EA044685B1 - ANTIBODIES TO GPRC5D, BISPECIFIC ANTIGEN-BINDING MOLECULES THAT BIND GPRC5D AND CD3, AND THEIR APPLICATIONS - Google Patents

Description

Cross-references to related inventions

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

List of sequences

The currently pending application contains a sequence listing provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of this listing, created on June 28, 2017, is named PRD3422USNP_SL.txt and is 57,673 bytes in size.

Field of technology

The invention provided herein relates to monoclonal antibodies that specifically bind G protein-coupled receptor class C, group 5, member D (GPRC5D), multispecific antibodies that specifically bind GPRC5D and cluster of differentiation 3 (CD3), and methods of production and use of the described antibodies.

Prerequisites for creating the invention

Multiple myeloma (MM) is the second most common hematologic malignancy and accounts for 2% of all cancer deaths. MM is a heterogeneous disease and is most often caused by chromosomal translocations, among others, t(11;14), t(4;14), t(8;14), del(13), del(17) (Drach et al. , (1998) Blood 92 (3):802-809; Gertz et al., (2005) Blood 106 (8):2837-2840; Facon et al., (2001) Blood 97(6): 1566-1571) . Patients with MM may experience various disease-related symptoms due to bone marrow infiltration, bone destruction, renal failure, immunodeficiency, and psychological pressure associated with a cancer diagnosis. As of 2006, the 5-year survival rate for MM was approximately 34%, indicating that MM is an intractable disease that currently has no cure.

G protein-coupled receptor class C group 5 member D (GPRC5D) is an orphan, atypical class C GPCR first identified in 2001 (Brauner-Osborne et al. Biochim Biophys Acta. 1518(3):237 -248, 2001). GPRC5D and other group 5 GPCRs have unusually short amino-terminal domains for class C receptors and are therefore predictably conformationally similar to class A receptors. In this respect, they are unique, having sequence homology to class C GPCRs and a predicted structural topology comparable to class A receptors The functional consequences of GPRC5D activation have not been described, and the ligand remains unknown. The gene has three exons and is located on chromosome 12p13.3 in humans. The GPRC5D receptor is highly conserved across species and has 92% identity with cynomolgus macaque GPRC5D.

GPRC5D mRNA is predominantly expressed in all malignant plasma cells obtained from patients with MM (Atamaniuk JA et al. Eur J Clin Invest 42(9) 953-960; 2012; Frigyesiblood and Cohen, et al. Hematology 18(6): 348- 35; 2013). GPRC5D expression varies among patients and correlates well 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).

This exclusive expression of GPRC5D on the plasma cell line indicates that it is an ideal target for antimyeloma antibodies.

Summary

Antibodies that specifically bind to GPRC5D, as well as antigen binding fragments thereof, are provided herein. Also described are related polynucleotides capable of encoding the proposed GPRC5D-specific antibodies and antigen binding fragments, cells expressing the proposed antibodies and antigen binding fragments, as well as corresponding vectors and detectably labeled antibodies and antigen binding fragments. Moreover, methods of using the proposed 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 cancers expressing GPRC5D; to determine whether to treat a patient's cancer; or to determine whether an individual is affected by a cancer expressing GPRC5D and, therefore, will respond to treatment with a GPRC5D-specific anticancer drug, such as the multispecific antibodies to GPRC5D and CD3 described herein.

Also provided herein are multispecific antibodies that immunospecifically bind to GPRC5D and CD3, as well as multispecific antigen binding fragments thereof. Also described are related polynucleotides capable of encoding the proposed multispecific antibodies to GPRC5DxCD3, cells expressing the proposed antibodies, as well as corresponding vectors and detectably labeled multispecific antibodies. Moreover, methods of using the proposed multispecific antibodies are described. For example, multispecific antibodies to GPRC5DxCD3 can be used to diagnose or monitor the progression, regression or stability of cancers expressing GPRC5D; to determine whether a patient should be treated for cancer; or to determine whether an individual is affected by a cancer expressing GPRC5D, and therefore will respond to treatment with a GPRC5D-specific anticancer drug, for example, the multispecific antibodies to GPRC5DxCD3 described herein.

GPRC5D-specific antibodies

Described herein are isolated antibodies and antigen binding fragments specific for GPRC5D. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments bind to human GPRC5D. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments bind to human GPRC5D and cynomolgus GPRC5D. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments bind to one or more residues of a polypeptide having the amino acid sequence of SEQ ID NO: 22. The GPRC5D-specific antibody or antigen-binding fragment can induce ADCC in vitro with an EC50 concentration of 28 nM or less.

In table 1 provides a summary of examples of some of the GPRC5D-specific antibodies described herein.

Table 1. CDR sequences of monoclonal antibodies generated to human GPRC5D

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 GC5B81 SYAIS (SEQ ID NO 1) GIIPIFGTANYAQ KFQG (SEQ ID NO 5) ESRWRGYKLD (SEQ ID NO 9) RASQSISSYLN (SEQ ID NO 13) AASSLQS (SEQ ID NO 16) QQSYSTPLT (SEQ ID NO 19) GC5B465 GC5B597 NYWMS (SEQ ID NO 2) GISYSGGSKYYAS SVKG (SEQ ID NO 6) AAFDFGRRAVRL D (SEQ ID NO 10) RASQSISSYLN (SEQ ID NO 13) AASSLQS (SEQ ID NO 16) QQSYSTPLT (SEQ ID NO 19) GC5B483 GC5B599 SYFIG (SEQ ID NO 3) IIYPGKSDTRYSP SFQG (SEQ ID NO 7) VYSFGGRHKALF DY (SEQ ID NO 11) RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B596 GYTMN (SEQ ID NO 4) LINPYNSDTNYAQK LQG (SEQ ID NO 8) VALRVALDY (SEQ ID NO 12) KASQNVATHVG (SEQ ID NO 15) SASYRYS (SEQ ID NO 18) QQYNRYPYT (SEQ ID NO 21) GC5B382 DYGMH (SEQ ID NO 61) AIKYSGGSTYYADS VKG (SEQ ID NO 67) RAESGPLDY (SEQ ID NO 72) KSSQSVLYSSNNNK NYLA (SEQ ID NO 98) WASTRES (SEQ ID NO 78) QQYYSTPLT (SEQ ID NO 80) GC5B379 NYWMS (SEQ ID NO 2) GISYSGGSKYYADS VKG (SEQ ID NO 28) AAWDFGRRAVRL DY (SEQ ID NO thirty) RASQSISSYLN (SEQ ID NO 13) AASSLQS (SEQ ID NO 16) QQSYSTPLT (SEQ ID NO 19) GC5B373 SYWIG (SEQ ID NO 27) IIYPGDSDTRYSP SFQG (SEQ ID NO 29) IGFYGRSFRIFD Y (SEQ ID NO 73) RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B376 SYWIG (SEQ ID NO 27) IIYPGDSDTRYSP SFQG (SEQ ID NO 29) VYSFGGRHKALFD Y (SEQ ID NO RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20)

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eleven) GC5B385 GYAMS (SEQ ID NO 62) AISGSGGSTYYADS VKG (SEQ ID NO 68) VDRSFGRSRYTL DY (SEQ ID NO 74) RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B370 GC5B598 SYGIS (SEQ ID NO 63) GIIPIFGNINYAQ KFQG (SEQ ID NO 69) VSRRFKRFAYYF DY (SEQ ID NO 75) KSSQSVLYSSNNNK NYLA (SEQ ID NO 98) WASTRES (SEQ ID NO 78) QQYYSTPLT (SEQ ID NO 80) GC5B602 GYSFTGYTMN (SEQ ID NO 64) LINPYNGDTN (SEQ ID NO 70) VALRVALDY (SEQ ID NO 12) KASQNVATHVG (SEQ ID NO 15) SASYRYS (SEQ ID NO 18) QQYNRYPYT (SEQ ID NO 21) GC5B603 SYAMS (SEQ ID NO 65) AISGSGGSTYYADS VKG (SEQ ID NO 68) SNFLPWFDY (SEQ ID NO 76) RASQSVRKSLA (SEQ ID NO 95) TASNRAT (SEQ ID NO 79) QQYFRAPIT (SEQ ID NO 81) GC5B601 GFSLTSYNVH (SEQ ID NO 66) VIWAGGSTNYNSA LMS (SEQ ID NO 71) DGIRLRFAY (SEQ ID NO 77) KASQNVATHVG (SEQ ID NO 15) SASYRYS (SEQ ID NO 18) QQYNRYPYT (SEQ ID NO 21)

In some embodiments, a CPK5O-specific antibody or antigen-binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2, and CDR3 of any one of the antibodies described in Table. 1. In some embodiments, a GPRC5D-specific antibody or antigen binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2 and CDR3 of any one of the antibodies described in Table. 1, and a light chain containing CDR1, CDR2 and CDR3 of any one of the antibodies described in table. 1. In some embodiments described herein, a GPRC5D-specific antibody or antigen-binding fragment thereof competes for binding to GPRC5D with an antibody or antigen-binding fragment containing a heavy chain containing CDR1, CDR2, and CDR3 of any one of the antibodies described in Table 1 , and a light chain containing CDR1, CDR2 and CDR3 of any one of the antibodies described in table. 1.

The IgG class in humans is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4. They have more than 95% homology in the amino acid sequence of the Fc regions, but have significant differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcgRs) on the surface of effector immune cells, such as natural killer cells and macrophages, leading to phagocytosis and lysis of target cells. In CDC, antibodies destroy target cells by triggering a cascade of complement reactions on the cell surface. Antibodies described herein include antibodies with the described variable domain features in combination with any of the IgG isotypes, including modified versions in which the Fc sequence is modified to affect various effector functions.

In many therapeutic antibody applications, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions may be deleterious and potentially pose a safety concern by causing toxicity unrelated to the mechanism of action. Effector functions can be altered by engineering Fc regions to reduce their binding to FcgR or complement factors. The binding of IgG to the activating (FcgRI, FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory (FcgRIIb) FcgR receptors or the first complement component (Clq) depends on residues located in the hinge region and the CH2 domain. Mutations have been introduced into IgGl, IgG2 and IgG4 to reduce or eliminate Fc functions. Antibodies described herein may include these modifications.

In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function compared to the parent Fc; (b) reduced affinity for Fcg RI, Fcg Rlla, Fcg Rllb, Fcg Rlllb and/or Fcg Rllla; (c) reduced affinity for FcgRI; (d) reduced affinity for FcgRIIa; (f) reduced affinity for FcgRIIb; (f) reduced affinity for Fcg Rlllb or (g) reduced affinity for FcgRIIIa.

In some embodiments, the antibodies or antigen-binding fragments are IgG or derivatives thereof, such as the IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, in which the antibody is of the IgGl isotype, the antibody contains substitution(s) L234A, L235A, and/or K409R in the Fc region. In some embodiments, in which the antibody is of the IgG4 isotype, the antibody contains substitutions S228P, L234A, and L235A in the Fc region. Antibodies described herein

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In addition to the described GPRC5D-specific antibodies and antigen-binding fragments, polynucleotide sequences capable of encoding the described antibodies and antigen-binding fragments are also provided. Also provided are vectors containing the disclosed polynucleotides, as well as cells expressing GPRC5D-specific antibodies or antigen binding fragments provided herein. In addition, cells capable of expressing the described vectors are described. These cells may be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf7 cells), yeast cells, plant cells, or bacterial cells (eg, E. coli). The described antibodies can also be produced by hybridoma cells.

Methods of using GPRC5D-specific antibodies

Methods of using GPRC5D-specific antibodies or antigen-binding fragments are also described. Specific antibodies for use in the methods described in this section include those having the set of CDRs described for the antibodies in table. 1. For example, these antibodies or antigen binding fragments can be used in the treatment of cancer by blocking interactions with the GPRC5D receptor or, when the antibody is conjugated to a toxin, directing the toxin to cancers expressing GPRC5D. In addition, these antibodies or antigen binding fragments can be used to detect the presence of GPRC5D in a biological sample, such as blood or serum; to quantify GPRC5D in a biological sample, such as blood or serum; for diagnosing cancers expressing GPRC5D; determining a method of treating an individual affected by cancer; or control the progression of cancer with GPRC5D expression in an individual. In some embodiments, the cancer expressing GPRC5D may be a lymphoma, such as multiple myeloma (MM). The described methods can be performed before the individual receives treatment for a cancer expressing GPRC5D, such as treatment with a multispecific antibody to GPRC5D and CD3. In addition, the described methods can be performed after the individual has received treatment for a cancer expressing GPRC5D, such as treatment with the multispecific anti-GPRC5D and CD3 antibody described herein.

Described methods for detecting GPRC5D in a biological sample include exposing the biological sample to one or more of the GPRC5D-specific antibodies or antigen binding fragments described herein.

Described methods for diagnosing GPRC5D-expressing cancer in an individual also include exposing a biological sample to one or more of the GPRC5D-specific antibodies or antigen-binding fragments described herein; however, the methods also include determining the amount of GPRC5D present in the sample; comparing the amount of GPRC5D present in a sample with a known standard or reference sample; and determining whether the individual's GPRC5D levels fall within the range of GPRC5D levels associated with cancer.

Also described herein are methods for controlling cancers expressing GPRC5D in an individual. The described methods include exposing a biological sample to one or more of the GPRC5D-specific antibodies or antigen-binding fragments described herein; determining the amount of GPRC5D present in the sample and bound to the antibody or antigen-binding fragment thereof; comparing the amount of GPRC5D present in a sample to either a known standard or reference sample or to the amount of GPRC5D in a similar sample previously obtained from an individual; and determining whether an individual's GPRC5D levels are indicative of disease progression, regression, or stable disease based on the difference in the amount of GPRC5D in the compared samples.

Samples obtained from individuals are biological samples, such as urine, blood, serum, plasma, saliva, ascites, circulating cells, circulating tumor cells, non-tissue-associated cells, tissue, surgically excised tumor tissue, biopsy materials, samples obtained by fine-needle aspiration biopsy or histological preparations.

The described GPRC5D-specific antibodies or antigen-binding fragments can be labeled using the methods described or other methods known to those skilled in the art. For example, the antibodies described herein, or antigen binding fragments thereof, can be labeled with a radiolabel, a fluorescent tag, an epitope tag, a biotin, a chromophore tag, an ECL tag, an enzyme, ruthenium, ¹ⁿ In-DOTA, ^11l In-diethylenetriaminepentaacetic acid (DTPA ), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine, or similar labels known in the art.

GPRC5D-specific antibody kits

Described herein are kits comprising the disclosed GPRC5D-specific antibodies or antigen binding fragments thereof. The kits described can be used to implement methods of using GPRC5D-specific antibodies or antigen binding fragments provided herein or other methods known to those skilled in the art.

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In some embodiments, the described kits may include antibodies or antigen binding fragments described herein and reagents for use in detecting the presence of GPRC5D in a biological sample. Accordingly, the disclosed kits may include one or more of the antibodies or antigen-binding fragments thereof described herein, a vessel for storing the antibody or fragment when not in use, instructions for use of the antibody or fragment, the antibody or fragment immobilized on a solid support, and/ or detectably labeled forms of the antibody or fragment as described herein.

Multispecific antibodies to GPRC5DxCD3

Redirecting T cells to GPRC5D-expressing MM cells via the TCR/CD3 complex represents an attractive alternative approach. The TCR/CD3 T cell complex consists of a TCR alpha (α)/beta ((β) or TCR gamma (γ)/delta (δ) heterodimer expressed on the cell surface together with CD3 constant subunits designated gamma (γ), delta (δ), epsilon (ε), zeta (ζ), and eta (η) Human CD3ε is described as UniProt P07766 (CD3E_HUMAN).

The anti-CD3ε antibody described in the state of the art is SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100). SP34 reacts with human and primate CD3. SP34 can be purchased from Pharmingen. An additional anti-CD3 antibody described in the state of the art is UCHT-1 (see WO 2000041474). An additional anti-CD3 antibody described in the state of the art is BC-3 (Fred Hutchinson Cancer Research Institute; used in the phase I/II GvHD studies, 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 CD3 ε chain (see Salmeron et al., (1991) J. Immunol. 147: 3047), and UCHT-1 and BC-3 recognizes an epitope consisting of ε- and γ chains. An antibody sequence identical to that of the SP34 antibody is described in WO 2008119565, WO 2008119566, WO 2008119567, WO 2010037836, WO 2010037837 and WO 2010037838. The sequence is 96% identical to the VH of the SP34 antibody. described in US 8236308 (WO 2007042261).

Described herein are isolated multispecific antibodies that bind to GPRC5D and CD3 (GPRC5DxCD3 multispecific antibodies) and their multispecific antigen binding fragments. In some embodiments, an isolated antibody or antigen-binding fragment thereof is provided that immunospecifically binds to GPRC5D.

In some embodiments, the GPRC5D-specific arm of the multispecific antibody binds human GPRC5D and cynomolgus GPRC5D. In some embodiments, the GPRC5D-specific arm of the multispecific anti-GPRC5DxCD3 antibodies or antigen-binding fragments binds to the extracellular domain of human GPRC5D. In preferred embodiments, the anti-GPRC5DxCD3 multispecific antibody or antigen binding fragment is a bispecific antibody or antigen binding fragment. In some embodiments, an isolated anti-GPRC5DxCD3 bispecific antibody is provided comprising: a) a first heavy chain (HC1); b) 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 immunospecifically binds GPRC5D, and HC2 and LC2 pair to form a second antigen binding site that immunospecifically binds CD3; or its bispecific fragment that binds GPRC5DxCD3. In another embodiment, an isolated cell expressing an antibody or bispecific binding fragment is provided. In some embodiments, the GPRC5D-binding arm (or GPRC5D-specific arm) of the anti-GPRC5DxCD3 multispecific antibody is derived from an anti-GPRC5D antibody described herein (eg, an antibody having the CDR sequences listed in Table 1).

In some embodiments, the GPRC5D-specific arm of the multispecific anti-GPRC5DxCD3 antibodies or antigen-binding fragments is an IgG or derivative thereof. In some embodiments, the CD3-binding arm (or CD3-specific arm) of the anti-GPRC5DxCD3 multispecific antibody is derived from the mouse monoclonal antibody SP34, a mouse IgG3/lambda isotype (K. R. Abhinandan and A. C. Martin, 2008. Mol. Immunol. 45, 38323839 ). In some embodiments, the CD3 binding arm of the anti-GPRC5DxCD3 multispecific antibody comprises one VH domain and one VL domain selected from Table 1. 2.

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Table 2. Heavy chains and light chains of CD3-specific antibody and ____________antigen-binding fragment____________

VH VL CD3B219 (SEQ ID NO:99): EVQLVESGGGLVQPGGSLRLSCAASGFTFN TYAMNWVRQAPGKGLEWVARIRSKYNNYAT YYAASVKGRFTISRDDSKNSLYLQMNSLKT EDTAVYYCARHGNFGNSYVSWFAYWGQGTL VTVSSASTKGPSVFPLAPCSRSTSESTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSWT VPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVWD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRWSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLV KGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFLLYSKLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK CD3B219 (SEQ ID NO:100): QTWTQEPSLTVSPGGTVTLTCRSSTGAVTTSN YANWVQQKPGQAPRGLIGGTNKRAPGTPARFSG SLLGGKAALTLSGVQPEDEAEYYCALWYSNLWV FGGGTKLTVLGQPKAAPSVTLFPPSSEELQANK ATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS

The IgG class in humans is divided into four isotypes: IgG1, IgG2, IgG3 and IgG4. They have more than 95% homology in the amino acid sequence of the Fc regions, but have significant differences in the amino acid composition and structure of the hinge region. The Fc region mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In ADCC, the Fc region of an antibody binds to Fc receptors (FcgRs) on the surface of effector immune cells, such as natural killer cells and macrophages, leading to phagocytosis and lysis of target cells.

In CDC, antibodies destroy target cells by triggering a cascade of complement reactions on the cell surface.

In many therapeutic antibody applications, Fc-mediated effector functions are not part of the mechanism of action. These Fc-mediated effector functions may be deleterious and potentially pose a safety concern by causing toxicity unrelated to the mechanism of action. Effector functions can be altered by engineering Fc regions to reduce their binding to FcgR or complement factors. The binding of IgG to the activating (FcgRI, FcgRIIa, FcgRIIIa and FcgRIIIb) and inhibitory (FcgRIIb) FcgR receptors or to the first complement component (C1q) depends on residues located in the hinge region and the CH2 domain. Mutations have been introduced into IgG1, IgG2 and IgG4 to reduce or eliminate Fc functions.

In one embodiment, the antibody comprises an Fc region having one or more of the following properties: (a) reduced effector function compared to the parent Fc; (b) reduced affinity for Fcg RI, Fcg RIIa, Fcg RIIb, Fcg RIIIb and/or Fcg RIIIa; (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.

In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is an IgG or a derivative thereof. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is IgG1 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG1 antibody from which the CD3 binding arm is derived contains substitutions L234A, L235A, and F405L in the Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived is IgG4 or a derivative thereof. In some embodiments, for example, the Fc region of the CD3-specific IgG4 antibody from which the CD3 binding arm is derived contains substitutions S228P, L234A, L235A, F405L, and R409K in the Fc region. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived binds CD3s on primary human T cells and/or primary cynomolgus T cells. In some embodiments, the CD3-specific antibody or antigen-binding fragment from which the CD3-specific arm of the multispecific antibody is derived activates primary human CD4+ T cells and/or primary cynomolgus CD4+ T cells.

Along with the described multispecific antibodies to GPRC5DxCD3, polynucleotide sequences capable of encoding the described multispecific anti

- 6 044685 bodies to GPRC5DxCD3. In some embodiments, an isolated synthetic polynucleotide encoding HC1, HC2, LC1, or LC2 of a GPRC5DxCD3 bispecific antibody or bispecific binding fragment is provided. Also provided herein are vectors containing the disclosed polynucleotides as well as cells expressing multispecific antibodies to GPRC5DxCD3. In addition, cells capable of expressing the described vectors are described. These cells may be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf7 cells), yeast cells, plant cells, or bacterial cells (eg, E. coli). The described antibodies can also be produced by hybridoma cells. In some embodiments, methods are provided for producing a GPRC5DxCD3 bispecific antibody or bispecific binding fragment by cell culture.

Also provided herein are pharmaceutical compositions comprising anti-GPRC5DxCD3 multispecific antibodies or antigen binding fragments and a pharmaceutically acceptable carrier.

Methods of using multispecific antibodies to GPRC5DxCD3

Methods of using multispecific antibodies to GPRC5DxCD3 or multispecific antigen binding fragments thereof are also described. For example, multispecific antibodies to GPRC5DxCD3 and multispecific antigen binding fragments thereof can be used in the treatment of cancer expressing GPRC5D in an individual in need thereof. In some embodiments, the cancer expressing GPRC5D is a lymphoma, such as multiple myeloma.

Described methods of treating GPRC5D expressing cancer in an individual in need of such treatment include administering to the individual a therapeutically effective amount of a disclosed multispecific anti-GPRC5DxCD3 antibody or antigen binding fragment thereof. In some embodiments, the individual is a mammal, preferably a human. In preferred embodiments, methods are provided for treating an individual affected by cancer by administering a therapeutically effective amount of a GPRC5DxCD3 bispecific antibody or bispecific antigen binding fragment to a patient in need of such treatment for a period of time sufficient to treat the cancer.

Further provided herein are methods of inhibiting the growth or proliferation of cancer cells by administering a therapeutically effective amount of a GPRC5DxCD3 bispecific antibody or a bispecific binding fragment to inhibit the growth or proliferation of cancer cells.

Also provided herein are methods of redirecting a T cell to a GPRC5D expressing cancer cell by administering a therapeutically effective amount of a GPRC5DxCD3 bispecific antibody or bispecific binding fragment to redirect the T cell to a cancer lesion.

Kits with GPRC5DxCD3-specific antibody

Disclosed herein are kits containing the described multispecific antibodies to GPRC5DxCD3. The described kits can be used to implement the methods of using multispecific antibodies to GPRC5DxCD3 proposed herein, or other methods known to those skilled in the art. In some embodiments, the described kits may include antibodies described herein and reagents for treating cancers expressing GPRC5D. Accordingly, the described kits may include one or more multispecific antibodies or multispecific antigen binding fragments thereof described herein, a container for storing the antibody or fragment when not in use, and/or instructions for use of the antibody or fragment, the antibody or fragment immobilized on a solid support, and/or detectably labeled form of the antibody or fragment as described herein.

Brief description of graphic materials

Fig. 1: Concentration-dependent binding profile of selected anti-GPRC5D monoclonal antibodies (mAbs) relative to HEK293 cells with human GPRC5D and the same untransfected cells. Three mAbs GC5B36, GC5B168, and GC5B205 were also observed to bind to untransfected (GPRC5D-null) HEK293 cells.

Fig. 2: Dose-dependent binding of bispecific antibodies to GPCR5DxCD3 to HEK293F cells with human GPRC5D.

Fig. 3: The binding profile of bispecific antibodies on MM1R and H929 cells was compared with human GPCR5D-overexpressing HEK293 cells and untransfected HEK293 cells by FACS.

Fig. 4A and 4B: T cell-mediated cytotoxicity and T cell activation of anti-GPCR5DxCD3 antibodies to cells expressing human GPRC5D.

Fig. 5A and 5B: T cell-mediated cytotoxicity and T cell activation of antibodies to

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GPCR5DxCD3 relative to cells expressing cynomolgus macaque GPRC5D.

Fig. 6A and 6B: Antibodies to GPRC5DxCD3 effectively kill H929 cells in the prophylactic NSG mouse model. GCDB32 (6A) and GCDB35 (6B) caused complete inhibition of tumor growth when using doses of 10 μg, while GCDB32 was also able to inhibit tumor growth 100% when using a dose of 1 μg.

Fig. 7A and 7B: Comparison of activity in the absence (7A) and presence (7B) of the Fc block.

Fig. 8A-8D: Binding of GCDB32 GCDB35, GCDB40 and GCDB43 to Fcy receptors.

Fig. 9A and 9B: Assessment of binding of hybridoma-derived mAbs by FACS.

Fig. 10A and 10B: T cell-mediated cytotoxicity of GPRC5DxCD3 bispecific antibodies against H929 cells.

Fig. 11A-11E: Binding of bispecific antibodies to GPRC5DxCD3 to cell lines positive (H929, MM1R, LP1, OPM2) and negative (NALM6) to GPRC5D.

Fig. 12A-12D: Bispecific antibodies to GPRC5DxCD3 are potent tumor inhibitors in vivo. All bispecific antibodies to GPRC5DxCD3 (GCDB32, shown in 12A, GCDB53, shown in 12B, GCDB61, shown in 12C, and GCDB72, shown in 12D) completely inhibited the growth of multiple myeloma tumor cells (H929) using doses of 10 and 1 μg. Differentiation was observed using a dose of 0.1 μg, and GCDB72 was observed to exhibit 80% tumor growth inhibition.

Fig. 13: GPRC5D+ MM1.R cell lines were stained for 60 min with various concentrations of leader antibodies to measure surface binding profiles (n=3). Phycoerythrin-labeled human IgG4Fc was used as a secondary signal capture antibody (Southern Biotech, clone HP6025). Binding is expressed as the normalized geometric mean of fluorescence intensity measured by FACS. Graphing and data fitting were performed in GraphPad Prism 6 using nonlinear slope regression (four parameters) and least squares fitting.

Fig. 14A and 14B: GPRC5D+ cell lines were stained for 60 min with various concentrations of antibodies FAB6300 and GC5M481 to measure surface binding profiles. Phycoerythrin-labeled human IgG4Fc was used as a secondary signal capture antibody (Southern Biotech, clone HP6025). Binding is expressed as histograms (black line indicates isotype and red line indicates GPRC5D-specific antibody (Figure 14A). Figure 14B shows the binding profile of leader molecules on GPRC5D+ multiple myeloma cell lines. The shaded area bounded by a dotted line indicates isotype control , and the solid line indicates binding of the leader molecule.

Fig. 15A and 15B: Frozen bone marrow-derived mononuclear cells obtained from two different MM patients were used to evaluate GPRC5DxCD3 bispecific antibody binding compared to an IgG4 isotype control, plasma cell cytotoxicity, and T-cell activation. For cytotoxicity assays, T cells from a normal healthy donor were added exogenously to patient MNC-BM samples and incubated with the four leader molecules for 48 hours. In all donor samples, the bispecific antibody to GPRC5DxCD3 bound to plasma cells in a dose-dependent manner, and the mean values were recorded on the Y axis. fluorescence intensity. There was a decrease in live plasma cells (CD138+) and a concomitant increase in CD25 expression on T cells in response to treatment with a bispecific antibody to GPRC5DxCD3.

