WO2021244552A1 - Anticorps bispécifique anti-pdl1 × kdr - Google Patents

Anticorps bispécifique anti-pdl1 × kdr Download PDF

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WO2021244552A1
WO2021244552A1 PCT/CN2021/097783 CN2021097783W WO2021244552A1 WO 2021244552 A1 WO2021244552 A1 WO 2021244552A1 CN 2021097783 W CN2021097783 W CN 2021097783W WO 2021244552 A1 WO2021244552 A1 WO 2021244552A1
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kdr
pdl1
terminus
linker2
light chain
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PCT/CN2021/097783
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Chinese (zh)
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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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Publication of WO2021244552A1 publication Critical patent/WO2021244552A1/fr

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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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • 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
    • 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
    • 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]

Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention discloses bispecific antibodies against PDL1 ⁇ KDR.
  • 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. .
  • VEGFR-2 Vascular Endothelial Growth Factor Receptor-2
  • VEGFR2 is mainly expressed in vascular endothelial cells, especially tumor vascular endothelial cells, and mainly combines with VEGF-C, D, and A to promote the survival of blood vessels.
  • VEGF specifically binds to the extracellular domain of VEGFR2, which can activate multiple downstream signaling pathways such as MAPK, PI3K, PKC, FAK, etc., and participate in endothelial cell budding, migration, vascular permeability, and tumor cell survival.
  • VEGFR-2 is closely related to many diseases such as tumors, psoriasis, rheumatoid arthritis, diabetic retinopathy, etc. Especially in tumor growth, metastasis and tumor multi-drug resistance. Therefore, VEGFR-2 has become an ideal target for the treatment of these diseases, especially tumors.
  • tumors are known to be rich in blood vessels, and clinical trial data show that targeting VEGFR-2 is essential to inhibit tumor angiogenesis.
  • anti-VEGFR-2 drugs used alone are often very short-lived, which may be closely related to excessive vascular pruning and extreme hypoxia. In 2018, the relationship between vascular normalization and immune response has been revealed. There are a large number of immunosuppressive cells and dysfunctional effector T cells in the tumor microenvironment.
  • immune checkpoint inhibitors and anti-angiogenesis drugs can significantly extend the treatment window for tumor vascular normalization, and the normalization of blood vessels weakens the tumor
  • the immunosuppressive process in the microenvironment increases the infiltration of T cells and ultimately promotes tumor regression.
  • 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 invention provides a bispecific antibody against PDL1 ⁇ KDR and its application.
  • the first object of the present invention is to provide a bispecific antibody against PDL1 ⁇ KDR.
  • 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 an anti-PDL1 ⁇ KDR bispecific antibody, comprising two polypeptide chains and two light chains selected from the group consisting of:
  • the polypeptide chain from N-terminus to C-terminus contains VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH -KDR-linker1-VL-KDR, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or
  • the polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus. -KDR-CL; or
  • the polypeptide chain includes VH-KDR-linker1-VL-KDR-linker2-VH-PDL1-CH1-CH2-CH3 or VL-KDR-linker1-VH-KDR-linker2-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 includes VH-KDR-CH1-CH2-CH3-linker2-VL-PDL1-linker1-VH-PDL1 or VH-KDR-CH1-CH2-CH3-linker2-VH from N-terminus to C-terminus -PDL1-linker1-VL-PDL1, the light chain includes VL-KDR-CL from N-terminus to C-terminus; or
  • the polypeptide chain includes VH-PDL1-linker1-VL-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL from N-terminus to C-terminus -KDR-CL;
  • VH-PDL1 is a heavy chain variable region that binds PD-L1
  • the VL-PDL1 is the light chain variable region that binds to PD-L1,
  • the VH-KDR is a heavy chain variable region that binds to KDR
  • VL-KDR is the light chain variable region that binds to KDR
  • linker1 and linker2 are each independently a flexible peptide linker
  • 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-KDR and the VL-KDR form an antigen binding site that specifically binds to the KDR.
  • the bispecific antibody has the activity of simultaneously binding to KDR and binding to PD-L1.
  • the flexible peptide linker includes 6-30 amino acids, preferably 10-25 amino acids.
  • the flexible peptide linker includes 2-6 G4S and/or G3S.
  • the Linker1 is 3-5 G4S.
