Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) has demonstrated great potential for patients with treatment-refractory metastatic melanoma. However, the need for interleukin-2 (IL-2) co-administration during TIL cell therapy limits patient eligibility and restricts treatment to intensive care units due to the risk of severe side effects. Instead, engineering TIL with membrane-bound interleukin-15 (mbIL15) has the potential to promote TIL expansion, antitumor activity, and persistence of CD8+ T cells, without the use of IL-2. cytoTIL15 cells express mbIL15 fused to a drug-responsive domain (DRD) that is regulated by the Food and Drug Administration-approved small-molecule drug acetazolamide (ACZ). As such, cytoTIL15 cells are manufactured with ACZ instead of IL-2, in the presence of engineered feeder cells. The cytoTIL15 cell product exhibits ACZ dose-dependent expansion and persistence in vitro and in vivo and potent tumor-killing activity in human melanoma models in the absence of IL-2. In patient-derived xenograft (PDX) tumors, spatial profiling revealed infiltrating cytoTIL15 cells to be highly cytotoxic and less exhausted than non-engineered TIL. This novel platform creates a powerful, IL-2-free TIL cell therapy with a potentially improved tolerability and safety profile, while allowing individualized pharmacologic regulation of the TIL product.

Adoptive cell therapies (ACT) have shown reduced efficacy against solid tumor malignancies compared to hematologic malignancies, partly due to the immunosuppressive nature of the tumor microenvironment (TME). ACT efficacy may be enhanced with pleiotropic cytokines that remodel the TME; however, their expression needs to be tightly controlled to avoid systemic toxicities. Here we show T cells can be armored with membrane-bound cytokines with surface expression regulated using drug-responsive domains (DRDs) developed from the 260-amino acid protein human carbonic anhydrase 2 (CA2). The CA2-DRD can be stabilized in vitro and in vivo with the FDA-approved small-molecule CA2 inhibitor acetazolamide (ACZ). We develop conditional degrons using library-based screening of mutants and show characterization of one DRD using crystallography and molecular dynamics (MD) simulations. Using protein-engineering solutions to increase the valency of DRDs fused to the cargo we have developed “modulation hubs” and show tight regulation of membrane-bound cytokines IL2, IL12, IL15, IL21, IL23, and IFNα in genetically engineered T cells. Finally, CA2-DRD regulated IL12 mediates regulated efficacy in a solid tumor model. Regulation of pleotropic cytokines potentially paves the way to safely use these powerful cytokines in ACT for cancer treatment. Drug-responsive domains developed from human carbonic anhydrase 2 and stabilized by acetazolamide tightly regulate expression and function of potent cytokines in engineered T cells for adoptive cell therapy

