WO2021244553A1 - Tetravalent bispecific antibody against pd-1 and egfr - Google Patents

Tetravalent bispecific antibody against pd-1 and egfr Download PDF

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WO2021244553A1
WO2021244553A1 PCT/CN2021/097784 CN2021097784W WO2021244553A1 WO 2021244553 A1 WO2021244553 A1 WO 2021244553A1 CN 2021097784 W CN2021097784 W CN 2021097784W WO 2021244553 A1 WO2021244553 A1 WO 2021244553A1
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cancer
pdl1
egfr
bispecific antibody
heavy chain
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PCT/CN2021/097784
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French (fr)
Chinese (zh)
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朱祯平
黄浩旻
赵杰
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三生国健药业（上海）股份有限公司
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Priority to CN202180038398.6A priority Critical patent/CN115698085A/en
Publication of WO2021244553A1 publication Critical patent/WO2021244553A1/en

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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
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention discloses a tetravalent bispecific antibody against PD-L1 and EGFR.
  • 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. .
  • Epidermal Growth Factor Receptor is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc.
  • the EGFR signaling pathway plays a role in the physiological processes of cell growth, proliferation, and differentiation. Important role.
  • the mutation or abnormal expression of EGFR plays an important role in the growth and development of tumors.
  • Anti-EGFR monoclonal antibody drugs have the functions of blocking tumor cell cycle progress, accelerating tumor cell apoptosis, inhibiting tumor angiogenesis, inhibiting tumor infiltration and metastasis, and enhancing the effect of radiotherapy and chemotherapy. The mechanism of action is clear, so it has attracted much attention in cancer treatment. .
  • Anti-tumor therapy targeting EGFR has become one of the most active areas in cancer research, and tremendous progress has been made. However, anti-tumor therapy targeting EGFR still has many shortcomings waiting to be perfected.
  • Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancers and other diseases. Although a large number of new bispecific antibody structures have been reported recently, the main technical difficulty in producing bispecific antibodies lies in obtaining the correct paired molecules. The current forms of bispecific antibodies all have mismatch problems, so one or more by-products or aggregates caused by mismatches will be produced, thereby affecting the yield, purity and physical and chemical stability of the target bispecific antibody This in turn affects the safety and effectiveness of bispecific antibodies in the body.
  • the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR and its application.
  • the first objective of the present invention is to provide a tetravalent bispecific antibody against PD-L1 and EGFR.
  • the second object of the present invention is to provide an isolated nucleotide encoding the tetravalent 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 objective of the present invention is to provide a method for preparing the tetravalent bispecific antibody.
  • the sixth object of the present invention is to provide a pharmaceutical composition containing the tetravalent bispecific antibody.
  • the seventh object of the present invention is to provide the use of the tetravalent 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 the treatment of cancer by the tetravalent bispecific antibody or the pharmaceutical composition.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR, comprising:
  • the polypeptide chain includes VH-PDL1-CH1-linker-VH-EGFR-CH1-CH2-CH3 or VH-EGFR-CH1-linker-VH-PDL1-CH1 from N-terminus to C-terminus -CH2-CH3, wherein the VH-PDL1 is a heavy chain variable region that binds PD-L1, the VH-EGFR is a heavy chain variable region that binds EGFR, and the CH1 is the first heavy chain constant region Domain, the CH2 is the second domain of the heavy chain constant region, and the CH3 is the third domain of the heavy chain constant region; and
  • the common light chain includes VL-CL from the N-terminus to the C-terminus, wherein the VL is the light chain variable region, the CL is the light chain constant region, and the polypeptide chain VH-PDL1-CH1 and the VH-EGFR-CH1 are respectively paired with the VL-CL of the common light chain, the VH-PDL1 and the VL form the PD-L1 antigen binding site, and the VH-EGFR and The VL forms an EGFR binding site;
  • each of the common light chains has an amino acid sequence as shown in SEQ ID NO:7.
  • the CH1 region of the heavy chain is selected from the CH1 domain of human IgG1 or the CH1 domain of human IgG4.
  • the heavy chain variable region VH-PDL1 that binds PD-L1 is shown in SEQ ID No:1;
  • the heavy chain variable region VH-EGFR that binds EGFR is shown in SEQ ID No: 9.
  • amino acid sequence of each of the polypeptide chains is the same.
  • the second aspect of the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR, which comprises two polypeptide chains and four common light chains, wherein the polypeptide chains have SEQ ID NO: 12 or The amino acid sequence shown in SEQ ID NO: 14, and the common light chain has the amino acid sequence shown in SEQ ID NO: 7.
  • the third aspect of the present invention provides an isolated nucleotide, which encodes the tetravalent bispecific antibody.
  • the nucleotide encodes the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is as shown in SEQ ID NO: 13 or SEQ ID NO: 15. As shown, the nucleotide sequence encoding the common light chain is shown in SEQ ID NO: 8.
  • the fourth aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotides.
  • the fifth aspect of the present invention provides a host cell, which contains the expression vector as described above.
  • the sixth aspect of the present invention provides a method for preparing the tetravalent bispecific antibody, the method comprising the following steps:
  • the seventh aspect of the present invention provides a pharmaceutical composition containing the tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also contains an anti-tumor agent.
  • 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 eighth aspect of the present invention provides the use of the above-mentioned tetravalent bispecific antibody, or its immunoconjugate, 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 Cancer, cervical cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
  • the ninth aspect of the present invention provides a method of treating cancer, comprising administering the above-mentioned tetravalent bispecific antibody, or its immunoconjugate, or the above-mentioned drug combination to a subject in need Things.
  • 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 Cancer, cervical cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
  • the tenth 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 tetravalent bispecific antibody against PD-L1 and EGFR.
  • the tetravalent bispecific antibody of the present invention does not require Fc modification, does not cause mismatch problems, has a simple preparation method, and has similar or even better biological activities and physical and chemical properties than monoclonal antibodies.
  • FIG. 1 is a schematic diagram of the structure of the bispecific antibody of the present invention, where VH-A represents the heavy chain variable region of Anti-PDL1 or Cetuximab, VH-B represents the heavy chain variable region of Cetuximab or Anti-PDL1, and VL represents The light chain variable region of the common light chain, CH1, CH2, and CH3 are the three domains of the heavy chain constant region, CL is the light chain constant region of the common light chain, and the line between the two heavy chains represents the disulfide bond.
  • VH-A represents the heavy chain variable region of Anti-PDL1 or Cetuximab
  • VH-B represents the heavy chain variable region of Cetuximab or Anti-PDL1
  • VL represents The light chain variable region of the common light chain
  • CH1, CH2, and CH3 are the three domains of the heavy chain constant region
  • CL is the light chain constant region of the common light chain
  • the line between the two heavy chains represents the disulfide bond.
  • the line between the heavy chain and the light chain also represents the disulfide bond
  • the line between CH1 and VH-A near the N-terminus of the polypeptide chain represents the artificially designed linker
  • the line between CH1 and CH2 near the C-terminus of the polypeptide chain Indicates the natural linker and hinge region of the antibody (if the heavy chain is of human IgG4 subtype, the hinge region will contain the S228P point mutation, according to the EU code).
  • Figure 2 shows the ELISA results of Cetuximab and Anti-PDL1 and their hybrid antibodies; among them, Figure 2A and Figure 2B show the ELISA plate coated with PD-L1-His and EGFR-ECD-hFc, respectively.
  • Figure 3 shows the ELISA results of PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1; among them, Figure 3A and Figure 3B show the ELISA plate coated with PD-L1-His and EGFR-ECD-hFc, respectively.
  • Figure 4 is the result of evaluating the functional activity of PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 in inhibiting the proliferation of A431 cells.
  • Figure 5 shows the HPLC-SEC profile of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1; wherein Figure 5A shows the HPLC-SEC profile of Anti-PDL1, and Figure 5B shows the HPLC-SEC profile of PDL1-Fab-Cetuximab-IgG1.
  • Figure 6 shows the NR-CE-SDS and R-CE-SDS maps of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1; among them, Figure 6A and Figure 6B show the NR-CE-SDS and R-CE of Anti-PDL1, respectively -SDS map, Figure 6C and Figure 6D show the NR-CE-SDS and R-CE-SDS maps of PDL1-Fab-Cetuximab-IgG1, respectively.
  • Figure 7 is the result of evaluating the ability of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 to enhance MLR; among them, Figure 7A shows the result of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 stimulated MLR to secrete IL-2, and Figure 7B shows Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 stimulate MLR to secrete IFN- ⁇ .
  • the bispecific antibody of the present invention has a "common light chain", which includes the light chain variable region of the anti-PD-L1 antibody and the human Kappa chain constant region.
  • the inventors unexpectedly discovered that the hybrid antibody formed by the "common light chain” and the heavy chain of the Cetuximab antibody can effectively bind to EGFR-ECD-hFc.
  • the bispecific antibody of the present invention can effectively inhibit the proliferation of human epidermal cancer cell A431, and has similar or even better biological activities and physical and chemical properties than monoclonal antibodies. The present invention has been completed on this basis.
  • the terms "Antibody (Ab)” and “Immunoglobulin G (Immunoglobulin G, IgG)” are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L ) And two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes (isotype) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3.
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain.
  • Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on.
  • the heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain.
  • the two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.
  • bispecific antibody 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.
  • humanized means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies.
  • framework residues can be changed to maintain binding affinity.
  • 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 antibodies interact with effector molecules or cells.
  • 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 an immunoglobulin that is inserted into an immunoglobulin domain to provide sufficient mobility for the light and heavy chain domains to fold into an immunoglobulin that exchanges dual variable regions. Or multiple amino acid residues.
  • the linker of the present invention connects the heavy chain variable region of Anti-PDL1 with the CH1 domain of human IgG4, and then connects the linker (preferably an artificial linker, the linker used here is three A series of GGGGS, SEQ ID NO: 11) is connected to the heavy chain variable region of Cetuximab.
  • linkers include monoglycine (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 a tetravalent bispecific antibody against PD-L1 and EGFR, including an anti-PD-L1 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.
  • 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.
  • the smaller the equilibrium dissociation constant the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • SPR Surface Plasmon Resonance
  • an BIACORE instrument an ELISA is used to measure the relative binding affinity of an antibody to the antigen.
  • the term "valency" refers to the presence of a specified number of antigen binding sites in an antibody molecule.
  • the bispecific antibody of the present invention has four antigen binding sites and is tetravalent.
  • the antigen binding site includes a heavy chain variable region (VH) and a light chain variable region (VL).
  • 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.
  • the term “common light chain” refers to a light chain comprising the same light chain variable region and light chain constant region, which can pair with the heavy chain of the first antibody that binds to the first antigen to form a specific binding first
  • the binding site of an antigen can also be paired with the heavy chain of the second antibody that binds to the second antigen to form a binding site that specifically binds to the second antigen.
  • the light chain variable region of the common light chain and the heavy chain variable region of the first antibody form the first antigen binding site
  • the light chain variable region of the common light chain and the heavy chain variable region of the second antibody form The second antigen binding site.
  • 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.
  • the form of DNA includes 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 tetravalent 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 present invention discloses The conformational integrity of the amino acid core sequence of the antibody that binds to human PD-L1 or its antigen-binding fragment or tetravalent bispecific antibody, while also protecting the protein's multifunctional groups from 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 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned tetravalent bispecific antibody of the present invention (or its conjugate) ) And pharmaceutically acceptable carriers or excipients.
  • 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.
  • 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 tetravalent bispecific antibody of the present invention can also be used with other therapeutic agents.
  • a safe and effective amount of the tetravalent bispecific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases it is not More than about 50 mg/kg body weight, preferably the dosage 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.
  • the protein expression and purification methods used in the following examples are described as follows: construct the target gene into the expression vector pcDNA4, and use PEI (Polyethylenimine) to transfer the constructed expression vector or combination of expression vectors into FreeStyle TM 293-F cells (later The abbreviation HEK293F, purchased from Thermo Fisher Scientific) was used to express antibodies or recombinant proteins.
  • HEK293F cells were cultured in Free Style 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days and then collected the cell supernatant, and then used ProteinA affinity chromatography or Purify antibodies or recombinant proteins by nickel affinity chromatography.
  • Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Due to changes such as post-translational modification, aggregation, and degradation, antibodies are heterogeneous in terms of biochemical and biophysical properties. When analyzing bispecific antibodies by separation techniques, variants, aggregates, and degraded fragments are usually observed, and their presence may compromise safety and effectiveness. In the process of antibody production and storage, aggregates, degraded fragments, and incompletely assembled molecules are prone to appear.
  • the present invention uses high-performance liquid chromatography-size exclusion chromatography (High-performance liquid chromatography-size exclusion chromatography, HPLC-SEC) to detect the content of the above-mentioned impurities in the sample.
  • the molecular weight of the aggregate is larger than the monomer, so the retention time of the corresponding peak is shorter; the molecular weight of the degraded fragment or the incompletely assembled molecule is smaller than the monomer, so the retention time of the corresponding peak is longer.
  • the chromatograph used for HPLC-SEC is Dionex Ultimate 3000; the mobile phase preparation method is as follows: take an appropriate amount of 20 mM sodium dihydrogen phosphate mother liquor, adjust the pH to 6.8 ⁇ 0.1 with 20 mM disodium hydrogen phosphate; injection volume: 20 ⁇ g; chromatographic column is TSK G3000SWXL , The specification is 7.8 ⁇ 300mm 5 ⁇ m; the flow rate is 0.5ml/min, the elution time is 30min; the column temperature is 25°C, the sample room temperature is 10°C; the detection wavelength is 214nm.
  • the present invention uses CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) to analyze the content of degraded fragments or incompletely assembled molecules in the sample.
  • CE is divided into two types: non-reducing and reducing.
  • the samples used for the former do not need to use the reducing agent DTT to destroy the disulfide bonds in the molecule during denaturation, while the samples used for the latter need to use the reducing agent DTT for denaturation.
  • the disulfide bond in the molecule is broken.
  • Non-reduced and reduced CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively.
  • the capillary electrophoresis instrument used was ProteomeLab TM PA800 plus (Beckman Coulter), equipped with UV 214nm detector, capillary model was Bare Fused-Silica Capillary, specification 30.7cm ⁇ 50 ⁇ m, effective length 20.5cm; other related reagents were purchased from Beckman Coulter.
  • the key parameters of the instrument are set as follows: the temperature of the capillary and the sample chamber is 20 ⁇ 2°C, and the separation voltage is 15kV.
  • Anti-PDL1 is a humanized monoclonal antibody against human PD-L1. Its heavy chain variable region and light chain variable region sequences (SEQ ID NO: 1 and 2) are derived from PCT/CN2020/090442. Connect the synthetic humanized heavy chain variable region with the human IgG1 heavy chain constant region (SEQ ID NO: 3) to obtain the full-length humanized heavy chain gene, named Anti-PDL1-HC (SEQ ID NO: 4 and 5); Connect the humanized light chain variable region with the human Kappa chain constant region (SEQ ID NO: 6) to obtain the full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NO: 7 and 8).
  • Cetuximab-VH and Cetuximab-VL are respectively connected to the human IgG1 heavy chain constant region (SEQ ID NO: 3) and human Kappa light chain constant region (SEQ ID NO: 6) to construct the heavy and light chains of the full-length Cetuximab antibody
  • the genes are named Cetuximab-HC and Cetuximab-LC, respectively.
  • Amino acid sequence of human IgG1 heavy chain constant region (SEQ ID NO: 3)
  • the amino acid sequence of the heavy chain of Anti-PDL1 (SEQ ID NO: 4)
  • the gene sequences of Cetuximab-HC and Cetuximab-LC were respectively constructed into pcDNA4 expression vector. Combine the expression vectors of Anti-PDL1-HC, Anti-PDL1-LC, Cetuximab-HC and Cetuximab-LC in the following manner: Anti-PDL1-HC+Anti-PDL1-LC, Cetuximab-HC+Cetuximab-LC, Anti-PDL1-HC+Cetuximab-LC and Cetuximab-HC+Anti-PDL1-LC, express and purified antibodies, the obtained antibodies are named Anti-PDL1, Cetuximab, Anti-PDL1-HC+Cetuximab-LC and Cetuximab-HC+ Anti-PDL1-LC.
  • the source of the gene encoding the extracellular region of PD-L1 is as described in WO2018/137576A1.
  • Using gene recombination technology connect the polyhistidine coding sequence to the end of the extracellular region coding gene of PD-L1, then clone the recombinant gene into pcDNA4 expression vector, express and purify the recombinant protein, and the resulting recombinant protein is named PD-L1 -His.
  • the extracellular region of human EGFR (sequence from NCBI, Accession: NP_005219) was connected to the end of the Fc segment coding sequence of human IgG1, and then the recombinant gene was cloned into the pcDNA4 expression vector to express and purify the recombinant protein.
  • the resulting recombinant protein was named EGFR-ECD-hFc.
  • the ELISA plate was coated with EGFR-ECD-hFc and PD-L1-His, and the coating concentration was 40ng/well and 10ng/well respectively.
  • PBST containing 1% bovine serum albumin KH 2 PO 4 0.2g, Na 2 HPO 4 ⁇ 12H 2 O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5ml, add pure water to 1L) ELISA plate.
  • Anti-PDL1 can effectively bind PD-L1-His with an EC50 of 0.0924nM; while Cetuximab, Anti-PDL1-HC+Cetuximab-LC and Cetuximab-HC+Anti-PDL1-LC cannot bind PD- L1-His.
  • both Cetuximab and Cetuximab-HC+Anti-PDL1-LC can effectively bind to EGFR-ECD-hFc, with EC50 of 0.2096nM and 0.2484nM, while Anti-PDL1 and Anti-PDL1-HC+Cetuximab-LC cannot Effectively bind EGFR-ECD-hFc.
  • Anti-PDL1-LC (SEQ ID NO: 7 and 8) is selected as the common light chain to construct a bispecific antibody.
  • the heavy chain variable region of Cetuximab is connected to the CH1 domain of human IgG4, and then an artificial linker (the linker used here is three series GGGGS, SEQ ID NO: 11) is connected to Anti-PDL1
  • the heavy chain variable region of human IgG1 is finally connected to the heavy chain constant region (CH1+CH2+CH3) of human IgG1.
  • This program is used to construct a long heavy chain gene containing two heavy chain variable regions and two CH1 domains Named Cetuximab-Fab-PDL1-IgG1 (SEQ ID NO: 14 and 15).
  • the above sequences were constructed into the pcDNA4 expression vector, the PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 expression vectors were combined with the Anti-PDL1-LC expression vector to express the purified antibodies, and the resulting antibodies were named as PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 (for brevity, only the name of the heavy chain is used as the name of the antibody).
  • amino acid sequence of PDL1-Fab-Cetuximab-IgG1 is shown below (SEQ ID NO: 12):
  • the ELISA detection method refers to that described in Example 1.2.
  • Anti-PDL1, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can effectively bind PD-L1-His, with EC50 of 0.2177nM, 0.2003nM and 0.3356nM, respectively.
  • Cetuximab-HC+Anti-PDL1-LC, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can effectively bind to EGFR-ECD-hFc, with EC50 of 0.2253nM, 0.2388nM and 0.1852nM.
  • A431( CRL-1555 TM ) is a human epidermal cancer cell line that overexpresses wild-type EGFR.
  • Anti-EGFR antibodies can inhibit the proliferation of A431 cells in vitro and in vivo.
  • This example evaluates the functional activity of the above antibodies in inhibiting the proliferation of A431 cells.
  • the method is as follows: Wash the A431 cells in the logarithmic growth phase twice with DMEM, centrifuge at 1000 rpm for 5 min; resuspend the cells to an appropriate density with DMEM containing 1% fetal bovine serum (fetal bovine serum and DMEM purchased from Gibco), and inoculate 96-well plate, 10 4 / 150 ⁇ l / hole; then 1% fetal bovine serum in DMEM in the above-described antibody was serially diluted; the diluted antibody was added to the 96 well plate inoculated A431 cells, 50 ⁇ l / Wells; incubate for 3 days at 37°C and 5% CO 2 in a cell incubator; add 20 ⁇ l of CCK-8 (purchased from Dojindo) solution to each well after 3 days and incubate for 4 hours in the incubator; read OD450 with a microplate reader ; GraphPad P
  • Cetuximab, Cetuximab-HC+Anti-PDL1-LC, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can effectively inhibit the proliferation of A431 cells, with IC50 of 0.6322nM and 0.5629nM, respectively , 0.7094nM and 0.9597nM.
  • Biacore 8K GE healthcare
  • a chip coupled with Protein A/G was used to capture various antibodies, and then the recombinant protein PD-L1-His (homemade) or EGFR-His (EGFR recombinant protein with His tag, purchased from Beijing Yiqiao Shenzhou) samples were injected, the binding-dissociation curve was obtained, and the next cycle was repeated after elution with 6M guanidine hydrochloride regeneration buffer; the data was analyzed by Biacore 8K Evaluation Software. The results are shown in Table 2.
  • Table 2-1 shows that the binding constant (Kon) and dissociation constant (Koff) of Anti-PDL1, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 to PD-L1 are very similar, and the equilibrium dissociation constant (KD) ) Is basically the same, KD is 9.66E-10, 6.46E-10 and 7.79E-10 respectively.
  • Table 2-2 shows that the binding constant (Kon) and dissociation constant (Koff) of Cetuximab, Cetuximab-HC+Anti-PDL1-LC and Cetuximab-Fab-PDL1-IgG1 to EGFR are very similar, and the equilibrium dissociation constant (KD) is also very similar.
  • KDs are 6.14E-10, 9.46E-10 and 9.57E-10 respectively; compared with the first three, the equilibrium dissociation constant (KD) of PDL1-Fab-Cetuximab-IgG1 for EGFR is slightly larger, which is 14.2 E-10.
  • the equilibrium dissociation constant (KD) is inversely proportional to the affinity.
  • Figure 5A shows the HPLC-SEC spectrum of Anti-PDL1, in which there are two obvious peaks, Peak1 and Peak2, which account for 0.2% and 99.8% (main peak) respectively.
  • Figure 5B shows the HPLC-SEC profile of PDL1-Fab-Cetuximab-IgG1, in which there are 3 obvious peaks, Peak1, Peak2 and Peak3, which account for 0.3%, 99.5% (main peak) and 0.2%, respectively.
  • Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 have similar main peaks.
  • Figure 6A and Figure 6B show the NR-CE-SDS and R-CE-SDS maps of Anti-PDL1, respectively
  • Figure 6C and Figure 6D show the NR-CE-SDS and R-CE-SDS of PDL1-Fab-Cetuximab-IgG1, respectively Atlas.
  • Anti-PDL1 NR-CE-SDS main peak Peak8 accounted for 98.11%
  • PDL1-Fab-Cetuximab-IgG1 NR-CE-SDS main peak Peak9 accounted for 97.14%.
  • the Mixed Lymphocyte Reaction (MLR) method used in this example is described as follows: Use Histopaque (purchased from Sigma) to isolate Peripheral Blood Mononuclear Cell (PBMC) from human blood, Then the monocytes in the PBMC were separated by the adherence method, and then IL-4 (25ng/ml) and GM-CSF (25ng/ml) were used to induce the monocytes to differentiate into dendritic cells. Seven days later, the above-induced dendritic cells were digested and collected. PBMCs were separated from the blood of other donors by the above method, and then CD4 + T cells were separated from the PBMCs with MACS magnet and CD4 MicroBeads (purchased from Miltenyi biotec).
  • MLR Mixed Lymphocyte Reaction
  • the induced dendritic cells (10 4 /well) and the isolated CD4 + T cells (10 5 /well) were mixed in proportion and then seeded into a 96-well plate, 150 ⁇ l per well; a few hours later, in the above 96 Add 50 ⁇ l of serially diluted antibody to the well plate; place the 96-well plate in a 37°C cell incubator and incubate for 3 days.
  • AIM-V medium purchased from Thermo Fisher Scientific
  • Double-antibody sandwich ELISA was used to detect the secretion of IL-2 and IFN- ⁇ (related paired antibodies were purchased from BD Biosciences). Read OD450 with a microplate reader (SpectraMax 190), graph with GraphPad Prism6 and calculate EC50.
  • the results of A and B are from the same MLR experiment.
  • both Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 can effectively stimulate MLR to secrete IL-2, and their EC50 is 0.1578nM and 0.409nM, respectively.
