range.
  • the present invention provides a bispecific antibody against PD1 ⁇ PDL1.
  • the experimental results show that the biantibody can better maintain the activity of the respective monoclonal antibodies, and can specifically bind PD-1 and PD-1 at the same time.
  • the two targets of PD-L1 have good physical and chemical properties.
  • Fig. 1 is a schematic diagram of the structure of the anti-PD1 ⁇ PDL1 double antibody of the present invention.
  • Figure 2A is the HPLC-SEC detection profile of the anti-PD1 ⁇ PDL1 double antibody of the present invention.
  • Figure 2B is the SDS-PAGE detection result of the anti-PD1 ⁇ PDL1 double antibody of the present invention.
  • Figure 3A is the result of ELISA detecting the binding of the anti-PD1 ⁇ PDL1 double antibody of the present invention to PD-1.
  • Figure 3B is the result of ELISA detecting the binding of the anti-PD1 ⁇ PDL1 double antibody of the present invention to PD-L1.
  • Fig. 4 is the result of dual-specific ELISA detecting the binding of the anti-PD1 ⁇ PDL1 double antibody of the present invention to PD-1 and PD-L1 at the same time.
  • Fig. 5A is the result of FACS detecting the binding of the anti-PD1 ⁇ PDL1 double antibody of the present invention to PD-1/CHO cells.
  • Fig. 5B is the result of FACS detecting the binding of the anti-PD1 ⁇ PDL1 double antibody of the present invention to N87-PDL1 cells.
  • Fig. 6 is the result of blocking PD1/PD-L1 activity on cells by the anti-PD1 ⁇ PDL1 double antibody of the present invention.
  • Figure 7A shows the results of anti-PD1 ⁇ PDL1 and rev2 blocking the PD1/PD-L1 signaling pathway on cells.
  • Figure 7B shows the results of anti-PD1 ⁇ PDL1 rev3, rev4, and rev5 blocking the PD1/PD-L1 signaling pathway on cells.
  • Figure 7C shows the results of anti-PD1 ⁇ PDL1 rev6, rev7, and rev8 blocking the activity of the PD1/PD-L1 signaling pathway on the cell.
  • the terms "Antibody (Ab)” and “Immunoglobulin G (Immunoglobulin G, IgG)” are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L ) And two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes (isotype) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain.
  • Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on.
  • the heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain.
  • the two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.
  • bispecific antibody double antibody refers to an antibody molecule that can specifically bind to two antigens (targets) or two epitopes at the same time.
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins.
  • the modifier "monoclonal" indicates the characteristics of the antibody, which is obtained from a substantially uniform antibody population, which should not be interpreted as requiring any special method to produce the antibody.
  • the terms "Fab” and “Fc” mean that papain can cleave an antibody into two identical Fab segments and one Fc segment.
  • the Fab segment is composed of the VH and CH1 of the heavy chain of the antibody and the VL and CL domains of the light chain.
  • the Fc segment can be a fragment crystallizable (Fc), which is composed of the CH2 and CH3 domains of the antibody.
  • the Fc segment has no antigen binding activity and is the site where the antibody interacts with effector molecules or cells.
  • scFv refers to a single chain antibody (single chain antibody fragment, scFv), which is formed by linking the variable region of the heavy chain of the antibody and the variable region of the light chain through a short peptide (linker) of 15-25 amino acids.
  • variable means that certain parts of the variable region of the antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three fragments called the complementarity-determining region (CDR) or hypervariable region in the variable region of the heavy chain and the variable region of the light chain.
  • CDR complementarity-determining region
  • FR frame region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)).
  • the terms "anti”, “binding”, and “specific binding” refer to the non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it is directed against.
  • the antibody binds to the antigen with an equilibrium dissociation constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or less.
  • KD refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • SPR Surface Plasmon Resonance
  • ELISA ELISA
  • epitope refers to a polypeptide determinant that specifically binds to an antibody.
  • the epitope of the present invention is a region of an antigen that is bound by an antibody.
  • expression vector can be pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, etc., and other vectors used in mammalian expression systems.
  • the expression vector includes those connected with appropriate transcription and translation regulatory sequences. Fusion DNA sequence.
  • the term "host cell” refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell.
  • mammalian or insect host cell culture systems can be used for the expression of the fusion protein of the present invention.
  • CHO Choinese hamster Ovary, Chinese Hamster Ovary
  • HEK293, COS BHK and derived cells of the above-mentioned cells are all suitable for the present invention.
  • the term "pharmaceutical composition” means that the bispecific antibody of the present invention can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical preparation composition so as to exert a more stable therapeutic effect. These preparations can ensure that the bispecific antibody disclosed in the present invention The conformational integrity of the amino acid core sequence of the sex antibody, while also protecting the multifunctional groups of the protein from its degradation (including but not limited to aggregation, deamination or oxidation).
  • pcDNA TM 3.4 vector purchased from Thermo fisher company, article number A14697;
  • CHO cells purchased from Thermofisher, catalog number A29133;
  • PD-1/PD-L1 Blockade Bioassay, Propagation model purchased from Promega, item number J1252;
  • Human gastric cancer cell line NCI-N87 purchased from the American Type Culture Collection (ATCC);
  • Anti-PD-L1 monoclonal antibody prepared according to the sequence in PCT/CN2020/090442;
  • Anti-PD-1 monoclonal antibody prepared according to the sequence in WO2018/137576;
  • HRP-labeled mouse anti-human Fab antibody purchased from sigma, catalog number A0293;
  • HRP-labeled anti-6 ⁇ His antibody purchased from abcam, catalog number ab178563;
  • Goat anti-human IgG-FITC purchased from sigma, item number F4143;
  • PBS purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number B548117;
  • BSA purchased from Shenggong Biological Engineering (Shanghai) Co., Ltd., catalog number A60332;
  • FBS purchased from Gibco, item number 10099;
  • TMB purchased from BD company, article number 555214;
  • Bio-Glo Luciferase Assay System purchased from Promega, item number G7940.
  • PCR instrument purchased from BioRad, article number C1000 Touch Thermal Cycler;
  • HiTrap MabSelectSuRe column purchased from GE, item number 11-0034-95;
  • Beckman Coulter CytoFLEX flow cytometer purchased from Beckman;
  • SpectraMax i3x microplate reader purchased from Molecular Devices.
  • the present invention adopts scFv (VL-linker1-VH, linker1 is 4 GGGGS) of anti-human PD-1 monoclonal antibody mAb1-25-Hu (sequence derived from WO2018/137576), connected in series through linker2 (3 GGGGS)
  • linker1 is 4 GGGGS
  • linker2 3 GGGGS
  • an anti-PD1 ⁇ PDL1 bispecific antibody was constructed, named anti-PD1 ⁇ PDL1 BsAb.
  • the structure is shown in Figure 1.
  • the present invention uses scFv2 (VH-linker1-VL, linker1 is 4 GGGGS) of anti-human PD-1 monoclonal antibody mAb1-25-Hu, connected in series with anti-human PD-L1 monoclonal antibody through linker2 (3 GGGGS)
  • the anti-PD1 ⁇ PDL1 bispecific antibody was constructed against the N-terminus of the heavy chain of M8 and named anti-PD1 ⁇ PDL1 Rev2.
