WO2021244553A1 - Anticorps bispécifique tétravalent contre pd-1 et egfr - Google Patents

Anticorps bispécifique tétravalent contre pd-1 et 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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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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Publication of WO2021244553A1 publication Critical patent/WO2021244553A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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.

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Abstract

La présente invention concerne un anticorps bispécifique tétravalent contre PD-1 et EGFR. L'anticorps bispécifique tétravalent de la présente invention ne nécessite pas de modification Fc, ne cause pas de problèmes de mésappariement, a un procédé de préparation simple, et présente des activités biologiques similaires ou encore meilleures, ainsi que des propriétés physiques et chimiques par comparaison avec celles d'anticorps monoclonaux.
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SIMON KRAH, CHRISTIAN SCHRöTER, CARLA ELLER, LAURA RHIEL, NICOLAS RASCHE, JAN BECK, CAROLIN SELLMANN, RALF GüNTHER, LARS: "Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display", PROTEIN ENGINEERING, DESIGN AND SELECTION, OXFORD JOURNAL, LONDON, GB, vol. 30, no. 4, 1 April 2017 (2017-04-01), GB , pages 291 - 301, XP055690141, ISSN: 1741-0126, DOI: 10.1093/protein/gzw077 *
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