WO2018176992A1 - Nouveau type d'anticorps bispécifique et son utilisation - Google Patents

Nouveau type d'anticorps bispécifique et son utilisation Download PDF

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WO2018176992A1
WO2018176992A1 PCT/CN2018/072572 CN2018072572W WO2018176992A1 WO 2018176992 A1 WO2018176992 A1 WO 2018176992A1 CN 2018072572 W CN2018072572 W CN 2018072572W WO 2018176992 A1 WO2018176992 A1 WO 2018176992A1
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antibody
amino acid
human igg1
seq
fragment
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刘志刚
刘玉兰
郝小勃
郭晶晶
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北京百特美博生物科技有限公司
北京智仁美博生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—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
    • C07K16/2809—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 the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • 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
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • 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
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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 application relates generally to the field of antibody drugs, and in particular, the present application provides novel bispecific antibodies and their medical and biological uses.
  • bispecific antibody is a type of artificial antibody that contains two different antigen binding sites. Bispecific antibodies are widely used in the field of biomedicine, especially in tumor immunotherapy.
  • Bispecific antibodies can be divided into dual signal blocking and mediating cell functional types. Typically, a cell-functional bispecific antibody is mediated as an anti-CD3 bispecific antibody that mediates T cell killing.
  • a cell-functional bispecific antibody is mediated as an anti-CD3 bispecific antibody that mediates T cell killing.
  • T cells to kill tumor cells.
  • the concept of using T cells to kill tumor cells has been proposed (Stearz at al. Nature 1985, 314: 628-631). It is generally believed that efficient activation of T cells requires a dual signal, the first signal is from the binding of the MHC-antigen complex on the antigen presenting cell to the T cell receptor TCR-CD3, and the second signal is the mutual stimulation of the T cell and the antigen presenting cell.
  • Non-antigen-specific costimulatory signals produced after action.
  • the bispecific antibody targeting CD3 can bind to the T cell surface CD3 molecule and the cancer cell surface antigen, thereby narrowing the distance between cytotoxic T cells (Tc or CTL) and cancer cells, and directing T cells to directly kill. Cancer cells are no longer dependent on the dual activation signal of T cells.
  • the bispecific antibody can be a trifunctional antibody, ie one arm targets the antigen on the tumor cell, the other arm targets the CD3 molecule of the T cell, and the Fc segment binds to the Fc receptor.
  • Such antibodies allow T cells, tumor cells, and effector cells that bind to the Fc domain of an antibody to form a complex (Muller and Kontermann, BioDrugs 2010; 24:89-98).
  • the CD3 molecule has four subunits of ⁇ , ⁇ , ⁇ , and ,, and has molecular weights of 18.9 kDa, 23.1 kDa, 20.5 kDa, and 18.7 kDa, respectively, including 171, 207, 182, and 164 amino acid residues.
  • the six polypeptide chains composed of four subunits bind tightly to the T cell receptor (TCR) to form a TCR-CD3 complex containing eight polypeptide chains, which conduct T cell activation signals and stabilize the TCR structure.
  • TCR T cell receptor
  • the intracellular portion of CD3 contains an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the TCR recognizes and binds to an antigenic peptide presented by MHC molecules, which phosphorylates the tyrosine protein kinase p561ck in T cells.
  • ITAM phosphorylation and binding to ZAP-70 are one of the important biochemical reactions of T cell activation early signaling processes. Therefore, the CD3 molecule has a function of transmitting an activation signal generated by the TCR recognition antigen.
  • bispecific antibodies have been limited due to early deficiencies in immunogenicity, structural stability, and antibody quality control.
  • improvements in upstream genetically engineered antibodies and downstream production techniques have overcome the shortcomings of traditional bispecific antibodies, thereby facilitating the entry of multiple new bispecific antibodies into clinical development.
  • a bispecific antibody of various structures was designed and developed.
  • bispecific antibody does not contain an Fc region.
  • the advantage of this type of structural antibody is that it has a small molecular weight and can be expressed in prokaryotic cells without considering the problem of proper assembly.
  • the disadvantage is that since there is no antibody Fc segment, the corresponding biological function cannot be mediated, and the half-life is short, so the clinical application is affected. Certain restrictions.
  • Such bispecific antibodies that have been reported so far include BiTE, DART, TrandAbs, bi-Nanobody and the like.
  • the BiTE (bispecific T-cell engager) series developed by Micromet in Germany is obtained by linking anti-CD3 scfv with different anti-tumor cell surface antigen scfv through peptides, and can simultaneously bind CD3 + T cells and tumor cells.
  • Such antibodies overcome production problems such as poor stability, low expression levels, and low solubility, with Blinatumomab being successfully marketed.
  • bispecific antibody retains the antibody Fc domain.
  • Such antibodies form IgG-like structures with Fc-mediated biological functions.
  • bispecific antibodies that have been reported so far include Triomabs, kish IgG, Cross-mab, ortho Fab IgG, DVD IgG, IgG scFv, scFv2-Fc and the like.
  • These bispecific antibodies comprising the Fc segment have the advantages of long half-life in vivo, ability to mediate ADCC and CDC, and the like.
