WO2024050797A1 - 结合bcma、gprc5d和cd3的多特异性抗体及其用途 - Google Patents

结合bcma、gprc5d和cd3的多特异性抗体及其用途 Download PDF

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WO2024050797A1
WO2024050797A1 PCT/CN2022/117979 CN2022117979W WO2024050797A1 WO 2024050797 A1 WO2024050797 A1 WO 2024050797A1 CN 2022117979 W CN2022117979 W CN 2022117979W WO 2024050797 A1 WO2024050797 A1 WO 2024050797A1
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variable region
bcma
heavy chain
chain variable
specifically binds
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PCT/CN2022/117979
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English (en)
French (fr)
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彭浩
李江美
吴国进
郝锋
胡稳奇
宁金鹰
李锋
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北京天广实生物技术股份有限公司
康源博创生物科技(北京)有限公司
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Priority to CN202280010224.3A priority Critical patent/CN118414351A/zh
Priority to PCT/CN2022/117979 priority patent/WO2024050797A1/zh
Priority to TW112126154A priority patent/TW202411254A/zh
Publication of WO2024050797A1 publication Critical patent/WO2024050797A1/zh

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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
    • 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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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

  • This application relates to a multispecific antibody that simultaneously targets BCMA, GPRC5D and CD3, and its use in the treatment of tumors and other diseases.
  • MM Multiple myeloma
  • the disease is characterized by the unrestricted proliferation of plasma cells in the bone marrow like tumor cells, and in most cases is accompanied by the secretion of monoclonal immunoglobulins, eventually causing organ damage. or tissue damage.
  • MM is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, kidney damage, etc.
  • MM mostly occurs in middle-aged and elderly people, and there are obvious differences in gender distribution.
  • the proportion of men affected is higher than that of women.
  • MM accounts for approximately 10-15% of hematological malignancies and is the third most common hematological tumor after leukemia and lymphoma.
  • the incidence rate in the United States is 9/100,000, while the incidence rate in my country is approximately 1/100,000, and is increasing year by year.
  • BCMA B cell maturation antigen
  • CD269 or TNFRSF17 B cell maturation antigen
  • TNFR tumor necrosis factor receptor
  • BCMA is mainly expressed on the surface of mature B lymphocytes and plasma cells, and is expressed in small amounts on hematopoietic stem cells or non-hematopoietic tissues. Its ligands include B-cell activating factor (BAFF) and proliferation-inducing factor (APRIL), the latter of which has a higher affinity for BCMA.
  • BAFF B-cell activating factor
  • APRIL proliferation-inducing factor
  • BCMA is also overexpressed on the surface of malignant plasma cells in the bone marrow of MM patients. BCMA can promote the survival of malignant plasma cells in the bone marrow environment.
  • the APRIL-BCMA signaling pathway of malignant plasma cells can promote the proliferation and apoptosis escape of malignant tumor cells, and also cause them to produce powerful immunosuppressive molecules, such as IL-10, PD-L1 and TGF- ⁇ (Tai Y-T et al., (2016) Blood. 127(25): 3225-3236).
  • BCMA Due to the selective expression/distribution characteristics of BCMA, that is, it is widely present on the surface of MM cells, but has low or no expression on other normal tissue cells, and the long serum half-life of BCMA, it is a promising candidate for MM and other plasma cell cancers. ideal target. Compared with CD138, which is specifically expressed in plasma cells but quickly disappears from the cell surface, BCMA is obviously a better biomarker for malignant plasma cells in MM.
  • GPRC5D member D of group C 5 of the G protein-coupled receptor family, is a C-type 7-transmembrane receptor protein whose ligand and signaling mechanism have not yet been determined.
  • GPRC5D was found to be a marker molecule for MM (Cohen Y et al., (2013) Hematology 18(6): 348-351). This protein is mainly expressed in cells with a plasma cell phenotype, including most malignant bone marrow plasma cells, and its expression distribution on CD138 + MM cells in bone marrow samples is very similar to BCMA, while in normal tissues, the protein of GPRC5D Expression is restricted to hair follicles (Smith EL et al., (2019) Sci Transl Med. 11(485):eaau7746).
  • GPRC5D mRNA expression in MM patients is associated with genetic aberrations such as Rb-1 deletion and poor prognosis (Atamaniuk J et al., (2012) Eur J Clin Invest. 42(9):953-960).
  • CD3 molecule is a marker molecule on the surface of T cells. It forms a TCR-CD3 complex with T cell receptor (TCR) and plays an important role in antigen recognition and immune signal transduction.
  • TCR is composed of ⁇ and ⁇ chains, or ⁇ and ⁇ chains. The intracellular domain of each chain has no signal transduction function. The T cell intracellular signaling pathway is entirely responsible for the CD3 protein.
  • the CD3 molecule is composed of one ⁇ chain, one ⁇ chain, two ⁇ and two ⁇ chains, and forms three dimers ⁇ , ⁇ , and ⁇ in the TCR/CD3 complex.
  • ⁇ , ⁇ , and ⁇ are all type I transmembrane proteins with extracellular functional domains similar to immunoglobulins.
  • ITAM immunoreceptor tyrosine activation motifs
  • Antibodies targeting CD3 can form bispecific molecules with functional groups targeting disease-related antigens (such as tumor-related antigens), establishing a physical connection between T cells and disease-related antigens, causing T cell activation and T cell-mediated induced killing of disease-related cells.
  • disease-related antigens such as tumor-related antigens
  • tumor-associated antigens can activate T cells and release supramolecular attack particles (SMAPs) containing more than 280 proteins to tumor cells after bringing them closer to tumor cells.
  • SMAPs supramolecular attack particles
  • the core region of SMAP contains perforin and granzyme. Perforin can pierce the outer membrane of tumor cells, while granzyme can induce tumor cell apoptosis ( Bálint et al., (2020)Science 368(6493):897-901).
  • CD3 antibodies accumulate CD3 on the surface of T cells, simulating the process of TCR recognizing MHC-antigen peptides, thereby activating the TCR complex signaling pathway of T cells, releasing cytokines such as IL-2, IFN-r, and TNF- ⁇ , and activating T cells. proliferation and differentiation.
  • the proliferation and differentiation of T cells is a double-edged sword in tumor treatment. On the one hand, it can produce more T cells to kill tumor cells, but on the other hand, it can also produce a great toxic reaction in the subject. That is, cytokine release syndrome ("CRS").
  • CRS cytokine release syndrome
  • CRS central nervous system
  • CRS is not unavoidable in the treatment of bispecific or multispecific antibodies targeting CD3.
  • severe CRS and CNS toxicity were frequently observed in clinical trials of the CD19/CD3 T cell bispecific agent blinatumomab.
  • Modification of the Fc region of CD3 monospecific antibodies is not sufficient to improve CRS caused by CD3 bispecific antibodies, because the binding of functional groups targeting disease-related antigens in the bispecific antibodies to target cells will cause cross-linking of the antibodies. , and thus cause T cells to release large amounts of cytokines.
  • BCMA targeting shows certain potential in MM treatment.
  • BCMA due to the certain heterogeneity in the expression of BCMA, not all tumor cells express BCMA even in the same patient's tumor sample, resulting in different treatment responses.
  • membrane-bound BCMA will be affected by ⁇ -secretase. It is shed from the cell surface due to the action, causing changes in the amount of BCMA on the cell surface (Brudno JN et al., (2016) J. Clin. Oncol 36(22): 2267-2280; Laurent SA et al., (2015) Nat Commun. 6:7333).
  • These problems may be alleviated to a certain extent by simultaneously targeting another marker molecule on MM cells, such as GPRC5D, FcRH5, etc.
  • BCMA and GPRC5D on MM tumor cells
  • a CD3 antigen-binding domain can be added to the bispecific antibody to recruit the patient's T cells to surround MM cells and activate T cells to eliminate MM cells.
  • CD3 bi/multispecific antibodies cannot be ignored.
  • multiple BCMA ⁇ CD3 bispecific antibodies (AMG701, Elranatamab, etc.) suspended clinical trials due to safety issues.
  • the inventor of the present application through ingenious design, constructed a GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody that has strong tumor killing power and causes a small toxic reaction, which has made a breakthrough and improved the current treatment of MM.
  • the pharmacodynamic-toxicity balance of CD3 bispecific antibodies including BCMA ⁇ CD3 biantibodies and GPRC5D ⁇ CD3 bispecific antibodies.
  • the construction of this kind of multi-specific antibody is by no means a simple random combination of various binding groups, but must consider the interaction between each binding group.
  • the antibody of this application it is not only necessary to adjust and optimize the types of tumor-specific antigens bound to the MM to overcome the escape of killing caused by tumor heterogeneity, but also to reduce the release of cytokines caused by activation of the CD3 pathway and find a better Good treatment window.
  • the application provides a multispecific antibody, which may comprise:
  • CD3 antigen binding domain i) CD3 antigen binding domain, ii) BCMA antigen binding domain, and iii) GPRC5D antigen binding domain.
  • the CD3 antigen-binding domain may be an agonistic antigen-binding domain, which specifically binds CD3 and activates the CD3 signaling pathway.
  • this CD3 antigen-binding domain does not bind to CD3 ⁇ or CD3 ⁇ alone, but only to the CD3 ⁇ & ⁇ complex.
  • This CD3 antigen-binding domain has almost no activation effect on the CD3 signaling pathway in the free state, that is, it hardly activates T cells, and only appears as a "cross-link" when the other two antigen-binding domains of the multispecific antibody bind to their respective antigens. Only when it is in this state can it have the ability to activate the CD3 signaling pathway.
  • the CD3 antigen-binding domain is an antibody that specifically binds CD3, or an antigen-binding portion thereof.
  • the BCMA antigen-binding domain can bind BCMA and optionally block BCMA-APRIL binding/interaction, that is, it can optionally be an antagonistic antigen-binding domain.
  • the BCMA antigen-binding domain binds to BCMA on target cells and is almost unaffected by soluble BCMA in the environment.
  • the BCMA antigen-binding domain is an antibody that specifically binds BCMA, or an antigen-binding portion thereof.
  • the GPRC5D antigen-binding domain can specifically bind to GPRC5D.
  • the GPRC5D antigen-binding domain is an antibody that specifically binds GPRC5D, or an antigen-binding portion thereof.
  • the multispecific antibody of the present application may comprise, for example, a CD3 antigen-binding domain, a BCMA antigen-binding domain, and a GPRC5D antigen-binding domain.
  • the multispecific antibody of the present application can be an IgG-like antibody, including a CD3 antigen-binding domain in the form of Fab or Fv, a GPRC5D antigen-binding domain in the form of Fab or Fv, and a BCMA antigen-binding domain in the form of a single-chain antibody (scFv).
  • a CD3 antigen-binding domain in the form of Fab or Fv a GPRC5D antigen-binding domain in the form of Fab or Fv
  • a BCMA antigen-binding domain in the form of a single-chain antibody (scFv).
  • the multispecific antibody may comprise i) a CD3 half-antibody, comprising a heavy chain variable region, a heavy chain constant region, a light chain variable region and optionally a light chain constant region, ii) a GPRC5D half-antibody, Comprising a heavy chain variable region, a heavy chain constant region, a light chain variable region, and optionally a light chain constant region, and iii) a BCMA antigen binding domain in the form of a scFv, comprising a heavy chain variable region, an optional first a linker, and a light chain variable region, wherein the CD3 half-antibody and the GPRC5D half-antibody can form an IgG full antibody, and the BCMA antigen-binding domain in scFv form can, optionally via a second linker, be combined with the heavy chain variable region of the CD3 half-antibody region, the N-terminus of the light chain variable region, the C-terminus of the heavy chain constant region,
  • Multispecific antibodies of the present application may include:
  • a first polypeptide chain comprising a heavy chain variable region that specifically binds BCMA, a light chain variable region that specifically binds BCMA, a heavy chain variable region that specifically binds CD3, and a heavy chain constant region
  • a second polypeptide chain comprising a light chain variable region that specifically binds CD3,
  • a third polypeptide chain comprising a heavy chain variable region that specifically binds GPRC5D, and a heavy chain constant region
  • a first polypeptide chain comprising a heavy chain variable region that specifically binds CD3, and a heavy chain constant region
  • a second polypeptide chain comprising a heavy chain variable region that specifically binds BCMA, a light chain variable region that specifically binds BCMA, and a light chain variable region that specifically binds CD3,
  • a third polypeptide chain comprising a heavy chain variable region that specifically binds GPRC5D, and a heavy chain constant region
  • the heavy chain variable region that specifically binds to BCMA and the light chain variable region that specifically binds to BCMA form the BCMA antigen-binding domain.
  • the heavy chain variable region in the first polypeptide chain that specifically binds to CD3 and the heavy chain variable region in the second polypeptide chain specifically bind to BCMA form the BCMA antigen-binding domain.
  • the CD3 antigen-binding domain is formed by a light chain variable region that binds to CD3, a heavy chain variable region that specifically binds to GPRC5D in the third polypeptide chain, and a light chain variable region that specifically binds to GPRC5D in the fourth polypeptide chain that forms the GPRC5D antigen. binding domain, and the heavy chain constant region of the first polypeptide chain is bound together with the heavy chain constant region of the third polypeptide chain.
  • the first polypeptide chain may comprise from N-terminus to C-terminus a heavy chain variable region that specifically binds BCMA, a light chain variable region that specifically binds BCMA, a heavy chain variable region that specifically binds CD3, and a heavy chain constant region, or From N-terminus to C-terminus, it includes a light chain variable region that specifically binds BCMA, a heavy chain variable region that specifically binds BCMA, a heavy chain variable region that specifically binds CD3, and a heavy chain constant region.
  • the third polypeptide chain can be from From N-terminus to C-terminus, it contains the heavy chain variable region that specifically binds GPRC5D, and the heavy chain constant region.
  • a multispecific antibody can comprise:
  • the first polypeptide chain from N-terminus to C-terminus, includes a heavy chain variable region that specifically binds to BCMA, a first linker, a light chain variable region that specifically binds to BCMA, a second linker, and a heavy chain that specifically binds to CD3.
  • the second polypeptide chain from the N-terminus to the C-terminus, includes a light chain variable region that specifically binds CD3, and a light chain constant region,
  • a third polypeptide chain from the N-terminus to the C-terminus, comprising a heavy chain variable region and a heavy chain constant region that specifically bind GPRC5D, and
  • the fourth polypeptide chain from N-terminus to C-terminus, includes a light chain variable region that specifically binds GPRC5D, and a light chain constant region.
  • the first polypeptide chain may comprise a heavy chain variable region that specifically binds to CD3 and a heavy chain constant region from the N terminus to the C terminus
  • the second polypeptide chain may comprise a heavy chain that specifically binds to BCMA from the N terminus to the C terminus.
  • variable region, the light chain variable region that specifically binds to BCMA, and the light chain variable region that specifically binds to CD3, or the light chain variable region that specifically binds to BCMA from the N-terminus to the C-terminus, and the heavy chain that specifically binds to BCMA can variable region, and a light chain variable region that specifically binds to CD3, and the third polypeptide chain may comprise a heavy chain variable region that specifically binds to GPRC5D, and a heavy chain constant region from the N-terminus to the C-terminus.
  • a multispecific antibody can comprise:
  • the first polypeptide chain from N-terminus to C-terminus, includes a heavy chain variable region that specifically binds CD3, and a heavy chain constant region,
  • the second polypeptide chain from N-terminus to C-terminus, includes a heavy chain variable region that specifically binds to BCMA, a first linker, a light chain variable region that specifically binds to BCMA, a second linker, and a light chain that specifically binds to CD3.
  • a third polypeptide chain from the N-terminus to the C-terminus, comprising a heavy chain variable region and a heavy chain constant region that specifically bind GPRC5D, and
  • the fourth polypeptide chain from N-terminus to C-terminus, includes a light chain variable region that specifically binds GPRC5D, and a light chain constant region.
  • the BCMA antigen-binding domain can be an antibody that specifically binds to BCMA or an antigen-binding portion thereof, and its heavy chain variable region can include VH-CDR1, VH-CDR2 and VH-CDR3 respectively as shown in SEQ ID NOs: 1-3, and light chain variable regions.
  • the chain variable region may include VL-CDR1, VL-CDR2 and VL-CDR3 as shown in SEQ ID NOs: 4-6 respectively.
  • the heavy chain variable region and the light chain variable region in the BCMA antigen-binding domain may comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91% with SEQ ID NOs: 7 and 8, respectively. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the amino acid sequence.
  • the CD3 antigen-binding domain can be an antibody that specifically binds to CD3 or an antigen-binding portion thereof, and its heavy chain variable region can include VH-CDR1, VH-CDR2 and VH-CDR3 as shown in SEQ ID NOs: 9-11, respectively, and light
  • the chain variable region may include VL-CDR1, VL-CDR2 and VL-CDR3 as shown in SEQ ID NOs: 12-14 respectively.
  • the heavy chain variable region and the light chain variable region in the CD3 antigen binding domain may comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91% with SEQ ID NOs: 15 and 16, respectively. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the amino acid sequence.
  • the GPRC5D antigen-binding domain can be an antibody that specifically binds to GPRC5D or an antigen-binding portion thereof, and its heavy chain variable region can include VH-CDR1, VH-CDR2, and VH-CDR3 as shown in SEQ ID NOs: 17-19, respectively.
  • the chain variable region may include VL-CDR1, VL-CDR2 and VL-CDR3 as shown in SEQ ID NOs: 20-22 respectively.
  • the heavy chain variable region and the light chain variable region in the GPRC5D antigen binding domain may comprise at least 85%, 86%, 87%, 88%, 89%, 90%, 91% with SEQ ID NOs: 23 and 24, respectively. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the amino acid sequence.
  • the heavy chain constant region in the first polypeptide chain and the third polypeptide chain may be a heavy chain constant region that weakly binds or does not bind FcR, preferably a heavy chain constant region that does not bind FcR, such as human IgG1 (N297A), human IgG1(L234A+L235A), human IgG1(L234A+L235A+P329G/A), human IgG1(L234A+L235A+N297A), human IgG1(L234A+L235A+N297A+P329G/A), human IgG2(V234A+V237A) , and human IgG1 (L234A+V235E) constant region, or human IgG4 constant region.
