A trispecific antibody targeting BCMA, GPRC5D, and CD3

Abstract

Provided herein are multispecific antibodies and multispecific antigen-binding fragments thereof that bind to BCMA, GPRC5D, and CD3. Also described are related polynucleotides capable of encoding the provided multispecific antibodies or multispecific antigen-binding fragments, cells expressing the provided multispecific antibodies or multispecific antigen-binding fragments, and related vectors and detectably labeled multispecific antibodies or multispecific antigen-binding fragments. In addition, methods of producing and using the provided multispecific antibodies and multispecific antigen-binding fragments are described. Further provided herein are antibodies and antigen-binding fragments thereof that bind to BCMA. Also described are related polynucleotides capable of encoding the provided BCMA-specific antibodies or antigen-binding fragments, cells expressing the provided BCMA-specific antibodies or antigen-binding fragments, and related vectors and detectably labeled BCMA-specific antibodies or antigen-binding fragments. In addition, methods of producing and using the provided BCMA-specific antibodies and antigen-binding fragments are described.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 149,921, filed February 16, 2021. The entire contents of the foregoing application are incorporated herein by reference in their entirety.

[0002] (Technical field) The disclosure provided herein relates to multispecific antibodies that bind to B-cell maturation antigen (BCMA), G-protein coupled receptor, class C, group 5, member D (GPRC5D), and cluster determinant 3 (CD3), monoclonal antibodies that bind to BCMA, and methods of making and using the described antibodies.

[0003] (background) Multiple myeloma (MM) is the second most common hematological malignancy, accounting for 2% of all cancer deaths. MM is a heterogeneous disease caused primarily by chromosomal rearrangements, particularly t(11;14), t(4;14), t(8;14), del(13), and del(17) (Drach et al., (1998) Blood 92(3):802-809; Gertz et al., (2005) Blood 106(8):2837-2840; Facon et al., (2001) Blood 97(6):1566-1571). Patients with MM may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and the psychological burden of a cancer diagnosis. As of 2006, the 5-year relative survival rate for MM was approximately 34%, highlighting that MM is a difficult-to-treat disease for which no therapeutic options currently exist.

[0004] B-cell maturation antigen (BCMA), also known as CD269 and TNFRSF17 (UniProt Q02223), is a member of the tumor necrosis receptor superfamily that is preferentially expressed on differentiated plasma cells [Laabi et al. (1992) EMBO J 11(11):3897-3904, Madry et al. (1998) Int Immunol 10(11):1693-1702]. BCMA is a non-glycosylated type I transmembrane protein involved in B-cell maturation, proliferation, and survival. BCMA is a receptor for two ligands of the TNF superfamily: APRIL (proliferation-inducing ligand, CD256, TNFSF13), a high-affinity ligand for BCMA, and BAFF (THANK, BlyS, B-lymphocyte stimulatory factor, TALL-1, and zTNF4), a low-affinity ligand for BCMA. APRIL and BAFF exhibit structural similarities and overlapping yet distinct receptor binding specificities. The negative regulator TACI also binds to both BAFF and APRIL. Cooperative binding of APRIL and BAFF to BCMA and / or TACI activates the transcription factor NF-κB, increases the expression of pro-survival Bcl-2 family members (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1), and downregulates the expression of pro-apoptotic factors (e.g., Bid, Bad, Bik, Bim, etc.), thus inhibiting apoptosis and promoting survival. This combined action promotes B cell differentiation, proliferation, survival, and antibody production (reviewed in Rickert RC et al., Immunol Rev (2011) 244(1):115-133). Consistent with this finding, BCMA also supports the proliferation and survival of malignant human B cells, including multiple myeloma (MM) cells. Novak et al. found that MM cell lines and freshly isolated MM cells express BCMA and TACI proteins on their cell surface and have variable expression of BAFF-R protein on their cell surface (Novak et al., (2004) Blood 103(2):689-694).

[0005] The use of anti-BCMA antibodies for the treatment of lymphoma and multiple myeloma is mentioned in WO 2002066516 and WO 2010104949. Antibodies against BCMA are described, for example, in Gras MP. et al. Int Immunol. 7 (1995) 1093-1106, WO 200124811 and WO 200124812. Nevertheless, although BCMA, BAFF-R, and TACI, i.e., B cell receptors belonging to the TNF receptor superfamily, and their ligands BAFF and APRIL, are the target of therapies to combat cancer, further options for the treatment of such medical conditions are needed.

[0006] G protein-coupled receptor class C group 5 member D (GPRC5D) is an orphan atypical class C GPCR first identified in 2001 (Brauner-Osborne et al. Biochim Biophys Acta. 1518(3):237-248, 2001). GPRC5D and other GPCRs in group 5 share a very short amino-terminal domain relative to class C receptors and are therefore predicted to have a similar three-dimensional structure to class A receptors. In this regard, they are unique due to their sequence homology to class C GPCRs and predicted structural topology similar to class A receptors. The functional significance of GPRC5D activation has not been described, and the ligand is still unknown. In humans, the gene has three exons and is located on chromosome 12p13.3. The GPRC5D receptor is highly conserved across species, sharing 92% identity with cynomolgus monkey GPRC5D.

[0007] The use of anti-GPRC5D antibodies for the treatment of multiple myeloma is mentioned in WO 2016090329, WO 2018017786, WO 2018147245, and WO 2019154890. Nevertheless, despite the fact that GPRC5D is a target of therapies for combating cancer, there is a need for further options for the treatment of such medical conditions.

[0008] GPRC5D mRNA is predominantly expressed in all malignant plasma cells from MM patients (Atamaniuk JA et al. Eur J Clin Invest 42(9)953-960; 2012, Frigyesi-blood and Cohen, et al. Hematology 18(6):348-35; 2013). GPRC5D expression varies among patients and correlates well with plasma cell burden and genetic abnormalities such as Rb-1 deficiency (Atamaniuk JA et al. Eur J Clin Invest 42(9)953-960; 2012).

[0009] Clonal resistance is a common mechanism in relapsed / refractory myeloma. Targeting two or more myeloma tumor antigens and engaging T cells can result in efficient killing of malignant plasma cells and minimal residual disease (MRD) negativity. Dual targeting of BCMA and GPRC5D can enhance antibody avidity and potency, maximize tumor eradication in the presence of heterogeneous cell populations, prevent tumor antigen escape (e.g., capture MM cells that do not express sufficient BCMA or GPRC5D alone), improve tumor efficacy, and reduce the potential for cytokine release syndrome (CRS).

[0010] Thus, there is a need for therapeutic antibodies that target both BCMA and GPRC5D for the treatment of multiple myeloma and / or related medical conditions.

[0011] (overview) In one aspect, provided herein are multispecific antibodies and multispecific antigen-binding fragments thereof that specifically bind to BCMA, GPRC5D, and CD3. In some embodiments, provided herein are trispecific antibodies and trispecific antigen-binding fragments thereof that bind or specifically bind to BCMA, GPRC5D, and CD3. Related polynucleotides capable of encoding the provided BCMAxGPRC5DxCD3 multispecific antibodies or multispecific antigen-binding fragments, cells expressing the provided antibodies or multispecific antigen-binding fragments, and related vectors and detectably labeled multispecific antibodies or multispecific antigen-binding fragments are also described. Additionally, methods of using the provided multispecific antibodies are described. For example, BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments can be used to treat cancer (e.g., BCMA- and / or GPRC5D-expressing cancers), and BCMAxGPRC5DxCD3 multispecific antibodies can be used to diagnose or monitor the progression, regression, or stability of BCMA- and / or GPRC5D-expressing cancers, to determine whether a patient needs to be treated for cancer, or whether a subject has a BCMA- and / or GPRC5D-expressing cancer and is therefore suitable for treatment with a BCMA- and / or GPRC5D-specific anti-cancer therapeutic, such as the BCMAxGPRC5DxCD3 multispecific antibodies described herein.

[0012] BCMA×GPRC5D×CD3 multispecific antibody Described herein is an isolated multispecific antibody that binds to BCMA, GPRC5D, and CD3 (a "BCMAxGPRC5DxCD3 multispecific antibody") and multispecific antigen-binding fragments thereof.

[0013] In preferred embodiments, the BCMAxGPRC5DxCD3 multispecific antibody or antigen-binding fragment is a trispecific antibody or antigen-binding fragment. In some embodiments, the isolated BCMAxGPRC5DxCD3 trispecific antibody or trispecific binding fragment thereof comprises (a) a first antigen-binding arm comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1), (b) a second antigen-binding arm comprising a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2), and (c) a third antigen-binding arm comprising a third heavy chain variable domain (VH3) and a third light chain variable domain (VL3). In some embodiments, the first antigen-binding arm binds to an epitope on CD3, the second antigen-binding arm binds to an epitope on GPRC5D, and the third antigen-binding arm binds to an epitope on BCMA.

[0014] In some embodiments, the first antigen-binding arm of the trispecific antibody or trispecific binding fragment thereof comprises a first heavy chain portion (HC1) comprising VH1 and a light chain portion comprising VL1. VH1 and VL1 form a first antigen-binding domain that binds to a first antigen. The second antigen-binding arm of the trispecific antibody or trispecific binding fragment thereof comprises a second heavy chain portion (HC2) comprising VH2. The VH2 of HC2 forms a second antigen-binding domain that binds to a second antigen. HC1 or HC2 is further linked to a third antigen-binding arm comprising VH3 that forms a third antigen-binding domain that binds to a third antigen. HC1 and HC2 each optionally comprise a fragment crystallizable (Fc) domain, where the Fc domain comprises constant heavy chain region 2 (CH2) and CH3. In some embodiments, the first antigen is Cluster of Differentiation 3 (CD3), the second antigen is B-cell maturation antigen (BCMA), and the third antigen is G-protein coupled receptor family C group 5 member D (GPRC5D). In some embodiments, the first antigen is Cluster of Differentiation 3 (CD3), the second antigen is G-protein coupled receptor family C group 5 member D (GPRC5D), and the third antigen is B-cell maturation antigen (BCMA). Some aspects of the BCMAxGPRC5DxCD3 trispecific antibody or trispecific binding fragment thereof are further described in the Detailed Description and Examples sections below.

[0015] In some embodiments, the BCMA-binding arm binds to human BCMA but not cynomolgus BCMA. In some embodiments, the BCMA-binding arm binds to an epitope comprising one or more residues from the BCMA extracellular domain (ECD). In some embodiments, the BCMA-binding arm (or "BCMA-specific arm") of the BCMAxGPRC5DxCD3 multispecific antibody is derived from a BCMA antibody described herein (e.g., from an antibody having the CDR sequences listed in Table 1).

[0016] In some embodiments, the GPRC5D-specific arm of the multispecific antibody binds to human GPRC5D but not to cynomolgus GPRC5D. In some embodiments, the GPRC5D-specific arm of the BCMAxGPRC5DxCD3 multispecific antibody or antigen-binding fragment binds to the extracellular domain of human GPRC5D. In some embodiments, the GPRC5D-binding arm (or "GPRC5D-specific arm") of the BCMAxGPRC5DxCD3 multispecific antibody is derived from a GPRC5D antibody described herein (e.g., from an antibody having the CDR sequences listed in Table 2).

[0017] Redirection of T lymphocytes to BCMA- and / or GPRC5D-expressing MM cells via the TCR / CD3 complex presents an attractive alternative approach. The TCR / CD3 complex of T lymphocytes consists of either TCR alpha (α) / beta (β) or TCR gamma (γ) / delta (δ) heterodimers coexpressed on the cell surface with gamma (γ), delta (δ), epsilon (ε), zeta (ζ), and eta (η)-tagged CD3 invariant subunits. In some embodiments, the multispecific antibodies or multispecific antigen-binding fragments described herein bind to CD3ε. In some embodiments, the CD3-binding arm (or "CD3-specific arm") of a BCMA×GPRC5D×CD3 multispecific antibody is derived from the monoclonal antibody CD3B376. In some embodiments, the CD3-binding arm (or "CD3-specific arm") of a BCMA×GPRC5D×CD3 multispecific antibody is derived from the monoclonal antibody SP34, a murine IgG3 / lambda isotype. (KRA Bhinandan and AC Martin, 2008. Mol. Immunol. 45, 3832-3839). In some embodiments, the CD3 binding arm of the BCMAxGPRC5DxCD3 multispecific antibody comprises the heavy chain CDRs and / or the light chain CDRs set forth in Table 3, or any one VH domain and / or any one VL domain selected from Table 3.

[0018] In some embodiments, the BCMA-, GPRC5D-, and / or CD3-specific arms or antigen-binding fragments of the BCMA×GPRC5D×CD3 multispecific antibody are IgG or derivatives thereof. In humans, the IgG class is divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. These share greater than 95% homology in the amino acid sequence of the Fc region, but exhibit major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcγR) on the surface of immune effector cells, such as natural killer cells and macrophages, resulting in the phagocytosis or lysis of target cells. In CDC, antibodies kill target cells by triggering the complement cascade on the cell surface.

[0019] For many therapeutic antibody applications, Fc-mediated effector functions are not responsible for the mechanism of action. These Fc-mediated effector functions can be harmful by causing extramechanistic toxicities and pose safety risks. Altered effector function can be achieved by genetically engineering the Fc region to reduce binding to FcγRs or complement factors. Binding of IgGs to activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) is determined by residues located in the hinge region and CH2 domain. Mutations introduced into IgG1, IgG2, and IgG4 reduce or silence Fc function.

[0020] In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function when compared to the parent Fc; (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb and / or FcγRIIIa; (c) reduced affinity for FcγRI; (d) reduced affinity for FcγRIIa; (e) reduced affinity for FcγRIIb; (f) reduced affinity for FcγRIIIb; or (g) reduced affinity for FcγRIIIa.

[0021] In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG or a derivative thereof. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG1 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG1 antibody from which the CD3-binding arm is derived comprises L234A, L235A, and D265S substitutions in the Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG4 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG4 antibody from which the CD3-binding arm is derived comprises S228P, L234A, L235A, F405L, and R409K substitutions in the Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived binds to CD3ε on primary human T cells and / or primary cynomolgus monkey T cells. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived activates primary human CD4+ T cells and / or primary cynomolgus monkey CD4+ T cells.

[0022] In addition to the described BCMAxGPRC5DxCD3 multispecific antibodies, polynucleotide sequences capable of encoding the described BCMAxGPRC5DxCD3 multispecific antibodies are also provided. In some embodiments, isolated synthetic polynucleotides are provided that encode one or more CDRs of the heavy chain variable domain and / or one or more CDRs of the light chain variable domain of each antigen-binding arm of a BCMAxGPRC5DxCD3 trispecific antibody or trispecific binding fragment. In some embodiments, isolated synthetic polynucleotides are provided that encode one or more heavy chain variable domains and / or one or more light chain variable domains of a BCMAxGPRC5DxCD3 trispecific antibody or trispecific binding fragment. In some embodiments, isolated synthetic polynucleotides are provided that encode one or more polypeptide chains of the first, second, and / or third antigen-binding arms of a BCMAxGPRC5DxCD3 trispecific antibody or trispecific binding fragment. Vectors containing the described polynucleotides are also provided, as are cells expressing BCMAxGPRC5DxCD3 multispecific antibodies. In another embodiment, isolated cells expressing multispecific antibodies or multispecific binding fragments are provided. Cells capable of expressing the disclosed vectors are also described. These cells can be mammalian cells (e.g., 293 cells, 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The described antibodies can also be produced by hybridoma cells. In some embodiments, methods are provided for producing BCMAxGPRC5DxCD3 trispecific antibodies or trispecific binding fragments by culturing cells.

[0023] Further provided herein is a pharmaceutical composition comprising a BCMAxGPRC5DxCD3 trispecific antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0024] Methods using BCMA x GPRC5D x CD3 multispecific antibodies Methods of using the described BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments thereof are also disclosed. For example, the BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments thereof may be useful for treating a GPRC5D- and / or BCMA-expressing cancer in a subject in need thereof. In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a lymphoma, e.g., multiple myeloma, including smoldering multiple myeloma (SMM). In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a relapsed or refractory form of lymphoma, e.g., a relapsed or refractory form of multiple myeloma.

[0025] The described methods of treating a GPRC5D- and / or BCMA-expressing cancer in a subject in need of such treatment comprise administering to the subject a therapeutically effective amount of the described BCMAxGPRC5DxCD3 multispecific antibody or multispecific antigen-binding fragment thereof. In some embodiments, the subject is a mammal, preferably a human. In preferred embodiments, methods are provided for treating a subject with cancer by administering a therapeutically effective amount of a BCMAxGPRC5DxCD3 trispecific antibody or trispecific antigen-binding fragment to a patient in need of treatment for a time sufficient to treat the cancer.

[0026] Further provided is a method for inhibiting the growth or proliferation of cancer cells by administering a therapeutically effective amount of a BCMAxGPRC5DxCD3 trispecific antibody or trispecific antigen-binding fragment to inhibit the growth or proliferation of cancer cells.

[0027] Also provided herein are methods for redirecting T cells to GPRC5D- and / or BCMA-expressing cancer cells by administering a therapeutically effective amount of a BCMAxGPRC5DxCD3 trispecific antibody or trispecific antigen-binding fragment to redirect T cells to the cancer.

[0028] Those skilled in the art will understand that methods of using the described BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments thereof can be defined in a pharmaceutical use format, for example in the form of BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments thereof for use in the treatment of diseases defined herein, in particular cancer. Those skilled in the art will also understand that methods of using the described BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments thereof can be defined in a so-called Swiss format, for example in the form of using BCMAxGPRC5DxCD3 multispecific antibodies and multispecific antigen-binding fragments for the manufacture of a medicament for the treatment of diseases defined herein, in particular cancer. This applies throughout the present disclosure.

[0029] BCMA×GPRC5D×CD3 specific antibody kit Kits are described herein that include the disclosed BCMA×GPRC5D×CD3 multispecific antibodies. The described kits can be used to perform methods using the BCMA×GPRC5D×CD3 multispecific antibodies provided herein, or other methods known to those skilled in the art. In some embodiments, the described kits may include the antibodies described herein and reagents for use in treating GPRC5D- and / or BCMA-expressing cancers. Thus, the described kits may include one or more of the multispecific antibodies or multispecific antigen-binding fragments thereof described herein, a container for housing the antibody or fragment when not in use, and / or instructions for using the antibody or fragment, an antibody or fragment immobilized on a solid support and / or a detectably labeled form of the antibody or fragment, as described herein.

[0030] BCMA specific antibody Also provided herein are antibodies and antigen-binding fragments thereof that bind to BCMA. Also described are related polynucleotides capable of encoding the provided BCMA-specific antibodies and antigen-binding fragments, cells expressing the provided antibodies and antigen-binding fragments, and related vectors, as well as detectably labeled antibodies and antigen-binding fragments. Additionally, methods of using the provided antibodies and antigen-binding fragments are described. For example, BCMA-specific antibodies and antigen-binding fragments can be used to treat cancer (e.g., BCMA-expressing cancers), diagnose or monitor the progression, regression, or stability of BCMA-expressing cancers, determine whether a patient needs to be treated for cancer, or determine whether a subject has a BCMA-expressing cancer and is therefore suitable for treatment with a BCMA-specific anti-cancer therapeutic, such as a multispecific antibody against BCMA and CD3 described herein. Some embodiments of BCMA-specific antibodies or antigen-binding fragments are further described in the Detailed Description and Examples sections below.

[0031] Methods using BCMA-specific antibodies Methods of using the described BCMA-specific antibodies or antigen-binding fragments are also disclosed. Particular antibodies for use in the methods discussed in this section include those with the set of CDRs described for the antibodies in Table 1 (e.g., BCMB519). For example, these antibodies or antigen-binding fragments may be useful in cancer treatment by interfering with BCMA-receptor interactions or, when conjugated to a toxin, by targeting the toxin to BCMA-expressing cancers. Furthermore, these antibodies or antigen-binding fragments may be useful for detecting the presence of BCMA in a biological sample, e.g., blood or serum; quantifying the amount of BCMA in a biological sample, e.g., blood or serum; diagnosing BCMA-expressing cancers; determining how to treat a subject with cancer; or monitoring the progression of a BCMA-expressing cancer in a subject. In some embodiments, the BCMA-expressing cancer may be a lymphoma, e.g., multiple myeloma (MM), including smoldering multiple myeloma (SMM). In some embodiments, the BCMA-expressing cancer is a relapsed or refractory form of lymphoma, e.g., a relapsed or refractory form of multiple myeloma. The described methods can be performed before the subject has received treatment for the BCMA-expressing cancer, e.g., treatment with a multispecific antibody against BCMA and CD3. Additionally, the described methods can be performed after the subject has received treatment for the BCMA-expressing cancer, e.g., treatment with a multispecific antibody against BCMA and CD3 described herein.

[0032] The described methods for detecting BCMA in a biological sample include exposing the biological sample to one or more of the BCMA-specific antibodies or antigen-binding fragments described herein.

[0033] Also described are methods of diagnosing BCMA-expressing cancer in a subject, which involve exposing a biological sample to one or more of the BCMA-specific antibodies or antigen-binding fragments described herein, wherein the method includes quantifying the amount of BCMA present in the sample, comparing the amount of BCMA present in the sample to a known standard or reference sample, and determining whether the subject's BCMA level falls within the range of BCMA levels associated with cancer.

[0034] Also described herein are methods for monitoring BCMA-expressing cancer in a subject. The methods described include exposing a biological sample to one or more of the BCMA-specific antibodies or antigen-binding fragments described herein, quantifying the amount of BCMA present in the sample that is bound to the antibody or antigen-binding fragment thereof, comparing the amount of BCMA present in the sample to either a known standard or reference sample or the amount of BCMA in a similar sample previously obtained from the subject, and determining whether the subject's BCMA levels indicate cancer progression, regression, or stable state based on differences in the amounts of BCMA in the compared samples.

[0035] The sample obtained from or derived from a subject is a biological sample, such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells, tissue, surgically resected tumor tissue, biopsy, fine needle aspirate sample, or histological preparation.

[0036] The BCMA-specific antibodies or antigen-binding fragments described herein can be labeled for use in the described methods or other methods known to those of skill in the art. For example, the antibodies or antigen-binding fragments thereof described herein can be labeled with a radiolabel, a fluorescent label, an epitope tag, biotin, a chromophore label, an ECL label, an enzyme, ruthenium, 111 In-DOTA, 111They may be labeled with In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and beta-galactosidase, or polyhistidine or similar such labels known in the art.

[0037] BCMA-specific antibody kit Kits containing the disclosed BCMA-specific antibodies or antigen-binding fragments thereof are described herein. The described kits can be used to perform methods using the BCMA-specific antibodies or antigen-binding fragments provided herein, or other methods known to those of skill in the art. In some embodiments, the described kits may include the antibodies or antigen-binding fragments described herein and reagents for use in detecting the presence of BCMA in a biological sample. Thus, the described kits may include one or more of the antibodies or antigen-binding fragments described herein, a container for housing the antibody or fragment when not in use, instructions for using the antibody or fragment, and an antibody or fragment immobilized on a solid support and / or a detectably labeled form of the antibody or fragment, as described herein. [Brief explanation of the drawings]