Fig. 16: NSG mice were subcutaneously implanted with human multiple myeloma cells MM.1S on day 0. Human PBMCs were inoculated intravenously on study day 7. PBS, GCDB72 (0.1 μg, 1 μg, 10 μg and 50 μg/animal (equivalent to 0.005; 0 05, 0.5, and 2.5 mg/kg, respectively)) and control null arm antibodies were administered intravenously on days 15, 18, 22, 24, 29, 32, and 36. Subcutaneous tumors were measured twice weekly and results were presented as the mean tumor volume expressed in mm ³ ± SPS for each group. Treatment with GCDB72 antibody at a dose of 1 μg (0.05 mg/kg) significantly inhibited subcutaneous tumor growth compared to PBS (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). Null arm control antibodies had negligible or no effect. Statistical significance was determined by 2-way analysis of variance (ANOVA) with multiple comparisons using Tukey's multiple comparison test using Graph Pad Prism software (version 6). Differences between groups were considered significant when the probability (p) value was <0.05.

Fig. 17: NSG mice were subcutaneously implanted with human multiple myeloma cells MM.1S on day 0. Human PBMCs were inoculated intravenously on study day 7. PBS, GCDB72 (0.1 μg, 1 μg, 10 μg and 50 μg/animal (equivalent to 0.005; 0 .05, 0.5, and 2.5 mg/kg, respectively)) and control null arm antibodies were administered intravenously on days 15, 18, 22, 24, 29, 32, and 36. Body weight is presented as absolute body weight from baseline treatment until the end of the study.

- 8 044685

Detailed Description of Illustrative Embodiments Definitions

Throughout the present specification and claims, various terms are used to relate to aspects of the specification. Such terms shall have their usual meaning in the art unless otherwise noted. Other specifically defined terms should be construed as defined herein.

When used in this specification and in the accompanying claims, the singular number includes the plural unless the content of the text clearly indicates otherwise. Thus, for example, a reference to a cell includes a combination of two or more cells and the like.

As used herein, the term about, when indicating a measurable quantity such as quantity, duration in time, etc., is considered to cover deviations of up to ± 10% from the specified value, as such deviations are acceptable for implementation of the described methods. Unless otherwise indicated, all numbers expressing amounts of ingredients, characteristics such as molecular weight, reaction conditions, etc., used in the description and claims are to be understood as modified in all cases by the term about. Accordingly, unless otherwise indicated, the numerical parameters set forth in the following description and the appended claims are approximate values that may vary depending on the desired properties that are desired to be obtained by the present invention. As a last resort, and not as an attempt to limit the application of the theory of equivalents to the scope of the claims, each numerical parameter should at least be considered in light of the number of significant figures presented and using standard rounding techniques.

Although the numerical ranges and parameters defining the broad scope of the invention are approximate, the numerical values indicated in the specific examples are presented as accurately as possible. However, any numerical value by its nature contains certain errors, inevitably resulting from the standard deviation found in the corresponding test measurements.

The term isolated means that a biological component (eg, a nucleic acid, peptide or protein) has been substantially separated from, obtained separately from, or purified from other biological components of the organism in which the component occurs naturally, i.e. from other chromosomal and extrachromosomal DNA, RNA and proteins. Thus, the nucleic acids, peptides and proteins that have been isolated include nucleic acids and proteins purified by standard purification methods. Isolated nucleic acids, peptides and proteins can be part of a composition and still be considered isolated if such composition is not part of the original environment of the nucleic acid, peptide or protein. The term also includes nucleic acids, peptides and proteins produced by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. As used herein, the term isolated antibody or antigen binding fragment means an antibody or antigen binding fragment substantially free of other antibodies or antigen binding fragments having different antigen specificities (e.g., an isolated antibody that specifically binds GPRC5D is substantially free of antibodies that specifically bind with antigens other than GPRC5D). However, an isolated antibody that specifically binds to an epitope, isoform, or variant of GPRC5D may be cross-reactive to other related antigens, for example, from other species (such as species homologues of GPRC5D).

The term polynucleotide, which is synonymous with the term nucleic acid molecule, nucleotides or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, a polynucleotide refers to three-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with the backbones modified for stability or other purposes. Modified bases include, for example, tritylated bases and atypical bases such as inosine. Various modifications can be made to DNA and RNA; therefore, the term polynucleotide covers the chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA found in viruses and cells. The term polynucleotide also covers relatively short chains of nucleic acids, often called oligonucleotides.

The meaning of a term essentially the same may differ depending on the context in which it is used. Due to the fact that in the natural sequence of light and heavy chains and codi

- 9 044685 variations are possible in their genes, it is expected that a certain level of variation may be encountered within the amino acid sequences or genes encoding the antibodies or antigen-binding fragments described herein, which have little or no effect on their unique binding properties (e.g., specificity and affinity ). This expectation is partly due to the degeneracy of the genetic code, as well as the evolutionary success of conservative variants of amino acid sequences that do not significantly change the nature of the encoded protein. Accordingly, in the context of nucleotide sequences, essentially the same means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identical, more preferably at least 97% identical, more preferably at least 98% identical, and more preferably at least 99% or more identical. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = number of identical positions/total number of positions χ 100), taking into account the number of gaps and the length of each gap introduced for optimal alignment of two sequences. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm described in E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is built into the ALIGN program (version 2.0), using the PAM120 residue weight table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage identity of two amino acid sequences can be determined using the algorithm , described in Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The degree of variation possible within the amino acid sequence of a protein without significantly affecting the function of the protein is much lower than in a nucleotide sequence, since the principles of degeneracy do not apply to amino acid sequences. Accordingly, in the context of an antibody or antigen binding fragment, substantially the same refers to an antibody or antigen binding fragments having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity described antibodies or antigen-binding fragments. Other embodiments include GPRC5D-specific antibodies or antigen-binding fragments that have a framework, backbone, or other non-binding regions that do not share significant identity with the antibodies and antigen-binding fragments described herein, but necessarily include one or more CDRs or other sequences necessary for binding to occur that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences described herein. A vector is a replicon, such as a plasmid, phage, cosmid or virus, into which another nucleotide segment can be operably inserted so that replication or expression of the segment occurs.

The term clone refers to a population of cells derived from a single cell or common precursor by mitosis. The term cell line refers to a clone of a primary cell capable of stable growth in vitro for many generations. In some of the examples presented herein, cells are transformed by transfecting DNA into them.

The terms express and produce are used interchangeably herein and refer to the biosynthesis of a gene product. These terms cover the transcription of a gene into RNA. These terms also cover the translation of RNA into one or more polypeptides and further cover all naturally occurring post-transcriptional and post-translational modifications. Expression or production of an antibody or antigen-binding fragment thereof may occur within the cytoplasm of a cell or in an extracellular environment, such as a cell culture growth medium.

The term treatment refers to any success or indication of success in reducing or alleviating the symptoms of an injury, pathology or condition, including any objective or individual measures such as relief of pain, remission, relief of symptoms or transformation of the condition into a form more easily tolerated by the patient, slowing the rate of progression of degenerative changes or loss of function, reversing the end point of degenerative changes to a form that is less distressing to the patient, improving the patient's general physical or mental condition, or prolonging survival. Treatment may be assessed by objective or individual measures, including the results of a physical examination, neurological examination, or psychiatric evaluation.

The term effective amount or therapeutically effective amount refers to an amount effective in doses and for periods of time necessary to achieve the desired

- 10 044685 therapeutic result. The therapeutically effective amount of an anti-GPRC5DxCD3 antibody may vary depending on factors such as the disease state, age, sex and weight of the patient and the ability of the antibody to elicit the desired response in the patient. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

Antibody refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD and IgY), including various monomeric, polymeric and chimeric forms, unless otherwise specified. In particular, the term antibody includes polyclonal antibodies, monoclonal antibodies (mAbs), and antibody-like polypeptides such as chimeric antibodies and humanized antibodies.

Antigen-binding moieties are any protein structure capable of exhibiting binding affinity for a specific antigen. Antigen-binding fragments include fragments obtained by any known method, such as enzymatic digestion, peptide synthesis and recombinant methods. Some antigen-binding fragments are composed of portions of intact antibodies that retain the antigen-binding specificity of the original antibody molecule. For example, antigen binding fragments may contain at least one variable region (heavy chain or light chain variable region) or one or more antibody CDR regions known to bind to a particular antigen. Examples of suitable antigen binding fragments include, but are not limited to, diabodies and single chain molecules, as well as Fab, F(ab')2, Fc, Fabc 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 and one light chain, monovalent fragment consisting of VL, VH, CL and CH1 domains, or a monovalent antibody described in WO 2007059782, divalent fragments containing two Fab fragments connected by a disulfide bridge in the hinge region, an Fd fragment consisting essentially of V.sub.H and C.sub.H1 domains; An Fv fragment consisting essentially of the VL and VH domains of one arm of an antibody, a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and is also called a domain antibody (Holt et al;Trends Biotechnol.2003 Nov;21(11):484-90); camelid antibody or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5(1):111-24); selected complementarity determining regions (CDRs), etc. All antibody isotypes can be used to obtain antigen-binding fragments. In addition, antigen binding fragments may include non-antibody protein scaffolds into which polypeptide segments can be successfully inserted in an orientation conferring affinity for a given antigen of interest, such as protein scaffolds. Antigen-binding fragments can be produced recombinantly or by enzymatic or chemical digestion of intact antibodies. The phrase antibody or antigen binding fragment thereof may be used to indicate that the antigen binding fragment comprises one or more amino acid segments of an antibody referred to in the phrase.

The terms CDR and the plural of CDR refer to the complementarity determining region (CDR), with three such regions defining the binding nature of the light chain variable region (CDRL1, CDRL2 and CDRL3), and three defining the binding nature of the heavy chain variable region (CDRH1, CDRH2 and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that include backbone or framework regions. The precise definition of the boundaries and extent of the CDR depends on various classification and numbering systems. Therefore, CDRs can be distinguished using Kabat, Chothia, contact or any other boundary definitions. Despite their different boundaries, each of these systems is characterized by a certain degree of overlap in that part that constitutes the so-called hypervariable region within the variable sequences. Thus, CDR definitions under such systems may differ in length and boundary regions relative to the adjacent frame region. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. NIH Publication No. 91-3242 (1991); Chothia et al., Canonical Structures for the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901 (1987); and MacCallum et al., Antibody-Antigen Interactions: Contact Analysis and Binding Site Topography, J. Mol. Biol. 262:732 (1996)), each of which is incorporated herein by reference in its entirety.

Typically, CDRs form a loop structure, which can be classified as a canonical structure. The term canonical structure refers to the backbone conformation adopted by the antigen-binding loops (CDRs). As a result of comparative structural studies, five of the six antigen-binding loops were found to have only a limited set of available conformations. Each canonical structure can be characterized using the torsion angles of the polypeptide backbone. Therefore, the corresponding loops between antibodies may have very similar three-dimensional structures, despite the high level of amino acid sequence variability in most regions of the loops (Chothia et al., Canonical Structures For the Hypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901 (1987) ; Chothia et al., Conformations

- 11 044685 of Immunoglobulin Hypervariable Regions, I 342:877 (1989); Martin and Thornton, Structural Families in Loops of Homologous Proteins: Automatic Classification, Modeling and Application to Antibodies, J. Mol. Biol. 263:800 (1996)), each of which is incorporated by reference in its entirety. Moreover, there is a certain relationship between the formed loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the chain length and the amino acid residues located at key positions in the loop, as well as within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of such key amino acid residues.

The term polypeptide is used interchangeably with the term protein and in its broadest sense refers to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. Subunits can be linked by peptide bonds. In another embodiment, the subunit may be linked by other linkages, such as ester, ester, etc. As used herein, the term amino acid refers to natural and/or artificial or synthetic amino acids, including glycine, as well as D and L optical isomers, amino acid analogues and peptidomimetics. A peptide consisting of three or more amino acids is usually called an oligopeptide if the peptide chain is short enough. If the peptide chain is long, the peptide is usually called a polypeptide or protein.

The terms specifically bind, or specifically bind, or derivatives thereof, when used with antibodies and antibody fragments, represent binding, through domains encoded by immunoglobulin genes or gene fragments, to one or more epitopes of the protein of interest and, preferably, the absence of binding to other molecules in the sample. containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1 x ^10'8 M, as measured by a surface plasmon resonance or cell binding assay. Phrases such as [antigen]-specific antibody (eg, GPRC5D-specific antibody) mean that said antibody specifically binds to said antigen.

The term polynucleotide, which is synonymous with nucleic acid molecule, nucleotides or nucleic acids, refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. Polynucleotides include, but are not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is a mixture of single- and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, a polynucleotide refers to three-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with the backbones modified for stability or other purposes. Modified bases include, for example, tritylated bases and atypical bases such as inosine. Various modifications can be made to DNA and RNA; therefore, the term polynucleotide covers the chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as the chemical forms of DNA and RNA found in viruses and cells. The term polynucleotide also covers relatively short chains of nucleic acids, often called oligonucleotides.

A vector is a replicon, such as a plasmid, phage, cosmid or virus, into which another nucleotide segment can be operably inserted so that replication or expression of the segment occurs.

As used herein, the term host cell can refer to any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In specific embodiments, the term host cell refers to a cell transfected with a nucleic acid molecule and the progeny or potential progeny of such cell. The descendants of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example due to mutations or environmental effects that may occur in subsequent generations, or integration of the nucleic acid molecule into the genome of the host cell. The terms expression and production are used interchangeably herein to refer to the biosynthesis of a gene product. These terms cover the transcription of a gene into RNA. These terms also cover the translation of RNA into one or more polypeptides and further cover all naturally occurring post-transcriptional and post-translational modifications.

Expression or production of an antibody or antigen-binding fragment thereof may occur within the cytoplasm of a cell or in an extracellular environment, such as a cell culture growth medium. The meaning of a term essentially the same may differ depending on the context in which it is used. Because variations are possible in the natural sequence of light and heavy chains and the genes encoding them, it is expected that a certain level of va

- 12 044685 variations within the amino acid sequences or genes encoding antibodies or antigen binding fragments described herein that have little or no effect on their unique binding properties (eg, specificity and affinity). This expectation is partly due to the degeneracy of the genetic code, as well as the evolutionary success of conservative variants of amino acid sequences that do not significantly change the nature of the encoded protein. Accordingly, in the context of nucleotide sequences, essentially the same means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identical, more preferably at least 97% identical, more preferably at least 98% identical, and more preferably at least 99% or more identical. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e. % homology = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap introduced for optimal alignment of two sequences. The percentage identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm described in E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), which is built into the ALIGN program (version 2.0), using the PAM120 residue weight table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percentage identity of two amino acid sequences can be determined using the algorithm , described in Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

The degree of variation possible within the amino acid sequence of a protein without significantly affecting the function of the protein is much lower than in a nucleotide sequence, since the principles of degeneracy do not apply to amino acid sequences. Accordingly, in the context of an antibody or antigen binding fragment, substantially the same refers to an antibody or antigen binding fragments having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity described antibodies or antigen-binding fragments. Other embodiments include GPRC5D-specific antibodies or antigen-binding fragments that have a framework, backbone, or other non-binding regions that do not share significant identity with the antibodies and antigen-binding fragments described herein, but necessarily include one or more CDRs or other sequences necessary for binding to occur that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences described herein.

The term individual means human and non-human animals, including all vertebrates, such as mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians and reptiles. In many embodiments of the described methods, the individual is a human.

As used herein, the term redirect or redirect refers to the ability of an anti-GPRC5DxCD3 antibody to effectively direct the activity of T cells from their inherent cognate specificity to reactivity against cells expressing GPRC5D.

As used herein, the term sample means a collection of similar fluids, cells or tissues (eg, surgically excised tumor tissue, biopsy material, including material obtained by fine needle aspiration biopsy) isolated from an individual, as well as fluids, cells or tissues located inside the individual's body. In some embodiments, the sample is a biological fluid. Biological fluids are typically liquids at physiological temperatures and may include naturally occurring fluids found within, sampled, expressed or otherwise extracted from an individual or biological source. Some biological fluids are derived from specific tissues, organs, or localized areas, and some other biological fluids may have a more global or systemic location in the body of an individual or biological source. Examples of biological fluids include blood, serum and serous fluids, plasma, lymph, urine, saliva, cyst contents, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, fluids from the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids obtained by washing from the bronchi, etc. Biological fluids may also include liquid solutions in contact with

- 13 044685 individual or biological source, for example cell or organ culture media, including cell or organ conditioned media, rinsing fluids, and the like. As used herein, the term sample covers materials removed from an individual's body or materials present in the individual's body.

The known standard may be a solution comprising a known amount or concentration of GPRC5D, where the solution may be a naturally occurring solution, such as a sample from a patient known to have early, mid or late stage cancer, advanced or stable cancer, or the solution may be a synthetic solution, for example an aqueous buffer, containing a known amount of GPRC5D diluted in it. Known standards described herein may include GPRC5D isolated from an individual, as well as recombinant or purified GPRC5D protein, or a GPRC5D concentration value associated with a disease state.

As used herein, the terms G protein-coupled receptor, family C, group 5, member D and GPRC5D specifically include human GPRC5D protein, for example, as described in Genbank catalog accession No. 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).

The term CD3 refers to the human CD3 protein complex, consisting of multiple subunits. Consisting of multiple subunits, the CD3 protein complex consists of 6 separate polypeptide chains. These include a CD3y chain (SwissProt accession number P09693), a CD3δ chain (SwissProt P04234), two CD3ε chains (SwissProt P07766) and one homodimer of the CD3 ζ chain (SwissProt 20963), with the complex associated with the α and β chains of T- cell receptor. The term CD3, unless otherwise specified, includes any variant, isoform, and species homolog of CD3 that is naturally expressed by cells (including T cells) or that can be expressed on cells transfected with genes or cDNA encoding those polypeptides.

The anti-GPRC5DxCD3 antibody is a multispecific antibody, optionally a bispecific antibody, that contains two different antigen binding regions, one that specifically binds to the GPRC5D antigen and the other that specifically binds to CD3.

The multispecific antibody may be a bispecific antibody, a diabody, or a similar molecule (see, for example, PNAS USA 90(14), 6444-8 (1993) for a description of the diabody). Bispecific molecules, diabodies, and the like provided herein can bind to any suitable target in addition to the GPRC5D portion. The term bispecific antibody should be understood as an antibody having two different antigen binding regions defined by different sequences of the antibody. This can be understood as binding to different targets, but also includes binding to different epitopes on the same target.

A reference sample is a sample that can be compared with another sample, such as a test sample, to characterize the comparison sample. A reference sample will have some characterized property that serves as a basis for comparison with the test sample. For example, the reference sample can be used as a reference value for GPRC5D concentrations, which serve as an indicator of the presence of cancer in an individual. The reference sample does not necessarily need to be analyzed in parallel with the test sample, therefore, in some cases, the reference sample may be a previously determined numerical value or range that characterizes a given condition, such as GPRC5D levels, which serve as indicators of the presence of cancer in an individual. The term also includes samples used for comparative purposes that are known to be associated with a physiological or disease state, such as a cancer that expresses GPRC5D but has an unknown amount of GPRC5D.

The term progress when used in the context of progression of cancers expressing GPRC5D includes a change in the state of the cancer from less severe to more severe. Progression may include an increase in the number or severity of tumors, the extent of metastasis, the rate at which the cancer grows or spreads, and the like. For example, progression of colon cancer includes progression of such cancer from a less severe to a more severe condition, such as progression from stage I to stage II, from stage II to stage III, and the like.

The term regression, when used in the context of regression of cancers expressing GPRC5D, includes a change in the status of the cancer from more severe to less severe. Reversal may include a reduction in the number or severity of tumors, the extent of metastasis, the rate at which the cancer grows or spreads, and the like. For example, reversal of colon cancer includes reversal of such cancer from a more severe to a less severe state, such as progression from stage III to stage II, from stage II to stage I, and the like.

The term stable, when used in the context of a stable GPRC5D-expressing cancer, describes a disease state that does not change or has not changed significantly over a clinically significant period of time to be considered an advanced or regressive cancer.

The embodiments described herein are not limited to specific methods, reagents, compounds, compositions or biological systems, which, of course, may vary.

GPRC5D-specific antibodies and antigen-binding fragments

Disclosed herein are isolated monoclonal antibodies or antigen binding fragments that specifically bind to GPRC5D. The general structure of an antibody molecule contains an antigen-binding domain, which includes heavy and light chains, and an Fc domain, which performs various functions, including complement fixation and binding to antibody receptors.

The described GPRC5D-specific antibodies or antigen-binding fragments include all isotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of the four-chain structure of immunoglobulins. The disclosed antibodies or antigen binding fragments also include the IgY isotype substantially found in chicken or turkey serum and in the yolk of chicken or turkey eggs.

GPRC5D-specific antibodies and antigen-binding fragments can be obtained from any species using recombinant methods. For example, the antibodies or antigen binding fragments may be murine, rat, goat, equine, porcine, bovine, chicken, rabbit, camel, donkey, human or chimeric variants thereof. For the purpose of administration to humans, antibodies or antigen-binding fragments obtained from non-human sources can be genetically or structurally modified to be less antigenic when administered to a human patient.

In some embodiments, the antibodies or antigen binding fragments are chimeric. As used herein, the term chimeric means an antibody or antigen-binding fragment thereof comprising at least some portion of at least one variable domain derived from the amino acid sequence of a non-human mammal, rodent or reptile antibody, while the remaining portions of the antibody or antigen-binding fragment thereof fragments are of human origin.

In some embodiments, the antibodies are humanized antibodies. Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab')2, or other antigen-binding antibody subsequences) containing a minimal sequence derived from a non-human animal immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced by residues from the complementarity determining region of a non-human species (donor antibody), such as a mouse, rat or rabbit having the desired specificity, affinity and ability. Typically, a humanized antibody will contain substantially all of at least one, and usually two, variable domains in which all or substantially all of the CDR regions correspond to those in an immunoglobulin from a non-human animal, and all or substantially all of the frame regions correspond to those of the human immunoglobulin sequence. The humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin.

The antibodies or antigen binding fragments described herein can take various forms but will include one or more antibody CDRs as shown in Table. 1.

Described herein are isolated antibodies and antigen binding fragments that immunospecifically bind to GPRC5D. In some embodiments, the GPRC5D-specific antibodies or antigen binding fragments are human IgG or derivatives thereof. Although the GPRC5D-specific antibodies or antigen binding fragments exemplified herein are human, the exemplified antibodies or antigen binding fragments can be chimerized.

In some embodiments, a GPRC5D-specific antibody or antigen binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2 and CDR3 of any one of the antibodies described in Table. 1. In some embodiments, a GPRC5D-specific antibody or antigen binding fragment thereof is provided comprising a heavy chain comprising CDR1, CDR2 and CDR3 of any one of the antibodies described in Table. 1, and a light chain containing CDR1, CDR2 and CDR3 of any one of the antibodies described in table. 1.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 1, a heavy chain CDR2 comprising SEQ ID NO: 5, and a heavy chain CDR3 comprising SEQ ID NO: 9. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D-specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 52. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 52, and a light chain variable domain substantially the same or identical to SEQ ID NO: 56. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 2, a heavy chain CDR2 containing SEQ ID NO: 6, and a heavy chain CDR3 comprising SEQ ID NO: 10. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D-specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 53. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 53, and a light chain variable domain substantially the same or identical to SEQ ID NO: 56. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 3, a heavy chain CDR2 comprising SEQ ID NO: 7, and a heavy chain CDR3 comprising SEQ ID NO: 11. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D-specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 54. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 54, and a light chain variable domain substantially the same or identical to SEQ ID NO: 57. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise 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, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D-specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 55. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 55, and a light chain variable domain substantially the same or identical to SEQ ID NO: 58. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 61, a heavy chain CDR2 comprising SEQ ID NO: 67, and a heavy chain CDR3 comprising SEQ ID NO: 72. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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, CDR2 light chain,

- 16 044685 containing SEQ ID NO: 78, and a light chain CDR3 containing SEQ ID NO: 80. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 82. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 82, and a light chain variable domain substantially the same or identical to SEQ ID NO: 92. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise 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, GPRC5D-specific antibodies and antigen binding fragments contain 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 comprising SEQ ID NO: 16, and a light chain CDR3 comprising SEQ ID NO: 19. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 83. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 83, and a light chain variable domain substantially the same or identical to SEQ ID NO: 56. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 29, and a heavy chain CDR3 comprising SEQ ID NO: 73. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 84. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 84, and a light chain variable domain substantially the same or identical to SEQ ID NO: 57. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 27, a heavy chain CDR2 comprising SEQ ID NO: 29, and a heavy chain CDR3 comprising SEQ ID NO: 11. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 85. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 85, and a light chain variable domain substantially the same or identical to SEQ ID NO: 57. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise 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, GPRC5D-specific antibodies and antigen-binding fragments contain CDR1 severe

- 17 044685 chain containing SEQ ID NO: 62, heavy chain CDR2 containing SEQ ID NO: 68, heavy chain CDR3 containing SEQ ID NO: 74, light chain CDR1 containing SEQ ID NO: 14, light chain CDR2 containing sEq ID NO: 17, and a light chain CDR3 comprising SEQ ID NO: 20. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 86. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 86, and a light chain variable domain substantially the same or identical to SEQ ID NO: 57. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 63, a heavy chain CDR2 containing SEQ ID NO: 69, and a heavy chain CDR3 comprising SEQ ID NO: 75. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 87. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 87, and a light chain variable domain substantially the same or identical to SEQ ID NO: 92. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise 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, GPRC5D-specific antibodies and antigen binding fragments contain 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. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 88. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments comprise a heavy chain variable domain substantially the same or identical to SEQ ID NO: 88, and a light chain variable domain substantially the same or identical to SEQ ID NO: 58. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 65, a heavy chain CDR2 comprising SEQ ID NO: 68, and a heavy chain CDR3 comprising SEQ ID NO: 76. In some embodiments, GPRC5D-specific antibodies and antigen binding fragments contain 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: 7-6, 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. This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 89. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 89, and a light chain variable domain substantially the same or identical to SEQ ID NO: 93. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments comprise a heavy chain CDR1 comprising SEQ ID NO: 66, a heavy chain CDR2 comprising

- 18 044685 containing SEQ ID NO: 71, and a heavy chain CDR3 containing SEQ ID NO: 77. In some embodiments, the GPRC5D-specific antibodies and antigen binding fragments contain a heavy chain CDR1 containing SEQ ID NO: 66, a 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 This GPRC5D specific antibody or antigen binding fragment may contain human framework sequences. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 91. In some embodiments, the GPRC5D-specific antibodies and antigen-binding fragments contain a heavy chain variable domain substantially the same or identical to SEQ ID NO: 91, and a light chain variable domain substantially the same or identical to SEQ ID NO: 94. The heavy chain variable domain and the light chain variable domain of the antibodies described in this paragraph are acceptable for inclusion in bispecific constructs, wherein one arm is an anti-GPRC5D antibody arm.

In some embodiments, the antibodies or antigen-binding fragments are IgG or derivatives thereof, such as the IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, in which the antibody is of the IgG1 isotype, the antibody comprises an IgG1 Fc region (SEQ ID NO. 60).

SEQ ID NO. 60

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

In some embodiments, in which the antibody is of the IgG4 isotype, the antibody contains substitutions S228P, L234A, and L235A in the Fc region (SEQ ID NO: 59).

SEQ ID NO. 59

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPK DTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLGK

Specific antibodies defined by the CDR and/or variable domain sequences described in the paragraphs above may include these modifications.

Also described are isolated polynucleotides encoding antibodies or antigen-binding fragments that immunospecifically bind to GPRC5D. Isolated polynucleotides capable of encoding the variable domain segments provided herein can be incorporated into the same or different vectors for the production of antibodies or antigen-binding fragments. An example of a polynucleotide sequence encoding the GPRC5D antibody is presented below:

Heavy chain sequence (SEQ ID NO:96):

atggcctgggtctggaccctgctgttcctgatggccgctgcccagagcatccaggcccaggtgc agetggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagetgcaaggc cagcggctacagcttcaccggctacaccatgaactgggtgcggcaggcccccggccagggcctg gagtggatggggcc tgatcaacccctacaacagcgacaccaactacgcccagaagctgcagggcc gggtgaccatgaccaccgacaccagcaccagcaccgcctacatggagctgcggagcctgcggag cgacgacaccgccgtgtactactgcgcccgggtggccctgcgggtggccctggactactggggc cagggcaccctggtgaccgtgagcagcgcctccacca agggcccatccgtcttccccctggcgc

- 19 044685 cctgctccaggagcacctccgagagcacagccgccctgggctgcctggtcaaggactacttccc cgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggct gtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctcca gcagcttgg gcacgaaaacctacacctgcaacgtagatcacaagcccagcaacaccaaggtggacaagagagt tgagtccaaatatggtcccccatgcccaccatgcccagcacctgaggccgccgggggaccatca gtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgt gcgtggt ggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgt ggaggtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgtaccgtgtggtc agcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtctcca acaaaggcctcccgt cctccatcgagaaaaccatctccaaagccaaagggcagccccgagagcc acaggtgtacaccctgcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgc ctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgac ggctccttcttcctctacagcaggct aaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggct ctgcacaaccactacacacagaagagcctctccctgtctctgggtaaatga

Light chain sequence (SEQ ID NO:90):

atgcgggtgctggcccagctgctgggactgctgctgctgtgcttccctggcgccagatgcgaca tccagatgacccagagccccagcagcctgagcgccagcgtgggcgaccgggtgaccatcacctg caaggccagccagaacgtggccacccacgtgggctggtaccagcagaagcccggcaaggccccc aagcggct gatctacagcgccagctaccggtacagcggcgtgcccagccggttcagcggcagcg gcagcggcaccgagttcaccctgaccatcagcaacctgcagcccgaggacttcgccacctacta ctgccagcagtacaaccggtacccctacaccttcggccagggcaccaagctggagatcaagcgt acggtggctgcaccatctgtct tcatcttcccgccatctgatgagcagttgaaatctggaactg cctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtgga taacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacc tacagcctcagcagcaccctgacgctgagcaaag cagactacgagaaacacaaagtctacgcct gcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttg a

Polynucleotides encoding recombinant antigen-binding proteins are also included within the scope of the description. In some embodiments, the described polynucleotides (and the peptides they encode) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence may include, without limitation, a restriction site or a translation initiation site.

