Chimeric antigen receptor dendritic cell (CAR-DC) for treatment of cancer
The current invention provides monocytic cells transfected with chimeric antigen receptor (CAR) to selectively home to tumors and upon homing differentiate into dendritic cells capable of activating immunity which is inhibitory to said tumor. In one embodiment of the invention, monocytic cells are transfected with a construct encoding an antigen binding domain, a transcellular or structural domain, and an intracellular signaling domain. In one specific aspect of the invention, the antigen binding domain interacts with sufficient affinity to a tumor antigen, capable of triggering said intracellular domain to induce an activation signal to induce monocyte differentiation into DC.
1. A method of making a population of chimeric antigen receptor (CAR) encoded CD14+ cells, the method comprising:
(a) extracting a leukapheresis sample or a PBMC sample from a human subject;
(b) isolating CD14+ cells from CD3+ cells in the leukapheresis sample from (a) or the PBMC sample from (a);
(c) after (b), culturing the isolated CD14+ cells ex vivo in the presence of (i) a growth factor, wherein the growth factor comprises recombinant human M-CSF and does not comprise GM-CSF and (ii) a cytokine that does not comprise IL-2,
thereby obtaining an ex vivo population of CD14+ cells; and
(d) introducing a recombinant polynucleic acid encoding a chimeric antigen receptor (CAR) into the ex vivo population of CD14+ cells, thereby obtaining the population of CAR encoded CD14+ cells;
wherein the CAR comprises (i) an extracellular domain comprising an antigen binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a CD3 zeta intracellular signaling domain.
2. The method of claim 1 , wherein isolating CD14+ cells from the CD3+ cells in the leukapheresis sample from (a) or the PBMC sample from (a) comprises enriching CD14+ cells that are also positive for CD16.
3. The method of claim 1 , wherein extracting comprises extracting a leukapheresis sample or a PBMC sample from a human subject with lymphoma or a solid tumor.
4. The method of claim 1 , wherein extracting comprises extracting a leukapheresis sample or a PBMC sample from a human subject with a breast cancer or a metastatic cancer.
5. The method of claim 1 , wherein extracting comprises extracting a leukapheresis sample or a PBMC sample from a human subject with an ErbB-2-expressing cancer.
6. The method of claim 1 , wherein the recombinant polynucleic acid is a viral vector.
7. The method of claim 6 , wherein introducing comprises transducing a viral vector encoding the CAR into the ex vivo population of CD14+ cells, thereby obtaining the population of CAR encoded CD14+ cells.
8. The method of claim 1 , wherein introducing comprises transfecting the ex vivo population of CD14+ cells with the recombinant polynucleic acid encoding the CAR.
9. The method of claim 1 , wherein the recombinant polynucleic acid is mRNA.
10. The method of claim 9 , wherein introducing comprises introducing an mRNA encoding the CAR into the ex vivo population of CD14+ cells, thereby obtaining the population of CAR encoded CD14+ cells.
11. The method of claim 1 , wherein the population of CAR encoded CD14+ cells is a population of virally transduced CAR encoded CD14+ cells.
12. The method of claim 1 , wherein the population of CAR encoded CD14+ cells comprises a viral component.
13. The method of claim 1 , wherein the population of CAR encoded CD14+ cells is a population of differentiated dendritic cells.
14. The method of claim 1 , wherein isolating CD14+ cells from CD3+ cells in the leukapheresis sample or the PBMC sample comprises isolating CD14+ monocytes from CD3+ cells in the leukapheresis sample or the PBMC sample, isolating CD14+ macrophages CD3+ cells in from the leukapheresis sample or the PBMC sample or isolating CD14+ dendritic cells from CD3+ cells in the leukapheresis sample or the PBMC sample.
15. The method of claim 1 , wherein the population of CAR encoded CD14+ cells is a population of cells that are positive for both CD14 and CD16.
16. The method of claim 1 , wherein the population of CAR encoded CD14+ cells is a population of CAR encoded CD14+ monocytes, a population of CAR encoded CD14+ macrophages or a population of CAR encoded CD14+ dendritic cells.
17. The method of claim 1 , wherein the population of CAR encoded CD14+ cells is phagocytic.
18. The method of claim 1 , wherein the population of CAR encoded CD14+ cells is a population of differentiated M1 macrophages.
19. The method of claim 1 , wherein the ex vivo population of CD14+ cells is a population of cells that are positive for both CD14 and CD16.
20. The method of claim 1 , wherein the intracellular domain comprises two or more signaling domains.
21. The method of claim 1 , wherein the transmembrane domain comprises a CD8a transmembrane domain or a TLR4 transmembrane domain.
22. The method of claim 1 , wherein the extracellular domain further comprises a CD8a hinge domain.
23. The method of claim 1 , wherein the antigen binding domain is a single domain antibody (sdAb) or a single chain variable fragment (scFv).
24. The method of claim 1 , wherein the antigen binding domain is an anti-HER2/neu binding domain.
25. A pharmaceutical composition comprising a population of chimeric antigen receptor (CAR) encoded CD14+ cells made according to the method of claim 1 .