Multispecific antibodies binding BCMA, GPRC5D, and CD3, and uses thereof

Abstract

This application relates to multispecific antibodies that simultaneously target BCMA, GPRC5D and CD3, and their use in the treatment of diseases such as tumors.

Description

[Technical Field]

[0001] The present disclosure relates to multispecific antibodies that simultaneously target BCMA, GPRC5D, and CD3, and their use in the treatment of diseases such as tumors. [Background technology]

[0002] Multiple myeloma (MM) is a disease involving the malignant proliferation of plasma cells. It is characterized by the uncontrolled proliferation of plasma cells in the bone marrow, acting as tumor cells, and the secretion of monoclonal immunoglobulins, which in most cases ultimately results in organ or tissue damage. For example, MM is often accompanied by multiple osteolytic lesions, hypercalcemia, anemia, and kidney disease. MM is commonly seen in middle-aged and older adults, and there is a clear gender difference in incidence, with more males than females. MM accounts for 10-15% of hematologic malignancies, making it the third most common hematologic malignancy after leukemia and lymphoma. The incidence rate is 9 per 100,000 in the United States and approximately 1 per 100,000 in China, and is increasing annually.

[0003] Despite recent advances in MM treatment, including chemotherapy, protease inhibitors, immunomodulatory agents, and CD38-targeted antibodies, the majority of patients do not respond to these therapies or respond only briefly to treatment. Furthermore, MM is largely incurable, and patients gradually develop resistance to treatment and experience relapse. Therefore, new drugs are urgently needed in this field.

[0004] Currently known antigens for MM targeted therapy are very limited, BCMA and GPRC5D being two of them.

[0005] BCMA and BCMA-targeted therapy B-cell maturation antigen (BCMA), also known as CD269 or TNFRSF17, is a type I transmembrane protein and a member of the tumor necrosis factor receptor (TNFR) superfamily. BCMA, along with the BAFF receptor (BAFFR) and transmembrane activator and calcium regulator and cyclophilin ligand interactor (TACI), which are also members of the TNFR superfamily, play an important role in the survival of B cells at different developmental stages (Rickert RC et al., (2011) Immunological Reviews 244(1):115-133). BCMA is primarily expressed on the surface of mature B lymphocytes and plasma cells, and is rarely expressed on hematopoietic stem cells or non-hematopoietic tissues. Its ligands include B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL), the latter of which has a higher BCMA-binding affinity.

[0006] BCMA overexpression has been observed on the surface of malignant plasma cells in the bone marrow of MM patients. BCMA promotes the survival of malignant plasma cells in the bone marrow, and APRIL-BCMA signaling in malignant plasma cells promotes malignant cell proliferation, evasion of apoptosis, and production of potent immune inhibitory molecules such as IL-10, PD-L1, and TGF-β (Tai YT et al., (2016) Blood.127(25):3225-3236). BCMA overexpression and activation have been shown to be associated with the progression of MM in many animal models and human patients (Tai YT et al., (2016) Supra; Sanchez E et al., (2016) Clin Cancer Res.22:3383-3397).

[0007] The selective expression / distribution pattern of BCMA, i.e., widespread presence on the surface of MM cells but very low or absent expression on cells in normal tissues, and the relatively long serum half-life of BCMA have made it an ideal therapeutic target for MM and other hematological malignancies. Compared with the CD138 molecule, which is uniquely expressed by plasma cells but rapidly lost from the cell surface, BCMA is clearly a better biomarker for malignant plasma cells in MM.

[0008] GPRC5D and GPRC5D-targeted therapy GPRC5D, G protein-coupled receptor class C group 5 member D, is a C-type seven-transmembrane receptor protein whose ligands and signaling mechanisms remain unknown.

[0009] GPRC5D has been found to be a marker for MM in studies of bone marrow samples from patients with MM, acute leukemia, and diffuse large B-cell lymphoma (Cohen Y et al., (2013) Hematology 18(6):348-351). This protein is primarily expressed in cells with a plasma cell phenotype, including the majority of malignant bone marrow plasma cells, and is associated with the detection of CD138 from bone marrow samples. + Its expression pattern in MM cells is very similar to that of BCMA. GPRC5D protein expression in normal tissues is restricted to hair follicles (Smith EL et al., (2019) Sci Transl Med. 11(485):eaau7746). Additionally, GPRC5D mRNA expression levels in MM patients correlate with genetic abnormalities, such as Rb-1 deletion, and poor prognosis (Atamaniuk J et al., (2012) Eur J Clin Invest. 42(9):953-960).

[0010] Due to such selective expression patterns, GPRC5D is a promising therapeutic target for plasma cell-associated diseases such as MM. A bispecific antibody against this target, JNJ-64407564, is currently in clinical trials.

[0011] T cells and CD3 The CD3 molecule is a marker on the surface of T cells. It forms a TCR-CD3 complex with the T cell receptor (TCR) and plays an important role in antigen recognition and immune signal transduction. The TCR molecule consists of an α chain and a β chain, or a γ chain and a δ chain. Because the intracellular domains of each chain do not have a signal transduction function, intracellular signaling in T cells is entirely dependent on the CD3 protein. The CD3 molecule is composed of a γ chain, a δ chain, two ε chains, and two ζ chains, which form three dimers in the TCR / CD3 complex: εγ, εδ, and ζζ. The ε, γ, and δ chains are all type I transmembrane proteins, each with an immunoglobulin-like extracellular domain. The ε, γ, δ, and ζ chains contain a total of 10 immunoreceptor tyrosine-based activation motifs (ITAMs) in their intracellular domains. Phosphorylation of these ITAMs enables these chains to bind to ZAP70, transmitting T cell activation signals to downstream components.

[0012] A CD3-targeting antibody can form a bispecific molecule with a functional moiety that targets a disease-associated antigen (such as a tumor-associated antigen) and physically link with a T cell bearing the disease-associated antigen, resulting in T cell activation and T cell-mediated disease-associated cell death. For example, a bispecific molecule targeting CD3 and a tumor-associated antigen can attract T cells to tumor cells such that the T cells are activated and release supramolecular attack particles (SMAPs) containing more than 280 proteins. SMAPs contain granzymes and perforin in their core region, where perforin can form holes on the plasma membrane of tumor cells and granzymes can induce tumor cell apoptosis (S. Balint et al., (2020) Science 368(6493): 897-901).

[0013] CD3 molecules aggregate on the surface of T cells through the action of anti-CD3 antibodies, mimicking the process of TCR recognition of MHC-antigen peptides, thereby inducing TCR complex signaling in T cells and releasing cytokines such as IL-2, IFN-γ, and TNF-α, which promote T cell proliferation and differentiation. T cell proliferation and differentiation have both advantages and disadvantages in tumor therapy. On the one hand, they produce more T cells to damage tumor cells, but on the other hand, they cause severe toxicity in subjects, i.e., cytokine release syndrome (CRS). Clinically, side effects associated with CRS include fatigue, vomiting, tachycardia, hypertension, and lower back pain, as well as central nervous system (CNS) reactions such as seizures, encephalopathy, cerebral edema, aseptic meningitis, and headache. For example, CRS has been observed in therapies using anti-CD3 monospecific antibodies such as OKT3, and is thought to be caused by antibody cross-linking upon binding to Fc receptors (FcRs) in the body (Herold KC et al., (2003) J Clin Invest. 111(3):409-418). Therefore, in later antibody development, the Fc region of anti-CD3 antibodies such as teplizumab was engineered to exhibit weak FcR binding ability.

[0014] Similarly, CRS cannot be avoided in therapies using CD3-targeting bispecific or multispecific antibodies. For example, in clinical trials of blinatumomab, a bispecific CD19 / CD3 T cell engager antibody, severe CRS and CNS toxicity were frequently observed. Modifications to the Fc region, as applied to monospecific anti-CD3 antibodies, are insufficient to alleviate bispecific anti-CD3 antibody-mediated CRS because the antigen-associated antigen-targeting functional portion of the bispecific antibody binds to target cells, resulting in antibody cross-linking and inducing massive cytokine release by T cells.

[0015] A trispecific antibody targeting GPRC5D, BCMA, and CD3 Targeting BCMA holds promise for MM treatment. However, due to heterogeneity in BCMA expression, not all tumor cells express BCMA, even within tumor samples from the same patient, resulting in different therapeutic outcomes. Furthermore, membrane-bound BCMA can be lost from the cell surface by the action of gamma-secretase, resulting in altered cell surface BCMA levels (Brudno JN et al., (2018) J. Clin. Oncol 36(22):2267-2280; Laurent SA et al., (2015) Nat Commun.6:7333). These issues may be partially resolved by targeting other markers on MM cells, such as GPRC5D or FcRH5. However, the expression distribution of BCMA is somewhat similar to that of GPRC5D, and it is unclear whether targeting both may result in more durable and stable MM tumor cell binding. In addition, an antigen-binding domain against CD3 may be added to such bispecific antibodies to induce T cell recruitment around MM cells in patients and activate the T cells to kill the MM cells. However, as mentioned above, attention should be paid to the toxicity caused by bispecific or multispecific anti-CD3 antibodies. In the first half of 2021, clinical trials of several BCMA × CD3 bispecific antibodies (e.g., AMG701, erlanatamab) were put on hold due to safety concerns.

[0016] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. Summary of the Invention

[0017] The inventors of the present application have constructed a well-designed trispecific GPRC5D×BCMA×CD3 antibody that retains potent anti-tumor activity, induces relatively low toxicity, and overcomes and alleviates the problem of balancing efficacy and toxicity of bispecific anti-CD3 antibodies, including BCMA×CD3 bispecific antibodies and GPRC5D×CD3 bispecific antibodies, currently used in MM treatment. The construction of such a multispecific antibody was far from a simple, random combination of various binding moieties, but rather took into consideration the interactions between the binding moieties. During the construction of the antibody of the present disclosure, the tumor-specific antigen species on MM cells to be bound were adjusted and optimized to avoid tumor evasion due to tumor heterogeneity, and CD3 signaling activation-mediated cytokine release was reduced to achieve a better treatment duration.