  • Bispecific antibodies eg, targeting CD3 and tumor surface antigens
  • the application provides a bispecific human IgG1 antibody comprising two Fc fragments having the same hinge region, the amino acid sequence of the hinge region being SEQ ID NO: 1 or SEQ ID NO: 28 and replacing the natural person
  • the sequence of positions 216-230 of the constant region of the IgG1 antibody, and the amino acid position of the antibody constant region were determined according to EU numbering.
  • the two Fc fragments comprise a mutation that is capable of ensuring heavy chain heteromerization.
  • one Fc fragment comprises the point mutations S354C and T366W and the other Fc fragment comprises the point mutations Y349C, T366S, L368A and Y407V.
  • the N-termini of the two Fc fragments are fused to a single-chain antibody (scfv) that recognizes different epitopes, respectively.
  • scfv single-chain antibody
  • an N-terminal fusion single chain antibody (scfv) of an Fc fragment, an N-terminal fusion Fab fragment of another Fc fragment, the single-chain antibody and the Fab fragment recognize different epitopes.
  • the N-terminus of the two Fc fragments respectively fuse Fab fragments that recognize different epitopes.
  • Fab fragments that recognize different epitopes comprise the same light chain.
  • one Fab fragment comprises a native human IgG1 antibody constant region CH1 fragment
  • the other Fab fragment comprises a mutated human IgG1 antibody constant region CH1 fragment
  • the mutated human IgG1 antibody constant region CH1 fragment comprises a point mutation G137E Any one, two or three of N203D and R214T, wherein the amino acid sequence of the CH1 fragment is the amino acid sequence of positions 118 to 215 of the constant region of the antibody.
  • the amino acid sequence of the native human IgG1 antibody constant region CH1 fragment is SEQ ID NO: 2.
  • the amino acid sequence of the mutated human IgG1 antibody constant region CH1 fragment is SEQ ID NO:30.
  • the CH2 fragments of both Fc fragments comprise the point mutations L234F, L235E and P331S, wherein the amino acid sequence of the CH2 fragment is the amino acid sequence at positions 231-340 of the constant region of the antibody. In some embodiments, the amino acid sequence of the CH2 fragment of both Fc fragments is SEQ ID NO:31.
  • the bispecific human IgGl antibody recognizes human immune cell surface antigen and tumor cell surface antigen.
  • the human immune cell surface antigen is CD3E.
  • the tumor surface antigen is selected from the group consisting of HER1, HER2, HER3, EpCAM, CEA, PSMA, CD19, CD20, CD22, CD38, and BCMA.
  • the application provides a pharmaceutical composition comprising the bispecific human IgGl antibody of the first aspect.
  • the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, excipient, diluent, and the like.
  • the present application provides the use of the bispecific human IgG1 antibody of the first aspect, or the pharmaceutical composition of the second aspect, for the preparation of a medicament for preventing or treating a tumor.
  • the present application provides a method of preventing or treating a tumor comprising administering to a subject in need thereof the bispecific human IgG1 antibody of the first aspect or the pharmaceutical composition of the second aspect.
  • the tumor expresses a tumor surface antigen to which the bispecific human IgGl antibody is directed.
  • the tumor is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, prostate cancer, pancreatic cancer, leukemia, multiple myeloma, and malignant lymphoma.
  • 1a-1d are schematic diagrams showing the structure of four different structural bispecific antibodies designed according to the present application.
  • Figure 2 shows the results of detection of various bispecific antibodies binding to both CD3E and HER2 antigens by ELISA.
  • Figure 3 shows the results of flow cytometry analysis of the binding of various bispecific antibodies to the surface HER2 binding of MDA-MB-453 human breast cancer cells.
  • Figure 4 shows the results of analyzing the binding of various bispecific antibodies to the surface CD3 of PBMC by flow cytometry.
  • Figures 5a-5g show the killing effect of different bispecific antibodies on tumor cells, wherein Figure 5a shows the killing effect of bispecific antibodies on HER2 positive cells; Figure 5b shows the killing of HER2-negative cells by bispecific antibodies Results; Figures 5c-5e show the effect of different hinge region structures of bispecific antibodies on killing activity; Figures 5f and 5g show the effect of different antigen binding designs of bispecific antibodies on killing activity.
  • Figures 6a-6c show activation of T cells by different bispecific antibodies mediated HER2 positive target cells (MDA-MB-453 human breast cancer cells), wherein Figures 6a and 6b show different bispecific antibody-activated T cells Results of expression of the early activation marker molecule CD69; Figure 6c shows the results of different bispecific antibodies inducing T cells to produce the cytokine IL-2.
  • SEQ ID NO: 1 is the amino acid sequence of the hinge region encompassed by the exemplary bispecific antibody of the present application, which is the hinge region of a native human IgG2 antibody.
  • SEQ ID NO: 2 is the amino acid sequence of the native human IgG1 antibody constant region CH1 fragment.
  • SEQ ID NO: 3 is the amino acid sequence of HCDR1 of an exemplary anti-human CD3E monoclonal antibody of the present application.
  • SEQ ID NO: 4 is the amino acid sequence of HCDR2 of an exemplary anti-human CD3E monoclonal antibody of the present application.
  • SEQ ID NO: 5 is the amino acid sequence of HCDR3 of an exemplary anti-human CD3E monoclonal antibody of the present application.
