WO2022174813A1 - 抗GPRC5DxBCMAxCD3三特异性抗体及其用途 - Google Patents

抗GPRC5DxBCMAxCD3三特异性抗体及其用途 Download PDF

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WO2022174813A1
WO2022174813A1 PCT/CN2022/076832 CN2022076832W WO2022174813A1 WO 2022174813 A1 WO2022174813 A1 WO 2022174813A1 CN 2022076832 W CN2022076832 W CN 2022076832W WO 2022174813 A1 WO2022174813 A1 WO 2022174813A1
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amino acid
acid sequence
seq
antibody
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French (fr)
Chinese (zh)
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徐伟
沈湾湾
李莉
王旋
朱陈娟
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Innovent Biologics Suzhou Co Ltd
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Innovent Biologics Suzhou Co Ltd
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Priority to EP22755586.9A priority Critical patent/EP4296286A4/en
Priority to US18/546,862 priority patent/US20250074996A1/en
Priority to JP2023550109A priority patent/JP2024510098A/ja
Priority to CA3211407A priority patent/CA3211407A1/en
Priority to CN202280015788.6A priority patent/CN116897166A/zh
Priority to AU2022223152A priority patent/AU2022223152B2/en
Publication of WO2022174813A1 publication Critical patent/WO2022174813A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • 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
    • C07K2317/90—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

  • T-cell bispecific antibodies have been used in tumor therapy. Such antibodies can form synapses between cytotoxic T lymphocytes and tumor cells, inducing tumor cell destruction.
  • BsAbs typically involve dual targeting of tumor-associated antigens (TAAs) and T-cell surface antigens (also known as T-cell engaging antigens, TEAs).
  • TAAs tumor-associated antigens
  • TEAs T-cell engaging antigens
  • BsAb-based therapeutic strategies typically rely on the distribution of tumor-associated antigens on the tumor cells to be treated. This results in treatment selectivity for the patient population and treatment limitations. Additionally, studies have shown that therapeutic modalities targeting a single TAA site may limit the effectiveness of treatment by causing disease recurrence due to tumor escape mechanisms.
  • the present invention provides a trispecific Y-type antibody molecule having the above-mentioned advantages, comprising an antigen binding site that specifically binds GPRC5D, BCMA and CD3, and comprising: two antibody arms, located in antibody arm C A stem at the end, and a conjugation member, wherein the conjugation member is conjugated to the antibody arm and/or the C-terminus of the stem.
  • the present invention also provides nucleic acids, vectors and host cells encoding the antibody molecules of the present invention; and uses of the antibody molecules of the present invention, especially methods and uses for the treatment of GPRC5D-positive and/or BCMA-positive tumors, such as multiple myeloma .
  • Figure 1 shows schematically: the structures of the designed trispecific antibody molecules of Format_1, 2, 6 and 7.
  • CDRs can also be determined based on having the same Kabat numbering positions as the reference CDR sequence.
  • a residue position in an antibody variable region refers to the numbering system according to the Kabat ( Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed . Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • affinity or "binding affinity” refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively).
  • KD dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • treatment refers to a clinical intervention intended to alter the natural course of disease in an individual being treated. Desired therapeutic effects include, but are not limited to, preventing disease occurrence or recurrence, reducing symptoms, reducing any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease state, and relieving or improving prognosis.
  • the antibody molecules of the invention are used to delay disease progression or to slow disease progression.
  • a high affinity CD3 antigen binding site is used, eg with a KD value of less than 50 nM.
  • the antigen binding specificity is cross-reactive with human and monkey CD3.
  • this technique involves introducing a "bump" at the interface of one chain and a corresponding "hole” at the interface of the other chain to be paired with, so that the bulge can be placed in the cavity.
  • a preferred interface comprises the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain to be paired with.
  • Bulges can be constructed by replacing small amino acid side chains from the interface of the CH3 domain of the heavy chain constant domain of one chain with larger side chains (eg tyrosine or tryptophan).
  • the same or similar to the bulge is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other chain to be paired size of the compensatory cavity.
  • Another optional interface is the CL domain of the Fab fragment described above, comprising the CL domain of the light chain and the CH1 domain of the heavy chain, which facilitates correct interaction between the two chains of the Fab fragment by building a bump-hole interaction. Heterodimerization.
  • Fv fragment is used herein to refer to an antibody fragment comprising a heavy chain variable domain VH and a light chain variable domain VL.
  • dsFv disulfide stabilized variable fragments
  • single-chain Fab or “scFab” is used to refer to a single-chain polypeptide formed by linking two chains of a Fab fragment through a linker.
  • an antibody of the invention may comprise a Fab fragment selected from: (i) a Fab consisting of a chain comprising VH-CH1 and a chain comprising VL-CL; and (ii) a Fab comprising a chain comprising A crossFab consisting of one chain of VH-CL and one chain comprising VL-CH1.
  • the antibodies of the invention may comprise a single-chain Fab fragment selected from the group consisting of: (i) a single-chain Fab comprising VH-CH1-linker-VL-CL; and (ii) a VH-CL - Linker-Single chain Fab of VL-CH1.
  • immunoglobulin constant domains can be selected according to the intended function of the antibody molecule.
  • the constant domain may be an IgA, IgD, IgE, IgG or IgM domain, especially an immunoglobulin constant domain of human IgG, eg, a constant domain of human IgGl, IgG2, IgG3 or IgG4, preferably of human IgGl constant domain.
  • a Fab fragment of an antibody may comprise the CH and CL constant domains from IgGl.
  • the Fc region of an antibody may comprise the CH2 and CH3 domains from IgGl.
  • the linker peptide comprises the amino acid sequence TS(G4S)n, wherein n is an integer equal to or greater than 1, eg, n is an integer of 2, 3, 4, 5, 6 or 7.
  • the linker peptide is a hinge region from an immunoglobulin, eg, a hinge region amino acid sequence comprising "CPPC”, eg, the amino acid sequence "EPKSCDKTHTCPPCP” (SEQ ID NO: 114) or "EPKSSDKTHTCPPCP” (SEQ ID NO: 114) NO: 115).
  • Y-type antibody molecules also encompass molecules that have the same or similar Y-type structure as IgG molecules, such as Y-type molecules with conjugation moieties on the antibody arms and/or stems of the antibody.
  • the antibody molecule of the present invention is a trispecific antibody molecule with a conjugation moiety.
  • the Y-type antibody molecules of the present invention preferably also comprise conjugation moieties that are conjugated to the arms and/or stems, said conjugation moieties comprising additional antigen binding domains such as Fab, scFv, sdAB , preferably scFv.
  • the antigen-binding domain in the conjugation member may confer antigen-binding specificity different from that of the antibody arm.
  • the two antibody arms of the Y-shaped molecule may comprise antigen-binding domains that provide a first and a second antigen-binding specificity, respectively, while the conjugation component of the Y-shaped molecule comprises an antigen-binding domain that provides a third antigen-binding specificity , and the first, second and third antigen binding specificities are different.
  • the two antibody arms of the Y-shaped molecule may comprise antigen-binding domains that provide a first antigen-binding specificity, while the conjugation component of the Y-shaped molecule comprises antigen-binding domains that provide second and third antigen-binding specificities, And the first, second and third antigen binding specificities are different.
  • the first, second and third antigen binding specificities are respectively for different antigen binding specificities selected from CD3, BCMA and GPRC5D.
  • the first and second antigen binding specificities can be directed against a different tumor associated antigen (TAA) selected from GPRC5D and BCMA, respectively;
  • the third antigen binding specificity can be directed against the T cell surface antigen (TEA) CD3.
  • the first (or second) and third antigen binding specificities can be directed against a different tumor associated antigen (TAA) selected from GPRC5D and BCMA, respectively, while the second (or first) antigen binding specificity can be directed against the T cell surface Antigen (TEA).
  • the present invention provides trispecific Y-type antibody molecules.
  • the Y-type antibody molecule of the present invention has the structure of the following formula:
  • M1 and M2 respectively represent the first antibody arm and the second antibody arm of the Y-shaped antibody molecule comprising the antigen binding site, wherein the antigen binding sites of the first antibody arm and the second antibody arm may be the same or different;
  • X1 denotes a conjugation moiety conjugated to the C-terminus of the antibody arm
  • X2 denotes a conjugation moiety conjugated to the C-terminus of the stem, wherein the conjugation moiety comprises an antigen-binding site, wherein the conjugation moiety is directly or via a linker peptide conjugated to an antibody molecule
  • the antibody specifically binds GPRC5D, BCMA and CD3 through the antibody arm and the conjugation member
  • the antibody arms (M1 and M2) of the antibody molecule comprise an antigen binding site selected from Fab or scFab (preferably Fab), and neither p and q are 0; wherein the antibody arms M1 and M2 bind the same the first antigen, the conjugation elements X1 and X2 bind the second antigen and the third antigen, respectively, wherein the first, second and third antigens are different from each other and independently selected from the group consisting of: GPRCR5D, BCMA and CD3,
  • conjugation part X1 is conjugated to the Fab light chains of both antibody arms M1 and M2 of the antibody molecule; and the conjugation part X2 is conjugated to the two Fc structures of the antibody molecule stem on the domain.
  • the first Fc domain and the second Fc domain comprise a hinge region having "CPPC" amino acid residues, whereby the antibody molecules of the invention are in the first and second Fc domains Interchain disulfide bonds are formed between them to facilitate the correct pairing of the polypeptide chains of the antibody molecules of the invention.
  • the antibody arm is fused to the N-terminus of the Fc region through the hinge region of the Fc region.
  • the conjugation member is preferably conjugated to the stem and/or arm of the antibody molecule via a flexible linker peptide.
  • the linker peptide is 5-15 amino acids in length, eg, 10, 12, 15 amino acids in length.
  • the present invention when one of p and q of Formula I is 0, the present invention provides an IgG-like antibody molecule having the structure:
  • M1 and M2 respectively comprise a Fab or scFab, preferably a Fab, that binds the first and second antigen,
  • the present invention provides IgG-like antibody molecules having the structure:
  • M1 and M2 respectively comprise a Fab or scFab, preferably a Fab, that binds the first and second antigen, and
  • X2 contains an scFv that binds a third antigen
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • M1 and M2 comprise Fabs that bind the first and second antigens, respectively, and
  • X2 contains an scFv that binds a third antigen
  • the present invention provides IgG-like antibody molecules having the structure:
  • M1 and M2 comprise a Fab or scFab, preferably a Fab, that binds the same first antigen, and
  • X1 and X2 comprise scFvs that bind the second and third antigens, respectively,
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • the present invention provides IgG-like antibody molecules having the structure:
  • M1 and M2 and X2 comprise scFvs that bind first, second, and third antigens, respectively,
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • Antibody molecules of the present invention provide antigen-binding sites that specifically bind to the first, second, and third antigens through the antibody arms and the antigen-binding sites of the conjugation member, wherein the first, second, and third antigens interact with each other. Not identical, and independently selected from the group consisting of the tumor-associated antigen GRPC5D and the tumor-associated antigen BCMA and the T-cell engagement antigen CD3.
  • the present invention provides an antibody molecule of formula I, wherein, when neither p and q are 0, the first antibody arm and the second antibody arm of the antibody provide a first antigen binding site, and are conjugated to The conjugation member on the C-terminus of the antibody arm provides the second antigen binding site, and the conjugation member conjugated to the antibody stem provides the third antigen binding site.
  • Antigen binding sites (including antigen binding sites that specifically bind GPRC5D, BCMA, and CD3) that can be used in the trispecific antibody molecules of the invention can be any antibody or antibody fragment comprising any antibody capable of binding an antigen of interest.
  • the antigen binding sites in the antibody arm and the conjugation moiety can independently be a scFv, dsFv, Fab, scFab, or sdAb.
  • the antigen binding site of the antibody arm is selected from: Fab and scFab, preferably Fab.
  • the antigen binding site of the conjugation member is selected from the group consisting of: scFv, dsFv and dsAb, preferably scFv, more preferably disulfide stabilized scFv (ie, dsscFv).
  • the antigenic structural site of the antibody arm is a Fab
  • the antigenic structural site of the conjugation member is a scFv, especially a dsscFv.
  • Fab_GPRC5D, Fab_BCMA and Fab_CD3 are used to denote the antigen binding site of the Fab form that binds GPRC5D, BCMA and CD3, respectively.