  • the present invention uses scFv2 (VH-linker1-VL, linker1 is 4 GGGGS) of anti-human PD-1 monoclonal antibody mAb1-25-Hu, connected in series with anti-human PD-L1 monoclonal antibody through linker2 (3 GGGGS)
  • the anti-M8 heavy chain C-terminus was used to construct an anti-PD1 ⁇ PDL1 bispecific antibody named anti-PD1 ⁇ PDL1 Rev3.
  • the present invention adopts the scFv (VL-linker1-VH, linker1 is 4 GGGGS) of anti-human PD-1 monoclonal antibody mAb1-25-Hu, connected in series with the anti-human PD-L1 monoclonal antibody through linker2 (3 GGGGS)
  • the anti-PD1 ⁇ PDL1 bispecific antibody was constructed against the N-terminus of the heavy chain of M8 and named anti-PD1 ⁇ PDL1 Rev4.
  • the present invention uses scFv3 (VL-linker1-VH, linker1 is 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8, connected in series with anti-human PD-1 monoclonal antibody mAb1-25 through linker2 (3 GGGGS) -Hu's heavy chain C-terminus, an anti-PD1 ⁇ PDL1 bispecific antibody was constructed, named anti-PD1 ⁇ PDL1 Rev5.
  • the present invention uses scFv4 (VH-linker1-VL, linker1 is 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8, connected in series with anti-human PD-1 monoclonal antibody mAb1-25 through linker2 (3 GGGGS) -Hu's heavy chain C-terminus, an anti-PD1 ⁇ PDL1 bispecific antibody was constructed, named anti-PD1 ⁇ PDL1 Rev6.
  • the present invention uses scFv4 (VH-linker1-VL, linker1 is 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8, connected in series with anti-human PD-1 monoclonal antibody mAb1-25 through linker2 (3 GGGGS) -Hu's heavy chain N-terminus, an anti-PD1 ⁇ PDL1 bispecific antibody was constructed, named anti-PD1 ⁇ PDL1 Rev7.
  • the present invention uses scFv3 (VL-linker1-VH, linker1 is 4 GGGGS) of anti-human PD-L1 monoclonal antibody M8, connected in series with anti-human PD-1 monoclonal antibody mAb1-25 through linker2 (3 GGGGS) -Hu's heavy chain N-terminus, an anti-PD1 ⁇ PDL1 bispecific antibody was constructed, named anti-PD1 ⁇ PDL1 Rev8.
  • the heavy chain and light chain expression vectors of the bispecific antibody and its corresponding monoclonal antibody were obtained by gene synthesis and conventional molecular cloning methods.
  • the corresponding amino acid sequences are shown in Table 1, and the CDRs are coded according to the Kabat rule.
  • SEQ ID NO: Sequence name 1 The amino acid sequence of H-CDR1 of the heavy chain complementarity determining region of anti-PD-L1 monoclonal antibody
  • the two polypeptide chains and light chain DNA fragments of anti-PD1 ⁇ PDL1 double antibody anti-PD1 ⁇ PDL1 BsAb were subcloned into pcDN3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells and/or 293E cells. After culturing for 5-7 days, the culture solution was filtered by high-speed centrifugation and microporous membrane, and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted with an eluent containing 100 mM citric acid, pH 3.5, and dialyzed to pH 7.4 PBS.
  • the purified protein was detected by HPLC.
  • the HPLC-SEC detection pattern of anti-PD1 ⁇ PDL1 double antibody anti-PD1 ⁇ PDL1 BsAb is shown in Figure 2A, and the purity of the double antibody monomer reached more than 96%.
  • the results of SDS-PAGE detection are shown in Figure 2B. Lanes 1 and 2 are reduced and non-reduced SDS-PAGE of anti-PD1 ⁇ PDL1 double antibody, and lanes 3 and 4 are reduced and non-reduced SDS-PAGE of anti-PD-L1 monoclonal antibody. .
  • the theoretical molecular weight of the double antibody is 197KD.
  • Example 3 Enzyme-linked immunosorbent assay (ELISA) to detect the affinity of anti-PD1 ⁇ PDL1 double antibodies to antigen
  • the PD1-ECD-hFc protein (according to the sequence provided by UniProt (SEQ ID NO: Q15116)) was synthesized into the extracellular domain gene with PBS buffer of pH 7.4 and added to its N The signal peptide sequence is added to the end, hFc is added to the C end, and the two restriction sites of EcoRI and HindIII are respectively constructed into the expression vector pcDNA3.4 or pTT5, and transfected into HEK-293E cells for expression and purification).
  • the experimental results are shown in Figure 3A.
  • the EC 50 of anti-PD-1 monoclonal antibody and anti-PD1 ⁇ PDL1 double antibody anti-PD1 ⁇ PDL1 BsAb binding to PD-1 antigen is 0.29nM and 0.30nM, respectively, and the affinity of the two is equivalent.
  • the PDL1-ECD-His protein (according to the sequence provided by NCBI (NCBI registration number NP_054862.1) was synthesized into PD-L1 with pH 7.4 PBS buffer The extracellular domain gene is added with a signal peptide sequence at its N-terminus, and a 6 ⁇ His tag at the C-terminus. It is constructed into the expression vector pcDNA3.4 or pTT5 through the two restriction sites of EcoRI and HindIII, and transfected into HEK-293E.
  • the PD1-ECD-hFc protein was diluted to 200ng/ml with pH7.4 PBS buffer, and then 100 ⁇ L/well was added to the ELISA plate Medium; incubate overnight at 4°C; wash the plate twice with PBST the next day; add PBST+1%BSA to each well for blocking, and block at 37°C for 1h; wash the plate twice with PBST; then add gradient dilutions of PBS+1%BSA
  • the antibody to be detected has an initial concentration of 100 nM, and 12 gradients of 3-fold dilution are made step by step.
  • the experimental results are shown in Figure 4.
  • the EC 50 of the anti-PD1 ⁇ PDL1 double antibody anti-PD1 ⁇ PDL1 BsAb is 0.36nM, while the anti-PD-1 monoclonal antibody and anti-PD-L1 monoclonal antibody have no ability to simultaneously bind to these two antigens. .
  • CHO stable transfected cells expressing PD-1 on the cell surface were used as target cells, and the binding affinity of anti-PD1 ⁇ PDL1 double antibodies to the cells was measured by flow cytometry.
  • the cells were washed three times with PBS containing 0.5% BSA, centrifuged at 300 g for 5 minutes each time, and the supernatant was discarded. Resuspend the cells in 0.5% BSA in PBS at a cell density of 1 ⁇ 10 6 cells/mL, and add 100 ⁇ L/well to a 96-well plate.
  • the anti-PD1 ⁇ PDL1 double antibody and the positive control anti-PD-1 monoclonal antibody were started at 200 nM, and 11 gradients were gradually diluted, and 100 ⁇ L/well was added to a 96-well plate, and incubated at 4°C for 1 hour. Wash the cells twice with PBS to remove unbound antibody to be tested. Then add 100 ⁇ L of goat anti-human IgG-FITC and incubate at 4°C for 30 minutes. Centrifuge at 300g for 5 minutes, and wash the cells twice with PBS to remove unbound secondary antibodies.
  • the EC 50 of the anti-PD-1 monoclonal antibody and the anti-PD1 ⁇ PDL1 double antibody anti-PD1 ⁇ PDL1 BsAb are 0.64 nM and 1.43 nM, respectively, and the affinity of the two is equivalent.
