Method
The present invention provides a method of preparing a population of genetically modified cells which comprise a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR) comprising: (i) providing a starting population of cells; (ii) depleting said starting population of cells which express a target antigen to form a depleted starting population; (iii) selecting for CD4+ and CD8+ cells in the depleted starting population to form a selected population of cells; and (iv) introducing into a cell in the selected population a nucleic acid sequence which encodes a CAR or transgenic TCR against the target antigen.
1 . A method of preparing a population of genetically modified cells which comprise a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR) comprising:
providing a starting population of cells;
depleting said starting population of cells which express a target antigen to form a depleted starting population;
selecting for CD4+ and CD8+ cells in the depleted starting population to form a selected population of cells; and
introducing into a cell in the selected population a nucleic acid sequence which encodes a CAR or transgenic TCR against the target antigen.
2 . A method according to claim 1 , wherein the CAR or transgenic TCR is introduced to a cytolytic immune cell.
3 . (canceled)
4 . A method according to claim 1 , wherein the starting population of cells comprises peripheral blood mononuclear cells (PBMCs).
5 - 6 . (canceled)
7 . A method according to claim 1 , wherein the depleted starting population comprises T cells.
8 . A method according to claim 1 , wherein the target antigen is TCR beta constant region 1 (TRBC1).
9 . A method according to claim 1 , wherein the target antigen is TCR beta constant region 2 (TRBC2).
10 . A method according to claim 1 , wherein the percentage of CAR or transgenic TCR target antigen positive cells is lower in the population of genetically modified cells than in the starting population.
11 - 14 . (canceled)
15 . A genetically modified cell comprising a CAR or transgenic TCR obtainable by the method of claim 1 .
16 . A population of genetically modified cells according to claim 15 .
17 - 18 . (canceled)
19 . A population of genetically modified cells according to claim 16 , wherein the genetically modified cells are less differentiated than genetically modified cells prepared without depleting cells which express the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.
20 . A population of genetically modified cells according to any claim 16 , wherein the genetically modified cells have increased expression of CD27 and/or CD62L compared with genetically modified cells prepared without depleting cells which express the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.
21 . A population of genetically modified cells according to claim 16 , wherein the genetically modified cells are more naive than genetically modified cells prepared without depleting cells which express the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.
22 . A population of genetically modified cells according to claim 16 , wherein said genetically modified cells are less exhausted compared with genetically modified cells prepared without depleting cells which express the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.
23 . A population of genetically modified cells according to claim 16 wherein said genetically modified cells have decreased expression of one or more exhaustion markers compared with genetically modified cells prepared without depleting cells which express the target antigen of the CAR or transgenic TCR and without selecting for CD4+ and CD8+ cells.
24 . A population of genetically modified cells according to claim 23 , wherein the one or more exhaustion marker(s) is selected from the group consisting of: PD1, Lag3 and Tim3.
25 . A pharmaceutical composition which comprises a population of genetically modified cells according to claim 16 .
26 . (canceled)
27 . A method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to claim 25 to a subject in need thereof.
28 . A method according to claim 27 , which comprises the following steps:
(i) providing a sample comprising a starting population of cells;
(ii) depleting said starting population of cells which express a target antigen;
(iii) introducing into a cell in the depleted starting population a nucleic acid sequence which encodes a CAR or transgenic TCR against the target antigen; and
(iv) administering the cells from (iii) to a subject.
29 - 35 . (canceled)
36 . A method of reducing the number of cells in a pharmaceutical composition which express a target antigen and express a CAR or transgenic TCR against the target antigen comprising:
providing a starting population of cells;
depleting said starting population of cells which express a target antigen;
introducing into a cell in the depleted starting population a nucleic acid which encodes a CAR or transgenic TCR against the target antigen; and
incorporating the cells into a pharmaceutical composition.
37 . A method according to claim 36 , wherein the number of cells which express a target antigen and express a CAR or transgenic TCR against the target antigen is reduced compared with a pharmaceutical composition produced without depleting the starting population of cells which express a target antigen and without selecting for CD4+ and CD8+ cells.
38 . (canceled)
39 . A method according to claim 36 , wherein the cells are activated prior to the introduction of a nucleic acid which encodes a CAR or transgenic TCR against the target antigen.
40 - 45 . (canceled)
46 . A method according to claim 36 , wherein the percentage of cells which express the target antigen is lower in the pharmaceutical composition than in the starting population of cells.