  • SEQ ID NO: 6 is the amino acid sequence of an exemplary anti-human CD3E monoclonal antibody and an anti-HER2 monoclonal antibody LCDR1 of the present application.
  • SEQ ID NO: 7 is the amino acid sequence of an exemplary anti-human CD3E monoclonal antibody and an anti-HER2 monoclonal antibody LCDR2 of the present application.
  • SEQ ID NO: 8 is the amino acid sequence of an exemplary anti-human CD3E monoclonal antibody and an anti-HER2 monoclonal antibody LCDR3 of the present application.
  • SEQ ID NO: 9 is the amino acid sequence of HCDR1 of an exemplary anti-HER2 monoclonal antibody of the present application.
  • SEQ ID NO: 10 is the amino acid sequence of HCDR2 of an exemplary anti-HER2 monoclonal antibody of the present application.
  • SEQ ID NO: 11 is the amino acid sequence of HCDR3 of an exemplary anti-HER2 monoclonal antibody of the present application.
  • SEQ ID NO: 12 is the amino acid sequence of the heavy chain variable region of an exemplary anti-human CD3E monoclonal antibody of the present application.
  • SEQ ID NO: 13 is the amino acid sequence of the light chain variable region of the exemplary anti-human CD3E monoclonal antibody and anti-HER2 monoclonal antibody of the present application.
  • SEQ ID NO: 14 is the amino acid sequence of the heavy chain variable region of an exemplary anti-HER2 monoclonal antibody of the present application.
  • SEQ ID NO: 15 is the amino acid sequence of an anti-HER2 scfv containing arm (anti-HER2 scfv-Fc fusion protein) of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 16 is the amino acid sequence of an anti-human CD3E scfv-containing arm (anti-human CD3E scfv-Fc fusion protein) of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 17 is the amino acid sequence of the heavy chain portion of an anti-HER2 Fab-containing arm of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 18 is the amino acid sequence of the light chain portion of an arm comprising an anti-HER2 Fab or an anti-human CD3E Fab of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 19 is the amino acid sequence of the heavy chain portion of an arm comprising an anti-human CD3E Fab of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 20 is the amino acid sequence of the heavy chain portion of an anti-HER2 Fab containing arm of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 21 is the amino acid sequence of an anti-HER2 scfv containing arm (anti-HER2 scfv-Fc fusion protein) of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 22 is the amino acid sequence of an anti-human CD3E scfv-containing arm (anti-human CD3E scfv-Fc fusion protein) of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 23 is the amino acid sequence of the heavy chain portion of an anti-HER2 Fab-containing arm of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 24 is the amino acid sequence of the heavy chain portion of an arm comprising an anti-human CD3E Fab of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 25 is the amino acid sequence of the heavy chain portion of an anti-HER2 Fab containing arm of an exemplary bispecific antibody of the present application.
  • SEQ ID NO:26 is the amino acid sequence of the heavy chain portion of an anti-HER2 Fab containing arm of an exemplary bispecific antibody of the present application.
  • SEQ ID NO:27 is the amino acid sequence of the heavy chain portion of an arm comprising an anti-human CD3E Fab of an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 28 is the amino acid sequence of the hinge region encompassed by an exemplary bispecific antibody of the present application, which is a variant of the hinge region of a native human IgG2 antibody.
  • SEQ ID NO:29 is the amino acid sequence of the hinge region comprised by the exemplary bispecific antibody of the present application, which is the hinge region of a native human IgGl antibody.
  • SEQ ID NO: 30 is the amino acid sequence of a CH1 fragment in a Fab fragment comprised by an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 31 is the amino acid sequence of a CH2 fragment in an Fc fragment comprised by an exemplary bispecific antibody of the present application.
  • SEQ ID NO: 32 is the amino acid sequence of the human CD3E extracellular domain (CD3E).
  • SEQ ID NO: 33 is the amino acid sequence of the human CD3D extracellular domain (CD3D).
  • SEQ ID NO: 34 is the amino acid sequence of the monkey CD3E extracellular domain (mfCD3E).
  • SEQ ID NO: 35 is the amino acid sequence of the monkey CD3D extracellular domain (mfCD3D).
  • SEQ ID NO: 36 is the amino acid sequence of mouse CD3E extracellular domain (mCD3E).
  • SEQ ID NO: 37 is the amino acid sequence of the mouse CD3D extracellular domain (mCD3D).
  • SEQ ID NO:38 is the amino acid sequence of the D1D2D3 portion (HER2) of the HER2 extracellular domain.
  • SEQ ID NO: 39 is the amino acid sequence of the His tag.
  • SEQ ID NO: 40 is the amino acid sequence of the Fc fragment (mFc) of murine antibody IgG2a.
  • SEQ ID NO: 41 is the amino acid sequence of an Fc fragment variant (FcK) of a human IgG1 antibody.
  • SEQ ID NO: 42 is the amino acid sequence of an Fc fragment variant (FcH) of a human IgG1 antibody.
  • SEQ ID NO: 43 is the amino acid sequence of the heavy chain constant region of a native human IgG1 antibody.
  • SEQ ID NO: 44 is the amino acid sequence of a variant (IgG1Hn) of the human IgG1 antibody heavy chain constant region.