  • N87-PDL1 is a stable transfected cell line constructed by transfecting NCI-N87 with PD-L1 using the lentiviral transfection method in our laboratory. After taking the N87-PDL1 in the logarithmic growth phase and digesting it with trypsin, it was washed three times with PBS containing 0.5% BSA, and centrifuged at 300 g for 5 minutes each time, and the supernatant was discarded. Resuspend the cells in 0.5% BSA in PBS at a cell density of 1 ⁇ 10 6 cells/mL, and add 100 ⁇ L/well to a 96-well plate.
  • Anti-PD1 ⁇ PDL1 double antibody blocks the activity of PD-1/PD-L1 at the cellular level
  • This experiment uses Promega's PD-1/PD-L1 Blockade Bioassay, Propagation model and method.
  • the experimental results are shown in Figure 6.
  • the IC 50 of anti-PD1 ⁇ PDL1 BsAb, anti-PD-L1 monoclonal antibody, and anti-PD-1 monoclonal antibody are 0.24nM, 0.42nM, 1.60nM, respectively, and the double antibody anti-PD1 ⁇ PDL1 BsAb
  • the IC 50 of the anti-PD-L1 monoclonal antibody is close to that of the anti-PD-1 monoclonal antibody.
  • the IC 50 of the anti-PD-1 monoclonal antibody is too large, but its high platform is slightly higher.
  • anti-PD1 ⁇ PDL1 rev2 is significantly worse than the positive control anti-PD-1 monoclonal antibody
  • anti-PD1 ⁇ PDL1 rev3, rev6 has the same activity as the positive control anti-PD-L1 monoclonal antibody
  • anti-PD1 ⁇ PDL1 rev4, rev5 is more active than the positive control anti-PD- L1 monoclonal antibody is slightly worse
  • the activity of anti-PD1 ⁇ PDL1 rev7, rev8 is significantly worse than the positive control anti-PD-L1 monoclonal antibody.
  • Example 2 Take 4 SD rats from each group, weighing about 200 g, and each rat was injected with the antibody prepared in Example 2 at a dose of 2 mg through the tail vein.
  • the blood collection time points after administration were: 0h, 3h, 24h, 48h, 96h, 168h, 336h, 504h. Blood was taken from the orbit, and the blood was centrifuged at 8000 rpm/min to obtain serum after spontaneous coagulation.
  • the serum drug concentration of anti-PD1 ⁇ PDL1 double antibody anti-PD1 ⁇ PDL1 BsAb is detected by the following method:
  • Protein A coats the ELISA plate, and detects the Fab fragment of the antibody. Coated with protein A, the coating amount was 100ng/well, overnight at 4°C; the next day, the plate was washed twice with PBST, and then blocked with PBS+2% BSA at 37°C for 2 hours. Wash the plate twice with PBST.
  • the standard of anti-PD1 ⁇ PDL1 double antibody starts from 1000ng/mL, and is diluted two-fold in 12 gradients. The rat serum samples are diluted 1000-4000 times.

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Abstract

提供一种抗PD1×PDL1的双特异性抗体及其制备方法和用途，实验结果显示该双特异性抗体能够较好的保持各自单抗的活性，并且能够同时特异性结合PD-1和PD-L1两个靶点，具有良好的理化性质。

Description

一种抗PD1×PDL1的双特异性抗体 技术领域
本发明涉及抗体领域，具体涉及一种抗PD1×PDL1的双特异性抗体及其制备方法和用途。
背景技术
人程序性细胞死亡受体-1(PD-1)是一种有288个氨基酸的I型膜蛋白，是已知的主要免疫检查点(Immune Checkpoint)之一(Blank et al,2005,Cancer Immunotherapy,54:307-314)。PD-1表达在已经激活的T淋巴细胞，它与配体PD-L1(程序性细胞死亡受体-配体1，programmed cell death-Ligand 1)和PD-L2(程序性细胞死亡受体-配体2，programmed cell death-Ligand 2)结合可以抑制T淋巴细胞的活性及相关的体内细胞免疫反应。PD-L2主要表达在巨噬细胞和树突状细胞，而PD-L1则广泛表达于B、T淋巴细胞及外周细胞如微血管上皮细胞，肺、肝、心等组织细胞中。大量研究表明，PD-1和PD-L1的相互作用不但是维持体内免疫系统平衡所必须，也是导致PD-L1表达阳性肿瘤细胞规避免疫监视的主要机制和原因。通过阻断癌细胞对PD-1/PD-L1信号通路的负调控，激活免疫系统，能够促进T细胞相关的肿瘤特异性细胞免疫反应，从而打开了一扇新的肿瘤治疗方法的大门——肿瘤免疫疗法。
PD-1(由基因Pdcd1编码)为与CD28和CTLA-4有关的免疫球蛋白超家族成员。研究成果显示，当PD-1与其配体(PD-L1和/或PD-L2)结合时会负调节抗原受体信号转导。目前已弄清鼠PD-1结构以及小鼠PD-1与人PD-L1的共结晶结构(Zhang，X.等，Immunity 20：337-347(2004)；Lin等，Proc.Natl.Acad.Sci.USA 105：3011-6(2008))。PD-1及类似的家族成员为I型跨膜糖蛋白，其含有负责配体结合的Ig可变型(V-型)结构域和负责结合信号转导分子的胞质尾区。PD-1胞质尾区含有两个基于酪氨酸的信号转导模体ITIM(免疫受体酪氨酸抑制作用模体)和ITSM(免疫受体酪氨酸转换作用模体)。