  • both Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 can effectively stimulate MLR to secrete IFN- ⁇ , and their EC50 is 0.1369nM and 0.08084nM, respectively.
  • the results show that Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 have equivalent functional activities.
  • the isotype control antibody is a human IgG1 antibody that has nothing to do with the target.

Abstract

The present invention provides a tetravalent bispecific antibody against PD-1 and EGFR. The tetravalent bispecific antibody of the present invention does not require Fc modification, does not cause mismatch issues, has a simple preparation method, and has similar or even better biological activities as well as physical and chemical properties compared with those of monoclonal antibodies.

Description

一种抗PD-L1和EGFR的四价双特异性抗体A tetravalent bispecific antibody against PD-L1 and EGFR 技术领域Technical field
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EGFR的四价双特异性抗体。The present invention relates to the field of antibodies. More specifically, the present invention discloses a tetravalent bispecific antibody against PD-L1 and EGFR.
背景技术Background technique
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***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***，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。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. A large number of studies have shown that the interaction of PD-1 and PD-L1 is not only necessary to maintain the balance of the immune system in the body, but also the main mechanism and reason that causes PD-L1 positive tumor cells to evade immune surveillance. By blocking the negative regulation of cancer cells on the PD-1/PD-L1 signaling pathway and activating the immune system, it can promote T cell-related tumor-specific cellular immune responses, thereby opening the door to a new tumor treatment method-- Tumor immunotherapy.
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 (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. 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***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、***、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***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。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-γ 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. USA 99: 12293-12297 (2002); Strome SE et al., Cancer Res., 63: 6501-6505) (2003)). Through immunohistochemical assessment of biopsies, the expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found in many primary human tumors. 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. .
表皮生长因子受体(Epidermal Growth Factor Receptor，EGFR)广泛分布于哺乳动物上皮细胞、成纤维细胞、胶质细胞、角质细胞等细胞表面，EGFR信号通路对细胞的生长、增殖和分化等生理过程发挥重要的作用。EGFR的突变或异常表达在肿瘤的生长、发展中扮演着重要作用。抗EGFR单克隆抗体药物具有阻滞肿瘤细胞周期进程、加速肿瘤细胞凋亡、抑制肿瘤血管生成、抑制肿瘤浸润和转移、增强放化疗效果等功能，作用机理清晰，因而在癌症治疗方面备受关注。以EGFR为靶点的抗肿瘤治疗已成为癌症研究中十分活跃的领域之一，并且取得了巨大的进展。但是以EGFR为靶点的抗肿瘤治疗依然还存在很多缺陷等着去完善。Epidermal Growth Factor Receptor (EGFR) is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, etc. The EGFR signaling pathway plays a role in the physiological processes of cell growth, proliferation, and differentiation. Important role. The mutation or abnormal expression of EGFR plays an important role in the growth and development of tumors. Anti-EGFR monoclonal antibody drugs have the functions of blocking tumor cell cycle progress, accelerating tumor cell apoptosis, inhibiting tumor angiogenesis, inhibiting tumor infiltration and metastasis, and enhancing the effect of radiotherapy and chemotherapy. The mechanism of action is clear, so it has attracted much attention in cancer treatment. . Anti-tumor therapy targeting EGFR has become one of the most active areas in cancer research, and tremendous progress has been made. However, anti-tumor therapy targeting EGFR still has many shortcomings waiting to be perfected.
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。虽然最近报道了大量新的双特异性抗体的构造形式，然而，生产双特异性抗体的主要技术难点在于获得正确配对的分子。目前现有的双特异性抗体的形式均存在错配的问题，因此会产生一种或多种错配导致的副产物或者聚集体，从而影响目的双特异性抗体的产率、纯度和理化稳定性，进而影响双特异性抗体在体内的安全性和有效性。Bispecific antibodies are gradually becoming a new class of therapeutic antibodies that can be used to treat various inflammatory diseases, cancers and other diseases. Although a large number of new bispecific antibody structures have been reported recently, the main technical difficulty in producing bispecific antibodies lies in obtaining the correct paired molecules. The current forms of bispecific antibodies all have mismatch problems, so one or more by-products or aggregates caused by mismatches will be produced, thereby affecting the yield, purity and physical and chemical stability of the target bispecific antibody This in turn affects the safety and effectiveness of bispecific antibodies in the body.
本领域迫切需要开发具有优良的用于治疗癌症等疾病的双特异性抗体。There is an urgent need in the art to develop bispecific antibodies that are excellent for the treatment of cancer and other diseases.
发明内容Summary of the invention
为了解决上述技术问题，本发明提供了一种抗PD-L1和EGFR的四价双特异性抗体及其应用。In order to solve the above technical problems, the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR and its application.
因此，本发明的第一个目的在于提供一种抗PD-L1和EGFR的四价双特异性抗体。Therefore, the first objective of the present invention is to provide a tetravalent bispecific antibody against PD-L1 and EGFR.
本发明的第二个目的在于提供一种编码所述的四价双特异性抗体的分离的核苷酸。The second object of the present invention is to provide an isolated nucleotide encoding the tetravalent bispecific antibody.
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。The third object of the present invention is to provide an expression vector containing the nucleotide.
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。The fourth object of the present invention is to provide a host cell containing the expression vector.
本发明的第五个目的在于提供所述的四价双特异性抗体的制备方法。The fifth objective of the present invention is to provide a method for preparing the tetravalent bispecific antibody.
本发明的第六个目的在于提供包含所述的四价双特异性抗体的药物组合物。The sixth object of the present invention is to provide a pharmaceutical composition containing the tetravalent bispecific antibody.
本发明的第七个目的在于提供所述的四价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。The seventh object of the present invention is to provide the use of the tetravalent bispecific antibody or the pharmaceutical composition in the preparation of drugs for the treatment of cancer.
本发明的第八个目的在于提供所述的四价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：The eighth object of the present invention is to provide a method for the treatment of cancer by the tetravalent bispecific antibody or the pharmaceutical composition. In order to achieve the above objective, the present invention provides the following technical solutions:
本发明的第一个方面提供了一种抗PD-L1和EGFR的四价双特异性抗体，包含：The first aspect of the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR, comprising:
(a)两条多肽链，所述多肽链从N末端到C末端包含VH-PDL1-CH1-接头-VH-EGFR-CH1-CH2-CH3或VH-EGFR-CH1-接头-VH-PDL1-CH1-CH2-CH3，其中，所述VH-PDL1为结合PD-L1的重链可变区，所述VH-EGFR为结合EGFR的重链可变区，所述CH1为重链恒定区的第一结构域，所述CH2为重链恒定区的第二结构域，所述CH3为重链恒定区的第三结构域；和(a) Two polypeptide chains, the polypeptide chain includes VH-PDL1-CH1-linker-VH-EGFR-CH1-CH2-CH3 or VH-EGFR-CH1-linker-VH-PDL1-CH1 from N-terminus to C-terminus -CH2-CH3, wherein the VH-PDL1 is a heavy chain variable region that binds PD-L1, the VH-EGFR is a heavy chain variable region that binds EGFR, and the CH1 is the first heavy chain constant region Domain, the CH2 is the second domain of the heavy chain constant region, and the CH3 is the third domain of the heavy chain constant region; and
(b)四条共同轻链，所述共同轻链从N末端到C末端包含VL-CL，其中，所述VL为轻链可变区，所述CL为轻链恒定区，所述多肽链的VH-PDL1-CH1和所述VH-EGFR-CH1分别与所述共同轻链的VL-CL配对，所述VH-PDL1和所述VL形成PD-L1抗原结合位点，所述VH-EGFR与所述VL形成EGFR结合位点；(b) Four common light chains, the common light chain includes VL-CL from the N-terminus to the C-terminus, wherein the VL is the light chain variable region, the CL is the light chain constant region, and the polypeptide chain VH-PDL1-CH1 and the VH-EGFR-CH1 are respectively paired with the VL-CL of the common light chain, the VH-PDL1 and the VL form the PD-L1 antigen binding site, and the VH-EGFR and The VL forms an EGFR binding site;
其中，所述的每个共同轻链具有如SEQ ID NO：7所示的氨基酸序列。Wherein, each of the common light chains has an amino acid sequence as shown in SEQ ID NO:7.
在另一优选例中，所述的重链的CH1区选自人IgG1的CH1结构域或人IgG4的CH1结构域。In another preferred example, the CH1 region of the heavy chain is selected from the CH1 domain of human IgG1 or the CH1 domain of human IgG4.
在另一优选例中，所述的结合PD-L1的重链可变区VH-PDL1如SEQ ID No:1所示；In another preferred embodiment, the heavy chain variable region VH-PDL1 that binds PD-L1 is shown in SEQ ID No:1;
所述的结合EGFR的重链可变区VH-EGFR如SEQ ID No:9所示。The heavy chain variable region VH-EGFR that binds EGFR is shown in SEQ ID No: 9.
在另一优选例中，每个所述的多肽链的氨基酸序列相同。In another preferred embodiment, the amino acid sequence of each of the polypeptide chains is the same.
本发明的第二个方面提供了一种抗PD-L1和EGFR的四价双特异性抗体，包含两条多肽链和四条共同轻链，其中，所述多肽链具有如SEQ ID NO：12或SEQ ID NO：14所示的氨基酸序列，所述共同轻链具有如SEQ ID NO：7所示的氨基酸序列。The second aspect of the present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR, which comprises two polypeptide chains and four common light chains, wherein the polypeptide chains have SEQ ID NO: 12 or The amino acid sequence shown in SEQ ID NO: 14, and the common light chain has the amino acid sequence shown in SEQ ID NO: 7.
本发明的第三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四价双特异性抗体。The third aspect of the present invention provides an isolated nucleotide, which encodes the tetravalent bispecific antibody.
在另一优选例中，所述的核苷酸编码所述多肽链和所述共同轻链，其中，编码所述多肽链的核苷酸序列如SEQ ID NO：13或SEQ ID NO：15所示，编码所述共同轻链的核苷酸序列如SEQ ID NO：8所示。In another preferred embodiment, the nucleotide encodes the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is as shown in SEQ ID NO: 13 or SEQ ID NO: 15. As shown, the nucleotide sequence encoding the common light chain is shown in SEQ ID NO: 8.
本发明的第四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。The fourth aspect of the present invention provides an expression vector, which contains the above-mentioned nucleotides.
本发明的第五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。The fifth aspect of the present invention provides a host cell, which contains the expression vector as described above.
本发明的第六个方面提供了所述的四价双特异性抗体的制备方法，所述方法包含以下步骤：The sixth aspect of the present invention provides a method for preparing the tetravalent bispecific antibody, the method comprising the following steps:
(a)在表达条件下，培养如上所述的宿主细胞，从而表达所述的四价双特异性抗体；(a) Culturing the host cell as described above under expression conditions to express the tetravalent bispecific antibody;
(b)分离并纯化(a)所述的四价双特异性抗体。(b) Isolation and purification of the tetravalent bispecific antibody described in (a).
本发明的第七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四价双特异性抗体和药学上可接受的载体。在另一优选例中，所述药物组合物中还含有抗肿瘤剂。The seventh aspect of the present invention provides a pharmaceutical composition containing the tetravalent bispecific antibody as described above and a pharmaceutically acceptable carrier. In another preferred embodiment, the pharmaceutical composition also contains an anti-tumor agent.
在另一优选例中，所述药物组合物中还含有抗肿瘤剂。In another preferred embodiment, the pharmaceutical composition also contains an anti-tumor agent.
在另一优选例中，所述药物组合物为单元剂型。In another preferred embodiment, the pharmaceutical composition is in a unit dosage form.
在另一优选例中，所述的抗肿瘤剂可以与所述双特异性抗体单独存在于独立的包装内，或所述抗肿瘤剂可以与所述双特异性抗体偶联。In another preferred embodiment, 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.
在另一优选例中，所述药物组合物的剂型包括胃肠给药剂型或胃肠外给药剂型。In another preferred embodiment, the dosage form of the pharmaceutical composition includes a dosage form for gastrointestinal administration or a dosage form for parenteral administration.
在另一优选例中，所述的胃肠外给药剂型包括静脉注射、静脉滴注、皮下注射、局部注射、肌肉注射、瘤内注射、腹腔内注射、颅内注射、或腔内注射。In another preferred embodiment, 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.
