WO2023273958A1 - 三特异性抗体、其制备方法和用途 - Google Patents

三特异性抗体、其制备方法和用途 Download PDF

Info

Publication number
WO2023273958A1
WO2023273958A1 PCT/CN2022/100105 CN2022100105W WO2023273958A1 WO 2023273958 A1 WO2023273958 A1 WO 2023273958A1 CN 2022100105 W CN2022100105 W CN 2022100105W WO 2023273958 A1 WO2023273958 A1 WO 2023273958A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
light chain
target
heavy chain
hexavalent
Prior art date
Application number
PCT/CN2022/100105
Other languages
English (en)
French (fr)
Inventor
赵杰
黄浩旻
朱祯平
Original Assignee
三生国健药业（上海）股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 三生国健药业（上海）股份有限公司 filed Critical 三生国健药业（上海）股份有限公司
Priority to EP22831776.4A priority Critical patent/EP4365205A1/en
Publication of WO2023273958A1 publication Critical patent/WO2023273958A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the field of antibodies, in particular to a trispecific antibody, its preparation method and use.
  • Anti-PD-1/PD-L1 monoclonal antibodies were the first cancer immunotherapy to gain widespread use. Subsequently, bispecific antibodies appeared in people's field of vision as cancer immunotherapy. One end of these antibodies can bind to the antigen on the surface of cancer cells, and the other end can bind to CD3 on the surface of T cells, recruiting and activating T cells to kill cancer cells (such as Amgen's Blinatumomab).
  • Blinatumomab is a bispecific antibody that simultaneously targets the CD19 antigen on the surface of cancer cells and the CD3 receptor on the surface of T cells. and double the lifetime. Such bispecific antibodies can effectively activate the anticancer activity of T cells.
  • T cells usually when T cells are activated, not only the CD3-mediated signaling pathway is activated, but also a costimulatory receptor-mediated signaling pathway called CD28 is also activated. This dual activation can ensure that T cells maintain longer anti-tumor activity.
  • Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.
  • Sanofi Sanofi
  • CD28 has been used as a co-stimulatory receptor in CAR-T therapy.
  • the receptors expressed by certain CAR-T cells contain costimulatory protein domains of CD3 and CD28.
  • Trispecific antibodies offer a third way forward for anticancer immunotherapy. Nature, doi: 10.1038/d41586-019-03495-3) stated that multiple myeloma Patients are in constant need of new treatments, as even treatments as effective as CAR-T can only bring temporary relief to most patients. Trispecific antibodies may provide a flexible platform to deliver precise combinations of immunomodulatory signals according to the tumor microenvironment, making them safer and more effective than combination therapy consisting of three monospecific antibodies.
  • Numab disclosed its trispecific antibody design method in US20180355024A1, which is characterized by multiple ScFv fragments and Fv.
  • Xencor disclosed its trispecific antibody design method in US20190352416A1, which is characterized by the use of heavy chain heterodimerization technology and ScFv fragments.
  • the three HIV-binding fragments are derived from three natural antibodies, and each natural antibody is potent. Neutralizes many HIV strains.
  • Using trispecific antibodies to treat cancer will be a very important conceptual breakthrough. Since the trispecific antibody is a flexible carrier that can specifically deliver the combination of immunomodulatory signals in the tumor microenvironment, it may be safer and more effective than the combination of multiple specific immunomodulatory mAbs. Therefore, this combination strategy is expected to improve the precision and efficacy of current immunotherapy and further expand the scope of application of immunotherapy.
  • Trispecific antibodies can simultaneously bind three different targets and are expected to provide a strong basis for the development of therapeutics against HIV infection, other infectious diseases, autoimmune diseases, and cancer.
  • the purpose of the present invention is to provide a trispecific antibody, its preparation method and use.
  • a hexavalent trispecific antibody is provided, and the hexavalent trispecific antibody is a dimer formed by two monomers, wherein each monomer comprises a heavy chain, a second a light chain and a second light chain;
  • the heavy chain includes, from N-terminal to C-terminal, an antibody heavy chain variable region element Z1 for the first target, an antibody heavy chain variable region element Z2 for the second target, and an antibody heavy chain for the third target in series from the N-terminal to the C-terminal variable region element Z3 and antibody heavy chain constant region Z4;
  • the first light chain and the second light chain cooperate with the heavy chain respectively, so that the hexavalent trispecific antibody specifically binds to the first target, the second target and the third target.
  • the first light chain includes, from N-terminus to C-terminus, antibody light chain variable region element Z5 for the first target and antibody light chain variable region element Z6 for the second target in tandem .
  • the second light chain includes, from the N-terminus to the C-terminus, an antibody light chain variable region element Z7 and an antibody light chain constant region Z8 directed against the third target in series.
  • the antibody heavy chain constant region Z4 includes a CH1 region, a CH2 region and a CH3 region.
  • the first light chain cooperates with the antibody heavy chain variable region element Z1 and the antibody heavy chain variable region element Z2; and the second light chain cooperates with the antibody heavy chain variable region element Z3 cooperates.
  • the second light chain further cooperates with the CH1 region in the constant region Z4 of the heavy chain of the antibody.
  • the antibody heavy chain constant region Z4 is derived from an antibody targeting the first target, an antibody targeting the second target, or an antibody targeting the third target.
  • the antibody heavy chain variable region element Z1, the antibody heavy chain variable region element Z2, the antibody heavy chain variable region element Z3 and the antibody heavy chain constant region Z4 are each independently fully human or humanized.
  • the antibody light chain variable region element Z5, the antibody light chain variable region element Z6, the antibody light chain variable region element Z7 and the antibody light chain constant region Z8 are each independently fully human or humanized.
  • the adjacent two of the antibody heavy chain variable region element Z1, antibody heavy chain variable region element Z2, antibody heavy chain variable region element Z3 and antibody heavy chain constant region Z4 Components can be connected directly or via joints.
  • the antibody light chain variable region element Z5 and the antibody light chain variable region element Z6 can be connected directly or through a linker.
  • the antibody light chain variable region element Z7 and the antibody light chain constant region Z8 may be connected directly or through a linker.
  • the joints include flexible joints and rigid joints.
  • the linker is a peptide linker with a length of 1-35 amino acids, preferably a peptide linker with a length of 6-30 amino acids.
  • the first target, the second target and the third target are PD-1, HER-2 and LAG-3.
  • the first target is PD-1
  • the second target is HER-2
  • the third target is LAG-3.
  • the first target is HER-2
  • the second target is PD-1
  • the third target is LAG-3.
  • the first target is PD-1
  • the second target is LAG-3
  • the third target is HER-2.
  • the first target is LAG-3
  • the second target is PD-1
  • the third target is HER-2.
  • the first target is HER-2
  • the second target is LAG-3
  • the third target is PD-1.
  • the first target is LAG-3
  • the second target is HER-2
  • the third target is PD-1.
  • the hexavalent trispecific antibody comprises a heavy chain, a first light chain and a second light chain; wherein each of the heavy chains has the structure of the following formula I:
  • Z1 is the heavy chain variable region element directed against the first target
  • Z2 is a heavy chain variable region element directed against a second target
  • Z3 is a heavy chain variable region element directed against a third target
  • Z4 is antibody heavy chain constant region CH1, CH2 and CH3;
  • the first light chain and the second light chain cooperate with the heavy chain respectively, so that the hexavalent trispecific antibody specifically binds to the first target, the second target and the third target.
  • the first light chain is coordinated with Z1 and Z2, and the second light chain is coordinated with Z3.
  • the first light chain is also connected to Z2 through a disulfide bond.
  • the second light chain is coordinated with Z3 and CH1, and the second light chain is also connected with CH1 through a disulfide bond.
  • the first light chain has the structure of the following formula II:
  • Z5 is the light chain variable region element for the first target
  • Z6 is the light chain variable region element for the second target.
  • the second light chain has the structure of the following formula III:
  • Z7 is the light chain variable region element for the third target and Z8 is the light chain constant region.
  • the first target is PD-1
  • the second target is HER-2
  • the third target is LAG-3.
  • the first target is HER-2
  • the second target is PD-1
  • the third target is LAG-3.
  • the first target is PD-1
  • the second target is LAG-3
  • the third target is HER-2.
  • the first target is LAG-3
  • the second target is PD-1
  • the third target is HER-2.
  • the first target is HER-2
  • the second target is LAG-3
  • the third target is PD-1.
  • the first target is LAG-3
  • the second target is HER-2
  • the third target is PD-1.
  • the first light chain has an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 17 or 19.
  • the second light chain has the amino acid sequence shown in SEQ ID NO: 13.
  • the heavy chain has an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 16 or 18.
  • the hexavalent trispecific antibody is a dimer.
  • the hexavalent trispecific antibody is a homodimer or a heterodimer.
  • the hexavalent trispecific antibody comprises two monomers, each monomer contains a heavy chain, a first light chain, and a second light chain, wherein each monomer has the following structure of formula IV :
  • VH A -L1-VH B -L2-VH C -CH1-CH2-CH3 is a heavy chain
  • VL A -L3-VL B is the first light chain
  • VL C -CL is the second light chain
  • VHA is the heavy chain variable region directed against the first target
  • VH B is the heavy chain variable region for the second target
  • VH C is the heavy chain variable region for a third target
  • CH1, CH2 and CH3 are antibody heavy chain constant regions CH1, CH2 and CH3, respectively;
  • VLA is the light chain variable region directed against the first target
  • VL B is the light chain variable region for the second target
  • VL C is the light chain variable region for the third target
  • CL is the light chain constant region of the antibody
  • L1, L2, and L3 are each independently a linker
  • ⁇ represents a disulfide bond or a covalent bond
  • the hexavalent trispecific antibody simultaneously binds to the first target, the second target and the third target.
  • the first target is HER-2
  • the second target is PD-1
  • the third target is LAG-3.
  • the first target is PD-1
  • the second target is LAG-3
  • the third target is HER-2.
  • the first target is LAG-3
  • the second target is PD-1
  • the third target is HER-2.
  • the first target is HER-2
  • the second target is LAG-3
  • the third target is PD-1.
  • the first target is LAG-3
  • the second target is HER-2
  • the third target is PD-1.
  • the hexavalent trispecific antibody comprises two monomers, each monomer contains a heavy chain, a first light chain, and a second light chain, wherein each monomer has the following structure of formula IV :
  • VH A -L1-VH B -L2-VH C -CH1-CH2-CH3 is a heavy chain
  • VL A -L3-VL B is the first light chain
  • VL C -CL is the second light chain
  • VHA is the heavy chain variable region of the anti - PD-1 antibody
  • VH B is the heavy chain variable region of an anti-HER-2 antibody
  • VH C is the heavy chain variable region of the anti-LAG-3 antibody
  • CH1, CH2 and CH3 are antibody heavy chain constant regions CH1, CH2 and CH3, respectively;
  • VL A is the light chain variable region of the anti-PD-1 antibody
  • VL B is the light chain variable region of an anti-HER-2 antibody
  • VL C is the light chain variable region of the anti-LAG-3 antibody
  • CL is the light chain constant region of the antibody
  • L1, L2, and L3 are each independently a linker
  • ⁇ represents a disulfide bond or a covalent bond
  • the hexavalent trispecific antibody simultaneously binds to PD-1, HER-2 and LAG-3.
  • the linker is a flexible peptide linker.
  • the flexible peptide linker includes 6-30 amino acids, preferably 10-25 amino acids.
  • the flexible peptide linker includes 2-6 G4S.
  • said L1, L2 or L3 are each independently 2-4 G4S.
  • the heavy chain constant region is the heavy chain constant region of an IgG1, IgG2, IgG3 or IgG4 antibody, or is derived therefrom.
  • the heavy chain constant region is selected from the heavy chain constant region of human IgG1 or human IgG4.
  • the heavy chain constant region of human IgG4 contains S228P mutation.
  • the light chain constant region is the light chain constant region of an IgG1, IgG2, IgG3 or IgG4 antibody, or is derived therefrom.
