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Tumor infiltrating lymphocyte (TIL) therapy has shown promising results in the treatment of metastatic melanoma. However, TIL therapy has conventionally required co-administration of IL2, which is associated with toxicity in patients. We previously showed that melanoma TILs engineered to express membrane-bound IL15 (mbIL15) under the control of the ligand acetazolamide (ACZ) can achieve IL2-independent expansion during manufacturing, antigen-independent persistence in vitro and anti-tumor efficacy in vivo. In the current study, we extend the cytoTIL15 cell therapy product concept to indications beyond melanoma including non-small cell lung cancers (NSCLC), triple-negative breast cancers (TNBC) and head and neck squamous cell carcinomas (HNSCC), tumor types which represent significant unmet medical needs, particularly in the post-checkpoint inhibitor refractory setting. TILs from primary NSCLC, HNSCC and TNBC were engineered to express mbIL15 in the presence of ACZ and expanded in the absence of IL2 using our proprietary rapid expansion protocol (REP). CytoTIL15 cells were predominantly CD8 positive, enriched for mbIL15 expression and maintained T cell receptor variable beta chain (TCRVβ) diversity throughout expansion. In vitro antigen- and cytokine-independent survival and polyfunctionality of cytoTIL15 cells was measured from cultures that included ACZ. To assess anti-tumor activity, cytoTIL15 cells were co-cultured with autologous patient-derived cell lines (PDc) or tumor digests from patient-derived xenografts (PDx), and cytotoxicity and IFNγ release into supernatant was measured. In vitro, cytoTIL15 cells + ACZ exhibited similar or increased polyfunctionality compared to unengineered TIL + IL2. Unlike unengineered TILs, cytoTIL15 cells + ACZ persisted in an antigen-free setting without IL2, were cytotoxic to autologous PDc and released IFNγ in response to autologous PDx tumor digest. Taken together, these data show that IL2-independent, fully functional cytoTIL15 cells can successfully be generated from tumors such as NSCLC, HNSCC & TNBC, which afflict large numbers of patients. Citation Format: Kyle D. Pedro, Rachel Burga, Alonso Villasmil Ocando, Meghan Langley, Gauri Kulkarni, Zheng Ao, Benjamin Primack, Theresa Ross, Violet Young, Jeremy Tchaicha, Michelle Ols, Jan Ter Meulen. IL15-engineered tumor infiltrating lymphocytes (cytoTIL15TM) exhibit activity against autologous tumor cells from multiple solid tumor indications without IL2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB096.

Standard tumor-infiltrating lymphocyte (TIL) therapy requires IL-2 administration to support TIL expansion and survival, but this cytokine is associated with T cell exhaustion and can result in severe toxicities that limit patient eligibility (1). To this end, we genetically engineered TIL to express membrane-bound IL-15 (mbIL15) under the control of Obsidian’s cytoDRIVE® technology (cytoTIL15࣪), which allows regulation of protein expression via a drug-responsive domain upon acetazolamide (ACZ) administration. IL-15 is a preferred cytokine over IL-2 to mediate TIL activation and expansion, because it does not result in CD8 T cell exhaustion or stimulate regulatory CD4 T cells, and enhances development of a memory T-cell phenotype. We have previously demonstrated IL-2-independent, 3-6-fold increased cytoTIL15 persistence in an antigen-independent setting relative to unengineered TIL therapy with IL-2 (uTIL) (2). Due to the challenge of generating autologous tumor/TIL-matched pairs and most importantly, to assess cytoTIL15 cell’s functional impact on anti-tumor growth across multiple donors, we developed an allogeneic patient-derived xenograft (PDX) model. To establish the model, different melanoma tumor digests were co-incubated in vitro with select HLA-A*02-matched, allogeneic melanoma TIL donors to assess their reactivity. Tumors were screened for expression of shared antigens, such as gp100 and MART1, and TIL donor TCRs were screened with tetramers. Once established, serially passaged tumor fragments were grown, measured, and randomized into groups to receive intravenous transfer of TIL (n=8/cohort). Mice receiving uTIL were treated with four saturating doses of recombinant IL-2, and mice receiving cytoTIL15 cells received either vehicle or oral 200 mg/kg ACZ daily for the entire study, without any IL-2. Three of four cytoTIL15 cell preparations from different donors dosed with ACZ achieved significant tumor growth inhibition compared to uTIL. Four mice developed complete responses as early as 17 days post cytoTIL15 cell transfer. The level of anti-tumor response was associated with increased frequency of MART1-reactive cytoTIL15 