Novel Anti-g protein-coupled receptor class c group 5 member d (GPRC5d) car molecules and uses thereof

Abstract

The present disclosure provides novel anti-GPRC5D chimeric antigen receptor (CAR) molecules, compositions, and their uses, where the CAR specifically recognizes and binds G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D) or fragments thereof. In particular the disclosed CAR molecules comprise anti-GPRC5D VHH nanobody directed at residues R154, M156, T162, and QI 65 of GPRC5D.

Description

[0001] NOVEL ANTI-G PROTEIN-COUPLED RECEPTOR CLASS C GROUP 5 MEMBER D (GPRC5D) CAR MOLECULES AND USES THEREOF TECHNOLOGICAL FIELD

[0002] The present disclosure relates to the field of immunotherapy. More specifically, the present disclosure relates to novel anti-G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D) CAR molecules, compositions, and uses thereof.

[0003] BACKGROUND ART

[0004] References considered to be relevant as background to the presently disclosed subject matter are listed below:

[0005] 1. Munshi, N. C., et al. N Engl J Med 384, 705-716 (2021).

[0006] 2. Hansen, D. K., et al.. J Clin Oncol 41, 2087-2097 (2023).

[0007] 3. Berdeja, J. G., et al. Lancet 398, 314-324 (2021).

[0008] 4. Martin, T., et al.. J Clin Oncol 41, 1265-1274 (2023).

[0009] 5. Brauner-Osborne, H., et al.. Biochim Biophys Acta 1518, 237-248 (2001).

[0010] 6. Atamaniuk, J., et al. Eur J Clin Invest 42, 953-960 (2012).

[0011] 7. Zhou, D., et al. J Hematol Oncol 17, 88 (2024).

[0012] 8. Rodriguez-Otero, P., et al. Blood Cancer J 14, 24 (2024).

[0013] 9. Mailankody, S., et al. N Engl J Med 387, 1196-1206 (2022).

[0014] 10. Zhang, M., et al. Lancet Haematol 10, e107-e116 (2023).

[0015] 11. Jurgens, E. M., et al. J Clin Oncol, JCO2401785 (2024).

[0016] 12. Pan, M., et al. Blood.2025030559 (2025).

[0017] 13. Jin L. et al., Clinical Trial Lancet Haematol. 2025 Oct;12(10):e798-e807

[0018] Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.BACKGROUND

[0019] Multiple myeloma (MM) is an incurable disease and the second most common hematological malignancy, accounting for about 1% of all cancers [Dimopoulos, M. A., et al. Hemasphere 5, e528 (2021)]. MM is a neoplasm of clonal plasma cells in the bone marrow. Its clinical evolution is characterized by relapses over time, with reduced progression-free survival following each relapse. According to the World Health Organization, the global incidence of MM was 187,952 new cases in the year 2022, and the mortality rate was 121,388 (PMID: 39658225). Therapy for MM presently involves novel agents such as proteasome inhibitors (Pls) and immunomodulators (IMiDs), as well as anti-CD38 antibodies that improve patients’ outcomes and overall survival (OS) [Cowan, A. J., et al. JAMA 327, 464-477 (2022)]. However, despite major advances in therapy, the vast majority of patients develop resistance to these drugs and pose a major treatment challenge. Advanced line therapies with second- and third-generation IMiDs and Pls, or with other newer agents, induce overall response rates (ORR) of 25-40%, with limited progression-free survival (PFS) [Gandhi, U. H., etal. Leukemia 33, 2266-2275 (2019); Kumar, S. K., etal. Leukemia 31, 2443-2448 (2017); Pick, M., et al. Eur J Haematol 100, 494-501 (2018)]. Patients resistant to over five agents (penta-refractory) have an extremely dismal prognosis with median PFS and OS of 3.9 and 6.0 months, respectively [Gil SK, et al., Blood Cancer J. 2022 Sep 23; 12(9): 138.]. In the past decade, chimeric antigen receptor T-cell (CAR T) therapy targeted to the B-cell maturation antigen (BCMA) has emerged as a highly promising immunotherapy for the treatment of relap sed / refractory MM (RRMM). Although BCMA CAR T shows excellent efficacy results with ORR of 73-97.7% and median PFS of 8.5-27.7 months [Ref. 1-4], the relapse rate is increasing worldwide.

[0020] G protein-coupled receptor class C group 5 member D (GPRC5D), an orphan receptor of mammalian G-protein coupled receptors (GPCRs) superfamily [Ref. 5], is a promising immunotherapy target for treatment of patients who relapse after anti-BCMA therapies [Ref. 6]. GPRC5D is detected in the plasma cells of MM patients and to a low extent in hair follicle, and its expression level is associated with poor prognosis multiple myeloma [Ref. 6]. The evolution of GPRC5D research initiated by 2001 (the initial cloning of the gene), however, only since 2019, pre-clinical and clinical studies evaluating GPRC5D-targeting immunotherapies were reported [Ref. 7],

[0021] According to a review [Ref. 8], there are five major clinical studies for MM therapy using GPRC5D as a target; one of them (the bispecific antibody, Talquetamab) has already beenapproved by the FDA. Currently, three of these studies use GPRC5D CAR T cells for the therapy of MM [Ref. 8]. More specifically, MCARH109 was the first trial developed [Ref. 9]. This construct is a second-generation CAR consisting of an anti-GPRC5D single-chain variable fragment, a CD28 hinge and transmembrane domain, a 4- IBB costimulatory domain, and a CD3 signaling domain [Smith, E. L., et al. Sci Transl Med 11(2019)]. OriCAR-017 is also a second-generation CAR construct consisting of two different anti-GPRC5D VHH nanobodies connected by a G4S linker, a CD8a hinge and transmembrane domain, a 4- IBB costimulatory domain, and a CD3 signaling domain [Ref. 10].

[0022] BMS-986393 is a GPRC5D-targeting autologous CAR-T therapy in the market that has the same CAR as MCARH109 [Ref. 11],

[0023] Since then, two main anti-GPRC5D CART cells were published: the RD118 ([Ref. 12], a fully human single-domain antibody fragment (VHH)-based GPRC5D CART and CT071 [Ref. 13], a fully human scFv- based GPRC5D CART.

[0024] GENERAL DESCRIPTION

[0025] The first aspect of the present disclosure relates to a chimeric antigen receptor (CAR) molecule comprising the following components: (i) at least one target-binding domain. The at least one target-binding domain specifically recognizes and binds G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), or any fragments thereof; (ii) at least one hinge region; (iii) at least one transmembrane domain; and (iv) at least one intracellular signal transduction domain. It should be noted that at least one of the target binding domains comprises at least one variable heavy chain only (VHH) nanobody, or any antigen binding fragment thereof. More specifically, the VHH nanobody of the disclosed CAR molecule is characterized by at least one of the following features. Tn some embodiments (a), the VHH nanobody specifically binds residues R154, Ml 56, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprises non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional oralternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the disclosed CAR molecule, comprises at least one Complementarity Determining Region 3 (CDR3) comprising an amino acid sequence as denoted by at least one of: AAGYGDCSDDGCYRGY (SEQ ID NO: 7), GILGG (SEQ ID NO: 10), DAGLSSGVEL (SEQ ID NO: 12), and / or RGPIGAPHIRSS (SEQ ID NO: 14), according to IMGT numbering, or any variant or derivatives thereof.

[0026] Another aspect of the present disclosure relates to a nucleic acid molecule comprising at least one nucleic acid sequence encoding at least one CAR molecule, or any cassette, vector or vehicle comprising the nucleic acid molecule. The CAR molecules encoded by the disclosed nucleic acid molecules comprise the following components. As component (i), at least one target-binding domain, wherein at least one of the target-binding domains specifically recognizes and binds GPRC5D, or any fragments thereof. Component (ii), comprises at least one hinge region. Component (iii) is at least one transmembrane domain. Component (iv) comprises at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one variable heavy chain only (VHH) nanobody, or any antigen binding fragment thereof. The VHH nanobody forming the target binding domain of the CAR molecule encoded by the disclosed nucleic acid molecule, is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprises noncontiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR molecule encoded by the nucleic acid molecule of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule encoded by the nucleicacid molecule of the present disclosure, comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, and / or SEQ ID NO: 14, or any variant or derivatives thereof.

[0027] Another aspect of the present disclosure relates to a genetically engineered cell expressing at least one CAR molecule, or a population of cells comprising at least one of the genetically engineered and / or modified cells. In some embodiments, the disclosed engineered cell is a hematopoietic cell. In yet some further embodiments, the hematopoietic cell is an immune cell. The CAR molecules expressed by the genetically engineered cells disclosed herein, comprise the following components: (i) at least one target-binding domain, wherein at least one of the target binding domains specifically recognizes and binds GPRC5D, or any fragments thereof; (ii) at least one hinge region; (iii) at least one transmembrane domain; and (iv) at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody of the CAR molecule expressed by the cells of the present disclosure is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprises non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR expressed by the cells of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule expressed by the cells of the present disclosure comprises at least one 3 CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, and / or SEQ ID NO: 14, or any variant or derivatives thereof.

[0028] Another aspect of the present disclosure relates to a composition comprising at least one of the following elements, acting as the active ingredient / s. Specifically, at least one CAR molecule, anynucleic acid molecule comprising at least one nucleic acid sequence encoding the CAR molecule, or any cassette, vector, vehicle or gene editing system comprising the nucleic acid molecule, and / or any genetically engineered cell expressing the CAR molecule of the present disclosure, or population of cells comprising at least one of the genetically engineered cell, and / or any combinations thereof. The CAR molecules of the disclosed compositions are any of the CAR molecules disclosed by the present disclosure. The compositions of the present disclosure further comprise at least one of pharmaceutically acceptable carrier / s, diluent / s, excipient / s and / or additive / s.

[0029] Another aspect of the present disclosure relates to a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathological disorder in a subject. The method comprises the step of administering to the subject an effective amount of at least one of: (A), at least one nucleic acid molecule encoding at least one CAR molecule; (B), at least one cassette, vector vehicle or gene editing system comprising the nucleic acid molecule of (A); (C), at least one genetically engineered cell expressing the CAR, or a population of the cells; (D), at least one VHH nanobody specific for GPRC5D; and (e), a composition comprising at least one of (A), (B), (C) and (D). More specifically, the CAR molecule comprising the following components: (i), at least one targetbinding domain, wherein at least one of the target binding domains specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. More specifically, at least one of the target binding domains of the CAR molecule used by the methods of the present disclosure comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody of the disclosed CAR molecule is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprises non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues QI,R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R1OOF, G1O1, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule used by the methods of the present disclosure, comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, and / or SEQ ID NO: 14, or any variant or derivatives thereof. Another aspect of the present disclosure relates to a therapeutically effective amount of at least one of: (A), at least one nucleic acid molecule encoding at least one CAR molecule; (B), at least one cassette, vector vehicle or gene editing system comprising said nucleic acid molecule of (A); (C), at least one hematopoietic cell expressing said CAR, or a population of said cells; (D), at least one VHH nanobody specific for GPRC5D; and (E), a composition comprising at least one of (A), (B), (C) and (D), for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onset of pathological disorder in a mammalian subject. The CAR molecules used herein comprise the following components: (i), at least one target-binding domain, wherein at least one of the target binding domains specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. At least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody of the CAR molecule used herein is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising noncontiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues QI, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y100E, R100F, G101, and Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the disclosed CAR molecule, comprises at least one CDR3 comprising the amino acid sequence asdenoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, and / or SEQ ID NO: 14, or any variant or derivatives thereof.

[0030] Another aspect of the present application relates to a method for targeted activation of a hematopoietic cell against a target cell expressing the GPRC5D protein and / or a tissue comprising the target cell. The method comprising the step of contacting the hematopoietic cell with an effective amount of at least one of: (A), at least one nucleic acid molecule encoding at least one CAR molecule; (B), at least one cassette, vector vehicle or gene editing system comprising said nucleic acid molecule of (A); and / or (C), a composition comprising at least one of (A) and (B). The CAR molecules encoded by the nucleic acid molecule used in the disclosed methods comprise the following components: (i), at least one target-binding domain, wherein at least one of the target binding domains specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. More specifically, at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody of the CAR molecules used by the methods of the present disclosure is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR molecule used by the methods of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the disclosed CAR molecule, comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, and / or SEQ ID NO: 14, or any variant and derivatives thereof.Another aspect of the present disclosure relates to an anti-GPRC5D VHH nanobody characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprises non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the present disclosure comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, and / or SEQ ID NO: 14, or any variant or derivatives thereof.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0033] Figure 1. SDS-PAGE of produced nanobodies

[0034] The nanobodies generated were run on a non-reduced SDS-PAGE gel and stained by Coomassie Blue. The expected protein sizes are: R4P1-A4 (127 AA; MW=13.8 kDa), R4P1-B1 (131 AA; MW=14.4 kDa), R4P1-D5 (125 AA; MW=13.5kDa), R4P1-H3 (121 AA; MW=13.1kDa).

[0035] Figure 2A-2C. Flow cytometer plots of 293T-GPRC5D cells incubated with the nanobodies Flow cytometer plots of 293T control cells (Fig.2A) or 293T-GPRC5D cells (Fig.2B-2C), stained with 10µg (Fig. 2A-2B) or 2µg (Fig. 2C) of R4P1-A4, R4P1-H3, R4P1-D5, or R4P1-B1.Figure 3A-3F. Flow cytometer overlay plots ofMMl. S or MM1. S-GPRC5D cells incubated with the nanobodies

[0036] Flow cytometer overlay plots of MM1. S cells (Fig. 3A-3C) or MM1. S-GPRC5D overexpressing cells (Fig. 3D-3F) incubated with anti-GPRC5D commercial antibody (Fig. 3A, 3D), or with 2, 10, or 20µg of B1 nanobody (Fig.3B, 3E) or with 20µg of H3 or D5 nanobodies (Fig.3C, 3F). Figure 4. Sensograms of nanobodies passed over a sensor surface

[0037] Sensograms of three independent repeats of solutions that passed over the sensor surface in concentrations of 0-7680 nM of Bl analyte. The Y-axis (Response, RU) represents resonance units proportional to the mass bound to the chip surface (1 RU ≈ 1 pg / mm2). The X-axis (Time, seconds) represents the duration of the experiment, showing association (binding) and dissociation (unbinding) phases.

[0038] Figure 5. Killing assay of NCI-H929-LUC cells co-cultured with CART cells

[0039] Six-days bioluminescence (BLI) signal detected by NCI-H929-luciferase (Luc) MM cells when co-cultured with different anti-GPRC5D CART cells with different antigen binding domains (Bl, D5 and H3 assigned as CARB 8a, CARD8a and CARH8a, respectively) compared to anti-BCMA HBI0101 positive control CART cells and to CARNC8a negative control CART cells. At day +2 and at day+6, co-culture wells were diluted 1:2 and 1:6, respectively, to avoid crowdedness. Figure 6A-6B. Cytotoxicity of various GPRC5D-expressing target cells in co-cultures with Bibased CART cells with different antigen binding domains

[0040] Averaged BLI (Fig.6A) or target cell count (CD3minus; Fig.6B) of three different MM cell lines (H929-LUC, MM1. S-LUC, or RPMI-LUC) co-cultured with CARB8a, CARD8a, and CARH8a produced from two AL patients' donors, normalized to BLI (Fig. 6A) or cell count (Fig. 6B) detected by MM cells in co-culture with negative control CART cells (K562-LUC; each CART was normalized to control CAR with the same hinge and TMD). The arrows under the X-axis indicate the time points by which the CART cells were re-exposed to a boost of 10,000 target cells (H929-LUC, MM1. S-LUC, or RPMI-LUC).

[0041] Figure 7 (panels I to IV). Illustration of Bl -based CAR derivatives' domains

[0042] I. B 1-based CARs with different hinge and transmembrane domains.

[0043] II. B1-based CARs with the addition of CH3 or CH2-CH3 spacers at the extracellular domain. III. Dual Bl and H3 CARs (both directions).

[0044] IV. Fourth-generation CARs with constitutive or inducible IL7 expression.Figure 8A-8B-. Cytotoxicity of various GPRC5D-expressing target cells co-cultured with B1-based CART cells with different spacers

[0045] Averaged BLI (Fig.8A) or target cell count (CD3minus; Fig.8B) of three different MM cell lines (H929-LUC, MM1. S-LUC, or RPMI-LUC) co-cultured with CARB8a28, CARB8a28-CH3, or CARB8a28-CH2CH3LALA produced from 2 AL patients' donors, normalized to BLI or cell count detected by MM cells co-cultured with negative control CART cells (K562-LUC; each CART was normalized to control CAR with the same hinge and TMD). The arrows under the X-axis indicate the time points by which the CART cells were re-exposed to a boost of 10,000 target cells (H929-LUC, MM1.S-LUC, or RPMI-LUC).

[0046] Figure 9A-9C. Effect of the different hinges and transmembrane domains on MM cells cytotoxicity and CART cell proliferation rate

[0047] Relative BLI (Fig. 9A) target cell count (Fig. 9B) and CART cells number (Fig. 9C) outputs of the different hinges and transmembrane domains Bl -based CART co-cultured with three different GPRC5D+ MM cells (H929-LUC, MM1.S-LUC, or RPMI-LUC; full lines) or GPRC5D-negative K562-LUC cells (dashed lines). CART cells were produced from leukapheresis of healthy, MM and 2 AL patients donors (n=4). BLI and target cell counts were normalized to the results of the negative control CART cells (each CART was normalized to control CAR with a same hinge and TMD). HBI0101 (BCMA-CART cells) are positive control CART effector cells. The arrows under the X-axis indicate the time points by which the CART cells were re-exposed to a boost of 10,000 target cells.

[0048] Figure 10. IL7 correlation with progression-free survival (PFS)

[0049] The X-axis represents the levels of IL7 in patient's serum prior to BCMA-CART therapy. Triangles represent patients in remission (ongoing) while the circles represent patients that relapsed at the indicated times (T-axis).

[0050] Figure 11A-11B. Combined effect of the CART molecules with constitutive expression of IL-7 on target-cell cytotoxicity and growth rate of CART cells

[0051] Fig. 11A. Averaged cell count of three different MM cells (H929-LUC, MM1.S-LUC, or RPMI-LUC; full lines) or GPRC5D-negative K562-LUC cells (dashed lines) co-cultured with anti-GPRC5D CART cells with (squares) or without (circles) constitutive interleukin-7 (IL-7) secretion, produced from leukapheresis of healthy, MM, and 2 AL patient donors (n=4) and normalized to cell count of target cells co-cultured with negative control CART cells (each CAR was normalized to control CAR with the same hinge and TMD). As a positive control, the countof target cells co-cultured with HBI0101 (BCMA-CART cells) and normalized to CARNC8a were presented (triangles). The arrows under the X-axis indicate the time points by which the CAR T cells were re-exposed to a boost of 10,000 target cells.

[0052] Fig. 11B. CAR T cell count by flow cytometer at the time points of 0, 3, 6, 9, 13, 16, and 20 days post the first exposure to target cells, extrapolated to the expected number without the routine dilution. Cell counts below 50 were not counted as they are below the threshold (target cells alone presented results below 50). When CART cells were not detected anymore, the line on the graph was disrupted. CART cell numbers of CD28-based CART cells produced from 4 donors (one healthy, two AL patients, and 1 MM patient) along the co-culture with three types of MM cells lines (NCI-H929, MM1. S and RPMI-8226) are presented. The arrows under the X-axis indicate the time points by which the CART cells were re-exposed to a boost of target cells.

[0053] Figure 12A-12B. Cytotoxicity of various target cells in co-culture with Bl and H3 dual CART cells

[0054] Averaged BLI (Fig. 12A) or target cell count (CD3 minus; Fig. 12B) of three different MM cells (H929-LUC, MM1. S-LUC, or RPMI-LUC) in co-culture with CARB28, CARBH28, and CARHB28 produced from 2 AL patients donors, normalized to BLI or cell count detected by MM cells in co-culture with negative control CART cells (each CAR was normalized to control CAR with the same hinge and TMD). The arrows under the X-axis indicate the time points by which the CAR T cells were re-exposed to a boost of 10,000 target cells (H929-LUC, MM1. S-LUC, or RPMI-LUC).

[0055] Figure 13A-13B. Eradication of primary malignant plasma cells by CARB28 and CARB28-IL7 Modulation of cleaved caspase-3 levels in CD 138+ malignant plasma cells purified from bone marrow of early diagnosed (BM-1; Fig.13A) or relapsed and refractory (BM-2; Fig.13B) multiple myeloma patients, post 24-hours of co-culturing with CARB28 or CARB28-IL7 (black dashed lines). As a negative control, the malignant plasma cells were co-cultured with CARNC28 or CARNC28-IL7 transduced T cells (Grey full histograms) and as a positive control, malignant plasma cells were co-incubated with the BCMA-CAR T (HBI0101; dashed line).

[0056] Figure 14A-14B. CARB28 and CARB28-IL7 activation by primary malignant plasma cells Expression of the T-cell activation markers 4-1BB (Fig. 14A) and CD25 (Fig. 14B) on CARB28 and CARB28-IL7 transduced T cells, originated from both healthy and MM patients leukapheresis and co-incubated with malignant plasma cells purified from MM patient as compared to their CAR controls, CARNC28 and CARNC28-IL7, respectively (P<0.01).Figure 15A-15B. Secretion of IL7 and interferon-y (IFN-y) by CARB28 and CARB28-IL7 cocultured with primary malignant plasma cells

[0057] IL-7 (Fig. 15A) and IFN-y (Fig. 15B) levels detected in the supernatant post 24 hours (circles) and 48 hours (squares) of co-culturing MM patients' plasma cells with CARB28 and CARB28-IL7 as compared to CARNC28 and CARNC28-IL7, respectively. Supernatants of CART cells alone are represented by empty circles (24 hours) or empty squares (48 hours).

[0058] Figure 16A-16D. Tumor and IL-7 monitoring in mice receiving CART-expressing IL-7 Fig. 16A-16B. MM IS Tumor volume was monitored in NSG mice, calculated by width x width x length / 2 (Fig. 16A) or by tumor BLI signal (Fig. 16B) post 15-days from intravenous CART injection.

[0059] Fig. 16C. CD4 / CD8 distribution of CARB28 cells in blood at day +12 compared with CARB-IL7.

[0060] Fig. 16D. IL-7 levels detected in plasma purified from mice blood.

[0061] Figure 17A-17C. Nucleic acid cassettes of CAR variants

[0062] Illustration of CAR vector domains of CARB28 (Fig. 17A), CARB28-IL7 (Fig. 17B), and inducible (I) CARB28-IL7 (Fig. 17C).

[0063] Figure 18A-18F GFP positivity and mean fluorescence intensity (MFI) determination in CARB28 cells at basal activation

[0064] CARB28 alone (Fig. 18A) or with GFP expression controlled by the regulatory element of the transcription factor NFAT (Fig. 18B), NFKB (Fig. 18C), NFkB-APl (Fig. 18D), NR4A (Fig.

[0065] 18E), and STAT5 (Fig. 18F) were measured by flow cytometer for GFP expression. The NFAT regulatory element was tested with a polyA (pA) element between the CAR sequence and the RE to better separate between the two genes.

[0066] Figure 19A-19H. GFP positivity and mean fluorescence intensity (MFI) determination in CARB28 cells after activation

[0067] CARB28 alone or with GFP expression controlled by the regulatory element of the transcription factor NFAT, NFKB, NFkB-APl, NR4A, and STAT5 were cultured alone (Fig. 19A, 19E), cocultured with GPRC5D-negative K562 cells (Fig. 19B, 19F), co-cultured with GPRC5D-positive NCI-H929 cells (Fig.19C, 19G), or stimulated by CD3 / CD28 beads (Fig. 19D, 19H) for 48 hours. Samples were taken at the time points of 0, 24, and 48 hours post culturing for GFP measurement by flow cytometer.Figure 20A-20D. Antibody structure, CDRs and paratope mapping via in silico Al-driven docking

[0068] Fig. 20A. Residues predicted to belong to the paratope of the Bl VHH variant, are marked on the nanobody sequence, as denoted by SEQ ID NO: 1. CDRs are defined with the Chothia numbering system, CDR1 (SEQ ID NO: 31, Chothia), is underlined with a yellow color, CDR2 (SEQ ID NO: 32, Chothia), is underlined with a light orange color, CDR3 (SEQ ID NO: 33, Chothia), is underlined with a dark orange color. The paratope residues are highlighted with light blue (low), dark blue (medium), light purple (high), dark purple (very high), reflecting the intensity of the predicted binding to the epitope as indicate.

[0069] Fig. 20B. An illustrative table of all residues of the Bl VHH nanobody of SEQ ID NO: 1, indicating the numbering of each residue according to Chothia numbering. CDR1 (SEQ ID NO: 31, Chothia), is highlighted with a yellow color, CDR2 (SEQ ID NO: 32, Chothia), is highlighted with a light orange color, CDR3 (SEQ ID NO: 33, Chothia), is highlighted with a dark orange color. The paratope residues are highlighted with light blue (low), dark blue (medium), light purple (high), dark purple (very high), reflecting the intensity of the predicted binding to the epitope as indicate.

[0070] Fig. 20C. A ribbon model of the B 1 VHH nanobody, presenting the three-dimensional structure. P-sheets are drawn as flat arrows, and loops / turns are drawn as thin ribbons or lines, specifically, the CDR1 (yellow), CDR2 (light orange) and CDR3 (dark orange) loops.

[0071] Fig. 20D. A ribbon model of the B 1 VHH nanobody, highlighting the paratope residues over the CDR1 (yellow), CDR2 (light orange) and CDR3 (dark orange) loops. Most of the paratope residues reside within CDR3.

[0072] Figure 21A-21C. Bl binding to GPRC5D variants

[0073] Flow cytometry density plot (Fig. 21A) and graphs of number of gated (Fig. 21B) or mean fluorescence intensity (MFI; Fig.21C) of 293T-expressing GPRC5D wild type or variant mutants, stained with Bl nanobody (Fig. 21A; second row, Fig. 21B-21C) or its isotype match (Fig. 21A; first row).

[0074] Figure 22A-22C. A refined epitope mapping via experimental validation

[0075] Fig. 22A. The epitope residues within the GPRC5D molecule demonstrated to be crucial for B 1 VHH nanobody binding (R154, M156, T162, Q165, of the GPRC5D, as denoted by SEQ ID NO: 140), marked with dark orange.Fig. 22B. Re-analyzed residues predicted to belong to the epitope are highlighted with light blue (low), dark blue (medium), light purple (high), dark purple (very high), reflecting the intensity of the predicted binding to the Bl VHH nanobody as indicate. The refined epitope is located in extracellular region C of the GPRC5D molecule.

[0076] Fig. 22C. The conformational epitope marked on the amino acid sequence of residues 1-268 of GPRC5D (SEQ ID NO: 140, residues 1-268 shown in the figure are denoted by SEQ ID NO: 145). The re-analyzed residues of the epitope are highlighted with light blue (low), dark blue (medium), light purple (high), dark purple (very high), reflecting the intensity of the predicted binding to the B 1 VHH nanobody as indicated. The refined epitope is located in extracellular region C of the GPRC5D molecule. The extracellular regions of GPRC5D are underlined A (yellow), B (light oranges), C (dark orange) and D (red).

[0077] Figure 23A-23D Annotated images from the Confirmation screens

[0078] Fig. 23A. Results of library and confirmation screen

[0079] The table presents all results of the library screen test. Summarizing of the library (L) and confirmation (C) screens positive spots. Library screen was performed in CARB28 in duplicates, whereas the confirmation screen was performed on both CARB28 and NT in duplicates. The spots were scaled to Very weak (v. weak), Weak, Medium and Strong binding. Results in black background represent CAR-dependent interaction while the results on white background represent CAR-independent interactions. Genes are categorized into plasma membrane (PM), Secreted (S), Tethered secreted (TS) and Heterodimer (HD) proteins.

[0080] Figs.23B-23D. Confirmation slides showing the Alexa-flour-647 fluorescently labelled CARB28-LV (Fig.23B) and control NT cells (Fig.23C) binding to HEK293 cells over-expressing ICOSLG, CD86, TNF, PVRa, SIRPA (isoform 4), CD40, ADA, SIRPA (isoform 2), CD44, CD244, PLXNBI, GPRC5D, MAG, CD80, CRTAM, CD70, TNFSF4, CD58, ICAM5, SIRPA (isoform 1), SPNS2, MFSD2B, CXCRL12-tathered, IGF2-tathered, PVRp, IGFl-tathered, EFNA4-tathered, TIGIT+PVR, TNFSF8 and EGFR after fixation in the confirmation screen. The interaction with GPRC5D is outlined by a framed box. (Fig.23D) ZsGreenl expression validating protein overexpression.

[0081] Figure 24A-24D. Tumor monitoring (GPRNSG4)

[0082] Fig. 24A. Tumor burden quantified by summing luminescence signals from dorsal and ventral images. Once the first mouse in each group reached a BLI of > 1010, the follow-up of the other mice in the specific group was terminated in this graph. Fig. 24B. Concentration of CART cellsdetected in peripheral blood during follow-up. Fig. 24C. Kaplan-Meier survival curve of mice.

[0083] Fig. 24D. Mice spleen weight comparison between different groups at the time of exclusion from study (due to either tumor BLI or safety issues (GVHD or weight loss).

[0084] Figure 25A-25F. Tumor monitoring (GPRNSG4)

[0085] Tumor (Fig. 25A-25C) and CART cells (Fig. 25D-25F) monitoring in mice following treatment injection of CARB28-LV (Fig. 25A, 25D), CARB28-RV (Fig. 25B, 25E) and HBI0101-RV (Fig.

[0086] 25C, 25F). N=6 (Females: superior triangle, circle and a square; Males: diamond, Hexagon and inferior triangle).

