Anti-gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof

Abstract

Provided herein are antibodies that specifically bind to GPRC5D. The present invention also describes related polynucleotides capable of encoding the provided GPRC5D-specific antibodies or antigen-binding fragments, cells expressing the provided antibodies or antigen-binding fragments, as well as related vectors and detectably labeled antibodies or antigen-binding fragments. In addition, methods of using the provided antibodies are also described. For example, the provided antibodies can be used to diagnose, treat, or monitor the progression, regression, or stability of a GPRC5D-expressing cancer; for determining whether a cancer patient should receive treatment; or for determining whether a subject has a GPRC5D-expressing cancer and is therefore amenable to treatment with a GPRC5D-specific anti-cancer therapeutic, such as a multi-specific antibody against GPRC5D and CD3 described herein.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 364,811, filed July 20, 2016. The entire contents of the above application are incorporated herein by reference.

[0003] sequence list

[0004] This application includes a sequence list that has been electronically filed in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on June 28, 2017, named PRD3422USNP_SL.txt, and is 57,673 bytes in size. Technical Field

[0005] The disclosure provided herein relates to a monoclonal antibody that specifically binds to G protein-coupled receptor C5 family subtype D (GPRC5D), a multispecific antibody that specifically binds to GPRC5D and cluster determinant 3 (CD3), and methods for producing and using said antibodies. Background Technology

[0006] Multiple myeloma (MM) is the second most common hematologic malignancy, accounting for 2% of all cancer deaths. MM is a heterogeneous disease and is primarily caused by chromosomal translocations, 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 affected by MM may experience a variety of disease-related symptoms due to bone marrow infiltration, bone destruction, kidney failure, immunodeficiency, and the psychological burden of cancer diagnosis. As of 2006, the 5-year relative survival rate for MM was approximately 34%, highlighting MM as a refractory disease with no current cure options.

[0007] G protein-coupled receptor C5 family subtype D (GPRC5D) is an orphan SARS-related class C GPCR first identified in 2001. -Osborne et al., Biochim Biophys Acta. 1518(3):237-248, 2001). GPRC5D and the other five GPCRs generally possess the shorter N-terminal domain of class C receptors and are therefore predicted to be conformationally similar to class A receptors. In this respect, they are unique, sharing sequence homology with class C GPCRs and having predicted structural topology equivalent to class A receptors. The functional outcomes of GPRC5D activation have not been described and the ligands remain unknown. Its gene has three exons and is located on human chromosome 12p13.3. The GPRC5D receptor is highly conserved across species and shares 92% identity with the cynomolgus monkey GPRC5D.

[0008] GPRC5D mRNA is mainly expressed in all malignant plasma cells of 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 is closely associated with plasma cell load and genetic aberrations such as Rb-1 deletion (Atamaniuk JA et al. Eur J Clin Invest 42(9)953-960; 2012).

[0009] The unique expression of GPRC5D in plasma cell lineages makes it an ideal target for anti-myeloma antibodies. Summary of the Invention

[0010] This article provides antibodies that specifically bind to GPRC5D and their antigen-binding fragments. It also describes the associated polynucleotides encoding the provided GPRC5D-specific antibodies and antigen-binding fragments, the cells expressing the provided antibodies and antigen-binding fragments, and the associated vectors and antibody-antigen-binding fragments with detectable labels. Furthermore, methods for using the provided antibodies and antigen-binding fragments are described. For example, GPRC5D-specific antibodies and antigen-binding fragments can be used to diagnose or monitor the progression, regression, or stability of GPRC5D-expressing cancers; to determine whether a cancer patient should receive treatment; or to determine whether a patient has GPRC5D-expressing cancer and is therefore suitable for treatment with GPRC5D-specific anticancer agents such as the multispecific antibodies against GPRC5D and CD3 described herein.

[0011] This article also provides a multispecific antibody that binds specifically to GPRC5D and CD3, and its multispecific antigen-binding fragment. This article also describes the associated polynucleotides encoding the provided GPRC5D×CD3 multispecific antibody, the cells expressing the provided antibody, and the associated vectors and detectable markers of the multispecific antibody. Furthermore, methods for using the provided multispecific antibody are described. For example, the GPRC5D×CD3 multispecific antibody can be used to diagnose or monitor the progression, regression, or stability of GPRC5D-expressing cancers; to determine whether a cancer patient should receive treatment; or to determine whether a patient has GPRC5D-expressing cancer and is therefore suitable for treatment with GPRC5D-specific anticancer agents such as the GPRC5D×CD3 multispecific antibody described herein.

[0012] GPRC5D specific antibody

[0013] This document describes a GPRC5D-specific isolation antibody and antigen-binding fragment. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment bind to human GPRC5D. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment bind to both human GPRC5D and cynomolgus monkey GPRC5D. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment bind to one or more residues of a polypeptide having the amino acid sequence of SEQ ID NO. 22. This GPRC5D-specific antibody or antigen-binding fragment is capable of EC50 at 28 nM or less. 50 ADCC was induced in vitro.

[0014] Table 1 provides a summary of some examples of GPRC5D-specific antibodies described in this article:

[0015] Table 1: CDR sequences of mAbs generated from human GPRC5D

[0016]

[0017]

[0018] In some embodiments, a GPRC5D-specific antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1. In some embodiments, a GPRC5D-specific antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1, and a light chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1. In some embodiments described herein, the GPRC5D-specific antibody or the antigen-binding fragment thereof competitively binds to GPRC5D with an antibody or antigen-binding fragment comprising a heavy chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1, and a light chain containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1.

[0019] Human IgG isoforms are classified into four types: IgG1, IgG2, IgG3, and IgG4. They share more than 95% homology in the amino acid sequence of their Fc region, but exhibit major differences in the amino acid composition and structure of their hinge region. The Fc region mediates effector functions such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cell cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to an Fc receptor (FcgR) on the surface of immune effector cells, such as natural killer cells and macrophages, leading to phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering a complement cascade reaction on the cell surface. The antibodies described herein include antibodies having the aforementioned features in combination with any IgG isoform, including modified forms in which the Fc sequence has been modified to achieve different effector functions.

[0020] For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions can be detrimental and may pose safety risks by inducing extramechanistic toxicity. Modification of effector functions can be achieved by altering the Fc region to weaken its binding to FcgR or complement factors. The binding of IgG to the first component of activating FcgR (FcgRI, FcgRIIa, FcgRIIIa, and FcgRIIIb) and inhibitory FcgR (FcgRIIb) or complement (C1q) depends on residues located in the hinge region and CH2 domain. Mutations have been introduced in IgG1, IgG2, and IgG4 to reduce or silence Fc function. Antibodies described herein may include these modifications.

[0021] In one embodiment, the antibody comprises an Fc region having one or more of the following characteristics: (a) reduced effector function compared to the parental Fc; (b) reduced affinity for FcgRI, FcgRIIa, FcgRIIb, FcgRIIIb and / or FcgRIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (e) reduced affinity for FcgRIIb; (f) reduced affinity for FcgRIIIb; or (g) reduced affinity for FcgRIIIa.

[0022] In some embodiments, the antibody or antigen-binding fragment is IgG or a derivative thereof, such as IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments where the antibody has an IgG1 isotype, the antibody contains L234A, L235A, and / or K409R substitutions in its Fc region. In some embodiments where the antibody has an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region. The antibodies described herein may include these modified forms.

[0023] In addition to the GPRC5D-specific antibody and antigen-binding fragment described herein, a polynucleotide sequence encoding the antibody and antigen-binding fragment is also provided. A vector comprising the polynucleotide is also provided, as well as cells expressing the GPRC5D-specific antibody or antigen-binding fragment provided herein. Cells capable of expressing the disclosed vector are also described. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as Escherichia coli). The antibody may also be produced by hybridoma cells.

[0024] Method using GPRC5D specific antibodies

[0025] Methods for using the GPRC5D-specific antibodies or antigen-binding fragments are also disclosed. Specific antibodies used in the methods discussed in this section include those having the group of CDRs described in Table 1. For example, these antibodies or antigen-binding fragments can be used to treat cancer by interfering with GPRC5D-receptor interactions or by having the antibody conjugate to a toxin, thereby targeting GPRC5D-expressing cancers. Furthermore, these antibodies or antigen-binding fragments can also be used to detect the presence of GPRC5D in biological samples such as blood or serum; to quantitatively analyze the amount of GPRC5D in biological samples such as blood or serum; to diagnose GPRC5D-expressing cancers; to determine methods for treating patients with cancer; or to monitor the progression of GPRC5D-expressing cancers in patients. In some embodiments, GPRC5D-expressing cancers may be lymphomas such as multiple myeloma (MM). The methods can be performed before the patient receives treatment for GPRC5D-expressing cancers, such as treatment with a multispecific antibody targeting GPRC5D and CD3. Furthermore, the method can be performed after the patient has received treatment for GPRC5D-expressing cancers, such as with a multispecific antibody targeting GPRC5D and CD3 as described herein.

[0026] The method for detecting GPRC5D in biological samples includes exposing the biological sample to one or more of the GPRC5D-specific antibodies or antigen-binding fragments described herein.

[0027] The method for diagnosing GPRC5D-expressing cancer in a treated subject further includes exposing a biological sample to one or more of the GPRC5D-specific antibodies or antigen-binding fragments described herein; however, the method also includes quantifying the amount of GPRC5D present in the sample; comparing the amount of GPRC5D present in the sample with a known standard or reference sample; and determining whether the treated subject's GPRC5D level falls within the range of cancer-associated GPRC5D levels.

[0028] This article also describes a method for monitoring GPRC5D-expressing cancers in treated subjects. The method includes exposing a biological sample to one or more of the GPRC5D-specific antibodies or antigen-binding fragments described herein; quantifying the amount of GPRC5D present in the sample bound by the antibody or its antigen-binding fragment; comparing the amount of GPRC5D present in the sample with the amount of GPRC5D in a known standard or reference sample or a similar sample previously obtained from the treated subject; and determining, based on the difference in the amount of GPRC5D in the compared samples, whether the treated subject's GPRC5D level indicates cancer progression, regression, or stable disease.

[0029] Samples obtained from or derived from the patient are biological samples, such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells, tissues, surgically removed tumor tissue, biopsies, fine-needle aspiration samples, or histological preparations.

[0030] The GPRC5D-specific antibody or antigen-binding fragment described herein can be labeled for use in the method described herein or other methods known to those skilled in the art. For example, the antibody or antigen-binding fragment described herein can be labeled with radiolabels, fluorescent labels, epitope tags, biotin, chromophore labels, ECL labels, enzymes, ruthenium, etc. 111 In-DOTA, 111 Labeled with In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and β-galactosidase, or polyhistidine or similar labels known in the art.

[0031] GPRC5D Specific Antibody Kit

[0032] This document describes a kit comprising the disclosed GPRC5D-specific antibody or antigen-binding fragment thereof. The kit can be used to implement methods using the GPRC5D-specific antibody or antigen-binding fragment provided herein or other methods known to those skilled in the art. In some embodiments, the kit may include the antibody or antigen-binding fragment described herein, as well as reagents for detecting the presence of GPRC5D in a biological sample. Therefore, the kit may include one or more of the antibodies or antigen-binding fragments described herein, as well as a container for holding the antibody or fragment when not in use, instructions for use of the antibody or fragment, the antibody or fragment attached to a solid support, and / or a detectable labeled form of the antibody or fragment as described herein.

[0033] GPRC5D×CD3 multispecific antibody

[0034] Redirecting T lymphocytes to GPRC5D-expressing MM cells via the TCR / CD3 complex represents an attractive alternative approach. The TCR / CD3 complex of T lymphocytes consists of TCRα / β or TCRγ / δ heterodimers co-expressed on the cell surface, along with CD3-labeled invariant subunits of γ, δ, ε, ζ, and η. Human CD3ε is described as UniProt P07766 (CD3E_HUMAN). The anti-CD3ε antibody described in the prior art is SP34 (Yang SJ, The Journal of Immunology, (1986) 137; 1097-1100). SP34 reacts with primate and human CD3. SP34 was purchased from Pharmingen. Another anti-CD3 antibody described in the prior art is UCHT-1 (see WO2000041474). Another anti-CD3 antibody described in the prior art is BC-3 (Fred Hutchinson Cancer Research Institute, for a phase I / II trial 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 ε chain of CD3 (see Salmeron et al., 1991, J. Immunol., Vol. 147, p. 3047), while UCHT-1 and BC-3 recognize epitopes contributed by both the ε and γ chains. Sequences of antibodies with the same sequence as antibody SP34 are mentioned in WO2008119565, WO2008119566, WO2008119567, WO2010037836, WO2010037837, and WO2010037838. US8236308 (WO2007042261) mentions a sequence that has 96% identity with the VH of antibody SP34.

[0035] This document describes a separation multispecific antibody (“GPRC5D×CD3 multispecific antibody”) that binds to GPRC5D and CD3, and its multispecific antigen-binding fragment. In some embodiments, a separation antibody or its antigen-binding fragment that specifically binds to GPRC5D is provided.

[0036] In some embodiments, the GPRC5D-specific arm of a multispecific antibody binds to human GPRC5D and cynomolgus monkey GPRC5D. In some embodiments, the GPRC5D-specific arm of a GPRC5D×CD3 multispecific antibody or antigen-binding fragment binds to the extracellular domain of human GPRC5D. In a preferred embodiment, the GPRC5D×CD3 multispecific antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment. In some embodiments, an isolated GPRC5D×CD3 bispecific antibody or its GPRC5D×CD3 bispecific binding fragment is provided comprising: a) a first heavy chain (HC1); b) a second heavy chain (HC2); c) a first light chain (LC1); and d) a second light chain (LC2), wherein HC1 and LC1 pair to form a first antigen-binding site that specifically binds to GPRC5D, and HC2 and LC2 pair to form a second antigen-binding site that specifically binds to CD3. In another embodiment, isolated cells expressing said antibody or bispecific binding fragment are provided. In some implementations, the GPRC5D binding arm (or “GPRC5D specific arm”) of the GPRC5D×CD3 multispecific antibody is derived from the GPRC5D antibody described herein (e.g., derived from an antibody having the CDR sequence listed in Table 1).

[0037] In some embodiments, the GPRC5D-specific arm of the GPRC5D×CD3 multispecific antibody or antigen-binding fragment is IgG or a derivative thereof. In some embodiments, the CD3-binding arm (or “CD3-specific arm”) of the GPRC5D×CD3 multispecific antibody is derived from mouse monoclonal antibody SP34, a mouse IgG3 / λ isotype (K.R. Abhinandan and A.C. Martin, 2008, Mol. Immunol., 45, 3832-3839). In some embodiments, the CD3-binding arm of the GPRC5D×CD3 multispecific antibody includes a VH domain and a VL domain selected from Table 2.

[0038] Table 2: Heavy and light chains of CD3-specific antibody-antigen binding fragments:

[0039]

[0040] Human IgGs are classified into four isotypes: IgG1, IgG2, IgG3, and IgG4. They share more than 95% homology in the amino acid sequence of their Fc region, but exhibit major differences in the amino acid composition and structure of their hinge region. The Fc region mediates effector functions such as antibody-dependent cell cytotoxicity (ADCC) and complement-dependent cell cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to an Fc receptor (FcgR) on the surface of immune effector cells, such as natural killer cells and macrophages, leading to phagocytosis or lysis of the target cell. In CDC, the antibody kills the target cell by triggering a complement cascade on the cell surface.

[0041] For many applications of therapeutic antibodies, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions can be detrimental and may pose safety risks by inducing extramechanistic toxicity. Modification of effector functions can be achieved by altering the Fc region to weaken its binding to FcgR or complement factors. The binding of IgG to the first component of activating FcgR (FcgRI, FcgRIIa, FcgRIIIa, and FcgRIIIb) and inhibitory FcgR (FcgRIIb) or complement (C1q) depends on residues located in the hinge region and CH2 domain. Mutations have been introduced in IgG1, IgG2, and IgG4 to reduce or silence Fc function.

[0042] In one embodiment, the antibody comprises an Fc region having one or more of the following characteristics: (a) reduced effector function compared to the parental Fc; (b) reduced affinity for FcgRI, FcgRIIa, FcgRIIb, FcgRIIIb and / or FcgRIIIa; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (e) reduced affinity for FcgRIIb; (f) reduced affinity for FcgRIIIb; or (g) reduced affinity for FcgRIIIa.

[0043] In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody is IgG or a derivative thereof. In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody is IgG1 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG1 antibody of the derived CD3-binding arm includes L234A, L235A, and F405L substitutions in its Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody is IgG4 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG4 antibody of the derived CD3-binding arm includes S228P, L234A, L235A, F405L, and R409K substitutions in its Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment of the CD3-specific arm of the derived multispecific antibody binds to CD3ε on primary human T cells and / or primary cynomolgus monkey T cells. In some implementations, CD3-specific antibodies or antigen-binding fragments derived from the CD3-specific arm of a multispecific antibody activate primary human CD4+ T cells and / or primary cynomolgus CD4+ T cells.

[0044] In addition to the GPRC5D×CD3 multispecific antibody, a polynucleotide sequence encoding the GPRC5D×CD3 multispecific antibody is also provided. In some embodiments, isolated and synthesized polynucleotides of HC1, HC2, LC1, or LC2 encoding a GPRC5D×CD3 bispecific antibody or a bispecific binding fragment are provided. A vector comprising the polynucleotide is also provided, as well as cells expressing the GPRC5D×CD3 multispecific antibody provided herein. Cells capable of expressing the disclosed vector are also described. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as Escherichia coli). The antibody may also be produced by hybridoma cells. In some embodiments, a method for producing a GPRC5D×CD3 bispecific antibody or a bispecific binding fragment by culturing cells is provided.

[0045] This article also provides pharmaceutical compositions containing GPRC5D×CD3 multispecific antibodies or antigen-binding fragments, as well as pharmaceutically acceptable carriers.

[0046] Method using GPRC5D×CD3 multispecific antibody

[0047] Methods for using the GPRC5D×CD3 multispecific antibody and its multispecific antigen-binding fragment are also disclosed. For example, the GPRC5D×CD3 multispecific antibody and its multispecific antigen-binding fragment can be used to treat GPRC5D-expressing cancers in patients in need of treatment. In some embodiments, the GPRC5D-expressing cancer is a lymphoma such as multiple myeloma.

[0048] The method of treating GPRC5D-expressing cancer in a subject in need of treatment includes administering a therapeutically effective amount of the GPRC5D×CD3 multispecific antibody or its multispecific antigen-binding fragment to the subject. In some embodiments, the subject is a mammal, preferably a human. In a preferred embodiment, a method is provided to treat a subject with cancer by administering a therapeutically effective amount of the GPRC5D×CD3 bispecific antibody or bispecific antigen-binding fragment to a patient in need for a time sufficient to treat the cancer.

[0049] This article also provides a method for inhibiting the growth or proliferation of cancer cells by administering a therapeutically effective amount of a GPRC5D×CD3 bispecific antibody or a bispecific binding fragment to inhibit the growth or proliferation of cancer cells.

[0050] This article also provides a method for redirecting T cells to GPRC5D-expressing cancer cells by administering a therapeutically effective amount of a GPRC5D×CD3 bispecific antibody or a bispecific binding fragment.

[0051] GPRC5D×CD3 Specific Antibody Kit

[0052] This document describes a kit comprising the disclosed GPRC5D×CD3 multispecific antibody. The kit can be used to implement methods using the GPRC5D×CD3 multispecific antibody provided herein or other methods known to those skilled in the art. In some embodiments, the kit may include the antibody described herein and reagents for treating GPRC5D-expressing cancers. Therefore, the kit may include one or more of the multispecific antibodies described herein or multispecific antigen-binding fragments thereof, as well as containers for containing the antibody or fragment when not in use and / or instructions for use of the antibody or fragment, the antibody or fragment attached to a solid support, and / or a detectable labeled form of the antibody or fragment as described herein. Attached Figure Description

[0053] Figure 1The selected anti-GPRC5D mAb exhibited concentration-dependent binding characteristics against human GPRC5D and untransfected HEK293 cells. Three mAbs, GC5B36, GC5B168, and GC5B205, were also observed to bind to untransfected (GPRC5D blank) HEK293 cells.

[0054] Figure 2 Dose-dependent binding of anti-GPCR5D×CD3 bispecific antibody to human GPRC5D HEK293F cells.

[0055] Figure 3 Using FACS, the binding characteristics of bispecific antibodies to MM1R and H929 cells were compared with those of overexpressing human GPCR5D HEK293 cells and untransfected HEK293 cells.