ACTs armored with potent cytokines, such as IL12, have shown promising activity in solid tumors. However, clinical application of IL12 has been hindered by significant systemic toxicities (Zhang Cancer Res 2015), dictating precise control over its expression. cytoDRiVE® technology allows pharmacologic regulation of mbIL12 expressed as a fusion protein with a drug-responsive domain (DRD) derived from human dihydrofolate reductase, stabilized by the small molecule, trimethoprim (TMP). Combining activation-dependent (spatial) control using nuclear factor of activated T cells response elements (NFAT-RE) with pharmacologic (temporal) regulation using the cytoDRiVE platform provides tight regulation of mbIL12, improving safety and enabling anti-tumor activity and survival in preclinical models (Smith SITC 2024). Here, we show that this novel spatiotemporal control 1) regulates downstream IL12 effects in the tumor microenvironment (TME), and 2) is efficacious in a liver model of colon cancer. Syngeneic studies were used to assess the efficacy and pharmacodynamics of murine transgenic CD8+ pmel TCR T cells (gp100) expressing either NFAT-RE-inducible secreted IL12 (NFAT-sec-IL12) or NFAT-RE and cytoDRiVE-regulated mbIL12 (NFAT-cytoDRiVE-mbIL12). B16-F10 and gp100-engineered MC38 tumor cells were implanted subcutaneously or intrahepatically, respectively. B16-F10 tumor-bearing mice were randomized and lymphodepleted 24 hours before cell product infusion. Flow cytometry and cytokine analyses characterized the distinct phenotypes of the infused cells and corresponding host immune cells. B16-F10 tumor-bearing mice receiving pmel T cells expressing NFAT-sec-IL12 displayed significant body weight loss, unlike mice infused with pmel T cells expressing NFAT-cytoDRiVE-mbIL12 (p<0.05), while both groups showed tumor control (Smith SITC 2024). On Day 7 post-ACT, systemic cytokines were higher in the NFAT-sec-IL12 than the NFAT-cytoDRiVE-mbIL12 group, including 30× higher IFNγ (p<0.05), as well as higher TNFα (p<0.0001), IL6 (p<0.01), and IL10 (p<0.0001). In the tumor, NFAT-cytoDRiVE-mbIL12 pmel T cells demonstrated TMP-regulated mbIL12 expression leading to a proinflammatory TME with increased bulk CD3+CD8+ cells, pmel T cells, an M1 macrophage shift and a 10× increase of IFNγ. Moreover, in an MC38-gp100+ tumor model, without lymphodepletion, NFAT-cytoDRiVE-mbIL12 pmel T cells + TMP led to complete and durable responses in an immune-suppressive TME. Evidence of liver toxicity, measured by assessing systemic alanine aminotransferase (ALT) levels, was significantly higher in the NFAT-sec-IL12 group versus the NFAT-cytoDRiVE-mbIL12 + TMP group in both models (p<0.01, p<0.05, respectively), with ALT levels in the NFAT-cytoDRiVE-mbIL12 groups at or near those of the untreated control tumor groups. In summary, integrating both activation-dependent and cytoDRiVE-mediated pharmacologic regulation to govern the spatiotemporal expression of IL12 is a strategy for enhancing the safety of an IL12-armored ACT. By overcoming the barrier of systemic toxicity, this approach has the potential to enable clinical application of this potent cytokine in the form of a cell therapy. Theresa Ross, Patricia Timpug, Sean G. Smith, Meghan Langley, Benjamin Primack, Violet Young, Dexue Sun, Dan Jun Li, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols, Jeremy Tchaicha. Spatiotemporally regulated expression of membrane-bound interleukin 12 (mbIL12) for armored adoptive cell therapy (ACT) shows strong antitumor activity in syngeneic solid tumor models without overt toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB025.

Abstract Introduction: Adoptive cellular therapies (ACT) have encountered challenges in solid tumors due in part to the immunosuppressive tumor microenvironment (TME). We have developed OBX-115, TIL engineered to express mbIL15 regulatable using the cytoDRiVE® platform, which allows for TIL expansion, persistence, and anti-tumor efficacy under control of the FDA-approved small-molecule ligand, acetazolamide (ACZ), eliminating the need for co-administration of IL2 (NCT05470283). LIGHT, a tumor necrosis factor family member, interacts with lymphotoxin beta receptor (LTbR) and herpes virus entry mediator (HVEM) found on various TME cell types, including stromal cells such as cancer associated fibroblasts (CAF). In preclinical studies, LIGHT expression within a tumor has been linked to the formation of tertiary lymphoid structures and vascular normalization (Ramachandran Cancer Cell 2023), both associated with better clinical outcomes (Sautès-Fridman Nat Rev Cancer 2019). We hypothesized that engineering TIL with regulatable mbIL15 and LIGHT expression could enhance their efficacy by modifying the TME. Methods: TIL from colorectal (CRC) and head and neck squamous cell carcinoma (HNSCC) were transduced with retroviral vectors to express regulatable mbIL15 and LIGHT. ACZ-induced surface expression of mbIL15 and LIGHT in expanded TIL was examined using flow cytometry. Functional signaling of LIGHT was assessed through co-culture with Jurkat-HVEM-NF-kappaB reporter cells and LTbR+ HUVEC cells. In vitro, engineered TIL were tested in stromal-rich tumor models (CRC and HNSCC) by co-culturing with autologous patient-derived tumor/CAF hybrid spheroids. In vivo, antigen-independent TIL persistence was assessed in NSG mice without exogenous IL2. Syngeneic studies were performed to assess the efficacy of adoptively transferred mbIL15 and LIGHT-engineered Pmel cells (CD8+ T cells transgenic for a gp100-specific T cell receptor) in a subcutaneous cold tumor model (B16-F10). Results: Engineered TIL were successfully expanded without exogenous IL2. ACZ-dependent mbIL15 and LIGHT expression were confirmed, validating co-regulation and functionality in vitro. TIL engineered with mbIL15 and LIGHT displayed significantly increased cytotoxicity against autologous tumor/CAF spheroids compared with TIL expressing mbIL15 alone (p<0.005) in CRC and HNSCC tumor/CAF hybrid models. TIL with mbIL15 and LIGHT expanded in vivo and persisted for ≥42 days without exogenous IL2 support. Moreover, Pmel cells engineered with mbIL15 and LIGHT demonstrated durable anti-tumor efficacy in B16-F10 tumor-bearing mice, which was greater than Pmel cells engineered with mbIL15 alone (p<0.01). Conclusions: These preclinical results suggest that TIL engineered with regulatable mbIL15 and LIGHT using the cytoDRiVE platform have the potential to address the high unmet clinical need in cold tumors with suppressive TME, which are currently not amenable to ACT. Citation Format: Balazs Koscso, Zheng Ao, Carmela Passaro, Nirzari Shah, Ngoc Ly, Patricia Timpug, Bulent A. Aksoy, Dexue Sun, Dan Jun Li, Kerri-Lynn Sheahan, Violet Young, Theresa Ross, Benjamin Primack, Meghan Langley, Jeremy Tchaicha, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB065.