[0087] Figure 26A-26C. CART cells characterization

[0088] Fig. 26A. Distribution of CART cells by differentiation status in final products prior to injection: naive / stem-cell memory (Tnaive / Tscm; CD62L+CD45RA+), central memory (Tcm; CD62L CD45RA ), effector memory (Tern; CD62L CD45RA ), and terminally differentiated effector memory RA+(Temra; CD62L CD45RA+) for CARB28-LV, CARB28-RV, HBI0101-RV, and HBI0101-TM.

[0089] Fig. 26B. Median fluorescence intensity (MFI) of CD62L.

[0090] Fig. 26C. Percentages of cells expressing CD27 and / or CD28.

[0091] Figure 27A-27F. CARB28 CART cells induce robust tumor regression in subcutaneous MM. IS xenografts

[0092] Tumor growth kinetics following treatment with saline (Fig. 27A), NT (Fig. 27B), PBMCs (Fig.

[0093] 27C), CARB28-RV (Fig. 27D), CARB28-LV (Fig. 27E), or Talquetamab (Fig. 27F). Mice were monitored for tumor volume over 90 days post-infusion (n = 5 mice per group).

[0094] Figure 28. Longitudinal bioluminescence imaging confirms CARB28-mediated tumor clearance

[0095] Representative IVIS bioluminescence images showing MM.1S-LUC tumor burden in each treatment group at weekly intervals (W0-W6). The radiance scale indicates signal intensity (p / s / cm2 / sr).

[0096] Figure 29A-29E. CART or CD3+ cell expansion in mice blood following CARB28 treatment Fig. 29A-29C. CAR-T (Fig. 29A-29B) or CD3+ (Fig. 29C) cell kinetics following infusion of CARB28-RV (Fig.29A), CARB28-LV (Fig.29B), or PBMCs accompanied by Talquetamab (Fig.

[0097] 29C).Fig.29D-29E. Cumulative CART cell levels categorized into Tnaive / SCM (CD62L+CD45RA+), Tcm (CD62L+CD45RA-), Tem (CD62L-CD45RA-), and Temra (CD62L-CD45RA+) in the blood of mice received CARB 28 -RV (Fig. 29D) or CARB20-LV (Fig. 29E).

[0098] Figure 30. CARB28 prolongs survival in MM.lS-bearing NSG mice

[0099] Kaplan-Meier survival analysis comparing treatment cohorts. Statistical analysis by log-rank (Mantel-Cox) test, p < 0.001.

[0100] DETAILED DESCRIPTION OF EMBODIMENTS

[0101] The inventors developed a novel GPRC5D CAR cells therapy focusing on the following strategies aiming to improve CART cell efficacy. More specifically, by designing a novel extracellular antigen binding domain (ABD), based on VHH nanobody, the present inventors provided optimized CAR molecules that target the GPRC5D expressed by diseased target cells.

[0102] The first aspect of the present disclosure relates to a chimeric antigen receptor (CAR) molecule comprising the following components: (i) at least one target-binding domain. At least one of the target binding domains specifically recognizes and binds G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), or any fragments thereof; (ii) at least one hinge region; (iii) at least one transmembrane domain; and (iv) at least one intracellular signal transduction domain. It should be noted that at least one of the target binding domains comprises at least one variable heavy chain only (VHH) nanobody, or any antigen binding fragment thereof. More specifically, the VHH nanobody of the CAR molecule of the present disclosure is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140, or any variants or isoforms thereof. In some embodiments, GPRC5D isoforms may include isoforms comprising the amino acid sequence as denoted by any one of SEQ ID NO: 141, 142, 143 and 146. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140, or any variants or isoforms thereof. In some specific additional or alternative embodiments, the epitope recognized by the VHH nanobody of the CAR molecule of the present disclosure comprises the amino acid sequence EY-TLIMTRGMMFVNMTPCQL-VD--VL, as denoted by SEQ ID NO: 144(specifically, TLIMTRGMMFVNMTPCQL (residues T149 to L166), as also presented by Figure 22C). Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. The paratope residues located over the VHH nanobody sequence (SEQ ID NO: 1) are shown in Figure 20A. The numbering of these residues (according to Chothia numbering), as well as the strength of their binding to the epitope of the GPRC5D target are illustrated in Figure 20B. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule of the present disclosure comprises at least one Complementarity Determining Region 3 (CDR3) comprising the amino acid sequence as denoted by at least one of: AAGYGDCSDDGCYRGY (SEQ ID NO: 7), GILGGE (SEQ ID NO: 10), DAGLSSGVEL (SEQ ID NO: 12), RGPIGAPHIRSS and / or (SEQ ID NO: 14), according to IMGT numbering, or any variant or derivatives thereof.

[0103] It should be understood that the at least one target-binding domain of the disclosed CAR molecules may be specified according to some embodiments, using CDR3, that is the main domain interacting with the target. The CDRS are indicated herein in accordance with IMGT numbering, that is commonly used for VHH nanobodies. However, it should be appreciated that the disclosed VHH nanobodies, may comprise CDR3 sequences identified by any other numbering methods used in the field of antibodies. For example, according to Chothia numbering, or alternatively, according to Kabat numbering, or according to any appropriate numbering methods. Accordingly, in some embodiments, the at least one target-binding domain of the disclosed CAR molecules may comprise at least one VHH nanobody comprising a CDR3 that comprise the amino acid sequence as denoted by any one of SEQ ID NO: 33 (VHH Bl, as also shown in Figure 20B), SEQ ID NO: 38 (VHH H3), SEQ ID NO: 43 (VHH D5), and / or SEQ ID NO: 48 (VHH A4), as defined according to Chothia numbering. In yet some further embodiments, the VHH molecule used as the target binding domain in the disclosed CAR molecule, may comprise a CDR3 that comprise the amino acid sequence as denoted by any one of SEQ ID NO: 33 (VHH Bl), SEQ ID NO: 38 (VHH H3), SEQ ID NO: 43 (VHH D5), and / or SEQ ID NO: 48 (VHH A4), as defined according to Kabat numbering.

[0104] In yet some specific embodiments, the VHH nanobody of the CAR molecule of the present disclosure comprises CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 5, CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 6, and CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 7, according to IMGT numbering. In somefurther embodiments, the VHH nanobody is designated Bl VHH nanobody. Still further, in some embodiments, the Bl VHH nanobody comprises CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 31, CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 32, and CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 33, according to Chothia numbering, as also disclosed in Figure 20A.

[0105] A " Chimeric Antigen Receptor (CAR)", as used herein, refers to a recombinant polypeptide comprising at least an extracellular target binding domain (also referred to herein as antigen binding domain), a transmembrane domain and an intracellular cytoplasmic signaling domain comprising a functional stimulatory domain. The receptors are chimeric because they couple, in a single receptor molecule, between extracellular target-binding capabilities and intracellular activating functions of a hematopoietic cell (e.g. a lymphocyte, such as T cell, B cell and NK cell or a myeloid cell such as a macrophage). CARs have been engineered to give the hematopoietic cells they are expressed in the new ability to recognize a specific antigen of interest, thereby facilitating an immune reaction against it. For example, the technology is used in immunotherapy for specifically recognizing specific cancer cells' antigens of interest in order to more effectively direct the immune cells towards those target cells and destroy them.

[0106] CAR, as used herein, relates to artificial receptors, such as T cell receptors (also known as chimeric T cell receptors, chimeric immuno-receptors). These are engineered receptors, which graft an arbitrary specificity onto an immune effector cell. Typically, these receptors are used to graft the specificity of a monoclonal antibody onto a T cell.

[0107] The initial design (also referred to as first generation) joined an antibody-derived scFv to the CD3ζ intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second-generation CARs added intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. More recent, third-generation CARs combine multiple signaling domains, such as CD27, CD28, 4-1BB, ICOS, or 0X40, to augment potency.

[0108] It should be understood that the CAR molecules of the present disclosure may be based on any CAR molecule of any generation as disclosed herein, provided that the target binding domain of the disclosed CAR molecules comprises the anti GPRC5D VHH nanobodies provided by the present disclosure. This includes, specifically, any antibodies or, nanobodies, or antigen-binding domains that recognize and bind the disclosed conformational epitope as discussed herein, oralternatively, that display the paratope as specified herein, or alternatively, that comprise CDR3 sequence as discussed by the present disclosure.

[0109] It should be understood that the disclosed CAR molecules may be further improved by adding at least one additional signaling domain.

[0110] The term "chimeric protein " relates to proteins created through the joining or fusing of two or more genes that originally coded for separate proteins. Translation of this chimeric / fusion gene results in single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant chimeric / fusion proteins are created artificially by recombinant DNA technology. Chimeric or chimera usually designates hybrid proteins made of polypeptides having different functions, sources, or physicochemical patterns.

[0111] The CAR of the present disclosure comprises at least one target-binding domain. " Target-binding domain" as used herein refers to a region of a molecule, such as an antibody or antibody fragment (e.g., scFv, Fab, or VHH nanobody), that specifically recognizes and binds to a target antigen. In the context of chimeric antigen receptors (CARs), the target-binding domain is typically derived from the variable region of an antibody and is responsible for conferring antigen specificity to the CAR molecule. In some specific embodiments, the at least one target-binding domain comprises at least one variable heavy chain only (VHH) nanobody. The term " VHH nanobody" refers to the single variable domain of the heavy chain of a heavy-chain-only antibody (HCAb). VHH nanobodies are naturally present in camelids (e.g., camels, llamas, and alpacas) and certain cartilaginous fish (e.g., sharks) [Tang, H., Gao, Y. & Han, J. Int J Mol Sci 24(2023); Bao, C., et al. Biomolecules 11(2021)]. Unlike conventional antibodies that consist of paired heavy and light chains, as detailed below, heavy-chain-only antibodies lack light chains, and their antigen-binding functionality is conferred solely by the variable domain of the heavy chain of a heavy-chain antibody (VHH), which comprises at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen or any epitope thereof.

[0112] VHH nanobodies are small (approximately 12–15 kDa), single-domain molecules that exhibit high specificity and affinity for their target antigens. Due to their compact size and structural stability, VHH nanobodies possess several advantageous properties, including high solubility, thermal and chemical stability, ease of engineering and production and enhanced tissue penetration. More specifically, VHH nanobodies are relatively small in size, and thus they are capable of reaching certain epitopes inaccessible to conventional antibodies (even intracellular epitopes). These antibodies display optimal stability, by reducing potential of VH-VL mispairing and VH-VHaggregation with high CAR expression levels. VHH nanobodies also display high capability to penetrate tissues.

[0113] In some embodiments, the VHH nanobody comprises at least one Complementarity Determining Region 3 (CDR3), and specifically bind to, or interact with GPRC5D or any epitope thereof, for example, the conformational epitope disclosed by the present disclosure. The term " Complementarity Determining Region 3 (CDR3)" refers to the third hypervariable loop within the variable domain of an antibody or T-cell receptor. CDR3 is typically the most diverse and variable of the CDRs and plays a crucial role in antigen recognition and binding specificity due to its central location and structural flexibility.

[0114] In some other embodiments of the present disclosure, the VHH nanobodies comprise the three CDRs (i.e., CDR1, CDR2, CDR3) and specifically bind to or interact with GPRC5D or any epitope thereof.

[0115] In some more specific embodiments, the VHH nanobody referred to as Bl comprises the CDR1 (GRTFSYYN), CDR2 (IKPSGRRV), and CDR3 (AAGYGDCSDDGCYRGY) amino acid sequences as denoted by SEQ ID NO: 5, 6, and 7 (according to IMGT numbering), respectively, or any variant or derivative thereof. According to some embodiments, the framework regions of the VHH of the disclosed CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 15 (FR1), SEQ ID NO: 16 (FR2), SEQ ID NO: 17 (FR3), and SEQ ID NO: 18 (FR4), according to IMGT numbering.

[0116] As indicated above, the VHH nanobodies used as the target binding domains of the disclosed CAR molecules, may be defined using other numbering methods to identify the CDR regions thereof. Accordingly, the Bl VHH nanobody may be further characterized in some embodiments as a nanobody having a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 31, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 32, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 33, according to Chothia numbering.

[0117] In yet some further embodiments, the Bl -VHH nanobody used for the disclosed CAR molecule, may comprise a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 34, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 35, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 33, according to Kabat numbering

[0118] In some other embodiments, the VHH nanobody referred to as H3 comprises the CDR1 (GFTFDDYA), CDR2 (ISWNGGST), and CDR3 (GILGGE) amino acid sequences as denoted bySEQ ID NO: 8, 9, and 10 (according to IMGT numbering), respectively, or any variants and derivatives thereof.

[0119] According to some embodiments, the framework regions of the VHH of the disclosed CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 19 (FR1), SEQ ID NO: 20 (FR2), SEQ ID NO: 21 (FR3), and SEQ ID NO: 22 (FR4), according to IMGT numbering. In some embodiments, the H3-VHH nanobodies used as the target binding domains of the disclosed CAR molecules, may be defined using other numbering methods to identify the CDR regions thereof. Accordingly, the H3 VHH nanobody may be further characterized in some embodiments as a nanobody having a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 36, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 37, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 38, according to Chothia numbering.

[0120] In yet some further embodiments, the H3-VHH nanobody used for the disclosed CAR molecule, may comprise a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 39, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 38, according to Kabat numbering.

[0121] In some additional embodiments, the VHH nanobody referred to as D5, comprises the CDR1 (GFTFDDFA), CDR2 (ISWNGGST), and CDR3 (DAGLSSGVEL) amino acid sequences as denoted by SEQ ID NO: 11, 9, and 12 (according to IMGT numbering), respectively, or any variants and derivatives thereof.

[0122] According to some embodiments, the framework regions of the VHH of the disclosed CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 23 (FR1), SEQ ID NO: 24 (FR2), SEQ ID NO: 25 (FR3), and SEQ ID NO: 26 (FR4), according to IMGT numbering. In some embodiments, the D5 VHH nanobody may be further characterized in some embodiments as a nanobody having a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 41, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 42, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 43, according to Chothia numbering.

[0123] In yet some further embodiments, the D5-VHH nanobody used for the disclosed CAR molecule, may comprise a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 44, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 45, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 43, according to Kabat numbering.In some further embodiments, the VHH nanobody referred to as A4, comprises the CDR1 (GFTFDDYA), CDR2 (ISWNAGST) and CDR3 (RGPIGAPHIRSS) amino acid sequence as denoted by SEQ ID NO: 8, 13, 14, (according to IMGT numbering) respectively, or any variant and derivatives thereof.

[0124] According to some embodiments, the framework regions of the VHH of the disclosed CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 27 (FR1), SEQ ID NO: 28 (FR2), SEQ ID NO: 29 (FR3), and SEQ ID NO: 30 (FR4), according to IMGT numbering. As indicated above, the VHH nanobodies used as the target binding domains of the disclosed CAR molecules, may be defined using other numbering methods to identify the CDR regions thereof. Accordingly, the A4 VHH nanobody may be further characterized in some embodiments as a nanobody having a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 46, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 48, according to Chothia numbering.

[0125] In yet some further embodiments, the A4-VHH nanobody used for the disclosed CAR molecule, may comprise a CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 49, a CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 50, and a CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 48, according to Kabat numbering.

[0126] In some embodiments, the VHH nanobody comprises the amino acid sequence as denoted by at least one of: SEQ ID NO: 1 (the Bl VHH), SEQ ID NO: 2 (the H3 VHH), SEQ ID NO: 3 (the D5 VHH), SEQ ID NO: 4 (the A4 VHH), or any variant and derivatives thereof. In some embodiments, the disclosed VHH nanobodies are encoded by a nucleic acid sequence comprising at least one of SEQ ID NO: 51, 52, 53 and 54, respectively.

[0127] It should be noted, however, that the present disclosure further encompasses any codon optimized nucleic acid sequence encoding the VHH nanobodies comprising the amino acid sequences of any one of SEQ ID NO: 1, 2, 3, and 4, or of any variants thereof. Such nucleic acid sequences may be referred to herein as variants of the nucleic acid sequences as denoted by any one of SEQ ID NO: 51, 52, 53, and 54, which encode the VHH Bl, H3, D5, and A4, respectively).

[0128] It should be understood that although at least one of the target binding domain / s of the disclosed CAR molecules comprises at least one VHH, the disclosed CDRs may be presented by any other antibody molecule or any antigen-binding fragments thereof. Moreover, in some alternative embodiments, the disclosed CAR may comprise in addition to the VHH indicated herein, alsoother antibodies (either specific for GPRCD5, and / or to other target molecules), or any antigen binding fragments thereof.

[0129] An "antibody” as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen or any epitope thereof. The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A typical antibody is composed of two immunoglobulin (Ig) heavy chains and two Ig light chains. In humans, antibodies are encoded by three independent gene loci, namely the immunoglobulin heavy locus (IgH) on chromosome 14, containing the gene segments for the immunoglobulin heavy chain, the immunoglobulin kappa (κ) locus (IgK) on chromosome 2, containing the gene segments for part of the immunoglobulin light chain and the immunoglobulin lambda (λ) locus (IgL) on chromosome 22, containing the gene segments for the immunoglobulin light chain.

[0130] At least one of the target binding domains of the CAR of the present disclosure specifically recognizes and binds GPRC5D, or any fragments thereof. It should be noted that the term “specifically recognizes and binds”, "specifically binds to an antigen”, “specifically immunoreactive with”, “specifically directed against” or “binding specificity”, when referring to an antigen or particular epitope, refers to a binding reaction which is determinative of the presence of the epitope in a heterogeneous population of proteins and other biologies. The term "epitope" is meant to refer herein to that portion of the GPRC5D capable of being bound by a VHH nanobody which can also be recognized by that nanobody. Epitopes or "antigenic determinants", is a specific molecular region of an antigen that is capable of being recognized and specifically bound by an antigen-binding molecule, including an antibody, an antigen-binding fragment thereof, a T-cellreceptor, or another antigen-binding protein. An epitope is typically formed by chemically active surface groupings of the antigen, such as amino acid residues, sugar side chains, or other molecular moieties, and is characterized by defined three-dimensional structural features and charge properties that mediate specific binding. An epitope may be linear, comprising a contiguous sequence of residues in the primary structure of the antigen, or conformational, comprising noncontiguous residues. Non-contiguous amino acid residues are residues that are not next to each other in the linear amino acid sequence (from the N-terminus to the C-terminus) but are brought into proximity when the protein folds into its three-dimensional structure. A conformational (discontinuous) epitope is an epitope formed by such non-contiguous amino acid residues that are distant in the primary sequence but are spatially adjacent in the folded protein and, together, constitute or contribute to) an antibody-binding site. In some specific embodiments, the VHH nanobody of the present disclosure specifically binds a conformational epitope in the GPRC5D. In more specific embodiments, the epitope recognized by the B 1 VHH nanobody of the present disclosure comprises residues EY-TLIMTRGMMFVNMTPCQL-VD— VL, as denoted by SEQ ID NO: 144 (TLIMTRGMMFVNMTPCQL (residues T149 to L166)), of the GPRC5D protein. For example, the disclosed epitope is a conformational epitope as it begins with the dipeptide EY, followed (after a one-residue gap) by an 18-amino-acid segment as set forth in SEQ ID NO: 144, followed (after a further one-residue gap) by the dipeptide VD, and then (after a two-residue gap) by the dipeptide VL. These sequence- separated elements are brought into close proximity in the folded protein, thereby forming a single antibody-binding surface.

[0131] Still further, as indicated above, a "target binding domain" or an "antigen-binding domain" of the disclosed CARs, can comprise or consist of an antigen-binding fragment of an antibody such as VHH. As shown in Figure 22C, the various residues of the conformational epitope are highlighted with light blue (low), dark blue (medium), light purple (high), dark purple (very high), reflecting the intensity of the predicted binding to the Bl VHH nanobody.

[0132] As mentioned above, the disclosed CAR molecules specifically recognize and bind G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), or any fragments thereof. GPRC5D, as used herein, is a transmembrane protein belonging to the G protein-coupled receptor (GPCR) superfamily, specifically classified under Class C GPCRs. These receptors are characterized by their seven transmembrane domains and their ability to transduce extracellular signals into intracellular responses via coupling with heterotrimeric G proteins. GPRC5D is encoded by the GPRC5D gene and is predominantly expressed in specific tissues, including the skin, immunecells, and bone marrow, with notable overexpression in certain pathological conditions, such as multiple myeloma. GPRC5D is thought to play a role in cellular signaling pathways related to immune regulation, inflammation, and bone remodeling. In some particular embodiments, the GPRC5D as used herein refers to the human GPRCD5. It should be understood that any isoform of GPRC5D, is applicable as a target for the disclosed CAR molecules of the present disclosure. In yet some further embodiments, the human GPRCD5 of the present disclosure comprises the amino acid sequence as denoted by any one of Q9NZD1l, NP_061124.1, NP_016875072.1, NP_054228435.1, or NP_054228436.1. In yet some further embodiments, the human GPRC5D as used herein is encoded by a nucleic acid sequence as denoted by NC_000012.12:cl2952170-12940575, NC_060936.1:cl2827419-12814207, or any codon optimized sequence thereof. Still further, in some embodiments, the human GPRC5D of the present disclosure comprises the amino acid sequence as denoted by any one of SEQ ID NO: 140, 141, 142, 143 or 146. In some embodiments, the human GPRC5D of the present disclosure is encoded by a nucleic acid sequence as denoted by any one of SEQ ID NO: 139 or 147.

[0133] Still further, in some embodiments, the target binding domain specifically recognizes and binds to a fragment of GPRC5D. The term "fragment” as used herein refers to a smaller portion or piece of GPRC5D that retains sufficient structural or functional features to be recognized and bound by the target binding domain. In more specific embodiments, the fragment or part of the target GPRC5D recognized by the disclosed nanobodies of the present CARs is comprised within the extracellular region of the molecule. As indicated by Figure 22C, the extracellular region of GPRC5D may comprise one or more domains that reside within residues 1 to 268 of the molecule, or any fragments thereof. In some embodiments, the extracellular region of GPRC5D comprises at least four domains specifically, domains A, B, C, and D. Domain A, comprises residues Ml to E27 of the amino acid sequence as denoted by SEQID NO: 140 (underlined as region A in yellow in Figure 22C), domain B comprises residues 181 to Y93 of the amino acid sequence as denoted by SEQ ID NO: 140 (underlined as region B in light orange in Figure 22C), domain C comprises residues T 145 to N 167 of the amino acid sequence as denoted by SEQ ID NO: 140 (underlined as region C in dark orange in Figure 22C), and domain D comprises residues R226 to D239 of the amino acid sequence as denoted by SEQ ID NO: 140 (underlined as region D in red in Figure 22C). In some specific embodiments, the fragment of the target GPRC5D recognized by the disclosed nanobodies of the present CARs is comprised within one or more of the disclosed domains. In some specific embodiments, the fragment of the target GPRC5D recognized by thedisclosed nanobodies of the present CARs is comprised within the extracellular domain C of the GPRC5D that contains residues T145 to N167. In yet some further embodiments, specifically residues R154, M156, T162, and Q165 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140, are recognized by the nanobodies of the present CARs. Accordingly, in some embodiments, the antigen-binding region of the CAR molecules of the present disclosure recognizes residues R154, M156, T162, and Q165 of GPRC5D. More specifically, the antigen-binding region recognizes a conformational epitope comprising residues E146 to Y147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the antigen-binding region of the CAR molecule comprises a VHH antibody (nanobody) paratope comprising residues QI, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y100E, R100F, G101, and Y102 of SEQ ID NO: 1, according to Chothia numbering. In yet further embodiments, the VHH comprises a CDR3 comprising the sequence set forth in SEQ ID NO: 7. In more specific embodiments, such VHH is the nanobody designated B 1 and comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0134] In some embodiments, the nanobody of the present disclosure (each one of the specified variants that recognize and binds the specified epitope as disclosed by the present disclosure) binds to the target molecule, GPRC5D, with an equilibrium dissociation constant (Kd) in the range of about 0.001 micromolar (pM) to about 100 pM, as determined by a binding assay such as surface plasmon resonance (SPR), flow cytometer test (as demonstrated in Example 3), or by biolayer interferometry (BLI) under defined conditions. In some embodiments, the Kd is at most about 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 2 pM, 1 pM, 0.5 pM, 0.1 pM, 0.05 pM, 0.01 pM, 0.005 pM, or 0.001 pM. In some embodiments, the Kd is at least about 0.001 pM, 0.005 pM, 0.01 pM, 0.05 pM, 0.1 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, 10 pM, 25 pM, 50 pM, or 100 pM.

[0135] In some embodiments, the B 1 nanobody of the present disclosure binds to GPRC5D or any epitope thereof, with a Kd in a range of about 1.0 pM to about 5.0 pM. In some embodiments, the Kd is selected from the group consisting of about 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, 1.5 pM, 1.6 pM, 1.7 pM, 1.8 pM, 1.9 pM, 2.0 pM, 2.1 pM, 2.2 pM, 2.3 pM, 2.4 pM, 2.5 pM, 2.6 pM, 2.7 pM, 2.8 pM, 2.9 pM, 3.0 pM, 3.1 pM, 3.2 pM, 3.3 pM, 3.4 pM, 3.5 pM, 3.6 pM, 3.7 pM, 3.8 pM, 3.9 pM, 4.0 pM, 4.1 pM, 4.2 pM, 4.3 pM, 4.4 pM, 4.5 pM, 4.6 pM, 4.7 pM, 4.8 pM, 4.9 pM, and 5.0 pM.

[0136] In some embodiments, the B 1 nanobody of the present disclosure binds to GPRC5D with a Kd in a range of about 2.9 pM to about 3.2 pM. In some embodiments, the Kd is about 2.9 pM, about3.0 μM, about 3.1 μM, or about 3.2 μM. Still further, the use of a target-binding domain (e.g., the nanobodies of the present disclosure), having an affinity characterized by a Kd in a range of about IpM to about 5pM, specifically, 2.9 pM to about 3.2 pM, for the CAR molecules of the present disclosure, and any cells expressing such CAR molecules, specifically, CAR T cells, may enhance the safety of such cells. More specifically, a CAR T cell expressing a target-binding domain with an affinity in the range of 1 pM to 5 pM, as detailed herein.

[0137] In some embodiments, the use of nanobodies, or CAR molecules comprising a nanobody-derived target-binding domain, having an affinity (e.g., Kd) of about 1 micromolar (pM) to about 5 pM for the target antigen can provide safety and selectivity advantages. Without being bound by theory, such moderate-affinity binding may reduce recognition of cells expressing low levels of the target antigen, thereby decreasing off-target binding and limiting on-target, off-tumor effects, while maintaining effective recognition and targeting of tumor cells that express higher levels of the target antigen. In some embodiments, this affinity range increases tumor specificity and improves the therapeutic window. In some embodiments, the reduced interaction with low-antigen-density cells further decreases chronic / tonic signaling in engineered immune cells (e.g., CAR-T or CAR-NK cells), thereby reducing persistent activation and mitigating functional exhaustion, which can improve durability and overall safety of the therapy.

[0138] As indicated above, in addition to the target-binding domain of (i), the disclosed CAR molecules comprise, as component (ii), at least one hinge region. A "hinge region," or hinge domain as used herein, refers to an extracellular flexible structure connecting the targeting moiety and the hematopoietic cell plasma membrane. These domains are generally derived from IgG subclasses (such as IgGl and IgG4), IgD, or alternatively from a co-receptor molecule (e.g., CD8 domains), or from co-stimulatory receptors, such as CD28.

[0139] Still further, the CAR molecule of the present disclosure comprises as component (iii), at least one transmembrane domain. A "transmembrane region", or transmembrane domain (TMD), also referred to herein as TM, of the disclosed CAR molecule, is a functional region of a protein that spans the phospholipid bilayer of a biological membrane, such as the plasma membrane of a cell. Integral membrane proteins typically comprise two or more such domains, alternating with intracellular and extracellular domains arranged on either side of the membrane. TMDs may consist predominantly of nonpolar amino acid residues and generally adopt an alpha- helix conformation. Amino acids of the transmembrane domains interact with the fatty acyl groups of the membrane phospholipids, thereby anchoring the protein in the membrane.In some embodiments, the hinge region may comprise between about 10 to 100, 20 to 90, 30 to 80, 30 to 70, or 30 to 60 amino acid residues, specifically, about 25 to 50 aa, specifically, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues.

[0140] In some embodiments, the TM region may comprise between about 30 to 15 amino acid residues, specifically, about 25 to 17 aa, specifically, 25, 24, 23, 22, 21, 20, 19, 18, or 17 amino acid residues. In some embodiments, the hinge region is derived from at least one of: a co-stimulatory receptor molecule, a co-receptor molecule and at least one immunoglobulin molecule.

[0141] In some other embodiments, the transmembrane domain is derived from at least one of: a costimulatory receptor molecule and / or at least one co-receptor molecule.

[0142] More specifically, a "co-stimulatory receptor" as used herein refers to a type of cell surface receptor that plays a critical role in modulating the activation, proliferation, and survival of immune cells (e.g., T cells). These receptors provide secondary signals that complement the primary activation signal delivered by the receptor (e.g., TCR) upon antigen recognition. Nonlimiting examples include CD28, ICOS, 4-1BB (CD137), 0X40 (CD134), CD27, SLAMF6 (CD352), HVEM (TNFRSF14), TIM-1, GITR (T cells); CD40, BAFF-R, SLAM receptors, TACI (B cells); DNAM-1 (CD226), NKG2D, CD244 (2B4) (NK cells); RANK, Dectin-1, TREM-2, CD80 / CD86 (B7-1 / B7-2), and TLR (macrophages).

