WO2023125728A1 - 抗gprc5d抗体及其应用 - Google Patents

抗gprc5d抗体及其应用 Download PDF

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WO2023125728A1
WO2023125728A1 PCT/CN2022/143042 CN2022143042W WO2023125728A1 WO 2023125728 A1 WO2023125728 A1 WO 2023125728A1 CN 2022143042 W CN2022143042 W CN 2022143042W WO 2023125728 A1 WO2023125728 A1 WO 2023125728A1
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amino acid
acid sequence
seq
sequence shown
hcdr
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French (fr)
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宁金鹰
彭浩
郝锋
贺锋
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康源博创生物科技(北京)有限公司
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biomedicine and relates to a novel anti-GPRC5D antibody or a functional fragment thereof.
  • the present invention also relates to the application of the antibody or its functional fragment.
  • Group 5 D member of the G protein-coupled receptor family class C is a retinoic acid-induced 40kDa protein with 7 transmembrane segments and a short N-terminal extracellular domain, which is expressed in keratinized epithelial cells, It is expressed in pancreas, kidney, small intestine, spleen and testis.
  • Science Translational Medicine published a research article targeting GPRC5D CAR-T in the treatment of multiple myeloma, initially showing the potential of GPRC5D as a new target for the treatment of multiple myeloma. Tissue expression profiling studies found that GPRC5D was highly expressed specifically in plasma cells of multiple myeloma.
  • diseases that have been proposed to be associated with GPRC5D include chromosome 13Q14 deletion syndrome, among others.
  • the present invention provides an antibody or fragment thereof against G protein-coupled receptor family class C group 5 D member (GPRC5D), and based on the antibody or fragment, provides the use thereof. In addition, based on the antibody or its fragment, the present invention also provides a bispecific antibody against GPRC5D and CD3.
  • GPRC5D G protein-coupled receptor family class C group 5 D member
  • the present invention provides the following technical solutions.
  • the present invention provides an antibody or fragment thereof capable of specifically binding to GPRC5D, especially human GPRC5D.
  • the antibody or fragment thereof provided by the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) ) comprising a combination of CDRs (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3) selected from the group consisting of:
  • HCDR-1 comprising the amino acid sequence (SDYAWN) shown in SEQ ID NO:28
  • HCDR-2 comprising the amino acid sequence (YISYSGSATYNPSLKS) shown in SEQ ID NO:29, comprising HCDR-2 shown in SEQ ID NO:30 HCDR-3 of the amino acid sequence (GGIAGRGRWGAMDY)
  • LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31
  • LCDR-2 comprising the amino acid sequence (SASYRDS) shown in SEQ ID NO:32
  • LCDR-3 comprising the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO:33
  • SDYAWN amino acid sequence
  • YISYSGSATYNPSLKS amino acid sequence
  • HCDR-2 shown in SEQ ID NO:30
  • LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31
  • LCDR-2 comprising the amino acid sequence (SASYRDS) shown in
  • HCDR-1 comprising the amino acid sequence (SDYAWN) shown in SEQ ID NO:28, HCDR-2 comprising the amino acid sequence (YISYSGSATYSPSLKS) shown in SEQ ID NO:34, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:30 HCDR-3 of the amino acid sequence (GGIAGRGRWGAMDY); and, LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31, LCDR-2 comprising the amino acid sequence (SASYRDS) shown in SEQ ID NO:32, LCDR-3 comprising the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO:33;
  • HCDR-1 comprising the amino acid sequence (SYTIQ) shown in SEQ ID NO:35
  • HCDR-2 comprising the amino acid sequence (YIIPSSGYTNYNQKFKD) shown in SEQ ID NO:36
  • HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:37
  • HCDR-3 of the amino acid sequence (NYGNWGFTY);
  • LCDR-1 comprising the amino acid sequence (KASQNVGSAVT) shown in SEQ ID NO:38
  • LCDR-2 comprising the amino acid sequence (SASNRYT) shown in SEQ ID NO:39
  • LCDR-3 comprising the amino acid sequence (QQYSNYPLT) shown in SEQ ID NO:40;
  • HCDR-1 comprising the amino acid sequence (YYVMH) shown in SEQ ID NO:41, HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:43 HCDR-3 of the amino acid sequence (GGVRRYFDY); and, LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46;
  • HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:47
  • HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42
  • HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:43
  • HCDR-3 of the amino acid sequence (GGVRRYFDY) HCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44
  • LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45
  • LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46; and
  • HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:47, HCDR-2 comprising the amino acid sequence (YINPYNAGTKYNEKFKG) shown in SEQ ID NO:48, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:43 HCDR-3 of the amino acid sequence (GGVRRYFDY); and, LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46.
  • the provided combinations of light and heavy chain CDRs are all derived from the antibody or fragment thereof of the present invention, and based on the amino acid sequence of the variable region contained in a given antibody or fragment thereof, those skilled in the art can routinely determine that it contains CDRs.
  • the KABAT definition method is used to define the CDRs in the amino acid sequence of the variable region.
  • the heavy chain variable region comprises a sequence selected from the group consisting of: shown in SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID
  • the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 18.
  • said antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region comprising a combination of sequences selected from:
  • amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:5; and, the amino acid sequence shown in SEQ ID NO:6 or the amino acid sequence shown in SEQ ID NO:6 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 6;
  • amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 13; and, the amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 14;
  • amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:8 or the amino acid sequence shown in SEQ ID NO:8 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:8;
  • amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 16;
  • amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and, the amino acid sequence shown in SEQ ID NO:10 or the amino acid sequence shown in SEQ ID NO:10 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 10;
  • amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 an amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 12; and
  • At least 75% identity is an identity of any percentage figure between 75% and 100%, such as 75%, 80%, 85%, 90%, or even 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% identity.
  • the antibody of the present invention or a fragment thereof comprises at least a heavy chain variable region and a light chain variable region, both of which include the above-mentioned CDRs and a spaced framework region (framework region), and the arrangement of each domain is: FR1- CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • at most 25% difference in amino acid sequence resulting from said "at least 75% identity" may exist in any framework region in the heavy chain variable region or light chain variable region, or in the present invention In any domain or sequence other than the heavy chain variable region and the light chain variable region in the antibody or fragment thereof. The differences may result from amino acid deletions, additions or substitutions at any position.
  • the antibody or fragment thereof of the present invention is an anti-GPRC5D antibody or fragment thereof; preferably, the antibody is mouse anti, rabbit anti, monoclonal antibody, chimeric antibody, partially or fully humanized antibody etc., or the fragment is a half antibody or an antigen-binding fragment of an antibody or half antibody, such as scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv; more preferably Preferably, the antibody is IgG.
  • the invention provides isolated and structurally characterized human antibodies, eg, human monoclonal antibodies, that specifically bind GPRC5D (eg, human GPRC5D).
  • the antibody or fragment thereof provided by the invention also comprises a constant region, such as a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and/or light chain constant region (CL ); preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region and/or a kappa or lambda type of IgG, IgA, IgM, IgD or IgE Light chain constant region.
  • a constant region such as a human or murine constant region, preferably a human or murine heavy chain constant region (CH) and/or light chain constant region (CL ); preferably, the antibody or fragment thereof comprises a heavy chain and a light chain; more preferably, the antibody or fragment thereof comprises a heavy chain constant region and/or a kappa or lambda type of IgG, IgA, IgM, IgD or IgE Light chain constant region.
  • the antibody is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody.
  • the heavy chain constant region of the monoclonal antibody is of IgG1 or IgG4 subtype, and the light chain constant region is of ⁇ type.
  • the heavy chain constant region of the monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 23 or an amino acid sequence at least 75% identical to the amino acid sequence shown in SEQ ID NO: 23; the monoclonal antibody
  • the light chain constant region of the antibody comprises the amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence at least 75% identical to the amino acid sequence set forth in SEQ ID NO:24.
  • the anti-human GPRC5D antibody of the present invention is a monoclonal antibody.
  • the anti-human GPRC5D antibody provided by the present invention is an immunoglobulin, for example, the type of the immunoglobulin is human IgA, IgD, IgE, IgG or IgM. Further preferably, the antibody is of human IgG1 or IgG4 subtype.
  • the antibodies or fragments thereof provided by the invention can also be used to construct bispecific antibodies against GPRC5D ⁇ CD3. Accordingly, the present invention also provides the following bispecific antibody constructs, as described below.
  • the present invention provides a bispecific antibody construct comprising a first binding domain that binds to a group 5 D member of the G protein-coupled receptor family class C (GPRC5D), and binds to The second binding domain of CD3.
  • GPRC5D G protein-coupled receptor family class C
  • the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) comprising a combination of CDRs selected from the group (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3):
  • HCDR-1 comprising the amino acid sequence (SDYAWN) shown in SEQ ID NO:28
  • HCDR-2 comprising the amino acid sequence (YISYSGSATYNPSLKS) shown in SEQ ID NO:29, comprising HCDR-2 shown in SEQ ID NO:30 HCDR-3 of the amino acid sequence (GGIAGRGRWGAMDY)
  • LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31
  • LCDR-2 comprising the amino acid sequence (SASYRDS) shown in SEQ ID NO:32
  • LCDR-3 comprising the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO:33
  • SDYAWN amino acid sequence
  • YISYSGSATYNPSLKS amino acid sequence
  • HCDR-2 shown in SEQ ID NO:30
  • LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31
  • LCDR-2 comprising the amino acid sequence (SASYRDS) shown in
  • HCDR-1 comprising the amino acid sequence (SDYAWN) shown in SEQ ID NO:28, HCDR-2 comprising the amino acid sequence (YISYSGSATYSPSLKS) shown in SEQ ID NO:34, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:30 HCDR-3 of the amino acid sequence (GGIAGRGRWGAMDY); and, LCDR-1 comprising the amino acid sequence (KASQNVGTNVA) shown in SEQ ID NO:31, LCDR-2 comprising the amino acid sequence (SASYRDS) shown in SEQ ID NO:32, LCDR-3 comprising the amino acid sequence (QQYKSYPLT) shown in SEQ ID NO:33;
  • HCDR-1 comprising the amino acid sequence (SYTIQ) shown in SEQ ID NO:35
  • HCDR-2 comprising the amino acid sequence (YIIPSSGYTNYNQKFKD) shown in SEQ ID NO:36
  • HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:37
  • HCDR-3 of the amino acid sequence (NYGNWGFTY);
  • LCDR-1 comprising the amino acid sequence (KASQNVGSAVT) shown in SEQ ID NO:38
  • LCDR-2 comprising the amino acid sequence (SASNRYT) shown in SEQ ID NO:39
  • LCDR-3 comprising the amino acid sequence (QQYSNYPLT) shown in SEQ ID NO:40;
  • HCDR-1 comprising the amino acid sequence (YYVMH) shown in SEQ ID NO:41, HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:43 HCDR-3 of the amino acid sequence (GGVRRYFDY); and, LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46;
  • HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:47
  • HCDR-2 comprising the amino acid sequence (YINPYNDGTKYNEKFKG) shown in SEQ ID NO:42
  • HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:43
  • HCDR-3 of the amino acid sequence (GGVRRYFDY) HCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44
  • LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45
  • LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46; and
  • HCDR-1 comprising the amino acid sequence (YYVIH) shown in SEQ ID NO:47, HCDR-2 comprising the amino acid sequence (YINPYNAGTKYNEKFKG) shown in SEQ ID NO:48, HCDR-2 comprising the amino acid sequence shown in SEQ ID NO:43 HCDR-3 of the amino acid sequence (GGVRRYFDY); and, LCDR-1 comprising the amino acid sequence (RASQDIGSNLN) shown in SEQ ID NO:44, LCDR-2 comprising the amino acid sequence (ATSSLDS) shown in SEQ ID NO:45, LCDR-3 comprising the amino acid sequence (LQYATFPYT) shown in SEQ ID NO:46.
  • said heavy chain variable region comprises a sequence selected from the group consisting of the sequences shown in SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:7, SEQ ID NO :15, the amino acid sequence of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:11 or SEQ ID NO:19 or an amino acid sequence having at least 75% identity to said amino acid sequence; and/or, all
  • the light chain variable region comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 10, SEQ ID NO: 18 , the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 20, or an amino acid sequence having at least 75% identity to said amino acid sequence.
