WO2021244328A1 - 一种抗pd-l1和her2的双特异性抗体 - Google Patents

一种抗pd-l1和her2的双特异性抗体 Download PDF

Info

Publication number
WO2021244328A1
WO2021244328A1 PCT/CN2021/095189 CN2021095189W WO2021244328A1 WO 2021244328 A1 WO2021244328 A1 WO 2021244328A1 CN 2021095189 W CN2021095189 W CN 2021095189W WO 2021244328 A1 WO2021244328 A1 WO 2021244328A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
cancer
her2
acid sequence
Prior art date
Application number
PCT/CN2021/095189
Other languages
English (en)
French (fr)
Inventor
朱祯平
黄浩旻
张学赛
李晴柔
Original Assignee
三生国健药业（上海）股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 三生国健药业（上海）股份有限公司 filed Critical 三生国健药业（上海）股份有限公司
Priority to CN202180032220.0A priority Critical patent/CN115605513B/zh
Publication of WO2021244328A1 publication Critical patent/WO2021244328A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C07K2317/52—Constant or Fc region; Isotype
    • 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

Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention discloses a bispecific antibody against PD-L1 and HER2.
  • 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-L1 has become a popular target for the development of tumor immunotherapy drugs. .
  • HER2/neu human epidermal growth factor receptor 2
  • erbB2 human epidermal growth factor receptor 2
  • HER2 has tyrosine protein kinase activity and is a member of the human epidermal growth factor receptor family. It is only expressed at low levels in a few normal tissues of adults .
  • studies have shown that HER2 is overexpressed in a variety of tumors. For example, there is overexpression in about 30% of breast cancer patients and 16% of gastric cancer patients. Overexpression of HER2 in tumors can significantly promote tumor angiogenesis, The growth of tumors and the enhancement of tumor invasion and metastasis are important indicators of poor prognosis for such patients.
  • Herceptin Herceptin, Trastuzumab/Trastuzumab, Genentech/Roche
  • Herceptin the first monoclonal antibody drug targeting HER2
  • the treatment of gastric cancer was approved by the FDA for marketing and used in HER2 overexpressing breast cancer.
  • 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 PD-L1 and HER2.
  • the first objective of the present invention is to provide a bispecific antibody against PD-L1 and HER2.
  • 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 for treating cancer with the bispecific antibody or the pharmaceutical composition.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides a bispecific antibody against PD-L1 and HER2, comprising two polypeptide chains and two light chains, wherein:
  • the polypeptide chain includes VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2 or VH-HER2-L2-VL-HER2-L1-VH from N-terminus to C-terminus -PDL1-CH1-CH2-CH3, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or
  • the polypeptide chain contains VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1 or VH-PDL1-L2-VL-PDL1-L1-VH from N-terminus to C-terminus -HER2-CH1-CH2-CH3, the light chain includes VL-HER2-CL from N-terminus to C-terminus;
  • the VH-PDL1 is a heavy chain variable region that binds PD-L1
  • the VL-PDL1 is a light chain variable region that binds PD-L1
  • the VH-HER2 is a heavy chain that binds HER2
  • the variable region, the VL-HER2 is a light chain variable region that binds to HER2
  • the L1 and L2 are (G 4 S)x, and x is 3, 4, 5 or 6,
  • the CH1-CH2 -CH3 is the heavy chain constant region
  • the CL is the light chain constant region
  • the VH-PDL1 and the VL-PDL1 form an antigen binding site that specifically binds to PD-L1, the VH-HER2 It forms an antigen binding site that specifically binds to HER2 with the VL-HER2.
  • said L1 is (G 4 S) 3 and said L2 is (G 4 S) 4 .
  • the VH-PDL1 includes the amino acid sequence of the heavy chain CDR shown in SEQ ID NO: 1-3, and the VL-PDL1 includes the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4-6.
  • the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 19
  • the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 20
  • the VH -HER2 has an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 23
  • said VL-HER2 has an amino acid sequence as shown in SEQ ID NO: 22 or SEQ ID NO: 24.
  • the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 31 or SEQ ID NO: 33, and the light chain has an amino acid sequence as shown in SEQ ID NO: 26; Or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34, and the light chain has the amino acid sequence shown in SEQ ID NO: 28; or the polypeptide chain has For example, the amino acid sequence shown in SEQ ID NO: 35 or SEQ ID NO: 36, the light chain has the amino acid sequence shown in SEQ ID NO: 30.
  • the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 34
  • the light chain has an amino acid sequence as shown in SEQ ID NO: 28, and the amino acid sequence is
  • the polypeptide chain shown in SEQ ID NO: 34 further includes mutations in the following amino acid residue positions: N76E and/or N213E.
  • the polypeptide chain has an amino acid sequence as shown in SEQ ID NO: 37 or SEQ ID NO: 38 or SEQ ID NO: 39, and the light chain has an amino acid sequence as shown in SEQ ID NO: 28 The amino acid sequence shown.
  • 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 second aspect of the present invention provides an isolated nucleotide, which encodes the bispecific antibody.
  • the third aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotides.
  • the fourth aspect of the present invention provides a host cell, which contains the expression vector as described above.
  • the fifth aspect of the present invention provides a method for preparing the bispecific antibody, the method comprising the following steps:
  • the sixth aspect of the present invention provides a pharmaceutical composition containing the bispecific antibody as described above and a pharmaceutically acceptable carrier.
  • 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 the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer Cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.
  • 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 the group consisting of: melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer Cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma and other neoplastic malignant diseases.
  • the present invention provides a bispecific antibody against PD-L1 and HER2.
  • the results of in vitro and in vivo experiments show that it can specifically bind to both PD-L1 and HER2 targets at the same time, which is similar to or even better than that of monoclonal antibodies. Excellent biological activity.
  • Figure 1 shows the result of ELISA to determine the affinity of the bispecific antibody to PDL1-ECD-his.
  • Figure 2 shows the results of ELISA to determine the affinity of the bispecific antibody to HER2-ECD-his.
  • Figure 2A shows the bispecific antibody combined with M8 and 607
  • Figure 2B shows the bispecific antibody combined with M8 and 612.
  • Figure 3 shows the result of the bispecific antibody competitively inhibiting the binding of PD-1 and PD-L1.
  • Figure 4 shows the results of determination of the binding activity of bispecific antibodies in inhibiting PD-1 and PD-L1 high-expressing cell lines.
  • Figure 5 shows the results of measuring the activity of bispecific antibodies in inhibiting the proliferation of BT474 cells.
  • Figure 6 shows the results of measuring the activity of bispecific antibodies to inhibit the proliferation of N87 cells.
  • Figure 7 shows the results of measuring the activity of bispecific antibodies in inhibiting the proliferation of BT474 cells.
  • Figure 8 shows the results of measuring the activity of bispecific antibodies to inhibit the proliferation of N87 cells.
  • Figure 9 shows the results of the bispecific antibody on the growth inhibition of gastric cancer cell NCI-N87 xenograft tumors.
  • Figure 10 shows the results of the growth inhibition of MC38-hPD-L1 xenografted tumors by bispecific antibodies.
  • Figure 11 shows the results of the affinity determination of the bispecific antibody PDL1scFv-607H mutant to PDL1-ECD-his.
  • Figure 12 shows the results of the affinity determination of the bispecific antibody PDL1scFv-607H mutant to HER2-ECD-his.
  • Figure 13 shows that the bispecific antibody PDL1scFv-607H mutant inhibits the binding activity of PD-1 and PD-L1 high-expressing cell lines.
  • Figure 14 shows the determination of the cell proliferation inhibitory activity of the bispecific antibody PDL1scFv-607H mutant on the HER2 high-expressing cell line.
  • Figure 15 shows the results of a mixed lymphocyte reaction (MLR) experiment measuring that bispecific antibodies promote the secretion of IL-2 by T cells.
  • MLR mixed lymphocyte reaction
  • Fig. 16 shows the results of the mixed lymphocyte reaction (MLR) experiment measuring that the bispecific antibody promotes the secretion of IFN- ⁇ from T cells.
  • MLR mixed lymphocyte reaction
  • the terms "Antibody (Ab)” and “Immunoglobulin G (Abbreviation IgG)” are heterotetrameric glycoproteins of about 150,000 Daltons with the same structural characteristics, which consist of two The same light chain (L) and two identical heavy chains (H) are composed. 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. There is a variable region (VH) at one end of each heavy chain, followed by a constant region. The constant region of the heavy chain is composed of three 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).
  • the general structural formula of the bispecific antibody of the present invention is as follows:
  • Ab is an antibody, which contains two heavy chains and two light chains;
  • the scFv is a single-chain antibody, which is formed by connecting the variable region of the heavy chain and the variable region of the light chain of another antibody through the linking sequence L2, and is connected to the N-terminus or C of the two heavy chains of the Ab through the linking sequence L1.
  • L1 and L2 are the connection sequence (G 4 S)x, and x can be 3, 4, 5, or 6.
  • the present invention uses anti-human PD-L1 antibody M8 (sequence derived from PCT/CN2020/090442), anti-human HER2 antibody 607 (prepared according to trastuzumab sequence, sequence derived from US5821337) and anti-human HER2 antibody 612 (sequence source)
  • the bispecific antibody was constructed by the combination of WO2020/025013A1), and the specific structure is shown in Table 1.
  • PDL1-HC represents the heavy chain of M8
  • PDL1-LC represents the light chain of M8
  • 607-VH represents the variable region of the heavy chain of 607
  • 607-VL represents the variable region of the light chain of 607, and the rest are similar.
  • the related sequence information of the bispecific antibody constructed in the present invention is shown in Table 2, where the CDR is encoded according to the Kabat rule.
