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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.

BackgroundAdoptive cell therapy with tumor-infiltrating lymphocytes (TILs) has demonstrated tremendous promise in clinical trials for patients with solid or metastatic tumors.1 However, current TIL therapy requires systemic administration of IL-2 to promote TIL survival, and IL-2-associated toxicities greatly limit patient eligibility and reduce the long-term clinical benefit of TIL therapy.2 3 Unlike IL-2, which promotes T cell exhaustion, IL-15 maintains antigen-independent TIL persistence through homeostatic proliferation and supports CD8+ T cell anti-tumor activity without stimulating regulatory T cells. We designed genetically engineered TILs to express a regulated form of membrane-bound IL-15 (mbIL15) for tunable long-term persistence, leading to enhanced efficacy and safety for the treatment of patients with solid tumors.MethodsObsidian’s cytoDRiVE™ platform includes small human protein sequences called drug responsive domains (DRD)s that enable regulated expression of a fused target protein under control of FDA-approved, bioavailable small molecule ligands. cytoTIL15 contains TILs engineered with mbIL15 under the control of a carbonic-anhydrase-2 DRD, controlled by the ligand acetazolamide (ACZ). After isolation from tumors, TILs were transduced and expanded in vitro through a proprietary TIL expansion process. cytoTIL15 were immunophenotyped and assessed for in vitro antigen-independent survival and co-cultured with tumor cells to assess polyfunctionality and cytotoxicity. In vivo TIL persistence and anti-tumor efficacy was evaluated through adoptive transfer of TILs into immunodeficient NSG mice, either naïve or implanted with subcutaneous patient-derived-xenograft (PDX) tumors.Results cytoTIL15 and conventional IL2-dependent TILs isolated from melanoma tumor samples expanded to clinically relevant numbers over 14 days. Throughout expansion, cytoTIL15 were enriched for CD8+ T cells and acquired enhanced memory-like characteristics, while maintaining diverse TCRVβ sub-family representation. cytoTIL15 demonstrated enhanced potency over conventional TILs, as measured by increased polyfunctionality and cytotoxicity against tumor and PDX lines in vitro (figure 1A). In a 10-day antigen-independent in vitro assay, cytoTIL15 persisted at greater frequencies than conventional TILs in the absence of IL-2 (figure 1B; *p30 days following adoptive cell transfer (figure 1C). Importantly, cytoTIL15 achieved significant tumor control in a human PDX model (figure 1D), which correlated with increased TIL accumulation in secondary lymphoid organs.Abstract 166 Figure 1cytoTIL15 demonstrate superior persistence. cytoTIL15 is an engineered TIL product expressing regulatable mbIL15. (A) cytoTIL15 demonstrate enhanced in vitro cytotoxicity after co-culture with melanoma tumor lines (representative data from 3 TIL donors). (B) cytoTIL15 have improved persistence in antigen- and IL2- independent culture conditions in vitro compared to conventional TILs cultured in the absence of IL-2 as well as (C) in vivo compared to conventional TILs supplemented with IL-2, when engrafted into NSG mice (in vitro: representative data from 1 TIL donor, performed in >3 replicate donors, in vivo: n=5/group, representative of 1 TIL donor, performed in >3 replicate donors). (D) cytoTIL15 (with 200mg/kg ACZ PO QD) demonstrate enhanced anti-tumor efficacy in a xenograft melanoma model as compared to conventional TILs (with 50000 IU IL-2 q8h BID, IP for 5 days) (n=8/group, representative of 1 TIL donor, performed in >2 replicate donors; ACT = adoptive cell transfer).ConclusionsTaken together, the superior persistence and potency of cytoTIL15 in the complete absence of IL-2 highlights the clinical potential of cytoTIL15 as a novel TIL product with enhanced safety and efficacy for patients with melanomas, and other solid tumors.AcknowledgementsThe authors wish to acknowledge the Cooperative Human Tissue Network for the their supply of human tumor tissue, and the MD Anderson Cancer Center for technical support; schematic created with BioRender.com.ReferencesChandran SS, Somerville RPT, Yang JC, Sherry RM, Klebanoff CA, Goff SL, Wunderlich JR, Danforth DN, Zlott D, Paria BC, Sabesan AC, Srivastava AK, Xi L, Pham TH, Raffeld M, White DE, Toomey MA, Rosenberg SA, Kammula US. Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study. Lancet Oncol 2017 Jun;18(6):792–802. doi: 10.1016/S1470-2045(17)30251-6. Epub 2017 Apr 7. PMID: 28395880; PMCID: PMC5490083.Yang JC. Toxicities associated with adoptive T-cell transfer for Cancer. Cancer J 2015;21:506–9.Schwartz RN, Stover L, Dutcher JP. Managing toxicities of high-dose interleukin-2. Oncology (Williston Park) 2002 Nov;16(11 Suppl 13):11–20. PMID: 12469935.