  • said first binding domain comprises a heavy chain variable region and a light chain variable region comprising a combination of sequences selected from:
  • amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:5; and, the amino acid sequence shown in SEQ ID NO:6 or the amino acid sequence shown in SEQ ID NO:6 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 6;
  • amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 13; and, the amino acid sequence shown in SEQ ID NO: 14 or the amino acid sequence shown in SEQ ID NO: 13 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 14;
  • amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:7; and, the amino acid sequence shown in SEQ ID NO:8 or the amino acid sequence shown in SEQ ID NO:8 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO:8;
  • amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 15; and, the amino acid sequence shown in SEQ ID NO: 16 or the amino acid sequence shown in SEQ ID NO: 16 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 16;
  • amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:9; and, the amino acid sequence shown in SEQ ID NO:10 or the amino acid sequence shown in SEQ ID NO:10 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 10;
  • amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO: 11; and, the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 12 An amino acid sequence having at least 75% identity to the amino acid sequence of SEQ ID NO: 12; or
  • the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) comprising a combination of CDRs selected from the group (HCDR-1, HCDR-2, HCDR-3; LCDR-1, LCDR-2, LCDR-3):
  • HCDR-1 comprising the amino acid sequence (TYAMN) shown in SEQ ID NO:49
  • HCDR-2 comprising the amino acid sequence (RIRSKYNNYATYYADSVKD) shown in SEQ ID NO:50, comprising the amino acid sequence shown in SEQ ID NO:51 ( HGNFGNSYVSYFAY) HCDR-3
  • LCDR-1 comprising the amino acid sequence (RSSTGAVTTSNYAN) shown in SEQ ID NO:52
  • LCDR-2 comprising the amino acid sequence (GTNKRAP) shown in SEQ ID NO:53, comprising the amino acid sequence shown in LCDR-3 of the amino acid sequence (ALWYSNLWV) of SEQ ID NO:54.
  • said heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence at least 75% identical to said amino acid sequence; and,
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 22 or an amino acid sequence having at least 75% identity to said amino acid sequence.
  • the bispecific antibody construct provided by the present invention comprises four polypeptide chains:
  • heavy chain 1 said heavy chain 1 comprising domains arranged according to VH-CH1-CH2-CH3 from the N-terminus to the C-terminus;
  • heavy chain 2 said heavy chain 2 comprising domains arranged according to VL-CH1-CH2-CH3 from N-terminus to C-terminus;
  • light chain 2 comprising domains arranged according to VH-CL from N-terminus to C-terminus;
  • VH and VL contained in the heavy chain 1 and light chain 1 are paired to form the first binding domain that binds GPRC5D; the VL and VH contained in the heavy chain 2 and light chain 2 are paired to form the second binding domain that binds CD3. binding domain.
  • the schematic diagram of the structure of the bispecific antibody construct provided by the present invention is shown in FIG. 1 .
  • the CH1-CH2-CH3 domain in the heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO: 25 or the amino acid sequence An amino acid sequence having at least 75% identity;
  • the CH1-CH2-CH3 domain in heavy chain 2 comprising the amino acid sequence shown in SEQ ID NO: 26 or an amino acid sequence having at least 75% identity to said amino acid sequence;
  • the CL domain in chain 1 comprises the amino acid sequence shown in SEQ ID NO:24 or an amino acid sequence at least 75% identical to said amino acid sequence;
  • the CL domain in light chain 2 comprises the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 75% identity to said amino acid sequence.
  • the present invention also provides a nucleic acid molecule, which comprises a heavy chain CDR, a light chain CDR, a light chain variable encoding an antibody or a fragment thereof provided in the present invention or a bispecific antibody construct provided in the present invention. region, heavy chain variable region, nucleotide sequence of heavy chain or light chain.
  • the nucleic acid molecule of the present invention can be cloned into a vector, and then transformed or transfected into a host cell.
  • the invention also provides a vector comprising a nucleic acid molecule of the invention.
  • the vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.
  • the vectors or nucleic acid molecules of the invention can be used to transform or transfect host cells. Therefore, in a further aspect, the present invention provides a host cell comprising, or transformed or transfected with, the nucleic acid molecule and/or vector of the present invention.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.
  • Antibodies or fragments thereof and bispecific antibody constructs provided by the present invention can be obtained by any method known in the art.
  • host cells provided herein are cultured under conditions that allow the host cells to express the heavy and light chains of the antibody.
  • the method further comprises the step of recovering the antibodies produced.
  • compositions such as a pharmaceutical composition, which comprises the antibody or fragment thereof, bispecific antibody construct, nucleic acid molecule, vector and/or host cells, and optional pharmaceutically acceptable excipients.
  • the present invention also provides the use of the antibody or its fragment, bispecific antibody construct, nucleic acid molecule, vector, host cell and/or composition in the preparation of a medicament for treating a disease
  • the disease is tumor or cancer.
  • the disease is a tumor or cancer associated with GPRC5D expression (eg high expression), such as lymphoma or myeloma, especially multiple myeloma.
  • the present invention also provides a method for treating diseases, the method comprising administering the antibody or fragment thereof, bispecific antibody construct, nucleic acid molecule, vector, host cell of the present invention to a subject in need thereof And/or the composition, the disease is tumor or cancer.
  • the disease is a tumor or cancer associated with GPRC5D expression (eg high expression), such as lymphoma or myeloma; the disease is especially multiple myeloma.
  • the subject is a mammal; more preferably, the subject is a human.
  • the present invention also provides the use of the antibody or its fragment, bispecific antibody construct, nucleic acid molecule, vector, host cell and/or composition in the preparation of reagents for diagnosing diseases or detecting the presence of the antigen GPRC5D , the disease is tumor or cancer.
  • the disease is a tumor or cancer associated with GPRC5D expression (eg high expression), such as lymphoma or myeloma, especially multiple myeloma.
  • the present invention provides a kit comprising an antibody or fragment thereof, a bispecific antibody construct, a nucleic acid molecule, a vector, a host cell and/or a composition of the present invention.
  • the kit can be used in the above-mentioned treatment, or in diagnosis or antigen detection.
  • the kits also contain other reagents for therapy, diagnosis or detection.
  • the kit is a kit for detecting the presence of GPRC5D in any biological sample using ELISA.
  • Figure 1 is a schematic diagram of the structure of an anti-human GPRC5D ⁇ CD3 bispecific antibody.
  • Figure 2 shows the FACS detection results of hybridoma cell supernatants binding to different cells.
  • Figure 3 shows the FACS detection results of hybridoma cell supernatants binding to different cells.
  • Figure 4 shows the FACS detection results of hybridoma cell supernatant combined with different cells, wherein 4-1: MM1R cells; 4-2: Nalm6 cells; 4-3: OPM2 cells; 4-4: NCI-H929 cells.
  • Figure 5 shows the FACS detection results of humanized antibody binding to human GPRC5D expressing cells, wherein 5-1: 9D7 (hz); 5-2: 26D1 (hz); 5-3: 24F5 (hz); 5-4: 6E97(hz).
  • Figure 6 shows the FACS detection results of humanized antibody binding to monkey GPRC5D expressing cells, wherein 6-1: 9D7 (hz); 6-2: 26D1 (hz); 6-3: 24F5 (hz); 6-4: 6E97(hz).
  • Figure 7 shows the FACS detection results of humanized antibody binding to different tumor cell lines, wherein 7-1: MM.1R cells; 7-2: MOLP8 cells.
  • Figure 8 shows the affinity test results of humanized antibody and GPRC5D, in which 8-1: GPRC5D ⁇ CD3 JNJ; 8-2: 9D7(hz); 8-3: 26D1(hz); 8-4: 24F5(hz) ;8-5: 6E97 (hz).
  • Figure 9 shows the detection results of humanized antibody-mediated ADCC, wherein 9-1: wild-type NCI-H929; 9-2: GPRC5D knockout NCI-H929.
  • FIG. 10 shows the detection results of bispecific antibody-mediated T cell killing, wherein 10-1: co-culture experiment 1; 10-2: co-culture experiment 2; 10-3: co-culture experiment 3.
  • Human GPRC5D protein Genbank accession number NM_018654;
  • Monkey GPRC5D protein Genbank accession number XM_005570193.2.
  • Embodiment 1 Preparation of hybridoma cells and acquisition of antibody protein
  • Cells expressing human GPRC5D (Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd. (kyinno company), Cat. No. KC-1583) were prepared in PBS containing 2% FBS to a cell concentration of 1 ⁇ 10 7 cells/ml Suspension.
  • the cell suspension was used as the immunogen to immunize mice, 5 mice were immunized subcutaneously, and 5 mice were immunized intramuscularly.
  • the adjuvant was quick antibody 5W water-soluble adjuvant.
  • the titer was measured 2 weeks after the booster immunization, and the titer was selected to be high.
  • the 2 mice were subjected to immune shock, serum was collected 3 days later, and spleen was obtained after dissection.
  • the isolated splenocytes were fused with the cultured myeloma cells in a 96-well plate, and selective medium was added for screening, the medium was changed after 7 days, and cell-based ELISA was detected after 10 days.
  • 293T cells expressing human GPRC5D (kyinno company, product number KC-1723) were used for ELISA detection, and cells with an OD value greater than 10 times that of the negative control (serum of normal mice) were selected for flow cytometry detection.
  • 293T cells expressing human GPRC5D (kyinno company, Cat. No. KC-1723) were used.
  • the resulting culture supernatant of the hybridoma cells was collected, centrifuged, concentrated, and then purified using a protein G affinity column. Then, the purified sample was diluted 10 times with 20mM citric acid + 100mM NaCl, pH 5.5 buffer solution, concentrated to an appropriate volume in an ultrafiltration concentrator tube, and dispensed into 1.5mL EP tubes, 200 ⁇ L/tube, -80°C save. At the same time, some samples were taken out for SDS-PAG and FACS identification to verify the purity and activity of the protein.
  • BXPC-3 cells expressing human GPRC5D (kyinno company, product number KC-1717) in PBS containing 2% FBS to a cell suspension with a cell concentration of 1 ⁇ 105 cells/ml, and add 96 cells at 100 ⁇ l/well Incubate overnight at 37°C. Discard the liquid in the wells, wash with washing solution 3 times at the same time, add 80 ⁇ l of 4% tissue fixation solution (Beyotime, product number P0099) to each well, fix at room temperature for 15 min, wash twice with PBS, dry in the air, add 250 ⁇ l to each well Blocking solution containing 2% BSA, incubated at 37°C for 1h, and washed three times with PBS.
  • tissue fixation solution (Beyotime, product number P0099)
  • the anti-human GPRC5D ⁇ CD3 bispecific antibody was constructed by using the GPRC5D-targeting domain and the CD3-targeting domain of Johnson & Johnson's Talquetamab (JNJ-64407564).
  • the structural diagram of the bispecific antibody is shown in Figure 1, in which the structural domains contained in each chain are as follows from the N-terminal to the C-terminal: heavy chain 1: VH-CH1-CH2-CH3
  • Heavy chain 2 VL-CH1-CH2-CH3
  • the obtained bispecific antibody was named "GPRC5D ⁇ CD3 JNJ”.
  • Embodiment 4 Hybridoma cell culture supernatant combined with human GPRC5D FACS detection
  • the 293T cells expressing human GPRC5D as above were prepared in PBS containing 2% FBS to a cell suspension with a cell concentration of 1 ⁇ 10 5 cells/ml.
  • sample tube Put 50 ⁇ l of cell suspension into each flow tube (sample tube), then add 50 ⁇ l of the culture supernatant of hybridoma cells to be tested and the anti-human GPRC5D ⁇ CD3 bispecific antibody GPRC5D ⁇ CD3 JNJ as a positive control antibody, Incubate at 4°C for 60 minutes. Add 1ml of flow buffer to each flow tube, centrifuge at 1200rpm for 5 minutes, discard the supernatant, and repeat the washing three times. At the same time, control tube 1 (no culture supernatant and secondary antibody below, only cell suspension) and control tube 2 (no culture supernatant, only cell suspension and secondary antibody below) were set.
  • Embodiment 5 Hybridoma cell supernatant combined with monkey GPRC5D FACS detection
  • Example 4 According to the operation described in Example 4, the cells expressing human GPRC5D were replaced with 293T cells expressing monkey GPRC5D (kyinno company, product number KC-1586), and the combination of the culture supernatant of hybridoma cells of the present invention and monkey GPRC5D was detected.
