NUCLEIC ACID CONSTRUCT FOR EXPRESSING MORE THAN ONE CHIMERIC ANTIGEN RECEPTOR
The present invention provides a nucleic acid construct comprising the following structure: A-X-B in which X is a nucleic acid sequence which encodes a cleavage site; and A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), each CAR comprising: (i) an antigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and (iv) an endodomain wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR and wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain; 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.
1 . A nucleic acid construct comprising the following structure:
A-X-B
in which
X is a nucleic acid sequence which encodes a cleavage site, and
A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), each CAR comprising:
(i) an antigen-binding domain,
(ii) a spacer,
(iii) a trans-membrane domain, and
(iv) an endodomain
wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR, wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain, 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 . The nucleic acid construct according to claim 1 , wherein the spacer of the first CAR has a different length and/or charge and/or size and/or configuration and/or glycosylation than the spacer of the second CAR, such that when the nucleic acid construct is expressed in a cell, and the first CAR and the second CAR bind their respective target antigens at the cell surface, the first CAR and second CAR become spatially separated on the cell membrane.
3 . (canceled)
4 . The nucleic acid construct according to claim 1 , wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain, which inhibitory CAR inhibits T-cell activation by the activating CAR in the absence of inhibitory CAR ligation but does not significantly inhibit T-cell activation by the activating CAR when the inhibitory CAR is ligated.
5 . The nucleic acid construct according to claim 4 , wherein the inhibitory endodomain comprises all or part of the endodomain from CD148 or CD45.
6 - 11 . (canceled)
12 . 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.
13 . The nucleic acid construct according to claim 1 wherein the first and/or second CAR comprises an intracellular retention signal 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.
14 - 20 . (canceled)
21 . 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.
22 . The nucleic acid construct according to claim 21 , wherein the signal peptide of the activating CAR comprises one or more mutation(s) such that it has fewer hydrophobic amino acids, such that when the nucleic acid construct is expressed in a cell, the relative expression level of the activating CAR at the cell surface is reduced in comparison with the inhibitory CAR.
23 . The nucleic acid construct according to claim 21 , wherein the hydrophobic amino acid(s) removed or replaced by mutation is/are selected from: Alanine Valine (A), Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Phenylalanine (P), Tyrosine (Y), and Tryptophan (W).
24 . The nucleic acid construct according to claim 21 , wherein the hydrophobic amino acid(s) removed or replaced by mutation is/are selected from: Valine (V), Isoleucine (I), Leucine (L), and Tryptophan (W).
25 . 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.
26 . A vector comprising a nucleic acid construct according to claim 1 .
27 . (canceled)
28 . A cell comprising a nucleic acid construct according to claim 1 .
29 . The cell according to claim 28 which is a T cell or a natural killer (NK) cell.
30 . A method for making a cell according to claim 28 , which comprises the step of introducing into a cell: a nucleic acid construct comprising the following structure:
A-X-B
in which
X is a nucleic acid sequence which encodes a cleavage site, and
A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), each CAR comprising:
(i) an antigen-binding domain,
(ii) a spacer,
(iii) a trans-membrane domain, and
(iv) an endodomain
wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR, wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain, 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.
31 . The method according to claim 30 , wherein the cell is from a sample isolated from a subject.
32 . A pharmaceutical composition comprising a plurality of cells according to claim 28 .
33 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 32 to a subject.
34 . The method according to claim 33 , 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
X is a nucleic acid sequence which encodes a cleavage site, and
A and B are nucleic acid sequences encoding a first and a second chimeric antigen receptor (CAR), each CAR comprising:
(i) an antigen-binding domain,
(ii) a spacer,
(iii) a trans-membrane domain, and
(iv) an endodomain
wherein the antigen binding domains of the first and second CARs bind to different antigens, wherein the spacer of the first CAR is different to the spacer of the second CAR, wherein one of the first or second CARs is an activating CAR comprising an activating endodomain and the other CAR is an inhibitory CAR comprising a ligation-off inhibitory endodomain, 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 the subject.
35 . The method according to claim 33 , wherein the disease is a cancer.
36 - 37 . (canceled)