PD-1在肿瘤的免疫逃避机制中起到了重要的作用。肿瘤免疫疗法，即利用人体自身的免疫系统抵御癌症，是一种突破性的肿瘤治疗方法，但是肿瘤微环境可保护肿瘤细胞免受有效的免疫破坏，因此如何打破肿瘤微环境成为抗肿瘤研究的重点。现有研究成果已确定了PD-1在肿瘤微环境中的作用：PD-L1在许多小鼠和人肿瘤中表达(并在大多数PD-L1阴性肿瘤细胞系中可由IFN-γ诱导)，并被推定为介导肿瘤免疫逃避的重要靶点(Iwai Y.等，Proc.Natl.Acad.Sci.U.S.A.99：12293-12297(2002)；Strome S.E.等，Cancer Res.，63：6501-6505(2003))。通过免疫组织化学评估活组织检查，已经在人的很多原发性肿瘤中发现 PD-1(在肿瘤浸润淋巴细胞上)和/或PD-L1在肿瘤细胞上的表达。这样的组织包括肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、结肠癌、神经胶质瘤、膀胱癌、乳腺癌、肾癌、食道癌、胃癌、口腔鳞状细胞癌、尿道上皮细胞癌和胰腺癌以及头颈肿瘤等。由此可见，阻断PD-1/PD-L1的相互作用可以提高肿瘤特异性T细胞的免疫活性，有助于免疫系统清除肿瘤细胞，因此PD-1和PD-L1成为开发肿瘤免疫治疗药物的热门靶点。
双特异性抗体是指能同时特异性结合两种抗原或两种表位的抗体分子。根据对称性，双特异性抗体可以分为结构对称的和不对称的分子。根据结合位点的多少，双特异性抗体可以分为二价、三价、四价和多价分子。双特异性抗体正在逐步成为一类新的治疗性抗体，可以用于治疗各种炎性疾病、癌症和其它疾病。
发明内容
本发明提供了一种抗PD1×PDL1的双特异性抗体。
因此，本发明的第一个目的在于提供一种抗PD1×PDL1的双特异性抗体。
本发明的第二个目的在于提供一种编码所述双特异性抗体的分离的核苷酸。
本发明的第三个目的在于提供一种包含所述核苷酸的表达载体。
本发明的第四个目的在于提供一种包含所述表达载体的宿主细胞。
本发明的第五个目的在于提供所述双特异性抗体的制备方法。
本发明的第六个目的在于提供包含所述双特异性抗体的药物组合物。
本发明的第七个目的在于提供所述的双特异性抗体或所述药物组合物在制备治疗癌症的药物中的用途。
本发明的第八个目的在于提供所述双特异性抗体或所述药物组合物用于治疗癌症的方法。
为了达到上述目的，本发明提供了以下技术方案：
本发明的第一个方面提供一种抗PD1×PDL1的双特异性抗体，所述双特异性抗体包含两条多肽链和两条轻链：
a)各所述多肽链从N末端至C末端依次包含VH-PDL1—CH1—CH2—CH3—linker2—VL-PD1—linker1—VH-PD1或VH-PDL1—CH1—CH2—CH3—linker2—VH-PD1—linker1—VL-PD1或VL-PD1—linker1—VH-PD1—linker2—VH-PDL1—CH1—CH2—CH3或VH-PD1—linker1—VL-PD1—linker2—VH-PDL1—CH1—CH2—CH3，各所述轻链从N末端至C末端依次包含VL-PDL1—CL；或
b)各所述多肽链从N末端至C末端依次包含VH-PD1—CH1—CH2—CH3—linker2 —VL-PDL1—linker1—VH-PDL1或VH-PD1—CH1—CH2—CH3—linker2—VH-PDL1—linker1—VL-PDL1或VL-PDL1—linker1—VH-PDL1—linker2—VH-PD1—CH1—CH2—CH3或VH-PDL1—linker1—VL-PDL1—linker2—VH-PD1—CH1—CH2—CH3，各所述轻链从N末端至C末端依次包含VL-PD1—CL，
其中，所述VH-PDL1为结合PD-L1的重链可变区，所述CH1-CH2-CH3为重链恒定区，所述VL-PD1为结合PD-1的轻链可变区，所述VH-PD1为结合PD-1的重链可变区，所述VL-PDL1为结合PD-L1的轻链可变区，所述CL为轻链恒定区。
所述抗PD1×PDL1的双特异性抗体中，所述VH-PDL1与所述VL-PDL1形成特异性结合PD-L1的抗原结合位点，所述VL-PD1与所述VH-PD1形成特异性结合PD-1的抗原结合位点。
根据本发明的优选实施例，所述抗PD1×PDL1的双特异性抗体还包括以下中的一项或多项：
1)所述VH-PDL1包含氨基酸序列如SEQ ID NO：1-3所示的重链CDR；
2)所述VL-PDL1包含氨基酸序列如SEQ ID NO：4-6所示的轻链CDR；
3)所述VH-PD1包含氨基酸序列如SEQ ID NO：7-9所示的重链CDR；
4)所述VL-PD1包含氨基酸序列如SEQ ID NO：10-12所示的轻链CDR。
在一种实施方式中，所述VH-PDL1包含氨基酸序列如SEQ ID NO：1所示的H-CDR1。
在一种实施方式中，所述VH-PDL1包含氨基酸序列如SEQ ID NO：2所示的H-CDR2。
在一种实施方式中，所述VH-PDL1包含氨基酸序列如SEQ ID NO：3所示的H-CDR3。
在一种实施方式中，所述VL-PDL1包含氨基酸序列如SEQ ID NO：4所示的L-CDR1。
在一种实施方式中，所述VL-PDL1包含氨基酸序列如SEQ ID NO：5所示的L-CDR2。
在一种实施方式中，所述VL-PDL1包含氨基酸序列如SEQ ID NO：6所示的L-CDR3。
在一种实施方式中，所述VH-PD1包含氨基酸序列如SEQ ID NO：7所示的H-CDR1。
在一种实施方式中，所述VH-PD1包含氨基酸序列如SEQ ID NO：8所示的H-CDR2。
在一种实施方式中，所述VH-PD1包含氨基酸序列如SEQ ID NO：9所示的H-CDR3。
在一种实施方式中，所述VL-PD1包含氨基酸序列如SEQ ID NO：10所示的L-CDR1。
在一种实施方式中，所述VL-PD1包含氨基酸序列如SEQ ID NO：11所示的L-CDR2。
在一种实施方式中，所述VL-PD1包含氨基酸序列如SEQ ID NO：12所示的L-CDR3。
根据本发明的优选实施例，所述VH-PDL1具有如SEQ ID NO：13所示的氨基酸序列，和/或，所述VL-PDL1具有如SEQ ID NO：14所示的氨基酸序列，和/或，所述VH-PD1具有如SEQ ID NO：15所示的氨基酸序列，和/或，所述VL-PD1具有如SEQ ID NO：16所示的 氨基酸序列。
所述linker2为3个G 4S，所述linker1为4个G 4S。
根据本发明，所述的重链恒定区包括IgG1、IgG2、IgG3或IgG4重链恒定区，所述的轻链恒定区包括κ或λ轻链恒定区。
根据本发明的优选实施例，所述多肽链具有如SEQ ID NO：17或SEQ ID NO：22或SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列，和/或，所述的轻链具有如SEQ ID NO：18所示的氨基酸序列；或所述多肽链具有如SEQ ID NO：25或SEQ ID NO：26或SEQ ID NO：27或SEQ ID NO：28所示的氨基酸序列，和/或，所述的轻链具有如SEQ ID NO：21所示的氨基酸序列。
本发明的第二个方面提供了一种分离的核苷酸，所述核苷酸编码所述双特异性抗体。
本发明所述核苷酸的制备方法为本领域常规的制备方法，较佳地包括以下制备方法：通过基因克隆技术例如PCR方法等，获得编码上述单克隆抗体的核苷酸，或者通过人工全序列合成的方法得到编码上述单克隆抗体的核苷酸。
本领域技术人员知晓，编码上述双特异性抗体的氨基酸序列的核苷酸序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该双特异性抗体基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。
本发明的第三个方面提供了一种表达载体，所述表达载体含有如上所述核苷酸。