Background We have previously demonstrated the successful generation of membrane-bound IL15 (mbIL15) engineered TIL (cytoTIL15™ therapy) from solid tumors, and acetazolamide (ACZ)-driven regulated expression of mbIL15 resulted in TIL persistence in an antigen-independent preclinical model (SITC 2021, 2022). Here, we evaluated the function of pharmacologically tunable mbIL15 in the setting of chronic antigen stimulation by melanoma tumor-associated antigens (TAAs), such as MART1. Methods CytoTIL15 cells were manufactured from metastatic melanoma TIL donors by introducing mbIL15 under the pharmacological control of a carbonic-anhydrase-2 (CA2) drug responsive domain (DRD) via ACZ, the stabilizing ligand, and expanded through a proprietary rapid expansion process (REP). ACZ-dependent IL15 expression and downstream signaling were assessed. In vitro, we employed peptide-loaded HLA-A*0201 T2 cells to present MART-1 to TIL for evaluation of TCR-based functionality. CytoTIL15 cells treated with 0–25 µM ACZ were stimulated with antigen twice weekly over 28 days, with routine assessments of cell health, phenotype, cytokine production, and gene expression. In vivo, antigen-independent cytoTIL15 cell persistence in response to ACZ doses was evaluated after adoptive transfer of the TIL into immunodeficient NSG mice. Results Compared to unengineered TIL, generation of cytoTIL15 therapy from melanoma-derived TIL led to an overall 2.3-fold enrichment of MART1-reactive TIL. CytoTIL15 cells exhibited ACZ-dependent expansion in response to repeat MART1 stimulation, with TIL reaching maximums of 2, 9, and 18-fold expansion for 0, 1, and 25µM of ACZ, respectively. Chronic antigen exposure revealed an ACZ-driven IL15-dependent enrichment of >80% MART1-reactive TIL, and an increase in effector cytokine production and polyfunctionality (IFNγ, IL2Rα, TNFα, IL2, Perforin, CD107a, Granzyme B). CytoTIL15 cells driven by ACZ demonstrated maintenance of a functional cytotoxic signature, which was enriched in the antigen-reactive cell population. Despite repeated antigen-stimulation, withdrawal of ACZ reduced cytokine production and persistence of the MART1-enriched cytoTIL15 cell population in vitro. In vivo studies further underscored ACZ-dependent tunability of cytoTIL15 cells, as increased ACZ doses enhanced TIL persistence (AUC: 41, 111, and 306%TIL*day for 0, 30, and 200mg/kg ACZ QD), and ACZ withdrawal after 8 days reduced TIL persistence by 1.7-fold. Conclusions The expansion and persistence of tumor specific cytoTIL15 cells in the setting of chronic antigen exposure was regulatable by ACZ-dependent mbIL15 expression. This concept supports clinical evaluation of OBX-115 in the relapsed metastatic melanoma setting without concurrent IL-2 administration (NCT05470283).

Abstract 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.