  • human IgG1 (N297A) human IgG1(L234A+L235A
  • human IgG1(L234A+L235A+P329G/A human I
  • One of the heavy chain constant region of the first polypeptide chain and the heavy chain constant region of the third polypeptide chain may be a heavy chain constant region with a knot structure, such as human IgG1 or IgG4 heavy chain with T366W mutation. chain constant region or functional fragment thereof.
  • the other one of the heavy chain constant region of the first polypeptide chain and the heavy chain constant region of the third polypeptide chain can be a heavy chain constant region with an acetyl structure, such as a human with T366S/L368A/Y407V mutation. IgG1 or IgG4 heavy chain constant region or functional fragment thereof.
  • one of the heavy chain constant region of the first polypeptide chain and the heavy chain constant region of the third polypeptide chain may be a heavy chain constant region with a knot structure and weak or no FcR binding.
  • the other one of the heavy chain constant region of the first polypeptide chain and the heavy chain constant region of the third polypeptide chain may be a heavy chain constant region with an acetyl structure and weak or no FcR binding.
  • the light chain constant region in the second polypeptide chain and/or the fourth polypeptide chain can be, for example, a human gamma light chain constant region.
  • the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 26.
  • the first linker and the second linker can be peptides of about 5-30 amino acids in length. In certain embodiments, the linker can be a peptide of 5-20 amino acids in length. In certain embodiments, the linker may be a GS linker, such as a GS linker shown in SEQ ID NO: 36 or 37.
  • the multispecific antibody of the present application in some embodiments, its first polypeptide chain, second polypeptide chain, third polypeptide chain, and fourth polypeptide chain can respectively comprise i) SEQ ID NOs: 27 , 28, 29 and 30, or ii) SEQ ID NOs: 27, 16, 29 and 24 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, An amino acid sequence with 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the multispecific antibody of the present application can i) specifically bind to BCMA (human and monkey BCMA), with slightly better binding force than prior art antibodies such as EM801, and is almost unaffected by soluble BCMA in the environment, ii) can bind to CD3 , the binding capacity is significantly lower than that of existing technology antibodies such as JNJ-64407564 (hereinafter referred to as JNJ) and EM801, and the CD3 signal is only activated when the multispecific antibody binds BCMA and/or GPRC5D and presents a "cross-linked" state.
  • BCMA human and monkey BCMA
  • JNJ-64407564 hereinafter referred to as JNJ
  • EM801 JNJ-64407564
  • the activation effect on T cells is weaker than JNJ and/or EM801, iii) can bind GPRC5D, and the binding force is equivalent to JNJ or Slightly better, vi) can kill tumor cells in vitro, and the killing power is stronger than JNJ and EM801, v) has anti-tumor effect in vivo, and the effect is significantly stronger than EM801, and is equivalent to JNJ, iv) is almost not observed at the test concentration In vivo toxicity.
  • the trispecific antibody of the present application can more comprehensively kill BCMA + tumor cells, PRGC5D + tumor cells, and BCMA + PRGC5D + tumor cells, and is more It can effectively deal with the heterogeneity of tumor cell surface antigen expression. That is, regardless of whether the expression of BCMA and PRGC5D on tumor cells is high or low, they can be targeted and killed by the trispecific antibody of the present application.
  • the activation of T cells by the multispecific antibody of the present application is weaker than that of JNJ and EM801; in the presence of tumor cells with high expression of GPRC5D and low expression of BCMA, The multispecific antibody of the present application has weaker activating power on T cells than JNJ; in the presence of tumor cells with high expression of BCMA and low expression of GPRC5D, the activating power of the multispecific antibody of the present application on T cells is weaker than that of EM801.
  • the trispecific antibody of the present application can not only kill tumor cells more comprehensively, but also produce lower toxic and side effects.
  • the present application also provides nucleic acid molecules encoding the multispecific antibodies of the present application, as well as expression vectors containing the nucleic acids, and host cells containing the expression vectors or integrating the nucleic acids into the genome.
  • the present application also provides methods for preparing multispecific antibodies using host cells containing the above expression vectors, including: (i) expressing the multispecific antibodies in the host cells, and (ii) isolating the multispecific antibodies from the host cells or their cultures. specific antibodies.
  • the present application also provides pharmaceutical compositions comprising a therapeutically effective amount of the multispecific molecule, nucleic acid molecule, expression vector, or host cell of the present application, and a pharmaceutically acceptable carrier.
  • the present application provides a method of treating or slowing down a tumor or cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of the present application.
  • Tumors or cancers associated with BCMA and/or PRGC5D including, but not limited to, multiple myeloma, and other hematologic malignancies such as plasmacytoma, plasma cell leukemia, macroglobulinemia, and solitary plasmacytoma of bone , and extramedullary plasmacytoma, etc.
  • the tumor or disease is multiple myeloma.
  • the tumor or disease is multiple myeloma, and the myeloma cells highly express both BCMA and GPRC5D.
  • the subject is a mammal, particularly a human.
  • the present application also provides the use of the multispecific antibodies, nucleic acid molecules, expression vectors, host cells, or pharmaceutical compositions of the present application in the preparation of a medicament for treating or alleviating tumors or cancers.
  • Figure 1 shows a schematic structural diagram of an exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody.
  • Figure 2 shows exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody (MBS314), BCMA ⁇ CD3 bispecific antibody (EM801), and GPRC5D ⁇ CD3 bispecific antibody (JNJ) against HEK293A/human BCMA (A), Binding activities of HEK293A/monkey BCMA (B), HEK293A/mouse BCMA (C), HEK293T/human GPRC5D (D), HEK293T/monkey GPRC5D (E), and human T cell line Jurkat (F).
  • MCS314 BCMA ⁇ CD3 bispecific antibody
  • JNJ GPRC5D ⁇ CD3 bispecific antibody
  • Figure 3 shows FACS analysis of BCMA and GPRC5D expression in three multiple myeloma cell lines (A), and an exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody “cross-linked” to Jurkat- Activation activity of CD3 signaling pathway in NFAT-Luc (B).
  • Figure 4 shows GPRC5D and BCMA expression in myeloma MM.1S cells (A), and growth curves of MM.1S tumors in mice treated with an exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody (B).
  • Figure 5 shows the FACS analysis pattern of bone marrow mononuclear cells isolated from fresh samples of relapsed and refractory multiple myeloma patients after treatment with an exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody and CD138+ staining (A), And the statistical graph of the percentage of CD138+ cells (B).
  • Figure 6 shows the body weight changes of C57BL/6 mice and CD3 humanized mice after injection of exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibodies, where B6 represents C57BL/6 mice and CD3 represents CD3 humanized mice. mouse.
  • Figure 7 shows a PK kinetic profile of an exemplary GPRC5D ⁇ BCMA ⁇ CD3 trispecific antibody in mice.
  • CD3 refers to cluster of differentiation 3, including gamma chain, delta chain, epsilon chain, and zeta chain, etc.
  • CD3 ⁇ or “CD3E” refers to the epsilon chain of CD3.
  • CD3 delta or “CD3D” refers to the delta chain of CD3.
  • CD3D&E or “CD3 ⁇ & ⁇ ” refers to the ⁇ complex formed by a ⁇ chain and an ⁇ chain. The above terms include variants, homologs, orthologs and paralogs.
  • BCMA refers to a B-cell maturation antigen selectively expressed on malignant plasma cells in large numbers, including variants, homologs, orthologs and paralogs.
  • Human BCMA refers to a BCMA protein with a human amino acid sequence, such as the amino acid sequence shown in SEQ ID NO: 31.
  • Monkey BCMA refers to a BCMA protein having a monkey amino acid sequence, for example, having the amino acid sequence shown in SEQ ID NO: 32.
  • Mae BCMA refers to a BCMA protein having a mouse amino acid sequence, for example, having the amino acid sequence shown in SEQ ID NO: 33.
  • GPRC5D refers to group C 5 member D of the G protein-coupled receptor family, which is a marker molecule for myeloma cells and includes variants, homologs, orthologs and paralogs.
  • Human GPRC5D refers to a GPRC5D protein with a human amino acid sequence, such as the amino acid sequence shown in SEQ ID NO: 34.
  • Monkey GPRC5D refers to a GPRC5D protein having a monkey amino acid sequence, for example, having the amino acid sequence shown in SEQ ID NO: 35.
  • antibody as used herein is intended to include IgG, IgA, IgD, IgE and IgM full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) thereof.
  • “Whole antibody” or “full-length antibody” refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains, which are connected by disulfide bonds; while a “half-antibody” It is half of a “whole antibody” and contains, for example, a heavy chain and a light chain, linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH ) and a heavy chain constant region.
  • VH heavy chain variable region
  • VH heavy chain constant region
  • the heavy chain constant region consists of three domains, namely CH1 , CH2 and CH3 .
  • Each light chain is composed of a light chain variable region (abbreviated as VL ) and a light chain constant region.
  • the light chain constant region consists of one domain, CL .
  • the VH and VL regions can also be divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conserved framework region (FR) regions.
  • CDRs complementarity determining regions
  • FR conserved framework region
  • Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody can mediate binding of immunoglobulins to host tissues or factors, including various immune system cells (eg, effector cells) and the first component (Clq) of the traditional complement system.
  • the antibody constant regions of the present application are contemplated to have weak or no binding to immune system cells and complement system proteins.
  • the antigen-binding function of antibodies can be implemented by fragments of full-length antibodies, including, but not limited to, (i) Fab fragments, monovalent fragments composed of V L , V H , CL and CH1 ; (ii) F(ab′) 2 fragment, a bivalent fragment containing two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of V H and CH1 ; (iv) An antibody single arm V L and V Fv fragments consisting of H ; (v) dAb fragments consisting of V H (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity determining regions (CDRs); and (vii) nano Antibody, a heavy chain variable region containing a single variable domain and two constant domains.
  • Fab fragments monovalent fragments composed of V L , V H , CL and CH1
  • F(ab′) 2 fragment a bivalent fragment containing two Fab
  • the “IgG-like antibody” herein refers to an antibody obtained by maintaining the basic configuration of an IgG antibody and adding some additional groups such as an antigen-binding domain.
  • a "functional fragment" of a heavy chain constant region refers to a heavy chain constant region that retains the ability to perform a desired function (e.g., bind to another heavy chain constant region or fragment, bind to an Fc receptor and/or a component of the complement system) fragment.
  • agonistic CD3 antibody used herein refers to a CD3 antibody that can bind to CD3 and activate or trigger the CD3 signaling pathway to promote the activation and proliferation of immune cells such as T cells.
  • the CD3 antibody of the present application stimulates the CD3 signaling pathway and causes the activity of immune cells only in the "cross-linked" state.
  • cross-linking refers to binding of a BCMA-targeting moiety of a multispecific antibody to BCMA, a PRGC5D-targeting moiety binding to PRGC5D, and/or (having binding capacity for FcR and/or complement system components).
  • the Fc part binds to Fc receptors and/or components of the complement system, thereby causing antibody aggregation or interaction.
  • antibody cross-linking can be formed by binding the antibody Fc to an anti-Fc secondary antibody.
  • free means that there will be no interaction between antibodies or between antibodies and other molecules to cause dimerization or multimerization.
  • the multispecific antibody of the present application will not stimulate the CD3 signaling pathway in the free state. , and therefore does not activate immune cells such as T cells.
  • antigenic BCMA antibody or “blocking BCMA antibody” refers to an antibody capable of binding BCMA and capable of blocking or inhibiting the BCMA signaling pathway initiated by the interaction of BCMA with its ligands such as BAFF or APRIL, especially APRIL.
  • Antagonistic BCMA antibodies can block the proliferation and escape of apoptosis of malignant tumor cells.
  • the BCMA antigen-binding domain in the multispecific antibody of the present application can be antagonistic, binding to BCMA, blocking BCMA-APRIL binding/interaction, and not triggering the BCMA signaling pathway.
  • FcR refers to a protein expressed on the surface of some cells, such as B lymphocytes, natural killer cells, macrophages, etc., which can be bound by the Fc part of the antibody, triggering phagocytosis and toxicity of target cells, etc., in the immune system play an important role in.
  • FcR includes Fc ⁇ receptor, Fc ⁇ receptor, and Fc ⁇ receptor.
  • Fc ⁇ receptor termed immunoglobulin superfamily, is the most important FcR that triggers phagocytosis of microorganisms, including Fc ⁇ RI (CD64), Fc ⁇ RIIA (CD32A), Fc ⁇ RIIB (CD32B), and Fc ⁇ RIIIA (CD16A), etc.
  • multispecific antibody or “polyantibody” refers to an antibody that specifically binds to more than two (eg, three) target molecules, or to two or more (eg, three) different epitopes on the same target molecule.
  • Multispecific molecules include antibodies of the present application that specifically bind BCMA, PRGC5D, and CD3.
  • Bispecific antibodies or “bisantibodies” in some cases refer to antibodies that specifically bind to two target molecules, or two epitopes on one target molecule.
  • a “monospecific” antibody refers to an antibody that specifically binds to a certain target molecule, especially an epitope on a certain target molecule.
  • telomere binding binds to BCMA As used herein, “specifically binds to BCMA,””specifically binds to CD3,” or “specifically binds to PRGC5D” refers to an antibody that binds to BCMA, CD3, or PRGC5D but does not substantially bind to non-BCMA, CD3, or PRGC5D proteins.
  • the antibody binds to human BCMA, CD3 or PRGC5D protein with "high affinity", which means that the K D value is below 5.0x10 -7 M.
  • substantially does not bind to proteins or cells means that it does not bind to proteins or cells, or does not bind to them with high affinity, that is, the K D of binding proteins or cells is above 1.0 x 10 -6 M.
  • EC 50 also called half-maximal effect concentration, refers to the antibody concentration that causes 50% of the maximum effect.
  • IC 50 also known as the half-inhibitory concentration, refers to the concentration of a drug or inhibitor required to inhibit half of a specified biological process.
  • subject includes any human or non-human animal.
  • non-human animals includes all vertebrates, such as mammals and non-mammalian species, such as non-human primates, sheep, dogs, cats, cattle, horses, chickens, amphibians, and reptiles, although mammals are preferred, Examples include non-human primates, sheep, dogs, cats, cows, and horses.
  • an effective amount refers to an amount of the antibody of the present application that is sufficient to achieve the desired results.
  • therapeutically effective amount refers to an amount of an antibody of the present invention sufficient to prevent or slow symptoms associated with a disease or condition (eg, cancer). The therapeutically effective amount is related to the disease being treated, and those skilled in the art can easily determine the actual effective amount.
  • the multispecific antibodies of the present application compared with prior art antibodies such as JNJ and EM801, have i) equivalent or higher BCMA binding capacity, ii) equivalent or higher PRGC5D binding capacity, iii) significantly lower CD3 Binding power, vi) equivalent or better in vitro tumor killing potency, v) equivalent or better in vivo tumor killing potency, and/or iv) equivalent or lower in vivo toxicity.
  • the trispecific antibody constructed by the inventor of this application through ingenious design not only has strong tumor killing power but also causes less toxic reactions. It can break through and improve the current CD3 bispecific antibodies in the treatment of MM, including BCMA ⁇ The balance of efficacy and toxicity of CD3 double antibodies and GPRC5D ⁇ CD3 double antibodies.
  • the trispecific antibody of the present application can more comprehensively kill BCMA + tumor cells, PRGC5D + tumor cells, and BCMA + PRGC5D + tumor cells, and is more It can effectively deal with the heterogeneity of tumor cell surface antigen expression. That is, regardless of whether the expression of BCMA and PRGC5D on tumor cells is high or low, they can be targeted and killed by the trispecific antibody of the present application.
  • the activation of T cells by the multispecific antibody of the present application is weaker than that of JNJ and EM801; in the presence of tumor cells with high expression of GPRC5D and low expression of BCMA, The multispecific antibody of the present application has weaker activating power on T cells than JNJ; in the presence of tumor cells with high expression of BCMA and low expression of GPRC5D, the activating power of the multispecific antibody of the present application on T cells is weaker than that of EM801.
  • the trispecific antibody of the present application can not only kill tumor cells more comprehensively, but also produce lower toxic and side effects.
  • the multispecific antibody of the present application has a killing advantage and produces less toxic and side effects.
  • the heavy chain variable region CDR and light chain variable region CDR of the monospecific antibody or antigen-binding fragment thereof used in the multispecific antibody of the present application are determined by the Kabat numbering system. It is well known in the art that heavy chain variable region and light chain variable region CDRs can be determined by, for example, Chothia, IMGT, AbM or Contact numbering systems/methods.
  • Multispecific antibodies of the present application also include bispecific antibodies.
  • the multispecific antibodies of the present application may comprise: i) CD3 antigen binding domain, ii) BCMA antigen binding domain, and iii) GPRC5D antigen binding domain.
  • the CD3 antigen-binding domain may be an agonistic antigen-binding domain, which specifically binds CD3 and activates the CD3 signaling pathway.
  • the BCMA antigen-binding domain can bind BCMA and optionally block BCMA-APRIL binding/interaction, that is, it can optionally be an antagonistic antigen-binding domain.
  • the GPRC5D antigen-binding domain can specifically bind to GPRC5D.
  • the multispecific antibody of the present application may comprise, for example, a CD3 antigen-binding domain, a BCMA antigen-binding domain, and a GPRC5D antigen-binding domain.
  • the multispecific antibody of the present application can be an IgG-like antibody, including a CD3 antigen-binding domain in the form of Fab or Fv, a GPRC5D antigen-binding domain in the form of Fab or Fv, and a BCMA antigen-binding domain in the form of a single-chain antibody (scFv).
  • a CD3 antigen-binding domain in the form of Fab or Fv a GPRC5D antigen-binding domain in the form of Fab or Fv
  • a BCMA antigen-binding domain in the form of a single-chain antibody (scFv).
  • the multispecific antibody may comprise i) a CD3 half-antibody, comprising a heavy chain variable region, a heavy chain constant region, a light chain variable region and optionally a light chain constant region, ii) a GPRC5D half-antibody, comprising a heavy chain variable region , a heavy chain constant region, a light chain variable region, and optionally a light chain constant region, and iii) a BCMA antigen binding domain in the form of a scFv, comprising a heavy chain variable region, an optional first linker, and a light chain variable region.
  • the CD3 half-antibody and the GPRC5D half-antibody can form an IgG full antibody
  • the BCMA antigen-binding domain in the form of scFv can, optionally via a second linker, be combined with the N-terminus and light chain variable region of the CD3 half-antibody.
