Combination therapies with a cell therapy expressing a GPRC5d-targeting car and related methods and uses

Abstract

Provided herein are methods and uses of combination therapies involving GPRC5D- targeted cell therapy comprising chimeric antigen receptors (CARs), which contain extracellular antigen-binding domains that bind to G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), and a combination agent, e.g. an immunomodulatory compound, for treating subjects with cancers such as multiple myeloma, and related methods, uses, and articles of manufacture.

Description

COMBINATION THERAPIES WITH A CELL THERAPY EXPRESSING A GPRC5D-TARGETING CAR AND RELATED METHODS AND USESCross-Reference to Related Applications

[0001] This application claims the benefit of U.S. provisional application No. 63 / 588,581 filed October 6, 2023, entitled “COMBINATION THERAPIES WITH A CELL THERAPY EXPRESSING A GPRC5D-TARGETING CAR AND RELATED METHODS AND USES”, the contents of which are incorporated by reference in its entirety.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042027840SeqList.XML, created September 30, 2024, which is 287,875 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to methods and uses of combination therapies involving a T cell therapy, e.g., a CAR T cell therapy, and an additional agent, e.g. an immunomodulatory compound, for treating subjects with cancers such as multiple myeloma, and related methods, uses, and articles of manufacture. The T cell therapy includes cells that express recombinant receptors such as chimeric antigen receptors (CARs) which contain extracellular antigen-binding domains that bind to G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D).Background

[0004] G-protein coupled receptor class C group 5 member D (GPRC5D) is a G-protein coupled receptor, the specific function of which has not yet been determined. The expression of GPRC5D is high in bone marrow samples of patients with multiple myeloma (MM) compared to the minimal expression of GPRC5D in bone marrow samples of patients with other hematological malignancies. Based on its expression, GPRC5D could be a marker of MM tumors and a therapeutic target. GPRC5D-binding chimeric antigen receptors (CARs), and cells expressing such CARs, are available. However, there remains a need for improved GPRC5D-binding CARs, engineered GPRC5D-CAR expressing targeting cells, and combination therapies. Provided herein are embodiments that meet such needs.Summary

[0005] Provided herein is a method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering to a subject having a RRMM a CAR-T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor classC group 5 member D (GPRC5D); and (2) administering to the subject the compound of Formulapharmaceutically acceptable salt thereof.

[0006] In some embodiments, the compound is administered in a dosing regimen that is initiated no earlier than 60 days after administration of the CAR T cell therapy. In some embodiments, the dosing regimen of the compound is a cycling regimen comprising more than one treatment cycle. In some embodiments, each treatment cycle is a 28-day cycle.

[0007] Provided herein is a method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); (2) administering to the subject the compound of Formula (I):pharmaceutically acceptable salt thereof, in a dosing regimen that is initiated after the CAR T cell administration, wherein the dosing regimen of the compound comprises a cycling regimen comprising more than one treatment cycle, wherein each treatment cycle is a 28-day treatment cycle.

[0008] In some embodiments, the initiation of administration of the compound is 28 days to 6 months after administering the CAR-T cell therapy to the subject. In some embodiments, the initiation of administration of the compound is no earlier than 60 days after the CAR-T celltherapy. In some of any embodiments, the initiation of administration of the compound is at or about 60 days after the CAR-T cell therapy.

[0009] In some of any embodiments, the first day of administration of the compound of the 28-day cycle is considered Day 1 (DI) of a cycle. In some of any embodiments, the dosing regimen comprises administration of the compound to the subject on Days 1-21 of the 28-day cycle. In some of any embodiments, the compound is administered daily. In some of any embodiments, the compound is administered at a dose from 0.1 mg to 1.0 mg daily. In some of any embodiments, the compound is administered at a dose from about 0.3 mg to 0.9 mg daily. In some of any embodiments, the compound is administered at a dose of about 0.60 mg daily.

[0010] In some of any embodiments, the compound is administered orally. In some of any embodiments, the compound is administered to the subject until disease progression. In some of any embodiments, the subject has not undergone previous treatment with the compound. In some of any embodiments, the dosing regimen of the immunomodulatory compound in combination with the CAR-T cell therapy is such that the subject does not develop a toxicity characterized by one or more of: (i) Any Grade 4 CRS and Grade CRS that does not resolve to Grade < 2 within 72 hours; (ii) any duration Grade 3 ICANS or neurotoxicity (NT); (iii) a higher than Grade 3 toxicity involving vital organs (eg, cardiac, pulmonary) of any duration; (iv) all other Grade 3 toxicities not attributable to underlying disease or lymphodepleting chemotherapy that do not resolve to < Grade 2 within 72 hours, other than a non-CRS toxicity of the liver in which liver amino transferase levels > 3x upper limit of normal (ULN) are accompanied by an elevation of total bilirubin to >2 x ULN without initial findings of cholestasis (e.g., elevated serum alkaline phosphatase) for which no other reason can be found to explain the combination of increased amino transferase to total bilirubin, such as viral hepatitis A, B, or C; pre-existing or acute liver disease; or another drug capable of causing the observed liver injury; (v) Grade 3 aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) increase lasting < 14 days or Grade 4 AST and / or ALT increase lasting < 7 days; (vi) certain treatment-emergent, isolated Grade 3 or 4, asymptomatic laboratory electrolyte abnormalities not directly related to vital organ toxicities that resolve, with or without intervention, to Grade < 2 in < 7 days; (vii) Grade 4 thrombocytopenia that does not resolve to Grade 3 or lower within 14 days or accompanied by clinically significant bleeding or Grade 3 thrombocytopenia with clinically significant bleeding; (viii) Grade 4 neutropenia that does not resolve to Grade 3 or lower within 14 days despite the use of granulocyte colony-stimulating factor (G-CSF); (ix) any Grade 5toxicity not related to disease progression; (x) any macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH); or (xi) any Grade > 3 thrombotic event.

[0011] Provided herein is a method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); and (2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof.

[0012] In some embodiments, the compound is administered in a dosing regimen that is initiated on the same day as the CAR T cell administration. In some embodiments, the compound is administered to the subject no earlier than 3 hours prior, no earlier than 2 hours prior, no earlier than 1 hour prior, no earlier than 0.5 hour prior, no later than 0.5 hour after, no later than 1 hour after, no later than 2 hours after, or no later than 3 hours after the CAR T cell administration. In some of any embodiments, at least one fixed dose of the compound is administered in an amount between 0.1 mg and 2.0 mg.

[0013] In some of any embodiments, the dosing regimen of the compound is a cycling regimen comprising more than one treatment cycle. In some embodiments, each treatment cycle is a 28-day cycle. In some of any embodiments, the dosing regimen of the compound comprises at least one treatment cycle characterized by administration of the compound at a first dose and at least one treatment cycle characterized by administration of the compound at a second dose, wherein the second dose is greater than the first dose.

[0014] Provided herein is a method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); (2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof, in a dosing regimen that is initiated on the same day as the CAR T cell administration; wherein the dosing regimen of the compound is a cycling regimen comprising more than one treatment cycle, wherein each treatment cycle is a 28-day treatment cycle; wherein at least one treatment cycle is characterized by administration of the compound at a first dose and at least one treatment cycle is characterized by administration of the compound at a second dose, wherein the second dose is greater than the first dose.

[0015] In some of any embodiments, the dosing regimen comprises administration of the compound to the subject on Days 1-21 of the 28-day cycle. In some of any embodiments, the compound of Formula (II) is administered daily. In some of any embodiments, the first dose of the compound is from about 0.3 mg to 0.9 mg daily. In some of any embodiments, the first dose of the compound is about 0.45 mg daily. In some of any embodiments, the first dose of the compound is about 0.75 mg daily. In some of any embodiments, the second dose of the compoundis from about 0.5 mg to 1.5 mg daily. In some of any embodiments, the second dose of the compound is about 1.0 mg daily.

[0016] In some of any embodiments, the dosing regimen comprises a first, second, third, fourth and fifth treatment cycles, wherein the each of the treatment cycles are characterized by daily administration of the first dose of the compound. In some embodiments, after the fifth treatment cycle, the dosing regimen comprises one or more additional treatment cycles characterized by daily administration of the second dose of the compound. In some of any embodiments, the compound is administered orally.

[0017] Provided herein is a method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); (2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof, to the subject, wherein administration of the compound is initiated on the same day as the CAR T cell administration and is administered at a daily dose on Days 1-21 of a 28-day treatment cycle for a plurality of treatment cycles, wherein: (i) the daily dose in the first, second, third, fourth and fifth treatment cycles is a first dose of 0.45 mg; and (ii) the daily dose in each successive treatment cycle is a second dose of 1.0 mg.

[0018] Provided herein is a method of treating a treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); (2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof, wherein administration of the compound is initiated on the same day as the CAR T cell administration and: is administered at a daily dose on Days 1-21 of a 28-day treatment cycle, wherein: (i) the daily dose in the first, second, third, fourth and fifth treatment cycles ois a first dose of 0.75 mg; and (iii) the daily dose in each successive treatment cycles is a second dose of 1.0 mg.

[0019] In some of any embodiments, the compound is administered to the subject until disease progression. In some of any embodiments, the subject has not undergone previous treatment with the compound. In some of any embodiments, the dosing regimen of the compound in combination with the CAR-T cell therapy is such that the subject does not develop a toxicity characterized by one or more of: (i) Any Grade 4 CRS and Grade CRS that does not resolve to Grade < 2 within 72 hours; (ii) any duration Grade 3 ICANS or neurotoxicity (NT);(iii) a higher than Grade 3 toxicity involving vital organs (eg, cardiac, pulmonary) of any duration; (iv) all other Grade 3 toxicities not attributable to underlying disease or lymphodepleting chemotherapy that do not resolve to < Grade 2 within 72 hours, other than a non-CRS toxicity of the liver in which liver amino transferase levels > 3x upper limit of normal (ULN) are accompanied by an elevation of total bilirubin to >2 x ULN without initial findings of cholestasis (e.g., elevated serum alkaline phosphatase) for which no other reason can be found to explain the combination of increased amino transferase to total bilirubin, such as viral hepatitis A, B, or C; pre-existing or acute liver disease; or another drug capable of causing the observed liver injury; (v) Grade 3 aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) increase lasting < 14 days or Grade 4 AST and / or ALT increase lasting < 7 days; (vi) certain treatment-emergent, isolated Grade 3 or 4, asymptomatic laboratory electrolyte abnormalities not directly related to vital organ toxicities that resolve, with or without intervention, to Grade < 2 in < 7 days; (vii) Grade 3 or 4 thrombocytopenia accompanied by clinically significant bleeding or Grade 4 thrombocytopenia that does not resolve to Grade 3 or lower within 42 days or accompanied by clinically significant bleeding; (viii) Grade 2 or higher infection with concurrent grade 3 or 4 neutropenia; (ix) any Grade 5 toxicity not related to disease progression; (x) any macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH); or (xi) any Grade > 3 thrombotic event.

[0020] In some of any embodiments, the CAR comprises (1) an extracellular antigenbinding domain that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D), (2) an immunoglobulin hinge spacer of at least 125 amino acids in length; (3) a transmembrane domain; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain and a costimulatory signaling region; wherein the extracellular antigen-binding domain comprises a VH region and a VL region, wherein: the VH region comprises the amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:1; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:2; the VH region comprises the amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:1; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 116 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 116; the VH region comprises the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:3; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:4 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:4; the VH region comprises the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:3; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 117 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 117; the VH region comprises the amino acid sequence set forth in SEQ ID NO:5 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:5; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:6; the VH region comprises the amino acid sequence set forth in SEQ ID NO:5 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:5; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 118 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 118; the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acidsequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:8; the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 119 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 119; the VH region comprises the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:9; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 10; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 9; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 120 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 120; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 11; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99%identity to SEQ ID NO: 12; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 11; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 121 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 121; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 13; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 14; or the VH region comprises the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 13; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 122 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 122.

[0021] In some embodiments, the extracellular antigen binding domain comprises a VH region and a VL region, wherein: the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:20, 21 and 17, respectively, and the VL region comprises the amino acid sequences of SEQ ID NOS:25, 26 and 27, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:35, 36, 32, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:40, 41 and 42, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:50, 51 and 47, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:55, 56 and 57, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acidsequences of SEQ ID NOS:65, 66 and 62, respectively, and the Vrregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:70, 71 and 72, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:80, 81 and 77, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:85, 86 and 87, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:80, 94 and 91, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:97, 98 and 99, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 107, 108 and 104, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 112, 26, 113, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 109, 110 and 111, respectively, and the VL region comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 114, 29 and 123, respectively.

[0022] In some of any embodiments, the extracellular antigen binding domain comprises a VH region and a VL region wherein: the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs:l and 2, respectively or the amino acid sequence set forth in SEQ ID NOs: 1 and 116, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs:3 and 4, respectively or the amino acid sequence set forth in SEQ ID NOs:3 and 117, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 5 and 6, respectively or the amino acid sequence set forth in SEQ ID NOs: 5 and 118, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 7 and 8, respectively or the amino acid sequence set forth in SEQ ID NOs: 7 and 119, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 9 and 10, respectively or the amino acid sequence set forth in SEQ ID NOs: 9 and 120, respective; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 11 and 12, respectively or the amino acid sequence set forth in SEQ ID Nos: 11 and 121, respectively; or the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 13 and 14 or the amino acid sequence set forth in SEQ ID Nos: 13 and 122, respectively.

[0023] In some of any embodiments, the extracellular antigen binding domain comprises the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequencesof SEQ ID NOS:65, 66 and 62, respectively, and the Vrregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:70, 71 and 72, respectively.

[0024] In some of any embodiments, the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 119 or or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 119.

[0025] In some of any embodiments, the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 119. In some of any embodiments, the single chain antibody fragment is or comprises a single chain variable fragment (scFv). In some of any embodiments, the VH region and the VL region are joined by a flexible linker. In some of any embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 127). In some of any embodiments, the VH region is carboxy-terminal to the VL region.

[0026] In some of any embodiments, the extracellular antigen-binding domain comprises the amino acid sequence selected from SEQ ID NOs: 131, 133, 137, and 141 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence selected from SEQ ID NOs: 131, 133, 137, and 141.

[0027] In some of any embodiments, the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 137. In some of any embodiments, the spacer comprises a sequence of a hinge region, a CH2 and CH3 region. In some of any embodiments, the spacer comprises an IgG4 / 2 chimeric hinge region or a modified IgG4 hinge region comprising at least one amino acid replacement compared to a human IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region.

[0028] In some of any embodiments, the spacer is or comprises (i) the sequence set forth in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 that has at least at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to SEQ ID NO: 162; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length.In some of any embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQID NO: 164.

[0029] In some of any embodiments, the cytoplasmic signaling domain of CD3(^ comprises the amino acid sequence set forth in SEQ ID NO: 176 or an amino acid that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 176. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4- IBB, or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling region comprises an intracellular signaling domain of 4-1BB.

[0030] In some of any embodiments, the costimulatory signaling region is or comprises the amino acid sequence set forth in SEQ ID NO: 179 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the sequence set forth in SEQ ID NO: 179. In some of any embodiments, the transmembrane domain is or comprises a transmembrane domain from CD4, CD28, or CD 8. In some of any embodiments, the transmembrane domain is or comprises a transmembrane domain derived from CD28.

[0031] In some of any embodiments, the CAR comprises: (1) an extracellular antigenbinding domain that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D), wherein the extracellular antigen-binding domain comprises: (i) a variable heavy chain (VH) region comprising an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and (ii) a variable light chain (VL) region comprising an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119; (2) a spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region, optionally that is about 228 amino acids in length; (3) a transmembrane domain from human CD28; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain and an intracellular signaling domain of a 4- IBB.

[0032] In some embodiments, the extracellular antigen-binding domain comprises the VH region amino acid sequence set forth in SEQ ID NO:7 and the VL region amino acid sequence set forth in SEQ ID NO: 119. In some of any embodiments, the extracellular antigen-binding domain comprises an scFv set forth in SEQ ID NO: 137. In some of any embodiments, the spacer is or comprises (i) the sequence set forth in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 that has at least at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to SEQ ID NO: 162; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length; or the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 164.

[0033] In some of any embodiments, the transmembrane domain is or comprises the amino acid sequence set forth in SEQ ID NO: 173 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO:173.

[0034] In some of any embodiments, the intracellular signaling region comprises (a) a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain comprising the amino acid sequence set forth in SEQ ID NO: 176 or an amino acid sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 176 and (b) and an intracellular signaling domain of a 4- IBB comprising the amino acid sequence set forth in SEQ ID NO: 179 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the sequence set forth in SEQ ID NO: 179.

[0035] In some of any embodiments, the intracellular signaling region is or comprises the sequences set forth in SEQ ID NO: 176 and SEQ ID NO: 179. In some of any embodiments, the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 183, or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 183.

[0036] In some of any embodiments, the chimeric antigen receptor is encoded by the nucleotide sequence set forth in SEQ ID NO: 182, or a nucleotide sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO:182.

[0037] In some of any embodiments, the dose of engineered cells comprises between about 1.0 x 107CAR-expressing T cells and 1.2 x 109CAR-expressing T cells, between about 1.0 x 107CAR-expressing T cells and 6.5 x 108CAR-expressing T cells, between about 1.5 x 107CAR-expressing T cells and 6.5 x 108CAR-expressing T cells, between about 1.5 x 107CAR- expressing T cells and 6.0 x 108CAR-expressing T cells, between about 2.5 x 107CAR- expressing T cells and 6.0 x 108CAR-expressing T cells, between about 5.0 x 107CAR- expressing T cells and 6.0 x 108CAR-expressing T cells, between about 1.25 x 107CAR- expressing T cells and 1.2 x 109CAR-expressing T cells, between about 1.5 x 107CAR- expressing T cells and 1.2 x 109CAR-expressing T cells, between about 5.0 x 107CAR- expressing T cells and 4.5 x 108CAR-expressing T cells, or between about 1.5 x 108CAR- expressing T cells and 3.0 x 108CAR-expressing T cells, each inclusive.

[0038] In some of any embodiments, the dose of engineered cells comprises at or about 1.5 x 107, at or about 2.5 x 107, at or about 5.0 x 107, at or about 7.5 x 107, at or about 1.5 x 108, at or about 2.25 x 108, at or about 3.0 x 108, at or about 4.5 x 108, at or about 6.0 x 108, at or about 8.0 x 108, or at or about 1.2 x 109CAR-expressing T cells. In some of any embodiments, the dose of engineered cells comprises at or about 7.5 x 107CAR-expressing T cells. In some of any embodiements, the dose of engineered cells comprises at or about 1.5 x 108CAR-expressing T cells.

[0039] In some of any embodiments, the method further comprises administering to the subject a lymphodepleting chemotherapy prior to administration of the CAR-T cell therapy. In some embodiments, the lymphodepleting therapy is completed within about 7 days prior to initiation of the administration of the dose of the CAR-expressing T cells. In some of any embodiments, the administration of the lymphodepleting therapy is completed within about 2 to 7 days prior to initiation of the administration of the dose of engineered T cells. In some of any embodiments, the lymphodepleting therapy comprises the administration of fludarabine and / or cyclopho sphamide .

[0040] In some of any embodiments, the lymphodepleting therapy comprises the administration of fludarabine and cyclophosphamide. In some of any embodiments, the lymphodepleting therapy comprises administration of cyclophosphamide at or about 200-400 mg / m2inclusive daily, optionally at or about 300 mg / m2daily. In some of any embodiments, the lymphodepleting therapy comprises administration of fludarabine at or about 20-40 mg / m2inclusive daily, optionally at or about 30 mg / m2daily. In some of any embodiments, the lymphodepleting therapy comprises administration of fludarabine and cyclophosphamide for 2-4 days, optionally for 3 days.

[0041] In some of any embodiments, the lymphodepleting therapy comprises the administration of bendamustine. In some of any embodiments, the lymphodepleting therapy comprises administration of bendamustine at or about 50-130 mg / m2inclusive daily, optionally at or about 90 mg / m2daily. In some of any embodiments, the lymphodepleting therapy comprises administration of bendamustine for 1-3 days, optionally for 2 days. In some of any embodiments, the subject does not develop a severe toxicity.Brief Description of the Drawings

[0042] FIG. 1 depicts an exemplary treatment regimen comprising anti-GPRC5D CAR T cell therapy and mezigdomide administration.

[0043] FIG. 2 depicts an exemplary treatment regimen comprising anti-GPRC5D CAR T cell therapy and iberdomide administration.Detailed Description

[0044] Among the provided embodiments are combination therapies involving administration of a T cell therapy (e.g., CAR-expressing T cells), and administration of a combination agent (e.g. an immunomodulatory compound). Among the provided embodiments are combination therapies for treating a subject having a cancer involving administration of a T cell therapy (e.g. CAR-T cells) and an additional agent (e.g. an immunomodulatory compound) for treating subjects with cancers or tumors such as multiple myeloma. In some embodiments, the methods provided herein are based on administration of a GPRC5D-directed CAR T cell therapy in which the CAR contains a GPRC5D-directed scFv antigen binding domain in combination with a combination agent including an immunomodulatory compound. Thus, in some embodiments, the methods and uses include administering to a subject T cells expressing genetically engineered (recombinant) cell surface receptors in adoptive cell therapy, which generally are chimeric receptors such as chimeric antigen receptors (CAR) recognizing an antigen expressed by, associated with and / or specific to the cell type from which it is derived, in combination with a an immunomodulatory compound.