[0038] [Figure 1A] FIG. 1A is a diagram of the BGCB463 trispecific antibody (FIG. 1A) and proposed mode of action (FIG. 1B). [Figure 1B] FIG. 1A is a diagram of the BGCB463 trispecific antibody (FIG. 1A) and proposed mode of action (FIG. 1B). [Figure 2A]BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with H929 wild-type (WT) (Figure 2A), H929-GPRC5D knockout (KO) (Figure 2B), H929-BCMA KO (Figure 2C), and H929-GPRC5D / BCMA KO (Figure 2D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. EC50 or EC90 values ​​are their averages. [Figure 2B] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with H929 wild-type (WT) (Figure 2A), H929-GPRC5D knockout (KO) (Figure 2B), H929-BCMA KO (Figure 2C), and H929-GPRC5D / BCMA KO (Figure 2D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. EC50 or EC90 values ​​are their averages. [Figure 2C] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with H929 wild-type (WT) (Figure 2A), H929-GPRC5D knockout (KO) (Figure 2B), H929-BCMA KO (Figure 2C), and H929-GPRC5D / BCMA KO (Figure 2D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. EC50 or EC90 values ​​are their averages. [Figure 2D]BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with H929 wild-type (WT) (Figure 2A), H929-GPRC5D knockout (KO) (Figure 2B), H929-BCMA KO (Figure 2C), and H929-GPRC5D / BCMA KO (Figure 2D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. EC50 or EC90 values ​​are their averages. [Figure 3A] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with MM.1R WT (Figure 3A), MM.1R-GPRC5D KO (Figure 3B), MM.1R-BCMA KO (Figure 3C), and MM.1R-GPRC5D / BCMA KO (Figure 3D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. [Figure 3B] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with MM.1R WT (Figure 3A), MM.1R-GPRC5D KO (Figure 3B), MM.1R-BCMA KO (Figure 3C), and MM.1R-GPRC5D / BCMA KO (Figure 3D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. [Figure 3C] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with MM.1R WT (Figure 3A), MM.1R-GPRC5D KO (Figure 3B), MM.1R-BCMA KO (Figure 3C), and MM.1R-GPRC5D / BCMA KO (Figure 3D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. [Figure 3D] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with MM.1R WT (Figure 3A), MM.1R-GPRC5D KO (Figure 3B), MM.1R-BCMA KO (Figure 3C), and MM.1R-GPRC5D / BCMA KO (Figure 3D) cells at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Curves are representative of five independent experiments. [Figure 4A] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with pan T cells (three random donors, Figures 4A-4C) at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Data are representative of two individual runs from three donors. [Figure 4B] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with pan T cells (three random donors, Figures 4A-4C) at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Data are representative of two individual runs from three donors. [Figure 4C] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with pan T cells (three random donors, Figures 4A-4C) at a starting concentration of 2 μM for 1 hour at 37°C. After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 and EC90 values. Data are representative of two individual runs from three donors. [Figure 5A] BGCB463 was incubated with pan T cells (three random donors, Figures 5A, 5B, and 5C) at a starting concentration of 2 μM for 1 hour at 4°C (circles) or 37°C (triangles). After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 values. [Figure 5B]BGCB463 was incubated with pan T cells (three random donors, Figures 5A, 5B, and 5C) at a starting concentration of 2 μM for 1 hour at 4°C (circles) or 37°C (triangles). After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 values. [Figure 5C] BGCB463 was incubated with pan T cells (three random donors, Figures 5A, 5B, and 5C) at a starting concentration of 2 μM for 1 hour at 4°C (circles) or 37°C (triangles). After secondary detection with AF647-labeled material, MFI signals were analyzed to generate EC50 values. [Figure 6A] BGCB463.003 (circles), BGCB463.004 (squares), and the negative control B23B251 (triangles) were tested on H929 (Figure 6A), MM.1R (Figure 6B), and pan T (Figure 6C) cells from three donors. Molecules were incubated at a starting concentration of 2 μM for 1 h at 37 °C. After secondary detection with AF647-labeled material, MFI signals were analyzed. [Figure 6B] BGCB463.003 (circles), BGCB463.004 (squares), and the negative control B23B251 (triangles) were tested on H929 (Figure 6A), MM.1R (Figure 6B), and pan T (Figure 6C) cells from three donors. Molecules were incubated at a starting concentration of 2 μM for 1 h at 37 °C. After secondary detection with AF647-labeled material, MFI signals were analyzed. [Figure 6C] BGCB463.003 (circles), BGCB463.004 (squares), and the negative control B23B251 (triangles) were tested on H929 (Figure 6A), MM.1R (Figure 6B), and pan T (Figure 6C) cells from three donors. Molecules were incubated at a starting concentration of 2 μM for 1 h at 37 °C. After secondary detection with AF647-labeled material, MFI signals were analyzed. [Figure 7A]H929 (Figure 7A), H929-GPRC5D KO (Figure 7B), H929-BCMA KO (Figure 7C), and H929-GPRC5D / BCMA KO (Figure 7D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1 hour, 3 hours, 5 hours, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG on ice for 30 minutes. The readout is the MFI of the AF647 signal. [Figure 7B] H929 (Figure 7A), H929-GPRC5D KO (Figure 7B), H929-BCMA KO (Figure 7C), and H929-GPRC5D / BCMA KO (Figure 7D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1 hour, 3 hours, 5 hours, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG on ice for 30 minutes. The readout is the MFI of the AF647 signal. [Figure 7C] H929 (Figure 7A), H929-GPRC5D KO (Figure 7B), H929-BCMA KO (Figure 7C), and H929-GPRC5D / BCMA KO (Figure 7D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1 hour, 3 hours, 5 hours, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG on ice for 30 minutes. The readout is the MFI of the AF647 signal. [Figure 7D] H929 (Figure 7A), H929-GPRC5D KO (Figure 7B), H929-BCMA KO (Figure 7C), and H929-GPRC5D / BCMA KO (Figure 7D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1 hour, 3 hours, 5 hours, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG on ice for 30 minutes. The readout is the MFI of the AF647 signal. [Figure 8A]MM.1R WT (FIG. 8A), MM.1R-GPRC5D KO (FIG. 8B), MM.1R-BCMA KO (FIG. 8C), and MM.1R-GPRC5D / BCMA KO (FIG. 8D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1, 3, 5, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG for 30 minutes on ice. The readout was the MFI of the AF647 signal. [Figure 8B] MM.1R WT (FIG. 8A), MM.1R-GPRC5D KO (FIG. 8B), MM.1R-BCMA KO (FIG. 8C), and MM.1R-GPRC5D / BCMA KO (FIG. 8D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1, 3, 5, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG for 30 minutes on ice. The readout was the MFI of the AF647 signal. [Figure 8C] MM.1R WT (FIG. 8A), MM.1R-GPRC5D KO (FIG. 8B), MM.1R-BCMA KO (FIG. 8C), and MM.1R-GPRC5D / BCMA KO (FIG. 8D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1, 3, 5, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG for 30 minutes on ice. The readout was the MFI of the AF647 signal. [Figure 8D] MM.1R WT (FIG. 8A), MM.1R-GPRC5D KO (FIG. 8B), MM.1R-BCMA KO (FIG. 8C), and MM.1R-GPRC5D / BCMA KO (FIG. 8D) cells were all incubated with three optimized concentrations of BGCB463 at 37°C for 1, 3, 5, and 24 hours. Cells were then washed and incubated with AF647-anti-human IgG for 30 minutes on ice. The readout was the MFI of the AF647 signal. [Figure 9A]BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with K562 cells expressing human GPRC5D (FIG. 9A), human BCMA (FIG. 9B), cynomolgus GPRC5D (FIG. 9C), or cynomolgus BCMA (FIG. 9D) at a starting concentration of 2 μM for 1 hour at 37° C. After secondary detection with AF647-anti-human IgG, MFI signals were analyzed to generate EC50 and EC90 values ​​(FIG. 9E). Data are representative of two independent experiments. [Figure 9B] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with K562 cells expressing human GPRC5D (FIG. 9A), human BCMA (FIG. 9B), cynomolgus GPRC5D (FIG. 9C), or cynomolgus BCMA (FIG. 9D) at a starting concentration of 2 μM for 1 hour at 37° C. After secondary detection with AF647-anti-human IgG, MFI signals were analyzed to generate EC50 and EC90 values ​​(FIG. 9E). Data are representative of two independent experiments. [Figure 9C] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with K562 cells expressing human GPRC5D (FIG. 9A), human BCMA (FIG. 9B), cynomolgus GPRC5D (FIG. 9C), or cynomolgus BCMA (FIG. 9D) at a starting concentration of 2 μM for 1 hour at 37° C. After secondary detection with AF647-anti-human IgG, MFI signals were analyzed to generate EC50 and EC90 values ​​(FIG. 9E). Data are representative of two independent experiments. [Figure 9D] BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with K562 cells expressing human GPRC5D (FIG. 9A), human BCMA (FIG. 9B), cynomolgus GPRC5D (FIG. 9C), or cynomolgus BCMA (FIG. 9D) at a starting concentration of 2 μM for 1 hour at 37° C. After secondary detection with AF647-anti-human IgG, MFI signals were analyzed to generate EC50 and EC90 values ​​(FIG. 9E). Data are representative of two independent experiments. [Figure 9E]BGCB463 (circles) or negative control B23B251 (triangles) were all incubated with K562 cells expressing human GPRC5D (FIG. 9A), human BCMA (FIG. 9B), cynomolgus GPRC5D (FIG. 9C), or cynomolgus BCMA (FIG. 9D) at a starting concentration of 2 μM for 1 hour at 37° C. After secondary detection with AF647-anti-human IgG, MFI signals were analyzed to generate EC50 and EC90 values ​​(FIG. 9E). Data are representative of two independent experiments. [Figure 10A] BGCB463 (circles) or negative control B23B251 (triangles) at a starting concentration of 10 nM were incubated with K562 cells expressing human or cynomolgus GPRC5D, or 100 nM for K562 cells expressing human / cynomolgus BCMA as target cells. Figure 10A: Human activation and cytotoxicity. Figure 10B: Cynomolgus activation and cytotoxicity. Human pan T cells (one donor) were added at an effector to target (E:T) ratio of 3:1 and incubated at 37°C for 72 hours. Cytotoxicity was determined using a live / dead fixable dye, and CD25 was measured using BV421-conjugated anti-CD25. [Figure 10B] BGCB463 (circles) or negative control B23B251 (triangles) at a starting concentration of 10 nM were incubated with K562 cells expressing human or cynomolgus GPRC5D, or 100 nM for K562 cells expressing human / cynomolgus BCMA as target cells. Figure 10A: Human activation and cytotoxicity. Figure 10B: Cynomolgus activation and cytotoxicity. Human pan T cells (one donor) were added at an effector to target (E:T) ratio of 3:1 and incubated at 37°C for 72 hours. Cytotoxicity was determined using a live / dead fixable dye, and CD25 was measured using BV421-conjugated anti-CD25. [Figure 11]H929 cells were incubated with increasing concentrations of BGCB463 or the negative control B23B251 for 1 hour at 37°C in the presence of AF647-anti-human IgG in the presence of medium (RPMI containing 10% FBS) or RPMI containing 90% human serum. BGCB463 binding was not affected by the presence of serum. [Figure 12] T cells were incubated with increasing concentrations of BGCB463 or negative control B23B251 for 1 hour at 37°C, in the presence of medium (RPMI containing 10% FBS) or RPMI containing 90% human serum, along with AF647-anti-human IgG. Data are representative of three donors tested in two experiments. BGCB463 binding is not affected by the presence of serum. [Figure 13A] H929-GFP cells were incubated with BGCB463 and human pan-T cells from three donors, and anti-CD25-AF647 was added at an E:T ratio of 3:1 to track T cell activation (Figure 13A). Percent cytolysis was calculated based on the GFP signal in treated versus untreated wells (Figure 13B). Incubation continued for approximately 140 hours, with images acquired every 3 hours. [Figure 13B] H929-GFP cells were incubated with BGCB463 and human pan-T cells from three donors, and anti-CD25-AF647 was added at an E:T ratio of 3:1 to track T cell activation (Figure 13A). Percent cytolysis was calculated based on the GFP signal in treated versus untreated wells (Figure 13B). Incubation continued for approximately 140 hours, with images acquired every 3 hours. [Figure 14A]T cell activation was tracked by adding anti-CD25-AF647 to H929-GFP cells at E:T ratios of 1:1 and 5:1 with BGCB463 or B23B251 (isotype control) molecules and human pan-T cells from four donors. Percent cell lysis was calculated based on the GFP signal in treated versus untreated wells. Incubation continued for approximately 140 hours, with images acquired every 3 hours. BGCB463, 1:1 ratio, cell lysis (Figure 14A). BGCB463, 1:1 ratio, total integrated intensity (Figure 14B). BGCB463, 5:1 ratio, cell lysis (Figure 14C). BGCB463, 5:1 ratio, total integrated intensity (Figure 14D). B23B251, 5:1 ratio, cell lysis (Figure 14E). B23B251, 5:1 ratio, total integrated intensity (Figure 14F). [Figure 14B] T cell activation was tracked by adding anti-CD25-AF647 to H929-GFP cells at E:T ratios of 1:1 and 5:1 with BGCB463 or B23B251 (isotype control) molecules and human pan-T cells from four donors. Percent cell lysis was calculated based on the GFP signal in treated versus untreated wells. Incubation continued for approximately 140 hours, with images acquired every 3 hours. BGCB463, 1:1 ratio, cell lysis (Figure 14A). BGCB463, 1:1 ratio, total integrated intensity (Figure 14B). BGCB463, 5:1 ratio, cell lysis (Figure 14C). BGCB463, 5:1 ratio, total integrated intensity (Figure 14D). B23B251, 5:1 ratio, cell lysis (Figure 14E). B23B251, 5:1 ratio, total integrated intensity (Figure 14F). [Figure 14C]T cell activation was tracked by adding anti-CD25-AF647 to H929-GFP cells at E:T ratios of 1:1 and 5:1 with BGCB463 or B23B251 (isotype control) molecules and human pan-T cells from four donors. Percent cell lysis was calculated based on the GFP signal in treated versus untreated wells. Incubation continued for approximately 140 hours, with images acquired every 3 hours. BGCB463, 1:1 ratio, cell lysis (Figure 14A). BGCB463, 1:1 ratio, total integrated intensity (Figure 14B). BGCB463, 5:1 ratio, cell lysis (Figure 14C). BGCB463, 5:1 ratio, total integrated intensity (Figure 14D). B23B251, 5:1 ratio, cell lysis (Figure 14E). B23B251, 5:1 ratio, total integrated intensity (Figure 14F). [Figure 14D] T cell activation was tracked by adding anti-CD25-AF647 to H929-GFP cells at E:T ratios of 1:1 and 5:1 with BGCB463 or B23B251 (isotype control) molecules and human pan-T cells from four donors. Percent cell lysis was calculated based on the GFP signal in treated versus untreated wells. Incubation continued for approximately 140 hours, with images acquired every 3 hours. BGCB463, 1:1 ratio, cell lysis (Figure 14A). BGCB463, 1:1 ratio, total integrated intensity (Figure 14B). BGCB463, 5:1 ratio, cell lysis (Figure 14C). BGCB463, 5:1 ratio, total integrated intensity (Figure 14D). B23B251, 5:1 ratio, cell lysis (Figure 14E). B23B251, 5:1 ratio, total integrated intensity (Figure 14F). [Figure 14E]T cell activation was tracked by adding anti-CD25-AF647 to H929-GFP cells at E:T ratios of 1:1 and 5:1 with BGCB463 or B23B251 (isotype control) molecules and human pan-T cells from four donors. Percent cell lysis was calculated based on the GFP signal in treated versus untreated wells. Incubation continued for approximately 140 hours, with images acquired every 3 hours. BGCB463, 1:1 ratio, cell lysis (Figure 14A). BGCB463, 1:1 ratio, total integrated intensity (Figure 14B). BGCB463, 5:1 ratio, cell lysis (Figure 14C). BGCB463, 5:1 ratio, total integrated intensity (Figure 14D). B23B251, 5:1 ratio, cell lysis (Figure 14E). B23B251, 5:1 ratio, total integrated intensity (Figure 14F). [Figure 14F] T cell activation was tracked by adding anti-CD25-AF647 to H929-GFP cells at E:T ratios of 1:1 and 5:1 with BGCB463 or B23B251 (isotype control) molecules and human pan-T cells from four donors. Percent cell lysis was calculated based on the GFP signal in treated versus untreated wells. Incubation continued for approximately 140 hours, with images acquired every 3 hours. BGCB463, 1:1 ratio, cell lysis (Figure 14A). BGCB463, 1:1 ratio, total integrated intensity (Figure 14B). BGCB463, 5:1 ratio, cell lysis (Figure 14C). BGCB463, 5:1 ratio, total integrated intensity (Figure 14D). B23B251, 5:1 ratio, cell lysis (Figure 14E). B23B251, 5:1 ratio, total integrated intensity (Figure 14F). [Figure 15A] Determination of soluble BCMA interference using patient sera: Donor 1 (Figure 15A), Donor 2 (Figure 15B), Donor 3 (Figure 15C), and normal serum (Figure 15D). Patient sera with known levels of sBCMA were incubated with directly labeled BGCB463-AF647 and B23B251-AF647 isotype controls on H929 cells for 1 hour at 37°C (90% serum vs. 10% media). 50% and 10% patient sera were diluted with normal serum to a final concentration of 50% and 10%, respectively. [Figure 15B]Determination of soluble BCMA interference using patient sera: Donor 1 (Figure 15A), Donor 2 (Figure 15B), Donor 3 (Figure 15C), and normal serum (Figure 15D). Patient sera with known levels of sBCMA were incubated with directly labeled BGCB463-AF647 and B23B251-AF647 isotype controls on H929 cells for 1 hour at 37°C (90% serum vs. 10% media). 50% and 10% patient sera were diluted with normal serum to a final concentration of 50% and 10%, respectively. [Figure 15C] Determination of soluble BCMA interference using patient sera: Donor 1 (Figure 15A), Donor 2 (Figure 15B), Donor 3 (Figure 15C), and normal serum (Figure 15D). Patient sera with known levels of sBCMA were incubated with directly labeled BGCB463-AF647 and B23B251-AF647 isotype controls on H929 cells for 1 hour at 37°C (90% serum vs. 10% media). 50% and 10% patient sera were diluted with normal serum to a final concentration of 50% and 10%, respectively. [Figure 15D] Determination of soluble BCMA interference using patient sera: Donor 1 (Figure 15A), Donor 2 (Figure 15B), Donor 3 (Figure 15C), and normal serum (Figure 15D). Patient sera with known levels of sBCMA were incubated with directly labeled BGCB463-AF647 and B23B251-AF647 isotype controls on H929 cells for 1 hour at 37°C (90% serum vs. 10% media). 50% and 10% patient sera were diluted with normal serum to a final concentration of 50% and 10%, respectively. [Figure 16] FIG. 1 is a diagram of the BGCB491 trispecific antibody. [Figure 17A]BGCB463, BGCB491, and three controls (BGCB482 (CD3 × Null × Null), BGCB483 (CD3 × Null × BCMA), and BGCB484 (CD3 × GPRC5D × Null)) were tested against BCMA+GPRC5D+ cells (Figure 17A) and BCMA-GPRC5D- cells (Figure 17B). Effector-to-target (E:T) ratio 3:1, n=1 T cell donor, 72-hour incubation, top concentration 53 nM, 1-5-fold dilutions. Both cytotoxicity and T cell activation were tested. [Figure 17B] BGCB463, BGCB491, and three controls (BGCB482 (CD3 × Null × Null), BGCB483 (CD3 × Null × BCMA), and BGCB484 (CD3 × GPRC5D × Null)) were tested against BCMA+GPRC5D+ cells (Figure 17A) and BCMA-GPRC5D- cells (Figure 17B). Effector-to-target (E:T) ratio 3:1, n=1 T cell donor, 72-hour incubation, top concentration 53 nM, 1-5-fold dilutions. Both cytotoxicity and T cell activation were tested. [Figure 18A] BGCB463 and BGCB491 depleted H929 WT, GPRC5D KO, and BCMA KO cell lines at both 72 and 96 hours after primary incubation (Figure 18A). The E:T ratio was 3:1, n=5 T cell donors; 72 (5) and 96 (4) hours of incubation, top concentration 532 mM, dilution gradient 1-5. Both antibodies showed increased T cell activation at both 72 and 96 hours in the same H929 WT, GPRC5D KO, and BCMA KO cells (Figure 18B). [Figure 18B]BGCB463 and BGCB491 depleted H929 WT, GPRC5D KO, and BCMA KO cell lines at both 72 and 96 hours after primary incubation (Figure 18A). The E:T ratio was 3:1, n=5 T cell donors; 72 (5) and 96 (4) hours of incubation, top concentration 532 mM, dilution gradient 1-5. Both antibodies showed increased T cell activation at both 72 and 96 hours in the same H929 WT, GPRC5D KO, and BCMA KO cells (Figure 18B). [Figure 19] BGCB463 and BGCB491 were tested in cytotoxicity and T cell activation assays using H929, RAMOS, and GRANTA-519 cell lines. E:T was 5:1, n=1 T cell donor; 72 hour incubation only, top concentration 53 nM; and 1-5 dilutions. [Figure 20] BGCB463 and BGCB491 were tested in a cytotoxicity and T cell activation whole blood assay using H292 cells. E:T ratio was 5:1, n=2 whole blood donors, 48 ​​hour incubation, top concentration 532 nM; and 1-5 dilutions. [Figure 21A] BGCB463 was shown to regress tumor growth in MM.1S and RPMI 8226 xenograft mouse models when administered at sufficiently high doses, i.e., either 2 ug or 10 ug in the MM.1S model (Figure 21A) or 5 ug or 10 ug in the RPMI 8226 model (Figure 21B). [Figure 21B] BGCB463 was shown to regress tumor growth in MM.1S and RPMI 8226 xenograft mouse models when administered at sufficiently high doses, i.e., either 2 ug or 10 ug in the MM.1S model (Figure 21A) or 5 ug or 10 ug in the RPMI 8226 model (Figure 21B). [Figure 22]BGCB463 and BGCB491 were shown to regress tumor growth in the H929 prophylactic xenograft mouse model. Compared to PBS controls, both the 0.5ug and 5ug doses slowed tumor growth in both the H929-BCMA KO and H929-GPRC5D KO models, with the higher 5ug concentration showing greater efficacy. [Figure 23] BGCB463 and BGCB491 were tested in the DRC pan T binding assay for 1 hour at 37° C. Two human pan T cell donors were used and the test molecules had a starting concentration of 2 uM with a 1:3 dilution and an 11-point DRC. [Figure 24A] BGCB463, BGCB491, and a null control were tested against H929 WT (Figure 24A), H929-BCMA KO (Figure 24B), H929-GPRC5D KO (Figure 24C), and H929-BCMA / GPRC5D double KO (Figure 24D) cells in a DRC series binding assay for 1 hour at 37°C. [Figure 24B] BGCB463, BGCB491, and a null control were tested against H929 WT (Figure 24A), H929-BCMA KO (Figure 24B), H929-GPRC5D KO (Figure 24C), and H929-BCMA / GPRC5D double KO (Figure 24D) cells in a DRC series binding assay for 1 hour at 37°C. [Figure 24C] BGCB463, BGCB491, and a null control were tested against H929 WT (Figure 24A), H929-BCMA KO (Figure 24B), H929-GPRC5D KO (Figure 24C), and H929-BCMA / GPRC5D double KO (Figure 24D) cells in a DRC series binding assay for 1 hour at 37°C. [Figure 24D] BGCB463, BGCB491, and a null control were tested against H929 WT (Figure 24A), H929-BCMA KO (Figure 24B), H929-GPRC5D KO (Figure 24C), and H929-BCMA / GPRC5D double KO (Figure 24D) cells in a DRC series binding assay for 1 hour at 37°C. [Figure 25]Representative binding profiles of BGCB491 to H929 WT, H929-BCMA KO cells, H929-GPRC5D KO cells, and / or H929-BCMA / GPRC5D double KO cells after 1 hour of incubation at 37°C. BGCB491 is indicated by a solid circle, and the negative control CD3xNullxNull BGCB510 is indicated by a solid circle. Curves are representative of five independent experiments. H929-GPRC5D-KO clone H15 is a heterozygous KO line with two copies of the gene, with a 30% and 61% distribution. H929-BCMA-KO clone J8 is a heterozygous KO line with two copies of the gene, with an equal 50% distribution. [Figure 26] Primary T cell binding profiles of BGCB491 to seven donor preparations after 1 hour incubation at 37°C. MFI (mean fluorescence intensity): mean fluorescence intensity. [Figure 27] Representative bioinformatics binding curve extrapolation using CD3B376 x Null control antibody (GCDB381). [Figure 28A]BGCB491-mediated (Figure 28A) cytotoxicity and (Figure 28B) T cell activation of BCMA-GPRC5D double-positive and BCMA-GPRC5D double-negative cell lines. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.00005-533.33 nM, x-axis) in the presence of pan-T cells from six donors (donor IDs: 20063323, 20063309, 20062062, 20063310, 20062105, and 20061963) and Fc blockers (2 mg / mL) and incubated for 72 hours. An optimal E:T ratio of 3:1 was tested in these studies (see Figures 33A-33B for E:T ratio evaluation). Plots were generated using Prism 8 by fitting a separate 4PL model to the observed data. The data points aligned closely along the generated fit curve, with little variability observed between T cell donors (6 donor averages were plotted). In Figure 28A, target cell death was measured and represented as percent cytotoxicity on the Y-axis. In Figure 28B, T cell activation was measured as the percent CD25+CD3+ T cells. [Figure 28B]BGCB491-mediated (Figure 28A) cytotoxicity and (Figure 28B) T cell activation of BCMA-GPRC5D double-positive and BCMA-GPRC5D double-negative cell lines. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.00005-533.33 nM, x-axis) in the presence of pan-T cells from six donors (donor IDs: 20063323, 20063309, 20062062, 20063310, 20062105, and 20061963) and Fc blockers (2 mg / mL) and incubated for 72 hours. An optimal E:T ratio of 3:1 was tested in these studies (see Figures 33A-33B for E:T ratio evaluation). Plots were generated using Prism 8 by fitting a separate 4PL model to the observed data. The data points aligned closely along the generated fit curve, with little variability observed between T cell donors (6 donor averages were plotted). In Figure 28A, target cell death was measured and represented as percent cytotoxicity on the Y-axis. In Figure 28B, T cell activation was measured as the percent CD25+CD3+ T cells. [Figure 29] BGCB 491-mediated cytotoxic activity and T cell activation of H929 CRISPR KO clones. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.00005-533.33 nM, x-axis) in the presence of pan-T cells from four healthy donors (donor IDs: 20063323, 20063309, 20062062, and 20063310) and Fc blocker (2 mg / mL) and incubated for 72 hours. A 3:1 E:T ratio was tested in these studies. Plots were generated by fitting a separate 4PL model to the observed data using Prism 8. Data points aligned closely along the generated fit curve, with little variability observed between T cell donors (four donor averages plotted). Target cell death was measured and expressed as percent cytotoxicity in the top row, and T cell activation measured as percent CD25 expressing CD3+ positive cells is shown in the bottom row (Y axis). [Figure 30] BGCB 491-mediated target cytotoxicity in whole blood spiked with H929 MM cells (MABEL assay). MABEL (minimum anticipated biological effect level), estimated minimum biological effect level BGCB 491, BCMA×GPRC5D×Null, and CD3×Null×Null were spiked at various concentrations (0.00005–533.33 nM, x-axis) into whole blood from seven healthy donors (donor IDs: 10145, 10403, 10420, 10083, 10463, 10493, and 10145; T cell source) in the presence of H929 target myeloma cells at an adjusted E:T ratio of 5:1 for 48 h. Plots were generated by fitting separate 4PL models to the observed data using Prism 8. The data points aligned closely along the generated fit curve, with little variability observed between T cell donors (means of 7 donors are plotted). Target cell death was measured and expressed as percent cytotoxicity, and T cell activation was measured as percent CD25+CD3+ cells. Teclistamab and talquetamab were tested as positive controls in this experiment. [Figure 31]Cytokine profile of BGCB491 from the MABEL assay. MSD, Meso Scale Discovery. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were spiked into whole blood from three healthy donors at various concentrations (0.00005–533.33 nM, x-axis). H929 cell supernatants from the cytotoxicity experiment described above (Figure 7; three T cell donors, donor IDs: 10083, 10463, and 10493) were collected and analyzed for cytokine levels using an MSD-based multiplex assay (catalog number: K15049D). Cytokine levels were measured as pg / mL. Plots were generated by fitting a separate 4PL model to the observed data using Prism 8. Data points aligned closely along the generated fit curve, with little variability observed between T cell donors (means of three donors were plotted). In this experiment, teclistamab and talquetamab were tested as positive controls. [Figure 32] Cytotoxic potency of BGCB491 against human primary MM CD138+ cells. MNC (mononuclear cells). Frozen bone marrow-derived mononuclear cells from four different myeloma patients (MM BM MNC-626, 649, 652, and 653) were used to evaluate the potential of BGCB491 for plasma cell depletion (top panel) and T cell activation (bottom panel). T cells from a normal healthy donor (donor ID: 20063309) were exogenously added to patient BM MNC samples (E:T ratio 1:1) and incubated with BGCB491, CD3xNullxNull, teclimab, or talquetamab (0.00005–533.33 nM, x-axis) for 48 hours. A loss of viable plasma cells (CD138+) and a concomitant upregulation of CD25 on these cells is observed in response to BGCB491 treatment. Variability in total initial plasma cell numbers was observed between donors. Individual target receptor density values ​​and percentages of the target-positive population are listed. [Figure 33A]BGCB491 cytotoxicity at various E:T ratios and incubation times (H929 cell line). (Figure 33A) BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were tested in the presence of pan T cells from one healthy donor (donor ID: 20063323) at four different E:T ratios (5:1, 3:1, 1:1, and 0.5:1) for 72 hours. (Figure 33B) BGCB491 was tested at E:T ratios of 3:1 and 1:1 using four different T cell donors (donor IDs: 20062105, 20063309, 20061963, and 20063310) for various time points (24 hours—circles, 48 ​​hours—upward triangles, 72 hours—downward triangles, 96 hours—hexagons, 120 hours—diamonds, 144 hours—stars, and 168 hours—squares). Target cell death and T cell activation were measured and expressed as percent cytotoxicity and percent CD25+CD3+ T cells. Plots were generated by fitting separate 4PL models to the observed data using Prism 8. Data points aligned closely along the generated fit curves, with little variability observed between T cell donors. Averages from one donor are plotted in (Figure 33A) and four donors in (Figure 33B). [Figure 33B]BGCB491 cytotoxicity at various E:T ratios and incubation times (H929 cell line). (Figure 33A) BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were tested in the presence of pan T cells from one healthy donor (donor ID: 20063323) at four different E:T ratios (5:1, 3:1, 1:1, and 0.5:1) for 72 hours. (Figure 33B) BGCB491 was tested at E:T ratios of 3:1 and 1:1 using four different T cell donors (donor IDs: 20062105, 20063309, 20061963, and 20063310) for various time points (24 hours—circles, 48 ​​hours—upward triangles, 72 hours—downward triangles, 96 hours—hexagons, 120 hours—diamonds, 144 hours—stars, and 168 hours—squares). Target cell death and T cell activation were measured and expressed as percent cytotoxicity and percent CD25+CD3+ T cells. Plots were generated by fitting separate 4PL models to the observed data using Prism 8. Data points aligned closely along the generated fit curves, with little variability observed between T cell donors. Averages from one donor are plotted in (Figure 33A) and four donors in (Figure 33B). [Figure 34A] The effect of BGCB491 on T cells in the absence of target cells. (Figure 34A) T cell activation assays were performed using six normal healthy donor T cells (donor IDs: 20063323, 20063309, 20062062, 20063310, 20062105, and 20061963) or (Figure 34B) six normal donor whole blood samples (donor IDs: 10274, 10402, 10427, 10470, 10500, and 10509). (Figure 34C) Nonspecific cytotoxicity was measured for various subsets. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.00005-533.33 nM, x-axis) for (Figure 34A) 72 hours and (Figures 34B, 34C) 48 hours. [Figure 34B]The effect of BGCB491 on T cells in the absence of target cells. (Figure 34A) T cell activation assays were performed using six normal healthy donor T cells (donor IDs: 20063323, 20063309, 20062062, 20063310, 20062105, and 20061963) or (Figure 34B) six normal donor whole blood samples (donor IDs: 10274, 10402, 10427, 10470, 10500, and 10509). (Figure 34C) Nonspecific cytotoxicity was measured for various subsets. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.00005-533.33 nM, x-axis) for (Figure 34A) 72 hours and (Figures 34B, 34C) 48 hours. [Figure 34C] The effect of BGCB491 on T cells in the absence of target cells. (Figure 34A) T cell activation assays were performed using six normal healthy donor T cells (donor IDs: 20063323, 20063309, 20062062, 20063310, 20062105, and 20061963) or (Figure 34B) six normal donor whole blood samples (donor IDs: 10274, 10402, 10427, 10470, 10500, and 10509). (Figure 34C) Nonspecific cytotoxicity was measured for various subsets. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.00005-533.33 nM, x-axis) for (Figure 34A) 72 hours and (Figures 34B, 34C) 48 hours. [Figure 35]Anticancer effects of BGCB491, teclistamab, and talquetamab in subcutaneous (SC) RPMI 8226 xenografts in T cell-humanized NSG mice (Study A). KO (knock-out), NSG, non-obese diabetic (NOD) severe combined immunodeficiency (scid) gamma, or NOD.CgPrkdcscidIl2rgtm1Wjl / SzJ; PBS (phosphate-buffered saline), phosphate-buffered saline; SEM (standard error of the mean). BGCB491 was administered intraperitoneally (IP) at 0.1, 0.4, 1, and 2.5 mg / kg to T cell-humanized NSG mice bearing established SC RPMI 8226 tumors. Teclistamab was administered intraperitoneally at 0.4, 1, and 2.5 mg / kg. Talquetamab was administered intraperitoneally at 2.5 mg / kg. Treatments were administered on days 19, 21, 25, 28, 31, 34, 38, and 41 (indicated by the black lines below the X-axis). Tumor volume was measured twice weekly, and the results were presented as the mean tumor volume ± SEM for each group. Data were graphed for each group where at least 70% of the animals remained in the study. [Figure 36]Anticancer effects of BGCB491, teclistamab, and talquetamab in subcutaneous (SC) H929-BCMA-KO and H929-GPRC5D-KO xenografts in T cell-humanized NSG mice (Study B). KO, knockout; NSG, nonobese diabetic (NOD) severe combined immunodeficiency (scid) gamma or NOD.CgPrkdcscidIl2rgtm1Wjl / SzJ; PBS, phosphate-buffered saline; SEM, standard error of the mean. BGCB491 was administered intraperitoneally (IP) at 0.025, 0.1, 0.25, and 0.5 mg / kg to T cell-humanized NSG mice bearing H929-BCMA-KO (left flank) and H929-GPRC5D-KO (right flank) tumors. Teclistamab and talquetamab were administered intraperitoneally at 0.1 and 0.25 mg / kg. Treatments were administered on days 1, 5, 9, 13, 16, 19, and 22 (indicated by the black lines below the X-axis). Tumor volumes were measured twice weekly, and the results were presented as the mean tumor volume ± SEM for each group. Data were graphed for each group where at least 70% of the animals remained in the study. [Figure 37] Individual BGCB491 serum concentration-time profiles (FIG. 37) and prediction model (FIG. 38) following a single intravenous (IV) 0.5 mg / kg dose in female cynomolgus monkeys. BQL (below quantification limit), below limit of quantification. Data points with concentrations below the lowest quantifiable concentration are not shown on the graph. [Figure 38] Individual BGCB491 serum concentration-time profiles (FIG. 37) and prediction model (FIG. 38) following a single intravenous (IV) 0.5 mg / kg dose in female cynomolgus monkeys. BQL (below quantification limit), below limit of quantification. Data points with concentrations below the lowest quantifiable concentration are not shown on the graph. [Figure 39A]1 shows mean (SD) BGCB491 concentration-time profiles and model predictions following a single dose of BGCB491 in male minipigs. BQL, below limit of quantification; SD (standard deviation), standard deviation. Data points with concentrations below the lowest quantifiable concentration are not shown on the graph. Group 1: Dose = 0.01 mg / kg, subcutaneous (SC). Group 2: Dose = 0.1 mg / kg, SC. ​​Group 3: Dose = 0.1 mg / kg, IV. [Figure 39B] 1 shows mean (SD) BGCB491 concentration-time profiles and model predictions following a single dose of BGCB491 in male minipigs. BQL, below limit of quantification; SD (standard deviation), standard deviation. Data points with concentrations below the lowest quantifiable concentration are not shown on the graph. Group 1: Dose = 0.01 mg / kg, subcutaneous (SC). Group 2: Dose = 0.1 mg / kg, SC. ​​Group 3: Dose = 0.1 mg / kg, IV. [Figure 40] Kinetic binding profile of BGCB463 to H929 WT and MM.1R WT cell lines over 24 hours of incubation at 37° C. MFI: Mean Fluorescence Intensity [Figure 41] BGCB491 binding to endogenous BCMA-GPRC5D-expressing cell lines. Geo, geometric. BGCB491, BCMAxGPRC5DxNull, and CD3xNullxNull were added at various concentrations (0.096–1,000 nM, X-axis) and incubated at 37°C for 1 hour. Plots were generated by fitting a separate 4PL model to the observed data using Prism 8. Binding intensity (Y-axis) is expressed as the geometric mean. [Figure 42]Dynamic cytotoxicity and T cell activation of BGCB491 and CD3B2271 (null control) using T cells from three T cell donors at (Figure 42A) 1:1 and (Figure 42B) 1:3 E:T ratios. CD25 measurements on the Incucyte are a measure of T cell number and CD25 expression (total integrated intensity). CD25 is a late marker of T cell activation, typically detectable 24 hours after activation and peaking at approximately 96 hours. It does not necessarily coincide with cytotoxicity in terms of timing. Percent cytolysis was calculated based on the green fluorescent protein (GFP) signal in treated versus untreated wells. [Figure 43] Percent cytolysis and T cell activation for BGCB491 and CD3B2271 (null control) at 72, 93, and 120 hours at an effector to target (E:T) ratio of 1:1. [Figure 44] Percent cytolysis and T cell activation of BGCB491 and CD3B2271 (null control) at 72, 93 and 120 hours at an E:T ratio of 1:3. [Figure 45]Anticancer effects of JNJ-79635322, teclistamab, and talquetamab in subcutaneous MM.1S xenografts in T cell-humanized NSG mice (Study C). NSG, nonobese diabetic (NOD) severe combined immunodeficiency (scid) gamma or NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ; PBS, phosphate-buffered saline; SEM, standard error of the mean. T cell-humanized NSG mice bearing established SC MM.1S tumors were administered BGCB491 intraperitoneally at 0.025, 0.1, 0.5, and 1 mg / kg. Talquetamab was administered intraperitoneally at 0.025 and 0.1 mg / kg. Teclistamar was administered intraperitoneally at 0.1 mg / kg and 0.5 mg / kg treatments were administered on days 15, 19, 22, 26, 30, 34, and 37 (indicated by the black line below the x-axis). Tumor volumes were measured twice weekly, and the results are presented as the mean tumor volume ± SEM for each group. Data were graphed for each group with at least 70% of animals remaining in the study. Animals were monitored post-treatment until signs of graft-versus-host disease (GvHD)-related morbidity appeared, at which point they were euthanized and the study was terminated on day 51. [Figure 46A] The BCMA lead (BCMB519) exhibits binding and cytotoxicity comparable to teclistamab with ideal scFv biophysical properties. [Figure 46B] The BCMA lead (BCMB519) exhibits binding and cytotoxicity comparable to teclistamab with ideal scFv biophysical properties. [Figure 47] The GPRC5d lead (GC5B680) demonstrates specific and improved binding compared to talquetamab with ideal scFv biophysical properties.