  • the light chain constant region is selected from the light chain constant region of human IgG1 or human IgG4.
  • the hexavalent trispecific antibody comprises two heavy chains, two first light chains and two second light chains,
  • the heavy chain is selected from the following group: the heavy chain shown in the amino acid sequence SEQ ID NO: 14, 16 or 18;
  • the first light chain is selected from the group consisting of the first light chain having an amino acid sequence as shown in SEQ ID NO: 15, 17 or 19; and/or
  • the second light chain is selected from the following group: the second light chain whose amino acid sequence is shown in SEQ ID NO: 13.
  • amino acid sequences of the heavy chain, the first light chain and the second light chain of the hexavalent trispecific antibody are shown in SEQ ID Nos: 14, 15 and 13, respectively.
  • amino acid sequences of the heavy chain, the first light chain and the second light chain of the hexavalent trispecific antibody are shown in SEQ ID Nos: 16, 17 and 13, respectively.
  • amino acid sequences of the heavy chain, the first light chain and the second light chain of the hexavalent trispecific antibody are shown in SEQ ID Nos: 18, 19 and 13, respectively.
  • an isolated nucleic acid molecule encoding the hexavalent trispecific antibody according to the first aspect of the present invention is provided.
  • the nucleic acid molecule encodes the heavy chain, the first light chain, and the second light chain, respectively.
  • an expression vector containing the nucleic acid molecule according to the second aspect of the present invention is provided.
  • a pharmaceutical composition which contains the hexavalent trispecific antibody as described in the first aspect of the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also contains antitumor agents.
  • the pharmaceutical composition is in unit dosage form.
  • the anti-tumor agent and the trispecific antibody may be present separately in an independent package, or the anti-tumor agent may be coupled to the trispecific antibody.
  • the dosage form of the pharmaceutical composition includes a gastrointestinal administration dosage form or a parenteral administration dosage form.
  • the dosage form for parenteral administration includes intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection, intracranial injection, or intracavitary injection.
  • the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, Cervical cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
  • a method for treating cancer comprising administering the hexavalent trispecific antibody according to the first aspect of the present invention, or an immunoconjugate thereof, to a subject in need, Or the pharmaceutical composition as described in the sixth aspect of the present invention.
  • the cancer is selected from the group consisting of melanoma, kidney cancer, prostate cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, esophageal cancer, squamous cell carcinoma of the head and neck, liver cancer, ovarian cancer, Cervical cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignant diseases.
  • an immunoconjugate comprising:
  • a conjugation moiety selected from the group consisting of a detectable label, drug, toxin, cytokine, radionuclide, or enzyme.
  • the conjugate part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computerized X-ray tomography) contrast agents, or capable of producing visible Detect enzymes, radionuclides, biotoxins, cytokines of products.
  • the immunoconjugate includes antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the immunoconjugate is used to prepare a pharmaceutical composition for treating tumors.
  • FIG. 1 is a schematic structural view of the trispecific antibody of the present invention, wherein, VH-A represents the heavy chain variable region of the first antibody, VH-B represents the heavy chain variable region of the second antibody, and VH-C represents the third Heavy chain variable region of an antibody; VL-A denotes the light chain variable region of the first antibody, VL-B denotes the light chain variable region of the second antibody, and VL-C denotes the light chain variable region of the third antibody.
  • CH1, CH2 and CH3 are the three domains of the heavy chain constant region, and CL is the light chain constant region.
  • the line segment between two heavy chains represents a disulfide bond
  • the line segment between a heavy chain and a light chain also represents a disulfide bond
  • the line segment between VH-B and VL-B represents an artificially designed disulfide bond.
  • the line segments between VH-A and VH-B, VH-B and VH-C, and VL-A and VL-B represent artificially designed linkers
  • the line segments between CH1 and CH2 represent 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).
  • Fig. 2 is the ELISA result of the trispecific antibody of the present invention.
  • Figure 2A, Figure 2B, and Figure 2C are the results of ELISA determination of the affinity of the trispecific antibody of the present invention to PD-1, HER-2, and LAG-3, respectively.
  • Fig. 3 is the HPLC-SEC pattern of monoclonal antibody 609-IgG4 and the trispecific antibody of the present invention.
  • Figure 3A represents the HPLC-SEC spectrum of monoclonal antibody 609-IgG4
  • Figure 3B represents the HPLC-SEC spectrum of 609-302-134-IgG4
  • Figure 3C represents the HPLC-SEC spectrum of 609-302-(44CC)-134-IgG4 Spectrum
  • Figure 3D shows the HPLC-SEC profile of 609-302-(105CC)-134-IgG4.
  • Fig. 4 is the HPLC-IEC pattern of monoclonal antibody 609-IgG4 and the trispecific antibody of the present invention.
  • Figure 4A represents the HPLC-IEC spectrum of monoclonal antibody 609-IgG4
  • Figure 4B represents the HPLC-IEC spectrum of 609-302-(44CC)-134-IgG4
  • Figure 4C represents the HPLC-IEC spectrum of 609-302-(105CC)-134-IgG4
  • the HPLC-IEC spectrum is the HPLC-IEC spectrum.
  • Fig. 5 is the HPLC-IEC pattern of monoclonal antibody 609-IgG4 and the trispecific antibody of the present invention.
  • Figure 5A and Figure 5B respectively represent the NR-CE-SDS and R-CE-SDS spectra of monoclonal antibody 609-IgG4
  • Figure 5C and Figure 5D represent the NR-CE-SDS and R - the collection of spectra of CE-SDS
  • Figure 5E and Figure 5F represent the collection of graphs of NR-CE-SDS and R-CE-SDS of 609-302-(44CC)-134-IgG4 respectively
  • Figure 5G and Figure 5H represent respectively 609-302 - NR-CE-SDS and R-CE-SDS profiles of (105CC)-134-IgG4.
  • Fig. 6 is a graph showing the functional activity of the trispecific antibody of the present invention to stimulate an immune response.
  • the hexavalent trispecific antibody of the present invention is a dimer formed by two monomers, wherein each monomer comprises a heavy chain, a first light chain and a second light chain. Specifically, based on the structure of the natural antibody, the structure of the first light chain and the heavy chain is designed and optimized, so that the trispecific antibody of the present invention retains the anti-PD-1, HER-2 and LAG-3 trispecific At the same time, it has similar or even superior biological activity and physicochemical properties to monoclonal antibodies. The present invention has been accomplished on this basis.
  • the term "antibody (Antibody, abbreviated Ab)” and “immunoglobulin G (Immunoglobulin G, abbreviated IgG)” are heterotetrameric glycoproteins with the same structural characteristics, which consist of two identical light chains (L ) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by a constant region consisting of three domains CH1, CH2, and CH3.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end.
  • the constant region of the light chain includes a domain CL; the constant region of the light chain is paired with the CH1 domain of the constant region of the heavy chain.
  • the variable region is paired with the variable region of the heavy chain.
  • the constant regions are not directly involved in the binding of antibodies to 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, IgG4 subtypes; the light chain constant region includes kappa (Kappa) or lambda (Lambda).
  • the heavy and light chains of an antibody are covalently linked together by a disulfide bond between the CH1 domain of the heavy chain and the CL domain of the light chain, and the two heavy chains of the antibody are linked together by an interpolypeptide disulfide formed between the hinge regions. bonded together covalently.
  • trispecific antibody refers to an antibody molecule that can specifically bind three antigens (targets) or three epitopes at the same time. Based on symmetry, trispecific antibodies can be divided into structurally symmetric and asymmetric molecules. According to the number of binding sites, trispecific antibodies can be divided into trivalent, tetravalent and multivalent molecules.
  • the term "monoclonal antibody (mAb)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few possible naturally occurring mutations. Monoclonal antibodies are highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which are typically a mixture of different antibodies directed against different antigenic determinants. In addition to their specificity, the benefit of monoclonal antibodies is that they can be synthesized by hybridoma culture without contamination by other immunoglobulins.
  • the modifier "monoclonal” indicates the identity of the antibody, which is obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring any particular method for producing the antibody.
  • humanized means that its CDRs are derived from non-human (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies.
  • framework region residues can be altered 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 fragment consists of the VH and CH1 of the heavy chain and the VL and CL domains of the light chain of an antibody.
  • the Fc segment is the crystallizable fragment (fragment crystallizable, Fc), which consists of the CH2 and CH3 domains of the antibody.
  • the Fc segment has no antigen-binding activity and is the site where the antibody interacts with effector molecules or cells.
  • variable means that certain parts of the variable regions in antibodies differ in sequence, which contribute to the binding and specificity of various specific antibodies to their specific antigens.
  • variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the heavy-chain variable region and the light-chain variable region.
  • CDRs complementarity-determining regions
  • FR frame region
  • the variable domains of native heavy and light chains each contain four FR regions that are roughly in a ⁇ -sheet configuration connected by three CDRs that form connecting loops, in some cases forming partial ⁇ -sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)).
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, referred to as FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain may thus 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 thus 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 human antibody FR derivative is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, or 96%. , 97%, 98%, or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • linker or “peptide linker” or refers to an insertion into an immunoglobulin domain that provides sufficient mobility for the domains of the light and heavy chains to fold into an exchanged dual variable region immunoglobulin. one or more amino acid residues.
  • preferred linkers refer to linkers L1, L2 and L3, wherein L1 connects the heavy chain variable region of the first antibody with the heavy chain variable region of the second antibody, and L2 connects the heavy chain variable region of the second antibody. The variable region and the heavy chain variable region of the third antibody, L3 connects the light chain variable region of the first antibody and the light chain variable region of the second antibody.
  • linkers examples 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 achieved in the linker.
  • the trispecific antibody of the present invention is a hexavalent trispecific antibody against PD-1, HER-2 and LAG-3, comprising anti-PD-1 antibody part, anti-HER-2 antibody part and anti-LAG-3 antibody part.
  • the sequence of the anti-PD-1 antibody of the present invention is as described in the patent application WO 2018/137576 A1, and those skilled in the art can also modify or transform the anti-PD-1 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, so as to further increase the affinity or structural stability of anti-PD-1, and obtain the modified or modified results through conventional assay methods.
  • the trispecific antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the trispecific antibody of the present invention, there are at most 10, preferably at most 8, and more preferably at most 5, preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservative variant polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the sequence information of the obtained antibody is shown in Table 1 below.
  • any amino acid sequence in the above amino acid sequence also includes at least one addition, deletion, modification and/or substitution (such as 1-5, 1-3, preferably 1-2, more preferably 1 ) amino acid derived sequences with PD-1, HER-2 and LAG-3 binding affinity.
  • the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90% %, optimally at least 95% of the amino acid sequence.
  • the number of amino acids added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, Optimally 1.