[0018] Thus, in a first aspect, the present application provides a multispecific antibody which may comprise i) an antigen-binding domain against CD3, ii) an antigen-binding domain against BCMA, and iii) an antigen-binding domain against GPRC5D.

[0019] The antigen-binding domain against CD3 may be an agonist antigen-binding domain that can specifically bind to CD3 and activate CD3 signaling. In particular, the antigen-binding domain against CD3 does not bind to CD3δ or CD3ε alone, but only to the CD3δ&ε complex. The antigen-binding domain against CD3 has almost no CD3 signaling activation activity, i.e., it does not substantially activate T cells when present as a free molecule, but retains the ability to activate CD3 signaling only when the other two antigen-binding domains in the multispecific antibody bind to their respective antigens, allowing for antibody "cross-linking." In a specific embodiment, the antigen-binding domain against CD3 is an antibody or antigen-binding portion thereof that specifically binds to CD3.

[0020] The antigen-binding domain against BCMA binds to BCMA and may optionally block BCMA-APRIL binding / interaction, i.e., may optionally be an antagonist antigen-binding domain. In particular, the antigen-binding domain against BCMA may bind to BCMA on target cells and is largely unaffected by soluble BCMA in the environment. In certain embodiments, the antigen-binding domain against BCMA is an antibody or antigen-binding domain thereof that specifically binds to BCMA.

[0021] An antigen-binding domain against GPRC5D can specifically bind to GPRC5D. In certain embodiments, an antigen-binding domain against GPRC5D can be an antibody or antigen-binding portion thereof that specifically binds to GPRC5D.

[0022] A multispecific antibody of the disclosure may comprise, for example, one antigen-binding domain to CD3, one antigen-binding domain to BCMA, and one antigen-binding domain to GPRC5D.

[0023] The multispecific antibody of the present disclosure may be an IgG-like antibody comprising an antigen-binding domain against CD3 in Fab or Fv format, an antigen-binding domain against GPRC5D in Fab or Fv format, and an antigen-binding domain against BCMA in single-chain variable fragment (ScFv) format.

[0024] In certain embodiments, a multispecific antibody may comprise i) an anti-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) an anti-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) an antigen-binding domain against BCMA in scFv format comprising a heavy chain variable region, an optional first linker, and a light chain variable region, wherein the anti-CD3 half antibody and the anti-GPRC5D half antibody may form a full-length IgG antibody, and the antigen-binding domain against BCMA in scFv format may be linked to the N-terminus of the heavy chain variable region, the N-terminus of the light chain variable region, the C-terminus of the heavy chain constant region, or the C-terminus of the light chain constant region of the anti-CD3 half antibody, optionally via a second linker. In certain embodiments, the antigen-binding domain against BCMA in scFv format may be linked to the N-terminus of the heavy chain variable region or the N-terminus of the light chain variable region of the anti-CD3 half antibody.

[0025] The multispecific antibodies of the present disclosure: i) a first polypeptide chain comprising a heavy chain variable region that specifically binds to BCMA, a light chain variable region that specifically binds to BCMA, a heavy chain variable region that specifically binds to CD3, and a heavy chain constant region; ii) a second polypeptide chain comprising a light chain variable region that specifically binds to CD3; iii) a third polypeptide chain comprising a heavy chain variable region and a heavy chain constant region that specifically binds to GPRC5D; and iv) a fourth polypeptide chain comprising a light chain variable region that specifically binds to GPRC5D; or alternatively, i) a first polypeptide chain comprising a heavy chain variable region and a heavy chain constant region that specifically binds to CD3; ii) 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; iii) a third polypeptide chain comprising a heavy chain variable region and a heavy chain constant region that specifically binds to GPRC5D; and iv) a fourth polypeptide chain comprising a light chain variable region that specifically binds to GPRC5D; and Here, the heavy chain variable region that specifically binds to BCMA and the light chain variable region that specifically binds to BCMA form an antigen-binding domain for BCMA, the heavy chain variable region that specifically binds to CD3 in the first polypeptide chain and the light chain variable region that specifically binds to CD3 in the second polypeptide chain form an antigen-binding domain for CD3, the heavy chain variable region that specifically binds to GPRC5D in the third polypeptide chain and the light chain variable region that specifically binds to GPRC5D in the fourth polypeptide chain form an antigen-binding domain for GPRC5D, and the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain are associated together.

[0026] 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, a light 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 may comprise, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region that specifically binds GPRC5D.

[0027] In certain embodiments, the multispecific antibody: i) a first polypeptide chain comprising, from N-terminus to C-terminus, a heavy chain variable region that specifically binds BCMA, a first linker, a light chain variable region that specifically binds BCMA, a second linker, a heavy chain variable region that specifically binds CD3, and a heavy chain constant region; or alternatively, a light chain variable region that specifically binds BCMA, a first linker, a heavy chain variable region that specifically binds BCMA, a second linker, a heavy chain variable region that specifically binds CD3, and a heavy chain constant region; ii) a second polypeptide chain comprising, from its N-terminus to its C-terminus, a light chain variable region that specifically binds CD3 and a light chain constant region; iii) a third polypeptide chain comprising, from the N-terminus to the C-terminus, a heavy chain variable region and a heavy chain constant region that specifically binds to GPRC5D; and iv) a fourth polypeptide chain comprising, from the N-terminus to the C-terminus, a light chain variable region and a light chain constant region that specifically binds to GPRC5D; It may comprise:

[0028] Alternatively, the first polypeptide chain may comprise, from N- to C-terminus, a heavy chain variable region that specifically binds CD3 and a heavy chain constant region. The second polypeptide chain may comprise, from N- to C-terminus, 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, or, from N- to C-terminus, a light chain variable region that specifically binds BCMA, a heavy chain variable region that specifically binds BCMA, and a light chain variable region that specifically binds CD3. The third polypeptide chain may comprise, from N- to C-terminus, a heavy chain variable region that specifically binds GPRC5D and a heavy chain constant region.

[0029] In certain embodiments, the multispecific antibody: i) a first polypeptide chain comprising, from its N-terminus to its C-terminus, a heavy chain variable region and a heavy chain constant region that specifically binds to CD3; ii) a second polypeptide chain comprising, from N-terminus to C-terminus, a heavy chain variable region that specifically binds BCMA, a first linker, a light chain variable region that specifically binds BCMA, a second linker, a light chain variable region that specifically binds CD3, and a light chain constant region; or alternatively, a light chain variable region that specifically binds BCMA, a first linker, a heavy chain variable region that specifically binds BCMA, a second linker, a light chain variable region that specifically binds CD3, and a light chain constant region. iii) a third polypeptide chain comprising, from the N-terminus to the C-terminus, a heavy chain variable region and a heavy chain constant region that specifically binds to GPRC5D; and iv) a fourth polypeptide chain comprising, from its N-terminus to its C-terminus, a light chain variable region and a light chain constant region that specifically bind to GPRC5D; may include:

[0030] The BCMA antigen-binding domain may be an antibody or antigen-binding portion thereof that specifically binds to BCMA, wherein the heavy chain variable region may comprise VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NOs: 1 to 3, respectively, and the light chain variable region may comprise VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 4 to 6, respectively. The heavy and light chain variable regions in the BCMA antigen-binding domain may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 7 and 8, respectively.

[0031] The CD3 antigen-binding domain may be an antibody or antigen-binding portion thereof that specifically binds to CD3, wherein the heavy chain variable region may comprise VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NOs: 9 to 11, respectively, and the light chain variable region may comprise VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 12 to 14, respectively. The heavy and light chain variable regions in the CD3 antigen-binding domain may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 15 and 16, respectively.

[0032] The antigen-binding domain for GPRC5D may be an antibody or an antigen-binding portion thereof that specifically binds to GPRC5D, wherein the heavy chain variable region may comprise VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NOs: 17 to 19, respectively, and the light chain variable region may comprise VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 20 to 22, respectively. The heavy chain variable region and light chain variable region in the antigen-binding domain for GPRC5D may comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 23 and 24, respectively.

[0033] The heavy chain constant regions in the first polypeptide chain and the third polypeptide chain may be heavy chain constant regions that exhibit weak or no FcR binding, preferably heavy chain constant regions that exhibit no FcR binding, such as the 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), or human IgG1(L234A+V235E) constant region, or a human IgG4 constant region.

[0034] With respect to the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain, one can be a heavy chain constant region with a knob mutation, such as a human IgG1 or IgG4 heavy chain constant region with a T366W mutation, or a functional fragment thereof. The other of the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can be a heavy chain constant region with a hole mutation, such as a human IgG1 or IgG4 heavy chain constant region with a T366S / L368A / Y407V mutation, or a functional fragment thereof. In a specific embodiment, one of the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain can be a heavy chain constant region with a knob mutation that exhibits weak or no FcR binding, such as a human IgG1 heavy chain constant region with L234A / L235A / N297A / T366W, e.g., as set forth in SEQ ID NO: 25 (X1=W, X2=L, X3=Y). In a specific embodiment, the other of the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain may be a heavy chain constant region with hole mutations that has weak or no binding to FcR, such as a human IgG1 heavy chain constant region with L234A / L235A / N297A / T366S / L368A / Y407V, e.g., as set forth in SEQ ID NO: 25 (X1=S, X2=A, X3=V).

[0035] 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. In a specific embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO:26.

[0036] The first linker and the second linker can be peptides of about 5 to 30 amino acids. In certain embodiments, the linker can be a peptide of 5 to 20 amino acids. In certain embodiments, the linker can be a GS linker, such as a GS linker comprising SEQ ID NO: 36 or 37.