  • ScFv_GPRC5D, ScFv_BCMA and ScFv_CD3 are used to denote the antigen binding sites of scFv forms that bind GPRC5D, BCMA and CD3, respectively.
  • the Fab antigen binding site comprised in the antibody molecule of the invention consists of two polypeptide chains comprising immunoglobulin VH, CH1, VL and CL domains, wherein the VH and VL are paired and the CH1 and CL pair to form the antigen binding site.
  • one chain comprises VH and CH1 from N-terminus to C-terminus (ie, VH-CH1) and the other chain comprises VL and CL from N-terminus to C-terminus (ie, VL-CL) .
  • one chain comprises VH and CL from N-terminus to C-terminus (ie, VH-CL) and the other chain comprises VL and CH1 from N-terminus to C-terminus (ie, VL-CH1 ).
  • the Fab constitutes the antigen binding site of the antibody arm of the antibody molecule of the invention.
  • the Fab may be fused to the N-terminus of the Fc domain of the antibody stem through the C-terminus of a VH-containing chain; or wherein the Fab may be fused to the Fc structure of the antibody stem through the C-terminus of a VL-containing chain N-terminal of the domain.
  • the Fab comprises a VH-CH1 chain and a VL-CL chain, and binds to the Fc region of the antibody stem through the C-terminus of the VH-CH1 chain.
  • the antibody molecule of the present invention comprises a conjugation moiety conjugated to an antibody arm
  • the conjugation moiety is conjugated to the C-terminus of a Fab chain that is not linked to the antibody stem.
  • the Fab chain that is not linked to the antibody stem is also referred to as the light chain of the Fab.
  • the antibody molecules of the invention have a conjugation moiety that is conjugated to the Fab light chain of the antibody arm.
  • the scFab antigen binding site comprised in the antibody molecule of the invention consists of one polypeptide chain comprising the VH, CH1, VL and CL domains of an immunoglobulin, wherein the VH and VL are paired and the CH1 and CL pair to form the antigen binding site.
  • the scFab comprises from N-terminus to C-terminus: VH-CH1, a linker, and VL-CL.
  • the scFab comprises from N-terminus to C-terminus: VL-CL, a linker, and VH-CH1.
  • the scFab comprises from N-terminus to C-terminus: VH-CL, a linker, and VL-CH1. In other embodiments, the scFab comprises from N-terminus to C-terminus: VL-CH1, a linker, and VH-CL. In some embodiments, the scFab constitutes the antigen binding site of the antibody arm of the antibody molecule of the invention. In some embodiments, the scFab is fused to the N-terminus of the Fc domain of the antibody stem via the C-terminus of its polypeptide chain.
  • the scFab comprises from N-terminus to C-terminus: VL-CL, linker, and VH-CH1, and binds to the Fc region of the antibody stem through the C-terminus of the CH1 domain.
  • the antibody molecule of the invention comprises a scFab antibody arm, the conjugation moiety of the antibody molecule is conjugated only at the C-terminus of the Fc domain of the antibody stem.
  • the Fab or scFab comprised in the antibody molecule of the invention comprises the CH1 domain from an IgG immunoglobulin, eg, the CH1 domain of IgG1, preferably the CH1 domain of human IgG1.
  • the CH1 domain comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity thereto.
  • the Fab or scFab comprised in the antibody molecule of the invention comprises a kappa light chain constant domain from an IgG immunoglobulin, eg, the CL ⁇ domain of IgG1, preferably the CL ⁇ domain of human IgG1.
  • the Fab or scFab comprises a kappa light chain constant domain having the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the same.
  • the Fab or scFab comprised in the antibody molecule of the invention comprises a lamda light chain constant region from an IgG immunoglobulin, eg, the CL lambda domain of IgGl, preferably the CL lambda domain of human IgGl.
  • the Fab comprises a lamda light chain constant domain having the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence that is at least 90%, 95%, 97%, 98%, or 99% identical to the same.
  • the antibody arms M1 and M2 of the antibody molecule of the invention comprise Fab or scFab (preferably Fab) that bind different antigens
  • the light chain constant domains of the Fab or scFab of antibody arm M1 and antibody arm M2 different from each other.
  • the Fab of antibody arm M1 comprises a kappa light chain constant domain
  • the Fab of antibody arm M2 comprises a lamda light chain variable domain
  • the Fab of antibody arm M1 comprises a lamda light chain constant domain
  • the antibody The Fab of arm M2 contains the kappa light chain variable domain.
  • the scFv antigen-binding site comprised in the antibody molecule of the invention consists of one polypeptide chain comprising the VH and VL domains of an immunoglobulin, wherein the VH and VL are linked by a linker to pair to form the antigen-binding site point.
  • the scFv is in a trans configuration comprising, from N-terminus to C-terminus: VH, linker, and VL (VH-linker-VL).
  • the scFv is in a cis configuration comprising, from N-terminus to C-terminus: VL, linker, and VH (VH-linker-VL).
  • the scFv constitutes the antigen binding site of the antibody arm of the antibody molecule of the invention and is fused to the N-terminus of the Fc domain of the antibody stem through the C-terminus of its polypeptide chain.
  • the scFv constitutes the antigen binding site of the conjugation moiety of the antibody molecule of the invention.
  • the scFv is fused to the C-terminus of the antibody stem and/or the C-terminus of the antibody arm through the N-terminus of its polypeptide chain.
  • the antibody arm comprises Fab
  • the conjugation part comprises scFv, wherein the scFv is fused to the C-terminus of the Fc of the antibody stem and/or the C-terminus of the Fab light chain of the antibody arm through the N-terminus of its polypeptide chain end.
  • the scFv antigen binding site comprised in the antibody molecule of the invention is a disulfide stabilized scFv, ie, a dsscFv.
  • the antigen binding site is a dsscFv
  • the disulfide bond introduced by mutation between the VH and VL variable domains of the scFv can be located between the following pairs of residues (the following positions are determined according to Kabat numbering):
  • cysteine substitutions can be introduced at residues 44 of VH and 100 of VL, thereby forming a disulfide bond between the two residues when said VH and VL are paired.
  • the GPRC5D antigen binding site that can be used in the antibody molecules of the invention can be any antibody or antibody fragment capable of binding GPRC5D.
  • the GPRC5D antigen binding site contained in the antibody arm or conjugation member may independently be a scFv, dsFv, Fab, scFab, or sdAb, and preferably has the scFv or Fab described above Structure of the antigen binding site.
  • the GPRC5D antigen binding site of the antibody arm is selected from: Fab and scFab, preferably Fab.
  • the GPRC5D antigen binding site of the conjugation member is selected from: scFv, dsFv and dsAb, preferably scFv, more preferably disulfide stabilized scFv (ie, dsscFv).
  • the antibody comprises an antibody arm that binds GPRC5D, and the GPRC5D antigenic structural site of the antibody arm is a Fab; in another more preferred embodiment, the antibody comprises a conjugation moiety that binds GPRC5D, and the The GPRC5D antigenic structural site of the co-part is scFv, especially dsscFv.
  • the antigen binding site that binds GPRC5D comprises:
  • the antigen binding site that binds to GPRC5D comprises a combination of CDR sequences selected from the group consisting of:
  • the antigen binding site e.g., scFv or Fab that binds GPRC5D
  • the antigen binding site comprises a variant of one of the combinations of CDR sequences, wherein the variant is at 1, 2, 3, 4, 5 or preferably 6
  • a total of at least one and no more than 5, 4, 3, 2 or 1 amino acid changes are included in the CDR regions, preferably the heavy chain CDR3 remains unchanged.
  • the antigen binding site that binds GPRC5D eg, the GPRC5D antigen binding site having the scFv or Fab structure described above, comprises a heavy chain variable domain VH selected from the group consisting of:
  • a heavy chain variable structure comprising the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area;
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 98 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 100 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area.
  • the antigen binding site that binds GPRC5D e.g., for example, the GPRC5D antigen binding site having the scFv or Fab structure described above, comprises a light chain variable domain VL selected from the group consisting of:
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 99 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area;
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 101 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto area.
  • the antigen binding site that binds to GPRC5D comprises a combination of VH and VL amino acid sequences selected from the group consisting of:
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith domain; or
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 27 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith domain; or
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith domain; or
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 98 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 99 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area;
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 100 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 101 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area.
  • the antigen binding site that binds GPRC5D comprises a combination of VH and VL sequences selected from the group consisting of:
  • the scFv when the antigen binding site that binds GPRC5D is a disulfide stabilized scFv, the scFv also comprises a cysteine substitution at position 44 of the VH domain and at position 100 of the VL domain Contains cysteine substitutions.
  • the BCMA antigen binding site that can be used in the antibody molecules of the invention can be any antibody or antibody fragment capable of binding BCMA.
  • the BCMA antigen binding site contained in the antibody arm or conjugation member may independently be a scFv, dsFv, Fab, scFab, or sdAb, and preferably has the scFv or Fab described above Structural antigenic structural sites.
  • the BCMA antigen binding site of the antibody arm is selected from: Fab and scFab, preferably Fab.
  • the BCMA antigen binding site of the conjugation member is selected from the group consisting of: scFv, dsFv and dsAb, preferably scFv, more preferably disulfide stabilized scFv (ie, dsscFv).
  • the antibody comprises an antibody arm that binds BCMA, and the BCMA antigenic structural site of the antibody arm is a Fab; in another more preferred embodiment, the antibody comprises a conjugation moiety that binds BCMA, and the The BCMA antigenic structural site of the co-part is scFv, especially dsscFv.
  • the antigen binding site that binds BCMA comprises: a heavy chain variable domain as set forth in SEQ ID NO:39 HCDR1, 2 and 3 sequences, and LCDR1, 2 and 3 sequences of the light chain variable domain as set forth in SEQ ID NO:40.
  • an antigen binding site that binds BCMA such as a BCMA antigen binding site having the scFv or Fab structure described above, comprises the following combination of CDR sequences:
  • an HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33;
  • an HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 34;
  • an HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35;
  • an LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 37;
  • said antigen binding site that binds BCMA comprises a variant of one of said combinations of CDR sequences, wherein said variant comprises at least one in 1, 2, 3, 4, 5 or preferably 6 CDR regions in total And no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), preferably the heavy chain CDR3 remains unchanged.
  • the antigen binding site that binds BCMA comprises or has at least 80% of the amino acid sequence shown in SEQ ID NO: 39 , 85%, 90%, 95%, 97%, 98% or 99% sequence identity of the heavy chain variable domain VH of the amino acid sequence.
  • the antigen binding site that binds BCMA comprises or has at least 80% of the amino acid sequence set forth in SEQ ID NO:40. , 85%, 90%, 95%, 97%, 98% or 99% sequence identity of the light chain variable domain VL of the amino acid sequence.
  • the antigen binding site that binds BCMA comprises or has at least 80% of the amino acid sequence shown in SEQ ID NO: 39 , 85%, 90%, 95%, 97%, 98% or 99% sequence identity of the heavy chain variable domain VH of the amino acid sequence; and comprising the amino acid sequence set forth in SEQ ID NO:40 or having at least Light chain variable domain VL of amino acid sequences of 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.
  • the antigen binding site that binds BCMA such as the BCMA antigen binding site having the scFv or Fab structure described above, comprises a VH domain having the amino acid sequence shown in SEQ ID NO:39 and a VH domain having the amino acid sequence shown in SEQ ID NO:40 The VL domain of the amino acid sequence shown.
  • the antigen binding site that binds BCMA is a disulfide stabilized scFv
  • the scFv also comprises a cysteine substitution at position 44 of the VH domain and at position 100 of the VL domain Contains cysteine substitutions.
  • the CD3 antigen binding site that can be used in the antibody molecules of the invention can be any antibody or antibody fragment capable of binding CD3.
  • the CD3 antigen binding site contained in the antibody arm or conjugation member may independently be an scFv, dsFv, Fab, scFab, or sdAb, and preferably has the scFv or Fab described above Structural antigenic structural sites.
  • the CD3 antigen binding site of the antibody arm is selected from: Fab and scFab, preferably Fab.
  • the CD3 antigen binding site of the conjugation member is selected from the group consisting of: scFv, dsFv and dsAb, preferably scFv, more preferably disulfide stabilized scFv (ie, dsscFv).
  • the antibody comprises an antibody arm that binds GPRC5D, and the CD3 antigenic structural site of the antibody arm is a Fab; in another more preferred embodiment, the antibody comprises a conjugation moiety that binds GPRC5D, and the The CD3 antigenic structural site of the co-part is scFv, especially dsscFv.