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.

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies, and more recently in multiple myeloma. However, CAR-T therapy has been less successful in treating solid tumors due to multiple obstacles, including the lack of robust CAR-T cell expansion, the immunosuppressive tumor microenvironment, and tumor escape due to the loss of targeted antigen. Engineering CAR-T cells to produce immunomodulatory factors such as Interleukin 12 (IL12) and Cluster of Differentiation 40 Ligand (CD40L) has been shown to enhance functional activity by driving T cell expansion, conferring resistance to immunosuppression, improving antigen presentation, and inducing antigen spread. However, the clinical utility of both IL12 and activators of the CD40 signaling pathway have been limited by systemic toxicity associated with their potent pharmacological activities. Providing precise tuning of the timing and level of expression of these immunomodulatory factors in CAR-T cells could significantly enhance safety and therapeutic efficacy, in particular against solid tumor malignancies. We describe here the implementation of ligand-controlled regulation of IL12 and CD40L in vitro and in vivo in engineered primary human T cells via the use of destabilizing domain (DD) technology. DDs are small protein domains that are misfolded and inherently unstable in the cell, but which can be reversibly stabilized by the binding of approved pharmacologic agents. This conditional stability of DDs can be readily conferred to any protein of interest by fusing it to the DD, thus providing fine-tuned, exogenous regulation of protein expression and function. We have identified mutations in several human protein substrates, including phosphodiesterase 5 (PDE5), dihydrofolate reductase (DHFR), and estrogen receptor (ER), which convey DD activity and can be regulated by FDA-approved drugs. We show that transduction of human T cells with either DD-IL12 or DD-CD40L fusion constructs yields low expression levels in the basal state and a rapid, dose-dependent induction of IL12 or CD40L protein in the presence of the corresponding stabilizing ligand. Moreover, kinetically precise, on-demand production of either factor from CAR-T cells can be achieved in mice by oral dosing of stabilizing drugs. A CD19 CAR-T Nalm6 mouse model has been established that measures potent CAR-T expansion and enhanced anti-tumor efficacy with armored immunomodulatory constructs. Studies are underway in this model, as well as in solid tumor models, to test for enhanced CAR-T activity via drug-induced activation of IL12 and CD40L towards the development of next generation cell therapies with more favorable efficacy and safety profiles. Citation Format: Michelle Ols, Michael Schebesta, Emily Brideau, Kutlu Elpek, Michelle Fleury, Jennifer Gori, Scott Heller, Dan Jun Li, Benjamin Primack, Christopher Reardon, Dhruv Sethi, Alex Storer, Dexue Sun, Karen Tran, Elizabeth Weisman, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah. CAR-Ts armored with small molecule-regulated IL12 or CD40L cassettes for enhanced activity against 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 LB-013.