  • substitutions include, in the trispecific antibody 609VH-302VH-134-IgG4 of the present invention, replacing the G at position 44 of the heavy chain variable region of 302 with C; replacing the The 100th Q in the light chain variable region of 302 is mutated to C;
  • the Q at position 105 of the 302 heavy chain variable region in 609VH-302VH-134-IgG4 was mutated to C; the Q at position 43 of the 302 light chain variable region in 609VL-302VL was mutated to C.
  • 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 its target antigen.
  • the antibody binds the antigen with an equilibrium dissociation constant (KD) of less than about 10" 7 M, eg, less than about 10" 8 M, 10" 9 M, 10" 10 M, 10" 11 M or less.
  • KD equilibrium dissociation constant
  • the term “KD” refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an 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
  • the term "valency" refers to the presence of a specified number of antigen-binding sites in an antibody molecule.
  • the trispecific antibody of the invention has six antigen-binding sites and is hexavalent.
  • 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.
  • An epitope of the present invention is a region of an antigen bound by an antibody.
  • first light chain refers to a light chain comprising the light chain variable region of an antibody directed against a different target.
  • said "first light chain” comprises the light chain variable region of an antibody directed against a first target and the light chain variable region of an antibody directed against a second target, which is capable of interacting with the heavy chain of an antibody directed against the first target.
  • the variable region and the antibody heavy chain variable region for the second target pair to form a first binding site that specifically binds the first target and a second binding site that specifically binds the second target.
  • the "first light chain” in the present invention comprises two antibody light chain variable regions directed against different targets connected by a linker or directly.
  • the "first light chain” in the present invention can be paired with the heavy chain at different positions through flexible linkers or bonds.
  • the "first light chain” in the present invention can be paired with the heavy chain of the trispecific antibody through a non-covalent bond or a covalent bond.
  • the "first light chain” in the present invention is paired with the heavy chain through a covalent bond (such as a disulfide bond).
  • the trispecific antibodies of the invention can be used alone, or can be combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, or a combination of any of the above.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • 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), phage, yeast plasmid or other vectors.
  • a viral vector such as adenovirus, retrovirus
  • phage phage
  • yeast plasmid yeast plasmid or other vectors.
  • Representative examples include but are not limited to: pTT5, pSECtag series, pCGS3 series, pcDNA series vectors, and other vectors used in mammalian expression systems.
  • Expression vectors include fusion DNA sequences linked to appropriate transcriptional and translational regulatory sequences.
  • recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • host cell refers to the cell that is suitable for expressing above-mentioned expression vector, can be eukaryotic cell, as mammalian or insect host cell culture system all can be used for the expression of fusion protein of the present invention, CHO (Chinese hamster Ovary, Chinese Hamster Ovary), HEK293, COS, BHK and derivative cells of the above cells are applicable to the present invention.
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • routes including (but not limited to): intravenous injection, intravenous infusion, subcutaneous injection, local injection, intramuscular injection, intratumoral injection, intraperitoneal injection (such as intraperitoneal injection) ), intracranial injection, or intracavitary injection.
  • the term "pharmaceutical composition” means that the hexavalent trispecific antibody of the present invention can form a pharmaceutical preparation composition together with a pharmaceutically acceptable carrier so as to exert a more stable therapeutic effect. Conformational integrity of the amino acid core sequence of an antibody or antigen-binding fragment thereof or a hexavalent trispecific antibody that binds human PD-1, 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 hexavalent trispecific antibody (or its conjugate) of the present invention ) and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to: saline, buffer, dextrose, 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, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the hexavalent trispecific antibodies of the invention can also be used with other therapeutic agents.
  • a safe and effective amount of the hexavalent trispecific antibody or immunoconjugate thereof is administered to the mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g/kg body weight, and in most cases not More than about 50 mg/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the invention also provides immunoconjugates based on the trispecific antibodies of the invention.
  • the immunoconjugate comprises the antibody, and an effector molecule to which the antibody is conjugated, preferably chemically.
  • the effector molecule is preferably a drug with therapeutic activity.
  • the effector molecule may be one or more of toxic proteins, chemotherapeutic drugs, small molecule drugs or radionuclides.
  • the effector molecules of the trispecific antibody of the present invention may be coupled through a coupling agent.
  • the coupling agent may be any one or more of non-selective coupling agents, coupling agents using carboxyl groups, peptide chains, and coupling agents using disulfide bonds.
  • the non-selective coupling agent refers to a compound that makes the effector molecule and the antibody form a covalent bond, such as glutaraldehyde and the like.
  • the coupling agent using carboxyl group can be any one or more of cis-aconitic anhydride coupling agents (such as cis-aconitic anhydride) and acyl hydrazone coupling agents (coupling site is acyl hydrazone).
  • antibodies such as Cys or Lys, etc.
  • imaging reagents such as chromophores and fluorescent groups
  • diagnostic reagents such as MRI contrast agents and radioisotopes
  • stabilizers such as glycol polymers
  • therapeutic agents eg, drugs, detection reagents, stabilizers
  • An antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate.
  • Functional agents eg, drugs, detection reagents, stabilizers
  • a functional agent can be attached to the antibody directly, or indirectly through a linker.
  • Antibodies can be coupled to drugs to form antibody-drug conjugates (ADCs).
  • ADCs comprise a linker between the drug and the antibody.
  • Linkers can be degradable or non-degradable linkers.
  • Degradable linkers are typically susceptible to degradation in the intracellular environment, eg, at the site of interest where the linker degrades, thereby releasing the drug from the antibody.
  • Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases, such as lysosomal proteases or endosomal proteases, or carbohydrate linkers, for example, that can be degraded by glucuronides.
  • Peptidyl linkers may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
  • Other suitable degradable linkers include, for example, pH-sensitive linkers (eg, linkers that hydrolyze at pH less than 5.5, eg, hydrazone linkers) and linkers that degrade under reducing conditions (eg, disulfide linkers).
  • Nondegradable linkers typically release the drug under conditions where the antibody is hydrolyzed by a protease.
  • the linker Before linking to the antibody, the linker has an active reactive group that can react with certain amino acid residues, and the connection is realized through the active reactive group.
  • Sulfhydryl-specific reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodo, bromo, or chloro); haloesters (e.g., iodo, bromo, or chloro ); halomethyl ketones (such as iodo, bromo or chloro), benzyl halides (such as iodo, bromo or chloro); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6- di-(mercurymethyl)dioxane with the counterion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate.
  • Linkers can include, for example, maleimide attached to the
  • the drug can be any cytotoxic, cytostatic or immunosuppressive drug.
  • the linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker.
  • a drug may have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group that can form a bond with a linker.
  • the drug has reactive reactive groups prior to attachment to the antibody.
  • Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors , Vinca alkaloids, etc.
  • particularly useful classes of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins, (camptothecins), duocarmycins/duocarmycins, etoposides, maytansines and maytansinoids (such as DM1 and DM4), taxanes ( taxanes), benzodiazepines, or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines (indolinobenzodiazepines) and oxazolidinbenzodiazepines (oxazolidinobenzodiazepines)) and vinca alkaloids (vinca alkaloids).
  • auristatins camptothecins, (camptothecins), duocarmycins/duocarmycins, etoposides
  • maytansines and maytansinoids such as DM1 and DM4
  • taxanes taxanes
  • drug-linker can be used to form ADC in one simple step.
  • bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine residue reacts with the reactive portion of the linker in a first step, and in a subsequent step, a functional group on the linker reacts with the drug, thereby forming the ADC.
  • the functional group on the linker is chosen to facilitate specific reaction with an appropriate reactive group on the drug moiety.
  • an appropriate reactive group on the drug moiety As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on drug moieties.
  • the drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and the alkynyl.
  • Other useful functional groups include, for example, ketones and aldehydes (suitable for reactions with hydrazides and alkoxyamines), phosphines (suitable for reactions with azides); isocyanates and isothiocyanates (suitable for reactions with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reactions with amines and alcohols).
  • ketones and aldehydes suitable for reactions with hydrazides and alkoxyamines
  • phosphines suitable for reactions with azides
  • isocyanates and isothiocyanates suitable for reactions with amines and alcohols
  • activated esters such as N-hydroxysuccinimide esters (suitable for reactions with amines and alcohols).
  • the present invention also provides a method for preparing ADC, which may further include: combining the antibody with the drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).
  • the methods of the invention comprise: conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the invention further comprise: attaching the antibody-linker conjugate to the drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.
  • the antibody drug conjugate ADC has the following molecular formula:
  • Ab is an antibody
  • LU is the linker
  • D is for drugs
  • the present invention provides a hexavalent trispecific antibody with a novel structure
  • the trispecific antibody of the present invention does not require Fc modification, does not cause mismatch problems, and has a simple and convenient preparation method.
  • the trispecific antibody of the present invention While retaining the anti-PD-1, HER-2 and LAG-3 trispecificity, the trispecific antibody of the present invention has similar or even superior biological activity and physicochemical properties to monoclonal antibodies.
  • the target gene is constructed into the expression vector pcDNA3.4, and the constructed expression vector or combination of expression vectors is transferred into FreeStyle TM 293-F Cells cells using PEI (Polyethyleneimine) (hereinafter referred to as HEK293F, purchased from Thermo Fisher Scientific) 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 the cell supernatant was harvested, and then protein A affinity Antibodies were purified by chromatography.
  • ELISA detection method used in the following examples is described as follows: the corresponding recombinant protein is used to coat the microwell plate, and the microplate is blocked with PBST containing 1% bovine serum albumin (PBST is phosphate buffer saline containing 0.05% Tween-20). orifice plate.
  • PBST phosphate buffer saline containing 0.05% Tween-20
  • the antibody to be tested was serially diluted, and then transferred to the microwell plate coated with the recombinant protein, incubated at room temperature for half an hour and then washed; an appropriate dilution of HRP (Horseradish Peroxidase)-labeled goat anti-human antibody (Fc specific, purchased from From Sigma), wash the plate after incubating at room temperature for half an hour; add 100 ⁇ l of TMB (3,3′,5,5′-Tetramethylbenzidine)-based chromogenic solution to each well, and incubate at room temperature for 1-5 min; add 50 ⁇ l of stop solution ( 2M H 2 SO 4 ) to terminate the reaction; the OD450 was read with a microplate reader (SpectraMax 190), and the graphing and data analysis were performed with GraphPad Prism7, and the EC50 was calculated.