  • Amino acid sequence of light chain variable region of anti-PD-L1 antibody twenty one Amino acid sequence of the variable region of the heavy chain of 607 twenty two Amino acid sequence of the light chain variable region of 607 twenty three Amino acid sequence of the variable region of the heavy chain of 612 twenty four Amino acid sequence of the light chain variable region of 612 25 Amino acid sequence of heavy chain of anti-PD-L1 antibody 26 Amino acid sequence of light chain of anti-PD-L1 antibody 27 Amino acid sequence of the heavy chain of 607 28 Amino acid sequence of the light chain of 607 29 Amino acid sequence of the heavy chain of 612 30 Amino acid sequence of the light chain of 612 31 Amino acid sequence of the polypeptide chain of PDL1H-607scFv 32 Amino acid sequence of the polypeptide chain of 607H-PDL1scFv 33 The amino acid sequence of the polypeptide chain of 607scFv-PDL1H 34 Amino acid sequence of the polypeptide chain
  • the pTT5 expression vector (purchased from NRC Biotechnology Research Institute) was constructed through the two restriction sites of EcoRI and HindIII to obtain the heavy chain of each bispecific antibody and its corresponding monoclonal antibody And light chain expression vector, the above heavy chain sequence expression vector and its corresponding light chain expression vector were co-transfected into HEK293F cells (purchased from Thermo Fisher, catalog number A14527), expressed and purified to obtain each bispecific antibody PDL1H-607scFv, 607H-PDL1scFv, 607scFv-PDL1H, PDL1scFv-607H, PDL1scFv-612H and 612H-PDL1scFv, and the corresponding monoclonal antibodies M8, 607, 612.
  • PDL1-ECD-his (NCBI registration number is NP_054862.1) and HER2-ECD-his protein (NCBI registration number is NP_004439.2) are prepared as follows: according to the sequence provided by NCBI, the PD-L1 and HER2 extracellular domain genes are synthesized and A signal peptide sequence was added to the N-terminus, and a 6 ⁇ His tag was added to the C-terminus. The two restriction sites of EcoRI and HindIII were respectively constructed into pTT5 expression vector, transfected into HEK-293F cells for expression and purified.
  • M8 was diluted to different concentrations and added to the ELISA wells, 100 ⁇ L/well, 3 replicate wells for each concentration, incubated for 1h at room temperature, washed with PBST 3 times, and diluted the secondary antibody (HRP-anti Human IgG Fc, purchased from Millipore, catalog number AP101P), add to ELISA wells, 100 ⁇ L/well, incubate at room temperature for 1h, wash 3 times with PBST, add TMB color developing solution, 100 ⁇ L/well, develop to the expected color, use 2M H 2 The color reaction was terminated by SO 4 , 50 ⁇ L/well, the reaction solution was shaken uniformly and the OD450nm was measured in a microplate reader, the data was analyzed, and the EC 50 was calculated.
  • HRP-anti Human IgG Fc purchased from Millipore, catalog number AP101P
  • 607scFv-PDL1H and PDL1H-607scFv are better, and the affinity is equivalent to 607 monoclonal antibody, PDL1scFv-607H and 607H-PDL1scFv are Relatively weak; in the bispecific antibody combination of 612 and M8, the affinity of PDL1scFv-612H and 612H-PDL1scFv to HER2-ECD-his is similar, and both are slightly weaker than that of 612 monoclonal antibody.
  • Example 3 Determination of the binding activity of bispecific antibodies to competitively inhibit PD-1 and PD-L1
  • PDL1-ECD-Fc Dilute the PDL1-ECD-Fc protein (the preparation method is the same as above, but replace the C-terminal label with adult Fc sequence) and coat it on a 96-well ELISA plate, 0.2 ⁇ g/well, overnight at 4°C, PBST (PBS containing 0.05% Tween20 ) Wash 3 times, prepare 2% BSA with PBS, 200 ⁇ L/well, block at room temperature for 2h, wash 2 times with PBST, use 500ng/ml biotin-labeled PD1-Fc (ie PD1- Fc-biotin, NCBI registration number is NP_005009.2, according to the registration information on NCBI to obtain the extracellular domain gene of PD-1, the cloning and preparation method is the same as PDL1-ECD-Fc) solution PDL1H-607scFv, 607H-PDL1scFv, 607scFv-PDL1H , PDL1s
  • the PD-1 high-expressing cell line PD-1 Effector Cells and the PD-L1 high-expressing cell line PD-L1 aAPC/CHO-K1 Cells were purchased from Promega (Cat. No.: J1252).
  • PDL1-aAPC/CHO-K1 cells in the logarithmic growth phase were trypsinized, centrifuged, counted, and resuspended in 10% FBS Ham/F12 medium (purchased from Thermo Fisher, Catalog No. 11765054), according to 100 ⁇ L/ Pour 40,000 cells per well, inoculate them into a 96-well plate with a transparent bottom on a white plate, and grow overnight at 37°C in a CO 2 cell incubator.
  • Example 5 Determination of cell proliferation inhibitory activity of bispecific antibodies against HER2 high-expressing cell lines
  • BT474 and N87 cells in the logarithmic growth phase were trypsinized, counted, and resuspended in a complete medium, namely RPMI1640 medium (purchased from Thermo Fisher, catalog number 61870036) containing 10% FBS, and 150 ⁇ L/well , Were inoculated into 96-well cell culture plates, breast cancer cells BT474 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHu143) 10,000 cells per well, and gastric cancer cells N87 (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP- 534) Pour 8000 cells in each well and incubate overnight at 37°C in a CO 2 cell incubator.
  • RPMI1640 medium purchased from Thermo Fisher, catalog number 61870036
  • TCHu143 breast cancer cells
  • gastric cancer cells N87 purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: SCSP- 534
  • each group is serially diluted 9 gradients, the highest concentration of each antibody is 400nM (final working concentration is 100nM), add 50 ⁇ L of the above antibody to a 96-well cell culture plate, each well The final volume is 200 ⁇ L, the non-administered group is set as a negative control, and the culture is continued for 5 days in a 37 °C cell incubator, and 2 replicate wells are made in parallel for each concentration.
  • CCK8 reaction solution diluted with complete medium at a ratio of 1:10, purchased from Dojindo, catalog number CK04, and incubate at 37°C.
  • PDL1scFv-607H and 607H-PDL1scFv have better inhibitory activity on the proliferation of BT474 cells; the activity of PDL1H-607scFv on the proliferation of N87 cells is the weakest, although PDL1scFv-607H, The IC 50 of 607H-PDL1scFv is comparable, but PDL1scFv-607H has the strongest maximum killing effect on N87 cells at high concentrations.
  • PDL1scFv-612H and 612H-PDL1scFv have the same inhibitory activity on cell proliferation.
  • Example 6 In vivo drug efficacy study based on gastric cancer cell NCI-N87 xenograft tumor
  • the gastric cancer cells NCI-N87 (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) in the logarithmic growth phase were digested by trypsin, collected by centrifugation and resuspended in RPMI1640 serum-free medium (purchased from Thermo Fisher, catalog number 61870036) to adjust the cells Concentration, mix the cell suspension with an equal volume of Matrigel so that the final cell concentration is 8*10 7 /mL. Aseptically, inoculate 100 ⁇ L of cell suspension into BALB/c nude mice (female, weighing 18-20 g, purchased from Beijing Weitong Lihua Biotechnology Co., Ltd.) subcutaneously on the right side of the rib.
  • the animals are randomly divided into 5 groups, each with 8 animals. Including solvent control group (Control), monoclonal antibody control group 607, and test sample group PDL1scFv-607H.
  • the administration was started on the day of grouping.
  • the dose of 607 was 20 mg/kg and the dose of PDL1scFv-607H was 27 mg/kg.
  • the dose was intraperitoneally administered twice a week for 7 consecutive times. Measure the tumor diameter twice a week, record, and calculate the tumor volume and tumor growth inhibition rate according to the following formula.
  • Example 7 In vivo pharmacodynamic study of MC38-hPD-L1 colon cancer animal model based on B-hPD-L1 humanized mice
  • B-hPD-L1 humanized mice and MC38-hPD-L1 colon cancer cells were both provided by Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.
  • the experimental results are shown in Figure 10.
  • the bispecific antibody PDL1scFv-607H and the monoclonal antibody M8 can effectively inhibit the growth of MC38-hPD-L1 transplanted tumors, and the tumor growth inhibition rate of PDL1scFv-607H on the 23rd day after administration 74%, the tumor growth inhibition rate of M8 is 89%.
  • the bispecific antibody PDL1scFv-607H had a slightly lower tumor growth inhibition rate than the monoclonal antibody reference M8 at the end of this experiment, there was no significant difference between the two and it was not statistically significant.
  • the specific asparagine (N) mutation on the scFv fragment can effectively improve the physicochemical properties of the bispecific antibody. Therefore, the present invention uses conventional molecular cloning technology to perform single-point or combined mutations of asparagine (N) on the scFv fragment of PDL1scFv-607H into glutamic acid (E), extract each mutant plasmid and associate it with the corresponding light chain Paired transfection HEK293F cell expression and purification to obtain the bispecific antibody PDL1scFv-607H mutant samples.
  • Example 10 The bispecific antibody PDL1scFv-607H mutant inhibits the binding activity of PD-1 and PD-L1 high-expressing cell lines
  • Example 11 Determination of the proliferation inhibitory activity of the bispecific antibody PDL1scFv-607H mutant on the cell line with high expression of HER2
  • Example 12 Mixed lymphocyte reaction (MLR) experiment to determine the activity of bispecific antibodies
  • PBMC peripheral blood mononuclear cells
  • CD14MicroBeads purchased from Miltenyi Biotec, catalog number 130-050-201
  • the DC cells were induced to mature by adding 1 ⁇ g/ml LPS (purchased from sigma, item number L6529-1MG) for 24 hours.
  • Pan T cell Isolation Kit purchased from Miltenyi Biotec, catalog number 130-096-535) to separate T cells from PBMC according to the instructions, and use PBS cells preheated at 37°C once to count the T cells for use.
  • mice-anti-human IL-2 protein purchased from BD Pharmingen, catalog number 555051
  • mouse-anti-human IFN- ⁇ purchased from BD Pharmingen, catalog number 551221
  • the protein was diluted to 4 ⁇ g/mL, respectively coated on the ELISA plate, 100 ⁇ L/well, placed in a wet box, 4°C, coated for 16h.
  • IL-2/IFN- ⁇ standard products Prepare standard products with RPMI1640 medium, make the initial concentration of IL-2 40ng/ml, and make the initial concentration of IFN- ⁇ 400ng/ml, respectively, carry out 2-fold dilutions, each A total of 12 gradients were diluted for each standard. Add 100 ⁇ l of standard diluent to each well, and set up 2 replicate wells for each concentration.
  • Streptavidin-HRP purchased from BD Pharmingen, catalog number 554066