[0056] Figure 4A and 4B Anti-GPCR5D×CD3 antibody induces T-cell cytotoxicity and T-cell activation in human GPRC5D-expressing cells.

[0057] Figure 5A and 5B Anti-GPCR5D×CD3 antibody against T-cell-mediated cytotoxicity and T-cell activation in cyno GPRC5D-expressing cells.

[0058] Figure 6A and 6B GPRC5D×CD3 Ab effectively killed H929 cells in a mouse model of NSG prevention. GCDB32 (6A) and GCDB35 (6B) caused complete tumor growth inhibition at a dose of 10 μg, and GCDB32 was also able to inhibit tumor growth 100% at a dose of 1 μg.

[0059] Figure 7A and 7B Comparison of effectiveness in the absence of (7A) and presence of (7B) Fc blocking.

[0060] Figures 8A-8D GCDB32, GCDB35, GCDB40 and GCDB43 bind to the Fcγ receptor.

[0061] Figure 9A and 9B FACS binding assessment of hybridoma-derived mAbs.

[0062] Figure 10A and 10B GPRC5D×CD3 bispecific antibody against T-cell-mediated cytotoxicity in H929 cells.

[0063] Figure 11A-11E : A bispecific antibody against GPRC5D×CD3 that binds to GPRC5D-positive (H929, MM1R, LP1, OPM2) and negative cell lines (NALM6).

[0064] Figure 12A-12D GPRC5D×CD3 bispecific Abs are potent tumor inhibitors in vivo. All GPRC5D×CD3 bispecific Abs (GCDB32 in 12A, GCDB53 in 12B, GCDB61 in 12C, and GCDB72 in 12D) completely inhibited the growth of multiple myeloma cells (H929) at doses of 10 μg and 1 μg. Differentiation was observed at a dose of 0.1 μg, and GCDB72 was observed to have 80% inhibition of tumor growth.

[0065] Figure 13 : GPRC5D + MM1.R cell lines were stained with various concentrations of leader antibodies for 60 minutes to measure surface binding characteristics (n=3). Phycoerythrin-labeled human IgG4Fc was used as a secondary antibody to capture the signal (Southern Biotech, clone HP6025). Binding is expressed as normalized geometric mean fluorescence intensity, as measured by FACS. Data were plotted and fitted in GraphPad Prism 6 using nonlinear regression with variable slopes (four parameters) and least squares fitting.

[0066] Figure 14A and 14B GPRC5D+ cell lines were stained with various concentrations of FAB6300 and GC5M481 antibodies for 60 minutes to measure surface binding characteristics. Phycoerythrin-labeled human IgG4Fc was used as a secondary antibody to capture the signal (SouthernBiotech, clone HP6025). Binding is represented by a bar graph (black lines represent isotypes, and red lines represent specific GPRC5D antibodies). Figure 14A ). Figure 14B The binding pattern of the leader molecule to the GPRC5D+ multiple myeloma cell line is shown. Dark dashed lines indicate isotype controls, and solid lines indicate leader molecule binding.

[0067] Figure 15A and 15BCompared to an IgG4 isotype control, frozen bone marrow-derived mononuclear cells from two different MM patients were used to assess GPRC5D×CD3 bispecific antibody binding, plasma cell cytotoxicity, and T-cell activation. For the cytotoxicity assay, exogenous T cells from healthy donors were added to patient BMMNC samples and incubated with four leader molecules for 48 hours. The GPRC5D×CD3 bispecific antibody bound to plasma cells in all donor samples in a dose-dependent manner, and mean fluorescence intensity was recorded on the Y-axis. It was noted that in response to GPRC5D×CD3 bispecific antibody treatment, live plasma cells (CD138)... + The cells are lost and CD25 on T cells is simultaneously upregulated.

[0068] Figure 16 On day 0, NSG mice were subcutaneously implanted with MM.1S human multiple myeloma cells. Human PBMCs were intravenously inoculated on day 7. On days 15, 18, 22, 24, 29, 32, and 36, mice were administered PBS, GCDB72 (0.1 μg, 1 μg, 10 μg, and 50 μg / animal (equivalent to 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, and 2.5 mg / kg, respectively) and a blank control antibody intravenously. Subcutaneous tumors were measured twice weekly, and results are expressed as mean tumor volume (in mm for each group). 3 (Mean ± SEM). Compared to PBS, GCDB72 antibody treatment significantly inhibited sc tumor growth at a dose of 1 μg (0.05 mg / kg) (TGI = 64%, p ≤ 0.0001). GCDB72 doses of 10 μg / animal (0.5 mg / kg) and 50 μg / animal (2.5 mg / kg) completely regressed tumor growth (p ≤ 0.0001). The blank control antibody had a negligible or no effect. Multiple comparisons were performed using Tukey's multiple comparison test with Graph Pad Prism software (version 6), and statistical significance was evaluated using two-way ANOVA. Differences between groups were considered significant when the probability value (p) ≤ 0.05.

[0069] Figure 17 On day 0, MM.1S human multiple myeloma cells were subcutaneously implanted into NSG. Human PBMCs were intravenously inoculated on day 7. On days 15, 18, 22, 24, 29, 32, and 36, animals were administered PBS, GCDB72 (0.1 μg, 1 μg, 10 μg, and 50 μg / animal (equivalent to 0.005 mg / kg, 0.05 mg / kg, 0.5 mg / kg, and 2.5 mg / kg, respectively), and a blank control antibody intravenously. Body weight is expressed as absolute body weight from the start of treatment to the end of the study. Detailed Implementation

[0070] definition

[0071] Various terms relating to various aspects of the specification are used throughout the specification and claims. Unless otherwise specified, such terms are given their ordinary meaning in the art. Other specifically defined terms should be understood in accordance with the definitions provided herein.

[0072] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0073] As used herein, the term "about," when referring to measurable values ​​such as quantity, time interval, etc., means to cover a variation of at most ±10% from the specified value, as such variation is suitable for performing the methods disclosed in this invention. Unless otherwise specified, all figures used in the specification and claims to represent the quantity of components, characteristics such as molecular weight, reaction conditions, etc., should in all cases be understood to be modified by the term "about." Therefore, unless stated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values, which may vary according to the desired characteristics sought to be obtained according to the invention. To a minimum and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should at least be interpreted according to the significant digits of the reported value and by applying customary rounding.

[0074] Although the numerical ranges and parameters used to illustrate the broad scope of the invention are approximate, the values ​​presented in specific embodiments are reported as precisely as possible. However, any numerical value inherently contains some error, which will necessarily arise from the standard deviation present in its respective test measurement method.

[0075] "Isolation" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, isolated, or purified from other biological components of the organism in which the component is naturally present (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins). Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. "Isolated" nucleic acids, peptides, and proteins may be part of a composition, and are still considered isolated if such a composition is not part of the environment of the nucleic acid, peptide, or protein itself. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids. As used herein, "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 to GPRC5D is substantially free of antibodies that specifically bind to antigens other than GPRC5D). However, isolated antibodies that specifically bind to epitopes, subtypes, or variants of GPRC5D may be cross-reactive with other related antigens, such as antigens from other species (e.g., GPRC5D species homologs).

[0076] The term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region 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 a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in many ways; therefore, "polynucleotide" includes chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides that are normally naturally occurring, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often referred to as oligonucleotides.

[0077] The meaning of "substantially identical" can vary depending on the context in which the term is used. Due to natural sequence variations that may exist between the heavy and light chains and the genes encoding them, a degree of variation is expected in the amino acid sequence or in the genes encoding the antibody or antigen-binding fragments described herein, with little or no impact on their unique binding properties (e.g., specificity and affinity). This expectation is partly due to the degeneracy of the genetic code and the successful evolution of conserved amino acid sequence variations, but this does not significantly alter the properties of the encoded protein. Therefore, in the context of nucleic acid sequences, "substantially identical" means that two or more sequences share at least 65% identity. Preferably, the term refers to a homology of at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% or higher. The percentage homology between two sequences is a function of the number of common positions shared by the sequences (i.e., homology % = number of common positions / total number of positions × 100). These parameters need to be introduced for optimal alignment of two sequences, taking into account the number of gaps and the length of each gap. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) (which has been incorporated into the ALIGN program (version 2.0)), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0078] The degree of variation that may occur in the amino acid sequence of a protein without substantially affecting its function is far less than that in the nucleic acid sequence, because the same degeneracy principle does not apply to amino acid sequences. Therefore, in the context of antibody or antigen-binding fragments, "substantially identical" means an antibody or antigen-binding fragment having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the stated antibody or antigen-binding fragment. Other embodiments include GPRC5D-specific antibody or antigen-binding fragments having a framework, scaffold, or other non-binding region that does not have significant identity with the antibody and antigen-binding fragments described herein, but does incorporate one or more CDRs or other sequences required to confer 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such sequences described herein. A vector is a replicon, such as a plasmid, bacteriophage, granule, or virus, in which another nucleic acid segment can be operatively inserted to cause replication or expression of that segment.

[0079] A "clone" is a population of cells derived from a single cell or common progenitor cell through mitosis. A "cell line" is a clone of primary cells that can stably grow for many generations in vitro. In some examples provided in this article, cells are transformed by transfecting them with DNA.

[0080] The terms “expression” and “production” are used synonymously herein and refer to the biosynthesis of gene products. These terms cover transcription from gene to RNA. They also cover translation from RNA to one or more polypeptides, and all naturally occurring post-transcriptional and post-translational modifications. The expression or production of antibodies or their antigen-binding fragments can occur in the cytoplasm of cells or in extracellular environments such as growth media for cell cultures.

[0081] The term "treatment" refers to any success or indication of success achieved in reducing or improving an injury, lesion, or condition, including any objective or subjective parameters such as symptom reduction, relief, weakening, or increased patient tolerance of the condition; slowing the rate of degeneration or decline; reducing the degree of failure at the degenerative endpoint; improving the physical or mental health of the treated individual; or prolonging survival. Treatment can be assessed through objective or subjective parameters, including the results of physical examination, neurological examination, or psychiatric evaluation.

[0082] "Effective dose" or "therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome within the required dose and time period. The therapeutic effective dose of GPRC5D×CD3 antibodies can vary depending on factors such as an individual's disease state, age, sex, weight, and the antibody's ability to elicit the desired response in an individual. Therapeutic effective dose is also the amount in which the beneficial therapeutic effect of the antibody or antibody fraction far outweighs any toxic or harmful effects.

[0083] Unless otherwise stated, “antibody” refers to all isotypes (IgG, IgA, IgE, IgM, IgD, and IgY) of immunoglobulins in various monomeric, polymeric, and chimeric forms. The term “antibody” specifically encompasses polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like peptides such as chimeric and humanized antibodies.

[0084] An "antigen-binding fragment" is any protein structure that exhibits binding affinity for a specific antigen. Antigen-binding fragments include those provided by any known technique such as enzyme cleavage, peptide synthesis, and recombinant technology. Some antigen-binding fragments consist of a portion of the 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 (heavy chain variable region 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, but are not limited to, bispecific antibodies and single-chain molecules, as well as Fab, F(ab')2, Fc, Fac, and Fv molecules; single-chain (Sc) antibodies; single antibody light chains; single antibody heavy chains; chimeric fusions of antibody chains or CDRs with 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 VL, VH, CL, and CH1 domains; or monovalent antibodies as described in WO2007059782; bivalent fragments comprising two Fab fragments linked by disulfide bonds in hinge regions; Fd fragments consisting essentially of V.sub.H and C.sub.H1 domains; Fv fragments consisting essentially of the VL and VH domains of a single arm of the antibody; dAb fragments consisting essentially of the VH domain (Ward et al., Nature 341, 544-546 (1989)), and also referred to as domain antibodies (Holt et al., Trends). Biotechnol., Nov 2003, 21(11):484-90); alpaca or nanobodies (Revets et al., Expert Opin Biol Ther., Jan 2005, 5(1):111-24); separation of complementarity-determining regions (CDRs), etc. All antibody isotypes can be used to generate antigen-binding fragments. In addition, antigen-binding fragments may include non-antibody protein frameworks that can be successfully incorporated into polypeptide segments in an orientation that confers affinity for a given antigen of interest (such as a protein scaffold). Antigen-binding fragments can be generated by recombinant methods or by enzymatic or chemical cleavage of the intact antibody. The phrase “antibody or antigen-binding fragment thereof” can be used to indicate that a given antigen-binding fragment is incorporated into one or more amino acid segments of the antibody mentioned in the phrase.

[0085] The term "CDR" refers to the complementarity-determining region (CDR), which consists of three binding features constituting the light chain variable region (CDRL1, CDRL2, and CDRL3) and three binding features constituting the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of antibody molecules and are separated by amino acid sequences containing scaffold or framework regions. The precisely defined boundaries and lengths of CDRs are constrained by different classification and numbering systems. Therefore, CDRs can be referred to by Kabat, Chothia, contact, or any other boundary definition. Despite the different boundaries, each of these systems has a degree of overlap in the aspects constituting so-called "hypervariate regions" within the variable sequences. Therefore, CDR definitions can differ in length and boundary regions relative to adjacent framework regions according to these systems. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 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 BindingSite Topography”, J.Mol.Biol. 262:732 (1996); each of these articles is incorporated herein by reference in full.

[0086] Typically, CDRs form ring structures that can be classified as canonical structures. The term "canonical structure" refers to the backbone conformation adopted by the antigen-binding (CDR) ring. From comparative structural studies, five out of six antigen-binding rings have been found to have only a limited library of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Therefore, corresponding loops between antibodies can have very similar three-dimensional structures, although most regions of the loop have high amino acid sequence variability (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”, I 342:877 (1989); Martin and Thornton, “Structural Families in Loops of Homologous Proteins: Automatic Classification, Modelling and Application to Antibodies”, J.Mol.Biol. 263:800 (1996); each of these articles is incorporated herein by reference in full). Furthermore, there is a relationship between the loop structure and the surrounding amino acid sequence. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop and within the conserved framework (i.e., outside the loop). Therefore, the assignment of a specific canonical class can be based on the presence or absence of these key amino acid residues.

[0087] The term "peptide" is used interchangeably with the term "protein" and, in its broadest sense, refers to a compound consisting of two or more subunit amino acids, amino acid analogs, or peptide mimics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to natural and / or non-natural amino acids or synthetic amino acids, including glycine and its D and L optical isomers, amino acid analogs, and peptide mimics. In cases where the peptide chain is short, peptides with three or more amino acids are typically referred to as oligopeptides. If the peptide chain is long, the peptide is typically referred to as a polypeptide or protein.

[0088] When used in the context of antibodies or antibody fragments, "specific binding" or "specifically binding," or derivatives thereof, means binding to one or more epitopes of the 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 mixed molecular population. Typically, antibodies are measured in quantities less than about 1 × 10⁻⁶, as measured by surface plasmon resonance assays or cell binding assays. -8 M of K d Binds to a homologous antigen. Phrases such as “[antigen] specific” antibody (e.g., GPRC5D specific antibody) are intended to express that the antibody specifically binds to the antigen.

[0089] The term "polynucleotide," synonymously referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA consisting of a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA consisting of a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Furthermore, "polynucleotide" refers to a triple-stranded region 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 a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and rare bases such as inosine. DNA and RNA can be modified in many ways; therefore, "polynucleotide" includes chemically modified, enzymatically modified, or metabolically modified forms of polynucleotides that are normally naturally occurring, as well as chemical forms of DNA and RNA specific to viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often referred to as oligonucleotides.

[0090] A vector is a replicon, such as a plasmid, bacteriophage, granule, or virus, in which another nucleic acid segment can be operatively inserted to cause replication or expression of that segment.

[0091] As used herein, the term "host cell" can be any type of cell, such as primary cells, cells in culture, or cells derived from a cell line. In a specific embodiment, the term "host cell" refers to cells transfected with nucleic acid molecules and the offspring or potential offspring of such cells. The offspring of such cells may differ from the parent cells transfected with nucleic acid molecules, for example, due to mutations or environmental influences that may occur in the offspring, or due to the integration of the nucleic acid molecules into the host cell genome. The terms "expression" and "production" are used synonymously herein and refer to the biosynthesis of a gene product. These terms cover transcription from gene to RNA. These terms also cover translation from RNA to one or more polypeptides, and also cover all naturally occurring post-transcriptional and post-translational modifications. The expression or production of antibodies or their antigen-binding fragments can occur in the cytoplasm of cells or in an extracellular environment such as a growth medium for cell cultures. The meaning of "substantially identical" may vary depending on the context in which the term is used. Due to the natural sequence variations that may exist between the heavy and light chains and the genes encoding them, some degree of variation is expected in the amino acid sequences or in genes encoding the antibody or antigen-binding fragments described herein, with little or no impact on their unique binding properties (e.g., specificity and affinity). This expectation is partly attributable to the degeneracy of the genetic code and the successful evolution of conserved amino acid sequence variations, but this does not significantly alter the properties of the encoded protein. Therefore, in the context of nucleic acid sequences, "substantially identical" means that two or more sequences share at least 65% identity. Preferably, the term refers to a homology of at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% or higher. The percentage homology between two sequences is a function of the number of common positions shared by the sequences (i.e., homology % = number of common positions / total number of positions × 100). These parameters need to be introduced for optimal alignment of two sequences, taking into account the number of gaps and the length of each gap. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) (which has been incorporated into the ALIGN program (version 2.0)), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.Furthermore, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0092] The degree of variation that may occur in the amino acid sequence of a protein without substantially affecting its function is far less than that in the nucleic acid sequence, because the same degeneracy principle does not apply to amino acid sequences. Therefore, in the context of antibody or antigen-binding fragments, "substantially identical" means an antibody or antigen-binding fragment having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the stated antibody or antigen-binding fragment. Other embodiments include GPRC5D-specific antibody or antigen-binding fragments having a framework, scaffold, or other non-binding region that does not have significant identity with the antibody and antigen-binding fragments described herein, but does incorporate one or more CDRs or other sequences required to confer 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with such sequences described herein.

[0093] The term "subject of treatment" refers to both humans and non-human animals, including all vertebrates such as mammals and non-mammals such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. In many embodiments of the method, the subject of treatment is a human.

[0094] As used herein, the term “redirection” or “reorientation” refers to the ability of a GPRC5D×CD3 antibody to efficiently exchange T cell activity from its inherent homology specificity to reactive cells expressing anti-GPRC5D.

[0095] As used herein, the term "sample" refers to a collection of fluids, cells, or tissues isolated from the patient (e.g., surgically removed tumor tissue, biopsy sections, including fine-needle aspiration tissue) and present in the patient. In some embodiments, the sample is a biological fluid. Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids present in, extracted from, expressed from, or otherwise extracted from the patient or biological source. Some biological fluids originate from a specific tissue, organ, or localized region, and some other biological fluids may be more systemic or present in the patient or biological source. Examples of biological fluids include blood, serum, and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites such as those associated with non-solid tumors, fluids from the pleura, pericardium, peritoneum, abdomen, and other body cavities, fluids collected via bronchial lavage, etc. Biofluids may also include liquid solutions that come into contact with the patient or biological source, such as cell and organ culture media, including cell or organ conditioned media, lavage solutions, etc. As used herein, the term "sample" encompasses substances removed from or present in the patient.

[0096] "Known standards" may be solutions containing a known amount or concentration of GPRC5D, wherein the solution may be a naturally occurring solution, such as a sample from a patient known to have early, intermediate, late, progressive, or static cancer; or the solution may be a synthetic solution, such as a buffered aqueous solution in which a known amount of GPRC5D is diluted. Known standards described herein may include GPRC5D isolated from treated subjects, recombinant or purified GPRC5D protein, or GPRC5D concentration values ​​associated with disease symptoms.

[0097] As used herein, the terms “G protein-coupled receptor C5 family subtype D” and “GPRC5D” specifically include human GPRC5D proteins, such as those described 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).

[0098] The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex consists of six distinct polypeptide chains. These chains include the CD3γ chain (SwissProt P09693), the CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766), and a CD3ζ chain homodimer (SwissProt 20963), and the complex associates with the T cell receptor α and β chains. Unless otherwise stated, the term "CD3" includes any CD3 variant, isotype, and species homolog that is naturally expressed by cells (including T cells) or can be expressed on cells transfected with genes or cDNA encoding those polypeptides.