  • monkey GPRC5D kyinno company, product number KC-1586
  • the detection results of FACS binding are shown in Table 3-1 to Table 3-3 and 3-1 to 3-3 in Figure 3; the positive control antibody is GPRC5D ⁇ CD3 JNJ.
  • human GPRC5D expressing cells were replaced with other cells: NALM6 (kyinno company, product number KC-0626), NCI-H929 (kyinno company, product number KC-0629), MM1R (kyinno company, product number KC -0619), OPM2 (kyinno company, product number KC-0631) cells, the combination of the culture supernatant of hybridoma cells of the present invention and human GPRC5D was detected.
  • NALM6 kyinno company, product number KC-0626
  • NCI-H929 kyinno company, product number KC-0629
  • MM1R kyinno company, product number KC -0619
  • OPM2 kyinno company, product number KC-0631
  • the detection results of FACS binding are shown in Table 4 and 4-1 to 4-4 in Figure 4; the positive control antibody is GPRC5D ⁇ CD3 JNJ.
  • RNA from the monoclonal cells extract RNA from the monoclonal cells, reverse transcribe into cDNA, amplify the cDNA, recover from the gel, and connect it to the sequencing vector for sequencing analysis.
  • sequence of an exemplary murine antibody heavy and light chain variable region obtained from a hybridoma cell is as follows, where CDRs (defined according to the KABAT method) are shown in bold and underlined.
  • A1 antibody human anti-9D7
  • human germline sequences were selected as templates for the heavy chain and light chain: IGHV4-39 and IGKV1-39.
  • Homology modeling was performed on the A1 antibody, and the structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the A1 antibody was finally obtained.
  • the sequence of the humanized antibody of the antibody is as follows:
  • A2 antibody human anti-26D1
  • human germline sequences were selected as templates for the heavy chain and light chain: IGHV1-46 and IGKV1-5.
  • Homology modeling was performed on the A2 antibody, and the structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the A2 antibody was finally obtained.
  • the VH except for the CDR region and 37I, 38K, 48I, 67R, 68A, 70L, 72A, 74K, 76S, 77N, 79A, 98N Except for original mouse amino acids, all other mouse amino acids were replaced with corresponding IGHV1-46 template human amino acids.
  • the amino acids of the original mouse were retained, and all other mouse amino acids were replaced with the corresponding IGKV1 -5 template human amino acids.
  • the obtained humanized sequence is as follows (wherein the heavy chain and light chain CDRs are shown in bold and underlined, which are obtained according to the KABAT definition method):
  • the sequence of the humanized antibody of the antibody is as follows:
  • A3 antibody human anti-24F5
  • human germline sequences were selected as templates for the heavy chain and light chain: IGHV1-46 and IGKV1-15.
  • Homology modeling was performed on the A3 antibody, and the structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the A3 antibody was finally obtained.
  • the amino acids of the original mouse were retained. All murine amino acids were replaced with corresponding IGKV1-15 template human amino acids.
  • the obtained humanized sequence is as follows (wherein the heavy chain and light chain CDRs are shown in bold and underlined, which are obtained according to the KABAT definition method):
  • the sequence of the humanized antibody of the antibody is as follows:
  • A4 antibody human anti-6E97
  • human germline sequences were respectively selected as templates for the heavy chain and light chain: IGHV1-46 and IGKV1-15.
  • Homology modeling was performed on the A4 antibody, and structural simulation of the Fab region was performed. After homology modeling calculations, the predicted Fab structure of the A4 antibody was finally obtained.
  • the VL retains the original mouse amino acids except for the CDR region and 4L, 36L, 42G, 43T, 44I, 46R, 66R, 69S, and 71Y , all other mouse amino acids were replaced with corresponding IGKV1-15 template human amino acids.
  • the obtained humanized sequence is as follows (wherein the heavy chain and light chain CDRs are shown in bold and underlined, which are obtained according to the KABAT definition method):
  • the sequence of the humanized antibody of the antibody is as follows:
  • sequence shown in SEQ ID NO:23 was used as the heavy chain constant region
  • sequence shown in SEQ ID NO:24 was used as the light chain constant region.
  • primers were redesigned and corresponding chimeras were synthesized.
  • the genes of synthetic antibody and humanized antibody were linked into eukaryotic expression vectors, transformed into DH5alpha competent cells, cultivated overnight in a constant temperature incubator at 37°C, and single-clonal strains were sequenced and identified. Select the strain with the correct sequence, shake the bacteria, extract the plasmid, transfect the mammalian expression cell 293F, place it in an incubator at 37°C and 5% CO2, and culture it for 7 days.
  • chimeric antibody is named "mouse antibody abbreviation (chi)"
  • humanized antibody is named "mouse antibody abbreviation (hz)”.
  • the experimental process refers to Example 4, using 293T cells expressing human GPRC5D (kyinno company, product number KC-1584), adding the antibody to be tested, the positive control antibody GPRC5D ⁇ CD3 JNJ or the negative control antibody (hIgG1), the concentration is shown in Figure 4.
  • set control tube 1 no antibody and secondary antibody, only cell suspension
  • control tube 2 no antibody, only cell suspension and secondary antibody
  • sample positive control antibody 9D7(hz) EC50( ⁇ g/mL) 0.4537 0.2830 MFI.Max 6590 8938 the the sample positive control antibody 26D1(hz) EC50( ⁇ g/mL) 0.3552 0.228
  • MFI.Max 8650 9560 the the sample positive control antibody 24F5(hz) EC50( ⁇ g/mL) 0.6986 0.6995 MFI.Max 9351 8908 the the the sample positive control antibody 6E97(hz) EC50( ⁇ g/mL) 0.3571 0.3508 MFI.Max 8941 14840
  • the experimental process refers to Example 4, using 293T cells expressing monkey (cyno) GPRC5D (kyinno company, product number KC-1586), adding the antibody to be tested, positive control antibody GPRC5D ⁇ CD3 JNJ or negative control antibody (hIgG1), the concentration is shown in Fig. 6. At the same time, set control tube 1 (no antibody and secondary antibody, only cell suspension) and control tube 2 (no antibody, only cell suspension and secondary antibody).
  • sample positive control antibody 9D7(hz) EC50( ⁇ g/mL) 13.96 0.3779 MFI.Max 656 4265 the the sample positive control antibody 26D1(hz) EC50( ⁇ g/mL) N/A N/A MFI.Max 334 12.3 the the sample positive control antibody 24F5(hz) EC50( ⁇ g/mL) 4.389 0.1666 MFI.Max 2110 3558 the the the sample positive control antibody 6E97(hz)
  • the experimental process refers to Example 4, using cells expressing other members of the 5 groups of G protein-coupled receptor family C: 293T-GPRC5A cells expressing human GPRC5A (kyinno company, Cat. No. KC-1888), 293T cells expressing human GPRC5B Cell 293T-GPRC5B (kyinno company, catalog number KC-1854), 293T cell 293T-GPRC5C expressing human GPRC5C (kyinno company, cat. JNJ or negative control antibody (hIgG1).
  • set control tube 1 no antibody and secondary antibody, only cell suspension
  • control tube 2 no antibody, only cell suspension and secondary antibody.
  • the experimental process refers to Example 4, and the cells used are replaced with multiple myeloma cells MM.1R (kyinno company, product number KC-0619) and MOLP8 (kyinno company, product number KC-0622), and the antibody to be tested and the positive control antibody GPRC5D are added ⁇ CD3 JNJ or negative control antibody (hIgG1). At the same time, set control tube 1 (no antibody and secondary antibody, only cell suspension) and control tube 2 (no antibody, only cell suspension and secondary antibody).
  • the antibody to be tested was prepared into a solution with a concentration of 10 ⁇ g/mL using PBS buffer, and the AHC chip was used to capture the antibody according to the instructions of Fortebio.
  • the human GPRC5D protein prepared in advance Kerijia Biology, Cat. No. GPR-HM05P
  • the specific conditions are: flow rate 30 ⁇ l/min; antigen-antibody binding time 200 seconds, dissociation time 500 seconds.
  • the measured results were fitted with the instrument-specific supporting software to analyze the affinity of the antibody to the antigen. The results are shown in Table 9 and 8-1 to 8-5 in Fig. 8.
  • the heavy chain variable region sequence SEQ ID NO.10 and the light chain variable region sequence SEQ ID NO.5 of the murine antibody were obtained from the patent US10066015B2.
  • the antibody sequence was humanized, and the obtained humanized sequence was as follows (wherein the heavy chain and light chain CDRs are shown in bold and underlined, which are obtained according to the KABAT definition method):
  • VH/HCDR-1/HCDR-2/HCDR-3 SEQ ID NO:21/49/50/51)
  • VL/LCDR-1/LCDR-2/LCDR-3 SEQ ID NO:22/52/53/54)
  • Example 8 an antibody comprising the above-mentioned humanized sequence was obtained, named SP34V1 (hz); similarly, a chimeric antibody was obtained by using the heavy and light chain variable region sequences of the above-mentioned murine antibody in the patent US10066015B2, Named SP34(chi).
  • the humanized antibody and chimeric antibody were prepared into a solution with a concentration of 10 ⁇ g/mL using PBS buffer, and the antibody was captured using an AHC chip according to the instructions of Fortebio.
  • the human CD3 protein prepared in advance Kerijia Biology, Cat. No. GPR-HM05P
  • the specific conditions are: flow rate 30 ⁇ l/min; antigen-antibody binding time 200 seconds, dissociation time 500 seconds.
  • the measured results were fitted with the instrument-specific supporting software to analyze the affinity of the antibody to the antigen. The results are shown in Table 11.
  • the bispecific antibody of the present invention was constructed, the only difference being that the VH in the heavy chain 1 and the VL in the light chain 1 were replaced by the humanized antibodies 9D7(hz) and 26D1(hz) of the present invention, respectively. ), 24F5 (hz) and 6E97 (hz) VH and VL, the VL in the heavy chain 2 and the VH in the light chain 2 were replaced by the humanized sequence against CD3 obtained in Example 15.
  • the anti-human GPRC5D ⁇ CD3 bispecific antibodies comprising the VH and VL of the humanized antibodies 9D7 (hz), 26D1 (hz), 24F5 (hz) and 6E97 (hz) of the present invention and the above sequence respectively were named " GPRC5D ⁇ CD3 9D7(hz)”, “GPRC5D ⁇ CD3 26D1(hz)”, “GPRC5D ⁇ CD3 24F5(hz)”, “GPRC5D ⁇ CD3 26D1(hz)”.
  • PBMC and CD3/CD28 magnetic beads were mixed in RPMI1640 containing 10 ng/ml IL-2 and 10% FBS at a quantity ratio of 2:1, cultured and expanded for 7 days to activate T cells.
  • Co-culture experiment 1 293T LDHA-Hibit-GPRC5D cells (kyinno company, product number KC-2151) were plated (96-well plate) one day in advance, and then 10,000 of the above T cells were added to each well, and positive control antibodies of different concentrations were added GPRC5D ⁇ CD3 JNJ and the bispecific antibody of the present invention (1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001, 0.0000001 ⁇ g/ml) were co-cultured in RPMI1640 containing 10ng/ml IL-2 and 10% FBS for 18 Hours. use HiBiT Extracellular Reagent (Promega, Cat. No.
  • N2420 detects LDHA-HIBIT released from dead cells in the supernatant, thereby detecting the ratio of killer cells.
  • two groups of controls were set up for each of the two antibodies: 293T LDHA-Hibit-GPRC5D cells and antibodies were added, but T cells were not added; 293T LDHA-Hibit cells (kyinno company), T cells and antibodies were added to exclude non-antibody mediated killing and antibody-mediated non-T cell killing. The results are shown in 10-1 in Fig. 10.
  • Co-culture experiment 2 NCI-H929 (expressing GPRC5D) and K562 cells (not expressing GPRC5D) were stained with CFSE and named CFSE-H929 and CFSE-K562, respectively.
  • Co-culture experiment 3 wild-type NCI-H929 (kyinno company, product number KC-0629) and GPRC5D knockout NCI-H929 cells (kyinno company, product number KC-2203) were stained with CFSE, named CFSE-H929 and CFSE respectively -H929GPRC5DKO.

