Lambda myeloma antigen chimeric antigen receptors and uses thereof

Abstract

The present disclosure relates to chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically recognises lambda myeloma antigen (LMA). The present disclosure also relates to a polynucleotide encoding the CAR, vectors, genetically modified cells and uses thereof.

Description

[0001] LAMBDA MYELOMA ANTIGEN CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

[0002] FIELD

[0003] The present disclosure relates to chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically recognises lambda myeloma antigen (LMA). The present disclosure also relates to a polynucleotide encoding the CAR, vectors, genetically modified cells and uses thereof.

[0004] RELATED APPLICATION DATA

[0005] The present application claims priority from Australian Patent Application No.

[0006] 2024903429 filed on 22 October 2024 entitled “Lambda Myeloma Antigen Chimeric Antigen Receptors and Uses Thereof’ and Australian Patent Application No. 2025903989 filed on 29 August 2025 entitled “Lambda Myeloma Antigen Chimeric Antigen Receptors and Uses Thereof’. The entire contents of which is hereby incorporated by reference.

[0007] SEQUENCE LISTING

[0008] The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0009] BACKGROUND

[0010] Lambda myeloma antigen (LMA) is found on the surface of malignant plasma cells in various proliferative diseases (e.g., multiple myeloma, lambda plasma cell dyscrasias (PCDs), amyloidosis, lymphoplasmacytoid and other B-cell malignancies). LMA is not present on normal B cells and is highly restricted to malignant plasma cells. Despite recent advances in therapy, LMA expressing malignancies, such as multiple myeloma and amyloidosis, remain incurable and treatment with monoclonal antibodies alone is not curative with incomplete eradication of the tumor leading to eventual relapse. The clinical course of such malignancies is typically characterised by an initial response to therapy, followed by repeated relapse with eventual resistance to all forms of treatment. Such malignancies are also associated with significant morbidity and disability both due to the disease itself and toxicity from available treatments.

[0011] Thus, there is a need in the art for new approaches for treating LMA expressing malignancies.

[0012] SUMMARY

[0013] The present disclosure is based on the inventors’ production of a modified single chain variable fragments (scFvs) specific to LMA, and subsequent production of LMA-targeting chimeric antigen receptors (CARs). Specifically, the inventors identified new variable light and heavy chains that specifically bind to lambda myeloma antigen (LMA), these variable light and heavy chains were modified and incorporated into LMA-CARs designated 7F12-CAR and 10B3-CAR. The inventors were also able to generate a functional bicistronic CAR (i.e., 7F12-P2A-10B3-CAR) demonstrating binding to lambda light chain (LLC).

[0014] Thus, the present disclosure is broadly directed to a CAR comprising an extracellular antigen binding domain comprising a modified scFv that specifically binds human LMA antigen and polynucleotides encoding the same.

[0015] The present disclosure is also broadly directed to a polynucleotide encoding a first CAR comprising a first extracellular antigen binding domain comprising a first modified scFv that specifically binds human LMA antigen and a second CAR comprising a second extracellular antigen binding domain comprising a second modified scFv that specifically binds human LMA antigen.

[0016] In an example, the present disclosure provides a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the polynucleotide comprises:

[0017] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the nucleotide sequence encoding the modified scFv comprises: a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and

[0018] b) a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;

[0019] (ii) a nucleotide sequence encoding a transmembrane domain; and

[0020] (iii)a nucleotide sequence encoding at least one co-stimulatory domain.

[0021] In another example, the present disclosure also provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises, in order from 5’ to 3’:

[0022] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the nucleotide sequence encoding the modified scFv comprises: a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and

[0023] b) a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;

[0024] (ii) a nucleotide sequence encoding a transmembrane domain; and

[0025] (iii)a nucleotide sequence encoding at least one co-stimulatory domain. The present disclosure also provides a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the polynucleotide comprises:

[0026] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that binds lambda myeloma antigen (LMA), wherein the nucleotide sequence encoding the modified scFv comprises:

[0027] a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and

[0028] b) a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27;

[0029] (ii) a nucleotide sequence encoding a transmembrane domain; and

[0030] (iii)a nucleotide sequence encoding at least one co-stimulatory domain.

[0031] The present disclosure also provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises, in order from 5’ to 3’:

[0032] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that binds lambda myeloma antigen (LMA), wherein the nucleotide sequence encoding the modified scFv comprises:

[0033] a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and

[0034] b) a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27;

[0035] (ii) a nucleotide sequence encoding a transmembrane domain; and

[0036] (iii)a nucleotide sequence encoding at least one co-stimulatory domain.

[0037] The present disclosure also provides a polynucleotide encoding a first chimeric antigen receptor (CAR) and second CAR, wherein the polynucleotide comprises:

[0038] (i) a nucleotide sequence encoding the first CAR comprising:

[0039] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a first modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), a nucleotide sequence encoding a first transmembrane domain, and a nucleotide sequence encoding a first at least one co-stimulatory domain, wherein the nucleotide sequence encoding the first modified scFv comprises: i. a first light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and

[0040] ii. a first heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;

[0041] (ii) a nucleotide sequence encoding the second CAR comprising:

[0042] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA, a nucleotide sequence encoding a second transmembrane domain, and a nucleotide sequence encoding a second at least one co-stimulatory domain, wherein the nucleotide sequence encoding the second modified scFv comprises:

[0043] i. a second light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and

[0044] ii. a second heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27; and

[0045] (iii)a nucleotide sequence encoding a cleavable domain operably linking the nucleotide sequence encoding the first CAR and the second nucleotide sequence encoding the second CAR.

[0046] The present disclosure also provides a polynucleotide encoding a first CAR and a second CAR, wherein the polynucleotide comprises, in order from 5’ to 3’

[0047] (i) a nucleotide sequence encoding the first CAR comprising:

[0048] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a first modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), a nucleotide sequence encoding a first transmembrane domain, and a nucleotide sequence encoding a first at least one co-stimulatory domain, wherein the nucleotide sequence encoding the first modified scFv comprises: i. a first light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and

[0049] ii. a first heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7; (ii)a nucleotide sequence encoding a cleavable domain operably linking the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR; and

[0050] (iii)a nucleotide sequence encoding the second CAR comprising:

[0051] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA, a nucleotide sequence encoding a second transmembrane domain, and a nucleotide sequence encoding a second at least one co-stimulatory domain, wherein the nucleotide sequence encoding the second modified scFv comprises:

[0052] i. a second light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and

[0053] ii. a second heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27.

[0054] The present disclosure also provides a polynucleotide encoding a first chimeric antigen receptor (CAR) and second CAR, wherein the polynucleotide comprises:

[0055] (i) a nucleotide sequence encoding the first CAR comprising:

[0056] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a first modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), a nucleotide sequence encoding a first transmembrane domain, and a nucleotide sequence encoding a first at least one co-stimulatory domain, wherein the nucleotide sequence encoding the first modified scFv comprises: iii. a first light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and

[0057] iv. a first heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;

[0058] (ii) a nucleotide sequence encoding the second CAR comprising:

[0059] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA, a nucleotide sequence encoding a second transmembrane domain, and a nucleotide sequence encoding a second at least one co-stimulatory domain, wherein the nucleotide sequence encoding the second modified scFv comprises: i. a second light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and

[0060] ii. a second heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27; and

[0061] (iii)a nucleotide sequence encoding a cleavable domain operably linking the nucleotide sequence encoding the first CAR and the second nucleotide sequence encoding the second CAR.

[0062] The present disclosure also provides a polynucleotide encoding a first CAR and a second CAR, wherein the polynucleotide comprises, in order from 5’ to 3’

[0063] (i) a nucleotide sequence encoding the first CAR comprising:

[0064] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a first modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), a nucleotide sequence encoding a first transmembrane domain, and a nucleotide sequence encoding a first at least one co-stimulatory domain, wherein the nucleotide sequence encoding the first modified scFv comprises: iii. a first light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and

[0065] iv. a first heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;

[0066] (ii)a nucleotide sequence encoding a cleavable domain operably linking the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR; and

[0067] (iii)a nucleotide sequence encoding the second CAR comprising:

[0068] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA, a nucleotide sequence encoding a second transmembrane domain, and a nucleotide sequence encoding a second at least one co-stimulatory domain, wherein the nucleotide sequence encoding the second modified scFv comprises:

[0069] i. a second light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and ii. a second heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27.

[0070] In one example:

[0071] (i) the transmembrane domain is a CD8a transmembrane domain; and / or

[0072] (ii)the at least one co-stimulatory domain is selected from the group consisting of a 4-1BB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD3Q signalling domain.

[0073] In one example, the transmembrane domain is a CD28 transmembrane domain.

[0074] In one example, the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3(^ signalling domain. For example, the at least one co-stimulatory domain comprises a 4-1BB signalling domain. In another example, the at least one co-stimulatory domain comprises a CD27 signalling domain. In a further example, the at least one co-stimulatory domain comprises an OX-40 signalling domain. In one example, the at least one co-stimulatory domain comprises a GITR signalling domain. In another example, the at least one co-stimulatory domain comprises a HVEM signalling domain. In a further example, the at least one co-stimulatory domain comprises a CD3^ signalling domain. In one example, the co-stimulatory domain comprises a 4-1BB signalling domain and a CD3 signalling domain. In a further example, the co-stimulatory domain consists of a 4-1BB signalling domain and a CD3(^ signalling domain.

[0075] In one example, the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region. For example, the hinge region is a CD8a hinge region.

[0076] In one example, the polynucleotide encoding the CAR of the present disclosure comprises from 5 ’ to 3 ’ :

[0077] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA) of the disclosure;

[0078] (ii) a nucleotide sequence encoding a CD8a hinge region;

[0079] (iii)a nucleotide sequence encoding a CD28 transmembrane domain;

[0080] (iv)a nucleotide sequence encoding a 4- IBB signalling domain; and

[0081] (v) a nucleotide sequence encoding a CD3^ signalling domain.

[0082] In one example, the polynucleotide encoding the CAR of the present disclosure comprises from 5 ’ to 3 ’ :

[0083] (i) a nucleotide sequence encoding the first CAR comprising: a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA) of the disclosure;

[0084] b) a nucleotide sequence encoding a CD8a hinge region;

[0085] c) a nucleotide sequence encoding a CD28 transmembrane domain;

[0086] d) a nucleotide sequence encoding a 4- IBB signalling domain; and

[0087] e) a nucleotide sequence encoding a CD3(^ signalling domain;

[0088] (ii) a nucleotide sequence encoding a cleavable domain; and

[0089] (iii)a nucleotide sequence encoding the second CAR comprising:

[0090] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA) of the disclosure;

[0091] b) a nucleotide sequence encoding a CD8a hinge region;

[0092] c) a nucleotide sequence encoding a CD28 transmembrane domain;

[0093] d) a nucleotide sequence encoding a 4- IBB signalling domain; and

[0094] e) a nucleotide sequence encoding a CD3(^ signalling domain.

[0095] In one example, the polynucleotide further comprises a signal peptide located 5’ to the antigen binding domain. For example, the signal peptide is a CD8a signal peptide.

[0096] In one example, the polynucleotide encoding the CAR of the present disclosure comprises from 5 ’ to 3 ’ :

[0097] (i) a nucleotide sequence encoding a CD8a signal peptide

[0098] (ii)a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA) of the disclosure;

[0099] (iii)a nucleotide sequence encoding a CD8a hinge region;

[0100] (iv)a nucleotide sequence encoding a CD28 transmembrane domain;

[0101] (v) a nucleotide sequence encoding a 4- IBB signalling domain; and

[0102] (vi)a nucleotide sequence encoding a CD3(^ signalling domain.

[0103] In one example, the polynucleotide encoding the CAR of the present disclosure comprises from 5 ’ to 3 ’ :

[0104] (i) a nucleotide sequence encoding a CD8a signal peptide

[0105] (ii) a nucleotide sequence encoding the first CAR comprising:

[0106] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA) of the disclosure;

[0107] b) a nucleotide sequence encoding a CD8a hinge region;

[0108] c) a nucleotide sequence encoding a CD28 transmembrane domain;

[0109] d) a nucleotide sequence encoding a 4- IBB signalling domain; and

[0110] e) a nucleotide sequence encoding a CD3(J signalling domain;

[0111] (iii)a nucleotide sequence encoding a cleavable domain; and (iv)a nucleotide sequence encoding the second CAR comprising:

[0112] a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA) of the disclosure;

[0113] b) a nucleotide sequence encoding a CD8a hinge region;

[0114] c) a nucleotide sequence encoding a CD28 transmembrane domain;

[0115] d) a nucleotide sequence encoding a 4- IBB signalling domain; and

[0116] e) a nucleotide sequence encoding a CD3 signalling domain.

[0117] In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4 and a VH comprising a sequence set forth in SEQ ID NO: 8.

[0118] In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24 and a VH comprising a sequence set forth in SEQ ID NO: 28.

[0119] In one example, the VL and the VH are covalently linked to each other via a linker.

[0120] In one example, the linker is a peptide linker comprising at least 2 amino acids in length For example, the linker is a GS linker. In one example, the linker is a GS linker and is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (GlyrSer s linker, a (Gly)s linker, a (Gly)e linker and a (GGGS)nlinker, wherein n=l, 2, 3 or 4. For example, the linker is a (GlyrSeQs linker. In one example, the (GlyrSeQr linker comprises a polypeptide encoded by a nucleotide sequence set forth in SEQ ID NO: 41.