  • the scFv form of the BCMA antigen binding domain is linked to the N-terminus of the heavy chain variable region of the CD3 half-antibody, or the N-terminus of the light chain variable region.
  • the BCMA antigen-binding domain in scFv form may comprise a heavy chain variable region, an optional first linker, and a light chain variable region from N-terminus to C-terminus; or a light chain variable region, an optional first linker, and Heavy chain variable region.
  • One of the heavy chain constant regions of the two half-antibodies can be a heavy chain constant region with a knot structure and weak or no binding to FcR, such as the human IgG1 heavy chain constant region with L234A/L235A/N297A/T366W
  • the other heavy chain constant region can be a heavy chain constant region with an acetal structure and weak or no binding to FcR, such as the human IgG1 heavy chain constant region with L234A/L235A/N297A/T366S/L368A/Y407V.
  • the first linker and the second linker may be composed of amino acids connected by peptide bonds, preferably 5-30 amino acids connected by peptide bonds, wherein the amino acids are selected from 20 naturally occurring amino acids. One or more of these amino acids may be glycosylated, as will be understood by those skilled in the art. In one embodiment, 5-30 amino acids may be selected from glycine, alanine, proline, asparagine, glutamine, serine and lysine. In one embodiment, the linker is composed of amino acids that are mostly sterically hindered by empty bonds, such as glycine and alanine. Exemplary linkers are polyglycine, especially poly(Gly-Ala), and polyalanine. Exemplary linkers in this application may be as shown in SEQ ID NOs: 19, 20, 21 or 22.
  • Linkers can also be non-peptidic linkers.
  • These alkyl linkers may also be substituted with any non-sterically hindered group such as lower alkyl (eg C 1-6 lower acyl), halogen (eg Cl, Br), CN, NH 2 , phenyl, etc.
  • the multispecific antibody of the present application contains one or more conservatively modified heavy chain and/or light chain variable region sequences or CDR1, CDR2 and CDR3 sequences with the antibody of the present application. It is known in the art that some conservative sequence modifications do not eliminate antigen binding properties.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as point mutations and PCR-mediated mutagenesis. Conservative amino acid substitutions are the replacement of amino acid residues with amino acid residues with similar side chains. Groups of amino acid residues with similar side chains are known in the art.
  • amino acid residue groups include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., lysine, arginine, histidine), e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains ( For example, tyrosine, phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains
  • one or more amino acid residues in the CDR region of an antibody of the present application can be replaced with other amino acid residues from the same side chain group, and the resulting antibody can be tested for retaining functionality using the functional assays described herein (i.e., testing of the above functions).
  • the multispecific antibody of the present application can be prepared into a genetically modified antibody by using an antibody having one or more V H /V L sequences of the antibody used in the present application as a starting material.
  • Antibodies can be modified by modifying one or more residues within one or both variable regions (i.e., VH and/or VL ) (e.g., in one or more CDR regions and/or one or more framework regions) to perform genetic modifications to improve binding affinity and/or increase similarity to antibodies naturally produced by certain species.
  • VH and/or VL variable regions
  • the backbone region is modified to provide a humanized antibody.
  • the antibody can be genetically modified by modifying residues in the constant region, for example to alter the antibody's effector function.
  • each heavy chain variable region and/or each light chain variable region contained therein may include VH-CDR1, VH-CDR2, VH-CDR3 in the present application, and/or VL-CDR1, VL-CDR2 and VL-CDR3, but contain different backbone sequences.
  • the inventors of the present application have discovered that when the antigen-binding domain is presented in scFv form, the stability may generally be weaker than that in whole antibody or Fab form. Therefore, in one embodiment of the present application, in order to solve the stability problems that may arise when the BCMA antigen-binding domain is used in the form of scFv in the construction of multispecific antibodies of the present application, based on computer structural simulation, BCMA heavy/light chain variable
  • the framework sequences of the region such as the amino acids of the FR3 and FR4 parts of the light chain variable region, were modified to increase the stability of the scFv form, reduce the formation of aggregates, and increase the monomer ratio.
  • the Fab form and scFv form before and after modification are equivalent in terms of affinity, BCMA-APRIL blocking activity and anti-tumor effect.
  • the inventor of the present application also found that the framework sequence of the BCMA heavy/light chain variable region can be changed through computer structural simulation, for example, by changing the hydrophilicity and hydrophobicity of the amino acids in the framework region sequence, thereby further improving the druggability of the multispecific antibody and thus improving the Industrial expression.
  • Genetically modified antibodies of the present application include those in which genetic modifications are made in the backbone residues of VH and/or VL to, for example, alter the properties of the antibody. Typically, these backbone modifications are used to reduce the immunogenicity of the antibody. For example, one approach is to "backmute" one or more backbone residues to the corresponding germline sequence. More specifically, antibodies that undergo somatic mutations may contain backbone residues that differ from the germline sequence from which the antibody was derived. These residues can be identified by comparing the antibody backbone sequence to the germline sequence from which the antibody was derived.
  • backbone modification involves mutating one or more residues of the backbone region, or even one or more CDR regions, to remove T cell epitopes, thereby reducing the possible immunogenicity of the antibody. This method is also called “deimmunization” and is described in more detail in US Patent Publication 20030153043.
  • the antibodies of the present application can be genetically engineered to include genetic modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation , Fc receptor binding, and/or antibody-dependent cytotoxicity.
  • the antibodies of the present application can be chemically modified (for example, one or more chemical functional groups can be added to the antibody), or modified to change its glycosylation to change one or more functional properties of the antibody.
  • the hinge region of CH1 is modified, such as by increasing or decreasing the number of cysteine residues in the hinge region. This method is further described in US Patent 5,677,425. Changing cysteine residues in the CH1 hinge region, for example, to promote the assembly of heavy and light chains or to increase/decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2 - CH3 linker region of the Fc-hinge fragment such that the antibody has reduced SpA binding relative to native Fc-hinge domain SpA binding. This method is described in more detail in US Patent 6,165,745.
  • the glycosylation of the antibody is modified.
  • deglycosylated antibodies can be prepared (i.e., antibodies lack glycosylation).
  • Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen.
  • Such glycosylation modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence.
  • one or more amino acid substitutions can be made to eliminate one or more variable region backbone glycosylation sites, thereby eliminating glycosylation at that position.
  • Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, US Patent Nos. 5,714,350 and 6,350,861.
  • antibodies with altered glycosylation types such as low-fucosyl antibodies with a reduced amount of fucose residues, or antibodies with an increased bisecting GlcNac structure, can be produced.
  • Altered glycosylation patterns have been shown to increase the ADCC activity of antibodies.
  • Such glycosylation modification can be performed, for example, by expressing the antibody in a host cell with an altered glycosylation system.
  • Cells with altered glycosylation systems are known in the art and can be used as host cells for expressing the recombinant antibodies of the present application to produce antibodies with altered glycosylation.
  • the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 ( ⁇ (1,6)-fucosyltransferase), so that antibodies expressed in the Ms704, Ms705, and Ms709 cell lines contain a Fucose is missing.
  • PEGylation pegylation
  • Antibodies can be PEGylated, for example, to increase the biological (eg, serum) half-life of the antibody.
  • PEGylate an antibody the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that attach one or more PEG groups to the antibody or antibody fragment. reaction.
  • PEG polyethylene glycol
  • the present application provides coding for the multispecific antibody of the present application such as BCMA heavy chain variable region-first linker-BCMA light chain variable region-second linker-CD3 heavy chain variable region-heavy chain constant region, Nucleic acid molecules such as CD3 light chain variable region-light chain constant region, PRGC5D heavy chain variable region-heavy chain constant region, PRGC5D light chain variable region-light chain constant region, etc.
  • Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure form.
  • a nucleic acid is "isolated” or “substantially pure” when purified by standard techniques from other cellular components or other contaminants such as other cellular nucleic acids or proteins.
  • the nucleic acid of the present application may be, for example, DNA or RNA, and may or may not contain intronic sequences.
  • the nucleic acid is a cDNA molecule.
  • Nucleic acids of the present application can be obtained using standard molecular biology techniques.
  • Preferred nucleic acid molecules of the present application may include those encoding VH and VL sequences or CDRs of CD3, BCMA, PRGC5D monospecific antibodies.
  • these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes.
  • a DNA fragment encoding VH or VL is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operably linked" means that two DNA fragments are joined together such that the amino acid sequences encoded by both DNA fragments are in reading frame.
  • the DNA fragment encoding VH and VL can be operably linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser) 3 , so that the VH and VL sequences can be expressed as a continuous single Chain proteins are expressed in which the VH and VL regions are connected through the flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al. (1990) Nature 348: 552-554).
  • a flexible linker such as the amino acid sequence (Gly4-Ser) 3
  • sequences encoding the CDRs, VH and VL, and linkers of CD3, BCMA, and PRGC5D antibodies can be obtained first, and then these sequences can be combined according to the desired form of the multispecific antibody.
  • sequences encoding a BCMA heavy chain variable region, a first linker, a BCMA light chain variable region, a second linker, a CD3 heavy chain variable region, and a heavy chain constant region can be operably linked together as desired.
  • the multispecific antibodies of the present application can be prepared by i) inserting the sequence encoding each polypeptide chain of the multispecific antibody into one or more expression vectors, wherein one or more expression vectors are operably linked to transcription and translation control sequences; ii) ) transducing or transfecting the host cell with an expression vector, and iii) expressing the polypeptide chain to form the multispecific antibody of the present application.
  • regulatory sequences includes promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody genes.
  • the expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell.
  • the antibody chain gene can be cloned into a vector such that the signal peptide is linked in reading frame to the amino terminus of the antibody chain gene.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin).
  • the expression vectors of the present application may carry other sequences, such as sequences that regulate replication of the vector in host cells (eg, origin of replication) and selectable marker genes.
  • the selectable marker gene confers drug resistance, such as G418, hygromycin, or methotrexate resistance, into the host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection/amplification of dhfr host cells) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • Expression vectors encoding different polypeptide chains of multispecific antibodies are transfected into host cells by standard techniques.
  • the term "transfection" in its various forms includes a variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, for example, electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, etc.
  • electroporation calcium phosphate precipitation
  • DEAE-dextrose transfection etc.
  • it is theoretically possible to express the antibodies of the present application in prokaryotic or eukaryotic host cells it is preferred that the antibodies are expressed in eukaryotic cells, and most preferably in mammalian host cells, because eukaryotic cells, especially mammalian cells, More likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.
  • expression vectors useful herein include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), transformable artificial chromosomes (TAC), mammalian artificial chromosomes (MAC), and artificial accessory chromosomes (HAEC).
  • YAC yeast artificial chromosomes
  • BAC bacterial artificial chromosomes
  • TAC transformable artificial chromosomes
  • MAC mammalian artificial chromosomes
  • HAEC artificial accessory chromosomes
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising one or more multispecific antibodies of the present application, a nucleic acid molecule encoding a multispecific antibody, an expression vector comprising the nucleic acid molecule, and/or comprising
  • the host cell of the nucleic acid molecule is formulated with a pharmaceutically acceptable carrier.
  • the composition may optionally include one or more other pharmaceutically active ingredients, such as another anti-tumor drug, or an immune-enhancing drug.
  • the pharmaceutical composition of the present application can be used in combination with, for example, another anti-cancer agent, or another immune enhancer.
  • compositions may contain any number of excipients.
  • Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solubilizers, coloring agents, flavoring agents, coatings, disintegrating agents, lubricants, Sweeteners, preservatives, isotonic agents and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).
  • compositions are suitable for oral, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or bolus).
  • the active ingredient may be encased in materials to protect it from acids and other natural conditions that may inactivate it.
  • Parenteral administration refers to methods other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intrasaccular, intraorbital, intracardiac, intradermal, Intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and bolus.
  • the antibodies of the present application may be administered by parenteral routes, such as topical, epidermal, or mucosal administration, such as intranasal, oral, vaginal, rectal, sublingual, or topical.
  • the pharmaceutical composition of the present application is administered orally.
  • compositions may be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.
  • the specific application of the pharmaceutical composition of the present application can be determined by medical workers, such as doctors, based on the specific conditions of the subject, such as gender, age, past medical history, etc.
  • a “therapeutically effective amount” of a multispecific antibody of the present application causes a reduction in the severity of disease symptoms or an increase in the frequency and duration of symptom-free periods.
  • a "therapeutically effective amount” preferably reduces tumor by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and more preferably at least Approximately 80% or even complete elimination of tumors.
  • compositions may be sustained release agents, including implants, and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • the antibodies of the present application are formulated to ensure appropriate in vivo distribution.
  • the antibodies can be formulated in liposomes, which can additionally contain targeting functional groups to enhance selective delivery to specific cells or organs.
  • the pharmaceutical composition of the present application has a variety of in vitro and internal and external applications, for example, it can be used for the treatment and alleviation of tumors or cancer.
  • the pharmaceutical composition of the present application can be used to treat or slow down tumors or cancers related to BCMA and/or PRGC5D, including, but not limited to, multiple myeloma, and other hematological malignancies, such as plasmacytoma and plasma cell leukemia. , macroglobulinemia, solitary plasmacytoma of bone, and extramedullary plasmacytoma.
  • the present application provides a combination therapy in which the pharmaceutical composition of the present application is administered together with one or more other antibody or non-antibody therapeutic agents, such as immune enhancers and the like.
  • combinations of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or as separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.
  • the order of the sequential administrations at each time point can be reversed or remain the same, and the sequential administrations can be combined with simultaneous administration or any combination thereof.
  • Example 1 Construction of cell lines stably expressing BCMA or GPRC5D
  • HEK293A cells were used to construct cell lines that stably overexpress human BCMA, monkey BCMA, and mouse BCMA respectively. Briefly speaking, the cDNA sequences encoding human BCMA, monkey BCMA, and mouse BCMA (the amino acid sequences are shown in SEQ ID NOs: 31, 32, and 33 respectively) were synthesized and cloned into pLV-EGFP(2A)- In the BamH1 and EcoR1 restriction sites of Puro vector (Beijing Yingmaoshengye Biotechnology Co., Ltd., China).
  • the obtained pLV-EGFP(2A)-Puro-human BCMA, pLV-EGFP(2A)-Puro-monkey BCMA, pLV-EGFP(2A)-Puro-mouse BCMA, psPAX and pMD2.G plasmids were passed through liposomes
  • the transfection method was transfected into HEK293T cells (Nanjing Kebai Company, China) to produce lentivirus.
  • the specific transfection method was completely consistent with the instructions of the Lipofectamine 3000 kit (Thermo Fisher Scientific, USA).
  • lentivirus was harvested from the cell culture medium (DMEM medium (Cat#: SH30022.01, Gibco) supplemented with 10% FBS (Cat#: FND500, Excell)) of HEK293T cells.
  • DMEM medium Cat#: SH30022.01, Gibco
  • FBS Cat#: FND500, Excell
  • HEK293A cells Nejing Kebai Company, China
  • HEK293A/human BCMA HEK293A/monkey BCMA
  • HEK293A/mouse BCMA cells referred to as HEK293A/mouse BCMA cells, respectively.
  • Transfected HEK293A cells were cultured in DMEM+10% FBS medium containing 0.2 ⁇ g/ml purinotoxin (Cat#: A11138-03, Gibco) for 7 days.
  • the expression of human and monkey BCMA was analyzed by FACS by flow cytometry using a commercially available BCMA antibody (PE-anti-human BCMA antibody, Cat#: 357503, Biolegend, USA).
  • expression of mouse BCMA was confirmed by FACS with a commercially available mouse BCMA antibody (anti-mouse BCMA antibody, Cat#: O88472, Novus, USA).
  • HEK293T cell lines that stably overexpress human and monkey GPRC5D respectively were constructed, using cDNA encoding human and monkey GPRC5D respectively (the amino acid sequences are shown in SEQ ID NOs: 34 and 35 respectively), and using GPRC5D antibodies (GPRC5D antibodies according to The amino acid sequences 10 and 11 in the published patent WO2022058445A1 were prepared, 20 ⁇ g/ml final concentration) to detect the expression of human and monkey GPRC5D.
  • Trispecific antibodies were constructed using an asymmetric whole-antibody assembly with a BCMA:CD3:GPRC5D antigen-binding domain ratio of 1:1:1.
  • the structure of an exemplary three-antibody is shown in Figure 1.
  • GS vector see ZL200510064335.0 for detailed information
  • MBS-314 pestle and MBS-314 mortar of the exemplary third antibody were constructed.
  • the long chain of MBS-314 contains BCMA scFv-linker-CD3 antibody heavy chain variable region-heavy chain constant region.
  • the amino acid sequence is shown in SEQ ID NO: 27, and the short chain contains the CD3 antibody light chain variable region- The light chain constant region, the amino acid sequence is shown in SEQ ID NO: 28, the long chain of MBS-314 contains the GPRC5D antibody heavy chain variable region - the heavy chain constant region, the amino acid sequence is shown in SEQ ID NO: 29, the short chain Contains the GPRC5D antibody light chain variable region-light chain constant region, and the amino acid sequence is shown in SEQ ID NO: 30.
  • the backbone of the light chain variable region (such as the fourth framework region) has been modified to make the scFv structure more stable.
  • Double enzyme digestion was performed on the two fragments, and the digested gene fragment was cloned into a vector containing the light chain constant region (SEQ ID NO: 26) to complete the assembly of two half-antibody short chain full-length gene expression vectors.
  • ClaI and HindIII were used to digest the short-chain full-length gene of the half-antibody, EcoRI and XhoI to digest the long-chain full-length gene of the half-antibody, HindIII and EcoRI to digest the pCMV-cofragment plasmid, and ClaI and XhoI to digest the GS-vector.
  • the four recovered DNA fragments were ligated, transformed, and single clones were selected for sequencing to obtain semi-antibody expression vectors containing the correct sequence, which were named MBS-314 pestle expression vector and MBS-314 mortar expression vector respectively.
  • Plasmids were extracted using Tiangen endotoxin-free plasmid maximal extraction kit (Cat#: DP117, Tiangen). Resuspend the plasmid in 5 mL of FreeStyle F17 cell culture medium. At the same time, take 3 times the volume of plasmid transfection reagent (PEI) and resuspend it in 5 mL of FreeStyle F17 cell culture medium. Slowly add PEI to the plasmid, mix thoroughly, and leave at room temperature for about 15 minutes.