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Abstract

提供一种抗G蛋白偶联受体家族C类的5组D成员(GPRC5D)的抗体或其片段。还提供所述抗体或其片段作为活性成分***或癌症的应用。此外，还提供一种包含所述抗体或其片段且针对GPRC5D和 CD3的双特异性抗体。

Description

抗GPRC5D抗体及其应用
相关申请的交叉引用
本专利申请要求于2021年12月31日提交的申请号为CN202111663745.2的中国发明专利申请的优先权权益，在此将其全部内容引入作为参考。
技术领域
本发明属于生物医药领域，涉及一种新的抗GPRC5D抗体或其功能性片段。本发明还涉及所述抗体或其功能性片段的应用。
背景技术
G蛋白偶联受体家族C类的5组D成员(GPRC5D)是一种维甲酸诱导的40kDa蛋白，具有7个跨膜段和1个短的N端胞外区，在角化上皮细胞、胰腺、肾脏、小肠、脾脏和睾丸等中均有表达。2019年3月，Science Translational Medicine期刊发表了靶向GPRC5D CAR-T治疗多发性骨髓瘤的研究文章，初步显示出了GPRC5D作为一个全新的靶点用于治疗多发性骨髓瘤的潜力。组织表达谱研究发现GPRC5D特异性地在多发性骨髓瘤的浆细胞高表达。另外，已提出与GPRC5D相关的疾病包括染色体13Q14缺失综合征等。
鉴于GPRC5D在包括多发性骨髓瘤的疾病中的显著作用，需要识别GPRC5D的更多抗体。
发明内容
针对上述技术问题，本发明提供一种抗G蛋白偶联受体家族C类的5组D成员(GPRC5D)的抗体或其片段，并基于该抗体或其片段，提供其用途。此外，基于该抗体或其片段，本发明还提供一种针对GPRC5D和CD3的双特异性抗体。
本发明提供以下技术方案。
一方面，本发明提供一种抗体或其片段，所述抗体或其片段能够特异性结合GPRC5D，特别是人GPRC5D。
具体而言，本发明提供的抗体或其片段包含重链可变区(VH)和轻链 可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR-1、HCDR-2、HCDR-3；LCDR-1、LCDR-2、LCDR-3)：
(1)包含示于SEQ ID NO:28的氨基酸序列(SDYAWN)的HCDR-1、包含示于SEQ ID NO:29的氨基酸序列(YISYSGSATYNPSLKS)的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列(GGIAGRGRWGAMDY)的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列(KASQNVGTNVA)的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列(SASYRDS)的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列(QQYKSYPLT)的LCDR-3；
(2)包含示于SEQ ID NO:28的氨基酸序列(SDYAWN)的HCDR-1、包含示于SEQ ID NO:34的氨基酸序列(YISYSGSATYSPSLKS)的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列(GGIAGRGRWGAMDY)的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列(KASQNVGTNVA)的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列(SASYRDS)的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列(QQYKSYPLT)的LCDR-3；
(3)包含示于SEQ ID NO:35的氨基酸序列(SYTIQ)的HCDR-1、包含示于SEQ ID NO:36的氨基酸序列(YIIPSSGYTNYNQKFKD)的HCDR-2、包含示于SEQ ID NO:37的氨基酸序列(NYGNWGFTY)的HCDR-3；和，包含示于SEQ ID NO:38的氨基酸序列(KASQNVGSAVT)的LCDR-1、包含示于SEQ ID NO:39的氨基酸序列(SASNRYT)的LCDR-2、包含示于SEQ ID NO:40的氨基酸序列(QQYSNYPLT)的LCDR-3；
(4)包含示于SEQ ID NO:41的氨基酸序列(YYVMH)的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列(YINPYNDGTKYNEKFKG)的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列(GGVRRYFDY)的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列(RASQDIGSNLN)的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列(ATSSLDS)的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列(LQYATFPYT)的LCDR-3；
(5)包含示于SEQ ID NO:47的氨基酸序列(YYVIH)的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列(YINPYNDGTKYNEKFKG)的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列(GGVRRYFDY)的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列(RASQDIGSNLN)的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列(ATSSLDS)的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列(LQYATFPYT)的LCDR-3；和
(6)包含示于SEQ ID NO:47的氨基酸序列(YYVIH)的HCDR-1、包含示于SEQ ID NO:48的氨基酸序列(YINPYNAGTKYNEKFKG)的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列(GGVRRYFDY)的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列(RASQDIGSNLN)的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列(ATSSLDS)的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列(LQYATFPYT)的LCDR-3。
在本发明的上下文中，所提供的轻重链CDR的组合均来自本发明的抗体或其片段，基于给定抗体或其片段包含的可变区氨基酸序列，本领域技术人员可以常规地确定其中包含的CDR。例如，根据本发明的具体实施方式，采用KABAT定义方法限定可变区氨基酸序列中的CDR。
在本发明提供的抗体或其片段中，优选地，所述重链可变区包含选自以下的序列：示于SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:11或SEQ ID NO:19的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述轻链可变区包含选自以下的序列：示于SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:12或SEQ ID NO:20的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
更优选地，所述抗体或其片段包含的重链可变区和轻链可变区包含选自以下的序列组合：
(1)示于SEQ ID NO:5的氨基酸序列或与示于SEQ ID NO:5的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:6的氨基酸序列或与示于SEQ ID NO:6的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)示于SEQ ID NO:13的氨基酸序列或与示于SEQ ID NO:13的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:14的氨基酸序列或与示于SEQ ID NO:14的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)示于SEQ ID NO:7的氨基酸序列或与示于SEQ ID NO:7的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:8的氨基酸序列或与示于SEQ ID NO:8的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)示于SEQ ID NO:15的氨基酸序列或与示于SEQ ID NO:15的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:16的氨基酸序列或与示于SEQ ID NO:16的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)示于SEQ ID NO:9的氨基酸序列或与示于SEQ ID NO:9的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:10的氨基酸序列或与示于SEQ ID NO:10的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)示于SEQ ID NO:17的氨基酸序列或与示于SEQ ID NO:17的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:18的氨基酸序列或与示于SEQ ID NO:18的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)示于SEQ ID NO:11的氨基酸序列或与示于SEQ ID NO:11的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:12的氨基酸序列或与示于SEQ ID NO:12的氨基酸序列具有至少75％同一性的氨基酸序列；和
(8)示于SEQ ID NO:19的氨基酸序列或与示于SEQ ID NO:19的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:20的氨基酸序列或与示于SEQ ID NO:20的氨基酸序列具有至少75％同一性的氨基酸序列。
在本发明的上下文中，“至少75％同一性”为75％至100％之间的任何百分比数字的同一性，例如75％、80％、85％、90％，甚至91％、92％、93％、94％、95％、96％、97％、98％、99％或甚至100％同一性。
特别地，本发明的抗体或其片段至少包含重链可变区和轻链可变区，二者均包括上述CDR以及间隔的框架区(framework region)，各个结构域的排列方式为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。进一步可选地，所述“至少75％同一性”导致的氨基酸序列上的至多25％差异可存在于重链可变区或轻链可变区中的任意框架区中，或者存在于本发明的抗体或其片段中重链可变区和轻链可变区以外的任意结构域或序列中。所述差异可以由任何位置的氨基酸缺失、添加或置换产生。
就抗原而言，本发明的抗体或其片段为抗GPRC5D的抗体或其片段；优选地，所述抗体为鼠抗、兔抗、单克隆抗体、嵌合抗体、部分或全部人源 化的抗体等任意形式，或者，所述片段为半抗体或者抗体或半抗体的抗原结合片段，例如scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；更优选地，所述抗体为IgG。根据本发明的具体实施方式，本发明提供特异性地结合GPRC5D(例如人GPRC5D)的经分离和结构表征的人抗体，例如人单克隆抗体。
除了可变区之外，本发明提供的抗体或其片段还包含恒定区，例如人或鼠的恒定区，优选包含人或鼠的重链恒定区(CH)和/或轻链恒定区(CL)；优选地，所述抗体或其片段包含重链和轻链；更优选地，所述抗体或其片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
根据本发明的具体实施方式，所述抗体为单克隆抗体，优选为鼠、嵌合或人源化的单克隆抗体。优选地，所述单克隆抗体的重链恒定区为IgG1或IgG4亚型，轻链恒定区为κ型。优选地，所述单克隆抗体的重链恒定区包含示于SEQ ID NO:23的氨基酸序列或者与示于SEQ ID NO:23的氨基酸序列具有至少75％同一性的氨基酸序列；所述单克隆抗体的轻链恒定区包含示于SEQ ID NO:24的氨基酸序列或者与示于SEQ ID NO:24的氨基酸序列具有至少75％同一性的氨基酸序列。
根据本发明的具体实施方式，本发明的抗人GPRC5D抗体为单克隆抗体。优选地，本发明提供的抗人GPRC5D抗体为免疫球蛋白，例如，所述免疫球蛋白的类型为人IgA、IgD、IgE、IgG或IgM。进一步优选地，所述抗体为人IgG1或IgG4亚型。
本发明提供的抗体或其片段还可用于构建抗GPRC5D×CD3的双特异性抗体。因此，如下文所述，本发明还提供如下双特异性抗体构建体。
本发明提供一种双特异性抗体构建体，所述抗体构建体包含结合G蛋白偶联受体家族C类的5组D成员(GPRC5D)的第一结合结构域，和结合T细胞表面上的CD3的第二结合结构域。
在本发明提供的双特异性抗体构建体中，所述第一结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR-1、HCDR-2、HCDR-3；LCDR-1、LCDR-2、LCDR-3)：
(1)包含示于SEQ ID NO:28的氨基酸序列(SDYAWN)的HCDR-1、包含示于SEQ ID NO:29的氨基酸序列(YISYSGSATYNPSLKS)的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列(GGIAGRGRWGAMDY)的 HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列(KASQNVGTNVA)的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列(SASYRDS)的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列(QQYKSYPLT)的LCDR-3；