[0121] In one example, the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 17.

[0122] In one example, the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 37.

[0123] In one example, the nucleotide sequence encoding the CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 43. In one example, the nucleotide sequence encoding the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 45. In one example, the nucleotide sequence encoding the CD3(^ signalling domain comprises a sequence set forth in SEQ ID NO: 47. In one example, the nucleotide sequence encoding the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 49.

[0124] In one example, the polynucleotide encoding the CAR comprises a sequence set forth in SEQ ID NO: 19. In one example, the polynucleotide encoding the CAR comprises a sequence set forth in SEQ ID NO: 39.

[0125] In one example, the nucleotide sequence encoding the first modified scFv comprises a sequence set forth in SEQ ID NO: 17 and the nucleotide sequence encoding the second modified scFv comprises a sequence set forth in SEQ ID NO: 37.

[0126] In one example, the cleavable domain is selected from the group consisting of a T2A domain, a P2A domain, E2A domain, a F2A domain and a furin domain. In one example, the nucleotide sequence encoding the P2A domain comprises a sequence set forth in SEQ IDNO: 51.

[0127] The present disclosure also provides a chimeric antigen receptor (CAR) encoded by a polynucleotide of the disclosure.

[0128] The present disclosure also provides a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the modified scFv comprises:

[0129] a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ IDNO: 11; and

[0130] b) a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15;

[0131] (ii) a transmembrane domain; and

[0132] (iii) at least one co-stimulatory domain.

[0133] The present disclosure also provides a chimeric antigen receptor (CAR) comprising: (i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the modified scFv comprises:

[0134] a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ IDNO: 31; and

[0135] b) a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35;

[0136] (ii) a transmembrane domain; and

[0137] (iii) at least one co-stimulatory domain.

[0138] The present disclosure also provides a chimeric antigen receptor (CAR) comprising, in order from N- to C-terminus:

[0139] (ii)an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the modified scFv comprises:

[0140] a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ IDNO: 11; and b) a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15;

[0141] (ii) a transmembrane domain; and

[0142] (iii) at least one co-stimulatory domain.

[0143] The present disclosure also provides a chimeric antigen receptor (CAR) comprising, in order from N- to C-terminus:

[0144] (ii)an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the modified scFv comprises:

[0145] a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ ID NO: 31; and

[0146] b) a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35;

[0147] (ii) a transmembrane domain; and

[0148] (iii) at least one co-stimulatory domain.

[0149] In one example, the CAR of the present disclosure comprises from N- to C- terminus: (i) the extracellular antigen binding domain comprising a modified scFv that specifically binds LMA of the disclosure;

[0150] (ii)the CD8a hinge region;

[0151] (iii)the CD28 transmembrane domain;

[0152] (iv)the 4- IBB signalling domain; and

[0153] (v)the CD3(^ signalling domain.

[0154] In one example, the CAR further comprises a signal peptide located at the N-terminus of the antigen binding domain. For example, the signal peptide is a CD8a signal peptide.

[0155] In one example, the CAR of the present disclosure comprises from N- to C-terminus: (i) the CD8a signal peptide;

[0156] (ii)the extracellular antigen binding domain comprising a modified scFv that specifically binds LMA of the disclosure;

[0157] (iii)the CD8a hinge region;

[0158] (iv)the CD28 transmembrane domain;

[0159] (v)the 4-1BB signalling domain; and

[0160] (vi)the CD3(^ signalling domain.

[0161] In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 12 and a VH comprising a sequence set forth in SEQ ID NO: 16; or

[0162] In one example, the modified scFv comprises a sequence set forth in SEQ ID NO: 18. In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 32 and a VH comprising a sequence set forth in SEQ ID NO: 36. In one example, wherein the modified scFv comprises a sequence set forth in SEQ ID NO: 38.

[0163] In one example, the linker comprises comprising a sequence set forth in SEQ ID NO: 42. In one example, the CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 44. In one example, the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 46. In one example, the CD3(^ signalling domain comprises a sequence set forth in SEQ ID NO: 48. In one example, the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 50. In one example, the CAR comprises a sequence set forth in SEQ ID NO: 20. In one example, the CAR comprises a sequence set forth in SEQ ID NO: 40.

[0164] The present disclosure provides a polynucleotide encoding the CAR of the present disclosure.

[0165] The present disclosure also provides a vector comprising the polynucleotide of the present disclosure.

[0166] In one example, the vector is a plasmid, a cosmid, a phage or a viral vector. For example, the vector is a plasmid. In another example, the vector is a cosmid. In a further example, the vector is a phage. In one example, the vector is a viral vector.

[0167] In one example, the viral vector is a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, a herpes simplex viral (HSV) vector or a retroviral vector. For example, the viral vector is a lentiviral vector. For example, the lentiviral vector is a self-inactivating lentiviral plasmid. In another example, the viral vector is an adeno-associated viral vector. In a further example, the viral vector is an adenoviral vector. In one example, the viral vector is a HSV vector. For example, a HSV type 1 vector. In another example, the viral vector is a retroviral vector.

[0168] The present disclosure also provides a genetically modified cell comprising the vector of the present disclosure.

[0169] The present disclosure also provides a population of genetically modified cells comprising the vector of the disclosure.

[0170] The present disclosure further provides a method of producing a genetically modified cell for adoptive cell therapy, the method comprising introducing a vector comprising the polynucleotide of the disclosure into the cell, thereby producing the genetically modified cell.

[0171] In one example, the cell is an immune cell. For example, the immune cell is selected from the group consisting of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, T cells, neutrophils, eosinophils and combinations thereof.

[0172] In one example, the cell is a T cell. For example, the T cell is a gamma delta (yb) T cell, a cytotoxic T cell, or a helper T cell. In another example, the cell is a NK cell.

[0173] The present disclosure provides a composition comprising a plurality of genetically modified cells (i.e., a population of genetically modified cells) of the disclosure for use as a medicament. The present disclosure also provides a method of treating an LMA-expressing malignant proliferative disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0174] The present disclosure further provides use of a population of genetically modified cells of the present disclosure in the manufacture of a medicament for treating an LMA-expressing malignant proliferative disease in a subject in need thereof.

[0175] The present disclosure provides a method of adjuvant therapy comprising administering to a subject having multiple myeloma, following or in combination with a primary treatment, an effective amount of a composition or a population of genetically modified cells as provided herein. The present disclosure provides an adjuvant method of treating multiple myeloma in a subject, the method comprising administering an effective amount of a composition or a population of genetically modified cells as provided herein in combination with an existing therapeutic protocol. In one example, the multiple myeloma is relapsed and / or refractory. In one example, the multiple myeloma is relapsed. In one example, the multiple myeloma is refractory.

[0176] The present disclosure provides a method of adjuvant therapy comprising administering to a subject having amyloidosis, following or in combination with a primary treatment, an effective amount of a composition or a population of genetically modified cells as provided herein. The present disclosure provides an adjuvant method of treating amyloidosis in a subject, the method comprising administering an effective amount of a composition or a population of genetically modified cells as provided herein in combination with an existing therapeutic protocol. In one example, the amyloidosis is relapsed and / or refractory. In one example, the multiple myeloma is relapsed. In one example, the amyloidosis is refractory.

[0177] The present disclosure provides a method of adjuvant therapy comprising administering to a subject having a plasma cell dyscrasias, following or in combination with a primary treatment, an effective amount of a composition or a population of genetically modified cells as provided herein. The present disclosure provides an adjuvant method of treating plasma cell dyscrasias in a subject, the method comprising administering an effective amount of a composition or a population of genetically modified cells as provided herein in combination with an existing therapeutic protocol. The present disclosure also provides a method of treating a plasma cell dyscrasias in a subject, the method comprising administering to the subject an effective amount of a composition or a population of genetically modified cells as provided herein.

[0178] The present disclosure also provides the use of a composition or a population of genetically modified cells as provided herein in the manufacture of a medicament for treating a plasma cell dyscrasias in a subject.

[0179] The present disclosure also provides a composition comprising a composition or a population of genetically modified cells as provided herein for use in treating a plasma cell dyscrasias in a subject.

[0180] In one example, the LMA-expressing malignancy is selected from the group consisting of: LMA-expressing malignancy is selected from the group consisting of: multiple myeloma, plasmacytoma, extramedullary plasmacytoma, amyloidosis, B-cell malignancies, smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstroms macroglobulinemia, mature B-cell malignancies and combinations thereof. In one example, the LMA-expressing malignancy is plasmacytoma. In one example, the LMA-expressing malignancy is extramedullary plasmacytoma. In one example, the LMA-expressing malignancy is B cell lymphoproliferative disorders (BLPD). In one example, the LMA-expressing malignancy is smoldering myeloma. In one example, the LMA-expressing malignancy is monoclonal gammopathy of undetermined significance (MGUS).

[0181] In one example, the LMA-expressing malignant proliferative disease is multiple myeloma. In one example, the LMA-expressing malignant proliferative disease is Waldenstroms macroglobulinemia. In one example, the LMA-expressing malignant proliferative disease is Waldenstroms amyloidosis. In one example, the LMA-expressing malignant proliferative disease is a mature B-cell malignancy. For example, the mature B-cell malignancy is a B-cell non-Hodgkin lymphoma (B-NHL) or a chronic B-cell leukemia (CBL). The B-NHL is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone lymphomas, Burkitt lymphoma, lymphoplasmacytoid lymphoma and combinations thereof. The CBL is selected from the group consisting of chronic lymphocytic leukemia / small lymphocytic lymphoma, hairy cell leukemia and combinations thereof. In one example, the B-cell malignancy is diffuse large B-cell lymphoma. In one example, the B-cell malignancy is follicular lymphoma. In one example, the B-cell malignancy is chronic lymphocytic leukemia / small lymphocytic lymphoma. In one example, the B-cell malignancy is mantle cell lymphoma. In one example, the B-cell malignancy is marginal zone lymphomas. In one example, the B-cell malignancy is Burkitt lymphoma. In one example, the B-cell malignancy is lymphoplasmacytoid lymphoma.

[0182] Particularly preferred embodiments are described herein, including in the independent claims.

[0183] BRIEF DESCRIPTION OF THE DRAWINGS

[0184] Figure 1 is a pictorial representation of a LV.CAR plasmid comprising an anti-LMA CAR cassette.

[0185] Figure 2 is a pictorial representation of a bicistronic LV.7F12-P2A-10B3-CAR plasmid comprising 7F12-P2A-10B3-CAR cassette.

[0186] Figure 3 is a series of graphical representations showing (A) LV.7F12-CAR transfected cells with 6 pg lambda light chain and Strep-PE; (B) LV.7F12-CAR transfected cells with 12.5 pg lambda light chain and Strep-PE; (C) LV.10B3-C AR transfected cells with 6 pg lambda light chain and Strep-PE; (D) LV.l OB 3 -CAR transfected cells with 12.5 pg lambda light chain and Strep-PE;

[0187] (E) LV.7F12-P2A-10B3-CAR transfected cells with 6 pg lambda light chain and Strep-PE; (F) LV.7F12-P2A-10B3-CAR transfected cells with 12.5 pg lambda light chain and Strep-PE; (G) Negative control, non-transfected cells with lambda light chain and Strep-PE.

[0188] Figure 4 is a graphical representation showing FACS titre of the three CAR constructs. Figure 5 is a graphical representation showing CAR expression in T cells across multiple donors over time. (A) Shows the percentage of CAR-positive CD3+T cells at Day 6 and Day 12 post-transduction and expansion. (B) Shows CAR expression levels in control, 7F12 and 10B3 groups at Days 1, 6, and 12.

[0189] Figure 6 is a graphical representation showing the distribution of CD4 and CD8 expression among CD3+CAR+T cells at Day 6 and Day 12 post-transduction. (A) 7F12.CAR; (B) 10B3.CAR.

[0190] Figure 7 is a graphical representation showing the proportion of memory T cell subsets within the CAR+T cell population across all donors on Day 12 post-expansion.

