Highlights d CD40L + CAR T cells kill antigen-negative tumor cells through CD40/CD40L interactions d CD40L + CAR T cells improve antitumor response compared with second-generation CAR T cells d CD40L + CAR T cells license APCs in vivo to aid in antitumor response d Licensed APCs prime non-CAR T cells to recognize tumor cells and produce cytokines

Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (TNFRSF17; BCMA) induces high overall response rates; however, relapse occurs commonly. A reservoir of multiple myeloma cells lacking sufficient BCMA surface expression (antigen escape) may be implicated in relapse. We demonstrate that simultaneous targeting of an additional antigen-here, G protein-coupled receptor class-C group-5 member-D (GPRC5D)-can prevent BCMA escape-mediated relapse in a model of multiple myeloma. To identify an optimal approach, we compare subtherapeutic doses of different forms of dual-targeted cellular therapy. These include; (i) parallel-produced and pooled mono-targeted CAR T cells, (ii) bicistronic constructs expressing distinct CARs from a single vector, and (iii) a dual-scFv "single-stalk" CAR design. When targeting BCMA-negative disease, bicistronic and pooled approaches had the highest efficacy, whereas for dual-antigen-expressing disease, the bicistronic approach was more efficacious than the pooled approach. Mechanistically, expressing two CARs on a single cell enhanced the strength of CAR T-cell/target cell interactions. SiGNifiCANCE: Myeloma frequently relapses post-CAR T-cell therapy; antigen escape-mediated relapse can be mitigated with upfront dual-targeting (BCMA/GPRC5D). A bicistronic vector encoding two CARs avoids the challenge of parallel manufacturing separate CAR T-cell products, while providing superior efficacy; this dual-targeted approach may enhance the durability of responses to cellular therapy for myeloma. See related commentary by Simon and Riddell iNtRODUctiON Treatment options for multiple myeloma have substantially improved over the last decade, resulting in improved overall survival (1, 2); however, despite this progress, patients are rarely cured. The natural history of multiple myeloma involves multiple relapses with progressively shorter durations of remission, until the patient develops refractory disease (3, 4). Addressing relapsed/refractory multiple myeloma (RRMM) necessitates the development of novel treatment approaches; one such approach under development, with early clinical data demonstrating unprecedented response

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable responses in B cell malignancies. However, many patients suffer from limited response and tumor relapse due to lack of persisting CAR T cells and immune escape. These clinical challenges have compromised the long-term efficacy of CAR T cell therapy and call for the development of novel CAR designs. We demonstrated that CAR T cells secreting a cytokine interleukin-36γ (IL-36γ) showed significantly improved CAR T cell expansion and persistence, and resulted in superior tumor eradication compared to conventional CAR T cells. The enhanced cellular function by IL-36γ was mediated through an autocrine manner. In addition, activation of endogenous antigen-presenting cells (APCs) and T cells by IL-36γ aided the formation of a secondary anti-tumor response which delayed the progression of antigen-negative tumor challenge. Together, our data provide preclinical evidence to support the translation of this design for an improved CAR T cell–mediated anti-tumor response.

designed the study, performed experiments, analyzed and interpreted data, and wrote the manuscript. C. Bebernitz performed experiments, analyzed data, and interpreted data. A. Lopez performed experiments. S. Rafiq designed experiments, interpreted data and wrote manuscript. R. Brentjens designed the study, interpreted data, and wrote the manuscript.

Kowa, Lilly, Merck, and Syros. He serves on the scientific advisory boards of Bridge Medicines, Earli, and Harpoon Therapeutics. RJB has licensed intellectual property to and collect royalties from BMS, Caribou and Sanofi and received research funding from BMS. RJB is a consultant to BMS, Atara Biotherapeutics Inc, Coimmune, Triumvira and was a consultant for Gracell Biotechnologies Inc but ended employment in the past 24 months. RJB is a member of the scientific advisory board for CoImmune and Triumvira, and has ownership interest (including patents) in IL-18. No potential conflicts of interest were disclosed by the other authors.