(2)包含示于SEQ ID NO:28的氨基酸序列(SDYAWN)的HCDR-1、包含示于SEQ ID NO:34的氨基酸序列(YISYSGSATYSPSLKS)的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列(GGIAGRGRWGAMDY)的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列(KASQNVGTNVA)的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列(SASYRDS)的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列(QQYKSYPLT)的LCDR-3；
(3)包含示于SEQ ID NO:35的氨基酸序列(SYTIQ)的HCDR-1、包含示于SEQ ID NO:36的氨基酸序列(YIIPSSGYTNYNQKFKD)的HCDR-2、包含示于SEQ ID NO:37的氨基酸序列(NYGNWGFTY)的HCDR-3；和，包含示于SEQ ID NO:38的氨基酸序列(KASQNVGSAVT)的LCDR-1、包含示于SEQ ID NO:39的氨基酸序列(SASNRYT)的LCDR-2、包含示于SEQ ID NO:40的氨基酸序列(QQYSNYPLT)的LCDR-3；
(4)包含示于SEQ ID NO:41的氨基酸序列(YYVMH)的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列(YINPYNDGTKYNEKFKG)的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列(GGVRRYFDY)的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列(RASQDIGSNLN)的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列(ATSSLDS)的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列(LQYATFPYT)的LCDR-3；
(5)包含示于SEQ ID NO:47的氨基酸序列(YYVIH)的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列(YINPYNDGTKYNEKFKG)的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列(GGVRRYFDY)的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列(RASQDIGSNLN)的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列(ATSSLDS)的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列(LQYATFPYT)的LCDR-3；和
(6)包含示于SEQ ID NO:47的氨基酸序列(YYVIH)的HCDR-1、包含示于SEQ ID NO:48的氨基酸序列(YINPYNAGTKYNEKFKG)的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列(GGVRRYFDY)的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列(RASQDIGSNLN)的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列(ATSSLDS)的LCDR-2、 包含示于SEQ ID NO:46的氨基酸序列(LQYATFPYT)的LCDR-3。
优选地，在所述第一结合结构域中，所述重链可变区包含选自以下的序列：示于SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:11或SEQ ID NO:19的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述轻链可变区包含选自以下的序列：示于SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:12或SEQ ID NO:20的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
更优选地，所述第一结合结构域包含的重链可变区和轻链可变区包含选自以下的序列组合：
(1)示于SEQ ID NO:5的氨基酸序列或与示于SEQ ID NO:5的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:6的氨基酸序列或与示于SEQ ID NO:6的氨基酸序列具有至少75％同一性的氨基酸序列；
(2)示于SEQ ID NO:13的氨基酸序列或与示于SEQ ID NO:13的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:14的氨基酸序列或与示于SEQ ID NO:14的氨基酸序列具有至少75％同一性的氨基酸序列；
(3)示于SEQ ID NO:7的氨基酸序列或与示于SEQ ID NO:7的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:8的氨基酸序列或与示于SEQ ID NO:8的氨基酸序列具有至少75％同一性的氨基酸序列；
(4)示于SEQ ID NO:15的氨基酸序列或与示于SEQ ID NO:15的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:16的氨基酸序列或与示于SEQ ID NO:16的氨基酸序列具有至少75％同一性的氨基酸序列；
(5)示于SEQ ID NO:9的氨基酸序列或与示于SEQ ID NO:9的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:10的氨基酸序列或与示于SEQ ID NO:10的氨基酸序列具有至少75％同一性的氨基酸序列；
(6)示于SEQ ID NO:17的氨基酸序列或与示于SEQ ID NO:17的氨基 酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:18的氨基酸序列或与示于SEQ ID NO:18的氨基酸序列具有至少75％同一性的氨基酸序列；
(7)示于SEQ ID NO:11的氨基酸序列或与示于SEQ ID NO:11的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:12的氨基酸序列或与示于SEQ ID NO:12的氨基酸序列具有至少75％同一性的氨基酸序列；或
(8)示于SEQ ID NO:19的氨基酸序列或与示于SEQ ID NO:19的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:20的氨基酸序列或与示于SEQ ID NO:20的氨基酸序列具有至少75％同一性的氨基酸序列。
在本发明提供的双特异性抗体构建体中，所述第二结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR-1、HCDR-2、HCDR-3；LCDR-1、LCDR-2、LCDR-3)：
包含示于SEQ ID NO:49的氨基酸序列(TYAMN)的HCDR-1、包含示于SEQ ID NO:50的氨基酸序列(RIRSKYNNYATYYADSVKD)的HCDR-2、包含示于SEQ ID NO:51的氨基酸序列(HGNFGNSYVSYFAY)的HCDR-3；和，包含示于SEQ ID NO:52的氨基酸序列(RSSTGAVTTSNYAN)的LCDR-1、包含示于SEQ ID NO:53的氨基酸序列(GTNKRAP)的LCDR-2、包含示于SEQ ID NO:54的氨基酸序列(ALWYSNLWV)的LCDR-3。
优选地，在所述第二结合结构域中，所述重链可变区包含示于SEQ ID NO:21的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO:22的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
进一步优选地，本发明提供的双特异性抗体构建体包含四个多肽链：
1)重链1，所述重链1从N端到C端包含按照VH-CH1-CH2-CH3排列的结构域；
2)重链2，所述重链2从N端到C端包含按照VL-CH1-CH2-CH3排列的结构域；
3)轻链1，所述轻链1从N端到C端包含按照VL-CL排列的结构域；
4)轻链2，所述轻链2从N端到C端包含按照VH-CL排列的结构域；
其中，重链1和轻链1中包含的VH和VL配对形成所述结合GPRC5D的第一结合结构域；重链2和轻链2中包含的VL和VH配对形成所述结合CD3的第二结合结构域。本发明提供的双特异性抗体构建体的结构示意图见图1。
根据本发明的具体实施方式，在本发明提供的双特异性抗体构建体中，重链1中的CH1-CH2-CH3结构域包含示于SEQ ID NO:25的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；重链2中的CH1-CH2-CH3结构域包含示于SEQ ID NO:26的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；轻链1中的CL结构域包含示于SEQ ID NO:24的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；轻链2中的CL结构域包含示于SEQ ID NO:27的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
另一方面，本发明还提供一种核酸分子，其包含编码本发明提供的抗体或其片段或者本发明提供的双特异性抗体构建体中包含的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链的核苷酸序列。
本发明的核酸分子可以被克隆到载体中，进而转化或转染宿主细胞。因此又一方面，本发明还提供一种载体，所述载体包含本发明的核酸分子。所述载体可以为真核表达载体、原核表达载体、人工染色体及噬菌体载体等。本发明的载体或核酸分子可以用于转化或转染宿主细胞。因此，还一方面，本发明提供一种宿主细胞，所述宿主细胞包含本发明的核酸分子和/或载体，或者所述宿主细胞被本发明的核酸分子和/或载体转化或转染。宿主细胞可以是任何原核或真核细胞，例如细菌或昆虫、真菌、植物或动物细胞。
本发明提供的抗体或其片段以及双特异性抗体构建体可以利用本领域已知的任何方法获得。例如，在允许本发明提供的宿主细胞表达所述抗体的重链和轻链的情况下，培养所述宿主细胞。任选地，所述方法还包括回收产生的抗体的步骤。
本发明提供的抗体或其片段、双特异性抗体构建体、核酸分子、载体、宿主细胞和/或可以被包含在组合物中，更特别地被包含在药物组合物例如药物制剂中，从而根据实际需要用于各种目的。因此，在又一方面，本发明还提供一种组合物，例如药物组合物，所述组合物包含本发明所述的抗体或其片段、双特异性抗体构建体、核酸分子、载体和/或宿主细胞，以及任选的药 学上可接受的辅料。
再一方面，本发明还提供所述抗体或其片段、双特异性抗体构建体、核酸分子、载体、宿主细胞和/或组合物在制备用于治疗疾病的药物中的用途，所述疾病为肿瘤或癌症。优选地，所述疾病为与GPRC5D表达(例如高表达)相关的肿瘤或癌症，例如淋巴瘤或骨髓瘤，特别是多发性骨髓瘤。
因此相应地，本发明还提供一种治疗疾病的方法，所述方法包括给有此需要的受试者施用本发明的抗体或其片段、双特异性抗体构建体、核酸分子、载体、宿主细胞和/或组合物，所述疾病为肿瘤或癌症。优选地，所述疾病为与GPRC5D表达(例如高表达)相关的肿瘤或癌症，例如淋巴瘤或骨髓瘤；所述疾病特别是多发性骨髓瘤。优选地，所述受试者为哺乳类动物；更优选地，所述受试者为人。
还一方面，本发明还提供所述抗体或其片段、双特异性抗体构建体、核酸分子、载体、宿主细胞和/或组合物在制备用于诊断疾病或检测抗原GPRC5D存在的试剂中的用途，所述疾病为肿瘤或癌症。优选地，所述疾病为与GPRC5D表达(例如高表达)相关的肿瘤或癌症，例如淋巴瘤或骨髓瘤，特别是多发性骨髓瘤。
又一方面，本发明提供一种试剂盒，所述试剂盒包含本发明的抗体或其片段、双特异性抗体构建体、核酸分子、载体、宿主细胞和/或组合物。所述试剂盒可以用于上述治疗，或者用于诊断或抗原检测中。取决于预期的应用方式，所述试剂盒还包含用于治疗、诊断或检测的其他试剂。例如，所述试剂盒为用于采用ELISA来检测任何生物学样本中GPRC5D存在的试剂盒。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为抗人GPRC5D×CD3双特异性抗体的结构示意图。
图2显示杂交瘤细胞上清与不同细胞结合的FACS检测结果。
图3显示杂交瘤细胞上清与不同细胞结合的FACS检测结果。
图4显示杂交瘤细胞上清与不同细胞结合的FACS检测结果，其中4-1：MM1R细胞；4-2：Nalm6细胞；4-3：OPM2细胞；4-4：NCI-H929细胞。
图5显示人源化抗体与人GPRC5D表达细胞结合的FACS检测结果，其中5-1：9D7(hz)；5-2：26D1(hz)；5-3：24F5(hz)；5-4：6E97(hz)。
图6显示人源化抗体与猴GPRC5D表达细胞结合的FACS检测结果， 其中6-1：9D7(hz)；6-2：26D1(hz)；6-3：24F5(hz)；6-4：6E97(hz)。
图7显示人源化抗体与不同肿瘤细胞系细胞结合的FACS检测结果，其中7-1：MM.1R细胞；7-2：MOLP8细胞。
图8显示人源化抗体与GPRC5D的亲和力检测结果，其中8-1：GPRC5D×CD3 JNJ；8-2：9D7(hz)；8-3：26D1(hz)；8-4：24F5(hz)；8-5：6E97(hz)。
图9显示人源化抗体介导ADCC的检测结果，其中9-1：野生型NCI-H929；9-2：GPRC5D敲除的NCI-H929。
图10显示双特异性抗体介导T细胞杀伤的检测结果，其中10-1：共培养实验1；10-2：共培养实验2；10-3：共培养实验3。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。其中：
人GPRC5D蛋白：Genbank登记号NM_018654；
猴GPRC5D蛋白：Genbank登记号XM_005570193.2。
实施例1杂交瘤细胞的制备以及抗体蛋白的获得
将表达人GPRC5D细胞(康源博创生物科技(北京)有限公司(kyinno公司)，货号KC-1583)在含2％FBS的PBS中制备成细胞浓度为1×10 7个细胞/ml的细胞悬液。
将该细胞悬液作为免疫原免疫小鼠，5只小鼠皮下免疫，5只小鼠肌肉免疫，佐剂采用quick antibody 5W水溶性佐剂，加强免疫以后2周测效价，选取效价高的2只小鼠进行免疫冲击，3天之后取血清，解剖后取脾脏。分离的脾细胞与培养的骨髓瘤细胞在96孔板中进行融合，同时加入选择性培养基进行筛选，7天后换液，10天后进行基于细胞的ELISA检测。ELISA检测使用表达人GPRC5D的293T细胞(kyinno公司，货号KC-1723)，选择OD值大于阴性对照(正常小鼠的血清)10倍以上的细胞再进行流式细胞术检测，流式细胞术中使用表达人GPRC5D的293T细胞(kyinno公司，货号KC-1723)。