  • Antibodies are high molecular weight proteins with highly complex secondary and tertiary structures. Antibodies are heterogeneous in biochemical and biophysical properties due to changes such as post-translational modifications, aggregation, and degradation. When trispecific antibodies are analyzed by separation techniques, variants, aggregates, and degraded fragments are commonly observed and their presence may compromise safety and efficacy. Aggregates, degraded fragments, and incompletely assembled molecules are prone to occur during antibody production and storage.
  • 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 chromatograph used for HPLC-SEC is Dionex Ultimate 3000; the mobile phase preparation method is as follows: take an appropriate amount of 20mM sodium dihydrogen phosphate mother liquor, adjust the pH to 6.8 ⁇ 0.1 with 20mM disodium hydrogen phosphate; injection volume: 20 ⁇ g; the 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 chamber temperature is 10°C; the detection wavelength is 214nm.
  • charge variants can be separated and analyzed based on their charge.
  • the commonly used analytical methods are cation exchange chromatography (CEX) and anion exchange chromatography (AEX).
  • CEX cation exchange chromatography
  • AEX anion exchange chromatography
  • Acidic species are variants that elute with a main peak earlier than CEX or later than AEX, while basic species are variants that elute with a main peak later than CEX or earlier than AEX.
  • the peaks corresponding to acidic species and basic species are called acidic peaks and basic peaks, respectively.
  • Charge variants are easily generated during antibody production and storage.
  • high-performance liquid chromatography-ion exchange chromatography HPLC-IEC was used to analyze the charge heterogeneity of the samples.
  • the chromatograph used for HPLC-IEC is Dionex Ultimate 3000; mobile phase A: 20mM PB pH6.3, mobile phase B: 20mM PB+200mM NaCl pH6.3, the mixing ratio of the two mobile phases changes with time according to the preset program , flow rate 1.0ml/min; chromatographic column: Thermo PropacTM WCX-10; column temperature 30°C, sample chamber temperature 10°C; injection volume: 20 ⁇ g; detection wavelength: 214nm.
  • the present invention uses CE-SDS (Capillary Electrophoresis-Sodium Dodecyl Sulfate) to analyze the content of degraded fragments or incompletely assembled molecules in a 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 intramolecular disulfide bonds during denaturation, while the samples used for the latter need to use the reducing agent DTT to destroy the disulfide bonds during denaturation.
  • Intramolecular disulfide bonds are broken.
  • Non-reducing and reducing CE-SDS are denoted as NR-CE-SDS and R-CE-SDS, respectively.
  • the Maurice CE-SDS analysis system used was purchased from ProteinSimple, equipped with a UV 214nm detector.
  • 609-IgG4 is an anti-PD-1 humanized monoclonal antibody, and its heavy chain and light chain amino acid sequences are from SEQ ID NO: 8 and SEQ ID in WO 2018/137576A1 NO: 10 (i.e. SEQ ID NOs in the present invention: 5 and 6).
  • the amino acid sequences of the heavy chain variable region and the light chain variable region of 609-IgG4 are shown in SEQ ID NO: 1 and 2.
  • the constant region of the heavy chain of 609-IgG4 is human IgG4 (SEQ ID NO: 3, the hinge region contains S228P mutation), and the constant region of the light chain is human Kappa (SEQ ID NO: 4).
  • 609-IgG4-HC and 609-IgG4-LC genes encoding the heavy chain and light chain of 609-IgG4 are named 609-IgG4-HC and 609-IgG4-LC, respectively.
  • the 609-IgG4-HC and 609-IgG4-LC genes were respectively constructed into the pcDNA3.4 expression vector, the two vectors were combined to express and purify the antibody, and the resulting antibody was named 609-IgG4.
  • the heavy chain variable region and the light chain variable region are connected to the human IgG1 heavy chain constant region (SEQ ID NO: 9) and the human Kappa light chain constant region (SEQ ID NO: 4), respectively, to obtain full-length heavy chain and Light chain amino acid sequence (SEQ ID NO: 10 and 11).
  • the genes encoding the full-length heavy chain and light chain are named 302-HC and 302-LC, respectively.
  • the coding genes of 302-HC and 302-LC were respectively constructed into the pcDNA3.4 expression vector, the two vectors were combined to express and purify the antibody, and the resulting antibody was named 302-IgG1.
  • Human IgG1 heavy chain constant region amino acid sequence (SEQ ID NO: 9):
  • the heavy chain amino acid sequence of 302-IgG1 (SEQ ID NO: 10):
  • 134-Hu-IgG4-C91S is an anti-human LAG-3 humanized monoclonal antibody, and its heavy chain and light chain amino acid sequences are from the SEQ ID NO in WO 2020/173378 A1: 32 and SEQ ID NO: 36 (i.e. SEQ ID NOs in the present invention: 12 and 13).
  • the heavy chain constant region of 134-IgG4 monoclonal antibody is human IgG4 (the hinge region contains S228P mutation), and the light chain constant region is human Kappa.
  • the genes encoding the heavy chain and light chain of 134-IgG4 are named 134-IgG4-HC and 134-IgG4-LC, respectively.
  • 134-IgG4-HC and 134-IgG4-LC were respectively constructed into the pcDNA3.4 expression vector, and the antibody was expressed and purified after the combination of the two vectors, and the resulting antibody was named 134-IgG4.
  • Amino acid sequence of 134 humanized heavy chain 134-IgG4-HC (SEQ ID NO: 12):
  • Amino acid sequence of 134 humanized light chain 134-IgG4-LC (SEQ ID NO: 13):
  • the heavy chain variable region of 609-IgG4 was connected to the heavy chain variable region of 302-IgG1 through an artificial linker (three GGGGS in series), and then connected to the human 134-IgG4 through an artificial linker (three GGGGS in series).
  • Heavy chain (hinge region contains S228P mutation) the long heavy chain gene containing three heavy chain variable regions constructed by this program is named 609VH-302VH-134-IgG4 (SEQ ID NO: 14).
  • the light chain variable region of 609 was connected to the light chain variable region of 302-IgG1 through an artificial linker (three GGGGS in series), and the long light chain gene containing two light chain variable regions constructed by this procedure was named is 609VL-302VL (SEQ ID NO: 15).
  • the coding genes of the above sequences were respectively constructed into the pcDNA3.4 expression vector, and the expression vectors of 609VH-302VH-134-IgG4, 609VL-302VL and 134-IgG4-LC were combined to express and purify the antibody, and the obtained antibody was named 609- 302-134-IgG4.
  • the G at position 44 of the 302 heavy chain variable region in 609VH-302VH-134-IgG4 was mutated to C, and the resulting sequence was named 609VH-302VH(44C)-134-IgG4; at the same time, the 609VL-302VL
  • the 100-position Q of the 302 light chain variable region was mutated to C, and the resulting sequence was named 609VL-302VL (100C);
  • the Q at position 105 of the 302 heavy chain variable region in 609VH-302VH-134-IgG4 was mutated to C, and the resulting sequence was named 609VH-302VH(105C)-134-IgG4; at the same time, the 609VL-302VL
  • the 43-position A of the 302 light chain variable region was mutated to C, and the resulting sequence was named 609VL-302VL (43C);
  • the coding genes of the above sequences were respectively constructed into pcDNA3.4 expression vectors, and after the corresponding vectors were combined (combination of 609VH-302VH-134-IgG4, 609VL-302VL and 134-IgG4-LC, 609VH-302VH(44C)-134- IgG4, 609VL-302VL (100C) and 134-IgG4-LC combination, 609VH-302VH (105C)-134-IgG4, 609VL-302VL (43C) and 134-IgG4-LC combination) expressed and purified antibodies, and the obtained antibodies were named respectively are 609-302-134-IgG4, 609-302-(44CC)-134-IgG4 and 609-302-(105CC)-134-IgG4.
  • Antibody name in the present invention is as shown in table 2:
  • Embodiment 1.3 ELISA measures relative affinity
  • HER2 extracellular segment recombinant protein with polyhistidine tag referred to as HER2-His, purchased from Sino Biological Inc.
  • PD-1 extracellular segment recombinant protein referred to as PD1-His, purchased from From Sino Biological Inc.
  • LAG-3 extracellular segment recombinant protein referred to as LAG3-His, purchased from ACROBiosystems coated microwell plate, the coating concentration was 20ng/well, 20ng/well and 10ng/well, respectively.
  • 609-302-134-IgG4, 609-302-(44CC)-134-IgG4 and 609-302-(105CC)-134-IgG4 can effectively bind PD-1, HER-2 and LAG-3, which indicates that they are trispecific antibodies, the EC50s are summarized in Table 3.
  • Embodiment 1.4 Characterization of physical and chemical properties
  • Figure 3A shows the HPLC-SEC pattern of monoclonal antibody 609-IgG4, the main peak accounts for 99.87%.
  • Figure 3B shows the HPLC-SEC spectrum of 609-302-134-IgG4, the main peak accounts for 98.24%.
  • Figure 3C shows the HPLC-SEC pattern of 609-302-(44CC)-134-IgG4, the main peak accounts for 98.4%.
  • Figure 3D shows the HPLC-SEC spectrum of 609-302-(105CC)-134-IgG4, the main peak accounts for 98.06%.
  • Figure 4A shows the HPLC-IEC spectrum of monoclonal antibody 609-IgG4, the main peak accounts for 71.45%.
  • Figure 4B shows the HPLC-IEC spectrum of 609-302-(44CC)-134-IgG4, the main peak accounts for 71.62%.
  • Figure 4C shows the HPLC-IEC spectrum of 609-302-(105CC)-134-IgG4, the main peak accounts for 66.12%.
  • Figure 5A and Figure 5B show the NR-CE-SDS and R-CE-SDS spectra of mAb 609-IgG4 respectively, the main peak Peak2.672 in the NR-CE-SDS spectrum accounts for 98.9%; the two peaks in the R-CE-SDS spectrum The two main peaks, Peak1.499 (corresponding to light chain) and Peak1.888 (corresponding to heavy chain), accounted for 31.3% and 67.7% respectively, and the sum of the two main peaks accounted for 99.0%.
  • Figure 5C and Figure 5D show the spectra of NR-CE-SDS and R-CE-SDS of 609-302-134-IgG4 respectively, and the two main peaks Peak1.525 and Peak2.893 in the NR-CE-SDS spectrum accounted for 18.6 % and 80.8%, 609VL-302VL did not form a covalent disulfide bond with 609VH-302VH-134-IgG4 in the 609-302-134-IgG4 molecule, so it is considered that the polypeptide chain corresponding to Peak1.525 is 609VL-302VL; R- In the CE-SDS spectrum, the two main peaks, Peak1.518 (corresponding to the light chain) and Peak2.158 (corresponding to the heavy chain), accounted for 34.8% and 62.5% respectively, and the sum of the two main peaks accounted for 97.3%.
  • Figure 5E and Figure 5F represent the spectra of NR-CE-SDS and R-CE-SDS of 609-302-(44CC)-134-IgG4 respectively, the main peak Peak3.018 in the NR-CE-SDS spectra accounts for 96.4%; R - In the CE-SDS spectrum, the two main peaks, Peak1.515 (corresponding to the light chain) and Peak2.159 (corresponding to the heavy chain), accounted for 34.9% and 62.0% respectively, and the sum of the two main peaks accounted for 96.9%.
  • Figure 5G and Figure 5H show the spectra of NR-CE-SDS and R-CE-SDS of 609-302-(105CC)-134-IgG4 respectively, and the main peak Peak3.020 in the NR-CE-SDS spectrum accounts for 91.8%; R - In the CE-SDS spectrum, the two main peaks, Peak1.508 (corresponding to the light chain) and Peak2.147 (corresponding to the heavy chain), accounted for 36.6% and 56.9% respectively, and the sum of the two main peaks accounted for 93.5%.
  • Example 2 Determination of the functional activity of the trispecific antibody of the present invention to stimulate the immune response
  • RPMI 1640 10% fetal bovine serum; 1% MEM Non-Essential Amino Acids Solution; 1% Sodium Pyruvate; 1% HEPES; 1 ⁇ 2-Mercaptoethanol; 1% Penicillin-Streptomycin; 1% GlutaMAX (above Media and supplements were purchased from Thermo Fisher Scientific). Freshly isolated human peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC.
  • PBMC Peripheral Blood Mononuclear Cell
  • PBMC purchased from Allcells, product number: PB005-C
  • PB005-C washed and resuspended with the above RPMI 1640 complete medium, and a certain amount of superantigen Staphylococcus aureus was added Enterotoxin B (staphylococcal enterotoxin B, SEB).
  • SEB was prepared in-house, the sequence was from Uniprot (Entry: P01552), expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography.
  • PBMC cell suspension Inoculate the PBMC cell suspension into a round-bottom 96-well cell culture plate, with 150 ⁇ l of suspension and 200,000 cells per well; add 50 ⁇ l of serially diluted related antibodies to the above 96-well plate; place the 96-well plate at 37°C Incubate for 4 days in the incubator. Take an appropriate amount of cell culture supernatant from the 96-well plate.
  • the IL-2 in the supernatant was detected by double antibody sandwich method (sandwich ELISA) (the paired antibodies for related detection were purchased from BD Biosciences).
  • OD450 was read with a microplate reader (SpectraMax 190), and GraphPad Prism7 was used for data analysis, graphing and calculation of EC 50 .
  • Antibody EC50 (nM) Top 609-IgG4 0.04091 5363 134-IgG4 0.8398 3369 609-302-(44CC)-134-IgG4 0.3217 10281 609-302-(105CC)-134-IgG4 0.1834 8795
  • 609-IgG4 exhibited a smaller EC 50 and a higher Top (high platform) than 134-IgG4, indicating that 609-IgG4 had higher functional activity than 134-IgG4.
  • 609-302-(44CC)-134-IgG4 and 609-302-(105CC)-134-IgG4 exhibited similar EC 50 and Top, indicating that the functional activities of these two trispecific antibodies are comparable.
  • 609-302-(44CC)-134-IgG4 and 609-302-(105CC)-134-IgG4 exhibited a higher Top, roughly when the antibody concentration was greater than 1nM (ie, the Log value was greater than 0 ), the ability of the two trispecific antibodies to stimulate PBMC to secrete IL-2 was significantly stronger than that of the monoclonal antibodies 609-IgG4 and 134-IgG4.