[0039] Detailed Description of the Preferred Embodiments definition Various terms relating to aspects of the present invention are used throughout the specification and claims. Unless otherwise indicated, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be construed in a manner consistent with the definition provided herein.

[0040] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0041] As used herein, the term "about" when referring to measurements, e.g., amounts, durations, etc., is intended to encompass deviations of up to ±10% from the specified value. Likewise, such deviations are appropriate for carrying out the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, as well as properties such as molecular weight and reaction conditions, used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, no attempt should be made to limit the application of the doctrine of equivalents to the scope of the claims, and each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0042] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0043] "Isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) has been substantially separated from, produced separately from, or purified away from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins, of the organism in which it naturally resides. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition, and are isolated even if such a composition is not part of the nucleic acid's, peptide's, or protein's natural environment. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, an "isolated" antibody or antigen-binding fragment is intended to mean an antibody or antigen-binding fragment that is substantially free of other antibodies or antigen-binding fragments with different antigen specificities (e.g., an isolated antibody that specifically binds BCMA is substantially free of antibodies that specifically bind to antigens other than BCMA). An isolated antibody that specifically binds to an epitope, isoform, or variant of BCMA or GPRC5D may, however, have cross-reactivity to other related antigens, for example from other species (e.g., BCMA or GPRC5D interspecies homologs).

[0044] A "polynucleotide," interchangeably referred to as a "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA (which may be single-stranded, or more typically, double-stranded, or a mixture of single- and double-stranded regions). In addition, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms characteristic of viral and cellular DNA and RNA. "Polynucleotide" also encompasses relatively short nucleic acid strands, often referred to as oligonucleotides.

[0045] The meaning of "substantially the same" can vary depending on the context in which the term is used. Because natural sequence variations may exist in heavy and light chains and the genes encoding them, some variation is expected to be found in the amino acid sequences described herein or in the genes encoding the antibodies or antigen-binding fragments, which has little or no effect on the inherent binding characteristics (e.g., specificity and affinity). Such expectation is due, in part, to the degeneracy of the genetic code and the evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the properties of the encoded proteins. Thus, in the context of nucleic acid sequences, "substantially the same" means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or more identity. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (i.e., percent homology = number of identical positions / total number of positions × 100). This consideration is necessary to derive optimal alignment of two sequences. The percent identity between two nucleotide or amino acid sequences may be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), which is incorporated into the ALIGN program (version 2.0), and which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Additionally, the percent identity between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0046] The degree of variation that can occur in the amino acid sequence of a protein without having a substantial effect on the function of the protein is much less than the degree of variation in a nucleic acid sequence, because the same principles of degeneracy do not apply to amino acid sequences. Thus, in the context of antibodies or antigen-binding fragments, "substantially the same" refers to antibodies or antigen-binding fragments that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the described antibodies or antigen-binding fragments. Other embodiments include antibodies or antigen-binding fragments that do not share significant identity with the antibodies and antigen-binding fragments described herein, but have framework, scaffold, or other non-binding regions that incorporate one or more CDRs or other sequences required to confer binding, and have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences described herein.

[0047] A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that can be grown stably in vitro for many generations. In some examples provided herein, the cells are transformed by transfecting the cells with DNA.

[0048] The terms "express" and "produce" are used interchangeably herein and refer to the biosynthesis of a gene product. These terms include transcription of a gene into RNA. These terms also include translation of RNA into one or more polypeptides, further encompassing all natural post-transcriptional and post-translational modifications. Expression or production of an antibody or antigen-binding fragment thereof can be within the cytoplasm of a cell or in an extracellular environment, such as the growth medium for a cell culture.

[0049] The term "treating" or "treatment" refers to any success or indication of success in attenuating or ameliorating an injury, pathology, or condition, including any objective or subjective parameter, such as remission, remission, reduction of symptoms or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, mitigating eventual debilitation from degeneration, improving the patient's physical or mental health, or prolonging survival. Treatment may be assessed by objective or subjective parameters, including the results of a physical examination, neurological examination, or psychiatric evaluation.

[0050] An "effective amount" or "therapeutically effective amount" means an amount effective, at the dosage and for the duration required, to achieve the desired therapeutic result. A therapeutically effective amount of a BCMAxGPRC5DxCD3 antibody can vary according to factors such as the individual's condition, age, sex, and weight, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0051] "Antibody," unless otherwise specified, refers to all immunoglobulin isotypes (IgG, IgA, IgE, IgM, IgD, and IgY), including various monomeric, polymeric, and chimeric forms. Polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides, such as chimeric and humanized antibodies, are specifically encompassed by the term "antibody."

[0052] The term "antigen-binding arm" refers to a portion of an antibody that comprises an antigen-binding domain that binds to an antigen (e.g., BCMA, GPRC5D, or CD3) and optionally comprises one or more other antibody regions (e.g., an Fc domain).

[0053] The term "antigen-binding fragment" refers to a fragment of an antigen-binding arm containing the antigen-binding domain. Antigen-binding fragments include those obtained by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. Some antigen-binding fragments consist of a portion of an intact antibody that retains the antigen-binding specificity of the parent antibody molecule. For example, an antigen-binding fragment may contain at least one variable region (either the heavy or light chain variable region) or one or more CDRs of an antibody known to bind to a specific antigen. Examples of suitable antigen-binding fragments include diabodies and single-chain molecules, as well as Fab, F(ab')2, Fc, Fabc, and Fv molecules, single-chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains or CDRs and other proteins, protein scaffolds, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy chain and one light chain, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or the monovalent antibodies described in WO2007059782, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region, Fd fragments consisting essentially of the VH and CH1 domains, Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, dAb fragments consisting essentially of the VH domain (Ward et al., Nature, 341:544-546, 1989), Trends Biotechnol. 2003 Nov.;21(11):484-90), camelids or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan.;5(1):111-24), as well as isolated complementarity determining regions (CDRs) and trispecific antibodies formed from antibody fragments. All antibody isotypes may be used to generate antigen-binding fragments.In addition, antigen-binding fragments may include proteinaceous frameworks other than antibodies, e.g., protein scaffolds, into which polypeptide segments may be advantageously incorporated in an orientation that confers affinity for a given antigen of interest. Antigen-binding fragments may be produced recombinantly or by enzymatic or chemical degradation of an intact antibody. The phrase "antibody or antigen-binding fragment thereof" may be used to indicate that a given antigen-binding fragment incorporates one or more amino acid segments of the antibody referenced within the phrase.

[0054] The term "antigen-binding domain" refers to the proteinaceous structure of an antigen-binding arm that exhibits binding affinity to a specific antigen. This proteinaceous structure is mediated by the complementarity-determining regions (CDRs) of the antigen-binding domain.

[0055] The terms "CDR" and its plural "CDRs" refer to the complementarity-determining regions (CDRs), three of which constitute the binding properties of the light chain variable region (CDRL1, CDRL2, and CDRL3) and three of which constitute the binding properties of the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences comprising scaffold or framework regions. The precise definitional boundaries and lengths of CDRs vary among various classification and numbering systems. Thus, CDRs may be referred to herein by the Kabat definition, the Chothia definition, the Contact definition, or any other boundary definition. Despite the different boundaries, each of these systems has some overlap in the elements that constitute the so-called "hypervariable regions" within the variable sequences. Thus, the definitions of CDRs according to these systems may differ in terms of length and boundary regions for the flanking framework regions. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987); and MacCallum et al., "Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography," J. Mol. Biol. 262:732 (1996)), each of which is incorporated herein by reference in its entirety.

[0056] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main chain conformation adopted by an antigen-binding (CDR) loop. Comparative structural studies have shown that five of the six antigen-binding loops have a very limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide main chain. Thus, corresponding loops among antibodies can have very similar three-dimensional structures, despite the high degree of amino acid sequence variability found in most of those loops (Chothia et al., "Canonical Structures For the Hypervariable Regions of Immunoglobulins," J. Mol. Biol. 196:901 (1987); Chothia et al., "Conformations of Immunoglobulin Hypervariable Regions," J. Mol. Biol. 196:901 (1987); Chothia et al., "Conformations of Immunoglobulin Hypervariable Regions," J. Mol. Biol. 1989:342:877 (1989); Martin and Thornton, "Structural Families in Loops of Homologous Proteins: Automatic Classification, Modeling and Application to Antibodies," J. Mol. Biol. 263:800 (1996), each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the loop structure adopted and the amino acid sequence surrounding it. The three-dimensional structure of a particular canonical class is determined by the length of the loop and the amino acid residues at key positions within the loop as well as those within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0057] The term "polypeptide" is used interchangeably with the term "protein" and, in its broadest sense, refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to any natural and / or unnatural amino acid or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics. Peptides of three or more amino acids are commonly referred to as oligopeptides when the peptide chain is short. When the peptide chain is long, the peptide is commonly referred to as a polypeptide or protein. As used herein, the term "Fc" refers to an antibody crystallizable domain fragment containing two constant heavy chain (CH) regions, CH2 and CH3. Herein, amino acid residues in the Fc region are typically numbered according to the EU numbering scheme (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969). As would be readily understood by one skilled in the art, these residues can be readily designated according to alternative numbering schemes, such as those of IMGT and Kabat (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication no. 91-3242, pp. 662, 680, 689 (1991)). For example, L234 according to the EU numbering scheme can also be represented as L247 according to Kabat.

[0058] When used in the context of an antibody or antibody fragment, "specifically binds" or "binds specifically," or derivatives thereof, refers to binding to one or more epitopes of a protein of interest via a domain encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene without preferentially binding to other molecules in a sample containing a variety of mixed molecules. Typically, an antibody binds to approximately 1×10 6 cells / min as measured by a surface plasmon resonance assay or a cell binding assay. -8 K less than M d and binds to its cognate antigen. Phrases such as "antigen-specific" antibody (e.g., BCMA-specific antibody) are meant to convey that the recited antibody specifically binds to the recited antigen. Whenever the term "binds" is used herein, it is intended to encompass "specifically binds," and these terms can be interchanged as appropriate.

[0059] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment thereof that has at least some portion of at least one variable domain derived from an antibody amino acid sequence of a non-human mammal, rodent, or reptile, while the remainder thereof is of human origin.

[0060] A "vector" is a replicon, such as a plasmid, phage, cosmid, or virus, into which another nucleic acid segment may be operatively inserted so as to bring about the replication or expression of the segment.

[0061] As used herein, the term "host cell" may refer to any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In certain embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due, for example, to mutations or environmental influences that may occur during the subsequent generation or integration of the nucleic acid molecule into the host cell genome. The terms "expression" and "production" are used interchangeably herein and refer to the biosynthesis of a gene product. These terms include transcription of a gene into RNA. These terms also include translation of RNA into one or more polypeptides, further encompassing all natural post-transcriptional and post-translational modifications.

[0062] The term "subject" refers to humans and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the methods described, the subject is a human.

[0063] As used herein, the term "redirecting" or "redirect" refers to the ability of BCMAxGPRC5DxCD3 antibodies to effectively convert the activity of T cells from their original allospecific activity to one that is reactive against GPRC5D and / or BCMA-expressing cells.

[0064] As used herein, the term "sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject (e.g., surgically removed tumor tissue, biopsies including fine needle aspirations), as well as fluids, cells, or tissues present within a subject. In some embodiments, the sample is a bodily fluid. Bodily fluids are typically liquid at physiological temperature and may include naturally occurring fluids present in, withdrawn from, or extracted from a subject or biological source. Some bodily fluids obtained from specific tissues, organs, or localized areas, and other bodily fluids may be more globally or systemically relevant in a subject or biological source. Examples of bodily fluids include blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites fluid, such as that associated with non-solid tumors, pleural, pericardial, peritoneal, abdominal, and other body cavity fluids, fluids collected by bronchial lavage, and the like. Body fluids may also include solutions that have come into contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media, lavage fluids, etc. As used herein, the term "sample" encompasses material removed from a subject or material present in a subject. Related aspects of the invention may optionally be practiced in vitro on isolated samples.

[0065] A "known standard" may be a solution in which the amount or concentration of GPRC5D and / or BCMA is known. In this case, the solution may be a naturally occurring solution, such as a sample from a patient known to have early-stage, intermediate-stage, late-stage, advanced, or static cancer, or the solution may be a synthetic solution, such as a buffered aqueous solution in which a known amount of GPRC5D and / or BCMA has been diluted. The known standard described herein may include GPRC5D and / or BCMA isolated from a subject, recombinant or purified GPRC5D and / or BCMA protein, or a GPRC5D and / or BCMA concentration value associated with a pathological condition.

[0066] As used herein, the terms "B cell maturation antigen" and "BCMA" include human B cell maturation antigen, also known as BCMA, CD269, and TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed on differentiated plasma cells. According to UniProt, the extracellular domain of human BCMA consists of amino acids 1-54 (or 5-51). As used herein, the term "antibody against BCMA, anti-BCMA antibody" relates to an antibody that specifically binds to BCMA.

[0067] The terms "G protein-coupled receptor family C group 5 member D" and "GPRC5D" specifically encompass the human GPRC5D protein as described, for example, in GenBank accession number BC069341, NCBI reference sequence: NP_061124.1 and UniProtKB / Swiss-Prot accession number Q9NZD1 (see also Brauner-Osborne, H. et al. 2001, Biochim. Biophys. Acta 1518, 237-248).

[0068] The terms "cluster of differentiation 3" and "CD3" refer to the human CD3 protein multisubunit complex, which is composed of six distinct polypeptide chains. These include the CD3 gamma chain (SwissProt P09693), the CD3 delta chain (SwissProt P04234), two CD3 epsilon chains (SwissProt P07766), and one CD3 zeta chain homodimer (SwissProt 20963), which associate with the T cell receptor alpha and beta chains. The term "CD3," unless otherwise specified, includes any CD3 variants, isoforms, and interspecies homologs that are naturally expressed by cells (including T cells) or that can be expressed on cells transfected with genes or cDNAs encoding these polypeptides.

[0069] A "BCMAxGPRC5DxCD3 antibody" is a multispecific antibody, optionally a trispecific antibody, which comprises three different antigen-binding arms, one of which binds to the antigen BCMA, one of which binds to the antigen GPRC5D, and one of which binds to CD3. A "BCMAxCD3 antibody" is a multispecific antibody, optionally a bispecific antibody, which comprises two different antigen-binding arms, one of which binds to the antigen BCMA and one of which binds to CD3. A "GPRC5DxCD3 antibody" is a multispecific antibody, optionally a bispecific antibody, which comprises two different antigen-binding arms, one of which binds to the antigen GPRC5D and one of which binds to CD3. The term "multispecific antibody" is used in the broadest sense herein and specifically includes antibodies with polyepitopic specificity. Multispecific antibodies include antibodies that comprise a heavy chain variable domain (V H ) and the light chain variable domain (V L ), an antibody comprising V H V L Antibodies with polyepitopic specificity, where the unit is two or more V L Domain and V H Each V H V L These include, but are not limited to, antibodies in which the units bind to different epitopes, antibodies with two or more single variable domains, each single variable domain binding to a different epitope, full-length antibodies, and antibodies comprising one or more antibody fragments, as well as antibodies comprising covalently or non-covalently linked antibody fragments.

[0070] Multispecific antibodies can be bispecific antibodies, trispecific antibodies, diabodies, or similar molecules (for a description of diabodies, see, e.g., PNAS USA 90(14), 6444-8 (1993)). The bispecific antibodies, trispecific antibodies, diabodies, etc. provided herein can bind to any suitable target in addition to a portion of BCMA or GPRC5D. The term "bispecific antibody" should be understood as an antibody having two different antigen-binding arms defined by different antibody sequences. The term "trispecific antibody" should be understood as an antibody having three different antigen-binding arms defined by different antibody sequences. This can be understood as binding to different targets, but includes binding to different epitopes within a single target as well.

[0071] A "reference sample" is a sample that can be compared with another sample, e.g., a test sample, to assess the characteristics of the compared sample. A reference sample has some characterized property that serves as a standard for comparison with a test sample. For example, a reference sample can be used as a benchmark for GPRC5D or BCMA levels that indicate a subject has cancer. A reference sample does not necessarily need to be analyzed in parallel with a test sample. Thus, in some examples, a reference sample may be a predetermined value or range for characterizing a given condition, e.g., a GPRC5D or BCMA level that is indicative of cancer in a subject. The term also includes samples used for comparison purposes that are known to be associated with a physiological state or pathology, e.g., a GPRC5D- or BCMA-expressing cancer, but in which the amount of GPRC5D or BCMA is unknown.

[0072] "Relapse" refers to the return of a disease or signs and symptoms of a disease after a period of improvement following prior treatment with a therapeutic agent.

[0073] "Refractory" refers to a disease that does not respond to treatment. A refractory disease can be resistant to treatment before or at the start of treatment, or a refractory disease can become resistant during treatment.

[0074] The term "progression," when used in the context of the progression of a GPRC5D- and / or BCMA-expressing cancer, includes a change in the cancer from a less severe to a more severe state. This can include an increase in the number or severity of tumors, the extent of metastasis, the rate at which the cancer grows or spreads, etc. For example, "progression of colon cancer" includes the progression of such cancer from a less severe to a more severe state, e.g., from stage I to stage II, from stage II to stage III, etc.

[0075] The term "regression," when used in the context of regression of a GPRC5D- and / or BCMA-expressing cancer, includes a change in the cancer from a more severe state to a less severe state. This could include a reduction in the number or severity of tumors, the extent of metastasis, the rate at which the cancer grows or spreads, etc. For example, "regression of colon cancer" includes the regression of such cancer from a more severe state to a less severe state, e.g., progression from stage III to stage II, from stage II to stage I, etc.

[0076] The term "stable," when used in the context of a stable GPRC5D- and / or BCMA-expressing cancer, is intended to describe a disease state that does not or has not changed significantly enough over a clinically relevant period of time to be considered a progressive or regressing cancer.

[0077] The embodiments described herein are not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary.

[0078] multispecific antibodies Provided herein are multispecific antibodies that bind to BCMA, GPRC5D, and CD3, as well as trispecific binding fragments thereof. Such antibodies or antibody fragments may allow for more specific targeting to particular subsets of cells compared to antibodies that target only one or two of these targets.

[0079] This can be achieved, for example, by generating a molecule comprising a first antigen-binding arm that binds CD3, a second antigen-binding arm that binds GPRC5D, and a third antigen-binding arm that binds BCMA. The antigen-binding arms can take any form that allows for specific recognition of the target; for example, the binding arms can be or include a heavy chain variable domain, an Fv (a combination of a heavy chain variable domain and a light chain variable domain), a single-chain Fv (scFv), an Fab, or a binding domain based on a fibronectin type III domain (fibronectin-derived or based on the consensus type III domain of fibronectin, such as the Centyrin molecule from Janssen Biotech, Inc., or tenascin-derived or based on the consensus type III domain of tenascin; see, e.g., WO 2010 / 051274 and WO 2010 / 093627). In certain embodiments, a trispecific antibody comprises three antigen-binding arms. In some embodiments, a trispecific antibody is composed of an antibody (e.g., in IgG format) in which an additional antigen-binding arm, e.g., in the form of a single-chain variable fragment, has been fused, e.g., to the N- or C-terminus of one of the heavy chains or one of the light chains of the antibody.

[0080] Thus, a trispecific molecule is provided that contains three different antigen-binding arms that bind to BCMA, GPRC5D, and CD3, respectively.

[0081] In some embodiments, the BCMAxGPRC5DxCD3 multispecific antibody (a) a first antigen-binding arm comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); (b) a second antigen-binding arm comprising a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2); and (c) a third antigen-binding arm comprising a third heavy chain variable domain (VH3) and a third light chain variable domain (VL3).

[0082] In some embodiments, the first antigen-binding arm binds to an epitope on CD3, the second antigen-binding arm binds to an epitope on GPRC5D, and the third antigen-binding arm binds to an epitope on BCMA.

[0083] In some embodiments, the first antigen-binding arm that binds CD3 comprises an HCDR1, HCDR2, and HCDR3 of a VH1 of SEQ ID NO: 8. In some embodiments, the first antigen-binding arm that binds CD3 comprises an LCDR1, LCDR2, and LCDR3 of a VL1 of SEQ ID NO: 7. In some embodiments, the first antigen-binding arm that binds CD3 comprises an HCDR1 comprising the amino acid sequence of GDSVFNNNAAWS (SEQ ID NO: 4), an HCDR2 comprising the amino acid sequence of RTYYRSKWLYD (SEQ ID NO: 5), and an HCDR3 comprising the amino acid sequence of GYSSSFDY (SEQ ID NO: 6). In some embodiments, the first antigen-binding arm that binds CD3 comprises an LCDR1 comprising the amino acid sequence of TGTSSNIGTYKFVS (SEQ ID NO: 1), an LCDR2 comprising the amino acid sequence of EVSKRPS (SEQ ID NO: 2), and an LCDR3 comprising the amino acid sequence of VSYAGSGTLL (SEQ ID NO: 3). In some embodiments, the first antigen-binding arm that binds CD3 has a VH1 of SEQ ID NO: 8. In some embodiments, the first antigen-binding arm that binds to CD3 has a VL1 of SEQ ID NO:7.

[0084] In some embodiments, the second antigen-binding arm that binds to GPRC5D comprises HCDR1, HCDR2, and HCDR3 of VH2 of SEQ ID NO: 16. In some embodiments, the second antigen-binding arm that binds to GPRC5D comprises LCDR1, LCDR2, and LCDR3 of VL2 of SEQ ID NO: 15. In some embodiments, the second antigen-binding arm that binds to GPRC5D comprises HCDR1 comprising the amino acid sequence of GFSLTNIRMSVS (SEQ ID NO: 12), HCDR2 comprising the amino acid sequence of HIFSNDEKS (SEQ ID NO: 13), and HCDR3 comprising the amino acid sequence of MRLPYGMDV (SEQ ID NO: 14). In some embodiments, the second antigen-binding arm that binds to GPRC5D comprises LCDR1 comprising the amino acid sequence of RSSQSLVHSDGNTYLS (SEQ ID NO: 9), LCDR2 comprising the amino acid sequence of KISNRFF (SEQ ID NO: 10), and LCDR3 comprising the amino acid sequence of MQATQFPHT (SEQ ID NO: 11). In some embodiments, the second antigen-binding arm that binds to GPRC5D has a VH1 of SEQ ID NO: 16. In some embodiments, the second antigen-binding arm that binds to GPRC5D has a VL1 of SEQ ID NO: 15.

[0085] In some embodiments, the third antigen binding arm that binds BCMA comprises HCDR1, HCDR2, and HCDR3 of VH3 of SEQ ID NO: 24. In some embodiments, the third antigen binding arm that binds BCMA comprises LCDR1, LCDR2, and LCDR3 of VL3 of SEQ ID NO: 23. In some embodiments, the third antigen binding arm that binds BCMA comprises an HCDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 20), an HCDR2 comprising the amino acid sequence of AISGSGGSTY (SEQ ID NO: 21), and an HCDR3 comprising the amino acid sequence of DEGYSSGHYYGMDV (SEQ ID NO: 22), and an LCDR1 comprising the amino acid sequence of RASQSISSSFLT (SEQ ID NO: 17). In some embodiments, the third binding domain that binds BCMA comprises an LCDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 18), and an LCDR3 comprising the amino acid sequence of QHYGSSPMYT (SEQ ID NO: 19). In some embodiments, the third antigen binding arm that binds BCMA has a VH1 of SEQ ID NO: 24. In some embodiments, the third antigen binding arm that binds to BCMA has a VL1 of SEQ ID NO: 23.

[0086] In some embodiments, the VH1 and VL1 of the antigen-binding arm that binds to the CD3 epitope are present in a diabody, Fab, Fab', F(ab')2, Fv, scFv, Fd, disulfide stabilized Fv fragment (dsFv), or disulfide stabilized diabody (dsdiabody).

[0087] In some embodiments, the VH2 and VL2 of the antigen-binding arms that bind to the GPRC5D epitope are present in a diabody, Fab, Fab', F(ab')2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (dsdiabody).

[0088] In some embodiments, the VH3 and VL3 of the antigen binding arms that bind to a BCMA epitope are present in a diabody, Fab, Fab', F(ab')2, Fv, scFv, Fd, disulfide stabilized Fv fragment (dsFv), or disulfide stabilized diabody (dsdiabody).

[0089] In some embodiments, the first antigen-binding arm of the BCMAxGPRC5DxCD3 multispecific antibody comprises a first heavy chain portion (HC1) comprising a VH1 and a light chain portion (LC) comprising a VL1, where the VH1 and VL1 pair to form a first antigen-binding domain that binds to a first antigen. In some embodiments, the HC1 comprises, from N-terminus to C-terminus, a VH1, a first heavy chain constant domain (CH1), and a first Fc domain. In some embodiments, the VH1 and CH1 of the HC1, together with the LC, form a fragment antigen-binding (Fab) domain.

[0090] In some embodiments, the VH1 of the first antigen-binding arm is linked to the VH3 of the third antigen-binding arm via a first Fc domain. In some embodiments, the first Fc domain of the first antigen-binding arm is linked to the third antigen-binding arm via a first linker (L1), thereby forming linked first and third antigen-binding arms. The linked first and third antigen-binding arms may comprise, from N-terminus to C-terminus, the VH1, CH1 domain, and Fc domain of the first antigen-binding arm, the first linker, and the third antigen-binding arm. In some embodiments, the third antigen-binding arm is a single-chain variable fragment (scFv) formed from the VH3 and VL3 of the third antigen-binding arm.

[0091] In some embodiments, the second antigen-binding arm of the BCMAxGPRC5DxCD3 multispecific antibody comprises a second heavy chain portion (HC2) that comprises a second heavy chain variable domain (VH2) that forms a second antigen-binding domain that binds to a second antigen. In some embodiments, the second binding arm comprises, from N- to C-terminus, a single-chain variable fragment (scFv) formed from VH2 and VL2, and a second Fc domain.