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 described GPRC5D-specific antibodies or antigen-binding fragments . In the context of the present invention, unless otherwise specified, the following notations are used to describe mutations: i) an amino acid change at a given position is written, for example, as S228P, which means a change from serine to proline at position 228; and ii) for specific variants, specific three-letter or one-letter codes are used, including Xaa and X codes, to designate any amino acid residue. Thus, a substitution of serine for arginine at position 228 is designated as S228P, and a substitution of any amino acid residue for serine at position 228 is designated as S228P. The deletion of serine at position 228 is designated S228*. Variants containing single or multiple amino acid substitutions, deletions or additions can be generated by those skilled in the art.

Such variants may include: (a) variants in which one or more amino acid residues are replaced by conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to or removed from the polypeptide, (c) variants in wherein one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused to another peptide or polypeptide, such as a fusion partner, a protein tag, or other chemical functional group capable of conferring beneficial properties on the polypeptide, such as an epitope for an antibody, polyhistidine sequence, biotin residue, etc. Antibodies or antigen binding fragments described herein may include variants in which amino acid residues of one type are replaced by corresponding residues of another type (at conserved or non-conserved positions). In other embodiments, amino acid residues at non-conservative positions are replaced with conservative or non-conservative residues. Techniques for obtaining such variants, including genetic (deletions, mutations, etc.), chemical and enzymatic, are known to those skilled in the art.

GPRC5D-specific antibodies or antigen-binding fragments described herein can be of several antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. In some embodiments, the antibody isotype is an IgG1, IgG2, IgG3, or IgG4 isotype, preferably an IgG1 or IgG4 isotype. The specificity of an antibody or antigen-binding fragment thereof is determined in large part by the amino acid sequence and the location of the CDRs. Therefore, CDRs from one isotype can be transferred to another isotype without changing antigen specificity. In an alternative embodiment, techniques have been established to cause hybridomas to switch from producing one antibody isotype to another (isotype switching) without changing antigen specificity. Accordingly, such antibody isotypes are included within the scope of the disclosed antibodies or antigen binding fragments.

In addition, vectors are provided containing the polynucleotides described herein. The vectors may be expression vectors. Therefore, recombinant expression vectors containing the sequence encoding the polypeptide of interest are considered to be within the scope of this specification. The expression vector may contain one or more additional sequences, such as, but not limited to, regulatory sequences (eg, promoter, enhancer), a selection marker, and a polyadenylation signal. Vectors for transforming a wide range of host cells are widely known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors included within the scope of the disclosure include synthetic, genomic or cDNA-derived nucleic acid fragments that encode at least one recombinant protein that can be operably linked to suitable regulatory elements. Such regulatory elements may include a transcription promoter, sequences encoding suitable sites for binding of mRNA to the ribosome, and sequences that control the termination of transcription and translation. Expression vectors, especially mammalian expression vectors, may also include one or more non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, other 5' or 3' flanking non-transcribed sequences, 5' or 3' non-translated sequences (eg, essential ribosome binding sites), polyadenylation site, splice donor and acceptor sites, or transcription termination sequences. In addition, an origin of replication may be inserted to provide the ability to replicate in the host cell.

Transcriptional and translational control sequences in expression vectors intended for transformation of vertebrate cells can be obtained from viral sources. Examples of vectors that can be constructed are described in Okayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the sequence encoding the antibody or antigen binding fragment is placed under the control of an effective constitutive promoter, such as, for example, the promoters of the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta actin, human myosin, human hemoglobin, human muscle creatine and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the described embodiments. Such viral promoters include, but are not limited to, cytomegalovirus (CMV) immediate-early promoter, SV40 early and late promoters, mouse mammary tumor virus (MMTV) promoter, Maloney leukemia virus (LTR) long terminal repeats, human immunodeficiency virus (HIV) , Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) and other retroviruses and the herpes simplex virus thymidine kinase promoter. In one embodiment, the coding sequence of a GPRC5D-specific antibody or antigen-binding fragment thereof is placed under the control of an inducible promoter, e.g., a metallothionein promoter, a tetracycline-inducible promoter, a doxycycline-inducible promoter, promoters containing one or more interferon-stimulated response elements (ISREs), promoters protein kinase R 2',5'-oligoadenylate synthases, Mx genes, ADAR1, etc.

The vectors described herein may contain one or more internal ribosome entry sites (IRES). Incorporation of an IRES sequence into fusion vectors may be useful for enhancing the expression of certain proteins. In some embodiments, the vector system may include one or more polyadenylation sites (eg, SV40), which may be upstream or downstream of any of the above nucleic acid sequences. Vector components can be stitched together or arranged to provide optimal

- 21 044685 spatial separation for expression of gene products (ie, by introducing spacer nucleotides between open reading frames (ORFs)), or otherwise located. Regulatory elements, such as the IRES motif, can also be positioned to provide optimal spacing for expression.

Vectors may contain selectable markers well known in the art. Selectable markers include, for example, positive and negative selection markers, antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, kanamycin resistance gene, tetracycline resistance gene, penicillin resistance gene, puromycin resistance gene , blasticidin resistance gene), glutamate synthase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection or bacterial purine nucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al., 7 Gene Ther. 1738-1743 (2000); Nucleotide sequence , encoding a selectable marker or cloning site, may be located upstream or downstream of the nucleotide sequence encoding the polypeptide or cloning site of interest.

The vectors described herein can be used to transform various cells with genes encoding the described antibodies or antigen binding fragments. For example, vectors can be used to generate cells that produce a GPRC5D-specific antibody or antigen binding fragment. Accordingly, another aspect of the invention describes host cells transformed with vectors containing a nucleotide sequence encoding an antibody or antigen binding fragment thereof that binds GPRC5D, for example, the antibodies or antigen binding fragments described and exemplified herein.

Numerous methods for introducing foreign genes into cells are known in the art and can be used to construct recombinant cells to implement the described methods in accordance with the various embodiments described and exemplified herein. The technique used must ensure stable transfer of the heterologous gene sequence into the host cell in such a way that the heterologous gene sequence can be inherited and expressed by the cell's progeny, and in such a way that the necessary development and physiological functions of the recipient cells are not disrupted. 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, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (for example, recombinant DNA viruses, recombinant RNA viruses), etc. (described in Cline, 29 Pharmac. Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells can also be used to transform cells.

Cells suitable for use in the expression of GPRC5D-specific antibodies or antigen binding fragments described herein preferably include eukaryotic cells, more preferably cells derived from a plant, rodent or human, such as, but not limited to, the NSO, CHO, CHOK1 cell lines , perC.6, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, Cv-1/EBNA, L cells, C127, 3T3, HeLa, NS1, myeloma cells Sp2/0, BHK, etc. In addition , antibody expression can be accomplished using hybridoma cells. Methods for producing hybridomas are well known in the art.

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. Recombination-positive cells are expanded and screened for subclones that exhibit a desired phenotype, such as high expression levels, enhanced growth characteristics, or the ability to produce proteins with desired biochemical properties, for example, due to protein modification or altered post-translational modifications. These phenotypes may be due to intrinsic properties of a given subclone or due to mutation. Mutations can be accomplished by the use of chemical agents, UV light, radiation, viruses, insertional mutagens, inhibition of DNA mismatch repair, or a combination of these methods.

Methods of using GPRC5D-specific antibodies for treatment

Provided herein are GPRC5D-specific antibodies or antigen-binding fragments thereof for use in therapy. In particular, these antibodies or antigen binding fragments can be used in the treatment of cancer, such as cancer expressing GPRC5D. Accordingly, the invention provides a method of treating cancer comprising administering an antibody described herein, for example, GPRC5D-specific antibodies or antigen binding fragments. For example, application can be achieved by blocking GPRC5D receptor interactions or when an antibody is conjugated to a toxin, thereby targeting the toxin to GPRC5D expressing cancers. In some embodiments, the cancer expressing GPRC5D includes lymphoma, such as multiple myeloma (MM). Antibodies for use in these methods include

- 22 044685 antibodies described above in this document, such as a GPRC5D-specific antibody or antigen binding fragment with the properties described in table. 1, for example, with sequences

CDRs or variable domains, which are also presented in the further description of these antibodies.

In some embodiments described herein, the immunoeffector properties of GPRC5D-specific antibodies can be enhanced or weakened by Fc modifications using techniques known to those skilled in the art.

For example, Fc fragment effector functions such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (eg, B cell receptor; BCR), etc., can be provided and/or controlled by modifying residues in the Fc fragment responsible for these actions.

Antibody-dependent cell-mediated cytotoxicity, or ADCC, is a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRc) (such as natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide component. Human IgG1 or IgG3 undergoes N-glycosylation at Asn297 by most glycans in the well-known 2-antennary forms G0, G0F, G1, G1F, G2 or G2F. Antibodies produced by unengineered CHO cells typically have a fucose glycan content of at least about 85%. Removal of central fucose from 2-antennary complex type oligosaccharides attached to Fc regions enhances antibody ADCC through improved Fc.gamma.RSha binding without altering antigen binding or CDC activity. Such mAbs can be produced by a variety of methods that have been shown to result in the successful expression of relatively highly defucosylated antibodies bearing Fco-oligosaccharides of the 2-antennary complex type, such as by controlling the osmolality of the culture medium (Konno et al., Cytotechnology 64 :249-65, 2012), use of the variant CHO cell line Lec13 as a host cell line (Shields et al., J Biol Chem 277:26733-2 6740, 2002), use of the variant CHO cell line EB66 as a host cell line (Olivier et al., MAbs; 2(4), 2010; electronic edition before print edition; PMID:20562582), using the rat hybridoma cell line YB2/0 as a host cell line (Shinkawa et al., J Biol Chem 278 :3466-3473, 2003), introduction of small interfering RNAs, especially to the alpha-1,6-fucosyltransferase (FUT8) gene (Mori et al., Biotechnol Bioeng 88:901-908, 2004), or coexpression of beta-1,4 -N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II or the effective alpha-mannosidase I inhibitor, kifunensin (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).

In some embodiments described herein, ADCC caused by antibodies to GPRC5D can also be enhanced by introducing certain substitutions into the Fc region of the antibody. Examples of substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378 or 430 (EC residue numbering), as described in US Pat. No. 6,737,056.

GPRC5D detection methods

Provided herein are methods 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 can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue associated cells (i.e., free cells), tissues (eg, surgical excised tumor tissue, biopsy materials, including those obtained using fine-needle aspiration biopsy), histological preparations, etc. In some embodiments, the described methods include detecting GPRC5D in a biological sample by contacting the sample with any of the GPRC5D-specific antibodies or antigen binding fragments thereof described herein.

In some embodiments, a sample can be contacted with more than one of the GPRC5D-specific antibodies or antigen binding fragments described herein. For example, a sample may be contacted with a first GPRC5D-specific antibody or antigen-binding fragment thereof and then contacted with a second GPRC5D-specific antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment are not the same the same antibody or antigen-binding fragment. In some embodiments, the first antibody or antigen-binding fragment thereof may be attached to a surface, such as the surface of a multiwell plate, chip, or similar substrate, before being contacted with a sample. In other embodiments, the first antibody or antigen-binding fragment thereof may not be attached or fixed to anything before contacting the sample.

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The disclosed GPRC5D-specific antibodies and antigen-binding fragments can be detectably labeled. In some embodiments, labeled antibodies and antigen binding fragments may facilitate detection of GPRC5D through the methods described herein. Many such marks are well known to those skilled in the art. For example, suitable tags include, but are not limited to, radioactive tags, fluorescent tags, epitope tags, biotin, chromophore tags, ECL tags, or enzymes. More specifically, the tags described include ruthenium, ⁿ¹ In-DOTA, ¹¹¹ In-diethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, polyhistidine (HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, Alexa Fluor® dyes, etc.

The described GPRC5D-specific antibodies and antigen-binding fragments can be used in various assays to detect GPRC5D in a biological sample. Some acceptable assays include, but are not limited to, Western blot, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) cytometry, or ELISA.

In some embodiments described herein, detection of GPRC5D-expressing cancer cells in an individual can be used to determine whether the individual can be treated with a therapeutic agent directed against GPRC5D.

GPRC5D is present at detectable concentrations in blood and serum samples. Thus, provided herein are methods for detecting GPRC5D in a sample obtained from blood, such as a serum sample, by contacting the sample with an antibody or antigen binding fragment thereof that specifically binds GPRC5D. The blood sample or derivative thereof may be diluted, fractionated, or otherwise processed to obtain a sample on which the described method can be performed. In some embodiments, GPRC5D can be detected in a blood sample or a derivative thereof using any number of assays known to those skilled in the art, such as, but not limited to, Western blot, radioimmunoassay, surface plasmon resonance, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) cytometry or ELISA.

Methods for diagnosing cancer

Provided herein are methods for diagnosing cancers expressing GPRC5D in an individual. In some embodiments, cancers expressing GPRC5D include lymphomas, such as multiple myeloma (MM). In some embodiments, as described above, detection of GPRC5D in a biological sample, such as a blood or serum sample, enables the diagnosis of cancer in the individual from whom the sample was obtained. Alternatively, in some embodiments, other samples, such as a histological sample, a fine needle aspiration biopsy sample, surgically excised tumor tissue, circulating cells, circulating tumor cells, can be used to assess whether the individual from whom the sample was obtained has cancer. and so on. In some embodiments, the individual from whom the sample is obtained may already be known to have cancer, but the type of cancer the individual has may not yet have been diagnosed, or the preliminary diagnosis may be inaccurate, and therefore, detection of GPRC5D in a biological sample, obtained from an individual may enable or clarify the diagnosis of cancer. For example, it may be known that an individual has cancer, but it may not be known or obvious whether the individual's cancer is a GPRC5D-expressing cancer.

In some embodiments, the described methods include assessing whether an individual has a cancer expressing GPRC5D by determining the amount of GPRC5D present in a biological sample obtained from the individual; 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 a sample obtained from the individual and the amount of GPRC5D in the control or reference sample is an indication that the individual has a cancer expressing GPRC5D. In another embodiment, the amount of GPRC5D observed in a biological sample obtained from an individual can be compared with levels of GPRC5D that are known to be associated with certain forms or stages of cancer, for the purpose of determining the form or stage of cancer in the individual. In some embodiments, the amount of GPRC5D in a sample obtained from an individual is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that immunospecifically binds to GPRC5D, such as the GPRC5D-specific antibodies described herein. The sample assessed for the presence of GPRC5D can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (eg, surgically excised tumor tissue, biopsy materials, including those obtained using fine-needle aspiration biopsy), histological preparations, etc. In some embodiments, cancers expressing GPRC5D include hematologic malignancies such as multiple myeloma (MM). In some embodiments, the individual is a human.

In some embodiments, a method for diagnosing a cancer expressing GPRC5D will include: contacting a biological sample from an individual with a GPRC5D-specific antibody or antigen-binding fragment thereof (e.g., derivatives of the antibodies and fragments presented in Table 1), determining the amount of GPRC5D present in a sample that has bound an antibody or an antigen binding fragment thereof, comparing the amount of GPRC5D present in the sample with a known standard or reference sample; and determining whether the individual's GPRC5D levels fall within the range of GPRC5D levels associated with cancer. In a further embodiment, the diagnostic method may be followed by the additional step of administering or administering a specific anticancer treatment. In another embodiment, the diagnostic method may be followed by the additional step of communicating the determination results to facilitate cancer treatment. In some embodiments, specific anticancer treatments may be directed against cancer types that express GPRC5D, such as the GPRC5DxCD3 multispecific antibodies described herein.

In some embodiments, the described methods include assessing whether an individual has a cancer expressing GPRC5D by determining the amount of GPRC5D present in a blood or serum sample obtained from the individual; 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 a sample obtained from the individual and the amount of GPRC5D in the control or reference sample is an indication that the individual has a cancer expressing GPRC5D.

In some embodiments, a control or reference sample may be obtained from an individual not affected by GPRC5D-expressing cancer. In some embodiments, a control or reference sample may be obtained from an individual affected by a cancer expressing GPRC5D. In some embodiments, in which the control or reference sample is obtained from an individual not affected by cancer expressing GPRC5D, the observed increase in the amount of GPRC5D in the test sample compared to the observed amount in the control or reference sample is an indication that the individual is affected by cancer with expression of GPRC5D. In some embodiments, in which the control sample is obtained from an individual not affected by cancer expressing GPRC5D, an observed decrease or similarity in the amount of GPRC5D in the test sample compared to the observed amount in the control or reference sample is an indication that the individual is not affected by cancer with GPRC5D expression. In some embodiments, in which the control or reference sample is obtained from an individual with a cancer expressing GPRC5D, the observed similarity in the amount of GPRC5D in the test sample compared to the observed amount in the control or reference sample is an indication that the individual has a cancer expressing GPRC5D. In some embodiments, in which the control or reference sample is obtained from an individual with cancer expressing GPRC5D, the observed decrease in the amount of GPRC5D in the test sample compared to the observed amount in the control or reference sample is an indication that the individual does not have cancer with expression of GPRC5D.

In some embodiments, the amount of GPRC5D in a sample obtained from an individual is assessed by contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds GPRC5D, such as the antibodies described herein. The sample assessed for the presence of GPRC5D can be obtained from a blood sample, a serum sample, circulating cells, circulating tumor cells, non-tissue-associated cells (ie, free cells), tissues (eg, surgically excised tumor tissue, biopsy materials , including those obtained using fine-needle aspiration biopsy), histological preparations, etc.

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

To implement the described diagnostic methods, various combinations of GPRC5D-specific antibodies and antigen-binding fragments can be used as the first and second antibodies and antigen-binding fragments. In some embodiments, cancers expressing GPRC5D include lymphomas, such as multiple myeloma (MM).

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In some embodiments, the amount of GPRC5D is determined by a method such as Western blot, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) cytometry, or ELISA.

In various embodiments of the described diagnostic methods, a control or reference sample is used. This sample may represent a positive or negative analytical control to ensure that the assay is performing as expected; for example, analytical controls of this kind can typically be used for immunohistochemical analyses. Alternatively, the sample may be a standardized reference for the amount of GPRC5D in a biological sample from a healthy individual. In some embodiments, the levels of GPRC5D observed in a test individual can be compared with the levels of GPRC5D observed in samples from individuals with a known diagnosis of cancer expressing GPRC5D. In some embodiments, the control individual may be affected by the specific cancer of interest. In some embodiments, the control individual is known to have an early stage cancer, which may or may not be a GPRC5D expressing cancer. In some embodiments, the control individual is known to have an intermediate stage cancer, which may or may not be a GPRC5D expressing cancer. In some embodiments, the control individual is known to have an advanced cancer, which may or may not be a GPRC5D-expressing cancer.

Ways to control cancer

Provided herein are methods for controlling cancers expressing GPRC5D in an individual. In some embodiments, cancers expressing GPRC5D include lymphomas, such as multiple myeloma (MM). In some embodiments, the described methods include assessing whether a cancer expressing GPRC5D is progressing, regressing, or remaining stable by determining the amount of GPRC5D present in a test sample obtained from an individual; and comparing the observed amount of GPRC5D with the amount of GPRC5D in a biological sample obtained in a similar manner from an individual at an earlier time point, the difference between the amount of GPRC5D in the test sample and the earlier sample being an indicator of whether the cancer is advanced, regressive, or stable. In this regard, an increased amount of GPRC5D in the test sample compared to an earlier pattern may indicate progression of cancer with GPRC5D expression. Conversely, decreased amounts of GPRC5D in the test sample compared to earlier patterns may indicate regression of cancers with GPRC5D expression.

Accordingly, a non-significant difference in the amount of GPRC5D in the studied sample compared with the earlier sample may indicate a stable course of cancer with GPRC5D expression. In some embodiments, the amount of GPRC5D in a biological sample obtained from an individual is assessed by contacting the sample with an antibody or fragment of such an antibody that specifically binds GPRC5D, such as the antibodies described herein. The sample assessed for the presence of GPRC5D can be obtained from urine, blood, serum, plasma, saliva, ascites fluid, circulating cells, circulating tumor cells, non-tissue-associated cells (i.e., free cells), tissues (eg, surgically excised tumor tissue, biopsy materials, including those obtained using fine-needle aspiration biopsy), histological preparations, etc. In some embodiments, the individual is a human.

In some embodiments, methods for monitoring cancers expressing GPRC5D will include: contacting a biological sample from an individual with a GPRC5D-specific antibody or antigen-binding fragment thereof (e.g., derivatives of the antibodies and fragments presented in Table 1), quantifying GPRC5D, present in a sample, comparing the amount of GPRC5D present in the sample with the amount of GPRC5D determined in a biological sample obtained in a similar manner from the same individual at an earlier point in time; and determining whether an individual's GPRC5D level has changed over time. An increased amount of GPRC5D in a test sample compared to the amount in an earlier sample may indicate cancer progression. Conversely, decreased amounts of GPRC5D in the test sample compared to earlier patterns may indicate regression of cancers with GPRC5D expression. Accordingly, a non-significant difference in the amount of GPRC5D in the study sample compared with the earlier sample may indicate a stable course of cancer with GPRC5D expression. In some embodiments, the levels of GPRC5D in a sample may be compared to a known standard or reference sample, alone or as a complement to the comparison to the levels of GPRC5D observed in the sample at an earlier point in time. In a further embodiment, the diagnostic method may be followed by the additional step of administering a specific anticancer treatment. In some embodiments, the specific anticancer treatment may be directed against cancers expressing GPRC5D.

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

To implement the described control methods, various combinations of antibodies and antigen-binding fragments can be used as the first and second antibodies or antigen-binding fragments, as described in table. 1. In some embodiments, cancers expressing GPRC5D include hematologic malignancies such as multiple myeloma (MM).

In some embodiments, the amount of GPRC5D is determined by a method such as Western blot, radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibrium dialysis, immunodiffusion, electrochemiluminescent (ECL) immunoassay, immunohistochemistry, fluorescence-activated cell sorting (FACS) cytometry, or ELISA.

GPRC5D detection kits

Proposed herein are kits for the detection of GPRC5D in a biological sample. These kits include one or more GPRC5D-specific antibodies described herein, or antigen binding fragments thereof, and instructions for use of the kit.

The proposed GPRC5D-specific antibody or antigen binding fragment may be in solution; be lyophilized; attached to a substrate, carrier or tablet; or may be detectably labeled.

The described kits may also include additional components used to carry out the methods described herein. By way of example, kits may contain means for obtaining a sample from an individual, a control or reference sample, such as a sample from an individual with slowly progressing cancer and/or an individual without cancer, one or more sample compartments, and/or instructional materials that describe the implementation of the methods inventions, and tissue-specific controls or standards.

Means for determining the level of GPRC5D may further include, for example, buffers and other reagents for use in the assay for determining the level of GPRC5D. The instructions may be, for example, printed instructions for performing the assay and/or instructions for assessing the level of GPRC5D expression.

The described kits may also include means for isolating a sample from an individual. These means may contain one or more pieces of equipment or reagents that can be used to obtain fluid or tissue from an individual. The means for obtaining a sample from an individual may also be means for isolating blood components, such as serum, from the blood sample. Preferably, the kit is intended for use in a human subject.

Multispecific antibodies

The binding domains of the anti-GPRC5D antibodies described herein recognize cells expressing GPRC5D on their surface. As noted above, GPRC5D expression may be an indicator of a cancer cell. More specific targeting of specific cell subpopulations can be achieved by producing bispecific molecules, such as antibodies or antibody fragments, that bind to GPRC5D and to another target such as CD3 and BCMA. This is achieved by producing a molecule that contains a first region that binds to GPRC5D and a second region that binds to another target antigen. Antigen binding regions are capable of taking any form that provides specific target recognition, for example, the binding region may be or may include a heavy chain variable domain, Fv (a combination of a heavy chain variable domain and a light chain variable domain), a domain-based binding domain fibronectin type III domain (such as a fibronectin domain, or a domain based on a consensus sequence of fibronectin type III domains, or a tenascin domain, or a domain based on a consensus sequence of tenascin type III domains, such as the centrin molecule from Janssen Biotech, Inc., see ., for example WO 2010/051274 and WO 2010/093627). Accordingly, bispecific molecules are provided that contain two antigen-binding regions that bind to GPRC5D and another antigen, respectively.

Some of the multispecific antibodies described herein contain two different antigen-binding regions that bind to GPRC5D and CD3, respectively. In a preferred embodiment, multispecific antibodies that bind to GPRC5D and CD3 (GPRC5DxCD3 multispecific antibodies) and multispecific antigen binding fragments thereof are provided. In some embodiments, the anti-GPRC5DxCD3 multispecific antibody comprises a first heavy chain (HC1) and a first light chain (LC1) that couple to form a first antigen binding site that specifically binds GPRC5D, and a second heavy chain (HC2) and a second light chain (LC2 ), which pair to form a second antigen-binding site that specifically binds to CD3. In preferred embodiments, the anti-GPRC5DxCD3 multispecific antibody is a bispecific antibody comprising a GPRC5D-specific arm that contains a first heavy chain (HC1) and a first light chain (LC1) that pair to form a first antigen-binding site that specifically binds CD3, and the CD3-specific arm, which contains a second heavy chain (HC2) and a second light chain (LC2), which pair to form a second antigen-binding site that specifically binds GPRC5D. In some embodiments, the bispecific antibodies of the invention include antibodies having the structure of a full-length antibody. As used herein, the term full-length antibody means an antibody having two full-length antibody heavy chains and two full-length antibody light chains. The heavy chain (HC) of a full-length antibody includes the heavy chain variable and constant domains VH, CH1, CH2 and CH3. The light chain (LC) of a full-length antibody includes the light chain variable and constant domains VL and CL. A full-length antibody may lack C-terminal lysine (K) in either one or both heavy chains. The term Fab arm or hemimolecule refers to a single heavy chain-light chain pair that specifically binds to an antigen. In some embodiments, one of the antigen binding domains is a non-antibody-based binding domain, such as a fibronectin type 3 domain-based binding domain, such as centrin.

The GPRC5D binding arm of the multispecific antibodies provided herein can be derived from any of the GPRC5D specific antibodies described above. In some exemplary embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises heavy chain CDR1, CDR2 and CDR3 derived from the antibody clone shown in Table 1. 1. In some exemplary embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 derived from the antibody clone shown in Table. 1. In some exemplary embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises the heavy chain CDR1, CDR2, and CDR3 of clone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B3 85, GC5B370, GC5B602 , GC5B603, GC5B599, GC5B601, GC5B598 or GC5B597.

In some example embodiments of such GPRC5D-binding arms, the first antigen binding region that binds GPRC5D comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 of clone GC5B81, GC5B465, GS5B483 or GC5B596. In some exemplary embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises a heavy chain variable domain derived from the antibody clone shown in Table 1. 1. In some exemplary embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises a heavy chain variable domain and a light chain variable domain derived from the antibody clone shown in Table. 1. In some exemplary embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises a heavy chain variable domain of clone GC5B81, GC5B465, GS5B483, or GC5B596. In some example embodiments of such GPRC5D binding arms, the first antigen binding region that binds GPRC5D comprises a heavy chain variable domain and a light chain variable domain of clone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B38 5, GC5B370, GC5B602 , GC5B603, GC5B599, GC5B601, GC5B598 or GC5B597.

In table 3 provides a list of bispecific antibodies to GPRC5DxCD3 having one pair of heavy and light chains specific for GPRC5D and another pair of heavy and light chains specific for CD3, wherein a specific antibody identifier is provided to describe the antigen-specific arms of the antibody used to provide the described embodiment.

Table 3

GPRC5D-specific arm=identifier AB CD 3-specific arm=identifier AB GC5B81 CD3B219 GC5B465 CD3B219 GC5B483 CD3B219 GC5B596 CD3B219 GC5B382 CD3B219 GC5B379 CD3B219 GC5B373 CD3B219

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In some embodiments of the bispecific antibodies, the GPRC5D binding arm also binds to cynomolgus GPRC5D, preferably its extracellular domain.

In some embodiments, the GPRC5D binding arm of the multispecific antibody is an IgG or a derivative thereof, such as the IgG1, IgG2, IgG3, and IgG4 isotypes. In some embodiments, in which the GPRC5D binding arm is of the IgG4 isotype, it contains substitution(s) S228P, L234A, and L235A in the Fc region.