[0045] Among the provided embodiments are methods of treating relapsed and / or refractory multiple myeloma (RRMM). In some embodiments, the method comprises the administration of a CAR-T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D) to a subject followed by the administration of an immunomodulatory compound. In some embodiments, the immunomodulatory compound is administered no earlier than 60 days after administration of the CAR T cell therapy.

[0046] In some embodiments, a combination therapy involving administration of CAR-T cells and an immunomodulatory compound may yield improved antimyeloma activity through at least 2 mechanisms. In some cases, the improved activity may be synergistic to each individual therapy. First, myeloma cells that escape CAR T cell killing may be susceptible to CELMoD- mediated cytotoxicity, yielding greater depth of tumor clearance, increased rates of clearance of MRD, and potentially greater durability of response. Second, in addition to myeloma cell killing, CELMoDs modulate the activity of normal immune cells, including increasing the proliferation and activation of T cells, while mitigating interleukin (IL)-6 induction.

[0047] Multiple myeloma (MM) is a hematological malignancy characterized by uncontrolled proliferation of monoclonal plasma cells in the bone marrow resulting in the overproduction of monoclonal immunoglobulin and immunosuppression (Al-Hujaily 2016; Dimopoulos, 2015). Adoptive T cell therapies, such as CAR-T cell therapies, have shown promise for treating multiple myeloma, with clinical efforts primarily focused on targeting the B cell maturation antigen (BCMA). However, although BCMA is expressed on many malignant plasma cells, expression levels, in some cases, can be heterogeneous. In some aspects, heterogeneity in target antigen expression can lead to variable or inconsistent response. In some aspects, it also has been observed that expression of BCMA on the cell surface varies over time due to gamma secretase-mediated shedding of the extracellular domain. Similar to observations with CD19 and CD22 CAR antigens, it has been reported that BCMA antigen down-regulation occurs in multiple myeloma (MM) patients who relapsed after BCMA-targeted T cell therapy (Brudno et al. (2018) J. Clin. Oncol., JCO2018778084,; Cohen et al. (2017) Blood 130:505). Also, in some contexts, recombinant receptors can exhibit antigen-independent activity or signaling (also known as “tonic signaling”), which could lead to undesirable effects, such as increased differentiation and / or exhaustion of T cells that express the recombinant receptor. In some aspects, such activities may limit the T cell’s activity, effect or potency. In some cases, during engineering and ex vivo expansion of the cells for recombinant receptor expression, thecells may exhibit phenotypes indicative of exhaustion, due to tonic signaling through the recombinant receptor. In some cases, alternative or additional MM-targeted T cell therapy approaches are needed.

[0048] The provided embodiments relate to GPRC5D as a CAR T cell target for multiple myeloma. GPRC5D (Uniprot Acc. No. Q9NZD1, e.g. set forth in SEQ ID NO:207) is a G protein coupled receptor class C, group 5 member D that belongs to the RAIG (retinoic acidinducible gene-1) family. It is a seven transmembrane helix 39kDa G-protein coupled receptor with two reported isoforms, with the isoform differences occurring in the intracellular C terminus of the protein. Results herein show that GPRC5D is expressed at high levels in multiple myeloma and, overall, it is expressed at low levels in most normal tissues.

[0049] In some embodiments, the methods include administration of the combination therapy to a subject selected or identified as having a certain prognosis or risk of multiple myeloma. In some embodiments, the methods, uses and articles of manufacture involve, or are used for treatment of subjects involving, selecting or identifying a particular group or subset of subjects, e.g., based on specific types of disease, diagnostic criteria, prior treatments and / or response to prior treatments, such as any group of subjects as described. In some embodiments, the methods involve treating a subject having relapsed following remission after treatment with, or become refractory to, one or more prior therapies; or a subject that has relapsed or is refractory (R / R) to one or more prior therapies, e.g., one or more lines of standard therapy. In some embodiments, the methods involve treating a subject having relapsed following remission after treatment with, or become refractory to, two or more prior therapies. In some embodiments, the subject has relapsed or is refractory (R / R) to two or more prior therapies, e.g., two or more lines of standard therapy. In some embodiments, the methods involve treating a subject having relapsed following remission after treatment with, or become refractory to, three or more prior therapies. In some embodiments, the subject has relapsed or is refractory (R / R) to three or more prior therapies, e.g., three or more lines of standard therapy.

[0050] In some aspects, the provided methods, compositions, uses and articles of manufacture achieve improved and superior responses to available therapies, including a T cell therapy alone or a combination agent (e.g. an immunomodulatory compound) alone. In some embodiments, the improved or superior responses are compared to current standard of care (SOC).

[0051] Also provided are combinations and articles of manufacture, such as kits, that contain a composition comprising the T cell therapy and / or a composition comprising the combinationagent (e.g. an immunomodulatory compound), and uses of such compositions and combinations to treat or prevent a GPRC5D-expressing cancer (e.g. multiple myeloma). In some embodiments, the cancer is a GPRC5D-expressing cancer. In some embodiments, the cancer is a BCMA-expressing cancer. In some embodiments, the cancer is associated with expression of both GPRC5D and BCMA.

[0052] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0053] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. Combination Therapy

[0054] Provided herein are methods of combination therapy that include administration and uses, such as therapeutic and prophylactic uses, of the GPRC5D-binding recombinant receptors (e.g., CARs) and / or compositions comprising the same, in combination with an immunomodulatory compound. In some embodiments, the GPRC5D-binding recombinant receptors is an anti-GPRC5D CAR that is administered to the subject in combination with the immunomodulatory compound. In some embodiments, the combination agent is an immunomodulatory compoundan immunomodulatory compound as described in Section I.B.

[0055] Also provided herein are methods of treating relapsed and / or refractory multiple myeloma (RRMM). In some embodiments, the method comprises the administration combination therapy, wherein the combination therapy comprises CAR-T cell therapy and an immunomodulatory compound. In some embodiments, the method comprises administration of a CAR-T cell therapy comprising a dose of engineered cells comprising T cells expressing a CAR that specifically binds human GPRC5D to a subject followed by the administration of an immunomodulatory compound. In some embodiments, the immunomodulatory compound is administered no earlier than 60 days after administration of the CAR T cell therapy. In some embodiments, the immunomodulatory compound is administered concurrently with the administration of the CAR T cell therapy.

[0056] In some embodiments, the GPRC5D-binding recombinant receptor (e.g., CAR) and / or engineered cells expressing said molecules (e.g., recombinant receptor) described herein are administered as part of a combination treatment or combination therapy, such as simultaneously with, sequentially with, or intermittently with, in any order, one or more additional therapeutic intervention involving administration of an immunomodulatory compound. Also provided are methods of combination treatment or combination therapy that includes GPRC5D-binding recombinant receptors (e.g., CARs), cells and / or compositions described herein and one or more additional therapeutic intervention involving administration of an immunomodulatory compound.

[0057] Among the provided embodiments are methods of administering and uses of a combination comprising GPRC5D-targeted cell therapy and an immunomodulatory compound. Such methods and uses include therapeutic methods and uses, for example, involving administration of the combination to a subject having a disease, condition, or disorder associated with GPRC5D such as a disease, condition, or disorder associated with GPRC5D expression, and / or in which cells or tissues express, e.g., specifically express, GPRC5D. In some embodiments, the combination is administered in an effective amount to effect treatment of the disease or disorder. Provided herein are uses of the GPRC5D-targeted cell therapy in combination with an immunomodulatory compound in such methods and treatments, and in the preparation of a medicament in order to carry out such therapeutic methods. In some embodiments, the methods are carried out by administering the combination to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. Also provided herein are use of the combination for the treatment of a disease or disorder associated with GPRC5D, such as use in a treatment regimen.

[0058] Also provided herein are uses of the anti-GPRC5D CAR cell therapy (e.g., T cell therapy) and the immunomodulatory compound as a combination therapy in such methods and treatments of a subject having a disease or disorder, and in the preparation of a medicament in order to carry out such therapeutic methods. Also provided herein are uses of the anti-GPRC5D CAR cell therapy (e.g., T cell therapy) for use in combination with the immunomodulatory compound in such methods and treatment of a subject having a disease or disorder, and in the preparation of a medicament in order to carry out such therapeutic methods in combination with the immunomodulatory compound. Also provided herein are uses of the immunomodulatory compound for use in combination with the anti-GPRC5D CAR cell therapy (e.g., T cell therapy)in such methods and treatment of a subject having a disease or disorder, and in the preparation of a medicament in order to carry out such therapeutic methods in combination with the anti- GPRC5D CAR cell therapy (e.g., T cell therapy).

[0059] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse event, effect, or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0060] As used herein, “adverse event” refers to any new untoward medical occurrence or worsening of a pre-existing medical condition occurring in a clinical investigation participant after signing of informed consent, whether or not considered related to the study intervention. An adverse event can therefore be any unfavorable and unintended sign (such as an abnormal laboratory test result), symptom, or disease temporally associated with the study intervention.

[0061] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or subject being treated. As sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0062] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease.

[0063] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody or composition or cell which suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody or composition or cell.

[0064] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0065] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0066] An “effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition, in the context of administration, refers to an amount effective, at dosages / amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.

[0067] A “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, binding molecule, antibody, cells, or composition refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and / or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the molecules, antibodies, cells, and / or compositions at effective amounts, e.g., therapeutically effective amounts.

[0068] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0069] As used herein, a “subject” or an “individual” is a mammal. In some embodiments, a “mammal” includes humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject is human.

[0070] Among the diseases to be treated is any disease or disorder associated with GPRC5D or any disease or disorder in which GPRC5D is specifically expressed and / or in which GPRC5D has been targeted for treatment (also referred to herein interchangeably as a “GPRC5D- associated disease or disorder”). Cancers associated with GPRC5D expression include hematologic malignancies such as myeloma, e.g., multiple myeloma. In some embodiments, the disease or disorder associated with GPRC5D is a B cell-related disorder or malignancy. In someembodiments the disease or disorder associated with GPRC5D is multiple myeloma or Waldenstrom’ Macroglobuhnemia. In certain embodiments, the disease or disorder is multiple myeloma. Among the provided embodiments are cells, populations, combinations, and compositions for administration to a subject having a relapsed and / or refractory multiple myeloma (RRMM), e.g., via adoptive cell therapy, such as adoptive T cell therapy.

[0071] In some embodiments, the methods may identify a subject who has, is suspected to have, or is at risk for developing a GPRC5D-associated disease or disorder. Hence, provided are methods for identifying subjects with diseases or disorders associated with GPRC5D expression and selecting them for treatment with a provided combination of GPRC5D-targeted cell therapy and an immunomodulatory compound.

[0072] For example, a subject may be screened for the presence of a disease or disorder associated with elevated GPRC5D expression, such as a GPRC5D-expressing cancer. In some embodiments, the methods include screening for or detecting the presence of a GPRC5D- associated disease, e.g., a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or disorder associated with elevated GPRC5D expression and assayed for the expression level of GPRC5D. In some aspects, a subject who tests positive for a GPRC5D-associated disease or disorder may be selected for treatment by the present methods, and may be administered a therapeutically effective amount of a combination comprising a GPRC5D-targeted cell therapy and an immunomodulatory compound.

[0073] In some embodiments, the subject has a history of relap sed / refractory multiple myeloma. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with a BCMA-specific antibody and / or cells expressing a BCMA-targeting chimeric receptor and / or other therapy, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT or or autologous HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another GPRC5D-targeted therapy. In some embodiments, the subject has not relapsed but is determined to be at risk for relapse, such as at a high risk of relapse, and thus the compound or composition is administered prophylactically, e.g., to reduce the likelihood of or prevent relapse.

[0074] In some embodiments, the subject is one that is eligible for a transplant, such as is eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT or autologous HSCT. In some of such embodiments, the subject has not previously received a transplant, despite being eligible, prior to administration of the combination comprising a GPRC5D-targeted cell therapy and an immunomodulatory compound.

[0075] In some embodiments, the subject is one that is not eligible for a transplant, such as is not eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT or or autologous HSCT. In some of such embodiments, such a subject is administered a combination comprising a GPRC5D-targeted cell therapy and an immunomodulatory compound according to the provided embodiments herein.

[0076] In some embodiments, prior to the initiation of administration of the combination comprising a GPRC5D-targeted cell therapy and an immunomodulatory compound, the subject has received one or more prior therapies. In some embodiments, the subject has received at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more prior therapies. In some embodiments, the subject has received at least 1, 2, 3, 4, or more prior therapies. In some embodiments, the subject has received at least 3 prior therapies. In some embodiments, the subject has received at least 2 prior therapies. In some embodiments, the subject has received at least 1 prior therapy. In some embodiments, the subject has received at least one, but not more than three, prior therapies. In some embodiments, the prior therapies include, but are not limited to, an immunomodulatory agent (e.g., thalidomide, lenalidomide, pomalidomide), a proteasome inhibitor (e.g., bortezomib, carfilzomib, ixaxomib), dexamethasone, an anti-CD38 antibody, and an autologous hematopoietic stem cell transplantation (HSCT). In some embodiments, induction with or without HSCT and with or without maintenance therapy is considered 1 regimen. In some embodiments, the prior therapy comprises a prior therapy targeting BCMA and / or GPRC5D.

[0077] In some aspects, the subject has relapsed following, or has been refractory to, one or more of, for example, each, individually, of the one or more prior therapies. In some embodiments, the subject has undergone at least 2 consecutive cycles of treatment for each regimen unless progressive disease was the best response to the regimen.

[0078] In some embodiments, the subject has undergone at least one complete cycle of treatment unless progressive disease was the best response to the treatment regimen. In some embodiments, the subject has been treated with at least one, but not greater than three prior antimyeloma treatment regimens, including a proteasome inhibitor and an immunomodulatory agent.

[0079] In some embodiments, the methods described herein may be used in the treatment of diseases, conditions, and disorders in which GPRC5D is expressed. In some embodiments, the disease or disorder associated with GPRC5D is a B cell-related disorder or malignancy. In some embodiments the disease or disorder associated with GPRC5D is multiple myeloma or Waldenstrom’ s Macroglobulinemia. In certain embodiments, the disease or disorder is multiplemyeloma. In some embodiments, the subject to be treated using the described method has a diagnosis of multiple myeloma with relapsed and / or refractory disease.

[0080] In some embodiments, the disease or disorder to be treated is multiple myeloma. In some embodiments, measurable disease criteria for multiple myeloma can include (1) serum M- protein 1 g / dL or greater; (2) Urine M-protein 200 mg or greater / 24 hour; (3) involved serum free light chain (sFLC) level 10 mg / dL or greater, with abnormal K to ratio. In some cases, light chain disease is acceptable only for subjects without measurable disease in the serum or urine.

[0081] In some embodiments, the Eastern Cooperative Oncology Group (ECOG) performance status indicator can be used to assess or select subjects for treatment, e.g., subjects who have had poor performance from prior therapies (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655). The ECOG Scale of Performance Status describes a patient’s level of functioning in terms of their ability to care for themselves, daily activity, and physical ability (e.g., walking, working, etc.). In some embodiments, an ECOG performance status of 0 indicates that a subject can perform normal activity. In some aspects, subjects with an ECOG performance status of 1 exhibit some restriction in physical activity but the subject is fully ambulatory. In some aspects, patients with an ECOG performance status of 2 is more than 50% ambulatory. In some cases, the subject with an ECOG performance status of 2 may also be capable of selfcare; see e.g., Sprensen et al., (1993) Br J Cancer 67(4) 773-775. In some embodiments, the subject that are to be administered according to the methods or treatment regimen provided herein include those with an ECOG performance status of 0 or 1.

[0082] In some embodiments, the administration can treat the subject despite the subject having become resistant to another therapy. In some embodiments, when administered to subjects according to the embodiments described herein, the combination is capable of achieving objective response (OR), in at least 50%, 60%, 70%, 80%, 90%, or 95% of subjects that were administered. In some embodiments, OR includes subjects who achieve stringent complete response (sCR), complete response (CR), very good partial response (VGPR), partial response (PR) and minimal response (MR). In some embodiments, when administered to subjects according to the embodiments described herein, the combination is capable of achieving stringent complete response (sCR), complete response (CR), very good partial response (VGPR) or partial response (PR), in at least 50%, 60%, 70%, 80%, or 85% of subjects that were administered. In some embodiments, when administered to subjects according to the embodiments described herein, combination is capable of achieving stringent complete response(sCR) or complete response (CR) in at least 20%, 30%, 40% 50%, 60% or 70% of subjects that were administered.

[0083] In some embodiments, treatment according to the provided methods can result in a lower rate and / or lower degree of toxicity, toxic outcome or symptom, toxicity-promoting profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity, such as severe CRS or severe neurotoxicity, for example, compared to administration of other therapies.A. GPRC5D-Targeted Cell Therapy

[0084] Provided in some aspects are GPRC5D-binding agents, such as recombinant receptors or chimeric antigen receptors that bind GPRC5D molecules and polynucleotides encoding GPRC5D binding cell surface proteins, such as recombinant receptors (e.g., CARs), and cells expressing such receptors. The GPRC5D-binding cell surface proteins generally contain antibodies (e.g., antigen-binding antibody fragments), and / or other binding peptides that specifically bind to GPRC5D, such as GPRC5D proteins, such as human GPRC5D protein. In some aspects, the agents bind to an extracellular portion of GPRC5D.

[0085] Among the provided polynucleotides are those that encode recombinant receptors, such as antigen receptors, that specifically bind GPRC5D. In some aspects, the encoded receptors, such as those containing GPRC5D-binding polypeptides, and compositions and articles of manufacture and uses of the same, also are provided.

[0086] Among the GPRC5D-binding polypeptides are antibodies, such as single-chain antibodies (e.g., antigen binding antibody fragments), or portions thereof. In some examples, the recombinant receptors are chimeric antigen receptors, such as those containing anti- GPRC5D antibodies or antigen-binding fragments thereof. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, such as those that can be introduced into cells for expression of the encoded recombinant GPRC5D- binding receptors.1. GPRC5D Chimeric Antigen Receptors

[0087] The provided GPRC5D-binding receptors generally contain an extracellular binding molecule and an intracellular signaling domain. Among the provided receptors are polypeptides containing antibodies, such as recombinant cell surface receptors containing anti-GPRC5D antibodies. Such receptors include chimeric antigen receptors that contain such antibodies.

[0088] Among the provided recombinant receptors are antigen receptors that include a GPRC5D-binding fragment. The recombinant receptors include antigen receptors that specifically bind to GPRC5D, such as antigen receptors containing the anti-GPRC5D antibodies, e.g., GPRC5D antigen-binding fragments. Among the antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Also provided are cells expressing the recombinant receptors and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with GPRC5D expression, e.g., multiple myeloma.

[0089] In some embodiments, the CAR comprises an extracellular antigen binding domain that specifically binds to GPRC5D, a spacer sequence between the extracellular antigen binding domain and a transmembrane domain, the transmembrane domain, and an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain and a costimulatory signaling region. In some embodiments, the spacer sequence is a hinge domaincontaining sequence. In some embodiments, the spacer sequence is an immunoglobulin hinge spacer of at least 125 amino acids in length. a. Extracellular antigen-binding domain

[0090] Among the chimeric receptors are chimeric antigen receptors (CARs). The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that includes, is, or comprises an anti-GPRC5D antibody such as an antibody fragment. Thus, the chimeric receptors, e.g., CARs, typically include in their extracellular portions one or more GPRC5D-binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable regions, and / or antibody molecules, such as those described herein.

[0091] The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, heavy chain variable (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof also referred to herein as “antigen-binding fragments.” The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.

[0092] The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known in the art to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR- H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR- L3). “Framework regions” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full- length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-E1, FR-E2, FR-E3, and FR-E4).

[0093] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Eazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745.” (“Contact” numbering scheme); Eefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657- 70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme).

[0094] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis ofcomplex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.

[0095] Table 1, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR-L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD2 - Al-Lazikani et al., (1997) JMB 273,927-948

[0096] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes or other known schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes,although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.

[0097] Likewise, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR- Hl, FR-H2, FR-H3, FR-H4), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method or other known schemes. In other cases, the particular amino acid sequence of a CDR or FR is given.

[0098] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable regions of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6thed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VH or VL domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0099] Among the antibodies included in the provided CARs are antibody fragments. An “antibody fragment” or “antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; heavy chain variable (VH) regions, single-chain antibody molecules such as scFvs and single-domain antibodies comprising only the VH region; and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding domain in the provided CARs is or comprises an antibody fragment comprising a variable heavy chain (VH) and a variable light chain (VL) region. In particular embodiments, the antibodies are single-chain antibody fragments comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, such as scFvs.