[0037] The first, second, third and fourth polypeptide chains in the multispecific antibodies of the present disclosure, in certain embodiments, comprise an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to i) SEQ ID NOs: 27, 28, 29 and 30, respectively; or ii) SEQ ID NOs: 27, 16, 29 and 24, respectively.

[0038] The multispecific antibodies of the present disclosure i) specifically bind to BCMA (human and monkey BCMA) with slightly better binding affinity than prior art antibodies such as EM801, while being largely unaffected by soluble BCMA in the environment; ii) bind to CD3 with significantly lower binding affinity than prior art antibodies such as JNJ-64407564 (hereinafter abbreviated as JNJ) and EM801, activate CD3 signaling only when the multispecific antibodies bind to BCMA and / or GPRC5D, allowing antibody "cross-linking"; and exhibit weaker T cell activation ability than JNJ and / or EM801 in the presence of certain tumor cells, such as tumor cells that highly express BCMA and GPRC5D; iii) bind to GPRC5D with similar or slightly higher binding affinity compared to JNJ; vi) cytotoxicity of tumor cells in vitro with higher cytotoxicity than JNJ and EM801; and v) cytotoxicity of tumor cells in vitro with higher cytotoxicity than JNJ and EM801. It has an antitumor effect in vivo that is clearly higher than that of EM801 and equivalent to that of JNJ, and iv) may induce little in vivo toxicity at the tested concentrations.

[0039] Compared to currently available bispecific BCMAxCD3 and bispecific GPRC5DxCD3 antibodies, the trispecific antibodies of the present disclosure inhibit BCMA+ tumor cells, GPRC5D + Tumor cells and BCMA + GPRC5D + Tumor cells may be killed more comprehensively and the problem of tumor antigen expression heterogeneity may be better addressed, i.e., tumor cells with high or low BCMA and GPRC5D expression levels may be targeted and killed by the trispecific antibodies of the present disclosure.

[0040] In addition, the ability of the multispecific antibody of the present disclosure to activate T cells is lower than that of JNJ and EM801 in the presence of tumor cells with high BCMA and GPRC5D expression; the ability of the multispecific antibody of the present disclosure to activate T cells is lower than that of JNJ in the presence of tumor cells with high GPRC5D expression and low BCMA expression; and the ability of the multispecific antibody of the present disclosure to activate T cells is lower than that of EM801 in the presence of tumor cells with high BCMA expression and low GPRC5D expression. In other words, the trispecific antibody of the present disclosure more comprehensively kills tumor cells and may cause fewer adverse side effects when addressing the issue of tumor antigen expression heterogeneity.

[0041] The present disclosure also provides nucleic acid molecules encoding the multispecific antibodies of the present disclosure, expression vectors comprising the nucleic acids, and host cells comprising the expression vectors or having the nucleic acids integrated into their genome. The present disclosure further provides methods for preparing multispecific antibodies using host cells comprising the above expression vectors, comprising the steps of (i) expressing the multispecific antibody in the host cells, and (ii) isolating the multispecific antibody from the host cells or their cell culture medium.

[0042] The present disclosure also provides a pharmaceutical composition comprising an effective amount of a multispecific antibody, nucleic acid molecule, expression vector, or host cell of the present disclosure and a pharmaceutically acceptable carrier.

[0043] In a second aspect, the present disclosure provides a method for treating or alleviating tumor or cancer in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition of the present disclosure.

[0044] The tumor or cancer may be associated with BCMA and / or GPRC5D, and includes, but is not limited to, multiple myeloma and other hematological malignancies such as plasmacytoma, plasma cell leukemia, macroglobulinemia, isolated plasmacytoma of bone, and extramedullary plasmacytoma.

[0045] In certain embodiments, the tumor or cancer is multiple myeloma.

[0046] In certain embodiments, the tumor or cancer is multiple myeloma, and the myeloma cells highly express BCMA and GPRC5D.

[0047] In certain embodiments, the subject is a mammal, particularly a human.

[0048] The present disclosure also provides the use of a multispecific antibody, a nucleic acid molecule, an expression vector, a host cell, or a pharmaceutical composition of the present disclosure in the preparation of a medicament for treating or alleviating a tumor or cancer.

[0049] All documents cited or referenced in this application (including, but not limited to, all references, patents, and published patent applications cited herein) ("documents cited herein"), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product mentioned herein, or any documents cited herein, are hereby incorporated by reference and may be utilized in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference. [Brief explanation of the drawings]

[0050] The following detailed description, provided by way of example and not intended to limit the invention to only the specific embodiments described, can be best understood in conjunction with the accompanying drawings, in which:

[0051] [Figure 1] FIG. 1 is a schematic diagram of the structure of an exemplary trispecific GPRC5D×BCMA×CD3 antibody.

[0052] [Figure 2]Binding activity of an exemplary trispecific GPRC5DxBCMAxCD3 antibody (MBS314), bispecific BCMAxCD3 antibody (EM801), and bispecific GPRC5DxCD3 antibody (JNJ) to HEK293A / human BCMA cells (A), HEK293A / monkey BCMA cells (B), HEK293A / mouse BCMA cells (C), HEK293T / human GPRC5D cells (D), HEK293T / monkey GPRC5D cells (E), and human Jurkat T cells (F) is shown.

[0053] [Figure 3] Shown are BCMA and GPRC5D expression on three multiple myeloma cell lines (A) and a FACS analysis chart of the activity of an exemplary trispecific GPRC5DxBCMAxCD3 antibody to activate CD3 signaling in Jurkat-NFAT-LUC cells after "cross-linking" with these three cell lines (B).

[0054] [Figure 4] GPRC5D and BCMA expression in MM.1S myeloma cells (A) and growth curves of MM.1S tumors in mice treated with an exemplary trispecific GPRC5DxBCMAxCD3 antibody (B) are shown.

[0055] [Figure 5] FIG. 1 shows a FACS analysis chart of monocytes isolated from a fresh sample from a patient with relapsed / refractory multiple myeloma treated with an exemplary trispecific GPRC5DxBCMAxCD3 antibody and stained for CD138+ (A), and a statistical chart showing the percentage of CD138+ cells (B).

[0056] [Figure 6] 1 shows the weight change of C57BL / 6 mice and CD3-humanized mice injected with an exemplary trispecific GPRC5DxBCMAxCD3 antibody, where B6 represents C57BL / 6 mice and CD3 represents CD3-humanized mice.

[0057] [Figure 7]1 is a line graph showing the PK of an exemplary trispecific GPRC5DxBCMAxCD3 antibody in mice. DETAILED DESCRIPTION OF THE INVENTION

[0058] To ensure that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0059] The term "CD3" refers to cluster of differentiation 3, which includes the gamma, delta, epsilon, and zeta chains. The terms "CD3ε" or "CD3E" refer to the epsilon chain of CD3. The terms "CD3δ" or "CD3D" refer to the delta chain of CD3. The terms "CD3D&E" or "CD3δ&ε" refer to the epsilon delta complex formed by the delta and epsilon chains. These terms include variants, homologs, orthologs, and paralogs.

[0060] The term "BCMA" refers to a B cell maturation antigen that is abundantly and selectively expressed on malignant plasma cells, and includes variants, homologs, orthologs, and paralogs. The term "human BCMA" refers to a BCMA protein having an amino acid sequence from a human, e.g., the amino acid sequence of SEQ ID NO: 31. The term "monkey BCMA" refers to a BCMA protein having an amino acid sequence from a monkey, e.g., the amino acid sequence of SEQ ID NO: 32. The term "mouse BCMA" refers to a BCMA protein having an amino acid sequence from a mouse, e.g., the amino acid sequence of SEQ ID NO: 33.

[0061] The term "GPRC5D" refers to G protein-coupled receptor class C group 5 member D, a marker molecule for myeloma cells, and includes variants, homologs, orthologs, and paralogs. The term "human GPRC5D" refers to a GPRC5D protein having an amino acid sequence from a human, for example, the amino acid sequence of SEQ ID NO: 34. The term "monkey GPRC5D" refers to a GPRC5D protein having an amino acid sequence from a monkey, for example, the amino acid sequence of SEQ ID NO: 35.

[0062] The term "optional" or "optionally" means the inclusion of some components or steps that are not mandatory or required, i.e., some components or steps are included in certain circumstances and not included in some other circumstances.

[0063] The term "antibody" as referred to herein includes IgG, IgA, IgD, IgE, and IgM whole antibodies, and any antigen-binding fragment (i.e., "antigen-binding portion") thereof. A "whole antibody" or "full-length antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, where the heavy and light chains are interconnected by disulfide bonds; whereas a "half antibody" is half of a "whole antibody," e.g., comprising one heavy chain and one light chain, where the heavy and light chains are interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H The heavy chain constant region comprises three domains: C H1 , C H2 , and C H3 Each light chain comprises a light chain variable region (referred to herein as V L The light chain constant region comprises one domain, C L Includes V H and V L The region can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V Lis composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The antibody constant regions of the present disclosure are designed to have weak or no binding to cells of the immune system and proteins of the complement system. The antigen-binding function of an antibody is determined by (i) the V L V H , C L , and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V H and C H1 (iv) a single-arm V fragment of an antibody; L and V H Fv fragment consisting of domains, (v) V H It has been shown that this can be performed by fragments of full-length antibodies, including, but not limited to, (i) dAb fragments consisting of domains (Ward et al., (1989) Nature 341:544-546); (ii) isolated complementarity-determining regions (CDRs); and (viii) nanobodies, which are heavy chain variable regions comprising a single variable domain and two constant domains. As used herein, "IgG-like antibodies" refer to antibodies that retain the basic structure of an IgG antibody and are obtained by additionally adding some portions, such as an antigen-binding domain. As used herein, a "functional fragment" of a heavy chain constant region refers to a fragment of a heavy chain constant region that retains the required function (e.g., binding to another heavy chain constant region or fragment, binding to an Fc receptor and / or a component of the complement system).

