PHARMACOLOGICAL COSTIMULATION OF ENGINEERED T CELLS

Abstract

Background: Chimeric antigen receptor (CAR) T cell therapy enlists genetic engineering to express a tumour targeting synthetic receptor on a patient’s T cells. While endogenous T cells rely on costimulatory signals for their development but not their effector function, CAR T cells are completely reliant on costimulatory domains incorporated within the CAR structure to promote T cell expansion and persistence in vivo, resulting in enhanced anti-tumour activity. Toxicities following CAR T cell infusion are linked to the magnitude and speed of the CAR T cell expansion, which is a function of signaling mediated through the costimulatory domain. Having a “druggable” method to provide costimulatory signals could be a strategy to: (i) tune in vivo T cell expansion, (ii) allow the physician to toggle costimulation during periods where it is advantageous to the therapeutic strategy, and (iii) enhance endogenous immune cell anti-tumour activity. A high throughput screen assessing 4000 biologically active compounds for their effect on engineered T cell proliferation identified Ferutinin, a natural small molecule with no known immunomodulatory activity. A small library of Ferutinin analogues (N=25) were synthesized to predict and optimize its biologically active molecular structure(s). Methods: We first assessed the impact of Ferutinin analogues on multiple classes of tumour re-targeting receptors TAC and DAP12 synthetic antigen receptor (SAR) engineered T cell function. We chose the TAC and DAP12 SAR receptor because they mediate T cell activation via natural signalling pathways and include no costimulatory domains. We then developed a high throughput screen (HTS) using luciferase as an indirect measure of TAC T cell number and screened >15,000 compounds with known biological activity at McMaster University’s Centre for Microbial and Chemical Biology. Finally, we validated the outcomes of the HTS. Results: Our studies did not find evidence of improved TAC or DAP12 SAR T cell function in the presence of Ferutinin analogues. The HTS did find classes of compounds with characterized costimulatory activity, including immunomodulatory drugs, phorbol esters and SMAC mimetics. 8- Geranyloxypsoralen and BAY 60 6583 demonstrated on-target enhancement of TAC T cell proliferation independent of T cell donor. Conclusions: These findings demonstrate the utility of small molecules as a method to enhance proliferation of engineered T cell products, recapitulating the effects of a costimulatory signal.