  • the antigen binding site that binds CD3, such as the CD3 antigen binding site having the scFv or Fab structure described above, comprises: a heavy chain variable domain as shown in SEQ ID NO:48 HCDR1, 2 and 3 sequences, and LCDR1, 2 and 3 sequences of the light chain variable domain as set forth in SEQ ID NO:49.
  • the CD3-binding antigen-binding site such as the CD3 antigen-binding site having the scFv or Fab structure described above, comprises: a heavy chain variable domain as shown in SEQ ID NO:50 HCDR1, 2 and 3 sequences of SEQ ID NO: 49, and LCDR1, 2 and 3 sequences of the light chain variable domain shown in SEQ ID NO:49.
  • the CD3-binding antigen-binding site such as the CD3 antigen-binding site having the scFv or Fab structure described above, comprises the following combinations of CDR sequences:
  • an HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 41;
  • an HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 42 or 47;
  • an HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 43;
  • an LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 45;
  • said antigen binding site that binds CD3 comprises a variant of one of said combinations of CDR sequences, wherein said variant comprises at least one in 1, 2, 3, 4, 5 or preferably 6 CDR regions in total And no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions), preferably the heavy chain CDR3 remains unchanged.
  • the CD3-binding antigen-binding site such as the CD3 antigen-binding site having the scFv or Fab structure described above, comprises or has at least the amino acid sequence set forth in SEQ ID NO: 48 or 50.
  • Heavy chain variable domain VHs of amino acid sequences of 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.
  • the CD3-binding antigen-binding site such as the CD3 antigen-binding site having the scFv or Fab structure described above, comprises or has at least 80% of the amino acid sequence set forth in SEQ ID NO: 49. , 85%, 90%, 95%, 97%, 98% or 99% sequence identity of the light chain variable domain VL of the amino acid sequence.
  • the CD3-binding antigen-binding site comprises or has at least the amino acid sequence set forth in SEQ ID NO: 48 or 50.
  • the antigen binding site that binds to CD3, such as the CD3 antigen binding site having the scFv or Fab structure described above, comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 48 or 50 and a VH domain having the amino acid sequence shown in SEQ ID NO: 48 or 50. : VL domain of the amino acid sequence shown in 49.
  • the antigen binding site that binds to CD3, such as the CD3 antigen binding site having the scFv or Fab structure described above comprises a VH domain having the amino acid sequence shown in SEQ ID NO:48 and a VH domain having the amino acid sequence shown in SEQ ID NO:48. VL domain of the amino acid sequence shown in 49.
  • the scFv when the antigen binding site that binds CD3 is a disulfide stabilized scFv, the scFv also comprises a cysteine substitution at position 44 of the VH domain and at position 100 of the VL domain Contains cysteine substitutions.
  • the antibody molecule of the present invention comprises a stem formed by a first Fc domain and a second Fc domain located at the C-terminus of the antibody arm.
  • the first and second Fc domains are the same.
  • the first Fc domain and the second Fc domain are different, pair and heterodimerize.
  • Fc domain fragments suitable for use in the antibody molecules of the present invention can be any antibody Fc domain.
  • the Fc domain of an antibody of the invention may comprise two or three constant domains, ie, a CH2 domain, a CH3 domain, and an optional CH4 domain.
  • the Fc domain of the antibody of the invention comprises from N-terminal to C-terminal: CH2-CH3, more preferably from N-terminal to C-terminal: hinge region-CH2-CH3.
  • the Fc domain may comprise second and third constant domains (CH2 and CH3 domains) derived from IgG, IgA and IgD class antibodies; or from IgM and IgE class antibodies The second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain).
  • the Fc domain of the antibody molecule is an Fc domain from IgG, eg, an Fc domain from IgGl, IgG2 or IgG4, preferably an Fc domain from human IgGl.
  • the antibody molecules of the invention may comprise modifications in the Fc domain that alter effector function.
  • one or more effector functions of an Fc domain of the invention have been reduced or eliminated relative to a wild-type Fc domain of the same isotype.
  • the effector function of the Fc domain can be reduced or eliminated by any method selected from the group consisting of altering the glycosylation of the Fc domain, using Fc isotypes that naturally have reduced or eliminated effector functions, and modifying the Fc domain amino acid sequence.
  • effector function is reduced or eliminated by reducing glycosylation of the Fc domain.
  • Various methods are known in the art that can be used to reduce glycosylation of the Fc domain, including but not limited to: producing antibody molecules of the invention in an environment that does not allow wild-type glycosylation; removing pre-existing glycosylation on the Fc domain; carbohydrate group; and modification of the Fc domain such that wild-type glycosylation does not occur.
  • the glycosylation of the Fc domain is reduced by modifying the Fc domain, such as introducing a mutation at position 297 of the Fc domain such that the wild-type asparagine residue at this position is interfered with by another sugar at this position Sylated amino acid substitutions, such as the N297A mutation.
  • effector function is reduced or eliminated by modifying the amino acid sequence of at least one Fc domain.
  • the Fc domain modification may be selected from the group consisting of introducing point mutations that impair binding of the Fc domain to one or more Fc receptors at one or more of the following positions: 238, 239, 248, 249, 252, 254 , 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373 , 376, 382, 388, 389, 414, 416, 419, 434, 435, 437, 438, and 439; or point mutations that impair C1q binding at positions selected from: 270, 322, 329, and 321.
  • the antibody molecules of the invention may also comprise modifications in the Fc domain that alter the binding affinity for one or more Fc receptors.
  • the Fc receptor is an Fc ⁇ receptor, in particular a human Fc ⁇ receptor.
  • the modification reduces the effector function of the antibody molecule of the invention.
  • the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the Fc domain of the antibody molecule of the invention comprises amino acid substitutions at positions 234 and 235 (EU numbering).
  • the amino acid substitutions are L234A and L235A (LALA mutation).
  • the Fc domain of the antibody molecule of the invention comprises an amino acid substitution at position 329 (EU numbering).
  • the amino acid substitution is P329G.
  • the antibody molecule of the present invention is an asymmetric antibody molecule (such as the antibody molecules of Format_2 and Format_7)
  • the antibody molecule of the present invention can be placed in the first and second
  • the Fc domain contains mutations that facilitate heterodimerization of the first Fc domain with the second Fc domain.
  • complementary Knob and Hole mutations can be introduced in the first Fc domain and the second Fc domain based on the Knob-in-Hole technology.
  • the present invention provides trispecific antibody molecules comprising in a first Fc domain and a second Fc domain, respectively, that facilitate pairing and heterodimerization of the first and second Fc domains
  • the first and second heterodimerization mutations of .
  • the first heterodimerization mutation on the first Fc domain comprises a Knob mutation and the second heterodimerization mutation in the second Fc domain comprises a Hole mutation complementary to said knob mutation; or , the first heterodimerization mutation on the first Fc domain comprises a Hole mutation, and the second heterodimerization mutation on the second Fc domain comprises a Knob mutation complementary to the hole mutation.
  • the Knob mutation is T366W and the complementary hole mutation is T366S/L368A/Y407V.
  • the present invention provides trispecific antibody molecules comprising:
  • a homodimeric Fc-region of human IgG4 subclass optionally with mutations P329G, S228P and L235E, or
  • one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises the mutations T366W and S354C, while the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C,
  • one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or
  • one Fc-region polypeptide comprises the mutations T366W and S354C, while the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C,
  • one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or
  • one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C, or
  • Fc-region polypeptide comprises the mutations T366W and S354C, while the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.
  • the Fc domains of the antibody molecules of the invention may also contain other mutations that facilitate purification of heteromultimeric antibodies.
  • the H435R mutation can be introduced into one of the first and second Fc domains (eg, a Hole-mutated Fc domain) to facilitate purification of heteromultimeric antibodies of interest using Protein A.
  • the antibody stem is linked to the antibody arm at the N-terminus of the Fc domain.
  • suitable linker peptides for linking the antibody arms and stem Fc domain fragments in the antibody molecules of the invention may be any flexible linker peptide known in the art.
  • the linker peptide may comprise a hinge region amino acid sequence from IgGl, or may comprise an amino acid sequence selected from the group consisting of (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, wherein n is an integer of at least 1.
  • the antibody stem is linked to the antibody arm by a hinge region from IgG, especially a hinge region from human IgGl.
  • a mutation such as C220S, can be introduced in the hinge region to facilitate the formation of the heteromultimeric antibody of interest.
  • the antibody arms M1 and M2 comprise Fab antigen-binding sites that bind the first antigen and the second antigen, respectively, and the conjugation member X2 comprises the scFv antigen-binding site that binds the third antigen, wherein
  • the first, second and third antigens are each different and are independently selected from: GPRC5D, BCMA and CD3.
  • the present invention provides trispecific Y-type antibody molecules having the structure:
  • M1 and M2 represent the first antibody arm and the second antibody arm of the antibody molecule, respectively, and M1 and M2 respectively comprise a Fab or scFab, preferably a Fab, that binds the first and second antigens, respectively,
  • Fc::Fc represents the stem of an antibody molecule, consisting of a paired and dimerized first and second Fc domains, wherein the first and second antibody arms, respectively, are directly or via a linking peptide (preferably the hinge region) Linked to the N-terminus of the first Fc domain and the second Fc domain;
  • X2 represents a conjugation moiety conjugated on the C-terminus of the stem, wherein the conjugation moiety comprises an scFv that binds a third antigen, wherein X2 is conjugated directly or via a linking peptide at the C-terminus of the first Fc domain, or conjugated At the C-terminus of the second Fc domain,
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • the present invention provides trispecific antibody molecules of Format-2 structure, wherein:
  • the Fab or scFv that binds GPRC5D can be any suitable Fab or scFv that specifically binds GPRC5D, such as the GPRC5D antigen binding site of the invention of the scFv or Fab structure described above, especially is a scFv or Fab having the VH and VL amino acid sequences described above.
  • the BCMA-binding Fab or scFv may be any suitable Fab or scFv that specifically binds BCMA, such as the BCMA antigen binding site of the invention of the scFv or Fab structure described above, especially is a scFv or Fab having the VH and VL amino acid sequences described above.
  • the antigen binding site that binds CD3 may be any suitable scFv or Fab that specifically binds CD3, such as the CD3 antigen binding site of the invention of the scFv or Fab structure described above , especially scFvs or Fabs having the VH and VL amino acid sequences described above.
  • the antibody molecule may comprise any of the antibody stem structures of the invention described above for Format_2.
  • the Format_2 asymmetric trispecific antibody molecule of the present invention, preferably, in order to promote the correct pairing of the polypeptide chains of the antibody molecule, the first Fc domain and the second Fc domain of the stem can be introduced into the first and second Fc domains.
  • first and/or second Fc domains of the Format_2 antibody molecules of the invention may preferably also contain mutations that affect antibody effector function, eg, mutations that reduce ADCC activity, such as LALA mutations.
  • the Format-2 trispecific antibody molecule of the invention comprises or consists of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain, wherein,
  • the first heavy chain polypeptide chain comprises from N-terminal to C-terminal: heavy chain variable domain VH, immunoglobulin CH1 domain, Fc domain;
  • the first light chain polypeptide chain comprises from the N-terminus to the C-terminus: the light chain variable domain VL, the immunoglobulin CL domain;
  • the second heavy chain polypeptide chain comprises from N-terminal to C-terminal: heavy chain variable domain VH, immunoglobulin CH1 domain, Fc domain;
  • the second light chain polypeptide chain comprises from N-terminal to C-terminal: light chain variable domain VL, immunoglobulin CL domain;
  • first heavy chain polypeptide chain or the second heavy chain polypeptide chain further comprises an scFv domain conjugated directly or preferably via a linking peptide (eg (G4S)2 or TS(G4S)2) at the C-terminus of its Fc domain;
  • a linking peptide eg (G4S)2 or TS(G4S)2
  • the VH-CH1 of the first heavy chain polypeptide chain is paired with the VL-CL of the first light chain polypeptide chain to form a first antibody arm (M1) that binds to the first antigen;
  • the VH-CH1 of the second heavy chain polypeptide chain is paired with the VL-CL of the second light chain polypeptide chain to form a second antibody arm (M1) that binds to the second antigen;
  • the Fc domain of the first heavy chain polypeptide chain pairs with the Fc domain of the second heavy chain polypeptide chain and dimerizes to form an antibody stem (Fc::Fc);
  • the scFv domain conjugated to the C-terminus of the polypeptide chain of the first heavy chain or the polypeptide chain of the second heavy chain forms a conjugation part (X2) that binds to the third antigen
  • first, second and third antigens are different and independently selected from the group consisting of: GPRC5D, BCMA, and CD3.