[0099] "GPRC5D×CD3 antibody" is a multispecific antibody, optionally a bispecific antibody, containing two distinct antigen-binding regions, one of which specifically binds to antigen GPRC5D and the other specifically binds to CD3. Multispecific antibodies can be bispecific antibodies, dimer antibodies, or similar molecules (for a description of dimers, see, for example, PNAS USA, 90(14), 6444-8(1993)). The bispecific antibodies, dimer antibodies, etc., described herein, can bind to any suitable target, except for a portion of GPRC5D. The term "bispecific antibody" should be understood as an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes within a single target.

[0100] A "reference sample" is a sample that can be compared with another sample, such as a test sample, to characterize the sample being compared. A reference sample will possess certain characteristic properties that serve as the basis for comparison with the test sample. For example, a reference sample can be used as a benchmark indicating the level of GPRC5D in a treated individual who has cancer. A reference sample does not necessarily have to be analyzed in parallel with the test sample; therefore, in some cases, a reference sample can be a previously determined value or range used to characterize a given condition, such as the level of GPRC5D indicating the level of GPRC5D in a treated individual who has cancer. The term also includes samples known to be associated with physiological states or disease conditions (such as GPRC5D-expressing cancers) but with unknown amounts of GPRC5D used for comparative purposes.

[0101] In the context of progression of GPRC5D-expressing cancers, the term "progression" includes a change in cancer from a less severe state to a more severe state. This can include an increase in the number or severity of the tumor, the extent of cancer cell metastasis, the rate of cancer growth or spread, etc. For example, "progression of colon cancer" includes the progression of this type of cancer from a less severe state to a more severe state, such as from stage I to stage II, from stage II to stage III, etc.

[0102] In the context of regression of GPRC5D-expressing cancers, the term "regression" includes a change in cancer from a more severe state to a less severe state. This can include a reduction in the number or severity of the tumor, the extent of cancer cell metastasis, the rate of cancer growth or spread, etc. For example, "regression of colon cancer" includes the regression of this type of cancer from a more severe state to a less severe state, such as progression from stage III to stage II, or from stage II to stage I.

[0103] In the context of stable GPRC5D-expressing cancers, the term "stable" is intended to describe a disease condition that has not or has not yet undergone significant changes within a clinically relevant time period to be considered a progressive or regressive cancer.

[0104] The implementation schemes described herein are not limited to specific methods, reagents, compounds, compositions, or biological systems, which can of course vary.

[0105] GPRC5D specific antibody and antigen binding fragment

[0106] This article describes isolated monoclonal antibodies or antigen-binding fragments that specifically bind to GPRC5D. The general structure of an antibody molecule includes an antigen-binding domain containing heavy and light chains as well as an Fc domain, and performs multiple functions (including complement fixation and binding to antibody receptors).

[0107] The GPRC5D specific antibody or antigen-binding fragment includes all isotypes of IgA, IgD, IgE, IgG, and IgM, as well as synthetic multimers with a four-stranded immunoglobulin structure. The antibody or antigen-binding fragment also includes the IgY isotype commonly found in hen or turkey serum and hen or turkey egg yolks.

[0108] GPRC5D-specific antibodies and antigen-binding fragments can be derived from any species through recombinant processes. For example, the antibody or antigen-binding fragment can be from mice, rats, goats, horses, pigs, cattle, chickens, rabbits, alpacas, donkeys, humans, or chimeric forms thereof. To make them suitable for human use, non-human antibodies or antigen-binding fragments can be genetically or structurally altered to reduce antigenicity when administered to human patients.

[0109] In some implementations, the antibody or antigen-binding fragment is chimeric. As used herein, the term "chimeric" means that at least some portions of at least one variable domain of the antibody or its antigen-binding fragment are derived from the antibody amino acid sequence of a non-human mammal, rodent, or reptile, while the remainder of the antibody or its antigen-binding fragment is derived from a human.

[0110] In some implementations, the antibody is a humanized antibody. A humanized antibody may be a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. To a large extent, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues in the complementarity-determining region (CDR) of the receptor are replaced by residues in the CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. Generally, a humanized antibody will contain at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those CDR regions of the non-human immunoglobulin, and all or substantially all of the frame regions are those frame regions of the human immunoglobulin sequence. A humanized antibody may contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the constant region of a human immunoglobulin.

[0111] The antibodies or antigen-binding fragments described herein can exist in various forms, but will include one or more of the antibody CDRs shown in Table 1.

[0112] This document describes an immunospecific antibody and antigen-binding fragment that binds to GPRC5D. In some embodiments, the GPRC5D-specific antibody or antigen-binding fragment is human IgG or a derivative thereof. Although the GPRC5D-specific antibody or antigen-binding fragment illustrated herein is human, the illustrated antibody or antigen-binding fragment may also be chimeric.

[0113] In some embodiments, GPRC5D-specific antibodies or antigen-binding fragments thereof are provided, comprising heavy chains containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1. In some embodiments, GPRC5D-specific antibodies or antigen-binding fragments thereof are provided, comprising heavy chains containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1, and light chains containing CDR1, CDR2, and CDR3 of any of the antibodies described in Table 1.

[0114] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:1, a heavy chain CDR2 containing SEQ ID NO:5, and a heavy chain CDR3 containing SEQ ID NO:9. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:1, a heavy chain CDR2 containing SEQ ID NO:5, a heavy chain CDR3 containing SEQ ID NO:9, a light chain CDR1 containing SEQ ID NO:13, a light chain CDR2 containing SEQ ID NO:16, and a light chain CDR3 containing SEQ ID NO:19. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:52. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:52 and a light chain variable domain substantially identical or the same as SEQ ID NO:56. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm being the anti-GPRC5D arm.

[0115] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:2, a heavy chain CDR2 containing SEQ ID NO:6, and a heavy chain CDR3 containing SEQ ID NO:10. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:2, a heavy chain CDR2 containing SEQ ID NO:6, a heavy chain CDR3 containing SEQ ID NO:10, a light chain CDR1 containing SEQ ID NO:13, a light chain CDR2 containing SEQ ID NO:16, and a light chain CDR3 containing SEQ ID NO:19. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:53. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:53 and a light chain variable domain substantially identical or the same as SEQ ID NO:56. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm being the anti-GPRC5D arm.

[0116] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:3, a heavy chain CDR2 containing SEQ ID NO:7, and a heavy chain CDR3 containing SEQ ID NO:11. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:3, a heavy chain CDR2 containing SEQ ID NO:7, a heavy chain CDR3 containing SEQ ID NO:11, a light chain CDR1 containing SEQ ID NO:14, a light chain CDR2 containing SEQ ID NO:17, and a light chain CDR3 containing SEQ ID NO:20. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:54. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:54 and a light chain variable domain substantially identical or the same as SEQ ID NO:57. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0117] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:4, a heavy chain CDR2 containing SEQ ID NO:8, and a heavy chain CDR3 containing SEQ ID NO:12. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:4, a heavy chain CDR2 containing SEQ ID NO:8, a heavy chain CDR3 containing SEQ ID NO:12, a light chain CDR1 containing SEQ ID NO:15, a light chain CDR2 containing SEQ ID NO:18, and a light chain CDR3 containing SEQ ID NO:21. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:55. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as that of SEQ ID NO:55 and a light chain variable domain substantially identical or the same as that of SEQ ID NO:58. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0118] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:61, a heavy chain CDR2 containing SEQ ID NO:67, and a heavy chain CDR3 containing SEQ ID NO:72. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:61, a heavy chain CDR2 containing SEQ ID NO:67, a heavy chain CDR3 containing SEQ ID NO:72, a light chain CDR1 containing SEQ ID NO:13, a light chain CDR2 containing SEQ ID NO:78, and a light chain CDR3 containing SEQ ID NO:80. The GPRC5D specific antibody or antigen-binding fragment may contain a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:82. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as that of SEQ ID NO:82 and a light chain variable domain substantially identical or the same as that of SEQ ID NO:92. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0119] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:2, a heavy chain CDR2 containing SEQ ID NO:28, and a heavy chain CDR3 containing SEQ ID NO:30. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:2, a heavy chain CDR2 containing SEQ ID NO:28, a heavy chain CDR3 containing SEQ ID NO:30, a light chain CDR1 containing SEQ ID NO:13, a light chain CDR2 containing SEQ ID NO:16, and a light chain CDR3 containing SEQ ID NO:19. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:83. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:83 and a light chain variable domain substantially identical or the same as SEQ ID NO:56. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm being the anti-GPRC5D arm.

[0120] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:27, a heavy chain CDR2 containing SEQ ID NO:29, and a heavy chain CDR3 containing SEQ ID NO:73. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:27, a heavy chain CDR2 containing SEQ ID NO:29, a heavy chain CDR3 containing SEQ ID NO:73, a light chain CDR1 containing SEQ ID NO:14, a light chain CDR2 containing SEQ ID NO:17, and a light chain CDR3 containing SEQ ID NO:20. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:84. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:84 and a light chain variable domain substantially identical or the same as SEQ ID NO:57. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm being the anti-GPRC5D arm.

[0121] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:27, a heavy chain CDR2 containing SEQ ID NO:29, and a heavy chain CDR3 containing SEQ ID NO:11. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:27, a heavy chain CDR2 containing SEQ ID NO:29, a heavy chain CDR3 containing SEQ ID NO:11, a light chain CDR1 containing SEQ ID NO:14, a light chain CDR2 containing SEQ ID NO:17, and a light chain CDR3 containing SEQ ID NO:20. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:85. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as that of SEQ ID NO:85 and a light chain variable domain substantially identical or the same as that of SEQ ID NO:57. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0122] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:62, a heavy chain CDR2 containing SEQ ID NO:68, and a heavy chain CDR3 containing SEQ ID NO:74. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:62, a heavy chain CDR2 containing SEQ ID NO:68, a heavy chain CDR3 containing SEQ ID NO:74, a light chain CDR1 containing SEQ ID NO:14, a light chain CDR2 containing SEQ ID NO:17, and a light chain CDR3 containing SEQ ID NO:20. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:86. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:86 and a light chain variable domain substantially identical or the same as SEQ ID NO:57. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm being the anti-GPRC5D arm.

[0123] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:63, a heavy chain CDR2 containing SEQ ID NO:69, and a heavy chain CDR3 containing SEQ ID NO:75. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:63, a heavy chain CDR2 containing SEQ ID NO:69, a heavy chain CDR3 containing SEQ ID NO:75, a light chain CDR1 containing SEQ ID NO:13, a light chain CDR2 containing SEQ ID NO:78, and a light chain CDR3 containing SEQ ID NO:80. The GPRC5D specific antibody or antigen-binding fragment may contain a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:87. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:87 and a light chain variable domain substantially identical or the same as SEQ ID NO:92. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0124] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:64, a heavy chain CDR2 containing SEQ ID NO:70, and a heavy chain CDR3 containing SEQ ID NO:12. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:64, a heavy chain CDR2 containing SEQ ID NO:70, a heavy chain CDR3 containing SEQ ID NO:12, a light chain CDR1 containing SEQ ID NO:15, a light chain CDR2 containing SEQ ID NO:18, and a light chain CDR3 containing SEQ ID NO:21. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:88. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:88 and a light chain variable domain substantially identical or the same as SEQ ID NO:58. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0125] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:65, a heavy chain CDR2 containing SEQ ID NO:68, and a heavy chain CDR3 containing SEQ ID NO:76. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain CDR1 containing SEQ ID NO:65, a heavy chain CDR2 containing SEQ ID NO:68, a heavy chain CDR3 containing SEQ ID NO:76, a light chain CDR1 containing SEQ ID NO:95, a light chain CDR2 containing SEQ ID NO:79, and a light chain CDR3 containing SEQ ID NO:81. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:89. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:89 and a light chain variable domain substantially identical or the same as SEQ ID NO:93. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm of which is an anti-GPRC5D arm.

[0126] In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises heavy chain CDR1 containing SEQ ID NO:66, heavy chain CDR2 containing SEQ ID NO:71, and heavy chain CDR3 containing SEQ ID NO:77. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises heavy chain CDR1 containing SEQ ID NO:66, heavy chain CDR2 containing SEQ ID NO:71, heavy chain CDR3 containing SEQ ID NO:77, light chain CDR1 containing SEQ ID NO:15, light chain CDR2 containing SEQ ID NO:18, and light chain CDR3 containing SEQ ID NO:21. The GPRC5D specific antibody or antigen-binding fragment may comprise a human frame sequence. In some embodiments, the GPRC5D specific antibody and antigen-binding fragment comprises a heavy chain variable domain that is substantially the same as or identical to that of SEQ ID NO:91. In some embodiments, the GPRC5D-specific antibody and antigen-binding fragment include a heavy chain variable domain substantially identical or the same as SEQ ID NO:91 and a light chain variable domain substantially identical or the same as SEQ ID NO:94. The heavy chain variable domain and light chain variable domain of the antibody discussed in this paragraph are suitable for inclusion in a bispecific construct, one arm being the anti-GPRC5D arm.

[0127] In some embodiments, the antibody or antigen-binding fragment is IgG or a derivative thereof, such as IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments where the antibody is an IgG1 isotype, the antibody comprises an IgG1 Fc region (SEQ ID NO. 60).

[0128] SEQ ID NO.60

[0129] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0130] In some embodiments in which the antibody has an IgG4 isotype, the antibody contains S228P, L234A, and L235A substitutions in its Fc region (SEQ ID NO. 59).

[0131] SEQ ID NO.59

[0132] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0133] Specific antibodies defined by the CDR and / or variable domain sequences discussed in the preceding paragraphs may contain these modifications.

[0134] This invention also discloses isolated polynucleotides encoding antibody or antigen-binding fragments that specifically bind to GPRC5D. Isolated polynucleotides capable of encoding the variable domain segments provided herein may be carried on the same or different vectors to generate antibody or antigen-binding fragments. Exemplary polynucleotide sequences encoding GPRC5D antibodies are shown below:

[0135] Heavy chain sequence (SEQ ID NO:96) :

[0136]

[0137] Light chain sequence (SEQ ID NO:90) :

[0138] atgcgggtgctggcccagctgctgggactgctgctgctgtgcttccctggcgccagatgcgacatccagatgacccagagccccagcagcctgagcgccagcgtgggcgaccgggtgaccatcacctgcaaggccagccagaacgtggccacccacgtgggctggtaccagcagaa gcccggcaaggcccccaagcggctgatctacagcgccagctaccggtacagcggcgtgcccagccggttcagcggcagcggcagcggcaccgagttcaccctgaccatcagcaacctgcagcccgaggacttcgccacctactactgccagcagtacaaccggtacccctacacct tcggccagggcaccaagctggagatcaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcg ggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttga

[0139] Polynucleotides encoding recombinant antigen-binding proteins are also within the scope of this disclosure. In some embodiments, the polynucleotide (and the peptide it encodes) comprises a leader sequence. Any leader sequence known in the art may be used. The leader sequence may include, but is not limited to, restriction sites or translation initiation sites.

[0140] The GPRC5D-specific antibodies or antigen-binding fragments described herein include variants having single or multiple amino acid substitutions, deletions, or additions that retain the biological properties (e.g., binding affinity or immune effector activity) of the GPRC5D-specific antibodies or antigen-binding fragments. In the context of this invention, unless otherwise stated, the following symbols are used to describe mutations: i) a substitution of an amino acid at a given position is written as, for example, S228P, indicating that serine at position 228 is replaced by proline; and ii) for a particular variant, a specific three-letter or single-letter code (including codes Xaa and X) is used to indicate any amino acid residue. Thus, a serine substitution for arginine at position 228 is represented as S228P, or any amino acid residue substitution for serine at position 228 is represented as S228P. In the case of a serine deletion at position 228, it is represented by S228*. Those skilled in the art can prepare variants having single or multiple amino acid substitutions, deletions, or additions.

[0141] These variants may include: (a) variants in which one or more amino acid residues are replaced by conserved or non-conserved amino acids; (b) variants in which one or more amino acids are added to or removed from the polypeptide; (c) variants in which one or more amino acids include substituents; and (d) variants in which the polypeptide is fused with another peptide or polypeptide (such as a fusion coupler, protein tag, or other chemical moiety) to impart useful properties to the polypeptide, such as, for example, antibody epitopes, multihistidine sequences, biotin moieties, etc. The antibody or antigen-binding fragments described herein may include variants in which amino acid residues from one species are replaced at conserved or non-conserved positions with corresponding residues from another species. In other embodiments, amino acid residues at non-conserved positions are replaced by conserved or non-conserved residues. Techniques for obtaining these variants, including genetic techniques (deletion, mutation, etc.), chemical techniques, and enzymatic techniques, are known to those skilled in the art.

[0142] The GPRC5D-specific antibodies or antigen-binding fragments described herein may include 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 the antibody or its antigen-binding fragment is primarily determined by the amino acid sequence and arrangement of the CDR. Therefore, a CDR of one isotype can be converted to another isotype without changing the antigen specificity. Alternatively, techniques have been developed to allow hybridomas to switch from producing one antibody isotype to producing another (isotype conversion) without changing the antigen specificity. Therefore, such antibody isotypes fall within the scope of the antibody or antigen-binding fragments described herein.

[0143] Vectors containing the polynucleotides described herein are also provided. The vector may be an expression vector. Therefore, recombinant expression vectors containing sequences encoding polypeptides of interest are also expected to be within the scope of this disclosure. Expression vectors may contain one or more additional sequences, such as, but not limited to, regulatory sequences (e.g., promoters, enhancers), selection markers, and polyadenylation signals. Vectors for transforming a variety of host cells are well known and include, but are not limited to, plasmids, phage particles, phages, baculoviruses, baculosomes, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and other bacterial, yeast, and viral vectors.

[0144] Recombinant expression vectors within the scope of this specification include synthetic, genomic, or cDNA-derived nucleic acid fragments that encode at least one recombinant protein operatively linked to a suitable regulatory element. Such regulatory elements may include a transcription promoter, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and translation. Expression vectors, particularly mammalian expression vectors, may also include one or more non-transcriptional 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-transcriptional sequences, 5' or 3' untranslated sequences (such as an essential ribosome binding site), polyadenylation sites, splicing donor and acceptor sites, or transcription termination sequences. An origin of replication conferring the ability to replicate in the host may also be incorporated.

[0145] The transcriptional and translational control sequences in the expression vector used for transforming vertebrate cells can be provided by a viral source. Exemplary vectors can be constructed as described by Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

[0146] In some embodiments, the antibody-coding sequence or the antigen-binding fragment-coding sequence is placed under the control of a potent constitutive promoter, such as promoters for genes including hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, β-actin, human myosin, human hemoglobin, human muscle creatine, etc. Furthermore, many viral promoters constitutively function in eukaryotic cells and are suitable for use with the embodiments described above. These viral promoters include, but are not limited to, the immediate early promoter of cytomegalovirus (CMV), early and late promoters of SV40, the mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) sequence of Maronnie leukemia virus, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), Raul's sarcoma virus (RSV), and other retroviruses, as well as the thymidine kinase promoter of herpes simplex virus. In one embodiment, the coding sequence of the GPRC5D specific antibody or its antigen-binding fragment is placed under the control of an inducible promoter (such as a metallothionein promoter, a tetracycline inducible promoter, a doxycycline inducible promoter, or a promoter containing one or more interferon-stimulated response elements (ISREs) (such as protein kinase R 2',5'-oligoadenylate synthase, the Mx gene, ADAR1, etc.).

[0147] The vectors described herein may contain one or more internal ribosome entry sites (IRES). The inclusion of IRES sequences in fusion vectors may enhance the expression of some proteins. In some embodiments, the vector system will include one or more polyadenylation sites (e.g., SV40), which may be upstream or downstream of any of the aforementioned nucleic acid sequences. Vector components may be sequentially linked, arranged in a manner that provides optimal spacing for gene product expression (i.e., by introducing “spacer” nucleotides between ORFs), or positioned in another manner. Regulatory elements such as IRES motifs may also be arranged to provide optimal spacing for expression.

[0148] The vector may contain selection markers well 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, blastomycin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial purine nucleoside phosphorylase genes for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). The nucleic acid sequence encoding the selection marker or cloning site may be upstream or downstream of the nucleic acid sequence encoding the polypeptide of interest or the cloning site.

[0149] The vectors described herein can be used to transform various cells with genes encoding the antibodies or antigen-binding fragments therein. For example, the vector can be used to generate GPRC5D-specific antibodies or cells that produce antigen-binding fragments. Thus, another aspect is characterized by host cells transformed with a vector containing a nucleic acid sequence encoding an antibody or its antigen-binding fragment that specifically binds to GPRC5D (such as the antibodies or antigen-binding fragments described and exemplified herein).

