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WO2025131075A1 - Anticorps multispécifiques anti-cd3 et anti-cd3, et utilisation - Google Patents

Anticorps multispécifiques anti-cd3 et anti-cd3, et utilisation Download PDF

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Publication number
WO2025131075A1
WO2025131075A1 PCT/CN2024/141038 CN2024141038W WO2025131075A1 WO 2025131075 A1 WO2025131075 A1 WO 2025131075A1 CN 2024141038 W CN2024141038 W CN 2024141038W WO 2025131075 A1 WO2025131075 A1 WO 2025131075A1
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seq
amino acid
antibody
variable region
sequence
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Chinese (zh)
Inventor
李帅
徐维丽
李理
李学廷
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Shanghai Junshi Biotechnology Co Ltd
Shanghai Junshi Biosciences Co Ltd
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Shanghai Junshi Biotechnology Co Ltd
Shanghai Junshi Biosciences Co Ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • T cells are a very important type of immune cell in the human body and play a key role in anti-tumor immunity.
  • the activation of T cells requires the participation of a series of cell membrane surface molecules, such as T cell receptor (T cell receptor, TCR), CD3 and CD28.
  • TCR is responsible for receiving stimulation from antigens, but lacks the functional structure to transmit signals into the cell.
  • TCR needs to form a complex (TCR/CD3 complex) with CD3 through non-covalent binding to complete the activation process of T cells.
  • the intracellular region of the CD3 molecule has an immunoreceptor tyrosine-based activation motif (ITAM), which can mediate the activation, proliferation and survival of T cells.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Bispecific antibodies are a class of bifunctional antibodies that can specifically bind to two antigens or different epitopes of the same antigen.
  • Bispecific T-cell engagers (BiTE) are representative non-IgG-like bispecific antibodies, which are formed by connecting two single-chain variable fragments (scFv) fragments that recognize T cell CD3 molecules and target cell antigens. They recruit T cells near tumor tissues, form immune synapses between tumor cells and T cells, and mediate T cell killing of tumor cells.
  • scFv single-chain variable fragments
  • CD3-based bispecific antibodies were approved for marketing, including Roche's CD3xCD20 bispecific antibody Mosunetuzumab, Johnson & Johnson's CD3xBCMA bispecific antibody Teclistamab, and CD3xGPRC5D bispecific antibody Talquetamab.
  • CD3 bispecific antibodies have shown very good therapeutic effects on blood tumors, the incidence of cytokine storm (CRS) is too high, and sometimes even a serious side effect.
  • CRS cytokine storm
  • the high incidence of CRS is related to the excessive activity of CD3 antibodies, so it is necessary to choose a CD3 antibody of appropriate strength for the development of bispecific antibodies.
  • the purpose of the present application is to provide an antibody or an antigen-binding fragment thereof that can specifically bind to CD3.
  • a multispecific antibody targeting CD3 and another antigen such as CD19
  • an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof or multispecific antibody of the present application is further provided, an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof or multispecific antibody of the present application, an expression vector and a host cell for expressing the antibody or antigen-binding fragment thereof or multispecific antibody of the present application, and the use of the antibody or antigen-binding fragment thereof or multispecific antibody of the present application.
  • the multispecific antibodies (eg, bispecific antibodies) of the present application activate T cells, thereby specifically killing tumor cells, and have significantly reduced cytokine release, thereby having significantly improved safety.
  • the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or the antigen-binding fragment thereof comprises: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29; a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 55, 56, 17, 2, 10, 24, 30 or 37; a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 18, 3, 11, 25, 31 or 38; a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 42, 43, 20, 5, 13 or 33; a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 21, 45, 46, 6, 14, 27, 34 or 44; and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 22, 7, 35 or 40.
  • a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29
  • a HCDR2 with an amino acid sequence as shown in SEQ ID NO
  • the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region:
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively;
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:18;
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;
  • (V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3;
  • HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11;
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively;
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31; or
  • HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38;
  • the light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22;
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:22 by 1, 2 or 3 amino acids;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22;
  • (V) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22 by 1, 2 or 3 amino acids;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7;
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35; or
  • (X) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • V a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively;
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:7, respectively;
  • VII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7, respectively;
  • VIII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or
  • (IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively.
  • the antibodies of the present invention are murine antibodies or chimeric antibodies.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:19, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:19; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:23, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23;
  • the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:4, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:8, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:8;
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:12; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15;
  • the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:26, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:28, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:28;
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:32, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:32; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:36, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:36; or
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:39, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:39; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:41, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:41.
  • the antibodies described herein are humanized antibodies.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52 or 53;
  • a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NO:47, 48, 49, 50, 51, 52 or 53; or
  • the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 58 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 51;
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 61 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 52 or 53.
  • the antibody or antigen-binding fragment thereof of the present invention wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the antigen-binding fragment of the present invention is selected from Fab, Fab', F(ab') 2 , Fv, scFv or sdAb.
  • the antibody or antigen-binding fragment thereof described in the present invention wherein the antibody or antigen-binding fragment thereof is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG4 subtype.
  • the antibody or antigen-binding fragment thereof described in the present invention wherein the antibody comprises a heavy chain constant region shown in the amino acid sequence of SEQ ID NO:63 and/or a light chain constant region shown in SEQ ID NO:62.
  • the present invention provides a multispecific antibody comprising an antigen binding domain targeting CD3 and at least one antigen binding domain targeting other antigens, wherein the antigen binding domain targeting CD3 is selected from the antibodies or antigen binding fragments thereof described herein, and the other antigen is selected from tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).
  • TAAs tumor-associated antigens
  • TSAs tumor-specific antigens
  • the multispecific antibody of the present invention is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.
  • the multispecific antibody of the present invention comprises the antigen binding domain targeting CD3 and the antigen binding domain targeting the tumor-associated antigen or tumor-specific antigen.
  • the tumor-associated antigen is selected from CD19.
