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T cells expressing a mesothelin (MSLN)-specific T cell receptor fusion construct (TRuC®), called TC-210, have demonstrated robust antitumor activity in preclinical models of mesothelioma, ovarian cancer, and lung cancer. However, they are susceptible to suppression by the programmed cell death protein 1 (PD-1)/programmed cell death protein ligand 1 (PDL1) axis and lack intrinsic costimulatory signaling elements. To enhance the function of anti-MSLN TRuC-T cells, chimeric switch receptors (CSRs) have been designed to co-opt the immunosuppressive PD-1/PD-L1 axis and to deliver a CD28-mediated costimulatory signal. Here, we report that coexpression of the PD1-CD28 CSR in TRuC-T cells enhanced T cell receptor signaling, increased Th1 effector cytokines, decreased Th2/Th17 cytokines, and sustained effector function in the presence of PD-L1 when compared with TC-210. AntiMSLN TRuC-T cells engineered to coexpress PD1-CD28 CSRs comprising the ectodomain of PD-1 and the intracellular domain of CD28 linked by the transmembrane domain of PD-1 were selected for integration into an anti-MSLN TRuC-T cell therapy product called TC-510. In vitro, TC-510 showed significant improvements in persistence and resistance to exhaustion upon chronic stimulation by tumor cells expressing MSLN and PDL1 when compared with TC-210. In vivo, TC-510 showed a superior ability to provide durable protection following tumor rechallenge, versus TC-210. These data demonstrate that integration of a PD1-CD28 CSR into TRuC-T cells improves effector function, resistance to exhaustion, and prolongs persistence. Based on these findings, TC-510 is currently being evaluated in patients with MSLN-expressing solid tumors.

Adoptive cell therapies have shown great promise in hematological malignancies. To realize the potential of T cell therapies in solid tumors, we have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes all TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin-targeting TRuC-T cells (aka TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the effector function and persistence of TRuC-T cells in the hostile tumor microenvironment, we generated TC-210 T cells that express a membrane-tethered IL15Rα-IL15 fusion protein. IL-15 is a common γ chain cytokine that promotes the differentiation, maintenance, and effector function of memory CD8+ T cell subsets and confers resistance to IL-2-mediated activation induced cell death (AICD). In vitro, the co-expression of the IL-15 fusion protein enhances T cell proliferation and persistence upon repeated stimulation with MSLN+ cancer cell lines, while exhaustion marker expression is decreased. Furthermore, IL-15 enhanced TC-210 T cells sustain a significantly higher TCF-1+ population. When tested in a mesothelioma xenograft mouse model, the presence of the IL-15 fusion protein increased tumor infiltration and persistence of TC-210 T cells. Altogether, the presented data support clinical studies that explore the impact of IL-15 enhancement on the persistence of TC-210 T cells and depth of response in patients with MSLN+ malignancies. Citation Format: Michelle Fleury, Courtney Anderson, Amy Watt, Holly Horton, Adam Zieba, Jian Ding, Robert Tighe, Robert Hofmeister, Derrick McCarthy, Dario Gutierrez. Expression of an IL-15 receptor fusion protein enhances the persistence of TRuC-T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P032.

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

Previously we have described the design and antitumor activity of T cell receptor fusion constructs (TRuC™) that tether an antibody-derived binder to one of the TCR subunits to achieve redirected T cell killing of tumor cells independent of HLA. Different from CAR-T cells, TRuCs are integrated into the full TCR complex and thus harness its full signaling capacity. The cell surface antigen CD70 represents a promising target for cancer immunotherapy for its selective overexpression in various hematological and solid tumor indications. Because the normal tissue expression of CD70 occurs on activated lymphocytes, including activated T cells, fratricide (self-killing) has been recognized as a significant challenge for CD70-targeted T cell therapies. To address this challenge, we discovered a diverse pool of fully human anti-CD70 scFv binders that were used to make TRuC-T cells and then functionally screened for fratricide-resistance in vitro. We successfully identified a CD70-targeted TRuC-T cell candidate that exhibits normal T cell expansion and an improved memory phenotype, clearly differentiating from fratricide-prone candidates, all while maintaining potent cytotoxicity and cytokine production against tumor cells expressing both low and high levels of CD70. In addition, our CD70-targeted TRuC-T cells showed significant anti-tumor efficacy in multiple xenograft mouse models with no evidence of in vivo fratricide. In summary, we have engineered a fratricide-resistant CD70-directed TRuC-T cell therapy that has the potential to treat a wide range of both hematologic and solid cancers. Citation Format: Jian Ding, Amy Watt, Erica Liu, Zieba Adam, Derrick McCarthy, Jessica Gierut, Brett Schrand, Michael Lofgren, Jason Lajoie, Vania Kenanova, Philippe Kiefer-Kwon, Holly Horton, Dario A. Gutierrez, Robert Hofmeister, Robert Tighe. Discovery and preclinical characterization of fratricide-resistant TRuC T-cells targeting CD70 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1528.