其中所述表达载体为本领域常规的表达载体，是指包含适当的调控序列，例如启动子序列、终止子序列、多腺苷酰化序列、增强子序列、标记基因和/或序列以及其他适当的序列的表达载体。所述表达载体可以是病毒或质粒，如适当的噬菌体或者噬菌粒，更多技术细节请参见例如Sambrook等，Molecular Cloning：A Laboratory Manual，第二版，Cold Spring Harbor Laboratory Press，1989。许多用于核酸操作的已知技术和方案请参见Current Protocols in Molecular Biology，第二版，Ausubel等编著。
本发明所述表达载体较佳地为pTT5，pDR1，pcDNA3.1，pcDNA3.4，pDHFF，pGM-CSF或pCHO1.0，更佳地为pcDNA3.4，pTT5。
本发明的第四个方面提供了一种宿主细胞，所述的宿主细胞含有如上所述的表达载体。
本发明所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核苷酸可被有效表达即可。其中所述宿主细胞包括原核表达细胞和真核表达细胞。具体的，所述宿主细胞例如为：COS、CHO(中国仓鼠卵巢，Chinese  Hamster Ovary)、NS0、sf9、sf21、DH5α、BL21(DE3)、E.coli TG1、BL21(DE3)细胞或者CHO-K1细胞、293E细胞或TG1。所述宿主细胞更佳地为CHO细胞和/或293E细胞。
本发明的第五个方面提供了所述双特异性抗体的制备方法，所述方法包含以下步骤：
(a)在表达条件下，培养如上所述的宿主细胞，从而表达所述双特异性抗体；
(b)分离并纯化(a)所述的双特异性抗体。
本发明所述的宿主细胞的培养方法、所述抗PD1×PDL1的双特异性抗体的分离和纯化方法为本领域常规方法，具体操作方法请参考相应的细胞培养技术手册以及双特异性抗体分离纯化技术手册。本发明中公开的抗PD1×PDL1的双特异性抗体的制备方法包括：在表达条件下，培养上述的宿主细胞，从而表达抗PD1×PDL1的双特异性抗体；分离和纯化所述的抗PD1×PDL1的双特异性抗体。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
可以利用亲和层析的方法对本发明公开的抗PD1×PDL1的双特异性抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的双特异性抗体。本发明的发明人对所得抗PD1×PDL1的双特异性抗体进行了检测实验，实验结果表明该双特异性抗体能很好地与PD-1、PDL-1结合，具有较高的亲和力。
本发明的第六个方面提供了一种药物组合物，所述药物组合物含有如上所述的双特异性抗体和药学上可接受的载体。
本发明提供的抗PD1×PDL1的双特异性抗体，可以和药学上可接受的载体一起组成药物组合物从而更稳定地发挥疗效。药物组合物具有一定的制剂形式可以保证本发明公开的抗PD1×PDL1的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
本发明的第七个方面提供了如上所述的双特异性抗体或如上所述的药物组合物在制备治疗癌症的药物中的用途。
根据本发明，所述癌症选自黑色素瘤、肾癌、肺癌、肝癌、胃癌、淋巴癌、乳腺癌、结直肠癌、白血病、前列腺癌、骨髓癌及其它赘生性恶性疾病中的一种或多种。
本发明所述抗PD1×PDL1的双特异性抗体可以单独使用或与其它抗肿瘤药物联合使用。
本发明所称的治疗癌症的药物，指具有抑制和/或治疗肿瘤的药物，可以包括伴随肿瘤相关症状发展的延迟和/或这些症状严重程度的降低，进一步还包括已存在的肿瘤伴随症状的减轻并防止其他症状的出现，还包括减少或防止肿瘤的转移等。
本发明中抗PD1×PDL1的双特异性抗体及其药物组合物在对包括人在内的动物给药时， 给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的第八个方面提供了一种治疗癌症的方法，包括向有需要的受试者施用如上所述的双特异性抗体或如上所述的药物组合物。
根据本发明，所述癌症选自黑色素瘤、肾癌、肺癌、肝癌、胃癌、淋巴癌、乳腺癌、结直肠癌、白血病、前列腺癌、骨髓癌及其它赘生性恶性疾病中的一种或多种。
所述抗PD1×PDL1的双特异性抗体及其药物组合物在向受试者施用时，给药剂量需为治疗有效量。所述治疗有效量是指在治疗癌症中有效果的量。具体的，所述抗PD1×PDL1的双特异性抗体及其药物组合物在向受试者施用时，给药剂量因病人的年龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。
本发明的有益效果如下：本发明提供了一种抗PD1×PDL1的双特异性抗体，实验结果显示该双抗能够较好的保持各自单抗的活性，并且能够同时特异性结合PD-1和PD-L1两个靶点，具有良好的理化性质。
附图说明
图1为本发明的抗PD1×PDL1双抗的结构示意图。
图2A为本发明的抗PD1×PDL1双抗的HPLC-SEC检测图谱。
图2B为本发明的抗PD1×PDL1双抗的SDS-PAGE检测结果。
图3A为ELISA检测本发明的抗PD1×PDL1双抗与PD-1的结合结果。
图3B为ELISA检测本发明的抗PD1×PDL1双抗与PD-L1的结合结果。
图4为双特异ELISA检测本发明的抗PD1×PDL1双抗同时与PD-1和PD-L1的结合结果。
图5A为FACS检测本发明的抗PD1×PDL1双抗与PD-1/CHO细胞的结合结果。
图5B为FACS检测本发明的抗PD1×PDL1双抗与N87-PDL1细胞的结合结果。
图6为本发明的抗PD1×PDL1双抗阻断细胞上PD1/PD-L1的活性结果。
图7A为anti-PD1×PDL1 rev2阻断细胞上PD1/PD-L1信号通路的活性结果。
图7B为anti-PD1×PDL1 rev3，rev4，rev5阻断细胞上PD1/PD-L1信号通路的活性结果。
图7C为anti-PD1×PDL1 rev6，rev7，rev8阻断细胞上PD1/PD-L1信号通路的活性结果。
具体实施方式
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“双特异性抗体(双抗)”是指能同时特异性结合两种抗原(靶点)或两种表位的抗体分子。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段。Fab段由抗体的重链的VH和CH1以及轻链的VL和CL结构域组成。Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“scFv”为单链抗体(single chain antibody fragment，scFv)，由抗体重链可变区和轻链可变区通过15～25个氨基酸的短肽(linker)连接而成。
本发明中，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。 它集中于重链可变区和轻链可变区中称为互补决定区(complementarity-determining region，CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(frame region，FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等，NIH Publ.No.91-3242，卷I，647-669页(1991))。
本发明中，术语“抗”、“结合”、“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。通常，抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M、10 -10M、10 -11M或更小的平衡解离常数(KD)结合该抗原。本发明中，术语“KD”是指特定抗体-抗原相互作用的平衡解离常数，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。例如，使用表面等离子体共振术(Surface Plasmon Resonance，缩写SPR)在BIACORE仪中测定抗体与抗原的结合亲和力或使用ELISA测定抗体与抗原结合的相对亲和力。
本发明中，术语“表位”是指与抗体特异性结合的多肽决定簇。本发明的表位是抗原中被抗体结合的区域。
