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Tumor infiltrating lymphocytes (TIL) have shown promising efficacy in immunologically “hot” solid tumors that have a high level of T cell infiltration, such as melanoma. However, current treatment regimens require high dose IL-2 administration to support TIL survival, which limits their clinical applications due to IL-2 related toxicity. Obsidian Therapeutics is engineering TIL with membrane bound IL-15 (mbIL15) to eliminate the dependence of TIL on exogenous IL-2, potentially enhancing the tolerability of TIL therapies. Because there is a high unmet medical need in patients with tumors colder than melanoma, we evaluated mbIL15-engineered TIL expansion and functionality from colorectal cancer (CRC) and sarcoma biopsies. Using an IL2-independent, proprietary rapid expansion process (REP) we successfully expanded mbIL15-engineered TIL from both CRC and sarcoma, despite significantly lower T-cell numbers in the tumor tissues (average of 12-15% CD45+ TIL versus 64% in melanoma). mbIL15-engineered TIL showed an enrichment for CD8+ T cells throughout the REP and high T cell receptor variable beta chain (TCR Vbeta) diversity. mbIL15-engineered TIL also exhibited lower immune checkpoint expression (LAG3/PD-1) and higher activation marker expression (CD25/CD27/CD28) when compared with unengineered TIL expanded from the same tumors using a conventional REP with IL-2. mbIL15-engineered TIL were polyfunctional, as defined by expression of more than one effector molecules (CD107a, perforin, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and granzyme b) in response to CD3/CD28 stimulation. To examine the cytotoxic function of mbIL15-engineered TIL, we developed autologous cell lines from the same tumors that were used to generate the TIL. Using whole exome and RNA sequencing we found that the autologous tumor cell lines maintained expression of conserved tumor antigens and HLA-expression when compared with the primary tumor. When co-cultured with the autologous tumor cell lines, mbIL15-engineered TIL secreted higher levels of IFN-γ and induced higher cytotoxicity as compared to unengineered TIL cultured with IL-2. Taken together, these data demonstrate that mbIL15-engineered TIL can successfully be expanded from comparatively “cold” tumors with low T-cell infiltration, such as CRC and sarcoma, while maintaining high TCR diversity and polyfunctionality and demonstrating higher cytokine production and cytotoxic activity against autologous tumor lines, compared to conventional TIL with IL2. Citation Format: Zheng Ao, Carmela Passaro, Bulent A. Aksoy, Balazs Koscso, Rachel Burga, Kyle Pedro, Natasha Ly, Nirzari Shah, Alonso V. Ocando, Gauri Kulkarni, Trisha Timpug, Seth M. Pollack, Jan ter Meulen, Michelle L. Ols. Engineering tumor infiltrating lymphocytes from sarcoma and colorectal tumors with membrane bound IL-15 for IL-2 independent expansion and enhanced cytotoxicity against autologous tumor cell lines [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 LB093.

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.

Significance Brain metastases are among the most severe complications of systemic breast cancer, and overexpression of the human epidermal growth factor receptor 2 (HER2) in breast cancer cells increases the incidence of brain metastases in patients. In this study, we engineered the human-derived, tumor cell tropic neural stem cells LM-NSC008 (LM008) to continuously secrete antibodies against HER2. These anti-HER2 antibodies impaired tumor cell proliferation by inhibiting the PI3K-Akt signaling pathway in HER2+ breast cancer cells in vitro. Importantly, our results demonstrate that the therapeutic combinatorial regimen consisting of LM-NSC008 anti-HER2 antibody-secreting cells and the HER2 kinase inhibitor tucatinib provide therapeutic benefit and prolong survival in preclinical models of HER2+ breast cancer brain metastases., Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood–brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation in vitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

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.