[0100] Single-domain antibodies (sdAbs) are antibody fragments comprising all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of anantibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.

[0101] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.

[0102] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0103] Among the anti-GPRC5D antibodies such as antibody fragments included in the provided CARs are human antibodies. A “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non- human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries. The term excludes humanized forms of non- human antibodies comprising non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.

[0104] Among the antibodies included in the provided CARs are those that are monoclonal antibodies, including monoclonal antibody fragments. The term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. Incontrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. The term is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody may be made by a variety of techniques, including but not limited to generation from a hybridoma, recombinant DNA methods, phage-display and other antibody display methods.

[0105] In some embodiments, the CAR includes a GPRC5D-binding portion or portions of the antibody molecule, such as a heavy chain variable (VH) region and / or light chain variable (VL) region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the provided GPRC5D-binding CARs contain an antibody, such as an anti-GPRC5D antibody, or an antigen-binding fragment thereof that confers the GPRC5D-binding properties of the provided CAR. In some embodiments, the antibody or antigen-binding domain can be any anti-GPRC5D antibody described or derived from any anti-GPRC5D antibody described (see, e.g., WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245, WO 2023 / 134718). Any of such anti-GPRC5D antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the anti-GPRC5D CAR contains an antigen-binding domain comprising a variable heavy (VH) and / or a variable light (VL) region derived from an antibody described in WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245, or WO 2023 / 134718.

[0106] In some embodiments, the antibody, e.g., the anti-GPRC5D antibody, or antigenbinding fragment, contains a heavy and / or light chain variable (VH or VL) region sequence as described, or a sufficient antigen-binding portion thereof. In some embodiments, the anti- GPRC5D antibody, e.g., antigen-binding fragment, contains a VH region sequence or sufficient antigen-binding portion thereof that contains a CDR-H1, CDR-H2 and / or CDR-H3 as described. In some embodiments, the anti-GPRC5D antibody, e.g., antigen-binding fragment, contains a VL region sequence or sufficient antigen-binding portion that contains a CDR-L1, CDR-L2 and / or CDR-L3 as described. In some embodiments, the anti-GPRC5D antibody, e.g., antigen-binding fragment, contains a VH region sequence that contains a CDR-H1, CDR-H2 and / or CDR-H3 as described and contains a VL region sequence that contains a CDR-L1, CDR-L2 and / or CDR-L3 as described. Also among the antibodies are those having sequences at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identical to such a sequence.

[0107] In some embodiments, the antibody or antibody fragment, in the provided CAR, has a VH region of any of the antibodies or antibody binding fragments described in any of WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245, and WO 2023 / 134718.

[0108] In some embodiments, the CAR contains an antibody or antigen-binding fragment thereof, that has a heavy chain variable (VH) region having the amino acid sequence selected from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13, or an amino acid sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the VH region amino acid selected from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13, or contains a CDR-H1, CDR-H2, and / or CDR-H3 present in such a VH sequence.

[0109] In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2, and / or CDR-H3 according to Chothia numbering. In some embodiments, the VH region of an antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR- H2, and / or CDR-H3 according to AbM numbering.

[0110] In some embodiments, the CAR contains an antibody or antigen-binding fragment thereof, that has a variable heavy chain (VH) region comprising a CDR-H1 comprising the amino acid sequence selected from SEQ ID Nos: 15, 18, 20, 22, 30, 33, 35, 37, 45, 48, 50, 52, 60, 63, 65, 67, 75, 78, 80, 82, 92, 102, 105, 107, and 109; (b) a CDR-H2 comprising the amino acid sequence selected from SEQ ID Nos: 16, 19, 21, 23, 31, 34, 36, 38, 46, 49, 51, 53, 61, 64, 66, 68, 76, 79, 81, 83, 90, 93, 94, 95, 103, 106, 108, and 110; and (c) a CDR-H3 comprising the amino acid sequence selected from SEQ ID Nos: 17, 24, 32, 39, 47, 54, 62, 69, 77, 84, 91, 96, 104, and 111.

[0111] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequence of SEQ ID Nos: 15, 16 and 17, respectively; SEQ ID Nos: 18, 19, and 17, respectively; SEQ ID Nos:20, 21 and 17, respectively; SEQ ID Nos:22, 23 and 24, respectively; SEQ ID Nos:30, 31, and 32, respectively; SEQ ID Nos:33, 34 and 32, respectively; SEQ ID Nos:35, 36 and 32, respectively; SEQ ID Nos:37, 38 and 39, respectively; SEQ ID Nos:45, 46 and 47, respectively; SEQ ID Nos:48, 49 and 47, respectively; SEQ ID Nos:50, 51 and 47, respectively; SEQ ID Nos:52, 53 and 54, respectively; SEQ ID Nos:60, 61 and 62, respectively; SEQ ID Nos:63, 64and 62, respectively; SEQ ID Nos:65, 66 and 62, respectively; SEQ ID Nos:67, 68 and 69, respectively; SEQ ID Nos:75, 76 and 77, respectively; SEQ ID Nos:78, 79 and 77, respectively; SEQ ID Nos:80, 81 and 77, respectively; SEQ ID Nos:82, 83 and 84, respectively; SEQ ID Nos:75, 90 and 91, respectively; SEQ ID Nos:92, 93 and 91, respectively; SEQ ID Nos:80, 94 and 91, respectively; SEQ ID Nos:82, 95 and 96, respectively; SEQ ID Nos: 102, 103 and 104, respectively; SEQ ID Nos: 105, 106 and 104, respectively; SEQ ID Nos: 107, 108 and 104, respectively; or SEQ ID Nos: 109, 110 and 111, respectively.

[0112] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence of SEQ ID Nos: 15, 16 and 17, respectively; SEQ ID Nos: 18, 19 and 17, respectively; SEQ ID Nos:20, 21 and 17, respectively; SEQ ID Nos:22, 23 and 24, respectively; SEQ ID Nos:30, 31 and 32, respectively; SEQ ID Nos:33, 34 and 32, respectively; SEQ ID Nos:35, 36 and 32, respectively; SEQ ID Nos:37, 38 and 39, respectively; SEQ ID Nos:45, 46 and 47, respectively; SEQ ID Nos:48, 49 and 47, respectively; SEQ ID Nos:50, 51 and 47, respectively; SEQ ID Nos:52, 53 and 54, respectively; SEQ ID Nos:60, 61 and 62, respectively; SEQ ID Nos:63, 64 and 62, respectively; SEQ ID Nos:65, 66 and 62, respectively; SEQ ID Nos:67, 68 and 69, respectively; SEQ ID Nos:75, 76 and 77, respectively; SEQ ID Nos:78, 79 and 77, respectively; SEQ ID Nos:80, 81 and 77, respectively; SEQ ID Nos:82, 83 and 84, respectively; SEQ ID Nos:75, 90 and 91, respectively; SEQ ID Nos:92, 93 and 91, respectively; SEQ ID Nos:80, 94 and 91, respectively; SEQ ID Nos:82, 95 and 96, respectively; SEQ ID Nos: 102, 103 and 104, respectively; SEQ ID Nos: 105, 106 and 104, respectively; SEQ ID Nos: 107, 108 and 104, respectively; SEQ ID Nos: 109, 110 and 111, respectively.

[0113] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, CDR-H2 and CDR-H3, respectively, comprising the amino acid sequence of a CDR- Hl, a CDR-H2, and a CDR-H3 contained within the VH region amino acid sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13.

[0114] In some embodiments of the antibody or antigen-binding fragment thereof provided herein, the VH region comprises any of the CDR-H1, CDR-H2 and CDR-H3 as described and comprises a framework region 1 (FR1), a FR2, a FR3 and / or a FR4 having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity, respectively, to a FR1, a FR2, a FR3 and / or a FR4 contained within the VH region amino acid sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region comprising the amino acid sequence set forth in any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13.

[0116] In some embodiments, the antibody or antibody fragment, in the provided CAR (e.g. an anti-GPRC5D CAR), comprising a VH region further comprises a light chain or a sufficient antigen binding portion thereof. For example, in some embodiments, the antibody or antigenbinding fragment thereof contains a VH region and a VL region, or a sufficient antigen-binding portion of a VH and VL region. In such embodiments, a VH region sequence can be any of the above described VH sequence. In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or an scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

[0117] In some embodiments, the antibody or antigen-binding fragment has a VL region described in any of WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245, and WO 2023 / 134718.

[0118] In some embodiments, the CAR contains an antibody or antigen-binding fragment thereof, that has a light chain variable (VL) region having the amino acid sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122, or an amino acid sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the VL region amino acid selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122, or contains a CDR-L1, CDR- L2, and / or CDR-L3 present in such a VL sequence. In some embodiments, the CAR contains an antibody or antigen-binding fragment thereof, that has a light chain variable (VL) region having the amino acid sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14, or an amino acid sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the VL region amino acid selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14, or contains a CDR-L1, CDR-L2, and / or CDR-L3 present in such a VL sequence. In some embodiments, the CAR contains an antibody or antigen-binding fragment thereof, that has a light chain variable (VL) region having the amino acid sequence selected from any one of SEQ ID Nos: 116, 117, 118, 119, 120, 121, or 122, or an amino acid sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about99% sequence identity to the VL region amino acid selected from any one of SEQ ID Nos: 116, 117, 118, 119, 120, 121, or 122, or contains a CDR-L1, CDR-L2, and / or CDR-L3 present in such a VL sequence.

[0119] In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and / or CDR-L3 according to Kabat numbering. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR-L2, and / or CDR-L3 according to Chothia numbering. In some embodiments, the VL region of an antibody or antigen-binding fragment thereof comprises a CDR-L1, CDR- L2, and / or CDR-L3 according to AbM numbering.

[0120] In some embodiments, the CAR contains an antibody or antigen-binding fragment thereof, that has a variable light chain (VL) region comprising a CDR-L1 comprising the amino acid sequence selected from SEQ ID Nos: 25, 28, 40, 43, 55, 58, 70, 73, 85, 88, 97, 100, 112, and 114; (b) a CDR-L2 comprising the amino acid sequence selected from SEQ ID Nos: 26, 29, 41, 44, 56, 59, 71, 74, 86, 89, 98, and 101; and (c) a CDR-L3 comprising the amino acid sequence selected from SEQ ID Nos: 27, 42, 57, 72, 87, 99, 113, and 115.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL region comprising a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequence of SEQ ID Nos:25, 26 and 27, respectively; SEQ ID Nos:28, 29 and 27, respectively; SEQ ID Nos:40, 41 and 42, respectively; SEQ ID Nos:43, 44 and 42, respectively; SEQ ID Nos:55, 56 and 57, respectively; SEQ ID Nos:58, 59 and 57, respectively; SEQ ID Nos:70, 71 and 72, respectively; SEQ ID Nos:73, 74 and 72, respectively; SEQ ID Nos:85, 86 and 87, respectively; SEQ ID Nos:88, 89 and 87, respectively; SEQ ID Nos:97, 98 and 99, respectively; SEQ ID Nos: 100, 101 and 99, respectively; SEQ ID Nos: 112, 26 and 113, respectively; SEQ ID Nos: 114, 29 and 115, respectively; SEQ ID Nos: 114, 29 and 123, respectively.

[0122] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL region comprising the amino acid sequence of SEQ ID Nos:25, 26 and 27, respectively; SEQ ID Nos:28, 29 and 27, respectively; SEQ ID Nos:40, 41 and 42, respectively; SEQ ID Nos:43, 44 and 42, respectively; SEQ ID Nos:55, 56 and 57, respectively; SEQ ID Nos:58, 59 and 57, respectively; SEQ ID Nos:70, 71 and 72, respectively; SEQ ID Nos:73, 74 and 72, respectively; SEQ ID Nos:85, 86 and 87, respectively; SEQ ID Nos:88, 89 and 87, respectively; SEQ ID Nos:97, 98 and 99, respectively; SEQ ID Nos: 100, 101 and 99, respectively; SEQ ID Nos: 112, 26 and 113, respectively; SEQ ID Nos: 114, 29 and 115, respectively; SEQ ID Nos: 114, 29 and 123, respectively.

[0123] In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, or 14. In some embodiments, the antibody or antigen-binding fragment thereof contains a CDR-L1, CDR-L2, and CDR-L3, respectively, contained within the VL region amino acid sequence selected from any one of SEQ ID Nos: 116, 117, 118, 119, 120, 121, or 122.

[0124] Among the CARs provided herein is a CAR in which the antibody, such as an anti- GPRC5D antibody, or antibody fragment, in the provided CAR, comprises a VH region amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence set forth in any of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13 and a VL region comprising an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120,121, or 122.

[0125] In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, a CDR-H3, respectively, comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence selected from any one of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13; and comprises a CDR- Ll, a CDR-L2, a CDR-L3, respectively, comprising the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3, respectively contained within the VL region amino acid sequence selected from any one of SEQ ID Nos: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or122.

[0126] In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprise the amino acid sequence of SEQ ID Nos: 1, 3, 5, 7, 9, 11, or 13 and the VL region of the antibody or antigen-binding fragment comprises the amino acid sequence 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122. In some embodiments, the VH and VL regions of the antibody or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID Nos: 1 and 2, respectively; SEQ ID Nos: 3 and 4, respectively; SEQ ID Nos: 5 and 6,respectively; SEQ ID Nos: 7 and 8, respectively; SEQ ID Nos: 9 and 10, respectively; SEQ ID Nos: 11 and 12, respectively; or SEQ ID Nos: 13 and 14, respectively, or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In other examples, the VH and VL regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID Nos: 1 and 116; SEQ ID Nos: 3 and 117; SEQ ID Nos: 5 and 118; SEQ ID Nos: 7 and 119; SEQ ID Nos: 9 and 120; SEQ ID Nos: 11 and 121; or SEQ ID Nos: 13 and 122, respectively or any antibody or antigen-binding fragment thereof that has at least 90% sequence identity to any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0127] For example, the VH and VL regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID Nos: 1 and 2; SEQ ID Nos: 3 and 4; SEQ ID Nos: 5 and 6; SEQ ID Nos: 7 and 8; SEQ ID Nos: 9 and 10; SEQ ID Nos: 11 and 12; or SEQ ID Nos: 13 and 14, respectively. In other examples, the VH and VL regions of the antibody or antigen-binding fragment thereof provided therein comprise the amino acid sequences selected from: SEQ ID Nos: 1 and 116; SEQ ID Nos: 3 and 117; SEQ ID Nos: 5 and 118; SEQ ID Nos: 7 and 119; SEQ ID Nos: 9 and 120; SEQ ID Nos: 11 and 121; or SEQ ID Nos: 13 and 122, respectively.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof, in the provided CAR, is a single-chain antibody fragment, such as a single chain variable fragment (scFv) or a diabody or a single domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the VH region. In some embodiments, the antibody or antigen binding fragment is an scFv comprising a heavy chain variable (VH) region and a light chain variable (VL) region. In some embodiments, the singlechain antibody fragment (e.g., scFv) includes one or more linkers joining two antibody domains or regions, such as a heavy chain variable (VH) region and a light chain variable (VL) region. The linker typically is a peptide linker, e.g., a flexible and / or soluble peptide linker. Among the linkers are those rich in glycine and serine and / or in some cases threonine. In some embodiments, the linkers further include charged residues such as lysine and / or glutamate, which can improve solubility. In some embodiments, the linkers further include one or more proline.

[0129] Accordingly, the provided CARs contain anti-GPRC5D antibodies that include single-chain antibody fragments, such as scFvs and diabodies, particularly human single-chain antibody fragments, typically comprising linker(s) joining two antibody domains or regions, such VH and VL regions. The linker typically is a peptide linker, e.g., a flexible and / or soluble peptide linker, such as one rich in glycine and serine.

[0130] In some aspects, the linkers rich in glycine and serine (and / or threonine) include at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% such amino acid(s). In some embodiments, they include at least at or about 50%, 55%, 60%, 70%, or 75%, glycine, serine, and / or threonine. In some embodiments, the linker is comprised substantially entirely of glycine, serine, and / or threonine. The linkers generally are between about 5 and about 50 amino acids in length, typically between at or about 10 and at or about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO: 125) or GGGS (3GS; SEQ ID NO: 126), such as between 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of a sequence set forth in SEQ ID NO: 127 (GGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 128 (GSTSGSGKPGSGEGSTKG). Exemplary linkers further include those having or consisting of the sequence set forth in SEQ ID NO: 129 (SRGGGGSGGGGSGGGGSLEMA). An exemplary linker includes those having or consisting of the sequence set forth in SEQ ID NO: 124 (GSRGGGGSGGGGSGGGGSLEMA).

[0131] Accordingly, in some embodiments, the provided embodiments include single-chain antibody fragments, e.g., scFvs, comprising one or more of the aforementioned linkers, such as glycine / serine rich linkers, including linkers having repeats of GGGS (SEQ ID NO: 126) or GGGGS (SEQ ID NO: 125), such as the linker set forth in SEQ ID NO: 124, 127 or 129.

[0132] In some embodiments, the VH region may be amino terminal to the VL region. In some embodiments, the VH region may be carboxy terminal to the VL region. In particular embedments, the fragment, e.g., scFv, may include a VH region or portion thereof, followed by the linker, followed by a VL region or portion thereof. In other embodiments, the fragment, e.g., the scFv, may include the VL region or portion thereof, followed by the linker, followed by the VH region or portion thereof.

[0133] In some aspects, an scFv of the extracellular antigen-binding domain of a CAR provided herein comprises the amino acid sequence selected from any one of SEQ ID Nos: 130-143, or has an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence selected from any one of SEQ ID Nos: 130-143.

[0134] In some aspects, an scFv of the extracellular domain of a CAR provided herein is encoded by the nucleotide sequence selected from any one of SEQ ID Nos: 144-157, or has a nucleotide sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the nucleotide sequence selected from any one of SEQ ID Nos: 144-157.

[0135] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:1; and contains a VL region comprising the sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:2. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:1 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:1; and contains a VL region comprising the sequence set forth in SEQ ID NO: 116 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 116. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 15, 16 and 17, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 25, 26, and 27, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 18, 19 and 17, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3comprising the amino acid sequence of SEQ ID NOS: 25, 26, and 27, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 20, 21 and 17, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 25, 26, and 27, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 22, 23 and 24, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 28, 29 and 27, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:1 and the VL region comprises the sequence set forth in SEQ ID NO:2. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:1 and the VL region comprises the sequence set forth in SEQ ID NO: 116. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 130 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 130. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 144 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 144. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 131 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 131. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 145 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 145.

[0136] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:3; and contains a VL region comprising thesequence set forth in SEQ ID NO:4 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:4. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:3; and contains a VL region comprising the sequence set forth in SEQ ID NO: 117 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 30, 31, 32, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS:40, 41 and 42, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 33, 34 and 32, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 40, 41 and 42, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 35, 36 and 32, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 40, 41 and 42, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 37, 38 and 39, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 43, 44 and 42, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:3 and the VL region comprises the sequence set forth in SEQ ID NO:4. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:3 and the VL region comprises the sequence set forth in SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 132 or a sequence of aminoacids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 132. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 146 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 146. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 133 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 133. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 147 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 147.

[0137] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:5 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:5; and contains a VL region comprising the sequence set forth in SEQ ID NO:6 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:6. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:5 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:5; and contains a VL region comprising the sequence set forth in SEQ ID NO: 118 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 118. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 45, 46, 47, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 55, 56 and 57, respectively. In some embodiments, theantibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 48, 49 and 47, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 55, 56 and 57, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 50, 51 and 47, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 55, 56 and 57, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 52, 53 and 54, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 58, 59 and 57, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:5 and the VL region comprises the sequence set forth in SEQ ID NO:6. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:5 and the VL region comprises the sequence set forth in SEQ ID NO: 118. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 134 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 134. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 148 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 148. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 135 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 135. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 149 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 149.

[0138] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:7 or anamino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and contains a VL region comprising the sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:8. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and contains a VL region comprising the sequence set forth in SEQ ID NO: 119 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 119. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 60, 61 and 62, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 70, 71 and 72, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 63, 64 and 62, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 70, 71 and 72, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 65, 66 and 62, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 70, 71 and 72, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 67, 68 and 69, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 73, 74 and 72, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:7 and the VL region comprises the sequence set forth in SEQ ID NO:8. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:7 and the VL region comprises thesequence set forth in SEQ ID NO: 119. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 136 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 136. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 150 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 150. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 137 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 137. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 151 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:151.

[0139] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:9; and contains a VL region comprising the sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 10. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:9; and contains a VL region comprising the sequence set forth in SEQ ID NO: 120 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 120. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has aCDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 75, 76 and 77, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 85, 86 and 87, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 78, 79 and 77, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 85, 86 and 87, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 80, 81 and 77, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 85, 86 and 87, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 82, 83 and 84, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 88, 89 and 87, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:9 and the VL region comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO:9 and the VL region comprises the sequence set forth in SEQ ID NO: 120. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 138 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 138. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 152 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 152. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 139 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 139. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 153 or a sequence of nucleotides at least at or about90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 153.