  • the antibody arm M1 binds GPCR5D
  • the second antibody arm M2 binds CD3
  • the conjugation member X2 binds BCMA
  • the first heavy chain polypeptide chain and the first light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of GPCRR5D,
  • the second heavy chain polypeptide chain and the second light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind the Fab antigen binding site of CD3,
  • the first heavy chain polypeptide chain when the conjugation part X2 is conjugated to the first heavy chain polypeptide chain, the first heavy chain polypeptide chain further comprises the amino acid sequence of the scFv that specifically binds BCMA at the C-terminus, or preferably, when the conjugation part X2 is conjugated
  • the second heavy chain polypeptide chain when on the second heavy chain polypeptide chain, the second heavy chain polypeptide chain further comprises the amino acid sequence of the scFv that specifically binds to BCMA at the C-terminus.
  • the antibody arm M1 binds BCMA
  • the second antibody arm M2 binds CD3
  • the conjugation member X2 binds GPCR5D
  • the first heavy chain polypeptide chain and the first light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of BCMA,
  • the second heavy chain polypeptide chain and the second light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind the Fab antigen binding site of CD3,
  • the first heavy chain polypeptide chain when the conjugation part X2 is conjugated to the first heavy chain polypeptide chain, the first heavy chain polypeptide chain further comprises an scFv amino acid sequence that specifically binds to GPCRR5D at the C-terminus, or preferably, when the conjugation part X2 is conjugated to When on the second heavy chain polypeptide chain, the second heavy chain polypeptide chain also includes the amino acid sequence of the scFv that specifically binds to GPRCR5D at the C-terminus.
  • the antibody arm M1 binds GPCR5D
  • the second antibody arm M2 binds BCMA
  • the conjugation member X2 binds CD3, whereby,
  • the first heavy chain polypeptide chain and the first light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of GPCRR5D,
  • the second heavy chain polypeptide chain and the second light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of BCMA,
  • the first heavy chain polypeptide chain when the conjugation part X2 is conjugated to the first heavy chain polypeptide chain, the first heavy chain polypeptide chain further comprises an scFv amino acid sequence that specifically binds to CD3 at the C-terminus, or when the conjugation part X2 is conjugated to the second heavy chain polypeptide chain.
  • the second heavy chain polypeptide chain when it is on the chain polypeptide chain, the second heavy chain polypeptide chain also contains the amino acid sequence of the scFv that specifically binds to CD3 at the C-terminus.
  • the Fab or scFv that specifically binds GPRC5D comprises VH and VL selected from the group consisting of:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 1-3 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 4-6;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 7-8 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 10-12;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 13-15 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 16-18;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 19-21 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 22-24;
  • the Fab and the scFv comprise a VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 25/26, SEQ ID NOs: 27/28, SEQ ID NOs: 29/30, and SEQ ID NOs: 29/30 NOs: 31/32,
  • the scFv when it is a dsscFv, it also comprises cysteine substitutions introduced into the VH and VL sequences, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence.
  • the Fab or scFv that specifically binds CD3 comprises VH and VL selected from the group consisting of:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs:41-43 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs:44-46;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 41, 47, 43 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 44-46;
  • the Fab and scFv comprise a VH and VL amino acid sequence pair selected from the group consisting of SEQ ID NOs:48/49, SEQ ID NOs:50/49, and more preferably SEQ ID NOs:48/49,
  • the scFv when it is a dsscFv, it also comprises cysteine substitutions introduced into the VH and VL sequences, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence.
  • the Fab or scFv that specifically binds BCMA comprises the following VH and VL:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 33-35 and VL comprising the LCDR1-3 sequences of SEQ ID NOs 36-38;
  • the Fab and scFv comprise the VH and VL amino acid sequence pair: SEQ ID NOs: 39/40,
  • the scFv when it is a dsscFv, it also comprises cysteine substitutions introduced into the VH and VL sequences, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence.
  • the first light chain polypeptide chain and the second light chain polypeptide chain comprise a kappa light chain constant domain CL ⁇ and a lamda light chain constant domain CL ⁇ , respectively.
  • the light chain constant domain CL ⁇ comprises or is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 105
  • the amino acid sequence of ; the light chain constant domain CL lambda comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto .
  • the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise the heavy chain constant region CH1 from human IgG1, preferably, comprise or have at least 90% of the amino acid sequence of SEQ ID NO: 104 , 92%, 95%, 97%, 98%, 99% or more identical amino acid sequences.
  • the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise an Fc domain containing knob or complementary hole mutations, respectively.
  • the Fc domain containing knob mutations comprises T366W; the Fc domain containing complementary hole mutations comprises T366S, L368A, Y407V mutations.
  • the Fc domain containing the hole mutation comprises, or has at least 90%, 92%, 95%, 97%, 98%, 99% amino acid sequence selected from the group consisting of SEQ ID NOs: 102 and 107 An amino acid sequence of or greater identity; the Fc domain containing the complementary knob mutation comprises, or has at least 90%, 92%, 95%, 97%, 98%, 99% or the amino acid sequence of SEQ ID NO: 103 therewith Amino acid sequences of higher identity.
  • the invention provides trispecific antibody molecules of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 67, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides trispecific antibody molecules of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides trispecific antibody molecules of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides trispecific antibody molecules of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77 or 78, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides trispecific antibody molecules of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the antibody arms M1 and M2 comprise Fab antigen-binding sites that bind the first antigen and the second antigen, respectively, and the conjugation part X1 comprises the scFv antigen-binding site that binds the third antigen, wherein the first, The second and third antigens are different and independently selected from: GPRC5D, BCMA and CD3.
  • the present invention provides trispecific Y-type antibody molecules having the structure:
  • M1 and M2 represent the first antibody arm and the second antibody arm of the antibody molecule, respectively, and M1 and M2 comprise Fabs that bind to the first and second antigens, respectively,
  • Fc::Fc represents the stem of an antibody molecule, consisting of a paired and dimerized first and second Fc domains, wherein the first and second antibody arms, respectively, are directly or via a linking peptide (preferably the hinge region) Linked to the N-terminus of the first Fc domain and the second Fc domain;
  • X1 represents a conjugation moiety conjugated to the C-terminus of the antibody arm, wherein the conjugation moiety comprises an scFv that binds a third antigen, wherein X1 is conjugated to the Fab light chains of the first and second antibody arms, either directly or via a linking peptide C terminal,
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • the present invention provides trispecific antibody molecules of Format-7 structure, wherein:
  • Antibody arm M1 and antibody arm M2 of the molecule comprising a BCMA-binding Fab and a CD3-binding Fab, respectively, and the conjugation part X1 comprising a GPRC5D-binding scFv, the molecule has the structure
  • the Fab or scFv that binds GPRC5D may be any suitable Fab or scFv that specifically binds GPRC5D, such as the GPRC5D antigen binding site of the invention of the scFv or Fab structure described above, especially is a scFv or Fab having the VH and VL amino acid sequences described above.
  • the BCMA-binding Fab or scFv may be any suitable Fab or scFv that specifically binds BCMA, such as the BCMA antigen binding site of the invention of the scFv or Fab structure described above, especially is a scFv or Fab having the VH and VL amino acid sequences described above.
  • the antigen binding site that binds CD3 may be any suitable scFv or Fab that specifically binds CD3, such as the CD3 antigen binding site of the invention of the scFv or Fab structure described above , especially scFvs or Fabs having the VH and VL amino acid sequences described above.
  • the antibody molecule may comprise any of the antibody stem structures of the invention described above for Format_7.
  • asymmetric trispecific antibody molecule of the present invention preferably, in order to promote the correct pairing of the polypeptide chains of the antibody molecule, the first Fc domain and the second Fc domain of the stem can be introduced into the first and second Fc domains.
  • the first and/or second Fc domain of the Format_7 antibody molecule of the invention may preferably also contain mutations that affect antibody effector function, eg, mutations that reduce ADCC activity, such as LALA mutations.
  • the Format-7 trispecific antibody molecule of the invention comprises or consists of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain, wherein,
  • the first heavy chain polypeptide chain comprises from N-terminal to C-terminal: heavy chain variable domain VH, immunoglobulin CH1 domain, Fc domain;
  • the first light chain polypeptide chain comprises from the N-terminus to the C-terminus: the light chain variable domain VL, the immunoglobulin CL domain;
  • the second heavy chain polypeptide chain comprises from N-terminal to C-terminal: heavy chain variable domain VH, immunoglobulin CH1 domain, Fc domain;
  • the second light chain polypeptide chain comprises from N-terminal to C-terminal: light chain variable domain VL, immunoglobulin CL domain;
  • first light chain polypeptide chain and the second light chain polypeptide chain further comprise an scFv domain conjugated directly or preferably via a linking peptide (eg (G4S)2 or TS(G4S)2) at the C-terminus of its CL domain;
  • a linking peptide eg (G4S)2 or TS(G4S)2
  • the VH-CH1 of the first heavy chain polypeptide chain is paired with the VL-CL of the first light chain polypeptide chain to form a first antibody arm (M1) that binds to the first antigen;
  • the VH-CH1 of the second heavy chain polypeptide chain is paired with the VL-CL of the second light chain polypeptide chain to form a second antibody arm (M1) that binds to the second antigen;
  • the Fc domain of the first heavy chain polypeptide chain pairs with the Fc domain of the second heavy chain polypeptide chain and dimerizes to form an antibody stem (Fc::Fc);
  • the scFv domains conjugated to the C-termini of the first light chain polypeptide chain and the second light chain polypeptide chain form a conjugation part (X1) that binds to the third antigen
  • first, second and third antigens are different and independently selected from the group consisting of: GPRC5D, BCMA, and CD3.
  • the antibody arm M1 binds GPCR5D
  • the second antibody arm M2 binds CD3
  • the conjugation member X1 binds BCMA
  • the first heavy chain polypeptide chain and the first light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of GPCRR5D,
  • the second heavy chain polypeptide chain and the second light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind the Fab antigen binding site of CD3,
  • first light chain polypeptide chain and the second light chain polypeptide chain further comprise the amino acid sequence of the scFv that specifically binds to BCMA at the C-terminus.
  • the antibody arm M1 binds BCMA
  • the second antibody arm M2 binds CD3
  • the conjugation member X1 binds GPCR5D
  • the first heavy chain polypeptide chain and the first light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of BCMA,
  • the second heavy chain polypeptide chain and the second light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind the Fab antigen binding site of CD3,
  • first light chain polypeptide chain and the second light chain polypeptide chain further comprise the amino acid sequence of the scFv that specifically binds to GPRCR5D at the C-terminus.
  • the antibody arm M1 binds GPCR5D
  • the second antibody arm M2 binds BCMA
  • the conjugation member X1 binds CD3, whereby,
  • the first heavy chain polypeptide chain and the first light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of GPCRR5D,
  • the second heavy chain polypeptide chain and the second light chain polypeptide chain respectively comprise at the N-terminus VH and VL amino acid sequences that specifically bind to the Fab antigen-binding site of BCMA,
  • first light chain polypeptide chain and the second light chain polypeptide chain further comprise the amino acid sequence of the scFv that specifically binds to CD3 at the C-terminus.
  • the Fab or scFv that specifically binds GPRC5D comprises VH and VL selected from the group consisting of:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 1-3 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 4-6;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 7-8 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 10-12;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 13-15 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 16-18;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 19-21 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 22-24;
  • the Fab and the scFv comprise a VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 25/26, SEQ ID NOs: 27/28, SEQ ID NOs: 29/30, and SEQ ID NOs: 29/30 NOs: 31/32,
  • the scFv when it is a dsscFv, it also comprises cysteine substitutions introduced into the VH and VL sequences, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence.
  • the Fab or scFv that specifically binds CD3 comprises VH and VL selected from the group consisting of:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs:41-43 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs:44-46;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 41, 47, 43 and VL comprising the LCDR1-3 sequences of SEQ ID NOs: 44-46;
  • the Fab and scFv comprise a VH and VL amino acid sequence pair selected from the group consisting of SEQ ID NOs:48/49, SEQ ID NOs:50/49, and more preferably SEQ ID NOs:48/49,
  • the scFv when it is a dsscFv, it also comprises cysteine substitutions introduced into the VH and VL sequences, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence.