挑选ELISA和FACS检测结果均为阳性的细胞，通过细胞有限稀释法进行亚克隆铺板，挑选单克隆细胞。将挑选出的单克隆细胞，取培养上清，进行ELISA检测及流式细胞术检测，挑选双阳的细胞扩大培养。
收集所得杂交瘤细胞的培养上清，离心、浓缩后使用G蛋白亲和柱纯化。然后，将纯化后的样品使用20mM柠檬酸+100mM NaCl，pH5.5缓冲溶液10倍稀释，超滤浓缩管浓缩至合适体积后，分装至1.5mL EP管中，200μL/管，-80℃保存。同时取出部分样品进行SDS-PAG和FACS鉴定，验证蛋白的纯度及活性。
实施例2杂交瘤细胞培养上清与人GPRC5D结合的ELISA检测
将表达人GPRC5D的BXPC-3细胞(kyinno公司，货号KC-1717)在含2％FBS的PBS中制备成细胞浓度为1×10 5个细胞/ml的细胞悬液，以100μl/孔加入96孔板中，置37℃培养过夜。弃去孔内的液体，同时用洗涤液洗3次，每孔加80μl 4％组织固定液(Beyotime，货号P0099)，于室温下固定15min，用PBS洗涤两次，晾干，每孔加250μl含2％BSA的封闭液，37℃孵育1h，PBS洗板三次。向每孔中加入50-100μl待检杂交瘤细胞的培养上清，同时设立阳性对照(加入融合小鼠的血清)、阴性对照(加入正常小鼠的血清)和空白对照(加入培养基)。在37℃孵育1-2小时，洗涤，拍干。然后向每孔中加入酶标二抗，为1:10000稀释的辣根过氧化物酶标记的羊抗小鼠IgG(SIGMA，货号A9044-2ml)，每孔50-100μl，37℃孵育1-2小时，洗涤，拍干。向每孔中加入新鲜配制的底物显色液TMB 50-100μl，37℃孵育10-30分钟。
加入2mol/L H 2SO 4终止反应，在酶联免疫阅读仪上读取OD值。
结果判定：以P/N>2:1(P代表阳性数值，N代表正常小鼠血清数值)为阳性。若阴性对照孔无色或接近无色，阳性对照孔明确显色，则可直接用肉眼观察结果。结果见表1。
表1杂交瘤细胞培养上清与人GPRC5D结合的ELISA检测结果
Figure PCTCN2022143042-appb-000001
Figure PCTCN2022143042-appb-000002
实施例3抗人GPRC5D×CD3双特异性抗体的构建
采用强生公司的Talquetamab(JNJ-64407564)中靶向GPRC5D的结构域与靶向CD3的结构域构建抗人GPRC5D×CD3双特异性抗体。该双特异性抗体的结构示意图见图1，其中各条链包含的结构域从N端至C端依次为：重链1：VH-CH1-CH2-CH3
>VH(SEQ ID NO:1)
Figure PCTCN2022143042-appb-000003
>CH1-CH2-CH3(SEQ ID NO:25)
Figure PCTCN2022143042-appb-000004
重链2：VL-CH1-CH2-CH3
>VL(SEQ ID NO:2)
Figure PCTCN2022143042-appb-000005
>CH1-CH2-CH3(SEQ ID NO:26)
Figure PCTCN2022143042-appb-000006
轻链1：VL-CL
>VL(SEQ ID NO:3)
Figure PCTCN2022143042-appb-000007
>CL(SEQ ID NO:24)
Figure PCTCN2022143042-appb-000008
轻链2：VH-CL
>VH(SEQ ID NO:4)
Figure PCTCN2022143042-appb-000009
>CL(SEQ ID NO:27)
Figure PCTCN2022143042-appb-000010
对于上述四条多肽链，设计引物，合成相应的编码基因，连入真核表达质粒中，将获得的四个重组表达质粒共转染HEK293F细胞，细胞培养5-7天后，收获分泌抗体的上清液；使用MabSelectSure亲和层析技术和SP阳离子交换层析技术，从细胞培养物上清中纯化双特异性抗体。
将得到的双特异性抗体命名为“GPRC5D×CD3 JNJ”。
实施例4杂交瘤细胞培养上清与人GPRC5D结合的FACS检测
将如上表达人GPRC5D的293T细胞在含2％FBS的PBS中制备成细胞浓度为1×10 5个细胞/ml的细胞悬液。
向每个流式管(样品管)中放入50μl细胞悬液，然后分别加入50μl待检杂交瘤细胞的培养上清和作为阳性对照抗体的抗人GPRC5D×CD3双特异性抗体GPRC5D×CD3 JNJ，4℃孵育60分钟。向每个流式管中加入1ml的流式缓冲液，1200rpm下离心5分钟，弃上清，重复洗涤三次。同时设置对照管1(不加入培养上清和下文的二抗，仅加入细胞悬液)和对照管2(不加入培养上清，仅加入细胞悬液和下文的二抗)。
然后向每个流式管中加入100μl流式缓冲液进行重悬，根据实验要求加入5μl PE标记的抗鼠Fc标签的二抗(Biolegend，货号409304)，4℃避光孵育30分钟，然后加入1ml流式缓冲液，室温下1200rpm离心5分钟，弃上清，重复洗涤三次。
再次向每个流式管中加入250μl流式缓冲液，重悬混匀，上机检测。检测结果见表2-1至表2-4和图2中的2-1至2-4。
表2-1
样品(以杂交瘤细胞编号示出) 26D1 阳性对照抗体
EC50(μg/mL) 0.7881 1.684
MFI.Max 11491 10980
表2-2
样品(以杂交瘤细胞编号示出) 26D1 9D7
EC50(μg/mL) 0.1688 0.1853
MFI.Max 9755 9785
表2-3
Figure PCTCN2022143042-appb-000011
表2-4
样品(以杂交瘤细胞编号示出) 9D7 57G4
EC50(μg/mL) 0.2793 0.1918
MFI.Max 9255 9227
实施例5杂交瘤细胞上清与猴GPRC5D结合的FACS检测
按照实施例4所述的操作，将人GPRC5D表达细胞换成表达猴GPRC5D的293T细胞(kyinno公司，货号KC-1586)，检测本发明的杂交瘤细胞的培养上清与猴GPRC5D的结合。
FACS结合的检测结果见表3-1至表3-3和图3中的3-1至3-3；阳性对照抗体为GPRC5D×CD3 JNJ。
表3-1
样品(以杂交瘤细胞编号示出) 26D1 阳性对照抗体
EC50(μg/mL) 12.31 13.07
MFI.Max 1317 726
表3-2
Figure PCTCN2022143042-appb-000012
表3-3
样品(以杂交瘤细胞编号示出) 9D7 57G4
EC50(μg/mL) 0.2293 0.1820
MFI.Max 6447 4779
实施例6杂交瘤细胞上清与不同细胞结合的FACS检测
按照实施例4所述的操作，将人GPRC5D表达细胞换成其他细胞：NALM6(kyinno公司，货号KC-0626)、NCI-H929(kyinno公司，货号KC-0629)、MM1R(kyinno公司，货号KC-0619)、OPM2(kyinno公司，货号KC-0631)细胞，检测本发明的杂交瘤细胞的培养上清与人GPRC5D的结合。
FACS结合的检测结果见表4和图4中的4-1至4-4；阳性对照抗体为GPRC5D×CD3 JNJ。
表4.杂交瘤细胞上清与细胞结合的FACS检测结果
Figure PCTCN2022143042-appb-000013
Figure PCTCN2022143042-appb-000014
实施例7本发明的鼠抗抗体的序列鉴定
选择筛选的杂交瘤细胞株，从单克隆细胞中提RNA，逆转录成cDNA，cDNA扩增，胶回收后连入测序载体中进行测序分析。
从杂交瘤细胞中获得的示例性鼠抗体重轻链可变区的序列如下，其中加粗且带下划线部分示出了CDR(根据KABAT方法定义得到)。
A1：
>9D7-H(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:5/28/29/30)
Figure PCTCN2022143042-appb-000015
>9D7-L(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:6/31/32/33)
Figure PCTCN2022143042-appb-000016
A2：
>26D1-H(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:7/35/36/37)
Figure PCTCN2022143042-appb-000017
>26D1-L(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:8/38/39/40)
Figure PCTCN2022143042-appb-000018
A3
>24F5-H(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:9/41/42/43)
Figure PCTCN2022143042-appb-000019
TLTVSS
>24F5-L(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:10/44/45/46)
Figure PCTCN2022143042-appb-000020
A4
>6E97-H(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:11/47/42/43/)
Figure PCTCN2022143042-appb-000021
>6E97-L(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:12/44/45/46)
Figure PCTCN2022143042-appb-000022
实施例8基于鼠抗体的嵌合抗体和人源化抗体的制备
对A1抗体(鼠抗9D7)，分别选取了如下人germline的序列作为重链与轻链的模板：IGHV4-39和IGKV1-39。对A1抗体进行同源建模，进行Fab区域的结构模拟。经过同源建模计算，最后得到预测的A1抗体的Fab结构。
通过对预测的Fab结构及重链与IGHV4-39序列进行比对分析，将该VH中除了CDR区及2V、49M、68I、72R为原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGHV4-39模板人的氨基酸。
通过对预测的Fab结构及轻链与IGKV1-39序列进行比对分析，将该VL中除了CDR区及42Q、43S、46A、60D保留原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGKV1-39模板人的氨基酸。得到的人源化序列如下(其中重链和轻链CDR以加粗和下划线示出，其根据KABAT定义方法得到)：
抗体的人源化抗体的序列如下：
>9D7-H人源化序列(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:13/28/34/30)
Figure PCTCN2022143042-appb-000023
Figure PCTCN2022143042-appb-000024
>9D7-L人源化序列(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:14/31/32/33)
Figure PCTCN2022143042-appb-000025
对A2抗体(鼠抗26D1)，分别选取了如下人germline的序列作为重链与轻链的模板：IGHV1-46和IGKV1-5。对A2抗体进行同源建模，进行Fab区域的结构模拟。经过同源建模计算，最后得到预测的A2抗体的Fab结构。
通过对预测的Fab结构及重链与IGHV1-46序列进行比对分析，将该VH中除了CDR区及37I、38K、48I、67R、68A、70L、72A、74K、76S、77N、79A、98N为原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGHV1-46模板人的氨基酸。
通过对预测的Fab结构及轻链与IGKV1-5序列进行比对分析，将该VL中除了CDR区及45R、60D、87F保留原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGKV1-5模板人的氨基酸。得到的人源化序列如下(其中重链和轻链CDR以加粗和下划线示出，其根据KABAT定义方法得到)：
抗体的人源化抗体的序列如下：
>26D1-H人源化序列(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:15/35/36/37)
Figure PCTCN2022143042-appb-000026
>26D1-L人源化序列(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:16/38/39/40)
Figure PCTCN2022143042-appb-000027
对A3抗体(鼠抗24F5)，分别选取了如下人germline的序列作为重链与轻链的模板：IGHV1-46和IGKV1-15。对A3抗体进行同源建模，进行Fab区域的结构模拟。经过同源建模计算，最后得到预测的A3抗体的Fab结构。
通过对预测的Fab结构及重链与IGHV1-46序列进行比对分析，将该VH中除了CDR区及48I、68A、70L、72S、74K、79A为原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGHV1-46模板人的氨基酸。
通过对预测的Fab结构及轻链与IGKV1-15序列进行比对分析，将该VL中除了CDR区及36L、42G、43T、44I、46R、66R、69S、71Y保留原 始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGKV1-15模板人的氨基酸。得到的人源化序列如下(其中重链和轻链CDR以加粗和下划线示出，其根据KABAT定义方法得到)：
抗体的人源化抗体的序列如下：
>24F5-H人源化序列(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:17/41/42/43)
Figure PCTCN2022143042-appb-000028
>24F5-L人源化序列(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:18/44/45/46)
Figure PCTCN2022143042-appb-000029
对A4抗体(鼠抗6E97)，分别选取了如下人germline的序列作为重链与轻链的模板：IGHV1-46和IGKV1-15。对A4抗体进行同源建模，进行Fab区域的结构模拟。经过同源建模计算，最后得到预测的A4抗体的Fab结构。
通过对预测的Fab结构及重链与IGHV1-46序列进行比对分析，将该VH中除了CDR区及48I、68A、70L、72S、74K、79A为原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGHV1-46模板人的氨基酸。
通过对预测的Fab结构及轻链与IGKV1-15序列进行比对分析，将该VL中除了CDR区及4L、36L、42G、43T、44I、46R、66R、69S、71Y保留原始鼠的氨基酸外，其它的鼠的氨基酸全部替换成相应的IGKV1-15模板人的氨基酸。得到的人源化序列如下(其中重链和轻链CDR以加粗和下划线示出，其根据KABAT定义方法得到)：
抗体的人源化抗体的序列如下：
