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 5 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 treating cancer comprising of: a) extracting a blood sample from a cancer patient; b) isolating monocytes from said blood sample; c) transfecting said monocytes from said blood sample with a construct encoding a chimeric antigen receptor; and d) infusing said transfected monocytes into said cancer patient.
2 . The method of claim 1 , wherein peripheral blood mononuclear cells (PBMC) are purified from said blood sample.
3 . The method of claim 2 , wherein said purification of said PBMC is performed using a density gradient.
4 . The method of claim 3 , wherein said density gradient is ficoll.
5 . The method of claim 1 , wherein said monocytes are isolated from said blood sample by isolation of cells expressing CD14.
6 . The method of claim 1 , wherein said monocytes are isolated from said blood sample by collection of cells adhering to a plastic vessel after a culture period of at least one hour.
7 . The method of claim 5 , wherein said CD14 expressing cells are isolated by magnetic activated cell sorting (MACS).
8 . The method of claim 5 , wherein said CD14 expressing cells are isolated by fluorescent activated cell sorting (FACS).
9 . The method of claim 1 , wherein said chimeric antigen receptor construct encodes a protein comprising of: a) an antigen binding domain; b) a transmembrane domain; and c) an intracellular domain.
10 . The method of claim 9 , wherein said antigen binding domain is an antibody or fragment thereof.
11 . The method of claim 9 , wherein said antigen binding domain is single chain antibody or fragment thereof.
12 . The method of claim 9 , wherein said antibody domain is a molecular entity possessing affinity towards a tumor antigen or a tumor endothelial antigen.
13 . The method of claim 1 , wherein said antibody binding domain possesses affinity towards a tumor endothelial cell antigen.
14 . The method of claim 13 , wherein said tumor endothelial cell antigen is selected from a group of antigens comprising of:
a) TEM-1;
b) ROBO-1-8;
c) VEGFR2;
d) CD109;
e) survivin; and
f) CD93.
15 . The method of claim 1 , wherein said antibody binding domain possesses affinity towards a tumor antigen.
16 . The method of claim 15 , wherein said tumor antigens are selected from a group comprising of: CLPP, 707-AP, AFP, ART-4, BAGE, MAGE, GAGE, SAGE, bcatenin/m, bcr-abl, CAMEL, CAP-1, CEA, CASP-8, CDK/4, CDC-27, Cyp-B, DAM-8, DAM-10, ELV-M2, ETV6, G250, Gp100, HAGE, HER-2/neu, EPV-E6, LAGE, hTERT, survivin, iCE, MART-1, tyrosinase, MUC-1, MC1-R, TEL/AML, and WT-1.
17 . The method of claim 1 , wherein said intracellular domain of said CAR is capable of inducing monocytic differentiation to DC.
18 . The method of claim 1 , wherein said intracellular domain of said CAR is capable of inducing monocytic differentiation to M1 macrophages.
19 . The method of claim 1 , wherein said intracellular domain of said CAR is capable of activating a toll like receptor signaling pathway.
20 . The method of claim 1 , wherein said intracellular domain of said CAR is capable of activating TLR4 signaling.
21 . The method of claim 1 , wherein said intracellular domain of said CAR is capable of inducing monocytic differentiation to DC.
22 . The method of claim 1 , wherein said intracellular domain of said CAR contains a signaling portion of the intracellular domain of the TLR-4 protein.
23 . The method of claim 1 , wherein said intracellular domain of said CAR contains an activator of molecular pathways endowing M1 phenotype is the functional portion of said TLR-4 protein which interacts with MyD88 at a sufficient affinity to trigger said MyD88 signal transduction.
24 . The method of claim 1 , wherein said intracellular domain of said CAR contains an activator of molecular pathways endowing DC phenotype is the functional portion of said TLR-4 protein which interacts with MyD88 at a sufficient affinity to trigger said MyD88 signal transduction.
25 . The method of claim 1 , wherein said activator of molecular pathways endowing M1 phenotype is the functional portion of said TLR-4 protein which interacts with TRAM and MAL at a sufficient affinity to trigger said TLR4 signal transduction.
26 . The method of claim 1 , wherein said activator of molecular pathways endowing DC phenotype is the functional portion of said TLR-4 protein which interacts with TRAM and MAL at a sufficient affinity to trigger said TLR4 signal transduction.