Background Solid tumors remain a challenging frontier for adoptive cellular therapies (ACT). Armoring T-cells with cytokines, such as interleukin 12 (IL-12), to remodel the tumor microenvironment (TME) has demonstrated preclinical efficacy against solid tumors. However, the clinical utility of IL-12 is limited by systemic toxicities, requiring tight control of expression. Herein, we show that T-cells armored with a small molecule-controlled membrane bound IL-12 (mbIL-12) drives regulation of pharmacodynamic markers and solid tumor efficacy in xenograft and syngeneic solid tumor models. Methods We regulated mbIL-12 expression using Obsidian's cytoDRIVE® technology. In this system, a drug responsive domain (DRD) is fused to a protein of interest. In the "off-state" the fusion protein is rapidly degraded by the proteasome. Adding a small molecule ligand stabilizes the complex, enabling expression (the "on-state"). Here, we use a DRD derived from carbonic anhydrase 2 and the FDA-approved inhibitor acetazolamide (ACZ) as a stabilizing ligand. Unlike most other regulation systems, cytoDRiVE® is both fully human, reducing immunogenicity, and induced pharmacologically, allowing on-demand control. Adding oligomerization domains increases the local density of DRDs to form modulation-hubs that further increase the regulation of mbIL-12. Cytokine levels were determined using flow cytometry and Meso Scale Discovery assays. Human mbIL-12 modulation hubs and CD19-CARs were transduced in peripheral blood T-cells and evaluated in vivo against subcutaneous Raji xenografts that form solid tumors in NSG mice after inoculation in Matrigel. Mouse constructs were evaluated in CD8 gp100 (PMEL) TCR transgenic cells against subcutaneous B16-F10 melanoma in C57BL6 mice. Results In the xenograft setting, ACZ dosing resulted in 35-fold regulation of IL-12 in the plasma and showed remarkable ACZ-dependent anti-tumor efficacy against large, solid Raji tumors at a 10x lower cell dose than unarmored CAR-Ts. In the immunocompetent PMEL/B16 model, IL-12 modulation-hub PMEL cells slowed tumor growth over unarmored PMEL cells and showed improved tolerability over secreted IL-12. Animals receiving IL-12 modulation-hub cells showed ACZ-dependent regulation of IL-12 and IFNγ in the plasma with levels 100-fold and 20-fold less, respectively, than with constitutive secreted IL-12 cells. This regulation led to functional impacts at the cellular level, including an increase in circulating antigen presenting cells. Conclusions The cytoDRiVE® platform enables enhanced regulation of IL-12 armored T-cells in multiple preclinical solid tumor models, potentiating an improved therapeutic window for IL12 in ACT. Ethics Approval All animals studies were IACUC approved.

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.

Background The clinical impact of tumor infiltrating lymphocytes (TIL) cell products is currently limited by suboptimal persistence and potency, as well as the need for high-dose adjuvant IL-2 treatment, which is associated with severe toxicities. Thus, we engineered an IL-2-independent TIL product, based on regulated expression of interleukin 15 (cytoTIL15TM cells), which has shown anti-tumor efficacy and persistence in human melanoma PDX models. Since the immuno-suppressive tumor microenvironment (TME) hinders cell therapies, we hypothesized that combining pleotropic cytokines of the interferon (IFN), IL-1, or TNF families with IL-15 would further enhance antitumor activity and that our cytoDRiVE® platform would allow pharmacologic control of these potent immune mediators. We tested constitutive and regulated combinations of a representative member of these cytokines with IL-15 in human TIL for in vitro polyfunctionality and in vivo antigen-independent persistence. We also engineered mouse pmel-TCR cells with cytokine combinations for evaluation in the syngeneic B16 melanoma model. Methods Human TIL were expanded and engineered with lentiviral vectors to express IL-15 with IFN-alpha, IL-18 (IL-1 family member) or undisclosed TNFSF-X (TNF superfamily member). Expanded TIL were immunophenotyped and assessed for polyfunctionality by flow cytometry after CD3/CD28 stimulation. Engineered TIL were transferred into NSG mice to assess antigen-independent TIL persistence in the absence of exogenous IL-2. Cytokines modified with our carbonic anhydrase 2 (CA2)-based cytoDRiVE® drug responsive domain (DRD) were evaluated for control of protein levels with the CA2 ligand, acetazolamide (ACZ). Cytokine expression was evaluated in flow cytometry and Meso Scale Discovery assays. To assess anti-tumor and TME remodeling capabilities, we used a syngeneic model with transduced pmel-TCR cells adoptively transferred into mice bearing B16 melanomas. Results Engineered TIL expressing both IL-15 and either IFN-alpha, IL-18 or TNFSF-X showed similar fold expansion, immunophenotype and polyfunctionality in vitro as TIL expressing only IL-15. Combination cytokine-expressing TIL showed similar in vivo antigen-independent persistence in the absence of IL-2 as TIL engineered with only IL-15. As compared to control pmel cells, sub-optimal cell doses of pmel T cells expressing both IL-15 and either IFN-alpha or, IL-18, showed improved efficacy and TME remodeling, while combining IL-15 with TNFSF-X resulted in significant tumor growth arrest of B16 melanoma tumors without escape. Conclusions While IL-15 drives expansion and persistence of cytoTIL15TM cells without IL-2, adding pleotropic and highly immune-stimulatory members of the IFN, IL-1 or TNF families may provide enhanced efficacy for patients with solid tumors marked by an immunosuppressive TME. Ethics Approval All animal studies were IACUC approved