  • PEI plasmid transfection reagent
  • the transfected HEK-293F cells were cultured in a 37°C, 5% CO 2 incubator with a rotation speed of 120 RPM. After 10-12 days, the cell culture supernatant was collected, centrifuged at 3500 rpm for 5 minutes, and filtered through a 0.22 ⁇ m filter to remove cell debris.
  • the half-antibody was enriched and purified by pre-equilibrated protein-A affinity column (Cat#: 17040501, GE, USA). Then use elution buffer (20mM citric acid, pH3.0-pH3.5) for elution. Afterwards, the antibodies were stored in PBS (pH 7.0), and the antibody concentration was detected by NanoDrop. The purified half-antibodies were further assembled.
  • the purified half-antibodies were assembled in vitro. Specifically, MBS-314 pestle and MBS-314 mortar half-antibodies were mixed at a molar ratio of 1:1, adjusted to pH 8.0 with Tris alkali buffer solution, a certain amount of reduced glutathione solution was added, and the reaction was performed at 25°C and stir on low speed overnight. After the reaction, adjust the pH value to 5.5 with 2M acetic acid solution. The reaction was terminated by removing the reducing agent by ultrafiltration. The assembled antibody was transferred to low-salt Tris buffer solution (pH 8.0) and filtered with a 0.2 ⁇ m filter membrane.
  • the assembled double antibody was named MBS314.
  • the purified antibody was identified by mass spectrometry as having a purity higher than 90% and was used for subsequent functional testing.
  • MBS314 antibody The binding affinity of MBS314 antibody to human CD3, human GPRC5D and human BCMA was determined by capture method (BIAcore 8K).
  • Mouse anti-human Fc segment antibody (Cat#: BR100839, cytiva) was coupled to the surface of chip CM5.
  • HBS-EP buffer (Cat#: BR-1006-69, GE Life Sciences) dilutes the antibody to be tested to 1 ⁇ g/mL to ensure that approximately 100RU of the antibody is captured by the anti-human Fc antibody.
  • Human BCMA antigen (Cat#: BCA-H522y, ACRO), human CD3D&E (Cat#: CT038-H2508H, Yiqiao Shenzhou) protein, and human GPRC5D antigen (Cat#: HM05P, Kaika Biotech) were buffered with HBS-EP The solution was diluted to 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, and 0.003900625 ⁇ g/ml. Flow each protein at different concentrations through the stationary phase surface. Regenerate the chip using 3M MgCl2 solution. Kinetic experiments were performed using the Kinetic Analysis Wizard in Biacore 8K control software.
  • JNJ (GPRC5D ⁇ CD3 bispecific antibody) and EM801 (BCMA ⁇ CD3 bispecific antibody) were used as positive controls.
  • JNJ was prepared according to the amino acid sequences 10, 11, 20, and 21 in the published patent WO2022058445A1;
  • EM801 was prepared according to WO2016020332A1 Amino acid sequences 43, 44, 45 and 46 were prepared. Fitting was performed according to Biacore 8K evaluation software to obtain the KD value of the detection antibody against the above antigens, which is summarized in Table 1.
  • MBS314 antibody has strong binding affinity to human CD3, human GPRC5D, and human BCMA. Among them, MBS314 has lower binding affinity than EM801 and JNJ for the CD3D&E complex.
  • Example 5 CD3 ⁇ BCMA ⁇ GPRC5D trispecific antibody and HEK293A/human BCMA cells, HEK293A/monkey BCMA cells, HEK293A/mouse BCMA cells, HEK293T/human GPRC5D cells cells, HEK293T/monkey GPRC5D cells and Jurkat cells
  • the binding activity of MBS314 to human BCMA, monkey BCMA, mouse BCMA, human GPRC5D, monkey GPRC5D and human CD3 complex expressed on the cell surface was detected by FACS.
  • trypsin digestion was used to collect HEK293A/human BCMA, HEK293A/monkey BCMA, HEK293A/mouse BCMA, HEK293T/human GPRC5D cells, HEK293T/monkey GPRC5D cells (prepared in Example 1), and Jurkat cells in good growth status. (Nanjing Kebai), centrifuged at 300 g for 5 min to remove the culture medium. Resuspend in PBS for counting and centrifuge again to remove the supernatant. Resuspend the cells in 2% FBS-PBS solution and adjust the cell concentration to 4 ⁇ 10 6 cells/ml. Add each cell to a U-shaped bottom 96-well plate at 50 ⁇ l/well.
  • MBS314 antibody has strong binding ability to HEK293A/human BCMA and HEK293A/monkey BCMA cells in a dose-dependent manner, but does not bind to mouse BCMA.
  • Figure 2 (D and E) shows that the MBS314 antibody binds to both HEK293T/human GPRC5D and HEK293T/monkey GPRC5D, and the binding activity of MBS314 to human GPCR5D and monkey GPCR5D is significantly higher than that of JNJ.
  • Figure 2F shows that MBS314 has lower CD3 complex binding activity compared to JNJ.
  • the activation of T cell signaling by the trispecific antibody of the present application is determined by the following assay.
  • the antibody binds to GPRC5D and/or BCMA on the surface of tumor cells and presents a "cross-linked" state, it can bind to CD3 on the surface of JURKAT-NFAT-Luc cells and activate NFAT signaling, causing JURKAT-NFAT-Luc cells to secrete luciferase , by detecting luciferase activity, the level of T cell activation mediated by the antibody can be determined.
  • hybridoma antibodies against GPRC5D and BCMA were used to detect the expression levels of GPRC5D and BCMA on the surface of multiple myeloma cells, and thereby select cells NCI-H929 (KC- 0629, Kangyuan Bochuang), cells with high GPRC5D expression and low BCMA expression, MOLP8 (KC-0622, Kangyuan Bochuang), and cells with low GPRC5D and high BMCA expression, KMS-11 (KC-0611, Kangyuan Bochuang). ), conduct subsequent T cell activation experiments.
  • NCI-H929 KC- 0629, Kangyuan Bochuang
  • MOLP8 KC-0622, Kangyuan Bochuang
  • KMS-11 KC-0611, Kangyuan Bochuang
  • the above myeloma cells (1 million/ml, 100,000/well) were suspended in 100 ⁇ l of PBS (containing 2% BSA), and incubated with GPRC5D hybridoma antibody or BCMA hybridoma antibody (20 ⁇ g/ml) at room temperature for half an hour. . After centrifugation and washing, PE-anti-mouse secondary antibody (1:400, biolegend, 405307) was added to the cells. After centrifugation and washing, the cells were suspended, 7-AAD was added, and the expression levels of GPRC5D and BCMA in tumor cells were analyzed by flow cytometry.
  • Figure 3(A) shows the flow cytometric analysis of the above three types of cells.
  • MBS314 can activate CD3 signaling on all three types of cells, even if the expression of GPRC5D or BCMA on tumor cells is low.
  • the CD3 signaling pathway caused by MBS314 is significantly lower than that of JNJ and EM801.
  • Myeloma cells MM.1S (KC-0620, Kangyuan Bochuang) expressing both GPRC5D and BCMA were subcutaneously injected into immunodeficient mice B-NDG (Biocyto), and human T cells were injected intravenously into the mice to establish In vivo pharmacodynamic model of humanized mouse tumors.
  • Figure 4(A) shows the expression of GPRC5D and BCMA in MM.1S cells.
  • myeloma cells MM.1S were revived, cultured in RPMI-1640 medium (containing 10% FBS), and passaged once every 3 to 4 days.
  • RPMI-1640 medium containing 10% FBS
  • ⁇ 1 ⁇ 10 8 PBMC were resuscitated, and each 1 ⁇ 10 6 cell was co-cultured with 20 ⁇ l CD3/CD28 immunomagnetic beads (Cat#: 11161D, GIBCO). T cells were expanded in vitro for about 8 days.
  • MM.1S tumor cells were collected, and the tumor cell density was adjusted to 2.5 ⁇ 10 7 /ml with a 1:1 mixture of Matrigel (GIBCO, 356234) and PBS, and the right abdomen of each NDG mouse was Inject 0.2ml subcutaneously, that is, 0.5 ⁇ 10 7 cells/mouse.
  • the expanded T cells were collected and suspended in PBS containing 0.1% FBS and 10ng/ml IL-2 at a density of approximately 0.5 ⁇ 10 8 /ml.
  • Each mouse was injected with 0.2ml T cells into the tail vein, which is approximately 1 ⁇ 10 7 cells.
  • the trispecific antibody of this application JNJ, EM801 or PBS is injected through the tail vein at a dose of 10 ⁇ g per mouse, twice a week for a total of 7 times. .
  • Figure 4(B) shows changes in tumor size of individual mice in each group. It can be seen that MBS314 can eliminate tumor cells in mice, and the effect is significantly better than EM801 and comparable to JNJ.
  • Example 8 The lethality of CD3 ⁇ BCMA ⁇ GPRC5D trispecific antibody to clinical tumor samples
  • Bone marrow mononuclear cells were isolated from fresh samples obtained from patients with relapsed and refractory multiple myeloma, and suspended in culture medium RPMI1640 (containing 10% FBS, 1% penicillin-streptomycin, 100ng/ ml IL-6, and 10ng/ml IL-2). Use the above culture medium to dilute the antibody with a starting concentration of 100 ⁇ g/ml and 10-fold gradient dilution for a total of 6 concentrations. In a U-bottomed 96-well, add 90 ⁇ l of cell suspension and 10 ⁇ l of gradient diluted antibodies of each concentration into each well.