[0150] Various techniques are known in the art for introducing foreign genes into cells, and for the purpose of implementing the methods described herein, these techniques can be used to construct recombinant cells according to various embodiments described and illustrated herein. The techniques used should enable the stable transfer of the heterologous gene sequence to the host cell such that the heterologous gene sequence is heritable and can be expressed by cell progeny, thereby preserving the necessary development and physiological functions of the recipient cell. Techniques that can be used include, but are not limited to, chromosome transfer (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection, protoplast fusion, microinjection, electroporation, liposome vectors), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses), etc. (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Cell transformation can also be achieved using calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells.

[0151] Cells suitable for expressing the GPRC5D-specific antibody or antigen-binding fragment described herein are preferably eukaryotic cells, more preferably plant, rodent, or human-derived cells, such as, but not limited to, NSO, 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. Furthermore, hybridoma cells can be used to express the antibody. Methods for generating hybridomas are well-established in the art.

[0152] Cells transformed with the expression vectors described herein can be selected or screened for recombinant expression of the antibodies or antigen-binding fragments described herein. Recombinant-positive cells are amplified and screened for subclones exhibiting the desired phenotype (such as high-level expression, enhanced growth properties, or the ability to produce proteins with the desired biochemical characteristics, for example, due to protein modification or altered post-translational modifications). These phenotypes may be due to inherent properties of a given subclone or due to mutations. Mutations can be achieved using chemicals, UV wavelength light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

[0153] Treatment using GPRC5D specific antibodies

[0154] This document provides GPRC5D-specific antibodies or antigen-binding fragments thereof for use in treatment. Specifically, these antibodies or antigen-binding fragments can be used to treat cancers, such as GPRC5D-expressing cancers. Therefore, the present invention provides a method of treating cancer comprising administering an antibody as described herein, such as a GPRC5D-specific antibody or antigen-binding fragment. For example, this use may target a toxin to GPRC5D-expressing cancer by interfering with GPRC5D-receptor interactions or where the antibody is conjugated to a toxin. In some embodiments, GPRC5D-expressing cancers include lymphomas such as multiple myeloma (MM). Antibodies used in these methods include those described above, such as GPRC5D-specific antibodies or antigen-binding fragments having the characteristics described in Table 1, such as the CDR or variable domain sequences in further discussion of these antibodies.

[0155] In some embodiments described herein, the immune effector properties of GPRC5D-specific antibodies can be enhanced or silenced via Fc modification using techniques known to those skilled 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 receptor; BCR) can be provided and / or controlled by modifying residues in the Fc that facilitate these activities.

[0156] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcRs) (such as natural killer (NK) cells, neutrophils, and macrophages) recognize binding antibodies on target cells and subsequently cause the target cells to lyse.

[0157] The ability of monoclonal antibodies to induce ADCC can be enhanced by modifying their oligosaccharide components. Most glycans of human IgG1 or IgG3 are N-glycosylated at Asn297 in the form of bibranched G0, G0F, G1, G1F, G2, or G2F. Antibodies produced from unengineered CHO cells typically contain at least 85% fucose glycan. Removing the core fucose from bibranched complex-type oligosaccharides linked to the Fc region can enhance antibody ADCC via improved Fc.γ.RIIIa binding without altering antigen binding or CDC activity. Such mAbs can be achieved using various methods reported to induce the successful expression of relatively high defucosylation antibodies with bibranched complex-type Fc oligosaccharides, such as controlling culture osmotic pressure (Konno et al., Cytotechnology, 64:249-65, 2012), using the variant CHO cell line Lec13 as the host cell line (Shields et al., J Biol Chem, 277:26733-26740, 2002), using the variant CHO cell line EB66 as the host cell line (Olivier et al., MAbs, 2(4), 2010; Epub ahead of print; PMID: 20562582), and using the rat hybridoma cell line YB2 / 0 as the host cell line (Shinkawa et al., J Biol Chem, 2012). Chem, 278:3466-3473; 2003), introduced small interfering RNA specifically targeting the α1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng, 88:901-908, 2004), or co-expressed β-1,4-N-acetylglucosamine transferase III and Golgi α-mannosidase II or potent α-mannosidase I inhibitors, chifrine (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).

[0158] In some embodiments described herein, ADCC induced by the GPRC5D antibody can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residues numbered according to the EU index), as described in U.S. Patent 6,737,056.

[0159] Methods for detecting GPRC5D

[0160] This document provides a method for detecting GPRC5D in a biological sample by contacting the sample with an antibody or antigen-binding fragment thereof described herein. As described herein, the sample may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissue (e.g., surgically removed tumor tissue, biopsies, including fine-needle aspiration tissue), histological preparations, etc. In some embodiments, the method includes detecting GPRC5D in the biological sample by contacting the sample with any GPRC5D-specific antibody or antigen-binding fragment thereof described herein.

[0161] In some embodiments, the sample may be contacted with more than one of the GPRC5D-specific antibodies or antigen-binding fragments described herein. For example, the sample may be contacted with a first GPRC5D-specific antibody or its antigen-binding fragment, and then with a second GPRC5D-specific antibody or its antigen-binding fragment, wherein 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 its antigen-binding fragment may be attached to a surface, such as a multi-well plate, chip, or similar substrate, before contacting the sample. In other embodiments, the first antibody or its antigen-binding fragment may not be attached to or connected to any object at all before contacting the sample.

[0162] The GPRC5D-specific antibody and antigen-binding fragment can be detectably labeled. In some embodiments, the labeled antibody and antigen-binding fragment, as described herein, can facilitate the detection of GPRC5D. Many such labels are readily known to those skilled in the art. For example, suitable labels include, but should not be considered limited to, radiolabeling, fluorescent labeling, epitope tags, biotin, chromophore labeling, ECL labeling, or enzymes. More specifically, the labels include ruthenium, 111 In-DOTA, 111 In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and β-galactosidase, polyhistidine (HIS tag), acridine dyes, anthocyanin dyes, fluorescent ketone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Dyes, etc.

[0163] The GPRC5D-specific antibody and antigen-binding fragment can be used in a variety of assays to detect GPRC5D in biological samples. Some suitable assays include, but should not be considered limited to, Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS), or ELISA assays.

[0164] In some of the embodiments described herein, detection of GPRC5D-expressing cancer cells in the patient can be used to determine whether the patient is eligible for treatment with a GPRC5D-targeting therapy.

[0165] GPRC5D is present in blood and serum samples at detectable levels. Therefore, this document provides a method for detecting GPRC5D in blood-derived samples, such as serum samples, by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. Blood samples or derivatives thereof may be diluted, fractionated, or otherwise processed to obtain samples to which the methods described herein can be performed. In some embodiments, GPRC5D in blood samples or derivatives thereof can be detected by any number of assays known in the art, such as, but not limited to, Western blot analysis, radioimmunoassay, surface plasmon resonance, immunofluorescence assay, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS), or ELISA assays.

[0166] Methods for diagnosing cancer

[0167] This document provides methods for diagnosing GPRC5D-expressing cancers in treated subjects. In some embodiments, GPRC5D-expressing cancers include lymphomas such as multiple myeloma (MM). In some embodiments, as described above, detecting GPRC5D in biological samples such as blood or serum samples provides the ability to diagnose cancer in treated subjects from which the sample was obtained. Alternatively, in some embodiments, other samples such as histological samples, fine-needle aspiration samples, excised tumor tissue, circulating cells, circulating tumor cells, etc., may also be used to assess whether treated subjects from which the sample was obtained have cancer. In some embodiments, it may be known that a treated subject from which the sample was obtained has cancer, but the type of cancer may not have been diagnosed or the preliminary diagnosis may be unclear; therefore, detecting GPRC5D in biological samples obtained from the treated subject can enable or confirm a diagnosis of cancer. For example, it may be known that a treated subject has cancer, but it may not be known or may be unclear whether the cancer is GPRC5D-expressing.

[0168] In some embodiments, the method involves assessing whether a patient has GPRC5D-expressing cancer by measuring the amount of GPRC5D present in a biological sample derived from the patient; and comparing the observed amount of GPRC5D with the amount of GPRC5D in a control or reference sample, wherein the difference between the amount of GPRC5D in the patient's sample and the amount of GPRC5D in the control or reference sample indicates that the patient has GPRC5D-expressing cancer. In another embodiment, the observed amount of GPRC5D in a biological sample obtained from the patient may be compared with GPRC5D levels known to be associated with certain forms or stages of cancer to determine the form or stage of cancer in the patient. In some embodiments, the amount of GPRC5D in a sample derived from the patient is assessed by contacting the sample with an antibody or antigen-binding fragment of GPRC5D that specifically binds to GPRC5D (such as the GPRC5D-specific antibody described herein). Samples for assessing the presence of GPRC5D can be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically removed tumor tissue, biopsies, including fine-needle aspiration tissue), histological preparations, etc. In some embodiments, GPRC5D-expressing cancers include hematologic malignancies such as multiple myeloma (MM). In some embodiments, the recipient is a human.

[0169] In some embodiments, a method for diagnosing GPRC5D-expressing cancers will involve: contacting a biological sample of a patient with a GPRC5D-specific antibody or its antigen-binding fragment (such as those derived from the antibodies and fragments provided in Table 1); quantitatively analyzing the amount of GPRC5D present in the sample bound by the antibody or its antigen-binding fragment; comparing the amount of GPRC5D present in the sample with a known standard or reference sample; and determining whether the patient's GPRC5D level falls within the range of cancer-associated GPRC5D levels. In another embodiment, the diagnostic method may be followed by an additional step of administering or delivering cancer-specific treatment. In yet another embodiment, the diagnostic method may be followed by an additional step of transmitting the assay results to facilitate cancer treatment. In some embodiments, the cancer-specific treatment may be targeted at GPRC5D-expressing cancers, such as the GPRC5D×CD3 multispecific antibody described herein.

[0170] In some embodiments, the method involves assessing whether a patient has GPRC5D-expressing cancer by measuring the amount of GPRC5D present in a blood or serum sample obtained from the patient; and comparing the observed amount of GPRC5D with the amount of GPRC5D in a control or reference sample, wherein the difference between the amount of GPRC5D in the sample from the patient and the amount of GPRC5D in the control or reference sample indicates that the patient has GPRC5D-expressing cancer.

[0171] In some embodiments, the control or reference sample may be derived from a treatment subject who does not have GPRC5D-expressing cancer. In some embodiments, the control or reference sample may be derived from a treatment subject who does have GPRC5D-expressing cancer. In some embodiments where the control or reference sample is derived from a treatment subject who does not have GPRC5D-expressing cancer, an increase in the amount of GPRC5D observed in the test sample compared to the observed amount of GPRC5D in the control or reference sample indicates that the evaluated treatment subject has GPRC5D-expressing cancer. In some embodiments where the control sample is derived from a treatment subject who does not have GPRC5D-expressing cancer, a decrease or approximation of the amount of GPRC5D observed in the test sample compared to the observed amount of GPRC5D in the control or reference sample indicates that the evaluated treatment subject does not have GPRC5D-expressing cancer. In some embodiments where the control or reference sample is derived from a patient with GPRC5D-expressing cancer, the observed amount of GPRC5D in the test sample is approximately the same as the observed amount of GPRC5D in the control or reference sample, indicating that the patient being evaluated has GPRC5D-expressing cancer. In some embodiments where the control or reference sample is derived from a patient with GPRC5D-expressing cancer, the observed amount of GPRC5D in the test sample is reduced compared to the observed amount of GPRC5D in the control or reference sample, indicating that the patient being evaluated does not have GPRC5D-expressing cancer.

[0172] In some implementations, the amount of GPRC5D in a sample derived from a treated subject is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds to GPRC5D (such as the antibody described herein). Samples for which the presence of GPRC5D is assessed may be derived from blood samples, serum samples, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically removed tumor tissue, biopsies, including fine-needle aspiration tissue), histological preparations, etc.

[0173] In various embodiments, the amount of GPRC5D is determined by contacting a sample with an antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. In some embodiments, the sample may be contacted with more than one type of antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. In some embodiments, the sample may be contacted with a first antibody or antigen-binding fragment thereof that specifically binds to GPRC5D, and then with a second antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. GPRC5D-specific antibodies or antigen-binding fragments, such as those described herein, may be used in this function.

[0174] Various combinations of GPRC5D-specific antibodies and antigen-binding fragments can be used to provide "first" and "second" antibodies or antigen-binding fragments to implement the diagnostic methods. In some embodiments, GPRC5D-expressing cancers include lymphomas such as multiple myeloma (MM).

[0175] In some implementations, the amount of GPRC5D is determined by Western blot analysis, radioimmunoassay, immunofluorescence assay, immunoprecipitation, balanced dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assay.

[0176] In various embodiments of the diagnostic method, a control sample or reference sample is used. This sample may be a positive or negative assay control to ensure the assay used functions correctly; for example, an assay control of this nature is typically used in immunohistochemical assays. Alternatively, the sample may be a standardized reference sample of GPRC5D levels in a biological sample from a healthy treatment subject. In some embodiments, the observed GPRC5D level in the test subject may be compared to the observed GPRC5D level in a sample from a treatment subject known to have GPRC5D-expressing cancer. In some embodiments, the control subject may have the specific cancer of interest. In some embodiments, the control subject is known to have early-stage cancer, which may or may not be a GPRC5D-expressing cancer. In some embodiments, the control subject is known to have intermediate-stage cancer, which may or may not be a GPRC5D-expressing cancer. In some embodiments, the control subject is known to have late-stage cancer, which may or may not be a GPRC5D-expressing cancer.

[0177] Methods for monitoring cancer

[0178] This document provides a method for monitoring GPRC5D-expressing cancers in treated subjects. In some embodiments, GPRC5D-expressing cancers include lymphomas such as multiple myeloma (MM). In some embodiments, the method involves assessing whether GPRC5D-expressing cancers are progressing, regressing, or remaining stable by measuring the amount of GPRC5D present in a test sample derived from the treated subject; and comparing the observed amount of GPRC5D with the amount of GPRC5D obtained from a biological sample from the treated subject in a similar manner at an earlier time point, wherein the difference between the amount of GPRC5D in the test sample and the earlier sample provides an indication of whether the cancer is progressing, regressing, or remaining stable. In this regard, an increase in the amount of GPRC5D in the test sample relative to the amount observed in the earlier sample may indicate progression of GPRC5D-expressing cancers. Conversely, a decrease in the amount of GPRC5D in the test sample relative to the amount observed in the earlier sample may indicate regression of GPRC5D-expressing cancers.

[0179] Therefore, a lack of significant difference in the amount of GPRC5D in the test sample compared to the amount observed in earlier samples may indicate that GPRC5D-expressing cancer is in a stable disease state. In some embodiments, the amount of GPRC5D in a biological sample derived from a treatment subject is assessed by contacting the sample with an antibody or antibody fragment thereof that specifically binds to GPRC5D (such as the antibody described herein). Samples for assessing the presence of GPRC5D may be derived from urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (e.g., surgically removed tumor tissue, biopsies, including fine-needle aspiration tissue), histological preparations, etc. In some embodiments, the treatment subject is a human.

[0180] In some implementations, methods for monitoring GPRC5D-expressing cancers involve: contacting a biological sample from a treated patient with a GPRC5D-specific antibody or its antigen-binding fragment (such as those derived from the antibodies and fragments provided in Table 1); quantifying the amount of GPRC5D present in the sample; comparing the amount of GPRC5D present in the sample with the amount of GPRC5D measured in a biological sample obtained in a similar manner from the same treated patient at an earlier time point; and determining whether the patient's GPRC5D level has changed over time. An increase in the amount of GPRC5D in the test sample relative to the amount observed in an earlier sample may indicate cancer progression. Conversely, a decrease in the amount of GPRC5D in the test sample relative to the amount observed in an earlier sample may indicate regression of GPRC5D-expressing cancer. Therefore, a lack of significant difference in the amount of GPRC5D in the test sample relative to the amount observed in an earlier sample may indicate a stable disease state in GPRC5D-expressing cancer. In some embodiments, the GPRC5D level of a sample may be compared alone with a known standard or reference sample, or the GPRC5D level of a sample may be compared with a known standard or reference sample in addition to the GPRC5D level observed in samples assessed at an earlier time point. In other embodiments, an additional step of administering cancer-specific treatment may follow the diagnostic method. In some embodiments, the cancer-specific treatment may target GPRC5D-expressing cancers.

[0181] In various embodiments, the amount of GPRC5D is determined by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. In some embodiments, the sample may be contacted with more than one type of antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. In some embodiments, the sample may be contacted with a first antibody or antigen-binding fragment thereof that specifically binds to GPRC5D, and then with a second antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. Antibodies such as those described herein may be used in this function.

[0182] Various combinations of antibodies and antigen-binding fragments described in Table 1 can be used to provide “first” and “second” antibodies or antigen-binding fragments to implement the described monitoring methods. In some embodiments, GPRC5D-expressing cancers include hematologic cancers such as multiple myeloma (MM).

[0183] In some implementations, the amount of GPRC5D is determined by Western blot analysis, radioimmunoassay, immunofluorescence assay, immunoprecipitation, balanced dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence activated cell sorting (FACS) or ELISA assay.

[0184] Kit for detecting GPRC5D

[0185] This article provides kits for detecting GPRC5D in biological samples. These kits include one or more of the GPRC5D-specific antibodies or antigen-binding fragments thereof described herein, along with instructions for use.

[0186] The provided GPRC5D specific antibody or antigen-binding fragment may be in the form of a solution; lyophilized; attached to a substrate, carrier, or plate; or detectably labeled.

[0187] The kit may also include additional components for carrying out the methods described herein. For example, the kit may include a device for obtaining samples from a patient, control or reference samples (e.g., samples from patients with slowly progressing cancer and / or patients without cancer), one or more sample chambers and / or explanatory materials describing the performance of the methods of the present invention, and tissue-specific controls or standards.

[0188] The apparatus for determining GPRC5D levels may also include, for example, buffer solutions or other reagents used in the assay for determining GPRC5D levels. Instructions for use may be, for example, printed instructions for performing the assay and / or instructions for evaluating GPRC5D expression levels.

[0189] The kit may also include means for separating samples from a patient. These means may include one or more devices or reagents that can be used to obtain fluids or tissues from a patient. The means for obtaining samples from a patient may also include means for separating blood components such as serum from a blood sample. Preferably, the kit is designed for use with human patients.

[0190] Multispecific antibodies

[0191] The binding domain of the anti-GPRC5D antibody described herein recognizes cells that express GPRC5D on their surface. As mentioned above, GPRC5D expression can indicate cancer cells. More specific targeting of specific cell subpopulations can be achieved by preparing bispecific molecules (such as antibodies or antibody fragments) that bind to GPRC5D and another target (such as CD3 and BCMA). This is achieved by preparing molecules comprising a first region binding to GPRC5D and a second binding region binding to another target antigen. The antigen-binding region can take any form that allows for specific recognition of the target; for example, the binding region may be or may contain a heavy chain variable domain, an Fv (a combination of heavy chain variable domains and light chain variable domains), or a type III fibronectin-based binding domain (such as a concordant sequence from fibronectin or based on a type III domain from fibronectin, or from tendinin or based on a type III domain from tendinin, such as Centyrin molecules from Janssen Biotech Ltd., see, for example, WO2010 / 051274 and WO2010 / 093627). Therefore, a bispecific molecule containing two different antigen-binding regions that bind to GPRC5D and another antigen respectively is provided.

[0192] Some of the multispecific antibodies described herein comprise two distinct antigen-binding regions, binding to GPRC5D and CD3 respectively. In a preferred embodiment, a multispecific antibody binding GPRC5D and CD3 (GPRC5D×CD3 multispecific antibody) and its multispecific antigen-binding fragment are provided. In some embodiments, the GPRC5D×CD3 multispecific antibody comprises a first heavy chain (HC1) and a first light chain (LC1) that pair to form a first antigen-binding site specifically binding to GPRC5D, and a second heavy chain (HC2) and a second light chain (LC2) that pair to form a second antigen-binding site specifically binding to CD3. In a preferred embodiment, the GPRC5D×CD3 multispecific antibody is a bispecific antibody comprising a GPRC5D-specific arm and a CD3-specific arm, wherein the GPRC5D-specific arm comprises a first heavy chain (HC1) and a first light chain (LC1) that pair to form a first antigen-binding site specifically binding to CD3, and the CD3-specific arm comprises a second heavy chain (HC2) and a second light chain (LC2) that pair to form a second antigen-binding site specifically binding to GPRC5D. In some embodiments, the bispecific antibody of the present invention comprises an antibody having a full-length antibody structure. As used herein, a “full-length antibody” refers to an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The full-length antibody heavy chain (HC) comprises a heavy chain variable domain VH and heavy chain constant domains CH1, CH2, and CH3. The full-length antibody light chain (LC) comprises a light chain variable domain VL and a light chain constant domain CL. A full-length antibody may lack a C-terminal lysine (K) in one or both heavy chains. The term “Fab arm” or “half-molecule” refers to a heavy chain-light chain pair that specifically binds to an antigen. In some embodiments, one of the antigen-binding domains is based on a non-antibody-based binding domain, such as a type 3 fibronectin-based binding domain, like Centyrin.