  • the multispecific antibody of the present invention is a bispecific antibody in the form of DuoBody, comprising:
  • peptide chain I-A which comprises the VL of the antigen binding domain targeting CD3 and the first light chain constant region (CL); preferably, the first light chain constant region is kappa;
  • peptide chain I-B which comprises the VH of the antigen binding domain targeting CD3 and the first heavy chain constant region (CH); preferably, the first heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4;
  • a peptide chain I-C comprising the VH of the antigen-binding domain targeting TAA or TSA and a second heavy chain constant region; preferably, the second heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; and
  • a peptide chain I-D which comprises the VL of the antigen binding domain targeting TAA or TSA and a second light chain constant region; preferably, the second light chain constant region is kappa.
  • the Fc domains contained in the peptide chains I-B and I-C respectively contain modifications (e.g., knob-into-hole modifications) to promote dimerization of I-B and I-C.
  • the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof
  • the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51, 47, 52 or 53 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51 or 47 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the multispecific antibody of the present invention is a common light chain bispecific antibody comprising:
  • peptide chain II-A which is a common light chain and comprises the VL and light chain constant region (CL) of the antigen binding domain targeting CD3; preferably, the light chain constant region is kappa;
  • peptide chain II-B which comprises the VH of the antigen binding domain targeting CD3 and the first heavy chain constant region (CH); preferably, the first heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; and
  • peptide chain II-C which comprises the VH of the antigen-binding domain targeting TAA or TSA and a second CH; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4.
  • the Fc domains comprised by the peptide chains II-B and II-C respectively comprise modifications to promote dimerization of II-B and II-C.
  • the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 47 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 57 or a variant thereof; or (2) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 47 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 58 or a variant thereof; or (3) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 51 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 58 or a variant thereof; or (4) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 52 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 53 or a variant thereof.
  • the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; or (1) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; or (2) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; the variants in any one of (1) to (7) have at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which they are derived.
  • the peptide chain II-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof; the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the present invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof as described herein.
  • the invention provides isolated nucleic acid molecules encoding the multispecific antibodies or polypeptide chains thereof described herein.
  • the present invention provides an expression vector comprising the isolated nucleic acid molecule described herein.
  • the present invention provides a host cell comprising an isolated nucleic acid molecule described herein or a vector (eg, an expression vector) described herein.
  • the present invention provides a method for preparing the antibody or antigen-binding fragment thereof, or the multispecific antibody described herein, the method comprising culturing the host cell described herein under conditions allowing protein expression, and recollecting the antibody or antigen-binding fragment thereof, or the multispecific antibody from the culture of the cultured host cell.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the multispecific antibody described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the host cell described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the isolated nucleic acid molecule described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein in the preparation of a medicament for preventing and/or treating a disease.
  • the present invention provides a method for preventing and/or treating a disease, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof described herein, the multispecific antibody described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the host cell described herein, or the pharmaceutical composition described herein.
  • the disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from ovarian cancer, breast cancer, gastric cancer, bile duct cancer, colorectal cancer, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, melanoma or colorectal cancer.
  • the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3.
  • anti-CD3 antibody refers to an antibody that can bind to a CD3 protein or a fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic agent and/or therapeutic agent targeting CD3.
  • the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or the antigen-binding fragment thereof comprises: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29; a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 55, 56, 17, 2, 10, 24, 30 or 37; a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 18, 3, 11, 25, 31 or 38; a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 42, 43, 20, 5, 13 or 33; a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 21, 45, 46, 6, 14, 27, 34 or 44; and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 22, 7, 35 or 40.
  • a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29
  • a HCDR2 with an amino acid sequence as shown in SEQ ID NO
  • the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and/or a light chain variable region:
  • the heavy chain variable region comprises:
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively;
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:18;
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3;
  • HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively;
  • HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively; or HCDR1, HCDR2 and HCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively;
  • the light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:22 by 1, 2 or 3 amino acids;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22;
  • (V) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22 by 1, 2 or 3 amino acids;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7;
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7;
  • LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35; or
  • (X) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • V a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively;
  • VI a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:7, respectively;
  • VII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7, respectively;
  • VIII a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or
  • (IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:19, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:19; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:23, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23;
  • the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:4, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:8, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:8;
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:12; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15;
  • the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:26, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:26; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:28, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:28;
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:32, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:32; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:36, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:36; or
  • the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:39, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:39; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:41, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:41.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 57, 58, 59, 60 or 61;
  • a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO:47, 48, 49, 50, 51, 52 or 53.
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NO: 57, 58, 59, 60 or 61;
  • the antibody or antigen-binding fragment thereof of the present invention comprises:
  • the heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 57 or 58 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 47;
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 61 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 52 or 53.
  • the antibodies or antigen-binding fragments thereof described in the present invention are selected from antibodies having the CDR and/or variable region sequences of antibody NP010-018 or humanized antibodies 18, 18-5, 18-8, 18-11, 18-12, 18-13 or 18-14 thereof, antibodies having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with their CDR or variable region sequences, and antibodies whose difference compared with their CDR or variable region sequences is only the deletion, addition or substitution of one or several amino acids.
  • antibodies having the CDR and/or variable region sequences of antibody NP010-018 or humanized antibodies 18, 18-5, 18-8, 18-11, 18-12, 18-13 or 18-14 thereof antibodies having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at
  • the antibody or antigen-binding fragment thereof described in the present invention is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.
  • the antibody molecules of the present invention are humanized. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633).
  • the antibodies of the present invention comprise a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene), and/or a light chain framework region (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene).
  • the heavy chain framework region and/or the light chain framework region optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to mouse residues.
  • the antibody molecules of the present invention are human antibodies or humanized antibodies.
  • Human antibodies or humanized antibodies can be prepared using various techniques known in the art.
  • the antibody molecules of the present invention also encompass antigen-binding fragments thereof, such as the following antibody fragments: Fab, Fab', F(ab') 2 , Fv, scFv or sdAb.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), which are based on the three-dimensional structure of the antibody and the topology of the CDR loops; and Kabat (Kabat et al., Sequences of Proteins of Immunoglobulins), which are based on the variability of antibody sequences. logical Interest, 4th edition, U.S.