In some embodiments of the bispecific antibodies, the second antigen-binding arm binds to human CD3. In some preferred embodiments, the CD3-specific arm of the GPRC5DxCD3 bispecific antibody is derived from a CD3-specific antibody that binds and activates primary human T cells and/or primary cynomolgus 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 comprising the N-terminal six amino acids of CD3ε. In some embodiments, the CD3-specific binding arm of the bispecific antibody is derived from the mouse monoclonal antibody SP34, a mouse IgG3/lambda isotype. In some embodiments, the CD3 binding arm comprises a CDR of the SP34 antibody. Such CD3 binding arms may bind CD3 with an affinity of 5 x 10' ⁷ M or less, for example 1 x 10' ⁷ M or less, 5 x 10' ⁸ M or less, 1 x 10' ⁸ M or less, 5 x 10' ⁹ M or less, or 1 x 10 ' ⁹ M or less. The CD3-specific binding arm may be a humanized version of the mouse monoclonal antibody SP34 arm. To humanize the anti-CD3 antibody from which the CD3-specific arm is derived, a human framework adaptation (HFA) can be used. In some embodiments of the bispecific antibodies, the CD3 binding arm comprises a pair of a heavy chain and a light chain selected from Table 1. 2.

In some embodiments, the CD3 binding arm is an IgG or a derivative thereof. In some embodiments, the CD3 binding arm is IgG1, IgG2, IgG3, or IgG4. In some embodiments, in which the CD3 binding arm is of the IgG4 isotype, it contains substitution(s) S228P, L234A, L235A, F405L, and R409K in the Fc region. In some embodiments, the antibodies or antigen-binding fragments bind to CD3ε on primary human T cells. In some embodiments, the antibodies or antigen-binding fragments bind to CD3ε on primary cynomolgus T cells. In some embodiments, the antibodies or antigen-binding fragments bind to CD3ε on primary human and cynomolgus T cells. In some embodiments, the antibodies or antigen binding fragments activate primary human CD3+ T cells. In some embodiments, the antibodies or antigen-binding fragments activate primary cynomolgus CD4+ T cells.

In some embodiments, a bispecific antibody to GPRC5DxCD3 is provided having a GPRC5D binding arm that contains the heavy chain of the antibody clone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598 or GC5B597. In some embodiments, a bispecific antibody to GPRC5DxCD3 is provided having a GPRC5D binding arm that contains the heavy chain and light chain of the antibody clone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B 370, GC5B602, GC5B603, GC5B599 , GC5B601, GC5B598 or GC5B597. In some embodiments, a GPRC5DxCD3 bispecific antibody is provided having a CD3 binding arm that contains the heavy chain of the CD3B219 antibody clone. In some embodiments, a bispecific antibody to GPRC5DχCD3 is provided having a CD3 binding arm that contains the heavy chain and light chain of the CD3B219 antibody clone. In some embodiments, a bispecific antibody to GPRC5DxCD3 is provided having a GPRC5D binding arm that contains the heavy chain of the antibody clone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382, GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598 or GC5B597, as well as the CD3-binding arm, which contains the heavy chain of the antibody clone CD3B219. In some embodiments, a GPRC5DxCD3 bispecific antibody is provided having a GPRC5D binding arm that

- 29 044685 contains the heavy chain and light chain of the antibody clone GC5B81, GC5B465, GS5B483, GC5B596, GC5B382,

GC5B379, GC5B373, GC5B376, GC5B385, GC5B370, GC5B602, GC5B603, GC5B599, GC5B601, GC5B598 or GC5B597, as well as the CD3 binding arm, which contains the heavy chain and light chain of the CD3B219 antibody clone.

An example of a bispecific antibody to GPRC5DxCD3 is given in table. 23.

Various bispecific antibody formats have been described and recently reviewed in Chames and Baty (2009) Curr Opin Drug Disc Dev 12: 276.

In some embodiments, the bispecific antibody of the present invention is a diabody, crossbody, or bispecific antibody produced through controlled exchange of Fab arms, such as those described herein.

In some embodiments, the bispecific antibodies include IgG-like molecules with complementary CH3 domains to enhance heterodimerization; recombinant dual-targeting IgG-like molecules, wherein each of the two sides of the molecule contains a Fab fragment or part of a Fab fragment of at least two different antibodies; IgG fusion molecules in which full-length IgG antibodies are fused to an additional Fab fragment or regions of the Fab fragment; Fc fusion molecules in which single chain Fv molecules or stabilized diabodies are fused to heavy chain constant domains, Fc regions or portions thereof; Fab fusion molecules, in which different Fab fragments are fused to each other; ScFv-based heavy chain antibodies and diabodies (eg, domain antibodies, nanobodies) in which different single-chain Fv molecules, or different diabodies, or different heavy chain antibodies (eg, domain antibodies, nanobodies) are fused to each other, or to another protein, or with a carrier molecule.

In some embodiments, IgG-like molecules with complementary CH3 domains include Triomab/Quadroma (Trion Pharma/Fresenius Biotechthe), ridge-to-groove (Genentech), CrossMAb (Roche), and electrostatically coupled (Amgen), LUZ-Y ( Genentech), chain exchange engineered domain antibody (SEEDbody) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A/S).

In some embodiments, the recombinant dual-targeting IgG-like molecules include dual-targeting (DT)-Ig molecules (GSK/Domantis), two-in-one antibody (Genentech), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star) and CovX-body (CovX/Pfizer).

In some embodiments, the IgG fusion molecules include double variable domain (DVD)-Ig molecules (Abbott), IgG-like bispecific molecule (InnClone/Eli Lilly), Ts2Ab (MedImmune/AZ), and BsAb (Zymogenetics) , HERCULES (Biogen Idec) and TvAb (Roche).

In some embodiments, the Fc fusion molecules include ScFv/Fc (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), dual affinity retargeting antibody (Fc-DART) (MacroGenics) and Dual(ScFv) fusions. .sub.2-Fab (National Research Center for Antibody Medicine, China).

In some embodiments, the Fab fusion bispecific antibodies include F(ab)2 (Medarex/AMGEN), dual action or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), divalent bispecific antibody (Biotecnol ) and Fab-Fv (UCB-Celltech). ScFv-based antibodies, diabodies and domain antibodies include, but are not limited to, bispecific T cell activator (BiTE) (Micromet), tandem diabody (Tandab) (Affimed), dual affinity retargeting antibodies (DART) (MacroGenics), single chain diabody ( Academic), TCR-like antibodies (AIT, ReceptorLogics), ScFv-human serum albumin fusion (Merrimack), COMBODY (Epigen Biotech), dual-targeting nanobodies (Ablynx), heavy chain-only dual-targeting domain antibodies.

Full-length bispecific antibodies of the invention can be created, for example, by exchanging Fab arms (or exchanging hemimolecules) between two monospecific divalent antibodies, introducing substitutions into the CH3 interface of the heavy chain in each hemimolecule that promote the formation of a heterodimer from two antibody hemimolecules having different specificities, or in vitro in a cell-free environment, or using co-expression. The Fab arm exchange reaction is the result of the isomerization reaction of the disulfide bond and the dissociation-association of CH3 domains. The disulfide bonds of the heavy chains in the hinge regions of the original monospecific antibodies are restored. The resulting free cysteines of one of the original monospecific antibodies form a disulfide bond of heavy chains with the cysteine residues of the second original molecule of the monospecific antibody, and at the same time the CH3 domains of the original antibodies are released and reformation occurs by dissociation-association. The CH3 domains of Fab arms can be designed to promote heterodimerization rather than homodimerization. The resulting product is a bispecific antibody having two Fab arms, or hemimolecules, each of which binds to a different epitope, i.e. an epitope on GPRC5D and an epitope on CD3.

The term homodimerization as used herein refers to the interaction of two heavy chains having identical CH3 amino acid sequences. In this document the term

- 30 044685 homodimer refers to an antibody having two heavy chains with identical CH3 amino acid sequences.

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

To create full-length bispecific antibodies, a projection-to-trench strategy can be used (see, for example, international publication No. WO 2006/028936). Briefly, selected amino acids forming the interface between the CH3 domains in human IgG can be mutated at positions that affect the interactions of the CH3 domains, promoting heterodimer formation. An amino acid with a short side chain (the valley) is inserted into the heavy chain of an antibody that specifically binds to the first antigen, and an amino acid with a long side chain (the overhang) is inserted into the heavy chain of an antibody that specifically binds to the second antigen. After co-expression of two antibodies, a heterodimer is formed as a result of the preferential interaction of the heavy chain with the trench and the heavy chain with the knob. Examples of CH3 substitution pairs that form a knob and a trough are the following (reported as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W/F405W, F405W/Y407A, T394W /Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions by introducing substitutions of positively charged residues on one CH3 surface and negatively charged residues on the other CH3 surface, as described in US Patent Publication No. US2010/0015133; US Patent Publication No. US 2009/0182127; US Patent Publication No. US 2010/028637 or US Patent Publication No. US 2011/0123532. In other strategies, heterodimerization can be promoted by the following substitutions (modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain is indicated): L351Y_F405AY407V/T394W, T366I_K392M_T394W/F405A_Y407V, T366L_K392M_T394W/F405A_ Y407V, L351Y_Y407A/T366A_K409F, L351Y_Y407A/ T366V K409F Y407A/T366A_K409F or T350V_L351Y_F405A

Y407V/T350V_T366L_K392L_T394W as described in US Patent Publication No. US 2012/0149876 or US Patent Publication No. US 2013/0195849.

In addition to the methods described above, the bispecific antibodies of the invention can be generated in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 regions of two monospecific homodimeric antibodies and forming a bispecific heterodimeric antibody from two parent monospecific homodimeric antibodies under reducing conditions, which promotes isomerization of the disulfide bond, in in accordance with the methods described in international patent publication No. WO 2011/131746. In these methods, a first monospecific divalent antibody (eg, an anti-GPRC5D antibody) and a second monospecific divalent antibody (eg, an anti-CD3 antibody) are designed to have certain substitutions in the CH3 domain that promote heterodimer stability; the antibodies are incubated together under reducing conditions sufficient to ensure that the cysteines in the hinge region undergo disulfide bond isomerization; thus, a bispecific antibody is obtained by exchanging Fab arms. Incubation conditions can be optimally returned to non-reducing conditions. Examples of reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and beta-mercaptoethanol, preferably the reducing agent is selected from the group consisting of: 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. For example, incubation for at least 90 minutes at a temperature of at least 20° C. in the presence of at least 25 mM 2-MEA or in the presence of at least 0.5 mM dithiothreitol at a pH level of 5-8, e.g. pH=7.0 or at pH=7.4.

In addition to the described multispecific antibodies to GPRC5DxCD3, polynucleotide sequences capable of encoding the described multispecific antibodies to GPRC5DxCD3 are also provided. Also provided herein are vectors containing the disclosed polynucleotides as well as cells expressing multispecific antibodies to GPRC5DxCD3. In addition, cells capable of expressing the described vectors are described. These cells may be mammalian cells (eg, 293F cells, CHO cells), insect cells (eg, Sf7 cells), yeast cells, plant cells, or bacterial cells (eg, E. coli). The described antibodies can also be produced by hybridoma cells.

Therapeutic composition and methods of treatment using multispecific antibodies and their multispecific antigen-binding fragments

Bispecific antibodies to GPRC5D described above, for example, bispecific antibodies to GPRC5DxCD3 described above, are useful in therapy. In particular, bispecific antibodies to GPRC5D are useful in the treatment of cancer. Also provided herein are therapeutic compositions for the treatment of a hyperproliferative disorder in mammals comprising a therapeutically effective amount of a multispecific antibody or multispecific antigen binding fragment described herein and a pharmaceutically acceptable carrier. In preferred embodiments, the multispecific antibody is a GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen binding fragment thereof, and more preferably a GPRC5DxCD3 bispecific antibody as described herein or a GPRC5DxCD3 bispecific antigen binding fragment thereof. In one embodiment, said pharmaceutical composition is for the treatment of cancers expressing GPRC5D, including (without limitation) the following cancers: B cell cancers expressing GPRC5D, such as multiple myeloma (MM); and other cancers not yet identified as having GPRC5D expression. Specific bispecific antibodies that may be used to treat cancers such as hematologic malignancies, including the specific cancers described above, include antibodies GC5B81, GC5B465, GS5B483, or GC5B596.

The pharmaceutical compositions provided herein contain: a) an effective amount of a multispecific antibody or antibody fragment of the present invention and b) a pharmaceutically acceptable carrier, which may be inert or physiologically active. In preferred embodiments, the multispecific antibody is a GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen binding fragment thereof, and more preferably a GPRC5DxCD3 bispecific antibody as described herein or a GPRC5DxCD3 bispecific antigen binding fragment thereof. As used herein, the term pharmaceutically acceptable carriers includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like, which are physiologically compatible. Examples of suitable carriers, diluents and/or excipients include one or more of water, saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, and the like, and any combination thereof. In many cases it will be preferable to include isotonic agents such as sugars, polyhydric alcohols or sodium chloride in the composition. In particular, suitable examples of an acceptable carrier include: (1) Dulbecco's phosphate-buffered saline solution, pH about 7.4, containing or not containing from about 1 to 25 mg/ml human serum albumin, (2) 0.9 % saline (0.9% w/v sodium chloride (NaCl)) and (3) 5% (w/v) dextrose; and it may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20®.

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

The compositions of the invention may be presented in various forms. These forms include, for example, liquid, semi-solid and solid dosage forms, but the preferred form depends on the intended route of administration and the therapeutic use. Typical preferred compositions are in the form of solutions suitable for injection or infusion. The preferred route of administration is parenteral (eg, intravenous, intramuscular, intraperitoneal, subcutaneous). In a preferred embodiment, the compositions of the invention are administered as an intravenous bolus or as a continuous infusion over a period of time. In another preferred embodiment, they are administered intramuscularly, subcutaneously, intra-articularly, intratumorally, peritumorally, into or near diseased tissue to produce local and systemic therapeutic effects.

Sterile compositions for parenteral administration can be prepared by introducing the antibody, antibody fragment or antibody conjugate of the present invention in the desired amount into a suitable solvent, followed by sterilization by microfiltration. The solvent or carrier may be water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., or a combination thereof. In many cases it will be preferable to include isotonic agents such as sugars, polyhydric alcohols or sodium chloride in the composition. These compositions may also contain auxiliary substances, in particular wetting, isotonicizing, emulsifying, dispersing and stabilizing agents. Sterile compositions for parenteral administration may also be prepared as sterile solid compositions that are dissolved at the time of use in sterile water or any other sterile injectable medium.

The multispecific antibody or antibody fragment can also be administered orally. As solid compositions for oral administration, tablets, pills, powders (gelatin capsules, sachets) or granules can be used. In these compositions, the active ingredient according to the invention is mixed in a stream of argon with one or more inert diluents such as starch, cellulose, sucrose, lactose or silica. These compositions may also contain substances other than diluents, for example, one or more lubricants such as magnesium stearate or talc, a colorant, a coating (sugar-coated tablet) or a glaze.

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

Doses depend on the desired effect, duration of treatment and route of administration used; they are essentially 5 to 1000 mg per day orally for adults, with single doses ranging from 1 to 250 mg of active substance. In general, the appropriate dosage will be determined by the physician based on age, body weight, and other factors specific to the individual being treated.

Also provided herein are methods of killing GPRC5D+ cells by administering to a patient in need thereof a multispecific antibody that binds said GPRC5D and is capable of recruiting T cells to kill said GPRC5D+ cells (ie, T cell redirection). Any of the multispecific antibodies or antibody fragments of the invention can be used for therapeutic purposes. For example, in one embodiment, a multispecific antibody to GPRC5DxCD3 can be used therapeutically to treat cancer in an individual.

In a preferred embodiment, the multispecific antibodies or antibody fragments of the invention are used to treat a hyperproliferative disorder in a mammal. In a more preferred embodiment, one of the above-described pharmaceutical compositions containing a multispecific antibody or antibody fragment of the invention is used for the treatment of a hyperproliferative disorder in a mammal. In one embodiment, the disorder is cancer. In particular, the cancer is a cancer that expresses GPRC5D, including but not limited to the following cancers: B-cell cancers that express GPRC5D, such as multiple myeloma (MM); and other cancers not yet identified as having GPRC5D expression. In preferred embodiments, the multispecific antibody is a GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen binding fragment thereof, and more preferably a GPRC5DxCD3 bispecific antibody as described herein or a GPRC5DxCD3 bispecific antigen binding fragment thereof.

Accordingly, the pharmaceutical compositions of the invention can be used for the treatment or prevention of various types of cancer, including (without limitation) the following: cancers expressing GPRC5D, including (without limitation) the following cancers: B cell/plasma cell cancers expressing GPRC5D, such as acute multiple myeloma (MM) or precancerous myelomas such as MGUS (monoclonal gammopathy of unknown origin) and TMM (smoldering multiple myeloma) and plasmacytoma; and other cancers not yet identified as having GPRC5D expression.

Likewise, herein further provided is a method of inhibiting the growth of selected populations of cells, comprising contacting GPRC5D-expressing target cells or tissue containing such cells with an effective amount of a multispecific antibody or antibody fragment of the present invention, alone or in combination with other cytotoxic or therapeutic agents. agents in the presence of peripheral blood mononuclear cells (PBMCs). In preferred embodiments, the multispecific antibody is a GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen binding fragment thereof, and more preferably a GPRC5DxCD3 bispecific antibody as described herein or a GPRC5DxCD3 bispecific antigen binding fragment thereof. In a preferred embodiment, the additional therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin 2. In a preferred embodiment, the additional therapeutic agent is a chemotherapeutic agent. The method of inhibiting the growth of selected populations of cells can be practiced in vitro, in vi vo or ex vi vo.

Examples of in vitro applications include treating autogenous bone marrow prior to transplantation into the same patient to destroy diseased or malignant cells; treatment of bone marrow before transplantation to destroy competent T cells and prevent graft-versus-host disease (GVHD); processing cell cultures to destroy all cells, with the exception of the desired variants that do not express the target antigen;

or for the purpose of eliminating variants expressing an undesired antigen. Conditions for non-clinical in vitro use can be easily determined by one skilled in the art.

An example of an ex vivo clinical application is the removal of tumor cells from bone marrow prior to autotransplantation in the treatment of cancer. Treatment can be carried out as described below. Bone marrow is collected from a patient or other individual and subsequently incubated in a medium containing serum to which the cytotoxic agent of the invention has been added. Concentrations range from about 10 to 1 µM over about 30 minutes to about 48 hours at about 37°C. The exact values of concentration and incubation time, i.e. doses can be easily determined by one skilled in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned to the patient by intravenous infusion according to known methods. In cases in which the patient receives other treatments, such as ablative chemotherapy or whole body irradiation, between the time the bone marrow is collected and the treated cells are reintroduced, the treated bone marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

In in vivo clinical use, a therapeutically effective amount of a multispecific antibody or antigen binding fragment is administered to an individual in need of such treatment. For example, multispecific antibodies to GPRC5DxCD3 and multispecific antigen binding fragments thereof can be used in the treatment of cancer expressing GPRC5D in an individual in need thereof. In some embodiments, the GPRC5D-expressing cancer includes B cell cancer, such as multiple myeloma (MM). In preferred embodiments, the multispecific antibody is a GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen binding fragment thereof, and more preferably a GPRC5DxCD3 bispecific antibody as described herein or a GPRC5DxCD3 bispecific antigen binding fragment thereof. In some embodiments, the individual is a mammal, preferably a human. In some embodiments, the multispecific antibody or antigen binding fragment is administered as a solution tested for sterility.

Dosage regimens for the above-described treatments and uses are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over a period of time, or the dose may be proportionally decreased or increased as indicated by the therapeutic situation. Parenteral compositions may be formulated in unit dosage units for ease of administration and uniform dosing.

Effective dosages and dosage regimens of multispecific antibodies and fragments depend on the disease or condition being treated and can be determined by one skilled in the art. A non-limiting example of a range for a therapeutically effective amount of a compound of the present invention is a range of about 0.001-10 mg/kg, for example about 0.001-5 mg/kg, for example about 0.001-2 mg/kg, for example about 0.001-1 mg/kg, for example about 0.001, about 0.01, about 0.1, about 1 or about 10 mg/kg.

A physician or veterinarian skilled in the art can readily determine and prescribe the required effective amount of the pharmaceutical composition. For example, a physician or veterinarian may begin with lower doses of the multispecific antibody or fragment contained in the pharmaceutical composition than necessary to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, an acceptable daily dose of the bispecific antibody of the present invention will be that amount of compound that is the lowest dose that produces a therapeutic effect. Administration may, for example, be parenteral, such as intravenous, intramuscular or subcutaneous. In one embodiment, the multispecific antibody or fragment can be administered by infusion at a weekly dose calculated in mg/m ² . Such doses, for example, can be calculated based on the mg/kg doses described above according to the following formula: dose (mg/kg) x 70: 1.8. Such administration can be repeated, for example, 1 to 8 times, for example 3 to 5 times. Administration may be by continuous infusion over a period of time ranging from 2 to 24 hours, such as 2 to 12 hours. In one embodiment, the multispecific antibody or fragment may be administered by slow continuous infusion over a long period of time, such as over 24 hours, to reduce side effects. toxic effects.

In one embodiment, the multispecific antibody or fragment can be administered in a weekly dosage calculated as a fixed dose administered up to eight times, such as four to six times, once a week. This regimen can be repeated one or more times as needed, for example after six months or twelve months. Such fixed doses, for example, could be based on the above doses in mg/kg based on an assumed body weight of 70 kg. The dose can be determined or adjusted by measuring the amount of the bispecific antibody of the present invention in the blood after administration, for example, by collecting a biological sample and using anti-idiotypic antibodies targeting the GPRC5D binding region of the multispecific antibodies of the present invention.

In one embodiment, the multispecific antibody or fragment can be administered as maintenance therapy, for example, once a week for a period of six months or more.

The multispecific antibody or fragment may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events as the cancer progresses, and/or reduce the risk of recurrence if the cancer is in remission.

Multispecific antibodies and fragments thereof described herein can also be administered as part of combination therapy, i.e. in combination with other therapeutic agents relevant for the treatment of the disease or condition. Accordingly, in one embodiment, the antibody drug is for combination with one or more additional therapeutic agents, such as a chemotherapeutic agent. In some embodiments, the other therapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin 2. Such combination administration may be simultaneous, separate, or sequential, in any order. When administered concomitantly, the agents may be administered as a single composition or as separate compositions, depending on the situation.

In one embodiment, a method is provided for treating a disorder in an individual involving cells expressing GPRC5D, the method comprising administering to the individual in need thereof a therapeutically effective amount of a multispecific antibody or fragment, such as the anti-GPRC5DxCD3 bispecific antibody described herein, and radiation therapy. In one embodiment, a method of treating or preventing cancer is provided, the method comprising administering to an individual in need thereof a therapeutically effective amount of a multispecific antibody or fragment, such as the anti-GPRC5DxCD3 antibody described herein, and administering radiation therapy. Radiation therapy is offered, which may include radiation or appropriate administration of radiopharmaceuticals to the patient. The radiation source may be external or internal to the patient being treated (radiation therapy may, for example, be external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that may be practically used for these 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.

Sets

Also provided herein are kits, for example, containing a disclosed multispecific antibody or antigen binding fragment thereof, and instructions for using the antibody or fragments to kill specific cell types. In preferred embodiments, the multispecific antibody is a GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen binding fragment thereof, and more preferably a GPRC5DxCD3 bispecific antibody as described herein or a GPRC5DxCD3 bispecific antigen binding fragment thereof. The instructions may include directions for use of the multispecific antibody or antigen binding fragment thereof in vitro, in vivo or ex vivo.

Typically, the kit has a compartment containing a multispecific antibody or antigen-binding fragment thereof. The multispecific antibody or antigen binding fragment thereof may be presented in lyophilized form, liquid form, or other form suitable for inclusion in a kit. The kit may also contain additional elements necessary for the practical application of the described method according to the kit instructions, for example, a sterilized solution for reconstituting the lyophilized powder, additional agents for combining with the multispecific antibody or antigen binding fragment thereof before administration to the patient, and instruments to facilitate the administration of the multispecific antibody or its antigen-binding fragment to the patient.

Diagnostic applications

The multispecific antibodies and fragments described herein can also be used for diagnostic purposes. Accordingly, diagnostic compositions containing the multispecific antibody or fragments described herein and their use are also provided. In preferred embodiments, the multispecific antibody is an anti-GPRC5DxCD3 multispecific antibody as described herein or a multispecific antigen-binding fragment thereof, and more preferably a bispecific anti-GPRC5DxCD3 antibody as described herein or a bispecific antibody thereof.

- 35 044685

GPRC5DxCD3 antigen binding fragment. In one embodiment, the present invention provides a cancer diagnostic kit comprising a container that contains a bispecific antibody to GPRC5DxCD3 and one or more reagents for detecting binding of the antibody to GPRC5D. Reagents may include, for example, fluorescent tags, enzymatic tags, or other detectable tags. Reagents may also include secondary or tertiary antibodies or enzymatic reaction reagents, wherein the enzymatic reactions produce a product that can be visualized. For example, the multispecific antibodies described herein, or antigen-binding fragments thereof, can be labeled with a radiolabel, a fluorescent tag, an epitope tag, biotin, a chromophore tag, an ECL tag, an enzyme, ruthenium, ¹ⁿ In-DOTA, ^11l In-diethylenetriaminepentaacetic acid ( DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine, or similar tags known in the art.

Embodiments

The following non-limiting embodiments are also provided herein.

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to GPRC5D and contains:

a) a heavy chain complementarity determining region 1 (CDR1) having the amino acid sequence SEQ ID NO: 1, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 5, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 9;

b) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 2, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 6, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 10;

c) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 3, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 7, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 11;

d) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 4, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 12;

e) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 61, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 67, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 72;

f) heavy chain CDR1 having the amino acid sequence SEQ ID NO: 2, heavy chain CDR2 having the amino acid sequence SEQ ID NO: 28, and heavy chain CDR3 having the amino acid sequence SEQ ID NO: 30;

g) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 27, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 29, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 73;

h) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 27, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 29, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 11;

i) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 62, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 68, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 74;

j) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 63, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 69, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 75;

k) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 64, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 70, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 12;

l) heavy chain CDR1 with amino acid sequence SEQ ID NO: 65, heavy chain CDR2 with amino acid sequence SEQ ID NO: 68 and heavy chain CDR3 with amino acid sequence SEQ ID NO: 76 or

m) a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 66, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 71, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 77.

2. The isolated antibody or antigen binding fragment of Embodiment 1, wherein:

a) said antibody comprising said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 1, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 5, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 9, up to -

- 36 044685 additionally contains a light chain CDR1 with the amino acid sequence SEQ ID NO: 13,

Light chain CDR2 having the amino acid sequence SEQ ID NO: 16 and light chain CDR3 having the amino acid sequence SEQ ID NO: 19;

b) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 2, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 6, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 10, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 13, light chain CDR2 having the amino acid sequence SEQ ID NO: 16 and light chain CDR3 having the amino acid sequence SEQ ID NO: 19;

c) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 3, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 7, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 11, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 14, light chain CDR2 having the amino acid sequence SEQ ID NO: 17 and light chain CDR3 having the amino acid sequence SEQ ID NO: 20;

d) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 4, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 8, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 12, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 15, light chain CDR2 having the amino acid sequence SEQ ID NO: 18 and light chain CDR3 having the amino acid sequence SEQ ID NO: 21;

e) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 61, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 67, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 72, further comprising a light CDR1 chains having amino acid sequence SEQ ID NO: 13, light chain CDR2 having amino acid sequence SEQ ID NO: 78, and light chain CDR3 having amino acid sequence SEQ ID NO: 80;

f) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 2, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 28, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 30, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 13, light chain CDR2 having the amino acid sequence SEQ ID NO: 6 and light chain CDR3 having the amino acid sequence SEQ ID NO: 19;

g) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 27, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 29, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 73, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 14, light chain CDR2 having the amino acid sequence SEQ ID NO: 17 and light chain CDR3 having the amino acid sequence SEQ ID NO: 20;

h) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 27, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 29, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 11, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 14, light chain CDR2 having the amino acid sequence SEQ ID NO: 17 and light chain CDR3 having the amino acid sequence SEQ ID NO: 20;

i) said antibody comprising said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 62, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 68, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 74, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 14, light chain CDR2 having the amino acid sequence SEQ ID NO: 17 and light chain CDR3 having the amino acid sequence SEQ ID NO: 20;

j) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 63, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 69, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 75, further comprising a light CDR1 chains having amino acid sequence SEQ ID NO: 13, light chain CDR2 having amino acid sequence SEQ ID NO: 78 and light chain CDR3 having amino acid sequence SEQ ID NO: 80;

k) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 61, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 67, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 72, further comprising a light CDR1 chain with amino acid sequence SEQ ID NO: 13, light chain CDR2 with amino acid sequence SEQ ID NO: 78 and light chain CDR3 with amino

- 37 044685 acid sequence SEQ ID NO: 80;

l) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 65, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 68, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 76, further comprising a light CDR1 chains having the amino acid sequence SEQ ID NO: 95, light chain CDR2 having the amino acid sequence SEQ ID NO: 79 and light chain CDR3 having the amino acid sequence SEQ ID NO: 81; or

m) said antibody containing said heavy chain CDR1 with the amino acid sequence SEQ ID NO: 66, said heavy chain CDR2 with the amino acid sequence SEQ ID NO: 71, and said heavy chain CDR3 with the amino acid sequence SEQ ID NO: 77, further comprising a light CDR1 chain having amino acid sequence SEQ ID NO: 15, light chain CDR2 having amino acid sequence SEQ ID NO: 18 and light chain CDR3 having amino acid sequence SEQ ID NO: 21.

