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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.

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. 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. Previously, we have described the discovery and preclinical efficacy of fratricide-resistant TRuC-T cells targeting CD70, a tumor antigen overexpressed in various solid and hematological malignancies. As a strategy to enhance T cell effector function and persistence in the hostile tumor microenvironment, we engineered anti-CD70 TRuC-T cells to co-express a membrane-bound IL15Ra-IL15 fusion protein (IL-15fu). IL-15 is a common ? 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).MethodsT cells were activated by CD3/CD28 stimulation and lentivirally transduced with a T2A-containing bicistronic vector encoding the anti-CD70 CD3?-TRuC and the IL-15fu proteins; the cells were further expanded for 9 days in media containing IL-7/IL-15. Surface co-expression of the TRuC and IL-15fu proteins and the T cell memory phenotype was assessed by flow cytometry. In vitro persistence was tested in a repeated stimulation assay in which T cells were challenged by addition of fresh CD70+ target cells every four days with longitudinal assessment of T-cell expansion, phenotype, cytokine production, and cytotoxicity. In vivo, the antitumor efficacy of the anti-CD70 TRuC/IL-15fu T cells was evaluated in MHC class I/II deficient NSG mice bearing human tumor xenografts.ResultsThe anti-CD70 TRuC and IL-15fu proteins showed high transduction efficiency and robust co-expression on the surface of T cells. The IL-15fu significantly increased the proportion of naïve cells within the TRuC-T cell product, most dramatically in the CD8+ subset. In vitro, TRuC-T cells bearing the IL-15fu showed greatly enhanced expansion and persistence upon repeated stimulation with CD70+ target cells. Moreover, the IL-15fu enhanced T-cell survival and persistence under unstimulated, cytokine-free conditions. In vivo, the anti-tumor activity of CD70-targeted TRuC-T cells was significantly improved by IL-15fu in multiple tumor models and was associated with enhanced intratumoral T-cell accumulation and a preferential expansion of CD8+ T cells.ConclusionsThe addition of the IL-15fu improved the phenotype, persistence, and anti-tumor activity of CD70-targeted TRuC-T cells, potentially increasing the likelihood of clinical benefit in patients with CD70 overexpressing solid and liquid cancers.Ethics ApprovalAll animal studies were conducted by TCR2 Therapeutics staff at the Charles River Laboratories CRADL facility under a protocol approved by the Charles River Laboratories Institutional Animal Care and Use Committee.

Obesity is a risk factor for a myriad of diseases including diabetes, cardiovascular dysfunction, cirrhosis and cancer and there is a need for new systems to study how excess adipose tissue relates to the onset of disease processes. This study provides proof of concept patient-specific tissue models of human white adipose tissue to accommodate the variability in human samples. Our 3D tissue engineering approach established lipolytic responses and changes in insulin-stimulated glucose uptake from small volumes of human lipoaspirate, making this methodology useful for patient specific sample source assessments of treatment strategies, drug responses, disease mechanisms and other responses that vary between patients. Mature unilocular cells were maintained ex vivo in silk porous scaffolds for up to a month of culture and imaged non-invasively with coherent anti-Stokes Raman scattering. Interestingly, differences in responsiveness between tissues were observed in terms of magnitude of lipolysis, ability to suppress lipolysis, differences in glucose uptake, and lipid droplet size. Body mass index (BMI) was not a factor in determining tissue responsiveness; rather, it is speculated that other unknown variables in the backgrounds of different patients (i.e. ethnicity, athleticism, disease history, lifestyle choices, etc.) likely had a more significant effect on the observed differences. This study reinforces the need to account for the variability in backgrounds and genetics within the human population to determine adipose tissue responsiveness. In the future, this tissue system could be used to inform individualized care strategies – enhancing therapeutic precision, improving patient outcomes, and reducing clinical costs.

Despite success in treating hematological malignancies, T cells expressing chimeric antigen receptors (CARs) have shown poor efficacy in solid tumor indications. The failure to initiate and elicit a complete TCR response is arguably a primary underlying factor preventing CAR-T cell success in solid tumor indications. Here, we present a novel T cell engineering platform: T Cell Receptor Fusion Constructs (TRuC™s), which target tumors independent of MHC. Unlike CARs, the constructs integrate into the TCR complex, harnessing the full potential of natural T cell activation, effector function and regulation. Here we describe preclinical evidence underscoring the efficacy of TRuC™-T cells re-programmed to target the solid tumor antigen mesothelin (TC-210). TC-210 has shown robust anti-tumor activity in cellular assays and animal models of lung, ovarian and MPM cancers. In these studies, TC-210 was compared head-to-head against mesothelin-targeting CAR-T cells (MSLN CAR-T cells) bearing the same mesothelin binder expressed on TC-210. Mesothelin-dependent T cell activation, expansion and tumor clearance by TC-210 was faster than that observed with MSLN CAR-T cells. In a study using Renilla-luciferase-labelled MSLN-CAR T cells and TC-210, TC-210 migrated and accumulated at a faster rate in mesothelioma tumors than MSLN-CAR T cells. This migration pattern correlated with an increased level of chemokine receptors and number of TC-210 expressing these receptors, including CXCR3. TC-210 T cells also showed long-term functional persistence capable of preventing relapse in a mesothelioma model. Metabolic profiling of TC-210 versus MSLN-CAR T cells showed that the observed persistence may be related to the metabolic profile of TC-210 T cells. Unlike MSLN-CAR-T cells, TC-210 showed increased levels of oxidative phosphorylation and mitochondrial reserve, attributes associated with long-term memory T cells. Finally, systemic cytokine levels in animals treated with TC-210 were lower than those observed in MSLN CAR-T cell treated animals. Together, these findings warrant the investigation of TC-210 in clinical trials as effective treatment for mesothelin-expressing tumors with potentially lower rates of adverse events. Citation Format: Jian Ding, Holly Horton, Seema Shah, Adam Zieba, Janani Krishnamurphy, Thomas Ashhurst, Ashley V. Menk, Patrick Baeuerle, Nicholas J. King, Gregory Delogoffe, Robert Hofmeister, Daniel R. Getts. Preclinical evaluation of TC-210, a mesothelin-specific T cell receptor (TCR) fusion construct (TRuC™) T cells for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2307.