  • SEQ ID NO: 45 is the amino acid sequence of a variant (IgG1Kn) of the human IgG1 antibody heavy chain constant region.
  • SEQ ID NO: 46 is the amino acid sequence of a variant (IgG1Hn-m3) of the human IgG1 antibody heavy chain constant region.
  • SEQ ID NO: 47 is the amino acid sequence of a variant (IgGlkn-m3) of the human IgGl antibody heavy chain constant region.
  • SEQ ID NO: 48 is the amino acid sequence of a variant (IgG1H3n-m3) of the human IgG1 antibody heavy chain constant region.
  • SEQ ID NO: 49 is the amino acid sequence of a variant (IgG1 kn1-m3) of the human IgG1 antibody heavy chain constant region.
  • SEQ ID NO: 50 is the amino acid sequence of a variant (IgG1H3n1-m3) of the human IgG1 antibody heavy chain constant region.
  • SEQ ID NO: 51 is the amino acid sequence of the human IgG1 antibody kappa subtype light chain constant region.
  • Fc fragment refers to a portion of an antibody heavy chain constant region, including a hinge region, a constant region CH2 fragment, and a CH3 fragment.
  • the Fc fragment is the amino acid sequence at positions 216-447 in the constant region of the antibody.
  • Fab fragment antigen binding
  • Fab portion refers to an antibody fragment capable of binding to an antigen produced by treatment of a complete antibody with papain, including the entire light chain (VL-CL). ), heavy chain variable region and CH1 fragment (VH-CH1).
  • single chain fragment variable refers to an antibody of a single-stranded structure generally constructed using genetic engineering techniques, comprising a heavy chain variable region (VH) and a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • a flexible linker is typically designed between the heavy chain variable region and the light chain variable region such that the heavy chain variable region and the light chain variable region can be folded into the correct conformation capable of binding the antigen.
  • bispecific antibody as used herein is an antibody having the ability to bind two different antigens, which may be composed of two Fc fragments and two antigen-binding portions fused thereto, each antigen-binding portion may be in the form of a Fab fragment. Or in the form of a single-chain antibody.
  • bispecific human IgGl antibody refers to a bispecific antibody based on a human IgGl antibody and which possesses the essential features and functions of a human IgGl antibody in addition to the altered structure described herein.
  • a bispecific antibody is described as having two "arms", for example, in the four structures shown in Figures 1a-1d, with the middle bounded, bispecific antibodies can be Divided into two arms.
  • the arm of the bispecific antibody can be composed of an Fc fragment and an antigen binding portion (Fab fragment or single chain antibody).
  • Fab fragment or single chain antibody For an arm consisting of an Fc fragment and a Fab fragment, the structure is similar to that of a normal antibody, and contains intact heavy and light chains.
  • CDRs complementarity determining regions
  • VH or VL There are two common definitions for the CDR sequences of VH or VL, namely the kabat definition and the Chothia definition.
  • CDR sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, CDR sequences are defined using Kabat.
  • variable region sequences of a given antibody the CDR region sequences in the variable region sequences can be analyzed in a variety of ways, for example, using the online software Abysis (http://www.abysis.org/).
  • the term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • a bispecific human IgG1 antibody comprising two Fc fragments having the same hinge region, the amino acid sequence of the hinge region being SEQ ID NO: 1 or SEQ ID NO: 28 and replacing the native human IgG1 antibody constant.
  • the 216-230 sequence of the region, the amino acid position of the antibody constant region is determined according to EU numbering.
  • SEQ ID NO: 1 is the hinge region of a native human IgG2 antibody
  • SEQ ID NO: 28 is a variant of the hinge region of a native human IgG2 antibody.
  • the structure of the bispecific antibody can be optimized from two perspectives: one is heavy chain heteromerization and the other is the correct assembly of the light and heavy chains.
  • the two Fc fragments comprise a mutation that is capable of ensuring heavy chain heteromerization.
  • KIH technology (knH-in-hole, KIH) is a strategy to solve heavy chain heteropolymerization.
  • the KIH technique refers to a structure which facilitates the pairing of heterologous half antibodies by modifying the amino acid sequence of the CH3 region, and can constitute a bispecific antibody while maintaining the structure of a normal antibody as much as possible.
  • the KIH technology utilized comprises one Fc fragment comprising point mutations S354C and T366W and the other Fc fragment comprising point mutations Y349C, T366S, L368A and Y407V.
  • Fc fragment comprising point mutations S354C and T366W
  • the other Fc fragment comprising point mutations Y349C, T366S, L368A and Y407V.
  • the N-termini of the two Fc fragments are fused to a single-chain antibody (scfv) that recognizes different epitopes, respectively.
  • scfv single-chain antibody
  • an N-terminal fusion of a single-chain antibody (scfv) of an Fc fragment, and an N-terminal fusion of a Fab fragment of another Fc fragment the single-chain antibody recognizes a different epitope than the Fab fragment.
  • the N-terminus of the two Fc fragments respectively fuse Fab fragments that recognize different epitopes.
  • the structure of the bispecific antibody thus formed is close to that of a natural antibody and is also a preferred embodiment.
  • Fab fragments that recognize different epitopes comprise the same light chain. This embodiment facilitates the correct assembly of the light and heavy chains and is also a preferred embodiment.