3. An isolated antibody or antigen binding fragment thereof that specifically binds to GPRC5D and contains a heavy chain variable region (VH) selected from the group consisting of SEQ ID NO: 52, 53, 54, 55, 82, 83, 84, 85, 86, 87, 88, 89 or 91.

4. The antibody of embodiment 3, wherein the antibody or antigen binding fragment thereof comprises a light chain variable region (VL) selected from the group consisting of SEQ ID NO: 56, 57, 58, 92, 93, or 94.

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

6. The antibody of Embodiment 5, wherein the VH chain region contains SEQ ID NO: 52, 53, or 83 paired with the VL chain region containing SEQ ID NO: 56.

7. The antibody of Embodiment 5, wherein the VH chain region contains SEQ ID NO: 54, 84, 85, 86, or 90 paired with the VL chain region containing SEQ ID NO: 57.

8. The antibody of Embodiment 5, wherein the VH chain region contains SEQ ID NO: 55 or 88 paired with the VL chain region containing SEQ ID NO: 58.

9. The antibody of Embodiment 5, wherein the VH chain region contains SEQ ID NO: 82 or 87 paired with the VL chain region containing SEQ ID NO: 92.

10. The antibody of Embodiment 5, wherein the VH chain region contains SEQ ID NO: 89 paired with the VL chain region containing SEQ ID NO: 93.

11. The antibody of Embodiment 5, wherein the VH chain region contains SEQ ID NO: 91 paired with the VL chain region containing SEQ ID NO: 94.

12. The antibody or antigen binding fragment of any one of embodiments 1-11 that binds to a polypeptide having the amino acid sequence of SEQ ID NO: 22.

13. The antibody or antigen binding fragment of any one of embodiments 1-12, which is a human antibody or antigen binding fragment.

14. The antibody or antigen binding fragment of any one of embodiments 1-13, which is recombinant.

15. The antigen binding fragment of any one of embodiments 1-14, which is a Fab fragment, a Fab2 fragment, or a single chain antibody.

16. The antibody or antigen binding fragment of any one of embodiments 1-15, which is of an IgG1, IgG2, IgG3, or IgG4 isotype.

17. The antibody or antigen binding fragment of any one of embodiments 1-9 is of the IgG1 or IgG4 isotype.

18. The antibody of embodiment 17, wherein the IgG1 has a K409R substitution in the Fc region.

19. The antibody of embodiment 17, wherein the IgG1 has an F405L substitution in the Fc region.

20. The antibody of embodiment 20, wherein the IgG4 has an F405L substitution and an R409K substitution in the Fc region.

21. The antibody of embodiment 16, further comprising an S228P substitution, an L234A substitution, and an L235A substitution in the Fc region.

22. The antibody or antigen binding fragment of any one of embodiments 1-14 that specifically binds human GPRC5D and cross-reacts with cynomolgus GPRC5D.

23. The antibody or antigen binding fragment of embodiment 17 that induces ADCC in vitro with an EC ₅₀ of less than about 28 nM.

24. An isolated cell expressing the antibody or antigen binding fragment of any one of embodiments 1-11.

25. The cell of embodiment 24, which is a hybridoma.

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26. The cell of embodiment 24, wherein the antibody is produced recombinantly.

27. Isolated bispecific antibody to GPRC5DxCD3, containing:

a) the first heavy chain (HC1);

b) second heavy chain (HC2);

c) the 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 CD3, and HC2 and LC2 pair to form a second antigen binding site that specifically binds GPRC5D, or a bispecific fragment thereof, binding GPRC5DxCD3.

28. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 27, wherein HC1 contains SEQ ID NO: 25 and LC1 contains SEQ ID NO: 26.

29. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 52 and LC2 contains SEQ ID NO: 56.

30. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 53 and LC2 contains SEQ ID NO: 56.

31. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 54 and LC2 contains SEQ ID NO: 57.

32. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 55 and LC2 contains SEQ ID NO: 58.

33. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 82 and LC2 contains SEQ ID NO: 92.

34. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 83 and LC2 contains SEQ ID NO: 56.

35. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 84 and LC2 contains SEQ ID NO: 57.

36. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 85 and LC2 contains SEQ ID NO: 57.

37. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 86 and LC2 contains SEQ ID NO: 57.

38. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 87 and LC2 contains SEQ ID NO: 92.

39. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 88 and LC2 contains SEQ ID NO: 58.

40. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 89 and LC2 contains SEQ ID NO: 93.

41. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 91 and LC2 contains SEQ ID NO: 94.

42. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 28, wherein HC2 contains SEQ ID NO: 85 and LC2 contains SEQ ID NO: 57.

43. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of any one of embodiments 27-42, which is of an IgG1, IgG2, IgG3, or IgG4 isotype.

44. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of Embodiment 43, which is of the IgG4 isotype.

45. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of embodiments 27-42, which binds GPRC5D on the surface of human myeloma cells.

46. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of embodiments 27-42, which binds GPRC5D on the surface of human multiple myeloma cells.

47. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of embodiments 27-42, which induces activation of human T cells in vitro at an EC ₅₀ of less than about 0.22 nM.

48. The anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of embodiments 27-42, which induces T cell-dependent cytotoxicity of GPRC5D-expressing cells in vitro at an EC ₅₀ of less than about 0.89 nM.

49. An isolated cell expressing the antibody or bispecific binding fragment of any one of embodiments 27-42.

50. The cell of embodiment 49, which is a hybridoma.

- 39 044685

51. The cell of embodiment 49, wherein the antibody or bispecific binding fragment is produced recombinantly.

52. A method of treating an individual suffering from cancer, the method comprising administering a therapeutically effective amount of a GPRC5DxCD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27-42 to a patient in need thereof for a time sufficient to treat the cancer.

53. A method of inhibiting the growth or proliferation of cancer cells, the method comprising administering a therapeutically effective amount of a GPRC5DxCD3 bispecific antibody or bispecific binding fragment according to any one of embodiments 27-42 to inhibit the growth or proliferation of cancer cells.

54. A method of redirecting T cells to a GPRC5D-expressing cancer cell, the method comprising administering a therapeutically effective amount of a GPRC5DxCD3 bispecific antibody or bispecific binding fragment of any one of embodiments 27-42 to redirect the T cell to the cancer lesion.

55. The method of embodiment 52, 53 or 54, wherein the cancer is a hematological malignancy.

56. The method of embodiment 55, wherein the hematologic malignancy is a B cell cancer expressing GPRC5D.

57. The method of embodiment 56, wherein the B cell cancer expressing GPRC5D is multiple myeloma.

58. The method of embodiment 52, comprising administering a second therapeutic agent.

59. The method of embodiment 58, wherein the second therapeutic agent is a chemotherapy drug or a targeted anticancer therapy.

60. The method of embodiment 59, wherein the chemotherapeutic agent is cytarabine, an anthracycline, histamine dihydrochloride, or interleukin 2.

61. The method of Embodiment 59, wherein the second therapeutic agent and the bispecific antibody are administered to the individual simultaneously, sequentially, or separately.

62. A pharmaceutical composition comprising a bispecific antibody to GPRC5DxCD3 or a bispecific binding fragment according to any one of embodiments 27-42 and a pharmaceutically acceptable carrier.

63. The method of producing a bispecific antibody to GPRC5DxCD3 or a bispecific binding fragment according to any one of embodiments 27-42 by culturing a cell according to any one of embodiments 49-51.

64. An isolated synthetic polynucleotide encoding HC1, HC2, LC1, or LC2 of the anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of any one of embodiments 27-42.

65. A kit comprising a GPRC5DxCD3 bispecific antibody or bispecific binding fragment as defined in any one of embodiments 27-42, and/or a polynucleotide as defined in embodiment 63, and packaging for the above components.

Examples

The following examples are provided as a supplement to the above description and in order to better understand the subject matter of the invention described herein. These examples should not be considered limiting of the described subject matter of the invention. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and in light of this, those skilled in the art will recognize various modifications and changes that should be included within the scope of the invention and that can be made without departing from the scope of the invention. inventions.

Example 1. Antigens

Due to difficulties in producing recombinant GPRC5D antigens, transfected cell lines expressing GPRC5D [human (SEQ ID NO: 22), cynomolgus (SEQ ID NO: 23) and mouse (SEQ ID NO: 24)] were generated using standard methods for use as whole cell antigens in studies in the field of antibody formation and characterization (Table 4).

Table 4. Cell lines expressing GPRC5D

Example 2: Generation of antibodies to GPRC5D using phage display technology

Two different approaches were used to generate antibodies to GPRC5D using phage: standard cell panning (negative selection) and FACS phage cell panning (competitive selection).

Standard cell phage panning (negative selection)

The de novo generation of custom phage libraries is described in detail (Shi et al (2010) J. Mol. Biol. 397:385-396; International Patent Publication No. WO 09/085462). These libraries were built on three human germline VH genes (IGHV1-69, 3-23, 5-51) and four human germline VL genes (A27, V3, L6, 012), designed to have wide diversity in terms of CDR- H3. Three de novo phage libraries (DNP00004 - 169 HC/LC mixture, DNP00005 - 323 HC/LC mixture and DNP00006 - 551 HC/LC mixture) demonstrating Fab variants or p1X phage coat protein variants were separated by panning on GPRC5D-extracting stable HEK293 cells G5 (target cells) during rounds 1, 3, 5, and during rounds 2 and 4, the previous round with amplified phage Fab-pIX was applied to background HEK293 cells (negative selection) (see Table 5). De novo phage Fab-pIX from round 1 bound to target cells was isolated for overnight amplification. The selected phage from round 1 was applied to the background cells during round 2, with unbound phage Fab-pIX isolated for phage amplification by negative selection overnight. Another series of positive and negative rounds of panning were performed - rounds 3 and 4. The final round was performed with the last round of negatively selected amplified phage divided into two panning samples, one for target cells and one for panning on background cells.

Table 5. Flowchart of negative selection for standard cell phage panning

Panning round Cells used in as an antigen 1 HEK293 GPRC5D human 2 HEK293 3 HEK293 GPRC5D human 4 HEK293 5 (separation) HEK293 GPRC5D human HEK293

Standard cell phage panning (background selection)

Fab-pIX phage display libraries were added to HEK293 cells after incubation under similar conditions as performed with the standard cell panning procedure mentioned above (Table 6). After three rounds, DNA corresponding to phage Fab-pIX bound to HEK293 was used to generate PCR amplicons for NGS. These NGS results will be used in additional dynamic subtraction analysis using NGS2.0 software to distinguish possible target-specific Fab candidates.

Table 6

Flowchart of background selection for standard cell phage panning

Panning round Cells used as antigen 1 HEK293 2 HEK293 3 HEK293

Cell phage panning FACS (competitive selection)

Three rounds of panning were performed by applying phage Fab-pIX to a mixture of target and background cells simultaneously (Table 7). For rounds 1 and 2, target cells with attached phage Fab-pIX were sorted using the GFP signal. Acid cell lysis followed by infection with E. coli was used to separate bound Fab-pIX phage from sorted cells. In the final round, a mixture of round 2 amplified Fab-pIX phage and cells was sorted into two populations: into a gate for target cells with GFP and into a gate for background cells without GFP. The bound phage Fab-pIX for both cell populations was isolated by acid lysis and infection with E. coli.

Table 7. Flowchart of competitive selection for cell phage panning by FACS

Panning round Cells used as antigen 1 Human GPRC5D_GFP and HEK293 2 Human GPRC5D_GFP and HEK293 3 Human GPRC5D_GFP and HEK293

Next generation sequencing (NGS) of phage panning results

Overnight glucose repression cultivation was applied to six samples of the final round of panning. These cultures were used to prepare a DNA minipreparation, Qiagen QIAspin DNA kit. Six DNA samples were used as PCR templates to generate amplicons that detected HCDR1 to HCDR3. Six amplicons purified the gel

-41 044685 by electrophoresis and pooled, keeping versions 2.1, 3.0 separate for standard cell panning, and fully pooled for FACS cell panning. These pooled gel electrophoresis purified amplicons were provided to Genewiz no NGS for study using MiSeq 1x300 technology. The files were transferred by Genewiz and uploaded to a local server (nas2.0). On this server, the NGS2.0 software application was used to download, read, and analyze sequence files. The top 88 sequences were selected for IgG conversion according to copy number (>50) and the ratio of target cell sequences (+) to background cell sequences (−) (ratio >5:1). Since NGS only detects the heavy chain variable sequence, full-length heavy chain constructs had to be inserted virtually on a computer into the appropriate scaffold regions. In addition, since only the heavy chain sequence was known, each candidate was paired with the four parental light chains (A27, B3, L6, O12). The final conversion of the candidates was carried out in the form of human IgG4PAA.

ELISA screening and standard sequencing

From the same DNA preparation used for NGS, restriction enzyme digestion and self-ligation were performed to excise the pIX gene to allow soluble Fab expression. Ninety-two colonies selected from each of the three panning samples were assessed for Fab expression by ELISA and sequenced for both heavy and light chain determination by Sanger. The final candidates with diversified sequence in the LCDR regions were cloned into mammalian expression plasmids.

Example 4. Preliminary description of the properties of antibodies to GPRC5D obtained using phage display technology

Linking with GPRC5D

Whole cell phage panning was performed using human cells with GPRC5D as the antigen as described above. After NGS analysis, only the heavy chain sequence was known for each candidate. Therefore, each heavy chain had to be paired with 4 parental light chains (A27, B3, L6, O12), resulting in 348 mAbs from 87 Hc sequences identified by NGS. These monoclonal antibodies were initially assessed for binding to cynomolgus macaque GPRC5D using FACS. The cynomolgus GPRC5D cell line was selected to perform the initial screen to maximize potential binding signal because the cynomolgus GPRC5D cell line had a higher expression level than the human GPRC5D cell line. Briefly, FACS screening was performed by normalizing the protein concentration to 1 μg/ml, and 100 μl of protein was mixed with 200,000 cells per well. The monoclonal antibody was allowed to incubate with cells for 1 hour at 4°C. The cells were then washed three times with PBS and 0.2% FBS. Anti-human secondary mAb conjugated to PE (Jackson, catalog no. 709-116-149) was then added as a development reagent. Cells and secondary antibody were incubated for 1 h at 4°C. The cells were then washed three times with PBS and 0.2% FBS. Cells were washed again with PBS and 0.2% FBS and then analyzed by FACSarry.

A significant number of desired antibodies were observed that bound to cynomolgus GPRC5D, including 15 mAbs with an MIF greater than 100,000, 23 mAbs with an MIF less than 100,000 but greater than 10,000, and 63 mAbs with an MIF less than 10,000 but greater than 1000 (Table 8).

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Table 8. FACS data for NGS-derived mAb binding to cynomolgus macaque GPRC5D. The heavy chain sequences identified by NGS analysis were paired with each parental Lc as shown. GC5M29 (PH9L3 light chain) was used as a control.

The isolated mAbs have a CIF > 1000.

mAb ID GPRC5D inAb GPRC5D GPRC5D mAh GPRC5D ator (ID) NS [PH9L1 (A27)] CIF [PH9L2 (VZ) ] CIF [PH9L3 (L6) ] CIF [PH9L4 (012)] CIF GC5H18 GC5B22 198 GC5B23 230 GC5B24 246 GC5B25 31 324 GC5H17 GC5B66 242 GC5B67 272 GC5B68 2682 GC5B69 17 416 GC5H20 GC5B114 199 GC5B115 266 GC5B116 253 GC5B117 5037 GC5H15 GC5B158 14 407 GC5B159 139 766 GC5B160 84 111 GC5B161 7856 GC5H13 GC5B202 89 948 GC5B203 9173 GC5B204 15 203 GC5MB205 52 663 GC5H23 GC5B242 242 GC5B243 116 641 GC5B244 249 GC5B245 469 GC5H14 GC5B282 487 GC5B283 341 GC5B284 249 GC5B285 87 116 GC5H22 GC5B326 195 GC5B237 265 GC5B328 248 GC5B329 292 GC5H24 GC5B26 194 GC5B27 265 GC5B28 229 GC5B29 590 GC5H19 GC5B70 214 GC5B71 229 GC5B72 2013 GC5B73 3827 GC5H33 GC5B118 178 GC5B119 222 GC5B120 880 GC5B121 43 924 GC5H36 GC5B162 86773 GC5B163 21,970 GC5B164 12 2043 GC5B165 1870 GC5H31 GC5B206 467 GC5B207 3636 GC5B208 481 GC5B209 1409 GC5H29 GC5B246 202 GC5B247 3037 GC5B248 2835 GC5B249 1373 GC5H34 GC5B286 239 GC5B287 163 990 GC5B288 259 GC5B289 271 GC5H35 GC5B330 202 GC5B331 552 GC5B332 191 978 GC5B333 1591

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GC5H26 GC5B30 204 GC5B31 230 GC5B32 553 GC5B33 270 GC5H27 GC5B74 238 GC5B75 365 GC5B76 829 GC5B77 317 GC5H25 GC5B122 153 GC5B123 237 GC5B124 6026 GC5B125 6197 GC5H30 GC5B166 1510 GC5B167 6266 GC5B168 32 482 GC5B169 327 GC5H47 GC5B210 1730 GC5B211 284 GC5B212 278 GC5B213 727 GC5H42 GC5B250 3969 GC5B251 213 233 GC5B252 63 295 GC5B253 574 GC5H37 GC5B290 8839 GC5B291 201 024 GC5B292 110 940 GC5B293 305 GC5H45 GC5B334 160 GC5B335 1434 GC5B336 3228 GC5B337 309 GC5H38 GC5B34 157 GC5B35 283 GC5B36 14 936 GC5B37 297 GC5H40 GC5B78 162 GC5B79 309 GC5B80 428 GC5B81 74 140 GC5H43 GC5B126 157 GC5B127 265 GC5B128 268 GC5B129 1283 GC5H49 GC5B170 1014 GC5B171 51,995 GC5B172 1434 GC5B173 332 GC5H62 GC5B214 2565 GC5B215 274 GC5B216 263 GC5B217 1589 GC5H60 GC5B254 161 GC5B255 5788 GC5B256 460 GC5B257 308 GC5H94 GC5B294 190 GC5B295 2312 GC5B296 745 GC5B298 261 GC5H69 GC5B338 188 GC5B339 260 GC5B340 1798 GC5B341 290 GC5H85 GC5B38 10 809 GC5B39 1112 GC5B40 110 654 GC5B41 4379 GC5H96 GC5B82 165 GC5B83 272 GC5B84 1547 GC5B85 3563 GC5H97 GC5B130 174 GC5B131 293 GC5B132 306 GC5B133 343 GC5H76 GC5B174 219 GC5B175 3631 GC5B176 244 GC5B177 324 GC5H68 GC5B218 21 420 GC5B219 310 GC5B220 777 GC5B221 762 GC5H79 GC5B256 714 GC5B257 1146 GC5B258 456 GC5B259 1129 GC5H71 GC5B298 185 GC5B299 1370 GC5B300 260 GC5B301 292 GC5H93 GC5B342 164 GC5B343 247 GC5B344 401 GC5B345 283 GC5H21 GC5B42 219 GC5B43 307 GC5B44 2694 GC5B45 1022 GC5H39 GC5B86 192 GC5B87 344 GC5B88 470 GC5B89 41

027

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GC5H50 GC5B134 764 GC5B135 340 GC5B136 600 GC5B137 2087 GC5H28 GC5B178 750 GC5B179 6708 GC5B180 1005 GC5B181 535 GC5H53 GC5B222 284 GC5B223 264 GC5B224 228 GC5B225 605 GC5H51 GC5B262 159 GC5B263 1013 GC5B267 238 GC5B268 309 GC5H64 GC5B302 184 GC5B303 329 GC5B304 240 GC5B305 1571 GC5H16 GC5B346 182 GC5B347 259 GC5B348 332 GC5B349 552 GC5H65 GC5B46 204 GC5B47 327 GC5B48 1320 GC5B49 392 GC5H32 GC5B90 335 GC5B91 340 GC5B92 1848 GC5B93 1780 GC5H54 GC5B138 193 GC5B139 354 GC5B140 323 GC5B141 292 GC5H99 GC5B182 196 GC5B183 6421 GC5B184 309 GC5B185 280 GC5H52 GC5B226 204 GC5B227 298 GC5B228 247 GC5B229 263 GC5H48 GC5B266 268 GC5B267 2160 GC5B268 235 GC5B269 281 GC5H44 GC5B306 204 GC5B307 313 GC5B308 302 GC5B309 621 GC5H46 GC5B350 198 GC5B351 287 GC5B352 4300 GC5B353 1708 GC5H56 GC5B50 229 GC5B51 36,679 GC5B52 386 GC5B53 446 GC5H55 GC5B94 255 GC5B95 609 GC5B96 411 GC5B97 350 GC5H98 GC5B142 194 GC5B143 407 GC5B144 284 GC5B145 483 GC5H61 GC5B186 543 GC5B187 1132 GC5B188 376 GC5B189 333 GC5H92 GC5B320 297 GC5B321 294 GC5B322 280 GC5B323 386 GC5H5 GC5B17 243 GC5B18 301 GC5B19 1048 GC5B20 384 GC5H59 GC5B98 272 GC5B99 388 GC5B100 661 GC5B101 528 GC5H66 GC5B354 223 GC5B355 852 GC5B356 374 GC5B357 500 GC5H80 GC5B54 299 GC5B55 415 GC5B56 422 GC5B57 487 GC5H87 GC5B102 383 GC5B103 460 GC5B104 3130 GC5B105 456 GC5H67 GC5B146 325 GC5B147 370 GC5B148 383 GC5B149 487 GC5H73 GC5B190 236 GC5B191 2556 GC5B192 409 GC5B193 387 17 GC5H86 GC5B230 885 GC5B231 543 GC5B232 494 GC5B233 620 GC5H70 GC5B270 390 GC5B271 660 GC5B272 389 GC5B273 450 GC5H90 GC5B314 219 GC5B315 334 GC5B316 363 GC5B317 403 GC5H95 GC5B358 211 GC5B359 344 GC5B360 414 GC5B361 449 GC5H74 GC5B58 294 GC5B59 448 GC5B60 477 GC5B61 548 GC5H78 GC5B106 267 GC5B107 440 GC5B108 474 GC5B109 497 GC5H77 GC5B150 312 GC5B151 452 GC5B152 412 GC5B153 709 GC5H91 GC5B194 296 GC5B195 448 GC5B196 491 GC5B197 483 128 195 134 eleven GC5H57 GC5B234 355 GC5B235 348 GC5B236 008 GC5B237 255 GC5H41 GC5B274 313 GC5B275 7431 GC5B278 435 GC5B279 542 49 GC5H58 GC5B318 2608 GC5B319 5791 GC5B320 616 GC5B321 4823 GC5H63 GC5B362 313 GC5B363 392 GC5B364 461 GC5B365 3613 GC5H72 GC5B62 326 GC5B63 463 GC5B64 485 GC5B65 694 208 140 104 GC5H75 GC5B110 487 GC5B111 692 GC5B112 269 GC5B113 136 GC5H81 GC5B154 351 GC5B155 412 GC5B156 440 GC5B157 555 GC5H82 GC5B198 285 GC5B199 593 GC5B200 499 GC5B201 485 GC5H83 GC5B238 3698 GC5B239 4370 GC5B240 4676 GC5B241 825 GC5H88 GC5B278 3659 GC5B279 481 GC5B280 425 GC5B281 633 GC5H89 GC5B332 299 GC5B333 561 GC5B334 4467 GC5B335 494 26 GC5H84 GC5B366 544 GC5B367 474 GC5B368 6313 GC5B369

116

The 40 mAbs with the highest binding affinity were selected for further determination.

- 45 044685 characterization, which consisted of repeating the binding assay to cynomolgus GPCR5D cells, as well as assessing binding to cells expressing human GPRC5D by FACS (Table 9). These data were analyzed to select 17 mAbs for purification and generation of bispecific antibodies to GPRC5DxCD3 (highlighted). Factors used to select mAbs for purification and generation of bispecific antibodies to GPRC5DxCD3 include binding specificity to human GPRC5D, cross-reactivity with cynomolgus GPRC5D, and Hc sequence diversity. For example, GC5H36 was paired with three different light chains and analyzed for binding (GC5B162, GC5B163, GC5B164). Only GC5B164 was nominated because this mAb was observed to have a higher CIF with human GPRC5D than GC5B162 or GC5B163.

Table 9. FACS binding data of NGS-derived mAbs to cynomolgus and human GPRC5D-expressing HEK293F cells. Untransfected HEK293F cells were used to assess the binding specificity of GPRC5D. TF7M1636 was used as an isotype control. The isolated mAbs were selected for subsequent analysis.

CIF GPRC5D

ID CIF GPRC5D CIF squirrel NS peptide ID Javanese person 293F toque GC5B38 GC5H85 6864 280 215 GC5B110 GC5H75 142 277 2543 235 GC5B158 GC5H15 4923 285 235 GC5B162 GC5H36 75 139 2981 230 GC5B202 GC5H13 30 470 2152 256 GC5B218 GC5H68 10 451 277 263 GC5B230 GC5H86 19 103 364 290 GC5B234 GC5H57 134 258 1657 261 GC5B51 GC5H56 22 661 667 480 GC5B159 GC5H15 129 370 1987 258 GC5B163 GC5H36 7605 840 310 GC5B171 GC5H49 24 133 1144 1321 GC5B235 GC5H57 158 011 2643 254 GC5B243 GC5H23 67,848 20 458 221 GC5B251 GC5H42 74 510 11 418 235 GC5B281 GC5H34 109 418 5317 570 GC5B291 GC5H37 77 798 10 427 880 GC5B32 GC5H26 3597 14,656 358 GC5B36 GC5H38 5353 2002 352 GC5B40 GC5H85 67 279 4510 310 GC5B112 GC5H75 104 875 470 433 GC5B160 GC5H15 40 881 635 276 GC5B164 GC5H36 78 672 12,664 343 GC5B168 GC5H30 41,779 28,885 1238 GC5B204 GC5H13 5090 390 212 GC5B236 GC5H57 78 546 1442 192 GC5B252 GC5H42 22 010 1498 204

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GC5B292 GC5H37 63,366 1872 480 GC5B320 GC5H58 63 345 22,891 231 GC5B332 GC5H35 106 515 13,348 253 GC5B25 GC5H18 22,079 705 431 GC5B69 GC5H17 1593 851 284 GC5B81 GC5H40 62,756 9986 288 GC5B89 GC5H39 31,613 1068 251 GC5B113 GC5H75 66 475 550 323 GC5B121 GC5H33 35 493 3061 299 GC5B205 GC5H13 63 610 25 199 260 GC5B237 GC5H57 2579 267 248 GC5B285 GC5H14 81 405 19,870 225 GC5B369 GC5H84 16 241 559 252 TF7M1636 956 2621 1538 Human 209 316 228 2nd aHTH-hGPRC5D 9001 379 70 isotype mouse 60 82 65 antibodies unpainted 44 50 33

The concentration-dependent binding profile of each of the selected mAbs against human GPRC5D-expressing HEK293 cells and untransfected HEK293 cells was determined by FACS (Fig. 1). All mAbs were observed to bind to human GPRC5D in a dose-dependent manner. Three mAbs GC5B36, GC5B168, and GC5B205 were also observed to bind to untransfected (GPRC5D null) HEK293 cells and were deprioritized due to this nonspecific interaction with cells.

The remaining 14 mAbs were selected to produce bispecific antibodies CD3B219 with an anti-CD3 arm and B23M46 with a null arm against RSV (Table 10).

Table 10. Relationship between GPRC5D mAb IDs and bispecific antibody IDs

x CD3 Protein ID shab ID bispecific. to GPRC5D x B23 zero ID bispecific. antibodies antibodies GC5B320 GCDB44 GCDB30 GC5B243 GCDB40 GCDB26 GC5B285 GCDB43 GCDB29 GC5B332 GCDB45 GCDB31 GC5B164 GCDB35 GCDB21 GC5B251 GCDB41 GCDB27 S5V81 GCDB32 GCDB18 GC5B235 GCDB38 GCDB24 GC5B110 GCDB34 GCDB20 GC5B202 GCDB36 GCDB22 GC5B234 GCDB37 GCDB23 GC5B252 GCDB42 GCDB28 GC5B236 GCDB39 GCDB25 GC5B89 GCDB33 GCDB19

One bispecific antibody, GCDB38, precipitated during recombination, and the other antibody, GCDB36, contained >10% aggregate. All other bispecific antibodies met standard release criteria, and the null arm bispecific antibodies were assayed for concentration-dependent binding to HEK293 cells expressing human GPRC5D (Fig. 2).