>6E97-H人源化序列(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:19/47/48/43)
Figure PCTCN2022143042-appb-000030
>6E97-L人源化序列(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:20/44/45/46)
Figure PCTCN2022143042-appb-000031
以SEQ ID NO:23所示序列作为重链恒定区，SEQ ID NO:24所示序列作为轻链恒定区，对于测序得到的针对GRRC5D靶点的抗体DNA序列，重 新设计引物，合成相应的嵌合抗体及人源化抗体的基因，连入真核表达载体中，转化DH5alpha感受态细胞，37℃恒温培养箱培养过夜，挑单克隆菌株测序鉴定。挑选序列正确的菌株，摇菌，大提质粒，转染哺乳动物表达细胞293F，置于37℃、5％CO2的培养箱中，表达培养7天。
收取上清，离心，过滤，选择protein G亲和层析柱进行纯化，纯化的抗体通过SDS-PAGE电泳检测纯度，用BCA蛋白检测试剂盒检测抗体浓度，分装，保存于-80℃冰箱中备用。其中，将嵌合抗体命名为“鼠抗体简称(chi)”，将人源化抗体命名为“鼠抗体简称(hz)”。
>CH1-CH3重链恒定区(SEQ ID NO:23)
Figure PCTCN2022143042-appb-000032
>CL1轻链恒定区(SEQ ID NO:24)
Figure PCTCN2022143042-appb-000033
实施例9人源化抗体与人GPRC5D结合的FACS检测
实验过程参照实施例4，使用表达人GPRC5D的293T细胞(kyinno公司，货号KC-1584)，加入待检抗体、阳性对照抗体GPRC5D×CD3 JNJ或阴性对照抗体(hIgG1)，浓度参见图4。同时设置对照管1(不加入抗体和二抗，仅加入细胞悬液)和对照管2(不加入抗体，仅加入细胞悬液和二抗)。
结果见表5和图5中的5-1至5-4。
表5.人源化抗体与细胞结合的FACS检测结果
样品 阳性对照抗体 9D7(hz)
EC50(μg/mL) 0.4537 0.2830
MFI.Max 6590 8938
     
样品 阳性对照抗体 26D1(hz)
EC50(μg/mL) 0.3552 0.228
MFI.Max 8650 9560
     
样品 阳性对照抗体 24F5(hz)
EC50(μg/mL) 0.6986 0.6995
MFI.Max 9351 8908
     
样品 阳性对照抗体 6E97(hz)
EC50(μg/mL) 0.3571 0.3508
MFI.Max 8941 14840
实施例10人源化抗体与猴GPRC5D结合的FACS检测
实验过程参照实施例4，使用表达猴(cyno)GPRC5D的293T细胞(kyinno公司，货号KC-1586)，加入待检抗体、阳性对照抗体GPRC5D×CD3 JNJ或阴性对照抗体(hIgG1)，浓度参见图6。同时设置对照管1(不加入抗体和二抗，仅加入细胞悬液)和对照管2(不加入抗体，仅加入细胞悬液和二抗)。
结果见表6和图6中的6-1至6-4。
表6.人源化抗体与细胞结合的FACS检测结果
样品 阳性对照抗体 9D7(hz)
EC50(μg/mL) 13.96 0.3779
MFI.Max 656 4265
     
样品 阳性对照抗体 26D1(hz)
EC50(μg/mL) N/A N/A
MFI.Max 334 12.3
     
样品 阳性对照抗体 24F5(hz)
EC50(μg/mL) 4.389 0.1666
MFI.Max 2110 3558
     
样品 阳性对照抗体 6E97(hz)
EC50(μg/mL) 3.265 0.1917
MFI.Max 2791 6246
实施例11人源化抗体与GPRC5A/B/C结合的FACS检测
实验过程参照实施例4，使用表达G蛋白偶联受体家族C类的5组其他成员的细胞：表达人GPRC5A的293T细胞293T-GPRC5A(kyinno公司，货号KC-1888)，表达人GPRC5B的293T细胞293T-GPRC5B(kyinno公司，货号KC-1854)，表达人GPRC5C的293T细胞293T-GPRC5C(kyinno公司，货号KC-1889)，按照2μg/mL浓度分别加入待检抗体、阳性对照抗体GPRC5D×CD3 JNJ或阴性对照抗体(hIgG1)。同时设置对照管1(不加入抗体和二抗，仅加入细胞悬液)和对照管2(不加入抗体，仅加入细胞悬液和二抗)。
结果见表7。
表7.人源化抗体与细胞结合的FACS检测结果(μg/mL)
Figure PCTCN2022143042-appb-000034
实施例12人源化抗体与肿瘤细胞系结合的FACS检测
实验过程参照实施例4，使用的细胞换为多发性骨髓瘤细胞MM.1R(kyinno公司，货号KC-0619)和MOLP8(kyinno公司，货号KC-0622)，加入待检抗体、阳性对照抗体GPRC5D×CD3 JNJ或阴性对照抗体(hIgG1)。同时设置对照管1(不加入抗体和二抗，仅加入细胞悬液)和对照管2(不加入抗体，仅加入细胞悬液和二抗)。
结果见表8和图7中的7-1和7-2。
表8.人源化抗体与肿瘤细胞系结合的FACS检测结果
Figure PCTCN2022143042-appb-000035
Figure PCTCN2022143042-appb-000036
实施例13人源化抗体与GPRC5D蛋白的亲和力检测
将待测抗体使用PBS缓冲液制备成浓度为10μg/mL溶液，按照Fortebio说明书，使用AHC芯片捕获抗体。使提前准备的人GPRC5D蛋白(恺佧生物，货号GPR-HM05P)(蛋白按照最高浓度为200nM，2倍稀释，6个梯度)流过芯片，具体条件为：流速30μl/min；抗原抗体结合时间200秒，解离时间500秒。测得的结果用仪器专用配套软件拟合，以分析抗体与抗原的亲和力。结果见表9和图8中的8-1至8-5。
表9.人源化抗体与GPRC5D蛋白的亲和力检测结果
抗体 KD(M) kon(1/Ms) kdis(1/s)
阳性对照抗体 9.47E-10 2.18E+04 2.06E-05
9D7(hz) <1.0E-12 2.26E+04 <1.0E-07
26D1(hz) 1.70E-09 1.85E+04 3.15E-05
24F5(hz) 1.36E-09 3.99E+04 5.42E-05
6E97(hz) 4.58E-10 4.55E+04 2.08E-05
实施例14人源化抗体介导ADCC的检测
在U底的96孔板中每孔加入20000个报告细胞Jurkat-NFAT-luc-CD16-V158(kyinno公司，货号KC-1507)，20000个野生型NCI-H929细胞(CFSE-H929，kyinno公司，货号KC-0629)或GPRC5D敲除的NCI-H929细胞(CFSE-H929GPRC5DKO，kyinno公司，货号KC-2203)，以及不同浓度的抗体(10，3.16，1，0.316，0.1，0.0316，0.01，0.00316μg/ml)。共培养6个小时，然后检测荧光素酶(luciferase)的信号。
结果见表10和图9中的9-1至9-2；阳性对照抗体为GPRC5D×CD3 JNJ。
表10.人源化抗体介导ADCC的检测结果
Figure PCTCN2022143042-appb-000037
Figure PCTCN2022143042-appb-000038
实施例15抗CD3抗体的获得和靶点亲和力的检测
从专利US10066015B2获得鼠源抗体的重链可变区序列SEQ ID NO.10和轻链可变区序列SEQ ID NO.5。对该抗体进行抗体序列的人源化，得到的人源化序列如下(其中重链和轻链CDR以加粗和下划线示出，其根据KABAT定义方法得到)：
>VH人源化序列(VH/HCDR-1/HCDR-2/HCDR-3：SEQ ID NO:21/49/50/51)
Figure PCTCN2022143042-appb-000039
>VL人源化序列(VL/LCDR-1/LCDR-2/LCDR-3：SEQ ID NO:22/52/53/54)
Figure PCTCN2022143042-appb-000040
按照实施例8所述，获得包含上述人源化序列的抗体，命名为SP34V1(hz)；同样，以专利US10066015B2中的上述鼠源抗体的重、轻链可变区序列，获得嵌合抗体，命名为SP34(chi)。
将该人源化抗体与嵌合抗体使用PBS缓冲液制备成浓度为10μg/mL溶液，按照Fortebio说明书，使用AHC芯片捕获抗体。使提前准备的人CD3蛋白(恺佧生物，货号GPR-HM05P)(蛋白按照最高浓度为200nM，2倍稀释，6个梯度)流过芯片，具体条件为：流速30μl/min；抗原抗体结合时间200秒，解离时间500秒。测得的结果用仪器专用配套软件拟合，以分析抗体与抗原的亲和力。结果见表11。
表11.抗体与CD3蛋白的亲和力检测结果
抗体 KD(M) kon(1/Ms) kdis(1/s)
SP34V1(hz) 1.80E-08 4.42E+05 7.95E-03
SP34(chi) 5.46E-09 5.01E+05 2.74E-03
实施例16本发明的双特异性抗体的构建
按照实施例3所述，构建本发明的双特异性抗体，区别仅在于将重链1中VH和轻链1中的VL分别替换为本发明的人源化抗体9D7(hz)、26D1(hz)、24F5(hz)和6E97(hz)的VH和VL，将重链2中VL和轻链2中的VH分别替换为实施例15中得到的针对CD3的人源化序列。
将分别包含本发明的人源化抗体9D7(hz)、26D1(hz)、24F5(hz)和6E97(hz)的VH和VL与如上序列的抗人GPRC5D×CD3双特异性抗体分别命名为“GPRC5D×CD3 9D7(hz)”、“GPRC5D×CD3 26D1(hz)”、“GPRC5D×CD3 24F5(hz)”、“GPRC5D×CD3 26D1(hz)”。
实施例17GPRC5D和CD3双抗介导T细胞杀伤实验
将PBMC和CD3/CD28磁珠以数量比2：1混合在含10ng/ml的IL-2和10％FBS的RPMI1640中，培养扩增7天，以激活T细胞。
共培养实验1：将293T LDHA-Hibit-GPRC5D细胞(kyinno公司，货号KC-2151)提前一天铺板(96孔板)，然后每孔加入10000个上述T细胞，以及分别加入不同浓度的阳性对照抗体GPRC5D×CD3 JNJ和本发明的双特异性抗体(1，0.1，0.01，0.001，0.0001，0.00001，0.000001，0.0000001μg/ml)，在含10ng/ml IL-2和10％FBS的RPMI1640中共培养18个小时。使用
Figure PCTCN2022143042-appb-000041
HiBiT Extracellular Reagent(Promega，货号N2420)检测上清中死亡细胞释放的LDHA-HIBIT，由此检测杀伤细胞比例。本实验针对2个抗体各设置了2组对照：加入293T LDHA-Hibit-GPRC5D细胞和抗体，但不加T细胞；加入293T LDHA-Hibit细胞(kyinno公司)、T细胞和抗体，以排除非抗体介导的杀伤和抗体介导的非T细胞杀伤。结果见图10中的10-1。
共培养实验2：NCI-H929(表达GPRC5D)和K562细胞(不表达GPRC5D)用CFSE进行染色，分别命名为CFSE-H929和CFSE-K562。在U底的96孔板中每孔加入10000个激活的T细胞，3000个CFSE-H929，以及分别加入不同浓度的阳性对照抗体GPRC5D×CD3 JNJ和本发明的双特异性抗体(1，0.1，0.01，0.001，0.0001，0.00001，0.000001，0.0000001μg/ml)，在含10ng/ml的IL-2和10％FBS的RPMI1640中共培养18个小时。然后用7-AAD染色， 流式检测，计数CFSE+7-AAD+细胞在CSFE+细胞中的比例为细胞的杀伤比例。本实验针对2个抗体各设置了2组对照：加入CFSE-H929细胞和抗体，但不加T细胞；加入CFSE-K562、T细胞和抗体，以排除非抗体介导的杀伤和抗体介导的非T细胞杀伤。结果见图10中的10-2。
共培养实验3：野生型NCI-H929(kyinno公司，货号KC-0629)和GPRC5D敲除的NCI-H929细胞(kyinno公司，货号KC-2203)用CFSE进行染色，分别命名为CFSE-H929和CFSE-H929GPRC5DKO。在U底的96孔板中每孔加入10000个激活的T细胞，3000个CFSE-H929，以及加入不同浓度的阳性对照抗体GPRC5D×CD3 JNJ和本发明的双特异性抗体(1，0.1，0.01，0.001，0.0001，0.00001，0.000001，0.0000001μg/ml)，在含10ng/ml的IL-2和10％FBS的RPMI1640中共培养18个小时。然后用7-AAD染色，流式检测，计数CFSE+7-AAD+细胞在CSFE+细胞中的比例为细胞的杀伤比例。本实验针对4个抗体各设置了2组对照：加入CFSE-H929细胞和抗体，但不加T细胞；加入CFSE-H929GPRC5DKO、T细胞和抗体，以排除非抗体介导的杀伤和抗体介导的非T细胞杀伤。结果见图10中的10-3。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。

Claims (19)

  1. 一种抗体或其片段，所述抗体或其片段包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR-1、HCDR-2、HCDR-3；LCDR-1、LCDR-2、LCDR-3)：
    (1)包含示于SEQ ID NO:28的氨基酸序列的HCDR-1、包含示于SEQ ID NO:29的氨基酸序列的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列的LCDR-3；
    (2)包含示于SEQ ID NO:28的氨基酸序列的HCDR-1、包含示于SEQ ID NO:34的氨基酸序列的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列的LCDR-3；
    (3)包含示于SEQ ID NO:35的氨基酸序列的HCDR-1、包含示于SEQ ID NO:36的氨基酸序列的HCDR-2、包含示于SEQ ID NO:37的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:38的氨基酸序列的LCDR-1、包含示于SEQ ID NO:39的氨基酸序列的LCDR-2、包含示于SEQ ID NO:40的氨基酸序列的LCDR-3；
    (4)包含示于SEQ ID NO:41的氨基酸序列的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列的LCDR-3；
    (5)包含示于SEQ ID NO:47的氨基酸序列的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列的LCDR-3；或
    (6)包含示于SEQ ID NO:47的氨基酸序列的HCDR-1、包含示于SEQ  ID NO:48的氨基酸序列的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列的LCDR-3。