  • the Linker2 is 2-4 G4S.
  • the linker1 is 4 G4S, and the linker2 is 3 G4S.
  • the anti-PDL1 ⁇ KDR bispecific antibody comprises two polypeptide chains and two light chains, wherein:
  • the polypeptide chain from N-terminus to C-terminus contains VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH -KDR-linker1-VL-KDR, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or
  • the polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus.
  • -KDR-CL The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus.
  • VH-PDL1 is a heavy chain variable region that binds PD-L1
  • the VL-PDL1 is the light chain variable region that binds to PD-L1,
  • the VH-KDR is a heavy chain variable region that binds to KDR
  • VL-KDR is the light chain variable region that binds to KDR
  • the linker1 and linker2 are each independently a flexible peptide linker; 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 forms an antigen binding site that specifically binds to PD-L1, and said VH-KDR and said VL-KDR form an antigen binding site that specifically binds to KDR.
  • the anti-PDL1 ⁇ KDR bispecific antibody comprises two polypeptide chains and two light chains, wherein:
  • the polypeptide chain from N-terminus to C-terminus contains VH-PDL1-CH1-CH2-CH3-linker2-VL-KDR-linker1-VH-KDR or VH-PDL1-CH1-CH2-CH3-linker2-VH -KDR-linker1-VL-KDR, the light chain includes VL-PDL1-CL from N-terminus to C-terminus; or
  • the polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus, and the light chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus.
  • -KDR-CL The polypeptide chain includes VL-PDL1-linker1-VH-PDL1-linker2-VH-KDR-CH1-CH2-CH3 from N-terminus to C-terminus.
  • VH-PDL1 is a heavy chain variable region that binds PD-L1
  • the VL-PDL1 is the light chain variable region that binds to PD-L1,
  • the VH-KDR is a heavy chain variable region that binds to KDR
  • VL-KDR is the light chain variable region that binds to KDR
  • the linker1 is 4 G4S
  • the linker2 is 3 G4S
  • the CH1-CH2-CH3 is the heavy chain constant region
  • the CL is the light chain constant region
  • the VH-PDL1 is the same as the
  • the VL-PDL1 forms an antigen binding site that specifically binds to PD-L1
  • the VH-KDR and the VL-KDR form an antigen binding site that specifically binds to KDR.
  • 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-KDR includes the heavy chain CDR shown in SEQ ID NO: 7-9, and the VL-KDR includes the amino acid sequence shown in SEQ ID NO: 10-12.
  • Light chain CDR is the amino acid sequence of the heavy chain CDR shown in SEQ ID NO: 1-3
  • the VL-PDL1 includes the amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4-6.
  • the VH-KDR includes the heavy chain CDR shown in SEQ ID NO: 7-9
  • the VL-KDR includes the amino acid sequence shown in SEQ ID NO: 10-12.
  • the VH-PDL1 has the amino acid sequence shown in SEQ ID NO: 13
  • the VL-PDL1 has the amino acid sequence shown in SEQ ID NO: 14
  • the VH -KDR has the amino acid sequence shown in SEQ ID NO: 15, and the VL-KDR has the amino acid sequence shown in SEQ ID NO: 16.
  • 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, SEQ ID NO: 18, SEQ ID NO: 24 or SEQ ID NO: 25, and the light chain has The amino acid sequence shown in SEQ ID NO: 19; or the polypeptide chain has the amino acid sequence shown in SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28, so The light chain has an amino acid sequence as shown in SEQ ID NO:21.
  • 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 pharmaceutical composition also contains an anti-tumor agent.
  • the pharmaceutical composition is in a unit dosage form.
  • the anti-tumor agent and the bispecific antibody may be separately present in a separate package, or the anti-tumor agent may be coupled to the bispecific antibody.
  • the dosage form of the pharmaceutical composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.
  • the parenteral administration dosage form includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavity injection.
  • 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: colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and Other neoplastic malignant diseases, etc.
  • the eighth aspect of the present invention provides a method of treating cancer, which comprises administering the above-mentioned bispecific antibody, or the immunoconjugate thereof, or the above-mentioned pharmaceutical composition to a subject in need.