  • the Fab or scFv that specifically binds BCMA comprises the following VH and VL:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 33-35 and VL comprising the LCDR1-3 sequences of SEQ ID NOs 36-38;
  • the Fab and scFv comprise the VH and VL amino acid sequence pair: SEQ ID NOs: 39/40,
  • the scFv when it is a dsscFv, it also comprises cysteine substitutions introduced into the VH and VL sequences, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence.
  • the first light chain polypeptide chain and the second light chain polypeptide chain comprise a kappa light chain constant domain CL ⁇ . In some embodiments, the first light chain polypeptide chain and the second light chain polypeptide chain comprise a lamda light chain constant domain CL ⁇ . In a specific embodiment, the light chain constant domain CL ⁇ comprises or is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 105 amino acid sequence. In a specific embodiment, the light chain constant domain CL ⁇ comprises or is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 106 Sexual amino acid sequence.
  • the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise the heavy chain constant region CH1 from human IgG1, preferably, comprise or have at least 90% of the amino acid sequence of SEQ ID NO: 104 , 92%, 95%, 97%, 98%, 99% or more identical amino acid sequences.
  • the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise an Fc domain containing a knob or complementary hole mutation, respectively.
  • the Fc domain containing knob mutations comprises T366W; the Fc domain containing complementary hole mutations comprises T366S, L368A, Y407V mutations.
  • the knob-mutated Fc domain comprises, or has at least 90%, 92%, 95%, 97%, 98%, 99% amino acid sequence selected from the group consisting of SEQ ID NOs: 102 and 107 An amino acid sequence of or greater identity; the Fc domain containing a complementary hole mutation comprises, or has at least 90%, 92%, 95%, 97%, 98%, 99% or the amino acid sequence of SEQ ID NO: 103 therewith Amino acid sequences of higher identity.
  • the invention provides trispecific antibody molecules of Format_7 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence that is at least 90%, 92%, 95%, 97%, 98%, 99% or more identical to it;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77 or 78, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides trispecific antibody molecules of Format_7 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:91, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides trispecific antibody molecules of Format_7 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain, and a second light chain polypeptide chain ,in,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the first light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 93 or 96, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto;
  • the second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 95 or 97, or an amino acid sequence at least 90%, 92%, 95%, 97%, 98%, 99% or more identical thereto.
  • the antibody molecule of the invention has the structure: (M1:M2)-Fc::Fc-(X2)q, wherein antibody arms M1 and M2 and conjugation member X2 comprise scFv antigen binding sites for binding a first antigen, a second antigen and a third antigen, respectively, wherein the first, second and third antigens are different and independently selected from: GPRC5D, BCMA and CD3.
  • the present invention provides trispecific Y-type antibody molecules having the structure:
  • M1 and M2 represent the first and second antibody arms of the antibody molecule, respectively, and M1 and M2 comprise scFvs that bind to the first and second antigens, respectively,
  • Fc::Fc represents the stem of an antibody molecule, consisting of a paired and dimerized first and second Fc domains, wherein the first and second antibody arms, respectively, are directly or via a linking peptide (preferably the hinge region) Linked to the N-terminus of the first Fc domain and the second Fc domain;
  • X2 represents a conjugation moiety conjugated on the C-terminus of the stem, wherein the conjugation moiety comprises an scFv that binds a third antigen, wherein X2 is conjugated directly or via a linking peptide at the C-terminus of the first Fc domain, or conjugated At the C-terminus of the second Fc domain,
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • the present invention provides trispecific antibody molecules of Format_1 structure, wherein:
  • the antibody arm M1 and the antibody arm M2 of the molecule comprise a BCMA-binding scFv and a CD3-binding scFv, respectively, and the conjugation component X2 comprises a GPRC5D-binding scFv, the molecule has the structure
  • the Format-1 trispecific antibody molecule of the invention comprises or consists of a first heavy chain polypeptide chain and a second heavy chain polypeptide chain, wherein,
  • the first heavy chain polypeptide chain comprises from the N-terminus to the C-terminus: the first scFv and the Fc domain;
  • the second heavy chain polypeptide chain comprises from the N-terminus to the C-terminus: a second scFv and an Fc domain;
  • first heavy chain polypeptide chain or the second heavy chain polypeptide chain further comprises a third scFv structure conjugated directly or preferably via a linking peptide (eg (G4S)2 or TS(G4S)2) at the C-terminus of its Fc domain area;
  • a linking peptide eg (G4S)2 or TS(G4S)2
  • the first scFv of the first heavy chain polypeptide chain forms a first antibody arm (M1) that binds to the first antigen;
  • the second scFv of the second heavy chain polypeptide chain forms a second antibody arm (M1) that binds the second antigen
  • the Fc domain of the first heavy chain polypeptide chain pairs with the Fc domain of the second heavy chain polypeptide chain and dimerizes to form an antibody stem (Fc::Fc);
  • the third scFv domain conjugated to the C-terminus of the first heavy chain polypeptide chain or the second heavy chain polypeptide chain forms a conjugation part (X2) that binds to the third antigen
  • first, second and third antigens are different and independently selected from the group consisting of: GPRC5D, BCMA, and CD3.
  • the invention provides a trispecific antibody molecule of Format_1 structure comprising or consisting of a first heavy chain polypeptide chain and a second heavy chain polypeptide chain, wherein,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the invention provides a trispecific antibody molecule of Format_1 structure comprising or consisting of a first heavy chain polypeptide chain and a second heavy chain polypeptide chain, wherein,
  • the first heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or greater identity thereto.
  • the antibody molecule of the invention has the structure: (M1:M2-(X2)p)-Fc::Fc-(X2)q, wherein antibody arms M1 and M2 comprising a Fab antigen-binding site that binds a first antigen, and the conjugation elements X1 and X2 comprise scFv antigen-binding sites that bind a second antigen and a third antigen, respectively, wherein the first, second, and third antigens are different, and independently of each other selected from: GPRC5D, BCMA and CD3.
  • the present invention provides trispecific symmetrical Y-type antibody molecules having the structure:
  • M1 and M2 represent the first antibody arm and the second antibody arm of the antibody molecule, respectively, and both M1 and M2 comprise a Fab that binds to the first antigen
  • Fc::Fc represents the stem of an antibody molecule, consisting of a paired and dimerized first and second Fc domains, wherein the first and second antibody arms, respectively, are directly or via a linking peptide (preferably the hinge region) Linked to the N-terminus of the first Fc domain and the second Fc domain;
  • X1 and X2 represent the conjugation part conjugated to the C-terminal of the Fab light chain of the antibody arm or the C-terminus of the stalk Fc domain, respectively, wherein the conjugation part X1 comprises the scFv that binds the second antigen, and the conjugation part X2 comprises an scFv that binds a third antigen, wherein X1 and X2 are conjugated to the arm or stem, either directly or through a linking peptide,
  • first, second and third antigens are different from each other and are independently selected from: GPRCR5D, BCMA and CD3.
  • the present invention provides trispecific antibody molecules of Format-6 structure, wherein:
  • Antibody arm M1 and antibody arm M2 of the molecule comprise a Fab that binds BCMA, conjugation part X1 comprises an scFv of CD3 and conjugation part X2 comprises an scFv that binds GPRC5D, the molecule has the structure: (Fab_BCMA:Fab_BCMA -(scFv_CD3) 2 )-Fc::Fc-(scFv_GPRC5D) 2 ; (eg, the molecule of Example Ts-F6-3,4).
  • the Format-6 trispecific antibody molecule of the invention comprises or consists of two identical heavy chain polypeptide chains and two identical light chain polypeptide chains, wherein,
  • the heavy chain polypeptide chain comprises from the N-terminus to the C-terminus: a heavy chain variable domain VH, an immunoglobulin CH1 domain, an Fc domain, and a first scFv domain;
  • the light chain polypeptide chain comprises from the N-terminus to the C-terminus: a light chain variable domain VL, an immunoglobulin CL domain and a second scFv domain;
  • first and second scFv domains of the heavy and light polypeptide chains are conjugated at their C-termini, either directly or preferably via a linking peptide (eg (G4S)2 or TS(G4S)2), respectively;
  • a linking peptide eg (G4S)2 or TS(G4S)2
  • VH-CH1 domain at the N-terminus of a heavy chain polypeptide chain is paired with the VL-CH1 domain at the N-terminus of a light chain polypeptide chain to form a first antibody arm (M1) that binds to the first antigen;
  • VH-CH1 domain at the N-terminus of the polypeptide chain of another heavy chain is paired with the VL-CH1 domain at the N-terminus of a light chain polypeptide chain to form a second antibody arm (M2) that binds to the first antigen;
  • Fc domains of two heavy chain polypeptide chains pair to form and dimerize to form an antibody stem (Fc::Fc);
  • the scFv domains conjugated to the C-termini of the two light chain polypeptide chains form a conjugation component (X1) that binds to the second antigen
  • the scFv domains conjugated to the C-termini of the two heavy chain polypeptide chains form a conjugation part (X2) that binds to the third antigen;
  • first, second and third antigens are different and independently selected from the group consisting of: GPRC5D, BCMA, and CD3.
  • the present invention provides trispecific antibody molecules of Format_6 structure comprising or consisting of two identical heavy chain polypeptide chains and two identical light chain polypeptide chains, wherein,
  • the heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82 or 83, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.
  • the present invention provides trispecific antibody molecules of Format_6 structure comprising or consisting of two identical heavy chain polypeptide chains and two identical light chain polypeptide chains, wherein,
  • the heavy chain polypeptide chain comprises: the amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or higher identity thereto;
  • the light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or 86, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.
  • the invention provides antibodies or antigen-binding fragments thereof that specifically bind GPRCR5D.
  • the GPRC5D antibody or antigen-binding fragment thereof of the invention comprises:
  • the GPRC5D antibody or antigen-binding fragment thereof of the invention comprises a combination of CDR sequences selected from the group consisting of:
  • the GPRC5D antibody molecule or antigen-binding fragment thereof of the invention comprises a combination of VH and VL amino acid sequences selected from the group consisting of:
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith domain; or
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 29 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith domain; or
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 31 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain
  • a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 32 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 98 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 99 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area;
  • a heavy chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 100 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto domain; and a light chain variable structure comprising the amino acid sequence set forth in SEQ ID NO: 101 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity therewith area.
  • the GPRC5D antibody molecule or antigen-binding fragment thereof of the present invention comprises a combination of VH and VL amino acid sequences selected from the group consisting of:
  • the GPRC5D antibodies of the invention are monospecific, bispecific, or trispecific antibodies.
  • the GPRC5D antibodies of the invention are bispecific antibodies that further comprise an antigen binding site that binds CD3, such as the CD3 antigen binding site of the invention described above.
  • the GPRC5D antibody of the invention is a trispecific antibody, further comprising an antigen-binding site that binds BCMA (eg, the BCMA antigen-binding site of the invention described above) and an antigen-binding site that binds CD3 ( For example, the CD3 antigen binding site of the invention described above).
  • the GPRC5D antibody of the invention is a bispecific antibody that binds GPRC5D and CD3,
  • the antibody comprises a pair of VH and VL amino acid sequences that bind to CD3 selected from the group consisting of:
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs:41-43 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs:44-46;
  • VH comprising the HCDR1-3 sequences of SEQ ID NOs: 47, 42, 43 and VL comprising the LCDR1-3 sequences of SEQ ID NOs: 44-46;
  • VH and VL amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 48/49, and SEQ ID NOs: 50/49,
  • introduced cysteine substitutions are included in the VH and VL sequences, e.g., cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence, whereby between the VH and VL sequences form disulfide bonds.
  • the present invention provides a bispecific antibody that binds GPRC5D and CD3, wherein: the antibody comprises a pair of VH and VL amino acid sequences that bind GPRC5D and a VH and VL amino acid that binds CD3 selected from the group consisting of a combination of sequence pairs,
  • the invention also provides variants of the above bispecific antibodies, wherein said variants comprise amino acid changes in said pair of VH and VL amino acid sequences that bind GPRC5D, and/or in said pair of VH and VL amino acid sequences that bind CD3 , eg, amino acid substitutions, deletions and/or insertions, wherein the amino acid changes do not affect the binding of the bispecific antibody to GPRC5D and CD3.