本发明中，术语“表达载体”可以为pTT5，pSECtag系列，pCGS3系列，pcDNA系列载体等，以及其它用于哺乳动物表达系统的载体等，表达载体中包括连接有合适的转录和翻译调节序列的融合DNA序列。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵巢，Chinese Hamster Ovary)，HEK293，COS，BHK以及上述细胞的衍生细胞均可适用于本发明。
本发明中，术语“药物组合物”是指本发明的双特异性抗体可以和药学上可以接受的载体一起组成药物制剂组合物从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱氨或氧化)。
以下实施例中使用的实验材料说明如下：
pcDNA TM 3.4
Figure PCTCN2021096396-appb-000001
vector：购自Thermo fisher公司，货号A14697；
CHO细胞：购自Thermo fisher公司，货号A29133；
293E细胞：来自NRC biotechnology Research Institute；
PD-1/PD-L1 Blockade Bioassay,Propagation model：购自Promega公司，货号J1252；
人胃癌细胞株NCI-N87：购自美国典型培养物保藏中心(ATCC)；
SD大鼠：购自上海灵畅生物科技有限公司。
以下实施例中使用的实验试剂说明如下：
抗PD-L1单抗：根据PCT/CN2020/090442中的序列制备；
抗PD-1单抗：根据WO2018/137576中的序列制备；
HRP标记的鼠抗人Fab抗体：购自sigma，货号A0293；
HRP标记的anti-6×His抗体：购自abcam，货号ab178563；
羊抗人IgG-FITC：购自sigma，货号F4143；
PBS：购自生工生物工程(上海)股份有限公司，货号B548117；
PBST：PBS+0.05％Tween 20；
BSA：购自生工生物工程(上海)股份有限公司，货号A60332；
FBS：购自Gibco，货号10099；
TMB：购自BD公司，货号555214；
Bio-Glo Luciferase Assay System：购自Promega，货号G7940。
以下实施例中使用的实验仪器说明如下：
PCR仪：购自BioRad，货号C1000 Touch Thermal Cycler；
HiTrap MabSelectSuRe柱：购自GE公司，货号11-0034-95；
Beckman Coulter CytoFLEX流式细胞仪：购自Beckman公司；
SpectraMax i3x酶标仪：购自Molecular Devices公司。
以下实施例、实验例是对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
实施例1 抗PD1×PDL1双抗分子的构建
本发明采用将抗人PD-1单抗mAb1-25-Hu(序列来源于WO2018/137576)的scFv(VL-linker1-VH，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-L1单抗M8(序列来源于PCT/CN2020/090442)的重链C-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 BsAb。结构如图1所示。
本发明采用了将抗人PD-1单抗mAb1-25-Hu的scFv2(VH-linker1-VL，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-L1单抗M8的重链N-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev2。
本发明采用了将抗人PD-1单抗mAb1-25-Hu的scFv2(VH-linker1-VL，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-L1单抗M8的重链C-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev3。
本发明采用了将抗人PD-1单抗mAb1-25-Hu的scFv(VL-linker1-VH，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-L1单抗M8的重链N-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev4。
本发明采用了将抗人PD-L1单抗M8的scFv3(VL-linker1-VH，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-1单抗mAb1-25-Hu的重链C-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev5。
本发明采用了将抗人PD-L1单抗M8的scFv4(VH-linker1-VL，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-1单抗mAb1-25-Hu的重链C-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev6。
本发明采用了将抗人PD-L1单抗M8的scFv4(VH-linker1-VL，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-1单抗mAb1-25-Hu的重链N-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev7。
本发明采用了将抗人PD-L1单抗M8的scFv3(VL-linker1-VH，linker1为4个GGGGS)，通过linker2(为3个GGGGS)，串联在抗人PD-1单抗mAb1-25-Hu的重链N-末端的方式，构建了抗PD1×PDL1双特异性抗体，命名为anti-PD1×PDL1 Rev8。
通过基因合成及常规的分子克隆方法获得双特异性抗体及其对应的单克隆抗体的重链和轻链表达载体，其对应的氨基酸序列如表1所示，其中CDR根据Kabat规则编码。
表1、本发明的抗体的序列信息
SEQ ID NO： 序列名称
1 抗PD-L1单抗的重链互补决定区H-CDR1的氨基酸序列
2 抗PD-L1单抗的重链互补决定区H-CDR2的氨基酸序列
3 抗PD-L1单抗的重链互补决定区H-CDR3的氨基酸序列
4 抗PD-L1单抗的轻链互补决定区L-CDR1的氨基酸序列
5 抗PD-L1单抗的轻链互补决定区L-CDR2的氨基酸序列
6 抗PD-L1单抗的轻链互补决定区L-CDR3的氨基酸序列
7 抗PD-1单抗的重链互补决定区H-CDR1的氨基酸序列
8 抗PD-1单抗的重链互补决定区H-CDR2的氨基酸序列
9 抗PD-1单抗的重链互补决定区H-CDR3的氨基酸序列
10 抗PD-1单抗的轻链互补决定区L-CDR1的氨基酸序列
11 抗PD-1单抗的轻链互补决定区L-CDR2的氨基酸序列
12 抗PD-1单抗的轻链互补决定区L-CDR3的氨基酸序列
13 抗PD-L1单抗的重链可变区的氨基酸序列
14 抗PD-L1单抗的轻链可变区的氨基酸序列
15 抗PD-1单抗的重链可变区的氨基酸序列
16 抗PD-1单抗的轻链可变区的氨基酸序列
17 anti-PD1×PDL1 BsAb的多肽链的氨基酸序列
18 抗PD-L1单抗的轻链的氨基酸序列
19 抗PD-L1单抗的重链的氨基酸序列
20 抗PD-1单抗的重链的氨基酸序列
21 抗PD-1单抗的轻链的氨基酸序列
22 anti-PD1×PDL1 Rev2的多肽链的氨基酸序列
23 anti-PD1×PDL1 Rev3的多肽链的氨基酸序列
24 anti-PD1×PDL1 Rev4的多肽链的氨基酸序列
25 anti-PD1×PDL1 Rev5的多肽链的氨基酸序列
26 anti-PD1×PDL1 Rev6的多肽链的氨基酸序列
27 anti-PD1×PDL1 Rev7的多肽链的氨基酸序列
28 anti-PD1×PDL1 Rev8的多肽链的氨基酸序列
实施例2 抗PD1×PDL1双抗的表达与纯化