  • the CDR of antibodies of the present invention can be determined by those skilled in the art according to any scheme (e.g., different assignment systems or combinations) of this area.
  • the CDR of antibodies of the present invention is defined by Kabat, Chothia, AbM, Contact, North or IMGT. In some embodiments, the CDR of antibodies of the present invention is defined by Kabat.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies using specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).
  • Antibodies with different specificities have different CDRs.
  • CDRs are different between antibodies, only a limited number of amino acid positions in CDRs are directly involved in antigen binding.
  • the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of a CDR.
  • the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.
  • the humanized antibodies of the present invention can be prepared by inserting mouse CDR regions into human germline framework regions using methods known in the art, such as Winter et al. U.S. Pat. No. 5,225,539 and Queen et al. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
  • the amino acid difference comprises an amino acid deletion, insertion or substitution.
  • the anti-CD3 antibodies or antigen-binding fragments thereof of the invention include those having amino acid sequences that have been mutated by amino acid deletion, insertion or substitution, but are still at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the above antibodies (particularly in the CDR regions depicted in the above sequences).
  • the antibodies of the invention have no more than 1, 2, 3, 4 or 5 amino acid mutations in the CDR regions by amino acid deletion, insertion or substitution when compared to the CDR regions depicted in the specific sequences.
  • the antibodies of the invention have no more than 1, 2, 3, 4 or 5 amino acid mutations in the framework regions by amino acid deletion, insertion or substitution when compared to the framework regions in the specific sequences.
  • the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, insertion or substitution, but still have at least about 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding region depicted in the sequence described above.
  • the CD3 antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody or a modified form thereof, as described in the following sections.
  • one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant.
  • the Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.
  • cysteine engineered antibodies such as "thioMAbs,” in which one or more residues of an antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-protein moieties known in the art and readily available.
  • Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
  • PEG poly
  • the reference antibody SP34 (Teclistamab-CD3) is selected from the heavy chain shown in the SEQ ID NO: 66 sequence and the light chain shown in the SEQ ID NO: 67 sequence.
  • the reference antibody blinatumumab is selected from the heavy chain shown in SEQ ID NO: 68 and the light chain shown in SEQ ID NO: 69.
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • the present invention provides a multispecific antibody, which comprises an antigen binding domain targeting CD3 and at least one antigen binding domain targeting other antigens, wherein the antigen binding domain targeting CD3 is selected from the antibodies or antigen binding fragments thereof described herein, and the other antigen is selected from tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).
  • TAAs tumor-associated antigens
  • TSAs tumor-specific antigens
  • the multispecific antibody of the present invention is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.
  • the multispecific antibody of the present invention comprises the antigen binding domain targeting CD3 and the antigen binding domain targeting the tumor-associated antigen or tumor-specific antigen.
  • the multispecific antibody of the present invention wherein the tumor-associated antigen or tumor-specific antigen is selected from CD19, CD20, CD22, EGFR, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CD H17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof.
  • the tumor-associated antigen is selected from CD19.
  • the multispecific antibody described in the present invention comprises the antigen binding domain targeting CD3 and the antigen binding domain targeting CD19;
  • the antigen binding domain targeting CD19 comprises a light chain variable region and a heavy chain variable region,
  • the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70 or a variant thereof, and
  • the light chain variable region comprises the sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the antigen binding domain targeting CD3 of such multispecific antibodies comprises HCDR1, HCDR2, HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 56, SEQ ID NO: 18 and LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 22, respectively.
  • such multispecific antibodies are bispecific antibodies in the form of a common light chain.
  • the common light chain of such multispecific antibodies comprises LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 42, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; or LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 22, respectively; or LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively; preferably, the common light chain comprises LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively.
  • the common light chain of such multispecific antibodies comprises the sequence shown in SEQ ID NO: 47, 51, 52 or 53.
  • the antigen binding domain targeting CD3 of the multispecific antibody of the present invention is Fab.
  • the multispecific antibody of the present invention further comprises an Fc domain.
  • the Fc domain is IgG, such as IgG1, IgG2, IgG3 or IgG4.
  • the Fc domain comprises a modification to promote dimerization of the first Fc monomer and the second Fc monomer.
  • the modification comprises a knob modification in one of the two monomers and a hole modification in the other of the two monomers to form a knob-into-hole modification.
  • the Fc domain may also be a native Fc sequence.
  • the multispecific antibody of the present invention is a DuoBody or a common light chain bispecific antibody.
  • the multispecific antibody of the present invention is a DuoBody, which comprises:
  • peptide chain I-A which comprises the VL of the antigen binding domain targeting CD3 and the first light chain constant region (CL); preferably, the first CL is kappa;
  • peptide chain I-B which comprises the VH of the antigen-binding domain targeting CD3 and the first heavy chain constant region (CH); preferably, the first CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;
  • a peptide chain I-C which comprises the VH and the second CH of the antigen-binding domain targeting TAA or TSA; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; and
  • a peptide chain I-D which comprises the VL of the antigen-binding domain targeting TAA or TSA and a second CL; preferably, the second CL is kappa.
  • the Fc domains comprised by the peptide chains I-B and I-C respectively comprise modifications to promote dimerization of I-B and I-C.
  • the Fc domains contained in the peptide chains I-B and I-C contain mutations that promote Fab arm exchange, such as a K409R mutation in the CH3 region of one of the Fc domains and a F405L mutation in the CH3 region of the other Fc domain.
  • the peptide chains I-B and I-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 63 and 72, respectively.
  • the peptide chains I-B and I-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 72 and 63, respectively.
  • the peptide chains I-A and I-D contain the light chain constant region sequence shown in SEQ ID NO:62.
  • the antigen binding domain targeting TAA or TSA is an antigen binding domain targeting CD19
  • the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof
  • the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the peptide chain I-A comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively
  • the peptide chain I-B comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively.