[0143] A "co-receptor molecule" as used herein refers to a cell surface protein that modulates the activity of a primary receptor by providing additional signaling inputs or enhancing the specificity and stability of receptor-ligand interactions. In the immune system, co-receptor molecules are particularly important in regulating the activation, differentiation, and effector functions of immune cells, such as T cells, B cells, and natural killer (NK) cells. Co-receptor molecules interact with the same ligand as the primary receptor or with separate ligands to facilitate proper receptor engagement and downstream signaling, ensuring efficient immune responses. In the context of chimeric antigen receptors (CARs), co-receptor molecules are intrinsic or extrinsic components that influence the activation and functional performance of CAR-engineered immune cells. Non limiting examples include CD4 and CD8 (T cells) for MHC molecule interaction; CD3 complex, LFA-1 (CD1 la / CD18), CCR7, and CXCR3 (T cells) for signal transduction and migration; CD19, CD21 (CR2), CD81 (TAPA-1), CD79a / CD79b (Iga / Igp), and CXCR5 (B cells) for BCR signaling and localization; CD16 (FcyRIII), KIR, NKG2A / NKG2C, 2B4 (CD244), and NKp30, NKp44, NKp46 (NK cells) for cytotoxic activity and regulation; Fey receptors (e.g., CD64, CD32), CD 14,scavenger receptors (e.g., CD36), CCR2, and Dectin-1 (macrophages) for phagocytosis and inflammation; and CD4, CD205 (DEC-205), Fc receptors (e.g., FcyRII, FcsRII), and CCR7 (dendritic cells) for antigen uptake and migration. In the context of the disclosed CAR molecules, co-receptors such as CD3 Complex, CD4, CD8, CD28, 4-1BB (CD137), and ICOS, may be applicable.

[0144] Still further, an "immunoglobulin molecule”, commonly referred to as an antibody, is a glycoprotein produced by B cells as part of the adaptive immune response. In the context of chimeric antigen receptors (CARs), parts of immunoglobulin molecules are often utilized to construct the hinge and transmembrane domains of the CAR structure. These domains play critical roles in maintaining the structural flexibility and functional stability of the CAR, ensuring effective signal transduction and antigen recognition by the engineered immune cell. IgG-based transmembrane domains and hinge regions, such as those derived from IgGl or IgG4, are occasionally used in CAR constructs. Other immunoglobulin-derived transmembrane domain and hinge regions may also be used for the CAR of the present disclosure.

[0145] As used herein, the term "derived from" refers to a component, sequence, or molecule that originates from, is obtained from, or is based on an original source. In some embodiments, the component, sequence, or molecule is modified from the original source. It may include, but is not limited to, an exact, a fragment, a variant, an analog, a mutant, an engineered version, or a synthetic construct that retains at least one structural or functional characteristic of the original source. In some embodiments, the at least one hinge region of the CAR molecules of the present disclosure, is derived from a co-stimulatory receptor molecule being the Cluster of Differentiation 28 (CD28) protein, or a co-receptor molecule being the Cluster of Differentiation 8 a (CD8a) protein, or any combination thereof.

[0146] In some other embodiments, the at least one transmembrane domain of the CAR molecules of the present disclosure is derived from a co-stimulatory receptor molecule, being the CD28 protein, or a co-receptor molecule, being the Cluster of Differentiation 8 a (CD8a) protein, or any combination thereof.

[0147] Cluster of Differentiation 28 (CD28) is a transmembrane glycoprotein expressed on the surface of some immune cells (e.g., T cells), belonging to the immunoglobulin superfamily. It functions as a critical costimulatory receptor that plays a central role in T cell activation, survival, and immune response regulation. CD28 is constitutively expressed, for example, on the majority of CD4+ T cells and a subset of CD8+ T cells.CD28 binds to its natural ligands, CD80 (B7- 1 ) and CD86 (B7-2), which are expressed on antigen-presenting cells (APCs). This interaction provides a secondary, costimulatory signal that is essential for full T cell activation in conjunction with T cell receptor (TCR) engagement with antigen-MHC complexes. In some embodiments the human CD28 is denoted by Uniprot accession # UP000005640.

[0148] The Cluster of Differentiation 8 (CD8), also known as Ly-2 or Leu-2 and T-cell surface glycoprotein CD8 alpha chain, is a two-chain transmembrane glycoprotein that is expressed on the surface of circulating T-cells. CD8 serves as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule but is specific for the class I MHC protein presented by antigen-presenting cells (APCs). There are two isoforms of the CD8 protein, alpha and beta, each encoded by a different gene. CD8 exists as a disulfide-linked dimer, either as an α / β heterodimer or an α / α homodimer. In some embodiments, CD8a as used herein refers to the human CD 8 a homodimer.

[0149] In yet some further specific embodiments, the hinge region of the CAR molecules of the present disclosure is derived from CD28 and comprises the amino acid sequence as denoted by SEQ ID NO: 73, and / or any variants or derivatives thereof. In yet some further embodiments, the hinge region may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 74. In yet some further specific embodiments, the hinge region of the CAR molecules of the present disclosure is derived from CD8a and comprises the amino acid sequence as denoted by SEQ ID NO: 69, and / or any variants and derivatives thereof. In yet some further embodiments, the hinge region may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 70. In yet some further specific embodiments, the transmembrane (TM) domain of the CAR molecules of the present disclosure is derived from the CD28 protein and comprises the amino acid sequence as denoted by SEQ ID NO: 75; and / or any variants and derivatives thereof. In yet some further embodiments the TM region may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 76.

[0150] In yet some further specific embodiments, the transmembrane domain (TM) of the CAR molecules of the present disclosure is derived from the CD8a protein and comprises the amino acid sequence as denoted by SEQ ID NO: 71, and / or any variants and derivatives thereof. In yet some further embodiments, the TM region may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 72.In certain embodiments, the CAR described in the present disclosure may comprise any combination of a hinge region and transmembrane (TM) domain. The term "combination" as used in this context refers to the deliberate joining, fusion, or integration of distinct regions (units) (e.g., hinge and TM) from the same protein or from separate proteins. For example, in some embodiments, the CAR molecule of the present disclosure may comprise a hinge region of CD8a and a TM region of CD28 (comprising the amino acid sequence as denoted by SEQ ID NO: 67, encoded by the nucleic acid sequence as denoted by SEQ ID NO: 68). In some other embodiments, the CAR molecule of the present disclosure may comprise a hinge region of CD28 and a TM region of CD8a. In some embodiments, the CAR molecule of the present disclosure may comprise a hinge region of CD28 and a TM region of CD28 (comprising the amino acid sequence as denoted by SEQ ID NO: 65, encoded by the nucleic acid sequence as denoted by SEQ ID NO: 66). In some further embodiments, the CAR molecule of the present disclosure may comprise a hinge region of CD8a and a TM region of CD8a (comprising the amino acid sequence as denoted by SEQ ID NO: 63, encoded by the nucleic acid sequence as denoted by SEQ ID NO: 64).

[0151] As indicated above, the CAR molecules of the present disclosure, further comprise as component (iv), at least one intracellular signal transduction domain. As used herein, the term "intracellular signal transduction domain", refers in some embodiments to the functional, intracellular portion of a receptor protein that acts to transmit the detected stimulatory information within the cell, thereby regulating the cellular activity through specific signaling pathways. According to some embodiments, this domain is an intracellular domain connected to the transmembrane domain, specifically, the TM domain used by the CAR molecules of the present disclosure.

[0152] In some embodiments, the at least one intracellular signal transduction domain may be an intracellular signal transduction domain that may be effective in any hematopoietic cell. In some specific embodiments, the intracellular signal transduction domain of the disclosed CAR molecules may be any domain effective in any immune cell. In yet some further embodiments, the intracellular signal transduction domain of the disclosed CAR molecules may be any domain effective in cell of the lymphoid lineage, or alternatively in any cell of the myeloid lineage. In some specific embodiments, the at least one intracellular signal transduction domain may be an intracellular signal transduction domain of at least one lymphocyte.

[0153] In some more specific embodiments, the at least one intracellular signal transduction domain may be an intracellular signal transduction domain of at least one of a T cell, a B cell and an NK cell.Accordingly, the disclosed CAR molecule may be in some embodiments a CAR T molecule, a CAR B molecule, or a CAR NK molecule.

[0154] Still further, the at least one intracellular signal transduction domain may be an intracellular signal transduction domain of a myeloid cell. In some more specific embodiments, the at least one intracellular signal transduction domain may be an intracellular signal transduction domain of a macrophage. Accordingly, the disclosed CAR molecule may be in some embodiments a CAR-macrophage molecule.

[0155] In some embodiments, the at least one intracellular signal transduction domain of the CAR molecules of the present disclosure is an intracellular T cell signal transduction domain. The term "intracellular T cell signal transduction domain" refers herein to the functional intracellular portion of a chimeric antigen receptor (CAR) that is specifically designed to transmit activating signals within a T cell upon antigen recognition. This domain is crucial for initiating and sustaining T cell activation, proliferation, and effector functions, such as cytokine production and target cell killing. It typically includes signaling motifs derived from T cell receptor (TCR) components and co-stimulatory molecules, which collectively orchestrate the downstream biochemical events necessary for a robust anti-tumor or anti-pathogen immune response.

[0156] Still further, in some embodiments, the at least one intracellular T cell signal transduction domain of the CAR molecule of the present disclosure comprises at least one tumor necrosis factor (TNF) receptor family member. The tumor necrosis factor receptor (TNFR) family members are membrane-bound or soluble receptors that interact with membrane-bound and / or soluble ligands of the TNF superfamily. The majority of the members of this TNF / TNFR superfamily are expressed by immune cells. Activation of the TNFR members via their ligands affects cell proliferation, survival, differentiation, and apoptosis of responding cells.

[0157] The TNF-like receptors are type I transmembrane proteins characterized by cysteine-rich domains (CRD) that are the hallmark of the TNFR superfamily. These pseudorepeats are defined by intrachain disulfides generated by highly conserved cysteine residues within the receptor chains. The members of the tumor necrosis factor (TNF) / tumor necrosis factor receptor (TNFR) superfamily are critically involved in the maintenance of homeostasis of the immune system. The biological functions of this system encompass beneficial and protective effects in inflammation and host defense, as well as a crucial role in organogenesis. At the same time, members of this superfamily are responsible for host-damaging effects in sepsis, cachexia, and autoimmune diseases. The TNFR superfamily includes, for example, TNFR1 (also sometimes referred to asp55 / p60), TNFR2 (also known as p75 / p80), and B-cell activating factor receptor (BAFFR). Still further, the tumor necrosis factor (TNF) family includes, for example, TNF alpha (TNFa), TNF beta (TNFP), CD40 ligand (CD40L), Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (is homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes), some of the most important cytokines involved in physiological processes, systemic inflammation, tumor lysis, apoptosis, and initiation of the acute phase reaction.

[0158] In some embodiments, a TNF receptor family member useful as a signal transduction intracellular domain of the CAR molecule of the present disclosure is the 4- IBB. Thus, in some embodiments, the CAR molecule of the present disclosure comprises an intracellular domain derived from the 4thlymphocyte activation molecule, BB antigen (4-1BB). The 4-1BB (also known as TNFRSF9, CD 137) is an activation-induced T cell costimulatory molecule and a TNFR superfamily member.

[0159] 4- IBB is expressed on a subset of resting CD8 T cells and is upregulated on both CD4 and CD8 T cells following activation. Upon binding to trimeric 4-1BBL on APCs, 4-1BB recruits TNFR-associated factor family members (TRAF1, TRAF2, and TRAF3) to its cytosolic region, forming the 4-1BB signalosome and leading to downstream activation of NF-KB, MAPK, and ERK. Agonistic stimulation of 4- IBB upregulates expression of the anti-apoptotic proteins Bcl-xLand Bfl-1. Still further, 4- IBB activation increases IL-2 and IFN-y in CD8 cells and IL-2 and IL-4 in CD4 cells. T cells expressing CARs that incorporate 4- IBB domains have been shown to express granzyme B, IFN-y, TNF-a, GM-CSF and the anti-apoptotic protein Bcl-xL. Still further, incorporation of the 4- IBB TM and cytoplasmic domain into a CAR leads to improved persistence and antitumor activity, as well as to prolonged T cell division.

[0160] In some embodiments, 4- IBB as used herein refers to the human 4- IBB. In some embodiments, the human 4-1BB, is as denoted by Uniprot accession # Q07011.

[0161] In yet some further optional embodiments, the at least one intracellular T cell signal transduction domain of the CAR-molecule of the present disclosure, further comprises at least one TCR molecule or any fragments thereof. More specifically, the T-cell receptor (TCR) is a protein complex found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. When the TCR engages with antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associatedenzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

[0162] The core TCR complex consists of two TCR chains and six cluster of differentiation 3 (CD3) chains. The human genome expresses four TCR genes known as TCRa, TCRp, TCRy, and TCR6, which form two distinct heterodimers: TCRa / TCRP or TCRy / TCR6. The majority of mature T cells express TCRa and TCRP isoforms, generally referred to as T cells (or aP T cells), while a small portion (0.5-5%) of T lymphocytes (y8 T cells) express TCRy and TCR6 isoforms. Both heterodimers form multiprotein complexes with CD3 6, y, s, and C, chains. However, in both complexes, three dimers of CD3 proteins, 6s and ys heterodimers and homodimers, are present. These CD3 proteins are associated with TCR via non-covalent hydrophobic interactions and are required for complete TCR localization on the cell surface. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules, transduce activation signals to the T cell.

[0163] In more specific embodiments, the CAR molecule of the present disclosure may comprise at least one region derived from at least one domain of the TCR, specifically, the cluster of differentiation 3 (CD3) zeta chain. Thus, in some embodiments, the CAR molecule of the present disclosure comprises an intracellular domain that further comprises a domain derived from the CD3-ζ. T-cell surface glycoprotein CD3 zeta chain also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247) is a protein encoded in human by the CD247 gene. More specifically, CD3 (cluster of differentiation 3) T-cell co-receptor helps to activate the cytotoxic T-cell. It consists of a protein complex and is composed of four distinct chains. In mammals, the complex contains a CD3y chain, a CD36 chain, and two CD3s chains. These chains associate with the T-cell receptor (TCR) and the ζ-chain (zeta-chain) to generate an activation signal in T lymphocytes. The TCR, ζ-chain, and CD3 molecules together constitute the TCR complex. T-cell receptor zeta, together with T-cell receptor alpha / beta and gamma / delta heterodimers and CD3-gamma, -delta, and -epsilon, forms the T-cell receptor-CD3 complex. The zeta-chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways and is thus included in the CAR T molecule of the present disclosure. In some embodiments, CD3 zeta, as used herein refers to the human CD3 zeta. In some embodiments, the human CD3 zeta is as denoted by Uniprot accession # P20963-1.

[0164] In some embodiments, the at least one intracellular T cell signal transduction domain comprises a domain derived from 4- IBB and a domain derived from the CD3 zeta chain. More specifically, insome embodiments, the T cell signal transduction domain of the disclosed CAR molecules comprise the amino acid sequence as denoted by SEQ ID NO: 77 (of the 4-1BB, encoded by a nucleic acid sequence as denoted by SEQ ID NO: 78) and / or comprise the amino acid sequence as denoted by SEQ ID NO: 79 (of the CD3 zeta, encoded by a nucleic acid sequence as denoted by SEQ ID NO: 80), or any derivatives or variants thereof.

[0165] Still further, it should be appreciated that the disclosed CAR molecules may comprise, in addition to the components described herein, also one or more spacers, as was also exemplified by the present disclosure. In some embodiments, the disclosed CAR molecules may comprise at least one CH3 spacer. In some embodiments, the disclosed CAR molecules may comprise at least one CH3 spacer that comprises the amino acid sequence as denoted by SEQ ID NO:85. In yet some further embodiments, the disclosed CAR molecules may comprise at least one CH3 spacer encoded by the nucleic acid sequence as denoted by SEQ ID NO: 86. Still further, in some embodiments, the disclosed CAR molecules may comprise at least one CH2CH3 spacer, that includes the LALA mutation (e.g., L234A and L235A). In some embodiments, the disclosed CAR molecules may comprise at least one CH2CH3LALA spacer that comprises the amino acid sequence as denoted by SEQ ID NO:87. In yet some further embodiments, the disclosed CAR molecules may comprise at least one CH2CH3LALA spacer encoded by the nucleic acid sequence as denoted by SEQ ID NO: 88.

[0166] In yet some further additional or alternative embodiments, the disclosed CAR molecules may comprise, in addition to the components described herein, also one or more linkers, as was also exemplified by the present disclosure. In some embodiments, the disclosed CAR molecules may comprise at least one G4S linker. In some embodiments, the disclosed CAR molecules may comprise at least one G4S linker that comprises the amino acid sequence as denoted by SEQ ID NO:89. In some embodiments, the linker is encoded by the nucleic acid sequence as denoted by SEQ ID NO: 90. In some additional embodiments, the disclosed CAR molecules may comprise a Whitlow linker. In some embodiments, the disclosed CAR molecules may comprise at least one Whitlow linker that comprises the amino acid sequence as denoted by SEQ ID NO:91. In some embodiments, the Whitlow linker is encoded by the nucleic acid sequence as denoted by SEQ ID NO: 92.

[0167] In some specific embodiments, the CAR molecule of the present disclosure comprises the amino acid sequence as denoted by at least one of: SEQ ID NOs: 110, 102 (a control CAR, designated CARNC8a), 104, 106, 108, 113 (a control CAR, designated CARNC28), 117, 119, 122, 124, 126,128 (a control CAR, designated CARNC8a28), 130 and / or 132, or any variants or derivatives thereof.

[0168] More specifically, in some embodiments, the CAR molecule of the present disclosure may be designated CARNC8a (a control CAR), and may comprise the SIP- Bl - CD8a hinge - CD8a TMD - 4- IBB - CD3 zeta (also referred to herein as variant#2). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 102 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 103).

[0169] In some embodiments, the CAR molecule of the present disclosure may be designated CARB8a, and may comprise SIP- Isotype-CNT - CD8a hinge - CD8a TMD - 4- IBB - CD3 zeta (also referred to herein as variant#3). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 104 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 105). In some embodiments, the CAR molecule of the present disclosure may be designated CARD8a, and may comprise SIP- D5 - CD8a hinge - CD8a TMD - 4- IBB -CD3 zeta (also referred to herein as variant#4). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 106 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 107).

[0170] In some embodiments, the CAR molecule of the present disclosure may be designated CARH8a and may comprise SIP- H3 - CD8a hinge - CD8a TMD - 4- IBB - CD3 zeta (also referred to herein as variant#5). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 108 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 109).

[0171] In some embodiments, the CAR molecule of the present disclosure may be designated CARB28, and may comprise SIP- Bl - CD28 hinge - CD28 TMD - 4- IBB - CD3 zeta (also referred to herein as variant#6). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 110 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 111).

[0172] In some embodiments, the CAR molecule of the present disclosure may be designated CARNC28 (a control CAR), and may comprise SIP- Isotype-CNT - CD28 hinge - CD28 TMD - 4- IBB - CD3 zeta (also referred to herein as variant#8). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 113 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 114).In some embodiments, the CAR molecule of the present disclosure may be designated CARBH28, and may comprise SIP- B1-H3 - CD28 hinge - CD28 TMD - 4-1BB - CD3 zeta (also referred to herein as variant#11). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 117 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 118).

[0173] In some embodiments, the CAR molecule of the present disclosure may be designated CARHB28, and may comprise SIP- H3-B1 - CD28 hinge - CD28 TMD - 4-1BB - CD3 zeta (also referred to herein as variant#12). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 119 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 120).

[0174] In some embodiments, the CAR molecule of the present disclosure may be designated CARB8a28, and may comprise SIP- Bl - CD8a hinge - CD28 TMD - 4-1BB - CD3 zeta (also referred to herein as variant#14). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 122 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 123).

[0175] In some embodiments, the CAR molecule of the present disclosure may be designated CARB8a28-CH3, and may comprise SIP- Bl - CD8a hinge-CH3 - CD28 TMD - 4-1BB - CD3 zeta (also referred to herein as variant#15). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 124 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 125).

[0176] In some embodiments, the CAR molecule of the present disclosure may be designated CARB8a28-CH2CH3LALA, and may comprise SIP- Bl - CD8a hinge-CH2CH3LALA - CD28 TMD - 4-1BB - CD3 zeta (also referred to herein as variant#16). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 126 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 127).

[0177] In some embodiments, the CAR molecule of the present disclosure may be designated CARNC8a28, and may comprise SIP- Isotype-CNT - CD8a hinge - CD28 TMD - 4- IBB - CD3 zeta (also referred to herein as variant#17). According to some embodiments, the CAR molecule may comprise the amino acid sequence as denoted by SEQ ID NO: 128 (encoded by the nucleic acid sequence as denoted by SEQ ID NO: 129).

[0178] In more specific embodiments, the CAR molecule is designated CARB28 and comprises the amino acid sequence as denoted by SEQ ID NO: 110. It should be further understood that thepresent disclosure encompasses any nucleic acid sequence that encodes a CAR molecule having the amino acid sequence set forth in SEQ ID NO: 110 or any variant thereof, for example, a nucleic acid sequence comprising SEQ ID NO: 111, as well as any codon-optimized version thereof. As used herein, a "codon-optimized" nucleic acid sequence refers to a nucleic acid sequence that has been modified (without changing the encoded amino acid sequence) to substitute one or more codons with synonymous codons that are preferred in a selected host cell or expression system, for example to improve expression, mRNA stability, translational efficiency, or overall protein yield.

[0179] In some embodiments, the present disclosure contemplates CAR molecules in which the antigenbinding domain comprises a combination of different antigen-recognition modules directed to the GPRC5D antigen. For example, the antigen-binding domain may comprise one nanobody or more than one nanobody or any antigen-binding fragment thereof, including without limitation a combination of two VHH nanobodies (or antigen-binding fragments thereof) arranged on the same CAR molecule (e.g., in tandem), such that the CAR presents multiple antigen-binding domains that bind the same target antigen. It should be noted that in some embodiments, expression of the disclosed CAR molecule by at least one cell of the T lineage results in at least one of: (i) increased specificity; (ii) reduced tonic signaling; (iii) reduced off-target activation; (iv) increased expression of activation markers in response to a specific stimulation; and (v) reduced expression of exhaustion markers in response to a specific stimulation, in an in vivo and / or in vitro / ex vivo setting.

[0180] The present disclosure provides CAR molecules that are composed of amino acid residues and are therefore, a polypeptide. The term "polypeptide" as used herein refers to amino acid residues, connected by peptide bonds. A polypeptide sequence is generally reported from the N-terminal end containing a free amino group to the C-terminal end containing a free carboxyl group and may include any polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that contains portions that occur in nature separately from one another (i.e., from two or more different organisms, for example, human and non-human portions). In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. More specifically, " Amino acid sequence" or "peptide sequence" is the order in which amino acid residues connected by peptide bonds, lie in the chainin peptides and proteins. The sequence is generally reported from the N-terminal end containing a free amino group to the C-terminal end containing an amide. Amino acid sequence is often called peptide, or protein sequence, if it represents the primary structure of a protein. However, one must discern between the terms " Amino acid sequence" or "peptide sequence" and "protein," since a protein is defined as an amino acid sequence folded into a specific three-dimensional configuration and that had typically undergone post-translational modifications, such as phosphorylation, acetylation, glycosylation, mannosylation, amidation, carboxylation, sulfhydryl bond formation, cleavage, and the like. It should therefore be understood that the present disclosure encompasses both polypeptides or amino-acid sequences, and a complete protein (e.g., the CAR molecules). It should be appreciated that the present disclosure encompasses the use of any variant or derivative of the polypeptides of the invention, specifically, any of the CAR molecules disclosed herein, any of the VHH nanobodies of the present disclosure or any fragments thereof.

[0181] The term "derivative” refers to homologues, variants, and analogues thereof. Protein orthologs or homologues having a sequence homology or identity to the proteins of interest in accordance with the invention, specifically, receptors, chimeras, and nanobodies described herein, may share at least 50%, at least 60%, and specifically 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, specifically as compared to the entire sequence of the proteins of interest in accordance with the invention, specifically, any one of SEQ ID NO: 1, 2, 3, 4, that are the nanobodies of the present disclosure, as well as any of the CAR molecules disclosed herein, specifically, any one of the CAR molecules that comprise the amino acid as denote by any one of SEQ ID NOs: 110, 104, 106, 108, 117, 119, 122, 124, 126, 130 and / or 132, or any variants or derivatives thereof and the control CARs disclosed herein, for example the CARs of SEQ ID NO: 102 (a control CAR, designated CARNC8a), 113 (a control CAR, designated CARNC28), 128 (a control CAR, designated CARNC8a28), or any variants or derivatives thereof..

[0182] With respect to CDRs, the term "derivative" refers herein to orthologs or homologues having a sequence homology or identity to the CDRs of interest that share at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, specifically as compared to the sequence of the CDRs of interest in accordance with the invention, specifically, any one of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 [according to IMGT numbering] and that retains the ability to bind the target antigen.

[0183] In some embodiments, derivatives refer to polypeptides, which differ from the polypeptides specifically defined in the present invention by insertions, deletions or substitutions of amino acidresidues. It should be appreciated that by the terms "insertion / s", "deletion / s" or "substitution / s", as used herein it is meant any addition, deletion or replacement, respectively, of amino acid residues to the polypeptides disclosed by the invention, specifically receptors, chimeras and nanobodies as indicated above, of between 1 to 50 amino acid residues, between 20 to 1 amino acid residues, and specifically, between 1 to 10 amino acid residues. More particularly, insertion / s, deletion / s or substitution / s may be of any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. It should be noted that the insertion / s, deletion / s or substitution / s encompassed by the invention may occur in any position of the modified peptide, as well as in any of the N' or C termini thereof.

[0184] With respect to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologues, and alleles of the invention. For example, substitutions may be made wherein an aliphatic amino acid (G, A, I, L, or V) is substituted with another member of the group, or substitution such as the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Each of the following eight groups contains other exemplary amino acids that are conservative substitutions for one another:

[0185] 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M).

[0186] More specifically, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., conservative amino acid replacements. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved. For example, nonpolar 'hydrophobic " amino acids are selected from the group consisting of Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (F), Tryptophan (W), Cysteine (C), Alanine (A), Tyrosine (Y), Histidine (H), Threonine (T), Serine (S), Proline (P), Glycine (G), Arginine (R), and Lysine (K); 'polar " amino acids are selected fromthe group consisting of Arginine (R), Lysine (K), Aspartic acid (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q); ‘positively charged” amino acids are selected from the group consisting of Arginine (R), Lysine (K), and Histidine (H); and wherein 'acidic " amino acids are selected from the group consisting of Aspartic acid (D), Asparagine (N), Glutamic acid (E), and Glutamine (Q).

[0187] " Variants” of the polypeptides of the present disclosure may have at least 80% sequence similarity or identity, often at least 85% sequence similarity or identity, 90% sequence similarity or identity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity or identity at the amino acid level, with the protein of interest, such as the various polypeptides of the invention, and that retain antigen-binding functionality, specifically, when the variant or derivative refers to the target binding domains. In case of derivatives or variants of the entire CAR molecule, any derivative or variant that retains the function of the CAR molecule in inducing targeted killing of target cells expressing the target GPRC5D molecule, and / or any particular function disclosed by the present disclosure.

[0188] Still further, it should be understood that the present disclosure further refers to variants in the context of the nucleic acid sequences that encode the CAR molecules of the present disclosure, the VHH molecules disclosed herein, and any parts or fragments thereof, and further include any codon optimized sequences thereof. More specifically, variants of a protein or peptide, such as the CDRs, receptors, chimeras and nanobodies of the present disclosure can also arise from differences in the nucleotide sequences that encode the same amino acid sequence due to the redundancy of the genetic code. This is known as codon degeneracy or the synonymous codon effect, where multiple codons can specify the same amino acid (e.g., GGT, GGC, GGA and GGG all encode glycine). While such variants do not alter the primary amino acid sequence of the protein and therefore do not affect its antigen-binding properties or functional characteristics, they may influence other factors. These include the efficiency of transcription, translation, and mRNA stability, which can vary depending on the organism or expression system used. This is particularly relevant in recombinant expression systems, where optimizing codon usage for the specific host organism (e.g., bacteria, yeast, or mammalian cells) can enhance protein yield and stability while preserving the functional integrity of the nanobody.

[0189] Another aspect of the present disclosure relates to a nucleic acid molecule comprising at least one nucleic acid sequence encoding at least one CAR molecule, or any cassette, vector or vehicle comprising the nucleic acid molecule. The CAR molecules encoded by the disclosed nucleic acidmolecule comprise the following components. As component (i), at least one target-binding domain, wherein at least one of the target binding domains specifically recognizes and binds GPRC5D, or any fragments thereof. Component (ii) comprises at least one hinge region. Component (iii) comprises at least one transmembrane domain. Component (iv) comprises at least one intracellular signal transduction domain. At least one of the target binding domains comprises at least one variable heavy chain only (VHH) nanobody, or any antigen binding fragment thereof. The VHH nanobody forming the target binding domain of the CAR molecule encoded by the disclosed nucleic acid molecule, is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising noncontiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR encoded by the nucleic acid molecule of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule encoded by the nucleic acid molecule of the present disclosure comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

[0190] In some embodiments, the target binding domain of the disclosed CAR molecules may comprise at least one VHH nanobody encoded by the nucleic acid sequence as denoted by SEQ ID NO: 51, 52, 53 and / or 54.

[0191] In some embodiments, the CAR molecule encoded by the disclosed nucleic acid molecule, is any of the CAR molecules of the present disclosure, specifically, as defined herein above.

[0192] In some specific embodiments, the CAR molecule encoded by the disclosed nucleic acid molecule, comprises the amino acid sequence as denoted by at least one of SEQ ID NO: 110, 102, 104, 106, 108, 113, 117, 119, 122, 124, 126, 128, 130, 132, and any variants or derivatives thereof.In some embodiments, the CAR molecules of the present disclosure may be encoded by a nucleic acid sequence as denoted by any one of SEQ ID NO: 111, 103, 105, 107, 109, 114, 118, 120, 123, 125, 127, 129, 131, and / or 133, respectively.

[0193] In some more specific embodiments, the CAR molecule encoded by the disclosed nucleic acid molecule, is designated CARB28, and comprises the amino acid sequence as denoted by SEQ ID NO: 110. Accordingly, in some embodiments, the nucleic acid molecule comprises the nucleic acid sequence as denoted by SEQ ID NO: 111.