  • the variant comprises a combination of amino acid sequences selected from the group consisting of,
  • the bispecific antibody of the invention comprises cysteine introduced into said pair of VH and VL amino acid sequences that bind to GPRC5D, and/or to said pair of VH and VL amino acid sequences that bind to CD3 Acid substitutions, eg, cysteine substitutions at position 44 in the VH sequence and 100 in the VL sequence, thereby forming a disulfide bond between the VH and VL.
  • the invention also provides fusion proteins and immunoconjugates (eg, conjugated to toxins or small chemical molecules) comprising the GPRC5D antibodies of the invention, as well as pharmaceutical compositions and drug combinations.
  • the antibody of the present invention may comprise another therapeutic agent, eg, another therapeutic agent that can be used for the intended use of the antibody of the present invention, eg, a chemotherapeutic agent, a radiotherapeutic agent, a tumor suppressor molecule.
  • the present invention provides a method for producing an antibody molecule of the present invention, the method comprising: culturing a host cell comprising a polypeptide chain encoding the antibody under conditions suitable for expression of the polypeptide chain; and The antibody is produced by assembling the polypeptide chain under conditions in which the polypeptide chain is assembled into the antibody molecule.
  • the antibody molecule production method of the present invention comprises the steps: (i) under conditions suitable for expressing the first heavy chain polypeptide chain and the first light chain polypeptide chain of the antibody molecule, culturing the a host cell encoding the first heavy chain and second light chain, producing the first parent protein, (ii) under conditions suitable for expressing the second heavy chain polypeptide chain and the second light chain polypeptide chain of the antibody molecule , culturing a host cell encoding the second heavy chain and the second light chain to produce the second parental protein, (iii) mixing the first parental protein and the second parental protein in an equimolar ratio, and placing them in an in vitro suitable
  • step (iii) comprises: adding glutathione (GSH) to the mixed solution of the purified first and second affinity proteins, wherein, preferably, the GHS/protein molar ratio is controlled at 500 -700 times.
  • step (iii) further comprises: performing natural oxidation for a period of time (eg, 2-3 hours) after removing GSH.
  • Polypeptide chains of antibody molecules of the invention can be obtained, for example, by solid state peptide synthesis (eg Merrifield solid phase synthesis) or recombinant production.
  • polynucleotides encoding any polypeptide chain and/or polypeptide chains of the antibody molecule are isolated and inserted into one or more vectors for further cloning and/or expression in host cells.
  • the polynucleotides can be readily isolated and sequenced using conventional methods.
  • a vector, preferably an expression vector, comprising one or more polynucleotides of the invention is provided.
  • Expression vectors can be constructed using methods well known to those skilled in the art.
  • Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).
  • the expression vector can be transfected or introduced into a suitable host cell.
  • a variety of techniques can be used to accomplish this, eg, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, biolistic, liposome-based transfection, or other conventional techniques.
  • host cells comprising one or more polynucleotides of the present invention are provided.
  • host cells comprising the expression vectors of the present invention are provided.
  • the term "host cell” refers to any kind of cellular system that can be engineered to produce the antibody molecules of the invention.
  • Host cells suitable for replication and to support expression of the antibody molecules of the invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors as desired, and large numbers of vector-containing cells can be grown for seeding large-scale fermenters to obtain sufficient quantities of the antibody molecules of the invention for clinical use.
  • Suitable host cells include prokaryotic microorganisms such as E.
  • coli eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like.
  • CHO Chinese hamster ovary cells
  • Mammalian cell lines suitable for suspension culture can be used.
  • Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63 and Sp2/0, etc.
  • suitable mammalian host cell lines for protein production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).
  • the host cell is a CHO, HEK293 or NSO cell.
  • Antibody molecules prepared as described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like.
  • the actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.
  • the purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.
  • the physical/chemical properties and/or biological activities of the antibody molecules provided herein can be identified, screened or characterized by a variety of assays known in the art.
  • the invention provides compositions, eg, pharmaceutical compositions, comprising an antibody molecule described herein formulated together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (eg, by injection or infusion).
  • the antibody molecule of the invention is the only active ingredient in the pharmaceutical composition.
  • a pharmaceutical composition may comprise an antibody molecule described herein and more than one therapeutic agent.
  • the invention also provides pharmaceutical combinations comprising the antibody molecules described herein and more than one therapeutic agent.
  • the therapeutic agent suitable for use in the pharmaceutical compositions and drug combinations of the present invention may be a therapeutic agent selected from any of the following classes (i)-(iii): (i) Drugs that enhance antigen presentation (eg, tumor antigen presentation) (ii) drugs that enhance effector cell responses (eg, B cell and/or T cell activation and/or mobilization); (iii) drugs that reduce immunosuppression; (iv) drugs that have tumor suppressive effects.
  • compositions of the present invention may be in a variety of forms. Such forms include, for example, liquid, semisolid, and solid dosage forms, such as liquid solutions (eg, injectable solutions and infusible solutions), dispersions or suspensions, liposomes, and suppositories.
  • liquid solutions eg, injectable solutions and infusible solutions
  • dispersions or suspensions e.g., liposomes, and suppositories.
  • the preferred form depends on the intended mode of administration and therapeutic use. Commonly preferred compositions are in the form of injectable solutions or infusible solutions.
  • the preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal (i.p.), intramuscular) injection.
  • the antibody molecule is administered by intravenous infusion or injection.
  • the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.
  • parenteral administration and “parenteral administration” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, Intradermal, intraperitoneal, transtracheal, subcutaneous injection and infusion.
  • compositions should generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or lyophilized forms.
  • Sterile injectable solutions can be prepared by incorporating the active compound (ie, the antibody molecule) in the required amount in an appropriate solvent followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients. Coatings such as lecithin and the like can be used.
  • proper fluidity of the solution can be maintained by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • the antibody molecules of the invention can be administered orally, eg, with an inert diluent or edible carrier.
  • the antibody molecules of the invention can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into a subject's diet.
  • the compounds can be incorporated with excipients and in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers It is used in the form of wafers and the like.
  • To administer an antibody molecule of the invention by a parenteral method of administration it may be desirable to coat or co-administer the antibody molecule with a material that prevents its inactivation.
  • Therapeutic compositions can also be administered using medical devices known in the art.
  • compositions of the present invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of the antibody molecule of the present invention.
  • a “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result, at the required dose and for the required period of time. The therapeutically effective amount may vary depending on a variety of factors, such as the disease state, the age, sex, and weight of the individual. A therapeutically effective amount is any amount in which the toxic or detrimental effects are outweighed by the therapeutically beneficial effects.
  • a “therapeutically effective amount” preferably inhibits a measurable parameter (eg, tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more, relative to an untreated subject. Preferably at least about 80%.
  • the ability of the antibody molecules of the invention to inhibit measurable parameters eg, tumor volume
  • prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result, at the required dose and for the required period of time. Typically, a prophylactically effective amount is less than a therapeutically effective amount because the prophylactic dose is administered in a subject prior to or at an earlier stage of the disease.
  • Kits comprising the antibody molecules described herein are also within the scope of the invention.
  • a kit may contain one or more other elements, including, for example, instructions for use; other reagents, such as labels or reagents for conjugation; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.
  • the present invention provides in vivo, in vitro uses and methods of application of the antibody molecules of the present invention.
  • the uses and methods of the invention involve applying the antibody molecules of the invention in vivo and/or in vitro to:
  • GPRC5D antigen with high affinity eg KD less than 10 nM, including cell surface expressed GPRC5D antigen
  • T cells eg CD4+ and/or CD8+ T cells
  • GPRC5D positive tumor cells eg CD4+ and/or CD8+ T cells
  • T cells eg CD4+ and/or CD8+ T cells
  • BCMA positive tumor cells eg CD4+ and/or CD8+ T cells
  • TNF- ⁇ TNF- ⁇ , IFN- ⁇ , and IL-2
  • GPRC5D-positive and/or BCMA-positive multiple myeloma such as treatment of BCMA-positive patient populations, GPRC5D-positive patient populations, GPRC5D-positive patient populations in which BCMA is lost after anti-BCMA molecule binding;
  • BCMA escape For avoiding tumor recurrence mediated by BCMA escape, such as multiple myeloma recurrence.
  • the antibody molecule of the invention or a pharmaceutical composition comprising the antibody molecule of the invention is used as a medicament for the treatment and/or prevention of a disease in an individual or as a diagnostic tool for a disease, preferably the individual is a mammal , more preferably a human.
  • the present invention provides methods and uses of applying the antibody molecules of the invention, especially trispecific antibody molecules, for the treatment of cancer, wherein the cancer may be selected, for example, from multiple myeloma, melanoma, B-cell lymphoma .
  • the cancer is multiple myeloma.
  • the trispecific antibody molecules of the invention may have a broader therapeutic cancer patient population than bispecific antibodies targeting BCMA and CD3 and bispecific antibodies targeting GPRC5D and CD3 .
  • the present invention provides methods of treating BCMA negative cancers or BCMA low expressing cancers using the trispecific antibody molecules of the present invention.
  • the present invention provides the use of the trispecific antibody molecules of the present invention for the treatment of GPRC5D and CD3 double positive cancers.
  • the invention provides diagnostic methods for detecting the presence of relevant antigens in biological samples, such as serum, semen or urine, or tissue biopsy samples (e.g., from hyperproliferative or cancerous lesions) in vitro or in vivo.
  • the diagnostic method comprises: (i) contacting a sample (and optionally a control sample) with an antibody molecule as described herein or administering the antibody molecule to a subject under conditions that allow the interaction to occur and (ii) The formation of complexes between the antibody molecule and the sample (and optionally, a control sample) is detected.
  • the formation of complexes indicates the presence of relevant antigens and may indicate applicability or need for treatment and/or prevention as described herein.
  • the relevant antigen is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment following a treatment interval.
  • Detection methods that can be used include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (eg, RT-PCR) or in vivo imaging techniques.
  • antibody molecules used in in vivo and in vitro detection methods are labeled, directly or indirectly, with detectable substances to facilitate detection of bound or unbound conjugates.
  • Suitable detectable substances include various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic (eg, nuclear magnetic resonance active) substances, and radioactive substances.
  • the level and/or distribution of the relevant antigen is determined in vivo, eg, non-invasively (eg, detectable by detection using a suitable imaging technique (eg, positron emission tomography (PET) scan)
  • a suitable imaging technique eg, positron emission tomography (PET) scan
  • PET positron emission tomography
  • the level of the relevant antigen is determined in vivo, for example, by detecting antibody molecules of the invention that are detectably labeled with a PET reagent (eg, 18F-fluorodeoxyglucose (FDG)). and/or distribution.
  • FDG 18F-fluorodeoxyglucose
  • the present invention provides diagnostic kits comprising the antibody molecules described herein and instructions for use.
  • the full-length sequences of human, monkey and mouse GPRC5D were inserted into the PEE17.4 plasmid (Lonza, GS Xceed Expression System) respectively, and the plasmid was transferred into the host cell GS-CHO by electroporation. Stable cell line overexpressing GPRC5D protein.
  • the full-length human GPRC5D sequence was inserted into the pCHO1.0 vector, which was transferred into the host cell 293F to construct an overexpression cell line 293F-huGPRC5D.
  • the full-length sequence of human GPRC5D was constructed into the pcDNA3.1 vector, and the plasmid was used to immunize Balb ⁇ c and Harbour mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), intramuscularly once every two weeks (each mouse mouse 50ug plasmid), immunized 3 times in total.
  • booster immunization was performed with the GS-CHO cell line overexpressing human GPRC5D, and intraperitoneal injection was performed once every two weeks (1 ⁇ 10 7 per mouse) for a total of 2 immunizations.
  • the spleen of the mouse was harvested to prepare a B lymphocyte suspension, which was mixed with SP2/0 myeloma cells (ATCC) at a ratio of 1:2 to 1:1 and electrofused. Transfer the fused cells from the electrode dish into a 50 ml centrifuge tube, dilute the cells with HAT medium to 1-2 ⁇ 10 4 cells/ml, and add 100 ⁇ l of cell suspension to each well of a 96-well plate.
  • the screening medium was changed on the 7th day after fusion, and the positive clones were screened by flow cytometry (FACS) detection after culturing on the 10th day (or longer, depending on the cell growth state).
  • FACS flow cytometry
  • the hybridoma cells that specifically express anti-GPRC5D antibody were screened by flow cytometry (FACS).