[0191] KEY TO SEQUENCE LISTING SEQ ID NO: 1 7F12 VL CDR1 nucleotide sequence

[0192] SEQ ID NO: 2 7F12 VL CDR2 nucleotide sequence

[0193] SEQ ID NO: 3 7F12 VL CDR3 nucleotide sequence

[0194] SEQ ID NO: 4 7F12 VL nucleotide sequence

[0195] SEQ ID NO: 5 7F12 VH CDR1 nucleotide sequence

[0196] SEQ ID NO: 6 7F12 VH CDR2 nucleotide sequence

[0197] SEQ ID NO: 7 7F12 VH CDR3 nucleotide sequence

[0198] SEQ ID NO: 8 7F12 VH nucleotide sequence

[0199] SEQ ID NO: 9 7F12 VL CDR1 amino acid sequence

[0200] SEQ ID NO: 10 7F12 VL CDR2 amino acid sequence

[0201] SEQ ID NO: 11 7F12 VL CDR3 amino acid sequence

[0202] SEQ ID NO: 12 7F12 VL amino acid sequence

[0203] SEQ ID NO: 13 7F12 VH CDR1 amino acid sequence

[0204] SEQ ID NO: 14 7F12 VH CDR2 amino acid sequence

[0205] SEQ ID NO: 15 7F12 VH CDR3 amino acid sequence

[0206] SEQ ID NO: 16 7F12 VH amino acid sequence

[0207] SEQ ID NO: 17 7F12 scFv nucleotide sequence

[0208] SEQ ID NO: 18 7F12 scFv amino acid sequence

[0209] SEQ ID NO: 19 7F12-CAR cassette nucleotide sequence

[0210] SEQ ID NO: 20 7F12-CAR cassette amino acid sequence

[0211] SEQ ID NO: 21 10B3 VL CDR1 nucleotide sequence

[0212] SEQ ID NO: 22 10B3 VL CDR2 nucleotide sequence

[0213] SEQ ID NO: 23 10B3 VL CDR3 nucleotide sequence

[0214] SEQ ID NO: 24 10B3 VL nucleotide sequence

[0215] SEQ ID NO: 25 10B3 VH CDR1 nucleotide sequence

[0216] SEQ ID NO: 26 10B3 VH CDR2 nucleotide sequence

[0217] SEQ ID NO: 27 10B3 VH CDR3 nucleotide sequence

[0218] SEQ ID NO: 28 10B3 VH nucleotide sequence

[0219] SEQ ID NO: 29 10B3 VL CDR1 amino acid sequence

[0220] SEQ ID NO: 30 10B3 VL CDR2 amino acid sequence SEQ ID NO: 31 10B3 VL CDR3 amino acid sequence SEQ ID NO: 32 10B3 VL amino acid sequence

[0221] SEQ ID NO: 33 10B3 VH CDR1 amino acid sequence

[0222] SEQ ID NO: 34 10B3 VH CDR2 amino acid sequence

[0223] SEQ ID NO: 35 10B3 VH CDR3 amino acid sequence

[0224] SEQ ID NO: 36 10B3 VH amino acid sequence

[0225] SEQ ID NO: 37 10B3 scFv nucleotide sequence

[0226] SEQ ID NO: 38 10B3 scFv amino acid sequence

[0227] SEQ ID NO: 39 10B3-CAR cassette nucleotide sequence

[0228] SEQ ID NO: 40 10B3-CAR cassette amino acid sequence

[0229] SEQ ID NO: 41 (Gly4Ser)s linker nucleotide sequence

[0230] SEQ ID NO: 42 (Gly4Ser)3 linker amino acid sequence

[0231] SEQ ID NO: 43 CD28 transmembrane domain nucleotide sequence SEQ ID NO: 44 CD28 transmembrane domain amino acid sequence SEQ ID NO: 45 4- IBB signalling domain nucleotide sequence SEQ ID NO: 46 4- IBB signalling domain amino acid sequence SEQ ID NO: 47 CD3 signalling domain nucleotide sequence

[0232] SEQ ID NO: 48 CD3(j signalling domain amino acid sequence SEQ ID NO: 49 CD8a hinge region nucleotide sequence

[0233] SEQ ID NO: 50 CD8a hinge region amino acid sequence

[0234] SEQ ID NO: 51 P2A domain nucleotide sequence

[0235] SEQ ID NO: 52 P2A domain amino acid sequence

[0236] SEQ ID NO: 53 LV.7F12-CAR plasmid nucleotide sequence

[0237] SEQ ID NO: 54 LV.10B3-CAR plasmid nucleotide sequence

[0238] SEQ ID NO: 55 LV.7F12-P2A-10B3-CAR plasmid nucleotide sequence SEQ ID NO: 56 EFla junction nucleotide sequence

[0239] SEQ ID NO: 57 Kozak nucleotide sequence

[0240] SEQ ID NO: 58 WPRE junction nucleotide sequence

[0241] SEQ ID NO: 59 LMA epitope 1 (lambda isotype 1)

[0242] SEQ ID NO: 60 LMA epitope 1 (lambda isotype 2 and 3)

[0243] SEQ ID NO: 61 LMA epitope 2 (lambda isotype 1 and 2)

[0244] SEQ ID NO: 62 LMA epitope 2 (lambda isotype 3)

[0245] SEQ ID NO: 63 T2A amino acid sequence

[0246] SEQ ID NO: 64 E2A amino acid sequence

[0247] SEQ ID NO: 65 F2A amino acid sequence

[0248] SEQ ID NO: 66 Furin amino acid sequence

[0249] Kabat numbering system was used to assign CDRs and framework regions. DESCRIPTION

[0250] General

[0251] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0252] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0253] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0254] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0255] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0256] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0257] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilised in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0258] The description and definitions of variable regions and parts thereof, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991. The term “EU numbering system of Kabat” will be understood to mean the numbering of an antibody heavy chain is according to the EU index as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgGl EU antibody.

[0259] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0260] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0261] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0262] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0263] Selected Definitions

[0264] As used herein, a “chimeric antigen receptor” or “CAR” refers to a protein or polypeptide comprising an extracellular domain capable of binding an antigen (i.e., an antigen binding domain linked to immune cell signalling domains). CARs are able to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, thus giving T cells expressing CARs the ability to recognise antigens independent of antigen processing. CARs do not dimerise with endogenous T cell receptor (TCR) a- and P-chains. T cells expressing a CAR are referred to herein as CAR T cells, CAR-T cells or CAR modified T cells, and these terms are used interchangeably herein. A “LMA-CAR” refers to a CAR having an extracellular binding domain specific for LMA.

[0265] The term “co- stimulatory domain” refers to any oligopeptide or polypeptide known to act as a domain that transmits signals to activate or inhibit biological processes within a cell.

[0266] As used herein, the term “transmembrane domain” refers to any three-dimensional structure formed by a sequence of amino acids which is thermodynamically stable in a biological membrane, e.g., a cell membrane.

[0267] The term “hinge region” refers to an amino acid sequence which provides for flexible linkage of the antigen binding and transmembrane domains of the CAR. As used herein, the terms “treating”, “treat” or “treatment” include any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition described herein, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., multiple myeloma or amyloidosis. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0268] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect treatment of a disease as herein described. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect a change associated with a disease as herein described. The effective amount may vary according to the disease to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the subject being treated. Typically, the effective amount will fall within a relatively broad range (e.g., a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number of cells. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

[0269] A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the cells of the present disclosure to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects.

[0270] As used herein, “adoptive cell therapy” (ACT) or “CAR-T cell therapy” refers to a treatment that involves the collection of immune cells from a subject, modifying the cells to express a CAR, growing or expanding these cells in vitro and reinfusing the modified cells back into the subject.

[0271] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0272] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions. The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0273] As used herein, the term “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. By convention, sequences are presented from the 5' end to the 3' end, unless otherwise specified.

[0274] The term “recombinant” shall be understood to mean the product of artificial genetic recombination. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

[0275] As used herein, the term “antigen binding domain” shall be taken to mean a structure formed by a protein that is capable of binding or specifically binding to an antigen. The antigen binding domain need not be a series of contiguous amino acids. For example, in a scFv the antigen binding domain is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always in the one or more of the CDRs in each variable region. In some examples, an antigen binding domain is a VH or a VL or a scFv.

[0276] As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. A “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

[0277] As used herein, “variable region” refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0278] As used herein, the term “complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of a variable region the presence of which are necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems in the performance of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al., J Mol Biol 273 : 927-948, 1997; the IMGT numbering system ofLefranc et al., Devel. And Compar. Immunol., 27: 55-77, 2003; or the AHO numbering system of Honnegher and Pliikthun J. Mol. Biol., 309: 657-670, 2001. For example, according to the numbering system of Kabat, VH framework regions (FRs) and CDRs are positioned as follows: residues 1-30 (FR1 ), 31-35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above. In one example, reference herein to a CDR (or a FR) is in respect of those regions according to the Kabat numbering system.

[0279] “Framework regions” (FRs) are those variable region residues other than the CDR residues. As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that the interaction between an antigen binding domain of the disclosure with an antigen is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. In addition, an antigen binding domain of the disclosure reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a CAR comprising an antigen binding domain of the disclosure binds to LMA with materially greater affinity (e.g., 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other antigens. Reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

[0280] For the purposes of clarification and as will be apparent to the skilled artisan based on the exemplified subject matter herein, reference to “affinity” in this specification is a reference to KD of a protein or scFv.

[0281] As used herein, the term “epitope” shall be understood to mean a region of LMA to which an antigen binding domain binds. This term is not necessarily limited to the specific residues or structure to which the antigen binding domain makes contact. For example, this term includes the region spanning amino acids contacted by the antigen binding domain and / or 5-10 or 2-5 or 1-3 amino acids outside of this region. In some examples, the epitope comprises a series of discontinuous amino acids that are positioned close to one another when the light chain is folded, i.e., a “conformational epitope”. For example, a conformational epitope comprises an amino acid sequence set forth in SEQ ID NO: 59 and 61, comprises an amino acid sequence set forth in SEQ ID NO: 60 and 61 or comprises an amino acid sequence set forth in SEQ ID NO: 60 and 62.

[0282] Chimeric antigen receptor (CAR)

[0283] The CAR of the present disclosure, or the polynucleotide encoding the CAR, comprises an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), a transmembrane domain and at least one co-stimulatory domain.

[0284] In one example, the CAR of the present disclosure comprises from N-terminus to C-terminus: a signal peptide, an extracellular antigen binding domain comprising a modified scFv that specifically binds LMA antigen, a transmembrane domain and at least one co-stimulatory domain.

[0285] In one example, the CAR of the present disclosure comprises from N-terminus to C-terminus: a CD8a signal peptide, an extracellular antigen binding domain comprising a modified scFv that specifically binds LMA, a CD8a hinge region, a CD28 transmembrane domain, a 4-1BB signalling domain and a CD3^ signalling domain.

[0286] In one example, the CAR of the present disclosure comprises a sequence set forth in SEQ ID NO: 20 or 40.

[0287] In one example, the CAR of the present disclosure comprises a sequence set forth in SEQ ID NO: 20. In one example, the CAR of the present disclosure comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 19.

[0288] In one example, the CAR of the present disclosure comprises a sequence set forth in SEQ ID NO: 39. In one example, the CAR of the present disclosure comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 40.

[0289] Polynucleotide sequences encoding the CARs described herein can be obtained using standard recombinant techniques and will be apparent to the skilled person. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesised using nucleotide synthesisers or PCR techniques.

[0290] Extracellular antigen binding domain

[0291] The present disclosure provides a CAR, or polynucleotide encoding the CAR, comprising an extracellular antigen binding domain comprising a modified scFv that specifically binds LMA.

[0292] The term “lambda myeloma antigen” or “LMA” is used in the context of the present disclosure to refer to a cell membrane antigen that is found on the surface of plasma cells such as malignant myeloma cells and, in some cases, non-malignant plasma cell precursors such as plasmablasts. LMA consists of lambda light chains expressed on the cell membrane. Various lambda light chain isotypes have been reported and these isotypes are defined by amino acid variances in the constant region of the molecule.

[0293] Lambda light chain isotypes are expressed at varying frequencies. For example, in subjects with multiple myeloma, about 14% express isotype 1, about 64% express isotype 2 and about 23% express isotype 3.

[0294] In an example, the conformational epitope of free lambda light chain isotype 1 comprises an amino acid sequence set forth in SEQ ID NO: 59 and 61.

[0295] In an example, the conformational epitope of free lambda light chain isotype 2 comprises an amino acid sequence set forth in SEQ ID NO: 60 and 61.

[0296] In an example, the conformational epitope of free lambda light chain isotype 3 comprises an amino acid sequence set forth in SEQ ID NO: 60 and 62.

[0297] These conformational epitopes on the lambda light chain are only available for binding when the lambda light chain is not associated with a heavy chain (i.e., free lambda light chain). Accordingly, antigen binding domains which specifically binds to these conformational epitopes do not bind to intact lambda-chain containing IgG, IgM, IgE or IgA. The conformational epitope of LMA is presented in lipid rafts of amyloidosis and myeloma clonal plasma cell membranes but are not found on normal plasma cells. In one example, the CAR according to the present disclosure does not bind intact immunoglobulin. Put another way, exemplary CARs do not recognise lambda light chains that are in association with Ig heavy chain such as in an intact Ig molecule.

[0298] In one example, the extracellular antigen binding domain comprising a modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ ID NO: 11.

[0299] In one example, the extracellular antigen binding domain comprising a modified scFv comprises a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15.

[0300] In one example, the extracellular antigen binding domain comprising a modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ ID NO: 11 and a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15.

[0301] In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 12 and / or a VH comprising a sequence set forth in SEQ ID NO: 16.

[0302] In one example, the modified scFv comprises a sequence set forth in SEQ ID NO: 18. In one example, the extracellular antigen binding domain comprising a modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ ID NO: 31.

[0303] In one example, the extracellular antigen binding domain comprising a modified scFv comprises a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35.

[0304] In one example, the extracellular antigen binding domain comprising a modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ ID NO: 31 and a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35.

[0305] In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 32 and / or a VH comprising a sequence set forth in SEQ ID NO: 36.

[0306] In one example, the modified scFv comprises a sequence set forth in SEQ ID NO: 38. In one example, a polynucleotide encodes the extracellular antigen binding domain. In one example, the nucleotide sequence encoding the extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3.

[0307] In one example, the nucleotide sequence encoding the extracellular antigen binding domain comprises a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7.

[0308] In one example, the nucleotide sequence encoding the extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3 and a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7.

[0309] In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4. In one example, the nucleotide sequence encoding the modified scFv comprises a VH comprising a sequence set forth in SEQ ID NO: 8. In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4 and a VH comprising a sequence set forth in SEQ ID NO: 8.

[0310] In one example, the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 17.

[0311] In one example, the nucleotide sequence encoding the extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23.

[0312] In one example, the nucleotide sequence encoding the extracellular antigen binding domain comprises a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27.