[0193] The GPRC5D binding arm of the multispecific antibody provided herein can be derived from any of the aforementioned GPRC5D specific antibodies. In some exemplary embodiments of such GPRC5D binding arms, the first antigen-binding region binding to GPRC5D comprises heavy chain CDR1, CDR2, and CDR3 derived from antibody clones as described in Table 1. In some exemplary embodiments of such GPRC5D binding arms, the first antigen-binding region binding to GPRC5D comprises heavy chain CDR1, CDR2, and CDR3, as well as light chain CDR1, CDR2, and CDR3, derived from antibody clones as described in Table 1. In some exemplary embodiments of this type of GPRC5D binding arm, the first antigen-binding region binding to GPRC5D comprises heavy chain CDR1, CDR2, and CDR3 of clones GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597.

[0194] In some exemplary embodiments of this type of GPRC5D binding arm, the first antigen-binding region binding to GPRC5D comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of clones GC5B81, GC5B465, GS5B483, or GC5B596. In some exemplary embodiments of this type of GPRC5D binding arm, the first antigen-binding region binding to GPRC5D comprises a heavy chain variable domain derived from antibody clones as described in Table 1. In some exemplary embodiments of this type of GPRC5D binding arm, the first antigen-binding region binding to GPRC5D comprises both heavy chain and light chain variable domains derived from antibody clones as described in Table 1. In some exemplary embodiments of this type of GPRC5D binding arm, the first antigen-binding region binding to GPRC5D comprises a heavy chain variable domain of clones GC5B81, GC5B465, GS5B483, or GC5B596. In some exemplary embodiments of this type of GPRC5D binding arm, the first antigen-binding region binding to GPRC5D includes a heavy chain variable domain and a light chain variable domain of clones GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597…

[0195] Table 3 provides a list of GPRC5D×CD3 bispecific antibodies having one heavy chain and light chain pair specific to GPRC5D and another heavy chain and light chain pair specific to CD3, with specific antibody IDs listed to describe the antigen-specific antibody arm used to generate the embodiments described.

[0196] Table 3 :

[0197] GC5B81 CD3B219 GC5B465 CD3B219 GC5B483 CD3B219 GC5B596 CD3B219 GC5B382 CD3B219 GC5B379 CD3B219 GC5B373 CD3B219 GC5B376 CD3B219 GC5B385 CD3B219 GC5B370 CD3B219 GC5B602 CD3B219 GC5B603 CD3B219 GC5B599 CD3B219 GC5B601 CD3B219 GC5B598 CD3B219 GC5B597 CD3B219

[0198] In some implementations of the bispecific antibody, the GPRC5D binding arm also binds to cynomolgus monkey GPRC5D, preferably its extracellular domain.

[0199] In some embodiments, the GPRC5D binding arm of the multispecific antibody is IgG or a derivative thereof, such as IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments where the GPRC5D binding arm has an IgG4 isotype, the binding arm contains S228P, L234A, and L235A substitutions in its Fc region.

[0200] In some embodiments of the bispecific antibody, the second antigen-binding arm binds to human CD3. In some preferred embodiments, the CD3-specific arm of the GPRC5D×CD3 bispecific 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 contacts an epitope containing the six N-terminal amino acids of CD3ε. In some embodiments, the CD3-specific binding arm of the bispecific antibody is derived from mouse monoclonal antibody SP34, a mouse IgG3 / λ isotype. In some embodiments, the CD3-binding arm contains the CDR of antibody SP34. Such CD3-binding arms can be 5 × 10 -7 M or lower, such as 1×10 -7 M or lower, 5×10 -8 M or lower, 1×10 -8 M or lower, 5×10 -9 M or lower, or 1×10 -9 M or lower affinity binds to CD3. The CD3-specific binding arm can be a humanized form of the arm of the mouse monoclonal antibody SP34. Human framework adaptation (HFA) can be used to humanize anti-CD3 antibodies from which the CD3-specific arm is derived. In some embodiments of bispecific antibodies, the CD3-binding arm comprises a heavy chain and light chain pair selected from Table 2.

[0201] 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 where the CD3 binding arm has an IgG4 isotype, the binding arm contains S228P, L234A, L235A, F405L, and R409K substitutions in its 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 monkey T cells. In some embodiments, the antibody or antigen-binding fragment binds to CD3ε on both primary and cynomolgus monkey 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 monkey CD4+ T cells.

[0202] In some embodiments, a GPRC5D×CD3 bispecific antibody with a GPRC5D binding arm is provided, the GPRC5D binding arm comprising the heavy chain of antibody clones GC5B81, GC5B465, GC5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597. ...In some embodiments, a GPRC5D×CD3 bispecific antibody is provided having a GPRC5D binding arm comprising the heavy and light chains of antibody clones GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597. In some embodiments, a GPRC5D×CD3 bispecific antibody is provided having a CD3 binding arm comprising the heavy chain of antibody clone CD3B219. In some embodiments, a GPRC5D×CD3 bispecific antibody is provided having a CD3 binding arm comprising both the heavy and light chains of antibody clone CD3B219. In some embodiments, a GPRC5D×CD3 bispecific antibody is provided having a GPRC5D binding arm and a CD3 binding arm, wherein the GPRC5D binding arm comprises the heavy chain of antibody clones GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597, and the CD3 binding arm comprises the heavy chain of antibody clone CD3B219. In some embodiments, a GPRC5D×CD3 bispecific antibody is provided having a GPRC5D binding arm and a CD3 binding arm, wherein the GPRC5D binding arm comprises the heavy and light chains of antibody clones GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598, or GC5B597, and the CD3 binding arm comprises the heavy and light chains of antibody clone CD3B219.

[0203] Exemplary GPRC5D×CD3 bispecific antibodies are provided in Table 23.

[0204] Different forms of bispecific antibodies have been described and recently reviewed by Chames and Baty in Curr OpinDrug Disc Dev, 2009, Vol. 12, p. 276.

[0205] In some embodiments, the bispecific antibodies of the present invention are bispecific antibodies, cross-linked antibodies, or bispecific antibodies obtained through controlled Fab arm exchange, as described in the present invention.

[0206] In some embodiments, bispecific antibodies include IgG-like molecules having complementary CH3 domains to force heterodimerization; recombinant IgG-like dual-targeting molecules, wherein each flanking element contains a portion or fragment of Fab fragments of at least two different antibodies; IgG fusion molecules, wherein a full-length IgG antibody is fused with an additional Fab fragment or a portion of a Fab fragment; Fc fusion molecules, wherein a single-chain Fv molecule or a stable bispecific antibody is fused with a heavy chain constant domain, Fc region, or a portion thereof; Fab fusion molecules, wherein different Fab fragments are fused together; and heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and bispecific antibodies, wherein different single-chain Fv molecules or different bispecific antibodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

[0207] In some implementations, IgG-like molecules with complementary CH3 domains include Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche), electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A / S).

[0208] In some implementations, recombinant IgG-like dual-targeting molecules include dual-targeting (DT)-Ig (GSK / Domantis), dual-antibody (Genentech), cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star), and CovX (CovX / Pfizer).

[0209] In some implementations, IgG fusion molecules include dual variable domain (DVD)-Ig (Abbott), IgG-like bispecific antibodies (InnClone / Eli Lilly), Ts2Ab (MedImmune / AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche).

[0210] In some implementations, Fc fusion molecules include ScFv / Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Fc-DART (MacroGenics), and Dual (ScFv).sub.2-Fab (National Research Center for Antibody Medicine--China).

[0211] In some implementations, Fab fusion bispecific antibodies include F(ab)2 (Medarex / AMGEN), Dual-Action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific antibodies (Biotecnol), and Fab-Fv (UCB-Celltech). ScFv-based, bispecific antibody domain-based antibodies include, but are not limited to, bispecific T-cell adaptors (BiTE) (Micromet), tandem bispecific antibodies (Tandab) (Affimed), dual-affinity retargeting molecules (DART) (MacroGenics), single-chain bispecific antibodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack) and COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), and antibodies that target only the heavy chain domain.

[0212] The full-length bispecific antibody of the present invention can be generated, for example, by Fab arm exchange (or half-molecule exchange) between two monospecific bivalent antibodies in the following manner: a substitution is introduced at the heavy chain CH3 junction in each half-molecule to facilitate the formation of heterodimers of two antibody half-molecules with different specificities in an in vitro cell-free environment or using co-expression. The Fab arm exchange reaction is the result of disulfide bond isomerization and CH3 domain dissociation-association. The heavy chain disulfide bonds in the hinge region of the parent monospecific antibody are reduced. The resulting free cysteine ​​of one of the parent monospecific antibodies forms an inter-heavy chain disulfide bond with the cysteine ​​residue of the second parent monospecific antibody molecule, while the CH3 domain of the parent antibody is released and reformed through dissociation-association. The CH3 domain of the Fab arm can be modified to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody with two Fab arms or half-molecules, each of which binds a different epitope, namely an epitope on GPRC5D and an epitope on CD3.

[0213] As used herein, “homodimerization” refers to the interaction between two heavy chains having the same CH3 amino acid sequence. As used herein, “homodimer” refers to an antibody having two heavy chains containing the same CH3 amino acid sequence.

[0214] As used herein, "heterodimerization" refers to the interaction between two heavy chains with different CH3 amino acid sequences. As used herein, "heterodimer" refers to an antibody having two heavy chains containing different CH3 amino acid sequences.

[0215] The “button” technique (see, for example, PCT International Dimer Antibody WO 2006 / 028936) can be used to generate full-length bispecific antibodies. In short, selected amino acids that form the CH3 domain boundaries in human IgG can be mutated at positions affecting CH3 domain interactions, thereby promoting heterodimer formation. Amino acids with small side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to the first antigen, and amino acids with large side chains (buttons) are introduced into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of the two antibodies, heterodimers are formed due to the preferential interaction between the heavy chains with “buttons” and those with “buttons.” Exemplary CH3 substitution pairs forming buttons and clasps (represented as modification positions in the first CH3 domain of the first heavy chain / modification positions in the second CH3 domain of the second heavy chain) are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V.

[0216] Other techniques may also be used, such as promoting heterodimerization of heavy chains by electrostatic interactions through the substitution of positively charged residues on one CH3 surface and negatively charged residues on another CH3 surface, as described in U.S. Patent Publication Nos. US2010 / 0015133, US2009 / 0182127, US2010 / 028637, or US2011 / 0123532. In other techniques, heterodimerization can be promoted by the following substitutions (represented as the modification position in the first CH3 domain of the first heavy chain / the modification position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366VK409FY407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W, as described in U.S. Patent Publication No. US2012 / 0149876 or U.S. Patent Publication No. US2013 / 0195849.

[0217] In addition to the methods described above, the bispecific antibody of the present invention can also be generated in an in vitro cell-free environment by: introducing an asymmetric mutation into the CH3 region of two monospecific homodimer antibodies, and forming a bispecific heterodimer antibody from the two parental monospecific homodimer antibodies under reducing conditions, thereby causing disulfide bond isomerization according to the method described in International Patent Publication No. WO2011 / 131746. In this method, a first monospecific bivalent antibody (e.g., anti-GPRC5D antibody) and a second monospecific bivalent antibody (e.g., anti-CD3 antibody) are modified to have certain substitutions at the CH3 domain that promote the stability of the heterodimer; these antibodies are incubated together under reducing conditions sufficient to cause disulfide bond isomerization of the cysteine ​​in the hinge region; thereby generating a bispecific antibody through Fab arm exchange. The incubation conditions are optimally reversible to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably those selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, the following conditions can be used: incubation for at least 90 minutes at a pH of 5-8, such as pH 7.0 or pH 7.4, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a temperature of at least 20°C.

[0218] In addition to the GPRC5D×CD3 multispecific antibody described herein, a polynucleotide sequence encoding the GPRC5D×CD3 multispecific antibody is also provided. A vector containing the polynucleotide is also provided, as well as cells expressing the GPRC5D×CD3 multispecific antibody provided herein. Cells capable of expressing the disclosed vector are also described. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as Escherichia coli). The antibody may also be produced by hybridoma cells.

[0219] Therapeutic compositions and methods of treatment using multispecific antibodies and their multispecific antigen-binding fragments

[0220] The GPRC5D bispecific antibodies described above, such as the GPRC5D×CD3 bispecific antibody described above, can be used in treatment. Specifically, GPRC5D bispecific antibodies can be used to treat cancer. This document also provides therapeutic compositions for treating hyperproliferative disorders in mammals, comprising a therapeutically effective amount of the multispecific antibody or multispecific antigen-binding fragment described herein and a pharmaceutically acceptable carrier. In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody or its multispecific antigen-binding fragment as described herein, more preferably a GPRC5D×CD3 bispecific antibody or its GPRC5D×CD3 bispecific antigen-binding fragment as described herein. In one embodiment, the pharmaceutical composition is used to treat GPRC5D-expressing cancers, including (but not limited to) GPRC5D-expressing B-cell cancers, such as multiple myeloma (MM); and other undetermined cancers expressing GPRC5D. Specific bispecific antibodies that can be used to treat cancers (such as hematologic cancers, including the specific cancers described above) include antibodies GC5B81, GC5B465, GS5B483, or GC5B596.

[0221] The pharmaceutical compositions provided herein comprise: a) an effective amount of the multispecific antibody or antibody fragment of the present invention, and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a GPRC5D×CD3 bispecific antibody or a GPRC5D×CD3 bispecific antigen-binding fragment thereof as described herein. As used herein, the term "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial agents, and antifungal agents. Examples of suitable carriers, diluents, and / or excipients include one or more of water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, etc., and any combination thereof. In many cases, it is preferred that the composition contains an isotonic agent, such as sugar, polyol, or sodium chloride. Specifically, relevant examples of suitable carriers include: (1) Duchenne phosphate-buffered saline with a pH of about 7.4, containing or without about 1 mg / mL to 25 mg / mL human serum albumin; (2) 0.9% saline (0.9% w / v sodium chloride (NaCl)); and (3) 5% (w / v) dextran; and may also contain antioxidants such as tryptophan and stabilizers such as Tween. .

[0222] The compositions described herein may also contain additional therapeutic agents necessary for the specific disorder being treated. Preferably, the multispecific antibody or antibody fragment and the complementary active compound will have complementary activities that will not adversely affect each other. In a preferred embodiment, the additional therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin-2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent.

[0223] The compositions of the present invention can be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, but the preferred form depends on the intended administration method and therapeutic application. Typical preferred compositions are in the form of injectable or infusionable solutions. Preferred administration methods are parenteral (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous). In one preferred embodiment, the compositions of the present invention are administered by intravenous bolus injection or by continuous infusion over a period of time. In another preferred embodiment, these compositions are injected via intramuscular, subcutaneous, intra-articular, intrasynovial, intratumoral, peritumoral, intralesional, or perilesional routes to exert local and systemic therapeutic effects.

[0224] Sterile compositions for parenteral administration can be prepared by incorporating the desired amount of the antibody, antibody fragment, or antibody conjugate of the present invention into a suitable solvent, followed by sterilization using microfiltration. Water, saline, phosphate-buffered saline, dextran, glycerol, ethanol, and combinations thereof can be used as solvents or mediators. In many cases, it is preferred that the composition contains an isotonic agent, such as sugar, polyol, or sodium chloride. These compositions may also contain adjuvants, particularly wetting agents, isotonic agents, emulsifiers, dispersants, and stabilizers. Sterile compositions for parenteral administration can also be prepared as sterile solid compositions that are soluble in sterile water or any other injectable sterile medium when used.

[0225] Multispecific antibodies or antibody fragments can also be administered orally. Tablets, pills, powders (gelatin capsules, small capsules), or granules can be used as solid compositions for oral administration. In these compositions, the active ingredient according to the invention is mixed with one or more inert diluents (such as starch, cellulose, sucrose, lactose, or silica) under an argon flow. 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.

[0226] As liquid compositions for oral administration, pharmaceutically acceptable solutions, suspensions, emulsions, syrups, and elixirs containing inert diluents (such as water, ethanol, glycerin, vegetable oil, or paraffin oil) may be used. These compositions may contain substances other than diluents, such as wetting, sweetening, thickening, flavoring, or stabilizing agents.

[0227] The dosage of these substances depends on the desired effect, duration of treatment, and route of administration; for adults, the usual oral dose is between 5 mg and 1000 mg, with unit doses ranging from 1 mg to 250 mg of the active substance. Generally, the physician will determine the appropriate dosage based on age, weight, and any other factors specific to the patient being treated.

[0228] This document also provides a method for killing GPRC5D+ cells by administering a multispecific antibody that binds to the GPRC5D and is capable of recruiting T cells to kill the GPRC5D+ cells (i.e., T cell redirection) to a patient in need of this. Any of the multispecific antibodies or antibody fragments of the present invention can be used therapeutically. For example, in one embodiment, the GPRC5D×CD3 multispecific antibody can be used therapeutically to treat cancer in a patient.

[0229] In a preferred embodiment, the multispecific antibody or antibody fragment of the present invention is used to treat hyperproliferative disorders in mammals. 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 hyperproliferative disorders in mammals. In one embodiment, the disorder is cancer. Specifically, the cancer is a GPRC5D-expressing cancer, including (but not limited to) the following cancers: GPRC5D-expressing B-cell cancers, such as multiple myeloma (MM); and other unidentified cancers expressing GPRC5D. In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a GPRC5D×CD3 bispecific antibody or a GPRC5D×CD3 bispecific antigen-binding fragment thereof as described herein.

[0230] Therefore, the pharmaceutical compositions of the present invention can be used to treat or prevent a variety of cancers, including (but not limited to) the following: GPRC5D-expressing cancers, including (but not limited to) the following cancers: GPRC5D-expressing B-cell / plasma cell cancers, such as acute multiple myeloma (MM) or precancerous myeloma such as MGUS (monoclonal gammopathy of undetermined significance) and SMM (suppressive multiple myeloma) and plasmacytoma; and other unidentified cancers expressing GPRC5D therein.

[0231] Similarly, this document also provides a method for inhibiting the growth of a selected cell population, comprising contacting GPRC5D-expressing target cells or tissues containing such target cells with an effective amount of the multispecific antibody or antibody fragment of the present invention alone in the presence of peripheral blood mononuclear cells (PBMCs), or contacting GPRC5D-expressing target cells or tissues containing such target cells with an effective amount of the multispecific antibody or antibody fragment of the present invention in combination with other cytotoxic agents or therapeutic agents. In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody or its multispecific antigen-binding fragment as described herein, more preferably a GPRC5D×CD3 bispecific antibody or its GPRC5D×CD3 bispecific antigen-binding fragment as described herein. In a preferred embodiment, the additional therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin-2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent. The method for inhibiting the growth of the selected cell population can be performed in vitro, in vivo, or ex vivo.

[0232] Examples of in vitro use include treating autologous bone marrow prior to transplantation into the same patient to kill diseased or malignant cells; treating bone marrow prior to transplantation to kill competent T cells and prevent graft-versus-host disease (GVHD); treating cell cultures to kill all cells except desired variants that do not express the target antigen; or killing variants that express undesired antigens. Those skilled in the art can readily determine the conditions for non-clinical in vitro use.

[0233] An example of clinical ex vivo use is the removal of tumor cells from bone marrow prior to autologous transplantation in cancer treatment. The treatment can be performed as follows: Bone marrow is collected from a patient or other individual and then incubated in a culture medium containing serum to which the cytotoxic agent of the present invention has been added. The concentration range is from about 10 μM to 1 μM, and incubation is performed at about 37°C for about 30 minutes to about 48 hours. Those skilled in the art can readily determine the exact conditions of concentration and incubation time, i.e., the dosage. After incubation, the bone marrow cells are washed with a culture medium containing serum, and these bone marrow cells are returned to the patient via intravenous infusion according to known methods. In cases where the patient is receiving other treatments (such as ablation chemotherapy or total body radiation therapy between bone marrow collection and re-infusion of the treated cells), the treated bone marrow cells are cryopreserved in liquid nitrogen using standard medical equipment.