  • one Fab fragment comprises a native human IgG1 antibody constant region CH1 fragment
  • the other Fab fragment comprises a mutated human IgG1 antibody constant region CH1 fragment
  • the mutated human IgG1 antibody constant region CH1 fragment comprises a point mutation G137E Any one, two or three of N203D and R214T, wherein the amino acid sequence of the CH1 fragment is the amino acid sequence of positions 118 to 215 of the constant region of the antibody.
  • the amino acid sequence of the native human IgG1 antibody constant region CH1 fragment is SEQ ID NO: 2.
  • the amino acid sequence of the mutated human IgG1 antibody constant region CH1 fragment is SEQ ID NO:30.
  • the introduction of the above mutations in a Fab fragment can change the charge characteristics of the heavy chain containing the mutation without changing the structure/function and immunogenicity of the antibody constant region (isoelectric point, pI ), which in turn facilitates the late purification of bispecific antibodies.
  • the three sites 137, 203 and 214 are located in the hydrophilic region of the CH1 domain (the surface of the domain), and mutations do not change the conformation of CH1.
  • the mutation involved is to change the basic amino acid to a neutral amino acid.
  • the above mutations are also naturally occurring in other subtypes of antibodies (such as IgG2, IgG4) and are not expected to cause significant immunogenicity problems.
  • the CH2 fragments of both Fc fragments comprise the point mutations L234F, L235E and P331S, wherein the amino acid sequence of the CH2 fragment is the amino acid sequence at positions 231-340 of the constant region of the antibody. In some embodiments, the amino acid sequence of the CH2 fragment of both Fc fragments is SEQ ID NO:31.
  • the bispecific human IgGl antibody recognizes human immune cell surface antigen and tumor cell surface antigen.
  • the human immune cell surface antigen is CD3E.
  • the anti-CD3E antibody binds to the CD3E subunit in the TCR receptor complex on the T cell surface and provides the first signal for T cell activation (similar to the binding of the MHC-peptide complex on antigen presenting cells to the TCR), favoring T cells. Activation.
  • the bispecific antibody comprising the antigen binding portion of CD3E can realize the enrichment of T cells around the tumor cells and improve the killing efficiency of T cells against tumor cells.
  • the tumor surface antigen is selected from the group consisting of HER1, HER2, HER3, EpCAM, CEA, PSMA, CD19, CD20, CD22, CD38, and BCMA.
  • the HER2 gene is located on the human 17q21 chromosome and encodes a transmembrane protein with a molecular weight of 185kD.
  • the protein has tyrosine kinase activity and exists in an inactive form under normal conditions. It is involved in regulating the normal differentiation of cells, usually only expressed in infancy, and only a few in adults. Low level expression in tissues.
  • the HER2 gene is a double-copy gene in normal cells and is activated by gene mutation, which results in up-regulation of transcription and increased protein expression.
  • HER2 is the second member of the human epidermal growth factor receptor (EGFR) family and belongs to the family of type I receptor tyrosine kinases, which are involved in the growth, differentiation and metabolism of many normal and abnormal epidermal cells. Important regulatory effects, the occurrence, development, and disease state of multiple tumors are associated with HER2.
  • the family has four receptors, named HER1, HER2, HER3 and HER4, which interact to form homo- or heterodimers, especially HER2 heterodimers, in the cell signaling process. Play an important role.
  • HER2 activation inhibits tumor cell apoptosis and promotes tumor cell proliferation; up-regulates VEGF/VPF, accelerates tumor angiogenesis, promotes tumor cell metastasis, and destroys tissue anti-invasive function (Artufel MV, Valero A C, Llado R R, et al. Clin Transl Oncol, 2005, 7. (11): 504-511).
  • Overexpression of HER2 protein plays an important role in inducing cell differentiation, proliferation and transformation, and promoting tumor metastasis, invasion and adhesion (Hynes N E, Stem D F. Biochem Biophys AcTa, 1994, 1198 (2-3): 165-184. ).
  • CEA cancer-embryonic antigen
  • colorectal cancer tissue which acts as an antigen to cause an immune response in patients. It is widely found in digestive system cancers of the origin of endoderm, and is also present in the digestive tract tissues of normal embryos, and may also be present in trace amounts in normal human serum.
  • Carcinoembryonic antigen is a broad-spectrum tumor marker that can reflect the existence of various tumors. It is a good tumor marker for the judgment of colorectal cancer, breast cancer and lung cancer, disease development, monitoring and prognosis. Things.
  • PSMA is a 110kDa type II transmembrane protein.
  • the gene is located on the chromosome break arm 11q and expressed in normal prostate epithelial and prostate tumor cells.
  • the expression level in tumor cells is up-regulated.
  • CD19 is a surface protein expressed in B lymphocytes and follicular dendritic cells. It belongs to the immunoglobulin (Ig) superfamily and is located on the short arm of chromosome 16 (16p11.2), encoding a 556 amino acid type I cross. Membrane glycoprotein, molecular weight 95KD. CD19 is expressed only in normal and malignant B cells and is hardly expressed in other tissues. Secondly, CD19 is not lost during the malignant transformation of B cells, and refractory/recurrent cases are still effective; further, CD19 is in hematopoietic stem cells and pro- B cells are not expressed, and B cells can be effectively replenished after treatment is stopped. Therefore, in recent years, various leukemia treatment strategies targeting CD19 have achieved good clinical effects, including CD3+CD19 bispecific antibody and CAR-T targeting CD19.