将抗PD1×PDL1双抗anti-PD1×PDL1 BsAb的两条多肽链和轻链的DNA片段分别亚克隆 到pcDN3.4载体中，抽提重组质粒共转染CHO细胞和/或293E细胞，细胞培养5-7天后，将培养液通过高速离心、微孔滤膜过滤后，上样至HiTrap MabSelectSuRe柱，用含有100mM柠檬酸，pH3.5的洗脱液洗脱蛋白，并透析至pH7.4的PBS。
将纯化后的蛋白用HPLC检测，抗PD1×PDL1双抗anti-PD1×PDL1 BsAb的HPLC-SEC检测图谱如图2A所示，双抗单体纯度达到96％以上。SDS-PAGE检测结果如图2B所示，泳道1与2为抗PD1×PDL1双抗的还原与非还原SDS-PAGE，泳道3与4为抗PD-L1单抗的还原与非还原SDS-PAGE。双抗理论分子量为197KD。
实施例3 酶联免疫吸附法(ELISA)检测抗PD1×PDL1双抗对抗原的亲和力
3.1与PD-1抗原的亲和力检测
为了检测抗PD1×PDL1双抗与PD-1抗原的亲和力，用pH7.4的PBS缓冲液将PD1-ECD-hFc蛋白(根据UniProt提供的序列(序列号Q15116)合成胞外域基因并在其N端加上信号肽序列，C末端加上hFc，通过EcoRI和HindIII两个酶切位点分别构建到表达载体pcDNA3.4或pTT5中，转染HEK-293E细胞表达并纯化获得)稀释至200ng/ml，然后100μL/孔加入ELISA板中；4℃孵育过夜；次日用PBST洗板两次；每孔加入PBST+1％BSA进行封闭，37℃封闭1h；用PBST洗板两次；然后加入用PBS+1％BSA梯度稀释的待检测抗体，抗PD-1单抗作为阳性对照，起始浓度为200nM，逐级3倍稀释12个梯度。37℃孵育1h；PBST洗板两次，加入二抗HRP标记的鼠抗人Fab，37℃再孵育40min；PBST洗板三次并拍干，每孔加入100μL TMB，室温(20±5℃)避光放置5分钟；每孔加入50μL的2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism进行数据分析，作图并计算EC 50。
实验结果如图3A所示，抗PD-1单抗和抗PD1×PDL1双抗anti-PD1×PDL1 BsAb与PD-1抗原结合的EC 50分别为0.29nM和0.30nM，两者亲和力相当。
3.2与PD-L1抗原的亲和力检测
为了检测抗PD1×PDL1双抗与PD-L1抗原的亲和力，用pH7.4的PBS缓冲液将PDL1-ECD-His蛋白(根据NCBI提供的序列(NCBI登记号为NP_054862.1)合成PD-L1胞外域基因并在其N端加上信号肽序列，C末端加上6×His标签，通过EcoRI和HindIII两个酶切位点分别构建到表达载体pcDNA3.4或pTT5中，转染HEK-293E细胞表达并纯化获得)稀释至1000ng/ml，然后100μL/孔加入ELISA板中；4℃孵育过夜；次日用PBST洗板两次；每孔加入PBST+1％BSA进行封闭，37℃封闭1h；用PBST洗板两次；然后加入用PBS+1％BSA梯度稀释的待检测抗体，抗PD-L1单抗作为阳性对照，起始浓度为200nM，逐级3倍稀释12 个梯度。37℃孵育1h；PBST洗板两次，加入二抗HRP标记的鼠抗人Fab，37℃再孵育40min；PBST洗板三次并拍干，每孔加入100μL TMB，室温(20±5℃)避光放置5分钟；每孔加入50μL2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism进行数据分析，作图并计算EC 50。
实验结果如图3B所示，抗PD-L1单抗及抗PD1×PDL1双抗anti-PD1×PDL1 BsAb与PD-L1抗原结合的EC 50为0.27nM和0.29nM，两者亲和力相当。
实施例4 双特异ELISA检测抗PD1×PDL1双抗同时结合两个抗原的能力
为了检测抗PD1×PDL1双抗同时结合PD-1抗原和PD-L1抗原的能力，用pH7.4的PBS缓冲液将PD1-ECD-hFc蛋白稀释至200ng/ml，然后100μL/孔加入ELISA板中；4℃孵育过夜；次日用PBST洗板两次；每孔加入PBST+1％BSA进行封闭，37℃封闭1h；用PBST洗板两次；然后加入用PBS+1％BSA梯度稀释的待检测抗体，起始浓度为100nM，逐级3倍稀释12个梯度。37℃孵育1h；PBST洗板两次，再加入pH7.4的PBS稀释的1000ng/ml的PDL1-ECD-His，100μL/孔加入ELISA板中。37℃孵育1h；PBST洗板两次，加入二抗HRP-anti-His，37℃再孵育40min；PBST洗板三次并拍干，每孔加入100μL TMB，室温(20±5℃)避光放置5分钟；每孔加入50μL 2M H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值，GraphPad Prism进行数据分析，作图并计算EC 50。
实验结果如图4所示，抗PD1×PDL1双抗anti-PD1×PDL1 BsAb的EC 50为0.36nM，而抗PD-1单抗和抗PD-L1单抗没有同时结合这两种抗原的能力。
实施例5 检测抗PD1×PDL1双抗对靶细胞的结合亲和力
细胞表面表达PD-1的CHO稳转细胞作为靶细胞，通过流式细胞仪测定抗PD1×PDL1双抗对该细胞的结合亲和力。用含有0.5％BSA的PBS洗涤三次细胞，每次300g离心5分钟，弃上清。0.5％BSA的PBS重悬细胞，细胞密度为1×10 6细胞/mL，100μL/孔加入96孔板。将抗PD1×PDL1双抗及阳性对照抗PD-1单抗200nM起始，逐级稀释11个梯度，100μL/孔加入96孔板，4℃孵育1h。PBS洗涤细胞两次以去除未结合的待检抗体。再加入100μL的羊抗人IgG-FITC，于4℃孵育30分钟。300g离心5分钟，PBS洗涤细胞两次以去除未结合的二抗。最后将细胞重悬在200μL PBS中，通过Beckman Coulter CytoFLEX流式细胞仪测定双抗对该细胞的结合亲和力。所得数据通过GraphPad Prism软件拟合分析。实验结果如图5A所示，抗PD-1单抗和抗PD1×PDL1双抗anti-PD1×PDL1 BsAb的EC 50分别为0.64nM和1.43nM， 两者亲和力相当。
N87-PDL1为本实验室采用慢病毒转染法给NCI-N87转染了PD-L1构建的稳转细胞株。取对数生长期的N87-PDL1用胰酶消化后，用含有0.5％BSA的PBS洗涤三次，每次300g离心5分钟，弃上清。0.5％BSA的PBS重悬细胞，细胞密度为1×10 6细胞/mL，100μL/孔加入96孔板。将抗PD1×PDL1双抗及阳性对照抗PD-L1单抗稀释为120nM，逐级稀释11个梯度，100μL/孔加入96孔板，与N87-PDL1细胞混合均匀。其余方法同上。实验结果如图5B所示，抗PD-L1单抗的EC 50为0.11nM，抗PD1×PDL1双抗anti-PD1×PDL1 BsAb的EC 50为0.15nM，两者亲和力相当。
实施例6 抗PD1×PDL1双抗阻断PD-1/PD-L1的细胞水平的活性
本实验采用Promega的PD-1/PD-L1 Blockade Bioassay,Propagation model及方法。
取对数期生长的PD-L1 aAPC/CHO-K1稳定细胞株，胰酶消化成单个细胞后转移到白色底透96孔板，100μL/孔，40000细胞/孔，置于37℃，5％CO 2，孵育过夜。取抗PD1×PDL1双抗、抗PD-L1单抗、抗PD-1单抗稀释成2×工作液浓度，起始浓度为100nM，逐级3倍梯度。取密度在1.4-2×10 6细胞/mL、细胞活率在95％以上的PD1效应细胞，胰酶消化成1.25×10 6细胞/ml的单细胞悬液。取前一天铺好的PD-L1 aAPC/CHO-K1细胞，弃掉上清，加入40μl梯度稀释的待检抗体工作液；再加入等体积的PD1效应细胞。置于37℃，5％CO 2，孵育6小时。每孔加入80μl检测试剂Bio-Glo。室温孵育10分钟后，用spectramax i3读取luminescence。用GraphPad Prism进行数据分析，作图并计算IC 50。
实验结果如图6所示，anti-PD1×PDL1 BsAb、抗PD-L1单抗、抗PD-1单抗的IC 50分别为0.24nM、0.42nM、1.60nM，双抗anti-PD1×PDL1 BsAb与抗PD-L1单抗的IC 50接近，抗PD-1单抗的IC 50偏大，但其高平台稍高。