[0234] For clinical in vivo use, a therapeutically effective amount of a multispecific antibody or antigen-binding fragment is administered to a recipient in need of treatment. For example, a GPRC5D×CD3 multispecific antibody and its multispecific antigen-binding fragment can be used to treat GPRC5D-expressing cancers in recipients in need of treatment. In some embodiments, the GPRC5D-expressing cancer is a B-cell cancer, such as multiple myeloma (MM). In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody as described herein or its multispecific antigen-binding fragment, more preferably a GPRC5D×CD3 bispecific antibody as described herein or its GPRC5D×CD3 bispecific antigen-binding fragment. In some embodiments, the recipient is a mammal, preferably a human. In some embodiments, the multispecific antibody or antigen-binding fragment is administered in the form of a solution tested for sterility.

[0235] The dosage regimen in the above-described treatment methods and uses can be adjusted to provide the best expected response (e.g., therapeutic response). For example, a single bolus injection may be administered, several fractionated doses may be administered over time, or the dose may be proportionally reduced or increased as instructed by an emergency indicative of the treatment situation. Parenteral compositions may be formulated into easily administered and consistent dosage units.

[0236] The effective dose and dosing regimen of the multispecific antibodies and fragments depend on the disease or condition to be treated and can be determined by those skilled in the art. Exemplary, non-limiting ranges for the therapeutically effective amount of the compounds of the present invention are about 0.001-10 mg / kg, such as about 0.001-5 mg / kg (e.g., about 0.001-2 mg / kg), such as about 0.001-1 mg / kg (e.g., about 0.001 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, or about 10 mg / kg).

[0237] A physician or veterinarian with ordinary skills in the art can readily determine and prescribe an effective amount of the desired pharmaceutical composition. For example, the physician or veterinarian may begin using a dose level of a multispecific antibody or fragment in the pharmaceutical composition that is lower than the level required to achieve the desired therapeutic effect, and then gradually increase the dose until the desired effect is achieved. Typically, the appropriate daily dose of the bispecific antibody of the present invention will be the amount of the lowest dose of the compound that effectively produces a therapeutic effect. Administration may be, for example, parenteral, such as intravenous, intramuscular, or subcutaneous. In one embodiment, the multispecific antibody or fragment can be administered at a dose of mg / m³. 2 The calculated weekly dose is administered by infusion. Such a dose can be based, for example, on the mg / kg dose provided above, according to the formula: dose (mg / kg) × 70: 1.8. Such administration can be repeated, such as 1 to 8 times, or 3 to 5 times. Administration can be carried out by continuous infusion over a period of 2 to 24 hours (e.g., 2 to 12 hours). In one embodiment, the multispecific antibody or fragment can be administered by slow, continuous infusion over a longer period (e.g., more than 24 hours) to reduce toxic side effects.

[0238] In one embodiment, the multispecific antibody or fragment can be administered as a fixed-dose weekly dose up to eight times, such as four to six times when administered once weekly. Such a regimen can be repeated once or more as needed, for example, after six or twelve months. Such a fixed dose can be, for example, based on the mg / kg dose provided above, where the body weight is estimated to be 70 kg. The dose can be determined or adjusted by measuring the amount of the bispecific antibody of the present invention in the blood upon administration, for example, by removing a biological sample, and using an anti-idiotype antibody targeting the GPRC5D antigen-binding region of the multispecific antibody of the present invention.

[0239] In one implementation, multispecific antibodies or fragments may be administered via maintenance therapy, such as once a week for six months or longer.

[0240] Multispecific antibodies or fragments can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events during cancer progression, and / or reduce the risk of recurrence after cancer remission.

[0241] The multispecific antibodies and fragments described herein can also be administered in combination therapy, i.e., in combination with other therapeutic agents associated with the disease or condition to be treated. Therefore, in one embodiment, the antibody-containing drug is used in combination with one or more additional therapeutic agents, such as chemotherapeutic agents. In some embodiments, the other therapeutic agents are cytarabine, anthracycline, histamine dihydrochloride, or interleukin-2. Such combination administration can be performed simultaneously, separately, or sequentially in any order. For simultaneous administration, these therapeutic agents can be administered as a composition or as a single composition, as appropriate.

[0242] In one embodiment, a method is provided for treating a disorder involving cells expressing GPRC5D in a patient, the method comprising administering a therapeutically effective amount of a multispecific antibody or fragment (such as the GPRC5D×CD3 bispecific antibody described herein) to a patient in need of treatment and radiotherapy. In another embodiment, a method is provided for treating or preventing cancer, comprising administering a therapeutically effective amount of a multispecific antibody or fragment (such as the GPRC5D×CD3 antibody described herein) to a patient in need of treatment and radiotherapy. Radiotherapy may include radiation or administration of an associated radiopharmaceutical to the patient. The radiation source may be external or internal to the patient being treated (radiation therapy may take the form of, for example, external beam radiotherapy (EBRT) or short-range radiotherapy (BT)). Radioactive elements that can be used to perform such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111.

[0243] Reagent test kit

[0244] This document also provides a kit comprising, for example, the multispecific antibody described herein or an antigen-binding fragment thereof, and instructions for using the antibody or fragment to kill a specific type of cell. In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a GPRC5D×CD3 bispecific antibody or a GPRC5D×CD3 bispecific antigen-binding fragment thereof as described herein. The instructions may include instructions for using the multispecific antibody or its antigen-binding fragment in vitro, in vivo, or ex vivo.

[0245] Typically, the kit will have a compartment containing a multispecific antibody or its antigen-binding fragment. The multispecific antibody or its antigen-binding fragment may be in lyophilized form, liquid form, or other forms suitable for inclusion in the kit. The kit may also include other elements necessary to perform the methods described in the kit's instructions, such as sterile solutions for reconstituted lyophilized powder, other reagents for combining with the multispecific antibody or its antigen-binding fragment prior to administration to a patient, and tools to facilitate administration of the multispecific antibody or its antigen-binding fragment to a patient.

[0246] Diagnostic uses

[0247] The multispecific antibodies and fragments described herein can also be used for diagnostic purposes. Therefore, diagnostic compositions comprising multispecific antibodies or fragments as defined herein and their uses are also provided. In a preferred embodiment, the multispecific antibody is a GPRC5D×CD3 multispecific antibody or a multispecific antigen-binding fragment thereof as described herein, more preferably a GPRC5D×CD3 bispecific antibody or a GPRC5D×CD3 bispecific antigen-binding fragment thereof as described herein. In one embodiment, the present invention provides a kit for diagnosing cancer, the kit comprising a container containing a bispecific GPRC5D×CD3 antibody and one or more reagents for detecting the binding of the antibody to GPRC5D. The reagents may include, for example, fluorescent tags, enzyme tags, or other detectable tags. The reagents may also include secondary or tertiary antibodies or reagents for an enzymatic reaction, wherein the enzymatic reaction produces a product that can be visualized. For example, the multispecific antibody or antigen-binding fragment thereof described herein may be a radiolabeled, fluorescently labeled, epitope-tagged, biotinylate, chromophore-labeled, ECL-labeled, enzyme, ruthenium, etc. 111 In-DOTA, 111 Labeled with In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and β-galactosidase, or polyhistidine or similar labels known in the art.

[0248] Implementation Plan

[0249] The disclosure provided herein also provides the following non-limiting embodiments.

[0250] 1. A separating antibody or antigen-binding fragment thereof that specifically binds to GPRC5D, said separating antibody or antigen-binding fragment comprising:

[0251] a. Heavy chain complementarity-determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO:1, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:9;

[0252] b. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:2, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:6, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:10;

[0253] c. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:3, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:7, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:11;

[0254] d. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:8, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:12;

[0255] e. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:61, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:72;

[0256] f. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:2, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:28, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:30;

[0257] g. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:27, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:73;

[0258] h. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:27, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:11;

[0259] i. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:62, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:68, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:74;

[0260] j. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:63, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:69, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:75;

[0261] k. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:64, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:70, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:12;

[0262] l. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:65, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:68, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:76; or

[0263] m. Heavy chain CDR1 having the amino acid sequence of SEQ ID NO:66, heavy chain CDR2 having the amino acid sequence of SEQ ID NO:71, and heavy chain CDR3 having the amino acid sequence of SEQ ID NO:77.

[0264] 2. The antibody or antigen-binding fragment is isolated according to the method described in Implementation Scheme 1, wherein...

[0265] a. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:1, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:5, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:9 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:13, the light chain CDR2 having the amino acid sequence of SEQ ID NO:16, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:19;

[0266] b. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:2, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:6, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:10, further comprising the light chain CDR1 having the amino acid sequence of SEQ ID NO:13, the light chain CDR2 having the amino acid sequence of SEQ ID NO:16, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:19;

[0267] c. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:3, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:7, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:11 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:14, the light chain CDR2 having the amino acid sequence of SEQ ID NO:17, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:20;

[0268] d. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:4, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:8, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:12, further comprising the light chain CDR1 having the amino acid sequence of SEQ ID NO:15, the light chain CDR2 having the amino acid sequence of SEQ ID NO:18, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:21;

[0269] e. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:61, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:72, further comprising the light chain CDR1 having the amino acid sequence of SEQ ID NO:13, the light chain CDR2 having the amino acid sequence of SEQ ID NO:78, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:80;

[0270] f. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:2, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:28, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:30 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:13, the light chain CDR2 having the amino acid sequence of SEQ ID NO:6, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:19;

[0271] g. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:27, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:73 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:14, the light chain CDR2 having the amino acid sequence of SEQ ID NO:17, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:20;

[0272] h. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:27, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:29, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:11 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:14, the light chain CDR2 having the amino acid sequence of SEQ ID NO:17, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:20;

[0273] i. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:62, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:68, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:74 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:14, the light chain CDR2 having the amino acid sequence of SEQ ID NO:17, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:20;

[0274] j. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:63, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:69, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:75 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:13, the light chain CDR2 having the amino acid sequence of SEQ ID NO:78, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:80;

[0275] k. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:61, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:67, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:72, further comprising the light chain CDR1 having the amino acid sequence of SEQ ID NO:13, the light chain CDR2 having the amino acid sequence of SEQ ID NO:78, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:80;

[0276] 1. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:65, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:68, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:76, further comprising the light chain CDR1 having the amino acid sequence of SEQ ID NO:95, the light chain CDR2 having the amino acid sequence of SEQ ID NO:79, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:81; or

[0277] m. The antibody comprising the heavy chain CDR1 having the amino acid sequence of SEQ ID NO:66, the heavy chain CDR2 having the amino acid sequence of SEQ ID NO:71, and the heavy chain CDR3 having the amino acid sequence of SEQ ID NO:77 further comprises the light chain CDR1 having the amino acid sequence of SEQ ID NO:15, the light chain CDR2 having the amino acid sequence of SEQ ID NO:18, and the light chain CDR3 having the amino acid sequence of SEQ ID NO:21.

[0278] 3. An isolation antibody or an antigen-binding fragment thereof, said isolation antibody or antigen-binding fragment thereof specifically binds to GPRC5D and includes a variable heavy (VH) chain region selected from SEQ ID NO: 52, 53, 54, 55, 82, 83, 84, 85, 86, 87, 88, 89, or 91.

[0279] 4. The antibody according to embodiment 3, wherein the antibody or its antigen-binding fragment comprises a variable light (VL) chain region selected from SEQ ID NO: 56, 57, 58, 92, 93, or 94.

[0280] 5. The antibody according to embodiment 3, wherein the antibody or its antigen-binding fragment comprises a VH region selected from SEQ ID NO: 52, 53, 54, 55, 82, 83, 84, 85, 86, 87, 88, 89, or 91 and a VL region selected from SEQ ID NO: 56, 57, 58, 92, 93, or 94.

[0281] 6. The antibody according to embodiment 5, wherein the VH chain region comprises SEQ ID NO:52, 53, or 83 paired with the VL chain region comprising SEQ ID NO:56.

[0282] 7. The antibody according to embodiment 5, wherein the VH chain region comprises SEQ ID NO:54, 84, 85, 86, or 90 paired with the VL chain region comprising SEQ ID NO:57.

[0283] 8. The antibody according to embodiment 5, wherein the VH chain region comprises SEQ ID NO:55 or 88 paired with the VL chain region comprising SEQ ID NO:58.

[0284] 9. The antibody according to embodiment 5, wherein the VH chain region comprises SEQ ID NO: 82 or 87 paired with the VL chain region comprising SEQ ID NO: 92.

[0285] 10. The antibody according to embodiment 5, wherein the VH chain region comprises SEQ ID NO:89 paired with the VL chain region comprising SEQ ID NO:93.

[0286] 11. The antibody according to embodiment 5, wherein the VH chain region comprises SEQ ID NO:91 paired with the VL chain region comprising SEQ ID NO:94.

[0287] 12. An antibody or antigen-binding fragment according to any one of embodiments 1 to 11, wherein the antibody or antigen-binding fragment binds to a polypeptide having the amino acid sequence SEQ ID NO:22.

[0288] 13. An antibody or antigen-binding fragment according to any one of embodiments 1 to 12, wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment.

[0289] 14. An antibody or antigen-binding fragment according to any one of embodiments 1 to 13, wherein the antibody or antigen-binding fragment is recombinant.

[0290] 15. An antigen-binding fragment according to any one of embodiments 1 to 14, wherein the antigen-binding fragment is a Fab fragment, a Fab2 fragment, or a single-chain antibody.

[0291] 16. An antibody or antigen-binding fragment according to any one of embodiments 1 to 15, wherein the antibody or antigen-binding fragment has an IgG1, IgG2, IgG3 or IgG4 isotype.

[0292] 17. An antibody or antigen-binding fragment according to any one of embodiments 1 to 9, wherein the antibody or antigen-binding fragment is an IgG1 or IgG4 isotype.

[0293] 18. The antibody according to embodiment 17, wherein the IgG1 has a K409R substitution in its Fc region.

[0294] 19. The antibody according to embodiment 17, wherein the IgG1 has an F405L substitution in its Fc region.

[0295] 20. The antibody according to embodiment 20, wherein the IgG4 has an F405L substitution and an R409K substitution in its Fc region.

[0296] 21. The antibody according to embodiment 16, wherein the antibody further comprises S228P substitution, L234A substitution and L235A substitution in its Fc region.

[0297] 22. An antibody or antigen-binding fragment according to any one of embodiments 1 to 14, wherein the antibody or antigen-binding fragment specifically binds to human GPRC5D and cross-reacts with cynomolgus monkey GPRC5D.

[0298] 23. An antibody or antigen-binding fragment according to any one of embodiments 17, wherein the antibody or antigen-binding fragment is expressed at an EC50 of less than about 28 nM. 50 In vitro induced ADCC.

[0299] 24. An isolated cell expressing an antibody or antigen-binding fragment according to any one of embodiments 1 to 11.

[0300] 25. The cell according to embodiment 24, wherein the cell is a hybridoma.

[0301] 26. The cells according to embodiment 24, wherein the antibody is recombinantly generated.

[0302] 27. A bispecific antibody for separating GPRC5D×CD3 or a bispecific binding fragment thereof, wherein the bispecific antibody for separating GPRC5D×CD3 or a bispecific binding fragment thereof comprises:

[0303] a) First heavy chain (HC1);

[0304] b) Second chain (HC2);

[0305] c) The first light chain (LC1); and

[0306] d) Second light chain (LC2),

[0307] The HC1 and LC1 pair to form a first antigen-binding site that specifically binds to CD3, and the HC2 and LC2 pair to form a second antigen-binding site that specifically binds to GPRC5D.

[0308] 28. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 27, wherein HC1 contains SEQ ID NO:25 and LC1 contains SEQ ID NO:26.

[0309] 29. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 comprises SEQ ID NO:52 and LC2 comprises SEQ ID NO:56.

[0310] 30. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:53 and LC2 contains SEQ ID NO:56.

[0311] 31. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:54 and LC2 contains SEQ ID NO:57.

[0312] 32. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:55 and LC2 contains SEQ ID NO:58.

[0313] 33. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:82 and LC2 contains SEQ ID NO:92.

[0314] 34. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:83 and LC2 contains SEQ ID NO:56.

[0315] 35. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:84 and LC2 contains SEQ ID NO:57.

[0316] 36. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:85 and LC2 contains SEQ ID NO:57.

[0317] 37. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:86 and LC2 contains SEQ ID NO:57.

[0318] 38. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:87 and LC2 contains SEQ ID NO:92.

[0319] 39. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:88 and LC2 contains SEQ ID NO:58.

[0320] 40. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:89 and LC2 contains SEQ ID NO:93.

[0321] 41. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:91 and LC2 contains SEQ ID NO:94.

[0322] 42. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 28, wherein HC2 contains SEQ ID NO:85 and LC2 contains SEQ ID NO:57.

[0323] 43. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27 to 42, wherein the antibody or bispecific binding fragment is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0324] 44. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiment 43, wherein the antibody or bispecific binding fragment is an IgG4 isotype.

[0325] 45. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiments 27 to 42, wherein the antibody or the bispecific binding fragment thereon binds to GPRC5D on the surface of human myeloma cells.

[0326] 46. ​​The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiments 27 to 42, wherein the antibody or the bispecific binding fragment thereof binds to GPRC5D on the surface of human multiple myeloma cells.

[0327] 47. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiments 27 to 42, wherein the antibody or bispecific binding fragment is expressed at an EC50 concentration of less than about 0.22 nM. 50 Induces in vitro activation of human T cells.

[0328] 48. The GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to embodiments 27 to 42, wherein the antibody or bispecific binding fragment is expressed at an EC50 concentration of less than about 0.89 nM. 50 Inducing T cell-dependent cytotoxicity in cells expressing GPRC5D in vitro.

[0329] 49. An isolated cell expressing an antibody or a bispecific binding fragment according to any one of embodiments 27 to 42.

[0330] 50. The cell according to embodiment 49, wherein the cell is a hybridoma.

[0331] 51. The cell according to embodiment 49, wherein the antibody or bispecific binding fragment is recombinantly generated.

[0332] 52. A method for treating a patient suffering from cancer, the method comprising:

[0333] The time required to administer a therapeutically effective amount of the GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27 to 42 to a patient in need of the cancer.

[0334] 53. A method for inhibiting the growth or proliferation of cancer cells, the method comprising:

[0335] Administer a therapeutically effective amount of the GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27 to 42 to inhibit the growth or proliferation of cancer cells.

[0336] 54. A method for redirecting T cells to GPRC5D-expressing cancer cells, the method comprising:

[0337] Administer a therapeutically effective amount of the GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27 to 42 to redirect T cells to cancer.

[0338] 55. The method according to embodiments 52, 53 or 54, wherein the cancer is a hematologic cancer.

[0339] 56. The method according to embodiment 55, wherein the hematologic cancer is a GPRC5D-expressing B-cell cancer.

[0340] 57. The method according to embodiment 56, wherein the GPRC5D-expressing B-cell cancer is multiple myeloma.

[0341] 58. The method according to embodiment 52, wherein the method includes administering a second therapeutic agent.

[0342] 59. The method according to embodiment 58, wherein the second therapeutic agent is a chemotherapeutic agent or a targeted anticancer therapy.

[0343] 60. The method according to embodiment 59, wherein the chemotherapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin-2.

[0344] 61. The method according to embodiment 59, wherein the second therapeutic agent is administered to the treated person simultaneously, sequentially, or separately from the bispecific antibody.

[0345] 62. A pharmaceutical composition comprising a GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27 to 42 and a pharmaceutically acceptable carrier.

[0346] 63. A method for generating a GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 49 to 51 by culturing cells according to any one of embodiments 27 to 42.

[0347] 64. An isolated synthetic polynucleotide, said isolated polynucleotide encoding HC1, HC2, LC1 or LC2 of a GPRC5D×CD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27 to 42.

[0348] 65. A kit comprising a GPRC5D×CD3 bispecific antibody or bispecific binding fragment as defined in any one of embodiments 27 to 42 and / or a polynucleotide and its packaging as defined in embodiment 63.

[0349] Example

[0350] The following embodiments are provided to supplement the existing disclosure and to provide a better understanding of the subject matter described herein. These embodiments should not be construed as limiting the described subject matter. Rather, it should be understood that the embodiments and implementations described herein are merely illustrative, and various modifications or alterations thereto will be apparent to those skilled in the art and are included within the true scope of the invention, and various modifications or alterations can be made without departing from the true scope of the invention.