[0092] In some embodiments, the VH2 of the second antigen-binding arm is linked to the VH3 of the third antigen-binding arm via a second Fc domain. In some embodiments, the second Fc domain of the second antigen-binding arm is linked to the third antigen-binding arm via a linker, thereby forming a linked second and third antigen-binding arm. The linked second and third antigen-binding arms may comprise, from N-terminus to C-terminus, a second antigen-binding domain, a second Fc domain, a first linker, and a third antigen-binding arm. In some embodiments, the third antigen-binding arm is a single-chain variable fragment (scFv) formed from the VH3 and VL3 of the third antigen-binding arm.

[0093] In a preferred embodiment, the BCMA x GPRC5D x CD3 multispecific antibody is a trispecific antibody comprising a CD3-specific arm comprising a first heavy chain portion (HC1) comprising a VH1 and a light chain portion (LC) comprising a VL1. The VH1 and VL1 domains pair to form a first antigen-binding domain that binds to CD3. The second antigen-binding arm of the trispecific antibody comprises a second heavy chain portion (HC2) with a VH2 that forms a second antigen-binding domain that binds to a second antigen. The HC1 of the CD3-specific binding arm or the HC2 of the second antigen-binding arm is linked to a third antigen-binding arm comprising a VH3 domain that forms a third antigen-binding domain that binds to a third antigen. In some embodiments, the second antigen is BCMA and the third antigen is GPRC5D. In some embodiments, the second antigen is GPRC5D and the third antigen is BCMA.

[0094] In one embodiment, the BCMAxGPRC5DxCD3 multispecific antibody is a trispecific antibody comprising a CD3-specific binding arm comprising an HC1 having a VH1 and an LC having a VL1. The VH1 and VL1 pair to form a first CD3-specific antigen-binding domain that binds to CD3. The second antigen-binding arm comprises a VH2 and a VL2 that form a second antigen-binding domain that binds to GPRC5D. The third antigen-binding arm is linked to the second antigen-binding arm and comprises a VH3 and a VL3 that form a third antigen-binding domain that binds to BCMA.

[0095] In one embodiment, the BCMAxGPRC5DxCD3 multispecific antibody is a trispecific antibody comprising a CD3-specific binding arm comprising an HC1 having a VH1 and an LC having a VL1. The VH1 and VL1 pair to form a first CD3-specific antigen-binding domain that binds to CD3. The second antigen-binding arm comprises a VH2 and a VL2 that form a second antigen-binding domain that binds to BCMA. The third antigen-binding arm comprises a VH3 and a VL3 linked to the second antigen-binding arm and that form a third antigen-binding domain that binds to GPRC5D.

[0096] In one embodiment, the BCMAxGPRC5DxCD3 multispecific antibody is a trispecific antibody comprising a CD3-specific binding arm comprising an HC1 having a VH1 and an LC having a VL1. The VH1 and VL1 pair to form a first CD3-specific antigen-binding domain that binds to CD3. The second antigen-binding arm comprises a VH2 and a VL2 that form a second antigen-binding domain that binds to GPRC5D. The third antigen-binding arm is linked to the first CD3-specific antigen-binding arm and comprises a VH3 and a VL3 that form a third antigen-binding domain that binds BCMA.

[0097] In one embodiment, the BCMAxGPRC5DxCD3 multispecific antibody is a trispecific antibody comprising a CD3-specific binding arm comprising an HC1 having a VH1 and an LC having a VL1. The VH1 and VL1 pair to form a first CD3-specific antigen-binding domain that binds to CD3. The second antigen-binding arm comprises a VH2 and a VL2 that form a second antigen-binding domain that binds to BCMA. The third antigen-binding arm is linked to the first CD3-specific antigen-binding arm and comprises a VH3 and a VL3 that form a third antigen-binding domain that binds to GPRC5D.

[0098] In some embodiments, an HC1 having a VH1 domain and an LC having a VL1 domain of a first antigen-binding arm form an antigen-binding fragment (Fab) comprising the first antigen-binding domain. In some embodiments, a VH2 and VL2 of a second antigen-binding arm form a single-chain variable fragment (scFv) comprising the second antigen-binding domain. In some embodiments, a VH3 and VL3 of a third antigen-binding arm form a single-chain variable fragment (scFv) comprising the third antigen-binding domain.

[0099] In one embodiment, the CD3 binding arm comprises an antigen binding fragment (Fab), the BCMA binding arm comprises a single chain variable fragment (scFv), and the GPRC5D binding arm comprises a single chain variable fragment (scFv).

[0100] In one embodiment, the CD3 binding arm comprises a single chain variable fragment (scFv), the BCMA binding arm comprises an antigen binding fragment (Fab), and the GPRC5D binding arm comprises a single chain variable fragment (scFv).

[0101] In one embodiment, the CD3 binding arm comprises a single chain variable fragment (scFv), the BCMA binding arm comprises a single chain variable fragment (scFv), and the GPRC5D binding arm comprises an antigen binding fragment (Fab).

[0102] In some embodiments, the CD3 binding arm of the trispecific antibody comprises an HC1 and an LC. The HC1 may comprise constant heavy chain regions (CH1, CH2, and CH3) and a VH1. The LC may comprise a VL1. The VH1 and VL1 combine to form the CD3 antigen-binding domain.

[0103] In some embodiments, the GPRC5D-binding arm of the trispecific antibody comprises HC2, which may comprise constant heavy chain regions (CH2 and CH3) and a single-chain variable fragment (scFv) attached to the N-terminus of the CH2 region, wherein the scFv comprises the GPRC5D antigen-binding domain.

[0104] In some embodiments, the trispecific antibody further comprises a BCMA antigen-binding arm that binds to the C-terminus of the CH3 region of the GPRC5D-binding arm to form a GPRC5D / BCMA-binding arm. In some embodiments, the BCMA antigen-binding arm comprises a second single-chain variable fragment (scFv). In some embodiments, the GPRC5D / BCMA arm may have the following structure: an scFv containing the GPRC5D-binding domain, CH2 and CH3 regions, and an scFv containing the BCMA-binding domain.

[0105] In some embodiments, the CD3 binding arm of the trispecific antibody comprises an HC1 and an LC. The HC1 may comprise constant heavy chain regions (CH1, CH2, and CH3) and a VH1. The LC may comprise a VL1. The VH1 and VL1 combine to form the CD3 antigen-binding domain.

[0106] In some embodiments, the BCMA binding arm of the trispecific antibody comprises HC2, which may comprise constant heavy chain regions (CH2 and CH3) and a single chain variable fragment (scFv) attached to the N-terminus of the CH2 region, wherein the scFv comprises a BCMA antigen binding domain.

[0107] In some embodiments, the trispecific antibody further comprises a GPRC5D antigen-binding arm linked to the C-terminus of the CH3 region of the CD3-binding arm to form a CD3 / GPRC5D-binding arm. In some embodiments, the BCMA antigen-binding arm comprises a second single-chain variable fragment (scFv). In some embodiments, the CD3 / GPRC5D arm may have the structure of a Fab containing the CD3-binding domain, CH2 and CH3 regions, and an scFv containing the GPRC5D-binding domain.

[0108] In some embodiments, the multispecific antibodies of the present invention include antibodies having a full-length antibody structure. As used herein, "full-length antibody" refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. A full-length antibody heavy chain (HC) comprises a heavy chain variable domain and constant domains, VH, CH1, CH2, and CH3. A full-length antibody light chain (LC) comprises a light chain variable domain and constant domains, VL and CL. A full-length antibody may lack the C-terminal lysine (K) in either one or both heavy chains. The term "Fab arm" or "half molecule" refers to a heavy-light chain pair that binds to an antigen. In some embodiments, one of the antigen-binding domains is a non-antibody-based binding domain, for example, a fibronectin type 3 domain, e.g., a binding domain based on centyrin.

[0109] BCMA-binding arm The BCMAxGPRC5DxCD3 multispecific antibodies described herein comprise an antigen-binding arm specific for BCMA. In some embodiments, the BCMA-binding arm binds to human BCMA. In some embodiments, the BCMA-binding arm binds to human BCMA and cynomolgus BCMA. In some embodiments, the BCMA-binding arm binds to human BCMA but not cynomolgus BCMA. In some embodiments, the BCMA-binding arm binds to an epitope comprising one or more residues from the BCMA extracellular domain (ECD). In some embodiments, the BCMA-binding arm binds to residues 17-26 of the BCMA BCMW37 chain (LLHACIPCQL (SEQ ID NO: 162)).

[0110] BCMA-binding arms are 5 × 10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M, or 5 x 10 -10 In one embodiment, the BCMA binding arm can bind to BCMA with an affinity of about 1 x 10 M or less. -10 M~1×10 -7 M. In one embodiment, the BCMA-binding arm binds to BCMA with an affinity of about 1 x 10 -10 M, approx. 2 x 10 -10 M, about 3 x 10 -10 M, approx. 4 x 10 -10 M, about 5 x 10 -10 M, about 6 x 10 -10 M, about 7 x 10 -10 M, about 8 x 10 -10 M, about 9 x 10 -10 M, about 1 x 10 -9 M, about 2 x 10 -9 M, about 3 x 10 -9 M, approx. 4 x 10 -9 M, about 5 x 10 -9 M, about 6 x 10 -9 M, about 7 x 10 -9 M, about 8 x 10 -9 M, or approximately 9 x 10 -9 M. In one embodiment, the BCMA-binding arm binds to BCMA with an affinity of about 1 x 10 -10 ~5×10 -10 M, about 1 x 10 -10 ~8×10 -10 M, approx. 2 x 10 -10 ~9×10 -10 M, about 3 x 10 -10 ~10×10 -10 M, approx. 4 x 10 -10 ~10×10 -10 M, or approximately 5 x 10 -10 ~10×10 -10M. In one embodiment, the BCMA-binding arm binds to BCMA with an affinity of about 8.4×10 as determined by surface plasmon resonance (SPR) assay. -10 M. In one embodiment, the BCMA-binding arm binds to BCMA with an affinity of about 2.1 x 10 as determined by surface plasmon resonance (SPR) assay. -10 Binds to BCMA with an affinity of M.

[0111] Table 1 provides a summary of examples of some BCMA-specific antibodies described herein.

[0112] [Table 1]

[0113] Characteristics of some BCMA-specific antibodies or antigen-binding fragments can be found, for example, in U.S. Pat. No. 10,072,088, the contents of which are incorporated herein by reference in their entirety.

[0114] In some embodiments, the BCMA binding arm comprises a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, the BCMA binding arm comprises a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1, and a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, the BCMA binding arm competes for binding to BCMA with an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1, and a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1.

[0115] In some embodiments, the BCMA binding arm comprises a heavy chain that comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, the BCMA binding arm comprises a light chain that comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, the BCMA binding arm comprises a heavy chain that comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1, and a light chain that comprises the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1.

[0116] In some embodiments, the BCMA binding arm comprises the heavy chain CDR1, CDR2, and CDR3 of clone BCMB519, BCMB69, BCMB117, BCMB123, BCMB128, BCMB129, BCMB176, or BCMB177. In some embodiments, the BCMA binding arm comprises the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone BCMB519, BCMB69, BCMB117, BCMB123, BCMB128, BCMB129, BCMB176, or BCMB177. In one embodiment, the BCMA binding arm comprises the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone BCMB519.

[0117] In some embodiments, the BCMA binding arm comprises a heavy chain variable domain derived from an antibody clone listed in Table 1. In some exemplary embodiments, the BCMA binding arm comprises a heavy chain variable domain and a light chain variable domain obtained from an antibody clone as listed in Table 1. In some exemplary embodiments, the BCMA binding arm comprises the heavy chain variable domain and light chain variable domain of clone BCMB519, BCMB69, BCMB117, BCMB123, BCMB128, BCMB129, BCMB176, or BCMB177. In one embodiment, the BCMA binding arm comprises the heavy chain variable domain and light chain variable domain of clone BCMB519.

[0118] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 21, and a heavy chain CDR3 comprising SEQ ID NO: 22. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 21, a heavy chain CDR3 comprising SEQ ID NO: 22, a light chain CDR1 comprising SEQ ID NO: 17, a light chain CDR2 comprising SEQ ID NO: 18, and a light chain CDR3 comprising SEQ ID NO: 19. The BCMA binding arm may comprise human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 24. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 24 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 23.

[0119] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 32, a heavy chain CDR2 comprising SEQ ID NO: 33, and a heavy chain CDR3 comprising SEQ ID NO: 34. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 32, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 34, a light chain CDR1 comprising SEQ ID NO: 39, a light chain CDR2 comprising SEQ ID NO: 40, and a light chain CDR3 comprising SEQ ID NO: 41. The BCMA binding arm may comprise human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 42. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 42 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 48.

[0120] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 38, a heavy chain CDR2 comprising SEQ ID NO: 33, and a heavy chain CDR3 comprising SEQ ID NO: 34. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 38, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 34, a light chain CDR1 comprising SEQ ID NO: 39, a light chain CDR2 comprising SEQ ID NO: 40, and a light chain CDR3 comprising SEQ ID NO: 41. The BCMA binding arm may comprise human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 43. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 43 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 48.

[0121] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 32, a heavy chain CDR2 comprising SEQ ID NO: 33, and a heavy chain CDR3 comprising SEQ ID NO: 37. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 32, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 37, a light chain CDR1 comprising SEQ ID NO: 39, a light chain CDR2 comprising SEQ ID NO: 40, and a light chain CDR3 comprising SEQ ID NO: 41. The BCMA binding arm may comprise human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 44. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 44 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 48.

[0122] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 32, a heavy chain CDR2 comprising SEQ ID NO: 36, and a heavy chain CDR3 comprising SEQ ID NO: 34. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 32, a heavy chain CDR2 comprising SEQ ID NO: 36, a heavy chain CDR3 comprising SEQ ID NO: 34, a light chain CDR1 comprising SEQ ID NO: 39, a light chain CDR2 comprising SEQ ID NO: 40, and a light chain CDR3 comprising SEQ ID NO: 41. The BCMA binding arm comprises human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 45. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 45 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 48.

[0123] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 38, a heavy chain CDR2 comprising SEQ ID NO: 33, and a heavy chain CDR3 comprising SEQ ID NO: 37. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 38, a heavy chain CDR2 comprising SEQ ID NO: 33, a heavy chain CDR3 comprising SEQ ID NO: 37, a light chain CDR1 comprising SEQ ID NO: 39, a light chain CDR2 comprising SEQ ID NO: 40, and a light chain CDR3 comprising SEQ ID NO: 41. The BCMA binding arm may comprise human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 46. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 46 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 48.

[0124] In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 38, a heavy chain CDR2 comprising SEQ ID NO: 164, and a heavy chain CDR3 comprising SEQ ID NO: 37. In some embodiments, the BCMA binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 38, a heavy chain CDR2 comprising SEQ ID NO: 164, a heavy chain CDR3 comprising SEQ ID NO: 37, a light chain CDR1 comprising SEQ ID NO: 39, a light chain CDR2 comprising SEQ ID NO: 40, and a light chain CDR3 comprising SEQ ID NO: 41. The BCMA binding arm may comprise human framework sequences. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 47. In some embodiments, the BCMA binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 47 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 48.

[0125] The BCMA-binding arm can be recombinantly derived from any species. For example, the BCMA-binding arm can be derived from mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in human administration, antigen-binding fragments of non-human origin may be genetically or structurally altered to render them less antigenic when administered to a human patient. In some embodiments, the BCMA-binding arm comprises an antigen-binding fragment that is chimeric.

[0126] In some embodiments, the BCMA-binding arm comprises a humanized antigen-binding fragment. A humanized antigen-binding fragment may be a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody or antigen-binding fragment is a human immunoglobulin (recipient antibody) or antigen-binding fragment in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. Generally, a humanized antibody or antigen-binding fragment will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody antigen-binding fragment can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0127] GPRC5D binding arm The BCMA×GPRC5D×CD3 multispecific antibodies described herein comprise an antigen-binding arm specific for GPRC5D. In some embodiments, the GPRC5D-binding arm binds to GPRC5D. In some embodiments, the GPRC5D-binding arm binds to human GPRC5D and cynomolgus monkey GPRC5D, preferably to the extracellular domain thereof. In some embodiments, the GPRC5D-binding arm binds to human GPRC5D but not to cynomolgus monkey GPRC5D. In some embodiments, the GPRC5D-binding arm binds to one or more residues of a polypeptide having the amino acid sequence of SEQ ID NO: 116.

[0128] Table 2 provides a summary of some examples of GPRC5D-specific antibodies described herein.

[0129] [Table 2]

[0130] Characteristics of some GPRC5D-specific antibodies or antigen-binding fragments can be found, for example, in U.S. Patent No. 10,562,968 and U.S. Patent Application Publication No. 2020 / 0231686, the contents of each of which are incorporated herein by reference in their entirety.

[0131] In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2. In some embodiments, the GPRC5D binding arm comprises a light chain variable domain comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any two of the antibodies listed in Table 2, and a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, the GPRC5D binding arm competes for binding to GPRC5D with an antibody or antigen binding comprising a heavy chain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2, and a light chain comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2.

[0132] In some embodiments, the GPRC5D binding arm comprises a heavy chain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2. In some embodiments, the GPRC5D binding arm comprises a light chain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2. In some embodiments, the GPRC5D binding arm comprises a heavy chain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2, and a light chain comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 2.

[0133] In some embodiments, the GPRC5D-binding arm comprises heavy chain CDR1, CDR2, and CDR3 derived from an antibody clone listed in Table 2. In some embodiments, the GPRC5D-binding arm comprises light chain CDR1, CDR2, and CDR3 derived from an antibody clone listed in Table 2. In some embodiments, the GPRC5D-binding arm comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 derived from an antibody clone listed in Table 2. In some embodiments, the GPRC5D binding arm comprises the heavy chain CDR1, CDR2, and CDR3 of clone GC5B680, GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597. In some embodiments, the GPRC5D binding arm comprises the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone GC5B680, GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597. In one embodiment, the GPRC5D binding arm comprises the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone GC5B680.

[0134] In some exemplary embodiments, the GPRC5D-binding arm comprises a heavy chain variable domain derived from an antibody clone listed in Table 2. In some exemplary embodiments, the GPRC5D-binding arm comprises a heavy chain variable domain and a light chain variable domain obtained from an antibody clone as listed in Table 2. In some exemplary embodiments, the GPRC5D-binding arm comprises the heavy chain variable domain and the light chain variable domain of clone GC5B680, GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597. In one embodiment, the GPRC5D-binding arm comprises the heavy chain variable domain and the light chain variable domain of clone GC5B680. In some embodiments, the GPRC5D-specific antibody clone has an EC 50 can induce ADCC in vitro.

[0135] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 12, a heavy chain CDR2 comprising SEQ ID NO: 13, and a heavy chain CDR3 comprising SEQ ID NO: 14. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 12, a heavy chain CDR2 comprising SEQ ID NO: 13, a heavy chain CDR3 comprising SEQ ID NO: 14, a light chain CDR1 comprising SEQ ID NO: 9, a light chain CDR2 comprising SEQ ID NO: 10, and a light chain CDR3 comprising SEQ ID NO: 11. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 16. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 16 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 15.

[0136] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:49, a heavy chain CDR2 comprising SEQ ID NO:53, and a heavy chain CDR3 comprising SEQ ID NO:57. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:49, a heavy chain CDR2 comprising SEQ ID NO:53, a heavy chain CDR3 comprising SEQ ID NO:57, a light chain CDR1 comprising SEQ ID NO:61, a light chain CDR2 comprising SEQ ID NO:64, and a light chain CDR3 comprising SEQ ID NO:67. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:97. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:97 and a light chain variable domain substantially the same as or identical to SEQ ID NO:101.

[0137] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:50, a heavy chain CDR2 comprising SEQ ID NO:54, and a heavy chain CDR3 comprising SEQ ID NO:58. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:50, a heavy chain CDR2 comprising SEQ ID NO:54, a heavy chain CDR3 comprising SEQ ID NO:58, a light chain CDR1 comprising SEQ ID NO:61, a light chain CDR2 comprising SEQ ID NO:64, and a light chain CDR3 comprising SEQ ID NO:67. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:98. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:98 and a light chain variable domain substantially the same as or identical to SEQ ID NO:101.

[0138] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:51, a heavy chain CDR2 comprising SEQ ID NO:55, and a heavy chain CDR3 comprising SEQ ID NO:59. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:51, a heavy chain CDR2 comprising SEQ ID NO:55, a heavy chain CDR3 comprising SEQ ID NO:59, a light chain CDR1 comprising SEQ ID NO:62, a light chain CDR2 comprising SEQ ID NO:65, and a light chain CDR3 comprising SEQ ID NO:68. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:99. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:99 and a light chain variable domain substantially the same as or identical to SEQ ID NO:102.

[0139] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 52, a heavy chain CDR2 comprising SEQ ID NO: 56, and a heavy chain CDR3 comprising SEQ ID NO: 60. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 52, a heavy chain CDR2 comprising SEQ ID NO: 56, a heavy chain CDR3 comprising SEQ ID NO: 60, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 66, and a light chain CDR3 comprising SEQ ID NO: 69. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 100. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 100 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 103.

[0140] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 70, a heavy chain CDR2 comprising SEQ ID NO: 77, and a heavy chain CDR3 comprising SEQ ID NO: 84. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 70, a heavy chain CDR2 comprising SEQ ID NO: 77, a heavy chain CDR3 comprising SEQ ID NO: 84, a light chain CDR1 comprising SEQ ID NO: 91, a light chain CDR2 comprising SEQ ID NO: 93, and a light chain CDR3 comprising SEQ ID NO: 95. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 104. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 104 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 113.

[0141] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:50, a heavy chain CDR2 comprising SEQ ID NO:78, and a heavy chain CDR3 comprising SEQ ID NO:85. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:50, a heavy chain CDR2 comprising SEQ ID NO:78, a heavy chain CDR3 comprising SEQ ID NO:85, a light chain CDR1 comprising SEQ ID NO:61, a light chain CDR2 comprising SEQ ID NO:64, and a light chain CDR3 comprising SEQ ID NO:67. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:105. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:105 and a light chain variable domain substantially the same as or identical to SEQ ID NO:101.

[0142] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 79, and a heavy chain CDR3 comprising SEQ ID NO: 86. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 79, a heavy chain CDR3 comprising SEQ ID NO: 86, a light chain CDR1 comprising SEQ ID NO: 62, a light chain CDR2 comprising SEQ ID NO: 65, and a light chain CDR3 comprising SEQ ID NO: 68. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 106. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 106 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 102.

[0143] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 79, and a heavy chain CDR3 comprising SEQ ID NO: 59. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 71, a heavy chain CDR2 comprising SEQ ID NO: 79, a heavy chain CDR3 comprising SEQ ID NO: 59, a light chain CDR1 comprising SEQ ID NO: 62, a light chain CDR2 comprising SEQ ID NO: 65, and a light chain CDR3 comprising SEQ ID NO: 68. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 107. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 107 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 102.

[0144] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 72, a heavy chain CDR2 comprising SEQ ID NO: 80, and a heavy chain CDR3 comprising SEQ ID NO: 87. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 72, a heavy chain CDR2 comprising SEQ ID NO: 80, a heavy chain CDR3 comprising SEQ ID NO: 87, a light chain CDR1 comprising SEQ ID NO: 62, a light chain CDR2 comprising SEQ ID NO: 65, and a light chain CDR3 comprising SEQ ID NO: 68. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 108. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 108 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 102.

[0145] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 73, a heavy chain CDR2 comprising SEQ ID NO: 81, and a heavy chain CDR3 comprising SEQ ID NO: 88. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 73, a heavy chain CDR2 comprising SEQ ID NO: 81, a heavy chain CDR3 comprising SEQ ID NO: 88, a light chain CDR1 comprising SEQ ID NO: 91, a light chain CDR2 comprising SEQ ID NO: 93, and a light chain CDR3 comprising SEQ ID NO: 95. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 109. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 109 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 113.

[0146] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 74, a heavy chain CDR2 comprising SEQ ID NO: 82, and a heavy chain CDR3 comprising SEQ ID NO: 60. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 74, a heavy chain CDR2 comprising SEQ ID NO: 82, a heavy chain CDR3 comprising SEQ ID NO: 60, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 66, and a light chain CDR3 comprising SEQ ID NO: 69. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 110. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 110 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 103.

[0147] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 75, a heavy chain CDR2 comprising SEQ ID NO: 80, and a heavy chain CDR3 comprising SEQ ID NO: 89. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 75, a heavy chain CDR2 comprising SEQ ID NO: 80, a heavy chain CDR3 comprising SEQ ID NO: 89, a light chain CDR1 comprising SEQ ID NO: 92, a light chain CDR2 comprising SEQ ID NO: 94, and a light chain CDR3 comprising SEQ ID NO: 96. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 111. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 111 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 114.

[0148] In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 83, and a heavy chain CDR3 comprising SEQ ID NO: 90. In some embodiments, the GPRC5D binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 76, a heavy chain CDR2 comprising SEQ ID NO: 83, a heavy chain CDR3 comprising SEQ ID NO: 90, a light chain CDR1 comprising SEQ ID NO: 63, a light chain CDR2 comprising SEQ ID NO: 66, and a light chain CDR3 comprising SEQ ID NO: 69. The GPRC5D binding arm can comprise human framework sequences. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 112. In some embodiments, the GPRC5D binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 112 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 115.

[0149] The GPRC5D-binding arm can be obtained by recombinant techniques from any species. For example, the GPRC5D-binding arm can be derived from mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, human, or chimeric versions thereof. For use in human administration, antigen-binding fragments of non-human origin may be genetically or structurally altered to be less antigenic when administered to a human patient. In some embodiments, the GPRC5D-binding arm comprises a chimeric antigen-binding fragment.

[0150] In some embodiments, the GPRC5D-binding arm comprises a humanized antigen-binding fragment. The humanized antigen-binding fragment may be a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody or antigen-binding fragment is a human immunoglobulin (recipient antibody) or antigen-binding fragment in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capacity. Generally, a humanized antibody or antigen-binding fragment will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody antigen-binding fragment can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0151] CD3-binding arm The BCMAxGPRC5DxCD3 multispecific antibodies described herein comprise an antigen-binding arm that binds to CD3. In some embodiments, the CD3-binding arm binds to CD3. In some preferred embodiments, the CD3-specific arm of the BCMAxGPRC5DxCD3 multispecific antibody is derived from a CD3-specific antibody that binds to and activates human primary T cells and / or cynomolgus monkey primary T cells. In some embodiments, the CD3-binding arm binds to an epitope at the N-terminus of CD3ε. In some embodiments, the CD3-binding arm binds to residues 22-35 of the CDR3ε chain (QDGNEEMGGITQTP (SEQ ID NO: 161)).

[0152] The CD3-binding arm is 5 × 10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8M or less, 1×10 -8 M or less, 5×10 -9 M or less, or 1 x 10 -9 In some embodiments, the CD3 binding arm can bind to CD3 with an affinity of about 1×10 M or less. -8 M, approx. 2 x 10 -8 M, about 3 x 10 -8 M, approx. 4 x 10 -8 M, about 5 x 10 -8 M, about 6 x 10 -8 M, about 7 x 10 -8 M, about 8 x 10 -8 M, about 9 x 10 -8 M, or approximately 1 x 10 -8 In some embodiments, the CD3 binding arm binds to CD3 with an affinity of about 1 x 10 -8 M ~ approx. 3×10 -8 M, approx. 2 x 10 -8 M ~ approx. 4×10 -8 M, about 1 x 10 -8 M ~ approx. 5×10 -8 M, approx. 2 x 10 -8 M ~ approx. 6×10 -8 M, or approximately 3 x 10 -8 M ~ approx. 8×10 -8 M. In one embodiment, the CD3 binding arm binds to CD3 with an affinity of about 2.5×10 -8 M. In one embodiment, the CD3 binding arm binds to CD3 with an affinity of about 3.1 x 10 -8 It binds to CD3 with an affinity of M. In some embodiments, the CD3 binding affinity is determined by a surface plasmon resonance (SPR) assay.

[0153] Human CD3ε is described in UniProt P07766 (CD3E_HUMAN). An anti-CD3ε antibody reported in the art is SP34 (Yang SJ, The Journal of Immunology (1986) 137:1097-1100). SP34 reacts with both primate and human CD3. SP34 is available from Pharmingen. A further anti-CD3 antibody described in the prior art is UCHT-1 (see WO 2000041474). A further anti-CD3 antibody described in the prior art is BC-3 (Fred Hutchinson Cancer Research Institute; used in Phase I / II trials of GvHD, Anasetti et al., Transplantation 54:844 (1992)). SP34 differs from UCHT-1 and BC-3 in that SP-34 recognizes an epitope present only on the epsilon chain of CD3 (Salmeron et al., (1991) J. Immunol. 147:3047), whereas UCHT-1 and BC-3 recognize epitopes contributed by both the epsilon and gamma chains. Antibody sequences identical to those of antibody SP34 are cited in WO 2008119565, WO 2008119566, WO 2008119567, WO 2010037836, WO 2010037837, and WO 2010037838. A sequence with 96% identity to the VH of antibody SP34 is cited in U.S. Pat. No. 8,236,308 (WO 2007042261).

[0154] In some embodiments, the CD3 binding arm contacts an epitope comprising the six N-terminal amino acids of CD3ε. In some embodiments, the CD3-specific binding arm of the multispecific antibody is derived from the mouse monoclonal antibody SP34, a mouse IgG3 / lambda isotype. In some embodiments, the CD3 binding arm comprises the CDRs of antibody SP34. Such CD3 binding arms may be 5x10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8M or less, 1×10 -8 M or less, 5×10 -9 M or less, or 1 x 10 -9 It can bind to CD3 with an affinity of M or less. The CD3-specific binding arm can be that of a humanized version of the mouse monoclonal antibody SP34. Human framework adaptation (HFA) can be used to humanize the anti-CD3 antibody from which the CD3-specific arm is obtained.