anti-PD1×PDL1 rev2，rev3，rev4，rev5，rev6，rev7，rev8阻断细胞上PD-1/PD-L1信号通路的活性，结果如图7A、7B和7C所示，anti-PD1×PDL1 rev2明显比阳性对照抗PD-1单抗差；anti-PD1×PDL1 rev3，rev6的活性与阳性对照抗PD-L1单抗相当；anti-PD1×PDL1 rev4,rev5的活性比阳性对照抗PD-L1单抗略差；anti-PD1×PDL1 rev7,rev8的活性比阳性对照抗PD-L1单抗明显较差。
实施例7 抗PD1×PDL1双抗的药代动力学研究
取每组4只SD大鼠，体重200g左右，每只大鼠通过尾静脉注射剂量为2mg的实施例 2制备的抗体。给药后取血时间点为：0h，3h，24h，48h，96h，168h，336h，504h。眼眶取血，血液自然凝固后8000rpm/min离心取血清。
抗PD1×PDL1双抗anti-PD1×PDL1 BsAb的血清中药物浓度采用以下方法检测：
1)protein A包被ELISA板，检测抗体Fab段。用protein A包被，包被量为100ng/孔，4℃过夜；次日PBST洗板两次，然后用PBS+2％BSA于37℃封闭2小时。PBST洗板两次。抗PD1×PDL1双抗的标准品从1000ng/mL起始，逐级两倍稀释12个梯度。大鼠血清样品1000-4000倍稀释。以上两组样品加入封闭后的ELISA板，孵育1小时；PBST洗板两次后加入HRP标记的鼠抗人Fab抗体，37℃放置30分钟；PBST洗板3次后，在吸水纸上尽量拍干残留液滴，每孔加入100μL的TMB，室温(20±5℃)避光放置5分钟；每孔加入50μL 2M的H 2SO 4终止液终止底物反应，酶标仪450nm处读取OD值。计算所得的半衰期为275.6小时。
2)PD1-ECD-hFc包被ELISA板，20ng/孔。其余方法同上。计算所得的半衰期为303.5小时。
3)PDL1-ECD-hFc包被ELISA板，100ng/孔。其余方法同上。计算所得的半衰期为307小时。
如表2-4所示，由以上三组ELISA结果，计算的半衰期结果相近，均为300小时左右，说明分析数据可靠。
表2、protein A包板ELISA结果
实验组 半衰期(小时) 最高浓度(μg/mL)
1 247.50433 170
2 316.48482 166
3 273.29592 150
4 265.20196 164
表3、PD1-ECD-hFc包板ELISA结果
实验组 半衰期(小时) 最高浓度(μg/mL)
1 299.46777 196
2 348.73796 202
3 295.3837 170
4 270.42874 174
表4、PDL1-ECD-hFc包板ELISA结果
实验组 半衰期(小时) 最高浓度(μg/mL)
1 280.15463 166
2 336 152
3 320.97912 146
4 291.3996 166
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (16)

  1. 一种抗PD1×PDL1的双特异性抗体，其特征在于，所述双特异性抗体包含两条多肽链和两条轻链：
    a)各所述多肽链从N末端至C末端依次包含VH-PDL1—CH1—CH2—CH3—linker2—VL-PD1—linker1—VH-PD1或VH-PDL1—CH1—CH2—CH3—linker2—VH-PD1—linker1—VL-PD1或VL-PD1—linker1—VH-PD1—linker2—VH-PDL1—CH1—CH2—CH3或VH-PD1—linker1—VL-PD1—linker2—VH-PDL1—CH1—CH2—CH3，各所述轻链从N末端至C末端依次包含VL-PDL1—CL；或
    b)各所述多肽链从N末端至C末端依次包含VH-PD1—CH1—CH2—CH3—linker2—VL-PDL1—linker1—VH-PDL1或VH-PD1—CH1—CH2—CH3—linker2—VH-PDL1—linker1—VL-PDL1或VL-PDL1—linker1—VH-PDL1—linker2—VH-PD1—CH1—CH2—CH3或VH-PDL1—linker1—VL-PDL1—linker2—VH-PD1—CH1—CH2—CH3，各所述轻链从N末端至C末端依次包含VL-PD1—CL，
    其中，所述VH-PDL1为结合PD-L1的重链可变区，所述CH1-CH2-CH3为重链恒定区，所述VL-PD1为结合PD-1的轻链可变区，所述VH-PD1为结合PD-1的重链可变区，所述VL-PDL1为结合PD-L1的轻链可变区，所述CL为轻链恒定区。
  2. 根据权利要求1所述的抗PD1×PDL1的双特异性抗体，其特征在于，所述VH-PDL1与所述VL-PDL1形成特异性结合PD-L1的抗原结合位点，所述VL-PD1与所述VH-PD1形成特异性结合PD-1的抗原结合位点。
  3. 根据权利要求1所述的抗PD1×PDL1的双特异性抗体，其特征在于，所述VH-PDL1包含氨基酸序列如SEQ ID NO：1-3所示的重链CDR；所述VL-PDL1包含氨基酸序列如SEQ ID NO：4-6所示的轻链CDR；所述VH-PD1包含氨基酸序列如SEQ ID NO：7-9所示的重链CDR；所述VL-PD1包含氨基酸序列如SEQ ID NO：10-12所示的轻链CDR。
  4. 根据权利要求1所述的抗PD1×PDL1的双特异性抗体，其特征在于，所述VH-PDL1具有如SEQ ID NO：13所示的氨基酸序列，和/或，所述VL-PDL1具有如SEQ ID NO：14所示的氨基酸序列，和/或，所述VH-PD1具有如SEQ ID NO：15所示的氨基酸序列，和/或，所述VL-PD1具有如SEQ ID NO：16所示的氨基酸序列。
  5. 根据权利要求1所述的抗PD1×PDL1的双特异性抗体，其特征在于，所述linker2为3个G 4S，所述linker1为4个G 4S。
  6. 根据权利要求1所述的抗PD1×PDL1的双特异性抗体，其特征在于，所述重链恒定区包括IgG1、IgG2、IgG3、IgG4重链恒定区，所述轻链恒定区包括κ或λ轻链恒定区。
  7. 根据权利要求1所述的抗PD1×PDL1的双特异性抗体，其特征在于，所述多肽链具有如SEQ ID NO：17或SEQ ID NO：22或SEQ ID NO：23或SEQ ID NO：24所示的氨基酸序列，和/或，所述轻链具有如SEQ ID NO：18所示的氨基酸序列；或所述多肽链具有如SEQ ID NO：25或SEQ ID NO：26或SEQ ID NO：27或SEQ ID NO：28所示的氨基酸序列，和/或，所述轻链具有如SEQ ID NO：21所示的氨基酸序列。
  8. 一种分离的核苷酸，其特征在于，所述核苷酸编码如权利要求1-7任一所述的双特异性抗体。
  9. 一种表达载体，其特征在于，所述表达载体包括如权利要求8所述的核苷酸。
  10. 一种宿主细胞，其特征在于，所述宿主细胞包括如权利要求9所述的表达载体。
  11. 如权利要求1-7任一所述的双特异性抗体的制备方法，其特征在于，所述制备方法包括以下步骤：
    a)在表达条件下，培养如权利要求10所述的宿主细胞，从而表达所述双特异性抗体；
    b)分离并纯化a)所得的双特异性抗体。
  12. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1-7任一所述的双特异性抗体和药学上可接受的载体。
  13. 权利要求1-7任一所述的双特异性抗体或权利要求12所述的药物组合物在制备治疗癌症的药物中的用途。
  14. 如权利要求13所述的用途，其特征在于，所述癌症选自黑色素瘤、肾癌、肺癌、肝癌、胃癌、淋巴癌、乳腺癌、结直肠癌、白血病、前列腺癌、骨髓癌及其它赘生性恶性疾病。
  15. 一种治疗癌症的方法，其特征在于，所述方法包括向受试者施用权利要求1-7任一所述的双特异性抗体或权利要求12所述的药物组合物。
  16. 如权利要求15所述的方法，其特征在于，所述癌症选自黑色素瘤、肾癌、肺癌、肝癌、胃癌、淋巴癌、乳腺癌、结直肠癌、白血病、前列腺癌、骨髓癌及其它赘生性恶性疾病。
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