[0194] In some embodiments, in addition to a nucleic acid sequence encoding at least one of the disclosed CAR molecules, the nucleic acid molecule of the present disclosure further comprises at least one nucleic acid sequence encoding and / or controlling at least one immunomodulatory agent. The term "controlling” in this context refers to the nucleic acid sequence's ability to regulate or influence the expression, activity, or function of the immunomodulatory agent. This regulation can occur at various levels, including transcriptional control (e.g., through promoters, enhancers, or responsive elements that dictate when and where the agent is produced), post-transcriptional control (e.g., through elements affecting mRNA stability or translation efficiency), or by encoding components that modulate the agent's activity once it is expressed. Essentially, the nucleic acid sequence contains instructions or elements that govern how the immunomodulatory agent behaves within the cell.

[0195] An "immunomodulatory agent" refers to a substance or molecule that alters or regulates the activity of the immune system. These agents can enhance or suppress immune responses, depending on their specific function and context. The inclusion of an immunomodulatory agent in an immune cell, specifically a CAR-T cell, is intended to enhance the cell's activity by modulating key immune pathways. Immunomodulatory agents can amplify the effector functions of the CAR-T cell, such as cytokine production, cytotoxicity, and proliferation, thereby improving its ability to target and eliminate cancer cells or other diseased cells. Additionally, these agents can enhance the persistence and survival of the CAR-T cells in the hostile tumor microenvironment, overcoming immune suppression and exhaustion to maintain robust and sustained therapeutic efficacy.

[0196] The term "immunomodulatory agent" encompasses a wide range of biologically active molecules, including for example cytokines (which mediate intercellular communication within the immune system), chemokines (which guide the migration of immune cells to sites of inflammation or injury), and antibodies (which can block or enhance specific immune responses). It also includesengineered molecules such as chimeric antigen receptors (CARs), which reprogram immune cells to target specific antigens, and Toll-Like Receptor (TLR) modulators, which activate or inhibit innate immune signaling pathways. Additionally, immune checkpoint inhibitors, which block inhibitory pathways to restore or amplify T cell activity, are an additional category of immunomodulatory agents used in cancer immunotherapy. Together, these diverse agents form a comprehensive toolbox for manipulating the immune system in a controlled and targeted manner. Thus, in some embodiments, the immunomodulatory agent encoded by or controlled by a nucleic acid sequence within the disclosed nucleic acid molecule comprises at least one of: a cytokine, a chemokine, an antibody, a CAR molecule, a Toll-Like Receptor (TLR) modulator, and an immune checkpoint inhibitor.

[0197] A "cytokine” refers to a broad category of small, secreted proteins that mediate intercellular communication within the immune system and other physiological processes. These molecules function by binding to specific receptors on the surface of target cells, triggering intracellular signaling cascades that influence gene expression and cellular behavior. Cytokines act in autocrine, paracrine, or endocrine manners, depending on their site of production and action. Cytokines are classified into several functional families based on their biological effects and structural similarities, including interleukins (ILs), such as IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL- 10, IL- 12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-27, IL-33, interferons (IFNs) such as IFN-α, IFN-β, IFN-γ, IFN-λ tumor necrosis factors (TNFs), such as TNF-α, TNF-β (lymphotoxin-α), CD40L (CD154), BAFF (B-cell activating factor), APRIL (A Proliferation-Inducing Ligand), RANKL (Receptor Activator of Nuclear Factor Kappa-B Ligand), colony-stimulating factors (CSFs), such as GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor), M-CSF (Macrophage Colony-Stimulating Factor), G-CSF (Granulocyte Colony-Stimulating Factor), IL-3 (Multi-CSF) and chemokines (for example: CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL19, CCL21, CXCL9 (MIG), CXCL10 (IP-10), CXCL11, CXCL12 (SDF-1), CX3CL1 (Fractalkine)). Each cytokine has a distinct but often overlapping role in orchestrating immune and inflammatory responses.

[0198] A "chemokine” refers to a small secreted protein that plays a critical role in the directed migration (chemotaxis) and activation of immune cells during immune surveillance, inflammation, and tissue repair. Chemokines bind to specific G protein-coupled receptors (GPCRs) on the surface of target cells, initiating intracellular signaling cascades that regulate cell movement, adhesion, and activation. These molecules are categorized into four structural subfamilies based on thearrangement of conserved cysteine residues in their sequences: CC, CXC, CX3C, and C chemokines.

[0199] Chemokines are essential for coordinating immune responses by guiding immune cells such as T cells, B cells, natural killer (NK) cells, macrophages, and dendritic cells to sites of infection, injury, or inflammation. For example, CXC chemokines like CXCL10 and CXCL11 recruit activated T cells and NK cells to inflamed tissues, while CC chemokines like CCL19 and CCL21 direct the migration of T cells and B cells to lymphoid organs. Beyond their chemotactic properties, some chemokines enhance immune cell activation and cytokine production, further amplifying the immune response.

[0200] As mentioned above, in some embodiments, the immunomodulatory agent encoded by, or controlled by, a nucleic acid sequence within the disclosed nucleic acid molecule, comprises a Toll-Like Receptor (TLR) modulator.

[0201] A " Toll-Like Receptor (TLR) modulator” refers to a compound or molecule that influences the activity of Toll-Like Receptors, which are key components of the innate immune system. TLRs are a family of pattern recognition receptors (PRRs) expressed on the surface or within endosomes of immune cells, such as macrophages, dendritic cells, T cells, and B cells. These receptors detect pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), triggering intracellular signaling pathways that activate immune responses. TLR modulators can either enhance (agonists) or suppress (antagonists) the activity of TLRs to achieve specific therapeutic outcomes. TLR agonists are used to stimulate immune responses by activating cytokine production, antigen presentation, and immune cell proliferation. Non limiting examples of TLR modulators include CpG oligodeoxynucleotides (CpG-ODNs), polyinosinic:polycytidylic acid (Poly(I:C)), Imiquimod (R-837), Resiquimod (R-848), Flagellin, Monophosphoryl Lipid A (MPLA).

[0202] In some other embodiments, the immunomodulatory agent encoded by or controlled by a nucleic acid sequence within the disclosed nucleic acid molecule comprises an immune checkpoint inhibitor.

[0203] An "immune checkpoint inhibitor” refers herein to an agent that blocks inhibitory pathways in the immune system, thereby enhancing the activity of immune cells such as T cells, B cells, and natural killer (NK) cells. These pathways, known as immune checkpoints, are normally involved in maintaining immune homeostasis and preventing excessive immune activation that could lead to autoimmunity. However, tumors and certain pathogens exploit these checkpoints to evadeimmune surveillance by dampening the immune response. By inhibiting these pathways, immune checkpoint inhibitors restore or amplify the immune system's ability to recognize and eliminate abnormal or infected cells.

[0204] Immune checkpoint inhibitors target key molecules such as programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). For example, anti-PD-1 and anti-PD-Ll antibodies block the interaction between PD-1 on T cells and PD-L1 on tumor cells, thereby reinvigorating exhausted T cells and promoting their cytotoxic activity. Similarly, anti-CTLA-4 antibodies enhance T cell priming and activation by preventing CTLA-4 -mediated suppression of co-stimulatory signals. In the present disclosure, immune checkpoint inhibitors can be delivered as nucleic acid-based constructs encoding the inhibitory-blocking agents. Non-limiting examples of immune checkpoint inhibitors include anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-TIGIT, anti-LAG-3, anti-TIM-3, and anti-BTLA. Immune checkpoint inhibitors include but are not limited to monoclonal antibodies, antibody fragments, fusion proteins, peptides, small molecules, and nucleic acid-based agents (e.g., siRNA, antisense RNA).

[0205] In some embodiments, the immunomodulatory agent / s encoded by, or controlled by, a nucleic acid sequence within the disclosed nucleic acid molecule, comprises at least one cytokine that enhances T cell cytotoxicity.

[0206] A "cytokine that enhances T cell cytotoxicity” as used herein refers to a signaling protein secreted by immune or other cells that specifically promotes the activation, proliferation, and functional enhancement of cytotoxic effect of T cells. These cytokines play a crucial role in modulating the immune response by augmenting the ability of T cells to recognize and eliminate infected or malignant cells. Key features of such cytokines include: (i) enhancement of T cell activation and expansion by amplifying T cell receptor (TCR) -mediated signaling, leading to increased proliferation and activation of CD8+ cytotoxic T cells; (ii) promotion of effector functions by boosting the production of cytotoxic molecules, such as perforin and granzyme, as well as pro-inflammatory cytokines like interferon-gamma (IFN-y), which are critical for effective target cell killing; (iii) support for T cell persistence by enhancing the survival and long-term functionality of cytotoxic T cells, ensuring sustained immune responses against persistent threats. Non limiting examples include the cytokines IL-7, IL-15, IL-2, IL-12, IL-18 and IL-21. In more specific embodiments, the at least one cytokine encoded by, or controlled by, a nucleic acid sequence within the disclosed nucleic acid molecule, is a member of the γc Cytokine Family. A "memberof the yc cytokine family” refers herein to a cytokine that signals through the common gamma chain (yc, also known as CD132), a shared receptor subunit utilized by multiple interleukins in this family. These cytokines are critical regulators of immune cell development, differentiation, survival, and function. The γc cytokine family includes several interleukins that play non-redundant roles in maintaining immune homeostasis and promoting effective immune responses. Non limiting examples include cytokines IL-7, IL-2, IL-15 and IL-2. As shown in Figure 10, a cytokine array performed on serum from patients before and during BCMA CART (HBI0101) therapy showed that the presence of IL-7 in the serum correlates with longer PFS in patients. The potency of adding constitutive interleukin-7 (IL-7) expression to the CART cell of the present disclosure was tested. IL-7 is produced by multiple stromal tissues, including epithelial cells in the thymus and bone marrow, but not by T cells [Fry, T. J. & Mackall, C. L. Blood 99, 3892-3904 (2002)]. IL-7 is a member of a family of cytokines that signal through the common cytokine gamma chain and is a critical modulator of low-affinity peptide-induced proliferation [Tan, J. T., et al. Proc Natl Acad Sci USA 98, 8732-8737 (2001); Schluns, K. S., Kieper, W. C., Jameson, S. C. & Lefrancois, L. Nat Immunol 1, 426-432 (2000)].

[0207] Thus, in some further specific embodiments, the at least one cytokine encoded by, or controlled by, a nucleic acid sequence within the disclosed nucleic acid molecule, is Interleukin -7 (IL-7). More specifically, "Interleukin-7 (IL-7)" refers herein to a cytokine of the γc cytokine family that is essential for lymphocyte development, survival, and homeostasis. It is produced by stromal cells in the bone marrow, thymus, and peripheral tissues and acts primarily through the IL-7 receptor (IL-7R), a heterodimer composed of IL-7Ra (CD127) and the common gamma chain (yc, CD132). IL-7 supports thymopoiesis, peripheral T cell survival, and homeostasis by activating signaling pathways such as JAK / STAT, PI3K / AKT, and MAPK. It plays a pivotal role in T cell development and immune recovery. In some embodiments, IL-7, as used herein is the human IL-7. In yet some further embodiments, the human IL-7 comprises the amino acid sequence as denoted by SEQ ID NO: 83, or any derivatives or variants thereof. In yet some further embodiments, the human IL-7 is encoded by a nucleic acid sequence comprising the sequence as denoted by SEQ ID NO: 84. Non-limiting embodiments for a nucleic acid molecule that, in addition to the nucleic acid sequence that encodes at least one of the CAR molecules of the present disclosure, further comprise a nucleic acid sequence encoding IL-7, include any one of the nucleic acid molecules that comprise SEQ ID NO: 112, 115, 116 [the control CARNC28-IL7], and 121, or any variants thereof.In some embodiments, the nucleic acid of the present disclosure comprises, in addition to a nucleotide sequence encoding one or more CAR molecules, one or more additional nucleotide sequences encoding one or more exogenous molecules configured to enhance the activity, persistence, safety, and / or functionality of the engineered cell, thereby providing an armored CAR (also referred to as an “armored CAR-T” or “armored CAR” cell). In some embodiments, such additional nucleotide sequences encode, by way of example, cytokines and / or cytokine variants (e.g., IL-12, IL-15, IL-18), chemokines, costimulatory ligands (e.g., 4-1BBL, CD40L), dominantnegative receptors, switch receptors, immune checkpoint inhibitors or blockers (e.g., a PD-1 dominant-negative receptor, PD-1 / CD28 switch receptor, or an anti-PD-1 scFv), safety switches (e.g., inducible caspase-9), trafficking and / or homing factors (e.g., chemokine receptors), enzymes that modulate the tumor microenvironment, and / or other pay loads that improve anti-tumor efficacy and / or reduce immunosuppression. In some embodiments, the nucleic acid is provided as a bicistronic or multicistronic construct (e.g., separated by an IRES and / or one or more 2A peptides) and / or as two or more separate nucleic acids introduced into the cell, such that the engineered cell expresses the CAR(s) and the additional molecule(s).

[0208] In some embodiments, the nucleic acid sequence encoding and / or controlling at least one immunomodulatory agent comprised within the nucleic acid molecules of the present disclosure is under the control of at least one regulatory element. Such a regulatory element allows and / or directs the expression of the at least one immunomodulatory agent in activated T cells. The term "regulatory element" refers to a specific DNA sequence or region within the nucleic acid molecule that interacts with cellular factors or external stimuli to regulate gene expression. These elements are crucial for controlling the timing, location, and / or intensity of gene expression in response to specific signals. Regulatory elements can include promoters, enhancers, silencers, and responsive elements that bind transcription factors. The phrase "allowing / directing the expression" describes the function of the regulatory element, which is to enable and guide the process by which genetic information encoded in the nucleic acid sequence is converted into a functional product, such as a protein (in this case, the immunomodulatory agent). This involves initiating, enhancing, or otherwise controlling the transcription of DNA into RNA and subsequent translation of RNA into protein, specifically ensuring that this process occurs in the designated cellular context (i.e., in activated T cells). The term "activated T cells" refers to T lymphocytes that have undergone a process of stimulation, typically through recognition of a specific antigen presented by an antigen-presenting cell, along with co-stimulatory signals. This activation leads to a series of cellularchanges, including proliferation, differentiation into effector cells (e.g., cytotoxic T lymphocytes or helper T cells), and the production of cytokines. These cells are in an active state, capable of mounting an immune response, as opposed to naive or resting T cells.

[0209] Still further, in some further embodiments, the nucleic acid sequence encoding and / or controlling at least one immunomodulatory agent comprised within the nucleic acid molecule of the present disclosure, is under the control of at least one regulatory element that comprises at least one responsive element for at least one transcription factor involved in immune regulation.

[0210] A "responsive element". as used herein, refers to a specific DNA sequence or region within the nucleic acid molecule that interacts with cellular factors or external stimuli to regulate gene expression, specifically, the expression of the immunomodulatory agent (e.g., IL-7). These elements are crucial for controlling the timing, location, and intensity of the expression in response to specific signals. Regulatory elements may include elements that operate in cis or in trans. Still further, in some embodiments, regulatory elements as used herein include promoters that initiate transcription and may be either inducible or constitutive. Regulatory elements in the context of the disclosed nucleic acid molecules further relate to enhancers that control transcriptional activity, often in a signal- specific manner. Response Elements (REs), in some embodiments, are elements that may bind transcription factors, thereby regulating translation of the attached coding region. The term "transcription factor” refers to a protein that binds to specific DNA sequences, thereby controlling the rate of transcription of genetic information from DNA to messenger RNA. Transcription factors play a critical role in gene expression by either promoting (activating) or blocking (repressing) the recruitment of RNA polymerase to specific genes, thus regulating when and how genes are turned on or off.

[0211] As mentioned above, the responsive element may bind at least one transcription factor involved in immune regulation. The term "involved” in this context indicates that the transcription factor plays an active and significant role in the process of immune regulation. It implies direct or indirect participation, contribution, or influence on how the immune system's functions are controlled and maintained. The term "immune regulation" refers to the processes and mechanisms by which the immune system controls its responses to maintain homeostasis, prevent autoimmunity, and effectively combat pathogens or abnormal cells. This involves a delicate balance of activating and inhibitory signals, cellular interactions, and molecular pathways that ensure appropriate immune activation when needed and suppression when the threat is cleared or to prevent excessive damage to host tissues.Transcription factors involved in immune regulation may be for example NF AT, STAT5, NFKB, API-NFKB and / or NR4A. Therefore, the responsive elements related to the present disclosure may be specific for at least one of NF AT, STAT5, NFKB, API-NFKB and / or NR4A.

[0212] In some embodiments, the nucleic acid molecules of the present disclosure comprise responsive elements specific for NFAT (Nuclear Factor of Activated T cells). NFAT is a family of transcription factors crucial for immune response regulation, development, and other physiological processes. Activated by calcium signaling through calcineurin, NFAT translocates from the cytoplasm to the nucleus, where it binds to specific NFAT-response elements (e.g., TGGAAA) to regulate genes involved in T-cell activation (e.g., IL-2), differentiation, and other immune cell functions. An example of such an RE may comprise the nucleic acid sequence as denoted by SEQ ID NO: 95. STAT5 (Signal Transducer and Activator of Transcription 5) is a transcription factor involved in regulating cell proliferation, survival, differentiation, and immune responses. It is part of the STAT family, which is activated downstream of cytokine and growth factor signaling pathways. An example of such an RE may comprise the nucleic acid sequence as denoted by SEQ ID NO: 96.

[0213] Still further, NF-KB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) is a family of transcription factors that regulate the expression of genes involved in immune responses, inflammation, cell proliferation, survival, and apoptosis. Activated by various stimuli such as cytokines, stress, infections, or receptor signaling (e.g., TLRs, TNFR), NF-KB is held inactive in the cytoplasm by IKB inhibitors. Upon activation, IKB is phosphorylated and degraded, allowing NF-KB to translocate into the nucleus, where it binds to specific KB sites in DNA to initiate transcription. An example of such an RE may comprise the nucleic acid sequence as denoted by SEQ ID NO: 99.

[0214] AP-1-NF-KB Interaction (API-NF-KB) refers to the cooperative or antagonistic crosstalk between two major transcription factor families: Activator Protein-1 (AP-1) and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-KB). Both play critical roles in regulating gene expression in response to external stimuli, such as cytokines, stress, and pathogens, and are key regulators of inflammation, immune responses, and cell survival. An example of such an RE may comprise the nucleic acid sequence as denoted by SEQ ID NO: 97.

[0215] NR4A refers to the Nuclear Receptor Subfamily 4 Group A (NR4A) family of orphan nuclear receptors, which includes NR4A1 (Nur77), NR4A2 (Nurr1), and NR4A3 (Nor1). Thesetranscription factors are rapidly induced in response to diverse stimuli, including stress, growth factors, cytokines, and metabolic signals. An example of such an RE may comprise the nucleic acid sequence as denoted by SEQ ID NO: 98.

[0216] Non- limiting embodiments for nucleic acid molecule that in addition to the nucleic acid sequence that encodes at least one of the CAR molecules of the present disclosure, further comprise a nucleic acid sequence encoding IL-7, under the control of at least one RE sequence, include any one of the nucleic acid molecules that comprise SEQ ID NO: 134, 135, 136, 137 and 138, or any variants thereof.

[0217] In some embodiments, the nucleic acid molecule of the present disclosure is designed in a manner that facilitates the targeted incorporation of the CAR encoding sequence into a target locus within the genome of a hematopoietic cell, specifically, an immune cell. More specifically, to a target locus in a T cell. In some embodiments, a target locus may be any of the TCR loci, for example, the beta TCR, or the alpha TCR. Accordingly, for a targeted insertion, the disclosed nucleic acid molecule (or any cassette thereof) may further include targeting sequences that may be homology arms and / or other recognition elements for recombinases and / or integrases that mediate the incorporation into a genomic locus.

[0218] Thus, in some embodiments, the nucleic acid molecule of the present disclosure is flanked on at least one of the 5' and 3' ends thereof by at least one of: (i) homology arms, for integration to a genomic target site by homologous recombination; and / or (ii) recognition sites for a site-specific nuclease, a site-specific integrase or a site-specific recombinase.

[0219] The term "flanked" as used herein means that the nucleic acid molecule is bordered or enclosed on one or both of its ends (specifically, the 5' and 3' ends) by the specified elements (homology arms, recognition sites). These flanking elements are positioned adjacent to the nucleic acid molecule and serve to facilitate its integration into a genomic target site. " Homology arms" as used herein refers to regions of DNA sequence that are identical or highly similar to sequences flanking a target site in a genome. In gene editing, these arms are included in a donor DNA construct to facilitate "homologous recombination ", a cellular repair pathway that uses a homologous template to accurately repair double-strand breaks. This process allows for the precise insertion of new genetic material (like the CAR-encoding sequence) into a specific genomic location. The term "genomic target site" refers to a specific, predetermined location within the host cell's genome where a nucleic acid sequence (such as the CAR-encoding sequence) is intended to be integratedor modified. The specificity of this site is crucial for ensuring that the CAR is expressed correctly and stably, and that its insertion does not disrupt essential host genes.

[0220] " Recognition sites" as used herein refers to specific DNA sequences that are recognized and bound by particular enzymes or proteins, such as site-specific nucleases, integrases, or recombinases. These sites act as molecular addresses, guiding the enzymes to precise locations in the DNA where they can perform their function, such as cleaving the DNA or mediating the insertion or rearrangement of genetic material.

[0221] As mentioned above, the invention provides a nucleic acid molecule comprising at least one nucleic acid sequence encoding at least one CAR molecule, or any cassette, vector, or vehicle comprising the nucleic acid molecule. The terms “nucleic acid”, “nucleic acid sequence”, or "polynucleotide" and “nucleic acid molecule” refer to polymers of nucleotides, and include but are not limited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), DNA / RNA hybrids including polynucleotide chains of regularly and / or irregularly alternating deoxyribosyl moieties and ribosyl moieties (i.e., wherein alternate nucleotide units have an —OH, then and — H, then an —OH, then an — H, and so on at the 2' position of a sugar moiety), and modifications of these kinds of polynucleotides, wherein the attachment of various entities or moieties to the nucleotide units at any position are included. The terms should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single- stranded (such as sense or antisense) and double- stranded polynucleotides. Preparation of nucleic acids is well known in the art. Still further, it should be understood that the invention encompasses as additional aspects thereof any vector or vehicle that comprises any of the nucleic acid molecule / s of the present disclosure or any cassettes described by the invention.

[0222] The nucleic acid molecule of the present disclosure comprises at least one nucleic acid sequence encoding at least one CAR molecule. The term "encoding” as used herein refers to the process by which the nucleotide sequence of a nucleic acid molecule (such as DNA or RNA) specifies the sequence of a corresponding functional biomolecule, such as a polypeptide, a peptide or a protein. The nucleotide sequence contains codons, triplets of nucleotides, that correspond to specific amino acids, dictating the primary structure of the encoded polypeptide, peptide or protein.

[0223] The present disclosure involves the provision of a nucleic acid molecule encoding the disclosed CAR, which in some embodiments may be, and / or comprised within, a cassette that is used in the methods, cells, compositions, and uses described in all aspects of the present disclosure. The term"cassette” or "nucleic acid cassette" refers to a polynucleotide sequence comprising at least one regulatory sequence operably linked to a sequence encoding a nucleic acid sequence encoding the CARs disclosed herein. All elements comprised within the cassette of the invention are operably linked together. The term "operably linked," as used in reference to a regulatory sequence and a structural nucleotide sequence, means that the nucleic acid sequences are linked in a manner that enables regulated expression of the linked structural nucleotide sequence. It should be appreciated that any nucleic acid cassette provided herein comprises the nucleic acid sequence encoding the CAR of the invention, or any homologs and variants thereof. According to some embodiments, such nucleic acid cassette may further comprise any control sequences that facilitate the transcription and / or translation of the CAR molecules of the present disclosure. Such sequences include, as non-limiting examples, a promoter sequence, specifically, a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain " TATA" boxes and " CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various vectors of the present disclosure.

[0224] In some embodiments, promoters applicable in the present disclosure may be either inducible or constitutive. In yet some further embodiments, a minimal promoter may be used; still further, an endogenous promoter or heterologous promoter are also applicable in the cassettes disclosed herein. In some embodiments, the cassettes of the present disclosure may further comprise a signal peptide leader, for example, a signal peptide leader derived from CD8, as used herein, specifically, comprising the amino acid sequence as denoted by SEQ ID NO: 57 (encoded by the nucleic acid SEQ ID NO: 58), and any derivatives and variants thereof. In some other embodiments, the signal peptide leader may be a signal peptide leader derived from immunoglobulins, comprising the amino acid sequence as denoted by SEQ ID NO: 59 or 61 (encoded by the nucleic acid SEQ ID NO: 60 or 62). In yet some further embodiments, the nucleic acid molecules encoding the CAR provided by the invention may further comprise at least one degron sequence, at least one 2A peptide sequence (e.g., amino acid SEQ ID NO: 81, encoded by the nucleic acid SEQ ID NO: 82)or a CHYSEL site, at least one mRNA stabilizing sequence, at least one stop codon (or termination codon), at least one 3 -frame stop codon sequence, at least one protein stabilizing sequence, at least one polyadenylation sequence (e.g., amino acid SEQ ID NO: 93, 94 or AATAAAGC), at least one transcription enhancer, splice donor and / or splice acceptor sites, and any transcription and / or translation element / s.

[0225] Still further, in some embodiments, the nucleic acid molecule / s of the present disclosure or any cassette used by the present disclosure may be comprised within a nucleic acid vector. In more specific embodiments, such vector may be any one of a viral vector, a non- viral vector and a naked DNA vector.

[0226] " Vectors”, as used herein, are nucleic acid molecules of particular sequence can be incorporated into a vehicle that is then introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art, including promoter elements that direct nucleic acid expression. Many vectors, e.g., plasmids, cosmids, minicircles, phage, viruses, etc., useful for transferring nucleic acids into target cells may be applicable in the present invention. The vectors comprising the nucleic acid(s) may be maintained episomally, e.g., as plasmids, minicircle DNAs, viruses such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g., retrovirus-derived vectors such as AAV, MMLV, HIV-1, ALV, etc. Vectors may be provided directly to the subject cells. In other words, the cells are contacted with vectors comprising the nucleic acid molecules, and / or cassettes of the invention that comprise the nucleic acid sequence encoding the immune effector of interest and the engineered CAR T disclosed herein such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. DNA can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV). More specifically, in some embodiments, the vector may be a viral vector. In yet some particular embodiments, such viral vector may be any one of recombinant adeno associated vectors (rAAV), single stranded AAV (ssAAV), self-complementary rAAV (scAAV), Simian Vacuolating Virus 40 (SV40) vector, Adenovirus vector, helper-dependent Adenoviral vector, retroviral vector and lentiviral vector. As indicated above, in some embodiments, viral vectors may be applicable in the present invention. The term "viralvector" refers to a replication competent or replication-deficient viral particle which are capable of transferring nucleic acid molecules into a host. The term "virus" refers to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism. The viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane. Examples of viruses useful in the practice of the present invention include baculoviridiae, parvoviridiae, picornoviridiae, herepesviridiae, poxviridiae, adenoviridiae, picotmaviridiae. The term recombinant virus includes chimeric (or even multimeric) viruses, i.e., vectors constructed using complementary coding sequences from more than one viral subtype.

[0227] In some embodiments, the nucleic acid molecules, and / or cassette of the invention may be comprised within a retroviral vector. A retroviral vector, as used herein consists of proviral sequences that can accommodate the nucleic acid molecule encoding the engineered CAR disclosed herein, to allow incorporation of both into the target cells. The vector may also contain viral and cellular gene promoters, to enhance expression of the nucleic acid molecule encoding the immune effector of interest and the engineered CAR disclosed herein in the target cells. Retroviral vectors stably integrate into the dividing target cell genome so that the introduced gene is passed on and expressed in all daughter cells. They contain a reverse transcriptase that allows integration into the host genome.

[0228] In some specific and non-limiting embodiments, the pMSGVl retroviral vector has been used for the CAR molecule of the present disclosure. More specifically, the pMSGVl retroviral vector contains a murine stem cell virus long-terminal repeat and RNA processing signals similar to the MFG class of retroviral vectors. The retroviral vector backbone used in the present disclosure, pMSGVl, is a derivative of the vector pMSGV (MSCV-based splice-gag vector) that utilizes a murine stem cell virus (MSCV) long terminal repeat (LTR) [Hawley et al., Gene Ther. 1:136-138 (1994)], and contains the extended gag region and env splice site from vector SFGtcLuc+ITE4 [Lindemann et al., Mol. Med. 1997;3:466-476 (1997)]. Vector pMSGV was generated from pMINV [Hawley et al., Ann. N. Y. Acad. Sci. 795:341-345. (1996)] by substituting a 756-bp Spel / Xhol fragment with a 798-bp Spel / Xhol fragment from SFGtcLuc+ITE4 and by replacing a 1955-bp XhoI / BamHI fragment containing a PGK-IRES-NEO cassette with a 47-bp Xho / BamHI poly linker containing unique Xhol, EcoRI, Sall, SacII, and BamHI sites. Vector pMSGVl was derived from pMSGV by replacing a 43 -bp PmBJXhoI fragment of pMSGV with a 76-bp Pmll / Xhol fragment from the vector Gcsap [Onodera et al., J. Virol. 72: 1769-1774 (1998)]. The latter modification incorporates a naturally occurring Kozak sequence to enhance translationalefficiency. In some alternative embodiments, the nucleic acid molecules, and / or cassette of the present disclosure may be comprised within an Adeno-associated virus (AAV). The term "adenovirus" is synonymous with the term "adenoviral vector". AAV is a single- stranded DNA virus with a small (~20nm) protein capsule that belongs to the family of parvoviridae, and specifically refers to viruses of the genus adenoviridiae. The term adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (AdllA and Ad IIP), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Due to its inability to replicate in the absence of helpervirus coinfections (typically Adenovirus or Herpesvirus infections) AAV is often referred to as dependovirus. AAV infections produce only mild immune responses and are considered to be nonpathogenic, a fact that is also reflected by lowered biosafety level requirements for the work with recombinant AAVs (rAAV) compared to other popular viral vector systems. Due to its low immunogenicity and the absence of cytotoxic responses AAV-based expression systems offer the possibility to express nucleic acid sequences encoding the immune effector of interest and the engineered CAR T disclosed herein for months in quiescent cells. Production systems for rAAV vectors typically consist of a DNA-based vector containing a transgene expression cassette, which is flanked by inverted terminal repeats. Construct sizes are limited to approximately 4.7-5.0 kb, which corresponds to the length of the wild-type AAV genome. rAAVs are produced in cell lines. The expression vector is cotransfected with a helper plasmid that mediates expression of the AAV rep genes which are important for virus replication and cap genes that encode the proteins forming the capsid. Recombinant adeno-associated viral vectors can transduce dividing and non-dividing cells, and different rAAV serotypes may transduce diverse cell types. These single-stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous Homologous Recombination without causing double strand DNA breaks in the host genome. It should be appreciated that many intermediate steps of the wild-type infection cycle of AAV depend on specific interactions of the capsid proteins with the infected cell. These interactions are crucial determinants of efficient transduction and expression of nucleic acid molecules encoding the immune effector of interest and the engineered CAR disclosed herein when rAAV is used asgene delivery tool. Indeed, significant differences in transduction efficacy of various serotypes for particular tissues and cell types have been described.