[0351] Example 1: Antigen

[0352] Since it is difficult to generate recombinant GPRC5D antigens, standard methods were used to generate transfected cell lines displaying GPRC5D [human (SEQ ID NO:22), cyno (SEQ ID NO:23) and mouse (SEQ ID NO:24)] for use as whole-cell antigens for antibody production and characterization studies (Table 4).

[0353] Table 4: GPRC5D expression cell lines

[0354] Human GPRC5D HEK293T CMV Neomycin Cyno GPRC5D HEK293F CMV Rice blast fungicide

[0355] Example 2: Generation of GPRC5D antibody using phage display

[0356] Two different methods were used to generate GPRC5D antibodies via phages: standard cell panning (negative selection) and FACS cell phage panning (competitive selection).

[0357] Standard cell phage panning (negative selection) :

[0358] In-house de novo phage libraries are detailed in Shi et al. (2010) J.Mol.Biol.397:385-396; International Patent Publication WO09 / 085462). These libraries are constructed based on three human VH germline genes (IGHV1-69, 3-23, 5-51) and four human VL germline genes (A27, B3, L6, O12) and are designed to have high CDR-H3 diversity. Three in-house de novo phage libraries (DNP00004-169HC / LC mixture, DNP00005-323HC / LC mixture, and DNP00006-551HC / LC mixture) targeting GPR are used to display Fab variants on the phage coat protein pIX. C5D-expressing HEK293G5 stable cells (target cells) were panned in rounds 1, 3, and 5. For rounds 2 and 4, Fab-pIX phages amplified in the previous round were applied to HEK293 background cells (negative selection) (see Table 5). Newly created Fab-pIX phages from round 1 that bound to target cells were recovered and amplified overnight. For round 2, the selected phages from round 1 were applied to background cells, and unbound Fab-pIX phages were recovered to amplify the negatively selected phages overnight. Another set of positive and negative rounds of panning—rounds 3 and 4—were performed. In the final round, the amplified phages from the previous round of negative selection were divided into two panning samples, one for target cells and one for background cells.

[0359] Table 5: Standard Cell Phage Screening Flowchart - Negative Selection

[0360]

[0361]

[0362] Standard cell phage screening (background selection) :

[0363] Following a similar environment and incubation time as the previously mentioned standard cell panning process, the Fab-pIX phage display library was added to HEK293 cells (Table 6). After three rounds, the DNA of the Fab-pIX phages corresponding to HEK293 binding was used to generate PCR amplicones for NGS. These NGS results will be used for additional dynamic subtraction analysis within the NGS 2.0 software to help identify potential target-specific Fab candidates.

[0364] Table 6: Standard Cell Phage Screening Flowchart - Background Selection

[0365] 1 HEK293 2 HEK293 3 HEK293

[0366] FACS phage screening (competitive selection)

[0367] Three rounds of panning were performed by simultaneously applying Fab-pIX phage to a mixture of target and background cells (Table 7). In rounds 1 and 2, target cells bound to Fab-pIX phage were sorted using GFP signaling. To capture the bound Fab-pIX phage from the sorted cells, acidic cell lysis was applied, followed by E. coli infection. In the final round, the mixture of Fab-pIX phage and cells amplified in round 2 was sorted into two populations: GFP-gated target cells and non-GFP-gated background cells. Fab-pIX phage bound to both cell populations was captured by acid lysis and E. coli infection.

[0368] Table 7: FACS Cell Phage Screening Flowchart - Competitive Selection

[0369] 1 Human GPRC5D_GFP&HEK293 2 Human GPRC5D_GFP&HEK293 3 Human GPRC5D_GFP&HEK293

[0370] Next-generation sequencing (NGS) based on phage panning

[0371] The six samples selected in the final round of panning were grown overnight under glucose inhibition. These cultures were used to prepare small-batch DNA (Qiagen QIAspin DNA Kit). The six DNA samples were used as PCR templates to generate amplicons for measurement from HCDR1 to HCDR3. The six amplicons were gel purified and pooled for standard cells and completely pooled for FACS cells by keeping the 2.1 and 3.0 formats separate. These pooled gel-purified amplicons were provided to the Genewiz NGS service for processing using MiSeq 1x300 technology. The files were transferred via Genewiz and uploaded to a local server (NAS2.0). Within this server, the sequence files were loaded, read, and analyzed using the NGS2.0 software application. The top 88 sequences, ranked by copy number (>50) and the ratio of target cell (+) to background cell (-) sequences (ratio >5:1), were selected for IgG conversion. Because only variable heavy chain sequences were determined by NGS, the full heavy chain construct had to be constructed electronically into an appropriate framework. Furthermore, since only the heavy chain sequence was known, each candidate was paired with one of four parental light chains (A27, B3, L6, O12). The candidates were ultimately converted into human IgG4PAA.

[0372] ELISA screening and standard sequencing

[0373] Restriction enzyme digestion and self-ligation were performed using the same DNA preparations used for NGS to excise the pIX gene, resulting in soluble Fab expression. Fab expression was assessed by ELISA on ninety-two clones selected from each of the three panned samples, and heavy and light chains were determined by Sanger sequencing. Final candidate clones with diverse sequences within LCDR were then incorporated into mammalian expression plasmids.

[0374] Example 4: Initial characterization of GPRC5D antibody obtained by phage display technology

[0375] GPRC5D combination :

[0376] As described above, whole-cell phage panning was performed using human GPRC5D cells as the antigen. Based on NGS analysis, only the heavy chain sequences of each candidate cell were known. Therefore, each heavy chain had to pair with four parental light chains (A27, B3, L6, O12), resulting in 348 mAbs generated from 87 Hc sequences identified by NGS. The binding of these mAbs to cyno GPRC5D cells was initially evaluated using FACS. The cyno GPRC5D cell line was selected for initial screening to maximize potential binding signals due to its higher expression levels compared to the human GPRC5D cell line. In brief, FACS screening was performed by normalizing the protein concentration to 1 μg / mL and mixing 100 μL of protein with 200,000 cells per well. The mAbs and cells were incubated at 4°C for 1 hour. The cells were then washed three times with PBS and 0.2% FBS. Subsequently, a second anti-human mAb conjugated with PE (Jackson catalog number 709-116-149) was added as the detection reagent. Cells were incubated with the second antibody at 4°C for 1 hour. The cells were then washed three times with PBS and 0.2% FBS. The cells were washed again with PBS and 0.2% FBS and subsequently analyzed on a FACSarry.

[0377] A large number of hits were observed that bind to cyno GPRC5D, including 15 mAbs with an MFI greater than 100,000, 23 mAbs with an MFI less than 100,000 but greater than 10,000, and 63 mAbs with an MFI less than 10,000 but greater than 1,000 (Table 8).

[0378] Table 8: FACS binding data of mAbs derived from NGS with cyno GPRC5D allowed for pairing of heavy chain sequences identified by NGS analysis with the parental Lc values ​​shown. GC5M29 was used as a control (PH9L3 light chain). The highlighted mAbs have an MFI > 1000.

[0379]

[0380]

[0381] Forty mAbs with the highest binding affinity were selected for additional characterization, including repeated binding studies on cynoGPCR5D cells and assessment of binding with human GPRC5D-expressing cells using FACS (Table 9). Analysis of these data led to the selection of 17 mAbs for purification and the generation of GPRC5D×CD3 bispecific antibodies (highlighted). Factors used to select mAbs for purification and GPRC5D×CD3 bispecific antibody generation included specificity for binding to human GPRC5D, cross-reactivity with cynoGPRC5D, and Hc sequence diversity. For example, GC5H36 was paired with three different light chains and binding was analyzed (GC5B162, GC5B163, GC5B164). Only GC5B164 was considered superior because it showed a higher MFI for human GPRC5D compared to GC5B162 or GC5B163.

[0382] Table 9: FACS binding data of NGS-derived mAbs to cyno GPRC5D and human GPRC5D-expressing HEK293F cells. Untransfected HEK293F cells were used to evaluate GPRC5D binding specificity. TF7M1636 was used as an isotype control. The highlighted mAbs were selected for further analysis.

[0383]

[0384]

[0385] The concentration-dependent binding characteristics of each selected mAb to human GPRC5D-expressing HEK293 cells and untransfected HEK293 cells were determined using FACS. Figure 1 All mAbs were observed to bind to human GPRC5D in a dose-dependent manner. Three mAbs, namely GC5B36, GC5B168, and GC5B205, were also observed to bind to untransfected (GPRC5D blank) HEK293 cells, but were not preferentially bound due to this non-specific interaction with the cells.

[0386] The remaining 14 mAbs were selected to generate bispecific antibodies with anti-CD3 arm CD3B219 and anti-RSV blank arm B23M46 (Table 10).

[0387] Table 10: Relationship between GPRC5D mAb ID and Bispecific ID

[0388]

[0389]

[0390] One bispecific antibody, GCDB38, precipitated during recombination, and another, GCDB36, contained >10% aggregates. All other bispecific antibodies were released according to standard release criteria, and the concentration-dependent binding of the bispecific antibody in the blank arm to human GPRC5D-expressing HEK293 cells was analyzed. Figure 2 ).

[0391] All bispecific antibodies were observed to bind in a dose-dependent manner. GC5B320 was included as a binding comparison to understand the binding differences between bivalent mAbs and monovalent bispecific antibodies. GCDB44 was included as a binding comparison to understand the binding differences between anti-CD3 bispecific antibodies and anti-RSV blank arm mAbs. When comparing mAb GC5B320 with bispecific antibodies GCDB30 and GCDB44, a decrease in the expected epigenetic binding affinity was observed. Furthermore, GCDB44 (anti-CD3 bispecific Ab) showed a slightly higher binding affinity against human GPRC5D cells compared to GCDB30 (anti-RSV blank arm bispecific Ab), suggesting that the anti-CD3 arm actively influences binding to this cell line.

[0392] The binding of the bispecific antibody group to MM1R and H929 cells endogenously expressing GPRC5D was also analyzed. Figure 3 Using FACS, the binding characteristics of bispecific antibodies to MM1R and H929 cells were compared with those of overexpressing human GPCR5D HEK293 cells and untransfected HEK293 cells. Bispecific antibodies were observed to bind to GPRC5D endogenously expressed in MM1R and H929 cells with a range of affinities. The highest binding was observed to human GPRC5D HEK cells, which, as a stable overexpression cell line, had a much higher receptor density than MM1R or H929 cells. GCDB37, GCDB38, GCDB39, and GCDB41 were less favorable, as lower binding affinities were observed to H929 and MM1R cells.

[0393] In vitro T-cell-dependent cytotoxicity

[0394] Then, using H929 and MM1R target cells, the potency of the bispecific antibody group in T-cell-mediated cytotoxicity assays was summarized. Figure 4A(See Table 11). In summary, target cells (H929, MM1.R, OPM2, LP-1, and Daudi or HEK parental and HEK+GPRC5D cells) were counted, 10 million cells were centrifuged at 1350 rpm for 3 minutes, and the cell pellet was resuspended in 1 mL of diluted CFSE solution (CellTrace CFSE proliferation staining agent reconstituted in 18 μL of sterile DMSO, with 1 μL of the solution diluted in 10 mL of sterile PBS) and incubated in the dark at room temperature for 8 minutes. After incubation, 1 mL of HIFBS was added to the cell suspension to quench excess CFSE. Cells were washed twice in RPMI-1640 containing 10% FBS. After recovery in 10 mL of RPMI, cells were counted and cell viability was recorded in a spreadsheet. Cells were diluted to 2.2 × 10^5 / mL and incubated at 37°C until use.

[0395] Pan-T cells from normal donors were thawed in a 37°C water bath and then centrifuged at 1350 rpm at 4°C for 3 minutes. The supernatant was discarded, and the cells were reconstituted in culture medium at a concentration of 1.1 × 10^6 / mL. 2 × 10^5 target cells were added to the wells of a 96-well U-shaped plate, followed by the addition of Fc blocking agent (to a final concentration of 2 mg / mL). All cell lines were incubated at room temperature for 10 minutes to block Fc receptor activity. 1 × 10^5 T cells (5:1 effector:target ratio) were added to each well. After mixing the target and T cells, 20 μl of GPRC5D×CD3 bispecific antibody dilution was added to each well. The GPRC5D×CD3 bispecific antibody was diluted to 800 μg / mL (10×) with PBS. Titration was prepared in a 96-well U-shaped plate using a 4-fold serial dilution in PBS. The last column was reserved as a separate PBS control. The plates were incubated at 37°C and 5% CO2 for 48 hours.

[0396] Two days later (48 hours), the plates were centrifuged, and 100 μL of the supernatant was stored at -80°C for cytokine release assays. Cells were washed in 200 μL of PBS and incubated for 20 min at room temperature in 50 μL of near-infrared Live / Dead staining agent (1:200 dilution) and anti-CD25 PE antibody (1:50 dilution). Cells were then washed once in 200 μL of FACS buffer and finally restored in 150 μL of FACS buffer. Target cytotoxicity (target %) and T cell activation CD25+ (live T cells %) were analyzed using FACSCanto II and FlowJo 7.6. Data were plotted and fitted in GraphPad Prism 6 using nonlinear regression with a variable slope (four parameters) function and least squares method.

[0397] Table 11: Mean EC50 calculated for T-cell-mediated cytotoxicity assessment of GPRC5D×CD3 bispecific antibody using H929 and MM1R target cells. 50 .

[0398]

[0399]

[0400] All bispecific antibodies were active in T-cell-mediated killing of H929 cells, and a range of potency was observed (Table 11). A similar rank was observed for both cell lines. However, lower EC50 was observed in MM1R cells. 50 Interestingly, binding affinity is not necessarily correlated with potency in T-cell-mediated cytotoxicity assays. For example, GCDB44 binds bispecific antibodies with the highest affinity but has the lowest potency in cytotoxicity assays. In contrast, GCDB43, which has a similar (albeit slightly lower) binding affinity, exhibits the strongest potency in cytotoxicity assays.

[0401] To assess functional cross-reactivity with cyno GPRC5D, T-cell-mediated cytotoxicity and T-cell activation in the bispecific antibody group were then analyzed using cyno GPRC5D HEK293 cell profiling. Figure 5A (and B). All bispecific antibodies were active in this assay, but limited potency was observed against cyno GPRC5D+ expressing cells.

[0402] Internal effects

[0403] To understand the in vivo potency of these GPRC5D×CD3 bispecific antibodies and then perform benchmarking analyses for in vivo studies, GCDB32 and GCDB35 were selected. Figure 5A &B) was tested in an H929 tumor prevention model. One week after injection of human PBMCs, H929 cells were engrafted into NSG mice. Bispecific antibody treatment was initiated simultaneously with H929 cell engraftment and continued every 2 or 3 days (q2d or q3d) at doses of 10 μg, 1 μg, and 0.1 μg / animal, for a total of five treatments. Ten mice were used in each group, and PBS was included as a vector control. Treatment was stopped on day 11, and on day 25 ( Figure 6A and B) or day 26 ( Figure 12A-D) The study was terminated. Except for GCDB35, which showed approximately 80% inhibition of tumor growth at a dose of 1 μg / animal, all GPRC5D×CD3 antibodies tested in this prophylactic model showed 100% inhibition of tumor growth at doses of 10 μg and 1 μg / animal. At doses ten times lower (0.1 μg / animal), these bispecific antibodies showed varying degrees of efficacy ranging from 10% to 80% tumor growth inhibition.

[0404] Fc inhibitors exhibit in vitro T-cell-dependent cytotoxicity.

[0405] To gain insight into the specificity of the GPRC5D targeting arm, the GPRC5D×CD3 bispecific antibody group was evaluated in a T-cell redirected cytotoxicity assay in the presence of an Fc blocker. This experiment is crucial for understanding the ability of bispecific antibodies to interact with the Fc portion of the bispecific antibody in in vitro assays, such as the specificity of the bispecific antibody to the target cell. Potency shifts were observed in several bispecific antibodies in the T-cell-mediated cytotoxicity assay, with the largest shifts observed in GCDB40 and GCDB34. Figures 7A-7B ).

[0406] Then, direct measurements were performed on the binding interactions between the four most potent bispecific antibodies (GCDB32, GCDB35, GCDB40, and GCDB43) and the Fcγ receptor. Figures 8A-8D Alpha Screen analysis was performed on each Fcγ receptor and the bispecific antibodies listed above. All samples were analyzed in duplicate. On FcγRI, the four bispecific antibodies behaved similarly to the B21M hIgG4 PAA. That is, their competitiveness did not exceed that of the matched isotype control. Similar differences were observed between the hIgG1 WT control and the four bispecific antibodies on FcγRIIIa, with GCD43 being the most similar to the IgG4PAA isotype control, while the other bispecific antibodies showed slightly higher affinity for FcγRIIIa. On FcγRIIa and FcγRIIb, the four bispecific antibodies competed in the following order: GCDB40 > GCDB32 > GCDB43 > GCDB35. GCDB40 was the most competitive or bound to FcγRIIa and FcγRIIb with the highest affinity. In fact, GCDB40 competed with both FcγRIIa and FcγRIIb to the same extent as hIgG1WT, confirming the potency shift observed in T-cell-mediated cytotoxicity assays when Fc blockers are included. GCDB40 was less advantageous due to unexpected interactions with FcγRIIa and FcγRIIb.

[0407] Competitive binding assay

[0408] The competitive binding of anti-GPRC5D mAbs to each other on human GPCR5D cells was evaluated. Briefly, cells were seeded at 50,000 cells / well in 50 μL of medium and allowed to settle at 37°C for 90 min. The wells were then blocked with 3% BSA at room temperature for 1 h. The mAbs were labeled with ruthenium(II) tri-bipyridine, N-hydroxysuccinimide (Ru-tag) according to standard procedure. In individual 96-well plates, 5 μM of the competitor mAb was incubated with 50 nM of the Ru-tag mAb. The blocking solution was removed from the cell plate, and 25 μL of the mAb solution was added. The plates were incubated at room temperature with shaking for 1 h. After washing the plates three times with PBS, 150 μL of MSD read buffer (surfactant-free) was added, and binding of the Ru-tag antibody was detected using an MSD plate reader.

[0409] All mAbs belonged to the same competition group, except that GC5B420 and GC5B421 were not fully competitive with GC5B81, GC5B285, and / or GC5B332 (blockage <70%) (Table 12). It is assumed that the binding of the two mAbs to GPRC5D may not spatially give a smaller extracellular domain of GPRC5D compared to the size of the mAb.

[0410] Table 12: Competitive binding epitope assessment of anti-GPRC5D mAb to each other on human GPCR5D cells (+ / - = blockade <70%).

[0411] GC5B81 + + + + + / - + / - GC5B164 + + + + + + GC5B285 + + + + + + / - GC5B332 + + + + + / - + GC5B420 + + + + + + GC5B421 + + + + + + GC5B243 + + + + + +

[0412] Example 4: Production of GPRC5D antibody using hybridoma technology

[0413] On days 0, 10, and 20, three Balb / c mice were immunized via percutaneous intradermal injection at the base of the tail with pCMV6-neo (CMV promoter) plasmid DNA expressing full-length human GPRC5D. On day 59, the mice were immunized by final intraperitoneal and intravenous injections of rat basophilic leukemia (RBL) cells overexpressing full-length human GPRC5D. On day 63, lymph nodes and spleens were harvested, B cells were enriched, and the cells were used to generate approximately 3500 mAb-secreting hybridomas.

[0414] Example 5: Initial characterization of GPRC5D antibody obtained by hybridoma technology

[0415] GPRC5D combination

[0416] Hybridomas that simultaneously bind to RBL and human GPRC5D-expressing RBL cells were screened using FACS. The background-regulated MFI ratio of each mAb binding to GPRC5D_RBL cells relative to untransfected RBL cells was calculated, and any sample with a binding ratio greater than 3 was considered potentially positive. Ninety-nine hybridomas had a ratio greater than 3 and were favorable for V-region clones. Thirty-one mAb sequences were identified, synthesized, expressed, and purified. Two of the 31 mAbs showed binding to RBL_GPRC5D cells beyond the background (…). Figure 9A and 8B GCDB390 and GCDB396 were selected for expression, purification, and generation of bispecific antibodies against CD3B219 on the CD3 arm, to produce bispecific antibodies GCDB46 and GCDB47, respectively.

[0417] T-cell-dependent cytotoxicity

[0418] GCDB46 and GCDB47 were evaluated in a T-cell-mediated cytotoxicity assay. Figure 10A and 10B Both bispecific antibodies were observed to be effective, and the reported EC values ​​were... 50 The values ​​were 0.67 nM and 0.1 nM, respectively. Based on these data, further lead optimization was performed on these two mAbs along with three phage-derived mAbs.