  • the supernatant was removed by centrifugation, and the cells were resuspended in 100 ⁇ l FITC-anti-human CD138 (1:100, Biolegend, 356508), and incubated on ice for 30 minutes.
  • the antibodies were removed by centrifugation, 7-AAD was added to the cells, and the proportion of CD138+ cells in viable cells was analyzed by FACS.
  • Figure 5(A) shows the FACS analysis chart
  • Figure 5(B) is a data statistical chart. The results showed that MBS314 was more lethal to tumor cells than both JNJ and EM801.
  • 9 B-hCD3EDG mice (CD3 humanized, Biocytogen) were divided into 3 groups, 9 C57BL/6 mice were divided into 3 groups, and MBS314, JNJ and PBS were injected through the tail vein respectively, 20mg/ kg, injected twice a week for a total of 3 injections. Measure body weight every day, calculate the percentage of body weight per day relative to day 0, and draw the body weight-experiment day correlation curve. The 10th day is the end point of the experiment.
  • 50-100 ⁇ l of anticoagulated blood is extracted, and the mouse blood routine is performed, and the heart, liver, spleen, kidney, bone marrow, brain, lung, and thymus are taken.
  • mice Female BALB/c mice aged 10-12 weeks were selected, and MBS314 was injected through the tail vein at 3.0mg/kg, 1.0mg/kg or 0.3mg/kg at 10 minutes, 1 hour, 6 hours, 1 day and 3 days. , blood was collected after 5 days, 7 days and 14 days. Blood samples are specifically collected through the orbit or tail vein (anticoagulation treatment is required after collection), and approximately 0.03ml is collected from each mouse each time. Take 10-30 ⁇ l of blood sample, add 4 times the volume of PBS, centrifuge at 10,000g for 5 minutes, take the supernatant, and store at -20°C for later use.
  • HRP-anti-human Fc (1:10000, Solarbio, SE101) was added and incubated at 37°C for 1 hour.
  • ELISA chromogenic solution 50 ⁇ l per well, develop color, and read the plate. Calculate the hourly antibody concentration in plasma based on the standard curve and plasma dilution factor.
  • Figure 7 shows the time-dependent curve of antibody concentration, in which the half-life of MBS314 injected at 3.0 mg/kg in mouse blood is about 4.7 days, that of MBS314 injected at 1.0 mg/kg is about 3.18 days, and that of MBS314 injected at 0.3 mg/kg It is about 4.97 days.
  • the results indicate that the blood half-life of MBS314 in mice is approximately 4-5 days.

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Abstract

本申请涉及一种同时靶向BCMA、GPRC5D和CD3的多特异性抗体，及其在肿瘤等疾病治疗中的用途。

Description

结合BCMA、GPRC5D和CD3的多特异性抗体及其用途 发明领域
本申请涉及一种同时靶向BCMA、GPRC5D和CD3的多特异性抗体，及其在肿瘤等疾病治疗中的用途。
背景技术
多发性骨髓瘤(MM)是浆细胞恶性增殖性疾病，其疾病特征是骨髓中的浆细胞像肿瘤细胞一样无限制地增殖，并且大部分情况下伴有单克隆免疫球蛋白分泌，最终导致器官或组织损伤。例如，MM常伴有多发性溶骨性损害、高钙血症、贫血、肾脏损害等。MM多发于中老年人，在性别分布上有明显差异，男性患病比例高于女性。MM约占血液系统恶性肿瘤的10-15％，是继白血病、淋巴瘤之后第三多发的血液系统肿瘤。美国发病率为9/10万，我国发病率约为1/10万，并呈逐年上升趋势。
近年来，尽管化疗、蛋白酶体抑制剂、免疫调节剂和CD38靶向抗体等治疗方法在MM领域取得了很大进展，但相当一部分的患者对这些疗法没有反应、或反应期很短。而且，MM几乎无法治愈，患者逐渐对既有疗法产生抗性，导致复发。因此，该领域对新药的需求仍然迫切。
当前已知可用于MM靶向疗法的特异性抗原非常有限，BCMA和GPRC5D是其中的两种。
BCMA和BCMA靶向疗法
B细胞成熟抗原，又称BCMA、CD269或TNFRSF17，是一种I型跨膜蛋白，为肿瘤坏死因子受体(TNFR)超家族的一员。其与同为TNFR超家族的BAFF受体(BAFFR)和跨膜激活剂及钙调亲环素配体相互作用分子(TACI)，在不同发育阶段的B细胞存活中发挥着重要的作用(Rickert R.C et al.，(2011)Immunological Reviews 244(1)：115-133)。BCMA主要表达于成熟B淋巴细胞及浆细胞表面，少量表达于造血干细胞或非造血组织。其配体包括B细胞活化因子(BAFF)和增殖诱导因子(APRIL)，其中后者的BCMA亲和性更高。
BCMA还在MM患者的骨髓恶性浆细胞的表面过表达。BCMA可促进恶性浆细胞在骨髓环境中的成活，恶性浆细胞的APRIL-BCMA信号通路会促进恶性肿瘤细胞的增殖、凋亡逃逸，还使其产生强效的免疫抑制分子，比如IL-10、PD-L1和TGF-β(Tai Y-T et al.，(2016)Blood.127(25)：3225-3236)。在大量的动物模型和人体患者中，BCMA的过表达和激活均表现出与MM进展的相关性(Tai Y-T et al.，(2016)Supra；Sanchez E et al.，(2016)Clin Cancer Res.22：3383-3397)。
由于BCMA的选择性表达/分布特点，即广泛存在于MM细胞表面，但在其他正常组织细胞上表达很低或不表达，以及BCMA的较长血清半衰期，使其成为MM和其他血浆细胞癌症的理想靶点。相比于特异表达于浆细胞却很快从细胞表面消失的CD138，BCMA显然是更好的MM恶性浆细胞的生物标记物。
GPRC5D和GPRC5D靶向疗法
GPRC5D，即G蛋白偶联受体家族C组5成员D，是一种C型7次跨膜受体蛋白，其配体和信号传导机制尚未确定。
在对MM、急性白血病和弥漫性大B细胞淋巴瘤患者的骨髓样本检测中，发现GPRC5D是MM的标记分子(Cohen Y et al.，(2013)Hematology 18(6)：348-351)。这种蛋白主要在具有浆细胞表型的细胞中表达，包括大多数的恶性骨髓浆细胞，且在骨髓样本CD138 +MM细胞上的表达分布与BCMA非常相似，而在正常组织中，GPRC5D的蛋白表达仅限于毛囊(Smith EL et al.，(2019)Sci Transl Med.11(485)：eaau7746)。此外，MM患者中的GPRC5D mRNA表达量与遗传畸变如Rb-1缺失以及较差的预后相关(Atamaniuk J et al.，(2012)Eur J Clin Invest.42(9)：953-960)。
GPRC5D的这种选择性表达，使得其成为有希望的浆细胞疾病例如MM的靶点。针对此靶点的双特异性抗体JNJ-64407564目前正处于临床试验中。
T细胞和CD3
CD3分子是T细胞表面的一种标记分子，与T细胞受体(TCR)组成TCR-CD3复合体，在抗原识别和免疫信号传导过程中具有重要的作用。TCR由α和β链组成，或由γ和δ链组成，各链的胞内域没有信号转导的功能，T细胞胞内信号通路完全由CD3蛋白负责。CD3分子由一条γ链、一条δ链、两条ε和两条ζ链构成，在TCR/CD3复合体中形成为三个二聚体εγ、εδ、和ζζ。ε、γ和δ均为I型跨膜蛋白，具有类似免疫球蛋白胞外功能域，ε、γ、δ和ζ的胞内域共含有10个免疫受体酪氨酸活化基序(ITAM)，当ITAM磷酸化后会与激酶ZAP70结合，向下游转导T细胞激活信号。
靶向CD3的抗体可以通过与靶向疾病相关抗原(如肿瘤相关抗原)的功能基团形成双特异性分子，建立T细胞与疾病相关抗原的物理连接，引起T细胞的活化和由T细胞介导的对疾病相关细胞的杀伤。例如，靶向CD3和肿瘤相关抗原的双特异性抗体在拉近T细胞与肿瘤细胞后，可活化T细胞，向肿瘤细胞释放包含有280多种蛋白的超分子攻击粒子(SMAP)。在SMAP的核心区带有穿孔素和颗粒酶，穿孔素可以刺穿肿瘤细胞的外膜，而颗粒酶会诱导肿瘤细胞凋亡(
Figure PCTCN2022117979-appb-000001
Bálint et al.，(2020)Science 368(6493)：897-901)。
CD3抗体通过在T细胞表面聚集CD3，模拟TCR识别MHC-抗原肽的过程，从而激活T细胞的TCR复合体信号通路，释放IL-2、IFN-r和TNF-α等细胞因子，激活T细胞的增殖和分化。T细胞的增殖和分化在肿瘤治疗中是一把双刃剑，一 方面可以产生更多的T细胞来杀伤肿瘤细胞，但在另一方面也会在受试者体内产生很大的毒性反应，即细胞因子释放综合征(“CRS”)。在临床上，与CRS相关的副作用不仅包括疲劳、呕吐、心动过速、高血压、背痛，还包括如癫痫发作、脑病、脑水肿、无菌性脑膜炎和头痛的中枢神经系统(CNS)反应。例如，在CD3单特异性抗体如OKT3的治疗中观察到CRS，且被认为是抗体在体内结合Fc受体(FcR)时形成的抗体交联而引起的(Herold KC et al.，(2003)J Clin Invest.111(3)：409-418)。因此，在后续的抗体研发中，CD3抗体的Fc区经改造而呈现弱的FcR结合力，比如Tepizumab。
同样地，在靶向CD3的双特异性或多特异性抗体的治疗中，也不免出现CRS。例如，在CD19/CD3T细胞双特异性药剂博纳吐单抗的临床试验中，频繁观察到重度CRS和CNS毒性。对于CD3单特异性抗体的Fc区改造，并不足以改善CD3双特异性抗体引起的CRS，因为双特异性抗体中靶向疾病相关抗原的功能基团与靶细胞的结合会引起抗体的交联，并从而引起T细胞释放大量的细胞因子。
靶向GPRC5D、BCMA和CD3的三特异抗体
BCMA靶向在MM治疗中表现出一定的潜力。然而，由于BCMA的表达呈现出一定的异质性，即使在同一个病人的肿瘤样本中也并非所有肿瘤细胞都表达BCMA，导致治疗反应不一，而且，膜结合BCMA会因为γ-分泌酶的作用而从细胞表面脱落，引起细胞表面BCMA量的变动(Brudno JN et al.，(2018)J.Clin.Oncol 36(22)：2267-2280；Laurent SA et al.，(2015)Nat Commun.6：7333)。这些问题可能可以通过同时靶向MM细胞上的另一标记分子如GPRC5D、FcRH5等来进行一定程度的缓解。然而，BCMA和GPRC5D在MM肿瘤细胞上的表达分布存在一定的相似性，同时靶向两者是否会带来更持久稳定的MM肿瘤细胞结合，存在着一定的不确定性。此外，可以在该双特异性抗体的基础上加上CD3抗原结合域，将患者的T细胞募集到MM细胞周围，激活T细胞消灭MM细胞。而与此同时，如上所述，CD3双/多特异性抗体的毒性不容忽视。2021年上半年，多个BCMA×CD3双特异抗体(AMG701、Elranatamab等)因为安全性问题暂停临床试验。
对于本申请中任何文件的引用，并不等同于承认这些文件是本申请的现有技术。
发明内容
本申请的发明人，通过巧妙的设计，构建出了一种既有强大的肿瘤杀伤力、又引起较小毒性反应的GPRC5D×BCMA×CD3三特异性抗体，其突破和改善了目前在MM治疗中CD3双特异性抗体，包括BCMA×CD3双抗和GPRC5D×CD3双抗，的药效毒性平衡问题。这种多特异性抗体的构建，绝不是简单随意地将各种结合基团相组合，而是要考虑各结合基团之间的相互作用。在本申请抗体的构建中，不仅需要对MM上结合的肿瘤特异性抗原的种类做调整和优化，克服肿瘤 异质性导致的杀伤逃逸，还需要减少CD3通路激活引起的细胞因子释放，找到更好的治疗窗口。
因而，在第一个方面，本申请提供一种多特异性抗体，其可以包含：
i)CD3抗原结合域、ii)BCMA抗原结合域、以及iii)GPRC5D抗原结合域。
CD3抗原结合域可以为激动型抗原结合域，其特异地结合CD3并激活CD3信号通路。特别地，该CD3抗原结合域不结合单独的CD3δ或CD3ε，而仅与CD3δ&ε复合体结合。该CD3抗原结合域在游离状态下几乎没有CD3信号通路激活作用，即几乎不会激活T细胞，而只有在多特异性抗体的另两个抗原结合域与其各自抗原结合而呈现为“交联”状态时，才具有激活CD3信号通路的能力。在一些实施方式中，CD3抗原结合域为特异结合CD3的抗体或其抗原结合部分。
BCMA抗原结合域可以结合BCMA，并任选地阻断BCMA-APRIL结合/相互作用，即可以任选地为拮抗型的抗原结合域。特别地，该BCMA抗原结合域与靶细胞上BCMA的结合，几乎不受环境中可溶性BCMA的影响。在一些实施方式中，BCMA抗原结合域为特异结合BCMA的抗体或其抗原结合部分。
GPRC5D抗原结合域可以与GPRC5D特异结合。在一些实施方式中，GPRC5D抗原结合域为特异结合GPRC5D的抗体或其抗原结合部分。
本申请的多特异性抗体可以包含例如1个CD3抗原结合域、1个BCMA抗原结合域、以及1个GPRC5D抗原结合域。
本申请的多特异性抗体可以为IgG样抗体，包含Fab或Fv形式的CD3抗原结合域，Fab或Fv形式的GPRC5D抗原结合域，以及单链抗体(scFv)形式的BCMA抗原结合域。
在一些实施方式中，多特异性抗体可以包含i)CD3半抗体，包含重链可变区、重链恒定区、轻链可变区和任选的轻链恒定区，ii)GPRC5D半抗体，包含重链可变区、重链恒定区、轻链可变区、和任选的轻链恒定区，以及iii)scFv形式的BCMA抗原结合域，包含重链可变区、任选的第一接头、和轻链可变区，其中，CD3半抗体和GPRC5D半抗体可以形成IgG全抗体，scFv形式的BCMA抗原结合域可以，任选地经由第二接头，与CD3半抗体的重链可变区的N端、轻链可变区的N端、重链恒定区的C端、或轻链恒定区的C端连接。在一些实施方式中，scFv形式的BCMA抗原结合域与CD3半抗体的重链可变区的N端、或轻链可变区的N端连接。
本申请的多特异性抗体可以包含：
i)第一多肽链，包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、特异结合CD3的重链可变区、和重链恒定区，
ii)第二多肽链，包含特异结合CD3的轻链可变区，
iii)第三多肽链，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
iv)第四多肽链，包含特异结合GPRC5D的轻链可变区；或者
i)第一多肽链，包含特异结合CD3的重链可变区、和重链恒定区，
ii)第二多肽链，包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、和特异结合CD3的轻链可变区，
iii)第三多肽链，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
iv)第四多肽链，包含特异结合GPRC5D的轻链可变区，
其中特异结合BCMA的重链可变区和特异结合BCMA的轻链可变区形成该BCMA抗原结合域，第一多肽链中特异结合CD3的重链可变区和第二多肽链中特异结合CD3的轻链可变区形成该CD3抗原结合域，第三多肽链中特异结合GPRC5D的重链可变区和第四多肽链中特异结合GPRC5D的轻链可变区形成该GPRC5D抗原结合域，且第一多肽链的重链恒定区与第三多肽链的重链恒定区结合在一起。
第一多肽链可以从N端到C端包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、特异结合CD3的重链可变区、和重链恒定区，或从N端到C端包含特异结合BCMA的轻链可变区、特异结合BCMA的重链可变区、特异结合CD3的重链可变区、和重链恒定区，第三多肽链可以从N端到C端包含特异结合GPRC5D的重链可变区、和重链恒定区。
在一些实施方式中，多特异性抗体可以包含：
i)第一多肽链，从N端到C端，包含特异结合BCMA的重链可变区、第一接头、特异结合BCMA的轻链可变区、第二接头、特异结合CD3的重链可变区、和重链恒定区，或包含特异结合BCMA的轻链可变区、第一接头、特异结合BCMA的重链可变区、第二接头、特异结合CD3的重链可变区、和重链恒定区，
ii)第二多肽链，从N端到C端，包含特异结合CD3的轻链可变区、和轻链恒定区，
iii)第三多肽链，从N端到C端，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
iv)第四多肽链，从N端到C端，包含特异结合GPRC5D的轻链可变区、和轻链恒定区。
或者，第一多肽链可以从N端到C端包含特异结合CD3的重链可变区、和重链恒定区，第二多肽链可以从N端到C端包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、和特异结合CD3的轻链可变区，或从N端到C端包含特异结合BCMA的轻链可变区、特异结合BCMA的重链可变区、和特异结合CD3的轻链可变区，第三多肽链可以从N端到C端包含特异结合GPRC5D的重链可变区、和重链恒定区。
在一些实施方式中，多特异性抗体可以包含：
i)第一多肽链，从N端到C端，包含特异结合CD3的重链可变区、和重链恒定区，
ii)第二多肽链，从N端到C端，包含特异结合BCMA的重链可变区、第一接头、特异结合BCMA的轻链可变区、第二接头、特异结合CD3的轻链可变区、和轻链恒定区，或包含特异结合BCMA的轻链可变区、第一接头、特异结合BCMA的重链可变区、第二接头、特异结合CD3的轻链可变区、和轻链恒定区
iii)第三多肽链，从N端到C端，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
iv)第四多肽链，从N端到C端，包含特异结合GPRC5D的轻链可变区、和轻链恒定区。