  • the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51, 47, 52 or 53 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51 or 47 or a variant thereof; the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof.
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51 or a variant thereof; the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or a variant thereof.
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 47 or a variant thereof; the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 57 or a variant thereof.
  • the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70
  • the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51
  • the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58.
  • the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70
  • the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71
  • the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 47
  • the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 57.
  • the present invention provides the following exemplary DuoBody:
  • the peptide chain I-A comprises the light chain sequence shown in SEQ ID NO:65; the peptide chain I-B comprises the heavy chain sequence shown in SEQ ID NO:64; the peptide chain I-C comprises the heavy chain sequence shown in SEQ ID NO:68; the peptide chain I-D comprises the light chain sequence shown in SEQ ID NO:69; or,
  • the peptide chain I-A comprises the light chain sequence shown in SEQ ID NO:84; the peptide chain I-B comprises the heavy chain sequence shown in SEQ ID NO:83; the peptide chain I-C comprises the heavy chain sequence shown in SEQ ID NO:68; and the peptide chain I-D comprises the light chain sequence shown in SEQ ID NO:69.
  • the multispecific antibody of the present invention is a common light chain bispecific antibody comprising:
  • peptide chain II-A which is a common light chain and comprises the VL and light chain constant region (CL) of the antigen binding domain targeting CD3; preferably, the CL is kappa;
  • peptide chain II-B which comprises the VH of the antigen-binding domain targeting CD3 and the first heavy chain constant region (CH); preferably, the first CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; and
  • peptide chain II-C which comprises the VH and the second CH of the antigen-binding domain targeting TAA or TSA; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;
  • the VL of the peptide chain II-A and the VH of the peptide chain II-B form a first binding site targeting CD3, and the VL of the peptide chain II-A and the VH of the peptide chain II-C form a second binding site targeting TAA or TSA.
  • the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise:
  • HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:16, SEQ ID NO:56 and SEQ ID NO:18, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or
  • the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise: HCDR1, HCDR2, HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, and LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively.
  • peptide chain II-A of the common light chain bispecific antibody comprises a VL sequence shown in SEQ ID NO: 47, 51, 52 or 53 or a variant thereof, preferably the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • peptide chain II-B of the common light chain bispecific antibody comprises a VH sequence shown in SEQ ID NO: 57, 58, 60 or 61 or a variant thereof, preferably the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise: (1) the VH sequence shown in SEQ ID NO: 57 or a variant thereof, the VL sequence shown in SEQ ID NO: 47 or a variant thereof; (2) the VH sequence shown in SEQ ID NO: 58 or a variant thereof, the VL sequence shown in SEQ ID NO: 47 or a variant thereof; (3) the VH sequence shown in SEQ ID NO: 58 or a variant thereof, the VL sequence shown in SEQ ID NO: 51 or a variant thereof; (4) The VH sequence shown in SEQ ID NO: 60 or a variant thereof, the VL sequence shown in SEQ ID NO: 52 or a variant thereof; (5) the VH sequence shown in SEQ ID NO: 60 or a variant thereof, the VL sequence shown in SEQ ID NO: 53 or a variant thereof; (6) the VH sequence shown in SEQ ID NO: 61 or a variant thereof, the VL sequence shown in SEQ ID
  • the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively contain: the VH sequence shown in SEQ ID NO: 61, and the VL sequence shown in SEQ ID NO: 53.
  • peptide chain II-C of the common light chain bispecific antibody comprises a VH of an antigen binding domain targeting CD19.
  • VH of peptide chain II-C of the common light chain bispecific antibody comprises a sequence shown in SEQ ID NO: 70 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.
  • the Fc domains contained in peptide chains II-B and II-C of the common light chain bispecific antibody optionally contain modifications to promote dimerization of I-B and I-C, respectively.
  • the Fc domains contained in peptide chains II-B and II-C contain a K409R mutation in the CH3 region of one of the Fc domains and a F405L mutation in the CH3 region of the other Fc domain.
  • the peptide chains II-B and II-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 63 and 72, respectively.
  • the peptide chains II-B and II-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 72 and 63, respectively.
  • the peptide chain II-A of the common light chain bispecific antibody comprises the light chain constant region sequence shown in SEQ ID NO:62.
  • the present invention provides an isolated nucleic acid molecule encoding any of the above antibodies or fragments thereof or any of their chains.
  • the isolated nucleic acid molecule may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and/or the heavy chain variable region of the antibody, or a polynucleotide encoding the amino acid sequence of the light chain and/or the heavy chain of the antibody.
  • the isolated nucleic acid molecule comprises a polynucleotide encoding each peptide chain of the multispecific antibody of the present invention.
  • the present invention provides a vector (e.g., an expression vector) comprising an isolated nucleic acid molecule as described herein, preferably, the vector is a eukaryotic expression vector.
  • the isolated nucleic acid molecule as described herein is contained in one or more vectors.
  • Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).
  • the present invention provides a host cell comprising an isolated nucleic acid molecule as described herein or an expression vector as described herein, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell (e.g., a CHO cell or a 293 cell). In another embodiment, the host cell is prokaryotic.
  • the present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These include Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has a high expression level.
  • ATCC American Type Culture Collection
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described herein, the multispecific antibody described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the host cell described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • composition refers to a preparation which permits the active ingredient contained therein to exist in biologically effective form, and which contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.
  • compositions containing the antibodies described herein can be prepared by mixing the antibodies of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized preparation.
  • any antibody provided herein can be used in a method of treatment. It should also be understood that when discussing "antibodies", compositions comprising antibodies are also included.
  • the antibodies of the present invention can be used in a therapeutically effective amount or a prophylactically effective amount in a method of treatment or prevention described in any embodiment of the present invention.
  • the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the isolated nucleic acid molecule described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein in the preparation of a medicament for preventing and/or treating a disease.
  • the present invention provides a method for preventing and/or treating a disease, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof described herein, the multispecific antibody described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the host cell described herein, or the pharmaceutical composition described herein.