[0419] Example 6: Hit Evaluation, Selection and Optimization

[0420] Based on the binding, function, cross-reactivity, and selectivity data summarized in Table 13, five GPRC5D bispecific antibodies were selected for lead optimization (GCDB32, GCDB43, GCDB35, GCDB46, and GCDB47).

[0421] Table 13: Lead optimization data for GPRC5D×CD3 bispecific antibodies: evaluation of binding, function, cross-reactivity and selectivity of bispecific antibodies.

[0422]

[0423] Leader optimization aims to address the risk of potential post-translational modification (PTM) sequences in GCDB32 (GC5B81 parent mAb), GCDB43 (GC5B285 parent mAb), and GCDB35 (GC5B164 parent mAb), as outlined in Table 14.

[0424] Table 14: Hc CDR sequences are shown as potential PTM sequences derived from phages, and potential PTM sequence probabilities are underlined.

[0425] (The SEQ ID NO for each listed sequence is provided in parentheses)

[0426]

[0427] All PTM variants analyzed for GC5B81 showed significantly reduced binding affinity, indicating the criticality of this residue (HCW102) for the complementary site (Table 15).

[0428] Table 15: CDR sequences and binding data of the GC5B81 PTM library: Mutation sites are underlined. Binding is classified as MFI>10,000=++; MFI>1,000=+; MFI<1,000

[0429] (The SEQ ID NO for each listed sequence is provided in parentheses)

[0430]

[0431] The combined study identified the mutation numbers of GC5B285 and GC5B164, which maintain binding to human GPRC5D (Tables 16 and 17).

[0432] Table 16: CDR sequences and binding data of the GC5B164 PTM library: Mutation sites are underlined. Binding is classified as MFI>10,000=++; MFI>1,000=+; MFI<1,000.

[0433] (The SEQ ID NO for each listed sequence is provided in parentheses)

[0434]

[0435]

[0436]

[0437] Table 17: CDR sequences and binding data of the GC5B285 PTM library: Mutation sites are underlined. Binding is classified as MFI>10,000=++; MFI>1,000=+; MFI<1,000.

[0438] (The SEQ ID NO for each listed sequence is provided in parentheses)

[0439]

[0440]

[0441] Based on this combined data, the selected mAb was generated as a GPRC5D×CD3 bispecific antibody, and T-cell-mediated cytotoxicity of H929 cells was evaluated (Table 18).

[0442] Table 18: Functional activity of selected GPRC5D PTM variants of GCDB164 and GCDB285

[0443]

[0444] A range of efficacies was observed, and T-cell-mediated cytotoxicity assays are not necessarily predicted by the observed binding affinity. For example, GC5B465 and GC5B463 bind human GPRC5D with similar affinity, differing only in two amino acids of their sequence (Table 17), and a 12.5-fold difference in their efficacies as GPRC5D×CD3 bispecific antibodies was observed (Table 18). Based on functional data, GC5B465 and GC5B483 were selected as the optimal sequences for GC5B285 (GCDB43 as CD3 bispecific) and GC5B164 (GCDB35 as CD3 bispecific) respectively.

[0445] Human frame adaptation was achieved for mouse hybridoma-derived GPCR5D mAb (GC5B390 and GC5B396, or GCDB46 and 47 as CD3 bispecific). Combined studies identified multiple frames of GC5B396 that maintains binding to human GPRC5D and one frame of GC5B390 (Table 19).

[0446] Table 19: Binding and functional data of human framework adaptation of hybridoma-derived anti-GPRC5D mAb libraries: binding is categorized as MFI>10,000=+++; MFI>5,000=++; MFI>1,000=+; MFI<1,000.

[0447]

[0448] Based on the combined data, several anti-GPCR5D mAbs were generated as CD3 bispecific antibodies, and T-cell-mediated cytotoxicity in H929 cells was assessed (Table 18). Functional analysis identified GCDB63, GCDB67, and GCDB69 as potent, fully humanized GPRC5D×CD3 bispecific antibodies. Based on these data, the corresponding anti-GPRC5D mAbs, namely GC5B515, GC5B532, and GC5B540, were selected as fully humanized sequences for GC5B390 and GC5B391.

[0449] Next, additional leader optimization of the fully humanized sequence was performed to address the risks of potential post-translational modifications in GC5B515, GC5B532, and GCDB540. The G56S mutation was generated in the heavy chain sequence to remove the potential deamidation risk of GC5B515 (Table 20).

[0450] Table 20: Binding and functional activity of selected GPRC5D PTM variants of GC5B532, GC5B540, and GCDB515:

[0451]

[0452] The heavy chains of GC5B532 and GC5B540 contain potential risks of isomerization and oxidation. M64K and G99A mutations were generated to mitigate these risks (Table 20). All tested variants with the G99A mutation showed a significant decrease in binding affinity, while the M64K and G56A variants were unaffected. Based on binding data, only GC5B596 continued functional evaluation and demonstrated potency as a CD3 bispecific molecule (GCDB72) in a T-cell-mediated cytotoxicity assay.

[0453] Therefore, four GPRC5D bispecific mAbs were selected for additional characterization: GCDB32, GCDB53, GCDB61, and GCDB72. Tables 21 and 22 below show the CDRs, as well as the heavy and light chain sequences, of the GPRC5D mAbs used to generate the bispecific molecules.

[0454] Table 21: Shows the CDR sequences of four GPRC5D mAb candidates that are functional when bound to human cyno GPRC5D and generated as CD3 bispecific molecules:

[0455]

[0456]

[0457] Table 22: Shows the heavy and light chain variable region sequences of four GPRC5D mAb candidates that are functional when bound to human and cyno GPRC5D and generated as CD3 bispecific molecules:

[0458]

[0459]

[0460] Example 7: Preparation of GPRC5D and CD3 antibodies in a bispecific form from IgG4S228P, L234A, and L235A

[0461] Four monospecific GPRC5D antibodies (see Table 21) are designated IgG4, with Fc substitutions for S228P, L234A, and L235A or S228P, L234A, L235A, F405L, and R409K (CD3 arms) (numbered according to the EU index). A monospecific anti-CD3 antibody, CD3B219, was also generated, comprising VH and VL regions having the heavy chain of SEQ ID NO:25 and the light chain of SEQ ID NO:26, and an IgG4 constant region with S228P, L234A, L235A, F405L, and R409K substitutions.

[0462] Monospecific antibodies were purified using standard methods with a Protein A column (HiTrap MabSelect SuRe column). After elution, the collected material was dialyzed against D-PBS (pH 7.2).

[0463] Bispecific GPRC5D×CD3 antibodies were generated by combining monospecific CD3 mAb and monospecific GPRC5D mAb in an in vitro Fab arm exchange (as described in WO2011 / 131746). Briefly, approximately 1–20 mg / mL of anti-GPRC5D / anti-CD3 antibody in PBS (pH 7–7.4) and 75 mM 2-mercaptoethanolamine (2-MEA) were mixed at a molar ratio of 1.08:1 and incubated at 25–37°C for 2–6 hours. The 2-MEA was then removed using standard methods via dialysis, percolation, tangential flow filtration, and / or rotary cell filtration.

[0464] The heavy and light chains of the GPRC5D×CD3 bispecific antibody are shown in Table 23 below.

[0465] Table 23: Heavy and light chain sequences of bispecific Ab IgG4-PAA

[0466]

[0467]

[0468]

[0469] Example 8: Functional characterization of GCDB32, GCDB53, GCDB61, and GCDB72

[0470] The binding of GCDB32, GCDB53, GCDB61, and GCDB72 to mouse GPRC5D was evaluated (Table 24). All four bispecific antibodies bound mouse GPRC5D with the observed range of binding affinities.

[0471] Table 24: Binding of anti-GPCR5D×CD3 antibody to mouse GPRC5D:

[0472]

[0473] In addition, cross-reactivity with cyno GPRC5D was assessed using a T-cell retargeting cytotoxicity assay with overexpressed human and cyno GPRC5D cell lines (Table 25). A GPRC5D×CD3 bispecific antibody was equivalent to human and cyno GPRC5D (GCDB32), while other bispecific molecules tested showed lower potency in inducing cyno GPRC5D cytotoxicity than human GPRC5D.

[0474] Table 25: Functional activity of the lead GPRC5D×CD3 antibody against human and cyno GPRC5D-expressing HEK cells:

[0475]

[0476] Additional characterization aims to understand in vitro binding ( Figure 11A-11E ) and efficacy (Table 26).

[0477] Table 26: T-cell-mediated cytotoxicity of lead GPRC5D×CD3 antibody against several human GPRC5D-expressing B cell lines:

[0478]

[0479] Although a range of binding affinities was observed, with GCDB61 being the strongest and GCDB72 & GCDB32 the weakest, their in vitro potency was very similar to the bispecific antibody group. However, according to ranking analysis, GCDB72 was the most effective across the various B cell lines analyzed. In vitro binding and potency assays using patient-derived MNCs yielded results more similar to in vitro assays (Table 27 and...). Figure 15A and 15B ).

[0480] Table 27: GPCR5D×CD3 bispecific antibody binding, T-cell-mediated cytotoxicity, and T-cell activation in MNCs derived from MM patients:

[0481]

[0482] GCDB61 exhibited the highest binding affinity to patient MNCs, while GCDB72 and GCDB32 showed the weakest binding affinity. Similarly, even with observed differences in binding affinity, all bispecific antibodies demonstrated sub-nanomolar efficacy in T-cell retargeting cytotoxicity assays. The molecules were virtually indistinguishable on the basis of in vivo and in vitro potency; however, in vivo data provided differentiation (…). Figure 12A-12D ).

[0483] One week after injection of human PBMCs, H929 cells were engrafted into NSG mice. Bispecific antibody treatment was initiated concurrently with H929 cell engraftment and administered at doses of 10 μg, 1 μg, and 0.1 μg / animal every 2 or 3 days (q2d or q3d) for a total of five treatments. Ten mice were used in each group, and PBS was included as a vector control. Treatment was stopped on day 11, and on day 26 (… Figure 12A -D) The study was terminated. All GPRC5D×CD3 bispecific antibodies tested in this prophylactic model showed 100% inhibition of tumor growth at doses of 10 μg and 1 μg / animal. Differentiation was observed at the lowest dose of 0.1 μg / animal, and GCDB72 showed superiority over the other bispecific antibodies tested, with 80% inhibition of tumor growth observed.

[0484] Example 9: GPRC5D antibody against GPRC5D + Binding characteristics of the MM1.R cell line

[0485] Using FACS to measure the GPRC5D antibody against GPRC5D + Binding affinity of human MM cell line (MM1.R, purchased from ATCC (American Center for Type Culture Collection)). Figure 13 This demonstrates that all lead antibodies bind in a dose-dependent manner to GPRC5D-expressing MM.1R cells, EC1N. 50 The values ​​ranged from 0.10 nm to 135 nm, and all except GC5B602 were significantly lower than the EC value of 121.7 nm. 50 The value of the commercial antibody FAB6300 (R&D Systems, catalog number FAB6300A, clone number 571961).

[0486] GPRC5D + MM1.R cell lines were stained with various concentrations of leader antibodies for 60 minutes to measure surface binding characteristics (n=3). Phycoerythrin-labeled human IgG4Fc was used as a secondary antibody to capture the signal (Southern Biotech, clone HP6025, catalog number 9200-09). Binding is expressed as normalized geometric mean fluorescence intensity, as measured by FACS. Data were plotted and fitted in GraphPad Prism 6 using nonlinear regression with variable slopes (four parameters) and least squares fitting.

[0487] Furthermore, compared to commercial antibodies, using three GPRC5D... + (JIM3, OPM-2, and MM.1R; cell lines, purchased from ATCC) Evaluation of the binding characteristics of GPRC5D mAb GC5M481 in multiple myeloma cell lines (JIM3, OPM-2, and MM.1R; cell lines, purchased from ATCC) Figure 14AAdditionally, profiling analysis of the cyno cross-reactivity of GPRC5D mAb (GC5M481) using cyno-GPRC5D-expressing Daudi cells showed a stronger binding compared to parental cells. Figure 14A In addition, the binding potential of five GPRC5D×CD3 bispecific antibodies (GCDB32, GCDB48, GCDB53, GCDB61, and GCDB72) in GPRC5D+ (JIM3, OPM-2, and MM1.R) cell lines was evaluated. Figure 14B The results show a significant combination, as evidenced by the shift in the histogram (solid black line) compared to the same type control (dashed line filled in gray).

[0488] Example 10: GCDB72 targeting subcutaneous MM.1S human multiple myeloma xenografts in PBMC-humanized NSG mice Antitumor efficacy of grafts

[0489] This in vivo study was conducted in PBMC humanized NSG mice to determine the efficacy of GCDB72 against the established MM.1S human multiple myeloma (MM) xenograft. On day 0 of the study, female NSG mice of similar weight and age were subcutaneously (sc) implanted with MM.1S human MM cells (1 × 10⁻¹⁰ cells per mouse in 200 μL PBS) via the right posterior rib area. 7 (cells). On the 7th day after tumor cell transplantation, 1×10⁻⁶ cells were injected intravenously via the lateral tail vein. 7 Personal PBMCs (dissolved in 200 μL PBS). Treatment began on day 15, when the average tumor volume was approximately 72–78 mm. 3 Mice were administered 0.1 μg (0.005 mg / kg), 1 μg (0.05 mg / kg), 10 μg (0.5 mg / kg), and 50 μg (2.5 mg / kg) of PBS or GCDB72 DuoBody antibody intravenously (iv). Blank DuoBody control, CD3× blank, and blank×GPRC5D were each administered at 10 μg per mouse. Treatments were administered approximately every three days (q3d) for a total of seven doses. Robust antitumor efficacy was observed with both high doses (10 μg and 50 μg) of GCDB72, with complete regression of MM.1Ssc tumors in 100% (n=10 out of 10) of the animals at the end of the study. Figure 16Furthermore, compared to PBS-treated tumors, a dose of 1 μg per mouse significantly inhibited tumor growth by 65% ​​(p ≤ 0.0001), while a dose of 0.1 μg had a smaller effect (TGI = 19.3%, p = 0.0023). The effect of CD3 × blank was not considered effective (TGI = 28%, p ≤ 0.0001), and blank × GPRC5D had a negligible effect of 3.1% TGI (p = 0.9971). TGI was measured on day 36, at which point at least 80% of animals in each group were surviving. Significant weight loss and / or mortality due to GVHD began to appear after day 36. Figure 17 The study was terminated on day 43, at which point 60% or fewer of the animals remained in the group.

[0490] Example 11: Antibody-dependent cell-mediated cytotoxicity (ADCC) of GPRC5D antibody

[0491] The anti-human GPRC5D mAb group was generated as an IgG1 mAb. In addition, a new anti-human GPRC5D mAb group was generated as described in Example 2. Tables 28 and 29 below show the CDR and heavy and light chain variable region sequences of the new GPRC5D mAbs. These new antibodies were used to generate bispecific CD3 molecules as described in Example 7, and were also introduced as IgG1 mAbs for ADCC activity assessment.

[0492] Table 28: CDR sequences of the new GPRC5D antibody group

[0493]

[0494]

[0495] Table 29: Heavy and light chain variable region sequences of the new GPRC5D antibody group

[0496]

[0497]

[0498]

[0499] ADCC activity against H929 cells (Tables 30 and 31). Briefly, multiple myeloma cells were labeled with calcein-AM at room temperature for 30 minutes and then washed twice with PBS at 0.2 × 10⁻⁶. 6 / mL resuspended in RPMI + 10% HI FBS. Thaw PBMCs and wash with PBS, then add 3 × 10⁹ / mL. 6Cells were resuspended in RPMI growth medium. 10,000 or 50,000 target cells were mixed with 100,000 or 2,500,000 PBMCs in the presence of antibody and incubated at 37°C for 3 hours in a CO2 incubator. After incubation, the plates were centrifuged at 200g for 4 minutes, and 100 μL of the supernatant was transferred to a new 96-well plate. Fluorescence intensity was measured at 485 / 535 nM. RFU values ​​were plotted to calculate the lysis percentage.

[0500] Table 30: Antibody-dependent cytotoxicity of anti-GPRC5D mAb with IgG1 Fc on H929 cells:

[0501]

[0502]

[0503] Table 31: Comparison of antibody-dependent cytotoxicity and T-cell-mediated cytotoxicity of H929 cells

[0504]

[0505] A range of potency was observed, from 2 pM to 27.7 nM. Binding affinity does not necessarily predict potency in ADCC assays. For example, GC5B382 and GC5B379 have similar binding affinity to human GPRC5D cells, but showed a 15× difference in cytotoxicity against H929 cells in ADCC assays. Similarly, cytotoxicity induction by GPRC5D×CD3 bispecific molecules does not predict potency in ADCC assays, as illustrated by GC5B370 and GC5B602. When formatted as a CD3 bispecific molecule, GC5B602 (GCDB63) showed sub-nanomolar potency against H929 cells, while GC5B370 was essentially inactivated as a CD3 bispecific molecule (GCDB41). The same v region resulted in opposite observations in ADCC assays when formatted as IgG1 mAb, with GC5B370 observed to be significantly more potent than GC5B602 (approximately 1100×).

[0506] Brief description of sequence lists

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

Claims

1. A separate antibody that specifically binds to GPRC5D, comprising a heavy chain and a light chain. The heavy chain includes a variable heavy chain (VH) region, the VH region being shown in the amino acid sequence of SEQ ID NO: 55; and The light chain includes a variable light chain (VL) region, the VL region being shown in the amino acid sequence of SEQ ID NO:

58. The antibody mentioned therein is an IgG4 isotype.

2. The isolated antibody of claim 1, wherein the heavy chain further comprises the amino acid sequence of SEQ ID NO:

59.

3. A GPRC5D x CD3 bispecific isolated antibody comprising a GPRC5D binding moiety, which includes a heavy chain and a light chain. The heavy chain includes a variable heavy chain (VH) region, the VH region being shown in the amino acid sequence of SEQ ID NO: 55; and The light chain includes a variable light chain (VL) region, the VL region being as shown in the amino acid sequence of SEQ ID NO: 58, and The CD3 binding region comprises the heavy chain of SEQ ID NO: 99 and the light chain of SEQ ID NO:

100. The bispecific antibody mentioned therein is an IgG4 isotype.

4. The isolated antibody of claim 3, wherein the GPRC5D binding heavy chain further comprises the amino acid sequence of SEQ ID NO:

59.

5. The isolated antibody of any one of claims 1-4, wherein the antibody binds to GPRC5D on the surface of human multiple myeloma cells.

6. The isolated antibody of any one of claims 3-4, wherein the antibody has an EC50 concentration of less than 0.22 nM. 50 Induces in vitro activation of human T cells.

7. The isolated antibody of any one of claims 3-4, wherein the antibody has an EC50 concentration of less than 0.89 nM. 50 Inducing T cell-dependent cytotoxicity in cells expressing GPRC5D in vitro.

8. An isolated cell expressing an antibody isolated according to any one of claims 1-4.

9. The isolated cells according to claim 8, wherein the antibody is recombinant.

10. Use of the isolated antibody according to any one of claims 1-4 in the preparation of a medicament for treating a patient with multiple myeloma.

11. The use according to claim 10, wherein the medicament further comprises a second therapeutic agent.

12. The use according to claim 11, wherein the second therapeutic agent is a chemotherapeutic agent or a targeted anticancer therapy.

13. The use according to claim 11, wherein the second therapeutic agent is cytarabine, anthracycline, histamine dihydrochloride, or interleukin-2.

14. The use according to claim 11, wherein the second therapeutic agent is formulated for use together with or separately from the antibody.

15. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1-4 and a pharmaceutically acceptable carrier.

16. A method for generating an isolated antibody according to any one of claims 1-4, the method comprising culturing cells expressing the isolated antibody.

17. An isolated synthetic polynucleotide, said polynucleotide encoding an isolated antibody according to any one of claims 1-4.

18. A kit for treating cancer, the kit comprising an isolated antibody and its packaging according to any one of claims 1-4, wherein the cancer is multiple myeloma.

19. A kit for treating cancer, the kit comprising the isolated synthetic polynucleotide and its packaging according to claim 17, wherein the cancer is multiple myeloma.

20. A kit for treating cancer, the kit comprising an isolated antibody according to any one of claims 1-4, an isolated synthetic polynucleotide encoding the isolated antibody, and packaging thereof, wherein the cancer is multiple myeloma.

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