NUCLEIC ACID CONSTRUCT FOR EXPRESSING MORE THAN ONE CHIMERIC ANTIGEN RECEPTOR

1 . A nucleic acid construct comprising the following structure:

A-X—B

in which

A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), and

X is a nucleic acid sequence which encodes a cleavage site,

wherein the first and second CAR recognise different antigens,

wherein the first and second CAR comprise or associate with activating endodomains, and

wherein

(a) the first and/or second CAR comprises an intracellular retention signal and/or

(b) the signal peptide of the first or second CAR comprises one or more mutation(s) such that it has fewer hydrophobic amino acids.

2 - 7 . (canceled)

8 . The nucleic acid construct according to claim 1 wherein the first and/or second CAR comprises an intracellular retention signal which directs the CAR to a membrane-bound intracellular compartment.

9 . The nucleic acid construct according to claim 1 wherein the first and/or second CAR comprises an intracellular retention signal which directs the CAR to a lysozomal, endosomal or Golgi compartment.

10 . The nucleic acid construct according to claim 1 wherein the first and/or second CAR comprises an intracellular retention signal which selected from the following group: an endocytosis signal; a Golgi retention signal; a trans-Golgi network (TGN) recycling signal; an endoplasmic reticulum (ER) retention signal; and a lysosomal sorting signal.

11 - 17 . (canceled)

18 . The nucleic acid construct according to claim 1 , wherein the signal peptide of the first or second CAR comprises one or more mutation(s) such that it has fewer hydrophobic amino acids than a) its wild-type sequence or b) the signal peptide of the other CAR.

19 . The nucleic acid construct according to claim 18 , wherein the hydrophobic amino acid(s) removed or replaced by mutation is/are selected from: Alanine (A), Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (P), Tyrosine (Y), and Tryptophan (W).

20 . The nucleic acid construct according to claim 19 , the hydrophobic amino acid(s) removed or replaced by mutation is/are selected from: Valine (V), Isoleucine (I), Leucine (L), and Tryptophan (W).

21 . The nucleic acid construct according to claim 1 , wherein the signal peptide of the first and second CAR differ in their number of hydrophobic amino acids and wherein one signal peptide comprises up to five more hydrophobic amino acids than the other signal peptide.

22 . The nucleic acid construct according to claim 1 comprising the following structure:

A-X—B—Y—C

in which

A, B and C are nucleic acid sequences encoding a first, second and third polypeptides of interest (POIs), and

X and Y are nucleic acid sequences which may be the same or different, each of which encodes a cleavage site,

wherein at least two of the POIs are chimeric antigen receptors (CARs) which

(a) comprise an intracellular retention signal and/or

(b) differ in the number of hydrophobic amino acids in their signal peptides.

23 . The nucleic acid construct according to claim 22 , wherein the at least two which are transmembrane proteins and which comprise an intracellular retention signal:

(a) comprise different intracellular retention signals and/or

(b) have the intracellular retention signal located at a different position in the CAR,

such that when the nucleic acid is expressed in a cell, there is differential relative expression of the at least two CARs at the cell surface.

24 . The nucleic acid construct according to claim 22 , wherein the third POI is a polypeptide which enables selection of transduced cells and/or enables cells expressing the polypeptide to be deleted.

25 . A vector comprising a nucleic acid construct according to claim 1 .

26 . (canceled)

27 . A cell comprising a nucleic acid construct according to claim 1 .

28 . The cell according to claim 27 which is a T cell or a natural killer (NK) cell.

29 . A method for making a cell according to claim 27 , which comprises the step of introducing into a cell: a nucleic acid construct comprising the following structure:

A-X—B

in which

A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), and

X is a nucleic acid sequence which encodes a cleavage site,

wherein the first and second CAR recognise different antigens,

wherein the first and second CAR comprise or associate with activating endodomains, and

wherein

(a) the first and/or second CAR comprises an intracellular retention signal and/or

(b) the signal peptide of the first or second CAR comprises one or more mutation(s) such that it has fewer hydrophobic amino acids.

30 . The method according to claim 29 , wherein the cell is from a sample isolated from a subject.

31 . A pharmaceutical composition comprising a plurality of cells according to claim 27 .

32 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 31 to a subject.

33 . A method according to claim 32 , comprising the following steps:

(i) isolating a T and/or NK cell-containing sample from a subject;

(ii) transducing or transfecting the T and/or NK cells with: a nucleic acid construct comprising the following structure:

A-X—B

in which

A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), and

X is a nucleic acid sequence which encodes a cleavage site,

wherein the first and second CAR recognise different antigens,

wherein the first and second CAR comprise or associate with activating endodomains, and

wherein

(a) the first and/or second CAR comprises an intracellular retention signal and/or

(b) the signal peptide of the first or second CAR comprises one or more mutation(s) such that it has fewer hydrophobic amino acids; and

(iii) administering the T and/or NK cells from (ii) to a subject.

34 . The method according to claim 32 , wherein the disease is a cancer.

35 - 36 . (canceled)