  • Ig immunoglobulin
  • CD20 antigen is a B cell differentiation antigen expressed only on the surface of pre-B cells and mature B cells. It is expressed in more than 95% of B-cell lymphomas but not in hematopoietic stem cells, plasma cells and other normal tissues. . Multiple monoclonal antibodies targeting CD20 (such as Rituxan) have been clinically successful for the treatment of multiple lymphomas.
  • CD38 is a glycoprotein localized on the membrane that catalyzes the synthesis and degradation of cyclic adenosine diphosphate (cADPR).
  • CD38 is a 45 kDa single-strand transmembrane glycoprotein with a global structure divided into a short N-terminal cytoplasmic tail, a single transmembrane domain and a C-terminally long extracellular domain.
  • CD38 is in most natural killer cells, T cells, B cells, and monocytes/macrophages. There is also a certain degree of expression on platelets and red blood cells.
  • the monoclonal antibody (daratumumab) targeting CD38 has been approved for the treatment of multiple myeloma.
  • BCMA B-cell maturation antigen
  • BCMA is a type III transmembrane protein composed of 185 amino acid residues. It belongs to the TNF receptor family and binds to its ligand B cell activating factor BAFF or the proliferation-inducing ligand APRIL to stimulate B cell proliferation. BCMA is normally expressed in mature B cells and plasma cells and is also widely expressed in multiple myeloma (MM), which is a very ideal immunotherapy target for multiple myeloma.
  • MM myeloma
  • the application provides a pharmaceutical composition comprising the bispecific human IgGl antibody of the first aspect.
  • the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier, excipient, diluent, and the like.
  • the pharmaceutical composition is for use in treating a tumor, such as a tumor that expresses a tumor surface antigen to which a bispecific human IgGl antibody is directed.
  • the pharmaceutical composition may further comprise a lubricant such as talc, magnesium stearate and mineral oil; a wetting agent; an emulsifier; a suspending agent; a preservative such as benzoic acid, sorbic acid and calcium propionate.
  • a lubricant such as talc, magnesium stearate and mineral oil
  • a wetting agent such as talc, magnesium stearate and mineral oil
  • an emulsifier such as talc, magnesium stearate and mineral oil
  • a suspending agent such as benzoic acid, sorbic acid and calcium propionate.
  • a preservative such as benzoic acid, sorbic acid and calcium propionate.
  • the pharmaceutical compositions of the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application can be delivered using any physiologically acceptable administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration. Drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use may be formulated in a lyophilized formulation or as an aqueous solution by mixing an agent having the desired purity with a pharmaceutically acceptable carrier, excipient, or the like, as appropriate. storage.
  • the present application provides the use of the bispecific human IgG1 antibody of the first aspect, or the pharmaceutical composition of the second aspect, for the preparation of a medicament for preventing or treating a tumor.
  • the present application provides a method of preventing or treating a tumor comprising administering to a subject in need thereof the bispecific human IgG1 antibody of the first aspect or the pharmaceutical composition of the second aspect.
  • the tumor expresses a tumor surface antigen to which the bispecific human IgGl antibody is directed.
  • the tumor is selected from the group consisting of breast cancer, gastric cancer, colorectal cancer, prostate cancer, pancreatic cancer, leukemia, multiple myeloma, and malignant lymphoma.
  • bispecific human IgG1 antibody displaying the present application a bispecific human IgG1 antibody targeting human CD3E and HER2 was prepared.
  • the inventors of the present application first prepared two monoclonal antibodies against human CD3E and HER2, which require the use of a plurality of different recombinant proteins, including extracellular to recombinant human CD3E.
  • CD3E SEQ ID NO: 32
  • human CD3D-extracellular domain CD3D, SEQ ID NO: 33
  • monkey CD3E extracellular domain mfCD3E, SEQ ID NO: 34
  • monkey CD3D extracellular domain mfCD3D, SEQ ID NO: 35
  • mouse CD3E extracellular domain mCD3E, SEQ ID NO: 36
  • mouse CD3D extracellular domain mCD3D, SEQ ID NO: 37
  • HER2 extracellular domain D1D2D3 moiety HER2, SEQ) ID NO: 38
  • CD3E forms a heterodimer with CD3D, so in order to prepare CD3E antigen with natural conformation, we simultaneously expressed CD3E and CD3D, and formed CD3D and CD3E by FcK+FcH heterodimer method.
  • Source dimers were used as antigens in this study.
  • These recombinant proteins have a large number of post-translational modifications (such as glycosylation or disulfide bonds), and thus the use of mammalian cell expression systems will be more conducive to maintaining the structure and function of the recombinant protein.
  • the non-antibody recombinant protein has a His-tag (SEQ ID NO: 39) at the C-terminus or an Fc-segment of the murine antibody IgG2a (mFc, SEQ ID NO: 40).