[0313] In one example, the nucleotide sequence encoding the extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23 and a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27,

[0314] In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24.

[0315] In one example, the nucleotide sequence encoding the modified scFv comprises a VH comprising a sequence set forth in SEQ ID NO: 28.

[0316] In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24 and a VH comprising a sequence set forth in SEQ ID NO: 28.

[0317] In one example, the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 37.

[0318] The skilled person will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv).

[0319] In one example, the linker is a peptide linker. For example, a peptide linker comprising at least 2 amino acids in length. In one example, the peptide linker is between 2 and 35 amino acids in length. For example, the linker is about 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length. In another example, the linker is between about 10 and 20 amino acids in length. For example, the peptide linker is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In one example, the peptide linker is 15 amino acids in length. In another example, the peptide linker is between about 20 and 35 amino acids in length. For example, the peptide linker is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length.

[0320] Suitable linkers for use in the present disclosure for linking the VH and VL will be apparent to the skilled person and / or disclosed herein. Exemplary linkers include GS linkers. For example, the linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3 linker, a (Gly)s linker, a (Gly)s linker and a (GGGS)n linker, wherein n=l, 2, 3 or 4. In one example, the linker is a (Gly4Ser)3 linker and comprises a sequence set forth in SEQ ID NO: 42. In one example, the linker is a (Gly4Ser)3 linker and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 41.

[0321] It will be apparent from the disclosure herein that the scFv is a modified scFv.

[0322] In one example, the modified scFv is a humanised scFv. The term “humanised scFv” shall be understood to refer to a scFv having an antigen binding domain or variable region derived from an antibody from a non-human species (e.g., murine) and the remaining antibody structure based upon the structure and / or sequence of a human antibody. In a humanised scFv, the antigen binding domain generally comprises the CDRs from the non-human antibody grafted onto appropriate FRs in the human variable regions and the antigen binding domains are modified by one or more amino acid substitutions. In some instances, FR residues of the human antibody are replaced by corresponding non-human residues. Methods for modifying non-human antibodies or parts thereof (e.g., variable regions) are known in the art. Modification can be performed following the method of US5225539, or US5585089. Other methods for modifying an antibody are not excluded.

[0323] The present disclosure provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds LMA of the present disclosure. The present disclosure provides a CAR encoded by a polynucleotide as described herein.

[0324] Bicistronic CAR Construct

[0325] The present disclosure also provides a CAR construct comprising multiple CARs (e.g., two, three, four, five, or more), each capable of binding to a single antigen, wherein each CAR is separated by a cleavable domain.

[0326] The present disclosure provides a CAR construct that can bind to two antigens (e.g., 7F12 and 10B3) simultaneously. In such examples, these CARs exhibit dual specificity for 7F12 and 10B3. As used herein, the phrases “dual specificity,” “dual specific,” “bispecific,” and “bivalent” refer to a CAR that can specifically bind to and immunologically recognise two different antigens, such that binding to at least one of the two antigens elicits an immune response. In one example, dual-specific CARs may be linked by cleavable domains.

[0327] The present disclosure provides a dual-specific CARs comprising antigen binding domain as described herein linked by cleavable domains.

[0328] In one example, the CAR construct of the present disclosure comprises from N-terminus to C-terminus: a signal peptide, a first extracellular antigen binding domain comprising a first modified scFv that specifically binds LMA antigen, a cleavable domain, a second extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA antigen, a transmembrane domain and at least one co-stimulatory domain.

[0329] In one example, the CAR construct of the present disclosure comprises from N-terminus to C-terminus: a CD8a signal peptide, a first extracellular antigen binding domain comprising a first modified scFv that specifically binds LMA antigen, a cleavable domain, a second extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA antigen, a CD8a hinge region, a CD28 transmembrane domain, a 4-1BB signalling domain and a CD3(^ signalling domain.

[0330] In one example, the first extracellular antigen binding domain comprising a first modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ ID NO: 11.

[0331] In one example, the first extracellular antigen binding domain comprising a first modified scFv comprises a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15. In one example, the first extracellular antigen binding domain comprising a first modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ ID NO: 11 and a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15.

[0332] In one example, the first modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 12 and / or a VH comprising a sequence set forth in SEQ ID NO: 16.

[0333] In one example, the first modified scFv comprises a sequence set forth in SEQ ID NO: 18. In one example, the second extracellular antigen binding domain comprising a second modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ ID NO: 31.

[0334] In one example, the second extracellular antigen binding domain comprising a second modified scFv comprises a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35.

[0335] In one example, the second extracellular antigen binding domain comprising a second modified scFv comprises a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ ID NO: 31 and a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35.

[0336] In one example, the second modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 32 and / or a VH comprising a sequence set forth in SEQ ID NO: 36.

[0337] In one example, the second modified scFv comprises a sequence set forth in SEQ ID NO: 38.

[0338] In one example, a polynucleotide encodes a first CAR comprising a first extracellular antigen binding domain and a second CAR comprising a second extracellular antigen binding domain.

[0339] In one example, the nucleotide sequence encoding the first extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3.

[0340] In one example, the nucleotide sequence encoding the first extracellular antigen binding domain comprises a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7.

[0341] In one example, the nucleotide sequence encoding the first extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3 and a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7.

[0342] In one example, the nucleotide sequence encoding the first modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4. In one example, the nucleotide sequence encoding the first modified scFv comprises a VH comprising a sequence set forth in SEQ ID NO: 8. In one example, the nucleotide sequence encoding the first modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4 and a VH comprising a sequence set forth in SEQ ID NO: 8

[0343] In one example, the nucleotide sequence encoding the first modified scFv comprises a sequence set forth in SEQ ID NO: 17.

[0344] In one example, the nucleotide sequence encoding the second extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23.

[0345] In one example, the nucleotide sequence encoding the second extracellular antigen binding domain comprises a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27.

[0346] In one example, the nucleotide sequence encoding the second extracellular antigen binding domain comprises a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23 and a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27,

[0347] In one example, the nucleotide sequence encoding the second modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24.

[0348] In one example, the nucleotide sequence encoding the second modified scFv comprises a VH comprising a sequence set forth in SEQ ID NO: 28. In one example, the nucleotide sequence encoding the second modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24 and a VH comprising a sequence set forth in SEQ ID NO: 28.

[0349] In one example, the nucleotide sequence encoding the second modified scFv comprises a sequence set forth in SEQ ID NO: 37.

[0350] In one example, the polynucleotide encoding the first CAR comprises a sequence set forth in SEQ ID NO: 19. In one example, the polynucleotide encoding the second CAR comprises a sequence set forth in SEQ ID NO: 37. In one example, the polynucleotide encoding the first CAR comprises a sequence set forth in SEQ ID NO: 19 and the polynucleotide encoding the second CAR comprises a sequence set forth in SEQ ID NO: 37.

[0351] In one example, cleavage of the cleavable domain releases each CAR, such as a first and second CAR, from the CAR construct so that each cleaved CAR is independently present on the surface of the T cell, retains antigenic specificity for its respective target, and can elicit an antigenspecific response. In one example, the CAR construct comprises two CARs that can be cleaved and released, thereby forming a bicistronic CAR. Without wishing to be bound by theory, the cleavable domains may be cleaved after full translation of the entire sequence or after translation of each CAR and its associated cleavable domain, such that a CAR is cleaved and released prior to translation of the next CAR in the sequence.

[0352] In one example, the cleavable domain comprises one or more of any suitable cleavable domain, including domains recognized by cleavage enzymes or domains that are self-cleaving. Suitable domains include, for example, the 2A domain, such as T2A and / or P2A, and furin cleavage sequences. Table X presents exemplary suitable cleavable domains.

[0353] In one example, the cleavable domain is selected from the group consisting of a T2A domain, a P2A domain, E2A domain, a F2A domain and a furin domain. In one example, the cleavable domain is a T2A domain. In one example, the 62A domain comprises a sequence set forth in SEQ ID NO: 63. In one example, the cleavable domain is a P2A domain. In one example, the P2A domain comprises a sequence set forth in SEQ ID NO: 52. In one example, the nucleotide sequence encoding the P2A domain comprises a sequence set forth in SEQ ID NO: 51. In one example, the cleavable domain is E2A domain. In one example, the E2A domain comprises a sequence set forth in SEQ ID NO: 64. In one example, the cleavable domain is a F2A domain. In one example, the F2A domain comprises a sequence set forth in SEQ ID NO: 65. In one example, the cleavable domain is a furin domain. In one example, the furin domain comprises a sequence set forth in SEQ ID NO: 66.

[0354] Table X.

[0355] Peptide Amino Acid Sequence*

[0356] T2A (GSG)EGRGSLLTCGDVEENPGP (SEQ ID

[0357]

[0358] NO: 63) P2A (GSG)ATNFSLLKQAGDVEENPGP (SEQ ID NO: 52)

[0359] E2A (GSG)QCTNY ALLKLAGDVESNPGP (SEQ ID NO: 64)

[0360] F2A (GSG)VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 65)

[0361]

[0362] Furin RKRR (SEQ ID NO: 66)

[0363] * The GSG residues may be added to improve cleavage efficiency.

[0364] In one example, the VL and the VH are joined to each other by a linker. The linker may be any of the linkers described herein with respect to other aspects of the disclosure. In one example, the VL and the VH are joined to each other by a linker comprising the amino acid sequence of SEQ ID NO: 42. Any linker sequence may be used as a spacer between the antigen binding domain and the transmembrane domain.

[0365] In one example, the first linker is a (Gly4Ser)3 linker and the nucleotide sequence encoding the first linker comprises a sequence set forth in SEQ ID NO: 41. In one example, the second linker is a (Gly4Ser)3 linker and the nucleotide sequence encoding the second linker comprises a sequence set forth in SEQ ID NO: 41. In one example, the first linker and the second linker is a (Gly4Ser)3 linker and the nucleotide sequence encoding the first linker and the second linker comprises a sequence set forth in SEQ ID NO: 41.

[0366] The present disclosure provides a CAR construct encoded by a polynucleotide as described herein.

[0367] Transmembrane Domain

[0368] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds LMA and a transmembrane domain.

[0369] It will be apparent to the skilled person that the transmembrane domain provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigen binding domain in the extracellular space and signalling domain (or co-stimulatory domain) inside the cell.

[0370] In one example, the transmembrane domain comprises or consists of a sequence of amino acids which forms a hydrophobic alpha helix or beta-barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of a transmembrane domain of a protein comprising a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g., using amino acid sequence analysis tools such as TMHMM (Krogh et al., J Mol Biol (2001) 305:567-580). In one example, the transmembrane domain is from a CD8 molecule or a CD28 molecule. In one example, the transmembrane domain is from a CD28 molecule. In one example, the transmembrane domain is a CD28 transmembrane domain. In one example, the transmembrane domain is a CD8 transmembrane domain. In one example, the transmembrane domain is a CD28 transmembrane domain and a CD8 transmembrane domain. In one example, the first transmembrane domain comprises a CD28 transmembrane domain. In one example, the second transmembrane domain comprises a CD28 transmembrane domain. In one example, the first transmembrane domain and the second transmembrane domain comprises a CD28 transmembrane domain.

[0371] In one example, the transmembrane domain is human. For example, the transmembrane domain is a human CD28.

[0372] An exemplary transmembrane domain of the present disclosure comprises a sequence set forth in SEQ ID NO: 44. In one example, the CD28 molecule comprises or consists of an amino acid sequence at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 44.

[0373] In one example, the transmembrane domain comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 43.

[0374] In one example, the nucleotide sequence encoding the CD28 transmembrane domain and comprises a sequence set forth in SEQ ID NO: 43. In one example, the nucleotide sequence encoding the first CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 43. In one example, the nucleotide sequence encoding the second CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 43. In one example, the nucleotide sequence encoding the first CD28 transmembrane domain and the second CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 43.

[0375] In one example, the transmembrane domain that can be directly or indirectly connected to the extracellular antigen binding domain.

[0376] In one example, the transmembrane domain is directly connected to the extracellular antigen binding domain.

[0377] In one example, the transmembrane domain is indirectly connected to the extracellular antigen binding domain. For example, the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region. In one example, the CAR further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0378] In one example, the first extracellular antigen binding domain is operably linked to the first transmembrane domain through a first hinge region. In one example, the second extracellular antigen binding domain is operably linked to the second transmembrane domain through a second hinge region. In one example, the first extracellular antigen binding domain is operably linked to the first transmembrane domain through a first hinge region and wherein the second extracellular antigen binding domain is operably linked to the second transmembrane domain through a second hinge region. It will be apparent to the skilled person that the hinge region not only provides separation between the antigen binding domain and the transmembrane domain but also allows the binding moiety to orient in different directions.

[0379] In one example, the hinge region is a CD8a hinge region. In one example, the first hinge region is a CD8a stalk hinge region. In one example, the second hinge region is a CD8a stalk hinge region. In one example, the first hinge region is a CD8a stalk hinge region and the second hinge region is a CD8a stalk hinge region. In one example, the hinge region is a CD8a hinge region and comprises a sequence set forth in SEQ ID NO: 50. In one example, the hinge region is a CD8a hinge region and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 49. In one example, the first VL and the first VH are covalently linked to each other via a first linker. In one example, the second VL and the second VH are covalently linked to each other via a second linker. In one example, the first VL and the first VH are covalently linked to each other via a first linker and the second VL and the second VH are covalently linked to each other via a second linker.