[0064] As used herein, the term "agonist anti-CD3 antibody" refers to an anti-CD3 antibody that can bind to CD3 and activate or induce CD3 signaling, which promotes the activation and proliferation of immune cells, such as T cells. The anti-CD3 antibodies of the present disclosure activate CD3 signaling and induce immune cell activity only when they are "crosslinked."

[0065] The term "cross-linking" or "cross-linking" refers to antibody aggregation or interaction induced by binding of the BCMA-targeting portion of a multispecific antibody to BCMA, binding of the GPRC5D-targeting portion to GPRC5D, and / or binding of the Fc region to an Fc receptor and / or a component of the complement system (in the case of an Fc region that retains its ability to bind to an FcR and / or a component of the complement system). In in vitro studies, antibody cross-linking can occur when an antibody Fc region binds to an anti-Fc secondary antibody. In contrast, the term "free" means that the antibodies do not interact with each other or with other molecules that could cause dimerization or multimerization. When "free," the multispecific antibodies of the present disclosure do not activate CD3 signaling or activate immune cells such as T cells.

[0066] The terms "antagonist anti-BCMA antibody" or "blocking anti-BCMA antibody" refer to an antibody that can bind to BCMA and block or inhibit BCMA signaling mediated by BCMA interaction with ligands such as BAFF or APRIL, particularly APRIL. Antagonist anti-BCMA antibodies may block malignant tumor cell proliferation and apoptosis evasion. The antigen-binding domain against BCMA in the multispecific antibodies of the present disclosure may be an antagonist that binds to BCMA and blocks BCMA-APRIL binding / interaction without inducing BCMA signaling.

[0067] The term "FcR" refers to a protein that can be bound by the Fc portion of an antibody and is expressed on the surface of certain cells, such as B lymphocytes, natural killer cells, and macrophages, and stimulates phagocytosis of target cells and their toxicity, thus playing an important role in the immune system. FcRs include Fcα receptors, Fcε receptors, and Fcγ receptors, where Fcγ receptors belong to the immunoglobulin superfamily and are the most important FcRs for inducing phagocytosis of microorganisms, and include FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), and FcγRIIIA (CD16A).

[0068] The term "multispecific" antibody refers to an antibody that specifically binds to two or more (e.g., three) target molecules or to two or more (e.g., three) different epitopes on the same target molecule. Multispecific molecules include antibodies of the present disclosure that specifically bind to BCMA, GPRC5D, and CD3. A "bispecific antibody" refers to an antibody that, in certain circumstances, specifically binds to two epitopes on two target cells or target molecules. In contrast, a "monospecific" antibody refers to an antibody that specifically binds to a particular target molecule, particularly an epitope on a particular target molecule.

[0069] As used herein, an antibody that "specifically binds to BCMA," "specifically binds to CD3," or "specifically binds to GPRC5D" refers to an antibody that binds to BCMA, CD3, or GPRC5D, but does not substantially bind to non-BCMA, non-CD3, or non-GPRC5D proteins. Preferably, the antibody binds with "high affinity," i.e., greater than 5.0 x 10 -7 K below M D and binds to human BCMA, CD3, or GPRC5D proteins.

[0070] The term "does not substantially bind" to proteins or cells means that the antibody does not bind to proteins or cells or does not bind with high affinity, i.e., less than 1.0 x 10 -6 K over M D This means that the molecule binds to a protein or cell.

[0071] The half-maximal effective concentration (EC 50 The term "antibody concentration" refers to the concentration of antibody that produces 50% of its maximum effect.

[0072] The half-maximal inhibitory concentration (IC 50 The term "inhibitory" refers to the concentration of a drug or inhibitor that inhibits a specific biological process by 50%.

[0073] The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals such as non-human primates, sheep, dogs, cats, cows, and horses are preferred.

[0074] The term "effective amount" refers to an amount of an antibody of the present disclosure sufficient to achieve a desired result. The term "therapeutically effective amount" refers to an amount of an antibody of the present disclosure sufficient to prevent or alleviate symptoms associated with a disease or condition (such as cancer). The therapeutically effective amount is relative to the disease being treated, where the actual effective amount can be readily determined by one skilled in the art.

[0075] Advantageous Properties of the Multispecific Antibodies of the Present Disclosure The multispecific antibodies of the present disclosure have i) equivalent or higher BCMA binding ability, ii) equivalent or higher GPRC5D binding ability, iii) significantly lower CD3 binding ability, vi) equivalent or better in vitro anti-tumor activity, v) equivalent or better in vivo anti-tumor efficacy, and / or iv) equivalent or lower in vivo toxicity when compared to prior art antibodies such as JNJ and EM801.

[0076] The inventors of the present application have constructed a trispecific GPRC5D×BCMA×CD3 antibody with an excellent design that retains potent tumor-killing ability, induces relatively low toxicity, and overcomes and alleviates the problem of the balance between efficacy and toxicity of bispecific anti-CD3 antibodies, including BCMA×CD3 bispecific antibodies and GPRC5D×CD3 bispecific antibodies, currently used in MM treatment. Compared with the currently available BCMA×CD3 bispecific antibodies and GPRC5D×CD3 bispecific antibodies, the trispecific antibody of the present disclosure has a high efficacy and toxicity of BCMA + Tumor cells, GPRC5D + Tumor cells and BCMA + GPRC5D + Tumor cells may be killed more comprehensively and the problem of tumor antigen expression heterogeneity may be better addressed, i.e., tumor cells may be targeted and killed by the trispecific antibodies of the present disclosure regardless of whether they express high or low levels of BCMA and GPRC5D on those cells.

[0077] In addition, the ability of the multispecific antibody of the present disclosure to activate T cells is lower than that of JNJ and EM801 in the presence of tumor cells with high BCMA and GPRC5D expression; the ability of the multispecific antibody of the present disclosure to activate T cells is lower than that of JNJ in the presence of tumor cells with high GPRC5D expression and low BCMA expression; and the ability of the multispecific antibody of the present disclosure to activate T cells is lower than that of EM801 in the presence of tumor cells with high BCMA expression and low GPRC5D expression. In other words, the trispecific antibody of the present disclosure can more comprehensively kill tumor cells and cause fewer adverse side effects when addressing the issue of tumor antigen expression heterogeneity. In particular, the multispecific antibody of the present disclosure has better cytotoxicity against tumors with high expression of both BCMA and GPRC5D, and causes relatively fewer adverse side effects.

[0078] The heavy and light chain variable region CDRs of the monospecific antibodies or antigen-binding fragments thereof used in the multispecific antibodies of the present disclosure are defined according to the Kabat numbering system. As is well known in the art, the heavy and light chain variable region CDRs can also be determined according to other systems, such as the Chothia, IMGT, AbM, or Contact numbering systems / methods.

[0079] Multispecific antibodies of the present disclosure include bispecific antibodies.

[0080] Multispecific antibodies of the present disclosure The multispecific antibody of the present disclosure may comprise: i) an antigen-binding domain against CD3, ii) an antigen-binding domain against BCMA, and iii) an antigen-binding domain against GPRC5D. The antigen-binding domain against CD3 may be an agonist antigen-binding domain that can specifically bind to CD3 and activate CD3 signaling. The antigen-binding domain against BCMA may bind to BCMA and optionally block BCMA-APRIL binding / interaction, i.e., optionally be an antagonist antigen-binding domain. The antigen-binding domain against GPRC5D may specifically bind to GPRC5D.

[0081] A multispecific antibody of the disclosure may comprise, for example, one antigen-binding domain to CD3, one antigen-binding domain to BCMA, and one antigen-binding domain to GPRC5D.

[0082] The multispecific antibody of the present disclosure may be an IgG-like antibody comprising an antigen-binding domain against CD3 in Fab or Fv format, an antigen-binding domain against GPRC5D in Fab or Fv format, and an antigen-binding domain against BCMA in single-chain variable fragment (ScFv) format.

[0083] The multispecific antibody may comprise: i) an anti-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) an anti-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) an antigen-binding domain against BCMA in scFv format comprising a heavy chain variable region, an optional first linker, and a light chain variable region, wherein the anti-CD3 half antibody and the anti-GPRC5D half antibody may form a full-length IgG antibody, and the antigen-binding domain against BCMA in scFv format may be linked to the N-terminus of the heavy chain variable region, the N-terminus of the light chain variable region, the C-terminus of the heavy chain constant region, or the C-terminus of the light chain constant region of the anti-CD3 half antibody, optionally via a second linker. In a specific embodiment, the antigen-binding domain against BCMA in scFv format may be linked to the N-terminus of the heavy chain variable region or the N-terminus of the light chain variable region of the anti-CD3 half antibody.

[0084] An antigen-binding domain against BCMA in scFv format may comprise, from N- to C-terminus, a heavy chain variable region, an optional first linker, and a light chain variable region; or a light chain variable region, an optional first linker, and a heavy chain variable region.

[0085] One heavy chain constant region in the two half antibodies can be a heavy chain constant region with knob mutations and weak or no FcR binding, such as a human IgG1 heavy chain constant region of L234A / L235A / N297A / T366W, and the other heavy chain constant region can be a heavy chain constant region with hole mutations and weak or no FcR binding, such as a human IgG1 heavy chain constant region of L234A / L235A / N297A / T366S / L368A / Y407V.

[0086] The first and second linkers can be composed of amino acids linked together by peptide bonds, preferably 5-30 amino acids linked by peptide bonds, where the amino acids are selected from the 20 naturally occurring amino acids. As will be appreciated by those skilled in the art, one or more of these amino acids can be glycosylated. In one embodiment, the 5-30 amino acids can be selected from glycine, alanine, proline, asparagine, glutamine, serine, and lysine. In one embodiment, the majority of the linker is composed of sterically unhindered amino acids, such as glycine and alanine. Exemplary linkers are polyglycines, particularly poly(Gly-Ala), and polyalanines. Exemplary linkers of the present disclosure can include the amino acid sequence of SEQ ID NO: 19, 20, 21, or 22.