  • the cancer is selected from the group consisting of: colorectal cancer, non-small cell lung cancer, gastric cancer, gastroesophageal junction adenocarcinoma, melanoma, lung cancer, liver cancer, lymphoma, leukemia, prostate cancer, bone marrow cancer and Other neoplastic malignant diseases, etc.
  • the ninth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
  • the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce Detect enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.) of the product.
  • the immunoconjugate includes an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.
  • the present invention provides a bispecific antibody against PDL1 ⁇ KDR.
  • Experimental results show that the biantibody can better maintain the activity of the respective monoclonal antibodies, and can specifically bind to the two targets of PD-L1 and KDR at the same time. Point, has good physical and chemical properties.
  • Figure 1 is a schematic diagram of the structure of the anti-PDL1 ⁇ KDR double antibody molecule of the present invention.
  • Figure 1A shows the anti-KDR scFv series connected to the C-terminal of anti-PDL1 mAb
  • Figure 1B shows the anti-PDL1 scFv series connected to the anti-KDR mAb. N-terminal.
  • Figure 2 shows the HPLC detection spectrum and SDS-PAGE detection results of the anti-PDL1 ⁇ KDR double antibody, among which Figure 2A is the HPLC detection spectrum and Figure 2B is the SDS-PAGE detection result.
  • Figure 3 shows the binding results of anti-PDL1 ⁇ KDR double antibodies to PD-L1 and KDR, respectively, by ELISA.
  • Figure 3A shows the binding results to PD-L1
  • Figure 3B shows the binding results to KDR.
  • Figure 4 shows the results of dual-specific ELISA detecting the binding of anti-PDL1 ⁇ KDR double antibodies to PD-L1 and KDR at the same time.
  • Figure 5 shows the results of FACS detection of the binding of anti-PDL1 ⁇ KDR double antibodies to N87-PDL1 cells.
  • Figure 6 shows the results of anti-PDL1 ⁇ KDR double antibodies blocking the activity of PD1/PD-L1 on cells.
  • Figure 7 shows the results of anti-PDL1 ⁇ KDR double antibodies blocking the binding of KDR and VEGF on cells.
  • Figure 8 shows the anti-PDL1 ⁇ KDR double antibody (anti-PDL1 ⁇ KDR rev3, rev4, rev5, rev6, rev7) blocking the activity of PD1/PD-L1 at the cellular level.
  • Figure 9 shows the anti-PDL1 ⁇ KDR double antibody (anti-PDL1 ⁇ KDR rev3, rev4, rev5, rev6, rev7) blocking the activity of KDR and VEGF binding on cells; where Figure 9A shows the resistance of anti-PDL1 ⁇ KDR rev3, rev4 Figure 9B shows the blocking activity of anti-PDL1 ⁇ KDR rev5 and rev6; Figure 9C shows the blocking activity of anti-PDL1 ⁇ KDR rev7.
  • the bispecific antibody of the present invention belongs to a homodimer.
  • the bispecific antibody of the present invention not only maintains the activities of the anti-KDR antibody and the anti-PD-L1 antibody, but can also bind to KDR and PD-L1 at the same time.
  • the bispecific antibody of the present invention can be developed as an antitumor drug with superior curative effect. On this basis, the inventor completed the present invention.
  • the terms "Antibody (Ab)” and “Immunoglobulin G (Abbreviation 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 refers to an antibody molecule that can specifically bind to two antigens (targets) 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.
  • 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 connecting the variable region of the heavy chain and the variable region of the light chain of the antibody through a linker of 15-25 amino acids. become.
  • 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)).
  • FR framework region
  • the light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
  • human framework region is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .
  • linker refers to the insertion of an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold to exchange one or more amino acid residues of the immunoglobulin with dual variable regions. base.
  • the preferred linker refers to the linker Linker1 and Linker2, where Linker1 connects the VH and VL of a single-chain antibody (scFv), and Linker2 is used to connect the scFv to the heavy chain of another antibody.
  • linkers include single glycine (Gly) or serine (Ser) residues, and the identity and sequence of amino acid residues in the linker can vary with the type of secondary structural elements that need to be implemented in the linker.
  • the bispecific antibody of the present invention is an anti-PDL1 ⁇ EGFR bispecific antibody, including an anti-PDL1 antibody part and an anti-EGFR antibody part.
  • the sequence of the anti-PD-L1 antibody of the present invention is as described in the patent application PCT/CN2020/090442.