[0155] Table 3 provides a summary of examples of some CD3-specific antibodies described herein.

[0156] [Table 3]

[0157] Characteristics of some CD3-specific antibodies or antigen-binding fragments can be found, for example, in U.S. Pat. Nos. 10,562,968 and 10,072,088, and U.S. Patent Application Publication No. 2019 / 0382481, the contents of each of which are incorporated herein by reference in their entirety.

[0158] In some embodiments, the CD3 binding arm comprises a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm comprises a light chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3, and a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm competes for binding to CD3 with an antibody or antigen-binding fragment comprising a heavy chain variable domain comprising the heavy chain CDR1, CDR2, and CDR3 of any three of the antibodies listed in Table 3, and a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1.

[0159] In some embodiments, the CD3 binding arm comprises a heavy chain that comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm comprises a light chain that comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm comprises a heavy chain that comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3, and a light chain that comprises the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3.

[0160] In some embodiments, the CD3 binding arm comprises the heavy chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm comprises the light chain CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments, the CD3 binding arm comprises a heavy chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3, and a light chain comprising the CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 3. In some embodiments of the multispecific antibody, the CD3 binding arm comprises a heavy and light chain pair selected from Table 3. In some embodiments, the GPRC5D binding arm comprises the heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clone CD3B376. In some embodiments, the GPRC5D binding arm comprises the heavy chain CDR1, CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 of clone CD3B219.

[0161] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:4, a heavy chain CDR2 comprising SEQ ID NO:5, and a heavy chain CDR3 comprising SEQ ID NO:6. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO:4, a heavy chain CDR2 comprising SEQ ID NO:5, a heavy chain CDR3 comprising SEQ ID NO:6, a light chain CDR1 comprising SEQ ID NO:1, a light chain CDR2 comprising SEQ ID NO:2, and a light chain CDR3 comprising SEQ ID NO:3. The CD3 binding arm may comprise human framework sequences. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:8. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO:8 and a light chain variable domain substantially the same as or identical to SEQ ID NO:7. In some embodiments, the CD3 binding arm comprises a heavy chain substantially the same as or identical to SEQ ID NO:26. In some embodiments, the CD3 binding arm comprises a heavy chain substantially the same as or identical to SEQ ID NO:26 and a light chain substantially the same as or identical to SEQ ID NO:27.

[0162] In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 117, a heavy chain CDR2 comprising SEQ ID NO: 118, and a heavy chain CDR3 comprising SEQ ID NO: 119. In some embodiments, the CD3 binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 117, a heavy chain CDR2 comprising SEQ ID NO: 118, a heavy chain CDR3 comprising SEQ ID NO: 119, a light chain CDR1 comprising SEQ ID NO: 123, a light chain CDR2 comprising SEQ ID NO: 124, and a light chain CDR3 comprising SEQ ID NO: 125. The CD3 binding arm may comprise human framework sequences. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 120. In some embodiments, the CD3 binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 120 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 125. In some embodiments, the CD3 binding arm comprises a heavy chain substantially the same as or identical to SEQ ID NO: 121. In some embodiments, the CD3 binding arm comprises a heavy chain substantially the same as or identical to SEQ ID NO:121 and a light chain substantially the same as or identical to SEQ ID NO:126.

[0163] In some embodiments, the heavy and / or light chain CDRs are derived from a known anti-CD3 antibody, such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, and WT-31.

[0164] In some embodiments, the CD3 binding arm is IgG or a derivative thereof. In some embodiments, the CD3 binding arm is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the CD3 binding arm has an IgG4 isotype, the binding arm contains S228P, L234A, L235A, F405L, and R409K substitutions in the Fc region. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary human T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary cynomolgus T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on primary human and cynomolgus T cells. In some embodiments, the antibody or antigen-binding fragment activates primary human CD3+ T cells. In some embodiments, the antibody or antigen-binding fragment activates primary cynomolgus CD4+ T cells.

[0165] In some embodiments, the trispecific antibodies described herein may adopt any format described in the art for trispecific antibodies. In some embodiments, the trispecific antibodies described herein are constructed based on a bispecific antibody format. This can be achieved by adding a third antigen-binding arm to the bispecific antibody. Various formats of bispecific antibodies have been described and are recently reviewed by Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276. In some embodiments, the trispecific antibody is a bispecific antibody, such as a diabody, crossbody, or bispecific antibody obtained by controlled Fab arm exchange, such as those described in the present disclosure.

[0166] In some embodiments, trispecific antibodies include IgG-like molecules with complementary CH3 domains that force heterodimer formation, recombinant IgG-like dual targeting molecules (the two sides of the molecule each comprise an Fab fragment or portion of an Fab fragment of at least two different antibodies); IgG fusion molecules (in which a full-length IgG antibody is fused to an extra Fab fragment or portion of an Fab fragment); Fc fusion molecules (in which a single-chain Fv molecule or stabilized diabody is fused to a heavy chain constant domain, Fc region, or portion thereof); Fab fusion molecules (in which different Fab fragments are fused together); ScFv and diabody-based and heavy chain antibodies (e.g., domain antibodies, nanobodies) (in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule).

[0167] In some embodiments, IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche) and electrostatically tuned (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), DuoBody (Genmab A / S), and other asymmetric variants (e.g., Zymeworks).

[0168] In some embodiments, recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one Antibody (Genentech), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX-body (CovX / Pfizer).

[0169] In some embodiments, IgG fusion molecules include Dual Variable Domain (DVD)-Ig (Abbott), IgG-like Bispecific (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

[0170] In some embodiments, Fc fusion molecules include ScFv / Fc Fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), and Dual(ScFv).sub.2-Fab (National Research Center for Antibody Medicine--China).

[0171] In some embodiments, Fab-fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), Bivalent Bispecific (Biotecnol), and Fab-Fv (UCB-Celltech). ScFv-, diabody-based antibodies and domain antibodies include, but are not limited to, Bispecific T Cell Engager (Bispecific T Cell Engager, BiTE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (Dual Affinity Retargeting Technology, DART) (MacroGenics), Single-chain Diabody (Academic), TCR-like Antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack), and COMBODY (Epigen Biotech), dual targeting nanobody (Ablynx), and dual targeting heavy chain only domain antibody.

[0172] Full-length trispecific antibodies of the present disclosure may be generated, for example, using Fab arm exchange (or half molecule exchange) between two monospecific bivalent antibodies, either in vitro in a cell-free environment or by co-expression, by introducing substitutions in the heavy chain CH3 interface in each half molecule to favor heterodimerization of two antibody half molecules with distinct specificities. The Fab arm exchange reaction is the result of disulfide bond isomerization and dissociation-association of the CH3 domains. Heavy chain disulfide bonds in the hinge region of the monospecific parent antibody are reduced. A free cysteine ​​occurring in one of the monospecific parent antibodies forms an intra-heavy chain disulfide bond with a cysteine ​​residue in the second monospecific parent antibody molecule, while the CH3 domains of the parent antibody open and reform upon dissociation-association. The CH3 domains of the Fab arms may be engineered to favor heterodimerization over homodimerization. The resulting product is a bispecific antibody with two Fab arms or half molecules, each binding to a different epitope, e.g., an epitope on BCMA (or GPRC5D) and an epitope on CD3. A third antigen-binding arm can then be introduced into the bispecific antibody, e.g., at the C-terminus of the first or second heavy chain, capable of binding to a third epitope, e.g., GPRC5D (or BCMA).

[0173] As used herein, "homodimerization" refers to the interaction of two heavy chains with identical CH3 amino acid sequences. As used herein, "homodimer" refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

[0174] As used herein, "heterodimerization" refers to the interaction of two heavy chains with non-identical CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains with non-identical CH3 amino acid sequences.

[0175] A "knob-in-hole" strategy (see, e.g., WO 2006 / 028936) may be used to generate full-length bispecific antibodies. Briefly, selected amino acids that form the interface of the CH3 domain in human IgG are mutated at positions that affect CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into the heavy chain of the antibody that binds to the first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of the antibody that binds to the second antigen. After coexpression of the two antibodies, a heterodimer forms as a result of the preferential interaction of the heavy chain with the "hole" and the heavy chain with the "knob." Exemplary pairs of CH3 substitutions that form knobs and holes are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain).

[0176] In some embodiments of the trispecific antibodies or trispecific binding fragments described herein, one of the Fc domains comprises the mutations T366S, L368A, and Y407V, and the other Fc domain comprises the mutation T366W. In some embodiments, the Fc domain of the first heavy chain portion (HC1) of the first antigen-binding arm (e.g., the CD3-binding arm) comprises the mutations T366S, L368A, and Y407V, and the Fc domain of the second heavy chain portion (HC2) of the second antigen-binding arm and / or third antigen-binding arm (e.g., the GPRC5D / BCMA-binding arm, or the BCMA-binding arm) comprises the mutation T366W. In some embodiments, the Fc domain of HC2 of the second antigen-binding arm and / or the third antigen-binding arm (e.g., the GPRC5D / BCMA-binding arm or the BCMA-binding arm) comprises the mutations T366S, L368A, and Y407V, and the Fc domain of HC1 of the first antigen-binding arm (e.g., the CD3-binding arm) comprises the mutation T366W.

[0177] Other strategies, such as promoting heavy chain heterodimer formation using electrostatic interactions by substituting positively charged residues on one CH3 surface and negatively charged residues on the second CH3 surface, may be used as described in U.S. Patent Application Publication No. 2010 / 0015133, U.S. Patent Application Publication No. 2009 / 0182127, U.S. Patent Application Publication No. 2010 / 028637, or U.S. Patent Application Publication No. 2011 / 0123532. In another strategy, heterodimer formation can be achieved using the following substitutions: L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A, as described in U.S. Patent Application Publication No. 2012 / 0149876 or U.S. Patent Application Publication No. 2013 / 0195849. It may be facilitated by Y407V / T350V_T366L_K392L_T394W (expressed as modification position in the first CH3 domain of the first heavy chain / modification position in the second CH3 domain of the second heavy chain) (Zymeworks).

[0178] In addition to the methods described above, trispecific antibodies of the present invention can also be generated in vitro in a cell-free environment by introducing asymmetric mutations into the CH3 regions of two parent monospecific homodimeric antibodies and forming a trispecific heterodimeric antibody from the two parent monospecific homodimeric antibodies under reducing conditions that allow disulfide bond isomerization, according to the method described in WO 2011 / 131746. In this method, a first monospecific bivalent antibody (e.g., an anti-GPRC5D antibody) and a second monospecific bivalent antibody (e.g., an anti-CD3 antibody) are engineered to have specific substitutions in the CH3 domains that promote heterodimer stability, and these antibodies are incubated together under reducing conditions sufficient to allow cysteines in the hinge regions to isomerize disulfide bonds, thereby generating a trispecific antibody by Fab arm exchange. The incubation conditions can optionally be returned to non-reducing conditions. Exemplary reducing agents that can be used include 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, incubation at a temperature of at least 20°C, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a pH of 5 to 8, e.g., pH 7.0 or 7.4, for at least 90 minutes may be used.

[0179] In some embodiments, the trispecific antibody or antigen-binding fragment is an IgG or a derivative thereof. In humans, the IgG class is divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. These share over 95% homology in the amino acid sequence of the Fc region but show major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcγR) on the surface of immune effector cells, such as natural killer cells and macrophages, resulting in the phagocytosis or lysis of target cells. In CDC, antibodies kill target cells by triggering the complement cascade on the cell surface. The antibodies described herein include antibodies having the described characteristics of the variable domains in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to provide different effector functions.

[0180] For many therapeutic antibody applications, Fc-mediated effector functions are not responsible for the mechanism of action. These Fc-mediated effector functions can be harmful by causing extramechanistic toxicity and pose safety risks. Altered effector function can be achieved by genetically engineering the Fc region to reduce binding to FcγRs or complement factors. Binding of IgGs to activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) is determined by residues located in the hinge region and CH2 domain. Mutations introduced into IgG1, IgG2, and IgG4 reduce or silence Fc function. The antibodies described herein may contain these modifications.

[0181] In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function when compared to the parent Fc; (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb and / or FcγRIIIa; (c) reduced affinity for FcγRI; (d) reduced affinity for FcγRIIa; (e) reduced affinity for FcγRIIb; (f) reduced affinity for FcγRIIIb; or (g) reduced affinity for FcγRIIIa.

[0182] In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the antibody has an IgG1 isotype, and the antibody comprises L234A, L235A, D265S, and / or K409R substitutions in its Fc region. In some embodiments, the antibody has an IgG4 isotype, and the antibody comprises S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may comprise these modifications.

[0183] In some embodiments, the Fc domains of the multispecific antibodies described herein each comprise one or more mutations selected from L234A, L235A, and D265S, hi some embodiments, the Fc domains of HC1 and HC2 each comprise the mutations L234A, L235A, and D265S.

[0184] In some embodiments, the Fc domain of one of the heavy chain portions of the multispecific antibodies described herein further comprises one or more mutations that reduce Fc binding to Protein A. In some embodiments, the Fc domain of one of the heavy chain portions comprises the mutation H435R and / or Y436F. In some embodiments, the Fc domain of HC2 of the second antigen-binding arm and / or the third antigen-binding arm (e.g., the GPRC5D / BCMA-binding arm, or the BCMA-binding arm) comprises the mutation H435R and / or Y436F.

[0185] In various embodiments, the third antigen-binding arm is operably linked to the Fc domain of the first or second antigen-binding arm via a linker. In some embodiments, the linker is a peptide linker and can include any naturally occurring amino acid. Exemplary amino acids that can be included in the linker are Gly, Ser, Pro, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The linker should be of sufficient length to link the third antigen-binding arm and the first or second antigen-binding arm in a manner that allows them to form the correct conformation relative to each other so that they retain the desired activity, such as binding to a third antigen (e.g., BCMA or GPRC5D).

[0186] In some embodiments of the trispecific antibodies described herein, HC1 comprises, from N-terminus to C-terminus, a VH1 of a first antigen-binding arm, a CH1 domain, an Fc domain, a linker, and a third antigen-binding arm.

[0187] In some embodiments of the trispecific antibodies described herein, HC2 comprises, from N-terminus to C-terminus, a second antigen-binding arm, an Fc domain, a linker, and a third antigen-binding arm.

[0188] In various embodiments, the scFvs used in the multispecific antibodies described herein comprise, from N- to C-terminus, a VH, a linker, and a VL (VH-L-VL) or a VL, a linker, and a VH (VL-L-VH). In some embodiments, the scFvs comprise, from N- to C-terminus, a VL, a linker, and a VH (VL-L-VH). In some embodiments, the scFvs comprise, from N- to C-terminus, a VH, a linker, and a VL (VH-L-VL).

[0189] The linker can be about 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to 40 amino acids in length. In some embodiments, the linker is about 10 to 35 amino acids in length. In some embodiments, the linker is about 10 to 30 amino acids in length. In some embodiments, the linker is about 10 to 25 amino acids in length. In some embodiments, the linker is about 10 to 20 amino acids in length. In some embodiments, the linker is about 15 to 20 amino acids in length. In some embodiments, the linker is 6 amino acids in length. In some embodiments, the linker is 7 amino acids in length. In some embodiments, the linker is 8 amino acids in length. In some embodiments, the linker is 9 amino acids in length. In some embodiments, the linker is 10 amino acids in length. In some embodiments, the linker is 11 amino acids in length. In some embodiments, the linker is 12 amino acids in length. In some embodiments, the linker is 13 amino acids in length. In some embodiments, the linker is 14 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 16 amino acids in length. In some embodiments, the linker is 17 amino acids in length. In some embodiments, the linker is 18 amino acids in length. In some embodiments, the linker is 19 amino acids in length. In some embodiments, the linker is 20 amino acids in length. In some embodiments, the linker is 21 amino acids in length. In some embodiments, the linker is 22 amino acids in length. In some embodiments, the linker is 23 amino acids in length. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker is 25 amino acids in length. In some embodiments, the linker is 26 amino acids in length. In some embodiments, the linker is 27 amino acids in length. In some embodiments, the linker is 28 amino acids in length. In some embodiments, the linker is 29 amino acids in length. In some embodiments, the linker is 30 amino acids in length. In some embodiments, the linker is 31 amino acids in length. In some embodiments, the linker is 32 amino acids in length.In some embodiments, the linker is 33 amino acids in length. In some embodiments, the linker is 34 amino acids in length. In some embodiments, the linker is 35 amino acids in length. In some embodiments, the linker is 36 amino acids in length. In some embodiments, the linker is 37 amino acids in length. In some embodiments, the linker is 38 amino acids in length. In some embodiments, the linker is 39 amino acids in length. In some embodiments, the linker is 40 amino acids in length. Exemplary linkers that can be used are GIy-rich linkers, GIy- and Ser-containing linkers, GIy- and Ala-containing linkers, Ala- and Ser-containing linkers, and other flexible linkers.

[0190] Other linker sequences can include portions of an immunoglobulin hinge region, CL, or CH1, derived from an immunoglobulin heavy or light chain isotype. Exemplary linkers that can be used are shown in Table 4. Additional linkers are described, for example, in WO 2019 / 060695.

[0191] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:25.

[0192] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:163.

[0193] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:127.

[0194] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:128.

[0195] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:129.

[0196] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:130.

[0197] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:131.

[0198] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:132.

[0199] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:133.

[0200] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:134.

[0201] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:135.

[0202] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:136.

[0203] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:137.

[0204] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:138.

[0205] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:139.

[0206] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:140.

[0207] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:141.

[0208] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:142.

[0209] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:143.

[0210] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:144.

[0211] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:145.

[0212] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:146.

[0213] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:147.

[0214] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:148.

[0215] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:149.

[0216] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:150.

[0217] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:151.

[0218] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:152.

[0219] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:153.

[0220] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:154.

[0221] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:155.

[0222] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:156.

[0223] In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:157.

[0224] [Table 4]

[0225] In one embodiment, HC1 of the BCMAxGPRC5DxCD3 trispecific antibody comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:26.

[0226] In one embodiment, the LC of the BCMAxGPRC5DxCD3 trispecific antibody comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:27.

[0227] In one embodiment, the GPRC5D / BCMA binding arm of the BCMAxGPRC5DxCD3 trispecific antibody comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:28.

[0228] In one embodiment, the CD3 / GPRC5D linked HC1 of the BCMAxGPRC5DxCD3 trispecific antibody comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:29.

[0229] In one embodiment, the LC of the BCMAxGPRC5DxCD3 trispecific antibody comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:30.

[0230] In one embodiment, the BCMA binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO:31.

[0231] In one embodiment, provided herein is an isolated trispecific antibody or trispecific binding fragment thereof, comprising: a) a CD3 binding arm comprising a heavy chain (HC1) and a light chain (LC); b) a GPRC5D / BCMA binding arm; HC1 comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO:26, LC comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO:27, and the GPRC5D / BCMA binding arm comprises an amino acid sequence that is substantially the same as or identical to SEQ ID NO:28.

[0232] In one embodiment, provided herein is an isolated trispecific antibody or trispecific binding fragment thereof comprising: a) a CD3 binding arm comprising a heavy chain (HC1) and a light chain (LC); b) a GPRC5D / BCMA binding arm; HC1 comprises the amino acid sequence of SEQ ID NO:26, LC comprises the amino acid sequence of SEQ ID NO:27, and the GPRC5D / BCMA binding arm comprises the amino acid sequence of SEQ ID NO:28.

[0233] In one embodiment, provided herein is an isolated trispecific antibody or trispecific binding fragment thereof, comprising: a) a CD3 binding arm comprising a heavy chain (HC1) and a light chain (LC), wherein the HC1 further comprises a GPRC5D binding arm; b) a BCMA-binding arm, The HC1 comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:29, the LC comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:30, and the BCMA binding arm comprises an amino acid sequence substantially the same as or identical to SEQ ID NO:31.

[0234] In one embodiment, provided herein is an isolated trispecific antibody or trispecific binding fragment thereof, comprising: a) a CD3 binding arm comprising a heavy chain (HC1) and a light chain (LC), wherein the HC1 further comprises a GPRC5D binding arm; b) a BCMA-binding arm, The HC1 comprises the amino acid sequence of SEQ ID NO: 29, the LC comprises the amino acid sequence of SEQ ID NO: 30, and the BCMA binding arm comprises the amino acid sequence of SEQ ID NO: 31.

[0235] In one embodiment, the BCMAxGPRC5DxCD3 trispecific antibody is BGCB463.

[0236] In one embodiment, the BCMAxGPRC5DxCD3 trispecific antibody is BGCB491.

[0237] In addition to the described BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments, polynucleotide sequences capable of encoding the described BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments are also provided. Vectors containing the described polynucleotides are also provided, as are cells expressing the BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments. Cells capable of expressing the disclosed vectors are also described. These cells may be mammalian cells (e.g., 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The described antibodies can also be produced by hybridoma cells. The described antibodies can also be recombinantly produced.

[0238] Polynucleotides encoding recombinant antigen-binding proteins are also within the scope of this disclosure. In some embodiments, the described polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be utilized. Leader sequences may include, but are not limited to, a restriction site or a translation initiation site.

[0239] The BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments described herein include variants with one or more amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments. In the context of the present invention, unless otherwise stated, the following notations are used to describe mutations: i) A substitution of an amino acid at a given position is written, for example, as K409R. K409R means a substitution of lysine at position 409 with arginine. ii) For specific variants, specific three-letter or one-letter codes are used to indicate the amino acid residue, including the codes Xaa and X. Thus, a substitution of arginine for lysine at position 409 is designated as K409R, or a substitution of any amino acid residue for lysine at position 409 is designated as K409X. In the case of a deletion of lysine at position 409, this deletion is replaced by K409R. * Those skilled in the art can generate variants having one or more amino acid substitutions, deletions or additions.

[0240] These variants may include (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, e.g., a fusion partner, protein tag, or other chemical moiety, that may confer useful properties to the polypeptide, such as an epitope for an antibody, a polyhistidine sequence, a biotin moiety, etc. The antibodies or antigen-binding fragments described herein may include variants in which an amino acid residue from one species is substituted for the corresponding residue in another species, either at a conserved or non-conserved position. In other embodiments, an amino acid residue at a non-conserved position is substituted with a conservative or non-conserved residue. Techniques for obtaining these variants, including genetic (deletion, mutation, etc.), chemical, and enzymatic techniques, are known to those of skill in the art.

[0241] The BCMA×GPRC5D×CD3 multispecific antibodies or antigen-binding fragments described herein can embody several antibody isotypes, such as IgM, IgD, IgG, IgA, and IgE. In some embodiments, the antibody isotype is IgG1, IgG2, IgG3, or IgG4, preferably IgG1 or IgG4. The specificity of an antibody or antigen-binding fragment thereof is primarily determined by the amino acid sequence and arrangement of its CDRs. Thus, the CDRs of one isotype can be changed to another isotype without changing the antigen specificity. Alternatively, a technology has been established to switch a hybridoma from one antibody isotype to another without changing the antigen specificity (isotype switching). Thus, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

[0242] The BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments described herein have an IC of at least 5.9 nM for APRIL binding. 50 The IC values ​​of the described BCMA×GPRC5D×CD3 multispecific antibodies or antigen-binding fragments can be 50 can be determined by various methods known in the art, such as ELISA-based methods or flow cytometry (FACS). 50 The assay for measuring APRIL has plate-bound BCMA in the presence and absence of a BCMAxGPRC5DxCD3 multispecific antibody or antigen-binding fragment, and various concentrations of APRIL are used. A BCMAxGPRC5DxCD3 multispecific antibody or antigen-binding fragment that blocks APRIL binding to BCMA is one that "blocks APRIL as measured by ELISA."

[0243] Vectors containing the polynucleotides described herein are also provided. The vectors may be expression vectors. Thus, recombinant expression vectors containing sequences encoding a polypeptide of interest are contemplated as being within the scope of the present disclosure. Expression vectors may contain one or more additional sequences, such as, but not limited to, control sequences (e.g., promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a wide range of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0244] Recombinant expression vectors within the scope of this description comprise a nucleic acid fragment of synthetic, genomic, or cDNA origin encoding at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements can include a transcriptional promoter, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control transcription and translation termination. Expression vectors, particularly mammalian expression vectors, can also contain one or more non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (e.g., essential ribosomal binding sites), a polyadenylation site, splice donor and acceptor sites, or a transcription termination sequence. An origin of replication that confers the ability to replicate in a host can also be incorporated.

[0245] Transcriptional and translational control sequences in expression vectors used to transform vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0246] In some embodiments, the coding sequence for the multispecific antibody or antigen-binding fragment is placed under the control of a strong constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the SV40 early and late promoters, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeats (LTRs) of Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In one embodiment, the coding sequence for the BCMAxGPRC5DxCD3 multispecific antibody or antigen-binding fragment thereof is placed under the control of an inducible promoter, e.g., a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, a promoter containing one or more interferon-stimulated response elements (ISREs), e.g., protein kinase R 2',5'-oligoadenylate synthetase, Mx gene, ADAR1, etc.

[0247] The vectors described herein may contain one or more internal ribosome entry sites (IRES). Inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing expression of some proteins. In some embodiments, the vector system will contain one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be closely linked, positioned to provide optimal spacing for expression of the gene product (i.e., by introducing "spacer" nucleotides between ORFs), or otherwise positioned. Regulatory elements, such as IRES motifs, may also be positioned to provide optimal spacing for expression.

[0248] The vector may contain a selection marker known in the art. Selection markers include positive and negative selection markers, such as antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, puromycin resistance gene, blasticidin resistance gene), glutamate synthase gene, HSV-TK or HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence or cloning site encoding the selection marker may be located upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[0249] The vectors described herein can be used to transform a variety of cells with genes encoding the described antibodies or antigen-binding fragments. For example, the vectors can be used to generate BCMAxGPRC5DxCD3 multispecific antibody or antigen-binding fragment-producing cells. Thus, another aspect features host cells transformed with a vector containing a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that specifically binds BCMA, GPRC5D, and CD3, such as the antibodies or antigen-binding fragments described and exemplified herein.

[0250] Numerous techniques for introducing foreign genes into cells are known in the art and can be used to generate recombinant cells for carrying out the methods described herein, according to various embodiments exemplified herein. The technique used must stably introduce the heterologous gene sequence into the host cell so that it is inheritable and expressible by the cell's progeny and does not impair essential growth and physiological functions of the recipient cell. Techniques that can be used include chromosomal transfer methods (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carriers), and viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) (Cline, 29 Pharmac. Ther. 69-92 (1985)). Cells can also be transformed using calcium phosphate precipitation of bacterial protoplasts and polyethylene glycol (PEG)-induced fusion with mammalian cells.

[0251] Cells suitable for use in expressing the BCMAxGPRC5DxCD3 multispecific antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin. These cells include, but are not limited to, NS0, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, and Sp2 / 0 myeloma cells, among others, and the BHK cell line. In addition, antibody expression may be achieved using hybridoma cells. Methods for generating hybridomas are well established in the art.

[0252] Cells transformed with the expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant positive cells are expanded and screened for subclones that exhibit the desired phenotype, e.g., high levels of expression, enhanced growth characteristics, or the ability to produce proteins with desired biochemical properties, e.g., due to protein modifications or altered post-translational modifications. These phenotypes may be due to the inherent properties of a given subclone or to mutations. Mutations may be caused by chemicals, UV light, irradiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.

[0253] Therapeutic compositions and methods of treatment using multispecific antibodies and multispecific antigen-binding fragments thereof The multispecific antibodies discussed above, such as the BCMAxGPRC5DxCD3 trispecific antibody discussed above, are useful for therapy. In particular, multispecific antibodies are useful for treating cancer. Also provided herein is a therapeutic composition for treating a hyperproliferative disorder in a mammal, comprising a therapeutically effective amount of a multispecific antibody or multispecific antigen-binding fragment described herein and a pharmaceutically acceptable carrier. In a preferred embodiment, the multispecific antibody is a BCMAxGPRC5DxCD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMAxGPRC5DxCD3 multispecific antibody or a BCMAxGPRC5DxCD3 multispecific antigen-binding fragment thereof as described herein. In one embodiment, such pharmaceutical compositions are for the treatment of GPRC5D- and / or BCMA-expressing cancers, including (but not limited to) the following: GPRC5D- and / or BCMA-expressing B-cell cancers, such as multiple myeloma (MM), including smoldering multiple myeloma (SMM), and other cancers currently determined to express GPRC5D and / or BCMA. In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a relapsed or refractory form of lymphoma, such as a relapsed or refractory form of multiple myeloma. Particular trispecific antibodies that may be used to treat cancers, e.g., hematological cancers, including certain cancers discussed above, include antibodies BGCB463 and BGCB491.

[0254] In some embodiments, the BCMAxGPRC5DxCD3 trispecific antibody or binding fragment thereof is utilized for the treatment of R / R multiple myeloma.

[0255] In some embodiments, the subject receiving the BCMAxGPRC5DxCD3 trispecific antibody or binding fragment thereof has received previous treatment. For example, the subject may have received one or more therapeutic agents, such as a proteasome inhibitor (PI) (e.g., marizomib (salinosporamide A), carfilzomib, ixazomib), an immunomodulatory drug (IMiD), a bispecific agent, a CAR-T therapy, and / or an anti-CD38 antibody, to treat multiple myeloma.