  • the cells to be tested (293F-huGPRC5D/GS-CHO-huGPRC5D) were counted, diluted to 1 ⁇ 10 6 cells/ml, and 100 ⁇ l/well was added to a U-bottom 96-well plate.
  • the cell culture medium was removed by centrifugation at 500 g for 5 min.
  • the culture supernatant of the hybridoma 96-well plate and the positive control antibody were added to the U-plate and the cells were resuspended, adding 100 ⁇ l to each well and standing on ice for 30 min.
  • the supernatant was removed by centrifugation at 500 g for 5 min, and the cells were washed once with PBS. 500g 5min to remove PBS. 100 ⁇ l of goat anti-mouse IgG FITC-labeled secondary antibody (Jackson Immunoresear, Cat#115-545-006, 1:500 dilution in PBS) was added to each well to which the hybridoma supernatant had been added; Antibody (Roche-5E11) wells were loaded with 100 ⁇ l of anti-human Fc PE-labeled secondary antibody (BioLegend, Cat #409304). Incubate on ice for 30 min in the dark. Remove the supernatant at 500 g for 5 min, and wash the cells once with PBS. Cells were resuspended in 50 ⁇ l 1 ⁇ PBS and detected by FACS.
  • the screened positive clones were re-screened with GS-cynoGPRC5D/GS-huGPRC5A cells by the same method as above, and a total of 82 hybridomas that bound to both human GPRC5D and monkey GPRC5D, but did not bind to human GPRC5A were obtained.
  • the candidate clones were subcloned in a conventional manner. After that, the above-mentioned high-throughput screening method is used to detect the target positive wells, and the cells are cryopreserved.
  • the antibody light and heavy chain gene sequences were extracted from the hybridoma candidate clone obtained in Example 1, and an adult mouse chimeric antibody was constructed from it.
  • RNA was extracted (Macherey-Nagel, Cat#740984.250).
  • the clones with correct and paired antibody light and heavy chain variable region sequences were selected, and their light and heavy chain variable region gene fragments were passed through the homologous recombinase ( Catalog number: C112-01) was ligated into pcDNA3.1 vector, in which the constant region was selected for IgG1 subtype, to obtain expression plasmids for light chain and heavy chain antibodies.
  • the light chain plasmid of the same antibody was then mixed with the heavy chain plasmid at a molar ratio of 1:1, and 293F cells were transfected with polyethyleneimine (PEI) (Polysciences, Cat#23966), and the cell viability was low after 5-7 days of culture. At 60%, the cell culture supernatant was collected and the monoclonal antibody was purified using a Protein A affinity column.
  • PEI polyethyleneimine
  • the affinity of anti-GPRC5D antibodies was detected by flow cytometry. Different concentrations of antibodies were incubated for 30 min with cells expressing human GPRC5D, including GS-CHO cells overexpressing human GPRC5D (huGPRC5D GS-CHO), multiple myeloma cells MM1.R, H929 and AMO-1.
  • a fluorescently labeled secondary antibody (APC-mouse anti-human IgG Fc antibody, Biolegend, Cat. No. 409306) was then added and detected by flow cytometry using Live/Dead yellow dead cell stain (Thermo Fisher, Cat. No. L34967). Cell fluorescence intensity, curve fitting using GraphPad Prism 8.0 and calculation of EC50 value to indicate the affinity of antibody binding to human GPRC5D.
  • HB15H1G1 (heavy chain variable region SEQ ID NO: 25, light chain variable region SEQ ID NO: 26) is a fully human antibody
  • ch5E12C4 (heavy chain variable region SEQ ID NO: 98, light chain variable region SEQ ID NO: 26) Region SEQ ID 99)
  • ch11B7 (heavy chain variable region SEQ ID 100, light chain variable region SEQ ID 101)
  • ch7F5D4 antibodies is a chimeric antibody.
  • Table 1 The results are shown in Table 1 below.
  • the screened ch5E12C4 and chimeric ch11B7 antibodies were humanized to form antibodies hz5E12.1.P1 and hz11B7.5.
  • Ts trispecific antibody
  • T-cell engager multispecific antibodies targeting GPRC5D, BCMA and CD3 at the same time can simultaneously bind to two types of tumor-associated antigens GPRC5D and BCMA on the surface of multiple myeloma cells (MM) and CD3 receptor on the surface of T cells.
  • 4 anti-GPRC5D antibodies (HB15H1B1, ch7F5D4, hz5E12.1.P1, hz11B7.5), 1 anti-BCMA antibody (ADI-38456) and two anti-CD3 antibodies with different affinity (lower affinity CD3 antibody hzsp34.87 Based on the higher affinity CD3 antibody hzsp34.24), a multispecific antibody targeting GPRC5D, BCMA and CD3 simultaneously was designed.
  • FIG. 1 As shown in Figure 1, four different formats of multispecific antibody molecules were designed: 2 example antibodies including 1+1+1 format 1 (TS-F1), 6 1+1+1 format 2 (TS-F2) 4 exemplary antibodies in 2+2+2 format 6 (TS-F6) and 5 exemplary antibodies in 1+1+2 format 7 (TS-F7) (see Table 4 below).
  • An example antibody design utilizes Fc "knob-in-hole” technology to resolve heavy chain mismatches of asymmetric IgG-like bispecific antibodies, where Fc is the heavy chain constant region of IgG1; while introducing attenuated effector function ( effector function) L234A, L235A (according to Kabat's "EU” numbering) amino acid mutation.
  • TS-F2 and TS-F7 are composed of 4 polypeptide chains to form a left-right asymmetric IgG-like tetramer
  • TS-F6 is composed of two polypeptide chains to form a left-right symmetrical IgG-like tetramer.
  • TS-F2 consists of a peptide chain 1# containing a heavy chain variable domain, an immunoglobulin CH1 domain and an Fc domain, and a peptide chain 2# containing a light chain variable domain and an immunoglobulin CL domain.
  • a peptide chain 3# comprising a heavy chain variable domain, an immunoglobulin CH1 domain, an Fc domain, and a single-chain antibody scFv connected by a synthetic linking peptide, and a light chain variable structure domain, the peptide chain 4# of the immunoglobulin CL domain.
  • the heavy chain variable domain of peptide #1 and the light chain variable domain of peptide 2# are paired to form the first antigen recognition site, and the heavy chain variable domain of peptide #3 and peptide 4# The light chain variable domains of the paired to form the second antigen recognition site, and the scFv to form the third antigen recognition site.
  • TS-F7 consists of a peptide chain 1# containing a heavy chain variable domain, an immunoglobulin CH1 domain and an Fc domain, one containing a light chain variable domain, an immunoglobulin CL domain, and a section artificially synthesized.
  • the peptide chain 2# of the single-chain antibody scFv connected by the connecting peptide a peptide chain 3# containing the heavy chain variable domain, the immunoglobulin CH1 domain, and the Fc domain, and a light chain variable structure.
  • Domain, immunoglobulin CL domain, a segment of single-chain antibody scFv peptide chain 4# composed of a synthetic linking peptide.
  • the heavy chain variable domain of peptide #1 and the light chain variable domain of peptide 2# are paired to form the first antigen recognition site, and the heavy chain variable domain of peptide #3 and peptide 4#
  • the variable domains of the light chain of 2 are paired to form a second antigen recognition site, and the two scFvs in peptide chain 2# and peptide chain 4# are two identical third antigen recognition sites.
  • TS-F6 consists of two identical peptide chains 1 and two identical peptide chains 2, wherein peptide chain 1# contains a heavy chain variable domain, an immunoglobulin CH1 domain, an Fc domain, and a section by artificial synthesis.
  • the heavy chain variable domain of peptide chain #1 and the light chain variable domain of peptide chain 2# are paired to form the first antigen recognition site, the scFv in peptide chain #1 is the second antigen recognition site, and the peptide chain The scFv in 2# is the third antigen recognition site.
  • TS-F1 consists of peptide chain 1 and peptide chain 2, wherein peptide chain 1# contains the first single chain antibody scFv and Fc domain; peptide chain 2# contains the second single chain antibody scFv and Fc domain.
  • the first scFv in peptide chain #1 forms the first antigen recognition site, and the scFv in peptide chain 2# forms the second antigen recognition site.
  • HB15H1B1, ch7F5D4, hz5E12.1.P1, hz11B7.5 are humanized antibodies
  • HB15H1B1 is a fully human antibody
  • ch7F5D4 is a chimeric antibody
  • hz5E12.1.P1 and hz11B7.5 are humanized antibodies
  • the amino acid sequences of the CDR regions, light chain variable regions and heavy chain variable regions of one anti-BCMA antibody (ADI-38456) and two anti-CD3 antibodies with different affinities (hzsp34.87 and hzsp34.24) are listed in the present application.
  • the "Sequence Listing" section lists the amino acid sequences of the CDR regions, light chain variable regions and heavy chain variable regions of the above-mentioned antibodies for the sequence numbers in Table 2.
  • GPRC5DxCD3 and BCMAxCD3 bispecific antibodies were also designed. As shown in Figure 2A, as a rational design, both ends of the bispecific antibody are in Fab form, forming an asymmetric Ig-like structure in 1+1 format.
  • the heavy chain mismatch of this asymmetric IgG-like bispecific antibody was resolved using Fc "knob-in-hole" technology, where Fc is the heavy chain constant region of IgG1; and the introduction of reduced effector function function) L234A, L235A (numbering according to Kabat's "EU") amino acid mutation.
  • the BCMAxCD3 bispecific antibody 46758 was also expressed.
  • one antibody arm of the bispecific antibody is a Fab that binds BCMA, and the other arm is a scFv that binds CD3, wherein the Fab is from the anti-BCMA parental antibody 38456, and the scFv is from a different previous antibody CD3 parental antibody.
  • the three polypeptide chains that make up the bispecific antibody are shown in SEQ ID NOs: 111-113.
  • the Roche-5E11 bispecific antibody (GPRC5DxCD3, 2:1 format, derived from patent WO2019/154890A1) was also expressed and purified. As shown in Figure 2C.
  • the bispecific antibody Roche_5E11 consists of the sequences SEQ ID NOs: 17, 18, 19 and 20 of WO 2019/154890A1 and contains two GPRC5D binding sites and one CD3 binding site.
  • Expi293F cells (purchased from Thermo Fisher scientific company) were subcultured in Expi293F cell culture medium (purchased from Thermo Fisher scientific company). The cell density was detected one day before transfection, and the cell density was adjusted to 3 ⁇ 10 6 cells/ml on the day of transfection by adjusting to 2 ⁇ 10 6 cells/ml with fresh Expi293 cell culture medium.
  • Opti-MEM medium purchased from Gibco
  • Opti-MEM medium purchased from Gibco
  • the recombinant plasmids containing the nucleotide sequences of the heavy chain and light chain of 200 mg were mixed well, and 30ug polyethyleneimine (PEI) (Polysciences) was added to each mL of transfection buffer, mixed well, and incubated at room temperature for 20 minutes.
  • the PEI/DNA mixture was gently poured into the Expi293F cell suspension, mixed well, and placed on a shaker for incubation under the conditions of 8% CO2, 36.5°C, and 120rpm.
  • the culture was collected, centrifuged at 4000 rpm for 30 minutes, and the cell supernatant was filtered through a 0.45 ⁇ M filter, and the parental protein was purified by affinity chromatography and ion exchange chromatography. For symmetrically structured IgG-like antibodies, the final molecule is purified.
  • the reaction solution of the overnight reaction was exchanged to remove GSH, and the exchange buffer could be the above-mentioned 0.2M PB solution (Ph6.0) for the recombination reaction without GSH. After 2-3 hours of natural oxidation, it can be purified by monoS (GE Cat. 17516801).
  • Roche-5E11 bispecific antibody was prepared according to the method disclosed in WO 2019/154890A1.
  • Symmetrical and asymmetrical antibodies prepared according to the methods described above are harvested and purified using affinity and ion exchange chromatography.
  • SEC Size exclusion chromatography
  • the purified bispecific antibody solution was centrifuged at 4500 rpm for 30 minutes in a 15ml ultrafiltration centrifuge tube, and the protein was diluted with PBS and then centrifuged at 4500 rpm for 30 minutes. Repeat this operation several times to replace the buffer. liquid.
  • the antibodies after the buffer exchange were combined, and the antibody concentration was measured.
  • the composition and content of multispecific antibodies were further characterized and quantified by the combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).
