Highlights d CD8 + T cell infiltration in tumors is associated with CCL5 and CXCL9 coexpression d CCL5 is expressed in tumor cells and CXCL9 is induced in APCs in response to IFN-g d CCL5 hi CXCL9 hi tumors are immunoreactive and respond to checkpoint blockade d Cancer cells negatively regulate CCL5 expression by epigenetic silencing mechanisms

Adoptive immunotherapies composed of T cells engineered to express a chimeric antigen receptor (CAR) offer an attractive strategy for treatment of human cancer. However, CARs have a fixed antigen specificity such that only one tumor-associated antigen (TAA) can be targeted, limiting the efficacy that can be achieved due to heterogeneous TAA expression. For this reason, a more generalized and effective application of CAR therapy would benefit from the capability to produce large panels of CARs against many known TAAs. In this study, we demonstrate a novel strategy to extend the recognition specificity potential of a bioengineered lymphocyte population, allowing flexible approaches to redirect T cells against various TAAs. Our strategy employs a biotin-binding immune receptor (BBIR) composed of an extracellular-modified avidin linked to an intracellular T cell signaling domain. BBIR T cells recognized and bound exclusively to cancer cells pre-targeted with specific biotinylated molecules. The versatility afforded by BBIRs permitted sequential or simultaneous targeting of a combination of distinct antigens. Together, our findings demonstrate that a platform of universal T cell specificity can significantly extend conventional CAR approaches, permitting the tailored generation of T cells of unlimited antigen specificity for improving the effectiveness of adoptive T cell immunotherapies for cancer.

Adoptive immunotherapy using lymphocytes genetically-modified to express a chimeric antigen receptor (CART) holds considerable promise for the treatment of cancer. However, CAR-based therapies may involve on-target toxicity against normal tissues expressing low amounts of the targeted tumor-associated antigen (TAA). To specify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy whereby T cell activation signal 1 (CD3ζ) is physically dissociated from costimulatory signal 2 (CD28) in two CARs of differing antigen specificity; mesothelin and a-folate receptor (FRa). Human T cells were genetically modified to co-express signal 1 (Anti-Meso scFv-CD3ζ) and signal 2 (Anti-FRa scFv-CD28) CARs in trans. Trans-signaling CART cells showed weak cytokine secretion against target cells expressing only one TAA in vitro, similar to first generation CART cells bearing CD3ζ only, but demonstrated enhanced cytokine secretion upon encountering natural or engineered tumor cells co-expressing both antigens, equivalent to that of second generation CART cells with dual signaling in cis. CART cells with dual specificity also showed potent anti-cancer activity and persistence in vivo which was superior to first generation CART cells and equivalent to second generation CARs. Importantly, second generation CART cells exhibited potent activity against cells expressing mesothelin alone, recapitulating normal tissue, whereas trans-signaling CART cells did not. Thus, a dual specificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CART cells against cancer while minimizing parallel reactivity against normal tissues bearing single antigen.