[0229] It is believed that a rate-limiting step for the AAV-mediated expression of transgenes is the formation of double- stranded DNA. Recent reports demonstrated the usage of rAAV constructs with a self-complementing structure (scAAV) in which the two halves of the single- stranded AAV genome can form an intra-molecular double- strand. This approach reduces the effective genome size usable for gene delivery to about 2.3kB but leads to significantly shortened onsets of expression in comparison with conventional single-stranded AAV expression constructs (ssAAV). Thus, in some embodiments, ssAAV may be applicable as a viral vector by the methods of the present disclosure.

[0230] In yet some further embodiments, HDAd vectors may be suitable for the CAR molecules, encoding sequences, cells, compositions and methods of the present disclosure. The Helper-Dependent Adenoviral (HDAd) vectors HD Ads have innovative features including the complete absence of viral coding sequences and the ability to mediate high level transgene expression with negligible chronic toxicity. HD Ads are constructed by removing all viral sequences from the adenoviral vector genome except the packaging sequence and inverted terminal repeats, thereby eliminating the issue of residual viral gene expression associated with early generation adenoviral vectors. HD Ads can mediate high efficiency transduction, do not integrate in the host genome, and have a large cloning capacity of up to 37 kb, which allows for the delivery of multiple transgenes or entire genomic loci, or large cis-acting elements to enhance or regulate tissue-specific transgene expression. One of the most attractive features of HDAd vectors is the long-term expression of the transgene. Still further, in some embodiments, SV40 may be used as a suitable vector by the methods of the invention. SV40 vectors (SV40) are vectors originating from modifications brought to Simian virus-40 an icosahedral papovavirus. Recombinant SV40 vectors are good candidates for gene transfer, as they display some unique features: SV40 is a well-known virus, non-replicative vectors are easy-to-make, and can be produced in titers of 10(12) lU / ml. They also efficiently transduce both resting and dividing cells, deliver persistent transgene expression to a wide range of cell types, and are non-immunogenic. Present disadvantages of rSV40 vectors for gene therapy are a small cloning capacity and the possible risks related to random integration of the viral genome into the host genome. In yet some alternative embodiments, lentiviral vectors may be used in the present invention. Lentiviral vectorsLentiviral vectors are derived from lenti viruses which are a subclass of Retroviruses. Commonly used retroviral vectors are "defective", i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising the nucleic acid molecules, vectors and / or cassette in accordance with the invention, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing the retroviral vectors comprising the nucleic acid molecules, vectors and / or cassette of the invention that contains the nucleic acids sequence encoding the CAR T of the invention, into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Still further, in other embodiments, the CAR-encoding nucleic acid sequence may be comprised within a retroviral vector. As used herein, a "retroviral vector" refers to a gene transfer vector derived from a non-lentiviral retrovirus (for example, a gammaretrovirus), engineered to deliver a heterologous nucleic acid (e.g., a CAR expression cassette) into a target cell. In typical embodiments, retroviral vectors are replication-incompetent and are produced in packaging cells that provide the viral proteins required for particle formation, while the vector genome carries the transgene and the cis-acting sequences required for reverse transcription, packaging, and transfer. In many embodiments, retroviral vectors are capable of mediating stable transgene expression by integration of vector-derived DNA into the host cell genome, and may be configured and selected to preferentially transduce actively dividing cells, depending on the retroviral system employed.

[0231] Nonviral vectors, in accordance with the invention, refer to all the physical and chemical systems except viral systems and generally include either chemical methods, such as cationic liposomes and polymers, or physical methods, such as gene gun, electroporation, particle bombardment, ultrasound utilization, and magnetofection. Efficiency of this system is less than viral systems in gene transduction, but their cost-effectiveness, availability, and more importantly reduced induction of immune system and no limitation in size of transgenic DNA compared with viral system have made them attractive also for gene delivery.For example, physical methods applied for in vitro and in vivo gene delivery are based on making transient penetrations in the cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy so that DNA entrance into the targeted cells is facilitated.

[0232] In more specific embodiments, the vector may be a naked DNA vector. More specifically, such a vector may be, for example, a plasmid, minicircle, or linear DNA. Naked DNA alone may facilitate the transfer of a gene (2-19 kb) into skin, thymus, cardiac muscle, and especially skeletal muscle and liver cells when directly injected. It also enables long-term expression. Although naked DNA injection is a safe and simple method, its efficiency for gene delivery is quite low.

[0233] Minicircles are modified plasmids in which a bacterial origin of replication (ori) was removed, and therefore they cannot replicate in bacteria. Linear DNA or Doggybone™ are double-stranded, linear DNA constructs that solely encode an antigen expression cassette, comprising an antigen, promoter, polyA tail, and telomeric ends. It should be appreciated that all DNA vectors disclosed herein may also be applicable for all nucleic acid molecules, vectors, and / or cassettes used in the methods and compositions of the invention, as described herein. Still further, it must be appreciated that the present disclosure further provides any vectors or vehicles that comprise any of the nucleic acid molecules, vectors, and / or nucleic acid cassettes disclosed by the invention, as well as any host cell expressing the nucleic acid molecules and / or nucleic acid cassettes disclosed by the present disclosure.

[0234] As mentioned, the disclosed nucleic acid molecule comprises at least one nucleic acid sequence encoding at least one CAR molecule of the present disclosure, or any cassette, vector or vehicle comprising said nucleic acid molecule. The term "vehicle” in this context refers to a delivery system or carrier designed to transport the nucleic acid molecule (encoding the CAR molecule) into a target cell. This can include various biological or synthetic constructs, such as viral particles (e.g., lentiviruses, adenoviruses), liposomes, nanoparticles, or other means that facilitate the introduction and expression of the genetic material within the cell. The primary function of the vehicle is to protect the nucleic acid molecule and enable its efficient uptake and delivery to the appropriate cellular compartment for CAR expression.

[0235] Another aspect of the present disclosure relates to a gene editing system comprising at least one nucleic acid molecule comprising at least one nucleic acid sequence encoding at least one CAR molecule, or any cassette, vector or vehicle comprising the nucleic acid molecule. The CAR molecule comprising the following components: (i) at least one target-binding domain, wherein at least one of the target binding domains specifically recognizes and binds GPRC5D, or anyfragments thereof; (ii) at least one hinge region; (iii) at least one transmembrane domain; and (iv) at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule of the present disclosure comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

[0236] The gene editing system further comprising at least one gene editing component or a nucleic acid sequence encoding the gene editing component.

[0237] As used herein, the term "gene editing system" refers to any system, platform, or set of molecular tools capable of altering, inserting, deleting, replacing, or otherwise modifying one or more nucleic acid sequences within the genome or epigenome of a cell. The gene editing system may function in a site- specific or sequence-directed manner, and may include one or more nucleic acid molecules, proteins, ribonucleoprotein complexes, or delivery vehicles. In certain embodiments, the gene editing system comprises at least one nucleic acid molecule encoding a gene editing component, or any cassette, vector, or delivery system containing or expressing such components. The gene editing system may be employed in ex vivo, in vivo, or in vitro settings.

[0238] A "gene editing component" refers to any molecular entity that contributes to the function of the gene editing system by enabling or facilitating site-specific or targeted modification of nucleic acids. The gene editing component may include, but is not limited to, nucleases, recombinases,integrases, transposases, deaminases, base editors, prime editors, or any functional domains thereof. Gene editing components may be delivered in the form of DNA, RNA, protein, or combinations thereof, and may be associated with additional elements such as guide RNAs, targeting domains, or regulatory sequences.

[0239] In some embodiments, a gene editing component of the gene editing system disclosed herein, may be any one of a site-specific nuclease, a class switch recombination, a site-specific integrase and a site- specific recombinase.

[0240] The term "site-specific nuclease" refers to an enzyme or protein complex capable of recognizing and cleaving DNA at one or more predetermined or target- specific sites. Site-specific nucleases typically include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated nucleases (such as Cas9, Casl2, Casl3), Meganucleases, or engineered variants thereof. Upon DNA cleavage, the resulting double-strand break or single-strand break may be repaired by cellular mechanisms, such as non-homologous end joining (NHEJ) or homology-directed repair (HDR), thereby enabling insertion, deletion, or replacement of genetic material.

[0241] In some other embodiments, the gene editing component of the disclosed gene editing system may be a class switch recombination.

[0242] The term "class switch recombination" (CSR) refers to a process of genetic recombination whereby a B cell changes the isotype or class of antibody it produces (e.g., from IgM to IgG, IgA, or IgE) without altering the antigen specificity of the antibody's variable region. In the context of gene editing systems, CSR may be harnessed or mimicked as a natural or engineered mechanism for controlled recombination or transcriptional regulation of gene loci, particularly within immune cells or in synthetic biology applications.

[0243] In some further embodiments, the gene editing component of the disclosed gene editing system may be a site- specific integrase.

[0244] The term "site-specific integrase" refers to an enzyme or protein that mediates the insertion of a nucleic acid sequence into a specific site within a target genome. Site-specific integrases include, but are not limited to, serine integrases (e.g., phiC31 integrase), tyrosine integrases (e.g., Cre, FLP), or engineered variants thereof. These enzymes recognize specific DNA sequences, often referred to as attachment sites, and catalyze unidirectional or bidirectional integration events, enabling stable incorporation of transgenes, gene circuits, or other regulatory elements.In some additional embodiments, the gene editing component of the disclosed gene editing system may be a site-specific recombinase.

[0245] The term "site-specific recombinase" refers to an enzyme or protein that catalyzes recombination between defined DNA recognition sites, resulting in excision, inversion, integration, or replacement of genetic material. Site-specific recombinases include, but are not limited to, Cre recombinase, FLP recombinase, Dre, Vika, or engineered recombinases with altered specificity. These enzymes can be used to control gene expression, excise selectable markers, or enable conditional gene activation or inactivation, and may operate independently or as part of a larger gene editing system.

[0246] More specifically, in some embodiments, the CAR encoding nucleic acid sequences (e.g., in a nucleic acid cassette) is inserted into the appropriate target genomic locus using a site-specific nuclease. The nuclease may be one of the following: CRISPR / Cas9 / Cpfl / CTc( 1 / 2 / 3), SpCas9, SaCas9, engineered CAS9, ZFN, TALEN, Homing endonuclease, Meganuclease, Mega-TALEN. The nuclease may be coded on a DNA vector such as a plasmid, a mini-circle or a viral vector. Alternatively, the mRNA coding for the nuclease may be delivered, or the nuclease may be delivered as a protein. A guide RNA may be provided or a DNA vector coding for a guide RNA. Integration catalyzed by a nuclease may utilize homologous arms flanking the DNA to be inserted or utilize recognition sites for the site-specific nuclease when such were coded preceding and or following the DNA to be inserted. Delivery of the nuclease or the vector coding for the nuclease can take place in vivo or ex vivo using autologous or allogeneic cells, as will be discussed herein after.

[0247] In some embodiments, a gene editing component useful in the systems of the present disclosure may be CRISPR / Cas.

[0248] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a bacterial immune system that has been modified for genome engineering. CRISPR-Cas systems fall into two classes. Class 1 systems use a complex of multiple Cas proteins to degrade foreign nucleic acids. Class 2 systems use a single large Cas protein for the same purpose. More specifically, Class 1 may be divided into types I, III, and IV, and Class 2 may be divided into types II, V, and VI.

[0249] It should be understood that the present disclosure contemplates the use of any of the known CRISPR systems, particularly any of the CRISPR systems disclosed herein. The CRISPR-Cas system has evolved in prokaryotes to protect against phage attack and undesired plasmidreplication by targeting foreign DNA or RNA. In bacterial immunity, the CRISPR-Cas system targets DNA molecules based on short homologous DNA sequences, called spacers, that have previously been extracted by the bacterium from the foreign pathogen sequence and inserted between repeats as a memory system. These spacers are transcribed and processed, and this RNA, named crRNA or guide-RNA (gRNA), guides CRISPR-associated (Cas) proteins to matching (and / or complementary) sequences within the foreign DNA, called protospacers, which are subsequently cleaved. The spacers, or other suitable constructs or RNAs, can be rationally designed and produced to target any DNA sequence. Moreover, this recognition element may be designed separately to recognize and target any desired target, including outside of a bacterium. In some specific embodiments, the CRISPR-Cas proteins used in the present disclosure may be from a CRISPR Class 2 system. In yet some further particular embodiments, such Class 2 system may be any one of CRISPR type II and type V systems. In certain embodiments, the Cas applicable in the present invention may be any Cas protein of the CRISPR type II system. The type II CRISPR-Cas systems include the 'HNH'-type system (Streptococcus-like; also known as the Nmeni subtype, for Neisseria meningitidis serogroup A str. Z2491, or CASS4), in which Cas9, a single, very large protein, seems to be sufficient for generating crRNA and cleaving the target DNA, in addition to the ubiquitous Casl and Cas2. Cas9 contains at least two nuclease domains, a RuvC-like nuclease domain near the amino terminus and the HNH (or McrA-like) nuclease domain in the middle of the protein. It should be appreciated that any type II CRISPR-Cas systems may be applicable in the present invention, specifically, any one of type II-A or B. Thus, in yet some further and alternative embodiments, at least one cas gene used in the methods and systems of the invention may be at least one cas gene of a type II CRISPR system (either type II-A or type II-B). In more particular embodiments, at least one cas gene of a type II CRISPR system used by the methods and systems of the invention may be the cas9 gene.

[0250] According to such embodiments, the CRISPR-Cas proteins used in the systems of the invention are CRISPR-associated endonuclease 9 (Cas9). Double- stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of "type II CRISPR-Cas" immune systems. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA: DNA complementarity to a target site (protospacer). After recognition between Cas9 and the target sequence, double- stranded DNA (dsDNA) cleavage occurs, creating double-strand breaks (DSBs).

[0251] CRISPR type II system as used herein requires the inclusion of two essential components: a “guide” RNA (gRNA) and a CRISPR-associated endonuclease (Cas9). The gRNA is an RNAmolecule composed of a “scaffold” sequence necessary for Cas9-binding (also named tracrRNA) and about 20 nucleotide long “spacer” or “targeting” sequence, which defines the genomic target to be modified. Guide RNA (gRNA), as used herein refers to a synthetic fusion or alternatively, annealing of the endogenous tracrRNA with a targeting sequence (also named crRNA), providing both scaffolding / binding ability for Cas9 nuclease and targeting specificity. Also referred to as “single guide RNA” or “sgRNA” or as a specificity conferring nucleic acid (SCNA).

[0252] In yet some further particular embodiments, the class 2 system in accordance with the invention, may be a CRISPR type V system. In a more specific embodiment, the RNA guided DNA binding protein nuclease may be CRISPR-associated endonuclease X (CasX) system or CRISPR-associated endonuclease 14 (Casl4) system or CRISPR-associated endonuclease F (CasF, also known as Casl2j) system. The type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing nuclease. As with type II CRISPR-Cas systems, CRISPR type V system as used herein requires the inclusion of two essential components: a gRNA and a CRISPR-associated endonuclease (CasX / Casl4 / CasF). The gRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for CasX / Casl4 / CasF-binding and about 20 nucleotide long “spacer” or “targeting” sequence, which defines the genomic target to be modified.

[0253] It should be noted that the gRNA used herein may comprise between about 3 nucleotides to about 100 nucleotides, specifically, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more. More specifically, between about 10 nucleotides to 70 nucleotides or more.

[0254] It should be noted that any CRISPR / Cas proteins may be used by the gene editing system / s of the present disclosure, in some embodiments of the present disclosure, the endonuclease may be a Cas9, CasX, Casl2, Casl3, Casl4, Cas6, Cpfl, CMS1 protein, or any variant thereof that is derived or expressed from Methanococcus maripaludis C7, Corynebacterium diphtheria, Corynebacterium efficiens YS-314, Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum (ATCC 13032), Corynebacterium glutamicum R, Corynebacterium kroppenstedtii (DSM 44385), Mycobacterium abscessus (ATCC 19977), Nocardia farcinica IFM10152, Rhodococcus erythropolis PR4, Rhodococcus jostii RFIA1, Rhodococcus opacus B4 (uid36573), Acidothermus cellulolyticus 11 B, Arthrobacter chlorophenolicus A6, Kribbella flavida (DSM 17836), Thermomono spora curvata (DSM43183), Bifidobacterium dentium Bdl, Bifidobacterium longum DJO10A, Slackia heliotrinireducens (DSM 20476), Persephonella marina EX H 1, Bacteroides fragilis NCTC 9434, Capnocytophaga ochracea (DSM 7271),Flavobacterium psychrophilum JIP02 86, Akkermansia muciniphila (ATCC BAA 835), Roseiflexus castenholzii (DSM 13941), Roseiflexus RSI, Synechocystis PCC6803, Elusimicrobium minutum Peil91, uncultured Termite group 1 bacterium phylotype Rs D17, Fibrobacter succinogenes S85, Bacillus cereus (ATCC 10987), Listeria innocua, Lactobacillus casei, Lactobacillus rhamnosus GG, Lactobacillus salivarius UCC118, Streptococcus agalactiae-5-A909, Streptococcus agalactiae NEM316, Streptococcus agalactiae 2603, Streptococcus dysgalactiae equisimilis GGS 124, Streptococcus equi zooepidemicus MGCS 10565, Streptococcus gallolyticus UCN34 (uid46061), Streptococcus gordonii Challis subst CHI, Streptococcus mutans NN2025 (uid46353), Streptococcus mutans, Streptococcus pyogenes Ml GAS, Streptococcus pyogenes MGAS5005, Streptococcus pyogenes MGAS2096, Streptococcus pyogenes MGAS9429, Streptococcus pyogenes MGAS 10270, Streptococcus pyogenes MGAS6180, Streptococcus pyogenes MGAS315, Streptococcus pyogenes SSI-1, Streptococcus pyogenes MGAS 10750, Streptococcus pyogenes NZ131, Streptococcus thermophiles CNRZ1066, Streptococcus thermophiles LMD-9, Streptococcus thermophiles LMG 18311, Clostridium botulinum A3 Loch Maree, Clostridium botulinum B Eklund 17B, Clostridium botulinum Ba4 657, Clostridium botulinum F Langeland, Clostridium cellulolyticum H10, Finegoldia magna (ATCC 29328), Eubacterium rectale (ATCC 33656), Mycoplasma gallisepticum, Mycoplasma mobile 163K, Mycoplasma penetrans, Mycoplasma synoviae 53, Streptobacillus, moniliformis (DSM 12112), Bradyrhizobium BTAil, Nitrobacter hamburgensis X14, Rhodopseudomonas palustris BisB18, Rhodopseudomonas palustris BisB5, Parvibaculum lavamentivorans DS-1, Dinoroseobacter shibae. DFL 12, Gluconacetobacter diazo trophicus Pal 5 FAPERJ, Gluconacetobacter diazo trophicus Pal 5 JGI, Azospirillum B510 (uid46085), Rhodo spirillum rubrum (ATCC 11170), Diaphorobacter TPSY (uid29975), Verminephrobacter eiseniae EF01 -2, Neisseria meningitides 053442, Neisseria meningitides alphal4, Neisseria meningitides Z2491, Desulfovibrio salexigens DSM 2638, Campylobacter jejuni doylei 269 97, Campylobacter jejuni 81116, Campylobacter jejuni, Campylobacter lari RM2100, Helicobacter hepaticus, Wolinella succinogenes, Tolumonas auensis DSM 9187, Pseudoalteromonas atlantica T6c, Shewanella pealeana (ATCC 700345), Legionella pneumophila Paris, Actinobacillus succinogenes 130Z, Pasteurella multocida, Francisella tularensis novicida U 112, Francisella tularensis holarctica, Francisella tularensis FSC 198, Francisella tularensis, Francisella tularensis WY96- 3418, or Treponema denticola (ATCC 35405).In some embodiments, the CAR encoding nucleic acid sequences (e.g. in a nucleic acid cassette) is inserted into the appropriate genomic locus using a site-specific recombinase / integrase. The recombinase / integrase may be one of the following: PhiC31, HK022, Cre, Flp, and more. The recombinase / integrase may be coded on a DNA vector such as a plasmid, a mini-circle or a viral vector. Alternatively, the mRNA coding for the recombinase / integrase may be delivered, or the recombinase / integrase may be delivered as a protein. Delivery of the nuclease or the vector coding for the recombinase / integrase can take place in vivo or ex vivo using autologous or allogeneic cells.

[0255] Another aspect of the present disclosure relates to a genetically engineered cell expressing at least one CAR molecule, or a population of cells comprising at least one of the genetically engineered and / or modified cell. In some embodiments, the disclosed engineered cell is a hematopoietic cell. In yet some further embodiments, the hematopoietic cell is an immune cell. The CAR molecules expressed by the genetically engineered cells disclosed herein, comprise the following components: (i) at least one target-binding domain, wherein at least one of the target binding domain specifically recognizes and binds GPRC5D, or any fragments thereof; (ii) at least one hinge region; (iii) at least one transmembrane domain; and (iv) at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR expressed by the cells of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule expressed by the cells of the present disclosure comprises at least one Complementarity Determining Region 3(CDR3) comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

[0256] In some embodiments, the CAR molecule expressed by the genetically engineered cell / s of the present disclosure is any of the CAR molecules disclosed herein, as defined above in connection with other aspects of the present disclosure.

[0257] In some other embodiments, the genetically engineered hematopoietic cell of the present disclosure is at least one cell of the lymphoid lineage, or at least one cell of the myeloid lineage. Still further, in some embodiments, the disclosed cell is a lymphocyte. In some further embodiments, the lymphocyte is a cell of the T lineage. In more specific embodiments, the cell of the T lineage is a T cell.

[0258] As indicated in the present aspect, the present invention provides a genetically engineered hematopoietic cell. A "hematopoietic cell" refers herein to any cell that originates from hematopoietic stem cells (HSCs), including myeloid cells (e.g., macrophages, monocytes, neutrophils, eosinophils, basophils, megakaryocytes, and erythrocytes) and lymphoid cells (e.g., T cells, B cells, and natural killer (NK) cells).

[0259] Hematopoietic cells encompass both progenitor cells, which are intermediates in the differentiation process, and fully differentiated cells, such as functional immune cells and red blood cells.

[0260] In some embodiments, the present invention provides an engineered cell that may be any lymphocyte, specifically, any lymphocyte of the T lineage. " Lymphocytes” are mononuclear nonphagocytic leukocytes found in the blood, lymph, and lymphoid tissues. They are divided on the basis of ontogeny and function into two classes, B and T lymphocytes, responsible for humoral and cellular immunity, respectively. Most are small lymphocytes 7-10 pm in diameter with a round or slightly indented heterochromatic nucleus that almost fills the entire cell and a thin rim of basophilic cytoplasm that contains few granules. When "activated” by contact with antigen, small lymphocytes begin macromolecular synthesis, the cytoplasm enlarges until the cells are 10-30 pm in diameter, and the nucleus becomes less completely heterochromatic; they are then referred to as large lymphocytes or lymphoblasts. These cells then proliferate and differentiate into B and T memory cells and into the various effector cell types: B cells into plasma cells and T cells into helper, cytotoxic, and suppressor cells.

[0261] As indicated by the present disclosure, the genetically engineered cells that express the CAR molecule disclosed herein may be cells of the T lineage. The " T lineage" refers to the developmental lineage of T cells, a subset of lymphocytes derived from hematopoietic stem cellsthat mature in the thymus and play key roles in adaptive immunity. A " T cell” or " T lymphocyte” as used herein is characterized by the presence of a T-cell receptor (TCR) on the cell surface. It should be noted that T cells include helper T cells ("effector T cells" or " Th cells"), cytotoxic T cells (" Tc," " CTL," or "killer T cell"), memory T cells, regulatory T cells, Natural Killer T cells, Mucosal Associated Invariant T cells, and Gamma Delta T cells.

[0262] More specifically, thymocytes are hematopoietic progenitor cells present in the thymus.

[0263] Thymopoiesis is the process in the thymus by which thymocytes differentiate into mature T lymphocytes. The thymus provides an inductive environment, which allows for the development and selection of physiologically useful T cells. The processes of beta-selection, positive selection, and negative selection shape the population of thymocytes into a peripheral pool of T cells that are able to respond to foreign pathogens and are immunologically tolerant towards self-antigens. Thymocytes are classified into a number of distinct maturational stages based on the expression of cell surface markers. The earliest thymocyte stage is the double negative (DN) stage (negative for both CD4 and CD8), which more recently has been better described as Lineage-negative, and which can be divided into four sub-stages. The next major stage is the double positive (DP) stage (positive for both CD4 and CD8). The final stage in maturation is the single positive (SP) stage (positive for either CD4 or CD8).

[0264] More specifically, the maturational stages of thymocytes may include the following substages: Double negative 1 (DN1) or ETP (Early T lineage Progenitor) is characterized by CD44+CD25-CD117+ defining surface markers, thymocytes are located in the cortex and proliferation, loss of B and myeloid potentials are observed; Double negative 2 (DN2) is characterized by CD44+CD25+CD117+ defining surface markers and thymocytes are located in the cortex; Double negative 3 (DN3) is characterized by CD44-CD25+ defining surface markers, thymocytes are located in the cortex and TCR-beta rearrangement and beta selection are observed; Double negative 4 (DN4) is characterized by CD44-CD25- defining surface markers and thymocytes are located in the cortex; Double positive is characterized by CD4+CD8+ defining surface markers, thymocytes are located in the cortex and TCR-alpha rearrangement, positive selection, negative selection are observed; Single positive is characterized by CD4+CD8- or CD4-CD8+ defining surface markers, thymocytes are located in the medulla and Negative selection is observed.

[0265] In human, circulating CD34+ hematopoietic stem cells (HSC) reside in bone marrow. They produce precursors of T lymphocytes, which seed the thymus (thus becoming thymocytes) and differentiate under influence of the Notch and its ligands. Early, double negative thymocytesexpress (and can be identified by) CD2, CD5 and CD7. Still during the double negative stage, CD34 expression stops and CD1 is expressed. Expression of both CD4 and CD8 makes them double positive and matures into either CD4+ or CD8+ cells. It should be appreciated that a cell of the T lineage as disclosed herein may be any of the thymocytes disclosed herein at any stage / sub stage and / or expressing any of the disclosed markers.

[0266] In some embodiments of the present disclosure, the cell is a T cell. For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4.sup.+ / CD8.sup.+ double positive T cells, CD4.sup.+ helper T cells, e.g., Th. sub.1 and Th. sub.2 cells, CD8.sup.+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cell may be a CD8.sup.+ T cell or a CD4.sup.+ T cell. The present disclosure therefore provides in some embodiments thereof, CAR T cells.

[0267] In an embodiment of the present disclosure, the cell is a natural killer (NK) cell. NK cells are a type of cytotoxic lymphocyte that plays a role in the innate immune system. NK cells are defined as large granular lymphocytes and constitute the third kind of cells differentiated from the common lymphoid progenitor which also gives rise to B and T lymphocytes. NK cells differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus. Following maturation, NK cells enter into the circulation as large lymphocytes with distinctive cytotoxic granules. NK cells are able to recognize and kill some abnormal cells, such as, for example, some tumor cells and virus-infected cells, and are thought to be important in the innate immune defense against intracellular pathogens. As described above with respect to T-cells, the NK cell can be any NK cell, such as a cultured NK cell, e.g., a primary NK cell, or an NK cell from a cultured NK cell line, or an NK cell obtained from a mammal. If obtained from a mammal, the NK cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. NK cells can also be enriched for or purified. The NK cell preferably is a human NK cell (e.g., isolated from a human). The present disclosure therefore provides in some embodiments thereof, CAR NK cells.Still further, in some alternative embodiments, the genetically engineered cells may be of the B lineage. Specifically, in some embodiments, the cells of the present disclosure may be B cells. The present disclosure therefore provides in some embodiments thereof, CAR B cells.

[0268] In some further alternative embodiments, the genetically engineered cells may be a cell of the myeloid lineage. In yet some further embodiments, cells of the myeloid lineage applicable in the present disclosure may be monocytes. In more specific embodiments, the genetically engineered cell may be a macrophage. The present disclosure therefore provides in some embodiments thereof, CAR Macrophage cells.

[0269] Also provided by an embodiment of the present disclosure is a population of cells comprising at least one host cell described herein, e.g., of the T lineage. The population of cells can be a heterogeneous population comprising the host cell comprising and / or genetically engineered by any of the nucleic acid sequences, cassettes, vectors and gene editing systems described herein, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the recombinant expression vector, cassette, gene editing system. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells that are genetically edited and / or comprising the nucleic acid sequences, cassettes and vectors as described herein.