[0380] Co-stimulatory Domains

[0381] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds LMA and at least one co-stimulatory domain.

[0382] It will be apparent to the skilled person that the co-stimulatory domain provides the costimulation signal necessary for enhancing immune cell activation and effector function.

[0383] In one example, the at least one co-stimulatory domain is at least one intracellular signalling domain. It will be apparent to the skilled person that the signalling domain comprises amino acid sequences required for activation of immune cell function.

[0384] In one example, the signalling domain comprises ITAM-containing sequence. An ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs comprise the amino acid sequence YXXL / I, wherein “X” denotes any amino acid. In ITAM-containing proteins, IT AM sequences are often separated by 6 to 8 amino acids. When phosphate groups are added to the tyrosine residue of an ITAM by tyrosine kinases, a signalling cascade is initiated within the cell.

[0385] Suitable co-stimulatory domains suitable for use in the present disclosure will be apparent to the skilled person and / or described herein. For example, the at least one co-stimulatory domain may be obtained or derived from a CD28 molecule, a CD3 zeta (Q molecule or modified versions thereof, a human Fc receptor gamma (FcRy) chain, a CD27 molecule, an OX-40 molecule, a 4-1BB molecule, a glucocorticoid-induced TNFR-related (GITR) molecule and / or a herpesvirus entry mediator (HVEM) molecule.

[0386] In one example, the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3^ signalling domain. In one example, the at least one co-stimulatory domain is a 4- IBB signalling domain. In one example, the at least one co-stimulatory domain is a CD27 signalling domain. In one example, the at least one co- stimulatory domain is an OX-40 signalling domain. In one example, the at least one co-stimulatory domain is a GITR signalling domain. In one example, the at least one co-stimulatory domain is a HVEM signalling domain. In one example, the at least one co-stimulatory domain is a CD3^ signalling domain.

[0387] For example, the co-stimulatory domain comprises the 4-1BB signalling domain and the CD3^ signalling domain. In another example, a CAR of the present disclosure comprise a CD28 co-stimulatory domain and a CD37 signalling domain.

[0388] In one example, the first at least one co-stimulatory domain is selected from the group consisting of a 4-1BB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3c signalling domain. In one example, the first at least one co-stimulatory domain is a 4- IBB signalling domain. In one example, the first at least one co-stimulatory domain is a CD27 signalling domain. In one example, the first at least one co-stimulatory domain is an OX-40 signalling domain. In one example, the first at least one co-stimulatory domain is a GITR signalling domain. In one example, the first at least one co-stimulatory domain is a HVEM signalling domain. In one example, the first at least one co-stimulatory domain is a CD3(^ signalling domain.

[0389] For example, the first co-stimulatory domain comprises the 4-1BB signalling domain and the CD3(^ signalling domain. In another example, a first CAR of the present disclosure comprise a CD28 co-stimulatory domain and a CD3(^ signalling domain.

[0390] In one example, the second at least one co-stimulatory domain is selected from the group consisting of a 4-1BB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3c signalling domain. In one example, the second at least one co-stimulatory domain is a 4-1BB signalling domain. In one example, the second at least one co-stimulatory domain is a CD27 signalling domain. In one example, the second at least one co-stimulatory domain is an OX-40 signalling domain. In one example, the second at least one co-stimulatory domain is a GITR signalling domain. In one example, the second at least one co-stimulatory domain is a HVEM signalling domain. In one example, the second at least one co-stimulatory domain is a CD3(^ signalling domain.

[0391] For example, the second co-stimulatory domain comprises the 4-1BB signalling domain and the CD3 signalling domain. In another example, a second CAR of the present disclosure comprise a CD28 co-stimulatory domain and a CD3^ signalling domain.

[0392] In one example, the first and the second at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3 signalling domain. In one example, the first and the second at least one co-stimulatory domain is a 4- IBB signalling domain. In one example, the first and the second at least one co-stimulatory domain is a CD27 signalling domain. In one example, the first and the second at least one co-stimulatory domain is an OX-40 signalling domain. In one example, the first and the second at least one co-stimulatory domain is a GITR signalling domain. In one example, the first and the second at least one co-stimulatory domain is a HVEM signalling domain. In one example, the first and the second at least one co-stimulatory domain is a CD3(^ signalling domain.

[0393] For example, the first and the second co-stimulatory domain comprises the 4-1BB signalling domain and the CD3q signalling domain. In another example, a first CAR of the present disclosure and a second CAR of the present disclosure comprise a CD28 co-stimulatory domain and a CD3q signalling domain.

[0394] A CD3(^ signalling domain may be used to enhance the effectiveness of a CAR T cell therapy. A CD3^ signalling domain may be included in the intracellular signaling domain of CARs to assist initiating T cell activation and proliferation upon antigen recognition. A CD3 signalling domain may be combined with one or more co-stimulatory domains.

[0395] A 4- IBB signalling domain may be used to enhance the effectiveness of a CAR T cell therapy. A 4-1BB signalling domain may be combined with one or more co-stimulatory domains. For example, a 4- IBB signalling domain may be used to amplify the initial activation signal provided by a CD3^ signalling domain leading to a more robust T cell response.

[0396] In one example, the at least one co-stimulatory domain is human. For example, the co-stimulatory domain comprises a human 4-1BB signalling domain and a human CD3(^ signalling domain.

[0397] In one example, the co-stimulatory domain comprises a human 4-1BB signalling domain and comprises or consists of an amino acid sequence at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 46. In one example, the co-stimulatory domain comprises a human 4- IBB signalling domain and comprises a sequence set forth in SEQ ID NO: 46. In one example, the co-stimulatory domain comprises a human 4- IBB signalling domain and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 45. In one example, the nucleotide sequence encoding the first 4-1BB signalling domain and the second 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 45. In one example, the nucleotide sequence encoding the first 4-1BB signalling domain. In one example, the second 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 45.

[0398] In one example, the co-stimulatory domain comprises a human CD3(^ signalling domain and comprises or consists of an amino acid sequence at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 48. In one example, the co-stimulatory domain comprises a human CD3(^ signalling domain and comprises a sequence set forth in SEQ ID NO: 48. In one example, the co-stimulatory domain comprises a human CD3(^ signalling domain and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 47. In one example, the nucleotide sequence encoding the first CD3(^ signalling domain. In one example, the nucleotide sequence encoding the second CD3(^ signalling domain comprises a sequence set forth in SEQ ID NO: 47. In one example, the nucleotide sequence encoding the first CD3(^ signalling domain and the second CD3 signalling domain comprises a sequence set forth in SEQ ID NO: 47. Vectors

[0399] The present disclosure also provides a vector comprising the polynucleotide sequences encoding the CAR described herein.

[0400] Suitable vectors and methods of preparing a vector will be apparent to the skilled person and / or described herein. For example, the vector is a plasmid, a cosmid, a phage or a viral vector. In one example, the viral vector is a lentiviral vector, an adeno-associated viral vector (AAV), an adenoviral vector, a herpes simplex viral (HSV) vector or a retroviral vector. In one example, example, the vector is a plasmid. In one example, example, the vector is a cosmid. In one example, example, the vector is a phage. In one example, example, the vector is a viral vector. In one example, the viral vector is a lentiviral vector. In one example, the viral vector is an AAV. In one example, the viral vector is an adenoviral vector. In one example, the viral vector is a HSV vector. In one example, the viral vector is a retroviral vector.

[0401] In one example, the viral vector is derived from the genome of an adenovirus (e g., Ad5), an adeno-associated virus (e.g., AAV2, 3, 5, 6, 8 or 9), a herpes simplex virus (e.g., HSV, HSV1), a retrovirus (e.g., avian leukosis virus (ALV), mouse mammary tumour virus (MMTV), murine leukemia virus (MLV), human T-lymphotropic virus (HTLV), Walleye dermal sarcoma virus (WDSV) or murine stem cell virus (MSCV)), a spumavirus (e.g., a human foamy virus (HFV) or simian foamy virus (SFV)), a lentivirus (e.g., simian immunodeficiency viruses (SIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), caprine arthritisencephalitis virus (CAEV), or the ovine visna-maedi virus (VMV)), an alphavirus (e g., Semliki forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis (VEE)), a flavivirus (e.g., Kunjin, West Nile, Dengue), a rhabdovirus (e.g., rabies), a measles virus, (e.g., MV-Edm), Newcastle disease virus, a poxvirus (e.g., Vaccinia virus) or a picornavirus (e g., Coxsackievirus).

[0402] In one example, the viral vector is derived from the genome of a retrovirus. For example, the viral vector is a retroviral vector.

[0403] In one example, the viral vector is derived from the genome of a lentivirus. For example, the viral vector is a lentiviral vector. In one example, the lentiviral vector is a pseudotyped lentiviral vector. In another example, the lentiviral vector is a self-inactivating lentiviral vector.

[0404] In one example, the viral vector is derived from the genome of human immunodeficiency virus (HIV). For example, the viral vector is derived from the genome of HIV-1. It will be apparent to the skilled person that to increase safety, the viral vector only contains HIV genes which are necessary for infection and gene delivery, whilst the genes necessary for replication and virulence factors have been removed. For example, the envelope protein of HIV- 1 is exchanged with that of another virus (e.g., VSV-G protein from Vesicular stomatitis Indiana virus (VSV)) to allow infection of a wide range of target cells.

[0405] Methods for the production of viruses, viral vectors and viral particles will be apparent to the skilled person and / or described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48: 362-377; Schweizer and Merten (2010) Curr. Gene Ther. 10: 474-486; and Rodrigues etal., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2: 15-40. For example, the methods generally involve introducing plasmids encoding the polynucleotide of the disclosure together with packaging components encoding the structural proteins required for virion assembly. These plasmids can be transiently transfected into the cells, or a producer cell line is created with stable integration of the plasmids with inducible promoters, in which lentivirus production can be induced. Once virus production has been induced, the release of the virus occurs by budding after successful assembly within the cells. The lentivirus (comprising the polynucleotide encoding the CAR of the disclosure introduced into its genome) is harvested from these producer cells and subsequently purified and concentrated for use in the downstream process, e.g., in the transduction of immune cells.

[0406] The skilled person will be aware of the cell types suitable for transfection with viral plasmids (e.g., producer cells, or ‘host’ cell). For example, mammalian cells are well known hosts for the production of viral vectors and are suitable for transfection with viral plasmids.

[0407] As used herein, “transfection”, “transformation”, or transduction” refer to the introduction of one or more exogenous polynucleotides into a cell by using physical or chemical methods.

[0408] The host or producer cell may be selected from any cell allowing production of e.g., the lentivirus. According to one example, the cell is selected from a human cell (HEK293T, HEK293FT, HEK293S, HEK29FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEKEBNA1-6E, HEK293MSR and HEK293A cells), a murine cell (NIH-3T3), a murine cell (Mpf), a canid cell (D17), and derivatives thereof. According to one example, the cell is selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells, and derivatives thereof.

[0409] The skilled person will be aware that in addition to the polynucleotide sequences encoding the CAR, the vector additionally comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators and internal ribosome entry sites (IRES), that provide for the expression of the polynucleotide sequence in the host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0410] Genetically Modified Cells

[0411] The present disclosure also provides a genetically modified cell comprising the vector of the disclosure.

[0412] As used herein, the term “cell” refers to any type of cell that can contain the expression vector. For example, the cell is modified to express an expression vector (e.g., a retroviral, lentiviral, adenoviral, adeno-associated viral vector). In one example, the cell is modified to express a CAR of the present disclosure.

[0413] Methods of genetically modifying cells with viral vectors will be apparent to the skilled person and / or described herein. For example, the lentivirus (comprising the polynucleotide encoding the CAR of the disclosure introduced into its genome) is harvested from the producer cells and applied to the cells to be modified, resulting in expression of the CAR of the disclosure by the modified cell.

[0414] In one example, the cell is a eukaryotic cell, for example, a plant, animal, fungi, or algae. In another example, the cell is a prokaryotic cell, for example bacteria or protozoa.

[0415] In one example, the cell is a cultured cell or a primary cell, i.e., isolated directly from an organism (e g., a human).

[0416] In one example, the cell is an adherent cell or a suspended cell, i.e., a cell that grows in suspension.

[0417] Suitable cells for use in the present disclosure will be apparent to the skilled person and / or are disclosed herein.

[0418] In one example, the cell is an immune cell. For example, the cell is an immune effector cell. In one example, the immune effector cell expresses FcyRIII and performs antibody-dependent cellular cytotoxicity (ADCC) effector function.

[0419] In one example, the immune effector cell is selected from the group consisting of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, T cells, neutrophils, eosinophils and combinations thereof.

[0420] In one example, the cell is of hematopoietic origin. For example, the cell is a neutrophil, an eosinophil, a basophil, a lymphocyte, or a monocyte. In one example, the lymphocyte is a T cell, a B cell, a NK cell or precursor thereof.

[0421] In one example, the cell expresses CD3 polypeptides. For example, the cell expresses a CD3y, a CD3e, a CD3(^ or CD35 polypeptide.

[0422] In one example, the cell expresses a T cell receptor (TCR) polypeptide. For example, a TCRa or TCRp polypeptide.

[0423] In one example, the cell expresses CD27, CD28, CD4 and / or CD8. For example, the cell expresses CD4 and / or CD8.

[0424] In one example, the cell is a T-cell. For example, the T-cell is a human T-cell. For example, isolated from a human. In one example, the T cells are autologous T cells. In one example, the T cells are allogeneic T cells.