  • the antibody heavy chain constant region may be derived from a human IgG1 antibody (SEQ ID NO: 43) or a variety of mutants of the IgG1 constant region, such as: IgG1Hn (SEQ ID NO: 44), IgG1Kn (SEQ ID NO: SEQ ID NO: :45), IgG1Hn-m3 (SEQ ID NO: 46), IgG1 kn-m3 (SEQ ID NO: 47), IgG1H3n-m3 (SEQ ID NO: 48), IgG1 kn1-m3 (SEQ ID NO: 49), IgG1H3n1- M3 (SEQ ID NO: 50), the light chain constant region is the kappa subtype (SEQ ID NO: 51).
  • the genes of the above various recombinant proteins were designed and synthesized based on the amino acid sequences of the recombinant proteins for various purposes of the Uniprot database.
  • the recombinant recombinant protein gene is cloned into a suitable eukaryotic expression vector (such as invitrogen pcDNA3.1, etc.) by conventional molecular biology techniques, and then subjected to liposome (such as invitrogen 293fectin, etc.) or other transfer.
  • the prepared recombinant protein expression plasmid is transfected into HEK293 cells (such as HEK293F of Invitrogen) by a staining reagent (such as PEI, etc.), and cultured in serum-free suspension culture for 3-5 days. The culture supernatant is then harvested by centrifugation or the like.
  • HEK293 cells such as HEK293F of Invitrogen
  • a staining reagent such as PEI, etc.
  • the His-tag fusion-expressed recombinant protein was subjected to one-step purification of the recombinant protein in the culture supernatant using a metal chelate affinity chromatography column (such as GE's HisTrap FF).
  • the recombinant protein and recombinant antibody expressed by mFc fusion were subjected to one-step purification using a ProteinA/G affinity chromatography column (e.g., Mabselect SURE, GE).
  • the recombinant protein storage buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (eg, Hitrap desaulting, GE, etc.). If necessary, the antibody samples can be sterilized by filtration and then stored separately at -20 °C.
  • a monoclonal antibody (designated C6G9+L1A7) targeting the tumor antigen HER2 and a monoclonal antibody (designated H10B7+L1A7) targeting human CD3E were obtained using recombinant antibody technology.
  • the heavy chain variable region sequence of C6G9+L1A7 is SEQ ID NO: 14, and the light chain variable region sequence is SEQ ID NO: 13.
  • the heavy chain variable region sequence of H10B7+L1A7 is SEQ ID NO: 12 and the light chain variable region sequence is SEQ ID NO: 13.
  • C6G9+L1A7 and H10B7+L1A7 have the same light chain.
  • the antibody functions and properties of each of C6G9+L1A7 and H10B7+L1A7 were experimentally confirmed.
  • Example 2 Based on two monoclonal antibodies prepared and validated in Example 1, a series of bispecific antibodies against human CD3E and HER2 were designed.
  • each arm of the bispecific antibody may comprise a Fab fragment or scfv
  • four structural bispecific antibodies were designed according to different combinations (see Figures 1a-1d).
  • an Fc fragment mutant (FcK, SEQ ID NO: 41 or FcH, SEQ ID NO: 42) of a human IgG1 antibody based on KIH (Knob-Into-Hole) technology was used, That is, an antigen binding region directed against human CD3E (derived from H10B7+L1A7) was fused to the N-terminus of Fc (FcK) containing a Knob mutation, and an antigen binding region directed against HER2 (derived from C6G9+L1A7) was fused to contain a Hole mutation. The N-terminus of Fc (FcH).
  • BsAb bispecific antibody
  • MAb monoclonal antibody
  • Antibody affinity assays were performed using GE's Biacore X100 plus. Amine coupling kit, human antibody capture kit, His capture kit, and CM5 chip and 10 ⁇ HBS-EP pH 7.4 and other related reagents and consumables are purchased. From GE Healthcare.
  • the affinity of different bispecific antibodies was determined by capture.
  • an anti-His antibody was conjugated to the surface of the CM5 chip, and the His-tagged CD3E antigen (CD3E-FcK-His/CD3D) was used.
  • -FcH The surface of the CM5 chip was captured as a stationary phase, and various anti-CD3E antibody proteins were subjected to a series of concentration gradients through the surface of the stationary phase by a single cycle method to determine the affinity of each antibody protein.
  • the anti-Fc antibody was conjugated to the surface of the CM5 chip, and the various anti-HER2 antibody proteins were diluted to a suitable concentration to capture CM5, respectively.
  • the surface of the chip was used as a stationary phase, and the recombinant HER2-His was subjected to a single cycle to set a series of concentration gradients flowing through the surface of the stationary phase to determine the affinity of different antibody proteins.
  • Example 1 The preparation of human CD3E and HER2 antigens used in this example is described in Example 1.
  • the simultaneous binding of the CD3E and HER2 antigens by the bispecific antibody prepared in Example 2 was detected by a conventional ELISA method.
  • the ELISA procedure was as follows: First, the ELISA plate was coated with CD3E-FcK/CD3D-FcH antigen, and the refrigerator was overnight at 4 ° C; Then, the blocking solution containing 1% BSA was blocked at 37 ° C for 1 hour; after washing, the bispecific antibody was added and incubated at 37 ° C for 1 hour; after washing, HER2-His antigen was added, and incubation was carried out at 37 ° C for 1 hour; after washing, HRP-anti-His IgG was added. Incubate at 37 ° C for 1 hour; after washing, HRP substrate solution was added for color analysis.