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供了一种抗PD-L1和HER2的双特异性抗体。本发明的双特异性抗体能够同时特异性结合PD-L1和HER2两个靶点，具有与单抗相似甚至更优的生物学活性。

Description

一种抗PD-L1和HER2的双特异性抗体 技术领域
本发明涉及抗体领域，更具体地，本发明公开了一种抗PD-L1和HER2的双特异性抗体。
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-L1成为开发肿瘤免疫治疗药物的热门靶点。
HER2/neu(人表皮生长因子受体2)，又称erbB2，具有酪氨酸蛋白激酶活性，是人表皮生长因子受体家族成员之一，只在成年人的少数正常组织中呈低水平表达。但研究表明，HER2在多种肿瘤中过表达，如在约30％的乳腺癌患者和16％的胃癌患者中均存在过度表达情况，HER2在肿瘤中的过表达可以显著促进肿瘤血管的新生、肿瘤的生长，并增强肿瘤的侵袭和转移能力，是这类患者预后较差的重要指征。因此，早在1998年，第一个靶向于HER2的单克隆抗体药物Herceptin(赫赛汀，Trastuzumab/曲妥珠单抗，Genentech/Roche)被FDA批准上市，并用于HER2过表达的乳腺癌和胃癌的治疗。
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PD-L1和HER2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PD-L1和HER2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的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的核苷酸的表达载体。
本发明的第四个目的在于提供一种包含所述的表达载体的宿主细胞。
本发明的第五个目的在于提供所述的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的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的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的双特异性抗体或所述的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了一种抗PD-L1和HER2的双特异性抗体，包含两条多肽链和两条轻链，其中：
(a)所述的多肽链从N末端至C末端包含VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2或VH-HER2-L2-VL-HER2-L1-VH-PDL1-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-PDL1-CL；或
(b)所述的多肽链从N末端至C末端包含VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1或VH-PDL1-L2-VL-PDL1-L1-VH-HER2-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-HER2-CL；
其中，所述的VH-PDL1为结合PD-L1的重链可变区，所述的VL-PDL1为结合PD-L1的轻链可变区，所述的VH-HER2为结合HER2的重链可变区，所述的VL-HER2为结合HER2的轻链可变区，所述的L1和L2为(G 4S)x，x为3、4、5或6，所述的CH1-CH2-CH3为重链恒定区，所述的CL为轻链恒定区，所述的VH-PDL1与所述的VL-PDL1形成特异性结合PD-L1的抗原结合位点，所述的VH-HER2与所述的VL-HER2形成特异性结合HER2的抗原结合位点。
根据本发明的优选实施例，所述的L1为(G 4S) 3，所述的L2为(G 4S) 4。
根据本发明的优选实施例，所述的VH-PDL1包含氨基酸序列如SEQ ID NO：1-3所示的重链CDR，所述的VL-PDL1包含氨基酸序列如SEQ ID NO：4-6所示的轻链CDR，所述的VH-HER2包含氨基酸序列如SEQ ID NO：7-9或SEQ ID NO：13-15所示的重链CDR，所述的VL-HER2包含氨基酸序列如SEQ ID NO：10-12或SEQ ID NO：16-18所示的轻链CDR。
根据本发明的优选实施例，所述的VH-PDL1具有如SEQ ID NO：19所示的氨基酸序列，所述的VL-PDL1具有如SEQ ID NO：20所示的氨基酸序列，所述的VH-HER2具有如SEQ ID NO：21或SEQ ID NO：23所示的氨基酸序列，所述的VL-HER2具有如SEQ ID NO：22或SEQ ID NO：24所示的氨基酸序列。
根据本发明的优选实施例，所述的多肽链具有如SEQ ID NO：31或SEQ ID NO：33所示的氨基酸序列，所述的轻链具有如SEQ ID NO：26所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：32或SEQ ID NO：34所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：35或SEQ ID NO：36所示的氨基酸序列，所述的轻链具有如SEQ ID NO：30所示的氨基酸序列。
根据本发明的优选实施例，所述的多肽链具有如SEQ ID NO：34所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列，且所述的氨基酸序列如SEQ ID NO：34所示的多肽链进一步包含以下氨基酸残基位点的突变：N76E和/或N213E。
根据本发明的优选实施例，所述的多肽链具有如SEQ ID NO：37或SEQ ID NO：38或SEQ ID NO：39所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列。
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。
本发明的第二个方面提供了一种分离的核苷酸，所述的核苷酸编码所述的双特异性抗体。
本发明的第三个方面提供了一种表达载体，所述的表达载体含有如上所述的核苷酸。
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。
本发明的第五个方面提供了所述的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，从而表达所述的双特异性抗体；
(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，所述癌症选自由以下组成的组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病等。
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，所述癌症选自由以下组成的组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病等。
有益效果：本发明提供了一种抗PD-L1和HER2的双特异性抗体，体外和体内实验结果显示其能够同时特异性结合PD-L1和HER2两个靶点，具有与单抗相似甚至更优的生物学活性。
附图说明
图1为ELISA测定双特异性抗体对PDL1-ECD-his的亲和力结果。
图2为ELISA测定双特异性抗体对HER2-ECD-his的亲和力结果，其中，图2A为M8与607组合的双特异性抗体，图2B为M8和612组合的双特异性抗体。
图3为双特异性抗体竞争性抑制PD-1与PD-L1的结合结果。
图4为双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性测定结果。
图5为双特异性抗体抑制BT474细胞增殖的活性测定结果。
图6为双特异性抗体抑制N87细胞增殖的活性测定结果。
图7为双特异性抗体抑制BT474细胞增殖的活性测定结果。
图8为双特异性抗体抑制N87细胞增殖的活性测定结果。
图9为双特异性抗体对胃癌细胞NCI-N87的异种移植瘤的生长抑制结果。
图10为双特异性抗体对MC38-hPD-L1移植瘤的生长抑制结果。
图11为双特异性抗体PDL1scFv-607H突变体对PDL1-ECD-his的亲和力测定结果。
图12为双特异性抗体PDL1scFv-607H突变体对HER2-ECD-his的亲和力测定结果。