Landscapes

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

Abstract

提供了一种六价三特异性抗体，该六价三特异性抗体为二个单体形成的二聚体，其中每个单体包含重链、第一轻链和第二轻链，重链从N端至C端包括串联的针对第一靶标的抗体重链可变区元件Z1、针对第二靶标的抗体重链可变区元件Z2、针对第三靶标的抗体重链可变区元件Z3以及抗体重链恒定区Z4；第一轻链从N端至C端包括串联的针对第一靶标的抗体轻链可变区元件Z5和针对第二靶标的抗体轻链可变区元件Z6；第二轻链从N端至C端包括串联的针对第三靶标的抗体轻链可变区元件Z7和轻链恒定区Z8；第一轻链和第二轻链分别与重链配合，使得六价三特异性抗体特异性结合于第一靶标、第二靶标和第三靶标。第一靶标、第二靶标和第三靶标可以是PD-1、HER-2和LAG-3。

Description

三特异性抗体、其制备方法和用途 技术领域
本发明涉及抗体领域，具体地，涉及一种三特异性抗体、其制备方法和用途。
背景技术
抗PD-1/PD-L1单克隆抗体是最早获得广泛使用的癌症免疫疗法。随后，双特异性抗体作为癌症免疫疗法出现在人们的视野里，这些抗体的一端可以与癌细胞表面的抗原结合，另一端与T细胞表面的CD3结合，招募并激活T细胞杀伤癌细胞(例如安进公司的Blinatumomab)。Blinatumomab是一款同时靶向癌细胞表面的CD19抗原和T细胞表面CD3受体的双特异性抗体，它在治疗晚期B细胞急性淋巴细胞白血病(B-ALL)患者时，能够将患者的缓解率和生存期翻倍。这类双特异性抗体能够有效激活T细胞的抗癌活性。
然而，通常T细胞被激活时，不但CD3介导的信号通路被激活，一种称为CD28的共刺激受体介导的信号通路也被激活。这种双重激活能够保证T细胞维持更长久的抗肿瘤活性。近日在Nature Cancer杂志上发表的一项研究中(参考文献：Wu et al.,(2019).Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.Nature Cancer,https://doi.org/10.1038/s 43018-019-0004-z)，赛诺菲(Sanofi)公司的研发团队开发出一款三特异性抗体，它不但能够与肿瘤相关抗原和T细胞CD3相结合，还能够与T细胞表面的CD28相结合，从而增强T细胞的抗癌活性。激活CD28受体能够刺激Bcl-xL蛋白的表达，Bcl-xL可以防止T细胞凋亡，从而延长T细胞的活性。CD28作为共刺激受体已经在CAR-T疗法中得到应用。某些CAR-T细胞表达的受体包含着CD3和CD28的共刺激蛋白域。在体外试验中，与已经获得批准的抗CD38单克隆抗体Daratumumab相比，三特异性抗体裂解的癌细胞比例提高了3-4倍。而且在小鼠的多发性骨髓瘤模型中，这款三特异性抗体也能够剂量依赖性缩小移植到小鼠体内肿瘤的大小。
研究人员仍然需要检验这一创新抗体的安全性，刺激T细胞活性的癌症免疫疗法的常见副作用是细胞因子释放综合征，这是由于T细胞被过度激活而造成的。在非人灵长类模型中，这款三特异性疗法表现出可控的安全性，不过它的安全性需要在人体和MM(多发性骨髓瘤)患者中得到验证。
在《Nature》发表的评论(参考文献：Garfall and June.(2019).Trispecific antibodies  offer a third way forward for anticancer immunotherapy.Nature,doi:10.1038/d41586-019-03495-3)表示，多发性骨髓瘤患者一直需要新的治疗手段，因为即使是像CAR-T这样有效的治疗手段，也只能给大多数患者带来暂时的缓解。三特异性抗体可能提供一种灵活的平台，根据肿瘤的微环境递送精准的免疫调节信号组合，从而比由三种单特异性抗体构成的组合疗法更为安全和有效。
此外，Numab在US20180355024A1中公开了其三特异性抗体的设计方法，其特点是由多个ScFv片段和Fv组成。Xencor公司在US20190352416A1中公开了其三特异性抗体的设计方法，其特点是使用了重链异源二聚化技术和ScFv片段。
除了上述产品和涉足公司外，另外还有一些早期研究曾发表在国际知名期刊上。在2017年，一项发表在《Science》期刊上的研究(参考文献：Liang Xu et al.Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques.Science,Published online:20 Sep 2017,doi:10.1126/science.aan8630.)显示，科学家们已在实验室开发出一种三特异性抗体，可以让猴子免受两种人猴嵌合免疫缺陷病毒(SHIV)菌株的感染。据介绍，这种三特异性抗体将广泛中和HIV抗体VRC01、PGDM1400和10E8v4的独特结构结合在一起，三种HIV结合片段源自于三种天然的抗体，每种天然的抗体都强效地中和很多HIV毒株。使用三特异性抗体治疗癌症将是非常重要的概念性突破。由于三特异性抗体是一个灵活的载体，可以在肿瘤微环境中特异性地传递免疫调节信号组合，这可能比多个特异性免疫调节单抗的组合更安全有效。因此，这种组合策略有望提高目前免疫疗法的精准性和疗效，并进一步扩大免疫疗法的适用范围。三特异性抗体能够同时结合三种不同靶标，有望为开发针对HIV感染、其他的传染病、自身免疫疾病和癌症的治疗方法提供有力基础。
因此，本领域需要开发新型有效的三特异性抗体。
发明内容
本发明的目的在于提供一种三特异性抗体、其制备方法和用途。
在本发明的第一方面，提供了一种六价三特异性抗体，所述的六价三特异性抗体为二个单体形成的二聚体，其中，每个单体包含重链、第一轻链和第二轻链；
所述的重链从N端至C端包括串联的针对第一靶标的抗体重链可变区元件Z1、针对第二靶标的抗体重链可变区元件Z2、针对第三靶标的抗体重链可变区元件Z3以及抗体重链恒定区Z4；
所述的第一轻链和第二轻链分别与所述重链配合，使得所述的六价三特异性抗体特异性结合于第一靶标、第二靶标和第三靶标。
在另一优选例中，所述的第一轻链从N端至C端包括串联的针对第一靶标的抗体轻链可变区元件Z5以及针对第二靶标的抗体轻链可变区元件Z6。
在另一优选例中，所述的第二轻链从N端至C端包括串联的针对第三靶标的抗体轻链可变区元件Z7以及抗体轻链恒定区Z8。
在另一优选例中，所述的抗体重链恒定区Z4包括CH1区、CH2区和CH3区。
在另一优选例中，所述的第一轻链与抗体重链可变区元件Z1和抗体重链可变区元件Z2配合；并且所述的第二轻链与抗体重链可变区元件Z3配合。
在另一优选例中，所述的第二轻链还进一步与抗体重链恒定区Z4中的CH1区配合。
在另一优选例中，所述的抗体重链恒定区Z4来源于靶向第一靶标的抗体、靶向第二靶标的抗体、或靶向第三靶标的抗体。
在另一优选例中，所述的抗体重链可变区元件Z1、抗体重链可变区元件Z2、抗体重链可变区元件Z3和抗体重链恒定区Z4各自独立地为全人的或人源化的。
在另一优选例中，所述的抗体轻链可变区元件Z5、抗体轻链可变区元件Z6、抗体轻链可变区元件Z7和抗体轻链恒定区Z8各自独立地为全人的或人源化的。
在另一优选例中，在所述的抗体重链可变区元件Z1、抗体重链可变区元件Z2、抗体重链可变区元件Z3和抗体重链恒定区Z4中相邻的两个元件可直接相连或通过接头连接。
在另一优选例中，所述的抗体轻链可变区元件Z5和抗体轻链可变区元件Z6可直接相连或通过接头连接。
在另一优选例中，所述的抗体轻链可变区元件Z7和抗体轻链恒定区Z8可直接相连或通过接头连接。
在另一优选例中，所述的接头包括柔性接头和刚性接头。
在另一优选例中，所述的接头为长度为1-35个氨基酸的肽接头，较佳地6-30个氨基酸的肽接头。
在另一优选例中，所述第一靶标、第二靶标和第三靶标为PD-1、HER-2和LAG-3。
在另一优选例中，第一靶标为PD-1，第二靶标为HER-2，第三靶标为LAG-3。
在另一优选例中，第一靶标为HER-2，第二靶标为PD-1，第三靶标为LAG-3。
在另一优选例中，第一靶标为PD-1，第二靶标为LAG-3，第三靶标为HER-2。
在另一优选例中，第一靶标为LAG-3，第二靶标为PD-1，第三靶标为HER-2。
在另一优选例中，第一靶标为HER-2，第二靶标为LAG-3，第三靶标为PD-1。
在另一优选例中，第一靶标为LAG-3，第二靶标为HER-2，第三靶标为PD-1。
在另一优选例中，所述六价三特异性抗体包含重链、第一轻链和第二轻链；其中，每个所述的重链具有下式I的结构：
Z1-Z2-Z3-Z4     (I)
Z1为针对第一靶标的重链可变区元件；
Z2为针对第二靶标的重链可变区元件；
Z3为针对第三靶标的重链可变区元件；
Z4为抗体重链恒定区CH1、CH2和CH3；
“-”各自独立地为键或接头(linker)；
其中，所述的第一轻链和第二轻链分别与重链配合，使得所述的六价三特异性抗体特异性结合于第一靶标、第二靶标和第三靶标。
在另一优选例中，所述的第一轻链与Z1和Z2配合，而第二轻链与Z3配合。
在另一优选例中，所述的第一轻链还与Z2通过二硫键连接。
在另一优选例中，所述的第二轻链与Z3和CH1配合，并且第二轻链还与CH1通过二硫键连接。
在另一优选例中，所述第一轻链具有下式II的结构：
Z5-Z6  (II)；
其中，Z5为针对第一靶标的轻链可变区元件；Z6为针对第二靶标的轻链可变区元件。
在另一优选例中，所述第二轻链具有下式III的结构：
Z7-Z8  (III)；
其中，Z7为针对第三靶标的轻链可变区元件和Z8为轻链恒定区。
在另一优选例中，第一靶标为PD-1，第二靶标为HER-2，第三靶标为LAG-3。
在另一优选例中，第一靶标为HER-2，第二靶标为PD-1，第三靶标为LAG-3。
在另一优选例中，第一靶标为PD-1，第二靶标为LAG-3，第三靶标为HER-2。
在另一优选例中，第一靶标为LAG-3，第二靶标为PD-1，第三靶标为HER-2。
在另一优选例中，第一靶标为HER-2，第二靶标为LAG-3，第三靶标为PD-1。
在另一优选例中，第一靶标为LAG-3，第二靶标为HER-2，第三靶标为PD-1。
在另一优选例中，所述的第一轻链具有选自下组的氨基酸序列：SEQ ID NO：15、17或19。
在另一优选例中，所述的第二轻链具有如SEQ ID NO：13所示的氨基酸序列。
在另一优选例中，所述重链具有选自下组的氨基酸序列：SEQ ID NO：14、16或18。
在另一优选例中，所述六价三特异性抗体为二聚体。
在另一优选例中，所述六价三特异性抗体为同源或异源二聚体。
在另一优选例中，所述六价三特异性抗体包含两个单体，每个单体含有重链和第一轻链、和第二轻链，其中每个单体具有下式IV结构：
Figure PCTCN2022100105-appb-000001
式中，
VH A-L1-VH B-L2-VH C-CH1-CH2-CH3为重链；
VL A-L3-VL B为第一轻链；
VL C-CL为第二轻链；
VH A为针对第一靶标的重链可变区；
VH B为针对第二靶标的重链可变区；
VH C为针对第三靶标的重链可变区；
CH1、CH2和CH3分别为抗体重链恒定区CH1、CH2和CH3；
VL A为针对第一靶标的轻链可变区；
VL B为针对第二靶标的轻链可变区；
VL C为针对第三靶标的轻链可变区；
CL为抗体的轻链恒定区；
L1、L2、L3各自独立地为接头(linker)；
“-”各自独立地为键；
“～”代表二硫键或共价键；
其中，所述的六价三特异性抗体同时结合于第一靶标、第二靶标和第三靶标。
在另一优选例中，第一靶标为HER-2，第二靶标为PD-1，第三靶标为LAG-3。
在另一优选例中，第一靶标为PD-1，第二靶标为LAG-3，第三靶标为HER-2。
在另一优选例中，第一靶标为LAG-3，第二靶标为PD-1，第三靶标为HER-2。
在另一优选例中，第一靶标为HER-2，第二靶标为LAG-3，第三靶标为PD-1。
在另一优选例中，第一靶标为LAG-3，第二靶标为HER-2，第三靶标为PD-1。
在另一优选例中，所述六价三特异性抗体包含两个单体，每个单体含有重链和第一轻链、和第二轻链，其中每个单体具有下式IV结构：
Figure PCTCN2022100105-appb-000002
式中，
VH A-L1-VH B-L2-VH C-CH1-CH2-CH3为重链；
VL A-L3-VL B为第一轻链；
VL C-CL为第二轻链；
VH A为抗PD-1抗体的重链可变区；
VH B为抗HER-2抗体的重链可变区；
VH C为抗LAG-3抗体的重链可变区；
CH1、CH2和CH3分别为抗体重链恒定区CH1、CH2和CH3；
VL A为抗PD-1抗体的轻链可变区；
VL B为抗HER-2抗体的轻链可变区；
VL C为抗LAG-3抗体的轻链可变区；
CL为抗体的轻链恒定区；
L1、L2、L3各自独立地为接头(linker)；
“-”各自独立地为键；
“～”代表二硫键或共价键；
其中，所述的六价三特异性抗体同时结合于PD-1、HER-2和LAG-3。
在另一优选例中，所述接头为柔性肽接头。
在另一优选例中，所述的柔性肽接头包括6-30个氨基酸，较佳地10-25个氨基酸。
在另一优选例中，所述的柔性肽接头包括2-6个G4S。
在另一优选例中，所述的L1、L2或L3各自独立地为2-4个G4S。
在另一优选例中，所述的重链恒定区为IgG1、IgG2、IgG3或IgG4抗体的重链恒定区，或由其衍生。
在另一优选例中，所述的重链恒定区选自人IgG1或人IgG4的重链恒定区。
在另一优选例中，所述的人IgG4的重链恒定区包含S228P突变。
在另一优选例中，所述的轻链恒定区为IgG1、IgG2、IgG3或IgG4抗体的轻链恒定区，或由其衍生。
在另一优选例中，所述的轻链恒定区选自人IgG1或人IgG4的轻链恒定区。
在另一优选例中，所述的六价三特异性抗体包含两条重链、两条第一轻链和两条第二轻链，
其中，所述重链选自下组：氨基酸序列SEQ ID NO：14、16或18所示的重链；
所述第一轻链选自下组：氨基酸序列如SEQ ID NO：15、17或19所示的第一轻链；和/或
所述的第二轻链选自下组：氨基酸序列如SEQ ID NO：13所示的第二轻链。
在另一优选例中，所述的六价三特异性抗体中的重链、第一轻链和第二轻链的氨基酸序列分别如SEQ ID No:14、15和13所示。
在另一优选例中，所述的六价三特异性抗体中的重链、第一轻链和第二轻链的氨基酸序列分别如SEQ ID No:16、17和13所示。
在另一优选例中，所述的六价三特异性抗体中的重链、第一轻链和第二轻链的氨基酸序列分别如SEQ ID No:18、19和13所示。
在本发明的第二方面，提供了一种分离的核酸分子，所述的核酸分子编码如本发明的第一方面所述的六价三特异性抗体。
在另一优选例中，所述的核酸分子分别编码所述重链和所述第一轻链、第二轻链。
在本发明的第三方面，提供了一种表达载体，所述的表达载体含有如本发明的第二方面所述的核酸分子。
在本发明的第四方面，提供了一种宿主细胞，所述的宿主细胞含有如本发明的第三方面所述的表达载体。
在本发明的第五方面，提供了本发明的第一方面所述的六价三特异性抗体的制备方法，所述方法包含以下步骤：
(a)在表达条件下，培养如本发明的第四方面所述的宿主细胞，从而表达所述的六价三特异性抗体；
(b)分离并纯化(a)所述的六价三特异性抗体。
在本发明的第六方面，提供了一种药物组合物，所述药物组合物含有如本发明的第一方面所述的六价三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。
在另一优选例中，所述药物组合物为单元剂型。
在另一优选例中，所述的抗肿瘤剂可以与所述三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三特异性抗体偶联。
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。
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。
在本发明的第七方面，提供了如本发明的第一方面所述的六价三特异性抗体或如本发明的第六方面所述的药物组合物在制备治疗癌症的药物中的用途。
在另一优选例中，所述癌症选自下组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病。
在本发明的第八方面，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本发明的第一方面所述的六价三特异性抗体、或其免疫偶联物、或如本发明的第六方面所述的药物组合物。
在另一优选例中，所述癌症选自下组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病。
在本发明的第九方面，提供了一种免疫偶联物，所述免疫偶联物包括：
(a)如本发明的第一方面所述的六价三特异性抗体；和
(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。
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。
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)。
在另一优选例中，所述的免疫偶联物用于制备治疗肿瘤的药物组合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于 篇幅，在此不再一一累述。
附图说明
图1为本发明的三特异性抗体的结构示意图，其中，VH-A表示第一抗体的重链可变区，VH-B表示第二抗体的重链可变区，VH-C表示第三抗体的重链可变区；VL-A表示第一抗体的轻链可变区，VL-B表示第二抗体的轻链可变区，VL-C表示第三抗体的轻链可变区。CH1、CH2和CH3是重链恒定区的三个结构域，CL是轻链恒定区。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，其中VH-B和VL-B之间的线段表示人工设计的二硫键。VH-A与VH-B、VH-B与VH-C以及VL-A与VL-B之间的线段表示人工设计的连接子，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)。
图2为本发明的三特异性抗体的ELISA结果。其中，图2A、图2B、图2C分别为ELISA测定本发明的三特异性抗体对PD-1、HER-2和LAG-3的亲和力结果。
图3为单抗609-IgG4及本发明的三特异性抗体的HPLC-SEC图谱。其中，图3A表示单抗609-IgG4的HPLC-SEC图谱；图3B表示609-302-134-IgG4的HPLC-SEC图谱；图3C表示609-302-(44CC)-134-IgG4的HPLC-SEC图谱；图3D表示609-302-(105CC)-134-IgG4的HPLC-SEC图谱。