[0087] The linker may also be a non-peptide linker. For example, alkyl linkers such as -NH-, -(CH2)sC(O)-, etc., where s=2 to 20, may be used. These alkyl linkers are preferably lower alkyl (e.g., C 1~6 It may be further substituted by any sterically unhindered group such as lower acyl), halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0088] conservative modification In another embodiment, a multispecific antibody of the present disclosure may comprise heavy chain variable region and / or light chain variable region or CDR1, CDR2, and CDR3 sequences with one or more conservative modifications, as understood in the art that certain conservative sequence modifications do not eliminate antigen-binding ability.

[0089] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the disclosure can be replaced with another amino acid residue from the same side chain family, and the resulting antibody can be tested for retained function (i.e., the functions described above) using the functional assays described herein.

[0090] Genetically engineered antibodies The multispecific antibodies of the present disclosure may be used in conjunction with the V H / V L Antibodies can be prepared using antibodies having one or more of the following sequences: H and / or V L), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions to improve binding affinity and / or increase similarity to naturally occurring antibodies in a particular species. For example, framework regions are modified to provide humanized antibodies. Additionally or alternatively, antibodies can be engineered by modifying residues in the constant region(s), for example, to alter the effector functions of the antibody.

[0091] Thus, each heavy chain variable region and / or each light chain variable region comprised in a multispecific antibody of the present disclosure may comprise the VH-CDR1, VH-CDR2, and VH-CDR3, and / or VL-CDR1, VL-CDR2, and VL-CDR3 of the present disclosure, although the framework sequences may differ.

[0092] The inventors of the present application discovered that when an antigen-binding domain is in scFv format, stability may be inferior to that of a full-length antibody or Fab format. Therefore, in one embodiment of the present disclosure, to resolve stability issues that may arise with an antigen-binding domain against BCMA in scFv format when used in the construction of a multispecific antibody, framework sequences in the anti-BCMA heavy / light chain variable regions, such as amino acids in the light chain variable region FR3 and FR4 portions, were modified based on computer modeling to enhance the stability of the ScFv and reduce the formation of aggregates and the increase in monomer levels. The Fab and scFv formats with or without modifications showed comparable binding affinity, BCMA-APRIL blocking activity, and anti-tumor efficacy.

[0093] The inventors of the present application have further discovered that the druggability, and therefore the industrial production level, of multispecific antibodies can be further improved by altering the framework sequences in the anti-BCMA heavy / light chain variable regions, for example by altering the hydrophilicity / hydrophobicity of amino acids in the framework regions.

[0094] The engineered antibodies of the present disclosure may be modified by VH and / or V L These include those in which modifications have been made to framework residues within the framework region. Typically, framework regions are modified to reduce the immunogenicity of an antibody. For example, one approach is to backmutate one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived.

[0095] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach, also known as "deimmunization," is described in further detail in U.S. Patent Application Publication No. 20030153043.

[0096] In addition to, or alternatively to, modifications made within the framework or CDR regions, antibodies of the disclosure can be engineered to contain modifications within 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 antigen-dependent cellular cytotoxicity. Furthermore, antibodies of the disclosure can be chemically modified (e.g., by attaching one or more chemical moieties to the antibody), again to alter one or more functional properties of the antibody, or can be modified to alter glycosylation.

[0097] In one embodiment, C H1 The hinge region of is modified such that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This technique is further described in U.S. Pat. No. 5,677,425. H1 The number of cysteine ​​residues in the hinge region of the is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease antibody stability.

[0098] In another embodiment, the Fc hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are made in the C of the Fc hinge fragment such that the antibody has weaker Staphylococcus aureus protein A (SpA) binding compared to native Fc hinge domain SpA binding. H2 -C H3 This approach is explained in more detail in US Patent No. 6,165,745.

[0099] In yet another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such glycomodifications can be achieved, for example, by altering one or more sites of glycosylation in the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at those sites. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Patent Nos. 5,714,350 and 6,350,861. Additionally, antibodies with altered glycosylation can be generated, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been shown to increase the ADCC ability of antibodies. Such glycosylation modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present disclosure, thereby producing antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), such that antibodies expressed in these cell lines lack the sugar fucose.

[0100] Another modification of antibodies herein is pegylation (PEGylation). Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment.

[0101] Nucleic acid molecules encoding antibodies of the present disclosure In another aspect, the present disclosure provides nucleic acid molecules encoding, for example, the anti-BCMA heavy chain variable region-first linker-anti-BCMA light chain variable region-second linker-anti-CD3 heavy chain variable region-heavy chain constant region, anti-GPRC5D heavy chain variable region-heavy chain constant region, or anti-GPRC5D light chain variable region-light chain constant region of a multispecific antibody of the present disclosure.

[0102] Nucleic acids can be present in whole cells, cell lysates, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it has been purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. Nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0103] Nucleic acid molecules of the present disclosure can be obtained using standard molecular biology techniques. Preferred nucleic acid molecules of the present disclosure are H and V L sequences encoding the CDRs of anti-CD3, anti-BCMA, and anti-GPRC5D monospecific antibodies. H and / or V L Once DNA fragments encoding the segments have been obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. L or V H A DNA fragment encoding 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 the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0104] To generate the scFv gene, V is synthesized as a continuous single chain protein. H and V LThe sequence is expressed as V L and V H V such that the domains can be linked by flexible linkers H and V L is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (see, e.g., 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).

[0105] For the multispecific antibodies of the present disclosure, sequences encoding the CDRs, VH, and VL of anti-CD3, anti-BCMA, and anti-GPRC5D antibodies are first obtained and then combined according to the required structure of the multispecific antibody. For example, sequences encoding an anti-BCMA heavy chain variable region, a first linker, an anti-BCMA light chain variable region, a second linker, an anti-CD3 heavy chain variable region, and a heavy chain constant region may be operably linked as needed.

[0106] Generation of antibodies of the present disclosure Multispecific antibodies of the disclosure can be produced by i) inserting sequences encoding the polypeptide chains of the multispecific antibody into one or more expression vectors operably linked to regulatory sequences that control transcription or translation; (ii) transducing or transfecting a host cell with the expression vectors; and (iii) expressing the polypeptide chains to form a multispecific antibody of the disclosure.

[0107] The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes.

[0108] The expression vector can encode a signal peptide that facilitates secretion of the polypeptide chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0109] In addition to the antibody polypeptide chain genes and control sequences, the expression vectors of the disclosure may carry additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and a selectable marker gene. For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0110] Expression vectors encoding the different polypeptide chains of a multispecific antibody can be transfected into host cells by standard techniques. The various forms of the term "transfection" encompass a wide range of techniques commonly used for the introduction of exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. While it is theoretically possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells is preferred, with mammalian host cells being most preferred, because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded, immunoreactive antibodies.

[0111] Expression vectors that can be used in the present application include, but are not limited to, plasmids, viral vectors, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), transformable artificial chromosomes (TACs), mammalian artificial chromosomes (MACs), and human artificial episomal chromosomes (HAECs). Pharmaceutical Composition

[0112] In another aspect, the present disclosure provides pharmaceutical compositions that may comprise one or more multispecific antibodies of the present disclosure, nucleic acid molecules encoding the multispecific antibodies, expression vectors comprising the nucleic acid molecules, and / or host cells comprising the nucleic acid molecules, formulated together with a pharmaceutically acceptable carrier. The pharmaceutical compositions may optionally comprise one or more additional pharmaceutically active ingredients, such as another anti-tumor drug or an immune-enhancing drug. The pharmaceutical compositions of the present disclosure may be used in combination with, for example, an additional anti-tumor drug or another immune-enhancing drug.

[0113] Pharmaceutical compositions may contain any number of excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersing or suspending aids, solubilizers, colorants, flavoring agents, coating agents, disintegrants, lubricants, sweeteners, preservatives, isotonicity 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).

[0114] The pharmaceutical compositions are suitable for oral, intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active ingredient may be coated with a material to protect it from the action of acids and other natural conditions that may inactivate it. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. Alternatively, the antibodies of the present disclosure may be administered via non-parenteral routes, such as topical, epidermal, or mucosal routes of administration, e.g., nasal, oral, vaginal, rectal, sublingual, or topical administration. Preferably, the pharmaceutical composition is administered orally.

[0115] The pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They may also be formulated as microemulsions, liposomes, or other ordered structures suitable to high drug concentration.

[0116] Dosage of the pharmaceutical compositions of the present disclosure can be determined by a medical practitioner, e.g., a physician, depending on the subject's particular circumstances, e.g., gender, age, medical history, and the like.

[0117] A "therapeutically effective amount" of a multispecific antibody of the present disclosure may result in a decrease in the severity of disease symptoms or an increase in the frequency and length of disease symptom-free periods. For example, in the case of a subject with a tumor, a "therapeutically effective amount" may preferably reduce tumor size by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, or even eliminate the tumor, compared to a control subject.

[0118] The pharmaceutical compositions can be in a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0119] In certain embodiments, antibodies of the present disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that therapeutic antibodies of the present disclosure cross the blood-brain barrier, they can be formulated in liposomes, which can additionally contain targeting moieties to enhance selective transport to specific cells or organs.

[0120] Uses and Methods of the Disclosure The pharmaceutical composition of the present disclosure has multiple in vitro and in vivo uses, for example, it can be used to treat or alleviate tumors or cancer.

[0121] The pharmaceutical compositions of the present disclosure may be used to treat or ameliorate BCMA and / or GPRC5D associated tumors or cancers, including, but not limited to, multiple myeloma and other hematological malignancies such as plasmacytoma, plasma cell leukemia, macroglobulinemia, isolated plasmacytoma of bone, and extramedullary plasmacytoma.

[0122] The present disclosure provides combination therapies in which a pharmaceutical composition of the present disclosure is co-administered with one or more additional antibodies or non-antibody agents, such as immune enhancers.