本发明的第八个方面提供了如上所述的四价双特异性抗体、或其免疫偶联物、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。The eighth aspect of the present invention provides the use of the above-mentioned tetravalent bispecific antibody, or its immunoconjugate, or the above-mentioned pharmaceutical composition in the preparation of a medicament for the treatment of cancer.
在另一优选例中，所述癌症选自由以下组成的组：黑素瘤、肾癌、***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、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病等。In another preferred embodiment, 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 Cancer, cervical cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
本发明的第九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四价双特异性抗体、或其免疫偶联物、或如上所述的药物组合物。The ninth aspect of the present invention provides a method of treating cancer, comprising administering the above-mentioned tetravalent bispecific antibody, or its immunoconjugate, or the above-mentioned drug combination to a subject in need Things.
在另一优选例中，所述癌症选自由以下组成的组：黑素瘤、肾癌、***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、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病等。In another preferred embodiment, 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 Cancer, cervical cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
本发明的第十个方面提供了一种免疫偶联物，所述免疫偶联物包括：The tenth aspect of the present invention provides an immunoconjugate, the immunoconjugate comprising:
(a)如本发明中第一方面所述的四价双特异性抗体；和(a) The tetravalent bispecific antibody as described in the first aspect of the present invention; and
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。(b) A coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
在另一优选例中，所述偶联物部分选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)。In another preferred embodiment, 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.
在另一优选例中，所述的免疫偶联物包括抗体－药物偶联物(ADC)。In another preferred embodiment, the immunoconjugate includes an antibody-drug conjugate (ADC).
在另一优选例中，所述的免疫偶联物用于制备***的药物组合物。In another preferred embodiment, the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.
有益效果：本发明提供了一种抗PD-L1和EGFR的四价双特异性抗体。本发明的四价双特异性抗体不需要进行Fc修饰，不会产生错配问题，制备方法简便，具有与单抗相似甚至更优的生物学活性和理化性质。Beneficial effects: The present invention provides a tetravalent bispecific antibody against PD-L1 and EGFR. The tetravalent bispecific antibody of the present invention does not require Fc modification, does not cause mismatch problems, has a simple preparation method, and has similar or even better biological activities and physical and chemical properties than monoclonal antibodies.
附图说明Description of the drawings
图1为本发明的双特异性抗体的结构示意图，其中，VH-A表示Anti-PDL1 或Cetuximab的重链可变区，VH-B表示Cetuximab或Anti-PDL1的重链可变区，VL表示共同轻链的轻链可变区，CH1、CH2和CH3是重链恒定区的三个结构域，CL是共同轻链的轻链恒定区，两条重链之间的线段表示二硫键，重链和轻链之间的线段也表示二硫键，靠近多肽链N末端的CH1和VH-A之间的线段表示人工设计的连接子，靠近多肽链C末端的CH1和CH2之间的线段表示抗体天然的连接子和铰链区(如果重链是人IgG4亚型，铰链区会含有S228P点突变，根据EU编码)。Figure 1 is a schematic diagram of the structure of the bispecific antibody of the present invention, where VH-A represents the heavy chain variable region of Anti-PDL1 or Cetuximab, VH-B represents the heavy chain variable region of Cetuximab or Anti-PDL1, and VL represents The light chain variable region of the common light chain, CH1, CH2, and CH3 are the three domains of the heavy chain constant region, CL is the light chain constant region of the common light chain, and the line between the two heavy chains represents the disulfide bond. The line between the heavy chain and the light chain also represents the disulfide bond, the line between CH1 and VH-A near the N-terminus of the polypeptide chain represents the artificially designed linker, and the line between CH1 and CH2 near the C-terminus of the polypeptide chain Indicates the natural linker and hinge region of the antibody (if the heavy chain is of human IgG4 subtype, the hinge region will contain the S228P point mutation, according to the EU code).
图2为Cetuximab和Anti-PDL1及其杂合抗体的ELISA结果；其中，图2A和图2B为分别用PD-L1-His和EGFR-ECD-hFc包被酶标板。Figure 2 shows the ELISA results of Cetuximab and Anti-PDL1 and their hybrid antibodies; among them, Figure 2A and Figure 2B show the ELISA plate coated with PD-L1-His and EGFR-ECD-hFc, respectively.
图3为PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1的ELISA结果；其中，图3A和图3B为分别用PD-L1-His和EGFR-ECD-hFc包被酶标板。Figure 3 shows the ELISA results of PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1; among them, Figure 3A and Figure 3B show the ELISA plate coated with PD-L1-His and EGFR-ECD-hFc, respectively.
图4为评估PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1抑制A431细胞增殖的功能活性的结果。Figure 4 is the result of evaluating the functional activity of PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 in inhibiting the proliferation of A431 cells.
图5为Anti-PDL1和PDL1-Fab-Cetuximab-IgG1的HPLC-SEC图谱；其中，图5A表示Anti-PDL1的HPLC-SEC图谱，图5B表示PDL1-Fab-Cetuximab-IgG1的HPLC-SEC图谱。Figure 5 shows the HPLC-SEC profile of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1; wherein Figure 5A shows the HPLC-SEC profile of Anti-PDL1, and Figure 5B shows the HPLC-SEC profile of PDL1-Fab-Cetuximab-IgG1.
图6为Anti-PDL1和PDL1-Fab-Cetuximab-IgG1的NR-CE-SDS和R-CE-SDS图谱；其中，图6A和图6B分别表示Anti-PDL1的NR-CE-SDS和R-CE-SDS图谱，图6C和图6D分别表示PDL1-Fab-Cetuximab-IgG1的NR-CE-SDS和R-CE-SDS图谱。Figure 6 shows the NR-CE-SDS and R-CE-SDS maps of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1; among them, Figure 6A and Figure 6B show the NR-CE-SDS and R-CE of Anti-PDL1, respectively -SDS map, Figure 6C and Figure 6D show the NR-CE-SDS and R-CE-SDS maps of PDL1-Fab-Cetuximab-IgG1, respectively.
图7为评估Anti-PDL1和PDL1-Fab-Cetuximab-IgG1增强MLR的能力的结果；其中，图7A表示Anti-PDL1和PDL1-Fab-Cetuximab-IgG1刺激MLR分泌IL-2的结果，图7B表示Anti-PDL1和PDL1-Fab-Cetuximab-IgG1刺激MLR分泌IFN-γ的结果。Figure 7 is the result of evaluating the ability of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 to enhance MLR; among them, Figure 7A shows the result of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 stimulated MLR to secrete IL-2, and Figure 7B shows Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 stimulate MLR to secrete IFN-γ.
具体实施方式detailed description
本发明人通过广泛而深入的研究，经过大量的筛选，成功获得一种抗PD-L1和EGFR的四价双特异性抗体。具体地，本发明的双特异性抗体具有“共同轻链”，其包含抗PD-L1抗体的轻链可变区和人Kappa链恒定区。本发明人意外发现，“共 同轻链”与Cetuximab抗体重链形成的杂合抗体居然能够有效结合EGFR-ECD-hFc。此外，本发明的双特异性抗体能够有效抑制人类表皮癌细胞A431的增殖，并具有与单抗相似甚至更优的生物学活性和理化性质。在此基础上完成了本发明。Through extensive and in-depth research and extensive screening, the inventors successfully obtained a tetravalent bispecific antibody against PD-L1 and EGFR. Specifically, the bispecific antibody of the present invention has a "common light chain", which includes the light chain variable region of the anti-PD-L1 antibody and the human Kappa chain constant region. The inventors unexpectedly discovered that the hybrid antibody formed by the "common light chain" and the heavy chain of the Cetuximab antibody can effectively bind to EGFR-ECD-hFc. In addition, the bispecific antibody of the present invention can effectively inhibit the proliferation of human epidermal cancer cell A431, and has similar or even better biological activities and physical and chemical properties than monoclonal antibodies. The present invention has been completed on this basis.
本发明中涉及的序列信息总结在表1中。The sequence information involved in the present invention is summarized in Table 1.
表1、本发明的抗体的序列信息Table 1. Sequence information of the antibody of the present invention
SEQ ID NO：SEQ ID NO: 序列名称 Sequence name
11 Anti-PDL1的重链可变区的氨基酸序列Amino acid sequence of the heavy chain variable region of Anti-PDL1
22 Anti-PDL1的轻链可变区的氨基酸序列Amino acid sequence of the light chain variable region of Anti-PDL1
33 人IgG1重链恒定区的氨基酸序列Amino acid sequence of human IgG1 heavy chain constant region
44 Anti-PDL1的重链的氨基酸序列Amino acid sequence of the heavy chain of Anti-PDL1
55 Anti-PDL1的重链的核苷酸序列Nucleotide sequence of the heavy chain of Anti-PDL1
66 人Kappa轻链恒定区的氨基酸序列Amino acid sequence of human Kappa light chain constant region
77 Anti-PDL1的轻链的氨基酸序列Amino acid sequence of the light chain of Anti-PDL1
88 Anti-PDL1的轻链的核苷酸序列The nucleotide sequence of the light chain of Anti-PDL1
99 Cetuximab的重链可变区的氨基酸序列Amino acid sequence of the heavy chain variable region of Cetuximab
1010 Cetuximab的轻链可变区的氨基酸序列Amino acid sequence of the light chain variable region of Cetuximab
1111 连接子(GGGGSGGGGSGGGGS)Linker (GGGGSGGGGSGGGGS)
1212 PDL1-Fab-Cetuximab-IgG1的氨基酸序列Amino acid sequence of PDL1-Fab-Cetuximab-IgG1
1313 PDL1-Fab-Cetuximab-IgG1的核苷酸序列The nucleotide sequence of PDL1-Fab-Cetuximab-IgG1
1414 Cetuximab-Fab-PDL1-IgG1的氨基酸序列Amino acid sequence of Cetuximab-Fab-PDL1-IgG1
1515 Cetuximab-Fab-PDL1-IgG1的核苷酸序列The nucleotide sequence of Cetuximab-Fab-PDL1-IgG1
术语the term
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。 每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。In the present invention, the terms "Antibody (Ab)" and "Immunoglobulin G (Immunoglobulin G, IgG)" are heterotetrameric glycoproteins with the same structural characteristics, which are composed of two identical light chains (L ) And two identical heavy chains (H). Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes (isotype) is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by a constant region. The heavy chain constant region is composed of three structural domains, CH1, CH2, and CH3. Each light chain has a variable region (VL) at one end and a constant region at the other end. The light chain constant region includes a structural domain CL; the light chain constant region is paired with the CH1 domain of the heavy chain constant region, and the light chain can be The variable region is paired with the variable region of the heavy chain. Constant regions are not directly involved in the binding of antibodies and antigens, but they exhibit different effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity) and so on. The heavy chain constant region includes IgG1, IgG2, IgG3, and IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda). The heavy and light chains of the antibody are covalently linked together by the disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain. The two heavy chains of the antibody are covalently linked together by the inter-polypeptide disulfide formed between the hinge regions. The bonds are linked together covalently.
本发明中，术语“双特异性抗体(或双抗)”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。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。In the present invention, the term "bispecific antibody (or dual 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.
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。In the present invention, 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.
本发明中，术语“人源化”是指其CDR来源于非人物种(优选小鼠)抗体，抗体分子中残余的部分(包括框架区和恒定区)来源于人抗体。此外，框架区残基可被改变以维持结合亲和性。In the present invention, the term "humanized" means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies. In addition, framework residues can be changed to maintain binding affinity.
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。In the present invention, 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 antibodies interact with effector molecules or cells.
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))。In the present invention, the term "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. However, the 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. The more conservative part of the variable region is called the frame region (FR). The variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a β-sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-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)).
如本文所用，术语“框架区”(FR)指***CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。As used herein, the term "framework region" (FR) refers to the amino acid sequence inserted between CDRs, that is, those parts of the light chain and heavy chain variable regions of immunoglobulins that are relatively conserved among different immunoglobulins in a single species . 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. Accordingly, 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). Preferably, 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%.
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。Knowing the amino acid sequence of the CDR, those skilled in the art can easily determine the framework regions FR1-L, FR2-L, FR3-L, FR4-L and/or FR1-H, FR2-H, FR3-H, FR4-H.
如本文所用，术语“人框架区”是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。As used herein, the term "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. .
如本文所用，术语“接头”或“连接子”是指***免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。As used herein, the term "linker" or "linker" refers to an immunoglobulin that is inserted into an immunoglobulin domain to provide sufficient mobility for the light and heavy chain domains to fold into an immunoglobulin that exchanges dual variable regions. Or multiple amino acid residues.