  • the disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from ovarian cancer, breast cancer, gastric cancer, bile duct cancer, colorectal cancer, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, melanoma or colorectal cancer.
  • the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration, etc.
  • CD3 refers to a part of the T cell receptor complex, which is composed of three different chains CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • concentration of CD3 on T cells such as by the fixation of anti-CD3 antibodies, leads to the activation of T cells, similar to the activation mediated by the T cell receptor, but independent of the specificity of the TCR clone.
  • the vast majority of anti-CD3 antibodies recognize the CD3 ⁇ chain.
  • the term refers to any natural CD3 from any vertebrate (including mammals such as primates (e.g., humans)) and rodents (e.g., mice and rats), unless otherwise specified.
  • CD3 refers to the full length or fragments thereof from humans and cynomolgus monkeys (such as mature fragments lacking signal peptides). In a preferred embodiment, CD3 refers to the full length or fragments thereof from mice/rat (such as mature fragments lacking signal peptides).
  • CD19 refers to the Cluster of Differentiation 19 protein (CD19), which is an antigenic determinant detectable on leukemic precursor cells.
  • Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot.
  • the amino acid sequence of human CD19 can be found in UniProt/Swiss-Prot Accession No. P15391, and the nucleotide sequence encoding human CD19 can be found in Accession No. NM_001178098.
  • CD19 is expressed on most B-lineage cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above.
  • “or” or “and/or” should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when terms are clearly indicated to the contrary, such as “only one” or “exactly one” or when “consisting of" is used in the claims, it will refer to only one number listed or one element of the list.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps introduced where necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity.
  • Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software.
  • One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.
  • immune response refers to the actions of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement), which result in the selective damage, destruction or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.
  • signal transduction pathway or “signal transduction activity” refers to a biochemical cause-effect relationship, usually initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another part of the cell.
  • the transmission involves specific phosphorylation of one or more tyrosine, serine or threonine residues on one or more proteins in a series of reactions that lead to signal transduction.
  • the penultimate process usually involves nuclear events, resulting in changes in gene expression.
  • activity or “biological activity”, or the terms “biological property” or “biological characteristic” are used interchangeably herein and include, but are not limited to, epitope/antigen affinity and specificity, the ability to neutralize or antagonize CD3 activity in vivo or in vitro, IC50 , the in vivo stability of the antibody, and the immunogenic properties of the antibody.
  • Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells.
  • antibody refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium.
  • isolated does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the binding compound described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule that contains at least four peptide chains when naturally present: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs).
  • CDRs complementary determining regions
  • FRs conservative framework regions
  • Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • antibody binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody.
  • antibody binding fragments include, but are not limited to, Fab, Fab', F(ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.
  • the binding fragment or derivative When the binding activity of the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody.
  • the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as “conservative variants” or “functional conservative variants” of antibodies).
  • binding compound refers to both antibodies and their binding fragments.
  • domain antibody is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the light chain.
  • two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody.
  • the two VH regions of a bivalent domain antibody can target the same or different antigens.
  • diabody refers to a small antibody fragment with two antigen binding sites, which comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen binding sites.
  • murine antibody or “hybridoma antibody” in the present disclosure refers to a monoclonal antibody against human CD3 prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with CD3 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated.
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from antibodies such as rodents ("parent antibodies”), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects than the parent rodent antibodies.
  • humanized antibody refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • a humanized antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat sugar chains. Similarly, a “mouse antibody” refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat sugar chains. Similarly, a "rat antibody” refers to an antibody that contains only rat immunoglobulin sequences.
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, such as to enhance or reduce effector function or binding to Fc receptors.
  • heavy chain constant region genes e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4
  • Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, such as Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • Knob into Hole structure refers to the mutation of the hydrophobic amino acids of CH3 of antibody Fc.
  • the side chain amino acids of one CH3 chain are mutated to form a relatively large hydrophobic amino acid (knob) to strengthen the hydrophobic force; the side chain amino acids of another CH3 are mutated to form a small amino acid (hole) to reduce steric hindrance; after the mutation, the CH3 with Knob and the CH3 with Hole form a Knob into Hole structure (KiH) in the form of hydrophobic interaction, which is conducive to the formation of heavy chain heterodimers; the KiH mutation mainly occurs in the internal hydrophobic amino acids of the spatial structure of the CH3 domain, and the amino acids exposed outside are almost unchanged after the mutation, so it does not affect the effector function of Fc and the immunogenicity caused.
  • epitope refers to a protein determinant that is capable of specific binding to an antibody.
  • Epitopes are usually composed of various chemically active surface molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that in the presence of denaturing solvents, the binding to the former but not to the latter is lost.
  • the term "common light chain” refers to a light chain comprising the same light chain variable region and light chain constant region, which can be paired with the heavy chain of a first antibody that binds to a first antigen to form a binding site that specifically binds to the first antigen, and can also be paired with the heavy chain of a second antibody that binds to a second antigen to form a binding site that specifically binds to the second antigen. Further, the light chain variable region of the common light chain forms a first antigen binding site with the heavy chain variable region of the first antibody, and the light chain variable region of the common light chain forms a second antigen binding site with the heavy chain variable region of the second antibody.
  • Duobody refers to a heterodimer comprising a heavy chain and a light chain from a first antibody and a heavy chain and a light chain from a second antibody paired therewith.
  • Duobody can be prepared by combining half of a first monospecific antibody comprising two heavy chains and two light chains with half of a second monospecific antibody comprising two heavy chains and two light chains.
  • the resulting heterodimer is a bispecific antibody.
  • each monospecific antibody includes a heavy chain constant region with a single point mutation in the CH3 domain.
  • the single point mutation in each monospecific antibody can be located at residues 366, 368, 370, 399, 405, 407 or 409 (according to EU numbering) in the CH3 domain of the heavy chain constant region.
  • a single point mutation is located at different residues in a monospecific antibody relative to another monospecific antibody.
  • one monospecific antibody may comprise the mutation F405L (EU numbering) and another monospecific antibody may comprise the mutation K409R (EU numbering).