  2. 根据权利要求1所述的抗体或其片段，其特征在于，所述重链可变区包含选自以下的序列：示于SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:9、SEQ ID NO:17、SEQ ID NO:11或SEQ ID NO:19的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述轻链可变区包含选自以下的序列：示于SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:12或SEQ ID NO:20的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
  3. 根据权利要求1或2所述的抗体或其片段，其特征在于，所述抗体或其片段包含的重链可变区和轻链可变区包含选自以下的序列组合：
    (1)示于SEQ ID NO:5的氨基酸序列或与示于SEQ ID NO:5的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:6的氨基酸序列或与示于SEQ ID NO:6的氨基酸序列具有至少75％同一性的氨基酸序列；
    (2)示于SEQ ID NO:13的氨基酸序列或与示于SEQ ID NO:13的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:14的氨基酸序列或与示于SEQ ID NO:14的氨基酸序列具有至少75％同一性的氨基酸序列；
    (3)示于SEQ ID NO:7的氨基酸序列或与示于SEQ ID NO:7的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:8的氨基酸序列或与示于SEQ ID NO:8的氨基酸序列具有至少75％同一性的氨基酸序列；
    (4)示于SEQ ID NO:15的氨基酸序列或与示于SEQ ID NO:15的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:16的氨基酸序列或与示于SEQ ID NO:16的氨基酸序列具有至少75％同一性的氨基酸序列；
    (5)示于SEQ ID NO:9的氨基酸序列或与示于SEQ ID NO:9的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:10的氨基酸 序列或与示于SEQ ID NO:10的氨基酸序列具有至少75％同一性的氨基酸序列；
    (6)示于SEQ ID NO:17的氨基酸序列或与示于SEQ ID NO:17的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:18的氨基酸序列或与示于SEQ ID NO:18的氨基酸序列具有至少75％同一性的氨基酸序列；
    (7)示于SEQ ID NO:11的氨基酸序列或与示于SEQ ID NO:11的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:12的氨基酸序列或与示于SEQ ID NO:12的氨基酸序列具有至少75％同一性的氨基酸序列；和
    (8)示于SEQ ID NO:19的氨基酸序列或与示于SEQ ID NO:19的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:20的氨基酸序列或与示于SEQ ID NO:20的氨基酸序列具有至少75％同一性的氨基酸序列。
  4. 根据权利要求1至3中任一项所述的抗体或其片段，其特征在于，所述抗体或其片段为抗GPRC5D的抗体或其片段；
    优选地，所述抗体为鼠抗、兔抗、单克隆抗体、嵌合抗体、部分或全部人源化的抗体等任意形式；或者，所述片段为半抗体或者抗体或半抗体的抗原结合片段，例如scFv、BsFv、dsFv、(dsFv) 2、Fab、Fab'、F(ab') 2或Fv；
    更优选地，所述抗体为IgG。
  5. 根据权利要求1至4中任一项所述的抗体或其片段，其特征在于，所述抗体或其片段还包含恒定区；
    优选地，所述抗体或其片段包含重链和轻链；更优选地，所述抗体或其片段包含IgG、IgA、IgM、IgD或IgE的重链恒定区和/或κ或λ型轻链恒定区。
  6. 一种双特异性抗体构建体，所述抗体构建体包含结合G蛋白偶联受体家族C类的5组D成员(GPRC5D)的第一结合结构域，和结合T细胞表面上的CD3的第二结合结构域，其中：
    所述第一结合结构域包含重链可变区(VH)和轻链可变区(VL)，所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR-1、HCDR-2、HCDR-3；LCDR-1、LCDR-2、LCDR-3)：
    (1)包含示于SEQ ID NO:28的氨基酸序列的HCDR-1、包含示于SEQ  ID NO:29的氨基酸序列的HCDR-2、包含示于SEQ ID NO:30的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列的LCDR-3；
    (2)包含示于SEQ ID NO:28的氨基酸序列的HCDR-1、包含示于SEQ ID NO:34的氨基酸序列的HCDR-2、SEQ ID NO:30的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:31的氨基酸序列的LCDR-1、包含示于SEQ ID NO:32的氨基酸序列的LCDR-2、包含示于SEQ ID NO:33的氨基酸序列的LCDR-3；
    (3)包含示于SEQ ID NO:35的氨基酸序列的HCDR-1、包含示于SEQ ID NO:36的氨基酸序列的HCDR-2、包含示于SEQ ID NO:37的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:38的氨基酸序列的LCDR-1、包含示于SEQ ID NO:39的氨基酸序列的LCDR-2、包含示于SEQ ID NO:40的氨基酸序列的LCDR-3；
    (4)包含示于SEQ ID NO:41的氨基酸序列的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列的LCDR-3；
    (5)包含示于SEQ ID NO:47的氨基酸序列的HCDR-1、包含示于SEQ ID NO:42的氨基酸序列的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列的LCDR-3；和
    (6)包含示于SEQ ID NO:47的氨基酸序列的HCDR-1、包含示于SEQ ID NO:48的氨基酸序列的HCDR-2、包含示于SEQ ID NO:43的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:44的氨基酸序列的LCDR-1、包含示于SEQ ID NO:45的氨基酸序列的LCDR-2、包含示于SEQ ID NO:46的氨基酸序列的LCDR-3。
  7. 根据权利要求6所述的双特异性抗体构建体，其特征在于，在所述第一结合结构域中，所述重链可变区包含选自以下的序列：示于SEQ ID NO:5、SEQ ID NO:13、SEQ ID NO:7、SEQ ID NO:15、SEQ ID NO:9、SEQ ID NO: 17、SEQ ID NO:11或SEQ ID NO:19的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和/或，所述轻链可变区包含选自以下的序列：示于SEQ ID NO:6、SEQ ID NO:14、SEQ ID NO:8、SEQ ID NO:16、SEQ ID NO:10、SEQ ID NO:18、SEQ ID NO:12或SEQ ID NO:20的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
  8. 根据权利要求6或7所述的双特异性抗体构建体，其特征在于，所述第一结合结构域包含的重链可变区和轻链可变区包含选自以下的序列组合：
    (1)示于SEQ ID NO:5的氨基酸序列或与示于SEQ ID NO:5的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:6的氨基酸序列或与示于SEQ ID NO:6的氨基酸序列具有至少75％同一性的氨基酸序列；
    (2)示于SEQ ID NO:13的氨基酸序列或与示于SEQ ID NO:13的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:14的氨基酸序列或与示于SEQ ID NO:14的氨基酸序列具有至少75％同一性的氨基酸序列；
    (3)示于SEQ ID NO:7的氨基酸序列或与示于SEQ ID NO:7的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:8的氨基酸序列或与示于SEQ ID NO:8的氨基酸序列具有至少75％同一性的氨基酸序列；
    (4)示于SEQ ID NO:15的氨基酸序列或与示于SEQ ID NO:15的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:16的氨基酸序列或与示于SEQ ID NO:16的氨基酸序列具有至少75％同一性的氨基酸序列；
    (5)示于SEQ ID NO:9的氨基酸序列或与示于SEQ ID NO:9的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:10的氨基酸序列或与示于SEQ ID NO:10的氨基酸序列具有至少75％同一性的氨基酸序列；
    (6)示于SEQ ID NO:17的氨基酸序列或与示于SEQ ID NO:17的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:18的氨基酸序列或与示于SEQ ID NO:18的氨基酸序列具有至少75％同一性的氨基酸序列；
    (7)示于SEQ ID NO:11的氨基酸序列或与示于SEQ ID NO:11的氨基 酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:12的氨基酸序列或与示于SEQ ID NO:12的氨基酸序列具有至少75％同一性的氨基酸序列；或
    (8)示于SEQ ID NO:19的氨基酸序列或与示于SEQ ID NO:19的氨基酸序列具有至少75％同一性的氨基酸序列；和，示于SEQ ID NO:20的氨基酸序列或与示于SEQ ID NO:20的氨基酸序列具有至少75％同一性的氨基酸序列。
  9. 根据权利要求6至8中任一项所述的双特异性抗体构建体，其特征在于，所述第二结合结构域包含重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)包含选自以下的CDR组合(HCDR-1、HCDR-2、HCDR-3；LCDR-1、LCDR-2、LCDR-3)：
    包含示于SEQ ID NO:49的氨基酸序列的HCDR-1、包含示于SEQ ID NO:50的氨基酸序列的HCDR-2、包含示于SEQ ID NO:51的氨基酸序列的HCDR-3；和，包含示于SEQ ID NO:52的氨基酸序列的LCDR-1、包含示于SEQ ID NO:53的氨基酸序列的LCDR-2、包含示于SEQ ID NO:54的氨基酸序列的LCDR-3。
  10. 根据权利要求6至9中任一项所述的双特异性抗体构建体，其特征在于，在所述第二结合结构域中，所述重链可变区包含示于SEQ ID NO:21的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列；和，所述轻链可变区包含示于SEQ ID NO:22的氨基酸序列或与所述氨基酸序列具有至少75％同一性的氨基酸序列。
  11. 根据权利要求6至10中任一项所述的双特异性抗体构建体，其特征在于，所述双特异性抗体构建体包含四个多肽链：
    1)重链1，所述重链1从N端到C端包含按照VH-CH1-CH2-CH3排列的结构域；
    2)重链2，所述重链2从N端到C端包含按照VL-CH1-CH2-CH3排列的结构域；
    3)轻链1，所述轻链1从N端到C端包含按照VL-CL排列的结构域；
    4)轻链2，所述轻链2从N端到C端包含按照VH-CL排列的结构域；
    其中，重链1和轻链1中包含的VH和VL配对形成所述结合GPRC5D的第一结合结构域；重链2和轻链2中包含的VL和VH配对形成所述结合CD3的第二结合结构域。
  12. 一种核酸分子，其包含编码权利要求1至5中任一项所述的抗体或其片段中或者权利要求6至11中任一项所述的双特异性抗体构建体中包含的重链CDR、轻链CDR、轻链可变区、重链可变区、重链或轻链的核苷酸序列。
  13. 一种载体，其包含权利要求12所述的核酸分子。
  14. 一种宿主细胞，其包含权利要求12所述的核酸分子和/或权利要求13所述的载体。
  15. 一种组合物，其包含权利要求1至5中任一项所述的抗体或其片段、权利要求6至11中任一项所述的双特异性抗体构建体、权利要求12所述的核酸分子、权利要求13所述的载体和/或权利要求14所述的宿主细胞；
    优选地，所述组合物为药物组合物，其还包含任选的药学上可接受的辅料。
  16. 权利要求1至5中任一项所述的抗体或其片段、权利要求6至11中任一项所述的双特异性抗体构建体、权利要求12所述的核酸分子、权利要求13所述的载体、权利要求14所述的宿主细胞和/或权利要求15所述的组合物在制备用于治疗疾病的药物中的用途，所述疾病为肿瘤或癌症；
    优选地，所述疾病为与GPRC5D表达(例如高表达)相关的肿瘤或癌症，例如淋巴瘤或骨髓瘤，特别是多发性骨髓瘤。
  17. 一种治疗疾病的方法，所述方法包括给有此需要的受试者施用权利要求1至5中任一项所述的抗体或其片段、权利要求6至11中任一项所述的双特异性抗体构建体、权利要求12所述的核酸分子、权利要求13所述的载体、权利要求14所述的宿主细胞和/或权利要求15所述的组合物，所述疾病为肿瘤或癌症；
    优选地，所述疾病为与GPRC5D表达(例如高表达)相关的肿瘤或癌症，例如淋巴瘤或骨髓瘤，特别是多发性骨髓瘤；
    优选地，所述受试者为哺乳类动物；更优选地，所述受试者为人。
  18. 权利要求1至5中任一项所述的抗体或其片段、权利要求6至11中任一项所述的双特异性抗体构建体、权利要求12所述的核酸分子、权利要求13所述的载体、权利要求14所述的宿主细胞和/或权利要求15所述的组合物在制备用于诊断疾病或检测抗原GPRC5D存在的试剂中的用途，所述疾病为肿瘤或癌症；
    优选地，所述疾病为与GPRC5D表达(例如高表达)相关的肿瘤或癌 症，例如淋巴瘤或骨髓瘤，特别是多发性骨髓瘤。
  19. 一种试剂盒，所述试剂盒包含权利要求1至5中任一项所述的抗体或其片段、权利要求6至11中任一项所述的双特异性抗体构建体、权利要求12所述的核酸分子、权利要求13所述的载体、权利要求14所述的宿主细胞和/或权利要求15所述的组合物。
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