在一具体实施例中，本发明的连接子将Anti-PDL1的重链可变区与人IgG4的CH1结构域相连，然后再通过连接子(优选人工连接子，在此使用的连接子是三个串联的GGGGS，SEQ ID NO：11)连接Cetuximab的重链可变区。In a specific embodiment, the linker of the present invention connects the heavy chain variable region of Anti-PDL1 with the CH1 domain of human IgG4, and then connects the linker (preferably an artificial linker, the linker used here is three A series of GGGGS, SEQ ID NO: 11) is connected to the heavy chain variable region of Cetuximab.
合适的连接子实例包括单甘氨酸(Gly)、或丝氨酸(Ser)残基，连接子中氨基酸残基的标识和序列可随着连接子中需要实现的次级结构要素的类型而变化。Examples of suitable linkers include monoglycine (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.
双特异性抗体Bispecific antibody
本发明的双特异性抗体是一种抗PD-L1和EGFR的四价双特异性抗体，包括抗PD-L1抗体部分以及抗EGFR抗体部分。The bispecific antibody of the present invention is a tetravalent bispecific antibody against PD-L1 and EGFR, including an anti-PD-L1 antibody part and an anti-EGFR antibody part.
优选地，本发明抗PD-L1抗体的序列如专利申请PCT/CN2020/090442中所述，本领域技术人员也可以通过本领域熟知的技术对本发明抗PD-L1抗体进行修饰或改造，例如添加、缺失和/或取代一个或几个氨基酸残基，从而进一步增加抗PD-L1的亲和力或结构稳定性，并通过常规的测定方法获得修饰或改造后的结果。Preferably, 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.
本发明中，术语“抗”、“结合”和“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。 In the present invention, 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. Generally, 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. In the present invention, the term "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. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. For example, Surface Plasmon Resonance (SPR) is used to measure the binding affinity of an antibody to an antigen in a BIACORE instrument or an ELISA is used to measure the relative binding affinity of an antibody to the antigen.
本发明中，术语“价”是指抗体分子中存在指定数量的抗原结合位点。优选的，本发明的双特异性抗体具有四个抗原结合位点，是四价的。本发明中，抗原结合位点包含重链可变区(VH)和轻链可变区(VL)。In the present invention, the term "valency" refers to the presence of a specified number of antigen binding sites in an antibody molecule. Preferably, the bispecific antibody of the present invention has four antigen binding sites and is tetravalent. In the present invention, the antigen binding site includes a heavy chain variable region (VH) and a light chain variable region (VL).
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“共同轻链”是指包含相同的轻链可变区和轻链恒定区的轻链，其能够与结合第一抗原的第一抗体的重链配对，形成特异性结合第一抗原的结合位点，也能够与结合第二抗原的第二抗体的重链配对，形成特异性结合第二抗原的结合位点。进一步的，共同轻链的轻链可变区与第一抗体的重链可变区形成第一抗原结合位点，共同轻链的轻链可变区与第二抗体的重链可变区形成第二抗原结合位点。In the present invention, the term "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. In the present invention, the term "common light chain" refers to a light chain comprising the same light chain variable region and light chain constant region, which can pair with the heavy chain of the first antibody that binds to the first antigen to form a specific binding first The binding site of an antigen can also be paired with the heavy chain of the second antibody that binds to the second antigen to form a binding site that specifically binds to the second antigen. Further, the light chain variable region of the common light chain and the heavy chain variable region of the first antibody form the first antigen binding site, and the light chain variable region of the common light chain and the heavy chain variable region of the second antibody form The second antigen binding site.
本发明的双特异性抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、或任何以上这些物质的组合结合或偶联。The 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.
编码核酸和表达载体Coding nucleic acid and expression vector
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。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. The form of DNA includes 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.
本发明中，术语“表达载体”指携带表达盒用于表达特定目的蛋白或其他物质的载体，如质粒、病毒载体(如腺病毒、逆转录病毒)、噬菌体、酵母质粒或其他载体。代表性的例子包括但并不限于：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***的载体等。表达载体中包括连接于合适的转录和翻译调节序列的融合DNA序列。In the present invention, 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. 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.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Once the relevant sequence is obtained, 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.
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。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.
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***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。In the present invention, the term "host cell" refers to a cell suitable for expressing the above-mentioned expression vector. It can be a eukaryotic cell. For example, mammalian or insect host cell culture systems can be used for the expression of the fusion protein of the present invention. CHO (Chinese hamster Ovary, Chinese Hamster Ovary), HEK293, COS, BHK and derived cells of the above-mentioned cells are all suitable for the present invention.
药物组合物和应用Pharmaceutical composition and application
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：静脉注射、静脉滴注、皮下注射、局部注 射、肌肉注射、瘤内注射、腹腔内注射(如腹膜内)、颅内注射、或腔内注射。The invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, 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.
本发明中，术语“药物组合物”是指本发明的四价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结合人PD-L1的抗体或其抗原结合片段或四价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)。In the present invention, the term "pharmaceutical composition" means that the tetravalent 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 present invention discloses The conformational integrity of the amino acid core sequence of the antibody that binds to human PD-L1 or its antigen-binding fragment or tetravalent bispecific antibody, while also protecting the protein's multifunctional groups from degradation (including but not limited to aggregation, deamination or oxidation) .
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的四价双特异性抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的四价双特异性抗体还可与其他治疗剂一起使用。The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned tetravalent bispecific antibody of the present invention (or its conjugate) ) And pharmaceutically acceptable carriers or excipients. 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. In addition, the tetravalent bispecific antibody of the present invention can also be used with other therapeutic agents.
使用药物组合物时，是将安全有效量的四价双特异性抗体或其免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When the pharmaceutical composition is used, a safe and effective amount of the tetravalent bispecific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases it is not More than about 50 mg/kg body weight, preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight. Of course, 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.
以下实施例中使用的蛋白表达和纯化方法说明如下：将目的基因构建到表达载体pcDNA4中，利用PEI(Polyethylenimine)将构建好的表达载体或表达载体的组合转入FreeStyle TM 293-F细胞(后文简称HEK293F，购自Thermo Fisher Scientific)中以表达抗体或重组蛋白，HEK293F细胞在Free Style 293 Expression Medium(购自Thermo Fisher Scientific)中培养5天后收取细胞上清，然后用ProteinA亲和层析或镍亲和层析纯化抗体或重组蛋白。 The protein expression and purification methods used in the following examples are described as follows: construct the target gene into the expression vector pcDNA4, and use PEI (Polyethylenimine) to transfer the constructed expression vector or combination of expression vectors into FreeStyle TM 293-F cells (later The abbreviation HEK293F, purchased from Thermo Fisher Scientific) was used to express antibodies or recombinant proteins. HEK293F cells were cultured in Free Style 293 Expression Medium (purchased from Thermo Fisher Scientific) for 5 days and then collected the cell supernatant, and then used ProteinA affinity chromatography or Purify antibodies or recombinant proteins by nickel affinity chromatography.
以下实施例中使用的理化性质检测方法说明如下：The physical and chemical property detection methods used in the following examples are described as follows:
HPLC-SECHPLC-SEC
抗体是高分子量蛋白质，具有高度复杂的二级和三级结构。由于翻译后修饰、 聚集和降解等变化，抗体在生物化学和生物物理特性方面是异质的。当通过分离技术分析双特异性抗体时，通常会观察到变体、聚集体和降解片段，它们的存在可能会损害安全性和有效性。在生产和存储抗体的过程中容易出现聚集体、降解片段和不完整组装的分子。本发明使用高效液相色谱-尺寸排阻色谱(High-performance liquid chromatography–size exclusion chromatography，HPLC-SEC)检测样品中上述杂质的含量。聚集体的分子量要大于单体，因此相应峰的保留时间较短；降解片段或不完整组装分子的分子量要小于单体，因此相应峰的保留时间较长。HPLC-SEC所用色谱仪为Dionex Ultimate 3000；流动相配制方法如下：取适量20mM磷酸二氢钠母液，用20mM磷酸氢二钠调节PH至6.8±0.1；进样量：20μg；色谱柱为TSK G3000SWXL，规格为7.8×300mm 5μm；流速0.5ml/min，洗脱时间30min；柱温25℃，样品室温度10℃；检测波长214nm。Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Due to changes such as post-translational modification, aggregation, and degradation, antibodies are heterogeneous in terms of biochemical and biophysical properties. When analyzing bispecific antibodies by separation techniques, variants, aggregates, and degraded fragments are usually observed, and their presence may compromise safety and effectiveness. In the process of antibody production and storage, aggregates, degraded fragments, and incompletely assembled molecules are prone to appear. The present invention uses high-performance liquid chromatography-size exclusion chromatography (High-performance liquid chromatography-size exclusion chromatography, HPLC-SEC) to detect the content of the above-mentioned impurities in the sample. The molecular weight of the aggregate is larger than the monomer, so the retention time of the corresponding peak is shorter; the molecular weight of the degraded fragment or the incompletely assembled molecule is smaller than the monomer, so the retention time of the corresponding peak is longer. The chromatograph used for HPLC-SEC is Dionex Ultimate 3000; the mobile phase preparation method is as follows: take an appropriate amount of 20 mM sodium dihydrogen phosphate mother liquor, adjust the pH to 6.8 ± 0.1 with 20 mM disodium hydrogen phosphate; injection volume: 20 μg; chromatographic column is TSK G3000SWXL , The specification is 7.8×300mm 5μm; the flow rate is 0.5ml/min, the elution time is 30min; the column temperature is 25℃, the sample room temperature is 10℃; the detection wavelength is 214nm.
CE-SDSCE-SDS
本发明使用CE-SDS(Capillary Electrophoresis-Sodium Dodecyl Sulfate)分析样品中降解片段或不完整组装的分子的含量。CE分为非还原和还原两种类型，用于前者的样品在变性时不需要用还原剂DTT将分子内的二硫键破坏，而用于后者的样品在变性时需要用还原剂DTT将分子内的二硫键破坏。非还原和还原CE-SDS分别记作NR-CE-SDS和R-CE-SDS。所用毛细管电泳仪为ProteomeLab TM PA800 plus(Beckman Coulter)，配备UV 214nm检测器，毛细管型号为Bare Fused-Silica Capillary，规格30.7cm×50μm，有效长度20.5cm；其它相关试剂购自Beckman Coulter。仪器关键参数设置如下：毛细管和样品室温度为20±2℃，分离电压为15kV。 The present invention uses CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) to analyze the content of degraded fragments or incompletely assembled molecules in the sample. CE is divided into two types: non-reducing and reducing. The samples used for the former do not need to use the reducing agent DTT to destroy the disulfide bonds in the molecule during denaturation, while the samples used for the latter need to use the reducing agent DTT for denaturation. The disulfide bond in the molecule is broken. Non-reduced and reduced CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively. The capillary electrophoresis instrument used was ProteomeLab TM PA800 plus (Beckman Coulter), equipped with UV 214nm detector, capillary model was Bare Fused-Silica Capillary, specification 30.7cm×50μm, effective length 20.5cm; other related reagents were purchased from Beckman Coulter. The key parameters of the instrument are set as follows: the temperature of the capillary and the sample chamber is 20±2℃, and the separation voltage is 15kV.
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***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。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The following examples and experimental examples are to further illustrate the present invention, and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.
实施例1 抗PD-L1和EGFR的双特异性抗体的构建Example 1 Construction of bispecific antibodies against PD-L1 and EGFR
实施例1.1序列Example 1.1 Sequence
Anti-PDL1是抗人PD-L1的人源化单抗，其重链可变区和轻链可变区序列(SEQ ID NO：1和2)来源于PCT/CN2020/090442。将合成的人源化重链可变区与人IgG1重链恒定区(SEQ ID NO：3)相连，获得全长的人源化重链基因，命名为Anti-PDL1-HC(SEQ ID NO：4和5)；将人源化轻链可变区与人Kappa链恒定区(SEQ ID NO：6)相连，获得全长的人源化轻链基因，命名为Anti-PDL1-LC(SEQ ID NO：7和8)。Anti-PDL1 is a humanized monoclonal antibody against human PD-L1. Its heavy chain variable region and light chain variable region sequences (SEQ ID NO: 1 and 2) are derived from PCT/CN2020/090442. Connect the synthetic humanized heavy chain variable region with the human IgG1 heavy chain constant region (SEQ ID NO: 3) to obtain the full-length humanized heavy chain gene, named Anti-PDL1-HC (SEQ ID NO: 4 and 5); Connect the humanized light chain variable region with the human Kappa chain constant region (SEQ ID NO: 6) to obtain the full-length humanized light chain gene, named Anti-PDL1-LC (SEQ ID NO: 7 and 8).