[0425] In one example, the T-cell is of any developmental stage. Stages of T cell differentiation include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells, from least to most differentiated. Methods of identifying T cells at different stages of differentiation will be apparent to the skilled person and / or described herein. For example, naive T cells express the cell surface markers CCR7+, CD62L+, CD45RO-, CD95-, stem central memory T cells (Tscm) express CCR7+, CD62L+, CD45RO-, CD95+, central memory T cells (Tcm) express CCR7+, CD62L+, CD45RO+, CD95+, effector memory T cells (Tern) express CCR7-, CD62L-, CD45RO+, CD95+, and terminal effector T cells (Teff) express CCR7-, CD62L-, CD45RO-, CD95+.

[0426] In one example, the T cell is a gamma delta (yb) T cell, a cytotoxic T cell, or a helper T cell. In another example, the T cell is a CD3+ / CD8+ T cell. In another example, the T cell is a CD4+ / CD8+ double positive T-cell, a CD4+ helper T-cell, (e.g., Th and Th2 cells), a CD8+ T- cell (e.g., a cytotoxic T-cell), a tumour infiltrating cell, a memory T-cell, or a naive T-cell. In one embodiment, the T-cell is a CD8+ T-cell or a CD4+ T-cell. Suitable T-cell lines for use in the present disclosure will be apparent to the skilled person and / or described herein. For example, suitable T cell lines are available from, e.g., the American Type Culture Collection (ATCC), and the German Collection of Microorganisms and Cell Cultures (DSMZ) and include, for example, Jurkat cells (ATCC TIB- 152), Sup-Tl cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.

[0427] In one example, the cell is a natural killer (NK) cell. In one example, the NK cell is a human NK cell. For example, the NK cell is isolated from a human. In one example, the NK cells are autologous NK cells. In one example, the NK cells are allogeneic NK cells. Exemplary NK cell lines suitable for use in the present disclosure are available from, e.g., the American Type Culture Collection (ATCC) and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), and derivatives thereof.

[0428] Methods of obtaining cells (e.g., T cells or NK cells) suitable for use in the present disclosure will be apparent to the skilled person and / or described herein.

[0429] In one example, the cell is a cultured cell, a primary cell, or a cell from a cultured cell line, or a cell obtained from a mammal. In one example, the cell is a cultured cell. In another example, the cell is a primary cell. In a further example, the cell is a cultured cell line. In one example, the cell is a cell obtained from a mammal (e.g., a human subject). In one example, the cell has been previously obtained from the subject (i.e., the human subject). In one example, the cells have been obtained from a tissue sample or biopsy.

[0430] It will be apparent to the skilled person that if obtained from a mammal, the cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. Cells for use in the present disclosure can also be enriched for or purified. Such methods may involve removing or substantially reducing the amount of, erythrocytes, platelets, serum and / or plasma in a sample. Methods disclosed herein may be performed on isolated immune cells, or a sample containing immune cells in addition to other cells.

[0431] In one example, the genetically modified cell of the disclosure is a CAR-expressing immune cell. In another example, the genetically modified cell of the disclosure is a CAR-T cell. In a further example, the genetically modified cell of the disclosure is a CAR-NK cell.

[0432] In one example, methods of producing a genetically modified cell of the disclosure comprises the stimulation and / or expansion of the immune cell. For example, stimulation and / or expansion of PBMCs or an immune cell population (e.g., T cells) from within a population of immune cells (e.g., PBMCs). In one example, a population of T-cells is expanded from within a population of PBMCs by stimulation of the T-cells within the population of PBMCs. In one example, stimulation and / or expansion of T cells involves stimulation of a population of PBMCs. In one example, a population of T-cells are expanded from within a population of tumor-infiltrating lymphocytes, by stimulation of the T-cells within the population of tumor-infiltrating lymphocytes. For example, in one example, stimulation and / or expansion of T cells involves stimulation of a population of tumor-infiltrating lymphocytes. In one example, a population of T-cells are expanded from within a population of T-cells (e.g., a population of T cells of heterogeneous specificity) obtained from a blood sample, a population of PBMCs, or from a population of tumor-infiltrating lymphocytes. In one example, the method involves the stimulation and / or expansion of T cells.

[0433] In one example, the cell is contacted with a transduction enhancer during a method of producing a genetically modified cell. For example, methods of producing a genetically modified cell of the disclosure comprise expanding and / or culturing the cell in the presence of a transduction enhancer. Transduction enhancers suitable for use in the present disclosure will be apparent to the skilled person and / or described herein. For example, transduction enhancers include, but are not limited to, cationic polymers (e.g., polybrene), fibronectin or fibronectin fragments (e.g., RetroNectin® or vitronectin), rapamycin and prostaglandin E2 (PGE2).

[0434] In one example, the transduction enhancer is a human fibronectin fragment.

[0435] In one example, the transduction enhancer is RetroNectin®. The skilled person will understand that RetroNectin® is a recombinant human fibronectin (CH-296) fragment which contains three functional domains: (1) an N-terminal cell-binding domain, which binds cells via VLA-5 integrin (this domain contains an RGDS motif and comprises 3 fibronectin type III domains 8, 9 and 10), (2) a heparin-binding domain, comprising 3 fibronectin type III domains 12, 13 and 14, and (3) a C-terminal CS-1 sequence, which binds cells via VLA-4 integrin.

[0436] In one example, the transduction enhancer is vitronectin.

[0437] Additional transduction enhancers will be apparent to the skilled person and are described, for example, in WO2024023245, hereby incorporated by reference in its entirety.

[0438] The present disclosure also provides at least one donor cell bank comprising genetically modified T cells expressing a CAR as described herein, wherein the scFv surface expression is present on at least 60% of live T cells.

[0439] The present disclosure also provides a population of genetically modified T cells expressing a CAR as described herein obtained from a single donor. Thus, the present disclosure also provides a population of genetically modified T cells expressing a CAR as described herein, wherein scFv surface expression is present on at least 60% of live T cells.

[0440] The present disclosure also provides a population of genetically modified T cells expressing a CAR as described herein obtained from multiple donors, wherein the donor samples are subsequently pooled. In one embodiment, the population of genetically modified T cells expressing a CAR as described herein obtained from multiple donors, wherein the donor samples are subsequently pooled and expanded as required. Thus, the present disclosure also provides a pooled population of genetically modified T cells expressing a CAR as described herein, wherein the scFv surface expression is present on at least 60% of live T cells.

[0441] In one example, the scFv surface expression is present on at least 65% of live T cells. In one example, the scFv surface expression is present on at least 70% of live T cells. In one example, the scFv surface expression is present on at least 75% of live T cells. In one example, the scFv surface expression is present on at least 80% of live T cells. In one example, the live T cells express CD4+, CD8+, CD4-CD8- and / or CD4+CD8+. In one example, at least 50% of the live T cells express CD4+. In one example, at least 55% of the live T cells express CD4+. In one example, at least 30% of the live T cells express CD8+. In one example, at least 35% of the live T cells express CD8+. In one example, at least 50% of the live T cells express CD4+ and at least 30% of the live T cells express CD8+.

[0442] In one example, the live T cells exhibit a naive T cell (Tn) phenotype, a central memory (Tcm) phenotype, an effector memory (Tem) phenotype, and an effector (Teff) phenotype. In one example, at least 60% of the live T cells exhibit a Tn phenotype. In one example, at least 65% of the live T cells exhibit a Tn phenotype. In one example, at least 70% of the live T cells exhibit a Tn phenotype. In one example, at least 75% of the live T cells exhibit a Tn phenotype. In one example, at least 80% of the live T cells exhibit a Tn phenotype. In one example, at least 85% of the live T cells exhibit a Tn phenotype. In one example, at least 10% of the live T cells exhibit a Teff phenotype.

[0443] In one example, the scFv surface expression is measured by flow cytometry. In one example, the scFv surface expression is measured by flow cytometry using a staining reagent specific to the linker region of the CAR. For example, the scFv surface expression is measured by flow cytometry using a staining reagent specific to a G4S linker.

[0444] Pharmaceutical Compositions

[0445] The present disclosure provides a composition comprising a population of genetically modified cells of the disclosure for use as a medicament. In one example, the present disclosure provides a composition for use in adoptive cell therapy. For example, adoptive T cell therapy.

[0446] Accordingly, the present disclosure provides a composition comprising a population of genetically modified cells of the disclosure and a pharmaceutically acceptable carrier.

[0447] It will be apparent to the skilled person that the population of genetically modified cells of the disclosure, e.g., population of genetically modified T cells, comprises a population of cells at different stages of differentiation.

[0448] Methods for preparing a compound into a suitable form for administration (e.g., a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990).

[0449] An appropriate pharmaceutical composition comprising a population of genetically modified cells to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. Exemplary carriers include water, saline, Ringer's solution, dextrose solution and <5% human serum albumin. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0450] The optimum concentration of the ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

[0451] In one example, the composition additionally comprises human serum albumin. In one example, the composition comprises <5% human serum albumin. For example, the composition comprises 5% human serum albumin. In another example, the composition comprises 2.5% human serum albumin. In a further example, the composition comprises 0.25% human serum albumin.

[0452] In one example, the composition additionally comprises dimethylsulfoxide (DMSO). For example, the composition comprises <7.5% DMSO. In one example, the composition comprises 7.5% DMSO. In another example, the composition comprises 5% DMSO.

[0453] In one example, the composition additionally comprises sodium chloride.

[0454] In one example, the composition additionally comprises dextrose. For example, the composition comprises 5% dextrose.

[0455] Upon formulation, compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The dosage ranges for the administration of the composition of the disclosure are those large enough to produce the desired effect. For example, the composition comprises an effective amount of the population of genetically modified cells. In one example, the composition comprises a therapeutically effective amount of the genetically modified cells.

[0456] The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

[0457] In one example, the composition is formulated for injection or infusion, e.g., via intravenous infusion.

[0458] In one example, the CAR-T cells are formulated at a dose of about 1.0 x 105to 6 x 108cells / kg. In one example, the CAR-T cells are formulated at a dose of about 0.2 to 5.0 x 106cells / kg. For example, the CAR-T cells are formulated at a dose of about 2 x 106cells / kg. In another example, the CAR-T cells are formulated at a dose of about 0.1 to 2.5 x io8cells / kg. In one example, the CAR-T cells are formulated at a dose of less than 1.0 x 108cells per subject. For example, the CAR-T cells are formulated at a dose of between 0.5-1.0 x 108cells per subject. In one example, the CAR-T cells are formulated at a dose of 0.6-6.0 x io8cells per subject. For example, the CAR-T cells are formulated at a dose of 2 x 108cells per subject. Methods of Treatment

[0459] The present disclosure provides methods of using the composition or a population of genetically modified cells of the disclosure as a medicament. For example, in a method of treating an LMA expressing malignancy in a subject in need thereof.

[0460] The present disclosure also provides a method of treating an LMA expressing malignancy ease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0461] The term “LMA expressing malignancy” as used herein refers to any proliferative disease that expresses LMA. Exemplary related pathologies include myeloma, plasmacytoma, amyloidosis and B-cell malignancies.

[0462] The terms “multiple myeloma” or “myeloma” are used in the context of the present disclosure to refer to cancer of plasma cells. In the context of the present disclosure, these terms encompass secretory myeloma, non-secretory myeloma, light chain only myeloma, smouldering myeloma and related pathologies. Subjects with multiple myeloma can be characterised into various subject populations. Exemplary populations are described in (Rajkumar et al. 2011). In an example, the multiple myeloma presents with amyloid deposits.

[0463] The term “amyloidosis” as used herein refers to a clonal plasma cell disorder associated with secretion of immunoglobulin free light chains. Approximately, 80% of patients produce a lambda isotype. Fragments of the light chain variable domain play a critical role in forming amyloid fibrils that deposit in peripheral organs leading to organ dysfunction.

[0464] In one example, the present disclosure encompasses methods of treating a B-cell malignancy wherein the malignant B-cells express LMA.

[0465] In another example, the methods of the present disclosure can be used to reduce lambda free light chain levels in a subject (i.e., reduce the amount of lambda light chain in a subject that is not expressed on the cell membrane, e.g., lambda light chain in serum).

[0466] In one example of any method described herein, a composition or population of genetically modified cells of the disclosure are for use in adoptive cell therapy.

[0467] Methods of adoptive cell therapy will be apparent to the skilled person and / or are described herein. For example, cells (e.g., immune cells) are obtained from a subject, e.g., by drawing a blood sample from which the cells are isolated. The cells are then genetically modified (e.g., with a CAR) and / or expanded, and then administered either to the same subject (in the case of adoptive therapy with autologous / autogeneic cells) or to a different subject (in the case of adoptive therapy of allogeneic cells).

[0468] Additional methods of adoptive cell therapy are described in Kalos and June, Immunity (2013) 39(l):49-60, and Davis et al., Cancer J. (2015) 21(6):486-491, both of which are hereby incorporated by reference in their entirety.

[0469] In one example, the method comprises isolating an immune cell, or generating / expanding a population of immune cells. In one example, the method comprises modifying an immune cell to comprise / express a CAR according to the present disclosure. In one example, the method comprises modifying an immune cell to comprise / express nucleic acid encoding a CAR according to the present disclosure.

[0470] In one example, the method comprises administering to a subject an immune cell modified to express / comprise a CAR according to the present disclosure (or modified to express / comprise a nucleic acid encoding the CAR).

