Cell

1 . A cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising an antigen-binding domain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22.

2 . A cell according to claim 1 , wherein each CAR comprises:

(i) an antigen-binding domain;

(ii) a spacer; and

(iii) a trans-membrane domain;

wherein the spacer of the first CAR is different to the spacer of the second CAR.

3 . A cell according to claim 2 , wherein the antigen-binding domain of the second CAR binds to an epitope on Ig domain 1, 2, 3 or 4 of CD22.

4 . A nucleic acid sequence encoding both the first and second chimeric antigen receptors (CARs) as defined in any of claims 1 to 3 .

5 . A nucleic acid sequence according to claim 4 , which has the following structure:

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR;

spacer1 is a nucleic acid sequence encoding the spacer of the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR;

spacer2 is a nucleic acid sequence encoding the spacer of the second CAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

which nucleic acid sequence, when expressed in a T cell, encodes a polypeptide which is cleaved at the cleavage site such that the first and second CARs are co-expressed at the T cell surface.

6 . A nucleic acid sequence according to claim 5 , wherein coexpr encodes a sequence comprising a self-cleaving peptide.

7 . A nucleic acid sequence according to claim 5 or 6 , wherein alternative codons are used in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination.

8 . A kit which comprises

(i) a first nucleic acid sequence encoding the first chimeric antigen receptor (CAR) as defined in any of claims 1 to 3 , which nucleic acid sequence has the following structure:

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR;

Spacer1 is a nucleic acid sequence encoding the spacer of the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR; and

(ii) a second nucleic acid sequence encoding the second chimeric antigen receptor (CAR) as defined in any of claims 1 to 3 , which nucleic acid sequence has the following structure:

AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR;

spacer2 is a nucleic acid sequence encoding the spacer of the second CAR; and

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR.

9 . A kit comprising: a first vector which comprises the first nucleic acid sequence as defined in claim 8 ; and a second vector which comprises the second nucleic acid sequence as defined in claim 8 .

10 . A kit according to claim 9 , wherein the vectors are integrating viral vectors or transposons.

11 . A vector comprising a nucleic acid sequence according to any of claims 4 to 7 .

12 . A retroviral vector or a lentiviral vector or a transposon according to claim 11 .

13 . A method for making a cell according to any of claim 1 to 3 , which comprises the step of introducing: a nucleic acid sequence according to any of claims 4 to 7 ; a first nucleic acid sequence and a second nucleic acid sequence as defined in claim 8 ; and/or a first vector and a second vector as defined in claim 9 or a vector according to claim 11 or 12 , into a cell.

14 . A method according to claim 13 , wherein the cell is from a sample isolated from a subject.

15 . A pharmaceutical composition comprising a plurality of cells according to any of claims 1 to 3 .

16 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 15 to a subject.

17 . A method according to claim 16 , which comprises the following steps:

(i) isolation of a cell-containing sample from a subject;

(ii) transduction or transfection of the cells with: a nucleic acid sequence according to any of claims 4 to 7 ; a first nucleic acid sequence and a second nucleic acid sequence as defined in claim 8 ; a first vector and a second vector as defined in claim 9 or 10 or a vector according to claim 11 or 12 ; and

(iii) administering the cells from (ii) to a the subject.

18 . A method according to claim 16 or 17 , wherein the disease is a cancer.

19 . A method according to claim 18 , wherein the cancer is a B cell malignancy.

20 . A pharmaceutical composition according to claim 15 for use in treating and/or preventing a disease.

21 . The use of a cell according to any of claims 1 to 3 in the manufacture of a medicament for treating and/or preventing a disease.

22 . A chimeric antigen receptor (CAR) comprising a CD19-binding domain which comprises

a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1-

(SEQ ID No. 15)

SYWMN;

CDR2-

(SEQ ID No. 16)

QIWPGDGDTNYNGKFK

CDR3-

(SEQ ID No. 17)

RETTTVGRYYYAMDY;

b) a light chain variable region (VL) having CDRs with the following sequences:

CDR1-

(SEQ ID No. 18)

KASQSVDYDGDSYLN;

CDR2-

(SEQ ID No. 19)

DASNLVS

CDR3-

(SEQ ID NO. 20)

QQSTEDPWT.

23 . A CAR according to claim 22 , wherein the CD19 binding domain comprises a VH domain having the sequence shown as SEQ ID No. 23, or SEQ ID NO 24; or a VL domain having the sequence shown as SEQ ID No 25, SEQ ID No. 26 or SEQ ID No. 40 a variant thereof having at least 90% sequence identity which retains the capacity to bind CD19.

24 . A CAR according to claim 22 , wherein the CD19 binding domain comprises the sequence shown as SEQ ID No 21, SEQ ID No. 22 or SEQ ID No. 39 or a variant thereof having at least 90% sequence identity which retains the capacity to bind CD19.

25 . A chimeric antigen receptor (CAR) comprising a CD22-binding domain which comprises

a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1-

(SEQ ID No. 27)

NYWIN;

CDR2-

(SEQ ID NO. 28)

NIYPSDSFTNYNQKFKD

CDR3-

(SEQ ID No. 29)

DTQERSWYFDV;

and

b) a light chain variable region (VL) having CDRs with the following sequences:

CDR1-

(SEQ ID No. 30)

RSSQSLVHSNGNTYLH;

CDR2-

(SEQ ID No. 31)

KVSNRFS

CDR3-

(SEQ ID NO. 32)

SQSTHVPWT.