从公开的文献资料(Magdelaine-Beuzelin C,Kaas Q,Wehbi V,et al.Structure–function relationships of the variable domains of monoclonal antibodies approved for cancer treatment[J].Critical reviews in oncology/hematology,2007,64(3):210-225.)中获得Cetuximab抗体的重链可变区和轻链可变区序列(SEQ ID NO：9和10)。由上海生工生物工程有限公司合成编码其重链可变区(Cetuximab-VH)和轻链可变区(Cetuximab-VL)的DNA。Cetuximab-VH和Cetuximab-VL分别与人IgG1重链恒定区(SEQ ID NO：3)和人Kappa轻链恒定区(SEQ ID NO：6)相连，构建成全长的Cetuximab抗体的重链和轻链基因，分别命名为Cetuximab-HC和Cetuximab-LC。From the published literature (Magdelaine-Beuzelin C, Kaas Q, Wehbi V, et al. Structure--function relationships of the variable domains of monoclonal antibodies approved for cancer treatment[J]. Critical (reviews in oncology/64hematology, 2007) 3): 210-225.) Obtain the heavy chain variable region and light chain variable region sequences of the Cetuximab antibody (SEQ ID NO: 9 and 10). The DNA encoding its heavy chain variable region (Cetuximab-VH) and light chain variable region (Cetuximab-VL) was synthesized by Shanghai Shenggong Bioengineering Co., Ltd. Cetuximab-VH and Cetuximab-VL are respectively connected to the human IgG1 heavy chain constant region (SEQ ID NO: 3) and human Kappa light chain constant region (SEQ ID NO: 6) to construct the heavy and light chains of the full-length Cetuximab antibody The genes are named Cetuximab-HC and Cetuximab-LC, respectively.
Anti-PDL1的重链可变区的氨基酸序列(SEQ ID NO：1)The amino acid sequence of the heavy chain variable region of Anti-PDL1 (SEQ ID NO: 1)
Figure PCTCN2021097784-appb-000001
Figure PCTCN2021097784-appb-000001
Anti-PDL1的轻链可变区的氨基酸序列(SEQ ID NO：2)The amino acid sequence of the light chain variable region of Anti-PDL1 (SEQ ID NO: 2)
Figure PCTCN2021097784-appb-000002
Figure PCTCN2021097784-appb-000002
人IgG1重链恒定区的氨基酸序列(SEQ ID NO：3)Amino acid sequence of human IgG1 heavy chain constant region (SEQ ID NO: 3)
Figure PCTCN2021097784-appb-000003
Figure PCTCN2021097784-appb-000003
Figure PCTCN2021097784-appb-000004
Figure PCTCN2021097784-appb-000004
Anti-PDL1的重链的氨基酸序列(SEQ ID NO：4)The amino acid sequence of the heavy chain of Anti-PDL1 (SEQ ID NO: 4)
Figure PCTCN2021097784-appb-000005
Figure PCTCN2021097784-appb-000005
人Kappa轻链恒定区的氨基酸序列(SEQ ID NO：6)The amino acid sequence of the constant region of the human Kappa light chain (SEQ ID NO: 6)
Figure PCTCN2021097784-appb-000006
Figure PCTCN2021097784-appb-000006
Anti-PDL1的轻链的氨基酸序列(SEQ ID NO：7)The amino acid sequence of the light chain of Anti-PDL1 (SEQ ID NO: 7)
Figure PCTCN2021097784-appb-000007
Figure PCTCN2021097784-appb-000007
Cetuximab的重链可变区的氨基酸序列(SEQ ID NO：9)The amino acid sequence of the heavy chain variable region of Cetuximab (SEQ ID NO: 9)
Figure PCTCN2021097784-appb-000008
Figure PCTCN2021097784-appb-000008
Cetuximab的轻链可变区的氨基酸序列(SEQ ID NO：10)The amino acid sequence of the light chain variable region of Cetuximab (SEQ ID NO: 10)
Figure PCTCN2021097784-appb-000009
Figure PCTCN2021097784-appb-000009
实施例1.2共同轻链的选择Example 1.2 Selection of common light chain
用BLAST对Anti-PDL1轻链可变区与Cetuximab轻链可变区的氨基酸序列进行对比分析，结果显示，两者之间完全相同的氨基酸占比75％(Identities)，性质相似的氨基酸占比89％(Positives)。Using BLAST to compare the amino acid sequence of Anti-PDL1 light chain variable region and Cetuximab light chain variable region, the results showed that the identical amino acids accounted for 75% (Identities), and the amino acids with similar properties accounted for 75%. 89% (Positives).
将Cetuximab-HC和Cetuximab-LC的基因序列分别构建到pcDNA4表达载体中。将Anti-PDL1-HC、Anti-PDL1-LC、Cetuximab-HC和Cetuximab-LC的表达载体按照下述方式进行组合：Anti-PDL1-HC+Anti-PDL1-LC、Cetuximab-HC+Cetuximab-LC、Anti-PDL1-HC+Cetuximab-LC和Cetuximab-HC+Anti-PDL1-LC，表达纯化抗体，所得的抗体分别命名为Anti-PDL1、Cetuximab、Anti-PDL1-HC+Cetuximab-LC和Cetuximab-HC+Anti-PDL1-LC。The gene sequences of Cetuximab-HC and Cetuximab-LC were respectively constructed into pcDNA4 expression vector. Combine the expression vectors of Anti-PDL1-HC, Anti-PDL1-LC, Cetuximab-HC and Cetuximab-LC in the following manner: Anti-PDL1-HC+Anti-PDL1-LC, Cetuximab-HC+Cetuximab-LC, Anti-PDL1-HC+Cetuximab-LC and Cetuximab-HC+Anti-PDL1-LC, express and purified antibodies, the obtained antibodies are named Anti-PDL1, Cetuximab, Anti-PDL1-HC+Cetuximab-LC and Cetuximab-HC+ Anti-PDL1-LC.
PD-L1的胞外区编码基因来源如WO2018/137576A1中所述。利用基因重组技术，在PD-L1的胞外区编码基因末端连接多聚组氨酸编码序列，然后将重组基因克隆到pcDNA4表达载体中，表达并纯化重组蛋白，所得重组蛋白命名为PD-L1-His。利用基因重组技术，在人EGFR的胞外区(序列来自NCBI，Accession：NP_005219)编码基因末端连接人IgG1的Fc段编码序列，然后将重组基因克隆到pcDNA4表达载体中，表达并纯化重组蛋白，所得重组蛋白命名为EGFR-ECD-hFc。用EGFR-ECD-hFc和PD-L1-His分别包被酶标板，包被浓度分别为40ng/孔和10ng/孔。用含有1％牛血清白蛋白的PBST(KH 2PO 4 0.2g，Na 2HPO 4·12H 2O 2.9g，NaCl 8.0g，KCl 0.2g，Tween-20 0.5ml，加纯水至1L)封闭酶标板。将待测抗体进行梯度稀释，然后转移到上述包被重组蛋白的酶标板中，室温孵育半小时后洗板；加入适当稀释的HRP(Horseradish Peroxidase)标记的羊抗人抗体(Fab-specific，购自Sigma)，室温孵育半小时后洗板；每孔加入100μl以TMB为底物的显色液(底物显色A液：醋酸钠·三水13.6g，柠檬酸·一水1.6g，30％双氧水0.3ml，纯水500ml；底物显色B液：乙二胺四乙酸二钠0.2g，柠檬酸·一水0.95g，甘油50ml，TMB 0.15g溶于3ml DMSO中，纯水500ml；使用前A和B液等体积混匀)，室温孵育1～5min；加50μl终止液(2M H 2SO 4)终止反应；酶标仪(SpectraMax 190)读取OD450，用GraphPad Prism6进行作图和数据分析，并计算EC50。 The source of the gene encoding the extracellular region of PD-L1 is as described in WO2018/137576A1. Using gene recombination technology, connect the polyhistidine coding sequence to the end of the extracellular region coding gene of PD-L1, then clone the recombinant gene into pcDNA4 expression vector, express and purify the recombinant protein, and the resulting recombinant protein is named PD-L1 -His. Using gene recombination technology, the extracellular region of human EGFR (sequence from NCBI, Accession: NP_005219) was connected to the end of the Fc segment coding sequence of human IgG1, and then the recombinant gene was cloned into the pcDNA4 expression vector to express and purify the recombinant protein. The resulting recombinant protein was named EGFR-ECD-hFc. The ELISA plate was coated with EGFR-ECD-hFc and PD-L1-His, and the coating concentration was 40ng/well and 10ng/well respectively. Use PBST containing 1% bovine serum albumin (KH 2 PO 4 0.2g, Na 2 HPO 4 ·12H 2 O 2.9g, NaCl 8.0g, KCl 0.2g, Tween-20 0.5ml, add pure water to 1L) ELISA plate. Dilute the antibody to be tested in a gradient, then transfer to the above-mentioned ELISA plate coated with recombinant protein, incubate at room temperature for half an hour and then wash the plate; add appropriately diluted HRP (Horseradish Peroxidase) labeled goat anti-human antibody (Fab-specific, (Purchased from Sigma), incubate for half an hour at room temperature and wash the plate; add 100μl of color developing solution with TMB as the substrate to each well (substrate color developing solution A: sodium acetate·trihydrate 13.6g, citric acid·monohydrate 1.6g, 30% hydrogen peroxide 0.3ml, pure water 500ml; Substrate color development solution B: 0.2g disodium edetate, 0.95g citric acid monohydrate, 50ml glycerol, 0.15g TMB dissolved in 3ml DMSO, 500ml pure water ；Mix the same volume of A and B before use), incubate at room temperature for 1～5min; add 50μl of stop solution (2M H 2 SO 4 ) to stop the reaction; read OD450 with a microplate reader (SpectraMax 190) and use GraphPad Prism6 for graphing And data analysis, and calculate EC50.
如图2A所示，Anti-PDL1能够有效结合PD-L1-His，EC50是0.0924nM；而Cetuximab、Anti-PDL1-HC+Cetuximab-LC和Cetuximab-HC+Anti-PDL1-LC均不能结合PD-L1-His。如图2B所示，Cetuximab和Cetuximab-HC+Anti-PDL1-LC均能有效结合EGFR-ECD-hFc，EC50为0.2096nM和0.2484nM，而Anti-PDL1和Anti-PDL1-HC+Cetuximab-LC不能有效结合EGFR-ECD-hFc。在此选择Anti-PDL1-LC(SEQ ID NO：7和8)作为共同轻链构建双特异性抗体。As shown in Figure 2A, Anti-PDL1 can effectively bind PD-L1-His with an EC50 of 0.0924nM; while Cetuximab, Anti-PDL1-HC+Cetuximab-LC and Cetuximab-HC+Anti-PDL1-LC cannot bind PD- L1-His. As shown in Figure 2B, both Cetuximab and Cetuximab-HC+Anti-PDL1-LC can effectively bind to EGFR-ECD-hFc, with EC50 of 0.2096nM and 0.2484nM, while Anti-PDL1 and Anti-PDL1-HC+Cetuximab-LC cannot Effectively bind EGFR-ECD-hFc. Here, Anti-PDL1-LC (SEQ ID NO: 7 and 8) is selected as the common light chain to construct a bispecific antibody.
