Abstract 3236: Rejuvenation of exhausted CAR-T cells with a targeted TLR7 agonist
Authors: Low Philip S. , Luo Qian , Napoleon John Victor , Zhang Boning 
Published: September 2020

Abstract
CAR T cell therapies have achieved remarkable responses in leukemias, but their potencies in solid tumors have been disappointing. Two proposed causes of CAR T cell failures in solid tumors are: 1) CAR-T cells may become dysfunctional or “exhausted” upon chronic exposure to tumor antigen and/or immunosuppressive factors such as MDSCs, TAMs, Tregs and inhibitory cytokines, and 2) cancer cells mutate to no longer express the tumor antigen recognized by the CAR. We have described a CAR T cell in which the CAR contains the usual activation motifs, but expresses an scFv against FITC instead of the usual scFv to a tumor antigen. The resulting CAR T cell kills and proliferates only when it encounters a cancer cell labeled with FITC. To achieve this FITC labeling, we construct multiple FITC-ligand conjugates (adaptor) by conjugating FITC to one of 10 different low molecular weight (&lt;1kDa) tumor targeting ligands that we have developed over the past 30 years. By bridging between the anti-FITC CAR T cell and the receptor-expressing cancer cell, the adapter mediates rapid CAR T cell engagement and killing of the cancer cell. Moreover, by administering a cocktail of different tumor-specific adapters, we have shown that we can eradicate heterogeneous solid tumors that have lost their primary tumor antigen; i.e. addressing the second cause of CAR T cell failure in solid tumors.
To address the first probable cause of CAR T cell failure, we exploit the natural endocytosis of the anti-FITC scFV CAR to deliver a FITC-drug conjugate into the CAR T cell, wherein the attached drug is a potent TLR7 agonist. By concentrating the TLR7 agonist within the CAR T cell, we demonstrate that an exhausted/dysfunctional CAR T cell can be rejuvenated to a functional and tumor-killing state, leading to renewed eradication of the solid tumor. For initial refinement of this strategy in vitro, we generated exhausted CAR T cells by their continuous transfer (every 12 hours) to fresh MDA-MB-231 cells in presence of FITC-folate adaptor while monitoring for appearance of exhaustion markers (PD-1+Tim3+LAG3+) and loss of cell cytotoxicity. We then documented the abilities of both nontargeted and FITC-targeted TLR7 agonists to reverse the above CAR T cell exhaustion. Then, to evaluate whether the same FITC-TLR7 agonists might reverse exhaustion/dysfunction in vivo, we treated NSG mice bearing KB tumors with the same anti-FITC CAR T cells and waited until the above hallmarks of CAR T cell dysfunction/exhaustion became prominent. Upon tail vein injection of the same FITC-TLR7 agonist, we found that the T cell exhaustion/dysfunction markers were reversed and tumor killing was restored. Taken together, we conclude that CAR T cell eradication of solid tumors can be achieved by use of a universal CAR T cell with multiple adapters that allow recognition of multiple tumor antigens plus administration of a CAR T cell targeted TLR7 agonist that can reverse exhaustion/dysfunction induced by the tumor microenvironment.
Citation Format: Philip S. Low, Qian Luo, John Victor Napoleon, Boning Zhang. Rejuvenation of exhausted CAR-T cells with a targeted TLR7 agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3236.