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.

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 (PLIN1, PPARy, 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.

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 was not a factor in determining tissue responsiveness; rather, it is speculated that other unknown variables in the backgrounds of different patients (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.

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.