实施例1.3双特异抗体的构建Example 1.3 Construction of bispecific antibodies
将Anti-PDL1的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS，SEQ ID NO：11)连接Cetuximab的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)， 通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为PDL1-Fab-Cetuximab-IgG1(SEQ ID NO：12和13)。相似地，将Cetuximab的重链可变区与人IgG4的CH1结构域相连，然后再通过人工连接子(在此使用的连接子是三个串联的GGGGS，SEQ ID NO：11)连接Anti-PDL1的重链可变区，最后再连接人IgG1的重链恒定区(CH1+CH2+CH3)，通过此程序构建成的含有两个重链可变区和两个CH1结构域的长重链基因命名为Cetuximab-Fab-PDL1-IgG1(SEQ ID NO：14和15)。Connect the heavy chain variable region of Anti-PDL1 with the CH1 domain of human IgG4, and then connect the heavy chain of Cetuximab through an artificial linker (the linker used here is three series GGGGS, SEQ ID NO: 11) The variable region is finally connected to the heavy chain constant region of human IgG1 (CH1+CH2+CH3). The long heavy chain gene constructed by this program containing two heavy chain variable regions and two CH1 domains is named PDL1 -Fab-Cetuximab-IgG1 (SEQ ID NO: 12 and 13). Similarly, the heavy chain variable region of Cetuximab is connected to the CH1 domain of human IgG4, and then an artificial linker (the linker used here is three series GGGGS, SEQ ID NO: 11) is connected to Anti-PDL1 The heavy chain variable region of human IgG1 is finally connected to the heavy chain constant region (CH1+CH2+CH3) of human IgG1. This program is used to construct a long heavy chain gene containing two heavy chain variable regions and two CH1 domains Named Cetuximab-Fab-PDL1-IgG1 (SEQ ID NO: 14 and 15).
将上述序列分别构建到pcDNA4表达载体中，将PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1表达载体分别与Anti-PDL1-LC表达载体组合，表达纯化抗体，所得的抗体分别命名为PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1(为简明起见，此处只取重链的名字作为抗体的名称)。The above sequences were constructed into the pcDNA4 expression vector, the PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 expression vectors were combined with the Anti-PDL1-LC expression vector to express the purified antibodies, and the resulting antibodies were named as PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 (for brevity, only the name of the heavy chain is used as the name of the antibody).
PDL1-Fab-Cetuximab-IgG1的氨基酸序列如下所示(SEQ ID NO：12)：The amino acid sequence of PDL1-Fab-Cetuximab-IgG1 is shown below (SEQ ID NO: 12):
Figure PCTCN2021097784-appb-000010
Figure PCTCN2021097784-appb-000010
Cetuximab-Fab-PDL1-IgG1的氨基酸序列如下所示(SEQ ID NO：14)：The amino acid sequence of Cetuximab-Fab-PDL1-IgG1 is shown below (SEQ ID NO: 14):
Figure PCTCN2021097784-appb-000011
Figure PCTCN2021097784-appb-000011
实施例2 ELISA测定相对亲和力Example 2 Determination of relative affinity by ELISA
ELISA检测方法参照实施例1.2中所述。The ELISA detection method refers to that described in Example 1.2.
如图3A所示，Anti-PDL1、PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1均能有效结合PD-L1-His，EC50分别是0.2177nM、0.2003nM和0.3356nM。如图3B所示，Cetuximab-HC+Anti-PDL1-LC、PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1均能有效结合EGFR-ECD-hFc，EC50分别是0.2253nM、0.2388nM和0.1852nM。上述结果显示，PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1既能够结合PD-L1又能结合EGFR，这说明它们是双特异性抗体。As shown in Figure 3A, Anti-PDL1, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can effectively bind PD-L1-His, with EC50 of 0.2177nM, 0.2003nM and 0.3356nM, respectively. As shown in Figure 3B, Cetuximab-HC+Anti-PDL1-LC, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can effectively bind to EGFR-ECD-hFc, with EC50 of 0.2253nM, 0.2388nM and 0.1852nM. The above results show that PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can bind to both PD-L1 and EGFR, indicating that they are bispecific antibodies.
实施例3 评估抑制A431细胞增殖的功能活性Example 3 Evaluation of the functional activity of inhibiting the proliferation of A431 cells
A431(
Figure PCTCN2021097784-appb-000012
CRL-1555 TM)是人类表皮癌细胞系，过量表达野生型EGFR。抗EGFR抗体能够在体外和体内抑制A431细胞的增殖。 A431(
Figure PCTCN2021097784-appb-000012
CRL-1555 ™ ) is a human epidermal cancer cell line that overexpresses wild-type EGFR. Anti-EGFR antibodies can inhibit the proliferation of A431 cells in vitro and in vivo.
本实施例评估上述抗体抑制A431细胞增殖的功能活性。方法如下：用DMEM将处于对数生长期的A431细胞洗2遍，1000rpm离心5min；用含1％胎牛血清的DMEM(胎牛血清和DMEM购自Gibco)将细胞重悬到适当密度，接种到96孔板中，10 4个/150μl/孔；然后在含1％胎牛血清的DMEM中将上述抗体进行梯度稀释；将稀释好的抗体加入上述接种A431细胞的96孔板中，50μl/孔；在37℃、5％CO 2细胞培养箱中孵育3天；3天后每孔加入20μl CCK-8(购自Dojindo)溶液，在培养箱中继续孵育4小时；用酶标仪读取OD450；GraphPad Prism6进行数据分析，作图并计算IC50。 This example evaluates the functional activity of the above antibodies in inhibiting the proliferation of A431 cells. The method is as follows: Wash the A431 cells in the logarithmic growth phase twice with DMEM, centrifuge at 1000 rpm for 5 min; resuspend the cells to an appropriate density with DMEM containing 1% fetal bovine serum (fetal bovine serum and DMEM purchased from Gibco), and inoculate 96-well plate, 10 4 / 150μl / hole; then 1% fetal bovine serum in DMEM in the above-described antibody was serially diluted; the diluted antibody was added to the 96 well plate inoculated A431 cells, 50μl / Wells; incubate for 3 days at 37°C and 5% CO 2 in a cell incubator; add 20μl of CCK-8 (purchased from Dojindo) solution to each well after 3 days and incubate for 4 hours in the incubator; read OD450 with a microplate reader ; GraphPad Prism6 performs data analysis, plots and calculates IC50.
如图4所示，Cetuximab、Cetuximab-HC+Anti-PDL1-LC、PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1均能有效抑制A431细胞的增殖，IC50分别是0.6322nM、0.5629nM、0.7094nM和0.9597nM。As shown in Figure 4, Cetuximab, Cetuximab-HC+Anti-PDL1-LC, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 can effectively inhibit the proliferation of A431 cells, with IC50 of 0.6322nM and 0.5629nM, respectively , 0.7094nM and 0.9597nM.
实施例4 Biacore测定亲和力Example 4 Biacore determination of affinity
在此通过Biacore 8K(GE healthcare)检测上述抗体与PD-L1或EGFR之间的亲和力。在Biacore 8K上，使用偶联有Protein A/G的芯片分别捕获各种抗体，再将重组蛋白PD-L1-His(自制)或EGFR-His(带有His标签的EGFR重组蛋 白，购自北京义翘神州)进样，得到结合-解离曲线，用6M盐酸胍再生缓冲液洗脱后重复下一个循环；利用Biacore 8K Evaluation Software对数据进行分析。结果如表2所示。Here, the affinity between the above antibodies and PD-L1 or EGFR is tested by Biacore 8K (GE healthcare). On Biacore 8K, a chip coupled with Protein A/G was used to capture various antibodies, and then the recombinant protein PD-L1-His (homemade) or EGFR-His (EGFR recombinant protein with His tag, purchased from Beijing Yiqiao Shenzhou) samples were injected, the binding-dissociation curve was obtained, and the next cycle was repeated after elution with 6M guanidine hydrochloride regeneration buffer; the data was analyzed by Biacore 8K Evaluation Software. The results are shown in Table 2.
表2-1.对PD-L1的结合和解离动力学参数以及平衡解离常数Table 2-1. Binding and dissociation kinetic parameters and equilibrium dissociation constants for PD-L1
Figure PCTCN2021097784-appb-000013
Figure PCTCN2021097784-appb-000013
表2-2.对EGFR的结合和解离动力学参数以及平衡解离常数Table 2-2. Binding and dissociation kinetic parameters and equilibrium dissociation constants for EGFR
Figure PCTCN2021097784-appb-000014
Figure PCTCN2021097784-appb-000014
表2-1显示，Anti-PDL1、PDL1-Fab-Cetuximab-IgG1和Cetuximab-Fab-PDL1-IgG1对PD-L1的结合常数(Kon)和解离常数(Koff)十分相近，平衡解离常数(KD)也基本相当，KD分别是9.66E-10、6.46E-10和7.79E-10。表2-2显示，Cetuximab、Cetuximab-HC+Anti-PDL1-LC和Cetuximab-Fab-PDL1-IgG1对EGFR的结合常数(Kon)和解离常数(Koff)十分相近，平衡解离常数(KD)也基本相当，KD分别是6.14E-10、9.46E-10和9.57E-10；与前三者相比，PDL1-Fab-Cetuximab-IgG1对EGFR的平衡解离常数(KD)略大，为14.2E-10。平衡解离常数(KD)与亲和力高低成反比。Table 2-1 shows that the binding constant (Kon) and dissociation constant (Koff) of Anti-PDL1, PDL1-Fab-Cetuximab-IgG1 and Cetuximab-Fab-PDL1-IgG1 to PD-L1 are very similar, and the equilibrium dissociation constant (KD) ) Is basically the same, KD is 9.66E-10, 6.46E-10 and 7.79E-10 respectively. Table 2-2 shows that the binding constant (Kon) and dissociation constant (Koff) of Cetuximab, Cetuximab-HC+Anti-PDL1-LC and Cetuximab-Fab-PDL1-IgG1 to EGFR are very similar, and the equilibrium dissociation constant (KD) is also very similar. Basically the same, KDs are 6.14E-10, 9.46E-10 and 9.57E-10 respectively; compared with the first three, the equilibrium dissociation constant (KD) of PDL1-Fab-Cetuximab-IgG1 for EGFR is slightly larger, which is 14.2 E-10. The equilibrium dissociation constant (KD) is inversely proportional to the affinity.
实施例5 物理化学性质的表征Example 5 Characterization of physicochemical properties
5.1 HPLC-SEC5.1 HPLC-SEC
图5A表示Anti-PDL1的HPLC-SEC图谱，其中存在2个明显的峰，分别是 Peak1和Peak2，占比分别为0.2％和99.8％(主峰)。图5B表示PDL1-Fab-Cetuximab-IgG1的HPLC-SEC图谱，其中存在3个明显的峰，分别是Peak1、Peak2和Peak3，占比分别为0.3％、99.5％(主峰)和0.2％。Anti-PDL1和PDL1-Fab-Cetuximab-IgG1的主峰占比相近。Figure 5A shows the HPLC-SEC spectrum of Anti-PDL1, in which there are two obvious peaks, Peak1 and Peak2, which account for 0.2% and 99.8% (main peak) respectively. Figure 5B shows the HPLC-SEC profile of PDL1-Fab-Cetuximab-IgG1, in which there are 3 obvious peaks, Peak1, Peak2 and Peak3, which account for 0.3%, 99.5% (main peak) and 0.2%, respectively. Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 have similar main peaks.
5.2 CE-SDS5.2 CE-SDS
图6A和图6B分别表示Anti-PDL1的NR-CE-SDS和R-CE-SDS图谱，图6C和图6D分别表示PDL1-Fab-Cetuximab-IgG1的NR-CE-SDS和R-CE-SDS图谱。Anti-PDL1的NR-CE-SDS主峰Peak8占比98.11％，PDL1-Fab-Cetuximab-IgG1的NR-CE-SDS主峰Peak9占比97.14％。Anti-PDL1的R-CE-SDS主峰Peak5(对应轻链)和Peak10(对应重链)分别占比32.62％和63.55％，两者峰面积之比为1:1.95，两者峰面积占比之和为96.17％；PDL1-Fab-Cetuximab-IgG1的R-CE-SDS主峰Peak4(对应轻链)和Peak11(对应重链)分别占比38.27％和57.37％，两者峰面积之比为2:3.0，两者峰面积占比之和为95.64％。NR-CE-SDS中，Anti-PDL1和PDL1-Fab-Cetuximab-IgG1的主峰占比十分相近；R-CE-SDS中，Anti-PDL1和PDL1-Fab-Cetuximab-IgG1的轻链和重链的峰面积之比均符合预期，两者的主峰占比之和十分相近。Figure 6A and Figure 6B show the NR-CE-SDS and R-CE-SDS maps of Anti-PDL1, respectively, and Figure 6C and Figure 6D show the NR-CE-SDS and R-CE-SDS of PDL1-Fab-Cetuximab-IgG1, respectively Atlas. Anti-PDL1 NR-CE-SDS main peak Peak8 accounted for 98.11%, and PDL1-Fab-Cetuximab-IgG1 NR-CE-SDS main peak Peak9 accounted for 97.14%. The main peaks of Anti-PDL1 R-CE-SDS Peak5 (corresponding to the light chain) and Peak10 (corresponding to the heavy chain) accounted for 32.62% and 63.55%, respectively, and the ratio of the two peak areas was 1:1.95, which is the ratio of the peak areas of the two The sum is 96.17%; Peak4 (corresponding to the light chain) and Peak11 (corresponding to the heavy chain) of the R-CE-SDS peaks of PDL1-Fab-Cetuximab-IgG1 account for 38.27% and 57.37%, respectively, and the ratio of the peak areas of the two is 2: 3.0, the sum of the ratio of the two peak areas is 95.64%. In NR-CE-SDS, the proportions of the main peaks of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 are very similar; in R-CE-SDS, the light and heavy chains of Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 The peak area ratios are in line with expectations, and the sum of the proportions of the main peaks of the two is very similar.