Background CytoTIL15® therapy is an IL2-independent, engineered TIL product which allows pharmacological control of membrane-bound IL15 (mbIL15). We have previously shown that cytoTIL15® TILs demonstrate enhanced persistence and anti-tumor efficacy in a human allogeneic melanoma PDX model. Here we use digital spatial profiling and single cell sequencing to characterize the RNA expression profile and phenotypic markers of tumor infiltrating immune cells as well as tumor cells in this model and compare the results to unengineered, IL2-dependent TIL. Methods cytoTIL15® therapy contains TILs engineered with mbIL15 under the control of a carbonic-anhydrase-2 drug responsive domain, regulated by the ligand acetazolamide (ACZ). cytoTIL15® cells were generated from human melanomas through a proprietary rapid expansion process. Expanded TILs were phenotyped and assayed for in vitro polyfunctionality, cytotoxicity, and frequency of tumor-associated antigen-specific TCR. In vivo phenotype and anti-tumor functionality was examined through adoptive transfer of TILs into NSG mice bearing subcutaneous, HLA-matched, patient-derived-xenograft (PDX) tumors expressing melanoma-associated antigen MART-1, in IACUC approved animal studies. Tumors, spleen, bone marrow, and blood were harvested 14-21 days following adoptive cell transfer and assessed by flow cytometry, GeoMx digital spatial profiling, and single cell sequencing for characterization of TIL and the tumor microenvironment (TME). Results cytoTIL15® cells demonstrated enrichment and reactivity for melanoma antigen-specific TCRs, while maintaining TCRβ diversity. Fifteen days post-ACT, tumors from animals treated with cytoTIL15® cells exhibited significantly (p=0.0175) higher frequency (3.4-fold) of TILs, in which MART-1 tetramer positive cells demonstrated increased T-cell factor 1 (TCF-1) and CD69 expression, and secreted significantly greater amounts of IFNγ and TNFα cytokines into the TME, compared to unengineered TILs with IL2. In addition, cytoTIL15® TILs had a distinct differential gene expression profile, demonstrating an increase in effector genes such as IL2RB, GZMB, GNLY and CCL5 and reduction in exhaustion-related genes such as EOMES. cytoTIL15® cells accumulating in the bone marrow exhibited a lower frequency of CD39+ terminally differentiated CD8+ T cells, while maintaining higher levels of memory phenotype makers. Conclusions In this allogeneic melanoma PDX model, cytoTIL15® cells showed a distinct profile of RNA expression and phenotypic markers, consistent with their increased persistence and anti-tumor efficacy. Interestingly, the subpopulation of cytoTIL15® cells reactive to tumor-associated antigen MART-1 displayed increased expression of TCF-1, which in melanoma patients has been associated with responses to immune checkpoint blockade, in addition to progression-free and overall survival. Ethics Approval All animal studies were IACUC approved.

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.

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; *p<0.05). cytoTIL15 adoptively transferred into naïve NSG mice demonstrated ACZ-dependent long-term persistence without antigen or exogenous IL-2, whereas conventional TILs were undetectable >30 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.

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.

Although poly (ADP-ribose) polymerase (PARP) inhibitors are active in homologous recombination (HR)-deficient cancers, their utility is limited by acquired resistance after restoration of HR. Here, we report that dinaciclib, an inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 5, and 9, additionally has potent activity against CDK12, a transcriptional regulator of HR. In BRCA-mutated triple-negative breast cancer (TNBC) cells and patient-derived xenografts (PDXs), dinaciclib ablates restored HR and reverses PARP inhibitor resistance. Additionally, we show that de novo resistance to PARP inhibition in BRCA1-mutated cell lines and a PDX derived from a PARP-inhibitor-naive BRCA1 carrier is mediated by residual HR and is reversed by CDK12 inhibition. Finally, dinaciclib augments the degree of response in a PARP-inhibitor-sensitive model, converting tumor growth inhibition to durable regression. These results highlight the significance of HR disruption as a therapeutic strategy and support the broad use of combined CDK12 and PARP inhibition in TNBC.