Quality of immunological synapse predicts effectiveness of chimeric antigen receptor (car) t cells

Abstract

Embodiments of the disclosure concern methods of determining the effectiveness of immune cells, such as T cells, with particular chimeric antigen receptors. In specific embodiments, a synapse between the CAR and the tumor antigen is measured for structure, signaling, and functionality by imaging. As such, the quality of the synapse is determined and positively correlates with effectiveness of the particular CAR immune cells.

1 .- 16 . (canceled) 
     
     
         17 . A system for determining the effectiveness of an immune cell, the system comprising:
 1) a lipid bilayer or a cell comprising at least one of
 a) one or more labeled tumor antigens; or 
 b) one or more accessory or costimulatory molecules; and 
   2) the immune cell expressing at least one of
 a) a binding moiety that targets the one or more tumor antigens; or 
 b) an endogenous immunoreceptor that targets the one or more accessory or costimulatory molecules, 
   wherein upon contacting the immune cell, the one or more labeled tumor antigens on the lipid bilayer or the cell binds to the binding moiety on the immune cell, thereby producing one or more complexes comprising an immunological synapse between the immune cell and the lipid bilayer or the cell, and   wherein the immunological synapse is examined horizontally in a high-throughput manner using an imaging module to determine the effectiveness of the immune cell.   
     
     
         18 . The system of  claim 17 , wherein the binding moiety is a part of a chimeric antigen receptor (CAR). 
     
     
         19 . The system of  claim 17 , wherein the binding moiety is a part of an engineered T-cell receptor (TCR). 
     
     
         20 . The system of  claim 17 , wherein the one or more accessory or costimulatory molecules comprises CD28 or LFA-1. 
     
     
         21 . The system of  claim 17 , wherein the lipid bilayer is a planar lipid bilayer. 
     
     
         22 . The system of  claim 17 , wherein the lipid bilayer is a glass-supported planar lipid bilayer. 
     
     
         23 . The system of  claim 17 , wherein the imaging module comprises microscopy. 
     
     
         24 . The system of  claim 23 , wherein the microscopy comprises confocal microscopy, epifluorescence microscopy, structured illumination microscopy (SIM), photoactivated localization microscopy (PALM), stochastical optical reconstruction microscopy (STORM), or stimulated emission depletion microscopy (STED). 
     
     
         25 . The system of  claim 17 , wherein the cell comprises a fixed cell or a live cell. 
     
     
         26 . The system of  claim 17 , wherein the immune cell comprises a fixed immune cell or a live immune cell. 
     
     
         27 . The system of  claim 17 , wherein the determining the effectiveness of the immune cell comprises measuring the quality of the immunological synapse between the lipid bilayer or the cell and the immune cell using the imaging of the immunological synapse. 
     
     
         28 . The system of  claim 26 , wherein the measuring comprises performing one or more of:
 (1) assaying for the distribution of the complexes in the lipid bilayer or the cell;   (2) assaying for the intensity of the complexes in the lipid bilayer or the cell;   (3) assaying for a phosphorylated form of the zeta chain of a T-cell receptor (TCR) or a chimeric antigen receptor (CAR), or another signaling molecule involved in T cell activation;   (4) assaying for the accumulation of F-actin at the complexes;   (5) assaying for the polarization of perforin at the complexes;   (6) assaying for the accumulation of zeta chain associated protein 70 (pZAP70) at the complexes; and   (7) assaying for the accumulation of lymphocyte specific protein tyrosine kinase (Lck) at the complexes,   
       wherein the quality of the immunological synapse measured in (1) to (7) predicts the effectiveness of the immune cell. 
     
     
         29 . The system of  claim 28 , wherein the assaying for the phosphorylated form of the zeta chain of the TCR or the CAR comprises assaying for the intensity and/or localization of a signal from the phosphorylated form of the zeta chain by exposing the complex to a labeled antibody to the phosphorylated form of the zeta chain. 
     
     
         30 . The system of  claim 28 , wherein the assaying for the accumulation of F-actin at the complexes comprises assaying for the intensity and/or localization of a signal from F-actin by exposing the complex to a labeled antibody or molecule to F-actin. 
     
     
         31 . The system of  claim 30 , wherein the labeled antibody or molecule comprises fluorophore-conjugated phalloidin. 
     
     
         32 . The system of  claim 28 , wherein the assaying for the polarization of perforin at the complexes comprises assaying for the intensity and/or localization of a signal from perforin by exposing the complex to a labeled antibody to perforin. 
     
     
         33 . The system of  claim 28 , wherein the assaying for the accumulation of pZAP70 at the complexes comprises assaying for the intensity and/or localization of a signal from pZAP70 by exposing the complex to a labeled antibody to pZAP70. 
     
     
         34 . The system of  claim 28 , wherein the assaying for the accumulation of Lck at the complexes comprises assaying for the intensity and/or localization of a signal from Lck by exposing the complex to a labeled antibody to Lck. 
     
     
         35 . The system of  claim 17 , wherein the effectiveness of the immune cell comprises the immune cell's cytotoxicity, cytokine secretion ability, proliferation ability, or homing ability to a tumor site.