  • cross-reactivity refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Therefore, “cross-reactivity” should be understood as an interspecies reaction with the same molecule X expressed in different species.
  • the cross-reactivity specificity of monoclonal antibodies recognizing human CD3, monkey, and/or murine CD3 (mouse or rat) can be determined by FACS analysis.
  • 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 be generally represented by the equilibrium dissociation constant ( KD ), which is the ratio of the dissociation rate constant and the association rate constant ( kdis and kon , respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method for measuring affinity is the ForteBio kinetic binding assay herein.
  • the term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD for binding to the protein or cell is 1.0 ⁇ 10-6 M or higher, more preferably 1.0 ⁇ 10-5 M or higher, more preferably 1.0 ⁇ 10-4 M or higher, 1.0 ⁇ 10-3 M or higher, more preferably 1.0 ⁇ 10-2 M or higher.
  • high affinity for IgG antibodies refers to a KD for an antigen of 1.0 ⁇ 10-6 M or less, preferably 5.0 ⁇ 10-8 M or less, more preferably 1.0 ⁇ 10-8 M or less, 5.0 ⁇ 10-9 M or less, and more preferably 1.0 ⁇ 10-9 M or less.
  • "high affinity” binding may vary.
  • “high affinity” binding for the IgM subtype refers to a KD of 10-6 M or less, preferably 10-7 M or less, and more preferably 10-8 M or less.
  • the ability to "compete for binding” refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CD3 and anti-CD3 antibody) to any detectable extent (e.g., inhibition of at least 85%, or at least 90%, or at least 95%).
  • antibody-dependent cellular cytotoxicity refers to a cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens to antibodies.
  • complement-dependent cytotoxicity refers to the effector function of IgG and IgM antibodies, which when bound to surface antigens initiate the classical complement pathway, including formation of the membrane attack complex and target cell lysis.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.
  • RNA ribonucleic acid
  • nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.
  • a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as sequences explicitly indicated.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • Construction refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner.
  • a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more
  • Recombinant constructs will typically include a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell.
  • a transcription initiation regulatory sequence that directs transcription of the polynucleotide in a host cell.
  • Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the invention.
  • Vector refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell).
  • plasmid refers to a circular double-stranded DNA loop into which additional DNA segments can be connected.
  • viral vector in which additional DNA segments can be connected to the viral genome.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors).
  • vectors After introduction into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors".
  • expression vector refers to a nucleic acid molecule that can replicate and express a target gene when transformed, transfected or transduced into a host cell.
  • the expression vector comprises one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host when necessary.
  • Response/reaction such as the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.
  • biochemical or physiological behavior e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
  • cyno or “cynomolgus monkey” refers to cynomolgus monkeys.
  • Administration "in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) administration and consecutive administration in either order.
  • Figure 1A-C Binding experiment of anti-human CD3E antibody to Jurkat cells.
  • Figure 3A-B Binding experiments of anti-CD3 humanized antibodies on Jurkat cells.
  • FIG. 5A-E Validation of the CD3xCD19 bispecific antibody in Duobody format and its in vitro killing experiment.
  • FIG. 10 In vivo anti-tumor activity experiment of CD3xBCMA common light chain bispecific antibody.
  • Table 1 The information of the sequences involved in the present invention is described in the table below:
  • the antibodies of the present invention can be prepared by a variety of methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.
  • Example 1 Production of anti-human CD3E monoclonal antibodies using the hybridoma method
  • the heterodimeric protein CD3E&G (Uniprot database, P07766-1&P09693) of the extracellular region of the recombinant human CD3 antigen protein was purchased from Acro Biosystems (Cat. No. CDG-H52W5) or the KLH-coupled CD3E peptide (sequence DGNEEMGGITQTPYKVSISGTTVILTC-KLH) was used to immunize 6- to 8-week-old Balb/C mice (purchased from Vital River) or Sprague-Dawley rats (purchased from Vital River).
  • the first immunization was performed with complete Freund's adjuvant at a dose of 50 ⁇ g antigen per mouse (or rat), and the second and subsequent immunizations were performed with incomplete Freund's adjuvant mixed with 25 ⁇ g protein antigen per mouse (or rat).
  • the mouse immunization time points were day 0, day 14, day 28, and day 49, respectively.
  • mice After immunization for 4 times, the tail blood of mice was collected and the titer of CD3E (Acro Biosystems, catalog number H5223) antibody in the serum was detected.
  • the mouse (or rat) with the highest CD3E antibody titer in the serum was selected, and its spleen cells were isolated and then fused with mouse myeloma cells SP2/0.
  • HAT selection medium After plating and culturing in HAT selection medium for 10 to 14 days, the wells with positive binding of culture supernatant to human CD3E were selected for subcloning, and then the monoclones that maintained positive binding to human CD3E after subcloning were selected, and the variable region sequence of anti-human CD3E antibody was obtained by Sanger sequencing.
  • the sequence composition of the light chain and heavy chain variable region and CDR (KABAT numbering rule) of anti-human CD3E antibody is shown in Table 2 below, and the specific amino acid sequence is shown in Table 1.
  • Table 2 Sequence composition of light chain and heavy chain variable regions of anti-human CD3E antibodies
  • variable region sequence of the anti-human CD3E antibody was subcloned into the human IgG4 (SEQ ID NO: 63)/Kappa (SEQ ID NO: 62) backbone, expressed in the HEK293 system and purified by Protein A to obtain the anti-human CD3E human-mouse chimeric antibodies NP010-007, NP010-012, NP010-018, NP010-023, NP010-025 and NP010-030.
  • the binding of anti-human CD3E antibody to Jurkat cells was determined by flow cytometry.