[0140] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 11; and contains a VL region comprising the sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 12. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 11; and contains a VL region comprising the sequence set forth in SEQ ID NO: 121 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:121. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 75, 90 and 91, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 97, 98 and 99, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 92, 93 and 91, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 97, 98 and 99, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 80, 94 and 91, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 97, 98 and 99, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 82, 95 and 96, respectively and a VL region that contains a CDRL1, a CDRL2and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 100, 101 and 99, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO: 11 and the VL region comprises the sequence set forth in SEQ ID NO: 12. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO: 11 and the VL region comprises the sequence set forth in SEQ ID NO: 121. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 140 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 140. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 154 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 154. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:141 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 141. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 155 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:155.

[0141] Among a provided anti-GPRC5D CAR is a CAR in which the antibody or antigenbinding fragment contains a VH region comprising the sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 13; and contains a VL region comprising the sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 14. In some embodiments, the provided CAR is a CAR in which the antibody or antigen-binding fragment contains a VH region comprising the sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about99% identity to SEQ ID NO: 13; and contains a VL region comprising the sequence set forth in SEQ ID NO: 122 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 122. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 102, 103 and 104, respectively and a VL region that has a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 112, 26, 113, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 105, 106 and 104, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 112, 26 and 113, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 107, 108 and 104, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 112, 26 and 113, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 109, 110, 111, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 114, 29 and 115, respectively. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains a VH region that has a CDRH1, a CDRH2 and a CDRH3 comprising the amino acid sequence of SEQ ID NOS: 109, 110, 111, respectively and a VL region that contains a CDRL1, a CDRL2 and a CDRL3 comprising the amino acid sequence of SEQ ID NOS: 114, 29 and 123, respectively. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO: 13 and the VL region comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, the VH region comprises the sequence set forth in SEQ ID NO: 13 and the VL region comprises the sequence set forth in SEQ ID NO: 122. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as an scFv. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 142 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 142. In some embodiments, the scFv isencoded by a nucleotide sequence set forth in SEQ ID NO: 156 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 156. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 143 or a sequence of amino acids at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 143. In some embodiments, the scFv is encoded by a nucleotide sequence set forth in SEQ ID NO: 157 or a sequence of nucleotides at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 157. b. Spacer

[0142] In some embodiments, the recombinant receptor such as a CAR comprising an antibody (e.g., antigen-binding fragment) provided herein, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof. In some embodiments, the portion of the immunoglobulin constant regon includes a hinge region, e.g., an IgG4 hinge region, and / or a CHI, CH2 or CH3 and / or Fc region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, such as antigen-binding domain e.g., scFv) and transmembrane domain. In some embodiments, the length of the spacer is adjusted to optimize the biophysical synapse distance between the CAR-expressing cell, such as a CAR-expressing cell, and the target of the CAR, such as a GPRC5D-expressing tumor cell. In some embodiments, the CAR is expressed by a T-cell, and the length of the spacer is adjusted to a length that is compatible for T-cell activation or to optimize CAR T-cell performance.

[0143] In some embodiments, the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer or as compared to an alternative spacer of a different length (e.g. shorter in length). In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. In some embodiments, the spacer is at least 100 amino acids in length, such as at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length. Exemplary spacers include those having at least about 10 to 300 amino acids,about 10 to 200 amino acids, about 50 to 175 amino acids, about 50 to 150 amino acids, about 10 to 125 amino acids, about 50 to 100 amino acids, about 100 to 300 amino acids, about 100 to 250 amino acids, about 125 to 250 amino acids, or about 200 to 250 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region is at least about 12 amino acids, at least about 119 amino acids or less, at least about 125 amino acids, at least about 200 amino acids, or at least about 220 amino acids, or at least about 225 amino acids in length.

[0144] In some embodiments, the spacer has a length of 125 to 300 amino acids in length, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length or 250 to 300 amino acids in length. In some embodiments, the spacer is at least or at least about or is or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids in length, or a length between any of the foregoing.

[0145] Exemplary spacers include an IgG hinge alone, an IgG hinge linked to one or more of a CH2 and CH3 domain, or IgG hinge linked to the CH3 domain. In some embodiments, the IgG hinge, CH2 and / or CH3 can be derived all or in part from IgG4 or IgG2, such as all or in part from humam IgG4 or human IgG2. In some embodiments, the spacer can be a chimeric polypeptide containing one or more of a hinge, CH2 and / or CH3 sequence(s) derived from IgG4, IgG2, and / or IgG2 and IgG4. In some embodiments, the hinge region comprises all or a portion of an IgG4 hinge region and / or of an IgG2 hinge region, wherein the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; the CH2 region comprises all or a portion of an IgG4 CH2 region and / or of an IgG2 CH2 region, wherein the IgG4 CH2 region is optionally a human IgG4 CH2 region and the IgG2 CH2 region is optionally a human IgG2 CH2 region; and / or the CH3 region comprises all or a portion of an IgG4 CH3 region and / or of an IgG2 CH3 region, wherein the IgG4 CH3 region is optionally a human IgG4 CH3 region and the IgG2 CH3 region is optionally a human IgG2 CH3 region. In some embodiments, the hinge, CH2 and CH3 comprises all or a portion of each of a hinge region, CH2 and CH3 from IgG4. In some embodiments, the hinge region is chimeric andcomprises a hinge region from human IgG4 and human IgG2; the CH2 region is chimeric and comprises a CH2 region from human IgG4 and human IgG2; and / or the CH3 region is chimeric and comprises a CH3 region from human IgG4 and human IgG2. In some embodiments, the spacer comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge comprising at least one amino acid replacement compared to human IgG4 hinge region; an human IgG2 / 4 chimeric CH2 region; and a human IgG4 CH3 region.

[0146] In some embodiments, the spacer can be derived all or in part from IgG4 and / or IgG2 and can contain mutations, such as one or more single amino acid mutations in one or more domains. In some examples, the amino acid modification is a substitution of a proline (P) for a serine (S) in the hinge region of an IgG4. In some embodiments, the amino acid modification is a substitution of a glutamine (Q) for an asparagine (N) to reduce glycosylation heterogeneity, such as an N177Q mutation at position 177, in the CH2 region, of the full-length IgG4 Fc sequence set forth in SEQ ID NO: 158 or an N176Q at position 176, in the CH2 region, of the full-length IgG2 Fc sequence set forth in SEQ ID NO: 159. In some embodiments, the spacer is or comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region. In some embodiments, the spacer is about 228 amino acids in length. In some embodiments, the spacer is set forth in SEQ ID NO: 162. In some embodiments, the spacer comprises the amino acid sequenceESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDG VEVHNAKTKPREEQFQSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 162)

[0147] In some embodiments, the spacer is encoded by a polynucleotide that has been optimized for codon expression and / or to eliminate splice sites such as cryptic splice sites. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 164. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 163. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 165. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO: 166.

[0148] Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-135, or international patent application publication number WO2014031687. In someembodiments, the nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity upon expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splice sites or reduce the likelihood of a splice event at a splice site.

[0149] In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 160, and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 167. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO:161. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 168. In some embodiments, the spacer has an amino acid sequence set forth in SEQ ID NO: 162, encoded by the polynucleotide sequence set forth in SEQ ID NO: 163, 164, 165 or 166 or a polynucleotide that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 163, 164, 165 or 166.

[0150] In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 162, encoded by a polynucleotide that has been optionally optimized for codon usage and / or to reduce RNA heterogeneity. Methods to reduce RNA heterogeneity, such as by removing cryptic splice donor and / or acceptor sites, are described below, such as in Section I.A.2.a.ii. Observations have shown that cryptic splice donor and / or acceptor sites are present in the spacer region of certain immunoglobulin spacers when present in a CAR. In some embodiments, the spacer in a provided CAR is encoded by a polynucleotide in which one or more cryptic splice donor and / or acceptor sites are eliminated and / or are modified to reduce heterogeneity of the RNA transcribed from the construct, such as mRNA, following expression in a cell. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 164 (also set forth in SEQ ID NO: 171). In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 165. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 166. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 172.

[0151] In some embodiments, the spacer contains a hinge domain from CD28. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 170, and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 169.

[0152] c. Transmembrane domain and intracellular signaling components

[0153] The antigen-recognition component generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. Thus, in some embodiments, a GPRC5D-binding molecule (e.g.. antibody or antigen binding fragment thereof) is linked to one or more transmembrane domains such as those described herein and intracellular signaling domains comprising one or more intracellular components such as those described herein. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0154] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane domains include those derived from (z.e. comprise at least the transmembrane domain(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and / or CD154. For example, the transmembrane domain can be a CD28 transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 173, encoded by the nucleic acid sequence set forth in SEQ ID NO: 174 or SEQ ID NO: 175. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s).

[0155] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet,is present and forms a linkage between the transmembrane domain and the intracellular signaling domain of the CAR.

[0156] The receptor, e.g., the CAR, generally includes an intracellular signaling region comprising at least one intracellular signaling component or components. In some embodiments, the receptor includes an intracellular component or signaling domain of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta (CD3-Q chain. Thus, in some aspects, the GPRC5D-binding antibody is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor y and CD8, CD4, CD25 or CD16.

[0157] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR stimulates and / or activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immuno stimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and / or any derivative or variant of such molecules, and / or any synthetic sequence that has the same functional capability.

[0158] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[0159] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling sequences), and those that act in an antigenindependent manner to provide a secondary or co- stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such classes of cytoplasmic signaling sequences.

[0160] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary stimulation and / or activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, the intracellular signaling region in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some embodiments the CD3 zeta comprises the sequence of amino acids set forth in SEQ ID NO: 176, encoded by the nucleic acid sequence set forth in SEQ ID NO: 177 or SEQ ID NO: 178.

[0161] In some embodiments, the CAR includes a signaling domain (e.g., an intracellular or cytoplasmic signaling domain) and / or transmembrane portion of a costimulatory molecule, such as a T cell costimulatory molecule. Exemplary costimulatory molecules include CD28, 4- IBB, 0X40, DAP10, and ICOS. For example, a costimulatory molecule can be derived from 4-1BB and can comprise the amino acid sequence set forth in SEQ ID NO: 179, encoded by the nucleotide sequence set forth in SEQ ID NO: 180 or SEQ ID NO: 181. In some aspects, the same CAR includes both the stimulatory or activating components (e.g., cytoplasmic signaling sequence) and costimulatory components.

[0162] In some embodiments, the stimulatory or activating components are included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, and costimulatory CARs, both expressed on the same cell (see WO 2014 / 055668). In some aspects, the GPRC5D-targeting CAR is the stimulatory or activating CAR; in other aspects, it is the costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than GPRC5D, whereby a stimulatory or an activating signal delivered through the GPRC5D -targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0163] In certain embodiments, the intracellular signaling region comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and 4- IBB (CD137; TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.

[0164] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and a stimulatory or an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3- zeta, CD28, and 4- IBB.

[0165] In some embodiments, provided embodiments of anti-GPRC5D CAR contains an extracellular antigen-binding domain containing any of the anti-GPRC5D antibody or antigenbinding fragments described herein, such as in Section I.A.l.a; a spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region, such as one that is about 228 amino acids in length, or a spacer set forth in SEQ ID NO: 162, such as encoded by the nucleotide sequence set forth in any of SEQ ID NOS: 163, 164, 165 or 166; a transmembrane domain, such as a transmembrane domain from a human CD28 ; and an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain and an intracellular signaling domain of a T cell costimulatory molecule. Also provided are polynucleotides encoding such a chimeric antigen receptor. In some embodiments, the transmembrane domain is or comprises the sequence set forth in SEQ ID NO: 173. In some embodiments, the intracellular signaling domain of a T cell costimulatory molecule is an intracellular signaling domain of human CD28, human 4- IBB or human ICOS or a signaling portion thereof. In particular embodiments, the intracellular signaling domain is an intracellular signaling domain of human 4-1BB. In some embodiments, the intracellular signaling domain is or comprises the sequence set forth in SEQ ID NO: 179. In some embodiments, the cytoplasmic signaling domain is a human CD3-zeta cytoplasmic signaling domain, such as set forth in SEQ ID NO: 176. In some embodiments, the intracellular signaling region comprises the sequences set forth in SEQ ID NO: 176 and SEQ ID NO: 179.

[0166] In some embodiments, provided embodiments of an anti-GPRC5D CARs has an amino acid sequence set forth in SEQ ID NO: 183, or an amino acid sequence that is at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 183. In some embodiments, provided embodiments of an anti-GPRC5D CAR is encoded by the nucleotide sequence set forth in SEQID NO: 182 or a nucleotide sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to the sequence set forth in any of SEQ ID NO: 182.

[0167] In some embodiments, provided embodiments of an anti-GPRC5D CARs has an amino acid sequence set forth in SEQ ID NO:208, or an amino acid sequence that is at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:208. In some embodiments, provided embodiments of an anti-GPRC5D CAR is encoded by the nucleotide sequence set forth in SEQ ID NO:209 or a nucleotide sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to the sequence set forth in any of SEQ ID NO:209.2. Nucleic Acids, Vectors and Methods for Genetic Engineering

[0168] In some embodiments, the cells, e.g., T cells, are genetically engineered to express a recombinant receptor. In some embodiments, the engineering is carried out by introducing polynucleotides that encode the recombinant receptor. Also provided are polynucleotides encoding a recombinant receptor, and vectors or constructs containing such nucleic acids and / or polynucleotides.

[0169] In some cases, the nucleic acid sequence encoding the recombinant receptor contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some cases, the nucleic acid sequence encoding the recombinant receptor, e.g., chimeric antigen receptor (CAR) contains a signal sequence that encodes a signal peptide.

[0170] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter that is operatively linked to control expression of the recombinant receptor. In some examples, the polynucleotide contains two, three, or more promoters operatively linked to control expression of the recombinant receptor.

[0171] In certain cases where nucleic acid molecules encode two or more different polypeptide chains, e.g., a recombinant receptor and a marker, each of the polypeptide chainscan be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, and each can be individually transferred or introduced into the cell for expression in the cell. In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are operably linked to the same promoter and are optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a self-cleaving peptide or a peptide that causes ribosome skipping, which optionally is a T2A, a P2A, an E2A or an F2A. In some embodiments, the nucleic acids encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the genome of the cell. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, such as by retroviral transduction, transfection, or transformation.

[0172] In some embodiments, such as those where the polynucleotide contains a first and second nucleic acid sequence, the coding sequences encoding each of the different polypeptide chains can be operatively linked to a promoter, which can be the same or different. In some embodiments, the nucleic acid molecule can contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tricistronic, see e.g., U.S. Patent No. 6,060,273). In some embodiments, transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products ((e.g. encoding the marker and encoding the recombinant receptor) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding the marker and encoding the recombinant receptor) separated from one another by sequences encoding a selfcleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins. In some cases, the peptide, such as a T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe, Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and system disclosed herein, without limitation, 2A sequencesfrom the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A) as described in U.S. Patent Publication No.20070116690.

[0173] Any of the recombinant receptors described herein can be encoded by polynucleotides containing one or more nucleic acid sequences encoding recombinant receptors, in any combinations or arrangements. For example, one, two, three or more polynucleotides can encode one, two, three or more different polypeptides, e.g., recombinant receptors. In some embodiments, one vector or construct contains a nucleic acid sequence encoding marker, and a separate vector or construct contains a nucleic acid sequence encoding a recombinant receptor, e.g., CAR. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is present downstream of the nucleic acid encoding the marker.

[0174] In some embodiments, the vector backbone contains a nucleic acid sequence encoding one or more marker(s). In some embodiments, the one or more marker(s) is a transduction marker, surrogate marker and / or a selection marker.

[0175] In some embodiments, the marker is a transduction marker or a surrogate marker. A transduction marker or a surrogate marker can be used to detect cells that have been introduced with the polynucleotide, e.g., a polynucleotide encoding a recombinant receptor. In some embodiments, the transduction marker can indicate or confirm modification of a cell. In some embodiments, the surrogate marker is a protein that is made to be co-expressed on the cell surface with the recombinant receptor, e.g. CAR. In particular embodiments, such a surrogate marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the surrogate marker is encoded on the same polynucleotide that encodes the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operably linked to a nucleic acid sequence encoding a marker, optionally separated by an internal ribosome entry site (IRES), or a nucleic acid encoding a selfcleaving peptide or a peptide that causes ribosome skipping, such as a 2A sequence, such as a T2A, a P2A, an E2A or an F2A. Extrinsic marker genes may in some cases be utilized in connection with engineered cell to permit detection or selection of cells and, in some cases, also to promote cell suicide.

[0176] Exemplary surrogate markers can include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and to not transduce or are notcapable of transducing a signal or a signal ordinarily transduced by the full-length form of the cell surface polypeptide, and / or do not or are not capable of internalizing. Exemplary truncated cell surface polypeptides including truncated forms of growth factors or other receptors such as a truncated human epidermal growth factor receptor 2 (tHER2), a truncated epidermal growth factor receptor (tEGFR, exemplary tEGFR sequence set forth in SEQ ID NO:210 or 211) or a prostate-specific membrane antigen (PSMA) or modified form thereof. tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the tEGFR construct and an encoded exogenous protein, and / or to eliminate or separate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434). In some aspects, the marker, e.g. surrogate marker, includes all or part (e.g., truncated form) of CD34, a NGFR, a CD19 or a truncated CD19, e.g., a truncated non-human CD19, or epidermal growth factor receptor (e.g., tEGFR).

[0177] In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), P-galactosidase, chloramphenicol acetyltransferase (CAT), P-glucuronidase (GUS) or variants thereof.

[0178] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene confers antibiotic resistance to a mammalian cell. In some embodiments, the selection marker is or comprises a Puromycin resistance gene, a Hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.

[0179] In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.

[0180] In some embodiments, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and / or dampen responses of the cells upon adoptive transfer and encounter with ligand.

[0181] In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., a T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in PCT Pub. No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 210 or 211 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 210 or 211.

[0182] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy, 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp. Hematol., 28(10): 1137-46; Alonso- Camino et al. (2013) Mol. Ther. Nucl. Acids., 2, e93; Park et al., Trends Biotechnol., 2011 November 29(11): 550-557.

[0183] In some embodiments, the vector is an adeno-associated virus (AAV).

[0184] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning thatthey are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and / or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0185] In some embodiments, the cells, e.g., T cells, may be transfected either during or after expansion e.g. with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection for the introduction of the gene of the desired receptor can be carried out with any suitable retroviral vector, for example. The genetically modified cell population can then be liberated from the initial stimulus (the anti-CD3 / anti-CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus e.g. via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in form of a peptide / MHC molecule, the cognate (cross-linking) ligand of the genetically introduced receptor (e.g. natural ligand of a CAR) or any ligand (such as an antibody) that directly binds within the framework of the new receptor (e.g. by recognizing constant regions within the receptor). See, for example, Cheadle et al, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0186] In some cases, a vector may be used that does not require that the cells, e.g., T cells, are activated. In some such instances, the cells may be selected and / or transduced prior to activation. Thus, the cells may be engineered prior to, or subsequent to culturing of the cells, and in some cases at the same time as or during at least a portion of the culturing.

[0187] Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and / or function of transferred cells; genes to provide a genetic marker for selection and / or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also the publications of PCT / US91 / 08442 and PCT / US 94 / 05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., US Patent No. 6,040,177, at columns 14-17.a. Polynucleotides

[0188] Also provided are polynucleotides encoding the chimeric antigen receptors and / or portions, e.g., chains, thereof. Among the provided polynucleotides are those encoding the anti- GPRC5D chimeric antigen receptors (e.g., antigen-binding fragment) described herein. The polynucleotides may include those encompassing natural and / or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications. The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.

[0189] In some cases, the polynucleotide encoding the GPRC5D-binding receptor contains a signal sequence that encodes a signal peptide, in some cases encoded upstream of the nucleic acid sequences encoding the GPRC5D-binding receptor, or joined at the 5’ terminus of the nucleic acid sequences encoding the antigen-binding domain. In some cases, the polynucleotide containing nucleic acid sequences encoding the GPRC5D-binding receptor, e.g., chimeric antigen receptor (CAR), contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptide include a signal peptide of the IgG kappa chain set forth in SEQ ID NO: 185, or encoded by the nucleotide sequence set forth in SEQ ID NO: 184or 186-189. In some aspects, a non-limiting exemplary signal peptide includes a signal peptide of a GMCSFR alpha chain set forth in SEQ ID NO: 191 and encoded by the nucleotide sequence set forth in SEQ ID NO: 190. In some aspects, a non-limiting exemplary signal peptide includes a signal peptide of a CD8 alpha signal peptide set forth in SEQ ID NO: 192. In some aspects, a non-limiting exemplary signal peptide includes a signal peptide of a CD33 signal peptide set forth in SEQ ID NO: 193. In some cases, the polynucleotide encoding the GPRC5D- binding receptor can contain nucleic acid sequence encoding additional molecules, such as a surrogate marker or other markers, or can contain additional components, such as promoters, regulatory elements and / or multicistronic elements. In some embodiments, the nucleic acid sequence encoding the GPRC5D-binding receptor can be operably linked to any of the additional components.