  • Those skilled in the art can also modify or transform the anti-PD-L1 antibody of the present invention through techniques well known in the art, such as adding , Deletion and/or substitution of one or several amino acid residues, thereby further increasing the affinity or structural stability of anti-PD-L1, and obtaining modified or modified results through conventional measurement methods.
  • “conservative variants of the bispecific antibody of the present invention” refer to at most 10, preferably at most 8, and more preferably at most 5 compared with the amino acid sequence of the bispecific antibody of the present invention. Optimally, at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Ser
  • Trp(W) Tyr Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu
  • 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.
  • bispecific antibodies of the present invention can be used alone, or can be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, or any combination of these substances.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the term "expression vector” refers to a vector carrying an expression cassette for expressing a specific target protein or other substances, such as a plasmid, a viral vector (such as adenovirus, retrovirus), a phage, a yeast plasmid or other vectors.
  • a viral vector such as adenovirus, retrovirus
  • a phage a yeast plasmid or other vectors.
  • Representative examples include, but are not limited to: pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, etc., and other vectors used in mammalian expression systems.
  • the expression vector includes fusion DNA sequences linked to appropriate transcription and translation regulatory sequences.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • 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 invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavity injection.
  • 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 pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bispecific antibody (or conjugate thereof) of the present invention, and A pharmaceutically acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, with physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the bispecific antibodies of the present invention can also be used with other therapeutic agents
  • a safe and effective amount of the bispecific antibody or its immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health status, etc., which are all within the skill range of a skilled physician.
  • pcDNA TM 3.4 vector purchased from Thermo fisher company, article number A14697;
  • CHO cells purchased from Thermofisher, catalog number A29133;
  • Human gastric cancer cell line NCI-N87 purchased from the American Type Culture Collection (ATCC);
  • PD-1/PD-L1 Blockade Bioassay, Propagation model purchased from Promega, item number J1252;
  • VEGF Bioassay, Propagation Model purchased from Promega, the article number is GA1082.
  • Anti-PD-L1 monoclonal antibody prepared according to the sequence in PCT/CN2020/090442;
  • CDR region sequence comes from Dan Lu et al. Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 The for Enhancement Biological of 2003. : 43496-43507.) in the clone number 3A10 sequence, other framework regions were obtained after mutation by the company;
  • HRP-labeled goat anti-human Fc antibody purchased from sigma, catalog number A0170;
  • FITC-labeled goat anti-human Fc antibody purchased from sigma, item number F9512;
  • 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 anti-human KDR monoclonal antibody scFv1 (VL-linker1-VH) is connected in series to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1 ⁇ KDR bispecific antibody named It is anti-PDL1 ⁇ KDR BsAb1.
  • the structure is shown in Figure 1A (the VL of scFv1 is connected to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).
  • the anti-human KDR monoclonal antibody scFv2 (VH-linker1-VL) is connected in series to the C-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1 ⁇ KDR bispecific antibody, which is named anti -PDL1 ⁇ KDR BsAb2.
  • the structure is shown in Figure 1A (the VH of scFv2 is connected to the C-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).
  • the anti-human PD-L1 monoclonal antibody scFv3 (VL-linker1-VH) is connected in series to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2 to construct an anti-PDL1 ⁇ KDR bispecific antibody, which is named anti -PDL1 ⁇ KDR BsAb3.
  • the structure is shown in Figure 1B (the VH of scFv3 is connected to the N-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).
  • the anti-human KDR monoclonal antibody scFv2 (VH-linker1-VL) is connected in series to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1 ⁇ KDR bispecific antibody named It is anti-PDL1 ⁇ KDR Rev3 (the VL of scFv2 is connected to the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).
  • the anti-human KDR monoclonal antibody scFv1 (VL-linker1-VH) is connected in series to the N-terminus of the heavy chain of the anti-human PD-L1 monoclonal antibody through linker2 to construct an anti-PDL1 ⁇ KDR bispecific antibody named It is anti-PDL1 ⁇ KDR Rev4 (the VH of scFv1 is connected to the N-terminus of the heavy chain of anti-human PD-L1 monoclonal antibody through linker2).
  • the present invention uses the scFv3 (VL-linker1-VH) of the anti-human PD-L1 monoclonal antibody, which is connected in series to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2, to construct an anti-PDL1 ⁇ KDR bispecific antibody, named as anti -PDL1 ⁇ KDR Rev5 (the VL of scFv3 is connected to the C-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).