All bispecific antibodies were observed to bind in a dose-dependent manner. GC5B320 was included as a binding comparison antibody to understand the binding difference between divalent mAbs and monovalent bispecific antibodies. GCDB44 was included as a binding comparison antibody to understand the difference in binding between the bispecific-47044685 anti-CD3 antibodies and the anti-RSV null arm mAb. An expected reduction in observed binding affinity was observed when mAb GC5B320 was compared with bispecific antibodies GCDB30 and GCDB44. In addition, GCDB44 (an anti-CD3 bispecific antibody) was observed to have a slightly higher binding affinity to human GPRC5D cells compared to GCDB30 (an anti-RSV null arm bispecific antibody), demonstrating that the anti-CD3 arm has a positive effect on binding to this cell line.

A panel of bispecific antibodies was also profiled for binding compared to MM1R and H929 cells, which endogenously express GPRC5D (Fig. 3). The binding profile of bispecific antibodies on MM1R and H929 cells was compared with human GPCR5D-overexpressing HEK293 cells and untransfected HEK293 cells by FACS. The bispecific antibodies were observed to bind to GPRC5D expressed endogenously on MM1R and H929 cells with a range of affinities. The highest binding was observed with human GPRC5D HEK cells, which as an overexpressed stable cell line have a much higher receptor density than MM1R or H929 cells. GCDB37, GCDB38, GCDB39, and GCDB41 were not selected due to the low binding affinity observed for H929 and MM1R cells.

T-cell-dependent cytotoxicity in vitro The panel of bispecific antibodies was then profiled for activity by a T-cell-mediated cytotoxicity assay using H929 and MM1R target cells (Fig. 4A and B, Table 11). Briefly, target cells (H929, MM1.R, OPM2, LP-1 and Daudi cells or parental HEK cells and HEK+GPRC5D cells) were counted and 10 million cells were centrifuged at 1350 rpm for 3 min, and cell the pellet was resuspended in 1 ml of diluted CFSE solution (CFSE CellTrace proliferation dye was reconstituted in 18 μl of sterile DMSO, and 1 μl of the solution was diluted in 10 ml of sterile PBS) and incubated at room temperature for 8 min in the dark. After incubation, 1 ml of HI FBS was added to the cell suspension to block excess CFSE. Cells were washed twice in RPMI-1640 using FBS 10%. After dilution in 10 ml RPMI, cells were counted and cell viability was recorded in the protocol. Cells were diluted to 2.2 x 10 ^L5 /ml and incubated at 37°C until use.

Pan T cells from normal donors were thawed at 37°C in a water bath, and then the cells were centrifuged at 1350 rpm at 4°C for 3 min. The supernatants were removed and reconstituted in a nutrient medium at a concentration of 1.1 x 10 l ⁶ /ml. 2x10L5 target cells were added to the wells of a 96-well U-bottom plate, followed by the addition of Fc blocker (to a final concentration of 2 mg/ml). All cell lines were incubated at room temperature for 10 min to block Fc receptor activity. 1x10L5 T cells were added to the wells (effector:target ratio 5:1). After mixing the target cells with the T cells, 20 μl of diluted anti-GPRC5DxCD3 bispecific antibodies was added to each well. Bispecific antibodies to GPRC5DxCD3 were diluted to a concentration of 800 μg/ml (10X) in PBS. Titration was performed using 4-fold serial dilutions in PBS in a 96-well U-bottom plate. The last column was left for only one FSB (carrier medium control). The plates were incubated at 37°C with 5% CO2 for 48 h.

After two days (8 h), the plates were centrifuged and 100 μl of supernatants were stored at −80°C for cytokine release assay. Cells were washed in 200 μL PBS and incubated in 50 μL NIR viable cell detection dye (1:200 dilution) and phycoerythrin (PE)-conjugated anti-CD25 antibody (1:50 dilution) for 20 min at room temperature. Cells were subsequently washed once in 200 μL FACS buffer and then diluted in 150 μL FACS buffer. Cells were analyzed using FACSCanto II and FlowJo 7.6 for target cytotoxicity (% targets) and CD25+ T cell activation (% live T cells). Graphing and data fitting were performed in GraphPad Prism 6 using nonlinear regression with a variable slope function (four parameters) using the method of least squares.

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Table 11. Mean EC ₅₀ value calculated from the assessment of T cell-mediated cytotoxicity of bispecific antibodies to GPRC5DxCD3 using target cells H929 and MM1R

EC50 GPRC5DxCD3 (nM)

Ranging ID bispecific. H929 (n=5) MM1R (n=3) (average) antibodies H929/MM1R GCDB32 0.39 ± 0.3 0, 12 ±0.1 2/1 GCDB33 4.29 ± 1.28 0.8 ±0.18 7/6 GCDB34 2.13 ± 0.93 0.77 ±0.24 5/5 GCDB35 1.39±0.84 0.74 ±0.32 4/4 GCDB36 5.2 ± 0.93 4.22 ±0.55 8/9 GCDB40 1.2 + 0.92 0.52 ±0.38 3/3 GCDB41 N/A 2.58 ±0.15 10/8 GCDB43 0.36 ± 0.41 0.21 ±0.29 1/2 GCDB44 10.29±2.65 4.41 ±0.29 9/10 GCDB45 2.25 + 1 0.91 ±0.93 6/7

All bispecific antibodies are active in T cell-mediated killing of H929 cells with the observed range of activity (Table 11). A similar rank order was observed for both cell lines. However, a lower EC ₅₀ was observed in MM1R cells. Interestingly, binding affinity does not necessarily correlate with activity in a T cell-mediated cytotoxicity assay. For example, in the cytotoxicity assay, GCDB44 was the bispecific antibody with the highest binding affinity but the lowest activity. While GCDB43 with similar, although slightly lower, binding affinity was the most active in the cytotoxicity assay.

To assess functional cross-reactivity with cynomolgus GPRC5D, a panel of bispecific antibodies was then profiled for T cell-mediated cytotoxicity and T cell activation using HEK293 cells with cynomolgus GPRC5D (Fig. 5A and B). All bispecific antibodies were active in this assay, although a range of activity was observed against cells expressing cynomolgus GPRC5D+.

Efficacy in vivo

An in vivo comparative study was then performed to understand the in vivo activity of these GPRC5DxCD3 bispecific antibodies. GCDB32 and GCDB35 (Fig. 5A and B) were selected for testing in the H929 prophylactic tumor model. H929 cells were implanted into NSG mice one week after injection of human PBMCs. Treatment with bispecific antibodies began simultaneously with implantation of H929 cells and continued every 2 or 3 days (once every 2 days or once every 3 days) at a dose of 10 μg, 1 mg and 0.1 μg/animal for a total of five treatments. Ten mice were used in each group, and PBS was included as a vehicle control. Treatment was stopped on day 11, and the study was stopped on day 25 (Fig. 6A and B) or day 26 (Fig. 12A-D). All anti-GPRC5DxCD3 antibodies tested in this prophylactic model showed 100% tumor growth inhibition at 10 and 1 μg/animal doses, with the exception of GCDB35, which demonstrated approximately 80% tumor growth inhibition at 1 μg/animal doses. When using a dose ten times lower (0.1 μg/animal), these bispecific antibodies demonstrated varying degrees of efficacy ranging from 10 to 80% tumor growth inhibition.

T cell-dependent cytotoxicity in vitro in the presence of an Fc blocker

To gain insight into the specificity of the targeting arm to GPRC5D, a panel of bispecific antibodies to GPRC5DxCD3 was then assessed using a T cell redirection cytotoxicity assay in the presence of Fc block. This experiment was critical to understanding specificity because the target cells for bispecific antibodies are B cells that express the ability of Fcy receptors to interact with the Fc portion of the bispecific antibody in an in vitro assay. In the T cell-mediated cytotoxicity assay, a shift in activity was observed for a number of bispecific antibodies, with the largest shift observed for GCDB40 and GCDB34 (Fig. 7A-B).

We then performed direct measurements of binding interactions between the four most active bispecific antibodies (GCDB32, GCDB35, GCDB40 and GCDB43) and Fcy receptors (Fig. 8A-D). One round of Alpha Screen analysis was performed for each of the Fcy receptors and bispecific antibodies listed above. All samples were analyzed in duplicate. At FcyRI, the four bispecific antibodies behave like B21M hIgG4 PAA. That is, they are not competitive to a greater extent than the corresponding isotype control. Similar differences were also observed between the hIgGI WT (wild type) control and the four FcyRIHa bispecific antibodies, with GCD43, like the IgG4PAA isotype control and other bispecific antibodies, having a slightly higher affinity for FcYRIIIa. For both FcyRIIa and FcYRIIb, the four bispecific antibodies compete in the following order: GCDB40 > GCDB32 > GCDB43 > GCDB35. GCDB40 is the most competitive or binds with the highest affinity to FcYRIIa and FcYRIIb. In fact, GCDB40 competes for FcYRIIa and FcYRIIb to the same extent as WT hIgG1, confirming the observed shift in activity observed in the T cell-mediated cytotoxicity assay when Fc blockade is included. Due to unexpected interactions with FcYRIIa and FcYRIIb, GCDB40 was not promoted.

Competitive Binning Analysis

Anti-GPRC5D mAbs were assessed for competitive binding to cells with human GPCR5D. Briefly, cells were seeded at a concentration of 50,000 cells/well in 50 μl of medium and allowed to settle for 90 min at 37°C. Wells were then blocked with 3% BSA for 1 h at room temperature.

Ruthenium(II) mAbs were labeled with tris-bipyridine, N-hydroxysuccinimide (Ru-tag) according to standard procedures. In a separate 96-well plate, 5 μM competitive monoclonal antibody was incubated with 50 nM Ru-labeled monoclonal antibody. The blocking solution was removed from the cell plate and 25 μl of mAb solution was added. The plates were incubated for 1 hour at room temperature with shaking. After washing the plates three times with PBS, 150 μl of MSD reading buffer (without surfactants) was added and Ru-labeled antibody binding was detected using an MSD plate reader.

All mAbs belong to the same competition group, only GC5B420 and GC5B421 do not fully compete with GC5B81, GC5B285 and/or GC5B332 (<70% blocked) (Table 12). It is speculated that simultaneous binding of two mAbs to GPRC5D may be sterically impossible given the small size of the extracellular domain of GPRC5D compared to the size of the mAb.

Table 12. Epitope binning during competitive binding of mAbs to GPRC5D. Anti-GPRC5D mAbs were assessed for competitive binding to human CGPCR5D cells (+/-=<70% blocking).

mAb ID GC5B81 GC5B164 GC5B285 GC5B332 GC5B420 GC5B421 GC5B81 + + + + +/- +/- GC5B164 + + + + + + GC5B285 + + + + + +/- GC5B332 + + + + +/- + GC5B420 + + + + + + GC5B421 + + + + + + GC5B243 + + + + + +

Example 4: Generation of Antibodies to GPRC5D Using Hybridoma Technology

Three Balb/c mice were immunized intradermally at the base of the tail with plasmid DNA pCMV6-neo (CMV promoter) expressing full-length human GPRC5D on days 0, 10, and 20. On day 59, mice received a final intraperitoneal and intravenous immunization with rat basophilic leukemia (RBL) cells. , overexpressing full-length human GPRC5D. On day 63, lymph nodes and spleens were collected, B cell enrichment was performed, and the cells were used to generate ~3500 mAb-secreting hybridomas.

Example 5. Preliminary description of the properties of antibodies to GPRC5D obtained using hybridoma technology

Linking with GPRC5D

Hybridomas were screened by FACS for binding to both RBL cells and RBL cells expressing human GPRC5D. The ratio of background-corrected SIF 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 selected for v-region cloning. Thirty-one mAb sequences were identified, synthesized, expressed, and purified. Two of the 31 mAbs demonstrated binding to RBL GPRC5D cells above background levels (Figures 9A and 8B). GCDB390 and GCDB396 were selected for expression, purification and production of bispecific antibodies with CD3B219 arm to CD3 to produce bispecific antibodies GCDB46 and GCDB47, respectively.

T cell dependent cytotoxicity

In the T cell-mediated cytotoxicity assay, GCDB46 and

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GCDB47 (Figs. 10A and 10B). It was noted that both bispecific antibodies were active, with the obtained EC ₅₀ values being 0.67 and 0.1 nM, respectively. Based on these data, both mAbs were selected for lead optimization along with three phage-derived mAbs.

Example 6: Evaluation, Selection and Optimization of Selected Antibodies

Five anti-GPRC5D bispecific antibodies were selected for lead optimization (GCDB32, GCDB43, GCDB35, GCDB46, GCDB47) based on the binding, function, cross-reactivity, and selectivity data presented in Table 1. 13.

Table 13. Leader optimization data for bispecific antibodies to GPRC5DxCD3. Bispecific antibodies were evaluated for binding, function, cross-reactivity and selectivity

GPRC5DxC D3 ID squirrel T cell-mediated killing of target cells Linking with GPRC5A, B, C H929 EC50 ± CO (nM) MM1R EC50 ± CO (nM) H929 without blocker EU by compare with EU blocker EC50 GPRC5D Cynomolgus (nM) GCDB32 No binding 0.43 ± 0.29 0.12 ± 0.01 2.4 0.19 GCDB43 No binding 0.39 ± 0.07 0.54 2.8 0.95 GCDB40 No binding 1.2 ± 0.83 0.52 ± 0.31 5.1 10.61 GCDB35 Low 1.39 ± 0.75 0.74 ± 0.26 2.7 4.65 GCDB34 No binding 2.01 ± 0.74 0.77 ± 0.09 6, 3 1.14 GCDB45 No binding 2.36 ± 0.84 0.91 ± 0.76 2.2 2.82 GCDB33 No binding 5.21 ± 1.21 0.8 ± 0.15 1.5 0.2 GCDB36 Low 5.57 ± 0.47 4.22 ± 0.29 3.35 GCDB46 No binding 0.67 GCDB47 No binding 0.1 GCDB48 No binding 0.17 Not active

Lead optimization was aimed at addressing potential risks associated with post-translational modification (PTM) sequences for GCDB32 (parent mAb GC5B81), GCDB43 (parent mAb GC5B285) and GCDB35 (parent mAb GC5B164), as described in Table. 14.

Table 14. Attenuation of potential labilities of PTM sequences for phage-derived antibodies. Hc CDR sequences are shown with potential PTM sequence labilities underlined (SEQ ID NO for each sequence described is in parentheses)_________________________________________________________________

ID GPCR5D ID Нс GPRC5D NS CDR1 NS CDR2 HCCDR3 GC5B81 GC5H40 SYAIS (1) GIIPIFGTANYAQKFQG (5) ESRWRGYKLD (9) GC5B285 GC5H14 NYWMS (2) GISYSGGSKYYADSVKG (28) AAWDFGRRAVRLDY (30) GC5B164 GC5H36 SYWIG (27) IIYPGDSDTRYSPSFQG (29) VYSFGGRHKALFDY (11)

All PTM variants analyzed for GC5B81 had a significant decrease in binding affinity, indicating the critical importance of this residue (HC W102) for the paratope (Table 15).

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Table 15. CDR sequences and binding data of the GC5B81 PTM library. The mutation site is underlined. Binding was classified as CIF > 10,000=++; CIF > 1,000=+; CIF <

1000 (SEQ ID NO for each described sequence is given in parentheses)

ID GPRC5D Heavy chain ID Mutation NS CDR1 NS CDR2 He CDR3 Human GPRC5D (FACS) GC5B427 GC5H199 W102Y SYAIS (1) GIIPIFGTANYAQKFQG (5) ESRYRGYKLDY (31) - GC5B428 GC5H198 W102V SYAIS (1) GIIPIFGTANYAQKFQG (5) ESVRRGYKLDY (32) - GC5B430 GC5H196 W102G SYAIS (1) GIIPIFGTANYAQKFQG (5) ESRGRGYKLDY (33) - GC5B431 GC5H195 W102A SYAIS (1) GIIPIFGTANYAQKFQG (5) ESRARGYKLDY (34) - GC5B429 GC5H197 W102F SYAIS (1) GIIPIFGTANYAQKFQG (5) ESRFRGYKLDY (35) -

Binding studies identified a number of mutations for both GC5B285 and GC5B164 that retained binding to human GPRC5D (Tables 16 and 17).

Table 16. CDR sequences and binding data of the GC5B164 PTM library. The mutation site is underlined. Binding was classified as CIF > 10,000=++; CIF > 1,000=+; CIF < 1000 (SEQ ID NO for each described sequence is given in parentheses)

ID GPRC5D Heavy chain ID Mutation He CDR1 He CDR2 He CDR3 Human GPR5D binding (FACS) GC5B471 GC5H278 D55A,W33Y SY YIG (36) IIYPGASDTRYSPSFQG (40) VYSFGGRHKALFDY (eleven) ++

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GC5B472 GC5H277 D55A, W33V SYVIG (37) IIYPGASDTRYSPSFQG (40) VYSFGGRHKALFDY (eleven) + GC5B473 GC5H276 D55A, W33F SYFIG (3) IIYPGASDTRYSPSFQG (40) VYSFGGRHKALFDY (eleven) ++ GC5B474 GC5H275 D55A, W33G SYGIG (38) IIYPGASDTRYSPSFQG (40) VYSFGGRHKALFDY (eleven) + GC5B475 GC5H274 D55A, W33A SYAIG (39) IIYPGASDTRYSPSFQG (40) VYSFGGRHKALFDY (eleven) + GC5B476 GC5H273 D55S, W33Y SYYIG (36) IIYPGSSDTRYSPSFQG (41) VYSFGGRHKALFDY (eleven) ++ GC5B477 GC5H272 D55S, W33V SYVIG (37) IIYPGSSDTRYSPSFQG (41) VYSFGGRHKALFDY (eleven) + GC5B478 GC5H271 D55S, W33F SYFIG (3) IIYPGSSDTRYSPSFQG (41) VYSFGGRHKALFDY (eleven) ++ GC5B479 GC5H270 D55S, W33G SYGIG (38) IIYPGSSDTRYSPSFQG (41) VYSFGGRHKALFDY (eleven) + GC5B480 GC5H269 D55S, W33A SYAIG (39) IIYPGSSDTRYSPSFQG (41) VYSFGGRHKALFDY (ID + GC5B481 GC5H268 D55K,W33Y SYYIG (36) IIYPGKSDTRYSPSFQG (7) VYSFGGRHKALFDY (eleven) ++ GC5B482 GC5H267 D55K, W33V SYVIG (37) IIYPGKSDTRYSPSFQG (7) VYSFGGRHKALFDY (eleven) ++ GC5B483 GC5H266 D55K, W33F SYFIG (3) IIYPGKSDTRYSPSFQG (7) VYSFGGRHKALFDY (eleven) ++ GC5B484 GC5H265 D55K, W33G SYGIG (38) IIYPGKSDTRYSPSFQG (7) VYSFGGRHKALFDY (eleven) + GC5B485 GC5H264 D55K, W33A SYAIG (39) IIYPGKSDTRYSPSFQG (7) VYSFGGRHKALFDY (eleven) + GC5B486 GC5H263 D55E, W33Y SYYIG (36) IIYPGESDTRYSPSFQG (42) VYSFGGRHKALFDY (eleven) ++ GC5B487 GC5H262 D55E, W33V SYVIG (37) IIYPGESDTRYSPSFQG (42) VYSFGGRHKALFDY (eleven) + GC5B488 GC5H261 D55E, W33F SYFIG (3) IIYPGESDTRYSPSFQG (42) VYSFGGRHKALFDY (ID ++ GC5B489 GC5H260 D55E, W33G SYGIG (38) IIYPGESDTRYSPSFQG (42) VYSFGGRHKALFDY (eleven) - GC5B490 GC5H259 D55E, W33A SYAIG (39) IIYPGESDTRYSPSFQG (42) VYSFGGRHKALFDY (eleven) + GC5B491 GC5H258 D55Y, W33Y SYYIG (36) IIYPGYSDTRYSPSFQG (43) VYSFGGRHKALFDY (eleven) ++ GC5B492 GC5H257 D55Y,W33V SYVIG (37) IIYPGYSDTRYSPSFQG (43) VYSFGGRHKALFDY (ID ++

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GC5B493 GC5H256 D55Y, W33F SYFIG (3) IIYPGYSDTRYSPSFQG (43) VYSFGGRHKALFDY (eleven) ++ GC5B494 GC5H255 D55Y, W33G SYGIG (38) IIYPGYSDTRYSPSFQG (43) VYSFGGRHKALFDY (eleven) + GC5B495 GC5H254 D55Y, W33A SYAIG (39) IIYPGYSDTRYSPSFQG (43) VYSFGGRHKALFDY (eleven) + GC5B496 GC5H253 W33Y SYYIG (36) IIYPGDSDTRYSPSFQG (29) VYSFGGRHKALFDY (eleven) ++ GC5B497 GC5H252 W33V SYVIG (37) IIYPGDSDTRYSPSFQG (29) VYSFGGRHKALFDY (eleven) ++ GC5B498 GC5H251 W33F SYFIG (3) IIYPGDSDTRYSPSFQG (29) VYSFGGRHKALFDY (ID ++ GC5B499 GC5H250 W33G SYGIG (38) IIYPGDSDTRYSPSFQG (29) VYSFGGRHKALFDY (eleven) - GC5B500 GC5H249 W33A SYAIG (39) IIYPGDSDTRYSPSFQG (29) VYSFGGRHKALFDY (eleven) - GC5B501 GC5H248 D55A SYWIG (27) IIYPGASDTRYSPSFQG (40) VYSFGGRHKALFDY (eleven) ++ GC5B502 GC5H247 D55S SYWIG (27) IIYPGSSDTRYSPSFQG (41) VYSFGGRHKALFDY (eleven) ++ GC5B503 GC5H246 D55K SYWIG (27) IIYPGKSDTRYSPSFQG (7) VYSFGGRHKALFDY (eleven) ++ GC5B504 GC5H245 D55E SYWIG (27) IIYPGESDTRYSPSFQG (42) VYSFGGRHKALFDY (eleven) ++ GC5B505 GC5H244 D55Y SYWIG (27) IIYPGYSDTRYSPSFQG (43) VYSFGGRHKALFDY (eleven) ++

Table 17. CDR sequences and binding data of the GC5B285 PTM library. The mutation site is underlined. Binding was classified as CIF > 10,000=++; CIF > 1,000=+; CIF < 1000 (SEQ ID NO for each described sequence is given in parentheses)

ID GPRC5D ID heavy chains Mutation He CDR1 He CDR2 He CDR3 Human GPRC5D (FACS) GC5B463 GC5H234 D62A, W101Y NYWMS (2) GISYSGGSKYYAASVKG (44) AAYDFGRRAVRLDY (48) ++ GC5B432 GC5H228 D62S, W101V NYWMS (2) GISYSGGSKYYASSVKG (6) AAVDFGRRAVRLDY (49) ++ GC5B465 GC5H227 D62S, W101F NYWMS (2) GISYSGGSKYYASSVKG (6) AAFDFGRRAVRLDY (97) ++ GC5B433 GC5H223 D62K, NYWMS GISYSGGSKYYAKSVKG AAVDFGRRAVRLDY ++

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W101V (2) (45) (49) GC5B434 GC5H222 D62K, W101F NYWMS (2) GISYSGGSKYYAKSVKG (45) AAFDFGRRAVRLDY (97) ++ GC5B435 GC5H219 D62E, W101Y NYWMS (2) GISYSGGSKYYAESVKG (46) AAYDFGRRAVRLDY (48) + GC5B436 GC5H217 D62E, W101F NYWMS (2) GISYSGGSKYYAESVKG (46) AAFDFGRRAVRLDY (97) + GC5B461 GC5H216 D62E, W101G NYWMS (2) GISYSGGSKYYAESVKG (46) AAGDFGRRAVRLDY (50) - GC5B462 GC5H215 D62E, W101A NYWMS (2) GISYSGGSKYYAESVKG (46) AAADFGRRAVRLDY (51) ++ GC5B437 GC5H214 D62Y, W101Y NYWMS (2) GISYSGGSKYYAYSVKG (47) AAYDFGRRAVRLDY (48) + GC5B438 GC5H213 D62Y, W101V NYWMS (2) GISYSGGSKYYAYSVKG (47) AAVDFGRRAVRLDY (49) + GC5B439 GC5H212 D62Y, W101F NYWMS (2) GISYSGGSKYYAYSVKG (47) AAFDFGRRAVRLDY (97) + GC5B440 GC5H211 D62Y, W101G NYWMS (2) GISYSGGSKYYAYSVKG (47) AAGDFGRRAVRLDY (50) - GC5B441 GC5H210 D62Y, W101A NYWMS (2) GISYSGGSKYYAYSVKG (47) AAADFGRRAVRLDY (51) + GC5B442 GC5H209 W101Y NYWMS (2) GISYSGGSKYYADSVKG (28) AAYDFGRRAVRLDY (48) + GC5B443 GC5H208 W101V NYWMS (2) GISYSGGSKYYADSVKG (28) AAVDFGRRAVRLDY (49) + GC5B444 GC5H207 W101F NYWMS (2) GISYSGGSKYYADSVKG (28) AAFDFGRRAVRLDY (97) + GC5B464 GC5H206 W101G NYWMS (2) GISYSGGSKYYADSVKG (28) AAGDFGRRAVRLDY (50) + GC5B445 GC5H205 W101A NYWMS (2) GISYSGGSKYYADSVKG (28) AAADFGRRAVRLDY (51) + GC5B446 GC5H204 D62A NYWMS (2) GISYSGGSKYYAASVKG (44) AAWDFGRRAVRLDY (thirty) + GC5B447 GC5H203 D62S NYWMS (2) GISYSGGSKYYASSVKG (6) AAWDFGRRAVRLDY (thirty) + GC5B448 GC5H202 D62K NYWMS (2) GISYSGGSKYYAKSVKG (45) AAWDFGRRAVRLDY (thirty) + GC5B449 GC5H201 D62E NYWMS (2) GISYSGGSKYYAESVKG (46) AAWDFGRRAVRLDY (thirty) + GC5B450 GC5H200 D62Y NYWMS (2) GISYSGGSKYYAYSVKG (47) AAWDFGRRAVRLDY (thirty) +

Based on these binding data, selected mAbs were generated as bispecific antibodies to GPRC5DxCD3 and assessed for T cell-mediated cytotoxicity of H929 cells (Table 18).

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Table 18. Functional activity of selected GPRC5D PTM variants for GCDB164 and GCDB285

ID of parent mAb to GPRC5D Squirrel ID AA GPRC5D ID GPRC5DxCD3 H929 EC ₅₀ (nM) Selected as leaders GC5B285 GC5B432 GCDB50 1.3 GC5B433 GCDB51 0.81 GC5B434 GCDB52 2.81 GC5B465 GCDB53 0.24 XX GC5B463 GCDB54 3, 09 GC5B164 GC5B471 GCDB57 3, 87 GC5B476 GCDB58 1.24 GC5B478 GCDB59 1.54 GC5B481 GCDB60 2.94 GC5B483 GCDB61 1.51 XX GC5B493 GCDB62 3, 1

In the T cell-mediated cytotoxicity assay, a range of activity was observed that was not necessarily predicted by the observed binding affinity. For example, GC5B465 and GC5B463, which bound with similar affinity to human GPRC5D, differ in sequence by only 2 amino acids (Table 17) and were observed to have a 12.5-fold difference in activity compared to bispecific antibodies to GPRC5DxCD3 (Table 17). 18). Based on the functional data, GC5B465 and GC5B483 were selected as optimized sequences for GC5B285 (GCDB43 as a bispecific anti-CD3 antibody) and GC5B164 (GCDB35 as a bispecific anti-CD3 antibody).

Human backbone adaptations were also performed for anti-GPCR5D mAbs derived from a mouse hybridoma (GC5B390 and GC5B396 or GCDB46 and 47 as CD3 bispecific antibodies, respectively). Binding studies identified a number of scaffolds for GC5B396 and one scaffold for GC5B390 that retained binding to human GPRC5D (Table 19).