[0123] The combination of therapeutic agents described herein can be administered simultaneously in a single composition in a pharmaceutically acceptable carrier, or simultaneously in separate compositions with each agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents can be administered sequentially.

[0124] Furthermore, when more than one dose of the combination therapy is administered sequentially, the order of sequential administration may be reversed or maintained in the same order at each time of administration, and sequential administration may be combined with simultaneous administration, or any combination thereof.

[0125] Various aspects and embodiments of the present disclosure are described with reference to the drawings and the following examples. Other aspects and embodiments will be apparent to those skilled in the art. All references described herein are incorporated herein by reference in their entirety. While the present application has been described with reference to exemplary embodiments, many equivalent variations and modifications will be apparent to those skilled in the art in light of this disclosure. Accordingly, the exemplary embodiments of the present disclosure are provided by way of illustration only, and not limitation. Various modifications may be made to the embodiments without departing from the spirit and scope of the present application.

[0126] example Example 1 Construction of cell lines stably expressing BCMA or GPRC5D Cell lines stably expressing human BCMA, monkey BCMA, and mouse BCMA were constructed using HEK293A cells. Briefly, cDNA sequences encoding human BCMA, monkey BCMA, and mouse BCMA (amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively) were synthesized, enzymatically digested, and then cloned into pLV-EGFP(2A)-Puro vectors (Beijing Inovogen, China) between the BamH1 and EcoR1 sites. Lentivirus was generated in HEK293T cells (Nanjing Cobioer, China) by lipofectinization of the resulting pLV-EGFP(2A)-Puro-human BCMA, pLV-EGFP(2A)-Puro-monkey BCMA, or pLV-EGFP(2A)-Puro-mouse BCMA vectors using the psPAX and pMD2.G plasmids according to the instructions of the Lipofectamine 3000 kit (Thermo Fisher Scientific, USA). Three days after transfection, lentivirus was collected from the cell culture medium of HEK293T cells (DMEM medium (Cat#: SH30022.01, Gibco)) supplemented with 10% FBS (Cat#: FND500, Excell). The lentivirus was then used to infect HEK293A cells (Nanjing Cobioer, China) to generate cells stably expressing human, monkey, or mouse BCMA (referred to as HEK293A / human BCMA cells, HEK293A / monkey BCMA cells, and HEK293A / mouse BCMA cells, respectively). These HEK293A cells were cultured for 7 days in DMEM containing 10% FBS and 0.2 μg / ml puromycin (Cat#: A11138-03, Gibco). Human and monkey BCMA expression was analyzed by FACS in flow cytometry using a commercially available anti-BCMA antibody (PE-anti-human BCMA antibody, Cat#: 357503, Biolegend, USA). Similarly, mouse BCMA expression was confirmed by FACS using a commercially available anti-mouse BCMA antibody (anti-mouse BCMA antibody, Cat#: O88472, Novus, USA).

[0127] Similarly, HEK293T cell lines stably expressing human or monkey GPRC5D were constructed using cDNA encoding human or monkey GPRC5D (amino acid sequences set forth in SEQ ID NOs: 34 and 35, respectively), and expression of human and monkey GPRC5D was tested using anti-GPRC5D antibodies (anti-GPRC5D antibodies were prepared using amino acid sequences 10 and 11 of patent application publication WO2022058445A1 at a final concentration of 20 μg / ml).

[0128] Example 2 Construction and expression of a trispecific CD3xBCMAxGPRC5D antibody The trispecific antibody was constructed as an asymmetric whole antibody with a 1:1:1 ratio of anti-BCMA:anti-CD3:anti-GPRC5D; the structure of an exemplary trispecific antibody is shown in Figure 1. Using the GS vector (see ZL200510064335.0 for details) as an expression vector, two half antibodies of the exemplary trispecific antibody, MBS-314-knob and MBS-314-hole, were constructed. The long chain of MBS-314-knob comprises anti-BCMA ScFv-linker-anti-CD3 heavy chain variable region-heavy chain constant region, and its amino acid sequence is set forth in SEQ ID NO: 27. The short chain comprises anti-CD3 light chain variable region-light chain constant region, and its amino acid sequence is set forth in SEQ ID NO: 28. The long chain of MBS-314-hole comprises an anti-GPRC5D heavy chain variable region-heavy chain constant region, the amino acid sequence of which is set forth in SEQ ID NO: 29, and the short chain comprises an anti-GPRC5D light chain variable region-light chain constant region, the amino acid sequence of which is set forth in SEQ ID NO: 30. In the BCMA-targeting ScFv, the framework of the light chain variable region (e.g., the fourth framework) was modified to stabilize the ScFv structure.

[0129] DNA sequences encoding the anti-BCMA ScFv-linker-anti-CD3 heavy chain variable region of the MBS-314-knob half antibody and the anti-GPRC5D heavy chain variable region of the MBS-314-hole half antibody were synthesized. These two fragments were digested with restriction endonucleases EcoRI and NheI, and the resulting DNA fragments were cloned into vectors containing the amino acid sequences of the respective heavy chain constant regions (SEQ ID NO: 25 (X1 = W, X2 = L, X3 = Y) and SEQ ID NO: 25 (X1 = S, X2 = A, X3 = V)), thereby constructing expression vectors with the full-length sequences of the two half antibodies. In addition, DNA sequences encoding the anti-CD3 light chain variable region of the MBS-314-knob half antibody and the anti-GPRC5D light chain variable region of the MBS-314-hole half antibody were synthesized. These two fragments were digested with restriction endonucleases ClaI and BsiWI, and the resulting DNA fragment was cloned into a vector containing the light chain constant region (SEQ ID NO: 26) to construct expression vectors with the full-length sequences of the two half-antibody short chains. The full-length sequences of the half-antibody short chains were digested with ClaI and HindIII, the full-length sequences of the half-antibody long chains were digested with EcoRI and XhoI, the pCMV co-fragment plasmid was digested with HindIII and EcoRI, and the GS vector was digested with ClaI and XhoI. The four DNA fragments were recovered, ligated, transformed, and monoclonal clones were selected for sequencing. Half-antibody expression vectors with the correct sequences, designated MBS-314-knob expression vector and MBS-314-hole expression vector, were obtained. Plasmids were extracted using the TianGen Endofree Maxi Plasmid Extraction Kit (Cat#: DP117, TianGen). The plasmid was resuspended in 5 ml of FreeStyle F17 cell culture medium, and a transfection agent (PEI) in a volume three times that of the plasmid was resuspended in 5 ml of FreeStyle F17 cell culture medium. The PEI was gradually added to the plasmid, mixed thoroughly, and left at room temperature for 15 minutes. The mixture obtained above was transfected into 100 ml of 293F cells (1 x 10 6The transfected HEK-293F cells were cultured at 37°C and 5% CO2 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 passed through a 0.22 μm film filter to remove cell debris. The half antibody was concentrated and purified using a pre-equilibrated protein A affinity column (Cat#: 17040501, GE, USA) and eluted with elution buffer (20 mM citric acid, pH 3.0-3.5). The antibody was stored in PBS (pH 7.0), and the antibody concentration was determined using a NanoDrop. Further construction was performed on the purified half antibody.

[0130] Example 3 Construction of a trispecific CD3×BCMA×GPRC5D antibody Purified half antibodies were assembled in vitro. Specifically, MBS-314-knob half antibodies and MBS-314-hole half antibodies were mixed at a 1:1 molar ratio, and a Tris buffer solution was added until the pH reached 8.0. A small amount of reduced glutathione solution was then added and the mixture was allowed to react overnight at 25°C with slow stirring. After the reaction was complete, 2M acetic acid solution was added to the mixture to adjust the pH to 5.5. The reducing agent was removed by ultrafiltration, and the reaction was terminated. The assembled antibodies were transferred to low-salt Tris buffer (pH 8.0) and filtered using a 0.2 μm film filter. First, an anion exchange column was equilibrated with low-salt Tris buffer (pH 8.0) and the sample was applied. The components that passed through the column were collected and rinsed with low-salt Tris buffer (pH 8.0) until the UV280 returned to baseline. The collected sample was adjusted to pH 5.5 using an acetic acid solution, concentrated to 1 ml using a 30 kDa ultrafiltration tube, and filtered using a 0.2 μm film filter. Next, a cation exchange column was equilibrated with a low-concentration acetate buffer (pH 5.5) and the sample was loaded. The column was then equilibrated using a low-concentration acetate buffer (pH 5.5) and a linear gradient elution was performed using a 20 CV 0-100% high-concentration acetate solution to collect the eluted components.

[0131] The constructed trispecific antibody was designated MBS314. The purified antibody, which had a purity of greater than 90% as determined by mass spectrometry, was further characterized below.

[0132] Example 4 Binding affinity of CD3×BCMA×GPRC5D to human CD3, human GPRC5D, and human BCMA The binding affinity of antibody MBS314 to human CD3, human GPRC5D, and human BCMA was measured using a capture method (BIAcore 8K). A mouse anti-human Fc antibody (Cat#: BR100839, Cytiva) was coupled to the surface of a CM5 chip. The antibody to be tested was diluted to 1 μg / ml using HBS-EP buffer (Cat#: BR-1006-69, GE Life Sciences) to ensure that approximately 100 RU of antibody was captured by the anti-human Fc antibody. Human BCMA antigen (Cat#: BCA-H522y, ACRO), human CD3D&E protein (Cat#: CT038-H2508H, Sino biological), and human GPRC5D antigen (Cat#: HM05P, Kactus Biosystems) were diluted with HBS-EP buffer to 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, and 0.003900625 μg / ml, respectively. Different concentrations of each protein were applied to the surface of the stationary phase. The chip was regenerated with 3 M MgCl2 solution. Kinetic assays were performed using the Kinetics Wizard in the BIAcore 8K control software. JNJ (bispecific GPRC5D x CD3 antibody) and EM801 (bispecific BCMA x CD3 antibody) were used as positive controls, where JNJ was prepared according to amino acid sequences 10, 11, 20, and 21 of WO2022058445A1, and EM801 was prepared according to amino acid sequences 43, 44, 45, and 46 of WO2016020332A1. Data were fitted in BIAcore 8K evaluation software to determine the K values ​​of the test antibodies against the above antigens. D The values ​​were determined and are summarized in Table 1.