  • the results are shown in Figure 2: The various bispecific antibodies were constructed to recognize both CD3E and HER2 antigens.
  • MDA-MB-453 cells were purchased from the Basic Medical Cell Center of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences and cultured in RPMI1640 medium.
  • PBMC were isolated from volunteer whole blood by Ficoll density gradient centrifugation and cultured in RPMI1640 medium.
  • the blood of normal volunteers (50 mL each) was collected, and the collected blood was provided by the inventors and colleagues as volunteers, and all volunteers had signed informed consent.
  • the inclusion criteria for volunteers are:
  • the age is greater than 18 years old;
  • the cultured cells were collected and evaluated for cell viability using trypan blue staining.
  • the viable cells were then adjusted to 3 x 10 6 cells/ml in PBS buffer containing 0.1% BSA, and 90 ⁇ l of the cell suspension was added to each well of a round bottom 96-well plate.
  • the cells were centrifuged (5 min, 350 g), washed with 150 ⁇ l/well of BSA-containing PBS staining buffer, resuspended and incubated with 100 ⁇ l/well of fluorescent dye-conjugated goat anti-human IgG antibody. Incubate for an additional 30 minutes at 4 °C. The cells were then washed by adding 150 ⁇ l/well PBS staining buffer and centrifuging at 350 g for 5 minutes. A second washing step was performed using 150 ⁇ l/well PBS staining buffer.
  • the samples were resuspended in 100 ⁇ l/well PBS staining buffer, and the samples were obtained and analyzed using a flow cytometer (BD Biosciences).
  • the results are shown in Figures 3 and 4:
  • the bispecific antibodies prepared in Example 2 were each capable of recognizing human CD3 expressed on human HER2 and PBMC expressed on MDA-MB-453 human breast cancer cells, respectively.
  • MDA-MB-453 human breast cancer cells HER2 positive cells
  • MDA-MB-468 human breast cancer cells HER2 negative cells, purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences
  • the cell density was adjusted to 2 ⁇ 10 5 /ml, 100 ⁇ l per well was seeded in a 96-well cell culture plate, and then the CD3+HER2 bispecific antibody prepared in Example 2 was added at a starting concentration of 3.3 nM in a 10-fold gradient.
  • different CD3+HER2 bispecific antibodies or control monoclonal antibodies MAb1 and MAb2 were adjusted to the same molarity.
  • PBMC cells effectors were added to the wells to obtain a final E:T ratio of 5:1.
  • separate target cells MDA-MB-453 human breast cancer cells or MDA-MB-468 human breast cancer cells
  • PBMC cells effector cells alone
  • medium alone were used as blank controls.
  • CytoTox Non-radioactive cytotoxicity test kit instructions (CytoTox Non-Radioactive Cytotoxicity Assay, promega, G1780) detects and analyzes the killing rate of bispecific antibody-mediated T cells against target cell MDA-MB-453 human breast cancer cells.
  • the results are shown in Figures 5a - 5g and Table 4:
  • the various bispecific antibodies (BsAbs) constructed in this application are effective in mediating T cell killing of HER2 positive target cells and cannot mediate T cell negative for HER2.
  • the killing of target cells, the form of the antigen binding portion in the bispecific antibody or the different selection of the sequence of the hinge region results in a certain difference in the activity of the bispecific antibody.
  • the bispecific antibody is capable of mediating T cells to achieve greater killing ability against tumor cells.
  • MDA-MB-453 human breast cancer cells expressing HER2 were harvested, counted, and cell viability assessed using trypan blue.
  • the MDA-MB-453 human breast cancer cells were adjusted to have a cell density of 2 ⁇ 10 5 /ml, and 100 ⁇ l per well was seeded in a 96-well cell culture plate, followed by serial dilutions of various CD3+HER2 bispecifics prepared in Example 2. antibody.
  • different CD3+HER2 bispecific antibodies or control monoclonal antibodies (MAb1 and MAb2) were adjusted to the same molarity.
  • human PBMC effector was added to the well to obtain a final E:T ratio of 5:1.
  • a separate target cell (MDA-MB-453 human breast cancer cell) control was also set. After incubation for 20 hours, the cells were pelleted by centrifugation (5 minutes, 350 g) and washed twice with 150 ⁇ l/well PBS buffer. Anti-human CD3 and human CD69 antibodies (eBioscience, 11-0037-42, 12-0699-42) were added and incubated for 30 minutes at 4 ° C in the dark. The cells were then washed twice with 150 ⁇ l/well PBS buffer, resuspended in 100 ⁇ l/well PBS buffer, and analyzed by flow cytometry (BD Biosciences C6) and compared for CD3 positive cell population after treatment with different samples. Difference in expression of the activation marker CD69.

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Abstract

L'invention concerne un anticorps IgG1 humain bispécifique, comprenant deux fragments Fc ayant les mêmes régions de charnière, la séquence d'acides aminés des régions de charnière étant SEQ ID NO : 1 ou SEQ ID NO : 28 et la séquence des positions 216-230 dans la région constante d'anticorps IgG1 humain naturel est remplacée, et la position d'acide aminé dans la région constante d'anticorps est déterminée selon la numérotation EU. L'invention concerne également l'utilisation de l'anticorps bispécifique.
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