[0256] Pharmaceutical compositions provided herein comprise a) an effective amount of a multispecific antibody or antibody fragment of the invention and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. In a preferred embodiment, the multispecific antibody is a BCMAxGPRC5DxCD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMAxGPRC5DxCD3 multispecific antibody or a BCMAxGPRC5DxCD3 multispecific antigen-binding fragment thereof as described herein. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, and the like that are physiologically compatible. Examples of suitable carriers, diluents, and / or excipients include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof. In many cases, it is preferable to include an isotonic agent, such as a sugar, polyalcohol, or sodium chloride, in the composition. Particularly relevant examples of suitable carriers include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without about 1 mg / mL to 25 mg / mL human serum albumin; (2) 0.9% saline (0.9% weight / volume sodium chloride (NaCl)); and (3) 5% (weight / volume) dextrose, which may also contain an antioxidant, e.g., tryptamine, and a stabilizer, e.g., Tween 20®.

[0257] The compositions herein may also contain additional therapeutic agents, if necessary for the particular disease being treated. Preferably, the multispecific antibody or antibody fragment and the auxiliary active compound have complementary activities that do not adversely affect each other. In some embodiments, the additional therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin-2. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof. In some embodiments, the additional therapeutic agent is an anti-CD38 agent, such as an anti-CD38 antibody (e.g., daratumumab). In some embodiments, the additional therapeutic agent is an immunomodulatory imide drug (IMiD), such as lenalidomide and pomalidomide. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, such as anti-PD-1 and anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT). In some embodiments, the additional therapeutic agent is an immune co-stimulatory agent, such as an agent that targets CD137 (e.g., a CD137 co-stimulatory bispecific antibody). In some embodiments, the additional therapeutic agent is a T cell enhancer, such as the addition of IL-2.

[0258] The compositions of the present invention may be in various forms. Such forms include, for example, liquid, semi-solid, and solid dosage forms, with the preferred form depending on the intended mode of administration and therapeutic application. Typically, preferred compositions are in the form of an injectable or infusible solution. A preferred mode of administration is parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the present invention are administered intravenously by bolus or continuous infusion over a period of time. In another preferred embodiment, the compositions are injected intramuscularly, subcutaneously, intra-articularly, intrasynovially, intratumorally, peritumorally, intralesionally, or perilesionally to exert local and systemic therapeutic effects.

[0259] Sterile compositions for parenteral administration can be prepared by incorporating the antibody, antibody fragment, or antibody conjugate of the present invention in the required amount in an appropriate solvent, followed by sterilization by microfiltration. As solvents or excipients, water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof can be used. In many cases, it is preferable to include an isotonic agent, such as a sugar, polyalcohol, or sodium chloride, in the composition. These compositions may also contain auxiliary agents, particularly wetting agents, isotonicity adjusting agents, emulsifying agents, dispersing agents, and stabilizing agents. Sterile compositions for parenteral administration can also be prepared in the form of sterile solid compositions that can be dissolved in sterile water or any other injectable sterile medium at the time of use.

[0260] Multispecific antibodies or antibody fragments may also be administered orally. Solid compositions for oral administration may be used in the form of tablets, pills, powders (gelatin capsules, sachets), or granules. In these compositions, the active ingredient according to the present invention is mixed with one or more inert diluents, such as starch, cellulose, sucrose, lactose, or silica, under an argon stream. These compositions may also contain substances other than diluents, such as one or more lubricants, such as magnesium stearate or talc, colorants, coatings (sugar-coated tablets), or glazes.

[0261] As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups, and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils, or paraffin oils may be used. These compositions may contain substances other than the diluents, such as wetting agents, sweeteners, thickeners, flavoring agents, or stabilizing products.

[0262] The dosage depends on the desired effect, the duration of treatment, and the route of administration used. The dosage is generally 5 mg to 1000 mg per day for adults orally, and the unit dosage ranges from 1 mg to 250 mg of active substance. Generally, the doctor will determine the appropriate dosage depending on the age, weight, and any other factors specific to the subject being treated.

[0263] Also provided herein is a method for killing GPRC5D and / or BCMA+ cells by administering to a patient in need thereof a multispecific antibody capable of binding to such GPRC5D and / or BCMA and recruiting T cells to kill such GPRC5D and / or BCMA+ cells (i.e., redirecting the T cells). Any of the multispecific antibodies or antibody fragments of the present invention may be used therapeutically. For example, in one embodiment, a BCMAxGPRC5DxCD3 multispecific antibody may be used therapeutically to treat cancer in a subject.

[0264] In a preferred embodiment, the multispecific antibody or antibody fragment of the present invention is used to treat a hyperproliferative disorder in a mammal. In a more preferred embodiment, one of the pharmaceutical compositions disclosed above containing the multispecific antibody or antibody fragment of the present invention is used to treat a hyperproliferative disorder in a mammal. In one embodiment, the disorder is cancer. In particular, the cancer is a GPRC5D- and / or BCMA-expressing cancer, including (but not limited to) a GPRC5D- and / or BCMA-expressing B-cell cancer, such as multiple myeloma (MM), including smoldering multiple myeloma (SMM), and other cancers currently determined to express GPRC5D and / or BCMA. In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a relapsed or refractory form of lymphoma, such as a relapsed or refractory form of multiple myeloma. In a preferred embodiment, the multispecific antibody is a BCMAxGPRC5DxCD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMAxGPRC5DxCD3 multispecific antibody or a BCMAxGPRC5DxCD3 multispecific antigen-binding fragment thereof as described herein.

[0265] Thus, the pharmaceutical compositions of the invention are useful for treating or preventing a variety of cancers or disorders, including, but not limited to, GPRC5D- and / or BCMA-expressing cancers, including GPRC5D- and / or BCMA-expressing B / plasma cell cancers, such as acute multiple myeloma (MM) or precancerous myelomas, such as MGUS (monoclonal gammopathy of undetermined significance) and SMM (smoldering multiple myeloma), and plasmacytoma; and other cancers that have not yet been determined to express GPRC5D and / or BCMA. In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a relapsed or refractory form of lymphoma, for example, a relapsed or refractory form of multiple myeloma.

[0266] Similarly, further provided herein is a method for inhibiting the growth of a selected cell population, comprising contacting GPRC5D- and / or BCMA-expressing target cells or tissues containing such target cells with an effective amount of a multispecific antibody or antibody fragment of the invention, either alone or in combination with other cytotoxic or therapeutic agents, in the presence of peripheral blood mononuclear cells (PBMCs). BCMAxGPRC5DxCD3, which blocks the binding of ligands (APIL, BAFF, and others) to BCMA and GPRC5D, can block BCMA- and GPRC5D-mediated signaling, resulting in target cell inhibition or cell death. In a preferred embodiment, the multispecific antibody is a BCMAxGPRC5DxCD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMAxGPRC5DxCD3 multispecific antibody or a BCMAxGPRC5DxCD3 multispecific antigen-binding fragment thereof as described herein.

[0267] In some embodiments, the methods described herein, comprising administering a multispecific antibody or a pharmaceutical composition comprising the same, further comprise administering another therapeutic agent. Suitable other therapeutic agents include, but are not limited to, an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune costimulator, a gamma secretase inhibitor, a T cell enhancer (e.g., IL-2 addition), tocilizumab, or any combination thereof. In some embodiments, the additional therapeutic agent is an anti-CD38 agent, such as an anti-CD38 antibody (e.g., daratumumab). In some embodiments, the additional therapeutic agent is an immunomodulatory imide drug (IMiD), such as lenalidomide and pomalidomide. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, such as anti-PD-1 and anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT). In some embodiments, the additional therapeutic agent is an immune costimulator, such as an agent targeting CD137 (e.g., a CD137 costimulatory bispecific antibody). The use of low-affinity CD137 binders with conditional agonism in the presence of T cell redirecting antibodies, such as the BCMAxGPRC5DxCD3 trispecific antibody described herein, may enhance anti-tumor activity and improve T cell persistence. In some embodiments, the additional therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin-2. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent. The methods for inhibiting the growth of selected cell populations can be performed in vitro, in vivo, or ex vivo.

[0268] Examples of in vitro uses include the treatment of autologous bone marrow prior to transplant into the same patient to kill diseased or malignant cells, and graft-versus-host disease (GVHD), the treatment of cell cultures to kill all cells except for desired variants that do not express the target antigen, or to kill variants expressing unwanted antigens. Non-clinical in vitro use conditions are readily determined by one of skill in the art.

[0269] An example of a clinical ex vivo use is the removal of tumor cells from bone marrow prior to autologous transplantation in cancer treatment. Treatment can be performed as follows: Bone marrow is harvested from a patient or other individual and then incubated in serum-containing medium supplemented with a cytotoxic agent of the present invention. The concentration ranges from about 10 μM to 1 μM at 37°C for about 30 minutes to about 48 hours. The exact conditions of concentration and time of incubation, i.e., dosage, can be easily determined by those skilled in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned to the patient via intravenous injection according to known methods. In situations where the patient is undergoing other treatments, such as a course of myeloablative chemotherapy or total body irradiation, the treated bone marrow cells are cryopreserved in liquid nitrogen between the time of bone marrow collection and the time of reinfusion of the treated cells using standard medical equipment.

[0270] For clinical in vivo use, a therapeutically effective amount of a multispecific antibody or antigen-binding fragment is administered to a subject in need of such an antibody or fragment. For example, BCMA×GPRC5D×CD3 multispecific antibodies and multispecific antigen-binding fragments thereof may be useful for treating GPRC5D- and / or BCMA-expressing cancers in subjects in need of such treatment. In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a B-cell cancer, e.g., multiple myeloma (MM), including smoldering multiple myeloma (SMM). In some embodiments, the GPRC5D- and / or BCMA-expressing cancer is a relapsed or refractory form of lymphoma, e.g., a relapsed or refractory form of multiple myeloma. In a preferred embodiment, the multispecific antibody is a BCMA×GPRC5D×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMA×GPRC5D×CD3 multispecific antibody or a BCMA×GPRC5D×CD3 multispecific antigen-binding fragment thereof as described herein. In some embodiments, the subject is a mammal, preferably a human. In some embodiments, the multispecific antibody or antigen-binding fragment is administered as a sterile-tested solution.

[0271] Dosage regimens in the above methods of treatment and use are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0272] Effective administration and dosing regimens for the multispecific antibodies and fragments depend on the disease or condition being treated and may be determined by one of skill in the art. An exemplary, non-limiting range for a therapeutically effective amount of a compound of the invention is about 0.001-10 mg / kg, e.g., about 0.001-5 mg / kg, e.g., about 0.001-2 mg / kg, e.g., about 0.001-1 mg / kg, e.g., about 0.001, about 0.01, about 0.1, about 1, or about 10 mg / kg.

[0273] A physician, pharmacist, or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start dosages of the multispecific antibodies or fragments utilized in the pharmaceutical composition at levels lower than required to achieve the desired therapeutic effect, and gradually increase such dosage until the desired effect is achieved. Generally, a suitable daily dose of the multispecific antibodies of the invention will be that amount of compound that is the lowest dose effective to produce a therapeutic effect. Administration may be, for example, parenteral, e.g., intravenous, intramuscular, intratumoral (e.g., bone marrow), or subcutaneous. In one embodiment, the multispecific antibodies or fragments are administered in a dose of at least 1 mg / m 2The multispecific antibody or fragment may be administered by infusion in weekly doses calculated as follows: Dose (mg / kg) x Body Weight (e.g., 50-100 kg). Such doses can be based on the mg / kg dose provided above, for example, according to the following: Dose (mg / kg) x Body Weight (e.g., 50-100 kg). Such administration may be repeated, for example, 1-8 times, for example, 3-5 times. Administration may be by continuous infusion over a period of 2-24 hours, for example, 2-12 hours. In one embodiment, the multispecific antibody or fragment may be administered by slow continuous infusion over an extended period, for example, more than 24 hours, to reduce toxic side effects.

[0274] In one embodiment, when given once a week, the multispecific antibody or fragment may be administered in weekly administrations calculated as up to eight, e.g., four to six, fixed doses. Such a regimen may be repeated one or more times as needed, e.g., after six or twelve months. Such fixed doses may be based, for example, on the mg / kg doses provided above, assuming a body weight of 50 to 100 kg. Doses may be determined or adjusted, for example, by collecting biological samples and measuring the blood levels of the bispecific antibody of the invention at the time of administration, using anti-idiotypic antibodies targeting the GPRC5D and / or BCMA antigen-binding arms of the multispecific antibody of the invention.

[0275] In one embodiment, the multispecific antibody or fragment may be administered as a maintenance treatment, such as once weekly for a period of six months or more.

[0276] The multispecific antibodies or fragments may also be administered prophylactically to reduce the risk of cancer progression, delay the onset of events in cancer progression, and / or reduce the risk of recurrence when the cancer goes into remission.

[0277] The multispecific antibodies and fragments thereof described herein may also be administered in combination therapy, i.e., combined with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, the antibody-containing medicament is intended for combination with one or more additional therapeutic agents, such as a chemotherapeutic agent, an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer (e.g., IL-2 addition), or any combination thereof. In some embodiments, the additional therapeutic agent is an anti-CD38 agent, such as an anti-CD38 antibody (e.g., daratumumab). In some embodiments, the additional therapeutic agent is an immunomodulatory imide drug (IMiD), such as lenalidomide and pomalidomide. In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor, such as anti-PD-1 and anti-T cell immunoreceptor with Ig and ITIM domains (TIGIT). In some embodiments, the additional therapeutic agent is an immune co-stimulatory agent, such as an agent that targets CD137 (e.g., a CD137 co-stimulatory bispecific antibody). In some embodiments, the other therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin-2. Such combined administration may be simultaneous, in any order, separately, or sequentially. For simultaneous administration, the agents may be administered as one composition or as separate compositions, as appropriate.

[0278] In one embodiment, a method is provided for treating a disorder involving cells expressing GPRC5D and / or BCMA in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody or fragment described herein, e.g., a BCMAxGPRC5DxCD3 multispecific antibody, and radiation therapy. In one embodiment, a method is provided for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a multispecific antibody or fragment described herein, e.g., a BCMAxGPRC5DxCD3 antibody, and radiation therapy. Radiation therapy may include the associated administration of radiation or a radiopharmaceutical to the patient. The source of radiation may be either external or internal to the patient being treated (radiation therapy may be in the form of, for example, external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used to practice such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, actinium-225, and indium-111.

[0279] kit Also provided herein are kits that include, for example, a described multispecific antibody or antigen-binding fragment thereof and instructions for using the antibody or fragment to kill a particular cell type. In a preferred embodiment, the multispecific antibody is a BCMAxGPRC5DxCD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMAxGPRC5DxCD3 multispecific antibody or a BCMAxGPRC5DxCD3 multispecific antigen-binding fragment thereof as described herein. The instructions may include directions for using the multispecific antibody or antigen-binding fragment thereof in vitro, in vivo, or ex vivo.

[0280] Typically, the kit has a compartment containing the multispecific antibody or antigen-binding fragment thereof. The multispecific antibody or antigen-binding fragment thereof may be in lyophilized, liquid, or other form amenable to inclusion in the kit. The kit may also contain additional components required to perform the methods described in the kit's instructions, such as sterile solutions for reconstituting the lyophilized powder, additional agents for combining with the multispecific antibody or antigen-binding fragment thereof prior to administration to a patient, and tools to assist in administering the multispecific antibody or antigen-binding fragment thereof to a patient.

[0281] Diagnostic Use The multispecific antibodies and fragments described herein may also be used for diagnostic purposes. For this reason, diagnostic compositions comprising the multispecific antibodies or fragments as defined herein and uses thereof are also provided. In a preferred embodiment, the multispecific antibody is a BCMA×GPRC5D×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a BCMA×GPRC5D×CD3 multispecific antibody or a BCMA×GPRC5D×CD3 multispecific antigen-binding fragment thereof as described herein. In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing a BCMA×GPRC5D×CD3 trispecific antibody and one or more reagents for detecting binding of the antibody to GPRC5D and / or BCMA. The reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for an enzymatic reaction, in which case the enzymatic reaction produces a product that can be visualized. For example, the multispecific antibodies or antigen-binding fragments thereof described herein can be labeled with a radiolabel, a fluorescent label, an epitope tag, biotin, a chromophore label, an ECL label, an enzyme, ruthenium, 111 In-DOTA, 111They may be labeled with In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and β-galactosidase, or polyhistidine, or similar such labels known in the art.

[0282] BCMA specific antibody Described herein are isolated antibodies and antigen-binding fragments specific for BCMA. In some embodiments, the BCMA-specific antibodies and antigen-binding fragments bind to human BCMA. The overall structure of a BCMA-specific antibody molecule can include an antigen-binding domain, which includes a heavy and light chain and an Fc domain, and which performs various functions, including complement fixation and binding antibody receptors.

[0283] In some embodiments, a BCMA-specific antibody or antigen-binding fragment thereof is provided, comprising a heavy chain (e.g., BCMB519) comprising CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1. In some embodiments, a BCMA-specific antibody or antigen-binding fragment thereof is provided, comprising a heavy chain (e.g., BCMB519) comprising CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1, and a light chain (e.g., BCMB519) comprising CDR1, CDR2, and CDR3 of any one of the antibodies listed in Table 1.

[0284] In some embodiments, BCMA-specific antibodies and antigen-binding fragments thereof comprise a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 21, and a heavy chain CDR3 comprising SEQ ID NO: 22. In some embodiments, the BCMA-binding arm comprises a heavy chain CDR1 comprising SEQ ID NO: 20, a heavy chain CDR2 comprising SEQ ID NO: 21, a heavy chain CDR3 comprising SEQ ID NO: 22, a light chain CDR1 comprising SEQ ID NO: 17, a light chain CDR2 comprising SEQ ID NO: 18, and a light chain CDR3 comprising SEQ ID NO: 19. The BCMA-binding arm may comprise human framework sequences. In some embodiments, the BCMA-binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 24. In some embodiments, the BCMA-binding arm comprises a heavy chain variable domain substantially the same as or identical to SEQ ID NO: 24 and a light chain variable domain substantially the same as or identical to SEQ ID NO: 23. The heavy and light chain variable domains of the antibodies discussed in this paragraph are suitable for inclusion in multispecific (bispecific or trispecific) constructs, in which one arm is an anti-BCMA arm. Exemplary trispecific constructs comprising the BCMA-specific antibodies or antigen-binding fragments thereof discussed in this paragraph are provided herein.

[0285] In some embodiments, BCMA-specific antibodies and antigen-binding fragments bind to human BCMA and cynomolgus BCMA. In some embodiments, BCMA-specific antibodies and antigen-binding fragments bind to human BCMA but not cynomolgus BCMA. In some embodiments, BCMA-specific antibodies and antigen-binding fragments bind to an epitope comprising one or more residues from the BCMA extracellular domain (ECD). In some embodiments, BCMA-specific antibodies and antigen-binding fragments bind to residues 17-26 of the BCMA BCMW37 chain (LLHACIPCQL (SEQ ID NO: 162)). Such BCMA-specific antibodies or antigen-binding fragments may be used in combination with BCMA-specific antibodies or antigen-binding fragments in a concentration of 5×10 -7 M or less, e.g., 1×10 -7 M or less, 5×10 -8 M or less, 1×10 -8 M or less, 5×10 -9 M or less, 1×10 -9 M, or 5 x 10-10 In one embodiment, the BCMA-specific antibody or antigen-binding fragment may bind to BCMA with an affinity of about 1×10 M or less. -10 M~1×10 -9 M. In one embodiment, the BCMA-binding arm binds to BCMA with an affinity of about 1 x 10 -10 M, approx. 2 x 10 -10 M, about 3 x 10 -10 M, approx. 4 x 10 -10 M, about 5 x 10 -10 M, about 6 x 10 -10 M, about 7 x 10 -10 M, about 8 x 10 -10 M, about 9 x 10 -10 M or approximately 1 x 10 -9 M. In one embodiment, the BCMA-specific antibody or antigen-binding fragment binds to BCMA with an affinity of about 8.4 x 10 as determined by surface plasmon resonance (SPR) assay. -10 M. In one embodiment, the BCMA-specific antibody or antigen-binding fragment binds to BCMA with an affinity of about 2.1 x 10 as determined by surface plasmon resonance (SPR) assay. -10 Binds to BCMA with an affinity of M.

[0286] In humans, the IgG class is divided into four isotypes: IgG1, IgG2, IgG3, and IgG4. These share greater than 95% homology in the amino acid sequence of the Fc region but exhibit major differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcγR) on the surface of immune effector cells, such as natural killer cells and macrophages, resulting in the phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering the complement cascade on the cell surface. The antibodies described herein include antibodies having the described characteristics of the variable domain in combination with any of the IgG isotypes, including modified versions in which the Fc sequence has been modified to confer different effector functions.

[0287] For many therapeutic antibody applications, Fc-mediated effector functions are not responsible for the mechanism of action. These Fc-mediated effector functions can be harmful by causing extramechanistic toxicity and pose safety risks. Altered effector function can be achieved by genetically engineering the Fc region to reduce binding to FcγRs or complement factors. Binding of IgGs to activating (FcγRI, FcγRIIa, FcγRIIIa, and FcγRIIIb) and inhibitory (FcγRIIb) FcγRs or the first component of complement (C1q) is determined by residues located in the hinge region and CH2 domain. Mutations introduced into IgG1, IgG2, and IgG4 reduce or silence Fc function. The antibodies described herein may contain these modifications.

[0288] In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function when compared to the parent Fc; (b) reduced affinity for FcγRI, FcγRIIa, FcγRIIb, FcγRIIIb and / or FcγRIIIa; (c) reduced affinity for FcγRI; (d) reduced affinity for FcγRIIa; (e) reduced affinity for FcγRIIb; (f) reduced affinity for FcγRIIIb; or (g) reduced affinity for FcγRIIIa.

[0289] In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, where the antibody has an IgG1 isotype, the antibody comprises L234A, L235A, D265S, and / or K409R substitutions in its Fc region. In some embodiments, where the antibody has an IgG4 isotype, the antibody comprises K409R, S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may comprise these modifications.

[0290] In some embodiments, the described antibodies have low nanomolar IC as measured by ELISA. 50 In some embodiments, the described antibodies have low micromolar IC50 concentrations as measured by ELISA. 50 It may be possible to inhibit BAFF binding with

[0291] In some embodiments, the described antibodies bind to BCMA-positive multiple myeloma cell lines.

[0292] In addition to the described BCMA-specific antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments are also provided. Vectors containing the described polynucleotides are also provided. Similarly, cells expressing the BCMA-specific antibodies and antigen-binding fragments provided herein are provided. Also described are cells capable of expressing the disclosed vectors. These cells can be mammalian cells (e.g., 293 cells, 293F cells, CHO cells), insect cells (e.g., Sf7 cells), yeast cells, plant cells, or bacterial cells (e.g., E. coli). The described antibodies can also be produced by hybridoma cells.

[0293] The BCMA-specific antibodies or antigen-binding fragments described include all isotypes: IgA, IgD, IgE, IgG, and IgM, as well as synthetic multimers of four-chain immunoglobulin structures. The described antibodies or antigen-binding fragments also include the IgY isotype, which is commonly found in hen or turkey serum and hen or turkey egg yolk.

[0294] BCMA-specific antibodies and antigen-binding fragments can be recombinantly derived from any species. For example, the antibodies or antigen-binding fragments can be mouse, rat, goat, horse, pig, cow, chicken, rabbit, camel, donkey, llama, human, or chimeric versions thereof. For use in human administration, antibodies or antigen-binding fragments of non-human origin can be genetically or structurally altered to make them less antigenic when administered to a human patient.

[0295] In some embodiments, the antibody or antigen-binding fragment is chimeric. As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment thereof that has at least some portion of at least one variable domain derived from a non-human mammalian, rodent, or reptilian antibody amino acid sequence, while the remainder is of human origin.

[0296] In some embodiments, the antibody is a humanized antibody. A humanized antibody may be a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequence of an antibody) that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin sequence. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0297] The antibodies or antigen-binding fragments described herein may exist in various forms, but comprise one or more of the antibody CDRs shown in Table 1 (eg, BCMB519).

[0298] Described herein are recombinant antibodies and antigen-binding fragments that bind to BCMA. In some embodiments, the BCMA-specific antibody or antigen-binding fragment is human IgG or a derivative thereof. The BCMA-specific antibodies or antigen-binding fragments exemplified herein are human, although the exemplified antibodies or antigen-binding fragments may be chimerized.

[0299] In some embodiments, the antibody or antigen-binding fragment is an IgG or derivative thereof, such as an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, where the antibody is an IgG1 isotype, the antibody comprises an IgG1 Fc region (SEQ ID NO: 158).

[0300] SEQ ID NO: 158 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0301] In some embodiments, where the antibody is an IgG1 isotype, the antibody contains L234A, L235A, and D265S substitutions in the Fc region (SEQ ID NO: 159).

[0302] SEQ ID NO: 159

[0303] [Table 5]

[0304] In some embodiments, where the antibody is an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in the Fc region (SEQ ID NO: 160).

[0305] SEQ ID NO: 160

[0306] [Table 6]

[0307] BCMA-specific antibodies defined by the CDR and / or variable domain sequences discussed in the above paragraphs may comprise these IgG Fc regions.

[0308] Also disclosed are isolated, synthetic polynucleotides encoding antibodies or antigen-binding fragments that bind BCMA. The isolated polynucleotides capable of encoding the variable domain segments provided herein may be included in the same or different vectors that produce the antibodies or antigen-binding fragments.

[0309] Polynucleotides encoding recombinant antigen-binding proteins are also within the scope of this disclosure. In some embodiments, the described polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be utilized. Leader sequences may include, but are not limited to, a restriction site or a translation initiation site.

[0310] The BCMA-specific antibodies or antigen-binding fragments described herein include variants with one or more amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the described BCMA-specific antibodies or antigen-binding fragments. In the context of the present invention, unless otherwise stated, the following notations are used to describe mutations: i) A substitution of an amino acid at a given position is written, for example, as K409R. K409R means a substitution of lysine at position 409 with arginine. ii) For specific variants, specific three-letter or one-letter codes are used to indicate the amino acid residue, including the codes Xaa and X. Thus, a substitution of arginine for lysine at position 409 is designated as K409R, or a substitution of any amino acid residue for lysine at position 409 is designated as K409X. In the case of a deletion of lysine at position 409, this deletion is designated as K409R. * Those skilled in the art can generate variants having one or more amino acid substitutions, deletions or additions.

[0311] These variants may include (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, e.g., a fusion partner, protein tag, or other chemical moiety, that may confer useful properties to the polypeptide, such as an epitope for an antibody, a polyhistidine sequence, a biotin moiety, etc. The antibodies or antigen-binding fragments described herein may include variants in which an amino acid residue from one species is substituted for the corresponding residue in another species, either at a conserved or non-conserved position. In other embodiments, an amino acid residue at a non-conserved position is substituted with a conservative or non-conserved residue. Techniques for obtaining these variants, including genetic (deletion, mutation, etc.), chemical, and enzymatic techniques, are known to those of skill in the art.

[0312] The BCMA-specific antibodies or antigen-binding fragments described herein can embody multiple antibody isotypes, such as IgM, IgD, IgG, IgA, and IgE. In some embodiments, the antibody isotype is IgG1, IgG2, IgG3, or IgG4, preferably IgG1 or IgG4. The specificity of an antibody or antigen-binding fragment thereof is primarily determined by the amino acid sequence and arrangement of its CDRs. Thus, the CDRs of one isotype can be changed to another isotype without changing the antigen specificity. Alternatively, a technology has been established to switch a hybridoma from one antibody isotype to another without changing the antigen specificity (isotype switching). Thus, such antibody isotypes are within the scope of the described antibodies or antigen-binding fragments.

[0313] The BCMA-specific antibodies or antigen-binding fragments described herein exhibit low nanomolar IC for APRIL binding. 50The IC values ​​of the described BCMA-specific antibodies or antigen-binding fragments can be 50 can be determined by various methods known in the art, such as ELISA-based methods or flow cytometry (FACS). 50 The assay for measuring APRIL has plate-bound BCMA in the presence and absence of a BCMA-specific antibody and various concentrations of APRIL are used. A BCMA antibody that blocks APRIL binding to BCMA is said to "block APRIL as measured by ELISA."

[0314] Vectors containing the polynucleotides described herein are also provided. The vectors may be expression vectors. Thus, recombinant expression vectors containing sequences encoding a polypeptide of interest are contemplated as being within the scope of the present disclosure. Expression vectors may contain one or more additional sequences, such as, but not limited to, control sequences (e.g., promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a wide range of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0315] Recombinant expression vectors within the scope of this description comprise a nucleic acid fragment of synthetic, genomic, or cDNA origin encoding at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements can include a transcriptional promoter, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control transcription and translation termination. Expression vectors, particularly mammalian expression vectors, can also contain one or more non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (e.g., essential ribosomal binding sites), a polyadenylation site, splice donor and acceptor sites, or a transcription termination sequence. An origin of replication that confers the ability to replicate in a host can also be incorporated.

[0316] Transcriptional and translational control sequences in expression vectors used to transform vertebrate cells can be provided by viral sources. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0317] In some embodiments, the coding sequence for the antibody or antigen-binding fragment is placed under the control of a strong constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, etc. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter, the SV40 early and late promoters, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeats (LTRs) of Moloney leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. In one embodiment, the coding sequence for the BCMA-specific antibody or antigen-binding fragment thereof is placed under the control of an inducible promoter, e.g., a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, a promoter containing one or more interferon-stimulated response elements (ISREs), e.g., protein kinase R 2',5'-oligoadenylate synthetase, Mx gene, ADAR1, etc.

[0318] The vectors described herein may contain one or more internal ribosome entry sites (IRES). Inclusion of an IRES sequence in a fusion vector may be beneficial for enhancing expression of some proteins. In some embodiments, the vector system will contain one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. The components of the vector may be closely linked, positioned to provide optimal spacing for expression of the gene product (i.e., by introducing "spacer" nucleotides between ORFs), or otherwise positioned. Regulatory elements, such as IRES motifs, may also be positioned to provide optimal spacing for expression.