  • the specific method is as follows: After washing Jurkat cells (Clone E6-1, from ATCC, catalog number TIB-152) twice with DPBS (Duluth's phosphate buffered saline), centrifuge at 1000rpm and harvest the cells, and adjust the cell density to 50,000 per well with FACS solution buffer (DPBS (Duluth's phosphate buffered saline) + 2% FBS (fetal bovine serum)). Incubate the chimeric antibody of the anti-human CD3E antibody with gradient dilution (starting concentration 15 ⁇ g/mL, 3-fold gradient dilution) with Jurkat cells on ice for 60 minutes.
  • SP34 is a positive control antibody (its heavy chain sequence is shown in SEQ ID NO:66, and its light chain sequence is shown in SEQ ID NO:67), which is expressed in-house by Junshi Biosciences.
  • Figures 1A-C show the binding of anti-human CD3E antibody to Jurkat cells.
  • This experiment uses the principle that the cell line Jurkat-NFAT-Luc is induced to express the NFAT reporter gene under the stimulation of anti-CD3 antibodies to detect the degree of activation of T cells by CD3 antibodies.
  • 40 ⁇ l of Jurkat-NFAT cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) (10,000 cells per well) were added to a 96-well cell culture plate (Corning, Cat. No. 3917), and then 40 ⁇ l of gradient diluted CD3 chimeric antibodies (starting concentration of 10 ⁇ g/ml, 3-fold dilution, a total of 8 concentration gradients) were added and incubated at 37°C for 6 hours.
  • the anti-human CD3E antibody NP010-018 was selected for humanization.
  • the CDR transplantation method used for humanization is briefly described as follows: the variable regions of the heavy and light chains of the mouse antibody are compared with the germline sequence, and the sequences with high similarity are selected as the humanization templates of the heavy and light chains, respectively, for CDR transplantation.
  • the VH and VL of known structural antibodies with high homology are homologously modeled to find the key amino acids of the Framework that maintain the binding activity of the mouse antibody, and the reference sites that need to restore the mutation are selected from them.
  • the light chain IGKV1-33*01 and IGKV1-39*02 were selected as humanized templates, and the light chain back mutation sites were 43P, 53N, 55Q, and 87S.
  • the heavy chain IGHV3-15*01 and IGHV3-72*01 were humanized templates, and the heavy chain back mutation sites were 35Y, 49A, and 81V.
  • the humanized light chains after CDR transplantation are NP010-Hz018-VL (IGKV1-33*01), NP010-Hz018-VL1 (IGKV1-33*01), NP010-Hz018-VL2 (IGKV1-33*01), NP010-Hz018-VL3 (IGKV1-33*01), NP010-Hz018-VL4 (IGKV1-33*01), NP010-Hz018-VL5 (IGKV1-39*02), and NP010-Hz018-VL6 (IGKV1-39*02), and their sequence compositions are shown in Table 3 below.
  • the humanized heavy chains after CDR transplantation are NP010-Hz018-VH (IGHV3-15*01), NP010-Hz018-VH1 (IGHV3-15*01), NP010-Hz018-VH3 (IGHV3-72*01), NP010-Hz018-VH4 (IGHV3-72*01), and NP010-Hz018-VH5 (IGHV3-72*01), and their sequence compositions are shown in Table 3 below.
  • the humanized antibody was named NP010-Hz018, and the number behind it represents the sequence number of the humanized antibody, such as NP010-Hz018-5, NP010-Hz018-8, and NP010-Hz018-14.
  • the humanized antibody sequence was cloned into the human IgG4 (SEQ ID NO: 63)/kappa (SEQ ID NO: 62) backbone (the sequence is shown below), and the humanized antibody was expressed and purified in the HEK293 expression system.
  • the light and heavy chain sequence compositions of the humanized antibodies are shown in Tables 6 and 7 below.
  • the two CD3xCD19 double antibodies in the present invention can mediate the killing of target cells, and the maximum killing ratio is close to 90%.
  • the killing activity of 18-8 double antibodies is significantly weaker, and the strength of its killing activity is related to the T cell binding activity at the CD3 end.
  • CD3xCD19 double antibodies can effectively stimulate CD4+ and CD8+ T cells to upregulate membrane surface activation molecules CD25 and CD69, among which 18xblinatumumab has a strong activation of T cells (Figure 5B-E).
  • the above results show that the Duobody form CD3 double antibodies of the present invention have moderate activation activity for T cells, which can reduce the side effects of cytokine storm while ensuring efficient killing.
  • Example 8 Preparation of CD3xBCMA bispecific antibody using Duobody method and verification of its in vitro killing activity
  • the present invention utilizes the Duobody in vitro recombination method of Genmab (see Example 1 of patent CN103097417B for details) to prepare CD3xBCMA bispecific antibodies.
  • the 18-8 clone was selected for CD3, Sp34 was used as a strong control, and Junshi's own antibody was selected for BCMA, numbered BCMA-005 (wherein the heavy chain variable region sequence is SEQ ID NO: 79, wherein the HCDR1 sequence of the heavy chain variable region is SEQ ID NO: 73, the HCDR2 sequence of the heavy chain variable region is SEQ ID NO: 74, the HCDR3 sequence of the heavy chain variable region is SEQ ID NO: 75, the light chain variable region sequence is SEQ ID NO: 80, wherein the LCDR1 sequence of the light chain variable region is SEQ ID NO: 76, the LCDR2 sequence of the light chain variable region is SEQ ID NO: 77, the LCDR3 sequence of the light chain variable region is SEQ ID NO: 79, the LCD
  • 18-8xBCMA-005 of the present invention can mediate T cell killing of target cell NCI-H929, with a maximum killing ratio of 80%.
  • the killing activity of 18-8xBCMA-005 is significantly weaker, with EC 50 of 9.6ng/mL and 780ng/mL, respectively.
  • 18-8xBCMA-005 can mediate the same maximum killing ratio (>90%) as sp34xBCMA-005.
  • CD69 is a marker for the early stage of T cell activation
  • CD25 is a marker for the late stage of T cell activation.
  • the degree of activation of T cells by CD3 dual antibodies can be obtained.
  • 18-8xBCMA-005 after 48 hours of co-culture with target cells, 18-8xBCMA-005 showed a dose-dependent upregulation of CD25 and CD69 expression on CD4+T cells.