[0190] Also provided herein are polynucleotide constructs encoding a CAR that are codon diverged. In some embodiments, a polynucleotide construct encodes a CAR capable of binding GPRC5D, such as any CAR as described herein. In some embodiments, cells express an anti- GPRC5D CAR as a therapeutic agent against multiple myeloma plasma cells. In some embodiments, the polynucleotide constructs are codon diverged to improve expression the CAR encoded by the polynucleotide.

[0191] In some embodiments, among CARs provided herein are those encoded by polynucleotides that are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and / or to modify, e.g., increase or render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor. In some embodiments, polynucleotides, encoding GPRC5D-binding cell surface proteins, are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, polynucleotides, encoding GPRC5D-binding cell surface proteins, are codon optimized, such as for expression in a mammalian, e.g., human, cell, such as in a human T cell. In some aspects, the modified polynucleotides result in in improved, e.g., increased or more uniform or more consistent level of, expression, e.g., surface expression, when expressed in a cell. Such polynucleotides can be utilized in constructs for generation of engineered cells that express the encoded GPRC5D- binding cell surface protein. Thus, also provided are cells expressing the recombinant receptors encoded by the polynucleotides provided herein and uses thereof in adoptive cell therapy, such as treatment of diseases and disorders associated with GPRC5D expression, e.g., multiple myeloma.

[0192] Also provided are cells, such as T cells, engineered to express a polynucleotide encoding a provided polynucleotide, including polynucleotides encoding a CAR, and compositions containing such cells. In some embodiments, the polynucleotide constructs are codon optimized for expression in a human cell. In some embodiments, one or more splice donor and / or acceptor sites in a polynucleotide construct is modified to reduce heterogeneity of the RNA transcribed from the construct, such as mRNA, following expression in a cell.(i) Codon Optimization

[0193] In some embodiments the polynucleotides are modified by optimization of the codons for expression in humans. In some aspects, codon optimization can be considered before and / or after the steps for splice site identification and / or splice site elimination, and / or at each ofthe iterative steps for reducing RNA heterogeneity. Codon optimization generally involves balancing the percentages of codons selected with the abundance, e.g., published abundance, of human transfer RNAs, for example, so that none is overloaded or limiting. In some cases, such balancing is necessary or useful because most amino acids are encoded by more than one codon, and codon usage generally varies from organism to organism. Differences in codon usage between transfected or transduced genes or nucleic acids and host cells can have effects on protein expression from the nucleic acid molecule. In some embodiments, to generate codon- optimized nucleic acid sequences, codons are chosen to select for those codons that are in balance with human usage frequency. The redundancy of the codons for amino acids is such that different codons code for one amino acid. In selecting a codon for replacement, it is desired that the resulting mutation is a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon (e.g., at the third position) can remain unchanged without affecting the amino acid sequence.(ii) Splice Sites

[0194] Provided herein are polynucleotides in which one or more potential splice donor and / or splice acceptor sites have been identified and the nucleic acid sequence at or near the one or more of the identified splice donor sites has been modified. In some embodiments, the resulting modified nucleic acid sequence(s) is / are then synthesized and used to transduce cells to test for splicing as indicated by RNA heterogeneity.

[0195] Also provided here are polynucleotides, such as those encoding any of the antibodies, receptors (such as antigen receptors such as chimeric antigen receptors) and / or GPRC5D- specific binding proteins provided herein, that are or have been modified to reduce heterogeneity or contain one or more nucleic acid sequences observed herein (such as by the optimization methods) to result in improved features of the polypeptides, such as the CARs, as compared to those containing distinct, reference, sequences or that have not been modified. Among such features include improvements in RNA heterogeneity, such as that resulting from the presence of one or more splice sites, such as one or more cryptic splice sites, and / or improved expression and / or surface expression of the encoded protein, such as increased levels, uniformity, or consistency of expression among cells or different therapeutic cell compositions engineered to express the polypeptides.

[0196] Also provided are polynucleotides that have been modified to eliminate splice sites, such as cryptic splice sites. Genomic nucleic acid sequences generally, in nature, in amammalian cell, undergo processing co-transcriptionally or immediately following transcription, wherein a nascent precursor messenger ribonucleic acid (pre-mRNA), transcribed from a genomic deoxyribonucleic acid (DNA) sequence, is in some cases edited by way of splicing, to remove introns, followed by ligation of the exons in eukaryotic cells. Consensus sequences for splice sites are known, but in some aspects, specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on the standard consensus sequences and are variably activated. Hence, variable splicing of pre-mRNA at cryptic splice sites leads to heterogeneity in the transcribed mRNA products upon expression in eukaryotic cells.

[0197] Polynucleotides generated for the expression of transgenes are typically constructed from nucleic acid sequences, such as complementary DNA (cDNA), or portions thereof, that do not contain introns. Thus, splicing of such sequences is not expected to occur. However, the presence of cryptic splice sites within the cDNA sequence can lead to unintended or undesired splicing reactions and heterogeneity in the transcribed mRNA. Such heterogeneity results in translation of unintended protein products, such as truncated protein products with variable amino acid sequences that exhibit modified expression and / or activity.

[0198] In some embodiments, eliminating splice sites, such as cryptic splice sites, can improve or optimize expression of a transgene product, such as a polypeptide translated from the transgene, such as an anti-GPRC5D CAR polypeptide. Splicing at cryptic splice sites of an encoded transgene, such as an encoded GPRC5D CAR molecule, can lead to reduced protein expression, e.g., expression on cell surfaces, and / or reduced function, e.g., reduced intracellular signaling. Provided herein are polynucleotides, encoding anti-GPRC5D CAR proteins that have been optimized to reduce or eliminate cryptic splice sites. Also provided herein are polynucleotides encoding anti-GPRC5D CAR proteins that have been optimized for codon expression and / or in which one or more sequence, such as one identified by the methods or observations herein regarding splice sites, is present, and / or in which an identified splice site, such as any of the identified splice sites herein, is not present. Among the provided polynucleotides are those exhibiting below a certain degree of RNA heterogeneity or splice forms when expressed under certain conditions and / or introduced into a specified cell type, such as a human T cell, such as a primary human T cell, and cells and compositions and articles of manufacture containing such polypeptides and / or exhibiting such properties. In some embodiments, the RNA heterogeneity of transcribed RNA is reduced by greater than or greaterthan about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more compared to a polynucleotide that has not been modified to remove cryptic splice sites and / or by codon optimization. In some embodiments, the provided polynucleotides encoding an anti-GPRC5D CAR exhibit RNA homogeneity of transcribed RNA that is at least 70%, 75%, 80%, 85%, 90%, or 95% or greater.

[0199] RNA heterogeneity can be determined by any of a number of methods provided herein or described or known. In some embodiments, RNA heterogeneity of a transcribed nucleic acid is determined by amplifying the transcribed nucleic acid, such as by reverse transcriptase polymerase chain reaction (RT-PCR) followed by detecting one or more differences, such as differences in size, in the one or more amplified products. In some embodiments, the RNA heterogeneity is determined based on the number of differently sized amplified products, or the proportion of various differently sized amplified products. In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is harvested from cells, expressing the transgene to be optimized, and amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using a primer specific to the 5' untranslated region (5' UTR), in some cases corresponding to a portion of the promoter sequence in the expression vector, located upstream of the transgene in the transcribed RNA, and a primer specific to the 3' untranslated region (3' UTR), located downstream of the expressed transgene in the transcribed RNA sequence or a primer specific to a sequence within the transgene. In particular embodiments, at least one primer complementary to a sequence in the 5' untranslated region (UTR) and at least one primer complementary to a sequence in the 3' untranslated region (UTR) are employed to amplify the transgene. The skilled artisan can resolve RNA, such as messenger RNA, and analyze the heterogeneity thereof by several methods. Non-limiting, exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field flow fractionation, and chromatography, such as size exclusion chromatography or liquid chromatography.

[0200] In some aspects, the presence of potential cryptic splice sites (splice donor and / or acceptor sites that are present in a transcript, such as a transgene transcript, can result in RNA heterogeneity of the transcript following expression in a cell. In some embodiments, the one or more potential splice sites that can be present in the transgene transcript, that are not desired and / or that may be created in a transgene transcript from various underlying sequences are identified, following codon optimization of a transcript and / or by mutation or mistake or error in transcription. In some aspects of the provided embodiments, the splice donor sites and spliceacceptor sites are identified independently. In some embodiments, the splice acceptor and / or donor site(s) is / are canonical, non-canonical, and / or cryptic splice acceptor and / or donor site(s).

[0201] In some embodiments, one or more potential splice site (e.g., canonical, non- canonical, and / or cryptic splice acceptor and / or donor site(s) or branch sites) in a polynucleotide, such as a polynucleotide encoding a transgene, such as a recombinant receptor, that may exhibit RNA heterogeneity, are identified and / or modified. Also provided are polypeptides having reduced numbers of such splice sites as compared to such reference polynucleotides.

[0202] In some aspects, identification of the one or more splice sites in a nucleic acid sequence is an iterative process. In some embodiments, splice sites can be identified using a splice site and / or codon optimization prediction tool, such as by submitting the starting or reference sequence encoding the transgene, such as a GPRC5D-binding receptor, e.g., anti- GPRC5D, to a database, a gene synthesis vendor or other source able to computationally or algorithmically compare the starting or reference sequence to identify or predict splice sites and / or for codon optimization and / or splice site removal. In some embodiments, after modifying the sequence for codon optimization and / or splice site removal, one or more further assessment of a sequence, such as a revised or modified nucleic acid sequence, is carried out to further evaluate for splice site removal, such as cryptic splice sites, using one or more other or additional splice site prediction tool(s).

[0203] In some aspects, RNA heterogeneity can be a result of the activity of the spliceosome present in a eukaryotic cell. In some aspects, splicing is typically carried out in a series of reactions catalyzed by the spliceosome. Consensus sequences for splice sites are known, but in some aspects, specific nucleotide information defining a splice site may be complex and may not be readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on the standard consensus sequences and are variably activated. Hence, variable splicing of pre-mRNA at cryptic splice sites leads to heterogeneity in the transcribed mRNA products following expression in eukaryotic cells. In some cases, within spliceosomal introns, a donor site (usually at the 5’ end of the intron), a branch site (near the 3’ end of the intron) and an acceptor site (3’ end of the intron) are required for a splicing event. The splice donor site can include a GU sequence at the 5’ end of the intron, with a large less highly conserved region. The splice acceptor site at the 3’ end of the intron can terminatewith an AG sequence.

[0204] In some embodiments, one or more splice donor and / or splice acceptor site(s), such as the potential splice donor and / or acceptor sites that may be involved in a cryptic splicing event that is not desired or that results in undesired RNA heterogeneity, is eliminated. In someembodiments, eliminating one or more splice sites comprises modifying one or more nucleotides (e.g., by substitution or replacement) in, at, containing or near the splice donor and / or acceptor sites that are candidates for removal. In some aspects, a particular nucleotide within a codon that is at, contains or is near the splice site is modified (e.g., substituted or replaced). In some aspects, the modification (such as substitution or replacement) retains or preserves the amino acid encoded by the particular codon at the site, at the same time removing the potential splice donor and / or acceptor sites.

[0205] In some embodiments, the codon at or near the splice site for modification comprises one or more codons that involve one or both of the two nucleotides at the potential splice site (in some cases referred to as “splice site codon”). When the potential splicing is predicted to occur between two nucleotides in a codon, the codon is the only splice site codon for this splice site. If the potential splicing is predicted to occur between two adjacent codons, for example, between the last nucleotide of the first codon and the first nucleotide of the next codon, the two codons are splice site codons. For example, for splice sites that are predicted to be at boundaries of two codons, the two adjacent codons can be candidates for nucleotide modification. In some embodiments, the one or more codons comprise one splice site codon. In some embodiments, the one or more codons comprise both splice site codons. In some embodiments, a potential splice donor site is eliminated by modifying one or both splice site codons. In some embodiments, a potential splice acceptor donor site is eliminated by modifying one or both splice site codons. In some embodiments, the one or both codons at the splice site is not modified, for example, when there are no synonymous codon for the splice site codon. In some embodiments, if there are no synonymous codons available for the particular splice site codon, one or more nucleotides in a nearby codon can be modified. In some embodiments, one or more codons that are modified include a splice site codon, wherein the modification comprises changing one or both nucleotides at the splice site to a different nucleotide or different nucleotides. In some embodiments, the splice donor site is eliminated by modifying one or both splice site codons., wherein the modification does not change one or two of the nucleotides of the at the splice site to a different nucleotide, but a nearby nucleotide, e.g., a part of a codon adjacent to the splice site, is modified. In some embodiments, the nearby or adjacent nucleotides that can be modified include modification of a nucleotide that is a part of a nearby or adjacent codon, such as a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the splice site codon.

[0206] In some cases, polynucleotides can be manually modified, while preserving the encoded amino acid sequence, to reduce the probability of a predicted splice site. In some embodiments, one or more of the predicted splice sites having at least 80%, 85%, 90%, or 95% probability of a splice site are manually modified to reduce the probability of the splicing event. In some embodiments, the one or more modification(s) is / are by nucleotide replacement or substitution of 1, 2, 3, 4, 5, 6 or 7 nucleotides. In some embodiments, the modification(s) is / are at the junction of the splice donor site or are at the junction of the splice acceptor site. In some embodiments, at least one of the one or more nucleotide modifications is within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues of the splice site junction of the splice acceptor and / or splice donor site. In some embodiments, libraries of modified nucleic acid sequences can be generated with reduced probability of cryptic splice sites. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor / acceptor pair. In particular embodiments, the splice donor sites and splice acceptor sites are evaluated independently, or individually, and not part as a splice donor / acceptor pair. In some embodiments, one or more predicted splice sites are not eliminated. In some embodiments, splice sites, such as known or predicted splice sites, within the promoter region of the transcript are not eliminated.

[0207] In some embodiments, one or more potential donor splice site is eliminated by modifying one or two splice site codons or one or more nearby or adjacent codons (for example, if a synonymous codon is not available for the splice site codon). In some embodiments, one or more potential acceptor splice site is eliminated by modifying one or two splice site codons or one or more nearby or adjacent codons (for example, if a synonymous codon is not available for the splice site codon). In some embodiments, the nearby or adjacent codon that is subject to modification include a codon that is within one, two, three, four, five, six, seven, eight, nine or ten codons upstream or downstream of the splice site codon, such as a codon that is within one, two or three codons from the splice site. In some embodiments, a potential branch site for splicing is removed or eliminated. In some aspects, a nucleotide within the codon at or near the branch site can be modified, e.g., substituted or replaced, to eliminate cryptic splicing and / or reduce RNA heterogeneity. In some embodiments, the modification of the one or more nucleotides can involve a substitution or replacement of one of the nucleotides that may be involved in splicing (such as at the splice donor site, splice acceptor site or splice branch site), such that the amino acid encoded by the codon is preserved, and the nucleotide substitution or replacement does not change the polypeptide sequence that is encoded by the polynucleotide. In some cases, the third position in the codon is more degenerate than the other two positions.Thus, various synonymous codons can encode a particular amino acid (see, e.g., Section I.A.2.a.i. above). In some embodiments, the modification includes replacing the codon with a synonymous codon used in the species of the cell into which the polynucleotide is introduced (e.g., human). In some embodiments, the species is human. In some embodiments, the one or more codon is replaced with a corresponding synonymous codons that the most frequently used in the species or synonymous codons that have a similar frequency of usage (e.g., most closest frequency of usage) as the corresponding codon (see, e.g., Section I.A.2.a.i. above).

[0208] In some aspects, a proposed modification can be further evaluated, for example, to assess whether the modification generates an undesired or additional restriction site in the polynucleotide. In some aspects, an additional restriction site may not be desired, and a further or a different modification (e.g., with a different nucleotide replacement at the same codon or a modification at a different position or codon) can be considered. In some aspects, particular restriction site, such as a designated restriction site, is avoided. In some aspects, if the modification does not substantially reduce the splice site prediction score, an additional or alternative modification can be proposed. In some embodiments, the splice site prediction score can be is reduced or lowered by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%, after one or more iteration of the methods.

[0209] In some embodiments, a provided polynucleotide encoding an anti-GPRC5D CAR provided herein, or a construct provided herein, includes modifications to remove one or more splice donor and / or acceptor site that may contribute to splice events and / or reduced expression and / or increased RNA heterogeneity. In some embodiments, provided polynucleotides are modified in one or more polynucleotides in the spacer region to eliminate or reduce splice events. Among potential splice donor and / or acceptor sites that are modified or not included in a provided CAR are set forth in SEQ ID NO: 194, 195, 196, 197, 198, or 199. In some embodiments, modified nucleotides of such sites to reduce or eliminate potential splice and / or donor sites are set forth in SEQ ID NO: 200, 201, 202, 203, 204, 205, or 206. In some embodiments, a provided polynucleotide encoding an anti-GPRC5D CAR, or other CAR, contains one or more nucleotide sequences set forth in SEQ ID NO: 200, 201, 202, 203, 204, 205, or 206. In some embodiments, a provided anti-GPRC5D CAR includes a spacer sequence that is encoded by the nucleotide sequence set forth in SEQ ID NO: 164. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 165. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 166. Insome embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 172.3. Cells and Preparation of Cells for Genetic Engineering

[0210] In some embodiments, the nucleic acids encoding the GPRC5D-targeted recombinant receptor (e.g., CAR) are introduced into cells, e.g., T cells, to that the cells are genetically engineered to express a recombinant receptor. In some embodiments, the nucleic acids are heterologous, z.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0211] The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and / or engineering them, and reintroducing them into the same subject, before or after cryopreservation.

[0212] Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, suchas stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0213] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0214] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0215] In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0216] In some embodiments, the cells for introduction of the recombinant receptor (e.g. CAR T cells, anti-GPRC5D CAR T cells) may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0217] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0218] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0219] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.

[0220] In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0221] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0222] In some embodiments, the provided methods involve leukapheresis of the subject approximately 4 to 6 weeks prior to lymphodepleting chemotherapy. In some embodiments, a leukapheresis collection is performed for each subject to obtain peripheral blood mononuclear cells (PBMCs) for the production of the CAR T cells (e.g., anti-GPRC5D CAR T cells).

[0223] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0224] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0225] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0226] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0227] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of orenrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0228] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0229] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+T cells, are isolated by positive or negative selection techniques.

[0230] For example, CD3+, CD28+T cells can be positively selected using anti-CD3 / anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0231] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (marker111811) on the positively or negatively selected cells, respectively.

[0232] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells. Such CD4+and CD8+populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0233] In some embodiments, CD8+cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. Insome embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood, 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TcM-enriched CD8+T cells and CD4+T cells further enhances efficacy.

[0234] In embodiments, memory T cells are present in both CD62L+and CD62L" subsets of CD8+peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L'CD8+and / or CD62L+CD8+fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0235] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+cell population or subpopulation, also is used to generate the CD4+cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0236] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD 14 and CD45RA or CD 19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0237] CD4+T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+T lymphocytes are CD45RO",CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+cells are CD62L+and CD45RO+. In some embodiments, effector CD4+cells are CD62L" and CD45RO".

[0238] In one example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0239] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0240] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[0241] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0242] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) areretained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0243] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0244] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and / or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0245] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0246] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and / or contamination. In one example, the system is a system asdescribed in International Patent Application, Publication Number W02009 / 072003, or US 20110003380 Al.

[0247] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0248] In some aspects, the separation and / or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[0249] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[0250] In certain embodiments, separation and / or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in someaspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0251] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS) -sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[0252] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence- activated cell sorting (FACS), including preparative scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0253] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and / or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in afreezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1 : 1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to -80° C. at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0254] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. The incubation and / or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[0255] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0256] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating or stimulating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti- CD28. In some embodiments, such agents and / or ligands may be, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL-2, IL- 15 and / or IL-7. In some aspects, the IL-2 concentration is at least about 10 units / mL.

[0257] In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,177 to Riddell et al., Klebanoff et a / .(2012) JImmunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0258] In some embodiments, the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0259] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0260] In embodiments, antigen-specific T cells, such as antigen- specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. Lor example, antigen- specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.4. Dose and Administration of anti-GPRC5D CAR T Cell Therapy

[0261] In embodiments of the provided methods and uses, a dose of cells of a provided GPRC5D-targeted cell therapy is administered to subjects in accord with the provided combination therapy methods. In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition in the subject. One may empirically determine the size or timing of the doses for a particular disease in view of the provided description.

[0262] Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. Lor example, adoptive T celltherapy methods are described, e.g., in US Pat. App. Pub. No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0263] In some embodiments, the methods include adoptive cell therapy, whereby genetically engineered cells expressing the provided recombinant receptors comprising a GPRC5D-binding molecule (e.g., CARs comprising anti-GPRC5D antibody or antigen-binding fragment thereof) are administered to subjects. Such administration can promote activation of the cells (e.g., T cell activation) in a GPRC5D-targeted manner, such that the cells of the disease or disorder are targeted for destruction.