  • the present invention uses the scFv4 (VH-linker1-VL) of the anti-human PD-L1 monoclonal antibody, which is connected in series to the C-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2, to construct an anti-PDL1 ⁇ KDR bispecific antibody, named as anti -PDL1 ⁇ KDR Rev6 (the VH of scFv4 is connected to the C-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).
  • the anti-human PD-L1 monoclonal antibody scFv4 (VH-linker1-VL) is connected in series to the N-terminus of the heavy chain of the anti-human KDR monoclonal antibody through linker2 to construct an anti-PDL1 ⁇ KDR bispecific antibody, which is named anti -PDL1 ⁇ KDR Rev7 (the VL of scFv4 is connected to the N-terminus of the heavy chain of anti-human KDR monoclonal antibody through linker2).
  • linker1 is 4 GGGGS
  • linker2 is 3 GGGGS.
  • the CDR region sequence of anti-human KDR monoclonal antibody comes from Dan Lu et al. 2 For Enhanced Neutralizing Activity. The Journal of Biological Chemistry, 2003,278:43496-43507.)
  • the sequence of clone number 3A10, the other framework regions are obtained after mutation of the company, and the source of the sequence of anti-human PD-L1 monoclonal antibody M8 In PCT/CN2020/090442.
  • the heavy chain and light chain expression vectors of each bispecific antibody and its corresponding monoclonal antibody were obtained through gene synthesis and conventional molecular cloning methods.
  • the corresponding amino acid sequences are shown in Table 1, and the CDRs are encoded according to Kabat rules.
  • the polypeptide chain of the anti-PDL1 ⁇ KDR double antibody in the above table refers to the heavy chain of the double antibody (formed by the scFv connected to the end of the heavy chain of the anti-human KDR monoclonal antibody through a linker).
  • the light chain of the double antibody does not contain Inside.
  • the polypeptide chain and light chain DNA fragments of the anti-PDL1 ⁇ KDR double antibody were respectively subcloned into pcDN3.4 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells and/or 293E cells. After 5-7 days of cell culture, the cells were cultured After the solution is filtered by high-speed centrifugation and vacuum filtration with a microporous membrane, the sample is loaded on a HiTrap MabSelect SuRe column, and the protein is eluted with an eluent containing 100 mM citric acid and pH 3.5, and the target sample is recovered and dialyzed to pH 7.4 PBS.
  • the purified protein was detected by HPLC.
  • the HPLC detection pattern of the anti-PDL1 ⁇ KDR double antibody is shown in Figure 2A, and the purity of the anti-PDL1 ⁇ KDR BsAb1 double antibody monomer reaches more than 96%.
  • the other two double antibodies (anti-PDL1 ⁇ KDR BsAb2, BsAb3) have similar profiles, and the monomer purity is above 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-PDL1 ⁇ KDR BsAb1, and lanes 3 and 4 are reduced and non-reduced SDS-PAGE of anti-PDL1 monoclonal antibody.
  • lanes 5 and 6 are the reduced and non-reduced SDS-PAGE of anti-PDL1 ⁇ KDR BsAb2
  • lanes 7 and 8 are the reduced and non-reduced SDS-PAGE of anti-PDL1 ⁇ KDR BsAb3.
  • the theoretical molecular weight of anti-PDL1 ⁇ KDR BsAb1 and anti-PDL1 ⁇ KDR BsAb2 is 197KD
  • the theoretical molecular weight of anti-PDL1 ⁇ KDR BsAb3 is 196KD.
  • Example 3 Enzyme-linked immunosorbent assay (ELISA) to determine the affinity of anti-PDL1 ⁇ KDR double antibodies to antigen
  • 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 the N-terminus and a 6 ⁇ His tag at the C-terminus. It is constructed into the expression vector through the two restriction sites of EcoRI and HindIII, and transfected into HEK-293E cells for expression and purification.
  • the EC 50 of anti-PD-L1 monoclonal antibody, anti-PDL1 ⁇ KDR BsAb1, anti-PDL1 ⁇ KDR BsAb2, and anti-PDL1 ⁇ KDR BsAb3 are 0.13nM, 0.13nM, 0.14nM, 0.15, respectively.