26 . A CAR according to claim 25 , wherein the CD22 binding domain comprises a VH domain having the sequence shown as SEQ ID No. 35, or SEQ ID NO 36; or a VL domain having the sequence shown as SEQ ID No 37, or SEQ ID No. 38 or a variant thereof having at least 90% sequence identity which retains the capacity to bind CD22.

27 . A CAR according to claim 25 , wherein the CD22 binding domain comprises the sequence shown as SEQ ID No 33 or SEQ ID No. 34 or a variant thereof having at least 90% sequence identity which retains the capacity to bind CD22.

28 . A cell according to any of claims 1 to 3 , wherein the first CAR is as defined in any of claims 22 to 24 and the second CAR is as defined in any of claims 25 to 27 .

29 . A nucleic acid sequence according to any of claims 4 to 7 , encoding a first CAR as defined in any of claims 22 to 24 and a second CAR as defined in any of claims 25 to 27 .

30 . A kit according to any of claims 8 to 10 , wherein the first nucleic acid sequence encodes a first CAR as defined in any of claims 22 to 24 and the second nucleic acid sequence encodes a second CAR as defined in any of claims 25 to 27 .

31 . A vector according to claim 11 or 12 , which comprises a nucleic acid sequence according to claim 29 .

32 . A cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising an intracellular signalling domain, wherein the intracellular signalling domain of the first CAR comprises a co-stimulatory domain; and the intracellular signalling domain of the second CAR comprises a TNF receptor family endodomain.

33 . A cell according to claim 32 , wherein the co-stimulatory domain is CD28 co-stimulatory domain.

34 . A cell according to claim 32 or 33 , wherein the TNF receptor family endodomain is OX-40 or 4-1BB endodomain.

35 . A cell according to any of claims 32 to 34 , wherein the intracellular signalling domain of the first and the second CAR also comprises an ITAM-containing domain.

36 . A cell according to claim 35 , wherein the first CAR has the structure:

in which:

AgB1 is the antigen-binding domain of the first CAR;

spacer1 is the spacer of the first CAR;

TM1 is the transmembrane domain of the first CAR;

costim is a co-stimulatory domain; and

ITAM is an ITAM-containing endodomain;

and the second CAR has the structure:

in which:

AgB2 is the antigen-binding domain of the second CAR;

spacer2 is the spacer of the second CAR;

TM2 is the transmembrane domain of the second CAR;

TNF is a TNF receptor endodomain; and

ITAM is an ITAM-containing endodomain.

37 . A nucleic acid sequence encoding both the first and second chimeric antigen receptors (CARs) as defined in any of claims 32 to 36 .

38 . A nucleic acid sequence according to claim 37 , which has the following structure:

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR;

spacer1 is a nucleic acid sequence encoding the spacer of the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

costim is a nucleic acid sequence encoding a co-stimulatory domain;

ITAM1 is a nucleic acid sequence encoding the ITAM-containing endodomain of the first CAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR;

spacer2 is a nucleic acid sequence encoding the spacer of the second CAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

TNF is a nucleic acid sequence encoding a TNF receptor endodomain;

ITAM2 is a nucleic acid sequence encoding the ITAM-containing endodomain of the second CAR;

which nucleic acid sequence, when expressed in a cell, encodes a polypeptide which is cleaved at the cleavage site such that the first and second CARs are co-expressed at the cell surface.

39 . A kit which comprises

(i) a first nucleic acid sequence encoding the first chimeric antigen receptor (CAR) as defined in any of claims 32 to 36 , which nucleic acid sequence has the following structure:

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain of the first CAR;

spacer1 is a nucleic acid sequence encoding the spacer of the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

costim is a nucleic acid sequence encoding a co-stimulatory domain;

ITAM1 is a nucleic acid sequence encoding the ITAM-containing endodomain of the first CAR;

and

(ii) a second nucleic acid sequence encoding the second chimeric antigen receptor (CAR) as defined in any of claims 32 to 36 , which nucleic acid sequence has the following structure:

AbB2-spacer2-TM2-TNF-ITAM2

AgB2 is a nucleic acid sequence encoding the antigen-binding domain of the second CAR;

spacer2 is a nucleic acid sequence encoding the spacer of the second CAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

TNF is a nucleic acid sequence encoding a TNF receptor endodomain; and

ITAM2 is a nucleic acid sequence encoding the ITAM-containing endodomain of the second CAR.

40 . A vector comprising a nucleic acid sequence according to claim 37 or 38 .

41 . A method for making a cell according to any of claims 37 to 38 , which comprises the step of introducing: a nucleic acid sequence according to claim 37 or 38 ; a first nucleic acid sequence and a second nucleic acid sequence as defined in claim 39 ; or a vector according to claim 40 , into a cell.

42 . A pharmaceutical composition comprising a plurality of cells according to any of claims 32 to 36 .

43 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 42 to a subject.

44 . A pharmaceutical composition according to claim 42 for use in treating and/or preventing a disease.

45 . The use of a cell according to any of claims 32 to 36 in the manufacture of a medicament for treating and/or preventing a disease.