[0471] In one example, the subject from which the immune cells (e.g., T cells) are isolated is the same subject to which the composition or population of genetically modified cells of the disclosure are administered. For example, the adoptive cell therapy is autologous / autogeneic cell therapy.

[0472] In one example, the subject from which the immune cells (e g., T cells) are isolated is a different subject to the subject to which the composition or population of genetically modified cells of the disclosure are administered. For example, the adoptive cell therapy is allogeneic cell therapy.

[0473] In one example, the method additionally comprises treating the genetically modified cells or the subject to induce / enhance expression of the CAR and / or to induce / enhance proliferation or survival of immune cells comprising / expressing the CAR. In one example, the method additionally comprises treating the genetically modified cells or the subject to induce / enhance expression of the CAR. In another example, the method additionally comprises treating the genetically modified cells or the subject to induce / enhance proliferation or survival of immune cells comprising / expressing the CAR.

[0474] In one example of the methods described herein, administration of the composition or population of genetically modified cells disclosed herein reduces the development / progression of an LMA expressing malignancy, alleviates the symptoms of an LMA expressing malignancy, or reduces the pathology of an LMA expressing malignancy. In one example, the methods prevent progression of the LMA expressing malignancy. For example, to prevent worsening of, or to slow the rate of development of, the LMA expressing malignancy. In one example, the methods lead to an improvement in the LMA expressing malignancy. For example, a reduction in the severity of symptoms of the LMA expressing malignancy e, or a reduction in some other correlate of the severity / activity of the LMA expressing malignancy. In one example, the methods prevent development of the LMA expressing malignancy to a later stage. For example, a chronic stage or metastasis.

[0475] In one example, the subject has, or suffers from, an LMA expressing malignancy. For example, the subject is in need of treatment (i.e., in need thereof).

[0476] In one example, the subject has been diagnosed with an LMA expressing malignancy. Methods of diagnosing an LMA expressing malignancy will be apparent to the skilled person and / or described herein.

[0477] In one example, the CAR-T cells are administered at a dose of about 1.0 x 105to 6 x 108positive viable T cells / kg. In one example, the CAR-T cells are administered at a dose of about 0.2 to 5.0 x 106positive viable T cells / kg. For example, the CAR-T cells are administered at a dose of about 2 x 106positive viable T cells / kg. In another example, the CAR-T cells are administered at a dose of about 0.1 to 2.5 x io8positive viable T cells / kg. In one example, the CAR-T cells are administered at a dose of less than 1.0 x 108positive viable T cells per subject. For example, the CAR-T cells are administered at a dose of between 0.5-1.0 x 108positive viable T cells per subject. In one example, the CAR-T cells are administered at a dose of 0.6-6.0 x 108positive viable T cells per subject. For example, the CAR-T cells are administered at a dose of 2 x 108positive viable T cells per subject.

[0478] In one example, one or more administrations of the genetically modified cells of the disclosure are provided to a subject in need thereof. The length of time between each administration may be of any suitable duration, including for example, 1-7 days, 1-4 weeks, 1-12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Multiple infusions within about a year may be employed, in some cases. In cases wherein more than one administration of cells is provided to the subject, the antigen to which the cells are targeted may or may not be the same antigen that was targeted with the cells utilised in earlier administration(s).

[0479] In one example, a composition of the present disclosure is administered in combination with an additional therapy useful for treating the malignant proliferative disease, e.g., cancer.

[0480] In one example, the additional therapy may be a standard of care therapy for treating the malignant proliferative disease, or for treating a complication associated with the malignant proliferative disease.

[0481] In one example, the standard of care therapy is administered prior to, concurrently or simultaneously, or after treatment with a composition of the present disclosure.

[0482] In one example, the composition of the disclosure is administered before the additional therapy. In one example, the composition of the disclosure is administered after the additional therapy. In one example, the composition of the disclosure is administered concurrently or simultaneously with the additional therapy. Standard of care therapies for treatment of the disease will be apparent to the skilled person and / or are described herein.

[0483] Kits

[0484] Another example of the disclosure provides kits containing compositions useful for treating an LMA expressing malignancy described herein.

[0485] In one example, the kit comprises (a) a container comprising a population of genetically modified cells as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating an LMA expressing malignancy in a subject in need thereof.

[0486] In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for treating the LMA expressing malignancy and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the population of genetically modified cells (e.g., CAR-T cells). The label or package insert indicates that the composition is used for treating of an LMA expressing malignancy in a subject eligible for treatment, with specific guidance regarding dosing amounts and intervals of compound and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0487] EXAMPLES

[0488] Example 1: Transgene constructs

[0489] The single-chain variable fragments (scFvs) were designed based on the nucleotide sequences coding for the heavy and light variable regions of 7F12 (SEQ ID NO: 8 and 4) or 10B3 (SEQ ID NO: 28 and 24) and a (Gly4Ser)3linker (SEQ ID NO: 41).

[0490] These candidate scFvs were cloned into CAR cassettes comprising a CD8a stalk, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain and the CD3(j signalling domain. These CAR cassettes were then cloned into a lentiviral plasmid comprising an EF-la promoter and a Kozak sequence (Figure 1).

[0491] A bicistronic LMA.CAR was generated based on the LV.7F12-CAR cassette and 10B3-CAR cassette as described in Example 1 and separated by a P2A ribosomal skip sequence (SEQ ID NO: 51). This 7F12-P2A-10B3-CAR cassette was cloned into a lentiviral plasmid comprising an EF-la promoter and Kozak sequence (Figure 2).

[0492] Example 2: Lentivirus production

[0493] HEK 293T cells at 30,000 cell / cm2were transfected in six well plates with 1 pl of the lentiviral plasmids (Table 1) and a DNA:polyethylenimine (PEI) ratio of 1 :3. The transfected HEK 293T cells were stained with 6pg or 12.5 pg lambda light chain and a fluorescent Strep-phycoerythrin (PE) conjugate protein at a 1:400 dilution. Successful cloning and CAR expression was validated using fluorescent activated cell sorting (FACS) and direct sequencing.

[0494] Figure 4 demonstrates that LV.7F12-CAR provides the highest FACS titre.

[0495] Table 1. Lentiviral plasmids

[0496] Plasmid LV.7F12-CAR LV.7F12-P2A-10B3-CAR LV.10B3-CAR (pg / cm2) (pg / cm2) (pg / cm2) Rev 0.037 0.037 0.037

[0497] VSV-G 0.015 0.015 0.015

[0498] Gag / Pol 0.024 0.024 0.024

[0499] Transfer 0.032 0.038* 0.032

[0500]

[0501] Total DNA 0.108 0.114 0.108

[0502] * Higher quantity used to maintain equivalent molar ratios Example 3: FACS functional and qPCR Genomic Titration Analysis

[0503] The HEK 293T cells that were transduced with the lentiviral plasmids (e.g., LV.7F12-CAR, LV.10B3-CAR and LV.7F12-P2A-10B3-CAR) in Example 2 were lysed, lambda light chain (LLC), Protein-L and / or G4S staining reagents were added and the results used to quantify expression. All CARs were successfully transferred and expressed and all reagents could detect the CAR as analysed using FACS (Tables 2 and 3).

[0504] Table 2. Genomic Titre and Functional Titre with LLC staining

[0505] Samples qPCR Titre (TU / mL) FACS Titre (TU / mL) qPCR / FACS Titration Standard 8.52 E + 06 7.42 E + 06 1.1

[0506] CellVec Control 1.89 E + 07 1.54 E + 06 1.2

[0507] 7F12-CAR 1.96 E + 07 1.74 E + 07 1.1

[0508] 7F12-P2A-10B3-CAR 2.66 E + 06 1.40 E + 06 1.9

[0509]

[0510] 10B3-CAR 1.55 E + 07 1.79 E + 06 9.3

[0511] Table 3. Genomic Titre and Functional Titre with LLC, Protein-L or G4S staining

[0512] Samples Staining qPCR Titre FACS Titre qPCR / FACS Reagent (TU / mL) (TU / mL)

[0513] 7F12-CAR LLC 2.16 E + 07 1.36 E + 07 1.6

[0514] Protein-L 1.70 E + 07 1.17 E + 07 1.5

[0515] G4S 2.10 E + 07 1.26 E + 07 1.7

[0516] 10B3-CAR LLC 2.56 E + 07 8.11 E + 05 31.6

[0517] Protein-L 2.48 E + 07 7.30 E + 06 3.4

[0518]

[0519] G4S 2.40 E + 07 8.18 E + 06 2.9

[0520] 7F12-CARwas detected by qPCR indicating HEK 293 genomic integration. FACS indicated that 7F12-CAR was detected by binding to biotinylated LLC, protein L (indicating binding to immunoglobulin light chain) and G4S antibody (specifically binds to the amino acids of the linker peptide between the VH and VL regions of the scFv, e.g., indicating successful scFv expression on cell surface).

[0521] 10B3-CARwas detected by qPCR indicating HEK 293 genomic integration. FACS indicated that 10B3-CAR scFv was detected by binding to biotinylated LLC (albeit at lower levels as compared to 7F12-CAR), protein L and G4S antibody.

[0522] 7F12-P2A-10B3-CAR was detected by qPCR indicating HEK 293 genomic integration. FACS indicated that 7F12-P2A-10B3-CAR was detected by binding to biotinylated LLC.

[0523] In summary, 7F12-CAR was both genomically integrated and functionally present as a CAR construct, while demonstrating the ability to bind to LLC. The 10B3-CARwas also genomically integrated and detected on the surface of the cell using indirect methods such as protein L and G4S, but it demonstrated less binding of the LLC. This was particularly unexpected as the 10B3 monoclonal antibody (e.g., comprising the same VH and VL regions as the 10B3-CAR) was demonstrated to bind with high affinity to all free LLC whereas, the 7F12 monoclonal antibody (e.g., comprising the same VH and VL regions as the 7F12-CAR) was demonstrated to bind with low affinity to only 50% of LLC.

[0524] The qPCR genomic titration was also used to directly compare number of positive cells and number of vector genomes per cell in the same population.

[0525] High ratios (e.g., 3 or higher) may indicate that the lentiviral plasmid genomes have been efficiently integrated into the target cell genome but that the expressed protein has not been efficiently detected by FACS (e.g., inefficient staining method, poor protein processing etc.). Low ratios (e.g., 0.8 and lower) may indicate detection of false positive by FACS titration.

[0526] The ratio between genomic titre and functional titre for LV.7F12-CAR confirmed both efficient expression and detection of the expressed protein upon plasmid genome integration (Tables 2 and 3). The ratios were comparable to the titration standard (Table 2). For 10B3-CAR the ratio indicates poor expression and / or detection of the expressed protein by FACS 7F12-P2A-10B3-CAR demonstrated a poor viral titre production.

[0527] Example 4: CAR Expression in Donor Cells

[0528] Normal donor T cells were seeded at a density of 30,000 cells / cm2and transduced with the lentiviral plasmids (e.g., LV.10B3-CAR and LV.7F12-P2A-10B3-CAR) described in Example 2. The resulting CAR-expressing T cells were expanded by co-culturing with autologous peripheral blood mononuclear cells (PBMCs) in the presence of interleukin- 15 (IL-15) at 10 ng / mL. Cultures were maintained for two weeks, with weekly replacement of PBMC feeder cells and replenishment of IL- 15 two to three times per week.

[0529] At the end of the culture period, T cells were harvested and assessed for CAR expression and T cell phenotype by FACS. CAR expression was measured using a G4S-specific staining reagent that binds to the linker region incorporated into the CAR construct. Both LV.10B3-CAR and LV.7F12-P2A-10B3-CAR constructs demonstrated robust surface expression on T cells, with sustained expression observed up to day 12 (Figure 5).

[0530] For intracellular cytokine staining, 2 x 105CAR T cells were stimulated with target cells at a 1 : 1 ratio for 5 hours. Monensin (2 pM) and Brefeldin A (1 pg / mL) (both from BD Biosciences) were added after 1 hour to inhibit cytokine secretion. CAR T cells stimulated non-specifically with phorbol 12-myristate 13-acetate (PMA, 50 ng / mL) and ionomycin (1 pg / mL) (Sigma-Aldrich) served as positive controls, while unstimulated cells served as negative controls.

[0531] Following stimulation, cells were harvested, washed, and surface- stained for CD3, CD4, and CD8. Cells were then fixed and permeabilised using Cytofix / Cytoperm™ buffer (BD Biosciences) and stained intracellularly with an anti-interferon gamma (IFN-y) antibody (BD Biosciences), followed by additional washes with Perm / Wash buffer. Samples were analysed on a FACSCanto™ II flow cytometer, with acquisition of at least 30,000 events per sample.

[0532] Figure 6 illustrates the distribution of T cell phenotypes among CD3+CAR+cells at two time points, Day 6 and Day 12. The majority of CAR-expressing cells were CD4+, followed by a substantial proportion of CD8+cells. Minor populations of CD4 CD8 and CD4+CD8+cells were also detected. These results indicate stable CAR expression for both 7F12.CAR and 10B3.CAR across key T cell subsets overtime.

[0533] Day 12 cultures were analysed for memory phenotype. It was found that both LV.10B3-CAR and LV.7F 12-P2A-10B3-CAR had a predominately naive T cells (TN) phenotype (93.3% of live cells for 7F12.CAR and 92% of live cells for 10B3.CAR) (Figure 7).

[0534] Example 5: Further experiments

[0535] Cytotoxicity assays using human myeloma cell lines and non-myeloma cell lines will be undertaken to determine targeting of LMA expressing cells. Cytotoxicity assays in the presence of free LLC will be undertaken determine the potential competition with serum light chains and if T cell exhaustion occurs.