BCMA抗原结合域可以为特异结合BCMA的抗体或其抗原结合部分，其重链可变区可以包含分别如SEQ ID NOs：1-3所示的VH-CDR1、VH-CDR2和VH-CDR3，轻链可变区可以包含分别如SEQ ID NOs：4-6所示的VL-CDR1、VL-CDR2和VL-CDR3。BCMA抗原结合域中的重链可变区和轻链可变区可以分别包含与SEQ ID NOs：7和8具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
CD3抗原结合域可以为特异结合CD3的抗体或其抗原结合部分，其重链可变区可以包含分别如SEQ ID NOs：9-11所示的VH-CDR1、VH-CDR2和VH-CDR3，轻链可变区可以包含分别如SEQ ID NOs：12-14所示的VL-CDR1、VL-CDR2和VL-CDR3。CD3抗原结合域中的重链可变区和轻链可变区可以分别包含与SEQ ID NOs：15和16具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
GPRC5D抗原结合域可以为特异结合GPRC5D的抗体或其抗原结合部分，其重链可变区可以包含分别如SEQ ID NOs：17-19所示的VH-CDR1、VH-CDR2和VH-CDR3，轻链可变区可以包含分别如SEQ ID NOs：20-22所示的VL-CDR1、VL-CDR2和VL-CDR3。GPRC5D抗原结合域中的重链可变区和轻链可变区可以分别包含与SEQ ID NOs：23和24具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
第一多肽链和第三多肽链中的重链恒定区可以为弱结合或不结合FcR的重链恒定区，优选为不结合FcR的重链恒定区，例如人IgG1(N297A)、人IgG1(L234A+L235A)、人IgG1(L234A+L235A+P329G/A)、人IgG1(L234A+L235A+N297A)、人IgG1(L234A+L235A+N297A+P329G/A)、人IgG2(V234A+V237A)、和人IgG1(L234A+V235E)恒定区，或者人IgG4恒定区。
第一多肽链的重链恒定区、和第三多肽链的重链恒定区的其中之一，可以为 带有杵结构的重链恒定区，例如带有T366W突变的人IgG1或IgG4重链恒定区或其功能片段。第一多肽链的重链恒定区、和第三多肽链的重链恒定区中的另一个，可以为带有臼结构的重链恒定区，例如带有T366S/L368A/Y407V突变的人IgG1或IgG4重链恒定区或其功能片段。在一些实施方式中，第一多肽链的重链恒定区、和第三多肽链的重链恒定区的其中之一，可以为带有杵结构且弱结合或不结合FcR的重链恒定区，如带有L234A/L235A/N297A/T366W的人IgG1重链恒定区，如SEQ ID NO：25(X1＝W、X2＝L、X3＝Y)所示。在一些实施方式中，第一多肽链的重链恒定区、和第三多肽链的重链恒定区中的另一个，可以为带有臼结构且弱结合或不结合FcR的重链恒定区，如带有L234A/L235A/N297A/T366S/L368A/Y407V的人IgG1重链恒定区，如SEQ ID NO：25(X1＝S、X2＝A、X3＝V)所示。
第二多肽链和/或第四多肽链中的轻链恒定区，可以为例如人γ轻链恒定区。在某些实施方式中，轻链恒定区包含SEQ ID NO：26所示的氨基酸序列。
第一接头和第二接头可以是约5-30氨基酸长度的肽。在某些实施方式中，接头可以是5-20氨基酸长度的肽。在某些实施方式中，该接头可以是GS接头，例如包含SEQ ID NO：36或37所示的GS接头。
本申请的多特异性抗体，在一些实施方式中，其第一多肽链、第二多肽链、第三多肽链、和第四多肽链可以分别包含与i)SEQ ID NOs：27、28、29和30，或ii)SEQ ID NOs：27、16、29和24具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
本申请的多特异性抗体，可以i)特异结合BCMA(人和猴BCMA)，结合力比现有技术抗体如EM801略好，且几乎不受环境中可溶BCMA的影响，ii)可以结合CD3，结合力显著低于现有技术抗体如JNJ-64407564(下文中简称为JNJ)和EM801，且仅在多特异性抗体结合BCMA和/或GPRC5D而呈现“交联”状态时，才激活CD3信号通路，且在某些肿瘤细胞例如同时高表达BCMA和PRGC5D的肿瘤细胞的存在时，对T细胞的激活作用要弱于JNJ和/或EM801，iii)可以结合GPRC5D，且结合力与JNJ相当或略好，vi)可以在体外杀伤肿瘤细胞，且杀伤力强于JNJ和EM801，v)具有体内抗肿瘤效果，且效果显著强于EM801，与JNJ相当，iv)在测试浓度下几乎没有观察到体内毒性。
相比于现有的BCMA×CD3双抗和GPRC5D×CD3双抗，本申请的三特异性抗体可以更全面地杀灭BCMA +肿瘤细胞、PRGC5D +肿瘤细胞、和BCMA +PRGC5D +肿瘤细胞，更好地应对肿瘤细胞表面抗原表达的异质性问题。即，不管肿瘤细胞上BCMA和PRGC5D的表达高还是低，都能被本申请的三特异性抗体靶向和杀灭。
此外，在存在高表达BCMA和GPRC5D的肿瘤细胞的情况下，本申请多特异性抗体对T细胞的激活力要弱于JNJ和EM801；在存在GPRC5D表达高BCMA 表达低的肿瘤细胞的情况下，本申请多特异性抗体对T细胞的激活力要弱于JNJ；在存在BCMA表达高GPRC5D表达低的肿瘤细胞的情况下，本申请多特异性抗体对T细胞的激活力要弱于EM801。也就是说，本申请的三特异性抗体在应对肿瘤细胞面抗原表达异质性的问题时，不仅能更全面地杀伤肿瘤细胞，而且产生的毒副作用更低。
本申请还提供编码本申请多特异性抗体的核酸分子，以及包含该核酸的表达载体，以及包含该表达载体或该核酸整合入基因组的宿主细胞。本申请还提供使用含有上述表达载体的宿主细胞来制备多特异性抗体的方法，包括：(i)在宿主细胞中表达多特异性抗体，以及(ii)从宿主细胞或其培养物中分离多特异性抗体。
本申请还提供药物组合物，其包含治疗有效量的本申请的多特异性分子、核酸分子、表达载体、或宿主细胞，以及药学上可接受的载体。
在第二个方面，本申请提供在受试者中治疗或减缓肿瘤或癌症的方法，包括向受试者施用有效量的本申请药物组合物。
肿瘤或癌症与BCMA和/或PRGC5D相关，包括，但不限于，多发性骨髓瘤，和其他血液系统恶性肿瘤，如浆细胞瘤、浆细胞白血病、巨球蛋白血症、孤立性骨浆细胞瘤、和髓外浆细胞瘤等。
在某些实施方式中，肿瘤或疾病为多发性骨髓瘤。
在某些实施方式中，肿瘤或疾病为多发性骨髓瘤，且骨髓瘤细胞同时高表达BCMA和GPRC5D。
在某些实施方式中，受试者为哺乳动物，特别是人。
本申请也提供本申请的多特异性抗体、核酸分子、表达载体、宿主细胞、或药物组合物在制备一种用于治疗或减缓肿瘤或癌症的药物中的用途。
在本申请中引用或提及的所有文件(包括但不限于本文引用的所有文献、专利、公开的专利申请)(“本申请引用文件”)，在本申请引用文件中引用或提及的所有文件，以及本申请或任意本申请引用文件中提及的任何产品的制造商手册、说明书、产品规格和产品页，均通过引用的方式并入本申请，且可能在实施本发明时采用。更具体而言，所有参考文件均通过引用的方式并入本申请，如同各文件通过引用的方式并入。在本文中提及的任何Genbank序列通过引用的方式并入本申请。
附图说明
以下以示例方式给出但不意在将本发明限制于具体实施方式的具体描述，可以结合附图更好地进行理解。
图1示出示例性GPRC5D×BCMA×CD3三特异性抗体的结构示意图。
图2示出示例性GPRC5D×BCMA×CD3三特异性抗体(MBS314)、BCMA×CD3双特异性抗体(EM801)、和GPRC5D×CD3双特异性抗体(JNJ)对 HEK293A/人BCMA(A)、HEK293A/猴BCMA(B)、HEK293A/小鼠BCMA(C)、HEK293T/人GPRC5D(D)、HEK293T/猴GPRC5D(E)、以及人T细胞系Jurkat(F)的结合活性。
图3示出三种多发性骨髓瘤细胞系的BCMA及GPRC5D表达的FACS分析图(A)，以及示例性GPRC5D×BCMA×CD3三特异性抗体经这三种细胞“交联”而对Jurkat-NFAT-Luc中CD3信号通路的激活活性(B)。
图4示出骨髓瘤MM.1S细胞的GPRC5D和BCMA表达情况(A)，以及MM.1S肿瘤在经示例性GPRC5D×BCMA×CD3三特异性抗体处理的小鼠体内的生长曲线(B)。
图5示出从复发难治多发性骨髓瘤病人的新鲜样品中分离的骨髓单核细胞，在经示例性GPRC5D×BCMA×CD3三特异性抗体处理和CD138+染色后的FACS分析图谱(A)，以及其中CD138+细胞百分比的统计图(B)。
图6示出C57BL/6小鼠和CD3人源化小鼠在注射示例性GPRC5D×BCMA×CD3三特异性抗体后的体重变化，其中B6代表C57BL/6小鼠，CD3代表CD3人源化小鼠。
图7示出示例性GPRC5D×BCMA×CD3三特异性抗体在小鼠体内的PK动力曲线图。
具体实施方式
为更好理解本申请，首先定义一些术语。其他定义则贯穿具体实施方式部分而列出。
术语“CD3”是指分化簇3，包含γ链、δ链、ε链和ζ链等。术语“CD3ε”或“CD3E”是指CD3的ε链。术语“CD3δ”或“CD3D”是指CD3的δ链。术语“CD3D&E”或“CD3δ&ε”是指δ链和ε链形成的εδ复合体。以上术语包括变体、同源物、直向同源物和平行同源物。
术语“BCMA”是指B细胞成熟抗原，大量选择性地表达于恶性浆细胞，包括变体、同源物、直向同源物和平行同源物。“人BCMA”是指具有人氨基酸序列的BCMA蛋白，例如具有SEQ ID NO：31所示的氨基酸序列。“猴BCMA”是指具有猴氨基酸序列的BCMA蛋白，例如具有SEQ ID NO：32所示的氨基酸序列。“小鼠BCMA”是指具有小鼠氨基酸序列的BCMA蛋白，例如具有SEQ ID NO：33所示的氨基酸序列。
术语“GPRC5D”是指G蛋白偶联受体家族C组5成员D，是骨髓瘤细胞的标记分子，包括变体、同源物、直向同源物和平行同源物。“人GPRC5D”是指具有人氨基酸序列的GPRC5D蛋白，例如具有SEQ ID NO：34所示的氨基酸序列。“猴GPRC5D”是指具有猴氨基酸序列的GPRC5D蛋白，例如具有SEQ ID NO：35所示的氨基酸序列。
本文中的术语“任选的”或“任选地”是指非强制或必要地包含某组分或步骤，即在某些情况下包含某组分或步骤，在另一些情况下不包含某组分或步骤。
本文中的术语“抗体”意在包括IgG、IgA、IgD、IgE和IgM全长抗体及其任何抗原结合片段(即，抗原结合部分)。“全抗体”或“全长抗体”是指包含至少两条重(H)链和两条轻(L)链的糖蛋白，重链和轻链由二硫键连接；而“半抗体”则是“全抗体”的一半，包含例如一条重链和一条轻链，重链和轻链由二硫键连接。各重链由重链可变区(简称V H)和重链恒定区构成。重链恒定区由三个结构域构成，即C H1、C H2和C H3。各轻链由轻链可变区(简称V L)和轻链恒定区构成。轻链恒定区由一个结构域C L构成。V H和V L区还可以划分为称作互补决定区(CDR)的高变区，其由较为保守的骨架区(FR)区分隔开。各V H和V L由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括多种免疫系统细胞(例如，效应细胞)和传统补体系统的第一组分(C1q)。本申请的抗体恒定区被涉及成具有弱结合或不结合免疫系统细胞和补体系统蛋白。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实施，包括，但不限于，(i)Fab片段，由V L、V H、C L和C H1构成的单价片段；(ii)F(ab′) 2片段，包含铰链区二硫桥连接的两个Fab片段的二价片段；(iii)由V H和C H1构成的Fd片段；(iv)由抗体单臂V L和V H构成的Fv片段；(v)由V H构成的dAb片段(Ward et al.，(1989)Nature 341：544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，一种包含单可变结构域和两个恒定结构域的重链可变区。本文中的“IgG样抗体”是指保持IgG抗体的基本构型，额外添加一些基团如抗原结合域而得到的抗体。本文中重链恒定区的“功能片段”是指重链恒定区中保留有执行所需功能(例如，与另一重链恒定区或片段结合，与Fc受体和/或补体系统成分结合)的片段。
本文中所用的术语“激动型CD3抗体”是指能够与CD3结合并激活或引发CD3信号通路从而促进免疫细胞如T细胞激活和增殖等的CD3抗体。本申请的CD3抗体仅在“交联”状态下才激发CD3信号通路，引起免疫细胞的活性。
本申请中的术语“交联”是指由多特异性抗体上靶向BCMA的部分与BCMA结合、靶向PRGC5D的部分与PRGC5D结合、和/或(在具有FcR和/或补体系统成分结合力的Fc部分的情况下)Fc部分与Fc受体和/或补体系统成分结合，而引起的抗体聚集或相互作用。在体外实验中，可以通过抗体Fc结合抗-Fc二抗而形成抗体交联。相对的，“游离”是指抗体之间或者抗体与其他分子之间不会产生相互作用而产生二聚化或者多聚化，本申请的多特异性抗体在游离状态下不会激发CD3信号通路，也因而不激活免疫细胞如T细胞。
术语“拮抗型BCMA抗体”或“阻断型BCMA抗体”是指能够结合BCMA且能够阻断或抑制由BCMA与其配体如BAFF或APRIL，特别是APRIL，相互作用 引发的BCMA信号通路的抗体。拮抗型BCMA抗体可以阻断恶性肿瘤细胞的增殖、凋亡逃逸等。本申请多特异性抗体中的BCMA抗原结合域可以为拮抗型的，与BCMA结合，阻断BCMA-APRIL结合/相互作用，且不引发BCMA信号通路。
术语“FcR”是指在一些细胞，如B淋巴细胞、天然杀伤细胞、巨噬细胞等表面表达的蛋白，可以被抗体的Fc部分其结合，引发对靶细胞的吞噬和毒性等，在免疫系统中发挥重要作用。FcR包括Fcα受体、Fcε受体、和Fcγ受体，其中Fcγ受体术语免疫球蛋白超家族，是引发对微生物的吞噬作用的最重要的FcR，包括FcγRI(CD64)、FcγRIIA(CD32A)、FcγRIIB(CD32B)、和FcγRIIIA(CD16A)等。
术语“多特异性”抗体或“多抗”是指特异性结合两个以上(如三个)靶分子、或同一靶分子上两个以上(如三个)不同表位的抗体。多特异性分子包括本申请中特异结合BCMA、PRGC5D和CD3的抗体。“双特异性抗体”或“双抗”在某些情况下是指特异性结合两个靶分子、或一个靶分子上两个表位的抗体。相对而言，“单特异性”抗体是指特异结合某一个靶分子，尤其是某一个靶分子上一个表位的抗体。
在本文中，“特异结合BCMA”、“特异结合CD3”、或“特异结合PRGC5D”是指与BCMA、CD3或PRGC5D结合但是基本不与非BCMA、CD3或PRGC5D蛋白结合的抗体。优选地，抗体以“高亲和力”结合人BCMA、CD3或PRGC5D蛋白，是指K D值为5.0x10 -7M以下。
术语“基本不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的K D为1.0 x 10 -6M以上。
术语“EC 50”，又叫半最大效应浓度，是指引起50％最大效应的抗体浓度。
术语“IC 50”，又称为半抑制浓度，是指对指定的生物过程抑制一半时所需的药物或抑制剂的浓度。
术语“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳类和非哺乳类，例如非人灵长类、羊、狗、猫、牛、马、鸡、两栖类、和爬行类，尽管优选哺乳动物，例如非人灵长类、羊、狗、猫、牛和马。
术语“有效量”是指足以达到预期结果而用到的本申请抗体的量。术语“治疗有效量”是指足以防止或减缓与疾病或病症(例如癌症)相关的症状的本申请抗体量。治疗有效量与被治疗的疾病相关，其中本领域技术人员可以方便地判别出实际的有效量。
本申请的多特异性抗体具有有益特征
本申请的多特异性抗体，与现有技术抗体如JNJ和EM801相比，具有i)相当或更高的BCMA结合力，ii)相当或更高的PRGC5D结合力，iii)显著更低的CD3结合力，vi)相当或更好的体外肿瘤杀伤力，v)相当或更好的体内肿瘤杀伤力，和/或iv)相当或更低的体内毒性。
本申请的发明人通过巧妙的设计构建出的三特异性抗体，既有强大的肿瘤杀 伤力、又引起较小毒性反应，可以突破和改善目前在MM治疗中CD3双特异性抗体，包括BCMA×CD3双抗和GPRC5D×CD3双抗，的药效毒性平衡问题。相比于现有的BCMA×CD3双抗和GPRC5D×CD3双抗，本申请的三特异性抗体可以更全面地杀灭BCMA +肿瘤细胞、PRGC5D +肿瘤细胞、和BCMA +PRGC5D +肿瘤细胞，更好地应对肿瘤细胞表面抗原表达的异质性问题。即，不管肿瘤细胞上BCMA和PRGC5D的表达高还是低，都能被本申请的三特异性抗体靶向和杀灭。
此外，在存在高表达BCMA和GPRC5D的肿瘤细胞的情况下，本申请多特异性抗体对T细胞的激活力要弱于JNJ和EM801；在存在GPRC5D表达高BCMA表达低的肿瘤细胞的情况下，本申请多特异性抗体对T细胞的激活力要弱于JNJ；在存在BCMA表达高GPRC5D表达低的肿瘤细胞的情况下，本申请多特异性抗体对T细胞的激活力要弱于EM801。也就是说，本申请的三特异性抗体在应对肿瘤细胞面抗原表达异质性的问题时，不仅能更全面地杀伤肿瘤细胞，而且产生的毒副作用更低。尤其在针对高表达BCMA和PRGC5D的肿瘤方面，本申请的多特异性抗体更具有杀伤优势，且产生的毒副作用较低。
本申请多特异性抗体中使用的单特异性抗体或其抗原结合片段的重链可变区CDR和轻链可变区CDR通过Kabat编号系统确定。领域内熟知，重链可变区和轻链可变区CDR可以通过例如Chothia、IMGT、AbM或Contact编号系统/方法确定。
本申请的多特异性抗体也包括双特异性抗体。
本申请的多特异性抗体
本申请的多特异性抗体可以包含：i)CD3抗原结合域、ii)BCMA抗原结合域、以及iii)GPRC5D抗原结合域。CD3抗原结合域可以为激动型抗原结合域，其特异地结合CD3并激活CD3信号通路。BCMA抗原结合域可以结合BCMA，并任选地阻断BCMA-APRIL结合/相互作用，即可以任选地为拮抗型的抗原结合域。GPRC5D抗原结合域可以与GPRC5D特异结合。
本申请的多特异性抗体可以包含例如1个CD3抗原结合域、1个BCMA抗原结合域、以及1个GPRC5D抗原结合域。
本申请的多特异性抗体可以为IgG样抗体，包含Fab或Fv形式的CD3抗原结合域，Fab或Fv形式的GPRC5D抗原结合域，以及单链抗体(scFv)形式的BCMA抗原结合域。
多特异性抗体可以包含i)CD3半抗体，包含重链可变区、重链恒定区、轻链可变区和任选的轻链恒定区，ii)GPRC5D半抗体，包含重链可变区、重链恒定区、轻链可变区、和任选的轻链恒定区，以及iii)scFv形式的BCMA抗原结合域，包含重链可变区、任选的第一接头、和轻链可变区，其中，CD3半抗体和GPRC5D半抗体可以形成IgG全抗体，scFv形式的BCMA抗原结合域可以，任选地经由第二接头，与CD3半抗体的重链可变区的N端、轻链可变区的N端、重链恒定区的C端、或轻链恒定区的C端连接。在一些实施方式中，scFv形式的BCMA抗原结 合域与CD3半抗体的重链可变区的N端、或轻链可变区的N端连接。
scFv形式的BCMA抗原结合域可以从N端到C端包含重链可变区、任选的第一接头、和轻链可变区；或轻链可变区、任选的第一接头、和重链可变区。
两个半抗体的重链恒定区的其中之一，可以为带有杵结构且弱结合或不结合FcR的重链恒定区，如带有L234A/L235A/N297A/T366W的人IgG1重链恒定区，另一个重链恒定区可以为带有臼结构且弱结合或不结合FcR的重链恒定区，如带有L234A/L235A/N297A/T366S/L368A/Y407V的人IgG1重链恒定区。
第一接头和第二接头可以由肽键连接的氨基酸构成，优选肽键连接的5-30个氨基酸，其中氨基酸选自20种天然存在的氨基酸。这些氨基酸中的一种或多种可以糖基化，如本领域技术人员所了解的。在一个实施方式中，5-30个氨基酸可以选自甘氨酸、丙氨酸、脯氨酸、天冬酰胺、谷氨酰胺、丝氨酸和赖氨酸。在一个实施方式中，接头由大部分有空键位阻的氨基酸构成，例如甘氨酸和丙氨酸。示例性的接头为多聚甘氨酸，特别是多聚(Gly-Ala)、以及多聚丙氨酸。本申请中的示例性接头可以如SEQ ID NOs：19、20、21或22所示。