[0319] The vector may contain a selection marker known in the art. Selection markers include positive and negative selection markers, such as antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, puromycin resistance gene, blasticidin resistance gene), glutamate synthase gene, HSV-TK or HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence or cloning site encoding the selection marker may be located upstream or downstream of the nucleic acid sequence or cloning site encoding the polypeptide of interest.

[0320] The vectors described herein can be used to transform a variety of cells with genes encoding the described antibodies or antigen-binding fragments. For example, the vectors can be used to generate BCMA-specific antibody or antigen-binding fragment-producing cells. Thus, another aspect features host cells transformed with a vector containing a nucleic acid sequence encoding an antibody or antigen-binding fragment thereof that binds BCMA, e.g., an antibody or antigen-binding fragment described and exemplified herein.

[0321] Numerous techniques for introducing foreign genes into cells are known in the art and can be used to generate recombinant cells for carrying out the methods described herein, according to various embodiments exemplified herein. The technique used must stably introduce the heterologous gene sequence into the host cell so that it is inheritable and expressible by the cell's progeny and does not impair essential growth and physiological functions of the recipient cell. Techniques that can be used include chromosomal transfer methods (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carriers), and viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) (Cline, 29 Pharmac. Ther. 69-92 (1985)). Cells can also be transformed using calcium phosphate precipitation of bacterial protoplasts and polyethylene glycol (PEG)-induced fusion with mammalian cells.

[0322] Cells suitable for use in expressing the BCMA-specific antibodies or antigen-binding fragments described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin. These cells include, but are not limited to, NS0, CHO, CHOK1, perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1 / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and BHK cell lines, among others. In addition, antibody expression may be achieved using hybridoma cells. Methods for generating hybridomas are well established in the art.

[0323] Cells transformed with the expression vectors described herein may be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant positive cells are expanded and screened for subclones that exhibit the desired phenotype, e.g., high levels of expression, enhanced growth characteristics, or the ability to produce proteins with desired biochemical properties, e.g., due to protein modifications or altered post-translational modifications. These phenotypes may be due to the inherent properties of a given subclone or to mutations. Mutations may be caused by chemicals, UV light, irradiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of such methods.

[0324] Methods of using BCMA-specific antibodies for treatment Provided herein are BCMA-specific antibodies or antigen-binding fragments thereof for use in therapy. In particular, these antibodies or antigen-binding fragments may be useful for treating cancer, e.g., BCMA-expressing cancers, or other BCMA-expressing disorders. Accordingly, the present invention provides methods of treating cancer comprising administering an antibody as described herein, e.g., a BCMA-specific antibody or antigen-binding fragment. For example, use may be by interfering with BCMA-receptor interaction, or, when such an antibody is conjugated with a toxin, by thus targeting the toxin to the BCMA-expressing cancer. In some embodiments, the BCMA-expressing cancer or disorder includes lymphomas such as multiple myeloma (MM), including smoldering multiple myeloma (SMM), or amyloidosis, plasma cell leukemia, and lupus. In some embodiments, the BCMA-expressing cancer is a relapsed or refractory form of lymphoma, e.g., a relapsed or refractory form of multiple myeloma. Antibodies for use in these methods include those previously described herein, e.g., BCMA-specific antibodies or antigen-binding fragments having the characteristics, e.g., CDR or variable domain sequences, listed in Table 1 and in further discussion of these antibodies (BCMB519).

[0325] In some embodiments described herein, the immune effector properties of BCMA-specific antibodies can be enhanced or silenced by Fc modification using techniques described herein and known to those of skill in the art. For example, Fc effector functions, such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptors, BCRs), can be provided and / or modulated by modifying residues of the Fc involved in these activities.

[0326] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells.

[0327] The ability of a monoclonal antibody to induce ADCC can be enhanced by manipulating its oligosaccharide components. Human IgG1 or IgG3 is N-glycosylated at Asn297, where the majority of glycans are in the known biantennary G0, G0F, G1, G1F, G2, or G2F forms. Antibodies produced by non-genetically engineered CHO cells typically have a glycan fucose content of at least about 85%. Removal of core fucose from biantennary complex-type oligosaccharides added to the Fc region enhances the ADCC of the antibody by improving FcγRIIIa binding, without altering antigen binding or CDC activity. Such mAbs have been reported to be successfully expressed as relatively highly defucosylated antibodies with biantennary and complex Fc oligosaccharides using various methods, such as controlling the culture osmolarity (Konno et al., Cytotechnology 64:249-65, 2012), applying the mutant CHO strain Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-26740, 2002), applying the mutant CHO strain EB66 as a host cell line (Olivier et al., MAbs;2(4), 2010, Epub ahead of print, PMID:20562582), and applying the rat hybridoma cell line YB2 / 0 as a host cell line (Shinkawa et al., J Biol Chem 278:3466-3473, 2003), introduction of small interfering RNA specific for the alpha-1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or co-expression of beta-1,4-N-acetylglucosaminyltransferase III with Golgi alpha-mannosidase II or the potent alpha-mannosidase I inhibitor kifunensine (Ferrara et al., J Biol Chem 281:5032-5036; 2006; Ferrara et al., Biotechnol Bioeng 93:851-861, 2006; Xhou et al., Biotechnol Bioeng 99:652-65, 2008).

[0328] In some embodiments described herein, the ADCC elicited by BCMA antibodies can also be improved by certain substitutions in the antibody Fc. Exemplary substitutions are at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residue numbering according to the EU index), as described, for example, in U.S. Patent No. 6,737,056.

[0329] How to detect BCMA Provided herein are methods for detecting BCMA in a biological sample by contacting the sample with an antibody or antigen-binding fragment thereof described herein. As described herein, the sample can be obtained from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspiration), histological preparation, etc. In some embodiments, the described methods comprise detecting BCMA in a biological sample by contacting the sample with any of the BCMA-specific antibodies or antigen-binding fragments thereof described herein.

[0330] In some embodiments, a sample may be contacted with two or more of the BCMA-specific antibodies or antigen-binding fragments described herein. For example, a sample may be contacted with a first BCMA-specific antibody or antigen-binding fragment thereof and then with a second BCMA-specific antibody or antigen-binding fragment thereof, where the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are not the same antibody or antigen-binding fragment. In some embodiments, the first antibody or antigen-binding fragment thereof may be immobilized to a surface, such as a multi-well plate, chip, or similar substrate, before contacting the sample. In other embodiments, the first antibody or antigen-binding fragment thereof may be unimmobilized or attached to anything before contacting the sample.

[0331] The described BCMA-specific antibodies and antigen-binding fragments may be detectably labeled. In some embodiments, labeled antibodies and antigen-binding fragments may facilitate detection of BCMA by the methods described herein. Many such labels are readily known to those of skill in the art. For example, suitable labels include, but should not be considered limited to, radiolabels, fluorescent labels, epitope tags, biotin, chromophore labels, ECL labels, or enzymes. More particularly, described labels include ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase, and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Alexafluor® dyes, and the like.

[0332] The described BCMA-specific antibodies and antigen-binding fragments may be used in a variety of assays to detect BCMA in biological samples. Some suitable assays include, but should not be considered limited to, Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) or ELISA assays.

[0333] In some embodiments described herein, detection of BCMA-expressing cancer cells in a subject can be used to determine whether the subject can be treated with a therapeutic agent that targets BCMA.

[0334] BCMA is present at detectable levels in blood and serum samples. Thus, provided herein are methods for detecting BCMA in a sample obtained from blood, such as a serum sample, by contacting the sample with an antibody or antigen-binding fragment thereof that binds to BCMA. The blood sample or a derivative thereof can be diluted, fractionated, or otherwise processed to generate a sample on which the described methods can be performed. In some embodiments, BCMA can be detected in a blood sample or a derivative thereof by any number of assays known in the art, including, but not limited to, Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA assay. In some embodiments, the method is an in vitro method.

[0335] Methods for diagnosing cancer or disorders Provided herein are methods for diagnosing a BCMA-expressing cancer or disorder in a subject. In some embodiments, the BCMA-expressing cancer or disorder includes lymphomas such as multiple myeloma (MM), including smoldering multiple myeloma (SMM), or amyloidosis, plasma cell leukemia, and lupus. In some embodiments, as described above, detecting BCMA in a biological sample, e.g., a blood sample or serum sample, provides the ability to diagnose cancer in the subject from whom the sample was obtained. Alternatively, in some embodiments, other samples, e.g., histological samples, fine needle aspirates, excised tumor tissue, circulating cells, circulating tumor cells, etc., may also be used to assess whether the subject from whom the sample was obtained has cancer. In some embodiments, the subject from whom the sample was obtained may already be known to have cancer, but the type of cancer the subject has may not have been diagnosed or may be unknown at preliminary diagnosis; therefore, detecting BCMA in a biological sample obtained from the subject may enable or clarify the diagnosis of cancer. For example, a subject may be known to have cancer, but it may not be known, or it may be unclear whether the subject's cancer expresses BCMA.

[0336] In some embodiments, the described methods involve assessing whether a subject has a BCMA-expressing cancer by determining the amount of BCMA present in a biological sample obtained from the subject and comparing the observed amount of BCMA to the amount of BCMA in a control or reference sample. In this case, a difference between the amount of BCMA in the sample obtained from the subject and the amount of BCMA in the control or reference sample is indicative of the subject having a BCMA-expressing cancer. In another embodiment, the amount of BCMA observed in the biological sample obtained from the subject can be compared to a level of BCMA known to be associated with a particular form or stage of cancer to determine the form or stage of the cancer in the subject. In some embodiments, the amount of BCMA in a sample obtained from the subject is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that binds BCMA, such as a BCMA-specific antibody described herein. Samples assessed for the presence of BCMA may be obtained from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspiration), histological preparations, etc. In some embodiments, the BCMA-expressing cancer or disorder includes hematological cancers such as multiple myeloma (MM), including smoldering multiple myeloma (SMM), or amyloidosis, plasma cell leukemia, and lupus. In some embodiments, the BCMA-expressing cancer is a relapsed or refractory form of lymphoma, such as a relapsed or refractory form of multiple myeloma. In some embodiments, the subject is a human.

[0337] In some embodiments, a method for diagnosing a BCMA-expressing cancer or disorder comprises contacting a subject's biological sample with a BCMA-specific antibody or antigen-binding fragment thereof (e.g., derived from the antibodies and fragments provided in Table 1 (e.g., BCMB519)), quantifying the amount of BCMA present in the sample bound by the antibody or antigen-binding fragment thereof, comparing the amount of BCMA present in the sample with a known standard or reference sample, and determining whether the subject's BCMA level falls within the level of BCMA associated with cancer. In further embodiments, the diagnostic method may be followed by the further step of administering or prescribing a BCMA-specific therapeutic agent. In another embodiment, the diagnostic method may be followed by the further step of communicating the result of the determination to facilitate treatment of the cancer or disorder. In some embodiments, the BCMA-specific treatment may target the BCMA-expressing cancer, such as the BCMAxCD3 multispecific antibodies described herein.

[0338] In some embodiments, the methods described involve assessing whether a subject is afflicted with a BCMA-expressing cancer by determining the amount of BCMA present in a blood or serum sample obtained from the subject and comparing the observed amount of BCMA to the amount of BCMA in a control or reference sample, where a difference between the amount of BCMA in the sample obtained from the subject and the amount of BCMA in the control or reference sample is indicative of the subject being afflicted with a BCMA-expressing cancer.

[0339] In some embodiments, the control or reference sample may be obtained from a subject not afflicted with a BCMA-expressing cancer or disorder. In some embodiments, the control or reference sample may be obtained from a subject afflicted with a BCMA-expressing cancer or disorder. In some embodiments, the control or reference sample is obtained from a subject not afflicted with a BCMA-expressing cancer or disorder, an observed increase in the amount of BCMA present in the test sample relative to the amount of BCMA observed in the control or reference sample is an indication that the subject being evaluated is afflicted with a BCMA-expressing cancer or disorder. In some embodiments, the control sample is obtained from a subject not afflicted with a BCMA-expressing cancer or disorder, an observed decrease or similarity in the amount of BCMA present in the test sample relative to the amount of BCMA observed in the control or reference sample is an indication that the subject being evaluated is not afflicted with a BCMA-expressing cancer or disorder. In some embodiments, the control or reference sample is obtained from a subject afflicted with a BCMA-expressing cancer or disorder, an observed increase in the amount of BCMA present in the test sample relative to the amount of BCMA observed in the control or reference sample is an indication that the subject being evaluated is afflicted with a BCMA-expressing cancer or disorder. In some embodiments, where the control or reference sample is obtained from a subject afflicted with a BCMA-expressing cancer or disorder, an observed increase in the amount of BCMA present in the test sample relative to the amount of BCMA observed in the control or reference sample is an indication that the subject being assessed is afflicted with a BCMA-expressing cancer or disorder.

[0340] In some embodiments, the amount of BCMA in a sample obtained from a subject is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that binds to BCMA, such as an antibody described herein. Samples assessed for the presence of BCMA may be obtained from blood samples, serum samples, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically resected tumor tissue, biopsies including fine needle aspirations), histological preparations, etc.

[0341] In various aspects, the amount of BCMA is determined by contacting the sample with an antibody or antigen-binding fragment thereof that binds to BCMA. In some embodiments, the sample may be contacted with more than one type of antibody or antigen-binding fragment thereof that binds to BCMA. In some embodiments, the sample may be contacted with a first antibody or antigen-binding fragment thereof that binds to BCMA, and then with a second antibody or antigen-binding fragment thereof that binds BCMA. BCMA-specific antibodies or antigen-binding fragments, such as those described herein, may be used in this capacity.

[0342] Various combinations of BCMA-specific antibodies and antigen-binding fragments can be used to provide "first" and "second" antibodies or antigen-binding fragments for practicing the described diagnostic methods. In some embodiments, the BCMA-expressing cancer or disorder comprises a lymphoma, e.g., multiple myeloma (MM), including smoldering multiple myeloma (SMM). In some embodiments, the BCMA-expressing cancer is a relapsed or refractory form of lymphoma, e.g., a relapsed or refractory form of multiple myeloma.

[0343] In certain embodiments, the amount of BCMA is determined by Western blot analysis, radioimmunoassay, immunofluorescence, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or ELISA assay.

[0344] In various embodiments of the described diagnostic methods, a control or reference sample is used. This sample may be a positive or negative assay control to ensure that the assay being used is functioning properly. For example, assay controls of this nature may be commonly used in immunohistochemistry assays. Alternatively, the sample may be a standardized reference for the amount of BCMA in biological samples from healthy subjects. In some embodiments, the observed BCMA level of the test subject may be compared to the BCMA level observed in a sample from a subject known to have a BCMA-expressing cancer. In some embodiments, the control subject may be afflicted with the particular cancer or disorder being targeted. In some embodiments, the control subject is known to have an early-stage cancer, which may or may not be a BCMA-expressing cancer. In some embodiments, the control subject is known to have an intermediate-stage cancer, which may or may not be a BCMA-expressing cancer. In some embodiments, the control subject is known to have a late-stage cancer, which may or may not be a BCMA-expressing cancer. In some embodiments, the method for diagnosing a cancer or disorder is an in vitro method.

[0345] Methods for monitoring cancer or disorders Provided herein are methods for monitoring a BCMA-expressing cancer or disorder in a subject. In some embodiments, the BCMA-expressing cancer or disorder includes lymphomas such as multiple myeloma (MM), including smoldering multiple myeloma (SMM), or amyloidosis, plasma cell leukemia, and lupus. In some embodiments, the BCMA-expressing cancer is a relapsed or refractory form of lymphoma, e.g., a relapsed or refractory form of multiple myeloma. In some embodiments, the described methods involve assessing whether the BCMA-expressing cancer or disorder is progressing, regressing, or remaining stable by determining the amount of BCMA present in a test sample obtained from the subject and comparing the observed amount of BCMA to the amount of BCMA in a similar biological sample obtained from the subject at an earlier time point. In this case, the difference between the amount of BCMA in the test sample and the amount of BCMA in the earlier sample provides an indication of whether the cancer is progressing, regressing, or remaining stable. In this regard, a test sample containing an increased amount of BCMA relative to the amount observed in an earlier sample may indicate progression of the BCMA-expressing cancer or disorder. Conversely, a test sample containing a decreased amount of BCMA compared to the amount observed in an earlier sample may indicate regression of the BCMA-expressing cancer or disorder.

[0346] Thus, a test sample having only a small difference in the amount of BCMA compared to the amount observed in an earlier sample may indicate a stable state for the BCMA-expressing cancer or disorder. In some embodiments, the amount of BCMA in a biological sample obtained from a subject is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that binds to BCMA, such as an antibody described herein. Samples assessed for the presence of BCMA may be obtained from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically resected tumor tissue, biopsy, including fine needle aspiration), histological preparations, and the like. In some embodiments, the subject is a human.

[0347] In some embodiments, a method for monitoring a BCMA-expressing cancer or disorder comprises contacting a subject's biological sample with a BCMA-specific antibody or antigen-binding fragment thereof (e.g., derived from the antibodies and fragments provided in Table 1 (e.g., BCMB519)), quantifying the amount of BCMA present in the sample, comparing the amount of BCMA present in the sample to the amount of BCMA determined to be present in a similarly obtained biological sample from the same subject at an earlier time point, and determining whether the subject's BCMA levels have changed over time. A test sample containing an increased amount of BCMA compared to the amount observed in the earlier sample may indicate cancer progression. Conversely, a test sample containing a decreased amount of BCMA compared to the amount observed in the earlier sample may indicate regression of the BCMA-expressing cancer or disorder. Thus, a test sample having only a small difference in the amount of BCMA compared to the amount observed in the earlier sample may indicate stable disease for the BCMA-expressing cancer or disorder. In some embodiments, the BCMA level of the sample may be compared to a known standard or reference sample, either alone or in addition to the BCMA level observed in the sample assessed at an earlier time point. In further embodiments, the diagnostic method may be followed by the further step of administering a BCMA-specific treatment, which in some embodiments may be targeted to BCMA-expressing cancers, such as the BCMAxCD3 multispecific antibodies described herein.

[0348] In various aspects, the amount of BCMA is determined by contacting the sample with an antibody or antigen-binding fragment thereof that binds to BCMA. In some embodiments, the sample may be contacted with more than one type of antibody or antigen-binding fragment thereof that binds to BCMA. In some embodiments, the sample may be contacted with a first antibody or antigen-binding fragment thereof that binds to BCMA, and then with a second antibody or antigen-binding fragment thereof that binds BCMA. Antibodies such as those described herein may be used in this capacity.

[0349] Various combinations of the antibodies and antigen-binding fragments described in Table 1 can be used to provide "first" and "second" antibodies or antigen-binding fragments for performing the described monitoring methods. In some embodiments, the BCMA-expressing cancer or disorder includes hematological cancers, such as acute myeloid leukemia (AML) or lymphomas (e.g., multiple myeloma (MM), smoldering multiple myeloma (SMM)), or amyloidosis, plasma cell leukemia, and lupus.

[0350] In certain embodiments, the amount of BCMA is determined by Western blot analysis, radioimmunoassay, immunofluorescence, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or ELISA assay. In some embodiments, the method is an in vitro method.

[0351] Kits for detecting BCMA Provided herein are kits for detecting BCMA in a biological sample, which kits comprise one or more of the BCMA-specific antibodies or antigen-binding fragments thereof described herein and instructions for using the kit.

[0352] The BCMA-specific antibodies or antigen-binding fragments provided may be in solution, lyophilized, immobilized on a substrate, carrier or plate, or detectably labeled.

[0353] The described kits may also include additional components useful for carrying out the methods described herein. By way of example, the kit may include means for obtaining a sample from a subject, a control or reference sample, e.g., a sample from a subject with a slowly progressing cancer and / or a subject without cancer, one or more sample compartments, and / or instructions describing the practice of the methods of the invention, and tissue-specific control or reference materials.

[0354] The means for determining the level of BCMA may further comprise, for example, buffers or other reagents for use in an assay to determine the level of BCMA. The instructions may be, for example, printed instructions for performing an assay and / or instructions for assessing the expression level of BCMA.

[0355] The described kits may also include means for isolating a sample from a subject. These means may include one or more items of equipment or reagents that can be used to obtain fluid or tissue from a subject. The means for obtaining a sample from a subject may also include means for isolating blood components, such as serum, from a blood sample. Preferably, the kit is designed for use with human subjects.

[0356] Embodiment The disclosure provided herein also provides the following non-limiting embodiments.

[0357] Embodiment 1. A trispecific antibody or trispecific binding fragment thereof, comprising: (a) a first antigen-binding arm comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); (b) a second antigen-binding arm comprising a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2); (c) a third antigen-binding arm comprising a third heavy chain variable domain (VH3) and a third light chain variable domain (VL3); A trispecific antibody or trispecific binding fragment thereof, wherein a first antigen-binding arm binds to an epitope on cluster of differentiation 3 (CD3), a second antigen-binding arm binds to an epitope on G protein-coupled receptor family C group 5 member D (GPRC5D), and a third antigen-binding arm binds to an epitope on B cell maturation antigen (BCMA).

[0358] Embodiment 2. The trispecific antibody or trispecific binding fragment of embodiment 1, wherein the VH1 and VL1 of the first antigen-binding arm are present in a diabody, Fab, Fab', F(ab')2, Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (dsdiabody), optionally in a Fab.

[0359] Embodiment 3. The trispecific antibody or trispecific binding fragment of embodiment 1 or 2, wherein the VH2 and VL2 of the second antigen-binding arm are present in a diabody, Fab, Fab', F(ab')2, Fv, scFv, Fv, Fd, a disulfide-stabilized Fv fragment (dsFv), or a disulfide-stabilized diabody (dsdiabody), optionally an scFv.

[0360] Embodiment 4. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 3, wherein the VH3 and VL3 of the third antigen-binding arm are present in an antibody fragment, diabody, Fab, Fab', F(ab'), Fv, scFv, Fd, disulfide-stabilized Fv fragment (dsFv), or disulfide-stabilized diabody (ds diabody), optionally an scFv.

[0361] Embodiment 5. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 4, wherein the first antigen-binding arm that binds to CD3 comprises heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of a heavy chain variable domain (VH1) of SEQ ID NO: 8, and light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of a light chain variable domain (VL1) of SEQ ID NO: 7.

[0362] Embodiment 6. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 5, wherein the first antigen-binding arm that binds to CD3 comprises an HCDR1 comprising the amino acid sequence of GDSVFNNNAAWS (SEQ ID NO: 4), an HCDR2 comprising the amino acid sequence of RTYYRSKWLYD (SEQ ID NO: 5), and an HCDR3 comprising the amino acid sequence of GYSSSFDY (SEQ ID NO: 6), and an LCDR1 comprising the amino acid sequence of TGTSSNIGTYKFVS (SEQ ID NO: 1), an LCDR2 comprising the amino acid sequence of EVSKRPS (SEQ ID NO: 2), and an LCDR3 comprising the amino acid sequence of VSYAGSGTLL (SEQ ID NO: 3).

[0363] Embodiment 7. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 6, wherein the first antigen-binding arm that binds to CD3 comprises a VH1 of SEQ ID NO:8 and a VL1 of SEQ ID NO:7.

[0364] Embodiment 8. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 7, wherein the second antigen-binding arm that binds to GPRC5D comprises heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of the heavy chain variable domain (VH2) of SEQ ID NO: 16, and light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of the light chain variable domain (VL2) of SEQ ID NO: 15.

[0365] Embodiment 9. A trispecific antibody or trispecific binding fragment according to any one of embodiments 1 to 8, wherein the second antigen-binding arm that binds to GPRC5D comprises an HCDR1 comprising the amino acid sequence of GFSLTNIRMSVS (SEQ ID NO: 12), an HCDR2 comprising the amino acid sequence of HIFSNDEKS (SEQ ID NO: 13), and an HCDR3 comprising the amino acid sequence of MRLPYGMDV (SEQ ID NO: 14), and an LCDR1 comprising the amino acid sequence of RSSQSLVHSDGNTYLS (SEQ ID NO: 9), an LCDR2 comprising the amino acid sequence of KISNRFF (SEQ ID NO: 10), and an LCDR3 comprising the amino acid sequence of MQATQFPHT (SEQ ID NO: 11).

[0366] Embodiment 10. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 9, wherein the second antigen-binding arm that binds to GPRC5D comprises a VH2 of SEQ ID NO: 16 and a VL2 of SEQ ID NO: 15.

[0367] Embodiment 11. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 10, wherein the third antigen-binding arm that binds to BCMA comprises heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3 of a heavy chain variable domain (VH3) of SEQ ID NO: 24, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of a light chain variable domain (VL3) of SEQ ID NO: 23.

[0368] Embodiment 12. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 11, wherein the third antigen-binding arm that binds to BCMA comprises an HCDR1 comprising the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 20), an HCDR2 comprising the amino acid sequence of AISGSGGSTY (SEQ ID NO: 21), and an HCDR3 comprising the amino acid sequence of DEGYSSGHYYGMDV (SEQ ID NO: 22), and an LCDR1 comprising the amino acid sequence of RASQSISSSFLT (SEQ ID NO: 17), an LCDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 18), and an LCDR3 comprising the amino acid sequence of QHYGSSPMYT (SEQ ID NO: 19).

[0369] Embodiment 13. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 12, wherein the third antigen-binding arm that binds to BCMA comprises a VH3 of SEQ ID NO: 24 and a VL3 of SEQ ID NO: 23.

[0370] Embodiment 14. The first antigen-binding arm that binds to CD3 comprises HCDR1, HCDR2, and HCDR3 of VH1 of SEQ ID NO: 8 and LCDR1, LCDR2, and LCDR3 of VL1 of SEQ ID NO: 7; the second antigen-binding arm that binds to GPRC5D comprises HCDR1, HCDR2, and HCDR3 of VH2 of SEQ ID NO: 16 and LCDR1, LCDR2, and LCDR3 of VL2 of SEQ ID NO: 15; 5. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 4, wherein the third antigen-binding arm that binds to BCMA comprises HCDR1, HCDR2, and HCDR3 of VH3 of SEQ ID NO: 24 and LCDR1, LCDR2, and LCDR3 of VL3 of SEQ ID NO: 23.

[0371] Embodiment 15. The first antigen-binding arm that binds to CD3 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 4, 5, 6, 1, 2, and 3, respectively; the second antigen-binding arm that binds to GPRC5D comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 12, 13, 14, 9, 10, and 11, respectively; 15. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 4 and 14, wherein the third antigen-binding arm that binds to BCMA comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 20, 21, 22, 17, 18, and 19, respectively.

[0372] Embodiment 16. The first antigen-binding arm that binds to CD3 comprises a VH1 of SEQ ID NO: 8 and a VL1 of SEQ ID NO: 7; the second antigen-binding arm that binds to GPRC5D comprises a VH2 of SEQ ID NO: 16 and a VL2 of SEQ ID NO: 15; 16. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 4, 14 and 15, wherein the third antigen binding arm that binds to BCMA comprises a VH3 of SEQ ID NO: 24 and a VL3 of SEQ ID NO: 23.

[0373] Embodiment 17. The trispecific antibody or trispecific binding fragment thereof of any one of embodiments 1 to 16, wherein the first antigen-binding arm comprises a fragment crystallizable (Fc) domain, and the second antigen-binding arm or the third antigen-binding arm comprises an Fc domain.

[0374] Embodiment 18. The trispecific antibody or trispecific binding fragment thereof of embodiment 17, wherein the Fc domain comprises one or more mutations that promote heterodimerization of the Fc domain.

[0375] Embodiment 19. The trispecific antibody or trispecific binding fragment of embodiment 18, wherein the mutations are selected from T366S, L368A, T366W, and Y407V (EU numbering).

[0376] Embodiment 20. The trispecific antibody or trispecific binding fragment of any one of embodiments 17 to 19, wherein the Fc domain further comprises one or more mutations that reduce Fc binding to an Fcγ receptor.

[0377] Embodiment 21. The trispecific antibody or trispecific binding fragment of embodiment 20, wherein the Fcγ receptor is FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB.

[0378] Embodiment 22. The trispecific antibody or trispecific binding fragment of embodiment 20 or 21, wherein the Fc domain comprises one or more mutations selected from L234A, L235A, and D265S (EU numbering).

[0379] Embodiment 23. The trispecific antibody or trispecific binding fragment of any one of embodiments 17 to 22, wherein the Fc domain further comprises one or more mutations that reduce Fc binding to Protein A.

[0380] Embodiment 24. The trispecific antibody or trispecific binding fragment of embodiment 23, wherein the Fc domain comprises the mutations H435R and / or Y436F (EU numbering).

[0381] Embodiment 25. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 24, wherein the first antigen-binding arm specifically binds to residues 22-35 of the CD3 epsilon chain (QDGNEEMGGITQTP (SEQ ID NO: 161)).

[0382] Embodiment 26. Approximately 1 x 10 -8 ~1×10 -7 26. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 25, wherein the first antigen-binding arm specifically binds to CD3 with an affinity of M.

[0383] Embodiment 27. Approximately 2 x 10 -8 ~4×10 -8 27. The trispecific antibody or trispecific binding fragment of embodiment 26, wherein the first antigen-binding arm specifically binds to CD3 with an affinity of M.

[0384] Embodiment 28. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 27, wherein the third antigen-binding arm specifically binds to residues 17-26 of the BCMA BCMW37 chain (LLHACIPCQL (SEQ ID NO: 162)).

[0385] Embodiment 29. The third antigen-binding arm comprises about 1 x 10 -10 ~1×10 -7 29. The trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 28, which specifically binds to BCMA with an affinity of M.