  • nM the affinity of the three double antibodies is equivalent to that of the monoclonal antibody, and the anti-PD-L1 monoclonal antibody platform is slightly higher, which may be due to the fact that the secondary antibody is anti-Fc. It has been experimentally proved that the ELISA of this sample is different due to the secondary antibody, and the result is slightly There are different. It can be considered that the affinity of the double antibody is not weaker than that of the monoclonal antibody.
  • the KDR-ECD-His protein (according to the sequence provided by UniProt (SEQ ID P35968)) was synthesized into the extracellular domain gene with pH 7.4 PBS buffer and added at its N-terminus.
  • the upper signal peptide sequence, the 6 ⁇ His tag is added to the C-terminus, the two restriction sites of EcoRI and HindIII are respectively constructed into the expression vector, and the HEK-293E cell is transfected to express and purified.
  • the antibody to be tested is diluted in %BSA, and anti-KDR monoclonal antibody is used as a positive control.
  • the initial concentration is 300nM, and 12 gradients are gradually diluted 3 times. Incubate at 37°C for 1h; wash the plate twice with PBST, add HRP-anti-Fab antibody, and incubate at 37°C for another 40min; wash the plate three times with PBST and pat dry, add 100 ⁇ l TMB to each well, and place in the dark at room temperature (20 ⁇ 5°C) 5 min; per well was added 50 ⁇ l 2M H 2 SO 4 stop solution to stop the substrate reaction, microplate read OD at 450nm, GraphPad Prism data analysis, plotting and calculation of EC 50. The experimental results are shown in Figure 3B.
  • the EC 50 of anti-KDR monoclonal antibody, anti-PDL1 ⁇ KDR BsAb1, anti-PDL1 ⁇ KDR BsAb2, and anti-PDL1 ⁇ KDR BsAb3 are 0.26nM, 0.23nM, 0.23nM, 0.41nM, respectively.
  • the affinity of anti-PDL1 ⁇ KDR BsAb1 and anti-PDL1 ⁇ KDR BsAb2 is slightly stronger than that of monoclonal antibodies. This may be due to the fact that the secondary antibody is anti-Fab. Experiments have shown that the ELISA of this sample is slightly different due to different secondary antibodies. . It can be considered that the affinity of the double antibody is not weaker than that of the monoclonal antibody.
  • the PDL1-ECD-hFc protein was replaced with a pH 7.4 PBS buffer (the C-terminus of the PDL1-ECD-His protein was replaced with the hFc tag) Dilute to 1 ⁇ g/ml, then add 100 ⁇ l/well to the ELISA plate; 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 1 hour; wash the plate twice with PBST Times; then add the antibody to be detected that is diluted with PBS+1% BSA, the initial concentration is 12nM, and 8 gradients of 3-fold dilution are gradually added.
  • the experimental results are shown in Figure 4.
  • the EC50 of anti-PDL1 ⁇ KDR BsAb1, anti-PDL1 ⁇ KDR BsAb2, and anti-PDL1 ⁇ KDR BsAb3 are 0.13nM, 0.14nM, 0.20nM, respectively.
  • anti-PDL1 ⁇ KDR BsAb3 is slightly weaker than the other two double antibodies, and the monoclonal antibody does not have the ability to bind these two antigens at the same time.
  • N87-PDL1 used the lentiviral transfection method in our laboratory to transfect NCI-N87 with a stable cell line constructed by PD-L1. 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.
  • the cells were resuspended in 200 ⁇ l PBS, and the binding affinity of the double antibody to the cells was determined by the Beckman Coulter CytoFLEX flow cytometer. The data obtained was fitted and analyzed by GraphPad Prism software.
  • the experimental results are shown in Figure 5.
  • the EC 50 of anti-PD-L1 monoclonal antibody is 0.13nM
  • the EC 50 of anti-PDL1 ⁇ KDR BsAb1 is 0.15nM
  • the EC 50 of anti-PDL1 ⁇ KDR BsAb2 is 0.15nM
  • the EC 50 of ⁇ KDR BsAb3 is 0.21 nM
  • the affinity of the three double antibodies is equivalent to that of the positive control anti-PD-L1 monoclonal antibody.