cells. On day 20 after TIL transfer, tumors and secondary lymphoid organs were collected (n=4/cohort). Tumors treated with cytoTIL15 cells + ACZ showed an 8-10-fold increased TIL infiltration compared to uTIL or cytoTIL15 cells + vehicle. Moreover, enhanced cytoTIL15 cell infiltration and anti-tumor activity was associated with increases in pro-inflammatory cytokines (e.g., IFNγ). Taken together, these data clearly demonstrate the superiority of cytoTIL15 cells over uTIL for controlling or eradicating melanoma tumor outgrowth and the utility of an allogeneic PDX model for comparative evaluation of tumor-antigen specific TIL reactivity. References: 1. Yang JC. Toxicities associated with adoptive T-cell transfer for Cancer. Cancer J. 2015. 2. Burga R. et al Genetically engineered tumor-infiltrating lymphocytes (cytoTIL15) exhibit IL-2-independent persistence and anti-tumor efficacy against melanoma in vivo. SITC 36th annual meeting 2021. Citation Format: Jeremy H. Tchaicha, Scott Lajoie, Rachel Burga, Theresa Ross, Benjamin Primack, Meghan Langley, Violet Young, Alonso Villasmil Ocando, Kyle Pedro, Jack Tremblay, Gauri Kulkarni, Mithun Khattar, Dhruv Sethi, Michelle Ols, Gabriel Helmlinger, Gary Vanasse, Shyam Subramanian, Jan ter Meulen. Allogeneic, IL-2-independent tumor-infiltrating lymphocytes expressing membrane-bound IL-15 (cytoTIL15࣪) eradicate tumors in a melanoma PDX model through recognition of shared tumor antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB212.

Interleukin 12 (IL12) is an attractive cancer immunotherapeutic known to be extremely potent against solid tumors preclinically. However, the clinical utility of IL12 has been limited by toxicities stemming from high systemic cytokine exposure. Thus, armoring cellular therapies such as chimeric antigen receptor T cells (CAR-Ts) or tumor infiltrating lymphocytes (TILs) with IL12 will require technologies that provide tight control of IL12 expression and localization. Herein, we describe a tightly regulated version of IL12 for use in cellular therapies. First, we show that tethering IL12 to the membrane increases the activity of IL12 at the tumor site in vivo while reducing potential systemic toxicities. Membrane bound (mbIL12) was compared to secreted IL12 in the syngeneic CD8 gp100 TCR transgenic PMEL model. In this model, PMEL T cells transduced with mbIL12 demonstrated similar reduction in B16 tumor outgrowth as secreted IL12. Like secreted IL12, mbIL12 enhanced the expansion of PMEL T cells and retained T cell extrinsic activities, such as activation of myeloid cells and remodeling the tumor microenvironment. However, mbIL12 showed reduced toxicity signals as compared to secreted IL12, including a reduction in serum IFNy. Likewise, in a xenograft system, CD19-CARTs expressing human mbIL12 demonstrated enhanced potency against Raji tumors. To enhance the safety of mbIL12 further we sought to regulate its expression using Obsidian’s cytoDRIVE® technology. This platform consists of small, fully human protein sequences called drug responsive domains (DRD)s, such as carbonic anhydrase 2, that enable regulation of expression of a fused target protein under the control of FDA-approved, orally bioavailable small molecule ligands, such as, acetazolamide. In the absence of ligand (the “off-state”), the fusion protein is unfolded and degraded. In the presence of ligand (the “on-state”), the DRD is stabilized, allowing for protein expression and function. Thus, the cytoDRiVE® system acts as a titratable and reversible rheostat for on-demand protein activity. While a single DRD can enable some regulation of mbIL12 levels, we found that adding a modulation hub that increases the multiplicity of DRDs greatly improves regulation. Indeed, these modifications enabled off-state levels of mbIL12 that were indistinguishable from untransduced controls in HEK and Jurkat cell lines as well as primary human CD19 CAR-T-cells using flow cytometry. These modifications also enhanced mbIL12 regulation, leading up to a 30-fold dynamic range of mbIL12 abundance. Regulated mbIL12 was demonstrated to be active by phosphorylation of STAT4 in bystander NK cells in vitro. Combining DRDs with modulation hubs and membrane tethering enables the control of highly potent, previously clinically intractable cytokines, such as IL12, for use in enhancing cell therapies. Citation Format: Sean Gregory Smith, James A. Storer, Dexue Sun, Dan Jun Li, Benjamin Primack, Theresa Ross, Scott LaJoie, Jeremy Tchaicha, Dhruv Sethi, Jan ter Meulen, Michelle Ols. Novel Drug-responsive domain (DRD)-based regulation technology enables tightly controlled activity of potent membrane-bound IL12 in adoptive cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB101.