图13为双特异性抗体PDL1scFv-607H突变体抑制PD-1和PD-L1高表达细胞株的结合活性。
图14为双特异性抗体PDL1scFv-607H突变体对HER2高表达细胞株细胞增殖抑制活性测定。
图15为混合淋巴细胞反应(MLR)实验测定双特异性抗体促进T细胞的IL-2的分泌的结果。
图16为混合淋巴细胞反应(MLR)实验测定双特异性抗体促进T细胞的IFN-γ的分泌的 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约150000道尔顿的异四聚糖蛋白，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、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PD-L1和HER2双特异性抗体的构建
本发明的双特异性抗体的结构通式如下：
1)Ab-L1-scFv；
2)scFv-L1-Ab；
其中Ab为抗体，包含两条重链和两条轻链；
scFv为单链抗体，是由另一个抗体的重链可变区和轻链可变区通过连接序列L2连接而成，并通过连接序列L1分别连接在Ab的两条重链的N末端或C末端；
L1和L2为连接序列(G 4S)x，x可以为3、4、5或6。
本发明使用抗人PD-L1抗体M8(序列来源于PCT/CN2020/090442)、抗人HER2抗体607(根据曲妥珠单抗序列制备，序列来源于US5821337)和抗人HER2抗体612(序列来源于WO2020/025013A1)的组合构建双特异性抗体，具体结构如表1所示。
表1、本发明的双特异性抗体的结构
名称 多肽链(N末端至C末端) 轻链
PDL1H-607scFv PDL1-HC-(G 4S) 3-607-VH-(G 4S) 4-607-VL PDL1-LC
607H-PDL1scFv 607-HC-(G 4S) 3-PDL1-VH-(G 4S) 4-PDL1-VL 607-LC
607scFv-PDL1H 607-VH-(G 4S) 4-607-VL-(G 4S) 3-PDL1-HC PDL1-LC
PDL1scFv-607H PDL1-VH-(G 4S) 4-PDL1-VL-(G 4S) 3-607-HC 607-LC
PDL1scFv-612H PDL1-VH-(G 4S) 4-PDL1-VL-(G 4S) 3-612-HC 612-LC
612H-PDL1scFv 612-HC-(G 4S) 3-PDL1-VH-(G 4S) 4-PDL1-VL 612-LC
其中，PDL1-HC代表M8的重链，PDL1-LC代表M8的轻链，607-VH代表607的重链可变区，607-VL代表607的轻链可变区，其余类似。
本发明构建的双特异性抗体相关的序列信息如表2所示，其中，CDR根据Kabat规则编码。
表2、本发明的抗体的序列信息
SEQ ID NO： 序列名称
1 抗PD-L1抗体的重链互补决定区H-CDR1的氨基酸序列
2 抗PD-L1抗体的重链互补决定区H-CDR2的氨基酸序列
3 抗PD-L1抗体的重链互补决定区H-CDR3的氨基酸序列
4 抗PD-L1抗体的轻链互补决定区L-CDR1的氨基酸序列
5 抗PD-L1抗体的轻链互补决定区L-CDR2的氨基酸序列
6 抗PD-L1抗体的轻链互补决定区L-CDR3的氨基酸序列
7 607的重链互补决定区H-CDR1的氨基酸序列
8 607的重链互补决定区H-CDR2的氨基酸序列
9 607的重链互补决定区H-CDR3的氨基酸序列
10 607的轻链互补决定区L-CDR1的氨基酸序列
11 607的轻链互补决定区L-CDR2的氨基酸序列
12 607的轻链互补决定区L-CDR3的氨基酸序列
13 612的重链互补决定区H-CDR1的氨基酸序列
14 612的重链互补决定区H-CDR2的氨基酸序列
15 612的重链互补决定区H-CDR3的氨基酸序列
16 612的轻链互补决定区L-CDR1的氨基酸序列
17 612的轻链互补决定区L-CDR2的氨基酸序列
18 612的轻链互补决定区L-CDR3的氨基酸序列
19 抗PD-L1抗体的重链可变区的氨基酸序列
20 抗PD-L1抗体的轻链可变区的氨基酸序列
21 607的重链可变区的氨基酸序列
22 607的轻链可变区的氨基酸序列
23 612的重链可变区的氨基酸序列
24 612的轻链可变区的氨基酸序列
25 抗PD-L1抗体的重链的氨基酸序列
26 抗PD-L1抗体的轻链的氨基酸序列
27 607的重链的氨基酸序列
28 607的轻链的氨基酸序列
29 612的重链的氨基酸序列
30 612的轻链的氨基酸序列
31 PDL1H-607scFv的多肽链的氨基酸序列
32 607H-PDL1scFv的多肽链的氨基酸序列
33 607scFv-PDL1H的多肽链的氨基酸序列
34 PDL1scFv-607H的多肽链的氨基酸序列
35 PDL1scFv-612H的多肽链的氨基酸序列
36 612H-PDL1scFv的多肽链的氨基酸序列
通过基因合成及常规的分子克隆方法，通过EcoRI和HindIII两个酶切位点构建到pTT5表达载体(购自NRC biotechnology Research Institute)，获得各双特异性抗体及其对应的单克隆抗体的重链和轻链的表达载体，将上述各重链序列表达载体与其对应的轻链表达载体共转染HEK293F细胞(购自Thermo Fisher，货号A14527)，表达并纯化获得各双特异性抗体PDL1H-607scFv、607H-PDL1scFv、607scFv-PDL1H、PDL1scFv-607H、PDL1scFv-612H和612H-PDL1scFv，以及对应的单抗M8、607、612。
实施例2 ELISA法测定双特异性抗体靶向HER2和PD-L1的亲和力
PDL1-ECD-his(NCBI登记号为NP_054862.1)和HER2-ECD-his蛋白(NCBI登记号为NP_004439.2)制备方法如下：根据NCBI提供的序列分别合成PD-L1和HER2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pTT5表达载体中，转染HEK-293F细胞表达并纯化获得。
将PDL1-ECD-his或HER2-ECD-his蛋白稀释并分别包被96孔ELISA板，0.05μg/孔，4℃包被过夜，PBST(PBS含0.05％Tween20)洗涤3次，用PBS配制2％BSA，200μL/孔，于室温封闭2h，PBST洗涤2次后，用PBST配制的1％BSA将PDL1H-607scFv、607H-PDL1scFv、607scFv-PDL1H、PDL1scFv-607H、PDL1scFv-612H、612H-PDL1scFv和M8稀释至不同浓度并加至ELISA孔，100μL/孔，每个浓度设置3个复孔，室温孵育1h，PBST洗涤3次，用PBST配制的1％BSA按照适当比例稀释二抗(HRP-anti human IgG Fc，购自Millipore公司，货号AP101P)，加入ELISA孔，100μL/孔，室温孵育1h，PBST洗涤3次后添加TMB显色液，100μL/孔，显色至预期颜色，用2M H 2SO 4终止显色反应，50μL/孔，使各反应液振荡均匀并于酶标 仪测定OD450nm，分析数据，计算EC 50。
实验结果分别如图1和图2所示，各双特异性抗体对PDL1-ECD-his和HER2-ECD-his的亲和力(EC 50)分别如表3和表4所示，可见各双特异性抗体与PDL1-ECD-his的亲和力与M8相当，均在同一个水平，PDL1scFv-607H稍优。而对HER2-ECD-his的亲和力，在607与M8的双特异性抗体组合中，则以607scFv-PDL1H和PDL1H-607scFv更优，与607单抗的亲和力相当，PDL1scFv-607H和607H-PDL1scFv则相对偏弱；在612与M8的双特异性抗体组合中，PDL1scFv-612H和612H-PDL1scFv对HER2-ECD-his的亲和力相当，均稍弱于612单抗。
表3、各双特异抗体对PDL1-ECD-his的EC 50
Figure PCTCN2021095189-appb-000001
表4、各双特异抗体对HER2-ECD-his的EC 50
Figure PCTCN2021095189-appb-000002
实施例3 双特异性抗体竞争性抑制PD-1与PD-L1结合活力测定