Cytokines are messenger molecules that act as regulators of innate and adaptive immunity by propagating cell-cell immune signaling. Several cytokines have been approved for the treatment of metastatic renal cell cancer, advanced melanoma, and hairy cell leukemia (HCL) and can be particularly effective when combined with adoptive cell therapy. However, systemic delivery or constitutive expression of cytokines even at moderate levels can potentially lead to significant toxicity. These hurdles to enabling cytokine-enhanced adoptive cell therapy motivated us to implement destabilizing domain (DD) technology for regulating cytokines in chimeric antigen receptor (CAR) reprogrammed T-cells. The ability of CAR-T-cells to traffic to tumor sites enables localized co-delivery of cytokines for enhanced CAR-T-cell antitumor activity while improving safety. Obsidian Therapeutics’ DD technology enables titratable and reversible regulation of a protein of interest with FDA-approved small-molecule drugs in a time- and dose-dependent manner. Protein-fused DDs are misfolded in the absence of a stabilizing small-molecule ligand and are rapidly degraded by the proteasome. However, the addition of ligand restores the folding, stability, and function of the DD-protein fusion. We have generated IL-12 and membrane-bound IL15-IL15Ra (mbIL15) fused to DDs such as FK506 binding protein (FKBP), Escherichia coli dihydrofolate reductase (ecDHFR), as well as human protein substrates (huDDs) with clinically approved ligands. DD-IL12 and DD-mbIL15 fusions displayed ligand-dependent regulation of cytokine secretion or cell surface expression, respectively, in cell lines and primary human T-cells. We then tested DD regulation of IL12 or mbIL15 in vivo by injecting T-cells engineered with cytokine-fused DDs into NSG mice, followed by oral administration of vehicle or corresponding ligand. Vehicle-treated mice displayed low level expression of the respective cytokines, whereas ligand treatment robustly induced the expression of target cytokine within 4-6 hours after treatment. Cytokine expression returned to baseline levels 24 hours following ligand administration. These data demonstrate the feasibility of exogenous control over transgene-derived protein expression in primary human T-cells for the development of next-generation CAR-T-cell products with enhanced efficacy and more favorable safety profiles. Citation Format: Karen Tran, Kutlu Elpek, Tucker Ezell, Scott Heller, Mara Inniss, Abhishek Kulkarni, Dan Jun Li, Grace Olinger, Michelle Ols, Christopher Reardon, Dexue Sun, Tariq Kassum, Michael Briskin, Celeste Richardson, Vipin Suri, Steven Shamah, Michael Gilman. Destabilizing domain technology facilitates exogenous regulation of IL15 and IL12 for adaptive T-cell therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A220.

Adoptive cell therapy with chimeric antigen receptor (CAR) modified T cells has demonstrated remarkable clinical efficacy in the treatment of certain B cell malignancies and more recently in multiple myeloma. However, in solid tumors, CAR-T therapy has been far less successful, likely due to the lack of robust CAR-T cell expansion, the immunosuppressive microenvironment, and clonal heterogeneity within these tumors. The trafficking of CAR-T cells to tumor sites provides an opportunity for selective delivery of cargo that can enhance CAR-T cell activity at the site of the tumor. Interleukin-12 (IL12) and membrane tethered interleukin-15 (mbIL15) have previously been shown to enhance CAR-T activity, yet the unregulated expression of either compromises safety and/or efficacy. The Obsidian Therapeutics platform equips engineered immune cells with new functionalities whose activity can be regulated via the administration of FDA-approved small-molecule drugs, putting dosing control back in the hands of the treating physician. We utilize small, fully human protein sequences called destabilizing domains (DDs) that confer reversible destabilization to a fused target protein. In the absence of ligand the fusion protein is degraded, whereas the presence of small molecule ligand restores expression and functionality. Furthermore, stabilization is titratable with small molecule ligand dose, providing finely tuned control over target protein expression and function. We have applied our technology to create DD-IL12 and DD-mbIL15 “cassettes” that provide exogenous regulation of cytokine activity when transduced into CAR-Ts for enhanced cellular function. We describe here the successful implementation of the DD technology in engineered primary human T cells and show small-molecule ligand controlled regulation of DD-IL12 and DD-mbIL15 protein expression in vivo in mouse models. These data demonstrate the feasibility of exogenous control over protein expression in primary human T cells for the development of next-generation CAR-T cell products with enhanced efficacy and more favorable safety profiles. Citation Format: Celeste Richardson, Kutlu Elpek, Michelle Ols, Tariq Kassum, Steve Shamah. Enhancing adoptive cell therapies through exogenous regulation [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 3580.