  • Anti-PDL1 ⁇ KDR double antibody blocks the activity of PD1/PD-L1 at the cellular level
  • This experiment uses Promega's PD-1/PD-L1 Blockade Bioassay, Propagation model and method.
  • the IC 50 of the anti-PD-L1 monoclonal antibody is 0.23 nM.
  • the IC 50 of anti-PDL1 ⁇ KDR BsAb1, anti-PDL1 ⁇ KDR BsAb2, anti-PDL1 ⁇ KDR BsAb3 are 0.27nM, 0.22nM, and 0.30nM, respectively, which are equivalent to monoclonal antibodies.
  • anti-PDL1 ⁇ KDR Rev3, Rev4, Rev5, Rev6, and Rev7 block the activity of PD1/PD-L1 signaling pathway on cells.
  • the results are shown in Figure 8.
  • Anti-PDL1 ⁇ KDR rev5 is significantly more single than the positive control anti-PD-L1 Anti-poor; anti-PDL1 ⁇ KDR rev6 is slightly worse than the positive control anti-PD-L1 monoclonal antibody; anti-PDL1 ⁇ KDR rev3, rev4, rev7 has no significant difference with the positive control anti-PD-L1 monoclonal antibody.
  • This experiment uses Promega's VEGF Bioassay, Propagation Model and method.
  • KDR/NFAT-RE HEK293 cells express KDR on the surface.
  • VEGF binds to the KDR on the cell surface, the signal is transmitted to the cell, and the fluorescent reporter gene is expressed, and the biological fluorescent signal can be detected.
  • the anti-KDR antibody is added to block the binding of VEGF to KDR on the cell surface, the fluorescence signal is weakened, and there is a dose-effect relationship with the concentration of KDR antibody within a certain range.
  • VEGF was diluted with assay buffer (DMED+10% FBS) to a 3x dilution, 60ng/mL, working concentration 20ng/mL, 25 ⁇ L/well was added to 96-well white transparent bottom plate. Dilute the antibody to be tested and the positive control with assay buffer (DMED+10% FBS) to 1000 nM 3 ⁇ dilution, and then dilute it four-fold step by step, and add 25 ⁇ L/well to 96-well white translucent bottom plate.
  • assay buffer DMED+10% FBS
  • the IC 50 of the anti-KDR monoclonal antibody is 3.10 nM.
  • the IC 50 of anti-PDL1 ⁇ KDR BsAb1, anti-PDL1 ⁇ KDR BsAb2, anti-PDL1 ⁇ KDR BsAb3 are 13.47nM, 6.09nM, 4.35nM, respectively, of which anti-PDL1 ⁇ KDR BsAb1 is slightly weaker than anti- PDL1 ⁇ KDR BsAb2 and anti-PDL1 ⁇ KDR BsAb3. There is little difference in the activity of the three groups of double antibodies and monoclonal antibodies.

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Abstract

La présente invention concerne un anticorps bispécifique anti-PDL1 × KDR. Les résultats expérimentaux montrent que l'anticorps bispécifique peut mieux maintenir l'activité de chaque anticorps monoclonal, peut se lier spécifiquement aux cibles PD-L1 et KDR, et présente de bonnes propriétés physiques et chimiques.
PCT/CN2021/097783 2020-06-02 2021-06-01 Anticorps bispécifique anti-pdl1 × kdr WO2021244552A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN109310755A (zh) * 2016-02-02 2019-02-05 卡德门企业有限公司 Pd-l1和kdr的双特异性结合蛋白
WO2020177627A1 (fr) * 2019-03-02 2020-09-10 上海一宸医药科技有限公司 Anticorps bispécifique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109310755A (zh) * 2016-02-02 2019-02-05 卡德门企业有限公司 Pd-l1和kdr的双特异性结合蛋白
WO2020177627A1 (fr) * 2019-03-02 2020-09-10 上海一宸医药科技有限公司 Anticorps bispécifique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG FENG, WANG KAI-QIN, WANG LAN: "Development in Bispecific Antibody", CHINESE JOURNAL OF PHARMACEUTICAL ANALYSIS, ZHONGGUO YAOXUEHUI, BEIJING, CN, vol. 39, no. 1, 31 January 2019 (2019-01-31), CN , pages 78 - 85, XP009522265, ISSN: 0254-1793, DOI: 10.16155/j.0254-1793.2019.01.10 *

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