[0133] It can be seen from Table 1 that MBS314 has strong binding affinity to human CD3, human GPRC5D, and human BCMA. MBS314 showed lower binding affinity to the CD3D&E complex than EM801 and JNJ. Table 1 Binding affinity of antibody MBS314 to human GPRC5D, human BCMA, and human CD3D&E [Table 1] Example 5 Binding ability of trispecific CD3×BCMA×GPRC5D antibodies to HEK293A / human BCMA cells, HEK293A / monkey BCMA cells, HEK293A / mouse BCMA cells, HEK293T / human GPRC5D cells, HEK293T / monkey GPRC5D cells, and Jurkat cells

[0134] The binding activity of MBS314 to the complex of human BCMA, monkey BCMA, mouse BCMA, human GPRC5D, monkey GPRC5D, and human CD3 expressed on the cell surface was measured by FACS.

[0135] Specifically, HEK293A / human BCMA cells (prepared in Example 1), HEK293A / monkey BCMA cells, HEK293A / mouse BCMA cells, HEK293T / human GPRC5D cells, HEK293T / monkey GPRC5D cells, and well-growing Jurkat cells (Nanjing Cobioer) were digested with trypsin, harvested, and centrifuged at 300 g for 5 minutes to remove the culture medium. The cells were resuspended in PBS, counted, and centrifuged again to remove the supernatant. The cells were resuspended in 2% FBS-PBS to a cell concentration of 4 x 10 6The antibody was diluted in 2% FBS-PBS and serially diluted fivefold starting at 40 μg / ml to obtain a total of 12 concentrations. 50 μl of antibody dilution was added to the 96-well plate, the wells were mixed, and the plate was incubated for 1 hour at room temperature. The plate was washed three times with 200 μl / well of 2% FBS-PBS. PE-labeled sheep anti-human IgG (H+L) secondary antibody (1:500 dilution) was added to the plate at 50 μl / well and incubated for 45 minutes at room temperature. The plate was washed three times with 200 μl / well of 2% FBS-PBS. Finally, 150 μl of 2% FBS-PBS was added to the plate to resuspend the cells, and the cells were then analyzed by flow cytometry. Data were processed using FlowJo and analyzed using GraphPad. The results are shown in Figure 2.

[0136] As shown in Figure 2 (A-C), antibody MBS314 potently bound to HEK293A / human BCMA cells and HEK293A / monkey BCMA cells in a dose-dependent manner, but did not bind to mouse BCMA. Figure 2 (D and E) show antibody MBS314 binding to HEK293T / human GPRC5D cells and HEK293T / monkey GPRC5D cells, with MBS314 binding activity to human GPRC5D and monkey GPRC5D significantly higher than JNJ. As shown in Figure 2 (F), MBS314 had relatively low CD3 complex binding activity compared to JNJ.

[0137] Example 6 Effect of trispecific CD3*BCMA*GPRC5D antibody on activation of T cell signaling The effect of the trispecific antibody of the present disclosure on T cell signaling activation was tested as follows: The antibody binds to GPRC5D and / or BCMA on the surface of tumor cells, and therefore, when present in a "crosslinked" state, it can bind to CD3 on the surface of Jurkat-NFAT-LUC cells and activate NFAT signaling so that Jurkat-NFAT-LUC cells can secrete luciferase. The level of antibody-mediated T cell activation can be determined by measuring luciferase activity.

[0138] First, the expression levels of GPRC5D and BCMA on the surface of multiple myeloma cells were measured using anti-GPRC5D and anti-BCMA antibodies produced by hybridoma technology (developed by KYINNO). NCI-H929 cells (KC-0629, KYINNO) with high GPRC5D and BCMA expression, MOLP8 cells (KC-0622, KYINNO) with high GPRC5D and low BCMA expression, and KMS-11 cells (KC-0611, KYINNO) with low GPRC5D and high BCMA expression were selected for subsequent T cell activation assays. Specifically, myeloma cells (1 billion / ml, 100,000 cells / well) were suspended in 100 μl of PBS (containing 2% BSA) and incubated with anti-GPRC5D or anti-BCMA antibodies (produced by hybridoma technology, 20 μg / ml) at room temperature for 30 minutes. 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 on tumor cells were measured by flow cytometry. Figure 3 (A) shows the flow cytometry analysis graphs of the three cells.

[0139] Myeloma cells, i.e., NCI-H929, MOLP8, and KSM-11 cells, and Jurkat-NFAT-LUC cells (Cat#: KC-1485, KYINNO), were collected and centrifuged at 400 g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to approximately 5 × 10 cells / well using culture medium (RPMI 1640 containing 10% FBS). 5The antibody was adjusted to 1 / ml in culture medium. Starting at 10 μg / ml, the antibody was serially diluted 3.16-fold. After uniformly distributing the cells, 45 μl of Jurkat-NFAT-LUC cells were added to a 96-well U-bottom plate along with 45 μl of H929, MOLP8, or KSM-11 cells. 10 μl of antibody was added to the plate at various concentrations and incubated in a 37°C incubator for 4–6 hours. The cells were then transferred to a 96-well, clear, flat-bottom, black-walled plate containing Bright-Glo solution (E2610, Promega). Luminescence was read using a microplate reader in multimode, and the luciferase signal was measured. The results are shown in Figure 3(B). Even when tumor cells expressed relatively low levels of GPRC5D or BCMA, MBS314 activated CD3 signaling in all three cell types. MBS314 induced significantly less CD3 signaling than JNJ and EM801 in tumor cells with high GPRC5D and BCMA expression.

[0140] Example 7 In vivo antitumor efficacy of a trispecific CD3×BCMA×GPRC5D antibody MM.1S myeloma cells (KC-0620, KYINNO) expressing both GPRC5D and BCMA were subcutaneously injected into immune-deficient B-NDG mice (BIOTYTOGEN), and human T cells were intravenously injected into these mice to establish a humanized mouse model for in vivo antitumor efficacy. Figure 4(A) shows GPRC5D and BCMA expression on MM.1S cells.

[0141] Approximately 2-3 weeks before tumor cell inoculation, MM.1S myeloma cells were regenerated and cultured in RPMI-1640 medium (containing 10% FBS) with cell passage every 3-4 days. Six days before tumor cell inoculation, approximately 1 × 10 8 For regenerating PBMCs and in vitro T cell expansion, 1 x 10 6 Such cells were co-cultured with 20 μl of CD3 / CD28 immunobeads (Cat#: 11161D, GIBCO) for approximately 8 days.

[0142] On day 0, MM.1S tumor cells were harvested and cultured at a tumor cell density of 2.5 × 10 using a 1:1 mixture of Matrigel (356234, GIBCO) and PBS. 7 Each NDG mouse was injected subcutaneously with 0.2 ml of such a mixture (i.e., 0.5 × 10 7 On day 2, the expanded T cells were harvested and approximately 0.5 × 10 8 Each mouse was injected with 0.2 ml of T cells (i.e., approximately 1 × 10 cells) via the tail vein at a density of 1 × 10 cells / ml suspended in PBS containing 0.1% FBS and 10 ng / ml IL-2. 7 cells). Tumor size is 50-100mm 3 On day 20, the mice were injected with a trispecific antibody of the present disclosure, JNJ, EM801, or PBS at a dose of 10 μg per mouse twice a week for a total of 7 injections via the tail vein.

[0143] Tumor size was measured every 3 days until day 40, and tumor volume was calculated as 0.5 × length × width. 2 It was calculated as:

[0144] Figure 4 (B) shows the change in tumor size in individual mice from each group. It can be seen that MBS314 can eliminate tumor cells in mice, and has an effect that is clearly better than EM801 but equivalent to JNJ.

[0145] Example 8 Lethal effect of trispecific CD3xBCMAxGPRC5D antibody on clinical tumor specimens Bone marrow monocytes were isolated from fresh specimens of patients with relapsed and refractory multiple myeloma and cultured at 5 × 10 cells / ml in RPMI 1640 medium (containing 10% FBS, 1% penicillin-streptomycin, 100 ng / ml IL-6, and 10 ng / ml IL-2). 5The cells were suspended at a density of 100 μg / ml. The antibodies were diluted in culture medium and serially diluted 10-fold starting at 100 μg / ml, resulting in a total of 6 concentrations. 90 μl of cell suspension and 10 μl of diluted antibody at various concentrations were added to a 96-well U-bottom plate. After 48 hours of culture, the plate was centrifuged to remove the supernatant, and the cells were resuspended in 100 μl FITC-anti-human CD138 (1:100, Biolegend, 356508) and incubated on ice for 30 minutes. The plate was centrifuged to remove unbound anti-CD138 antibody, and 7-AAD was added to the cells and subjected to FACS to detect CD138. + The ratio of cells to viable cells was analyzed.

[0146] Figure 5(A) shows the FACS analysis graph, and Figure 5(B) shows the statistical chart. The results showed that the lethal effect of MBS314 on tumor cells was higher than that of JNJ and EM801.

[0147] Example 9 In vivo toxicity of trispecific CD3×BCMA×GPRC5D antibodies The trispecific CD3xBCMAxGPRC5D antibody of the present disclosure was tested for its in vivo adverse side effects in wild-type or CD3-humanized mice.