[0536] Release of interferon gamma (TFNy) and tumour necrosis factor alpha (TNFa) by the CARs will be investigated to ensure the intracellular domains are activated by scFv binding to LMA on the cell surface. The stability of the CAR constructs will also be determined during T cell proliferation.

Claims

1. CLAIMS:

1. A polynucleotide encoding a chimeric antigen receptor (CAR), wherein the polynucleotide comprises:3.(i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the nucleotide sequence encoding the modified scFv comprises: a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and4.b) a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;5.(ii) a nucleotide sequence encoding a transmembrane domain; and6.(iii)a nucleotide sequence encoding at least one co-stimulatory domain.

2. The polynucleotide of claim 1, wherein the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4 and / or a VH comprising a sequence set forth in SEQ ID NO: 8.

3. The polynucleotide of claim 1 or 2, wherein the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 17.

4. A polynucleotide encoding a chimeric antigen receptor (CAR), wherein the polynucleotide comprises:10.(i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that binds lambda myeloma antigen (LMA), wherein the nucleotide sequence encoding the modified scFv comprises:11.a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and12.b) a heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27;13.(ii) a nucleotide sequence encoding a transmembrane domain; and14.(iii)a nucleotide sequence encoding at least one co-stimulatory domain.

5. The polynucleotide of claim 4, wherein the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24 and / or a VH comprising a sequence set forth in SEQ ID NO: 28.

6. The polynucleotide of claim 4 or 5, wherein the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 37.

7. The polynucleotide of any one of claims 1 to 6, wherein:17.(i) the transmembrane domain comprises a CD28 transmembrane domain; and / or18.(ii)the at least one co-stimulatory domain is selected from the group consisting of a 4-1BB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD3Q signalling domain.

8. The polynucleotide of any one of claims 1 to 7, wherein the co-stimulatory domain comprises the 4-1BB signalling domain and the CD3(^ signalling domain.

9. The polynucleotide of any one of claims 1 to 8, wherein the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region.

10. The polynucleotide of claim 9, wherein the hinge region is a CD8a stalk hinge region.

11. The polynucleotide of any one of claims 1 to 10, wherein the VL and the VH are covalently linked to each other via a linker.

12. The polynucleotide of claim 11, wherein the linker is a peptide linker comprising at least 2 amino acids in length.

13. The polynucleotide of claim 11 or 12, wherein the linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3 linker, a (Gly)s linker, a (Gly)e linker and a (GGGS)n linker, wherein n=l, 2, 3 or 4.

14. The polynucleotide of any one of claims 11 to 13, wherein the linker is a (Gly4Ser)3 linker and the nucleotide sequence encoding the linker comprises a sequence set forth in SEQ ID NO: 41.

15. The polynucleotide of any one of claims 7 to 14, wherein:27.(i) the nucleotide sequence encoding the CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 43; (ii)the nucleotide sequence encoding the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 45; and / or28.(iii)the nucleotide sequence encoding the CD3 signalling domain comprises a sequence set forth in SEQ ID NO: 47.

16. The polynucleotide of any one of claims 9 to 16, wherein the nucleotide sequence encoding the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 49.

17. The polynucleotide of any one of claims 1 to 3 and 7 to 16, wherein the polynucleotide encoding the CAR comprises a sequence set forth in SEQ ID NO: 19.

18. The polynucleotide of any one of claims 4 to 16, wherein the polynucleotide encoding the CAR comprises a sequence set forth in SEQ ID NO: 39.

19. A polynucleotide encoding a first chimeric antigen receptor (CAR) and a second CAR, wherein the polynucleotide comprises:33.(i) a nucleotide sequence encoding the first CAR comprising:34.a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a first modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), a nucleotide sequence encoding a first transmembrane domain, and a nucleotide sequence encoding a first at least one co-stimulatory domain, wherein the nucleotide sequence encoding the first modified scFv comprises: i. a first light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 1, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 2, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 3, and35.ii. a first heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 5, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 6, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 7;36.(ii) a nucleotide sequence encoding the second CAR comprising:37.a) a nucleotide sequence encoding an extracellular antigen binding domain comprising a second modified scFv that specifically binds LMA, a nucleotide sequence encoding a second transmembrane domain, and a nucleotide sequence encoding a second at least one co-stimulatory domain, wherein the nucleotide sequence encoding the second modified scFv comprises:38.i. a second light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 21, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 22, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 23, and ii. a second heavy chain variable region (VH) comprising: a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 25, a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 26, and a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 27; and39.(iii)a nucleotide sequence encoding a cleavable domain operably linking the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR.

20. The polynucleotide of claim 19, wherein:41.(i) the nucleotide sequence encoding the first modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 4 and / or a VH comprising a sequence set forth in SEQ ID NO: 8; and / or42.(ii)the nucleotide sequence encoding the second modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 24 and / or a VH comprising a sequence set forth in SEQ ID NO: 28.

21. The polynucleotide of claim 19 or 20, wherein the nucleotide sequence encoding the first modified scFv comprises a sequence set forth in SEQ ID NO: 17 and / or the nucleotide sequence encoding the second modified scFv comprises a sequence set forth in SEQ ID NO: 37.

22. The polynucleotide of any one of claims 19 to 21, wherein:45.(i) the first transmembrane domain and / or the second transmembrane domain comprises a CD28 transmembrane domain; and / or46.(ii)the first at least one co-stimulatory domain and / or the second at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD30 signalling domain.

23. The polynucleotide of any one of claims 19 to 22, wherein the first at least one co-stimulatory domain and / or the second at least one co-stimulatory domain comprises the 4-1BB signalling domain and the CD3(^ signalling domain.

24. The polynucleotide of any one of claims 19 to 23, wherein the first extracellular antigen binding domain is operably linked to the first transmembrane domain through a first hinge region and / or wherein the second extracellular antigen binding domain is operably linked to the second transmembrane domain through a second hinge region.

25. The polynucleotide of claim 24, wherein the first hinge region and / or the second hinge region is a CD8a stalk hinge region.

26. The polynucleotide of any one of claims 19 to 25, wherein the first VL and the first VH are covalently linked to each other via a first linker and / or the second VL and the second VH are covalently linked to each other via a second linker.

27. The polynucleotide of claim 26, wherein the first linker and / or the second linker is a peptide linker comprising at least 2 amino acids in length.

28. The polynucleotide of claim 26 or 27, wherein the first linker and / or the second linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)i linker, a (Gly)s linker, a (Gly)e linker and a (GGGS)n linker, wherein n=l, 2, 3 or 4.

29. The polynucleotide of any one of claims 26 to 28, wherein the first linker and / or the second linker is a (GlyrSer)! linker and the nucleotide sequence encoding the first linker and / or the second linker comprises a sequence set forth in SEQ ID NO: 41.

30. The polynucleotide of any one of claims 22 to 29, wherein:54.(i) the nucleotide sequence encoding the first CD28 transmembrane domain and / or the second CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 43;55.(ii)the nucleotide sequence encoding the first 4-1BB signalling domain and / or the second 4- 1BB signalling domain comprises a sequence set forth in SEQ ID NO: 45; and / or (iii)the nucleotide sequence encoding the first CD3^ signalling domain and / or the second CD3(^ signalling domain comprises a sequence set forth in SEQ ID NO: 47.

31. The polynucleotide of any one of claims 25 to 30, wherein the nucleotide sequence encoding the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 49.

32. The polynucleotide of any one of claims 19 to 31, wherein the polynucleotide encoding the first CAR comprises a sequence set forth in SEQ ID NO: 19 and / or the polynucleotide encoding the second CAR comprises a sequence set forth in SEQ ID NO: 37.

33. The polynucleotide of any one of claims 19 to 32, wherein the cleavable domain is selected from the group consisting of a T2A domain, a P2A domain, E2A domain, a F2A domain and a furin domain.

34. The polynucleotide of claim 33, wherein the nucleotide sequence encoding the P2A domain comprises a sequence set forth in SEQ ID NO: 51.

35. A chimeric antigen receptor (CAR) encoded by the polynucleotide of any one of claims 136. A chimeric antigen receptor (CAR) comprising:61.(i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the modified scFv comprises:62.a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 9; a CDR2 comprising a sequence set forth in SEQ ID NO: 10; and a CDR3 comprising a sequence set forth in SEQ ID NO: 11; and63.b) a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 13; a CDR2 comprising a sequence set forth in SEQ ID NO: 14; and a CDR3 comprising a sequence set forth in SEQ ID NO: 15;64.(ii) a transmembrane domain; and65.(iii) at least one co-stimulatory domain.

37. The CAR of claim 36, wherein the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 12 and / or a VH comprising a sequence set forth in SEQ ID NO: 16.

38. The CAR of claim 36 or 37, wherein the modified scFv comprises a sequence set forth in SEQ ID NO: 18.

39. A chimeric antigen receptor (CAR) comprising:69.(i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds lambda myeloma antigen (LMA), wherein the modified scFv comprises:70.a) a light chain variable region (VL) comprising: a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 29; a CDR2 comprising a sequence set forth in SEQ ID NO: 30; and a CDR3 comprising a sequence set forth in SEQ ID NO: 31; and71.b) a heavy chain variable region (VH) comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 33; a CDR2 comprising a sequence set forth in SEQ ID NO: 34; and a CDR3 comprising a sequence set forth in SEQ ID NO: 35;72.(ii) a transmembrane domain; and73.(iii) at least one co-stimulatory domain.

40. The CAR of claim 39, wherein the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 32 and / or a VH comprising a sequence set forth in SEQ ID NO: 36.

41. The CAR of claim 39 or 40, wherein the modified scFv comprises a sequence set forth in SEQ ID NO: 38.

42. The CAR of any one of claims 36 to 41, wherein:76.(i) the transmembrane domain is a CD28 transmembrane domain; and / or77.(ii)the at least one co-stimulatory domain is selected from the group consisting of a 4-1BB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD3Q signalling domain.

43. The CAR of any one of claims 36 to 42, wherein the co-stimulatory domain comprises the 4- IBB signalling domain and the CD3(^ signalling domain.

44. The CAR of any one of claims 36 to 43, wherein the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region.

45. The CAR of claim 44, wherein the hinge region is a CD8a stalk hinge region.

46. The CAR of any one of claims 36 to 45, wherein the VL and the VH are covalently linked to each other via a linker, optionally wherein the linker is a peptide linker comprising at least 2 amino acids in length.

47. The CAR of claims 45 or 46, wherein the linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3linker, a (Gly)s linker, a (Gly)e linker and a (GGGS)n linker, wherein n=l, 2, 3 or 4.

48. The CAR of any one of claims 45 to 47, wherein the linker is a (Gly4Ser)3linker and comprises a sequence set forth in SEQ ID NO: 42.

49. The CAR of any one of claims 36 to 48, wherein:85.(i) the CD28 transmembrane domain comprises a sequence set forth in SEQ ID NO: 44; (ii)the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 46; and / or (iii)the CD3 signalling domain comprises a sequence set forth in SEQ ID NO: 48.

50. The CAR of any one of claims 44 to 49, wherein the CD8ot hinge region comprises a sequence set forth in SEQ ID NO: 50.

51. The CAR of any one of claims 36 to 38 and 42 to 50, wherein the CAR comprises a sequence set forth in SEQ ID NO: 20.

52. The CAR of any one of claims 39 to 50, wherein the CAR comprises a sequence set forth in SEQ ID NO: 40.

53. A polynucleotide encoding the CAR of any one of claims 36 to 52.

54. A vector comprising the polynucleotide of any one of claims 1 to 34 or 53.

55. The vector of claim 54, wherein the vector is a plasmid, a cosmid, a phage or a viral vector.

56. The vector of claim 55, wherein the viral vector is a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, a herpes simplex viral (HSV) vector or a retroviral vector.

57. A genetically modified cell comprising the vector of any one of claims 54 to 56.

58. A method of producing a genetically modified cell for adoptive cell therapy, the method comprising introducing a vector comprising the polynucleotide of any one of claims 1 to 34 or 53 into a cell, thereby producing the genetically modified cell.

59. The genetically modified cell of claim 57 or the method of claim 58, wherein the cell is an immune cell.

60. The genetically modified cell or the method of claim 59, wherein the immune cell is selected from the group consisting of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, T cells, neutrophils, eosinophils and combinations thereof.

61. The genetically modified cell of any one of claims 57, 59 and 60, or the method of any one of claims 58 to 60, wherein the cell is a T cell.

62. The genetically modified cell of any one of claims 57 and 59 to 61, or the method of any one of claims 58 to 61, wherein the T cell is a gamma delta (y5) T cell, a cytotoxic T cell, or a helper T cell.

63. A composition comprising a population of genetically modified cells of any one of claims 57 and 59 to 62, for use as a medicament.

64. A method of treating an LMA-expressing malignant proliferative disease in a subject in need thereof, the method comprising administering a composition of claim 63 or a population of genetically modified cells of any one of claims 57 and 59 to 62.

65. Use of a population of genetically modified cells of any one of claims 57 and 59 to 62 in the manufacture of a medicament for treating an LMA-expressing malignant proliferative disease in a subject in need thereof.

66. The method of claim 64 or the use of claim 65, wherein the LMA-expressing malignant proliferative disease is selected from the group consisting of multiple myeloma, Waldenstroms macroglobulinemia, mature B-cell malignancies, amyloidosis and combinations thereof.

Images