将PDL1-ECD-Fc蛋白(制备方法同上，只是将C端标签换成人Fc序列)稀释并包被96孔ELISA板，0.2μg/孔，4℃包被过夜，PBST(PBS含0.05％Tween20)洗涤3次，用PBS配制2％BSA，200μL/孔，于室温封闭2h，PBST洗涤2次后，用含1％BSA的PBST配制的500ng/ml生物素标记的PD1-Fc(即PD1-Fc-biotin，NCBI登记号为NP_005009.2，根据NCBI上登记信息获取PD-1的胞外域基因，克隆及制备方法同PDL1-ECD-Fc)溶液将PDL1H-607scFv、607H-PDL1scFv、607scFv-PDL1H、PDL1scFv-607H、PDL1scFv-612H、612H-PDL1scFv和M8稀释至不同浓度并加至ELISA孔，100μL/孔，每个浓度设置3个复孔，室温孵育1h，PBST洗涤3次，用PBST配制的1％BSA按照适当比例稀释二抗(HRP标记的streptavidin，购自Sigma，货号S4672-5MG)，加入ELISA孔，100μL/孔，室温孵育1h，PBST洗涤3次后添加TMB显色液，100μL/孔，显色至预期颜色，用2M H 2SO 4终止显色反应，50μL/孔，使各反应液振荡均匀并于酶标仪测定OD450nm，分析数据，计算EC 50。
实验结果如图3所示，各双特异性抗体竞争性抑制PD-1与PD-L1结合的活力(IC 50)如表5所示，可见各双特异性抗体竞争抑制PD-1与PD-L1活性相当。
表5、各双特异抗体竞争性抑制PD-1与PD-L1结合的IC 50
Figure PCTCN2021095189-appb-000003
实施例4 双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性
PD-1高表达细胞系PD-1 Effector Cells和PD-L1高表达细胞系PD-L1 aAPC/CHO-K1 Cells 均购自Promega公司(货号：J1252)。
1)将处于对数生长期的PDL1-aAPC/CHO-K1细胞用胰酶消化，离心，计数，用10％FBS Ham/F12培养基(购自Thermo Fisher，货号11765054)重悬，按照100μL/孔，每孔铺40000个细胞，接种至白板透明底96孔板中，于CO 2细胞培养箱37℃生长过夜。
2)用含1％FBS的RPMI1640培养基(购自Thermo Fisher，货号61870036)分别配制最高浓度为200nM的M8、607H-PDL1scFv、PDL1scFv-607H、PDL1H-607scFv、607scFv-PDL1H、PDL1scFv-612H、612H-PDL1scFv，并按照3倍梯度稀释(工作浓度最高剂量为100nM)，同时以1％FBS的RPMI1640培养基稀释PD-1 Effector Cells至1.25×10 6个/mL。
3)弃净上述培养有PDL1-aAPC/CHO-K1细胞的96孔板上清，加入40μL稀释好的PD-1 Effector Cell悬液以及40μL稀释好的抗体，轻拍孔板摇匀，于37℃细胞培养箱继续培养6h。
4)按照80μL/孔加入已恢复至室温的Bio-Glo Luciferase Assay Reagent(购自Promega，货号：G7941)，室温孵育10min。
5)于多功能酶标仪中测定其发光强度，通过GraphPad Prism 6分析数据。
实验结果如图4所示，各双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性的IC 50如表6所示，可见PDL1H-607scFv、607scFv-PDL1H活性相对较优，PDL1scFv-612H和PDL1scFv-607H次之。
表6、各双特异抗体抑制PD-1和PD-L1高表达细胞株的结合的IC 50
Figure PCTCN2021095189-appb-000004
实施例5 双特异性抗体对HER2高表达细胞株细胞增殖抑制活性测定
1)将处于对数生长期的BT474和N87细胞用胰酶消化，计数，用完全培养基，即含10％FBS的RPMI1640培养基(购自Thermo Fisher，货号61870036)重悬，按照150μL/孔，分别接种至96孔细胞培养板中，乳腺癌细胞BT474(购自中国科学院细胞库，目录号：TCHu143)每孔铺10000个，胃癌细胞N87(购自中国科学院细胞库，目录号：SCSP-534)每孔铺8000个，于CO 2细胞培养箱37℃培养过夜。
2)用完全培养基按照3倍梯度稀释待测抗体，各组均连续稀释9梯度，各抗体最高浓度为400nM(最终工作浓度为100nM)，加入50μL上述抗体至96孔细胞培养板，每孔终体积为200μL，设置未给药组作为阴性对照，于37℃细胞培养箱继续培养5d，每个浓度平行做2个复孔。
3)弃净细胞培养上清液，按照100μL/孔加入CCK8反应液(以完全培养基按1：10比例稀释，购自Dojindo，货号CK04)，于37℃孵育。
4)待细胞培养孔颜色显色至预期深浅，于多功能酶标仪中在450nm波长处测定其吸光度，通过GraphPad Prism 6分析数据。
5)按照下述公式计算细胞存活率及生长抑制率：
存活率＝(OD给药-OD空白)/(OD对照-OD空白)×100％。生长抑制率＝1-存活率。
实验结果分别如图5、图6、图7和图8所示，各双特异性抗体对HER2高表达细胞株BT474和N87增殖抑制的IC 50分别如表7、表8、表9、表10所示，可见，对于607和M8的双抗组合，PDL1scFv-607H、607H-PDL1scFv对BT474细胞增殖的抑制活性更优；对N87细胞增殖抑制活性，PDL1H-607scFv活性最弱，虽然PDL1scFv-607H、607H-PDL1scFv的IC 50相当，但在高浓度下PDL1scFv-607H对N87细胞的最大杀伤效应最强。对于612和M8的双抗组合，则PDL1scFv-612H和612H-PDL1scFv对细胞增殖抑制活性相当。
表7、双特异性抗体抑制BT474细胞增殖的IC 50
Figure PCTCN2021095189-appb-000005
表8、双特异性抗体抑制N87细胞增殖的IC 50
Figure PCTCN2021095189-appb-000006
表9、双特异性抗体抑制BT474细胞增殖的IC 50
Figure PCTCN2021095189-appb-000007
表10、双特异性抗体抑制N87细胞增殖的IC 50
Figure PCTCN2021095189-appb-000008
实施例6 基于胃癌细胞NCI-N87异种移植瘤的体内药效研究
通过胰酶消化对数生长期的胃癌细胞NCI-N87(购自中科院典型培养物保藏委员会细胞库)，离心收集并重悬于RPMI1640无血清培养基(购自Thermo Fisher，货号61870036)中以调整细胞浓度，将细胞悬液与等体积基质胶混合，使细胞终浓度为8*10 7/mL。无菌操作，接种100μL细胞悬液至BALB/c裸小鼠(雌性，体重18-20g，购自北京维通利华生物科技有限公司)右侧肋部皮下。待肿瘤生长至100-200mm 3，将动物随机分为5组，每组8只动物。包括溶剂对照组(Control)，单抗对照组607，受试样品组PDL1scFv-607H。分组当天开始给药，607给药剂量为20mg/kg，PDL1scFv-607H给药剂量为27mg/kg，每周通过腹腔给药两次，连续给药7次。每周测量两次肿瘤直径，记录，并按照下述公式计算肿瘤体积和肿瘤生长抑制率。
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×长×宽 2。
肿瘤生长抑制率(TGI)＝(1-实验组肿瘤体积/溶剂对照组肿瘤体积)×100％。
实验结果如图9所示，双特异性抗体PDL1scFv-607H和单抗607均可有效抑制胃癌细胞 NCI-N87的异种移植瘤的生长，且给药后的第25天PDL1scFv-607H的肿瘤生长抑制率为57％，明显优于单抗对照品607的40％。
实施例7 基于B-hPD-L1人源化小鼠的MC38-hPD-L1结肠癌动物模型的体内药效研究
收集体外培养的MC38-hPD-L1结肠癌细胞，并用PBS重悬至浓5*10 5/0.1mL浓度，按照0.1mL/只接种于B-hPD-L1人源化小鼠的右侧皮下。当平均肿瘤体积达到大约138mm 3时，挑选个体肿瘤体积合适的小鼠入组实验组，并分配到3个实验组，即溶剂对照组(Control)、单抗对照组M8和受试样品组PDL1scFv-607H，每组8只小鼠。分组当天通过腹腔注射，开始给药，每两天给药1次，连续给药8次，其中M8给药剂量为20mg/kg，PDL1scFv-607H给药剂量为27.5mg/kg。每周测量两次肿瘤直径，记录，并按照下述公式计算肿瘤体积和肿瘤生长抑制率。
肿瘤体积(tumor volume,TV)的计算公式为：TV＝1/2×长×宽 2。
肿瘤生长抑制率(TGI)＝(1-实验组肿瘤体积/溶剂对照组肿瘤体积)×100％。
本实验中B-hPD-L1人源化小鼠和MC38-hPD-L1结肠癌细胞均由百奥赛图(北京)医药科技股份有限公司提供。