Obesity is a rising issue especially in the United States that can lead to heart problems, type II diabetes, and respiratory problems. Since the 1970s, obesity rates in the United States have more than doubled in adults and children. Recent evidence suggests that exposure to certain chemicals, termed "obesogens," in utero may alter metabolic processes, predisposing individuals to weight gain. There is a need to develop a three-dimensional human tissue system that is able to model the effects of obesogens in vitro in order to better understand the impact of obesogens on early development. Human embryonic-derived stem cells in three-dimensional collagen embedded silk scaffolds were exposed to three different obesogens: Bisphenol A (BPA), Bisphenol S (BPS), and Tributyltin (TBT). The exposed tissues accumulated triglycerides and increased expression of adipogenic genes (Perilipin (PLIN1), peroxisome proliferator-activated receptor gamma (PPARy), fatty acid binding protein 4 (FABP4)) compared to equivalent control cultures with no obesogen exposure. These cultures were also compared to human adult stem cell cultures, which did not respond the same upon addition of obesogens. These results demonstrate the successful development of a representative tissue model of in utero obesogen exposures. This tissue system could be used to determine mechanisms of action of current obesogens and to screen other potential obesogens.

There is a critical need for monitoring physiologically relevant, sustainable, human adipose tissues in vitro to gain new insights into metabolic diseases. To support long term culture, a 3D silk scaffold assisted culture system was developed that maintained mature unilocular adipocytes ex vivo in co-culture with pre-adipocytes, endothelial cells, and smooth muscle cells obtained from small volumes of liquefied adipose samples. Without the silk scaffold, adipose tissue explants could not be sustained in long term culture (3 months) due to their fragility. Adjustments to media components were used to tune lipid metabolism and proliferation, in addition to responsiveness to an inflammatory stimulus. Interestingly, patient specific responses to TNFα stimulation were observed, providing a proof of concept translational technique for patient specific disease modeling in the future. In summary, our novel 3D scaffold assisted approach is required for establishing physiologically relevant, sustainable, human adipose tissue systems from small volumes of lipoaspirate, making this methodology of great value to studies of metabolism, adipokine-driven diseases, and other diseases where the roles of adipocytes are only now becoming uncovered.

T cells expressing chimeric antigen receptors (CARs) have demonstrated remarkable clinical benefit in certain hematological malignancies albeit with side effect such as cytokine release syndrome, on Target off tumor response and limited efficacy in solid tumors. Here, we present the preclinical evaluation of a novel T cell engineering platform designed to overcome potential hurdles of CARs and other T cell receptor modalities. Specifically, we have generated novel T Cell Receptor Fusion Constructs (TRuC™s) that fuse binder domains to subunits of the T cell receptor. These constructs when introduced using lentiviral technology, integrate into endogenous TCR, reprograming T cells to target tumor antigen in a non-major histocompatibility complex (MHC) restricted fashion and harnessing the full spectrum of TCR signaling. TRuC™ variants were constructed by recombinant fusion of an scFv or sdAb to various TCR subunits via a flexible linker sequence. Likewise, CD28ζ and 41BBζ CARs were generated using the same binders for side-by-side comparison. We demonstrated that TRuC™ variants can effectively reprogram T cells to recognize tumor surface antigens in a non-MHC-restricted fashion. TRuC™s are distinct from CARs in their ability to activate T cells through the entire TCR without additional costimulatory domains. In vitro, TRuC™ T cells were equally potent as CAR T cells in eliminating tumor cells. Compared to CAR-T cells, TRuC-T cells produced lower cytokine levels and proliferated less. Despite these difference, TRuC™ T cells were more efficient in clearing tumors in subcutaneous Raji and MSTO-211H mesothelioma models. Our findings support the development of TRuC-T cells for the treatment of solid tumors. Citation Format: Ekta Patel, Jian Ding, Nikolaus Thorausch, Janani Krishnamurthy, Rashmi Choudhary, Solly Weiler, Bonnie Le, Patrick Tavares, Adam Zieba, Justin Quinn, Yan Wang, Wolfgang Schamel, Irene Scarfo, Marcela Maus, Patrick Baeuerle, Dainel Getts, Robert Hofmeister. Characterization of a novel class of engineered (TCR) fusion constructs (TRuCTMs) aimed to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3584.