T cell receptor-deficient T cell compositions
The invention is directed to modified T cells, methods of making and using isolated, modified T cells, and methods of using these isolated, modified T cells to address diseases and disorders. In one embodiment, this invention broadly relates to TCR-deficient T cells, isolated populations thereof, and compositions comprising the same. In another embodiment of the invention, these TCR-deficient T cells are designed to express a functional non-TCR receptor. The invention also pertains to methods of making said TCR-deficient T cells, and methods of reducing or ameliorating, or preventing or treating, diseases and disorders using said TCR-deficient T cells, populations thereof, or compositions comprising the same.
1. A method of producing one or more compositions comprising human T cells which express a chimeric antigen receptor (CAR), wherein said human T cells are derived from primary human T cells and wherein said method comprises:
(i) obtaining one or more compositions comprising isolated primary human T cells, each of said compositions being comprised of primary human T cells isolated from at least one human donor;
(ii) modifying the primary human T cells comprised in said one or more compositions comprising primary human T cells in order to functionally impair or reduce expression of one or more components of the TCR complex endogenously expressed by said human primary T cells;
(iii) further modifying the primary human T cells in the one or more compositions comprising human T cells by introducing a nucleic acid which encodes a desired CAR polypeptide which comprises a non-TCR receptor which comprises (1) a ligand binding domain which binds to an antigen expressed by cancer cells and (2) a signaling domain;
thereby producing one or more compositions comprising human T cells which express at least one CAR that specifically binds to cancer cells.
2. The method of claim 1 , wherein reduced expression of the targeted TCR component or the functional impairment or reduced expression of the endogenous TCR by the T cells in said one or more compositions obtained after effecting steps (i)-(iii) is confirmed by effecting one or more of the following procedures: (a) detecting the level of expression of said targeted TCR component in T cells comprised in said one or more compositions, (b) detecting the proliferation of T cells obtained in said one or more compositions in the presence of HLA-mismatched PMBCs and (c) detecting the level of gamma interferon produced by T cells comprised in said one or more compositions in response to HLA-mismatched donor cells.
3. The method of claim 1 wherein said one or more compositions comprising human T cells which express a CAR are derived from different human donors.
4. The method of claim 1 wherein said one or more compositions human T cells which express a CAR are derived from the same human donor.
5. The method of claim 1 wherein said one or more components of the TCR complex include TCR-α, TCR-β, TCR-γ, TCR-δ, CD3-γ, CD3-δ, CD3-ϵ, and CD3-ζ.
6. The method of claim 1 wherein in step (i) the functional impairment or reduced expression of the one or more components of the TCR complex is effected by introducing into the primary human T cells at least one short hairpin RNA (shRNA) which targets a nucleic acid that encodes at least one component of a functional TCR expressed by said primary human T cells.
7. The method of claim 6 wherein said one or more components of the TCR complex include TCR-α, TCR-β, TCR-γ, TCR-δ, CD3-γ, CD3-δ, CD3-ϵ, and CD3-ζ.
8. The method of claim 1 wherein the compositions are in a form whereby they may be stored and can be used for future therapy.
9. The method of claim 6 wherein the compositions are in a form whereby they may be stored and can be used for future therapy.
10. The method of claim 1 , wherein the primary human T cells express CD4 or CD8.
11. The method of claim 6 , wherein the primary human T cells express CD4 or CD8.
12. The method of claim 1 , wherein the primary human T cells of (i) comprise human peripheral blood mononuclear cells or cytotoxic T cells.
13. The method of claim 6 , wherein the primary human T cells of (i) comprise human peripheral blood mononuclear cells or cytotoxic T cells.
14. The method of claim 1 , wherein the ligand binding domain in the CAR is obtained from NKG2D, NKG2A, NKG2C, NKG2F, LLT1, AICL, CD26 or NKRP1.
15. The method of claim 1 , wherein the ligand binding domain in the CAR is obtained from an anti-tumor chimeric antigen receptor or anti-tumor antibody.
16. The method of claim 1 , wherein the signaling domain in the CAR is obtained from CD3 ζ.
17. The method of claim 1 , wherein the chimeric receptor binds to (i) MIC-A, MIC-B, an estrogen receptor, a progesterone receptor, RON or one or more members of the ULBP family.
18. The method of claim 17 , wherein the one or more members of the ULBP family include Rae-1, H-60, HCMV UL18, Rae-1β, ULBP2, and ULBP3.
19. The method of claim 1 , further comprising engineering the primary human T cells to express another receptor which initiates a signal to T cells.
20. The method of claim 1 , wherein the chimeric receptor comprises a ligand binding domain from NKG2D and a signaling domain from CD3 ζ.
21. The method of claim 1 , wherein the chimeric receptor is chNKG2D.
22. The method of claim 6 , wherein the ligand binding domain is a NKG2D ligand binding domain and the signaling domain is a CD3 ζ signaling domain.