[0270] The present disclosure provides cells, specifically, of the T lineage that were genetically engineered to express the CAR T molecules disclosed herein. It should be, however, noted that the present disclosure thus encompasses any host cell, specifically, a T host cell comprising, transfected by, transformed by and / or engineered and / or edited by the nucleic acid sequence, cassette or vector disclosed herein.

[0271] The term "host cell" includes a cell into which a heterologous (e.g., exogenous) nucleic acid or protein has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also is used to refer to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation orenvironmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell". As used herein, a "genetically engineered cell" or a "genetically modified cell" has been "transformed" or "transfected" by exogenous or heterologous DNA, e.g., the nucleic acid molecule / s of the invention or any cassette, vector and / or gene editing system of the invention, when such DNA has been introduced inside the cell. The transforming DNA may be integrated (covalently linked) into the genome of the cell. With respect to the present invention, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. It should be appreciated that in some embodiments, the host cells of the invention may be any engineered cells of the invention or any cell population comprising, at least in part, the engineered cells of the invention. Still further, the invention further encompasses any population of cells comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9% or more, specifically, 100%) specifically, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9% or more, specifically, 100% of the host cells, specifically, the genetically engineered cells of the invention. It should be appreciated that the CAR T cells of the present disclosure may express one or more CAR molecules, for example, the CAR molecules as disclosed herein. Still further, as indicated above and demonstrated by the present examples (e.g., Figure 11), the CAR expressing cells (e.g., CAR T, CAR NK) of the present disclosure may express in addition to at least one CAR molecule, any additional immunomodulatory agent (e.g., a cytokine such as IL-7). However, beyond cytokine- secreting CART, the CAR T cells of the present disclosure may be designed as armored CAR T cells such that in addition to the at least one CAR molecule disclosed herein, the cells may be further engineered with cell-intrinsic or membrane-tethered modules that reprogram T-cell signaling and persistence. These include (i) chimeric switch receptors, such as PD1-CD28 or TGFβ-41BB, which convert inhibitory tumor-microenvironment cues into costimulatory signals; (ii) co-stimulatory receptors (CCRs) that provide additional CD28 or 4- IBB signaling upon recognition of a second antigen, improving activation under low antigen density and enabling " AND gate" specificity; and (iii) cell-intrinsic exhaustion-resistance programs, such as c-Jun overexpression, which preserve function and durability during chronic antigen exposure. In some embodiments, “improving activation under low antigen density” refers to increasing, enhancing,restoring, or otherwise maintaining activation and / or effector function of an engineered immune cell (e.g., a CAR-T cell or CAR-NK cell) when the target antigen is present on a cell surface at relatively low copy number or low surface density (for example, relative to a high-antigen-density tumor cell), as compared to a reference engineered immune cell and / or a reference binding domain. In some embodiments, “enabling AND gate specificity” refers to configuring a cell therapy or engineered receptor system such that a desired activation state, effector function, or therapeutic response is triggered only when two (or more) independent target-associated inputs are present on the same target cell and / or in the same microenvironment. By way of example, an AND gate may be implemented using (i) a first recognition module (e.g., a first CAR, TCR, or synNotch receptor) that provides a priming, costimulatory, or conditional expression signal in response to a first antigen, and (ii) a second recognition module (e.g., a second CAR) that provides an activation signal in response to a second antigen, such that full activation occurs only upon concurrent engagement of both antigens. In some embodiments, AND gate specificity reduces off-target activity by limiting full activation in the presence of only one antigen and favors selective targeting of cells (e.g., tumor cells) that co-express the two antigens.

[0272] Accordingly, it should be appreciated that in some embodiments, the disclosed genetically engineered cell expressing at least one CAR molecule of the present disclosure may express one or more CAR molecules directed to one or more target molecules expressed by one or more target cells. In some embodiments, the genetically engineered cell expresses a single CAR molecule. In some embodiments, the genetically engineered cell expresses two, three, four, five, or more CAR molecules. In some embodiments, the two or more CAR molecules are directed to the same antigen (e.g., the same target molecule) via different antigen-binding domains (e.g., different antibodies, nanobodies, scFvs, or other binding moieties) that bind the same antigen at the same or different epitopes, thereby providing, for example, redundancy and / or enhanced coverage against antigen escape. In some embodiments, the two or more CAR molecules are directed to different antigens, including antigens co-expressed on the same target cell, and / or antigens expressed on different target cells, thereby enabling, for example, multi-target recognition, improved tumor selectivity, and / or broader targeting of heterogeneous cell populations. In some embodiments, the CAR molecules are provided in any suitable combination or architecture, including co-expression of separate CARs, bicistronic or multicistronic constructs, tandem CARs, dual-signaling CARs, and / or conditional logic configurations (e.g., AND-gated and / or OR-gated designs).Another aspect of the present disclosure relates to a composition comprising at least one of the following elements, acting as the active ingredient / s of the disclosed compositions. Specifically, at least one CAR molecule, any nucleic acid molecule comprising at least one nucleic acid sequence encoding the CAR molecule, or any cassette, vector, vehicle or gene editing system comprising the nucleic acid molecule, and / or any genetically engineered cell expressing the CAR molecule of the present disclosure, or population of cells comprising at least one of the genetically engineered cell, and / or any combinations thereof. The CAR molecules of the disclosed compositions comprise the following components: (i), at least one target-binding domain, wherein at least one of the target binding domain specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. The at least one of the target binding domains of the CAR molecules of the disclosed compositions comprise at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule of the present disclosure comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

[0273] The compositions of the present disclosure further comprise at least one of pharmaceutically acceptable carrier / s, diluent / s, excipient / s and / or additive / s.

[0274] In some embodiments, the compositions of the present disclosure comprise any of the CAR molecules disclosed herein, and / or any of the nucleic acid molecules of the present disclosureand / or the genetically engineered cells disclosed by the present disclosure as defined herein above in connection with other aspects of the present disclosure.

[0275] The pharmaceutical compositions of the present disclosure can be administered and dosed by the methods of the invention, in accordance with good medical practice, systemically, for example, by parenteral administration, e.g., intrathymic, into the bone marrow, and intravenous administration. It should be noted, however, that the present disclosure may further encompass additional administration modes. In other examples, the pharmaceutical composition can be introduced to a site by any suitable route, including intraperitoneal, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g., oral, intranasal, or intraocular administration.

[0276] Local administration to the area in need of treatment may be achieved, for example, by local infusion during surgery, topical application, direct injection into the specific organ (bone marrow, spleen, lymph nodes), etc. More specifically, the compositions used in any of the methods of the present disclosure, described herein, may be adapted for administration by parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal, and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example, by bringing into association the active ingredient with the carrier(s) or excipient(s).

[0277] More specifically, pharmaceutical compositions used to treat subjects in need thereof according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients, specifically, the CAR T, nucleic acid molecule / s of the invention or any cassette / s thereof, or any genetically engineered cell of the disclosure, with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may contain further substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions of the present inventionalso include, but are not limited to, emulsions and liposome-containing formulations. It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question. Still further, pharmaceutical preparations are compositions that include one or more nucleic acid molecules, vectors and / or cassette and / or cells of the present in a pharmaceutically acceptable vehicle. " Pharmaceutically acceptable vehicles" may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term "vehicle", when referred to the compositions in the present aspect, refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Pharmaceutical compositions may be formulated into preparations in solid, semisolid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the nucleic acid molecule / s encoding the CARs of the invention or any genetically engineered cells, and systems of the invention, can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. The active agent may be systemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation. The active agent may be formulated for immediate activity, or it may be formulated for sustained release.

[0278] Another aspect of the present disclosure relates to a method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathological disorder in a subject. The method comprising the step of administering to the subject an effective amount of at least one of: (A), at least one nucleic acid molecule encoding at least one CAR molecule; (B), at least one cassette, vector vehicle or gene editing system comprising the nucleic acid molecule of (A); (C), at least one genetically engineered cell expressing the CAR, or a population of the cells; (D), at least one VHH nanobody specific for GPRC5D; and (E), a composition comprising at least one of (A), (B), (C) and (D).-11 - More specifically, the CAR molecule comprising the following components: (i), at least one targetbinding domain, wherein at least one of the target binding domain specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR used by the methods of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule used by the methods of the present disclosure comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

[0279] In some embodiments, the VHH nanobody of the CAR molecule used by the disclosed methods, or encoded by the nucleic acid molecules or the engineered cells used by the disclosed methods may comprise the amino acid sequence as denoted by at least one of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or any variant or derivatives thereof (encoded by SEQ ID NO: 51, 52, 53 and 54, respectively).

[0280] In some other embodiments, the at least one hinge region of the CAR molecules used in the disclosed methods is derived from at least one of: a co-stimulatory receptor molecule, a co-receptor molecule and at least one immunoglobulin molecule.

[0281] In some further embodiments, the at least one hinge region of the CAR used in the disclosed methods is derived from a co-stimulatory receptor molecule being the CD28 protein, or a coreceptor molecule being the CD 8 a protein, or any combination thereof.In some more specific embodiments of the disclosed methods, the hinge region is derived from CD28. Accordingly, in some specific embodiments, the hinge region comprises the amino acid sequence as denoted by SEQ ID NO: 73, and / or any variants or derivatives thereof. In some alternative and specific embodiments, the hinge region is derived from CD8a. Accordingly, in some specific embodiments, the hinge region comprises the amino acid sequence as denoted by SEQ ID NO: 69, and / or any variants or derivatives thereof.

[0282] In some embodiments, the transmembrane domain of the CAR used in the disclosed methods is derived from at least one of: a co-stimulatory receptor molecule and / or at least one co-receptor molecule.

[0283] In some further embodiments, the at least one transmembrane domain of the CAR used in the disclosed methods is derived from a co-stimulatory receptor being the CD28 protein. Alternatively, the transmembrane domain is derived from a co-receptor molecule being the CD8a protein, or any combination thereof.

[0284] In some more specific embodiments, the at least one transmembrane domain of the CAR used in the disclosed methods is derived from the CD28 protein. Accordingly, the transmembrane domain comprises the amino acid sequence as denoted by SEQ ID NO: 75; and / or any variants or derivatives thereof. In some alternative embodiments, the transmembrane domain of the CAR used in the disclosed methods is derived from the CD8a protein. Accordingly, such transmembrane domain comprises the amino acid sequence as denoted by SEQ ID NO: 71, and / or any variants or derivatives thereof.

[0285] In some embodiments, the at least one intracellular signal transduction domain of the CAR used in the disclosed methods, is a T cell signal transduction domain.

[0286] In some further embodiments, the T cell signal transduction domain of the CAR used in the disclosed methods, comprises at least one domain of TNF receptor family member. In some additional embodiments, the T cell signal transduction domain comprises at least one domain of a TCR molecule.

[0287] In some embodiments, the at least one TNF receptor family member comprises the 4- IBB. In some other embodiments, the TCR molecule comprises a CD3 zeta chain.

[0288] In further specific embodiments, the at least one intracellular signal transduction domain of the CAR used in the disclosed methods, comprises the amino acid sequence as denoted by SEQ ID NO: 77 and / or by SEQ ID NO: 79, or any derivatives or variants thereof.In some embodiments, the CAR molecules used in the disclosed methods, comprise the amino acid sequence as denoted by at least one of SEQ ID NO: 110, 102, 104, 106, 108, 113, 117, 119, 122, 124, 126, 128, 130, 132, or any variants or derivatives thereof.

[0289] In more specific embodiments, the CAR molecule used in the disclosed methods, is designated CARB28, and comprises the amino acid sequence as denoted by at least one of SEQ ID NO: 110, encoded by the nucleic acid sequence as denoted by SEQ ID NO: 111.

[0290] In some embodiments of the disclosed methods, the expression of the CAR by at least one cell of the T lineage of the subject results in at least one of: (i) increased specificity; (ii) reduced tonic signaling; (iii) reduced off-target activation; (iv) increased expression of activation markers in response to a specific stimulation; (v) reduced expression of exhaustion markers in response to a specific stimulation; (vi) increased survival; (vii) reduced relapse rate; and (viii) long-term effect; in the subject.

[0291] In some embodiments, “increased specificity” refers to a higher degree of selective recognition and / or effector activity toward intended target cells (e.g., tumor cells expressing a target antigen) relative to non-target cells, as measured by one or more of (a) increased killing of target cells relative to non-target cells, (b) increased cytokine release in the presence of target cells relative to non-target cells, (c) a higher ratio of on-target activity to off-target activity, and / or (d) reduced on-target, off-tumor activity against cells expressing low levels of the target antigen.

[0292] In some embodiments, “reduced tonic signaling” refers to decreased basal, ligand-independent signaling through the CAR in the absence of cognate antigen stimulation, as compared to a reference CAR and / or reference engineered T cell. Tonic signaling may be assessed by measuring basal phosphorylation of signaling proteins (e.g., CD3 zeta, ZAP-70, ERK), basal expression of activation markers (e.g., CD69 and / or CD25), baseline cytokine secretion, and / or spontaneous proliferation in antigen-free culture conditions.

[0293] In some embodiments, “reduced off-target activation” refers to a reduction in activation and / or effector responses of the engineered T-lineage cell that occur in response to non-cognate antigens, unintended epitopes, or non-target cells (including normal tissues), as compared to a reference CAR and / or reference engineered T cell. Off-target activation may be assessed by decreased cytokine release, decreased degranulation (e.g., CD107a), decreased killing, and / or decreased activation marker expression when co-cultured with antigen-negative cells or irrelevant antigenexpressing cells.In some embodiments, “increased expression of activation markers in response to a specific stimulation” refers to an increase, relative to a reference, in the level and / or frequency of expression of one or more activation markers on the engineered T-lineage cell after exposure to cognate antigen and / or antigen-positive target cells (the “specific stimulation”). Exemplary activation markers include CD69, CD25, 4-1BB (CD137), 0X40 (CD134), and / or CD71, and may be quantified by flow cytometry and / or other suitable assays at one or more defined time points following stimulation.

[0294] In some embodiments, “reduced expression of exhaustion markers in response to a specific stimulation” refers to a decrease, relative to a reference, in the level and / or frequency of expression of one or more exhaustion-associated markers on the engineered T-lineage cell following repeated stimulation and / or prolonged exposure to cognate antigen and / or antigen-positive target cells. Exemplary exhaustion markers include PD-1, TIM-3, LAG-3, TIGIT, CTLA-4, and / or TOX, and may be assessed by flow cytometry, transcript levels, and / or functional readouts (e.g., preserved cytokine production upon restimulation).

[0295] In some embodiments, “increased survival” refers to an increase in overall survival and / or progression-free survival of the subject relative to a reference (e.g., standard of care, placebo, or baseline), as assessed by clinical outcome measures known in the art.

[0296] In some embodiments, “reduced relapse rate” refers to a lower frequency, probability, and / or incidence of disease recurrence after an initial response or remission, relative to a reference (e.g., standard of care or historical control), as assessed over a defined follow-up period using accepted clinical criteria.

[0297] In some embodiments, “long-term effect” refers to sustained therapeutic benefit over an extended period following administration of the engineered cells, including durable response, durable disease control, and / or persistence of the engineered cells and / or their functional activity, as measured by clinical endpoints and / or by detection of the engineered cells (e.g., CAR transgene copies, CAR-positive cells, and / or functional responsiveness) at one or more time points posttreatment.

[0298] In some embodiments, the nucleic acid molecules used by the disclosed methods and / or the genetically engineered cell / s used by the disclosed methods, and / or the compositions used by the disclosed methods, are any of the nucleic acid molecules, the engineered cells and any of the compositions of the present disclosure, specifically, any of those as defined herein before or after in connection with other aspects of the present disclosure.In some specific embodiments, the disclosed therapeutic methods involve an ex vivo step of genetically engineering at least one hematopoietic cell, specifically, an immune cell, to express the CAR molecules of the present disclosure. According to such specific embodiments, the subject is administered with at least one genetically engineered cell expressing the CAR molecule. In some specific embodiments, the subject is administered with at least one hematopoietic cell genetically engineered with the at least one nucleic acid molecules of the present disclosure, or with any cassette or any vector or vehicle comprising the cassette. In some further embodiments, the subject is administered with a population of these cells.

[0299] Accordingly, for such ex vivo therapeutic method, the genetically engineered cell used, is according to some embodiments, of an autologous source, specifically, obtained from the treated subject, genetically engineered ex vivo, and transferred back to the subject. In some alternative embodiments, the genetically engineered cell is of an allogeneic source.

[0300] As indicated herein above, the present disclosure provides method allowing in vivo as well as ex-vivo or in vitro genetic engineering of cells of the T lineage to express the CAR T molecules of the present invention. In case the engineering of the cells is performed ex vivo or in vitro, the engineered cells are transferred back to the subject, by adoptive transfer.

[0301] The term 'adoptive transfer” as herein defined applies to all the therapies that consist of the transfer of components of the immune system, specifically cells that are already capable of mounting a specific immune response. In such option, the targeted insertion of the nucleic acid sequence encoding the CAR Ts disclosed herein, is performed in cells of an autologous or allogeneic source, that are then administered to the subject, specifically, by adoptive transfer. In some embodiments, the cells that express, comprise, are transduced with, or transfected with the nucleic acid molecule / s of the invention or any cassette provided by the invention may be cells of an autologous source. The term "autologous” when relating to the source of cells, refers to cells derived or transferred from the same subject that is to be treated by the methods of the invention. The term "allogeneic” when relating to the source of cells, refers to cells derived or transferred from a different subject, referred to herein as a donor, of the same species.

[0302] It should be, however, appreciated that in some alternative embodiments, the present disclosure provides therapeutic methods involving an in vivo manipulation of hematopoietic cells (e.g., T cells) to express the disclosed CAR molecules. In such embodiments, the subject is administered with a nucleic acid molecule comprising a nucleic acid sequence encoding the disclosed CAR molecules, or any cassette thereof, or any vector comprising the at least one cassette. In someembodiments, the vector may be a viral vector, and / or a non-viral vector, and / or a naked DNA vector.

[0303] In some embodiments, the insertion of the nucleic acid sequence that encodes the CAR molecule into the genome of a cell of the T lineage in the subject (either in vivo or ex vivo) is mediated by a site-specific nuclease. The nuclease is at least one PEN, wherein the PEN comprises at least one CRISPR / Cas protein system. The method further comprises the step of administering to the subject at least one of: (a), at least one CRISPR / Cas protein, or any nucleic acid molecule encoding the Cas protein; and (b), at least one nucleic acid sequence comprising at least one gRNA that targets the insertion of the nucleic acid sequence encoding the CAR into a genomic sequence, or any nucleic acid sequence encoding the gRNA; or any kit, composition or vehicle comprising at least one of (a) and (b).

[0304] Still further, in some embodiments, the methods of the present disclosure are applicable for any pathologic disorder, specifically, a disorder associated with overexpression of the GPRC5D protein.

[0305] A disorder associated, either directly or indirectly, with overexpression of the GPRC5D protein refers to any pathological condition in which the levels of the GPRC5D protein are abnormally elevated, contributing to disease progression. It should be appreciated that the term "associated with", as used herein when referring to a disorder and a molecular or cellular characteristic (such as the overexpression of the GPRC5D protein), refers to any biological, pathological, functional, or clinical relationship between the disorder and the specified characteristic. This association may be causal, correlative, predictive, contributory, or indicative, and may be identified based on experimental data, clinical evidence, statistical correlation, or mechanistic understanding. In particular, a disorder "associated with overexpression of the GPRC5D protein" includes any condition in which GPRC5D protein levels are elevated in comparison to a normal, healthy state, whether as a driver, biomarker, or consequence of the disorder. Such disorders may be characterized by excessive proliferation or survival of GPRC5D-expressing cells, leading to the disruption of normal tissue function. Overexpression of GPRC5D can also affect signaling pathways that promote tumor growth, metastasis, and immune evasion. Examples for such disorders include plasma cell malignancy and neoplastic disorders of B cells as disclosed below. However, it should be appreciated that in some embodiments, the disorder may be any proliferative disorder, or any neoplastic disorder. The methods of the present disclosure may be applicable in someembodiments for any neoplasms, either benign neoplasms, in situ neoplasms, or malignant neoplasms.

[0306] In some other embodiments, the methods of the present disclosure are applicable for at least one immune-related disorder. The term "immune-related disorder" refers to any condition or disease that involves dysfunction or dysregulation of the immune system. These disorders can arise from an overactive immune response (e.g., autoimmune diseases, allergies, inflammatory conditions), an underactive or deficient immune response (e.g., immunodeficiencies), or an immune response directed against abnormal cells (e.g., certain cancers). In the context of the present disclosure, an immune-related disorder is specifically associated with increased expression of the GPRC5D marker.

[0307] In yet some embodiments, such immune-related disorder may be at least one plasma cell pathology. Still further, in some additional embodiments, the immune-related disorder is at least one of: at least one proliferative disorder, at least one autoimmune disease, a deposition disorder, any plasma cell-mediated disorder and any B cell-mediated disorder.

[0308] The term "proliferative disorder" refers to any condition characterized by an abnormal increase in cell number due to excessive cell division, decreased cell death, or both. This uncontrolled growth can lead to the formation of tumors (neoplasms) and is a hallmark of various diseases, including cancer.

[0309] The term "autoimmune disease" refers to a condition in which the body's immune system mistakenly attacks its own healthy cells and tissues, leading to inflammation and damage. Normally, the immune system defends the body against foreign invaders like bacteria and viruses, but in autoimmune diseases, it fails to distinguish between self and non-self.

[0310] The term "deposition disorder" refers to a group of conditions characterized by the abnormal accumulation of substances within tissues or organs, which can impair their normal function. These substances can be proteins, lipids, minerals, or other metabolic products that are either overproduced, improperly processed, or not adequately cleared by the body.

[0311] The term "plasma cell-mediated disorder" refers to a group of diseases characterized by the abnormal proliferation or dysfunction of plasma cells. Plasma cells are a type of white blood cell that develops from B lymphocytes and are responsible for producing large amounts of antibodies. In these disorders, the plasma cells may produce abnormal antibodies or accumulate in excessive numbers, leading to various pathological effects.The term " B cell-mediated disorder" refers to a group of diseases characterized by the abnormal function, proliferation, or regulation of B lymphocytes (B cells). B cells are a type of white blood cell central to the adaptive immune system, primarily responsible for producing antibodies and presenting antigens. Dysregulation of B cells can lead to a wide range of conditions, including autoimmune diseases, immunodeficiencies, and B cell malignancies.

[0312] In some further embodiments, the disclosed methods are applicable for treating at least one proliferative disorder is any plasma cell malignancy. In some more specific embodiments, such plasma cell malignancy is multiple myeloma (MM) and any related conditions. In some embodiments, the deposition disorder is amyloidosis, and any related conditions.

[0313] The term "plasma cell malignancy" refers to a type of cancer that originates from plasma cells, which are specialized white blood cells responsible for producing antibodies. In plasma cell malignancies, these plasma cells become cancerous, proliferate uncontrollably, and accumulate in the bone marrow or other tissues, leading to various symptoms and organ damage. Multiple myeloma is the most common example of a plasma cell malignancy.

[0314] In yet some further alternative embodiments, the CAR molecule of the present disclosure may be applicable for an inflammatory disorder, an autoimmune disorder, an infectious disease caused by a pathogen, a neurodegenerative disease, a congenital disorder, an allergic condition, a cardiovascular disease, immuno deficiency (acquired or inherited) and a metabolic condition.

[0315] Specific examples for disorders applicable for the present disclosure or any aspects thereof, is disclosed herein after in connection with other aspects of the invention.

[0316] In some embodiments, the disclosed methods are applicable for treating at least one plasma-cell pathology. Plasma-cell pathology refers herein to any disorder which involves plasma cells. For example, multiple myeloma, myelomatosis and medullary plasmacytoma are bone marrow-based, malignant disorders of postgerminal center B-cells that is characterized by a clonal proliferation of plasma cells, with associated serum and / or urine monoclonal proteins.

[0317] In some embodiments, the plasma-cell pathology may comprise any one of Multiple Myeloma (MM), Amyloidosis (AL), Monoclonal Gammopathy of Undetermined Significance (MGUS), Plasmacytoma (PL), and / or Waldenstrom Macroglobulinemia (WM).

[0318] Still further, Monoclonal Gammopathy of Undetermined Significance (MGUS), as used herein, is an asymptomatic preneoplastic plasma cell disorder that is characterized by serum M-protein lessthan 30 g / L, bone marrow clonal plasma cells less than 10 percent, and absence of plasma cell myeloma-related end-organ damage (hypercalcemia, renal insufficiency).

[0319] In some embodiments, Plasmacytoma (PL) is a plasma cell dyscrasia in which a plasma cell tumor grows within soft tissue or within the axial skeleton.

[0320] Waldenstrom Macroglobulinemia (WM), as used herein, is a neoplastic disorder affecting lymphoplasmacytoid cells and plasma cells. It is characterized by having high levels of a circulating antibody, immunoglobulin M (IgM), which is made and secreted by the cells involved in the disease. WM is an "indolent lymphoma" (characterized by slow growth and spread) and a type of lymphoproliferative disease which shares clinical characteristics with the indolent nonHodgkin lymphomas. It is commonly classified as a form of plasma cell dyscrasia, similar to other plasma cell dyscrasias that, for example, lead to multiple myeloma.

[0321] Multiple Myeloma (MM) and Amyloidosis (AL) will be described in more detail hereinafter. As indicated above, the therapeutic methods of the present disclosure may be applicable for any disorder that is associated with the expression (e.g., modulated expression, specifically, overexpression) of the GPRC5D protein. Examples for such disorders include neoplastic disorders of B cells as disclosed above. However, it should be appreciated that in some embodiments, the disorder may be any proliferative disorder, or any neoplastic disorder that involves overexpression of GPRC5D. The methods of the present disclosure may be applicable in some embodiments for any neoplasms, either benign neoplasms, in situ neoplasms, or malignant neoplasms.

[0322] As used herein to describe the present invention, “proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the methods, compositions and kits of the present invention may be applicable for a patient suffering from any one of non-solid and solid tumors.

[0323] Malignancy, as contemplated in the present invention may be any one of lymphomas, leukemia, myeloma, carcinomas, melanomas and sarcomas. Therefore, in some embodiments any of the methods of the invention (provided that they involve, directly or indirectly, overexpression of GPRC5D), systems and compositions disclosed herein, may be applicable for any of the malignancies disclosed by the present disclosure.

[0324] More specifically, myeloma as mentioned herein is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies. Collections of abnormal cellsaccumulate in bones, where they cause bone lesions, and in the bone marrow where they interfere with the production of normal blood cells. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. Hypercalcemia (high calcium levels) is often encountered.

[0325] Lymphoma is a cancer in the lymphatic cells of the immune system. Typically, lymphomas present as a solid tumor of lymphoid cells. These malignant cells often originate in lymph nodes, presenting as an enlargement of the node (a tumor). It can also affect other organs in which case it is referred to as extranodal lymphoma. Non limiting examples for lymphoma include Hodgkin's disease, non-Hodgkin's lymphomas and Burkitt's lymphoma.

[0326] Leukemia refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease-acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non-increase in the number of abnormal cells in the blood-leukemic or aleukemic (subleukemic).

[0327] Still further, carcinoma as used herein, refers to an invasive malignant tumor consisting of transformed epithelial cells. Alternatively, it refers to a malignant tumor composed of transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges.

[0328] Melanoma as used herein, is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin but are also found in other parts of the body, including the bowel and the eye. Melanoma can occur in any part of the body that contains melanocytes.

[0329] Sarcoma is a cancer that arises from transformed connective tissue cells. These cells originate from embryonic mesoderm, or middle layer, which forms the bone, cartilage, and fat tissues. This is in contrast to carcinomas, which originate in the epithelium. The epithelium lines the surface of structures throughout the body, and is the origin of cancers in the breast, colon, and pancreas. It should be understood that the CAR molecules, nucleic acid molecules, cells, gene editing system / s, compositions, and methods of the present disclosure are applicable for any type and / or stage and / or grade of any of the malignant disorders discussed herein or any metastasis thereof.Still further, it must be appreciated that the methods, compositions, and systems of the invention may be applicable for invasive as well as non-invasive cancers. When referring to "non-invasive" cancer, it should be noted as a cancer that does not grow into or invade normal tissues within or beyond the primary location. When referring to "invasive cancers," it should be noted as cancer that invades and grows in normal, healthy adjacent tissues.

[0330] Still further, in some embodiments, the methods, compositions and systems of the present disclosure are applicable for any type and / or stage and / or grade of any metastasis, metastatic cancer or status of any of the cancerous conditions disclosed herein.

[0331] As used herein, the term "metastatic cancer" or "metastatic status" refers to a cancer that has spread from its primary site to another place in the body. A tumor formed by metastatic cancer cells, originating from primary tumors or other metastatic tumors that spread using the blood and / or lymph systems, is referred to herein as a metastatic tumor or a metastasis.