[0148] Specifically, nine B-hCD3EDG mice (CD3-humanized or human CD3, BIOTYTOGEN) were assigned to three groups, and nine C57BL / 6 mice were assigned to three groups. These mice were injected via the tail vein with 20 mg / kg of MBS314, JNJ, or PBS twice a week for a total of three injections. Body weight was measured daily, and the ratio of the weight on that day to the weight on day 0 was calculated to plot a weight curve during the study. On day 10, the final day of the study, 50–100 μl of blood was collected from each wild-type or CD3-humanized mouse. Anticoagulant was added, and routine blood tests were performed. The heart, liver, spleen, kidney, bone marrow, brain, lung, and thymus were harvested, and the pancreas was cut into cubes with sides of 0.3–0.5 cm or shorter using a blade. The cubes were fixed in a volume of 10% neutral buffered formalin solution at least 10 times larger than the tissue, embedded in paraffin, sectioned, and stained with HE to observe tissue damage.

[0149] The test results showed that after injection of high doses of MBS314, no obvious weight loss (Figure 6) or obvious tissue damage (results not shown) was observed in wild-type and CD3-humanized mice, indicating that MBS314 has no obvious adverse side effects.

[0150] Example 10 Pharmacokinetics (PK) of a trispecific CD3xBCMAxGPRC5D antibody in mice MBS314 was injected into 10-12 week-old female BALB / c mice via the tail vein at 3.0 mg / kg, 1.0 mg / kg, or 0.3 mg / kg. Blood samples were collected 10 minutes, 1 hour, 6 hours, 1 day, 3 days, 5 days, 7 days, and 14 days later. Specifically, 0.03 ml of blood was collected per mouse via the retroorbital sinus or tail vein (anticoagulant was added after sampling). A 4-fold larger volume of PBS was added to each 10-30 μl blood sample, followed by centrifugation at 10,000 g for 5 minutes. The supernatant was collected and stored at -20°C for further use.

[0151] 50 ng / well of BCMA-mFc (prepared by KYINNO) was added and coated onto an ELISA plate and left overnight at 4°C. On the second day, 200 μl of blocking solution (2% BSA) was added to the plate and incubated at 37°C for 2 hours. The plasma samples prepared above were diluted 1:50, 1:100, and 1:500 with PBS containing 2% BSA, respectively. MBS314 was serially diluted 3.16-fold starting from 1 μg / ml with PBS containing 2% BSA, obtaining a total of 12 concentrations for standard curve preparation. 50 μl of plasma sample and 50 μl of MBS314 were added to the ELISA plate and incubated at 37°C for 1 hour. After washing, 50 μl of HRP-anti-human Fc (1:10,000, Solarbio, SE101) was added to the plate and incubated at 37°C for 1 hour. 50 μl of ELISA color developing solution was added per well to the plate, and the absorbance was read. The antibody concentration in the plasma was determined according to the standard curve and plasma dilution ratio.

[0152] Figure 7 shows the antibody concentration-time curves, showing that the half-life of MBS314 in mouse blood was approximately 4.7 days when injected at 3.0 mg / kg, approximately 3.18 days when injected at 1.0 mg / kg, and approximately 4.97 days when injected at 0.3 mg / kg. The results indicated that the serum half-life of MBS314 in mice was approximately 4 to 5 days.

[0153] Exemplary sequences in this application are listed below.

[0154] [Table 2] JPEG2025532460000004.jpg199162JPEG2025532460000005.jpg210162JPEG2025532460000006.jpg196162

[0155] While this application has been described in one or more embodiments, it is understood that the invention is not limited to those embodiments, and that the description is intended to encompass all other alternatives, modifications, and equivalents within the spirit and scope of the appended claims. All references cited herein are incorporated by reference in their entirety.

Claims

1. i) an antigen-binding domain against CD3; ii) an antigen-binding domain against BCMA, and iii) an antigen-binding domain for GPRC5D A multispecific antibody comprising:

2. i) a first polypeptide chain comprising a heavy chain variable region that specifically binds to BCMA, a light chain variable region that specifically binds to BCMA, a heavy chain variable region that specifically binds to CD3, and a heavy chain constant region; ii) a second polypeptide chain comprising a light chain variable region that specifically binds to CD3; iii) a third polypeptide chain comprising a heavy chain variable region that specifically binds to GPRC5D and a heavy chain constant region; and iv) a fourth polypeptide chain comprising a light chain variable region that specifically binds to GPRC5D; or i) a first polypeptide chain comprising a heavy chain variable region that specifically binds to CD3, and a heavy chain constant region; ii) 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; iii) a third polypeptide chain comprising a heavy chain variable region that specifically binds to GPRC5D and a heavy chain constant region; and iv) a fourth polypeptide chain comprising a light chain variable region that specifically binds to GPRC5D; Equipped with wherein the heavy chain variable region that specifically binds to BCMA and the light chain variable region that specifically binds to BCMA form an antigen-binding domain for BCMA, the heavy chain variable region that specifically binds to CD3 in the first polypeptide chain and the light chain variable region that specifically binds to CD3 in the second polypeptide chain form the antigen-binding domain for CD3, the heavy chain variable region that specifically binds to GPRC5D in the third polypeptide chain and the light chain variable region that specifically binds to GPRC5D in the fourth polypeptide chain form the antigen-binding domain for GPRC5D, and the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain are associated together. The multispecific antibody of claim 1.

3. the first polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to BCMA, the light chain variable region that specifically binds to BCMA, the heavy chain variable region that specifically binds to CD3, and the heavy chain constant region; or, from N-terminus to C-terminus, the light chain variable region that specifically binds to BCMA, the heavy chain variable region that specifically binds to BCMA, the heavy chain variable region that specifically binds to CD3, and the heavy chain constant region, the third polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds GPRC5D, and the heavy chain constant region; or the first polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to CD3, and the heavy chain constant region; the second polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to BCMA, the light chain variable region that specifically binds to BCMA, and the light chain variable region that specifically binds to CD3; or, from N-terminus to C-terminus, the light chain variable region that specifically binds to BCMA, the heavy chain variable region that specifically binds to BCMA, and the light chain variable region that specifically binds to CD3, the third polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region that specifically binds to GPRC5D, and the heavy chain constant region; The multispecific antibody of claim 2.

4. i) the first polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to BCMA, a first linker, the light chain variable region that specifically binds to BCMA, a second linker, the heavy chain variable region that specifically binds to CD3, and the heavy chain constant region; or the light chain variable region that specifically binds to BCMA, a first linker, the heavy chain variable region that specifically binds to BCMA, a second linker, the heavy chain variable region that specifically binds to CD3, and the heavy chain constant region; ii) the second polypeptide chain comprises, from N-terminus to C-terminus, the light chain variable region that specifically binds CD3, and a light chain constant region; iii) the third polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to GPRC5D, and the heavy chain constant region; iv) the fourth polypeptide chain comprises, from N-terminus to C-terminus, the light chain variable region that specifically binds GPRC5D, and a light chain constant region; or i) the first polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds CD3 and the heavy chain constant region; ii) the second polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to BCMA, a first linker, the light chain variable region that specifically binds to BCMA, a second linker, the light chain variable region that specifically binds to CD3, and a light chain constant region; or the light chain variable region that specifically binds to BCMA, a first linker, the heavy chain variable region that specifically binds to BCMA, a second linker, the light chain variable region that specifically binds to CD3, and a light chain constant region; iii) the third polypeptide chain comprises, from N-terminus to C-terminus, the heavy chain variable region that specifically binds to GPRC5D, and the heavy chain constant region; iv) the fourth polypeptide chain comprises, from the N-terminus to the C-terminus, the light chain variable region that specifically binds to GPRC5D, and a light chain constant region; The multispecific antibody of claim 3.

5. the antigen-binding domain against CD3 is an antibody or antigen-binding portion thereof that specifically binds to CD3, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NOs: 9 to 11, respectively, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 12 to 14, respectively; The antigen-binding domain against BCMA is an antibody or antigen-binding portion thereof that specifically binds to BCMA, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NOs: 1 to 3, respectively, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 4 to 6, respectively; and / or The antigen-binding domain against GPRC5D is an antibody or an antigen-binding portion thereof that specifically binds to GPRC5D, wherein the heavy chain variable region comprises VH-CDR1, VH-CDR2, and VH-CDR3 of SEQ ID NOs: 17 to 19, respectively, and the light chain variable region comprises VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 20 to 22, respectively; The multispecific antibody of claim 1.

6. the heavy chain variable region and the light chain variable region in the antigen-binding domain against CD3 comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 15 and 16, respectively; the heavy chain variable region and the light chain variable region in the antigen-binding domain against BCMA comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 7 and 8, respectively; and / or The heavy chain variable region and the light chain variable region in the antigen-binding domain against GPRC5D comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 23 and 24, respectively. The multispecific antibody of claim 5.

7. the heavy chain constant region of the first polypeptide chain and the heavy chain constant region of the third polypeptide chain are human heavy chain constant regions that exhibit weak or no binding to Fc receptors and / or complement system proteins; and / or one of the heavy chain constant region in the first polypeptide chain and the heavy chain constant region in the third polypeptide chain has a knob mutation and the other has a hole mutation; The multispecific antibody of claim 2.

8. The multispecific antibody of claim 4 , wherein the first linker and / or the second linker comprise the amino acid sequences of SEQ ID NOs: 36 and 37.

9. 3. The multispecific antibody of claim 2, wherein the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain comprise amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NOs: 27, 28, 29 and 30, respectively.

10. A nucleic acid molecule encoding the multispecific antibody of any one of claims 1 to 9.

11. An expression vector comprising the nucleic acid molecule of claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10 or the expression vector of claim 11.

13. 13. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 1 to 9, a nucleic acid molecule according to claim 10, an expression vector according to claim 11, or a host cell according to claim 12, and a pharmaceutically acceptable carrier.

14. 14. Use of the pharmaceutical composition of claim 13 in the preparation of a medicament for treating a BCMA and / or GPRC5D-related disease.

15. 15. The use according to claim 14, wherein the BCMA and / or GPRC5D-related disease is multiple myeloma.

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