[0386] Embodiment 30. The third antigen-binding arm comprises about 2 x 10 -10 ~9×10 -10 30. The trispecific antibody or trispecific binding fragment of embodiment 29, which specifically binds to BCMA with an affinity of M.

[0387] Embodiment 31. A trispecific antibody or trispecific binding fragment comprising a first antigen-binding arm that binds to an epitope on cluster of differentiation 3 (CD3), a second antigen-binding arm that binds to an epitope on G protein-coupled receptor family C group 5 member D (GPRC5D), and a third antigen-binding arm that binds to an epitope on B cell maturation antigen (BCMA), the first antigen-binding arm comprises a heavy chain (HC1) polypeptide and a light chain (LC) polypeptide; A trispecific antibody or trispecific binding fragment thereof, wherein the trispecific antibody or trispecific binding fragment thereof comprises a single polypeptide comprising a second antigen-binding arm and a third antigen-binding arm.

[0388] Embodiment 32. The trispecific antibody or trispecific binding fragment of embodiment 31, wherein HC1 of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 26.

[0389] Embodiment 33. The trispecific antibody or trispecific binding fragment of embodiment 32, wherein the LC of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 27.

[0390] Embodiment 34. The trispecific antibody or trispecific binding fragment of embodiment 32 or 33, wherein the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 28.

[0391] Embodiment 35. The trispecific antibody or trispecific binding fragment of embodiment 31, wherein the first antigen-binding arm comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 26 and an LC comprising the amino acid sequence of SEQ ID NO: 27, and the polypeptide comprising the second and third antigen-binding arms comprises the amino acid sequence of SEQ ID NO: 28.

[0392] Embodiment 36. A trispecific antibody or trispecific binding fragment comprising a first antigen-binding arm that binds to an epitope on cluster of differentiation 3 (CD3), a second antigen-binding arm that binds to an epitope on G protein-coupled receptor family C group 5 member D (GPRC5D), and a third antigen-binding arm that binds to an epitope on B cell maturation antigen (BCMA), the first antigen-binding arm comprises a heavy chain (HC1) polypeptide and a light chain (LC) polypeptide, the heavy chain (HC1) polypeptide further comprising a second antigen-binding arm; A trispecific antibody or trispecific binding fragment thereof, wherein the trispecific antibody or trispecific binding fragment thereof further comprises a single polypeptide comprising a third antigen-binding arm.

[0393] Embodiment 37. The trispecific antibody or trispecific binding fragment of embodiment 36, wherein HC1 of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 29.

[0394] Embodiment 38. The trispecific antibody or trispecific binding fragment of embodiment 36 or 37, wherein the LC of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 30.

[0395] Embodiment 39. The trispecific antibody or trispecific binding fragment of any one of embodiments 36 to 38, wherein the single polypeptide comprising the third antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 31.

[0396] Embodiment 40. The trispecific antibody or trispecific binding fragment of embodiment 36, wherein the first antigen-binding arm comprises an HC1 comprising the amino acid sequence of SEQ ID NO: 29 and an LC comprising the amino acid sequence of SEQ ID NO: 30, and the single polypeptide comprising the third antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 31.

[0397] Embodiment 41. The trispecific antibody or trispecific binding fragment thereof of any one of embodiments 1 to 40, wherein the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0398] Embodiment 42. The trispecific antibody or trispecific binding fragment thereof of any one of embodiments 1 to 41, wherein the antibody or antigen-binding fragment thereof is of the IgG1 isotype.

[0399] Embodiment 43. A synthetic polynucleotide encoding the trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 42.

[0400] Embodiment 44. A pharmaceutical composition comprising the trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 42 and a pharmaceutically acceptable carrier.

[0401] Embodiment 45. The pharmaceutical composition of embodiment 44, wherein the pharmaceutical composition further comprises a second therapeutic agent.

[0402] Embodiment 46. The pharmaceutical composition of embodiment 45, wherein the second therapeutic agent comprises an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof.

[0403] Embodiment 47. A cell expressing the trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 42.

[0404] Embodiment 48. The cell of embodiment 47, wherein the cell is a hybridoma.

[0405] Embodiment 49. The cell of embodiment 47, wherein the trispecific antibody is recombinantly produced.

[0406] Embodiment 50. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 42, or a pharmaceutical composition of any one of embodiments 43 to 46.

[0407] Embodiment 51 The method of embodiment 50, wherein the trispecific antibody or trispecific binding fragment, or pharmaceutical composition is administered for a time sufficient to treat cancer.

[0408] Embodiment 52. A method for inhibiting the growth or proliferation of cancer cells, comprising administering to such cells an effective amount of the trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 42, or the pharmaceutical composition of any one of embodiments 43 to 46, wherein said effective amount is sufficient to inhibit the growth or proliferation of said cancer cells.

[0409] Embodiment 53. The method of embodiment 52, wherein the cancer cells are in a subject and the trispecific antibody or trispecific binding fragment or pharmaceutical composition is administered to the subject.

[0410] Embodiment 54 The method of embodiment 52, wherein said administering is performed ex vivo.

[0411] Embodiment 55. A method of directing T cells against BCMA and / or GPRC5D-expressing cancer cells in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a trispecific antibody or trispecific binding fragment of any one of embodiments 1 to 42, or a pharmaceutical composition of any one of embodiments 43 to 46.

[0412] Embodiment 56 The method of embodiment 55, wherein said therapeutically effective amount is sufficient to direct said T cell response against said cancer cells.

[0413] Embodiment 57. The method of any one of embodiments 50 to 56, wherein the cancer is a blood cancer.

[0414] Embodiment 58. The method of embodiment 57, wherein the hematological cancer is a BCMA- and / or GPRC5D-expressing B-cell cancer.

[0415] Embodiment 59. The method of embodiment 58, wherein the BCMA- and / or GPRC5D-expressing B-cell cancer is multiple myeloma.

[0416] Embodiment 60. The method of embodiment 59, wherein the BCMA- and / or GPRC5D-expressing B-cell cancer is smoldering multiple myeloma (SMM).

[0417] Embodiment 61. The method of any one of embodiments 50-60, wherein the cancer is a recurrent, refractory, or malignant cancer, or any combination thereof.

[0418] Embodiment 62. The method of any one of embodiments 50-51, 53, and 55-61, wherein the subject has undergone prior treatment.

[0419] Embodiment 63. The method of embodiment 62, wherein the pretreatment comprises a proteasome inhibitor, an immunomodulatory agent, a CD38 antibody, a bispecific agent, a CAR-T therapy, or any combination thereof.

[0420] Embodiment 64. The method of any one of embodiments 50-63, further comprising administering a second therapeutic agent.

[0421] Embodiment 65. The method of embodiment 64, wherein the second therapeutic agent is a chemotherapeutic agent or a targeted anti-cancer treatment.

[0422] Embodiment 66. The method of embodiment 65, wherein the chemotherapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin 2 (IL-2).

[0423] Embodiment 67. The method of embodiment 64, wherein the second therapeutic agent is an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof.

[0424] Embodiment 68. The method of any one of embodiments 50-51, 53, and 55-67, wherein the trispecific antibody or trispecific binding fragment, or pharmaceutical composition is administered to the subject intravenously, intramuscularly, intraperitoneally, and / or subcutaneously.

[0425] Embodiment 69. The method of any one of embodiments 50-51, 53, and 55-68, wherein the trispecific antibody or trispecific binding fragment, or pharmaceutical composition is administered subcutaneously to the subject.

[0426] Embodiment 70. A method for producing a trispecific antibody or trispecific binding fragment described in any one of embodiments 1 to 42, comprising culturing a cell described in any one of embodiments 47 to 49 and isolating the trispecific antibody or trispecific binding fragment.

[0427] Embodiment 71. A kit comprising (i) a trispecific antibody or trispecific binding fragment according to any one of embodiments 1 to 42 and / or a polynucleotide according to embodiment 43, and (ii) packaging therefor.

[0428] Embodiment 72. An antibody or antigen-binding fragment thereof that binds to BCMA, comprising a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of GFTFSSYAMS (SEQ ID NO: 20), a heavy chain CDR2 having the amino acid sequence of AISGSGGSTY (SEQ ID NO: 21), and a heavy chain CDR3 having the amino acid sequence of DEGYSSGHYYGMDV (SEQ ID NO: 22).

[0429] Embodiment 73. The antibody or antigen-binding fragment of embodiment 72, further comprising a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of RASQSISSSFLT (SEQ ID NO: 17), a light chain CDR2 having the amino acid sequence of GASSRAT (SEQ ID NO: 18), and a light chain CDR3 having the amino acid sequence of QHYGSSPMYT (SEQ ID NO: 19).

[0430] Embodiment 74. The antibody or antigen-binding fragment of embodiment 72 or 73, comprising a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 24.

[0431] Embodiment 75. The antibody or antigen-binding fragment of any one of embodiments 72 to 74, comprising a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 23.

[0432] Embodiment 76. The antibody or antigen-binding fragment of any one of embodiments 72 to 75, wherein the antibody or antigen-binding fragment specifically binds to residues 17 to 26 of the BCMA BCMW37 chain (LLHACIPCQL (SEQ ID NO: 162)).

[0433] Embodiment 77. The antibody or antigen-binding fragment is about 1 x 10 -10 ~1×10 -7 77. The antibody or antigen-binding fragment of any one of embodiments 72 to 76, which specifically binds to BCMA with an affinity of M.

[0434] Embodiment 78. The antibody or antigen-binding fragment is about 2 x 10 -10 ~9×10 -10 78. The antibody or antigen-binding fragment of embodiment 77, which specifically binds to BCMA with an affinity of M.

[0435] Embodiment 79. The antibody or antigen-binding fragment of any one of embodiments 72 to 78, wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment.

[0436] Embodiment 80. The antibody or antigen-binding fragment of any one of embodiments 72 to 79, wherein the antibody or antigen-binding fragment is recombinant.

[0437] Embodiment 81. The antigen-binding fragment of any one of embodiments 72 to 80, wherein the antigen-binding fragment is a Fab fragment, a Fab2 fragment, or a single-chain antibody.

[0438] Embodiment 82. The antibody or antigen-binding fragment thereof of any one of embodiments 72 to 81, wherein the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 isotype.

[0439] Embodiment 83. The antibody or antigen-binding fragment thereof of any one of embodiments 72 to 82, wherein the antibody or antigen-binding fragment thereof is of the IgG or IgG4 isotype.

[0440] Embodiment 84. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of embodiments 72 to 83 and a pharmaceutically acceptable carrier.

[0441] Embodiment 85. The pharmaceutical composition of embodiment 84, wherein the pharmaceutical composition further comprises a second therapeutic agent.

[0442] Embodiment 86. The pharmaceutical composition of embodiment 85, wherein the second therapeutic agent comprises an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof.

[0443] Embodiment 87. A cell expressing the antibody or antigen-binding fragment of any one of embodiments 72 to 83.

[0444] Embodiment 88. The cell of embodiment 87, wherein the cell is a hybridoma.

[0445] Embodiment 89. The cell of embodiment 87, wherein the antibody is recombinantly produced.

[0446] Embodiment 90. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment described in any one of embodiments 72 to 83, or a pharmaceutical composition described in any one of embodiments 84 to 86.

[0447] Embodiment 91 The method of embodiment 90, wherein the antibody or antigen-binding fragment, or pharmaceutical composition is administered for a time sufficient to treat cancer.

[0448] Embodiment 92. A method for inhibiting the growth or proliferation of cancer cells, comprising administering to such cells an effective amount of the antibody or antigen-binding fragment described in any one of embodiments 72 to 83, or the pharmaceutical composition described in any one of embodiments 84 to 86, wherein said effective amount is sufficient to inhibit the growth or proliferation of such cancer cells.

[0449] Embodiment 93 The method of embodiment 92, wherein the cancer cells are in a subject and the antibody or antigen-binding fragment or pharmaceutical composition is administered to the subject.

[0450] Embodiment 94 The method of embodiment 92, wherein said administering is ...

Claims

1. 1. A trispecific antibody or trispecific binding fragment thereof, comprising: (a) a first antigen-binding arm comprising a first heavy chain variable domain (VH1) and a first light chain variable domain (VL1); (b) a second antigen-binding arm comprising a second heavy chain variable domain (VH2) and a second light chain variable domain (VL2); (c) a third antigen-binding arm comprising a third heavy chain variable domain (VH3) and a third light chain variable domain (VL3); A trispecific antibody or trispecific binding fragment thereof, wherein the first antigen-binding arm binds to an epitope on cluster of differentiation 3 (CD3), the second antigen-binding arm binds to an epitope on G protein-coupled receptor family C group 5 member D (GPRC5D), and the third antigen-binding arm binds to an epitope on B cell maturation antigen (BCMA).

2. The trispecific antibody or trispecific binding fragment of claim 1, (a) the VH1 and the VL1 of the first antigen-binding arm are present in a Fab, a diabody, a Fab', F(ab')2, Fv, scFv, Fd, a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (dsdiabody), optionally a Fab; (b) the VH2 and the VL2 of the second antigen-binding arm are present in a scFv, a diabody, a Fab, Fab', F(ab')2, Fv, Fv, Fd, a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (dsdiabody), optionally in a scFv; and / or (c) A trispecific antibody or trispecific binding fragment, wherein the VH3 and the VL3 of the third antigen binding arm are present in an scFv, an antibody fragment, a diabody, Fab, Fab', F(ab')2, Fv, Fd, a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (dsdiabody), optionally in an scFv.

3. The trispecific antibody or trispecific binding fragment of claim 1 or claim 2, comprising: (a) (i) the first antigen-binding arm that binds to CD3 comprises heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of a heavy chain variable domain (VH1) of SEQ ID NO: 8, and light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of a light chain variable domain (VL1) of SEQ ID NO: 7; (ii) the first antigen-binding arm that binds to CD3 comprises an HCDR1 comprising the amino acid sequence of GDSVFNNNAAWS (SEQ ID NO: 4), an HCDR2 comprising the amino acid sequence of RTYYRSKWLYD (SEQ ID NO: 5), and an HCDR3 comprising the amino acid sequence of GYSSSFDY (SEQ ID NO: 6); and an LCDR1 comprising the amino acid sequence of TGTSSNIGTYKFVS (SEQ ID NO: 1), an LCDR2 comprising the amino acid sequence of EVSKRPS (SEQ ID NO: 2), and an LCDR3 comprising the amino acid sequence of VSYAGSGTLL (SEQ ID NO: 3); (iii) the first antigen-binding arm that binds to CD3 comprises a VH1 of SEQ ID NO:8 and a VL1 of SEQ ID NO:7; (iv) the second antigen-binding arm that binds to GPRC5D comprises heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of a heavy chain variable domain (VH2) of SEQ ID NO: 16, and light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of a light chain variable domain (VL2) of SEQ ID NO: 15; (v) the second antigen-binding arm that binds to GPRC5D comprises an HCDR1 comprising the amino acid sequence of GFSLTNIRMSVS (SEQ ID NO: 12), an HCDR2 comprising the amino acid sequence of HIFSNDEKS (SEQ ID NO: 13), and an HCDR3 comprising the amino acid sequence of MRLPYGMDV (SEQ ID NO: 14); and an LCDR1 comprising the amino acid sequence of RSSQSLVHSDGNTYLS (SEQ ID NO: 9), an LCDR2 comprising the amino acid sequence of KISNRFF (SEQ ID NO: 10), and an LCDR3 comprising the amino acid sequence of MQATQFPHT (SEQ ID NO: 11); (vi) the second antigen-binding arm that binds to GPRC5D comprises a VH2 of SEQ ID NO: 16 and a VL2 of SEQ ID NO: 15; (vii) the third antigen-binding arm that binds BCMA comprises heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of a heavy chain variable domain (VH3) of SEQ ID NO: 24, and light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of a light chain variable domain (VL3) of SEQ ID NO: 23; (viii) the third antigen-binding arm that binds BCMA comprises an HCDR1 comprising the amino acid sequence of GFTFSYAMS (SEQ ID NO: 20), an HCDR2 comprising the amino acid sequence of AISGSGGSTY (SEQ ID NO: 21), and an HCDR3 comprising the amino acid sequence of DEGYSSGHYYGMDV (SEQ ID NO: 22); and an LCDR1 comprising the amino acid sequence of RASQSISSSFLT (SEQ ID NO: 17), an LCDR2 comprising the amino acid sequence of GASSRAT (SEQ ID NO: 18), and an LCDR3 comprising the amino acid sequence of QHYGSSPMYT (SEQ ID NO: 19); and / or (ix) the third antigen-binding arm that binds BCMA comprises a VH3 of SEQ ID NO: 24 and a VL3 of SEQ ID NO: 23; or (b) (i) the first antigen-binding arm that binds CD3 comprises HCDR1, HCDR2, and HCDR3 of VH1 of SEQ ID NO: 8, and LCDR1, LCDR2, and LCDR3 of VL1 of SEQ ID NO: 7; the second antigen-binding arm that binds to GPRC5D comprises HCDR1, HCDR2, and HCDR3 of VH2 of SEQ ID NO: 16 and LCDR1, LCDR2, and LCDR3 of VL2 of SEQ ID NO: 15; the third antigen-binding arm that binds to BCMA comprises HCDR1, HCDR2, and HCDR3 of VH3 of SEQ ID NO:24, and LCDR1, LCDR2, and LCDR3 of VL3 of SEQ ID NO:23; (ii) the first antigen-binding arm that binds to CD3 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 4, 5, 6, 1, 2, 3, respectively; the second antigen-binding arm that binds to GPRC5D comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 12, 13, 14, 9, 10, and 11, respectively; and / or the third antigen-binding arm that binds BCMA comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 20, 21, 22, 17, 18, and 19, respectively; and / or (iii) the first antigen-binding arm that binds to CD3 comprises a VH1 of SEQ ID NO:8 and a VL1 of SEQ ID NO:7; the second antigen-binding arm that binds to GPRC5D comprises a VH2 of SEQ ID NO: 16 and a VL2 of SEQ ID NO: 15; A trispecific antibody or trispecific binding fragment, wherein the third antigen binding arm that binds to BCMA comprises a VH3 of SEQ ID NO:24 and a VL3 of SEQ ID NO:

23.

4. The trispecific antibody or trispecific binding fragment thereof according to any one of claims 1 to 3, the first antigen-binding arm comprises a fragment crystallizable (Fc) domain, and the second antigen-binding arm or the third antigen-binding arm comprises an Fc domain; Optionally, (a) the Fc domain of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 158; (b) the Fc domain comprises one or more mutations that promote heterodimerization of the Fc domain; Optionally, (i) the mutation is selected from T366S, L368A, T366W, and Y407V (EU numbering); (c) the Fc domain further comprises one or more mutations that reduce Fc binding to an Fcγ receptor; Optionally, (i) the Fcγ receptor is FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB, and / or (ii) the Fc domain comprises one or more mutations selected from L234A, L235A, and D265S (EU numbering); and / or (d) the Fc domain further comprises one or more mutations that reduce Fc binding to Protein A. Optionally, A trispecific antibody or trispecific binding fragment, wherein the Fc domain comprises the mutation H435R and / or Y436F (EU numbering).

5. The trispecific antibody or trispecific binding fragment of claim 1, (a) the first antigen-binding arm specifically binds to residues 22-35 (QDGNEEMGGITQTP (SEQ ID NO: 161)) of the CD3 epsilon chain; (b) the first antigen-binding arm specifically binds to CD3 with an affinity of about 1×10 −8 to 1×10 −7 M; Optionally, the first antigen-binding arm specifically binds to CD3 with an affinity of about 2×10 −8 to 4×10 −8 M; (c) the third antigen-binding arm specifically binds to residues 17-26 of the BCMA BCMW37 chain (LLHACIPCQL (SEQ ID NO: 162)); and / or (d) the third antigen-binding arm specifically binds to BCMA with an affinity of about 1×10 −10 to 1×10 −7 M; Optionally, A trispecific antibody or trispecific binding fragment, wherein said third antigen-binding arm specifically binds to BCMA with an affinity of about 2×10 −10 to 9×10 −10 M.

6. The trispecific antibody or trispecific binding fragment of claim 1, a first antigen-binding arm that binds to an epitope on cluster of differentiation 3 (CD3), a second antigen-binding arm that binds to an epitope on G protein-coupled receptor family C group 5 member D (GPRC5D), and a third antigen-binding arm that binds to an epitope on B cell maturation antigen (BCMA); the first antigen-binding arm comprises a heavy chain (HC1) polypeptide and a light chain (LC) polypeptide, the heavy chain (HC1) polypeptide further comprising the second antigen-binding arm; The trispecific antibody or trispecific binding fragment thereof further comprises a single polypeptide comprising the third antigen-binding arm.

7. The trispecific antibody or trispecific binding fragment of claim 6, comprising: (a) (i) the HC1 of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO:29; (ii) the LC of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 30, and / or (iii) the single polypeptide comprising the third antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 31; or (b) a trispecific antibody or trispecific binding fragment, wherein the first antigen binding arm comprises an HC1 comprising the amino acid sequence of SEQ ID NO:29 and an LC comprising the amino acid sequence of SEQ ID NO:30, and the single polypeptide comprising the third antigen binding arm comprises the amino acid sequence of SEQ ID NO:

31.

8. The trispecific antibody or trispecific binding fragment of claim 1, A polypeptide comprising the amino acid sequence of SEQ ID NO:29, comprising the second antigen-binding arm and the HC of the first antigen-binding arm; a polypeptide comprising the amino acid sequence of SEQ ID NO: 30, comprising the LC of the first antigen-binding arm; and A trispecific antibody or trispecific binding fragment comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:31, said trispecific antibody or trispecific binding fragment comprising said third antigen-binding arm.

9. The trispecific antibody or trispecific binding fragment of claim 1, a first antigen-binding arm that binds to an epitope on cluster of differentiation 3 (CD3), a second antigen-binding arm that binds to an epitope on G protein-coupled receptor family C group 5 member D (GPRC5D), and a third antigen-binding arm that binds to an epitope on B cell maturation antigen (BCMA); the first antigen-binding arm comprises a heavy chain (HC1) polypeptide and a light chain (LC) polypeptide; The trispecific antibody or trispecific binding fragment thereof comprises a single polypeptide comprising the second antigen-binding arm and the third antigen-binding arm.

10. The trispecific antibody or trispecific binding fragment of claim 9, comprising: (a) (i) the HC1 of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 26; (ii) the LC of the first antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 27, and / or (iii) the polypeptide comprising the second antigen-binding arm and the third antigen-binding arm comprises the amino acid sequence of SEQ ID NO: 28; or (b) a trispecific antibody or trispecific binding fragment, wherein the first antigen binding arm comprises an HC1 comprising the amino acid sequence of SEQ ID NO:26 and an LC comprising the amino acid sequence of SEQ ID NO:27, and the polypeptide comprising the second antigen binding arm and the third antigen binding arm comprises the amino acid sequence of SEQ ID NO:

28.

11. The trispecific antibody or trispecific binding fragment of claim 1, comprising: A polypeptide comprising the amino acid sequence of SEQ ID NO:26, comprising the HC of the first antigen-binding arm; a polypeptide comprising the amino acid sequence of SEQ ID NO:27, comprising the LC of the first antigen-binding arm; and A trispecific antibody or trispecific binding fragment comprising a polypeptide comprising the amino acid sequence of SEQ ID NO:28, the trispecific antibody or trispecific binding fragment comprising the second antigen-binding arm and the third antigen-binding arm.

12. The trispecific antibody or trispecific binding fragment of any one of claims 1 to 11, (a) the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 (human) isotype; and / or (b) A trispecific antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof is of the (human) IgG1 isotype.

13. An antibody or antigen-binding fragment thereof that binds to BCMA, comprising a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence of GFTFSYAMS (sequence number 20), a heavy chain CDR2 having the amino acid sequence of AISGSGGSTY (sequence number 21), and a heavy chain CDR3 having the amino acid sequence of DEGYSSGHYYGMDV (sequence number 22).

14. The antibody or antigen-binding fragment of claim 13, (a) a light chain complementarity determining region 1 (CDR1) having the amino acid sequence of RASQSISSSFLT (SEQ ID NO: 17), a light chain CDR2 having the amino acid sequence of GASSRAT (SEQ ID NO: 18), and a light chain CDR3 having the amino acid sequence of QHYGSSPMYT (SEQ ID NO: 19); (b) a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 24; (c) a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 23; (d) the antibody or antigen-binding fragment specifically binds to residues 17-26 of the BCMA BCMW37 chain (LLHACIPCQL (SEQ ID NO: 162)); (e) the antibody or antigen-binding fragment specifically binds to BCMA with an affinity of about 1×10 −10 to 1×10 −7 M; Optionally, the antibody or antigen-binding fragment specifically binds to BCMA with an affinity of about 2×10 −10 to 9×10 −10 M; (f) the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment. (g) the antibody or antigen-binding fragment is recombinant. (h) the antigen-binding fragment is a Fab fragment, a Fab2 fragment, or a single chain antibody. (i) the antibody or antigen-binding fragment thereof is of the IgG1, IgG2, IgG3, or IgG4 isotype; and / or (j) An antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is of the IgG1 or IgG4 isotype.

15. A pharmaceutical composition comprising the trispecific antibody or trispecific binding fragment of any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 13 or claim 14 and a pharma- ceutically acceptable carrier.

17. The pharmaceutical composition of claim 15 or claim 16, the pharmaceutical composition further comprises a second therapeutic agent; Optionally, the second therapeutic agent comprises an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof.

18. A cell expressing a trispecific antibody or trispecific binding fragment according to any one of claims 1 to 12, or an antibody or antigen-binding fragment according to claim 13 or claim 14.

19. The cell of claim 18, (a) the cell is a hybridoma, or (b) a cell in which the trispecific antibody is recombinantly produced.

20. The pharmaceutical composition according to any one of claims 15 to 17 for treating cancer in a subject in need thereof.

21. The pharmaceutical composition according to any one of claims 15 to 17, for inhibiting the growth or proliferation of cancer cells.

22. 22. The pharmaceutical composition of claim 21, wherein the cancer cells are in a subject and the pharmaceutical composition is administered to the subject.

23. 16. The pharmaceutical composition of claim 15 for redirecting T cells against BCMA and / or GPRC5D expressing cancer cells in a subject in need thereof.

24. The pharmaceutical composition according to any one of claims 20 to 23, (a) the cancer is a blood cancer; Optionally, the hematological cancer is a BCMA and / or GPRC5D-expressing B cell cancer; Optionally, the BCMA and / or GPRC5D expressing B cell cancer is multiple myeloma; For example, the BCMA and / or GPRC5D-expressing B cell cancer is smoldering multiple myeloma (SMM); (b) the cancer is recurrent, refractory, or malignant cancer, or any combination thereof; (c) the subject has received prior treatment; Optionally, the pretreatment comprises a proteasome inhibitor, an immunomodulatory agent, a CD38 antibody, a bispecific agent, a CAR-T therapy, or any combination thereof; (d) further comprising administering a second therapeutic agent; Optionally, (i) the second therapeutic agent is a chemotherapeutic agent or a targeted anti-cancer treatment; Optionally, the chemotherapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin 2 (IL-2); or (ii) the second therapeutic agent is an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof; (e) the trispecific antibody or trispecific binding fragment, or the pharmaceutical composition, is administered to the subject intravenously, intramuscularly, intraperitoneally, and / or subcutaneously; and / or (f) the trispecific antibody or trispecific binding fragment, or the pharmaceutical composition, is administered subcutaneously to the subject. Pharmaceutical compositions.

25. A method for inhibiting the growth or proliferation of cancer cells, comprising administering to said cells ex vivo an effective amount of a trispecific antibody or trispecific binding fragment of any one of claims 1 to 12, an antibody or antigen-binding fragment of claim 13 or claim 14, or a pharmaceutical composition of any one of claims 15 to 17, wherein said effective amount is sufficient to inhibit the growth or proliferation of said cancer cells.

26. The method of claim 25, comprising: (a) the cancer is a blood cancer; Optionally, the hematological cancer is a BCMA and / or GPRC5D-expressing B cell cancer; Optionally, the BCMA and / or GPRC5D expressing B cell cancer is multiple myeloma; For example, the BCMA and / or GPRC5D-expressing B cell cancer is smoldering multiple myeloma (SMM); (b) the cancer is recurrent, refractory, or malignant cancer, or any combination thereof; (c) the subject has received prior treatment; Optionally, the pretreatment comprises a proteasome inhibitor, an immunomodulatory agent, a CD38 antibody, a bispecific agent, a CAR-T therapy, or any combination thereof; (d) the method further comprises administering a second therapeutic agent; Optionally, (i) the second therapeutic agent is a chemotherapeutic agent or a targeted anti-cancer treatment; Optionally, the chemotherapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin 2 (IL-2); or (ii) the second therapeutic agent is an anti-CD38 agent, an immunomodulatory imide drug (IMiD), an immune checkpoint inhibitor, an immune co-stimulator, a gamma secretase inhibitor, a T cell enhancer, or any combination thereof; method.

27. A method for producing a trispecific antibody or trispecific binding fragment according to any one of claims 1 to 12, or an antibody or antigen-binding fragment according to claim 13 or claim 14, comprising culturing a cell according to claim 18 or claim 19, and isolating the trispecific antibody or trispecific binding fragment, or the antibody or antigen-binding fragment.

28. A synthetic polynucleotide encoding a trispecific antibody or trispecific binding fragment of any one of claims 1 to 12, or an antibody or antigen-binding fragment of claim 13 or claim 14.

29. 29. A kit comprising (i) a trispecific antibody or trispecific binding fragment according to any one of claims 1 to 12, an antibody or antigen-binding fragment according to claim 13 or claim 14, and / or a polynucleotide according to claim 28, and (ii) packaging therefor.

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