实验结果如图10所示，双特异性抗体PDL1scFv-607H和单抗M8均可有效抑制MC38-hPD-L1移植瘤的生长，且给药后的第23天PDL1scFv-607H的肿瘤生长抑制率为74％，M8的肿瘤生长抑制率为89％。虽然，双特异性抗体PDL1scFv-607H在本实验结束时对肿瘤的生长抑制率略低于单抗对照品M8，但二者无显著差异，不具有统计学意义。
实施例8 双特异性抗体PDL1scFv-607H突变体的制备
根据本申请人之前的研究结果(参照中国专利申请202110085824.3)，通过对scFv片段上特异性天冬酰胺(N)的突变可有效的改善双特异性抗体的理化性质。因此，本发明利用常规的分子克隆技术对PDL1scFv-607H的scFv片段上天冬酰胺(N)分别进行单点或组合突变成谷氨酸(E)，抽提各突变质粒并与对应的轻链配对转染HEK293F细胞表达并纯化获得双特异性抗体PDL1scFv-607H各突变体样品。排除不表达、或突变后对抗体活性有较大影响以及突变后理化性质变得更差的位点，最终确定PDL1scFv-607H的多肽链上的第76位N、第213位N突变成E后对样品的理化性质有明显的改善作用，将上述突变体对应命名为PDL1scFv-607H-VHN76E、PDL1scFv-607H-VLN76E和PDL1scFv-607H-VHN76E-VLN76E，各自对应的多肽链序列如SEQ ID NO：37、SEQ ID NO：38和SEQ ID NO：39所示。
PDL1scFv-607H各突变体通过一步proteinA纯化后，其超高效液相色谱(UPLC)检测结果如表11所示。
表11、PDL1scFv-607H各突变体经一步proteinA纯化后的UPLC检测结果
Figure PCTCN2021095189-appb-000009
Figure PCTCN2021095189-appb-000010
实施例9 双特异性抗体PDL1scFv-607H突变体对PD-L1和HER2的亲和力的亲和力测定
实验方法参照实施例2。
实验结果分别如图11和图12所示，各双特异性抗体对PDL1-ECD-his和HER2-ECD-his的亲和力(EC 50)分别如表12和表13所示，可见各突变体与PDL1-ECD-his的亲和力与PDL1scFv-607H和M8相当，未见明显衰减。各突变体与HER2-ECD-his的亲和力与PDL1scFv-607H相当，稍弱于单抗607。
表12、PDL1scFv-607H各突变体对PDL1-ECD-his结合的EC 50
Figure PCTCN2021095189-appb-000011
表13、PDL1scFv-607H各突变体对HER2-ECD-his结合的EC 50
Figure PCTCN2021095189-appb-000012
实施例10 双特异性抗体PDL1scFv-607H突变体抑制PD-1和PD-L1高表达细胞株的结合活性
实验方法参照实施例4。
实验结果如图13所示，PDL1scFv-607H各突变双特异性抗体抑制PD-1和PD-L1高表达细胞株的结合活性与PDL1scFv-607H和M8相当，未见突变后活性下降，其各自IC 50详见表14。
表14、PDL1scFv-607H各突变体抑制PD-1和PD-L1高表达细胞株的结合的IC 50
Figure PCTCN2021095189-appb-000013
实施例11 双特异性抗体PDL1scFv-607H突变体对HER2高表达细胞株细胞增殖抑制活性测定
实验方法参照实施例5。
实验结果如图14所示，PDL1scFv-607H各突变双特异性抗体对HER2高表达细胞株BT474的增殖抑制作用与PDL1scFv-607H活性相同，其各自的IC 50分别如表15所示。
表15、PDL1scFv-607H各突变体抑制BT474细胞增殖的IC 50
Figure PCTCN2021095189-appb-000014
实施例12 混合淋巴细胞反应(MLR)实验测定双特异性抗体的活性
实验步骤如下：
1、树突状细胞的诱导
1)取适当数量的PBMC(外周血单核细胞，购自上海赛笠生物科技有限公司)与适当体积的CD14MicroBeads(购自MiltenyiBiotec，货号130-050-201)混合，混匀后4℃冰箱中放置15min；然后按照生产商提供的说明用MACS磁性细胞分选装置将CD14+单核细胞分离出来。
2)用含10％FBS的RPMI1640培养基(购自Thermo Fisher，货号61870036)重悬CD14+单核细胞至浓度5×10 5cells/mL，分别加入50ng/mL的GM-CSF(购自R&D，货号215-GM-050)和IL-4(购自R&D，货号：204-IL-050)进行诱导。
3)第四天，含10％FBS、50ng/mL GM-CSF和50ng/mL IL-4的RPMI1640培养基对上述细胞进行半换液。
4)第七天，通过添加1μg/ml LPS(购自sigma，货号L6529-1MG)作用24h诱导DC细胞成熟。
2、T细胞的分离和给药
1)利用Pan T cell Isolation Kit(购自MiltenyiBiotec，货号130-096-535)根据说明书要求从PBMC中分离T细胞，并用37℃预热的PBS细胞T细胞一次，计数，备用。
2)收集上述诱导成熟的DC细胞(mDC)，并用含2％FBS的PBS洗涤3次，计数备用。
3)将待测抗体用含10％FBS的RPMI1640培养基按照3倍梯度进行稀释，最高终浓度为300nM。
4)取上述稀释好的抗体按照50μL/孔加入到96-孔U形底培养板中，将mDC和T细胞按照2×10 5/mL和1×10 6/mL的浓度比例进行混匀，并取100μL细胞混合液加入到上述96-孔U形底培养板中。
5)于37℃,5％CO 2培养箱培养3d，收集50μL细胞培养上清用于IL-2检测；培养5d，收集120μL细胞培养上清用于IFN-γ检测。
3、IL-2和IFN-γ分泌水平检测
1)用ELISA包被液将mouse-anti-human IL-2蛋白(购自BD Pharmingen，货号555051)稀释至2.5μg/mL和mouse-anti-human IFN-γ(购自BD Pharmingen，货号551221)蛋白稀释至4μg/mL，分别包被ELISA板，100μL/孔，置于湿盒中，4℃，包被16h。
2)用PBST洗涤ELISA板三次，去除未结合抗原，并将ELISA板于吸水纸上拍干，除去多余的液体，然后用PBS配制的2％BSA，200μL/孔，于室温封闭1-2h。
3)配制IL-2/IFN-γ标准品：用RPMI1640培养基配制标准品，使IL-2初始浓度为40ng/ml，使IFN-γ初始浓度为400ng/ml，分别进行2倍稀释，每个标准品共稀释12个梯度。每孔加入100μl的标准品稀释液，每个浓度设2个复孔。
4)用PBST洗涤一次，洗除多余的封闭液，并将ELISA板拍干，除去多余的液体，将培养5d的细胞上清用PBST配制的1％BSA稀释10倍加入到mouse-anti-human IFN-γ包被板；将45μL 3d的细胞上清及65μL 5d的细胞上清的上清混合后，取100μL混合液加入到mouse-anti-human IL-2包被板，室温孵育1h。
5)洗除未结合的或非特异性结合的一抗，用含有1％BSA的PBST(抗体稀释液)将biotin-mouse-anti-human IL-2(购自BD Pharmingen，货号555040)和biotin-mouse-anti-human IFN-γ(购自BD Pharmingen，货号554550)二抗分别稀释1000倍，加入ELISA板，100μL/孔，室温孵育1h。随后PBST洗涤三次，用抗体稀释液将Streptavidin-HRP(购自BD Pharmingen，货号554066)稀释5000倍，加入ELISA板，100μL/孔，室温孵育1h。
6)用PBST洗涤五次，并将ELISA板于吸水纸上拍干，除去多余的液体，加入TMB显色液，100μL/孔，显色至合适深浅，加入2M H2SO4，50μL/孔，以终止显色，并于多功能酶标仪中在450nm波长处测定其吸光度，分析数据。
实验结果分别如图15和图16所示，可见突变体PDL1scFv-607H-VHN76E可显著促进T细胞的IL-2和IFN-γ的分泌，并且在高浓度下对IL-2分泌的促进作用优于PDL1scFv-607H，其各自的EC 50分别如表16和17所示。
表16、PDL1scFv-607H及突变体促进IL2分泌的EC 50
Figure PCTCN2021095189-appb-000015
表17、PDL1scFv-607H及突变体促进IFN-γ分泌的EC 50
Figure PCTCN2021095189-appb-000016