[0264] In some embodiments, the methods include administration of the cells or a composition containing the cells to a subject, tissue, or cell, such as one having, at risk for, or suspected of having the disease, condition or disorder. In some embodiments, the cells, populations, and compositions are administered to a subject having the particular disease or condition to be treated, e.g., via adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells or compositions are administered to the subject, such as a subject having or at risk for the disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptom of the disease or condition, such as by lessening tumor burden in a GPRC5D-expressing cancer.

[0265] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0266] In some embodiments, the cell therapy, e.g., adoptive cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0267] In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a primate, such as a human. In some embodiments, the subject, to whom the cells, cell populations, or compositions are administered, is a non-human primate. In some embodiments, the non-human primate is a monkey (e.g., cynomolgus monkey) or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a nonprimate mammal, such as a rodent (e.g., mouse, rat, etc.). In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and / or for assessing toxic outcomes such as cytokine release syndrome (CRS).

[0268] The GPRC5D-binding recombinant receptors (e.g., CARs) and cells expressing the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjunctival injection, subconjunctival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.

[0269] For the prevention or treatment of disease, the appropriate dosage of the binding molecule, recombinant receptor or cell may depend on the type of disease to be treated, the type of binding molecule or recombinant receptor, the severity and course of the disease, whether the binding molecule or recombinant receptor is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the recombinant receptor or cell, and the discretion of the attending physician. The compositions and molecules and cells are in some embodiments suitably administered to the patient at one time or over a series of treatments.

[0270] In some embodiments, the dose and / or frequency of administration is / are determined based on efficacy and / or response. In some embodiments, efficacy is determined by evaluating disease status. Exemplary methods for assessing disease status include: measurement of M protein in biological fluids, such as blood and / or urine, by electrophoresis and immunofixation;serum and urine immunofixation (IFE) tests; quantification of sFLC (K and X) in blood; skeletal survey; and uPEP (urine protein electrophoresis) and sPEP (serum protein electrophoresis) tests. In some embodiments, disease status can be evaluated by identified by radiographic or bone marrow assessments.

[0271] In some examples, dose and / or frequency of administration is determined by the expansion and persistence of the recombinant receptor or cell in the blood and / or bone marrow. In some embodiments, dose and / or frequency of administration is determined based on the antitumor activity of the recombinant receptor or engineered cell. In some embodiments antitumor activity is determined by the overall response rate (ORR) and / or International Myeloma Working Group (IMWG) Uniform Response Criteria (see Kumar et al. (2016) Lancet Oncol 17(8):e328-346). In some embodiments, response is evaluated using minimal residual disease (MRD) assessment. In some embodiments, MRD can be assessed by methods such as flow cytometry and high-throughput sequencing, e.g., deep sequencing. In some embodiments, response is evaluated based on the duration of response following administration of the recombinant receptor or cells. In some examples, dose and / or frequency of administration can be based on toxicity. In some embodiments, dose and / or frequency can be determined based on health-related quality of life (HRQoL) of the subject to which the recombinant receptor and / or cells is / are administered. In some embodiments, dose and / or frequency of administration can be changed, i.e., increased or decreased, based on any of the above criteria.

[0272] Subjects administered the provided CAR-expressing T cells are monitored for adverse events including any unfavorable and unintended sign, symptom, or disease temporally associated with the T cell administration or the combination of the CAR-expressing T cells and a combination agent. Adverse events include clinical risks that have been observed in subjects treated with a monotherapy of the CAR-expressing T cells. Clinical risks associated with the administration of the CAR-expressing T cells may include, but are not limited to, cytokine release syndrome (CRS); neurologic toxicity; tumor lysis syndrome (TLS); infusion-related reaction; infections; hematologic toxicity (e.g., thrombocytopenia, neutropenia, febrile neutropenia); second primary malignancy (SPM); macrophage activation syndrome / hemophagocytic lymphohistiocytosis; hair follicle, tongue, or skin inflammation; uncontrolled T-cell proliferation; replication-competent lentivirus; predominant clonal expansion and potential risk of insertional oncogenesis; advers reactions to excipients; graft versus host disease; and plasma-cell aplasia / hypogammaglobulinemia. Straegies for clinical risk mitigation include interruption and dose modification guidelines; prophylaxis, monitoring, andmanagement of infection / toxicity; premdication, exclusion criteria; inclusion criteria; SPM surveillance; pause of the study; treatment with supportive care; and management as per institutional standard of care. In some embodiments, toxicity and / or side-effects of treatment can be monitored and used to adjust dose and / or frequency of administration of the CAR- expressing T cells, combinations, and / or compositions. For example, adverse events and laboratory abnormalities can be monitored and used to adjust dose and / or frequency of administration. Any of such events adverse events may establish dose-limiting toxicities and warrant decrease in dose and / or a termination of treatment.

[0273] In some embodiments, the percentage of viable cells in the CAR-expressing T cell composition is greater than 80%. In some embodiments, the percentage of viable cells in the composition is greater than 90%. In some embodiments, the percentage of viable cells in the composition is greater than 95%. In some embodiments, the percentage of viable cells in the composition is greater than 96%. In some embodiments, the percentage of viable cells in the composition is greater than 97%. In some embodiments, the percentage of viable cells in the composition is greater than 98%. In some embodiments, the percentage of viable cells in the composition is greater than 99%.

[0274] Once the cells are administered to a mammal (e.g., a human), the biological activity of the engineered cell populations and / or antibodies in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and / or secretion of certain cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0275] In certain embodiments, engineered cells are modified in any number of ways, such that their therapeutic or prophylactic efficacy is increased. For example, the engineered CAR expressed by the population in some embodiments are conjugated either directly or indirectly through a linker to a targeting moiety. The practice of conjugating compounds, e.g., the CAR or TCR, to targeting moieties is known in the art. See, for instance, Wadwa et al., J. Drug Targeting, 3(2): 111 (1995), and U.S. Patent 5,087,616.

[0276] In some embodiments, the methods also include, prior to administration of the T cell therapy, administration of a lymphodepleting therapy to the subject, e.g. such as cyclophosphamide, fludarabine, bendamustine, or combinations thereof. a. Compositions and Formulations

[0277] In some embodiments, the dose of cells of the cell therapy, such as a T cell therapy comprising cells engineered with a recombinant antigen receptor, e.g. CAR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accord with the provided methods and / or with the provided articles of manufacture or compositions.

[0278] In some embodiments, the cell therapy, such as engineered T cells (e.g. CAR T cells), are formulated with a pharmaceutically acceptable carrier. In some aspects, the choice of carrier is determined in part by the particular cell or agent and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0279] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0280] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells or agents, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0281] The pharmaceutical composition in some embodiments contains cells in amounts effective to treat the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.

[0282] The cells may be administered using standard administration techniques, formulations, and / or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. With respect to cells, administration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheteradministration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0283] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the agent or cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

[0284] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0285] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.

[0286] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. b. Dosing

[0287] In some embodiments, the methods comprise administering a dose of the engineered cells or a composition comprising a dose of the engineered cells. In some embodiments, the engineered cells or compositions containing engineered cells can be used in a treatment regimen, wherein the treatment regimen comprises administering a dose of the engineered cells or a composition comprising a dose of the engineered cells. In some embodiments, the dose cancontain, for example, a particular number or range of recombinant receptor-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), such as any number of such cells described herein. In some embodiments, a composition containing a dose of the cells can be administered. In some aspects, the number, amount or proportion of CAR-expressing cells in a cell population or a cell composition can be assessed by detection of a surrogate marker, e.g., by flow cytometry or other means, or by detecting binding of a labelled molecule, such as a labelled antigen, that can specifically bind to the binding molecules or receptors provided herein.

[0288] In certain embodiments, in the context of genetically engineered cells containing the binding molecules or recombinant receptors, a subject is administered the range of about one million to about 100 billion cells and / or that amount of cells per kilogram of body weight, such as, e.g., about 1 million to about 50 billion cells (e.g., about 5 million cells, about 10 million, about 12.5 million, about 15 million, about 20 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 10 million cells, about 12.5 million cells, about 15 million cells, 20 million cells, about 25 million cells, about 30 million cells, about 40 million cells, about 50 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about120 million cells, about 150 million cells, about 250 million cells, about 300 million cells, about350 million cells, about 450 million cells, about 500 million cells, about 600 million cells, about650 million cells, about 800 million cells, about 900 million cells, about 1 billion cells, about 1.2 billion cells, or about 3 billion cells) or any value in between these ranges and / or per kilogram of body weight. Again, dosages may vary depending on attributes particular to the disease or disorder and / or patient and / or other treatments. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0289] In some embodiments, the provided methods implement flat dosing, e.g. total number of CAR+ cells, total number of CAR+CD8+ T cells and / or CAR+CD4+ T cells, such as to administer a precise or fixed dose of such cell type(s) to each of a group of subjects treated, including subjects of variable weight. Thus, the provided methods include methods in which the dose of cells is a flat dose of cells or fixed dose of cells such that the dose of cells is not tied to or based on the body surface area or weight of a subject. In some embodiments, such methodsminimize or reduce the chance of administering too many cells to the subject, which may increase the risk of a toxic outcome associated with administration of the CAR-T cells.

[0290] In some embodiments, for example, where the subject is a human, the dose includes more than about 1 x 106total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) and fewer than about 2 x 109total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 2.5 x 107to about 1.2 x 109such cells, such as 2.5 x 107, 5 x 107, 7.5 x 107, 1.5 x 108, 3 x 108, 4.5 x 108, 8 x 108, or 1.2 x 109total such cells, or the range between any two of the foregoing values. In some embodiments, for example, where the subject is a human, the dose includes more than about 1 x 106total CAR- expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) and fewer than about 2 x 109total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 1.0 x 107to about 1.2 x 109such cells, such as 1.0 x 107, 1.25 x 107, 1.5 x 107, 2.0 x 107, 2.5 x 107, 5 x 107, 7.5 x 107, 1.5 x 108, 2.25 x 108, 3 x 108, 4.5 x 108, 6.0 x 108, 8 x 108,or 1.2 x 109total such cells, or the range between any two of the foregoing values. In some embodiments, for example, where the subject is a human, the dose includes more than about 1 x 106total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) and fewer than about 2 x 109total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of about 1.0 x 107to about 6.5 x 108such cells, about 1.5 x 107to about 6.0 x 108such cells, about 1.5 x 107to about 6.5 x 108such cells, about 2.5 x 107to about 6.0 x 108such cells, or about about 5.0 x 107to about 6.0 x 108such cells. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0291] In some embodiments, the dose of the cell therapy is a dose of CAR-expressing cells (e.g., T cells). In some embodiments, the dose of the cell therapy is the total number of viable CAR-expressing (CAR+) T cells. In some embodiments, the dose of the cell therapy is the total number of viable CD3+ CAR-expressing (CAR+) T cells.

[0292] In some embodiments, the dose of genetically engineered cells comprises between at or about 2.5 x 107CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), and at or about 1.2 x 109CAR-expressing T cells, total T cells, or total PBMCs, between at or about 5.0 x 107CAR-expressing T cells and at or about 4.5 x 108CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), between at or about 7.0 x 107CAR-expressing T cells and at or about 2.0 x 108CAR-expressing T cells, total T cells, or total PBMCs, each inclusive. In some embodiments, the number is with reference to the total number of CD3+ or CD8+, in some cases also CAR-expressing (e.g. CAR+) cells. Insome embodiments, the dose comprises a number of cell from or from about 2.5 x 107to or to about 1.2 x 109CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 5.0 x 107to or to about 4.5 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, or from or from about 7 x 107to or to about 2.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+CAR-expressing cells, each inclusive. In some embodiments, the number of cells is the number of such cells that are viable cells. In some embodiments, the number of cells is the number of CD3+ CAR-expressing cells that are viable cells.

[0293] In some embodiments, the dose of genetically engineered cells comprises between at or about 1.0 x 107CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), and at or about 1.2 x 109CAR-expressing T cells, total T cells, or total PBMCs, between at or about 2.0 x 107CAR-expressing T cells and at or about 4.5 x 108CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), between at or about 7.0 x 107CAR-expressing T cells and at or about 2.0 x 108CAR-expressing T cells, total T cells, or total PBMCs, each inclusive. In some embodiments, the number is with reference to the total number of CD3+ or CD8+, in some cases also CAR-expressing (e.g. CAR+) cells. In some embodiments, the dose comprises a number of cell from or from about 1.0 x 107to or to about 1.2 x 109CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 1.5 x 107to or to about 1.2 x 109CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 2.5 x 107to or to about 1.2 x 109CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 1.5 x 107to or to about 8.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 2.5 x 107to or to about 8.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR- expressing cells, from or from about 1.5 x 107to or to about 6.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 2.5 x 107to or to about 6.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from about 5.0 x 107to or to about 6.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR- expressing cells, from or from about 5.0 x 107to or to about 4.5 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, or from or from about 7.0 x 107to or to about 2.0 x 108CD3+ or CD8+ total T cells or CD3+ or CD8+CAR-expressing cells, each inclusive. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0294] In some embodiments, exemplary doses include about 5.0 x 107, 7.5 x 107, 1.5 x 108, 3.0 x 108or 4.5 x 108CAR-expressing T cells. In some embodiments, the exemplary dose is in a range of between 1.0 x 107and 1.5 x 107, between 1.25 x 107and 2.5 x 107, between 2.0 x 107,and 3.5 x 107, between 3.0 x 107and 4.5x 107, between 4.0 x 107and 7.5 x 107, between 5.0 x 107and 2.25 x IO8, between 1.5 x IO8and 4.5 x IO8, or between 3.0 x IO8and 6.0 x IO8CAR- expressing T cells. In some embodiments, exemplary doses include about 1.0 x 107, 1.25 x 107,1.5 x 107, 2.0 x 107, 2.0 x 107, 2.5 x 107, 3.0 x 107, 3.5 x 107, 4.0 x 107, 4.5x 107, 5.0 x 107, 7.5 x107, 1.5 x IO8, 2.25 x IO8, 3.0 x IO8, 4.5 x IO8, or 6.0 x IO8CAR-expressing T cells. In some aspects, particular response to the treatment, e.g., according to the methods provided herein, can be assessed based on the International Myeloma Working Group (IMWG) Uniform Response Criteria (see Kumar et al. (2016) Lancet Oncol 17(8):e328-346). In some embodiments, exemplary doses to achieve particular outcomes, such as OR, includes about 7.5 x 107CAR- expressing T cells. In some embodiments, exemplary doses to achieve particular outcomes, such as OR, includes about 1.5 x 108CAR-expressing T cells. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0295] In some embodiments, the dose of genetically engineered cells comprises 2.5 x 107CAR-expressing cells. In some embodiments, the dose of genetically engineered cells comprises7.5 x 107CAR-expressing cells. In some embodiments, the dose of genetically engineered cells comprises 7.5 x 107CAR-expressing T cells. In some embodiments, the dose of genetically engineered cells comprises 1.5 x 108CAR-expressing cells. In some embodiments, the dose of genetically engineered cells comprises 1.5 x 108CAR-expressing T cells. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0296] In some embodiments, the T cells of the dose include CD4+ T cells, CD8+ T cells or CD4+ and CD8+ T cells.

[0297] In some embodiments, for example, where the subject is human, the CD8+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 106and at or about 2 x 109total CAR-expressing CD8+cells, e.g., in the range of at or about 5 x 107to at or about 4.5 x 108such cells, such as at or about 2.5 x 107, at or about 5 x 107, at or about 7.5 x 107, at or about 1.5 x 108, at or about 3 x 108, at or about 4.5 x 108, at or about 8 x108, or at or about 1.2 x 109total such cells, or the range between any two of the foregoing values. In some embodiments, the number of cells is the number of such cells that are viable cells.

[0298] In the context of adoptive cell therapy, administration of a given “dose” of CAR- expressing cells encompasses administration of the given amount or number of cells as a single composition and / or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a splitdose or as a plurality of compositions, provided in multiple individual compositions or infusions, over a specified period of time, such as over no more than 3 days. Thus, in some contexts, the dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, however, the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.

[0299] Thus, in some aspects, the cells of the dose are administered in a single pharmaceutical composition. In some embodiments, the cells of the dose are administered in a plurality of compositions, collectively containing the cells of the dose.

[0300] In some embodiments, the term “split dose” refers to a dose that is split so that it is administered over more than one day. This type of dosing is encompassed by the present methods and is considered to be a single dose.

[0301] Thus, the dose of cells may be administered as a split dose, e.g., a split dose administered over time. For example, in some embodiments, the dose may be administered to the subject over 2 days or over 3 days. Exemplary methods for split dosing include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not spread over more than 3 days.

[0302] In some embodiments, cells of the dose may be administered by administration of a plurality of compositions or solutions, such as a first and a second, optionally more, each containing some cells of the dose. In some aspects, the plurality of compositions, each containing a different population and / or sub-types of cells, are administered separately or independently, optionally within a certain period of time. For example, the populations or subtypes of cells can include CD8+and CD4+T cells, respectively, and / or CD8+- and CD4+- enriched populations, respectively, e.g., CD4+and / or CD8+T cells each individually including cells genetically engineered to express the recombinant receptor. In some embodiments, the administration of the dose comprises administration of a first composition comprising a dose of CD8+T cells or a dose of CD4+T cells and administration of a second composition comprising the other of the dose of CD4+T cells and the CD8+T cells.

[0303] In some embodiments, the administration of the dose, e.g., administration of the plurality of cell compositions, involves administration of the cell compositions separately. In some aspects, the separate administrations are carried out simultaneously, or sequentially, in any order. In particular embodiments, the separate administrations are carried out sequentially by administering, in any order, a first composition comprising a dose of CD8+T cells or a dose of CD4+T cells and a second composition comprising the other of the dose of CD4+T cells and the CD8+T cells. In some embodiments, the dose comprises a first composition and a second composition, and the first composition and second composition are administered within 48 hours of each other, such as no more than 36 hours of each other or not more than 24 hours of each other. In some embodiments, the first composition and second composition are administered 0 to 12 hours apart, 0 to 6 hours apart or 0 to 2 hours apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out no more than 2 hours, no more than 1 hour, or no more than 30 minutes apart, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes apart. In some embodiments, the initiation and / or completion of administration of the first composition and the completion and / or initiation of administration of the second composition are carried out no more than 2 hours, no more than 1 hour, or no more than 30 minutes apart, no more than 15 minutes, no more than 10 minutes or no more than 5 minutes apart. In some embodiments, the first composition and the second composition are administered less than 2 hours apart.

[0304] In some composition, the first composition, e.g., first composition of the dose, comprises CD4+T cells. In some composition, the first composition, e.g., first composition of the dose, comprises CD8+T cells. In some embodiments, the first composition is administered prior to the second composition. In particular embodiments, the CD8+ T cells are administered prior to the CD4+ T cells.

[0305] In some embodiments, the dose or composition of cells includes a defined or target ratio of CD4+cells expressing a recombinant receptor (e.g. CAR) to CD8+cells expressing a recombinant receptor (e.g. CAR) and / or of CD4+cells to CD8+cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1, such as approximately 1:1. In some aspects, the administration of a composition or dose with the target or desired ratio of different cell populations (such as CD4+:CD8+ratio or CAR+CD4+:CAR+CD8+ratio, e.g., 1:1) involves the administration of a cell composition containing one of the populations and then administration of a separate cell compositioncomprising the other of the populations, where the administration is at or approximately at the target or desired ratio. In some aspects, administration of a dose or composition of cells at a defined ratio leads to improved expansion, persistence and / or antitumor activity of the T cell therapy.

[0306] In some embodiments, for example, where the subject is human, the CD8+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 106and at or about 2 x 109total recombinant receptor (e.g., CAR)-expressing CD8+cells, e.g., in the range of at or about 1 x 107to at or about 4.5 x 108such cells, such as at or about 1.0 x107, at or about 1.25 x 107, at or about 1.5 x 107, at or about 2.0 x 107, at or about 2.5 x 107, at or about 5 x 107, at or about 7.5 x 107, at or about 1.5 x 108, at or about 3 x 108, at or about 4.5 x108, at or about 6.0 x 108, at or about 8 x 108, or at or about 1.2 x 109total such cells, or the range between any two of the foregoing values.

[0307] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, three months, six months, 1 year or more. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values.

[0308] In some embodiments, the engineered cells for administration or composition of engineered cells for administration, exhibits properties indicative of or consistent with cell health. In some embodiments, at or about or at least at or about 70, 75, 80, 85, or 90% CAR+ cells of such dose exhibit one or more properties or phenotypes indicative of cell health or biologically active CAR cell, such as absence expression of an apoptotic marker.

[0309] In particular embodiments, the phenotype is or includes an absence of apoptosis and / or an indication the cell is undergoing the apoptotic process. Apoptosis is a process of programmed cell death that includes a series of stereotyped morphological and biochemical events that lead to characteristic cell changes and death, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay. In some aspects, early stages of apoptosis can be indicated by activation of certain caspases, e.g., 2, 8, 9, and 10. In some aspects, middle to late stages of apoptosis are characterized by further loss of membrane integrity, chromatin condensation and DNA fragmentation, include biochemical events such as activation of caspases 3, 6, and 7.