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Table 19. Binding data and functional data for human scaffold adaptation of hybridoma-derived GPCR5D mAb libraries. The binding was classified as SIF >

000=+++; CIF > 5,000=++; CIF > 1,000=+; CIF < 1000. ________

ID parental mAb to GPRC5D ID AA mAb GPRC5D Heavy chain ID Light chain ID Human GPRC5D (FACS) ID GPRC5D xCD3 H929 EC50 (nM) GPRC5D B396 GC5B541 GC5H241 GC5L58 + GC5B540 GC5H240 GC5L58 + + + GCDB69 0.58 GC5B539 GC5H242 GC5L58 + + GC5B538 GC5H243 GC5L58 + + GC5B537 GC5H241 GC5L57 + GC5B536 GC5H240 GC5L57 + + + GCDB68 2.51 GC5B535 GC5H242 GC5L57 + GC5B534 GC5H243 GC5L57 + + GC5B533 GC5H241 GC5L56 + GC5B532 GC5H240 GC5L56 + + + GCDB67 0.61 GC5B531 GC5H242 GC5L56 + + GC5B530 GC5H243 GC5L56 + + + GCDB66 1.41 GC5B529 GC5H241 GC5L55 + + GC5B528 GC5H240 GC5L55 + + + GCDB65 1, 01 GC5B527 GC5H242 GC5L55 - GC5B526 GC5H243 GC5L55 + + GCDB64 1.5 GPRC5D B390 GC5B525 GC5H279 GC5L53 - GC5B524 GC5H237 GC5L53 - GC5B523 GC5H238 GC5L53 - GC5B522 GC5H236 GC5L53 - GC5B521 GC5H279 GC5L52 - GC5B520 GC5H237 GC5L52 - GC5B519 GC5H238 GC5L52 - GC5B518 GC5H236 GC5L52 - GC5B517 GC5H279 GC5L51 + GC5B516 GC5H237 GC5L51 + GC5B515 GC5H238 GC5L51 + + GCDB63 0.52 GC5B514 GC5H236 GC5L51 + GC5B513 GC5H279 GC5L50 - GC5B512 GC5H237 GC5L50 + GC5B511 GC5H238 GC5L50 + GC5B510 GC5H236 GC5L50 + GC5B509 GC5H279 GC5L49 - GC5B508 GC5H237 GC5L49 + GC5B507 GC5H238 GC5L49 + GC5B506 GC5H236 GC5L49 +

Based on binding data, several monoclonal antibodies to GPCR5D were generated as bispecific antibodies to CD3 and evaluated for T cell-mediated cytotoxicity of H929 cells (Table 18). Functional analysis identified GCDB63, GCDB67, and GCDB69 as active fully humanized bispecific antibodies to GPRC5Dx POPs. Based on these data, the corresponding mAbs to GPRC5D, GC5B515, GC5B532, and GC5B540, were selected as fully humanized sequences for GC5B390 and GC5B391.

Additional lead optimization of fully humanized sequences was then performed to address potential risks associated with post-translational sequence modification for GC5B515, GC5B532 and GCDB540. The G56S mutation was generated in the heavy chain sequence to eliminate the potential risk of deamidation for GC5B515 (Table 20).

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Table 20. Binding and functional activity of selected GPRC5D PTM variants for _____________GC5B532, GC5B540 and GCDB515______________

ID parental mAb to GPRC5D GC5B540 ID AA mAb GPRC5D GC5B590 GC5B592 GC5B594 Mutation M64K G99A M64K, G99A Human GPRC5D (FACS) + + ID H929 GPRC5Dx EC50 CD3 (nM) GC5 B532 GC5B591 M64K + + + GC5B593 G99A + GC5B595 M64K, G99A + GC5 B515 GC5B596 G56S + + GCDB72 0.15

The heavy chains for GC5B532 and GC5B540 are subject to both a potential risk of isomerization and a potential risk of oxidation. The M64K and G99A mutations were obtained to reduce this risk (Table 20). All variants tested with the G99A mutation had a significant reduction in binding affinity, whereas the M64K and G56A variants were not affected. Based on binding data alone, GC5B596 was functionally evaluated and demonstrated activity as an anti-CD3 bispecific antibody (GCDB72) in a T cell-mediated cytotoxicity assay.

Thus, four bispecific mAbs to GPRC5D were selected for further characterization: GCDB32, GCDB53, GCDB61, and GCDB72. Below in the table. 21 and 22 show the CDR sequences and the heavy and light chain sequences of the anti-GPRC5D mAbs used to prepare the bispecific molecules.

Table 21. CDR sequences for 4 anti-GPRC5D mAb candidates that demonstrated binding to human and cynomolgus GPRC5D and that were functional when produced as anti-CD3 antibodies.

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 GC5B81 SYAIS (SEQ ID NO 1) GIIPIFGTANYAQKFQ G (SEQ ID NO 5) ESRWRGYKLD (SEQ ID NO 9) RASQSISSYLN (SEQ ID NO 13) AASSLQS (SEQ ID NO 16) QQSYSTPLT (SEQ ID NO 19) GC5B465 NYWMS (SEQ ID NO 2) GISYSGGSKYYASSVK G (SEQ ID NO 6) AAFDFGRRAVRLD (SEQ ID NO 10) RASQSISSYLN (SEQ ID NO 13) AASSLQS (SEQ ID NO 16) QQSYSTPLT (SEQ ID NO 19) GC5B48 3 SYFIG (SEQ ID NO 3) IIYPGKSDTRYSPSFQ G (SEQ ID NO 7) VYSFGGRHKALFDY (SEQ ID NO 11) RASQSVSSYLA (SEQ ID NO 14) DAS N RAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B59 6 GYTMN (SEQ ID NO 4) LINPYNSDTNYAQKLQ G (SEQ ID NO 8) VALRVALDY (SEQ ID NO 12) KASQNVATHVG (SEQ ID NO 15) SASYRYS (SEQ ID NO 18) QQYNRYPYT (SEQ ID NO 21)

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Table 22. Heavy and light chain variable region sequences of 4 mAb candidates

GPRC5D, which demonstrated binding to human and cynomolgus GPRC5D and which were functional when produced as bispecific antibodies to CD3.

mAb ID AA Amino acid subsequence VH SEQ ID NO: Amino acid VL sequence SEQ ID NO GC5B81 QVQLVQSGAEVKKPGSSVKV SCKASGGTFSSYAISWVRQA PGQGLEWMGGIIPIFGTANY AQKFQGRVTITADE STS TAY MELSSLRSEDTAVYYCARES RWRGYKLDYWGQGTLVTVSS 52 DIQMTQSPSSLSASVGDRVTIT CRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYC QQSYSTPLTFGQGTKVEIK 56 GC5B465 EVQLLESGGGLVQPGGSLRL SCAASGFTFSNYWMSWVRQA PGKGLEWVSGISYSGGSKYY AS SVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCAKAA FDFGRRAVRLDYWGQGTLVT VSS 53 DIQMTQSPSSLSASVGDRVTIT CRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYC QQSYSTPLTFGQGTKVEIK 56 GC5B483 EVQLVQSGAEVKKPGESLKI SCKGSGYSFTSYFIGWVRQM PGKGLEWMGIIYPGKSDTRY S PS FQGQVTISADKSIS TAY LQWSSLKASDTAMYYCARVY SFGGRHKALFDYWGQGTLVT VSS 54 EIVLTQSPATLSLSPGERATLS CRASQSVSSYLAWYQQKPGQAP RLLIYDASNRATGIPARFSGSG SGTDFTLTISSLEPEDFAVYYC QQRSNWPLTFGQGTKVEIK 57 GC5B596 QVQLVQSGAEVKKPGASVKV SCKASGYSFTGYTMNWVRQA PGQGLEWMGLINPYNSDTNY AQKLQGRVTMTTDTSTSTAY ME LRS LRS DDTAVYYCARVA LRVALDYWGQGTLVTVSS 55 DIQMTQSPSSLSSASVGDRVTIT CKASQNVATHVGWYQQKPGKAP KRLIYSASYRYSGVPSRFSGSG SGTEFTLTISNLQPEDFATYYC QQYNRYPYTFGQGTKLEIK 58

Example 7. Preparation of antibodies to GPRC5D and CD3 in bispecific format in IgG4 S228P, L234A, L235A

Four monospecific antibodies to GPRC5D (see Table 21) were expressed as IgG4, having substitutions S228P, L234A in the Fc regions, as well as L235A or S228P, L234A, L235A, F405L and R409K (CD3 arm) (numbering according to the EU index) . A monospecific anti-CD3 antibody CD3B219 was also generated containing the VH and VL regions, having a heavy chain of SEQ ID NO: 25 and a light chain of SEQ ID NO: 26 and an IgG4 constant region with substitutions S228P, L234A, L235A, F405L and R409K.

Monospecific antibodies were purified using standard methods using a protein A column (HiTrap MabSelect SuRe column). After elution, the pools were dialyzed into D-PBS, pH 7.2.

Bispecific antibodies to GPRC5DxCD3 were generated by combining a monospecific mAb to CD3 and a monospecific mAb to GPRC5D during in-vitro Fab arm exchange (as described in WO 2011/131746). Briefly, about 1-20 mg/ml anti-GPRC5D/CD3 antibody at a molar ratio of 1.08:1 in PBS, pH 7-7.4, and 75 mM 2-mercaptoethanolamine (2-MEA) were mixed together and incubated at 25 37°C for 2-6 hours, after which 2-MEA was removed by dialysis, diafiltration, tangential flow filtration and/or vortex cell filtration using standard methods.

The heavy and light chains of bispecific antibodies to GPRC5DxCD3 are presented below in table. 23.

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Table 23. Sequences of heavy and light chains of IgG4-PAA bispecific antibodies

Antibody Amino acid sequence GCDB32 Heavy chain 1 CD3B219 (SEQ ID NO:2 5) EVQLVE S GGGLVQPGGS LRLS CAAS GET ENTYAMNWVRQ APGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSK NSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQ GTLVTVSS Light chain 1 CD3B219 (SEQ ID NO:2 6) Q T WT QE P S L T VS P GG T VT L T CRS S T GAVT T S N YANWVQ QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS GVQPEDEAEYYCALWYSNLWVFGGGTKLTVL Heavy chain 2 GC5B81 (SEQ ID NO:52) QVQLVQS GAEVKKPGS SVKVS CKAS GGT FS SYAISWVRQ APGQGLEWMGG11РIFGTANYAQKFQGRVTITADE S T S T AYMELSSLRSEDTAVYYCARESRWRGYKLDYWGQGTLVT VSS Light chain 2 GC5B81 (SEQ ID NO:5 6) DIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQK PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQSYSTPLTFGQGTKVEIK GCDB53 Heavy chain 1 CD3B219 (SEQ ID NO:2 5) EVQLVE S GGGLVQPGG S LRL S CAAS G FT FNTYAMNWVRQ APGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSK NSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQ GTLVTVSS Light chain 1 CD3B219 (SEQ ID NO:2 6) Q TWT QE P S L TVS P GG TVT L T CRS S T GAVT T S N YANWVQ QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS GVQPEDEAEYYCALWYSNLWVFGGGTKLTVL Heavy chain 2 GC5B465 (SEQ ID NO:53) EVQLLESGGGLVQPGGSLRLSCAASGTFFSNYWMSWVRQ APGKGLEWVSGISYSGGSKYYASSVKGRFTISRDNSKNT LYLQMNSLRAEDTAVYYCAKAAFDFGRRAVRLDYWGQGT LVTVSS Light chain 2 GC5B465 (SEQ ID NO:5 6) DIQMTQSPSSLSSASVGDRVTITCRASQSISSYLNWYQQK PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQSYSTPLTFGQGTKVEIK GCDB61 Heavy chain 1 CD3B219 (SEQ ID NO:2 5) EVQLVE S GGGLVQPGG S LRL S CAAS G FT FNTYAMNWVRQ APGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSK NSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQ GTLVTVSS Light chain 1 CD3B219 (SEQ ID NO:2 6) Q TWT QE P S L TVS P GG TVT L T CRS S T GAVT T S N YANWVQ QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS GVQPEDEAEYYCALWYSNLWVFGGGTKLTVL Heavy chain 2 GC5B483 (SEQ ID NO:54) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYFIGWVRQ MPGKGLEWMGIIYPGKSDTRYS PS FQGQVTISADKS1ST AYLQWSSLKASDTAMYYCARVYSFGGRHKALFDYWGQGT LVTVSS

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Light chain 2 GC5B483 (SEQ ID NO:57) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQK PGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSL EPEDFAVYYCQQRSNWPLTFGQGTKVEIK GCDB72 Heavy chain 1 CD3B219 (SEQ ID NO:25) EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQ APGKGLEWVARIRSKYNNYATYYAASVKGRFTISRDDSK NSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQ GTLVTVSS Light chain 1 CD3B219 (SEQ ID NO:2 6) QTWTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQ QKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLS GVQPEDEAEYYCALWYSNLWVFGGGTKLTVL Heavy chain 2 GC5B596 (SEQ ID NO:55) QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQ APGQGLEWMGLINPYNSDTNYAQKLQGRVTMTTDTSTSTST AYMELRSLRSDDTAVYYCARVALRVALDYWGQGTLVTVS S Light chain 2 GC5B596 (SEQ ID NO:58) DIQMTQSPSSLSSASVGDRVTITCKASQNVATHVGWYQQK PGKAPKRLIYSASYRYSGVPSRFSGSGSGTEFTLTISNL QPEDFATYYCQQYNRYPYTFGQGTKLEIK

Example 8. Functional characteristics of GCDB32, GCDB53, GCDB61 and GCDB72

GCDB32, GCDB53, GCDB61 and GCDB72 were assessed for binding to mouse GPRC5D (Table 24). All four antibodies bound to mouse GPRC5D with a range of binding affinities observed.

Table 24. Binding of antibodies to GPCR5DxCD3 to mouse GPRC5D

ID GPRC5D><CD3 Binding of FACS to mouse GPRC5D (SIF) GCDB32 1151 GCDB53 2658 GCDB61 3552 GCDB72 481

Cross-reactivity with cynomolgus GPRC5D was also assessed using a T-cell redirection cytotoxicity assay on overexpressed human and cynomolgus GPRC5D cell lines (Table 25). One bispecific antibody to GPRC5DxCD3 was equally active against human and cynomolgus GPRC5D (GCDB32), whereas the other bispecific antibodies tested were less active in inducing cytotoxicity for cynomolgus GPRC5D compared to human GPRC5D.

Table 25. Functional activity of leader antibodies to GPRC5DxCD3 against HEK cells ___________expressing human and cynomolgus GPRC5D___________

ID GPRC5DxCD3 Human HEK GPRC5D cells (EC ₅₀ nM) Cynomolgus HEK GPRC5D cells (EC ₅₀ nM) GCDB32 0.04 0.07 GCDB53 0.08 0.36 GCDB61 0.03 1.22 GCDB72 0.03 3, 41

Additional characterization was aimed at understanding in vitro binding (Fig. 11A-E) and activity (Table 26).

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Table 26. T cell-mediated cytotoxicity of several human GPRC5D-expressing B cell lines to anti-leader antibodies. GPRC5DxCD3

ID GPRC5DxC D3 H929 cells, EC ₅₀ , nM (Rank order) MM1R EU ₅₀ , nM (Rank order) ORM2 EC ₅₀ , nM (Rank order) LP-1 EC ₅₀ , nM (Rank order) GCDB32 0.45 (3) 0.04 (1) 0.98 (2) 1.02 (2) GCDB53 0.69 (4) 0.28 (4) 2.83 (4) 1.58 (3) GCDB61 0.39 (2) 0.06 (3) 1.46 (3) 1.67 (4) GCDB72 0.22 (1) 0.04 (1) 0.77 (1) 0.7 (1)

Although a range of binding affinities was observed, with the strongest binding antibody being GCDB61 and the weakest binding being GCDB72 and GCDB32, the in vitro activities were very similar to the panel of bispecific antibodies. However, based on rank order analysis, GCDB72 was the most active among the various B cell lines analyzed. Ex vivo binding and activity experiments using patient-derived MNCs yielded largely similar results to in vitro assays (Table 27 and Figures 15A and 15B).

Table 27. Anti-GPCR5DxCD3 bispecific antibodies binding, T cell-mediated cytotoxicity, and T cell activation of MNC-derived . from a patient with MM

Patient MMZOZVM ID YES GPRC5DXC D3 GCDB32 GCDB53 GCDB61 EC ₅₀ binding nM 16.1 3.98 5.53 ec ₅₀ cytotoxicity nM 0.36 0.42 0.89 ec ₅₀ cell activation nM 0. 18 0. 17 0.22 GCDB72 27.5 0.30 0, 12 MM305VM GCDB32 70.4 0.26 0.16 GCDB53 10.2 0.26 0.19 GCDB61 6, 93 0.6 0.22 GCDB72 65, 9 0.28 0, 12

GCDB61 had the highest binding affinity to patient MNCs, whereas GCDB72 and GCDB32 were the weakest in binding. Again, although differences in binding affinities were observed, all bispecific antibodies demonstrated subnanomolar potency in T cell redirection cytotoxicity assays. These molecules were virtually indistinguishable based on in vitro and ex vivo activity values, but the in vivo data provided differentiation (Figure 12A-D).

H929 cells were implanted into NSG mice one week after injection of human PBMCs. Treatment with bispecific antibodies began simultaneously with implantation of H929 cells and continued every 2 or 3 days (once every 2 days or once every 3 days) at a dose of 10 μg, 1 mg and 0.1 μg/animal for a total of five treatments. Ten mice were used in each group, and PBS was included as a vehicle control. Treatment was stopped on day 11, and the study was stopped on day 26 (Fig. 12A-D). All GPRC5DxCD3 bispecific molecules tested in this prophylactic model showed 100% inhibition of tumor growth when using a dose of 10 and 1 μg/animal. Differentiation was observed at the lowest dose, 0.1 μg/animal, with GCDB72 demonstrating superiority over other bispecific antibodies tested, with 80% tumor growth inhibition observed.

Example 9. Binding profile of anti-GPRC5D antibody on the GPRC5D+ MM1.R cell line

The binding affinities of anti-GPRC5D antibodies were measured on the human MM GPRC5D+ cell line (MM1.R, purchased from ATCC (American Type Culture Collection)) using FACS. In fig. 13 shows that all leader antibodies bound to MM.1R cells expressing GPRC5D in a dose-dependent manner with EC ₅₀ concentration values ranging from 0.10 nM to 135 nM, with all values except GC5B602 being significantly lower than commercial values. antibody FAB6300 (R&D Systems, catalog number FAB6300A, clone number 571961), which gave an EC value _{of 50,121.7} nM.

GPRC5D+ MM1.R cell lines were stained for 60 min with various concentrations of leader antibodies to measure surface binding profiles (n=3). Phycoerythrin-labeled human IgG4Fc was used as a secondary signal capture antibody (Southern

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Biotech, clone HP6025, catalog no. 9200-09). Binding is expressed as the normalized geometric mean of fluorescence intensity measured by FACS. Graphing and data fitting were performed in GraphPad Prism 6 using nonlinear slope regression (four parameters) and least squares fitting.

In addition, the binding profile of the GPRC5D mAb GC5M481 was assessed in comparison with a commercial antibody using three GPRC5D+ multiple myeloma cell lines (JIM3, OPM-2 and MM.1R; cell lines purchased from ATCC) (Fig. 14A). In addition, mAb to GPRC5D (GC5M481) was profiled for cross-reactivity using Daudi cells expressing cynomolgus GPRC5D, which showed strong binding compared to parental cells (Fig. 14A). Also, five bispecific antibodies to GPRC5DxCD3 (GCDB32, GCDB48, GCDB53, GCDB61 and GCDB72) when assessed for binding capacity using GPRC5D+ cell lines (JIM3, OPM-2 and MM1.R) (Fig. 14B) showed a significant degree of binding, as seen in shift on the histogram (black solid line), compared with the isotype control (dashed line limiting the gray zone).

Example 10 Antitumor efficacy of GCDB72 against subcutaneous xenografts of human multiple myeloma cells MM.1S in PBMC-humanized NSG mice

This in vivo study was conducted to determine the effectiveness of GCDB72 against adapted MM.1S human multiple myeloma (MM) cell xenografts in PBMC humanized NSG mice. Weight- and age-matched female NSG mice were implanted subcutaneously (s.c.) with human MM MM cells. 1S ( ^1x107 cells in 200 μl PBS per mouse) into the right dorsal posterior inguinal region on study day 0. On day 7 after tumor cell implantation, ^1x107 human PBMCs (in 200 μl PBS) were injected intravenously through the lateral tail vein. Treatments began on day 15, when the average tumor volume was approximately 72-78 mm ³ , each mouse was injected intravenously with PBS or DuoBody GCDB72 antibody at a dose of 0.1 μg (0.005 mg/kg), 1 μg (0 .05 mg/kg), 10 mcg (0.5 mg/kg) and 50 mcg (2.5 mg/kg). Each DuoBody null control, CD3 x null and null x GPRC5D, was administered at a dose of 10 μg to each mouse. Treatments were carried out approximately every three days (once every 3 days) - a total of seven doses. High antitumor efficacy was observed for the two highest doses (10 μg and 50 μg) of GCDB72, with subcutaneous MM.1S tumors completely regressing in 100% (10 of 10 per group) of animals by the end of the study (Figure 16). Moreover, a dose of 1 μg in each mouse significantly inhibited tumor growth by 65% (p < 0.0001) compared to tumors treated with PBS, while a dose of 0.1 μg had a negligible effect (TGI = 19.3%, p =0.0023). The effect of CD3 x null antibody was not considered effective (TGI = 28%, p < 0.0001), and null x GPRC5D had a negligible effect of 3.1% TGI, p = 0.9971. TGI was determined on day 36 when the group was at least 80% viable. Significant weight loss and/or mortality began to appear after day 36 due to GVHD (Fig. 17). The study was stopped on day 43 when 60% of the animals or less remained in the groups.

Example 11 Antibody-dependent cell-mediated cytotoxicity (ADCC) of anti-GPRC5D antibodies

A panel of monoclonal antibodies to human GPRC5D was generated as IgG1 mAb. In addition, a new panel of mAbs to human GPRC5D was obtained as described in example 2. Below in table. 28 and 29 show the heavy and light chain variable region sequences of the new mAbs to GPRC5D. These new antibodies were used to generate CD3 bispecific molecules as described in Example 7, and they were also included as an IgG1 mAb to evaluate ADCC activity.

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Table 28. CDR sequences of the new panel of antibodies to GPRC5D

ID HC-CDR1 HC-CDR2 HC-CDR3 LC-CDR1 LC-CDR2 LC-CDR3 GC5B382 DYGMH (SEQ ID NO 61) AIKYSGGSTYYADS VKG (SEQ ID NO 67) RAESGPLDY (SEQ ID NO 72) KSSQSVLYSSNN KNYLA (SEQ ID NO 98) WASTRES (SEQ ID NO 78) QQYYSTPLT (SEQ ID NO 80) GC5B379 NYWMS (SEQ ID NO 2) GISYSGGSKYYADS VKG (SEQ ID NO 28) AAWDFGRRAVRLD Y (SEQ ID NO thirty) RASQSISSYLN (SEQ ID NO 13) AASSLQS (SEQ ID NO 16) QQSYSTPLT (SEQ ID NO 19) GC5B373 SYWIG (SEQ ID NO 27) IIYPGDSDTRYSPS FQG (SEQ ID NO 29) IGFYGRSFRIFDY (SEQ ID NO 73) RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B376 SYWIG (SEQ ID NO 27) IIYPGDSDTRYSPS FQG (SEQ ID NO 29) VYSFGGRHKALFD Y (SEQ ID NO eleven) RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B385 GYANS (SEQ ID NO 62) AISGSGGSTYYADS VKG (SEQ ID NO 68) VDRSFGRSRYTLD Y (SEQ ID NO 74) RASQSVSSYLA (SEQ ID NO 14) DASNRAT (SEQ ID NO 17) QQRSNWPLT (SEQ ID NO 20) GC5B370 GC5B597 SYGIS (SEQ ID NO 63) GIIPIFGNINYAQK FQG (SEQ ID NO 69) VSRRFKRFAYYFD Y (SEQ ID NO 75) KSSQSVLYSSNN KNYLA (SEQ ID NO 98) WASTRES (SEQ ID NO 78) QQYYSTPLT (SEQ ID NO 80) GC5B602 GYSFTGYTMN (SEQ ID NO 64) LINPYNGDTN (SEQ ID NO 70) VALRVALDY (SEQ ID NO 12) KASQNVATHVG (SEQ ID NO 15) SASYRYS (SEQ ID NO 18) QQYNRYPYT (SEQ ID NO 21) GC5B603 SYAMS (SEQ ID NO 65) AISGSGGSTYYADS VKG (SEQ ID NO 68) SNFLPWFDY (SEQ ID NO 76) RASQSVRKSLA (SEQ ID NO 95) TASNRAT (SEQ ID NO 79) QQYFRAPIT (SEQ ID NO 81) GC5B601 GFSLTSYNVH (SEQ ID NO 66) VIWAGGSTNYNSA LMS (SEQ ID NO 71) DGIRLRFAY (SEQ ID NO 77) KASQNVATHVG (SEQ ID NO 15) SASYRYS (SEQ ID NO 18) QQYNRYPYT (SEQ ID NO 21)

Table 29. Sequences of the variable region of the heavy and light chain of the new panel of antibodies to GPRC5D

mAb ID AA Amino acid sequence of VH SEQ ID NO: Amino acid sequence VL SEQ ID NO GC5B382 EVQLLESGGGLVQPGGSLRLSC AAS G FT FS DYGMHWVRQAPGKG LEWVSAIKYSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKRAESGPGLDYWG QGTLVTVSS 82 DIVMTQSPDSLAVSLGERA TINCKS S QSVLYS SNNKNY LAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTD FTLTISSLQAEDVAVYYCQ QYYSTPLTFGQGTKVEIK 92 GC5B379 EVQLLESGGGLVQPGGSLRLSC AASGTFFSNYWMSWVRQAPGKG LEWVSGISYSGGSKYYADSVKG RFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKAAWDFGRRAVRL DYWGQGTLVTVSS 83 DIQMTQSPSSLSASVGDRV TITCRASQSISSYLNWYQQ KPGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQSYSTP LTFGQGTKVEIK 56 GC5B373 EVQLVQSGAEVKKPGESLKISC KGSGYSFTSYWIGWVRQMPGKG 84 EIVLTQSPATLSLSPGERA TLSCRASQSVSSYLAWYQQ 57

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LEWMGIIYPGDSDTRYSPSFQG QVTISADKSIS TAYLQWS S LKA SDTAMYYCARIGFYGRSFRIED YWGQGTLVTVSS KPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQRSNWP LTFGQGTKVEIK GC5B376 EVQLVQSGAEVKKPGESLKISC KGSGYSFTSYWIGWVRQMPGKG LEWMGIIYPGDSDTRYSPSFQG QVTISADKSIS TAYLQWS S LKA S DTAMYYCARVYSFGGRHKAL F DYWGQGTLVTVSS 85 EIVLTQSPATLSLSPGERA TLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQRSNWP LTFGQGTKVEIK 57 GC5B385 EVQLLESGGGLVQPGGSLRLSC AASGFTFSGYAMSWVRQAPGKG LEWVSAISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKVDRSFGRSRYTL DYWGQGTLVTVSS 86 EIVLTQSPATLSLSPGERA TLSCRASQSVSSYLAWYQQ KPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQRSNWP LTFGQGTKVEIK 57 GC5B370 GC5B597 QVQLVQSGAEVKKPGSSVKVSC KASGGTFSSYGISWVRQAPGQG LEWMGGIIPIFGNINYAQKFQG RVTITADESTSTAYMELSSLRS EDTAVYYCARVSRRFKRFAYYF DYWGQGTLVTVSS 87 DIVMTQSPDSLAVSLGERA TINCKS S QSVLYS SNNKNY LAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTD FTLTISSLQAEDVAVYYCQ QYYSTPLTFGQGTKVEIK 92 GC5B602 QVQLVQSGAEVKKPGASVKVSC KASGYSFTGYTMNWVRQAPGQG LEWMGLINPYNGDTNYAQKLQG RVTMTTDTSTSTAYMELRSLRS DDTAVYYCARVALRVALDYWGQ GTLVTVSS 88 DIQMTQSPSSSLSASVGDRV TITCKASQNVATHVGWYQQ KPGKAPKRLIYSASYRYSG VPSRFSGSGSGTEFTLTIS NLQPEDFATYYCQQYNRYP YTFGQGTKLEIK 58 GC5B603 EVQLLESGGGLVQPGGSLRLSC AASGFFSSYAMSWVRQAPGKG LEWVSAISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRA EDTAVYYCAKSNFLPWFDYWG QGTLVTVSS 89 EIVLTQSPATLSLSPGERA TLSCRASQSVRKSLAWYQQ KPGQAPRLLIYTASNRATG IPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQQYFRAP ITFGQGTKVEIKK 93 GC5B601 QVTLKESGPVLVKPTETLTLTC TVSGFSLTSYNVHWIRQPPGKA LEWLAVIWAGGSTNYNSALMSR LTISKDTSKSQWLTMTNMRAE DTATYYCARDGIRLRFAYWGQG TLVTVSS 91 EIVMTQSPATLSVSPGERA TLSCKASQNVATHVGWYQQ KPGQAPRLLIYSASYRYSG IPARFSGSGSGTEFTLTIS SLQSEDFAVYYCQQYNRYP YTFGQGTKLEIK 94

ADCC activity against H929 cells (Tables 30 and 31). Briefly, multiple myeloma cells were labeled with calcein-AM for 30 min at room temperature and resuspended at a concentration of 0.2 x 10 ⁶ /ml in RPMI + 10% HI FBS after two washes in PBS. PBMCs were thawed and resuspended after washing in PBS at a concentration of 3x10 ⁶ cells per ml in RPMI nutrient 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 for 3 hours in a CO ₂ incubator at 37°C. After the incubation plate was centrifuged at 200 g for 4 min, 100 μl of the supernatant was transferred to a new 96-well plate and fluorescence intensity was measured at 485/535 nm. Values in relative fluorescence units (RFU) were plotted to calculate the percentage of lysis.