实施例6 评估增强MLR的能力Example 6 Evaluation of the ability to enhance MLR
本实施例中使用的混合淋巴细胞反应(Mixed Lymphocyte Reaction，MLR)的方法说明如下：用Histopaque(购自Sigma)从人血液中分离出外周血单个核细胞(Peripheral Blood Mononuclear Cell，缩写PBMC)，然后通过贴壁法将PBMC中的单核细胞分离出来，然后用IL-4(25ng/ml)和GM-CSF(25ng/ml)诱导单核细胞分化成树突状细胞。七天之后，消化收集上述诱导的树突状细胞。用上述方法从另外供体的血液中分离出PBMC，然后用MACS磁铁和CD4 MicroBeads(购自Miltenyi biotec)从PBMC中分离CD4 +T细胞。将诱导的树突状细胞(10 4/孔)和分离出的CD4 +T细胞(10 5/孔)按比例混匀后接种到96孔板中，每孔150μl；数小时后，在上述96孔板中加入50μl梯度稀释的抗体；将96孔板置于37℃细胞培养箱中孵育3天。上述实验过程中使用AIM-V培养基(购自Thermo Fisher  Scientific)培养细胞。然后按照标准操作流程检测IL-2和IFN-γ的分泌。使用双抗夹心ELISA检测IL-2和IFN-γ的分泌(相关配对抗体购自BD Biosciences)。用酶标仪(SpectraMax 190)读取OD450，用GraphPad Prism6进行作图并计算EC50。 The Mixed Lymphocyte Reaction (MLR) method used in this example is described as follows: Use Histopaque (purchased from Sigma) to isolate Peripheral Blood Mononuclear Cell (PBMC) from human blood, Then the monocytes in the PBMC were separated by the adherence method, and then IL-4 (25ng/ml) and GM-CSF (25ng/ml) were used to induce the monocytes to differentiate into dendritic cells. Seven days later, the above-induced dendritic cells were digested and collected. PBMCs were separated from the blood of other donors by the above method, and then CD4 + T cells were separated from the PBMCs with MACS magnet and CD4 MicroBeads (purchased from Miltenyi biotec). The induced dendritic cells (10 4 /well) and the isolated CD4 + T cells (10 5 /well) were mixed in proportion and then seeded into a 96-well plate, 150μl per well; a few hours later, in the above 96 Add 50μl of serially diluted antibody to the well plate; place the 96-well plate in a 37°C cell incubator and incubate for 3 days. During the above experiment, AIM-V medium (purchased from Thermo Fisher Scientific) was used to culture the cells. Then detect the secretion of IL-2 and IFN-γ according to standard operating procedures. Double-antibody sandwich ELISA was used to detect the secretion of IL-2 and IFN-γ (related paired antibodies were purchased from BD Biosciences). Read OD450 with a microplate reader (SpectraMax 190), graph with GraphPad Prism6 and calculate EC50.
A和B的结果来自同一份MLR实验体。如图7A所示，Anti-PDL1和PDL1-Fab-Cetuximab-IgG1均能有效刺激MLR分泌IL-2，它们的EC50分别是0.1578nM和0.409nM。另外，如图7B所示，Anti-PDL1和PDL1-Fab-Cetuximab-IgG1均能有效刺激MLR分泌IFN-γ，它们的EC50分别是0.1369nM和0.08084nM。该结果显示，Anti-PDL1和PDL1-Fab-Cetuximab-IgG1的功能活性相当。其中同型对照抗体为与靶点无关的人IgG1抗体。The results of A and B are from the same MLR experiment. As shown in Figure 7A, both Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 can effectively stimulate MLR to secrete IL-2, and their EC50 is 0.1578nM and 0.409nM, respectively. In addition, as shown in Figure 7B, both Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 can effectively stimulate MLR to secrete IFN-γ, and their EC50 is 0.1369nM and 0.08084nM, respectively. The results show that Anti-PDL1 and PDL1-Fab-Cetuximab-IgG1 have equivalent functional activities. The isotype control antibody is a human IgG1 antibody that has nothing to do with the target.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (17)

  1. 抗PD-L1和EGFR的四价双特异性抗体，其特征在于，包含：A tetravalent bispecific antibody against PD-L1 and EGFR, characterized in that it contains:
    (a)两条多肽链，所述多肽链从N末端到C末端包含VH-PDL1-CH1-接头-VH-EGFR-CH1-CH2-CH3或VH-EGFR-CH1-接头-VH-PDL1-CH1-CH2-CH3，其中，所述VH-PDL1为结合PD-L1的重链可变区，所述VH-EGFR为结合EGFR的重链可变区，所述CH1为重链恒定区的第一结构域，所述CH2为重链恒定区的第二结构域，所述CH3为重链恒定区的第三结构域；和(a) Two polypeptide chains, the polypeptide chain includes VH-PDL1-CH1-linker-VH-EGFR-CH1-CH2-CH3 or VH-EGFR-CH1-linker-VH-PDL1-CH1 from N-terminus to C-terminus -CH2-CH3, wherein the VH-PDL1 is a heavy chain variable region that binds PD-L1, the VH-EGFR is a heavy chain variable region that binds EGFR, and the CH1 is the first heavy chain constant region Domain, the CH2 is the second domain of the heavy chain constant region, and the CH3 is the third domain of the heavy chain constant region; and
    (b)四条共同轻链，所述共同轻链从N末端到C末端包含VL-CL，其中，所述VL为轻链可变区，所述CL为轻链恒定区，所述多肽链的VH-PDL1-CH1和所述VH-EGFR-CH1分别与所述共同轻链的VL-CL配对，所述VH-PDL1和所述VL形成PD-L1抗原结合位点，所述VH-EGFR与所述VL形成EGFR结合位点；(b) Four common light chains, the common light chain includes VL-CL from the N-terminus to the C-terminus, wherein the VL is the light chain variable region, the CL is the light chain constant region, and the polypeptide chain VH-PDL1-CH1 and the VH-EGFR-CH1 are respectively paired with the VL-CL of the common light chain, the VH-PDL1 and the VL form the PD-L1 antigen binding site, and the VH-EGFR and The VL forms an EGFR binding site;
    其中，所述的每个共同轻链具有如SEQ ID NO：7所示的氨基酸序列。Wherein, each of the common light chains has an amino acid sequence as shown in SEQ ID NO:7.
  2. 如权利要求1所述的四价双特异性抗体，其特征在于，所述的重链的CH1区选自人IgG1的CH1结构域或人IgG4的CH1结构域。The tetravalent bispecific antibody of claim 1, wherein the CH1 region of the heavy chain is selected from the CH1 domain of human IgG1 or the CH1 domain of human IgG4.
  3. 如权利要求1所述的四价双特异性抗体，其特征在于，所述的结合PD-L1的重链可变区VH-PDL1如SEQ ID No:1所示；所述的结合EGFR的重链可变区VH-EGFR如SEQ ID No:9所示。The tetravalent bispecific antibody of claim 1, wherein the heavy chain variable region VH-PDL1 that binds to PD-L1 is shown in SEQ ID No:1; the heavy chain that binds to EGFR The chain variable region VH-EGFR is shown in SEQ ID No: 9.
  4. 如权利要求1所述的四价双特异性抗体，其特征在于，每个所述的多肽链的氨基酸序列相同。The tetravalent bispecific antibody of claim 1, wherein the amino acid sequence of each of the polypeptide chains is the same.
  5. 抗PD-L1和EGFR的四价双特异性抗体，其特征在于，包含两条多肽链和四条共同轻链，其中，所述多肽链具有如SEQ ID NO：12或SEQ ID NO：14所示的氨基酸序列，所述共同轻链具有如SEQ ID NO：7所示的氨基酸序列。A quadrivalent bispecific antibody against PD-L1 and EGFR, which is characterized by comprising two polypeptide chains and four common light chains, wherein the polypeptide chains have the following SEQ ID NO: 12 or SEQ ID NO: 14 The amino acid sequence of the common light chain has the amino acid sequence shown in SEQ ID NO: 7.
  6. 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1-5任一所述的四价双特异性抗体。An isolated nucleotide, characterized in that the nucleotide encodes the tetravalent bispecific antibody according to any one of claims 1-5.
  7. 如权利要求6所述的核苷酸，其特征在于，所述的核苷酸编码所述多肽链和所述共同轻链，其中，编码所述多肽链的核苷酸序列如SEQ ID NO：13或SEQ ID NO：15所示，编码所述共同轻链的核苷酸序列如SEQ ID NO：8所示。7. The nucleotide of claim 6, wherein the nucleotide encodes the polypeptide chain and the common light chain, wherein the nucleotide sequence encoding the polypeptide chain is as SEQ ID NO: 13 or SEQ ID NO: 15, and the nucleotide sequence encoding the common light chain is shown in SEQ ID NO: 8.
  8. 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6或7所述的核苷酸。An expression vector, characterized in that the expression vector contains the nucleotide according to claim 6 or 7.
  9. 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8所述的 表达载体。A host cell, characterized in that the host cell contains the expression vector according to claim 8.
  10. 如权利要求1-5任一所述的四价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：The method for preparing a tetravalent bispecific antibody according to any one of claims 1 to 5, wherein the method comprises the following steps:
    (a)在表达条件下，培养如权利要求9所述的宿主细胞，从而表达所述的四价双特异性抗体；(a) Culturing the host cell according to claim 9 under expression conditions, thereby expressing the tetravalent bispecific antibody;
    (b)分离并纯化(a)所述的四价双特异性抗体。(b) Isolation and purification of the tetravalent bispecific antibody described in (a).
  11. 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1-5任一所述的四价双特异性抗体和药学上可接受的载体。A pharmaceutical composition, characterized in that it contains the tetravalent bispecific antibody according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.
  12. 如权利要求1-5任一所述的四价双特异性抗体、或其免疫偶联物、或如权利要求11所述的药物组合物在制备治疗癌症的药物中的用途。Use of the tetravalent bispecific antibody according to any one of claims 1 to 5, or an immunoconjugate thereof, or the pharmaceutical composition according to claim 11 in the preparation of a medicine for the treatment of cancer.
  13. 如权利要求12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、***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、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病。The use according to claim 12, wherein 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, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
  14. 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1-5任一所述的四价双特异性抗体、或其免疫偶联物、或如权利要求11所述的药物组合物。A method for treating cancer, which is characterized in that it comprises administering the tetravalent bispecific antibody according to any one of claims 1 to 5, or an immunoconjugate thereof, or according to claim 11, to a subject in need The pharmaceutical composition.
  15. 如权利要求14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、***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、***、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病。The method of claim 14, wherein 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, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
  16. 一种免疫偶联物，其特征在于，所述免疫偶联物包括：An immunoconjugate, characterized in that, the immunoconjugate comprises:
    (a)如权利要求1中所述的四价双特异性抗体；和(a) The tetravalent bispecific antibody as claimed in claim 1; and
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。(b) A coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
  17. 如权利要求16所述的免疫偶联物的用途，其特征在于，用于制备***的药物组合物。The use of the immunoconjugate according to claim 16, characterized in that it is used to prepare a pharmaceutical composition for treating tumors.
PCT/CN2021/097784 2020-06-02 2021-06-01 Tetravalent bispecific antibody against pd-1 and egfr WO2021244553A1 (en)

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