[0310] In particular embodiments, the phenotype is negative expression of one or more factors associated with programmed cell death, for example pro-apoptotic factors known toinitiate apoptosis, e.g., members of the death receptor pathway, activated members of the mitochondrial (intrinsic) pathway, such as Bcl-2 family members, e.g., Bax, Bad, and Bid, and caspases. In certain embodiments, the phenotype is the absence of an indicator, e.g., an Annexin V molecule or by TUNEL staining, that will preferentially bind to cells undergoing apoptosis when incubated with or contacted to a cell composition. In some embodiments, the phenotype is or includes the expression of one or more markers that are indicative of an apoptotic state in the cell. In some embodiments, the phenotype is lack of expression and / or activation of a caspase, such as caspase 3. In some aspects, activation of caspase-3 is indicative of an increase or revival of apoptosis. In certain embodiments, caspase activation can be detected by known methods. In some embodiments, an antibody that binds specifically to an activated caspase (i.e., binds specifically to the cleaved polypeptide) can be used to detect caspase activation. In particular embodiments, the phenotype is or includes active caspase 3-. In some embodiments, the marker of apoptosis is a reagent that detects a feature in a cell that is associated with apoptosis. In certain embodiments, the reagent is an annexin V molecule.

[0311] In some embodiments, the compositions containing the engineered cells for administration contain a certain number or amount of cells that exhibit phenotypes indicative of or consistent with cell health. In some of any embodiments, less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express a marker of apoptosis, optionally Annexin V or active Caspase 3. In some of any embodiments, less than 5%, 4%, 3%, 2% or 1% of the CAR-expressing T cells in the dose of engineered T cells express Annexin V or active Caspase 3.

[0312] In some embodiments the cells administered are immune cells engineered to express the GPRC5D-binding recombinant receptor, e.g., CAR. In some embodiments the immune cells are T cells. In some embodiments, the administered cells are CD4+ T cells. In some embodiments the administered cells are CD8+ T cells. In some embodiments, the administered cells are a combination of CD4+ and CD8+ T cells, such as CAR T cells. In some examples the ratio of CD4+ cells to CD8+ cells (CD4:CD8) is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1.B. Immunomodulatory Compound

[0313] In some embodiments, the GPRC5D-binding recombinant receptors (e.g., CAR) is administered to the subject in combination with an immunomodulatory compound that is a astructural or functional analog or derivative of thalidomide or is an inhibitor of E3 ubiquitin ligase.

[0314] In some embodiments, the immunomodulatory compound binds to cereblon (CRBN). In some embodiments, the immunomodulatory compound binds to the CRBN E3 ubiquitinligase complex. In some embodiments, the immunomodulatory compound binds to CRBN and the CRBN E3 ubiquitin-ligase complex. In some embodiments, the immunomodulatory compound up-regulates the protein or gene expression of CRBN. In some aspects, CRBN is the substrate adaptor for the CRL4CRBNE3 ubiquitin ligase, and modulates the specificity of the enzyme. In some embodiments, binding to CRBN or the CRBN E3 ubiquitin ligase complex inhibits E3 ubiquitin ligase activity. In some embodiments, the immunomodulatory compound induces the ubiqutination of KZF1 (Ikaros) and IKZF3 (Aiolos) and / or induces degradation of IKZF1 (Ikaros) and IKZF3 (Aiolos). In some embodiments, the immunomodulatory compound induces the ubiquitination of casein kinase 1A1 (CKla) by the CRL4CRBNE3 ubiquitin ligase. In some embodiments, the ubiquitination of CKla results in CKla degradation.

[0315] In some embodiments, the immunomodulatory compound is a cereblon E3 ligase modulator (CELMoD) agent. The molecular target for such compounds has been identified as the protein Cereblon (CRBN), a substrate receptor of the Cullin 4 RING E3 ubiquitin ligase complex. In some embodiments, the compound is able to bind CRBN and modulate its function. Binding to a hydrophobic tri-tryptophan pocket within CRBN promotes the recruitment, ubiquitination, and subsequent proteasomal degradation of several protein substrates, including Aiolos (IKZF3) and Ikaros (IKZF1). In some embodiments, the compound is a compound that modulates CRBN, the binding of which induces ubiquitination of the transcription factors Aiolos and Ikaros, increasing their proteasome dependent degradation and / or augmenting T cell function. In some embodiments, the compound is able to induce loss of Aiolos and Ikaros in cells.

[0316] In addition to its cell autonomous activity against malignant B cells, E3 ligase modulatory compounds, such as Compound A and Compound B described herein, also exert costimulatory effects on immune cells such T and NK-cells. This activity also has been shown to be through CRBN mediated degradation of Aiolos and Ikaros, which are negative regulators of activation molecules and cytokines such as interleukin-2 (IL-2) expression. (Gandhi, Br J Haematol. 2014 Mar;164(6):811-21, Krbnke, Oncoimmunology, 2014; 3(7): e941742.).

[0317] In some embodiments, the immunomodulatory compound is an inhibitor of the Ikaros (IKZF1) transcription factor. In some embodiments, the immunomodulatory compoundenhances ubiquitination of Ikaros. In some embodiments, the immunomodulatory compound enhances the degradation of Ikaros. In some embodiments, the immunomodulatory compound down-regulates the protein or gene expression of Ikaros. In some embodiments, administration of the immunomodulatory compound causes a decrease in Ikaros protein levels.

[0318] In some embodiments, the immunomodulatory compound is an inhibitor of the Aiolos (IKZF3) transcription factor. In some embodiments, the immunomodulatory compound enhances ubiquitination of Aiolos. In some embodiments, the immunomodulatory compound enhances the degradation of Aiolos. In some embodiments, the immunomodulatory compound down-regulates the protein or gene expression of Aiolos. In some embodiments, administration of the immunomodulatory compound causes a decrease in Aiolos protein levels.

[0319] In some embodiments, the immunomodulatory compound is an inhibitor of both the Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors. In some embodiments, the immunomodulatory compound enhances ubiquitination of both Ikaros and Aiolos. In some embodiments, the immunomodulatory compound enhances the degradation of both Ikaros and Aiolos. In some embodiments, the immunomodulatory compound enhances ubiquitination and degradation of both Ikaros and Aiolos. In some embodiments, administration of the immunomodulatory compound causes both Aiolos protein levels and Ikaros protein levels to decrease.

[0320] In some embodiments, the immunomodulatory compound is a Selective cytokine inhibitory drug (SelCID). In some embodiments, the immunomodulatory compound inhibit the activity of phosphodiesterase-4 (PDE4). In some embodiments, the immunomodulatory compound suppresses the enzymatic activity of the CDC25 phosphatases. In some embodiments, the immunomodulatory compound alters the intracellular trafficking of CDC25 phosphatases.

[0321] In some embodiments, the immunomodulatory compound in the combination therapy is thalidomide (2-(2,6-dioxopiperidin-3-yl)-lH-isoindole- l,3(2H)-dione) or an analog or derivative of thalidomide. In certain embodiments, a thalidomide derivative includes structural variants of thalidomide that have a similar biological activity. In some embodiments, the immunomodulatory compound is a derivative of thalidomide. In some embodiments, the immunomodulatory compound is a structural and / or functional analogue of thalidomide. In some embodiments, the immunomodulatory compound is lenalidomide, pomalidomide, avadomide, or a pharmaceutically acceptable salt thereof. Exemplary thalidomide derivatives include, but are not limited to lenalidomide (REVLIMMUNOMODULATORY COMPOUND™; Celgene Corporation), pomalidomide (also known asACTIMMUNOMODULATORY COMPOUND™ or POMALYST™ (Celgene Corporation)), CC-1088, CDC-501, and CDC- 801, and the compounds disclosed in U.S. Pat. Nos. 5,712,291; 7,320,991; and 8,716,315; U.S. Appl. No. 2016 / 0313300; and PCT Pub. Nos. WO 2002 / 068414 and WO 2008 / 154252.

[0322] In some embodiments, the immunomodulatory compound is avadomide, which also is known as 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is an enantiomer or a mixture of enantiomers of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2, 6-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph of 3-(5-amino-2-methyl-4- oxo-4H-quinazolin-3-yl)-piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is a solvate of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is a hydrate of 3-(5-amino-2-methyl- 4-oxo-4H-quinazolin-3-yl)-piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is a pharmaceutically acceptable salt of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3- yl)-piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is a polymorph of 3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is 3-(5-amino-2-methyl-4-oxo-4H-quinazolin- 3 -y 1) -piperidine-2, 6-dione .

[0323] In some embodiments, the immunomodulatory compound is lenalidomide, which also is known as 3-(4-amino-l -oxo- l,3-dihydro-2H-isoindol-2-yl)piperidine-2, 6-dione, or is an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. In some embodiments, lenalidomide is 2,6- Piperidinedione, 3-(4-amino-l,3-dihydro-l-oxo-2H-isoindol-2-yl)-, 3-(4-Amino-l-oxo-l,3- dihydro-2H-isoindol-2-yl)-2,6-piperidinedione, 3-(4- Amino- 1 -oxo- 1,3-dihy dro-2H-isoindol-2- yl)-2,6-piperidinedione, 3-(4- Amino- 1 -oxo- 1,3-dihy dro-2H-isoindol-2-yl)piperidin-2,6-dion, 3- (4-amino-l -oxo- l,3-dihydro-2H-isoindol-2-yl)piperidine-2, 6-dione, 3-(4-amino-l-oxo-l,3- dihydro-2H-isoindol-2-yl)piperidine-2, 6-dione, all of which can be used interchangeably, or is an enantiomer or a mixture of enantiomers thereof, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph thereof. In some embodiments, the immunomodulatory compound is (R)-3-(4-amino-l -oxo- 1,3-dihy dro-2H-isoindol-2- yl)piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is (.S')-3-(4- amino-1 -oxo- 1,3-dihy dro-2H-isoindol-2-yl)piperidine-2, 6-dione. In some embodiments, the immunomodulatory compound is a mixture of (R)-3-(4-amino- 1 -oxo- 1,3-dihy dro-2H-isoindol-2-yl)piperidine-2, 6-dione and (S)-3-(4-amino-l-oxo-l,3-dihydro-2H-isoindol-2-yl)piperidine-2,6- dione.

[0324] In some embodiments, the immunomodulatory compound is pomalidomide, which is also known as 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione, or is an enantiomer or a mixture of enantiomers thereof; or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, clathrate, or polymorph thereof. In some embodiments, the immunomodulatory compound is an enantiomer or a mixture of enantiomers of 4-amino-2-(2,6-dioxopiperidin-3- yl)isoindoline- 1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione. In some embodiments, the immunomodulatory compound is (7?)-4-amino-2-(2,6-dioxopiperidin-3- yl)isoindoline- 1,3-dione and / or (S)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione, or a pharmaceutically acceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorph of (7?)-4- amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1 ,3-dione and / or (5')-4-amino-2-(2,6- dioxopiperidin-3-yl)isoindoline- 1,3-dione. In some embodiments, the immunomodulatory compound is a solvate of (7?)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione and / or (S)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione. In some embodiments, the immunomodulatory compound is a hydrate of (7?)-4-amino-2-(2,6-dioxopiperidin-3- yl)isoindoline- 1,3-dione and / or (S)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione. In some embodiments, the immunomodulatory compound is a pharmaceutically acceptable salt of (7?)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1 ,3-dione and / or (5')-4-amino-2-(2,6- dioxopiperidin-3-yl)isoindoline- 1,3-dione. In some embodiments, the immunomodulatory compound is (7?)-4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline- 1,3-dione, (5')-4-amino-2- (2, 6-dioxopiperidin-3-yl)isoindoline- 1,3-dione, or a mixture thereof in any ratio.

[0325] In some embodiments, the immunomodulatory compound is Compound A having the chemical name S)-4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)- l-oxoisoindolin-4- yl)oxy)methyl)benzyl)piperazin-l-yl)-3-fluorobenzonitrile and / or that has the structure of Formula (I), or a pharmaceutically acceptable salt thereof (e.g., a hydrobromide salt of Formula (I). In some embodiments, Compound A is CC-92480 (also called mezigdomide).

[0326] In some embodiments, the immunomodulatory compound is Compound B having the chemical name S)-3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-l-oxo-l,3-dihydro-isoindol-2-yl]- piperidine-2, 6-dione and / or that has the structure of Formula (II) or an enantiomer, mixture of enantiomers, tautomer, isotopolog, or pharmaceutically acceptable salt thereof. In some embodiments, Compound B is CC-220 (also called iberdomide).

[0327] The provided methods are based on observations that immunomodulatory compounds, including CelMods such as Compound A and Compound B, improve T cell function, including functions related to the ability to produce one or more cytokines, cytotoxicity, expansion, proliferation, and persistence of T cells. In some aspects, the provided methods enhance or modulate proliferation and / or activity of T cell activity associated with administration of the T cell therapy (e.g. CAR-expressing T cells). It is found that such methods and uses provide for or achieve improved or greater T cell functionality, and thereby improved anti-tumor efficacy.

[0328] In some embodiments, in connection with potentiating T cell function, immunomodulatory compounds, e.g. Compound A or Compound B, may also exhibit effects to reverse, delay, or prevent T cell exhaustion, including by increasing T cell signaling and / or altering one or more genes that are differentially regulated following chronic (long-term) stimulation. Thus, while in some cases agents that increase or potentiate T cell activity may drive the cells to an exhausted state, it is found herein that activity of such immunomodulatory compounds, e.g. Compound A or Compound B, to exert a potentiating effect on T cell activity is decoupled from T cell exhaustion. In some embodiments, the provided methods involving administration of such immunomodulatory compounds, e.g. Compound A or Compound B, is capable of potentiating activity of naive T cells and delaying, limiting, reducing, inhibiting or preventing exhaustion. In some embodiments, the administration of such immunomodulatory compounds, e.g. Compound A or Compound B, exhibit activity to rescue T cells from T cell exhaustion, such as by restoring or partially restoring one or more T cell activities after a cell has shown features of exhaustion .In some aspects, exposure of T cells that have been chronically stimulated and exhibit features of exhausted T cells, to an immunomodulatory compound described herein, such as Compound A or Compound B are able to recover activity or have their activity restored or partially restored.

[0329] In particular aspects, provided combination therapy methods involving administration of a GPRC5D-taregeted cell therapy in combination with an immunomodulatory compound, such as a structural or functional analog or derivative of thalidomide and / or an inhibitor of E3 ubiquitin ligase, e.g. Compound A or Compound B, promotes continued function and / or survival of cells of a T cell therapy (e.g. CAR-T cells) after activation, including after encounter with antigen. In some aspects, administration of the immunomodulatory compound, such as Compound A or Compound B, increases the ability of such T cells to persist or function long-term, such as by preventing exhaustion or cell death. In some embodiments, the providedcombination therapy methods may thus also achieve improved or more durable responses as compared to certain alternative methods, such as in particular groups of subjects treated.

[0330] In some embodiments, the methods are particularly advantageous in subjects in which the cells of the T cell therapy exhibit weak expansion, have become exhausted, exhibit a reduced or decreased persistence in the subject and / or in subjects that have a cancer that is resistant or refractory to other therapies, is an aggressive or high-risk cancer, and / or that is or is likely to exhibit a relatively lower response rate to a CAR-T cell therapy administered without the immunomodulatory compound compared to another type of cancer or compared to administration with a different CAR-T cell therapy. In some aspects, the provided methods increase overall response and / or survival by or more than 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold or more compared to an alternative treatment, such as compared to a monotherapy involving administration of the T cell therapy (e.g. CAR-T cell) or immunomodulatory compound (e.g. Compound A or Compund B) alone.

[0331] In particular embodiments, the immunomodulatory compound in accord with the provide combination therapy methods is used in an effective amount to exhibit a T cell modulatory effect. Yet, the immunomodulatory compound also is administered in a dosing regimen that, while improving one or more outcomes or functional attributes of the cell therapy, does not affect one or more side effects or unwanted adverse event. Ikaros is expressed in immature stages of myeloid differentiation and regulates early neutrophil differentiation (Dumortier et al. (2003) Blood 101:2219). Thus, in some cases, depletion of Ikaros, such as by administration of an immunomodulatory compound, e.g. Compound A or Compound B, to subjects can, in some instances, result in neutropenia. In some embodiments, the dosing regimen of the immunomodulatory compound is one that is not expected to cause severe toxicity, such as Grade 3 neutropenia and dermatitis.

[0332] The immunomodulatory compound can be administered prior to, subsequently to, during, simultaneously or near simultaneously, sequentially and / or intermittently with administration of the T cell therapy, e.g., administration of T cells expressing a chimeric antigen receptor (CAR).

[0333] In some embodiments, the method involves initiating the administration of the immunomodulatory compound prior to administration of the T cell therapy. In other embodiments, the method involves initiating the administration of the immunomodulatory compound after administration of the T cell therapy. In some embodiments, the dosage schedulecomprises initiating the administration of the immunomodulatory compound concurrently or simultaneously with the administration of the T cell therapy.

[0334] In some embodiments, the immunomodulatory compound is administered in a cycle. In some embodiments, the cycle comprises an administration period in which the immunomodulatory compound is administered followed by a rest period during which the immunomodulatory compound is not administered. In some embodiments, the cycle is a 21 -day cycle or is a 28-day cycle. In some embodiments, the dosing regimen of the immunomodulatory compound is characterized by a plurality of treatment cycles.

[0335] In some embodiments, in each treatment cycle in which the immunomodulatory compound is administered, the immunomodulatory compound is administered several times a day, twice a day, daily, every other day, three times a week, twice a week, or once a week. In some embodiments, in each treatment cycle in which the immunomodulatory compound is administered, the immunomodulatory compound is administered daily.

[0336] In certain embodiments, the immunomodulatory compound is administered once daily for 14 days over a 21 day treatment cycle, e.g., once daily for 14 consecutive days over a 21 day treatment cycle. In certain embodiments, the immunomodulatory compound is administered once daily for 21 days over a 28 day treatment cycle, e.g., once daily for 21 consecutive days over a 28 day treatment cycle. In some embodiments, the immunomodulatory compound is administered during the administration period for no more than 14 consecutive days. In some embodiments, the immunomodulatory compound is administered during the administration period for no more than 21 consecutive days.

[0337] In some embodiments of the methods provided herein, the immunomodulatory compound, e.g., Compound A or Compound B, and the T cell therapy are administered simultaneously or near simultaneously.

[0338] In some embodiments, administration of the immunomodulatory compound on each day it is administered is administered at a dose of from or from about 0.1 mg to about 100 mg, from or from about 0.1 mg to 50 mg, from or from about 0.1 mg to 25 mg, from or from about 0.1 mg to 10 mg, from or from about 0.1 mg to 5 mg, from or from about 0.1 mg to 1 mg, from or from about 0.2 mg to 1 mg, from or from about 0.3 mg to 1 mg, from or from about 0.4 mg to 1 mg, from or from about 0.5 mg to 1 mg, from or from about 0.6 mg to 1 mg, from or from about 0.7 mg to 1 mg, from or from about 0.8 mg to 1 mg, from or from about 0.9 mg to 1 mg, from or from about 1 mg to 100 mg, from or from about 1 mg to 50 mg, from or from about 1 mg to 25 mg, from or from about 1 mg to 10 mg, from or from about 1 mg to 5 mg, from orfrom about 5 mg to 100 mg, from or from about 5 mg to 50 mg, from or from about 5 mg to 25 mg, from or from about 5 mg to 10 mg, from or from about 10 mg to 100 mg, from or from about 10 mg to 50 mg, from or from 10 mg to 25 mg, from or from about 25 mg to 100 mg, from or from about 25 mg to 50 mg or from or from about 50 mg to 100 mg, each inclusive. In some embodiments, the amount is a once daily amount of the immunomodulatory compound.

[0339] In any of the provided embodiments, the immunomodulatory compound may be administered orally. In some embodiments, the immunomodulatory compound is administered as a tablet or capsule.

[0340] In some embodiments, dosages, such as daily dosages, are administered in one or more divided doses, such as 2, 3, or 4 doses, or in a single formulation. The immunomodulatory compound can be administered alone, in the presence of a pharmaceutically acceptable carrier, or in the presence of other therapeutic agents.

[0341] Exemplary dosing regimens are provided in the following subsections.1. Dosage Regimen of Compound A (e.g, Formula (I))

[0342] In some embodiments, the immunomodulatory compound for use in the provided combination therapy is Compound A having the chemical name S)-4-(4-(4-(((2-(2,6- dioxopiperidin-3-yl)-l-oxoisoindolin-4-yl)oxy)methyl)benzyl)pip...

Claims

Claims1. A method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising:(1) administering to a subject having a RRMM a CAR-T cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); and(2) administering to the subject the compound of Formula (I):pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the compound is administered in a dosing regimen that is initiated no earlier than 60 days after administration of the CAR T cell therapy.

3. The method of claim 2, wherein the dosing regimen of the compound is a cycling regimen comprising more than one treatment cycle.