[0332] Thus, in some embodiments, malignancies that may find utility in the present invention can comprise but are not limited to hematological malignancies (including lymphoma, leukemia, myeloproliferative disorders, Acute lymphoblastic leukemia; Acute myeloid leukemia), hypoplastic and aplastic anemia (both virally induced and idiopathic), myelodysplastic syndromes, all types of paraneoplastic syndromes (both immune mediated and idiopathic) and solid tumors (including GI tract, colon, lung, liver, breast, prostate, pancreas and Kaposi's sarcoma. The invention may be applicable as well for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extrahepatic bile ducts, ampulla of Vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma, Adrenocortical carcinoma; AIDS -related cancers; AIDS -related lymphoma; Anal cancer; Appendix cancer; Astrocytoma, childhood cerebellar or cerebral; Basal cell carcinoma; Bile duct cancer, extrahepatic; Bladder cancer; Bone cancer, Osteosarcoma / Malignant fibrous histiocytoma; Brainstem glioma; Brain tumor; Brain tumor, cerebellar astrocytoma; Brain tumor, cerebral astrocytoma / malignant glioma; Brain tumor, ependymoma; Brain tumor, medulloblastoma; Brain tumor, supratentorial primitiveneuroectodermal tumors; Brain tumor, visual pathway and hypothalamic glioma; Breast cancer; Bronchial adenomas / carcinoids; Burkitt lymphoma; Carcinoid tumor, childhood; Carcinoid tumor, gastrointestinal; Carcinoma of unknown primary; Central nervous system lymphoma, primary; Cerebellar astrocytoma, childhood; Cerebral astrocytoma / Malignant glioma, childhood; Cervical cancer; Childhood cancers; Chronic lymphocytic leukemia; Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon Cancer; Cutaneous T-cell lymphoma; Desmoplastic small round cell tumor; Endometrial cancer; Ependymoma; Esophageal cancer; Ewing's sarcoma in the Ewing family of tumors; Extracranial germ cell tumor, Childhood; Extragonadal Germ cell tumor; Extrahepatic bile duct cancer; Eye Cancer, Intraocular melanoma; Eye Cancer, Retinoblastoma; Gallbladder cancer; Gastric (Stomach) cancer; Gastrointestinal Carcinoid Tumor; Gastrointestinal stromal tumor (GIST); Germ cell tumor: extracranial, extragonadal, or ovarian; Gestational trophoblastic tumor; Glioma of the brain stem; Glioma, Childhood Cerebral Astrocytoma; Glioma, Childhood Visual Pathway and Hypothalamic; Gastric carcinoid; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin lymphoma; Hypopharyngeal cancer; Hypothalamic and visual pathway glioma, childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi sarcoma; Kidney cancer (renal cell cancer); Laryngeal Cancer; Leukemias; Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia); Leukemia, acute myeloid (also called acute myelogenous leukemia); Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia); Leukemia, chronic myelogenous (also called chronic myeloid leukemia); Leukemia, hairy cell; Lip and Oral Cavity Cancer; Liver Cancer (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphomas; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-Cell; Lymphoma, Hodgkin; Lymphomas, Non- Hodgkin (an old classification of all lymphomas except Hodgkin's); Lymphoma, Primary Central Nervous System; Marcus Whittle, Deadly Disease; Macroglobulinemia, Waldenstrom; Malignant Fibrous Histiocytoma of Bone / Osteosarcoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular (Eye); Merkel Cell Carcinoma; Mesothelioma, Adult Malignant; Mesothelioma, Childhood; Metastatic Squamous Neck Cancer with Occult Primary; Mouth Cancer; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplastic Syndromes; Myelodysplastic / Myeloproliferative Diseases; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Adult Acute; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple (Cancer of the Bone-Marrow); Myeloproliferative Disorders, Chronic; Nasal cavity and paranasal sinus cancer;Nasopharyngeal carcinoma; Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Oral Cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer (Surface epithelial- stromal tumor); Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell; Paranasal sinus and nasal cavity cancer; Parathyroid cancer; Penile cancer; Pharyngeal cancer; Pheochromocytoma; Pineal astrocytoma; Pineal germinoma; Pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood; Pituitary adenoma; Plasma cell neoplasia / Multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, childhood; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary syndrome; Skin cancer (nonmelanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma - see Skin cancer (nonmelanoma); Squamous neck cancer with occult primary, metastatic; Stomach cancer; Supratentorial primitive neuroectodermal tumor, childhood; T-Cell lymphoma, cutaneous (Mycosis Fungoides and Sezary syndrome); Testicular cancer; Throat cancer; Thymoma, childhood; Thymoma and Thymic carcinoma; Thyroid cancer; Thyroid cancer, childhood; Transitional cell cancer of the renal pelvis and ureter; Trophoblastic tumor, gestational; Unknown primary site, carcinoma of, adult; Unknown primary site, cancer of, childhood; Ureter and renal pelvis, transitional cell cancer; Urethral cancer; Uterine cancer, endometrial; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, childhood; Vulvar cancer; Waldenstrom macroglobulinemia and Wilms tumor (kidney cancer).

[0333] As indicated above, the CAR molecules of the present disclosure and any cells, specifically, cells of the T lineage genetically engineered to express the CAR molecules provided by the present disclosure, may be applicable for any disorder that involves B cells.

[0334] Thus, in some specific embodiments, the methods of the present disclosure are applicable for treating proliferative disorder, specifically, any B cell malignancy.

[0335] In some embodiments, B cell malignancies include myeloma, specifically, multiple myeloma, as well as any type of lymphoma, including non-Hodgkin lymphomas as well as Hodgkin lymphomas.

[0336] More specifically, B-cell lymphomas make up most of the non-Hodgkin lymphomas (NHL). The methods of the present disclosure are therefore applicable for any type of lymphoma, specificallyaffecting B lymphocytes. The most common types of B-cell lymphomas applicable in the present disclosure, include, but are not limited to Diffuse large B-cell lymphoma (DLBCL), as well as to any subtype thereof (primary mediastinal B-cell lymphoma), Follicular lymphoma, diffuse large B-cell lymphoma, Chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), Marginal zone lymphomas, Extranodal marginal zone B-cell lymphoma, also known as mucosa-associated lymphoid tissue (MALT) lymphoma, Nodal marginal zone B-cell lymphoma, Splenic marginal zone B-cell lymphoma, Burkitt lymphoma, Lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), Hairy cell leukemia (also called Chronic Lymphocytic Leukemia), Primary central nervous system (CNS) lymphoma, and Primary intraocular lymphoma (lymphoma of the eye).

[0337] Still further, in some embodiments, B cell-mediated disorder applicable in the present disclosure may be Hodgkin's lymphoma. As used herein, Hodgkin's lymphoma (formerly known as Hodgkin's disease) is a cancer of the immune system that is marked by the presence of a multinucleated cell type called Reed-Sternberg cells. The two major types of Hodgkin's lymphoma include classical Hodgkin's lymphoma and nodular lymphocyte-predominant Hodgkin's lymphoma and are both treatable by the compositions and methods disclosed herein.

[0338] In some embodiments, the therapeutic methods of the present disclosure may be applicable for a B cell malignancy such as multiple myeloma (MM) and any related conditions.

[0339] Multiple myeloma (MM), also known as plasma cell myeloma and simple myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, bleeding, frequent infections, and anemia may occur. Complications may include amyloidosis. The cause of multiple myeloma is unknown. Risk factors include obesity, radiation exposure, family history, and certain chemicals. Multiple myeloma may develop from monoclonal gammopathy of undetermined significance that progresses to smoldering myeloma. The abnormal plasma cells produce abnormal antibodies, which can cause kidney problems and overly thick blood. The plasma cells can also form a mass in the bone marrow or soft tissue. When only one tumor is present, it is called a plasmacytoma; more than one is called multiple myeloma. Multiple myeloma is diagnosed based on blood or urine tests finding abnormal antibodies, bone marrow biopsy finding cancerous plasma cells, and medical imaging finding bone lesions. Another common finding is high blood calcium levels. Because many organs can be affected by myeloma, the symptoms and signs vary greatly. The common symptoms of multiple myeloma are indicatedas CRAB: C = calcium (elevated), R = renal failure, A = anemia, B = bone lesions. Myeloma has many other possible symptoms, including opportunistic infections (e.g., pneumonia) and weight loss. Multiple myeloma is considered treatable, but generally incurable. Monoclonal gammopathy of undetermined significance (MGUS) increases the risk of developing multiple myeloma. MGUS transforms to multiple myeloma at the rate of 1% to 2% per year, and almost all cases of multiple myeloma are preceded by MGUS.

[0340] Smoldering multiple myeloma increases the risk of developing multiple myeloma. Individuals diagnosed with this premalignant disorder develop multiple myeloma at a rate of 10% per year for the first 5 years, 3% per year for the next 5 years, and then 1% per year. Obesity is related to multiple myeloma with each increase of body mass index by five increasing the risk by 11%. Studies have reported a familial predisposition to myeloma. Hyperphosphorylation of a number of proteins, the paratarg proteins, a tendency that is inherited in an autosomal dominant manner, appears a common mechanism in these families. This tendency is more common in African-American with myeloma and may contribute to the higher rates of myeloma in this group. Rarely, Epstein-Barr virus (EBV) is associated with multiple myeloma, particularly in individuals who have an immunodeficiency due to e.g. HIV / AIDS, organ transplantation, or a chronic inflammatory condition such as rheumatoid arthritis. EBV-positive multiple myeloma is classified by the World Health Organization as one form of the Epstein-Barr virus-associated lymphoproliferative diseases and termed Epstein-Barr virus-associated plasma cell myeloma. EBV-positive disease is more common in the plasmacytoma rather than multiple myeloma form of plasma cell cancer. Tissues involved in EBV+ disease typically show foci of EBV+ cells with the appearance of rapidly proliferating immature or poorly differentiated plasma cells. The cells express products of EBV genes such as EBER1 and EBER2. While the EBV contributes to the development and / or progression of most Epstein-Barr virus-associated lymphoproliferative diseases, its role in multiple myeloma is not known. However, people who are EBV-positive with localized plasmacytoma(s) are more likely to progress to multiple myeloma compared to people with EBV-negative plasmacytoma(s). This suggest that EBV may have a role in the progression of plasmacytomas to systemic multiple myeloma. It should be thus understood that the methods of the present disclosure may be applicable for any type or stage of MM or any stage, background, source or type as disclosed herein.

[0341] In some particular embodiments, the methods of the invention are applicable to protein misfolding disorders or deposition disorders, for example proteopathies. The present disclosure provides insome embodiments thereof, therapeutic methods applicable for subjects suffering from any proteopathy, specifically, amyloidosis.

[0342] Proteopathy refers to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Often the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a gain of toxic function) or they can lose their normal function. The proteopathies (also known as proteinopathies, protein conformational disorders, or protein misfolding diseases) may further include such diseases as Creutzfeldt-Jakob disease and other prion diseases, Alzheimer's disease, Parkinson's disease, amyloidosis, multiple system atrophy, and a wide range of other disorders. In some specific embodiments, the proteopathy or protein-misfolding disorder may be Amyloidosis.

[0343] Thus, in some embodiments, the therapeutic methods of the present disclosure may be applicable for treating amyloidosis, and any related conditions.

[0344] Specifically, Amyloidosis is a group of diseases in which abnormal proteins, known as amyloid fibrils, build up in tissue. Symptoms depend on the type and are often variable. They may include diarrhea, weight loss, feeling tired, enlargement of the tongue, bleeding, numbness, feeling faint with standing, swelling of the legs, or enlargement of the spleen.

[0345] There are about 30 different types of amyloidosis, each due to a specific protein misfolding. Some are genetic while others are acquired. They are grouped into localized and systemic forms. The four most common types of systemic disease are light chain (AL), inflammation (AA), dialysis (Aβ₂M), and hereditary and old age (ATTR). It should be understood that the CAR molecules, nucleic acid molecules, cells, gene editing system / s, compositions and methods of the present disclosure, may be applicable for any type of amyloidosis, specifically, any type discussed in the present disclosure.

[0346] Additional examples of protein misfolding diseases relevant to the methods of the present disclosure may include any disorder that involves directly or indirectly GPRC5D expression, specifically, overexpression. Such disorders, include but are not limited to Alzheimer's disease, Cerebral β-amyloid angiopathy, Retinal ganglion cell degeneration in glaucoma, Prion diseases (multiple), Parkinson's disease and other synucleinopathies (multiple), Tauopathies (multiple) Frontotemporal lobar degeneration (FTLD), Amyotrophic lateral sclerosis (ALS), Huntington's disease and other trinucleotide repeat disorders (multiple), Familial British dementia, Familial Danish dementia, Hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHWA-I),Alexander disease, Pelizaeus-Merzbacher disease, Seipinopathies, Familial amyloidotic neuropathy, Senile systemic amyloidosis, Serpinopathies (multiple), AL (light chain) amyloidosis (primary systemic amyloidosis), AH (heavy chain) amyloidosis, AA (secondary) amyloidosis, Type II diabetes, Aortic medial amyloidosis, ApoAI amyloidosis, ApoAII amyloidosis, ApoAIV amyloidosis, Familial amyloidosis of the Finnish type (FAF), Lysozyme amyloidosis, Fibrinogen amyloidosis, Dialysis amyloidosis, Inclusion body myositis / myopathy, Cataracts, Retinitis pigmentosa with rhodopsin mutations, Medullary thyroid carcinoma, Cardiac atrial amyloidosis, Pituitary prolactinoma, Hereditary lattice corneal dystrophy, Cutaneous lichen amyloidosis, Mallory bodies, Corneal lactoferrin amyloidosis, Pulmonary alveolar proteinosis, Odontogenic (Pindborg) tumor amyloid, Seminal vesicle amyloid, Apolipoprotein C2 amyloidosis, Apolipoprotein C3 amyloidosis, Lect2 amyloidosis, Insulin amyloidosis, Galectin-7 amyloidosis (primary localized cutaneous amyloidosis), Corneodesmosin amyloidosis, Enfuvirtide amyloidosis, Cystic fibrosis, Sickle cell disease.

[0347] In yet some further embodiments, since amyloidosis is also classified as a deposition disorder, the methods of the invention may be also applicable for any deposition disorder. Deposition disorder, as used herein is any disorder involving or characterized by deposition of insoluble extracellular protein fragments, or any other metabolite, that have been rendered resistant to digestion, thereby interfering and impairing tissue or organ function and may lead to organ failure.

[0348] Still further, as discussed hereinabove, according to some embodiments, the methods of the invention may be used for the treatment of a patient suffering from any autoimmune disorder. In some specific embodiments, the methods of the invention may be used for treating an autoimmune disease such as, for example, but not limited to, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, fatty liver disease, Lymphocytic colitis, Ischaemic colitis, Diversion colitis, Behcet's syndrome, Indeterminate colitis, rheumatoid arthritis, Graft versus Host Disease (GvHD), Eaton-Lambert syndrome, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM) and NIDDM, multiple sclerosis (MS), myasthenia gravis, plexus disorders (e.g., acute brachial neuritis), polyglandular deficiency syndrome, primary biliary cirrhosis, scleroderma, thrombocytopenia, thyroiditis (e.g., Hashimoto's disease), Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, arthritis, alopecia areata, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, ankylosing spondylitis,pemphigus, bullous pemphigoid, dermatitis herpetiformis, psoriatic arthritis, reactive arthritis, and ankylosing spondylitis, inflammatory arthritis, including juvenile idiopathic arthritis, gout and pseudogout, as well as arthritis associated with colitis or psoriasis, Pernicious anemia, some types of myopathy, and Lyme disease (Late).

[0349] Another aspect of the present disclosure relates to a therapeutically effective amount of at least one of: (a), at least one nucleic acid molecule encoding at least one CAR molecule; (b), at least one cassette, vector vehicle or gene editing system comprising said nucleic acid molecule of (a); (c), at least one hematopoietic cell expressing said CAR, or a population of said cells; (d), at least one VHH nanobody specific for GPRC5D; and (e), a composition comprising at least one of (a), (b), (c) and (d), for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onset of pathological disorder in a mammalian subject. The CAR molecules used herein comprise the following components: (i), at least one target-binding domain, wherein at least one of the target binding domain specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody of the CAR molecule used herein is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising noncontiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule of the present disclosure comprises at least one Complementarity Determining Region 3(CDR3) comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant and derivatives thereof.

[0350] In some embodiments, the CAR molecule and / or the nucleic acid molecule and / or the genetically engineered cell, and / or the composition as used in the present aspect are any one of the CAR molecules, encoding nucleic acid molecules, cells thereof or any composition disclosed by the present disclosure as defined above, in connection with other aspects of the present disclosure. In numerous embodiments, the one or more CAR molecules of the present disclosure, as well as nucleic acid molecules, engineered cells expressing the one or more CARs (e.g., CAR T cells), and any compositions of the present invention may be administered by the disclosed methods and uses, as part of a combination therapy, i.e., in combination with one or more additional therapeutic agents. In some specific embodiments, a combination therapy in connection with the present disclosure comprises administration of two or more populations of engineered cells (e.g., T cells), wherein the engineered cells collectively express two or more CAR molecules directed against the same or different target molecules expressed by a single target cell type or by multiple target cell types. In some embodiments, the two or more CAR molecules are directed to the same target molecule (e.g., via different antigen-binding domains and / or different epitopes). In some embodiments, the two or more CAR molecules are directed to different target molecules coexpressed on the same target cell, thereby enabling multi-antigen targeting. In some embodiments, the two or more CAR molecules are directed to target molecules expressed on different target cell types, thereby enabling broader coverage of heterogeneous cell populations. Combination therapy may include administration of a single pharmaceutical dosage formulation comprising at least one composition of the invention and additional therapeutic agent(s); as well as administration of at least one composition of the invention and one or more additional agent(s) in its own separate pharmaceutical dosage formulation. Further, where separate dosage formulations are used, compositions of the invention and one or more additional agents can be administered concurrently or at separately staggered times, i.e., sequentially. Still further, the concurrent or separate administrations may be carried out by the same or different administration routes. Thus, in some further embodiments, the CAR molecules, nucleic acid sequences, cassettes, systems, and cells of the present disclosure may be applicable in boosting the immune response of a subject suffering from an immune-related disorder, specifically, any disorder involving B cells or B cell malignancies, and may be used in combined treatment with any therapeutic agent, for example, a chemotherapeutic agent.As used herein, a 'chemotherapeutic agent” or 'chemotherapeutic drug” (also termed chemotherapy) refers to a drug treatment intended for eliminating or destroying (killing) cancer cells or cells of any other proliferative disorder. The mechanism underlying the activity of some chemotherapeutic drugs is based on destroying rapidly dividing cells, as many cancer cells grow and multiply more rapidly than normal cells. As a result of their mode of activity, chemotherapeutic agents also harm cells that rapidly divide under normal circumstances, for example, bone marrow cells, digestive tract cells, and hair follicles. Damaging normal cells results in the common side effects of chemotherapy: myelosuppression (decreased production of blood cells, hence also immunosuppression), mucositis (inflammation of the lining of the digestive tract), and alopecia (hair loss).

[0351] Various types of chemotherapeutic drugs are available. A chemotherapeutic drug may be used alone or in combination with another chemotherapeutic drug or with other forms of cancer therapy, in addition to the CAR T of the present disclosure, for example, other biological drugs (antibodies, ligands, receptors), radiation therapy, or surgery.

[0352] Chemotherapeutic drugs affect cell division or DNA synthesis and function and can be generally classified into several groups, based on their structure or biological function. More specifically, chemotherapeutic agents that are classified as alkylating agents, anti-metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents such as DNA-alkylating agents, anti-tumor antibiotic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonist agents, protein kinase inhibitors, HMG-CoA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triplehelix DNAs, nucleic acids aptamers, and molecularly-modified viral, bacterial or exotoxic agents. It should be appreciated that any combination therapy disclosed herein, using any of the indicated compounds with the CAR T, DNA cassettes, systems and cells of the present disclosure, together with any of the therapeutic agents discussed above, is encompassed by the present disclosure. As described herein above, the invention provides in some aspects thereof therapeutic and prophylactic methods. It is to be understood that the terms "treat”, “treating”, “treatment" or forms thereof, as used herein, mean preventing, ameliorating or delaying the onset of one or more clinical indications of disease activity in a subject having a pathologic disorder. Treatment refers to therapeutic treatment. Those in need of treatment are subjects suffering from pathologic disorder / s. Specifically, providing a "preventive treatment" (to prevent) or a "prophylactictreatment" is acting in a protective manner, to defend against or prevent something, especially a condition or disease.

[0353] The term “treatment or prevention,” as used herein, refers to the complete range of therapeutically positive effects of administering to a subject, including inhibition, reduction of, alleviation of, and relief from, an immune-related condition and illness, immune-related symptoms or undesired side effects or immune-related disorders. More specifically, treatment or prevention of relapse or recurrence of the disease includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms, and / or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying metabolic causes of symptoms. It should be appreciated that the terms "inhibition," "moderation," “reduction," "decrease," or "attenuation," as referred to herein, relate to the retardation, restraining, or reduction of a process by any one of about 1% to 99.9%, specifically, about 1% to about 5%, about 5% to 10%, about 10% to 15%, about 15% to 20%, about 20% to 25%, about 25% to 30%, about 30% to 35%, about 35% to 40%, about 40% to 45%, about 45% to 50%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 75% to 80%, about 80% to 85%, about 85% to 90%, about 90% to 95%, about 95% to 99%, or about 99% to 99.9%, 100% or more.

[0354] With regards to the above, it is to be understood that, where provided, percentage values such as, for example, 10%, 50%, 120%, 500%, etc., are interchangeable with "fold change" values, i.e., 0.1, 0.5, 1.2, 5, etc., respectively. The term "amelioration" as referred to herein, relates to a decrease in the symptoms, and improvement in a subject's condition brought about by the compositions and methods according to the invention, wherein said improvement may be manifested in the forms of inhibition of pathologic processes associated with the immune-related disorders described herein, a significant reduction in their magnitude, or an improvement in a diseased subject physiological state.

[0355] The term "inhibit" and all variations of this term is intended to encompass the restriction or prohibition of the progress and exacerbation of pathologic symptoms or a pathologic process progress, said pathologic process symptoms or process are associated with. The term "eliminate" relates to the substantial eradication or removal of the pathologic symptoms and possibly pathologic etiology, optionally, according to the methods of the invention described herein. The terms "delay", "delaying the onset", "retard" and all variations thereof are intended to encompassthe slowing of the progress and / or exacerbation of a disorder associated with the immune-related disorders and their symptoms slowing their progress, further exacerbation or development, so as to appear later than in the absence of the treatment according to the invention.

[0356] As indicated above, the methods and compositions provided by the present invention may be used for the treatment of a “pathological disorder,” specifically, immune-related disorders as specified by the invention, which refers to a condition in which there is a disturbance of normal functioning, any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with that person. It should be noted that the terms "disease," "disorder," "condition," and "illness" are used interchangeably herein. It should be appreciated that any of the methods and compositions described by the invention may be applicable for treating and / or ameliorating any of the disorders disclosed herein or any condition associated therewith.

[0357] It is understood that the interchangeably used terms "associated", “linked” and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, “disease”, “disorder”, “condition”, “pathology” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.

[0358] The present invention relates to the treatment of subjects or patients, in need thereof. By “patient” "subject" or “subject in need” it is meant any organism who may be affected by the above-mentioned conditions, and to whom the therapeutic and prophylactic methods herein described are desired, including any vertebrate, specifically mammals such as humans, domestic and nondomestic mammals such as canine and feline subjects, bovine, simian, equine and rodents, specifically, murine subjects. More specifically, the methods of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, livestock, equine, canine, and feline subjects, most specifically humans. It should be appreciated that the invention may be applicable for any vertebrates, for example, avian subjects, and fish.

[0359] The compositions of the invention may comprise an effective amount of the nucleic acid molecules, and / or cassette thereof, or of any vector thereof, or of any cell comprising the same, or any CAR molecule as described by the invention. The term "effective amount” or" therapeutically effective amount" relates to the amount of an active agent present in a composition, specifically, the nucleic acid molecules, vectors, and / or cassette and / or the genetically engineered cells of the invention as described herein that is needed to provide a desired level of active agent in the bloodstream or at the site of action in an individual (e.g., the thymus or bone marrow) to be treated to give an anticipated physiological response when such composition is administered. The precise amount will depend upon numerous factors, e.g., the active agent, the activity of the composition, the delivery device employed, the physical characteristics of the composition, intended patient use (i.e., the number of doses administered per day), patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein. An “effective amount" of the genetically engineered CAR cells disclosed herein, or any nucleic acid molecule / s of the invention or any cassette of the invention and gene editing systems thereof, or any genetically engineered cell, can be administered in one administration, or through multiple administrations of an amount that totals an effective amount, preferably within a 24-hour period. It can be determined using standard clinical procedures for determining appropriate amounts and timing of administration. It is understood that the "effective amount" can be the result of empirical and / or individualized (case-by-case) determination on the part of the treating healthcare professional and / or individual. Still further, administration and doses are determined by good medical practice of the attending physician and may depend on the age, sex, weight, and general condition of the subject in need.

[0360] It should be appreciated that the effective amount as discussed herein is applicable for each and every embodiment of each and every aspect of the present disclosure, specifically, for any of the CAR molecule, CAR cells expressing the CAR molecule of the present disclosure, and / or any nucleic acid sequence encoding the disclosed CAR molecule, or any construct comprising the same, any dosage forms thereof, dosage unit forms thereof, compositions, kits, uses and methods thereof.

[0361] Another aspect of the present application relates to a method for targeted activation of a hematopoietic cell against a target cell expressing the GPRC5D protein and / or a tissue comprising the target cell. The method comprising the step of contacting the hematopoietic cell with an effective amount of at least one of: (A), at least one nucleic acid molecule encoding at least one CAR molecule; (B), at least one cassette, vector vehicle or gene editing system comprising said nucleic acid molecule of (A); and / or (C), a composition comprising at least one of (A) and (B).The CAR molecules encoded by the nucleic acid molecule used in the disclosed methods comprise the following components: (i), at least one target-binding domain, wherein at least one of the target binding domain specifically recognizes and binds GPRC5D, or any fragments thereof; (ii), at least one hinge region; (iii), at least one transmembrane domain; and (iv), at least one intracellular signal transduction domain. The at least one of the target binding domains comprises at least one VHH nanobody, or any antigen binding fragment thereof. The VHH nanobody of the disclosed CAR molecules is characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the CAR of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule of the present disclosure comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

[0362] As described above, the method of the present disclosure is for targeted activation of a hematopoietic cell. " Targeted activation" refers to the deliberate and specific triggering of effector cells activation (i.e. immune cells), when they encounter a target cell or tissue expressing a designated target protein. In the case of the present disclosure, the designated target protein is GPRC5D. This approach is designed to ensure that T-cell activation occurs only in the presence of specific antigens or markers (e.g. GPRC5D), minimizing off-target effects and enhancing therapeutic precision.

[0363] The method of the invention involves the step of contacting the nucleic acid molecule and / or cassette provided by the methods of the invention with the hematopoietic cells. In someembodiments, the step of contacting the hematopoietic cell with the at least one nucleic acid cassette, is performed in vivo, in vitro or ex vivo.

[0364] The term "contacting" means to bring, put, incubate or mix together. More specifically, in the context of the present invention, the term "contacting" includes all measures or steps, which allow the positioning of the nucleic acid cassettes of the present invention such that they are in direct or indirect contact with the hematopoietic cell / s.

[0365] To induce DNA integration either in vitro or in vivo, the nucleic acid cassette of the invention may be provided to and / or contacted with the target cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which may be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The nucleic acid cassette may be provided to the target cells one or more times, e.g. one time, twice, three times, or more than three times, and the cells allowed to incubate with the nucleic acid cassette for some amount of time following each contacting event e.g. 16-24 hours. Still further, in some embodiments, the contacting step of the hematopoietic cell / s (e.g., of T lineage) with the at least one nucleic acid cassette, is performed in vivo in a subject suffering from at least one pathological disorder. According to such embodiments, the method further comprises administering to the subject an effective amount of the nucleic acid cassette, a vector comprising the nucleic acid cassette, a gene editing system comprising the nucleic acid molecule, or any composition thereof.

[0366] In yet some alternative embodiments of the disclosed methods, the step of contacting the hematopoietic cell (e.g., of T lineage) with the at least one nucleic acid cassette is performed in vitro or ex vivo to obtain genetically engineered cells of the hematopoietic lineage, or a population of these cells.

[0367] In other embodiments, the method is applicable for targeted activation of a hematopoietic cell against a target cell expressing the GPRC5D protein and / or a tissue comprising the target cell in a mammalian subject suffering from a pathological disorder. The method further comprises the step of introducing the genetically engineered cells to the subject.

[0368] In some embodiments, the hematopoietic cells used for engineering are of autologous or allogeneic source.In some other embodiments, the pathological disorder, from which the mammalian subject is suffering, is at least one disorder associated with overexpression of the GPRC5D protein.

[0369] In some further embodiments, the disorder associated with overexpression of the GPRC5D protein is at least one immune-related disorder.

[0370] In some additional embodiments, the immune-related disorder is at least one of: at least one proliferative disorder, at least one deposition disorder and / or at least one autoimmune disease. In some embodiments, the method of the present disclosure utilizes a CAR molecule and / or a nucleic acid molecule, and / or a cell and / or a composition as defined above.

[0371] Another aspect of the present disclosure relates to an anti-GPRC5D VHH nanobody characterized by at least one of the following features. In some embodiments (a), the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D. The GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140. In yet some additional or alternative embodiments (b), the VHH nanobody specifically binds a conformational epitope on GPRC5D. In more specific embodiments, the epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D. More specifically, the residues refer to GPRC5D that comprises the amino acid sequence as denoted by SEQ ID NO: 140. In some specific embodiments, the epitope recognized by the VHH nanobody of the present disclosure comprises the amino acid sequence as denoted by SEQ ID NO: 144. Still further, in some additional or alternative embodiments (c), the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering. In yet some additional or alternative embodiments (d), the VHH nanobody of the CAR molecule of the present disclosure comprising at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variants or derivatives thereof.

[0372] In some embodiments, the disclosed VHH nanobodies comprise the amino acid sequence as denoted by any one of SEQ ID NOs: 1, 2, 3, or 4. In yet some further embodiments, the VHH nanobodies may be encoded by a nucleic acid sequence as denoted by any one of SEQ ID NOs: 51, 52, 53, or 54.

[0373] In some embodiments, the nanobody of the present disclosure (e.g., B 1) is incorporated into and / or used as the antigen-binding domain of any chimeric antigen receptor (CAR) format, including CARs of any generation (e.g., first-, second-, third-, fourth-, and / or fifth-generation CARs). In some embodiments, the nanobody is incorporated into a universal CAR system, including adapter-based and / or switchable CAR platforms, wherein target recognition is mediated by an adaptor molecule that bridges the universal CAR to a target antigen. In some embodiments, the nanobody is included in, fused to, or otherwise provided as part of an adaptor molecule configured to direct a universal CAR-expressing cell to a target antigen.