  • 18-8xBCMA-005 induced an upregulation of CD25 and CD69 expression (see Figures 6E and 6F).
  • 18-8xBCMA-005 in the present invention compared with the sp34xBCMA-005 control molecule, 18-8xBCMA-005 in the present invention induced significantly lower levels of cytokine release, such as INF- ⁇ , TNF- ⁇ , IL-2, IL-4 and IL-10. While achieving the same maximum killing ratio, 18-8xBCMA-005 has a significantly lower level of cytokine release, which may be safer in clinical practice.
  • Example 10 In vitro binding activity of common light chain CD3xBCMA bispecific antibodies
  • CD3 humanized molecules (18, 18-5, 18-8, 18-11, 18-12, 18-13, 18-14) were matched to the heavy chains of anti-BCMA antibodies (BCMA-41) (whose sequence composition is shown in Table 8 below), and 7 anti-BCMA antibodies were obtained.
  • the CD3 humanized molecules and anti-BCMA antibodies were recombined in vitro to finally produce 7 common light chain CD3xBCMA bispecific antibodies. Since the light chains of 18 and 18-5 are exactly the same, the light chains of 18-11 and 18-13 are the same, and the light chains of 18-12 and 18-14 are the same, one of the molecules was selected to detect the binding activity of the common light chain CD3xBCMA bispecific antibody to the BCMA end.
  • a 96-well U-bottom cell culture plate 50 ⁇ l of NCI-H929 cells (total number of cells 1x10 5 ) highly expressing BCMA molecules were added, and then 50 ⁇ l of gradient diluted CD3xBCMA dual antibody (starting and ending concentration was 15 ⁇ g/ml, 5-fold dilution, a total of 8 concentration gradients) were added and incubated in the 96-well plate at 4°C for 30 minutes. After the incubation, the plate was washed twice with flow staining solution (BD, catalog number 554658) to remove antibodies that did not bind to cells.
  • flow staining solution BD, catalog number 554658
  • PBMC Human peripheral blood mononuclear cells
  • PBMNC50C Human peripheral blood mononuclear cells
  • human total T cells were isolated and purified using a total T cell purification kit (Miltenyi Biotec, catalog number 130-096-535). Then 50 ⁇ l of purified human T cells (1x10 5 cells per well) were added to a 96-well U-bottom cell culture plate, and then 50 ⁇ l of gradient diluted CD3xBCMA common light chain dual antibody (starting and ending concentration was 200nM, 3-fold dilution, a total of 8 concentration gradients) was added and incubated in a 96-well plate at 4°C for 30 minutes.
  • PBMC peripheral blood mononuclear cells
  • the plate was washed twice with flow staining solution (BD, catalog number 554658) to remove antibodies that were not bound to cells. Then 50 ⁇ l of PE-labeled Goat Anti-Human IgG secondary antibody (Biolegend, catalog number 398004, 1:1000 dilution) was added and incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow cytometry staining solution, the cells were resuspended in 150 ⁇ l staining solution, and the flow cytometry was detected. The data were analyzed using Flowjo software.
  • flow staining solution BD, catalog number 554658
  • Example 11 In vitro killing activity of common light chain CD3xBCMA bispecific antibodies
  • PBMC Human peripheral blood mononuclear cells
  • PBMNC50C Human peripheral blood mononuclear cells
  • Purified total human T cells 100,000 cells per well
  • NCI-H929 cells 20,000 cells per well
  • pre-labeled with cell-trace violet Invitrogen, catalog number C34557
  • gradiently diluted CD3xBCMA common light chain bispecific antibody starting concentration of 66.7 nM, 3-fold dilution, a total of 8 concentration gradients
  • the common light chain bispecific antibody can effectively stimulate CD4+ and CD8+ T cells to upregulate the membrane surface activation molecules CD25 and CD69, among which 18-8xBCMA has the weakest activation on T cells, and 18-14xBCMA has the strongest stimulation on T cells, but it is still weaker than the control molecule Teclistamab ( Figure 9B-E).
  • the above results show that the common light chain form CD3 bispecific antibody of the present invention has moderate activation activity on T cells, which can reduce the side effects of cytokine storm while ensuring efficient killing.
  • Example 12 In vivo anti-tumor activity of CD3xBCMA common light chain bispecific antibody
  • luciferase substrate D-luciferin potassium salt (Fubaike Bio, catalog number 12505) was injected into each mouse through the tail vein, and then the luciferase substrate signal was detected on a small animal in vivo imager (Guangzhou Bolu Teng Biotechnology Co., Ltd., model Aniview100) to determine the size of the tumor, and the mice were randomly divided into 5 groups according to the size of the tumor, with 5 animals in each group. They were:
  • 18-8xBCMA41, 18xBCMA41 and 18-14xBCMA41 common light chain bispecific antibodies were injected into the tail vein at a dose of 10 ⁇ g per mouse, and Teclistamab was used as the control antibody for this experiment. After that, the drug was administered once every 5 days for a total of 4 times.
  • the size of tumor growth was measured by tail vein injection of D-luciferin potassium salt (3 mg per animal), and then the luciferase substrate signal was detected on the AniView100 small animal in vivo imager. The larger the signal value, the faster the tumor growth.

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Abstract

L'invention concerne un anticorps se liant spécifiquement au CD3 humain ou à un fragment de liaison à l'antigène de celui-ci, et un anticorps multispécifique ciblant CD3 et d'autres antigènes (par exemple, des antigènes associés à une tumeur). L'invention concerne également des molécules d'acide nucléique codant pour les anticorps, des vecteurs et des cellules hôtes pour exprimer les anticorps, ainsi que des méthodes de traitement et de diagnostic et des utilisations des anticorps ou du fragment de liaison à l'antigène de ceux-ci.
PCT/CN2024/141038 2023-12-21 2024-12-20 Anticorps multispécifiques anti-cd3 et anti-cd3, et utilisation Pending WO2025131075A1 (fr)

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