接头也可以是非肽类接头。例如，可以使用烷基接头，例如-NH-、-(CH 2)s-C(O)-，其中s＝2-20。这些烷基接头还可以经任何非空间位阻基团例如低级烷基(例如C 1-6低级酰基)、卤素(例如Cl、Br)、CN、NH 2、苯基等进行取代。
保守修饰
在另一实施方式中，本申请的多特异抗体，包含与本申请抗体存在一个或多个保守修饰的重链和/或轻链可变区序列或CDR1、CDR2和CDR3序列。本领域知道，一些保守序列修改不会使抗原结合性消失。
本文所用的术语“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和删除。可以通过领域内已知的标准技术，例如点突变和PCR介导的突变，将修饰引入本申请抗体中。保守氨基酸替换是氨基酸残基用具有相似侧链的氨基酸残基进行替换。具有相似侧链的氨基酸残基组在领域内已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，本申请抗体的CDR区中的一个或多个氨基酸残基可以用同侧链组的其他氨基酸残基替换，且得到的抗体可以使用本文所述的功能检测对其进行保留功能(即，上述的功能)的测试。
基因修饰的抗体
本申请的多特异性抗体，可以以具备本申请所用抗体的一个或多个V H/V L序 列的抗体作为起始材料，制备成基因修饰的抗体。抗体可以通过修饰一个或两个可变区(即，V H和/或V L)内(例如，在一个或多个CDR区和/或一个或多个骨架区)的一个或多个残基来进行基因修饰，以改善结合亲和力和/或增加与某些物种天然产生的抗体的相似性。例如，骨架区经修饰成提供人源化的抗体。此外，或者抗体可以通过修饰恒定区中的残基进行基因修饰，例如改变抗体的效应功能。
因此，本申请的多特异性抗体，其中所包含的各重链可变区和/或各轻链可变区可以包含本申请中的VH-CDR1、VH-CDR2、VH-CDR3，和/或VL-CDR1、VL-CDR2和VL-CDR3，但含有不同的骨架序列。
本申请的发明人发现，当抗原结合域呈现为scFv形式时，稳定性通常可能会弱于全抗体或Fab形式。因此，在本申请的一个实施方式中，为了解决BCMA抗原结合域以scFv形式应用于本申请多特异性抗体构建时可能导致的稳定性问题，基于计算机结构模拟，对BCMA重/轻链可变区的框架序列，例如轻链可变区的FR3和FR4部分的氨基酸进行了改造，以增加scFv形式的稳定性，减少聚体的形成，增加单体比例。改造前后的Fab形式和scFv形式在亲和力、BCMA-APRIL阻断活性以及抗肿瘤效果方面相当。
本申请的发明人还发现，可以通过计算机结构模拟改变BCMA重/轻链可变区的框架序列，例如改变框架区序列氨基酸的亲疏水性，从而进一步改善多特异性抗体的成药性，从而提高在产业上的表达量。
本申请的基因改造抗体包括在V H和/或V L的骨架残基中做出基因修饰以例如改变抗体特性的那些。通常而言，这些骨架修饰用来降低抗体的免疫原性。例如，一种方法是将一个或多个骨架残基“回复突变”成相应的种系序列。更加具体而言，经历体细胞突变的抗体可能包含不同于得到抗体的种系序列的骨架残基。这些残基可以通过将抗体骨架序列与得到抗体的种系序列相比较而识别出来。
另一类的骨架修饰包括对骨架区的、或者甚至一个或多个CDR区的一个或多个残基进行突变，以去除T细胞表位，从而减少抗体的可能导致的免疫原性。该方法也称为“去免疫化”，在美国专利公开20030153043中有更加详细的描述。
此外，作为骨架或CDR区内修饰之外的另一种选择，本申请的抗体可以基因改造成在Fc区包括基因修饰，通常来改变抗体的一个或多个功能特性，例如血清半衰期、补体结合、Fc受体结合、和/或抗体依赖的细胞毒性。此外，本申请的抗体可以进行化学修饰(例如，可以向抗体附加一个或多个化学功能基团)，或者修饰成改变其糖基化，来改变抗体的一个或多个功能特性。
在一个实施方式中，C H1的铰链区进行修饰，改变，例如增加或减少铰链区的半胱氨酸残基的数量。该方法在美国专利5,677,425中进一步描述。改变C H1铰链区的半胱氨酸残基，来例如促进重链轻链的组装或增加/降低抗体的稳定性。
在另一个实施方式中，对抗体的Fc铰链区进行突变，以降低抗体的生物半衰期。更加具体地，将一个或多个氨基酸突变引入Fc铰链片段的C H2-C H3连接区， 从而抗体相对于天然Fc-铰链结构域SpA结合而言，具有减弱的SpA结合力。该方法在美国专利6,165,745中有更详细的描述。
在另一实施方式中，修饰抗体的糖基化。例如，可以制备去糖基化的抗体(即，抗体缺少糖基化)。可以改变糖基化，来例如增加抗体对抗原的亲和性。这样的糖化修饰可以通过例如改变抗体序列中的一个或多个糖基化位点来达成。例如，可以做出一个或多个氨基酸替换，以消除一个或多个可变区骨架糖基化位点，从而消除该位置的糖基化。这样的去糖基化可以增加抗体对抗原的亲和性。参见，例如美国专利5,714,350和6,350,861。此外，可以制备具有改变的糖基化类型的抗体，例如岩藻糖残基量减少的低岩藻糖基抗体，或者具有增加的平分型GlcNac结构的抗体。改变的糖基化形式被证明能增加抗体的ADCC活性。这样的糖化修饰可以通过例如在糖基化系统改变的宿主细胞中表达抗体而进行。具有改变的糖基化系统的细胞在领域中已知，且可以用作表达本申请重组抗体的宿主细胞，以制备具有改变的糖基化的抗体。例如，细胞系Ms704、Ms705和Ms709缺少岩藻糖基转移酶基因FUT8(α(1，6)-岩藻糖基转移酶)，从而在Ms704、Ms705和Ms709细胞系中表达的抗体在其糖中缺失岩藻糖。
本文抗体的另一修饰是聚乙二醇化(PEG化)。抗体可以PEG化，例如来增加抗体的生物(例如，血清)半衰期。为使抗体PEG化，抗体或其片段通常与聚乙二醇(PEG)，例如PEG的反应性酯或醛类衍生物，在使一个或多个PEG基团附于抗体或抗体片段的条件下反应。
编码本申请抗体的核酸分子
在另一方面，本申请提供编码本申请多特异抗体中如BCMA重链可变区-第一接头-BCMA轻链可变区-第二接头-CD3重链可变区-重链恒定区、CD3轻链可变区-轻链恒定区、PRGC5D重链可变区-重链恒定区、PRGC5D轻链可变区-轻链恒定区等的核酸分子。
核酸可以存在整细胞中，在细胞裂解液中，或处于部分纯化或基本纯的形式。当通过标准技术从其他细胞组分或其他污染物例如其他细胞核酸或蛋白中纯化出来后，核酸是“分离的”或“基本纯的”。本申请的核酸可以为例如DNA或RNA，且可能包含或可能不包含内含子序列。在优选实施方式中，核酸是cDNA分子。
本申请的核酸可以使用标准的分子生物学技术获得。优选的本申请核酸分子可以包括编码CD3、BCMA、PRGC5D单特异性抗体的V H和V L序列或CDR的那些。一旦获得了编码V H和V L的DNA片段，这些DNA片段可以进一步通过标准的重组DNA技术进行操作，例如将可变区基因转变为全长抗体链基因、Fab片段基因或scFv基因。在这些操作中，编码V H或V L的DNA片段与编码另一蛋白的另一DNA片段，例如抗体恒定区或柔性接头，可操作地连接。术语“可操作地连接”是指两个DNA片段连接在一起，从而两个DNA片段编码的氨基酸序列都在阅读框内。
为创建scFv基因，编码V H和V L的DNA片段可以可操作地与编码柔性接头例如编码氨基酸序列(Gly4-Ser) 3的另一片段连接，从而V H和V L序列可以作为连续的单链蛋白进行表达，其中V H和V L区域通过该柔性接头连接(参见，例如Bird et al.，(1988)Science 242：423-426；Huston et al.，(1988)Proc.Natl.Acad.Sci.USA 85：5879-5883；McCafferty et al..(1990)Nature 348：552-554)。
对于本申请中的多特异性抗体，可以先获得编码CD3、BCMA、和PRGC5D抗体的CDR、VH和VL、以及接头等的序列，然后根据所需的多特异抗体的形式来组合这些序列。例如，编码BCMA重链可变区、第一接头、BCMA轻链可变区、第二接头、CD3重链可变区、和重链恒定区的序列可以按需可操作地连接在一起。
本申请抗体的制备
本申请的多特异抗体的制备可以通过i)将编码多特异抗体的各多肽链的序列插入一个或多个表达载体，其中一个或多个表达载体与转录和翻译调控序列可操作地连接；ii)用表达载体转导或转染宿主细胞，以及iii)表达多肽链以形成本申请的多特异抗体。
术语“调控序列”包括控制抗体基因转录或翻译的启动子、增强子和其他表达控制元件(例如，多腺苷酸化信号)。
表达载体可以编码促进抗体链从宿主细胞分泌的信号肽。抗体链基因可以克隆到载体中，从而信号肽在阅读框内连接到抗体链基因的氨基端。信号肽可以是免疫球蛋白信号肽或异源信号肽(即，来自非免疫球蛋白的信号肽)。
除抗体链基因和调控序列外，本申请的表达载体可以携带其他序列，例如调控载体在宿主细胞中复制的序列(例如，复制起始点)和可选择标记物基因。例如，通常可选择标记物基因赋予已导入载体的宿主细胞以药物抗性，例如G418、潮霉素、或氨甲喋呤抗性。优选的可选择标记物基因包括二氢叶酸还原酶(DHFR)基因(用于dhfr宿主细胞的氨甲喋呤选择/扩增)和neo基因(用于G418选择)。
编码多特异性抗体不同多肽链的表达载体通过标准技术转染到宿主细胞中。多个形式的术语“转染”包括多种常用于将外源DNA导入原核或真核宿主细胞的技术，例如，电穿孔、磷酸钙沉淀、DEAE-右旋糖转染等。尽管在原核或真核宿主细胞中表达本申请抗体在理论上是可行的，优选抗体在真核细胞中表达，最优选在哺乳动物宿主细胞中表达，因为真核细胞，特别是哺乳动物细胞，比原核细胞更可能组装并分泌适当折叠且有免疫活性的抗体。
本申请可用的表达载体的例子包括但不限于质粒、病毒载体、酵母人工染色体(YAC)、细菌人工染色体(BAC)、可转化人工染色体(TAC)、哺乳动物人工染色体(MAC)和人工附加染色体(HAEC)。
药物组合物
在另一方面，本申请提供一种药物组合物，其包含本申请的一种或多种多特异性抗体、编码多特异性抗体的核酸分子、包含该核酸分子的表达载体、和/或包 含该核酸分子的宿主细胞，与药学上可接受的载体配制在一起。组合物可以任选地包含一种或多种其他药学上的有效成分，例如另一抗肿瘤药物、或免疫增强药物。本申请的药学组合物可以与例如另一抗癌剂、或另一免疫增强剂联合使用。
药学组合物可以包含任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠或乳化剂、固体粘合剂、分散或混悬剂、增溶剂、染色剂、矫味剂、涂层、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂及其组合。合适赋形剂的选择和使用在Gennaro，ed.，Remington：The Science and Practice of Pharmacy，20th Ed.(Lippincott Williams&Wilkins 2003)中有教导。
药物组合物适合于经口、静脉内、肌内、皮下、肠道外、脊柱或表皮施用(例如通过注射或推注)。基于施用途径的不同，有效成分可以包在材料中，以保护其不受酸和可能使其失活的其他自然条件的影响。“肠道外施用”是指不同于肠道和局部外用的方式，通常通过注射进行，包括但不限于静脉内、肌内、动脉内、膜内、囊内、眶内、心脏内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬脑膜上和胸骨内注射和推注。或者，本申请的抗体可以通过非肠道外路径施用，例如外用、表皮施用或粘膜施用，例如鼻内、经口、阴道、直肠、舌下、或局部外用。优选地，本申请的药物组合物经口施用。
药物组合物可以为无菌水溶液或分散液的形式。它们也可以配制在微乳剂、脂质体或其他适于高浓度药物的有序结构中。
对于本申请药物组合物的施用，具体可以由医学工作者，如医生，根据受试者的具体情况，如性别、年龄、既往病史等进行确定。
“治疗有效量”的本申请多特异性抗体，引起疾病症状严重程度的降低、或无症状期频率和持续时间的增加。例如，对于肿瘤患者，“治疗有效量”优选地，与对照受试者相比，将肿瘤减少至少约20％、更优选至少约40％，甚至更优选至少约60％，且更优选地至少约80％，甚至完全消除肿瘤。
药物组合物可以是缓释试剂，包括植入体、和微胶囊递送系统。可以使用生物可降解、生物相容的聚合物，例如乙烯-醋酸乙烯、聚酸酐、聚乙醇酸、胶原蛋白、聚原酸酯、和聚乳酸。
在某些实施方式中，本申请的抗体以经配制，以确保合适的体内分布。例如，为确保本申请的治疗抗体穿越血脑屏障，抗体可以配制在脂质体中，其还可以额外地包含靶向功能基团，以增强对特定细胞或器官的选择性输送。
本申请的用途和方法
本申请的药物组合物具有多种体外和外内应用，例如可以用于肿瘤或癌症的治疗和缓解。
本申请的药物组合物可以用于治疗或减缓与BCMA和/或PRGC5D相关的肿瘤或癌症，包括，但不限于，多发性骨髓瘤，和其他血液系统恶性肿瘤，如浆细胞瘤、浆细胞白血病、巨球蛋白血症、孤立性骨浆细胞瘤、和髓外浆细胞瘤。
本申请提供本申请药物组合物与一种或多种其他抗体或非抗体类治疗剂一起施用的联合疗法，如免疫增强剂等。
本文讨论的治疗剂的组合可以作为在药学可接受载体中的单一组合物同时施用，或者作为分开的组合物同时施用，其中各药剂处于药学可接受载体中。在另一个实施方式中，治疗剂的组合可以按序施用。
此外，如果进行多次联合疗法施用，且药剂按序施用，则在各时间点的按序施用的次序可以反转或保持相同，按序施用可以与同时施用或其任何组合相结合。
本申请的各方面和实施方式将参照附图和以下实施例进行讨论。其他方面和实施方式对于本领域技术人员是清楚的。在本文中描述的所有文献通过引用的方式全部并入本文。尽管本申请已经结合示例性实施方式进行了描述，很多等同修改和变化在给出本申请时对于本领域技术人员是清楚的。因而，本申请的示例性实施方式是示例性的，非限制性的。可以对所述实施方式做出多种变化，而不脱离本申请的宗旨和范围。
实施例
实施例1.稳定表达BCMA或GPRC5D的细胞株的构建
使用HEK293A细胞来构建分别稳定过表达人BCMA、猴BCMA、小鼠BCMA的细胞系。简单而言，合成编码人BCMA、猴BCMA、小鼠BCMA(氨基酸序列分别如SEQ ID NOs：31、32和33所示)的cDNA序列，并经酶切分别克隆到pLV-EGFP(2A)-Puro载体(北京英茂盛业生物科技有限公司，中国)的BamH1和EcoR1酶切位点中。将得到的pLV-EGFP(2A)-Puro-人BCMA、pLV-EGFP(2A)-Puro-猴BCMA、pLV-EGFP(2A)-Puro-小鼠BCMA与psPAX和pMD2.G质粒通过脂质体转染的方式转染到HEK293T细胞(南京科佰公司，中国)中，产生慢病毒，具体转染方法与Lipofectamine 3000试剂盒(Thermo Fisher Scientific，美国)说明书步骤完全一致。转染三天后，从HEK293T细胞的细胞培养基(DMEM培养基(Cat#：SH30022.01，Gibco)，补充有10％FBS(Cat#：FND500，Excell))中收获慢病毒。然后用慢病毒转染HEK293A细胞(南京科佰公司，中国)，得到稳定表达人、猴或小鼠BCMA细胞(分别称为HEK293A/人BCMA、HEK293A/猴BCMA、HEK293A/小鼠BCMA细胞)。转染的HEK293A细胞培养在含有0.2μg/ml嘌呤毒素(Cat#：A11138-03，Gibco)的DMEM+10％FBS培养基中7天。人和猴BCMA的表达使用市售可得的BCMA抗体(PE-抗人BCMA抗体，Cat#：357503，Biolegend，美国)通过流式分析仪经FACS分析。相似地，小鼠BCMA的表达通过市售可得的小鼠BCMA抗体(抗小鼠BCMA抗体，Cat#：O88472，Novus，美国)经FACS确证。
类似地，构建分别稳定过表达人和猴GPRC5D的HEK293T细胞系，使用分别编码人和猴GPRC5D(氨基酸序列分别如SEQ ID NOs：34和35所示)的cDNA，且使用GPRC5D抗体(GPRC5D抗体根据公开专利WO2022058445A1中的氨基酸序列10和11制备，20μg/ml终浓度)来检测人和猴GPRC5D的表达。
实施例2.CD3×BCMA×GPRC5D三特异性抗体的构建和表达
采用BCMA∶CD3∶GPRC5D抗原结合域为1∶1∶1的不对称全抗体组装形式进行三特异性抗体的构建，示例性三抗的结构如图1所示。以GS载体(具体信息见ZL200510064335.0)为表达载体，构建示例性三抗的2个半抗体MBS-314杵和MBS-314臼。其中，MBS-314杵的长链包含BCMA scFv-接头-CD3抗体重链可变区-重链恒定区，氨基酸序列如SEQ ID NO：27所示，短链包含CD3抗体轻链可变区-轻链恒定区，氨基酸序列如SEQ ID NO：28所示，MBS-314臼的长链包含GPRC5D抗体重链可变区-重链恒定区，氨基酸序列如SEQ ID NO：29所示，短链包含GPRC5D抗体轻链可变区-轻链恒定区，氨基酸序列如SEQ ID NO：30所示。在靶向BCMA的scFv中，轻链可变区的骨架(例如第四框架区)做了一定的改造，以使scFv的结构更加稳定。
全基因合成编码MBS-314杵半抗体中的BCMA scFv-接头-CD3抗体重链可变区、以及MBS-314臼半抗体中GPRC5D抗体重链可变区的DNA序列，利用限制性内切酶EcoRI和NheI分别对上述两个片段进行双酶切，并将酶切后的基因片段克隆至含各自重链恒定区(氨基酸序列分别如SEQ ID NO：25(X1＝W、X2＝L、X3＝Y)、SEQ ID NO：25(X1＝S、X2＝A、X3＝V)所示)的载体中，完成2个半抗长链全长基因表达载体的装配。另外，全基因合成编码MBS-314杵半抗体中CD3抗体轻链可变区、以及MBS-314臼半抗体中GPRC5D抗体轻链可变区的DNA序列，利用限制性内切酶ClaI和BsiWI分别对2个片段进行双酶切，并将酶切后的基因片段克隆至含轻链恒定区(SEQ ID NO：26)的载体中，完成2个半抗体短链全长基因表达载体的装配。分别采用ClaI和HindIII酶切半抗体短链全长基因，EcoRI和XhoI酶切半抗体长链全长基因，HindIII和EcoR I酶切pCMV-cofragment质粒，ClaI和XhoI酶切GS-载体。将回收的4个DNA片段进行连接、转化并挑取单克隆进行测序，获得含有正确序列的半抗表达载体，分别命名为MBS-314杵表达载体和MBS-314臼表达载体。采用天根无内毒素质粒大提试剂盒(Cat#：DP117，天根)提取质粒。采用5mL FreeStyle F17细胞培养基重悬质粒，同时吸取3倍质粒体积的转染试剂(PEI)重悬在5mL FreeStyle F17细胞培养基中。将PEI缓慢地加入到质粒中，充分混合，室温放置约15min。将上述混合液加入到100ml 293F细胞中(细胞密度为1×10 6/mL)。转染后的HEK-293F细胞在37℃、5％CO 2的培养箱中以120RPM转速培养。10-12天后，收集细胞培养上清，3500rpm离心5分钟，并通过0.22μm滤膜过滤除去细胞碎片。半抗体通过预平衡的protein-A亲和柱(Cat#：17040501，GE，美国)来富集纯化。后用洗脱缓冲液(20mM柠檬酸，pH3.0-pH3.5)进行洗脱。之后，抗体保存在PBS(pH 7.0)中，并通过NanoDrop检测抗体浓度。纯化的半抗进行进一步组装。
实施例3.CD3×BCMA×GPRC5D三特异性抗体的组装
将纯化的半抗体通过体外方式进行组装。具体地，将MBS-314杵和MBS-314 臼半抗体以1∶1的摩尔比混合，用Tris碱缓冲溶液调节至pH 8.0，添加一定量的还原型谷胱甘肽溶液，在25℃反应并低速搅拌过夜。反应结束后用2M醋酸溶液调节pH值至5.5。通过超滤去除还原剂，终止反应。装配后的抗体调至低盐Tris缓冲溶液(pH 8.0)中，用0.2μm的过滤膜过滤。首先采用低盐Tris缓冲溶液(pH8.0)平衡阴离子层析柱，然后将样品装载到阴离子层析柱，收集流穿组分，随后使用低盐Tris缓冲溶液(pH8.0)进行冲洗直至UV280趋向于基线。用醋酸溶液将收集的流穿样品pH调至5.5。将阴离子收集的样品用30kDa超滤管浓缩至1mL，用0.2μm的过滤膜过滤。采用低浓度乙酸盐缓冲溶液(pH 5.5)平衡阳离子层析柱，将样品上样到阳离子层析柱中。上样完毕后，再用低浓度乙酸盐缓冲溶液(pH5.5)平衡柱子，然后进行线性梯度洗脱，0-100％高浓度醋酸盐(pH 5.5)，20CV，收集洗脱组分。
