Chimeric antigen receptor dendritic cell (car-dc) for treatment of cancer

Abstract

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 modified cell comprising a chimeric antigen receptor (CAR),
 wherein the CAR comprises
 an anti-HER2/neu antigen binding domain, 
 a transmembrane domain, and 
 an intracellular domain of a stimulatory and/or co-stimulatory molecule, and 
   wherein the modified cell is a macrophage or a monocyte.   
     
     
         2 . The modified cell of  claim 1 , wherein the CAR further comprises a hinge domain. 
     
     
         3 . The modified cell of  claim 2 , wherein the hinge domain comprises a CD8α hinge domain. 
     
     
         4 . The modified cell of  claim 1 , wherein the transmembrane domain comprises a CD8α transmembrane domain or a TLR4 transmembrane domain. 
     
     
         5 . The modified cell of  claim 1 , wherein the intracellular domain comprises two or more signaling domains. 
     
     
         6 . The modified cell of  claim 1 , wherein the intracellular domain comprises a CD3 zeta intracellular domain, an Fc6RI intracellular domain, an FcγRI intracellular domain or a TLR4 intracellular domain. 
     
     
         7 . The modified cell of  claim 1 , wherein the antigen binding domain is selected from a group consisting of monoclonal antibody, humanized antibody, single domain antibody, single chain variable fragment, and antigen-binding fragments thereof. 
     
     
         8 . The modified cell of  claim 1 , wherein the modified cell expresses one or more M1 markers, wherein the one or more M1 markers comprise HLA DR. 
     
     
         9 . The modified cell of  claim 1 , wherein the modified cell is a human cell. 
     
     
         10 . The modified cell of  claim 1 , wherein the CAR further comprises one or more linker domains linking the transmembrane domain to the antigen binding domain. 
     
     
         11 . The modified cell of  claim 1 , further comprising an agent, wherein the agent is selected from the group consisting of a nucleic acid a peptide and any combination thereof. 
     
     
         12 . The modified cell of  claim 1 , further comprising one or more viral components. 
     
     
         13 . A pharmaceutical composition comprising the modified cell of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         14 . A method of making a pharmaceutical composition for cancer immunotherapy, the pharmaceutical composition comprising:
 a modified cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an anti-HER2/neu antigen binding domain, a transmembrane domain, and an intracellular domain of a stimulatory and/or co-stimulatory molecule, and wherein the modified cell is a macrophage or a monocyte;   the method comprising:   (i) extracting a blood sample from a subject with cancer;   (ii) purifying peripheral blood mononuclear cells (PBMCs) from the blood sample;   (iii) isolating a population of macrophages and/or monocytes from the PBMCs, thereby obtaining isolated population of macrophages and/or monocytes;   (iv) introducing a polynucleic acid encoding the CAR into the isolated monocytes, thereby obtaining a population of macrophages and/or monocytes encoding a CAR,   (v) culturing the population of macrophages and/or monocytes encoding a CAR in the presence of a cytokine; and   (vi) preparing a pharmaceutical composition comprising the population of macrophages and/or monocytes encoding a CAR from (v), wherein the pharmaceutical composition is suitable for infusing into the subject with cancer.   
     
     
         15 . The method of  claim 14 , wherein the intracellular domain is capable of activating TLR4 signaling. 
     
     
         16 . The method of  claim 14 , wherein the intracellular domain contains an intracellular signaling domain of FcγRI, CD3-zeta, TLR-4, TRIF, TRADD, MYD99 or MYD88. 
     
     
         17 . The method of  claim 14 , wherein the cytokine is selected from the group consisting of IL-2, M-CSF, EGF, and VEGF. 
     
     
         18 . The method of  claim 14 , wherein the purification of the PBMCs is performed using a density gradient. 
     
     
         19 . The method of  claim 14 , wherein isolating comprises isolating a population of macrophages and/or monocytes from the PBMCs by isolation of cells expressing CD14. 
     
     
         20 . The method of  claim 14 , wherein introducing comprises transducing with a viral vector encoding the CAR.