[0374] In some embodiments, the nanobody of the present disclosure is incorporated into any bi-valent or multi-valent molecule, including multimeric binding constructs and / or antibodies. By way of example, the nanobody may be used to generate bispecific or multispecific antibodies, and / or incorporated into bi-specific or multispecific CAR molecules (including tandem CARs) directed against the same target or different targets, thereby enabling dual targeting, improved coverage of heterogeneous tumors, and / or enhanced selectivity (e.g., logic-gated targeting) depending on the configuration.

[0375] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0376] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Thus, as used herein the term "about" refers to ± 10 %.

[0377] The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and / or steps, and / or parts, but only if the additional ingredients and / or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly,the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging / ranges between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0378] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0379] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0380] Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[0381] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.EXAMPLES

[0382] Experimental procedures

[0383] Reagents

[0384] The antibodies used in all experiments are listed in Table 1.

[0385] Table 1: Antibodies and reagents used for flow cytometer

[0386] Antibody / Reagent Catalog Number Manufacturer Percent of transduction and CART characterization MonoRab™ Rabbit Anti-Camelid VHH

[0387] Cocktail [[iFluor 488] A02021-200 Genescript G4S linker AF488 CST-50515S Cell Signaling SYTOX Blue Dead Cell Stain S34857 Invitrogen CD45RA-ECD IM2711U BC Beckman coulter CD62L-AF700 304820 Biolegend CD28-APC BLG-377609 Biolegend CD4 -APC-Cy7 300518 Biolegend CD8-Krome Orange B00067 Beckman coulter CD3-BV780 417-0038-42 eBioScience CART and Plasma cell count

[0388] MonoRab™ Rabbit Anti-Camelid VHH

[0389] Cocktail [[iFluor 488] A02021-200 Genescript CD138-Pacific Blue B37788 Beckman coulter CD3-Krome orange B00068 Beckman coulter CART cells activation

[0390] MonoRab™ Rabbit Anti-Camelid VHH

[0391] Cocktail [iFluor 488] A02021-200 Genescript 4-1BB-ECD B76262 Beckman coulter CD25-APC750 B13979 Beckman coulter Caspasc-3 killing assay

[0392] Cleaved Capspase-3 V450 560627 BD horizon Cell Trace Far-red BC-B37788 Beckman coulter VHH nanobody binding

[0393] MonoRab™ Rabbit Anti-Camelid VHH

[0394] Cocktail [[iFluor 488] A02021-200 Genescript MonoRab™ Rabbit Anti-Camelid VHH

[0395] Cocktail [iFluor 647] A02019-200 Genescript Alpaca VHH Isotype Control Antibody FWK30510-1 Invitrogen Human GPRC5D Allophycocyanin MAb

[0396] (Clone 57196) FAB6300A R& D Mouse IgG2B Allophycocyanin Isotype

[0397]

[0398] Control IC0041A R& DViral vectors

[0399] CARB20-LV generation:

[0400] CARB28 GOI is inserted into a lentiviral plasmid backbone driven by human-EFl-a promoter. The lentiviral plasmid is derived from the human immunodeficiency virus (HIV), belongs to the retroviridae family of viruses.

[0401] Plasmids transiently co-transfer into HEK cells for lentiviral vector packaging with additional three plasmids suitable for the 3rd generation 4 packaging system. Supernatant from transfected HEK cells were harvest, underwent a multi-step purification process.

[0402] CARB20-RV generation:

[0403] This retroviral vector is the pMSGVl vector previously described and used in multiple clinical trials to modify human T-cells to express anti-tumor TCRs or CARs (Morgan 2006, Robbins 2015, Brudno 2018). It is a derivative of the murine stem cell virus (MSCV)-based splice-gag vector (pMSGV) which uses a MSCV long terminal repeat to drive expression of the chimeric receptor. The virus used for transduction was produced by PG 13 mouse fibroblast cells which had been previously transduced with retroviral supernatant of Pheonix-ECO cells which had been transfected by the plasmid of insert (MSGV1).

[0404] Procedures

[0405] CART cell generation

[0406] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy or patient (multiple myeloma or light chain amyloidosis) leukapheresis products by density gradient centrifugation (Cytvia premium grade) and underwent T cell stimulation by anti-CD3 soluble antibody (OKT3, Miltenyi) and 300IU / mL IL-2 (Proleukin, Novartis) or by TransAct and lOng / mL of IL-7 and IL-15 in AIM-V CTS medium (Gibco) supplemented with 1-5% AB serum (Grifols Bio Supplies Inc.) with or without ImM GlutaMAX (Gibco) or with X-Vivo media (Lonza) without any supplements for two days. On day 2, stimulated cells were transduced with retrovirus either by vectofusin directly or by retroviral supernatant by spinoculation (1000g, 10 mins) on RetroNectin (10µg / mL, Takara Bio) - pre-coated non-tissue culture six-well plates on which retroviral supernatant was previously precipitated by centrifugation (2000g, 32°C, 2 hours). Alternatively, stimulated cells at day +2 are transduced with lentivirus at multiplicity of infection (MOI) of 1-20either alone or by using LentiBoost enhancer. On day 3, transduced cells were suspended and expanded with Flasks or GRex devices (Wilson Wolf) for 3-10 days.

[0407] Flow cytometry

[0408] Percent of transduction, CART and plasma cell count, and CAR T cell activation assays were performed as follows: IxlO5cells or the entire co-cultures cellular fraction were washed twice with PBS (Biological Industries) + 2% FBS (heat inactivated Gibco) (FACS buffer) and then incubated with antibody mixtures as detailed in Table 1, for 20 minutes on ice, washed twice with FACS buffer, and resuspended with the same buffer for acquiring by flow cytometer. For percent of transduction assay, Sytox (Invitrogen) was added before sample acquisition as per manufacturer’s instructions. Samples are acquired on a Dx-Flex cytometer (Beckman Coulter), and the data is processed with Kaluza Analysis software 2.1.

[0409] For VHH nanobody binding assay, IxlO6cells were washed twice with ice-cold PBS+0.3% protease free BSA (BSA buffer) and incubated with 2µg or 10µg Bl, H3, D5 or A4 nanobodies for Ih at room temperature. Cells were then washed twice with BSA buffer and incubated with 1:1000 dilution of secondary antibody for Ih at room temperature in the dark. Cells were then washed twice with BSA buffer and resuspended in BSA buffer for FACS analysis.

[0410] Caspase-3 killing assay

[0411] CD 138 positive cells were isolated (by using EasySep human CD 138 positive Selection kit II, STEMCELL) from BM mononuclear cells (BM-MNCs), purified from MM patients' bone marrow aspiration. Then CD 138+ cells were labeled by Far-red Cell-Trace (Invitrogen) and co-cultured either with CARB28, CARB28-IL7, HBI0101 or with their control CART cells at an E: T ratio of 1:4 s for 1-2 days. Cellular fractions were washed twice in FACS buffer and stained with a- VHH, a-41BB and a-CD25 for CART cell activation assay, or fixed and permeabilized with Fix / Perm solution (BD), and then intracellularly stained with anti-active (cleaved) caspase-3 antibody (BD Pharmingen). The percent of cleaved caspase-3 in cell-trace-i- gated cells was determined using Dx-Flex flow cytometer (Beckman Coulter).

[0412] Cell supernatants post 1-2 days of co-incubation were collected and the secretion of IFN-y and IL7 was quantified by ELISA (R& D) according to manufacturer's instructions.

[0413] CART cell cytotoxicity assay

[0414] Assessment of CART cytotoxicity was performed by co-culturing CART cells with GPRC5D-expressing human myeloma NCI-H929, MM1. S and RPMI-8226 MM cell line or with GPRC5D-negative K562 CML cell line (ATCC) genetically modified to express the firefly luciferase. Theco-culturing was with an effector to target (E: T) ratio of 1:4 for 3-4 days and then re-challenged with additional 10,000 target cells every 3-4 days. Co-cultured cells were analyzed every 3-4 days until days 14-20. The traceability of remaining target cells was performed by two methods:

[0415] 1. Bioluminescence (BLI) measurement (GloMax, Promega) following addition of luciferin (Vivo Gio, Promega) to the cell co-culture, and an incubation of 10 minutes at 37°C. 2. CART and multiple myeloma cell count by staining the cells with a-CD3, a-VHH and a- CD138 and acquiring lOOuL of resuspended cells for 30 minutes by Dx-Flex flow cytometer.

[0416] The resulting BLI or cell count was normalized to the BLI and cell count detected by CAR negative control cells (with antigen-binding domain against egg white lysozyme as denoted by SEQ ID NO: 55 and encoded by SEQ ID NO: 56). CARB8a, CARB28, and CARB8a28 were normalized to NC8a, NC28, and P-NC28, respectively. HBI0101 was normalized to CARNC8a, and CARB8a-IL7, CARB28-IL7, and CARB8a28-IL7 were normalized to CARNC28-IL7 (isotype-matched CAR that constitutively secretes IL7).

[0417] OFF target binding interactions (Retrogenix Cell Microarray Technology)

[0418] Pre-screen

[0419] Slides were spotted with expression vectors encoding both ZsGreenl and human GPRC5D, PVR, CD244, TNFSF8, TNFSF4, ICOSLG, CD86 and EGFR, and used to reverse-transfect HEK293 cells. Cells were then fixed. Next, 2 x 106, 4 x 106or 6 x 106cells / slide of fluorescently test CAR T cells or untransduced control T cells labelled with red cell dye were added to the above slides. Binding to target-expressing cells and untransfected cells was assessed by fluorescence imaging.

[0420] Library screen

[0421] 6108 expression vectors, encoding both ZsGreenl and a full-length human plasma membrane protein, secreted or a cell surface-tethered human secreted protein, plus a further 403 human heterodimers were individually arrayed in duplicate across cell microarray slides (‘slide- sets’). An expression vector (pIRES -hEGFR-IRES -ZsGreenl) was spotted in quadruplicate on every slide and was used to ensure that a minimal threshold of transfection efficiency had been achieved or exceeded on every slide. HEK293 cells were used for reverse transfection / expression. Fluorescently labelled CARB28-transduced with lentivirus (LV) cells were added to each slide after cell fixation giving a final concentration of 4 x 106cells / slide. Detection of binding was performed by fluorescence imaging as before. The test CART cells were screened against 2replicate slide-sets. Fluorescent images were analyzed and quantitated (for transfection) using ImageQuant software (GE Healthcare, Version 8.2).

[0422] A protein interaction is defined as a duplicate spot showing a raised signal compared to background levels. This is achieved by visual inspection using the images gridded on the ImageQuant software. Interactions were classified as ‘strong, medium, weak or very weak’, depending on the intensity of the duplicate spots.

[0423] The term ‘significant’ in the context of this EXAMPLE does not refer to statistical significance. A significant interaction is defined as a signal of weak intensity or greater.

[0424] Confirmation screen

[0425] To confirm the interactions and assess specificity, vectors encoding all interactions identified in the Library screen, plus a control vector encoding EGFR, were arrayed and expressed in HEK293 cells on new slides. Confirmation screens and analyses were carried out as for Library screening, except that identical slides were treated with 4 x 106cells / slide of CARB28-LV or 4 x 106cells / slide of NT (untransduced control T cells) (n=2 slides per treatment). Binding to targetexpressing cells and untransfected cells was again assessed by fluorescence imaging.

[0426] Testing the potency of CARB28 in-vivo

[0427] Subcutaneous model

[0428] Subcutaneous injection of the MM cell line was designed to model poor-prognosis extramedullary myeloma presentation. Accordingly, a minimal effective CAR T-cell dose of about 5xl06was administered based on prior HBI0101 murine study expertise. In the initial study, mice received subcutaneous injections of NCI-H929 or MM IS cells followed by intravenous CAR T-cell administration after 5 or 14 days, respectively. Negative control mic...

Claims

1. CLAIMS:

1. A chimeric antigen receptor (CAR) molecule comprising:(i) at least one target-binding domain, wherein the at least one of said target binding domain specifically recognizes and binds G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D), or any fragments thereof;(ii) at least one hinge region;(iii) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;and wherein the at least one of said target binding domain comprises at least one variable heavy chain only (VHH) nanobody, or any antigen binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one Complementarity Determining Region 3 (CDR3) comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

2. The CAR molecule according to claim 1, wherein said VHH nanobody comprises CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 5, CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 6, and CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 7.

3. The CAR molecule according to any one of claims 1 and 2, wherein said at least one hinge region is derived from at least one of: a co- stimulatory receptor molecule, a co-receptor molecule and at least one immunoglobulin molecule.

4. The CAR molecule according to any one of claims 1 to 3, wherein said at least one hinge region is derived from a co-stimulatory receptor molecule, being the Cluster of Differentiation 28 (CD28) protein, or a co-receptor molecule, being the Cluster of Differentiation 8a (CD8a) protein, or any combination thereof.

5. The CAR molecule according to any one of claims 1 to 4, wherein:(i) said hinge region is derived from CD28 and comprises the amino acid sequence as denoted by SEQ ID NO: 73, and / or any variants or derivatives thereof; or(ii) said hinge region is derived from CD8a and comprises the amino acid sequence as denoted by SEQ ID NO: 69, and / or any variants or derivatives thereof.

6. The CAR molecule according to any one of claims 1 to 5, wherein said transmembrane domain is derived from at least one of: a co-stimulatory receptor molecule and / or at least one co-receptor molecule.

7. The CAR molecule according to any one of claims 1 to 6, wherein said at least one transmembrane domain is derived from a co-stimulatory receptor molecule being the CD28 protein, or a co-receptor molecule being the CD8a protein, or any combination thereof.

8. The CAR molecule according to claim 7, wherein:(i) said at least one transmembrane domain is derived from the CD28 protein and comprises the amino acid sequence as denoted by SEQ ID NO: 75; and / or any variants or derivatives thereof; or(ii) said at least one transmembrane domain is derived from the CD8a protein and comprises the amino acid sequence as denoted by SEQ ID NO: 71, and / or any variants or derivatives thereof.

9. The CAR molecule according to any one of claims 1 to 8, wherein at least one of said intracellular signal transduction domains is an intracellular T cell signal transduction domain.

10. The CAR molecule according to claim 9, wherein said at least one intracellular T cell signal transduction domain comprises at least one domain of a tumor necrosis factor (TNF) receptor family member, and optionally, at least one domain of a T cell receptor (TCR) molecule.

11. The CAR molecule according to claim 10, wherein at least one of: said at least one TNF receptor family member is 4-1BB, and wherein said TCR molecule comprises a CD3 zeta chain.

12. The CAR molecule according to any one of claims 9 to 11, wherein said at least one intracellular T cell signal transduction domain comprises the amino acid sequence as denoted by SEQ ID NO: 77 and / or by SEQ ID NO: 79, or any derivatives or variants thereof.

13. The CAR molecule according to any one of claims 1 to 12, wherein said CAR molecule comprises the amino acid sequence as denoted by at least one of: SEQ ID NOs: 110, 104, 106, 108, 117, 119, 122, 124, 126, 130, 132, or any variants or derivatives thereof.

14. The CAR molecule according to any one of claims 1 to 13, wherein said CAR molecule is designated CARB28, and comprises the amino acid sequence as denoted by SEQ ID NO: 110.

15. A nucleic acid molecule comprising at least one nucleic acid sequence encoding at least one CAR molecule, or any cassette, vector comprising said nucleic acid molecule, said CAR comprising:(i) at least one target-binding domain, wherein at least one of said target-binding domains specifically recognizes and binds GPRC5D, or any fragments thereof;(ii) at least one hinge region;(iii) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;wherein at least one of said target-binding domains comprises at least one VHH nanobody, or any antigen-binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivatives thereof.

16. The nucleic acid molecule according to claim 15, wherein said CAR molecule is as defined by any one of claims 2 to 14.

17. The nucleic acid molecule according to any one of claims 15 and 16, wherein said CAR molecule comprises the amino acid sequence as denoted by at least one of SEQ ID NO: 110, 104, 106, 108, 117, 119, 122, 124, 126, 130, 132, or any variants or derivatives thereof.

18. The nucleic acid molecule according to any one of claims 15 to 17, wherein said CAR molecule is designated CARB28, and comprises the amino acid sequence as denoted by SEQ ID NO: 110, said nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 111.

19. The nucleic acid molecule according to claim 18, further comprising at least one nucleic acid sequence encoding and / or controlling at least one immunomodulatory agent.

20. The nucleic acid molecule according to claim 19, wherein said immunomodulatory agent comprises at least one of: a cytokine, a chemokine, an antibody, a CAR molecule, a Toll-Like Receptor (TLR) modulator, and an immune checkpoint inhibitor.

21. The nucleic acid molecule according to any one of claims 19 and 20, wherein said immunomodulatory agent comprises at least one cytokine that enhances T cell cytotoxicity.

22. The nucleic acid molecule according to any one of claims 20 and 21, wherein said at least one cytokine is a member of the γc Cytokine Family.

23. The nucleic acid molecule according to any one of claims 20 to 22, wherein said at least one cytokine is Interleukin-7 (IL-7).

24. The nucleic acid molecule according to any one of claims 19 to 23, wherein said nucleic acid sequence encoding and / or controlling at least one immunomodulatory agent is under the control of at least one regulatory element directing the expression of said at least one immunomodulatory agent to activated T cells.

25. The nucleic acid molecule according to claim 24, wherein said regulatory element comprises at least one responsive element for at least one transcription factor involved in immune regulation.

26. The nucleic acid molecule according to any one of claims 15 to 25, wherein said nucleic acid molecule is flanked on at least one of the 5' and 3' ends thereof by at least one of: (i) homology arms, for integration to a genomic target site by homologous recombination; and / or (ii) recognition sites for a site-specific nuclease, a site-specific integrase or a site-specific recombinase.

27. A genetically engineered cell expressing at least one CAR molecule, or a population of cells comprising at least one said genetically engineered cell, wherein the genetically engineered cell is at least one hematopoietic cell, said CAR comprising:(i) at least one target-binding domain, wherein at least one of said target-binding domains specifically recognizes and binds GPRC5D, or any fragments thereof;(ii) at least one hinge region;(iii) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;wherein at least one of said target-binding domains comprises at least one VHH nanobody, or any antigen-binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivative thereof.

28. The genetically engineered cell according to claim 27, wherein said CAR molecule is as defined by any one of claims 1 to 14, and / or wherein said engineered cell comprises or expresses at least one nucleic acid molecule as defined by any one of claims 15 to 26.

29. The genetically engineered cell according to any one of claims 27 to 28, wherein said hematopoietic cell is a lymphocyte.

30. The genetically engineered cell according to claim 29, wherein said lymphocyte is a cell of the T lineage.

31. The genetically engineered cell according to claim 30, wherein said cell of the T lineage is a T cell.

32. A composition comprising at least one of: at least one CAR molecule, any nucleic acid molecule comprising at least one nucleic acid sequence encoding at least one CAR molecule, or any cassette, vector, vehicle or gene editing system comprising said nucleic acid molecule, and / or any genetically engineered cell expressing said CAR molecule or a population of cells comprising at least one of said genetically engineered hematopoietic cell, and / or any combinations thereof, said CAR comprising:(i) at least one target-binding domain, wherein at least one of said target-binding domains specifically recognizes and binds GPRC5D, or any fragments thereof;(ii) at least one hinge region;(i) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;wherein at least one of said target-binding domains comprises at least one VHH nanobody, or any antigen-binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102, of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivative thereof; the composition further comprising at least one of pharmaceutically acceptable carrier / s, diluent / s, excipient / s, and additive / s.

33. The composition according to claim 32, wherein said CAR molecule is as defined in any one of claims 1 to 14, said nucleic acid molecule is as defined in any one of claims 15 to 26, and said cell is as defined in any one of claims 27 to 31.

34. A method for treating, preventing, ameliorating, inhibiting or delaying the onset of a pathological disorder in a subject, said method comprising the step of administering to said subject an effective amount of at least one of:(A) at least one nucleic acid molecule encoding at least one CAR molecule;(B) at least one cassette, vector, vehicle, or gene editing system comprising said nucleic acid molecule of (A);(C) at least one genetically engineered cell expressing said CAR, or a population of said cells; (D) at least one VHH nanobody specific for GPRC5D; and(E) a composition comprising at least one of (A), (B), (C) and (D);said CAR comprising:(i) at least one target-binding domain, wherein at least one of said target-binding domains specifically recognizes and binds GPRC5D or any fragments thereof;(ii) at least one hinge region;(iii) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;wherein at least one of said target-binding domains comprises at least one VHH nanobody, or any antigen-binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102 (according to Chothia numbering), of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivative thereof.

35. The method according to claim 34, wherein said VHH nanobody comprises CDR1 comprising the amino acid sequence as denoted by SEQ ID NO: 5, CDR2 comprising the amino acid sequence as denoted by SEQ ID NO: 6, and CDR3 comprising the amino acid sequence as denoted by SEQ ID NO: 7.

36. The method according to any one of claims 34 and 35, wherein said at least one hinge region is derived from at least one of: a co- stimulatory receptor molecule, a co-receptor molecule and at least one immunoglobulin molecule.

37. The method according to any one of claims 34 to 36, wherein said at least one hinge region is derived from a co-stimulatory receptor molecule being the CD28 protein, or a co-receptor molecule being the CD 8 a protein, or any combination thereof.

38. The method according to any one of claims 34 to 37, wherein:(i) said hinge region is derived from CD28, and comprises the amino acid sequence as denoted by SEQ ID NO: 73, and / or any variants or derivatives thereof; or(ii) said hinge region is derived from CD8a, and comprises the amino acid sequence as denoted by SEQ ID NO: 69, and / or any variants or derivatives thereof.

39. The method according to any one of claims 34 to 38, wherein said transmembrane domain is derived from at least one of: a co-stimulatory receptor molecule and / or at least one co-receptor molecule.

40. The method according to any one of claims 34 to 39, wherein said at least one transmembrane domain is derived from a co-stimulatory receptor being the CD28 protein, or a co-receptor molecule being the CD 8 a protein, or any combination thereof.

41. The method according to claim 40, wherein:(i) said at least one transmembrane domain is derived from the CD28 protein and comprises the amino acid sequence as denoted by SEQ ID NO: 75; and / or any variants or derivatives thereof; or(ii) said at least one transmembrane domain is derived from the CD8a protein and comprises SEQ ID NO: 71, and / or any variants or derivatives thereof.

42. The method according to any one of claims 34 to 41, wherein said at least one intracellular signal transduction domain is a T cell signal transduction domain.

43. The method according to claim 42, wherein said T cell signal transduction domain comprises at least one domain of TNF receptor family member, and optionally, at least one domain of a TCR molecule.

44. The method according to claim 43, wherein at least one of said at least one TNF receptor family member comprises 4-1BB, and wherein said TCR molecule comprises a CD3 zeta chain.

45. The method according to any one of claims 34 to 44, wherein said at least one intracellular signal transduction domain comprises the amino acid sequence as denoted by SEQ ID NO: 77 and / or by SEQ ID NO: 79, or any derivatives or variants thereof.

46. The method according to any one of claims 34 to 45, wherein said CAR molecule comprises the amino acid sequence as denoted by at least one of SEQ ID NOs: 110, 104, 106, 108, 117, 119, 122, 124, 126, 130, 132, and any variants or derivatives thereof.

47. The method according to any one of claims 34 to 46, wherein said CAR molecule is designated CARB28, and comprises the amino acid sequence as denoted by SEQ ID NO: 110, said nucleic acid molecule comprising the nucleic acid sequence as denoted by SEQ ID NO: 111.

48. The method according to any one of claims 34 to 47, wherein said nucleic acid molecule is as defined in any one of claims 15 to 26, said cell is as defined in any one of claims 27 to 31, and said composition is as defined in any one of claims 32 to 33.

49. The method according to any one of claims 34 to 48, wherein said subject is administered with at least one genetically engineered cell expressing said CAR molecule, and / or a cell genetically engineered with said at least one nucleic acid cassette or any vector or vehicle comprising said cassette, or with a population of said cells, or any composition thereof.

50. The method according to claim 49, wherein said at least one hematopoietic cell is of an autologous or of an allogeneic source.

51. The method according to any one of claims 34 to 50, wherein said subject is administered with a nucleic acid vector comprising at least one cassette comprising at least one nucleic acid sequence encoding said CAR molecule, wherein said vector is any one of a viral vector, a non-viral vector, and a naked DNA vector.

52. The method according to any one of claims 34 to 51, wherein said pathological disorder is at least one disorder associated with overexpression of the GPRC5D protein.

53. The method according to claim 52, wherein said at least one disorder associated with overexpression of the GPRC5D protein comprises at least one immune-related disorder.

54. The method according to claim 53, wherein said immune-related disorder is at least one of: at least one proliferative disorder, at least one autoimmune disease, a deposition disorder, a plasma cell-mediated disorder and a B cell-mediated disorder.

55. The method according to claim 54, wherein said proliferative disorder is a plasma cell malignancy.

56. The method according to claim 55, wherein said plasma cell malignancy is multiple myeloma (MM) and any related conditions.

57. The method according to claim 54, wherein said deposition disorder is amyloidosis or any related conditions.

58. A therapeutically effective amount of at least one of:(A) at least one nucleic acid molecule encoding at least one CAR molecule;(B) at least one cassette, vector vehicle or gene editing system comprising said nucleic acid molecule of (A);(C) at least one genetically engineered cell expressing said CAR, or a population of said cells; (D) at least one VHH nanobody specific for GPRC5D; and(E) a composition comprising at least one of (A), (B), (C) and (D); for use in a method for treating, preventing, ameliorating, inhibiting or delaying the onset of pathological disorder in a mammalian subject, said CAR comprising:(i) at least one target-binding domain, wherein at least one of said target-binding domains specifically recognizes and binds GPRC5D, or any fragments thereof;(ii) at least one hinge region;(iii) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;wherein at least one of said target-binding domains comprises at least one VHH nanobody, or any antigen-binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) the VHH nanobody specifically binds residues R154, M156, T162, and Q165of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102 (according to Chothia numbering), of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivative thereof.

59. The effective amount for use according to claim 58, wherein said CAR is as defined in any one of claims 1 to 14, said nucleic acid molecule is as defined in any one of claims 15 to 26, said cell is as defined in any one of claims 27 to 31, and said composition is as defined in any one of claims 32 to 33.

60. A method for targeted activation of a hematopoietic cell against a target cell expressing the GPRC5D protein and / or a tissue comprising said target cell, the method comprising the step of contacting said cell with an effective amount of at least one of:(A) at least one nucleic acid molecule encoding at least one CAR molecule;(B) at least one cassette, vector vehicle or gene editing system comprising said nucleic acid molecule of (A); and(C) a composition comprising at least one of (A) and (B);wherein the CAR comprises:(i) at least one target-binding domain, wherein at least one of said target binding domain specifically recognizes and binds GPRC5D, or any fragments thereof;(ii) at least one hinge region;(iii) at least one transmembrane domain; and(iv) at least one intracellular signal transduction domain;wherein at least one of said target-binding domains comprises at least one VHH nanobody, or any antigen-binding fragment thereof, and wherein said VHH nanobody is characterized by at least one of:(a) the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102 (according to Chothia numbering), of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprises at least one CDR3 comprising the amino acid sequence as denoted by at least one of: SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivative thereof.

61. The method according to claim 60, wherein the step of contacting said hematopoietic cell with said at least one nucleic acid cassette, is performed in vivo, in vitro or ex vivo.

62. The method according to claim 61, wherein contacting said hematopoietic cell with said at least one nucleic acid cassette, is performed in vivo in a subject suffering from at least one pathological disorder, the method further comprising administering to said subject an effective amount of any one of said nucleic acid cassette, a vector comprising said nucleic acid cassette, a gene editing system comprising said nucleic acid molecule, or any composition comprising thereof.

63. The method according to claim 61, wherein contacting said cell with said at least one nucleic acid cassette is performed in vitro or ex vivo to obtain genetically engineered cells of the hematopoietic lineage, or a population of said cells.

64. The method according to claim 63, wherein the method is for targeted activation of a hematopoietic cell against a target cell expressing the GPRC5D protein and / or a tissue comprising said target cell in a subject suffering from a pathological disorder, and wherein said method further comprises the step of introducing said genetically engineered cells to said subject.

65. The method according to any one of claims 63 to 64, wherein said hematopoietic cells are of autologous or allogeneic source.

66. The method according to any one of claims 62 to 65, wherein said pathological disorder is at least one disorder associated with overexpression of the GPRC5D protein.

67. The method according to claim 66, wherein said disorder associated with overexpression of the GPRC5D protein is at least one immune-related disorder.

68. The method according to claim 67, wherein said immune-related disorder is at least one of: at least one proliferative disorder, at least one deposition disorder and / or at least one autoimmune disease.

69. The method according to any one of claims 60 to 68, wherein said CAR is as defined in any one of claims 1 to 14, said nucleic acid molecule is as defined in any one of claims 15 to 26, said cell is as defined in any one of claims 27 to 31, and said composition is as defined in any one of claims 32 to 33.

70. An anti-GPRC5D VHH nanobody characterized by at least one of:(a) the VHH nanobody specifically binds residues R154, M156, T162, and Q165 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140; (b) the VHH nanobody specifically binds a conformational epitope on GPRC5D, said epitope comprising non-contiguous amino acid residues E146 toY147, T149 to L166, V168 to D169, and V172 to L173 of GPRC5D, wherein said GPRC5D comprises the amino acid sequence as denoted by SEQ ID NO: 140;(c) the VHH nanobody comprises a paratope comprising residues Q1, R27, T28, Y31, Y32, Y96, G97, D98, C99, D100B, C100D, Y10E, R100F, G101, Y102 of SEQ ID NO: 1, according to Chothia numbering; and(d) the VHH nanobody comprising at least one CDR3 comprising the amino acid sequence as denoted by at least one of SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or any variant or derivative thereof.

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