组装好的双抗命名为MBS314，纯化后的抗体经过质谱鉴定纯度高于90％，用于后续功能检测。
实施例4.CD3×BCMA×GPRC5D与人CD3、人GPRC5D以及人BCMA的结合 亲和力
通过捕获法(BIAcore 8K)测定MBS314抗体与人CD3、人GPRC5D以及人BCMA的结合亲和力。将鼠抗人Fc段的抗体(Cat#：BR100839，cytiva)偶联至芯片CM5的表面。HBS-EP缓冲液(Cat#：BR-1006-69，GE Life Sciences)稀释待测抗体至1μg/mL，保证约100RU抗体被抗人Fc的抗体捕获。人BCMA抗原(Cat#：BCA-H522y，ACRO)、人CD3D&E(Cat#：CT038-H2508H，义翘神州)蛋白、以及人GPRC5D抗原(Cat#：HM05P，恺佧生物)分别用HBS-EP缓冲液稀释至0.5、0.25、0.125、0.0625、0.03125、0.015625、0.0078125、和0.003900625μg/ml。将不同浓度的各蛋白分别流经固定相表面。采用3M MgCl 2溶液对芯片进行再生。用Biacore 8K控制软件中的动力学分析Wizard进行动力学实验。JNJ(GPRC5D×CD3双特异性抗体)和EM801(BCMA×CD3双特异性抗体)用作阳性对照，其中，JNJ根据公开专利WO2022058445A1中的氨基酸序列10、11、20、和21制备；EM801根据WO2016020332A1中的氨基酸序列43、44、45和46制备。根据Biacore 8K评估软件进行拟合，获得检测抗体对上述抗原的KD值，总结于表1中。
从表1可知，MBS314抗体与人CD3、人GPRC5D、以及人BCMA都有着很强的结合亲和力。其中，针对CD3D&E复合体，MBS314相比于EM801和JNJ具有更低的结合亲和力。
表1.MBS314抗体与人GPRC5D、人BCMA以及人CD3D&E的结合亲和力
抗体 抗原 ka(1/Ms) kd(1/s) K D(M)
JNJ 人GPRC5D 9.30E+05 3.92E-03 4.21E-09
MBS314 人GPRC5D 4.93E+05 1.74E-03 3.53E-09
MBS314 人BCMA 6.42E+05 2.83E-04 4.41E-10
EM801 人BCMA 2.28E+06 2.27E-03 9.96E-10
MBS314 人CD3D&E 3.11E+04 7.74E-03 2.49E-07
JNJ 人CD3D&E 4.44E+05 1.14E-02 2.56E-08
EM801 人CD3D&E 1.85E+05 7.57E-03 4.10E-08
实施例5.CD3×BCMA×GPRC5D三特异性抗体与HEK293A/人BCMA细胞、 HEK293A/猴BCMA细胞、HEK293A/小鼠BCMA细胞、HEK293T/人GPRC5D细 胞、HEK293T/猴GPRC5D细胞以及Jurkat细胞的结合力
通过FACS检测MBS314对细胞表面表达的人BCMA、猴BCMA、鼠BCMA、人GPRC5D、猴GPRC5D以及人CD3复合体的结合活性。
具体地，利用胰酶消化收集生长状态良好的HEK293A/人BCMA、HEK293A/猴BCMA、HEK293A/小鼠BCMA、HEK293T/人GPRC5D细胞、HEK293T/猴GPRC5D细胞(实施例1中制备)、以及Jurkat细胞系(南京科佰)，300g离心5min去除培养基。PBS重悬计数后再次离心去除上清。利用2％FBS-PBS溶液重悬细胞，调整细胞浓度为4×10 6个/ml。将各细胞加入U型底96孔板中，50μl/孔。用2％FBS-PBS稀释检测抗体，从40μg/ml开始进行5倍梯度稀释，共设置12个稀释梯度。在上述96孔板中加入50μl抗体稀释液，吹打混匀，室温孵育1h。2％FBS-PBS洗板，200μL/孔，洗涤3次。向板中加入PE标记的山羊抗人IgG(H+L)二抗，1∶500稀释，50μl/孔，室温结合45min。2％FBS-PBS洗板，200μL/孔，洗涤3次。最后向板中加入150μL 2％FBS-PBS以重悬细胞，并经流式细胞仪检测，FlowJo软件进行数据处理，GraphPad软件进行数据分析，实验结果显示在图2中。
如图2(A-C)所示，MBS314抗体与HEK293A/人BCMA以及HEK293A/猴BCMA细胞有很强的结合力，并且呈剂量依赖关系，但是不结合小鼠BCMA。图2(D和E)显示，MBS314抗体与HEK293T/人GPRC5D和HEK293T/猴GPRC5D均结合，且MBS314与人GPCR5D和猴GPCR5D结合活性都显著高于JNJ。图2F显示，与JNJ相比，MBS314具有较低的CD3复合体结合活性。
实施例6.CD3×BCMA×GPRC5D三特异性抗体对T细胞的信号活化
通过以下检测来确定本申请的三特异性抗体对T细胞信号的活化。当该抗体结合肿瘤细胞表面的GPRC5D和/或BCMA而呈现“交联”状态时，其可结合JURKAT-NFAT-Luc细胞表面的CD3并激活NFAT信号，使JURKAT-NFAT-Luc细胞分泌荧光素酶，通过检测荧光素酶的活性，可确定由该抗体介导的T细胞激活水平。
首先，使用GPRC5D和BCMA的杂交瘤抗体(康源博创研发)，检测多发性骨髓瘤细胞表面的GPRC5D和BCMA表达水平，并从而挑选出GPRC5D和BCMA均高表达的细胞NCI-H929(KC-0629，康源博创)、GPRC5D高表达而BCMA低表达的细胞MOLP8(KC-0622，康源博创)、以及GPRC5D低表达BMCA高表达的细胞KMS-11(KC-0611，康源博创)，进行后续的T细胞激活实验。将上述骨髓瘤细胞(1百万/ml、10万/孔)悬浮于100μl的PBS(含2％BSA)中，分别和GPRC5D杂交瘤抗体或BCMA杂交瘤抗体(20μg/ml)室温孵育半小时。离心洗涤后，向细胞加入PE-抗-小鼠二抗(1∶400，biolegend，405307)，离心洗涤后，悬浮细胞，加入7-AAD，流式分析肿瘤细胞的GPRC5D和BCMA的表达水平。图3(A)示出上述三种细胞的流式分析图。
收集骨髓瘤细胞NCI-H929、MOLP8和KSM-11细胞，以及JURKAT-NFAT-LUC细胞(康源博创，Cat#：KC-1485)，400g离心5分钟，去除上清。用培养基(含10％FBS的RPMI1640)将细胞密度调整为～5×10 5/ml。同时，抗体用培养基进行3.16倍梯度稀释，起始浓度10μg/ml。充分混匀细胞，在U底96孔板中加入45μl JURKAT-NFAT-Luc、45μl H929、MOLP8或KSM-11细胞，以及10μl各浓度的抗体，在37℃培养箱中培养4～6小时。之后，将细胞转移到96孔透明平底黑壁板，加入Bright-Glo(promega，E2610)溶液，经多模式酶标仪读取冷光值，检测荧光素酶信号。实验结果如图3(B)所示，MBS314在所有三种细胞上均能激活CD3信号，即便肿瘤细胞上的GPRC5D或BCMA表达量较低。在肿瘤细胞同时高表达GPRC5D和BCMA时，MBS314引起的CD3信号通路要显著低于JNJ和EM801。
实施例7.CD3×BCMA×GPRC5D三特异性抗体的体内抗肿瘤药效
将同时表达GPRC5D和BCMA的骨髓瘤细胞MM.1S(KC-0620，康源博创)皮下注射至免疫缺陷小鼠B-NDG(百奥赛图)，同时对小鼠静脉注射人T细胞，建立人源化小鼠肿瘤体内药效模型。图4(A)显示出MM.1S细胞的GPRC5D和BCMA表达情况。
在肿瘤细胞接种前约2～3个星期，复苏骨髓瘤细胞MM.1S，培养条件为RPMI-1640培养基(含10％FBS)，3到4天传代一次。肿瘤细胞接种前6天，复苏～1×10 8PBMC，每1×10 6细胞与20μl CD3/CD28免疫磁珠(Cat#：11161D，GIBCO)共培养，体外扩增T细胞约8天。
在第0天，收集MM.1S肿瘤细胞，用基质胶(matrigel，GIBCO，356234)与PBS的1∶1混合物将肿瘤细胞密度调整为2.5×10 7/ml，每只NDG小鼠右侧腹部皮下注射0.2ml，即0.5×10 7细胞/小鼠。第2天，收集扩增的T细胞，悬浮于含0.1％FBS和10ng/ml IL-2的PBS中，密度为约0.5×10 8/ml。每只小鼠尾静脉注射0.2ml T细胞，即约1×10 7细胞。肿瘤长到50-100mm 3时，即第20天时，经尾静脉注射本申请的三特异性抗体、JNJ、EM801或PBS，剂量分别为每只小鼠10μg，每周注射2次，共7次。
每3天测量一次肿瘤大小，直至第40天，肿瘤体积计算为＝0.5×长×宽 2。
图4(B)示出各组中小鼠个体的肿瘤大小变化。可以看出，MBS314能在小鼠体内清除肿瘤细胞，效果明显好于EM801，与JNJ相当。
实施例8.CD3×BCMA×GPRC5D三特异性抗体对临床肿瘤样本的杀伤力
从复发难治多发性骨髓瘤病人获得的新鲜样品中分离骨髓单核细胞，以5×10 5/ml的密度悬浮于培养基RPMI1640(含10％FBS、1％青霉素-链霉素、100ng/ml IL-6、以及10ng/ml IL-2)。用以上培养基稀释抗体，起始浓度100μg/ml，10倍梯度稀释，共6个浓度。在U底96孔中，每孔加入90μl细胞悬液、以及10μl梯度稀释后的各浓度的抗体。培养48小时后，离心去除上清，细胞重悬于100μl FITC-抗人CD138(1∶100，Biolegend，356508)，冰上孵育30分钟。离心去除抗体，对细胞加入7-AAD，FACS分析成活细胞中CD138+细胞的比例。
图5(A)示出FACS的分析图谱，图5(B)为数据统计图。结果表明，MBS314对肿瘤细胞的杀伤力，比JNJ和EM801都更强。
实施例9.CD3×BCMA×GPRC5D三特异性抗体的体内毒性
在野生型或CD3人源化的小鼠中，检测本申请三特异性抗体的体内毒副作用。
具体地，将9只B-hCD3EDG小鼠(CD3人源化，百奥赛图)分成3组，将9只C57BL/6小鼠分成3组，分别经尾静脉注射MBS314、JNJ和PBS，20mg/kg，每周注射2次，共注射3次。每天测量体重，计算每天相对于第0天的体重百分比，绘制体重-实验天数相关曲线。第10天为实验终点，对野生型和CD3人源化小鼠，抽取抗凝血50-100μl，做小鼠血常规，并取心、肝、脾、肾、骨髓、脑、肺、和胸腺，胰腺用刀片切取0.3-0.5em或更小体积见方，用10倍体积以上的10％中性福尔马林液固定，做石蜡切片，HE染色观察组织损伤。
实验结果表明，野生型小鼠和CD3人源化小鼠在注射高剂量的MBS314后，都未见体重明显下降(图6)以及明显的组织损伤(结果未示出)，说明MBS314没有明显的毒副作用。
实施例10.CD3×BCMA×GPRC5D三特异性抗体在小鼠体内的PK动力学
选用10-12周龄的雌性BALB/c小白鼠，通过尾静脉注射MBS314，3.0mg/kg、1.0mg/kg或0.3mg/kg，在10分钟、1小时、6小时、1天、3天、5天、7天和14天后采血。血样具体地经眼眶或尾静脉采集(采集后需做抗凝处理)，每只小鼠每次采集约0.03ml。取10-30μl血样，加入4倍体积的PBS，10000g离心5分钟，取上清，-20℃保存备用。
对ELISA板加入50ng/孔的BCMA-mFc(康源博创制备)，4℃包被过夜。第二天，加入200μl封闭液(2％BSA)，37℃孵育2小时。之后将上述制备的血浆样品用含2％BSA的PBS稀释50倍、100倍和500倍；另MBS314用PBS(含2％BSA)进行3.16倍梯度稀释，起始浓度1μg/mL，共12个浓度，用于标准曲线的制作。向ELISA板中分别加入50μl血浆样品和50μl MBS314，37℃孵育1小时。洗涤 后，加入50μl HRP-抗人Fc(1∶10000，Solarbio，SE101)，37℃孵育1小时。加入ELISA显色液，50μl每孔，显色，读板。根据标准曲线和血浆稀释倍数，计算小时血浆中的抗体浓度。
图7示出抗体浓度的时间相关曲线，其中以3.0mg/kg注射的MBS314在小鼠血液中的半衰期约为4.7天，1.0mg/kg注射的为约3.18天，而0.3mg/kg注射的为约4.97天。该结果表明，MBS314在小鼠体内的血液半衰期为约4-5天。
本申请的示例性序列如下所示。
Figure PCTCN2022117979-appb-000002
Figure PCTCN2022117979-appb-000003
Figure PCTCN2022117979-appb-000004
Figure PCTCN2022117979-appb-000005
Figure PCTCN2022117979-appb-000006
                          ***
尽管本申请已经结合一个或多个实施方式进行了描述，应当理解的是，本发明不限于这些实施方式，且上述描述意在涵盖包含在所附权利要求的精神和范围内的所有其他可选形式、修饰和等同物。本文中应用的文献均通过引用的方式全部并入本文。

Claims (15)

  1. 一种多特异性抗体，包含
    i)CD3抗原结合域，
    ii)BCMA抗原结合域，以及
    iii)GPRC5D抗原结合域。
  2. 如权利要求1所述的多特异性抗体，包含
    i)第一多肽链，包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、特异结合CD3的重链可变区、和重链恒定区，
    ii)第二多肽链，包含特异结合CD3的轻链可变区，
    iii)第三多肽链，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
    iv)第四多肽链，包含特异结合GPRC5D的轻链可变区；或者
    i)第一多肽链，包含特异结合CD3的重链可变区、和重链恒定区，
    ii)第二多肽链，包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、和特异结合CD3的轻链可变区，
    iii)第三多肽链，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
    iv)第四多肽链，包含特异结合GPRC5D的轻链可变区，
    其中特异结合BCMA的重链可变区和特异结合BCMA的轻链可变区形成该BCMA抗原结合域，第一多肽链中特异结合CD3的重链可变区和第二多肽链中特异结合CD3的轻链可变区形成该CD3抗原结合域，第三多肽链中特异结合GPRC5D的重链可变区和第四多肽链中特异结合GPRC5D的轻链可变区形成该GPRC5D抗原结合域，且第一多肽链的重链恒定区与第三多肽链的重链恒定区结合在一起。
  3. 如权利要求2所述的多特异性抗体，其中，
    第一多肽链从N端到C端包含特异结合BCMA的重链可变区、特异结合BCMA的轻链可变区、特异结合CD3的重链可变区、和重链恒定区，或从N端到C端包含特异结合BCMA的轻链可变区、特异结合BCMA的重链可变区、特异结合CD3的重链可变区、和重链恒定区，
    第三多肽链从N端到C端包含特异结合GPRC5D的重链可变区、和重链恒定区；或者
    第一多肽链从N端到C端包含特异结合CD3的重链可变区、和重链恒定区，
    第二多肽链从N端到C端包含特异结合BCMA的重链可变区、特异结合 BCMA的轻链可变区、和特异结合CD3的轻链可变区，或从N端到C端包含特异结合BCMA的轻链可变区、特异结合BCMA的重链可变区、和特异结合CD3的轻链可变区，
    第三多肽链从N端到C端包含特异结合GPRC5D的重链可变区、和重链恒定区。
  4. 如权利要求3所述的多特异性抗体，其中，
    i)第一多肽链，从N端到C端，包含特异结合BCMA的重链可变区、第一接头、特异结合BCMA的轻链可变区、第二接头、特异结合CD3的重链可变区、和重链恒定区，或包含特异结合BCMA的轻链可变区、第一接头、特异结合BCMA的重链可变区、第二接头、特异结合CD3的重链可变区、和重链恒定区，
    ii)第二多肽链，从N端到C端，包含特异结合CD3的轻链可变区、和轻链恒定区，
    iii)第三多肽链，从N端到C端，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
    iv)第四多肽链，从N端到C端，包含特异结合GPRC5D的轻链可变区、和轻链恒定区；或者
    i)第一多肽链，从N端到C端，包含特异结合CD3的重链可变区、和重链恒定区，
    ii)第二多肽链，从N端到C端，包含特异结合BCMA的重链可变区、第一接头、特异结合BCMA的轻链可变区、第二接头、特异结合CD3的轻链可变区、和轻链恒定区，或包含特异结合BCMA的轻链可变区、第一接头、特异结合BCMA的重链可变区、第二接头、特异结合CD3的轻链可变区、和轻链恒定区
    iii)第三多肽链，从N端到C端，包含特异结合GPRC5D的重链可变区、和重链恒定区，以及
    iv)第四多肽链，从N端到C端，包含特异结合GPRC5D的轻链可变区、和轻链恒定区。
  5. 如权利要求1所述的多特异性抗体，其中，
    CD3抗原结合域为特异结合CD3的抗体或其抗原结合部分，其重链可变区包含分别如SEQ ID NOs：9-11所示的VH-CDR1、VH-CDR2和VH-CDR3，轻链可变区包含分别如SEQ ID NOs：12-14所示的VL-CDR1、VL-CDR2和VL-CDR3，
    BCMA抗原结合域为特异结合BCMA的抗体或其抗原结合部分，其重链可变区包含分别如SEQ ID NOs：1-3所示的VH-CDR1、VH-CDR2和VH-CDR3，轻链可变区包含分别如SEQ ID NOs：4-6所示的VL-CDR1、VL-CDR2和VL-CDR3，和/或
    GPRC5D抗原结合域为特异结合GPRC5D的抗体或其抗原结合部分，其重链可变区包含分别如SEQ ID NOs：17-19所示的VH-CDR1、VH-CDR2和VH-CDR3，轻链可变区包含分别如SEQ ID NOs：20-22所示的VL-CDR1、VL-CDR2和VL-CDR3。
  6. 如权利要求5所述的多特异性抗体，其中，
    CD3抗原结合域中的重链可变区和轻链可变区分别包含与SEQ ID NOs：15和16具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列，
    BCMA抗原结合域中的重链可变区和轻链可变区分别包含与SEQ ID NOs：7和8具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列，和/或
    GPRC5D抗原结合域中的重链可变区和轻链可变区分别包含与SEQ ID NOs：23和24具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
  7. 如权利要求2所述的多特异性抗体，其中第一多肽链中的重链恒定区和第三多肽链中的重链恒定区为弱结合或不结合Fc受体和/或补体系统蛋白的人抗体重链恒定区，和/或
    第一多肽链中的重链恒定区和第三多肽链中的重链恒定区的其中之一带有杵结构，另一带有臼结构。
  8. 如权利要求4所述的多特异性抗体，其中第一接头和/或第二接头包含SEQ ID NOs：36和37所示的氨基酸序列。
  9. 如权利要求2所述的多特异性抗体，其中第一多肽链、第二多肽链、第三多肽链、和第四多肽链分别包含与SEQ ID NOs：27、28、29和30具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的氨基酸序列。
  10. 一种核酸分子，编码权利要求1-9中任一项所述的多特异性抗体。
  11. 一种表达载体，包含权利要求10所述的核酸分子。
  12. 一种宿主细胞，包含权利要求10所述的核酸分子、或权利要求11所述的表达载体。
  13. 一种药物组合物，包含权利要求1-9中任一项所述的多特异性抗体、权利要求10所述的核酸分子、权利要求11所述的表达载体、或权利要求12所述的宿主细胞，以及药学上可接受的载体。
  14. 权利要求13所述的药物组合物在制备用于治疗与BCMA和/或GPRC5D相关的疾病的药物中的用途。
  15. 如权利要求14所述的用途，其中与BCMA和/或GPRC5D相关的疾病为多发性骨髓瘤。
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