  • trispecific antibodies antigen binding sites from humanized antibodies hz5E12.1.P1 and hz11B7.5 were examined for binding affinity to GPRC5D.
  • different concentrations of trispecific antibodies were incubated with huGPRC5D GS-CHO cells for 30 minutes, then fluorescently labeled secondary antibodies were added, and the fluorescence intensity of cells was detected by flow cytometry (BD).
  • Curves were fitted with GraphPad Prism 8.0 and EC50 values were calculated to show the affinity of the antibody for binding to human GPRC5D. The results are shown in Table 5 below.
  • the affinity of the anti-BCMA and anti-CD3 ends was detected by Biofilm Layer Optical Interferometry (BLI).
  • BLI Biofilm Layer Optical Interferometry
  • the binding kinetics of the exemplary antibodies to human BCMA, monkey BCMA, human CD3E and monkey CD3E were determined.
  • Soak probe sensors (Fortebio, Cat. No. 18-5019) coupled to streptavidin protein (SA) or anti-human-Fc (AHC) in 200ul SD buffer (1x PBS, 0.1% BSA, 0.05% tween-20).
  • SA streptavidin protein
  • AHC anti-human-Fc
  • Example antibodies and biotinylated or Fc-tagged BCMA and CD3 D&E heterodimeric antigens were diluted in SD buffer, respectively.
  • 200 ⁇ l of SD buffer and each diluted sample (100 nM) were added to a 96-well black plate (Greiner, Cat. No. 655209). Probes and samples were placed in Octet (Fortebio, Red96e).
  • Open Data Acquisition 10.0 select "New Kinetic Experiment" arrange the board according to the sample position, select the sensor position, and set the running steps and time: Baseline 60s, Loading 250s, Baseline100s, Association 600s and Dissociation 600s.
  • the experimental speed was 1000 rpm and the temperature was 30 °C.
  • Exemplary antibodies were detected using human BCMA antigen (ACRO Biosystems) and monkey BCMA antigen (ACRO Biosystems) in experiments performed as described in the assays above.
  • the results of affinity detection with human BCMA are shown in Table 6; the results of affinity detection with monkey BCMA are shown in Table 7.
  • Exemplary antibodies were tested for affinity using human CD3D&E heterodimer antigen (ACRO Biosystems) and monkey CD3D&E heterodimer antigen (ACRO Biosystems) in experiments performed as described in the assays above and the results are shown in Table 8, Table 8. 9 shown.
  • the GPRC5DXCD3 bispecific antibodies, hz5E12.1-P1/SP34.24 and hz5E12.1-P1/SP34.87, were also examined for binding affinity to human/monkey CD3D&E.
  • Example 7 Jurkat cell activation experiment mediated by anti-GPRC5DxBCMAxCD3 antibody
  • the activation activity of anti-GPRC5DxBCMAxCD3 antibody on CD3E downstream signaling pathway was detected by Jurkat-NFAT-Luc reporter system.
  • the anti-GPRC5DxBCMAxCD3 polyclonal antibody binds to GPRC5D and/or BCMA on the surface of multiple myeloma (MM) cell lines, and at the same time binds to CD3E on the surface of Jurkat-NFAT-Luc cells (Jurkat), it can pass tumor-associated antigens (GPRC5D and/or BCMA) or BCMA)-dependent CD3 cross-linking, stimulates CD3E downstream signaling in Jurkat-NFAT-Luc cells. Therefore, the level of T cell activation mediated by exemplary antibodies was assessed by the Luciferase reporter system.
  • samples were diluted first: antibodies were diluted to 100 nM concentration in RPMI medium 1640 (5% FBS), followed by 5-fold serial dilutions. Then prepare the cells: Jurkat cells and tumor cells were centrifuged at 300g for 5min, the supernatant was discarded, resuspended in RPMI medium1640 (5% FBS), counted (Countstar), and the cell density was adjusted to: Jurkat NFAT 2x10 6 cell/ml, tumor cells Adjust according to the ratio of effector cells to target cells. In a 96-well white cell culture plate, 45 ⁇ l of tumor cells were added to each well, 30 ⁇ l of serially diluted samples were added, and 45 ⁇ l of Jurkat NFAT cells were added.
  • the 96-well plate was then placed in a 37°C 5% CO2 incubator to continue culturing. After culturing for a period of time, the cell culture plate was taken out, placed at room temperature for 5 minutes, 80ul Bio-Glo (Promega, G7940) was added to each well, incubated in the dark for 10 minutes, and the fluorescence value was read on SpectraMax i3 (MOLECULAR DEVICES). Curves were fitted using GraphPad Prism 8.0 and EC50 values were calculated to compare the activating activity of example antibodies on T cells. The results are shown as the fold ratio of the readings of the experimental group to the readings of the control hIgG1 group.
  • TS-F2-1 and TS-F2-2 can significantly increase the expression of luciferase in Jurkat cells with the increase of antibody concentration (Figure 3A and 3B); and their agonistic activity is higher than that of Ts-F1 and control group GPRC5DxCD3 double Anti- and BCMAxCD3 dual antibodies.
  • Table 10 The specific EC50s of the tested antibodies are shown in Table 10.
  • TS-F2-1 and TS-F2-2 both consist of three binding specificities from HB15H1G1 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody), but HB15H1G1 and 38456 binding specificities Sex is in a different position.
  • TS-F2-3 can significantly increase the expression of luciferase in Jurkat cells with the increase of antibody concentration (Fig.
  • TS-F6-1 to -4 The specific EC50s of the tested antibodies are shown in Table 11.
  • Exemplary antibodies TS-F2-3 and TS-F6 consist of three binding specificities from ch7F5D4 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody).
  • huGPRC5D-overexpressing GS-CHO huGPRC5D-GS-CHO cells and Jurkat cells were co-cultured for 16 hours and exemplified antibodies were added to mimic T cell activation mediated by GPRC5D-only multiple myeloma cells.
  • the ratio of effector cells (Jurkat cells) to target cells (huGPRC5D-GS-CHO cells) was 50:1.
  • the experimental results show that TS-F2-3 can significantly increase the expression of luciferase in Jurkat cells with the increase of antibody concentration (Figure 4D); and its agonistic activity is higher than that of Ts-F6 antibody and the control group GPRC5DxCD3 double antibody and BCMAxCD3 double antibody (46758).
  • the specific EC50 is shown in Table 11.
  • 8226 cells were co-cultured with Jurkat cells (5:1 ratio of effector cells to tumor cells) for 5 hours, and
  • Both TS-F2-4 and TS-F2-5 can significantly increase the expression of luciferase in Jurkat cells with the increase of antibody concentration (Figure 5A, 5B, 5C); and their agonistic activity is higher than that of the control group GPRC5DxCD3, BCMAxCD3 double antibody (38456/hzsp34.24) and 46758 double antibody.
  • the specific EC50 is shown in Table 12.
  • example antibody TS-F2-4 consists of hz5E12.1.P1 (GPRC5D antibody), hzsp34.24 (high affinity CD3 antibody) and 38456 (BCMA antibody), and the example antibody TS-F2-5 consists of hz5E12.1.P1 ( GPRC5D antibody), hzsp34.87 (low affinity CD3 antibody) and 38456 (BCMA antibody).
  • TS-F2-4 and TS-F2-5 can significantly increase the expression of luciferase in Jurkat cells with the increase of antibody concentration (Figure 5E); and their agonistic activity is higher than that of the control group GPRC5DxCD3 double antibody and BCMAxCD3 Double antibody 46758.
  • the specific EC50 is shown in Table 13.
  • 8226 cells were co-cultured with Jurkat cells (5:1 ratio of effector cells to tumor cells) for 5 hours, and
  • TS-F7-1 and TS-F7-2 can significantly increase the expression of luciferase in Jurkat cells with the increase of antibody concentration (Figure 6A, 6B, 6C, 6D); and their agonistic activity is higher than that of the control group GPRC5DxCD3 double antibody and BCMAxCD3 dual antibody 46758.
  • the specific EC50 is shown in Table 14.
  • example antibody TS-F7-1 consists of hz5E12.1.P1 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody), and the example antibody TS-F7-2 consists of hz11B7.5 (GPRC5D antibody), hzsp34.87 (CD3 antibody) and 38456 (BCMA antibody) composition.
  • TS-F7-3 and TS-F7-4 could significantly increase Jurkat cytofluorescein with increasing antibody concentration enzyme expression (Figure 7); but its agonistic activity was lower than that of Ts-F2-3.
  • TS-F7-3 and TS-F7-4 consist of ch7F5D4 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody), hzsp34.24 antibody forms a single chain antibody when TS-F7-3 VL is in the front, VH is in the back, TS-F7-4 is VH in the front, VL in the back.
  • Antibody-mediated killing ability of T cells against human multiple myeloma cells was detected by lactate dehydrogenase (LDH) assay under co-culture conditions of PBMC and GPRC5D and/or BCMA positive MM cells.
  • LDH lactate dehydrogenase
  • the anti-GPRC5DxBCMAxCD3 polyclonal antibody binds to GPRC5D and/or BCMA on the surface of MM cells, and at the same time binds to CD3E on the surface of primary T cells, it can cross-link with T cells in a tumor-associated antigen (GPRC5D and/or BCMA)-dependent manner to stimulate T cell activation mediates tumor cell killing.
  • Multi-factor detection kits Human Th1/Th2/Th17, BD were used to simultaneously detect the levels of multiple cytokines; at the same time, the percentage of CD69 positive cells in T cells was detected by flow cytometry (BD).
  • the PBMC cells were taken out from the liquid nitrogen, quickly dissolved in a 37-degree water bath, added to 9 ml of serum-free medium, centrifuged at 300 g for 4 min, discarded the supernatant, resuspended in 10% FBS phenol red-free 1640 medium, and adjusted the cell density. was 4 ⁇ 10 6 cells/ml. A certain amount of log-phase H929 cells were collected, centrifuged, and the tumor cell density was adjusted to 2 ⁇ 10 5 cells/ml with 10% FBS phenol red-free 1640 medium. Antibodies were diluted to 100 nM in 10% FBS phenol red-free 1640 medium according to the table above, with 5-fold serial dilutions from 5 nM.
  • ⁇ L of tumor cells 100 ⁇ L of tumor cells, 50 ⁇ L of serially diluted samples and 50 ⁇ L of PBMC cells were added to a 96-well cell culture plate (U shape). At the same time, control wells were set, and the medium was supplemented to 200 ⁇ L/well. Incubate in a 37°C CO2 incubator for 16h. After culturing for 16 h, the cell culture plate was taken out and placed at room temperature for 5 min. 10% Lysis Solution was added to each well of the TM well, and the cells were lysed in the dark for 15 min. Centrifuge the cell culture plate at 400 g for 5 min, and transfer 50 ⁇ l of the supernatant to a new flat-bottom 96-well plate.
  • Example 9 Antitumor efficacy of exemplary antibodies in H929 tumor-bearing humanized mouse model
  • mice Female NOG mice (35-41 days old) were purchased from Beijing Weitongda Laboratory Animal Technology Co., Ltd. The grade is SPF grade. Mice were acclimated and quarantined for 7 days upon arrival before the study began.
  • the H929 cells were routinely subcultured for subsequent in vivo experiments, the cells were collected by centrifugation, and the H929 cells were dispersed with PBS. The right back and abdomen of NOG mice were shaved and inoculated with H929 cells, 5 ⁇ 10 6 cells/cell, and the inoculation volume was 200ul/cell. On the fifth day after inoculation of H929 cells, mice were intravenously injected with PBMC cells, 5 ⁇ 10 6 cells/mice, and the inoculation volume was 200ul/mice.
  • the tumor growth curve is shown in Figure 12, and exemplary antibodies can significantly inhibit the growth of H929 cells. At the same time, no weight loss was found in the administered mice groups.
  • Example 10 Expression levels of GPRC5D and BCMA on different multiple myeloma surfaces
  • MM cells were stained with saturated concentrations of BCMA antibody and GPRC5D antibody, and BCMA and GPRC5D molecules on the surface of different multiple myeloma cell lines were quantified by flow cytometry.
  • the trispecific antibody of the present invention can cover both GPRC5D-positive tumor patients and BCMA-positive tumor patients, thereby improving the coverage rate for patients with multiple tumors; Loss of a single antigen such as BCMA leads to tumor recurrence, improving therapeutic efficacy.

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