图4为单抗609-IgG4及本发明的三特异性抗体的HPLC-IEC图谱。其中，图4A表示单抗609-IgG4的HPLC-IEC图谱；图4B表示609-302-(44CC)-134-IgG4的HPLC-IEC图谱；图4C表示609-302-(105CC)-134-IgG4的HPLC-IEC图谱。
图5为单抗609-IgG4及本发明的三特异性抗体的HPLC-IEC图谱。其中，图5A和图5B分别表示单抗609-IgG4的NR-CE-SDS和R-CE-SDS图谱；图5C和图5D分别表示609-302-134-IgG4的NR-CE-SDS和R-CE-SDS的图谱；图5E和图5F分别表示609-302-(44CC)-134-IgG4的NR-CE-SDS和R-CE-SDS的图谱；图5G和图5H分别表示609-302-(105CC)-134-IgG4的NR-CE-SDS和R-CE-SDS的图谱。
图6为本发明的三特异性抗体的刺激免疫反应的功能活性图。
具体实施方式
本发明人经过广泛而深入地研究，经过大量的筛选，首次成功获得一种结构新颖的抗PD-1、HER-2和LAG-3的六价三特异性抗体。本发明的六价三特异性抗体为二个单体形成的二聚体，其中，每个单体包含重链、第一轻链和第二轻链。具体地，以天然抗体的结构为基础，设计并优化了第一轻链以及重链的结构，使在本发明三特异性 抗体在保留抗PD-1、HER-2和LAG-3三特异性的同时，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。
术语
本发明中，术语“抗体(Antibody，缩写Ab)”和“免疫球蛋白G(Immunoglobulin G，缩写IgG)”是有相同结构特征的异四聚糖蛋白，其由两条相同的轻链(L)和两条相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型(isotype)的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是恒定区，重链恒定区由三个结构域CH1、CH2、以及CH3构成。每条轻链的一端有可变区(VL)，另一端有恒定区，轻链恒定区包括一个结构域CL；轻链的恒定区与重链恒定区的CH1结构域配对，轻链的可变区与重链的可变区配对。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体依赖的细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)等。重链恒定区包括IgG1、IgG2、IgG3、IgG4亚型；轻链恒定区包括κ(Kappa)或λ(Lambda)。抗体的重链和轻链通过重链的CH1结构域和轻链的CL结构域之间的二硫键共价连接在一起，抗体的两条重链通过铰链区之间形成的多肽间二硫键共价连接在一起。
本发明中，术语“三特异性抗体(或三抗)”是指能同时特异性结合三种抗原(靶点)或三种表位的抗体分子。根据对称性，三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，三特异性抗体可以分为三价、四价和多价分子。
本发明中，术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体，即该群体中包含的单个抗体是相同的，除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且，与常规多克隆抗体制剂(通常是具有针对不同抗原决定簇的不同抗体的混合物)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们可以通过杂交瘤培养来合成，不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性，是从基本均一的抗体群中获得的，这不应被解释成需要用任何特殊方法来生产抗体。
本发明中，术语“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。
本发明中，术语“Fab”和“Fc”是指木瓜蛋白酶可将抗体裂解为两个完全相同的Fab段和一个Fc段。Fab段由抗体的重链的VH和CH1以及轻链的VL和CL结构域组 成。Fc段即可结晶片段(fragment crystallizable，Fc)，由抗体的CH2和CH3结构域组成。Fc段无抗原结合活性，是抗体与效应分子或细胞相互作用的部位。
本发明中，术语“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))。
如本文所用，术语“框架区”(FR)指插入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％。
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。
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。
如本文所用，术语“接头”或“肽接头”或是指插入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。在本发明中，优选的接头是指接头L1、L2和L3，其中，L1连接第一抗体的重链可变区与第二抗体的重链可变区，L2连接第二抗体的重链可变区与第三抗体的重链可变区，L3连接第一抗体的轻链可变区与第二抗体的轻链可变区。
合适的接头实例包括单甘氨酸(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接头中需要实现的次级结构要素的类型而变化。
三特异性抗体
本发明的三特异性抗体是一种抗PD-1、HER-2和LAG-3的六价三特异性抗体，包括抗PD-1抗体部分、抗HER-2抗体部分和抗LAG-3抗体部分。
优选地，本发明抗PD-1抗体的序列如专利申请WO 2018/137576 A1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-1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-1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。
在本发明中，本发明的三特异性抗体还包括其保守性变异体，指与本发明三特异性抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
在本发明的一个优选实施例中，所获得的抗体的序列信息如下表1所示。
表1.本发明的抗体的序列信息
Figure PCTCN2022100105-appb-000003
其中，上述氨基酸序列中任意一种氨基酸序列还包括经过添加、缺失、修饰和/或取代至少一个(如1-5个、1-3个，较佳地1-2个，更佳地1个)氨基酸的具有PD-1、HER-2和LAG-3结合亲和力的衍生序列。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4 或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
在本发明中，优选的取代包括，在本发明的三特异性抗体609VH-302VH-134-IgG4中，将302重链可变区的第44位的G替换为C；将609VL-302VL中的302轻链可变区的100位Q突变成C；
将609VH-302VH-134-IgG4中的302重链可变区的105位的Q突变成C；将609VL-302VL中的302轻链可变区的43位Q突变成C。
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。
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，本发明的三特异性抗体具有六个抗原结合位点，是六价的。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)。
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。
如本文所用，术语“第一轻链”是指包含针对不同靶标的抗体的轻链可变区的轻链。具体地，所述“第一轻链”包含针对第一靶标的抗体的轻链可变区和针对第二靶标的抗体的轻链可变区，其能够与针对第一靶标的抗体重链可变区和针对第二靶标的抗体重链可变区配对，形成特异性结合第一靶标的第一结合位点和特异性结合第二靶标的第二结合位点。
优选的，本发明中的“第一轻链”包含通过接头或直接连接的两个针对不同靶标的抗体轻链可变区。进一步的，本发明中的“第一轻链”可以通过柔性接头或键在不同的位置与重链配对。具体地，本发明中的“第一轻链”可以通过非共价键或共价键与三特异性抗体的重链配对。较佳地，本发明中的“第一轻链”通过共价键(如二硫键)与重链配对。
本发明的三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。
编码核酸和表达载体
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。
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系统的载体等。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。
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。
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。
本发明中，术语“宿主细胞”是指适用于表达上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本发明的融合蛋白的表达，CHO(中国仓鼠卵巢，Chinese Hamster Ovary)，HEK293，COS，BHK以及上述细胞的衍生细胞均可适用于本发明。
药物组合物和应用
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。
本发明中，术语“药物组合物”是指本发明的六价三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-1的抗体或其抗原结合片段或六价三特异性抗体的氨基酸核心序列的构象完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、 脱氨或氧化)。
本发明的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％)的本发明上述的六价三特异性抗体(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，本发明的六价三特异性抗体还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的六价三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。
免疫偶联物
本发明还提供了基于本发明三特异性抗体的免疫偶联物。
典型地，所述免疫偶联物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。
本发明三特异性抗体所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。
抗体可以偶联药物从而形成抗体药物偶联物(antibody-drug conjugate，ADC)。典 型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。
连接到抗体之前，接头具有能够和某些氨基酸残基反应的活性反应基团，连接通过活性反应基团实现。巯基特异性的活性反应基团是优选的，并包括：例如马来酰亚胺类化合物，卤代酰胺(例如碘、溴或氯代的)；卤代酯(例如碘、溴或氯代的)；卤代甲基酮(例如碘、溴或氯代)，苄基卤代物(例如碘、溴或氯代的)；乙烯基砜，吡啶基二硫化物；汞衍生物例如3,6-二-(汞甲基)二氧六环，而对离子是醋酸根、氯离子或者硝酸根；和聚亚甲基二甲基硫醚硫代磺酸盐。接头可以包括，例如，通过硫代丁二酰亚胺连接到抗体上的马来酰亚胺。
药物可以是任何细胞毒性，抑制细胞生长或者免疫抑制的药物。在实施方式中，接头连接抗体和药物，而药物具有可以和接头成键的功能性基团。例如，药物可以具有可以和连接物成键的氨基，羧基，巯基，羟基，或者酮基。在药物直接连接到接头的情况下，药物在连接到抗体之前，具有反应的活性基团。
有用的药物类别包括，例如，抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱等。特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))和长春花生物碱(vinca alkaloids)。
在本发明中，药物-接头可以用于在一个简单步骤中形成ADC。在其它实施方式中，双功能连接物化合物可以用于在两步或多步方法中形成ADC。例如，半胱氨酸残 基在第一步骤中与接头的反应活性部分反应，并且在随后的步骤中，接头上的功能性基团与药物反应，从而形成ADC。
通常，选择接头上功能性基团，以利于特异性地与药物部分上的合适的反应活性基团进行反应。作为非限制性的例子，基于叠氮化合物的部分可以用于特异性地与药物部分上的反应性炔基基团反应。药物通过叠氮和炔基之间的1,3-偶极环加成，从而共价结合于接头。其它的有用的功能性基团包括，例如酮类和醛类(适合与酰肼类和烷氧基胺反应)，膦(适合与叠氮反应)；异氰酸酯和异硫氰酸酯(适合与胺类和醇类反应)；和活化的酯类，例如N-羟基琥珀酰亚胺酯(适合与胺类和醇类反应)。这些和其它的连接策略，例如在《生物偶联技术》，第二版(Elsevier)中所描述的，是本领域技术人员所熟知的。本领域技术人员能够理解，对于药物部分和接头的选择性反应，当选择了一个互补对的反应活性功能基团时，该互补对的每一个成员既可以用于接头，也可以用于药物。
本发明还提供了制备ADC的方法，可进一步地包括：将抗体与药物-接头化合物，在足以形成抗体偶联物(ADC)的条件下进行结合。
在某些实施方式中，本发明方法包括：在足以形成抗体-接头偶联物的条件下，将抗体与双功能接头化合物进行结合。在这些实施方式中，本发明方法还进一步地包括：在足以将药物部分通过接头共价连接到抗体的条件下，将抗体接头偶联物与药物部分进行结合。
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：
Figure PCTCN2022100105-appb-000004
其中：
Ab是抗体，
LU是接头；
D是药物；
而且下标p是选自1到8的值。
本发明的主要优点包括：
(1)本发明提供了一种结构新颖的六价三特异性抗体；
(2)本发明的三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。(3)本发明的三特异性抗体在保留抗PD-1、HER-2和LAG-3三特异性的同时，具有与单抗相似甚至更优的生物学活性和理化性质。
下面结合具体实施例，进一步陈述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
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pcDNA3.4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 Cells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Protein A亲和层析纯化抗体。
以下实施例中使用的ELISA检测方法说明如下：用相应的重组蛋白包被微孔板，用含有1％牛血清白蛋白的PBST(PBST为含0.05％Tween-20的磷酸盐缓冲液)封闭微孔板。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微孔板中，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(Fc specific，购自Sigma)，室温孵育半小时后洗板；每孔加入100μl以TMB(3,3′,5,5′-Tetramethylbenzidine)为底物的显色液，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 Prism7进行作图和数据分析，并计算EC50。
以下实施例中使用的理化性质检测方法说明如下：
HPLC-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三特异性抗体时，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。
HPLC-IEC
许多翻译后修饰(例如N糖基化、C末端赖氨酸残基修饰、N末端谷氨酰胺或谷氨酸环化、天冬酰胺脱酰胺化、天冬氨酸异构化和氨基酸残基氧化等)会直接或间接地引起抗体表面电荷的改变，导致电荷异质性的产生。基于所带电荷可对电荷变体进行分离和分析，常用的分析方法有阳离子交换色谱法(cation exchange chromatography，CEX)和阴离子交换色谱法(anionexchange chromatography，AEX)。当通过基于色谱法的方法分析时，酸性种类(acidic species)和碱性种类(basic species)基于它们相对于主峰(main peak)的保留时间来定义。酸性种类是早于CEX的主峰或晚于AEX的主峰洗脱出来的变体，而碱性种类是晚于CEX的主峰或早于AEX的主峰洗脱出来的变体。酸性种类和碱性种类所对应的峰分别称作酸性峰和碱性峰。在生产和存储抗体的过程中容易产生电荷变体。在此使用高效液相色谱-离子交换色谱(High-performance liquid chromatography-ionexchange chromatography，HPLC-IEC)分析样品的电荷异质性。HPLC-IEC所用色谱仪为Dionex Ultimate 3000；流动相A：20mM PB pH6.3，流动相B：20mM PB+200mM NaCl pH6.3，两种流动相混合的比例按照预先设置的程序随时间而改变，流速1.0ml/min；色谱柱：Thermo PropacTM WCX-10；柱温30℃，样品室温度10℃；进样量：20μg；检测波长：214nm。
CE-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Maurice CE-SDS分析系统购自ProteinSimple，配备UV 214nm检测器。