Claims (17)

  1. 抗PD-L1和HER2的双特异性抗体，其特征在于，包含两条多肽链和两条轻链，其中：
    (a)所述的多肽链从N末端至C末端包含VH-PDL1-CH1-CH2-CH3-L1-VH-HER2-L2-VL-HER2或VH-HER2-L2-VL-HER2-L1-VH-PDL1-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-PDL1-CL；或
    (b)所述的多肽链从N末端至C末端包含VH-HER2-CH1-CH2-CH3-L1-VH-PDL1-L2-VL-PDL1或VH-PDL1-L2-VL-PDL1-L1-VH-HER2-CH1-CH2-CH3，所述的轻链从N末端至C末端包含VL-HER2-CL；
    其中，所述的VH-PDL1为结合PD-L1的重链可变区，所述的VL-PDL1为结合PD-L1的轻链可变区，所述的VH-HER2为结合HER2的重链可变区，所述的VL-HER2为结合HER2的轻链可变区，所述的L1和L2为(G 4S)x，x为3、4、5或6，所述的CH1-CH2-CH3为重链恒定区，所述的CL为轻链恒定区，所述的VH-PDL1与所述的VL-PDL1形成特异性结合PD-L1的抗原结合位点，所述的VH-HER2与所述的VL-HER2形成特异性结合HER2的抗原结合位点。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述的L1为(G 4S) 3，所述的L2为(G 4S) 4。
  3. 如权利要求1所述的双特异性抗体，其特征在于，所述的VH-PDL1包含氨基酸序列如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的重链CDR，所述的VL-PDL1包含氨基酸序列如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的轻链CDR，所述的VH-HER2包含氨基酸序列如SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9或如SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的重链CDR，所述的VL-HER2包含氨基酸序列如SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12或如SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的轻链CDR。
  4. 如权利要求3所述的双特异性抗体，其特征在于，所述的VH-PDL1具有如SEQ ID NO：19所示的氨基酸序列，所述的VL-PDL1具有如SEQ ID NO：20所示的氨基酸序列，所述的VH-HER2具有如SEQ ID NO：21或SEQ ID NO：23所示的氨基酸序列，所述的VL-HER2具有如SEQ ID NO：22或SEQ ID NO：24所示的氨基酸序列。
  5. 如权利要求4所述的双特异性抗体，其特征在于，所述的多肽链具有如SEQ ID NO：31或SEQ ID NO：33所示的氨基酸序列，所述的轻链具有如SEQ ID NO：26所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：32或SEQ ID NO：34所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列；或所述的多肽链具有如SEQ ID NO：35或SEQ ID NO：36所示的氨基酸序列，所述的轻链具有如SEQ ID NO：30所示的氨基酸序 列。
  6. 如权利要求5所述的双特异性抗体，其特征在于，所述的多肽链具有如SEQ ID NO：34所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列，且所述的氨基酸序列如SEQ ID NO：34所示的多肽链进一步包含以下氨基酸残基位点的突变：N76E和/或N213E。
  7. 如权利要求6所述的双特异性抗体，其特征在于，所述的多肽链具有如SEQ ID NO：37或SEQ ID NO：38或SEQ ID NO：39所示的氨基酸序列，所述的轻链具有如SEQ ID NO：28所示的氨基酸序列。
  8. 如权利要求1所述的双特异性抗体，其特征在于，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。
  9. 一种分离的核苷酸，其特征在于，所述的核苷酸编码如权利要求1-8中任一项所述的双特异性抗体。
  10. 一种表达载体，其特征在于，所述的表达载体含有如权利要求9所述的核苷酸。
  11. 一种宿主细胞，其特征在于，所述的宿主细胞含有如权利要求9所述的表达载体。
  12. 如权利要求1-8中任一项所述的双特异性抗体的制备方法，其特征在于，所述方法包含以下步骤：
    (a)在表达条件下，培养如权利要求11所述的宿主细胞，从而表达所述的双特异性抗体；
    (b)分离并纯化(a)所述的双特异性抗体。
  13. 一种药物组合物，其特征在于，所述药物组合物含有如权利要求1-8中任一项所述的双特异性抗体和药学上可接受的载体。
  14. 如权利要求1-8中任一项所述的双特异性抗体或如权利要求13所述的药物组合物在制备治疗癌症的药物中的用途。
  15. 如权利要求14所述的用途，其特征在于，所述癌症选自由以下组成的组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。
  16. 一种治疗癌症的方法，其特征在于，包括向有需要的受试者施用如权利要求1-8中任一项所述的双特异性抗体或如权利要求13所述的药物组合物。
  17. 如权利要求16所述的方法，其特征在于，所述癌症选自由以下组成的组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤、白血病、淋巴瘤及其它赘生性恶性疾病。
PCT/CN2021/095189 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体 WO2021244328A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180032220.0A CN115605513B (zh) 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010487636.9 2020-06-02
CN202010487636.9A CN113754775A (zh) 2020-06-02 2020-06-02 一种抗pd-l1和her2的双特异性抗体