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Table 30. Antibody-dependent cytotoxicity of H929 cells for monoclonal antibodies to

GPRC5D with IgG1 Fc

Squirrel ID IgG4 GPRC5D Squirrel ID IgGl GPRC5D GC5B243 GC5B382 GC5B285 GC5B379 GC5B332 GC5B373 GC5B164 GC5B376 GC5B320 GC5B385 GC5B251 GC5B370 GC5B515 GC5B602 GC5B420 GC5B603 GC5B483 GC5B599 GC5B540 GC5B601 GC5B465 GC5B598 GC5B251 GC5B597

EC ₅₀ cytotoxicity H929 (pM) % lysis 1346.5 29, 14 87.4 17.3 244.2 32.94 22.0 32.21 944.0 29.73 24.2 8 27,721.7 20 3.4 16 7.4 14 169, 6 14 2.0 12 705, 1 10

Table 31. Comparison of antibody-dependent cytotoxicity and T cell-mediated cytotoxicity of H929 cells

ADCC CD3 redirection Squirrel ID IgGl GPRC5D ID DuoBody to GPRC5D><CD3 EC50 cytotoxicity H929 (pM) EC50 T cell-mediated cytotoxicity H929 (nM) GC5B382 GCDB40 1346.5 1.2 ± 0.83 GC5B379 GCDB43 87.4 0.39 + 0.07 GC5B373 GCDB45 244.2 2.36±0.84 GC5B376 GCDB35 22.0 1.39 ± 0.75 GC5B385 GCDB44 944.0 > 10 GC5B370 GCDB41 24.2 > 10 GC5B602 GCDB72 27,721.7 0.15 GC5B603 GCDB48 3.4 0.17 GC5B599 GCDB61 7.4 1.51 GC5B601 GCDB69 169, 6 0.58 GC5B598 GCDB53 2.0 0.24

A range of activity values from 2 pM to 27.7 nM was observed. Binding affinity did not necessarily predict efficacy in the ADCC assay. For example, GC5B382 and GC5B379 had similar binding affinities to human GPRC5D cells, but they had a 15-fold difference in cytotoxicity to H929 cells in the ADCC assay. Likewise, cytotoxic induction as a bispecific antibody to GPRC5DxCD3 did not predict activity in the ADCC assay, as demonstrated by GC5B370 and GC5B602. In the CD3 bispecific antibody format, GC5B602 (GCDB63) had subnanomolar activity against H929 cells, whereas GC5B370 in the CD3 bispecific antibody format (GCDB41) was essentially inactive. The same v-regions in IgG1 mAb format produced the opposite result in the ADCC assay, with GC5B370 observed to be more active (~1100x) compared to GC5B602.

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Brief description of the sequence list

SEQ ID NO: Type View Description Subsequence GC5B81, GC5B427, GC5B428, GC5B430, 1 PRT Human SYAIS GC5B431 AND GC5B429- HCDR1 GC5B379, GC5B598, GC5B465, GC5B285, GC5B463, GC5B432, GC5B433, GC5B434, GC5B435, GC5B436, GC5B461, GC5B462, GC5B437, GC5B438, 2 PRT Human NYWMS GC5B439, GC5B440, GC5B441, GC5B442, GC5B443, GC5B444, GC5B464, GC5B445, GC5B446, GC5B447, GC5B448, GC5B449 and GC5B450-HCDR1 GC5B483, GC5B473, GC5B478, GC5B488, 3 PRT Human SYFIG GC5B493 and GC5B498 -HCDR1 4 PRT Human GC5B596-HCDR1 GYTMN GC5B81, GC5B427, GC5B428, GC5B430, 5 PRT Human GIIPIFGTANYAQKFQG GC5B431 and GC5B429- HCDR2 GC5B598, GC5B465, 6 PRT Human GC5B432 AND GC5B447- GISYSGGSKYYASSVKG HCDR2 7 PRT Human GC5B599, GC5B483, IIYPGKSDTRYSPSFQG

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GC5B481, GC5B482, GC5B484, GC5B485 AND GC5B503-HCDR2 8 PRT Human GC5B596-HCDR2 LINPYNSDTNYAQKLQG 9 PRT Human GCB581-HCDR3 ESRWRGYKLD 10 PRT Human GC5B598, GCB5465- HCDR3 AAFDFGRRAVRLD eleven PRT Human GC5B376,GC5B599, GC5B483, GC5B164, GC5B471, GC5B472, GC5B473, GC5B474, GC5B475, GC5B476, GC5B477, GC5B478, GC5B479, GC5B480, GC5B481, GC5B482, GC5B484, GC5B485, GC5B486, GC5B487, GC5B488, GC5B489, GC5B490, GC5B491, GC5B492, GC5B493, GC5B494, GC5B495, GC5B496, GC5B497, GC5B498, GC5B499, GC5B500, GC5B501, GC5B502, GC5B503, GC5B504 AND GC5B505 -HCDR3 VYSFGGRHKALFDY 12 PRT Human GC5B602, GCB596- HCDR3 VALRVALDY 13 PRT Human GC5B382, GC5B379, GC5B370, GC5B598, GC5B597, GC5B81, GC5B465-LCDR1 RASQSISSYLN 14 PRT Human GC5B373, GC5B376, GC5B385, GC5B599, RASQSVSSYLA

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GC5B483-LCDR1 15 PRT Human GC5B605, GC5B601, GC5B596-LCDR1 KASQNVATHVG 16 PRT Human GC5B81, GC5B465, GC5B379, GC5B598- LCDR2 AASSLQS 17 PRT Human GC5B373, GC5B376, GC5B385, GC5B599, GC5B483-LCDR2 DASNRAT 18 PRT Human GC5B602, GC5B601, GC5B596-LCDR2 SASYRYS 19 PRT Human GC5B379, GC5B598, GC5B81, GC5B465- LCDR3 QQSYSTPLT 20 PRT Human GC5B373, GC5B376, GC5B385, GC5B599, GC5B483-LCDR3 QQRSNWPLT 21 PRT Human GC5B602, GC5B601, GC5B596-LCDR3 QQYNRYPYT 22 PRT Human GPRC5D MYKDCIESTGDYFLLCDAEGP WGIILESLAILGIVVTILLLL AFLFLMRKIQDCSQWNVLPTQ LLFLLSVLGLFGLAFAFIIEL NQQTAPVRYFLFGVLFALCES CLLAHASNLVKLVRGCVS FSW TTILCIAIGCSLLQIIIATEY VTLIMTRGMMFVNMTPCQLNV DFWLLVYVLFLMALTFFVSK AT FCGPC ENWKQHGRLIFITV LFSIIIWWWISMLLRGNPQF QRQPQWDDPWCIALVTNAWV FLLLYIVPELCILYRSCRQEC PLQGNACPVTAYQHSFQVENQ ELSRARDSDGAEEDVALTSYG TPIQPQTVDPTQECFIPQAKL SPQQDAGGV 23 PRT Cynomolgus macaque GPRC5D MYKDCIESTGDYFLPCDSEGP WGIVLESLAILGIWTILLLL AFLFLMRKIQDCSQWNVLPTQ LLFLLSVLGLFGLAFAFIIQL NQQTAPVRYFLFGVLFALCFS CLLAHASNLVKLVRGRVS FSW TTILCIAIGCSLLQVIΙΑΙΕΥ

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VTLIMTRGMMFVHMTPYQLNV DFWLLVYVLFLMALTFFVSK AT FCGPCENWKQHGRLIFITV LFSIIIWWWISMLLRGNPQF QRQPQWDDPWCIALVTNAWV FLLLYIVPELCILYRSCRQEC PSQGHACPVTAYQRSFQVENQ ELSRARDSDGAEEDVALTSFG TPIQPQTVDPTQECFIPRAKL SPQQ DAGV 24 PRT Mouse GPRC5D MYEDCVKSTEDYYLFCDNEGP WAIVLESLAVIGIWTILLLL AFL FLMRKVQDC S QWNVL R T Q FLFLLAVLGLFGLTFAFIIQL NHQTAPVRY FL FGVL FAIC FS CLLAHASNLVKLVRGRVS FCW TTILFIAIGVSLLQTIIAIEY VTLIMTRGLMFEHMTPYQLNV DFVCLLLIYVLFLMALTFF VSK AT FCGPCENWKQHGRLIFATV LVS111 WWW ISMLLRGNPQL QRQPHWDDAVIСIGLVTNAWV FLLIYIIPELSILYRSCRQEC PTQGNVCQVPVYQRS FRMDTQ EPTRARDSDGAQEDVALTAYG TPIQLQSADPSREYLIPSATL SPQQDAGL 25 PRT Human HEAVY CHAIN CD3B219 EVQLVESGGGLVQPGGSLRLS CAAS GFT ΕΝT YAMNWVRQAPG KGLEWVARIRSKYNNYATYYA ASVKGRFTISRDDSKNSLYLQ MNSLKTEDTAVYYCARHGNFG NSYVSWFAYWGQGTLVTVSS 26 PRT Human LIGHT CHAIN CD3B219 QTWTQEPSLTVSPGGTVTLT CRSSTGAVTTSNYANWVQQKP GQAPRGLIGGTNKRAPGTPAR FSGSLLGGKAALTLSGVQPED EAEYYCALWYSNLWVFGGGTK LTVL 27 PRT Human GC5B373, GC5B376, GC5B164, GC5B501, GC5B502, GC5B503, GC5B504 AND GC5B505 -HCDR1 SYWIG

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28 PRT Human GC5B379,GC5B285, GC5B442, GC5B443, GC5B444, GC5B464 AND GC5B445-HCDR2 GISYSGGSKYYADSVKG 29 PRT Human GC5B373, GC5B376, GC5B164, GC5B496, GC5B497, GC5B498, GC5B499 AND GC5B500- HCDR2 IIYPGDSDTRYSPSFOG thirty PRT Human GC5B379, GC5B285, GC5B446, GC5B447, GC5B448, GC5B449 AND GC5B450-HCDR3 AAWDFGRRAVRLDY 31 PRT Human GC5B427-HCDR3 ESRYRGYKLDY 32 PRT Human GC5B428-HCDR3 ESVRRGYKLDY 33 PRT Human GC5B430-HCDR3 ESRGRGYKLDY 34 PRT Human GC5B431-HCDR3 ESRARGYKLDY 35 PRT Human GC5B429HCDR3-VH ESRFRGYKLDY 36 PRT Human GC5B471, GC5B476, GC5B486,GC5B481, GC5B491 and GC5B496, -HCDR1 SYYIG 37 PRT Human GC5B497, GC5B472, GC5B477, GC5B482, GC5B487 AND GC5B492- HCDR1 SYVIG 38 PRT Human GC5B474, GC5B479, GC5B484, GC5B489, GC5B494 AND GC5B499- HCDR1 SYGIG 39 PRT Human GC5B475, GC5B480, GC5B485, GC5B490, GC5B495 AND GC5B500- HCDR1 SYAIG 40 PRT Human GC5B471, GC5B472, IIYPGASDTRYSPSFQG

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GC5B473, GC5B474, GC5B475 AND GC5B501- HCDR2 41 PRT Human GC5B476, GC5B477, GC5B478, GC5B479, GC5B480 AND GC5B502 -HCDR2 IIYPGSSDTRYSPSFQG 42 PRT Human GC5B486, GC5B487, GC5B488, GC5B489, GC5B490 AND GC5B504- HCDR2 IIYPGESDTRYSPSFQG 43 PRT Human GC5B491, GC5B492, GC5B493, GC5B494, GC5B495 AND GC5B505- HCDR2 IIYPGYSDTRYSPSFQG 44 PRT Human GC5B463 AND GC5B446- HCDR2 GISYSGGSKYYAASVKG 45 PRT Human GC5B433, GC5B434 AND GC5B448-HCDR2 GISYSGGSKYYAKSVKG 46 PRT Human GC5B435, GC5B436, GC5B461, GC5B462 AND GC5B449-HCDR2 GISYSGGSKYYAESVKG 47 PRT Human GC5B437, GC5B438, GC5B439, GC5B440, GC5B441 AND GC5B450- HCDR2 GISYSGGSKYYAYSVKG 48 PRT Human GC5B463, GC5B435, GC5B437 And GC5B442-HCDR3 AAYDFGRRAVRLDY 49 PRT Human GC5B432, GC5B433, GC5B438 And GC5B443-HCDR3 AAVDFGRRAVRLDY 50 PRT Human GC5B461, GC5B440 And GC5B464-HCDR3 AAGDFGRRAVRLDY 51 PRT Human GC5B462, GC5B441 AAADFGRRAVRLDY

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And GC5B445-HCDR3 52 PRT Human GC5B81-VH QVQLVQSGAEVKKPGSSVKVS CKAS GGT FS S YAISWVRQAPG QGLEWMGGIIPIFGTANYAQK FQGRVTITADESTSTAYMELS S LRS E DTAVYYCARES RWRGY KLDYWGQGTLVTVSS 53 PRT Human GC5B598, GC5B465- VH EVQLLESGGGLVQPGGSLRLS CAASGTFFSNYWMSWVRQAPG KGLEWVSGISYSGGSKYYASS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKAAFDFGR RAVRLDYWGQGTLVTVS S 54 PRT Human GC5B599, GC5B483- VH EVQLVQS GAEVKKP GE S LKIS CKGSGYSFTSYFIGWVRQMPG KGLEWMGIIYPGKSDTRYSPS FQGQVTISADKSIS TAYLQWS SLKASDTAMYYCARVYSFGGR HKAL FDYWGQGT LVTVS S 55 PRT Human GC5B596-VH QVQLVQSGAEVKKPGASVKVS CKASGYSFTGYTMNWVRQAPG QGLEWMGLINPYNSDTNYAQK LQGRVTMTTDTSTSTAYMELR S LRS DDTAVYYCARVALRVAL DYWGQGTLVTVSS 56 PRT Human GC5B379, GC5B598, GC5B81 and GC5465-VL DIQMTQSPSSLSASVGDRVTI TCRASQSISSYLNWYQQKPGK APKLLIYAASSLQSGVPSRFS GSGSGTDFTLTISSLQPEDFA TYYCQQSYSTPLTFGQGTKVE IK 57 PRT Human GC5B373, GC5B376, GC5B385, GC5B599, GC5B483-VL EIVLTQSPATLSLSPGERATL SCRASQSVSSYLAWYQQKPGQ APRLLIYDASNRATGIPARFS GSGSGTDFTLTISSLEPEDFA

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VYYCQQRSNWPLTFGQGTKVE IK 58 PRT Human GC5B596, GC5B602- VL DIQMTQSPSSLSASVGDRVTI TCKASQNVATHVGWYQQKPGK APKRLIYSASYRYSGVPSRFS GSGSGTEFTLTISNLQPEDFA TYYCQQYNRYPYTFGQGTKLE IK 59 PRT Human IgG4PAA ASTKGPSVFPLAPCSRSTSES TAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTKTYTCN VDHKPSNTKVDKRVESKYGPP CPPCPAPEAAGGPSVFLFPPK PKDTLMISRTPEVTCVWDVS QEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRWSVLTV L HQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK 60 PRT Human IgGl ASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLY SLSSWTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVW DVS HE D PEVK FNWYVDGVEVH NAKTKPREEQYNSTY RWSVL TVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENN

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YKTPPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 61 PRT Human GC5B382-HCDR1 DYGMH 62 PRT Human GC5B385-HCDR1 GYAMS 63 PRT Human GC5B370, GC5B597- HCDR1 SYGIS 64 PRT Human GC5B602-HCDR1 GYSFTGYTMN 65 PRT Human GC5B603-HCDR1 SYAMS 66 PRT Human GC5B601-HCDR1 GFSLTSYNVH 67 PRT Human GC5B382-HCDR2 AIKYSGGSTYYADSVKG 68 PRT Human GC5B603, GC5B385- HCDR2 AISGSGGSTYYADSVKG 69 PRT Human GC5B370, GC5B597- HCDR2 GIIPIFGNINYAQKFQG 70 PRT Human GC5B602-HCDR2 LINPYNGDTN 71 PRT Human GC5B601-HCDR2 VIWAGGSTNYNSALMS 72 PRT Human GC5B382-HCDR3 RAESGPLDY 73 PRT Human GC5B373-HCDR3 IGFYGRSFRIFDY 74 PRT Human GC5B385-HCDR3 VDRSFGRSRYTLDY 75 PRT Human GC5B370, GC5B597- HCDR3 VSRRFKRFAYYFDY 76 PRT Human GC5B603-HCDR3 SNFLPWFDY 77 PRT Human GC5B601-HCDR3 DGIRLRFAY 78 PRT Human GC5B382, GC5B370, GC5B597-LCDR2 WASTRES 79 PRT Human GC5B603-LCDR2 TASNRAT 80 PRT Human GC5B382, GC5B370, GC5B597-LCDR3 QQYYSTPLT 81 PRT Human GC5B603-LCDR3 QQYFRAPIT 82 PRT Human GC5B382-VH EVQLLESGGGLVQPGGSLRLS CAASGETFSDYGMHWVRQAPG

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KGLEWVSAIKYSGGSTYYADS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKRAESGPG LDYWGQGTLVTVSS 83 PRT Human GC5B379-VH EVQLLESGGGLVQPGGSLRLS CAASGTFFSNYWMSWVRQAPG KGLEWVSGISYSGGSKYYADS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKAAWDFGR RAVRLDYWGQGTLVTVSS 84 PRT Human GC5B376-VH EVQLVQS GAEVKKPGE S LKIS CKGSGYSFTSYWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPS FQGQVTISADKSIS TAYLQWS SLKASDTAMYYCARIGFYGRS FRIFDYWGQGTLVTVSS 85 PRT Human GC5B376-VH EVQLVQSGAEVKKPGESLKIS CKGSGYSFTSYWIGWVRQMPG KGLEWMGIIYPGDSDTRYSPS FQGQVTISADKSIS TAYLQWS SLKASDTAMYYCARVYSFGGR HKALFDYWGQGTLVTVSS 86 PRT Human GC5B385-VH EVQLLESGGGLVQPGGSLRLS CAASGFTFSGYAMSWVRQAPG KGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKVDRSFGR SRYTLDYWGQGTLVTVSS 87 PRT Human GC5B370, GC5B597- VH QVQLVQSGAEVKKPGSSVKVS CKASGGTFSSYGISWVRQAPG QGLEWMGGIIPIFGNINYAQK FQGRVTITADE STS TAYME L S SLRSEDTAVYYCARVSRRFKR FAYYFDYWGQGTLVTVSS 88 PRT Human GC5B602-VH QVQLVQSGAEVKKPGASVKVS

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CKASGYSFTGYTMNWVRQAPG QGLEWMGLINPYNGDTNYAQK LQGRVTMTTDTSTSTAYMELR S LRS DDTAVYYCARVALRVAL DYWGQGTLVTVSS 89 PRT Human GC5B603-VH EVQLLESGGGLVQPGGSLRLS CAASGFFSSYAMSWVRQAPG KGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMN SLRAEDTAVYYCAKSNFLPW FDYWGQGTLVTVSS 90 DNA Human Light chain GC5B596 atgcgggtgctggcccagctg ctgggactgctgctgctgtgc ttccctggcgccagatgcgac atccagatgacccagagcccc agcagcctgagcgccagcgtg ggcgaccgggtgaccatcacc tgcaaggccagccagaacgtg gccacccacgtgggctggtac cagcagaagcccggcaaggcc c ccaagcggctgatctacagc gccagctaccggtacagcggc gtgcccagccggttcagcggc agcggcagcggcaccgagttc accctgaccatcagcaacctg cagcccgaggacttcgccacc tactactgccagcagtacaac cggtacccctacaccttcggc cagggcaccaagctggagatc aagcgtacggtggctg cacca tctgtcttcatcttcccgcca tctgatgagcagttgaaatct ggaactgcctctgttgtgtgc ctgctgaataacttctatccc agagaggccaaagtacagtgg

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aaggtggataacgccctccaa tcgggtaactcccaggagagt gtcacagagcaggacagcaag gacagcacctacagcctcagc agcaccctgacgctgagcaaa gcagactacgagaaacacaaa gtctacgcctgcgaagtcacc catcagggcctgagctcgccc gtcacaaagagcttcaacagg ggagagtg ttga 91 PRT Human GC5B601-VH QVTLKESGPVLVKPTETLTLT CTVSGFSLTSYNVHWIRQPPG KALEWLAVIWAGGSTNYNSAL MSRLTISKDTSKSQWLTMTN MRAEDTATYYCARDGIRLRFA YWGQGTLVTVSS 92 PRT Human GC5B382, GC5B597, GC5B370-VL DIVMTQSPDSLAVSLGERATI NCKS S QSVLYS SNNKNYLAWY QQKPGQPPKLLIYWASTRESG VPDRFSGSGGTDFTLTISSL QAEDVAVYYCQQYYSTPLTFG QGTKVEIK 93 PRT Human GC5B603-VL EIVLTQSPATLSLSPGERATL SCRASQSVRKSLAWYQQKPGQ APRLLIYTASNRATGIPARFS GSGSGTDFTLTISSLEPEDFA VYYCQQYFRAPIT FGQGTKVE IKK 94 PRT Human GC5B601-VL EIVMTQSPATLSVSPGERATL SCKASQNVATHVGWYQQKPGQ APRLLIYSASYRYSGIPARFS GSGSGTEFTLTISSLQSEDFA VYYCQQYNRYPYTFGQGTKLE IK 95 PRT Human GC5B603-LCDR1 RASQSVRKSLA 96 DNA Human Heavy chain GC5B596 atggcctgggtctggaccctg ctgttcctgatggccgctgcc cagagcatccaggcccaggtg cagctggtgcagagcggcgcc gaggtgaagaagcccggcgcc agcgtgaaggtgagctgcaag gccagcggctacagcttcacc ggctacaccatgaactgggtg

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cggcaggcccccggccagggc ctggagtggatgggcctgatc aacccctacaacagcgacacc aactacgcccagaagctgcag ggccgggtgaccatgaccacc gacaccagcaccagcaccgcc tacatggagctgcggagcctg cggagcgacgacaccgccgtg tactactgcgcccgggtggcc ctgcgggtggccct ggactac tggggccagggcaccctggtg accgtgagcagcgcctccacc aagggcccatccgtcttcccc ctggcgccctgctccaggagc acctccgagagcacagccgcc ctgggctgcctggtcaaggac tacttccccgaaccggtgacg gtgtcgtggaactcaggcgcc ctgaccagcggcgtgcacacc t tcccggctgtcctacagtcc tcaggactctactccctcage agcgtggtgaccgtgccctcc agcagcttgggcacgaaaacc tacacctgcaacgtagatcac aagcccagcaacaccaaggtg gacaagagagttgagtccaaa tatggtcccccatgcccacca tgcccagcacctgaggccgcc gggggaccatcagt cttcctg ttccccccaaaacccaaggac actctcatgatctcccggacc cctgaggtcacgtgcgtggtg gtggacgtgagccaggaagac cccgaggtccagttcaactgg tacgtggatggcgtggaggtg

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Claims (28)

cataatgccaagacaaagccg cgggaggagcagttcaacagc acgtaccgtgtggtcagcgtc ctcaccgtcctgcaccaggac tggctgaacggcaagggagtac aagtgcaaggtctccaacaaa ggcctcccgtcctccatcgag aaaaccatctccaaagccaaa gggcagccccgagagccacag gtgt acaccctgcccccatcc caggaggatgaccaagaac caggtcagcctgacctgcctg gtcaaaggcttctaccccagc gacatcgccgtggagtgggag agcaatgggcagccggagaac aactacaagaccacgcctccc gtgctggactccgacggctcc ttcttcctctacagcaggcta accgtggacaagagcaggtgg cagg aggggaatgtcttctca tgctccgtgatgcatgaggct ctgcacaaccactacacacag aagagcctctccctgtctctg ggtaaatga CLAIM 1. An isolated antibody or antigen-binding fragment thereof that binds to GPRC5D, comprising a heavy chain CDR1 having the amino acid sequence SEQ ID NO: 4, a heavy chain CDR2 having the amino acid sequence SEQ ID NO: 8, and a heavy chain CDR3 having the amino acid sequence SEQ ID NO: 12 , light chain CDR1 having the amino acid sequence SEQ ID NO: 15, light chain CDR2 having the amino acid sequence SEQ ID NO: 18, and light chain CDR3 having the amino acid sequence SEQ ID NO: 21. 2. The isolated antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 55 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: : 58. 3. The isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to a polypeptide having the amino acid sequence of SEQ ID NO: 22. 4. The isolated antibody or antigen binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen binding fragment thereof is a human antibody or antigen binding fragment. 5. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is recombinant. 6. An isolated antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is a Fab fragment, a Fab2 fragment or a single chain antibody. 7. An isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, which is of the IgG1, IgG2, IgG3 or IgG4 isotype. 8. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof is of the IgG1 or IgG4 isotype. 9. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein IgG1 contains a K409R substitution in the Fc region. 10. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein IgG1 contains the F405L substitution in the Fc region. 11. The isolated antibody or antigen-binding fragment thereof according to claim 8, wherein IgG1 contains the F405L substitution and the R409K substitution in the Fc region. 12. The isolated antibody or antigen-binding fragment thereof according to claim 11, wherein the antibody or antigen-binding fragment thereof further comprises an S228P substitution, an L234A substitution, and a L235A substitution in - 80 044685 area Fc. 13. The isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof specifically binds human GPRC5D and cross-reacts with cynomolgus GPRC5D. 14. The isolated antibody or antigen binding fragment thereof according to any one of claims 1 to 13, wherein the antibody or antigen binding fragment thereof induces ADCC in vitro with an EC50 of less than about 28 nM. 15. A recombinant cell containing a nucleic acid molecule encoding an isolated antibody or an antigen-binding fragment thereof according to any one of claims 1 to 14. 16. Isolated bispecific antibody to GPRC5DxCD3 or its antigen-binding fragment containing: a) the first heavy chain (HC1); b) second heavy chain (HC2); c) the first light chain (LC1) and d) a second light chain (LC2), wherein the pair of HC1 and LC1 forms a first antigen binding site that specifically binds CD3, and the pair of HC2 and LC2 forms a second antigen binding site that specifically binds GPRC5D, wherein HC1 contains the amino acid sequence of SEQ ID NO: 25, LC1 contains the amino acid sequence of SEQ ID NO: 26, and where HC2 contains the amino acid sequence of SEQ ID NO: 55 and LC2 contains the amino acid sequence of SEQ ID NO: 58. 17. The isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16, wherein the bispecific antibody or bispecific binding fragment thereof is of an IgG1, IgG2, IgG3 or IgG4 isotype. 18. The isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment according to claim 16, wherein the bispecific antibody or bispecific binding fragment thereof is of the IgG4 isotype. 19. The isolated GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16, wherein the GPRC5D binding region of the bispecific antibody comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 55, a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 58, and an Fc domain containing the amino acid sequence of SEQ ID NO: 59. 20. The isolated GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16, wherein the CD3 binding region of the bispecific antibody comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 25, a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 26, and an Fc domain containing the amino acid sequence of SEQ ID NO: 59, where the Fc domain further contains the substitution F405L and R409K. 21. The isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16, wherein the bispecific antibody or bispecific binding fragment thereof binds GPRC5D on the surface of human myeloma cells. 22. The isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16, wherein the bispecific antibody or bispecific binding fragment binds GPRC5D on the surface of human multiple myeloma cells. 23. The isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16, wherein the bispecific antibody or bispecific binding fragment thereof induces activation of a human T cell in vitro at an EC50 of less than about 0.22 nM. 24. The isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment of claim 16, wherein the antibody or bispecific binding fragment thereof induces T cell-dependent cytotoxicity of GPRC5D-expressing cells in vitro with an EC50 of less than about 0.89 nM. 25. A recombinant cell containing a nucleic acid molecule encoding a bispecific antibody to GPRC5DxCD3 or a bispecific binding fragment thereof according to any one of claims 16-24. 26. A method of treating an individual suffering from hematologic malignancy, wherein the method comprises administering a therapeutically effective amount of an isolated anti-GPRC5DxCD3 bispecific antibody or bispecific binding fragment thereof according to claim 16 to a patient in need thereof for a time sufficient to treat the cancer. 27. A method of inhibiting the growth or proliferation of hematologic malignancy cells, wherein the method comprises administering a therapeutically effective amount of an isolated anti-GPRC5DxCD3 bispecific antibody or a bispecific binding fragment thereof according to claim 16 to a patient in need thereof for a time sufficient to treat the cancer. 28. A method of redirecting T cells to a GPRC5D-expressing cancer cell, wherein the method comprises administering a therapeutically effective amount of an isolated bispecific -