实施例1构建抗PD-1、HER-2和LAG-3的三特异性抗体
实施例1.1序列
mAb1-25-Hu(后文简称为609-IgG4)是抗PD-1人源化单克隆抗体，其重链和轻链氨基酸序列来自于WO 2018/137576A1中的SEQ ID NO：8和SEQ ID NO：10(即本发明中的 SEQ ID NO：5和6)。609-IgG4的重链可变区和轻链可变区的氨基酸序列如SEQ ID NO：1和2所示。609-IgG4重链恒定区为人IgG4(SEQ ID NO：3，铰链区含S228P突变)，轻链恒定区为人Kappa(SEQ ID NO：4)。在此，将609-IgG4重链和轻链的编码基因分别命名为609-IgG4-HC和609-IgG4-LC。将609-IgG4-HC和609-IgG4-LC基因分别构建到pcDNA3.4表达载体中，两种载体组合后表达并纯化抗体，所得抗体命名为609-IgG4。
609-IgG4重链可变区氨基酸序列(SEQ ID NO：1)：
Figure PCTCN2022100105-appb-000005
609-IgG4轻链可变区氨基酸序列(SEQ ID NO：2)：
Figure PCTCN2022100105-appb-000006
609-IgG4重链恒定区氨基酸序列((SEQ ID NO：3)：
Figure PCTCN2022100105-appb-000007
609-IgG4轻链恒定区氨基酸序列(SEQ ID NO：4)：
Figure PCTCN2022100105-appb-000008
609-IgG4重链氨基酸序列(SEQ ID NO：5)：
Figure PCTCN2022100105-appb-000009
609-IgG4轻链氨基酸序列(SEQ ID NO：6)：
Figure PCTCN2022100105-appb-000010
从公开的文献(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Trastuzumab(抗HER-2人源化单克隆抗体)的重链可变区和轻链可变区的氨基酸序列(SEQ ID NO：7和8)。该重链可变区和轻链可变区分别与人IgG1重链恒定区(SEQ ID NO：9)和人Kappa轻链恒定区(SEQ ID NO：4)相连，获得全长的重链和轻链氨基酸序列(SEQ ID  NO：10和11)。在此，将全长重链和轻链的编码基因分别命名为302-HC和302-LC。将302-HC和302-LC的编码基因分别构建到pcDNA3.4表达载体中，两种载体组合后表达并纯化抗体，所得抗体命名为302-IgG1。
302-IgG1的重链可变区氨基酸序列(SEQ ID NO：7)：
Figure PCTCN2022100105-appb-000011
302-IgG1的轻链可变区氨基酸序列(SEQ ID NO：8)：
Figure PCTCN2022100105-appb-000012
人IgG1重链恒定区氨基酸序列(SEQ ID NO：9)：
Figure PCTCN2022100105-appb-000013
302-IgG1的重链氨基酸序列(SEQ ID NO：10)：
Figure PCTCN2022100105-appb-000014
302-IgG1的轻链氨基酸序列(SEQ ID NO：11)：
Figure PCTCN2022100105-appb-000015
134-Hu-IgG4-C91S(后文简称为134-IgG4)是抗人LAG-3人源化单克隆抗体，其重链和轻链氨基酸序列来自于WO 2020/173378 A1中的SEQ ID NO：32和SEQ ID NO：36(即本发明中的SEQ ID NO：12和13)。134-IgG4单抗的重链恒定区为人IgG4(铰链区含有S228P突变)，轻链恒定区为人Kappa。在此，将134-IgG4重链和轻链的编码基因分别命名为134-IgG4-HC和134-IgG4-LC。将134-IgG4-HC和134-IgG4-LC的基因分别构建到pcDNA3.4表达载体中，两种载体组合后表达并纯化抗体，所得抗体命名为134-IgG4。
134人源化重链134-IgG4-HC的氨基酸序列(SEQ ID NO：12)：
QVQLVQSGAEVKKPGASVKVSCKASGYTLT AYYMNWVRQAPGQSLEWIG VINPYNGD SSYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCAR DDGYYRWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(其中下划线部分为重链互补决定区)
134人源化轻链134-IgG4-LC的氨基酸序列(SEQ ID NO：13)：
DIQMTQSPSSLSASVGDRVTITC RASQDIGSRLNWLQQKPGKSIKRLIY ATSSLESGVPSRFSGSRSGSDYTLTISSLQPEDFATYYC LQSGSSPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(其中下划线部分为轻链互补决定区)。
实施例1.2三特异性抗体的构建
将609-IgG4的重链可变区通过人工连接子(三个串联的GGGGS)连接302-IgG1的重链可变区，再通过人工连接子(三个串联的GGGGS)连接人134-IgG4的重链(铰链区含有S228P突变)，通过此程序构建成的含有三个重链可变区的长重链基因命名为609VH-302VH-134-IgG4(SEQ ID NO：14)。将609的轻链可变区通过人工连接子(三个串联的GGGGS)连接302-IgG1的轻链可变区，通过此程序构建成的含有两个轻链可变区的长轻链基因命名为609VL-302VL(SEQ ID NO：15)。
将上述序列的编码基因分别构建到pcDNA3.4表达载体中，将609VH-302VH-134-IgG4、609VL-302VL和134-IgG4-LC的表达载体组合，表达并纯化抗体，所得抗体命名为609-302-134-IgG4。
609VH-302VH-134-IgG4氨基酸序列：
Figure PCTCN2022100105-appb-000016
609VL-302VL氨基酸序列：
Figure PCTCN2022100105-appb-000017
为增强三特异性抗体的稳定性，在此尝试在609VH-302VH-134-IgG4和609VL-302VL 之间引入一对二硫键。根据文献(Cho,Hyun-Soo&Mason,Karen&Ramyar,Kasra&Stanley,Ann&Gabelli,Sandra&Denney,Dan&Leahy,Daniel.(2003).Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.Nature.421.756-60.10.1038/nature01392.)报道的Trastuzumab的晶体结构(相关信息见https://www.rcsb.org/structure/1N8Z)，在此选择处于VH和VL界面上的并且空间上相近的氨基酸残基对并突变成半胱氨酸，这些配对的氨基酸残基分别是：VH上的44位氨基酸和VL上的100位氨基酸，VH上的105位氨基酸和VL上的43位氨基酸(上述氨基酸残基按照Kabat规则编码)。
在此，将609VH-302VH-134-IgG4中的302重链可变区的44位的G突变成C，所得序列命名为609VH-302VH(44C)-134-IgG4；同时将609VL-302VL中的302轻链可变区的100位Q突变成C，所得序列命名为609VL-302VL(100C)；
609VH-302VH(44C)-134-IgG4氨基酸序列：
EVKLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKRLEWVATISGGGRYTYYPDTVKGRFTISRDNAKNSHYLQMNSLRAEDTAVYFCASPYGGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGK CLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTLTAYYMNWVRQAPGQSLEWIGVINPYNGDSSYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDDGYYRWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：16，其中下划线处为突变位点)
609VL-302VL(100C)氨基酸序列：
EIVLTQSPATLSLSPGERATLSCRASQSISNFLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNSWPHTFGQGTKVEIKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG CGTKVEIK(SEQ ID NO：17，其中下划线处为突变位点)
在此，将609VH-302VH-134-IgG4中的302重链可变区的105位的Q突变成C，所得序列命名为609VH-302VH(105C)-134-IgG4；同时将609VL-302VL中的302轻链可变区的43位A突变成C，所得序列命名为609VL-302VL(43C)；
609VH-302VH(105C)-134-IgG4氨基酸序列：
EVKLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKRLEWVATISGGGRYTYYPDTVKGRFTISRDNAKNSHYLQMNSLRAEDTAVYFCASPYGGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYY CSRWGGDGFYAMDYWG CGTLVTVSSGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTLTAYYMNWVRQAPGQSLEWIGVINPYNGDSSYNQKFKGRATLTVDKSTSTAYMELSSLRSEDTAVYYCARDDGYYRWYFDVWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO：18，其中下划线处为突变位点)
609VL-302VL(43C)氨基酸：
EIVLTQSPATLSLSPGERATLSCRASQSISNFLHWYQQKPGQAPRLLIKYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYFCQQSNSWPHTFGQGTKVEIKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK CPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK(SEQ ID NO：19，其中下划线处为突变位点)
将上述序列的编码基因分别构建到pcDNA3.4表达载体中，对应的载体组合后(609VH-302VH-134-IgG4、609VL-302VL和134-IgG4-LC组合，609VH-302VH(44C)-134-IgG4、609VL-302VL(100C)和134-IgG4-LC组合，609VH-302VH(105C)-134-IgG4、609VL-302VL(43C)和134-IgG4-LC组合)表达并纯化抗体，所得抗体分别命名为609-302-134-IgG4、609-302-(44CC)-134-IgG4和609-302-(105CC)-134-IgG4。
本发明中的抗体名称如表2所示:
表2.本发明的抗体名称
Figure PCTCN2022100105-appb-000018
实施例1.3 ELISA测定相对亲和力
在此分别用带有多聚组氨酸标签的HER2胞外段重组蛋白(记作HER2-His，购自Sino Biological Inc.)、PD-1胞外段重组蛋白(记作PD1-His，购自Sino Biological Inc.)和LAG-3胞外段重组蛋白(记作LAG3-His，购自ACROBiosystems)包被微孔板，包被浓度分别为20ng/孔、20ng/孔和10ng/孔。
表3.ELISA测定相对亲和力的EC 50结果
Figure PCTCN2022100105-appb-000019
如图2A-C所示，609-302-134-IgG4、609-302-(44CC)-134-IgG4和609-302-(105CC)-134-IgG4均能够有效结合PD-1、HER-2和LAG-3，这表明它们是三特异性抗体，EC 50汇总于表3。
实施例1.4物理化学性质的表征
1.4.1 HPLC-SEC
图3A表示单抗609-IgG4的HPLC-SEC图谱，主峰占比99.87％。
图3B表示609-302-134-IgG4的HPLC-SEC图谱，主峰占比98.24％。
图3C表示609-302-(44CC)-134-IgG4的HPLC-SEC图谱，主峰占比98.4％。
图3D表示609-302-(105CC)-134-IgG4的HPLC-SEC图谱，主峰占比98.06％。
上述结果表明，经过一步Protein A亲和层析纯化之后，三特异性抗体的SEC纯度可以达到98％以上，尺寸异质性接近单克隆抗体。
1.4.2 HPLC-IEC
图4A表示单抗609-IgG4的HPLC-IEC图谱，主峰占比71.45％。
图4B表示609-302-(44CC)-134-IgG4的HPLC-IEC图谱，主峰占比71.62％。
图4C表示609-302-(105CC)-134-IgG4的HPLC-IEC图谱，主峰占比66.12％。
上述结果表明，经过一步Protein A亲和层析纯化之后，三特异性抗体的IEC主峰可 以达到66％以上，电荷异质性与单克隆抗体相当。
1.4.3 CE-SDS
图5A和图5B分别表示单抗609-IgG4的NR-CE-SDS和R-CE-SDS图谱，NR-CE-SDS图谱中主峰Peak2.672占比98.9％；R-CE-SDS图谱中两个主峰Peak1.499(对应轻链)和Peak1.888(对应重链)分别占比31.3％和67.7％，两主峰占比之和为99.0％。
图5C和图5D分别表示609-302-134-IgG4的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中两个主峰Peak1.525和Peak2.893分别占比18.6％和80.8％，在609-302-134-IgG4分子中609VL-302VL没有与609VH-302VH-134-IgG4形成共价二硫键，因此认为Peak1.525对应的多肽链为609VL-302VL；R-CE-SDS图谱中两个主峰Peak1.518(对应轻链)和Peak2.158(对应重链)分别占比34.8％和62.5％，两主峰占比之和为97.3％。
图5E和图5F分别表示609-302-(44CC)-134-IgG4的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中主峰Peak3.018占比96.4％；R-CE-SDS图谱中两个主峰Peak1.515(对应轻链)和Peak2.159(对应重链)分别占比34.9％和62.0％，两主峰占比之和为96.9％。图5G和图5H分别表示609-302-(105CC)-134-IgG4的NR-CE-SDS和R-CE-SDS的图谱，NR-CE-SDS图谱中主峰Peak3.020占比91.8％；R-CE-SDS图谱中两个主峰Peak1.508(对应轻链)和Peak2.147(对应重链)分别占比36.6％和56.9％，两主峰占比之和为93.5％。
上述结果表明，经过一步Protein A亲和层析纯化之后，三特异性抗体609-302-(44CC)-134-IgG4的NR-CE-SDS和R-CE-SDS纯度均可以达到96％以上，与单抗609-IgG4最为相近。
实施例2测定本发明的三特异性抗体刺激免疫反应的功能活性
在RPMI 1640中加入以下添加剂：10％胎牛血清；1％MEM Non-Essential Amino Acids Solution；1％Sodium Pyruvate；1％HEPES；1‰2-Mercaptoethanol；1％Penicillin-Streptomycin；1％GlutaMAX(上述培养基和添加剂购自Thermo Fisher Scientific)。用上述RPMI 1640完全培养基将新鲜分离的人外周血单个核细胞(Peripheral Blood Mononuclear Cell，PBMC。PBMC购自Allcells，货号：PB005-C)洗涤并重悬，加入一定量的超抗原金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B，SEB)。SEB为实验室内部制备，序列来自Uniprot(Entry:P01552)，使用大肠杆菌表达，用Ni-NTA亲和层析法纯化。将PBMC细胞悬液接种到圆底96孔细胞培养板中，每孔150μl悬液和20 万个细胞；在上述96孔板中加入50μl梯度稀释的相关抗体；将96孔板置于37℃细胞培养箱中孵育4天。从96孔板中取适量细胞培养上清。用双抗体夹心法(sandwich ELISA)检测上清中的IL-2(相关检测用的配对抗体购自BD Biosciences)。用酶标仪(SpectraMax 190)读取OD450，用GraphPad Prism7进行数据分析、作图并计算EC 50。
表4.本发明的三特异性抗体的功能活性参数
抗体 EC 50(nM) Top
609-IgG4 0.04091 5363
134-IgG4 0.8398 3369
609-302-(44CC)-134-IgG4 0.3217 10281
609-302-(105CC)-134-IgG4 0.1834 8795
如图6和表4所示，609-IgG4比134-IgG4表现出更小的EC 50和更高的Top(高平台)，表明609-IgG4功能活性比134-IgG4高。609-302-(44CC)-134-IgG4和609-302-(105CC)-134-IgG4展现出相近的EC 50和Top，说明这两种三特异性抗体的功能活性相当。与609-IgG4相比，609-302-(44CC)-134-IgG4和609-302-(105CC)-134-IgG4展现出更高的Top，大致在抗体浓度大于1nM时(即Log值大于0时)，两种三特异性抗体刺激PBMC分泌IL-2的能力明显强于单克隆抗体609-IgG4和134-IgG4。
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。