Publications (1)

Publication Number Publication Date
WO2021244328A1 true WO2021244328A1 (zh) 2021-12-09

Family

ID=78782246

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/095189 WO2021244328A1 (zh) 2020-06-02 2021-05-21 一种抗pd-l1和her2的双特异性抗体

Country Status (3)

Country Link
CN (2) CN113754775A (zh)
TW (1) TW202146457A (zh)
WO (1) WO2021244328A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114409797A (zh) * 2021-12-24 2022-04-29 合肥天港免疫药物有限公司 重组抗体及其应用
EP4032913A1 (en) * 2020-05-15 2022-07-27 Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd An antibody that binds to human pd-l1
WO2023071676A1 (zh) * 2021-11-01 2023-05-04 达石药业(广东)有限公司 一种抗her2/抗pd-l1双功能抗体及其应用
WO2023236844A1 (zh) * 2022-06-10 2023-12-14 三优生物医药(上海)有限公司 靶向her2和pd-l1的双特异性抗体及其制备方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821337A (en) * 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO2017196867A1 (en) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anti-pd-l1 antibodies
WO2018002339A1 (en) * 2016-07-01 2018-01-04 Alligator Bioscience Ab Bispecific antibodies directed against ox40 and a tumor-associated antigen
CN110790840A (zh) * 2018-08-01 2020-02-14 三生国健药业（上海）股份有限公司 结合人her2的抗体、其制备方法和用途
CN111196856A (zh) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 抗her2/pd1双特异性抗体

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5821337A (en) * 1991-06-14 1998-10-13 Genentech, Inc. Immunoglobulin variants
WO2017196867A1 (en) * 2016-05-09 2017-11-16 Igm Biosciences, Inc. Anti-pd-l1 antibodies
WO2018002339A1 (en) * 2016-07-01 2018-01-04 Alligator Bioscience Ab Bispecific antibodies directed against ox40 and a tumor-associated antigen
CN110790840A (zh) * 2018-08-01 2020-02-14 三生国健药业（上海）股份有限公司 结合人her2的抗体、其制备方法和用途
CN111196856A (zh) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 抗her2/pd1双特异性抗体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MITTAL DEEPAK, VIJAYAN DIPTI, NEIJSSEN JOOST, KREIJTZ JOOST, HABRAKEN MAURICE M. J. M., VAN EENENNAAM HANS, VAN ELSAS ANDREA, SMYT: "Blockade of ErbB2 and PD-L1 using a bispecific antibody to improve targeted anti-ErbB2 therapy", ONCOIMMUNOLOGY, vol. 8, no. 11, 2 November 2019 (2019-11-02), pages e1648171, XP055877778, DOI: 10.1080/2162402X.2019.1648171 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4032913A1 (en) * 2020-05-15 2022-07-27 Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd An antibody that binds to human pd-l1
WO2023071676A1 (zh) * 2021-11-01 2023-05-04 达石药业(广东)有限公司 一种抗her2/抗pd-l1双功能抗体及其应用
CN114409797A (zh) * 2021-12-24 2022-04-29 合肥天港免疫药物有限公司 重组抗体及其应用
CN114409797B (zh) * 2021-12-24 2024-02-20 合肥天港免疫药物有限公司 重组抗体及其应用
WO2023236844A1 (zh) * 2022-06-10 2023-12-14 三优生物医药(上海)有限公司 靶向her2和pd-l1的双特异性抗体及其制备方法和应用

Also Published As

Publication number Publication date
CN115605513B (zh) 2024-03-19
TW202146457A (zh) 2021-12-16
CN115605513A (zh) 2023-01-13
CN113754775A (zh) 2021-12-07

Similar Documents

Publication Publication Date Title
CN113227146B (zh) 密蛋白18.2结合部分和其用途
JP6224759B2 (ja) 抗ｂ７−ｈ３抗体
WO2021244328A1 (zh) 一种抗pd-l1和her2的双特异性抗体
JP6124377B2 (ja) 新規抗ｄｒ５抗体
WO2018137576A1 (zh) 抗pd-1单克隆抗体及其制备方法和应用
CN111454357B (zh) 一种含有抗体的肿瘤治疗剂的开发和应用
US20220162331A1 (en) Anti-cd73 monoclonal antibody and application thereof
CN110577597A (zh) 一种阻断CD47和SIRPα相互作用的抗体
WO2020025013A1 (zh) 结合人her2的抗体、其制备方法和用途
WO2021170082A1 (zh) 抗cd47/抗pd-l1抗体及其应用
JP7547581B2 (ja) 腫瘍治療薬及びその応用
JP7397897B2 (ja) ヒトｐｄ－ｌ１に結合する抗体
WO2022194201A1 (zh) 一种靶向cldn18.2的抗体或其抗原结合片段及其应用
US20220127374A1 (en) ANTIBODIES AGAINST hEPCR
JP2024504124A (ja) 新規の抗グレムリン１抗体
WO2021244554A1 (zh) 抗pdl1×egfr的双特异性抗体
WO2022184162A1 (zh) 针对NKp46的抗体及其应用
US20210024638A1 (en) Anti-egfr/anti-4-1bb bispecific antibody and use thereof
WO2021244392A1 (zh) 一种抗pd1×pdl1的双特异性抗体
WO2021244553A1 (zh) 一种抗pd-l1和egfr的四价双特异性抗体
WO2021244371A1 (zh) 一种抗pd-l1/vegf融合蛋白
WO2019174637A1 (zh) 一种针对tim-3的全人源化抗体分子、抗原结合片段及其医药用途
WO2023241629A1 (zh) 抗cldn18.2抗体、其药物组合物及用途
WO2022268168A1 (zh) 靶向lag-3和pd-l1的新型双特异抗体及其应用
WO2022184155A1 (zh) 抗ctla-4抗体及其应用

Legal Events

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

Ref document number: 21816748

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21816748

Country of ref document: EP

Kind code of ref document: A1