4. The method of claim 3, wherein each treatment cycle is a 28-day cycle.

5. A method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising:(1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D);(2) administering to the subject the compound of Formula (I):pharmaceutically acceptable salt thereof, in a dosing regimen that is initiated after the CAR T cell administration, wherein the dosing regimen of the compound comprises a cycling regimen comprising more than one treatment cycle, wherein each treatment cycle is a 28-day treatment cycle.

6. The method of claim 5, wherein the initiation of administration of the compound is 28 days to 6 months after administering the CAR-T cell therapy to the subject.

7. The method of claim 5 or 6, wherein the initiation of administration of the compound is no earlier than 60 days after the CAR-T cell therapy.

8. The method of any of claims 1-7, wherein the initiation of administration of the compound is at or about 60 days after the CAR-T cell therapy.

9. The method of any one of claims 4-8, wherein the first day of administration of the compound of the 28-day cycle is considered Day 1 (DI) of a cycle.

10. The method of any one of claims 4-9, wherein the dosing regimen comprises administration of the compound to the subject on Days 1-21 of the 28-day cycle.

11. The method of any one of claims 1-10, wherein the compound is administered daily.

12. The method of any of claims 1-11, wherein the compound is administered at a dose from 0.1 mg to 1.0 mg daily.

13. The method of any one of claims 1-12, wherein the compound is administered at a dose from about 0.3 mg to 0.9 mg daily.

14. The method of any one of claims 1-13, wherein the compound is administered at a dose of about 0.60 mg daily.

15. The method of any one of claims 1-14, wherein the compound is administered orally.

16. The method of any one of claims 1-15, wherein the compound is administered to the subject until disease progression.

17. The method of any one of claims 1-16, wherein the subject has not undergone previous treatment with the compound.

18. The method of any one of claims 1-17, wherein the dosing regimen of the immunomodulatory compound in combination with the CAR-T cell therapy is such that the subject does not develop a toxicity characterized by one or more of:(i) Any Grade 4 CRS and Grade CRS that does not resolve to Grade < 2 within 72 hours;(ii) any duration Grade 3 ICANS or neurotoxicity (NT);(iii) a higher than Grade 3 toxicity involving vital organs (eg, cardiac, pulmonary) of any duration;(iv) all other Grade 3 toxicities not attributable to underlying disease or lymphodepleting chemotherapy that do not resolve to < Grade 2 within 72 hours, other than a non-CRS toxicity of the liver in which liver amino transferase levels > 3x upper limit of normal (ULN) are accompanied by an elevation of total bilirubin to >2 x ULN without initial findings of cholestasis (e.g., elevated serum alkaline phosphatase) for which no other reason can be found to explain the combination of increased amino transferase to total bilirubin, such as viral hepatitis A, B, or C; pre-existing or acute liver disease; or another drug capable of causing the observed liver injury;(v) Grade 3 aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) increase lasting < 14 days or Grade 4 AST and / or ALT increase lasting < 7 days;(vi) certain treatment-emergent, isolated Grade 3 or 4, asymptomatic laboratory electrolyte abnormalities not directly related to vital organ toxicities that resolve, with or without intervention, to Grade < 2 in < 7 days;(vii) Grade 4 thrombocytopenia that does not resolve to Grade 3 or lower within 14 days or accompanied by clinically significant bleeding or Grade 3 thrombocytopenia with clinically significant bleeding;(viii) Grade 4 neutropenia that does not resolve to Grade 3 or lower within 14 days despite the use of granulocyte colony- stimulating factor (G-CSF);(ix) any Grade 5 toxicity not related to disease progression;(x) any macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH); or(xi) any Grade > 3 thrombotic event.

19. A method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising:(1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D); and(2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof.

20. The method of claim 19, wherein the compound is administered in a dosing regimen that is initiated on the same day as the CAR T cell administration.

21. The method of claim 19 or 20, wherein the compound is administered to the subject no earlier than 3 hours prior, no earlier than 2 hours prior, no earlier than 1 hour prior, no earlier than 0.5 hour prior, no later than 0.5 hour after, no later than 1 hour after, no later than 2 hours after, or no later than 3 hours after the CAR T cell administration.

22. The method of any one of claims 19-21, wherein at least one fixed dose of the compound is administered in an amount between 0.1 mg and 2.0 mg.

23. The method of any one of claims 20-22, wherein the dosing regimen of the compound is a cycling regimen comprising more than one treatment cycle.

24. The method of claim 23, wherein each treatment cycle is a 28-day cycle.

25. The method of claim 23 or 24, wherein the dosing regimen of the compound comprises at least one treatment cycle characterized by administration of the compound at a firstdose and at least one treatment cycle characterized by administration of the compound at a second dose, wherein the second dose is greater than the first dose.

26. A method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising:(1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D);(2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof, in a dosing regimen that is initiated on the same day as the CAR T cell administration; wherein the dosing regimen of the compound is a cycling regimen comprising more than one treatment cycle, wherein each treatment cycle is a 28-day treatment cycle; wherein at least one treatment cycle is characterized by administration of the compound at a first dose and at least one treatment cycle is characterized by administration of the compound at a second dose, wherein the second dose is greater than the first dose.

27. The method of any one of claims 20-26, wherein the dosing regimen comprises administration of the compound to the subject on Days 1-21 of the 28-day cycle.

28. The method of any one of claims 20-27, wherein the compound of Formula (II) is administered daily.

29. The method of any of claims 25-28, wherein the first dose of the compound is from about 0.3 mg to 0.9 mg daily.

30. The method of any one of claims 25-29, wherein the first dose of the compound is about 0.45 mg daily.

31. The method of any one of claims 25-29, wherein the first dose of the compound is about 0.75 mg daily.

32. The method of any one of claims 25-31, wherein the second dose of the compoundis from about 0.5 mg to 1.5 mg daily.

33. The method of any one of claims 25-32, wherein the second dose of the compound is about 1.0 mg daily.

34. The method of any one of claims 25-33, wherein the dosing regimen comprises a first, second, third, fourth and fifth treatment cycles, wherein the each of the treatment cycles are characterized by daily administration of the first dose of the compound.

35. The method of claim 34, wherein after the fifth treatment cycle, the dosing regimen comprises one or more additional treatment cycles characterized by daily administration of the second dose of the compound.

36. The method of any one of claims 19-35, wherein the compound is administered orally.

37. A method of treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising:(1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D);(2) administering the compound of Formula (II):pharmaceutically acceptable salt thereof, to the subject, wherein administration of the compound is initiated on the same day as the CAR T cell administration and is administered at a daily dose on Days 1-21 of a 28-day treatment cycle for a plurality of treatment cycles, wherein:(i) the daily dose in the first, second, third, fourth and fifth treatment cycles is a first dose of 0.45 mg; and(ii) the daily dose in each successive treatment cycle is a second dose of 1.0 mg.

38. A method of treating a treating a relapsed and / or refractory multiple myeloma (RRMM), the method comprising:(1) administering to a subject having a RRMM a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D);(2) administering the compound of Formula (II):(II), or a pharmaceutically acceptable salt thereof, wherein administration of the compound is initiated on the same day as the CAR T cell administration and: is administered at a daily dose on Days 1-21 of a 28-day treatment cycle, wherein:(i) the daily dose in the first, second, third, fourth and fifth treatment cycles ois a first dose of 0.75 mg; and(iii) the daily dose in each successive treatment cycles is a second dose of 1.0 mg.

39. The method of any one of claims 19-38, wherein the compound is administered to the subject until disease progression.

40. The method of any one of claims 19-39, wherein the subject has not undergone previous treatment with the compound.

41. The method of any one of claims 19-40, wherein the dosing regimen of the compound in combination with the CAR-T cell therapy is such that the subject does not develop a toxicity characterized by one or more of:(i) Any Grade 4 CRS and Grade CRS that does not resolve to Grade < 2 within 72 hours;(ii) any duration Grade 3 ICANS or neurotoxicity (NT);(iii) a higher than Grade 3 toxicity involving vital organs (eg, cardiac, pulmonary) of any duration;(iv) all other Grade 3 toxicities not attributable to underlying disease or lymphodepleting chemotherapy that do not resolve to < Grade 2 within 72 hours, other than a non-CRS toxicity of the liver in which liver amino transferase levels > 3x upper limit of normal (ULN) are accompanied by an elevation of total bilirubin to >2 x ULN without initial findings of cholestasis (e.g., elevated serum alkaline phosphatase) for which no other reason can be found to explain the combination of increased amino transferase to total bilirubin, such as viral hepatitis A, B, or C; pre-existing or acute liver disease; or another drug capable of causing the observed liver injury;(v) Grade 3 aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) increase lasting < 14 days or Grade 4 AST and / or ALT increase lasting < 7 days;(vi) certain treatment-emergent, isolated Grade 3 or 4, asymptomatic laboratory electrolyte abnormalities not directly related to vital organ toxicities that resolve, with or without intervention, to Grade < 2 in < 7 days;(vii) Grade 3 or 4 thrombocytopenia accompanied by clinically significant bleeding or Grade 4 thrombocytopenia that does not resolve to Grade 3 or lower within 42 days or accompanied by clinically significant bleeding;(viii) Grade 2 or higher infection with concurrent grade 3 or 4 neutropenia;(ix) any Grade 5 toxicity not related to disease progression;(x) any macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH); or(xi) any Grade > 3 thrombotic event.

42. The method of any one of claims 1-41, wherein the CAR comprises (1) an extracellular antigen-binding domain that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D), (2) an immunoglobulin hinge spacer of at least 125 amino acids in length; (3) a transmembrane domain; and (4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain and a costimulatory signaling region; wherein the extracellular antigen-binding domain comprises a VH region and a VL region, wherein: the VH region comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:1; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:2 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:2; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:1; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 116 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 116; the VH region comprises the amino acid sequence set forth in SEQ ID NOG or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NOG; and the VL region comprises the amino acid sequence set forth in SEQ ID NOG or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NOG;the VH region comprises the amino acid sequence set forth in SEQ ID NO:3 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:3; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 117 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 117; the VH region comprises the amino acid sequence set forth in SEQ ID NO:5 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:5; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:6 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:6; the VH region comprises the amino acid sequence set forth in SEQ ID NO:5 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:5; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 118 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 118; the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and the VL region comprises the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:8; the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 119 or an amino acid sequence having at least at or about90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 119; the VH region comprises the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:9; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 10; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 9; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 120 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 120; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 11; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 12 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 12; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 11; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 121 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:121; the VH region comprises the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about98%, or at or about 99% identity to SEQ ID NO: 13; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 14; or the VH region comprises the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 13; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 122 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 122.

43. The method of claim 42, wherein the extracellular antigen binding domain comprises a VH region and a VL region, wherein: the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:20, 21 and 17, respectively, and the VL region comprises the amino acid sequences of SEQ ID NOS:25, 26 and 27, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:35, 36, 32, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:40, 41 and 42, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:50, 51 and 47, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:55, 56 and 57, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:65, 66 and 62, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:70, 71 and 72, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:80, 81 and 77, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:85, 86 and 87, respectively;the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:80, 94 and 91, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:97, 98 and 99, respectively; the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS: 107, 108 and 104, respectively, and the VL region comprises a CDR- Ll, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 112, 26, 113, respectively; or the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:109, 110 and 111, respectively, and the V region comprises a CDR- Ll, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS: 114, 29 and 123, respectively.

44. The method of claim 42 or 43, wherein the extracellular antigen binding domain comprises a VH region and a VL region wherein: the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs:l and 2, respectively or the amino acid sequence set forth in SEQ ID NOs: 1 and 116, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs:3 and 4, respectively or the amino acid sequence set forth in SEQ ID NOs:3 and 117, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 5 and 6, respectively or the amino acid sequence set forth in SEQ ID NOs: 5 and 118, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 7 and 8, respectively or the amino acid sequence set forth in SEQ ID NOs: 7 and 119, respectively; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 9 and 10, respectively or the amino acid sequence set forth in SEQ ID NOs: 9 and 120, respective; the VH region and the VL region comprise the amino acid sequence set forth in SEQ ID NOs: 11 and 12, respectively or the amino acid sequence set forth in SEQ ID Nos: 11 and 121, respectively; orthe VH region and the VL region comprise the amino acid sequence set forth inSEQ ID NOs:13 and 14 or the amino acid sequence set forth in SEQ ID Nos: 13 and 122, respectively.

45. The method of any of claims 42-44, wherein the extracellular antigen binding domain comprises the VH region comprises a CDR-H1, CDR-H2, and CDR-H3 comprising the amino acid sequences of SEQ ID NOS:65, 66 and 62, respectively, and the Vuregion comprises a CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences of SEQ ID NOS:70, 71 and 72, respectively.

46. The method of any of claims 42-45, wherein: the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO:7; and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 119 or or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% identity to SEQ ID NO: 119.

47. The method of any of claims 42-46, wherein: the VH region comprises the amino acid sequence set forth in SEQ ID NO:7 and the VL region comprises the amino acid sequence set forth in SEQ ID NO: 119.

48. The method of any of claims 42-47, wherein the single chain antibody fragment is or comprises a single chain variable fragment (scFv).

49. The method of any of claims 42-48, when the VH region and the VL region are joined by a flexible linker.

50. The method of claim 49, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 127).

51. The method of any of claims 42-50, wherein the VH region is carboxy-terminal to the VL region.

52. The method of any of claims 42-51, wherein the extracellular antigen-binding domain comprises the amino acid sequence selected from SEQ ID NOs: 131, 133, 137, and 141 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence selected from SEQ ID NOs: 131, 133, 137, and 141.

53. The method of any of claims 42-52, wherein the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 137.

54. The method of any of claims 42-53, wherein the spacer comprises a sequence of a hinge region, a CH2 and CH3 region.

55. The method of any of claims 42-54, wherein the spacer comprises an IgG4 / 2 chimeric hinge region or a modified IgG4 hinge region comprising at least one amino acid replacement compared to a human IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region.

56. The method of any of claims 42-55, wherein the spacer is or comprises (i) the sequence set forth in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 that has at least at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to SEQ ID NO: 162; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length.

57. The method of any one of claims 42-56, wherein the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 164.

58. The method of any of claims 42-57, wherein the cytoplasmic signaling domain of CD3(^ comprises the amino acid sequence set forth in SEQ ID NO: 176 or an amino acid that hasat least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 176.

59. The method of claim 58, wherein the costimulatory signaling region comprises an intracellular signaling domain of CD28, 4- IBB, or ICOS, or a signaling portion thereof.

60. The chimeric antigen receptor of any of claims 58 or 59, wherein the costimulatory signaling region comprises an intracellular signaling domain of 4- IBB.

61. The method of any of claims 58-60, wherein the costimulatory signaling region is or comprises the amino acid sequence set forth in SEQ ID NO: 179 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the sequence set forth in SEQ ID NO: 179.

62. The method of any of claims 42-61, wherein the transmembrane domain is or comprises a transmembrane domain from CD4, CD28, or CD8.

63. The method of any of claims 42-62, wherein the transmembrane domain is or comprises a transmembrane domain derived from CD28.

64. The method of any one of claims 42-63, wherein the CAR comprises:(1) an extracellular antigen-binding domain that specifically binds human G-protein coupled receptor class C group 5 member D (GPRC5D), wherein the extracellular antigenbinding domain comprises:(i) a variable heavy chain (VH) region comprising an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and(ii) a variable light chain (VL) region comprising an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, ator about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 119;(2) a spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric CH2 region; and an IgG4 CH3 region, optionally that is about 228 amino acids in length;(3) a transmembrane domain from human CD28; and(4) an intracellular signaling region comprising a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain and an intracellular signaling domain of a 4-1BB.

65. The method of claim 64, wherein: the extracellular antigen-binding domain comprises the VH region amino acid sequence set forth in SEQ ID NO:7 and the VL region amino acid sequence set forth in SEQ ID NO: 119.

66. The method of claim 64 or 65, wherein the extracellular antigen-binding domain comprises an scFv set forth in SEQ ID NO: 137.

67. The method of any one of claims 64-66, wherein: the spacer is or comprises (i) the sequence set forth in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 that has at least at or about 95%, at or about 96%, at or about 97%, at or about 98% or at or about 99% sequence identity to SEQ ID NO: 162; or (iii) a contiguous portion of (i) or (ii) that is at least 125 amino acids in length; or the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO: 164.

68. The method of any of claims 42-67, wherein the transmembrane domain is or comprises the amino acid sequence set forth in SEQ ID NO: 173 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 173.

69. The method of any of claims 42-68, wherein the intracellular signaling region comprises (a) a cytoplasmic signaling domain of a CD3-zeta (CD3Q chain comprising the amino acid sequence set forth in SEQ ID NO: 176 or an amino acid sequence that has at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 176 and (b) and an intracellular signaling domain of a 4-1BB comprising the amino acid sequence set forth in SEQ ID NO: 179 or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to the sequence set forth in SEQ ID NO: 179.

70. The method of any of claims 42-69, wherein the intracellular signaling region is or comprises the sequences set forth in SEQ ID NO: 176 and SEQ ID NO: 179.

71. The method of any of claims 1-70, wherein the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 183, or an amino acid sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 183.

72. The method of any of claims 1-70, wherein the chimeric antigen receptor is encoded by the nucleotide sequence set forth in SEQ ID NO: 182, or a nucleotide sequence having at least at or about 90%, at or about 91%, at or about 92%, at or about 93%, at or about 94%, at or about 95%, at or about 96%, at or about 97%, at or about 98%, or at or about 99% sequence identity to SEQ ID NO: 182.

73. The method of any of claims 1-72, wherein the dose of engineered cells comprises between about 1.0 x 107CAR-expressing T cells and 1.2 x 109CAR-expressing T cells, between about 1.0 x 107CAR-expressing T cells and 6.5 x 108CAR-expressing T cells, between about 1.5 x 107CAR-expressing T cells and 6.5 x 108CAR-expressing T cells, between about 1.5 x 107CAR-expressing T cells and 6.0 x 108CAR-expressing T cells, between about 2.5 x 107CAR-expressing T cells and 6.0 x 108CAR-expressing T cells, between about 5.0 x 107CAR-expressing T cells and 6.0 x 108CAR-expressing T cells, between about 1.25 x 107CAR-expressing T cells and 1.2 x 109CAR-expressing T cells, between about 1.5 x 107CAR- expressing T cells and 1.2 x 109CAR-expressing T cells, between about 5.0 x 107CAR- expressing T cells and 4.5 x 108CAR-expressing T cells, or between about 1.5 x 108CAR- expressing T cells and 3.0 x 108CAR-expressing T cells, each inclusive.

74. The method of any of claims 1-73, wherein the dose of engineered cells comprises at or about 1.5 x 107, at or about 2.5 x 107, at or about 5.0 x 107, at or about 7.5 x 107, at or about 1.5 x 108, at or about 2.25 x 108, at or about 3.0 x 108, at or about 4.5 x 108, at or about 6.0 x 108, at or about 8.0 x 108, or at or about 1.2 x 109CAR-expressing T cells.

75. The method of any of claims 1-74, wherein the dose of engineered cells comprises at or about 7.5 x 107CAR-expressing T cells.

76. The method of any of claims 1-74, wherein the dose of engineered cells comprises at or about 1.5 x 108CAR-expressing T cells.

77. The method of any one of claims 1-76, wherein the method further comprises administering to the subject a lymphodepleting chemotherapy prior to administration of the CAR-T cell therapy.

78. The method of claim 77, wherein the lymphodepleting therapy is completed within about 7 days prior to initiation of the administration of the dose of the CAR-expressing T cells.

79. The method of claim 77 or 78, wherein the administration of the lymphodepleting therapy is completed within about 2 to 7 days prior to initiation of the administration of the dose of engineered T cells.

80. The method of any one of claims 77-79, wherein the lymphodepleting therapy comprises the administration of fludarabine and / or cyclophosphamide.

81. The method of any one of claims 77-80, wherein the lymphodepleting therapy comprises the administration of fludarabine and cyclophosphamide.

82. The method of any one of claims 77-81, wherein the lymphodepleting therapy comprises administration of cyclophosphamide at or about 200-400 mg / m2inclusive daily, optionally at or about 300 mg / m2daily.

83. The method of any one of claims 77-81, wherein the lymphodepleting therapy comprises administration of fludarabine at or about 20-40 mg / m2inclusive daily, optionally at or about 30 mg / m2daily.

84. The method of any one of claims 77-83, wherein the lymphodepleting therapy comprises administration of fludarabine and cyclophosphamide for 2-4 days, optionally for 3 days.

85. The method of any one of claims 77-80 wherein the lymphodepleting therapy comprises the administration of bendamustine.

86. The method of any one of claims 77-80 and 85, wherein the lymphodepleting therapy comprises administration of bendamustine at or about 50-130 mg / m2inclusive daily, optionally at or about 90 mg / m2daily.

87. The method of any one of claims 77-80, 85, and 86, wherein the lymphodepleting therapy comprises administration of bendamustine for 1-3 days, optionally for 2 days.

88. The method of any one of claims 1-87, wherein the subject does not develop a severe toxicity.

Images