Claims (15)

  1. 一种六价三特异性抗体，其特征在于，所述的六价三特异性抗体为二个单体形成的二聚体，其中，每个单体包含重链、第一轻链和第二轻链；
    所述的重链从N端至C端包括串联的针对第一靶标的抗体重链可变区元件Z1、针对第二靶标的抗体重链可变区元件Z2、针对第三靶标的抗体重链可变区元件Z3以及抗体重链恒定区Z4；
    所述的第一轻链和第二轻链分别与所述重链配合，使得所述的六价三特异性抗体特异性结合于第一靶标、第二靶标和第三靶标。
  2. 如权利要求1所述的六价三特异性抗体，其特征在于，包含重链、第一轻链和第二轻链；其中，每个所述的重链具有下式I的结构：
    Z1-Z2-Z3-Z4  (I)
    Z1为针对第一靶标的重链可变区元件；
    Z2为针对第二靶标的重链可变区元件；
    Z3为针对第三靶标的重链可变区元件；
    Z4为抗体重链恒定区CH1、CH2和CH3；
    “-”各自独立地为键或接头(linker)；
    其中，所述的第一轻链和第二轻链分别与重链配合，使得所述的六价三特异性抗体特异性结合于第一靶标、第二靶标和第三靶标；所述第一轻链具有下式II的结构：
    Z5-Z6  (II)；
    其中，Z5为针对第一靶标的轻链可变区元件；Z6为针对第二靶标的轻链可变区元件；
    所述第二轻链具有下式III的结构：
    Z7-Z8  (III)；
    其中，Z7为针对第三靶标的轻链可变区元件；Z8为轻链恒定区。
  3. 如权利要求1或2所述的六价三特异性抗体，其特征在于，所述第一靶标、第二靶标和第三靶标为PD-1、HER-2和LAG-3。
  4. 如权利要求1-3中任一项所述的六价三特异性抗体，其特征在于，所述的接头为长度为1-35个氨基酸的肽接头，较佳地6-30个氨基酸的肽接头。
  5. 如权利要求1-4中任一项所述的六价三特异性抗体，其特征在于，所述六价三特异性抗体包含两个单体，每个单体含有重链和第一轻链、和第二轻链，其中每个单体具有下式IV结构：
    Figure PCTCN2022100105-appb-100001
    式中，
    VH A-L1-VH B-L2-VH C-CH1-CH2-CH3为重链；
    VL A-L3-VL B为第一轻链；
    VL C-CL为第二轻链；
    VH A为抗PD-1抗体的重链可变区；
    VH B为抗HER-2抗体的重链可变区；
    VH C为抗LAG-3抗体的重链可变区；
    CH1、CH2和CH3分别为抗体重链恒定区CH1、CH2和CH3；
    VL A为抗PD-1抗体的轻链可变区；
    VL B为抗HER-2抗体的轻链可变区；
    VL C为抗LAG-3抗体的轻链可变区；
    CL为抗体的轻链恒定区；
    L1、L2、L3各自独立地为接头(linker)；
    “-”各自独立地为键；
    “～”代表二硫键或共价键；
    其中，所述的六价三特异性抗体同时结合于PD-1、HER-2和LAG-3。
  6. 如权利要求1-5中任一项所述的六价三特异性抗体，其特征在于，所述接头为柔性肽接头；其中，所述的柔性肽接头包括6-30个氨基酸，较佳地10-25个氨基酸。
  7. 如权利要求1-6中任一项所述的六价三特异性抗体，其特征在于，所述的六价三特异性抗体包含两条重链、两条第一轻链和两条第二轻链，
    其中，所述的六价三特异性抗体中的重链、第一轻链和第二轻链的氨基酸序列分别如SEQ ID No:14、15和13所示；或
    所述的六价三特异性抗体中的重链、第一轻链和第二轻链的氨基酸序列分别如SEQ ID No:16、17和13所示；或
    所述的六价三特异性抗体中的重链、第一轻链和第二轻链的氨基酸序列分别如SEQ ID No:18、19和13所示。
  8. 一种分离的核酸分子，其特征在于，所述的核酸分子编码如权利要求1-7中任一项所述的六价三特异性抗体。
  9. 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8所述的核酸分 子。
  10. 一种宿主细胞，其特征在于，所述的宿主细胞含有如权利要求9所述的表达载体。
  11. 如权利要求1-7中任一项所述的六价三特异性抗体的制备方法，其特征在于，所述方法包含以下步骤：
    (a)在表达条件下，培养如权利要求10所述的宿主细胞，从而表达所述的六价三特异性抗体；
    (b)分离并纯化(a)所述的六价三特异性抗体。
  12. 一种药物组合物，其特征在于，所述药物组合物含有如权利要求1-7中任一项所述的六价三特异性抗体和药学上可接受的载体。
  13. 如权利要求1-7中任一所述的六价三特异性抗体或如权利要求12所述的药物组合物在制备治疗癌症的药物中的用途。
  14. 如权利要求13所述的用途，其特征在于，所述癌症选自下组：黑素瘤、肾癌、前列腺癌、胰腺癌、乳腺癌、结肠癌、肺癌、食道癌、头颈鳞状细胞癌、肝癌、卵巢癌、宫颈癌、甲状腺癌、成胶质细胞瘤、神经胶质瘤及其它赘生性恶性疾病。
  15. 一种免疫偶联物，其特征在于，所述免疫偶联物包括：
    (a)如权利要求1-7中任一项中所述的六价三特异性抗体；和
    (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。
PCT/CN2022/100105 2021-06-29 2022-06-21 三特异性抗体、其制备方法和用途 WO2023273958A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP22831776.4A EP4365205A1 (en) 2021-06-29 2022-06-21 Trispecific antibody and preparation method therefor and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110726683.9 2021-06-29
CN202110726683.9A CN115536749A (zh) 2021-06-29 2021-06-29 三特异性抗体、其制备方法和用途

Publications (1)

Publication Number Publication Date
WO2023273958A1 true WO2023273958A1 (zh) 2023-01-05

Family

ID=84691286

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/100105 WO2023273958A1 (zh) 2021-06-29 2022-06-21 三特异性抗体、其制备方法和用途

Country Status (4)

Country Link
EP (1) EP4365205A1 (zh)
CN (1) CN115536749A (zh)
TW (1) TW202305008A (zh)
WO (1) WO2023273958A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018137576A1 (zh) 2017-01-24 2018-08-02 三生国健药业（上海）股份有限公司 抗pd-1单克隆抗体及其制备方法和应用
US20180355024A1 (en) 2015-06-15 2018-12-13 Numab Innovation Ag Hetero-dimeric multi-specific antibody format
US20190352416A1 (en) 2014-12-22 2019-11-21 Xencor, Inc. Trispecific antibodies
WO2020173378A1 (zh) 2019-02-28 2020-09-03 三生国健药业（上海）股份有限公司 结合人lag-3的抗体、其制备方法和用途
CN111836829A (zh) * 2018-02-01 2020-10-27 默沙东公司 抗pd-1/lag3双特异性抗体
CN111836832A (zh) * 2018-01-08 2020-10-27 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法
CN112204052A (zh) * 2018-04-05 2021-01-08 诺华股份有限公司 针对癌症的三特异性结合分子及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352416A1 (en) 2014-12-22 2019-11-21 Xencor, Inc. Trispecific antibodies
US20180355024A1 (en) 2015-06-15 2018-12-13 Numab Innovation Ag Hetero-dimeric multi-specific antibody format
WO2018137576A1 (zh) 2017-01-24 2018-08-02 三生国健药业（上海）股份有限公司 抗pd-1单克隆抗体及其制备方法和应用
CN111836832A (zh) * 2018-01-08 2020-10-27 南京传奇生物科技有限公司 多特异性抗原结合蛋白及其使用方法
CN111836829A (zh) * 2018-02-01 2020-10-27 默沙东公司 抗pd-1/lag3双特异性抗体
CN112204052A (zh) * 2018-04-05 2021-01-08 诺华股份有限公司 针对癌症的三特异性结合分子及其用途
WO2020173378A1 (zh) 2019-02-28 2020-09-03 三生国健药业（上海）股份有限公司 结合人lag-3的抗体、其制备方法和用途

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Uniprot", Database accession no. P01552
CHO, HYUN-SOMASON, KARENRAMYAR, KASRASTANLEY, ANNGABELLI, SANDRADENNEY, DANLEAHY, DANIEL: "Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab", NATURE, vol. 421, 2003, pages 756 - 60, XP009113017, DOI: 10.1038/nature01392
GARFALLJUNE: "Trispecific antibodies offer a third way forward for antagonists alike", NATURE, 2019
LIANG XU ET AL.: "Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in Macaques", SCIENCE, 20 September 2017 (2017-09-20)
MAGDELAINE-BEUZELIN CKAAS QWEHBI V ET AL.: "Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment", CRITICAL REVIEWS IN ONCOLOGY/HEMATOLOGY, vol. 64, no. 3, 2007, pages 210 - 225, XP022344458, DOI: 10.1016/j.critrevonc.2007.04.011
MAGDELAINE-BEUZELIN, C. KAAS, Q. WEHBI, V. OHRESSER, M. JEFFERIS, R. LEFRANC, M.P. WATIER, H.: "Structure-function relationships of the variable domains of monoclonal antibodies approved for cancer treatment", CRITICAL REVIEWS IN ONCOLOGY/HEMATOLOGY, vol. 64, no. 3, 14 November 2007 (2007-11-14), AMSTERDAM, NL , pages 210 - 225, XP022344458, ISSN: 1040-8428, DOI: 10.1016/j.critrevonc.2007.04.011 *
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
WU ET AL.: "Trispecific antibiotics enhance the therapeuti c efficacy of tutor directed T cells through T cell receiver co stimulation", NATURE CANCER, 2019, Retrieved from the Internet <URL:https://doi.org/10.1038/s43018-019-0004-z>

Also Published As

Publication number Publication date
TW202305008A (zh) 2023-02-01
CN115536749A (zh) 2022-12-30
EP4365205A1 (en) 2024-05-08

Similar Documents

Publication Publication Date Title
EP2788379B1 (en) Pdgf receptor beta binding polypeptides
TWI593707B (zh) Ｃｄ３３抗體類及彼等於治療癌症之用途
US20220356246A1 (en) Anti-ROR1 antibodies and preparation method and uses thereof
CN109496149B (zh) 抗体及其药物缀合物的制剂
US11834498B2 (en) Biparatopic FR-alpha antibodies and immunoconjugates
KR20220008841A (ko) 인간 면역글로불린 힌지 영역인 두 개의 결합 도메인 사이의 링커 또는 이의 변이체를 포함하는 활성화 가능한 이중특이성 항체 및 이의 용도
US11814441B2 (en) Bi-specific antigen-binding polypeptides
CN112996809A (zh) 结合4-1bb和肿瘤相关抗原的抗体构建体以及其用途
EP4215549A1 (en) Anti-4-1bb-anti-pd-l1 bispecific antibody, and pharmaceutical composition and use thereof
JP2021515542A (ja) Ｐｄ１結合剤
JP2021516537A (ja) Ｃ−ｍｅｔ結合剤
TW202122421A (zh) 抗ｐｄ－ｌ１抗體及抗體藥物結合物
KR20210063782A (ko) c-kit에 대한 항체 및 이의 용도
WO2022179039A1 (zh) 抗人cd73抗体及其应用
CN113461821B (zh) 抗cd3人源化抗体
WO2023273958A1 (zh) 三特异性抗体、其制备方法和用途
WO2022068775A1 (zh) 抗pd-l1抗体及其用途
CN115698085A (zh) 一种抗pd-l1和egfr的四价双特异性抗体
US20240182560A1 (en) Biparatopic fr-alpha antibodies and immunoconjugates
US20230265202A1 (en) Antibody constructs binding 4-1bb and folate receptor alpha and uses thereof
JP2024505673A (ja) 電荷対を有する二重特異性抗体及びその使用
KR20240004837A (ko) 항-5t4 항체 및 이의 용도

Legal Events

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

Ref document number: 22831776

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023579715

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 18575782

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2022831776

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022831776

Country of ref document: EP

Effective date: 20240129