Introduction: Liver myeloid-derived suppressor cells (L-MDSC) can suppress genetically-modified T cells (GMT). STAT3 is a regulator of myeloid progenitors and promotes MDSC expansion. We hypothesized an increase in L-MDSC STAT3 activity in the setting of liver metastases (LM), contributing to suppression of GMT. Methods: LM were established in C57BL/6 mice by splenic injection of CEA+ MC38 tumor cells. L-MDSC were isolated with anti-CD11b immunomagnetic beads and flow cytometry used for phenotypic analysis. Ex vivo blockade of STAT3 was performed with cucurbitacin I (JSI-124) and Celastrol. Serum STAT3 and phospohorylated STAT3 (pSTAT3) levels were quantified by ELISA. Results: Both STAT3 and pSTAT3 levels were elevated (1.9-fold, p=0.005 and 1.6-fold, p=0.019) in serum of mice with established LM. L-MDSC isolated from LM-mice contained more pSTAT3 than normal mice (4.9% vs. 1.1%, p=0.007). Having previously demonstrated PDL-1-dependent suppression of anti-CEA GMT by L-MDSC, we asked if increased STAT3 activity contributed to immunosuppression. Blockade of STAT3 led to decreased PDL-1 expression on L-MDSC [52.7% vs. 9.9% (JSI-124 p=0.002) vs. 15.4% (Celastrol p=0.002)], likely hampering PDL-1 dependent immunosuppression. Conclusion: We demonstrated increased STAT3 and pSTAT3 in L-MDSC of mice with LM, and STAT3-dependent PDL-1 expression. Blockade of STAT3 is a potential clinical strategy for preventing L-MDSC PDL-1 expression and suppression of GMT via the PD-1/PDL-1 axis.

Introduction: The immunosuppressive environment in the liver limits the potency of immunotherapy for liver metastases (LM). Myeloid-derived suppressor cells (MDSC) expand in response to LM and mediate T cell suppression via indoleamine 2,3-dioxygenase (IDO). We hypothesized that IDO inhibition rescues genetically modified T cell (GMT) performance through blockade of MDSC-mediated suppression. Methods: Anti-CEA GMT were produced from activated murine splenocytes. C57BL/6 mice were injected via spleen with CEA+ MC38 tumor cells, and MDSC isolated by CD11b+ immunomagnetic beads. MDSC were evaluated by flow cytometry and ex vivo co-culture with CFSE-labeled GMT stimulated by CEA+ tumor, with and without IDO-blockade. Results: IDO expression was demonstrated in MDSC isolated from tumor-bearing (15.7-46.2%) and control livers (15.1%-37.7%). Increased IDO expression was dependent on STAT3 activity in the setting of LM, as blockade with cucurbitacin I (JSI-124) led to downregulated IDO expression on MDSC (48% vs. 21.5%, p=0.005). Liver MDSC suppressed GMT proliferation two-fold (p=0.004). IDO blockade with competitive inhibitor 1-methyltryptophan (1-MT) reversed GMT suppression, resulting in a five-fold increase in GMT division (p=0.016). Conclusions: Liver MDSC express IDO, and IDO represents a mechanism through which MDSC suppress anti-CEA GMT. Our results indicate that blockade of IDO activity may be a viable strategy for enhancing GMT targeted immunotherapy for liver metastases.

Background: We and others have found that liver metastases (LM) mediate tolerogenic effects. B cells make up a significant proportion of liver lymphocytes but their function in the normal liver and in the setting of LM is poorly defined. Therefore, we attempted to define the features of HBC in the setting of LM. Methods: We have used a murine model of CEA+ colorectal LM to study the effects of malignancy on HBC. Tumor cells were injected into the portal circulation of C57Bl/6 mice to induce LM, and HBC were analyzed after two weeks. Results: 50% of mice with LM secreted anti-CEA antibodies, suggesting that the tumor is recognized by B cells in our model. HBC comprised 45±10% of lymphocytes in normal livers, and their frequency was reduced in mice with LM (25±3%, p=0.03). A significant reduction in the frequency of IgM+IgDlo transitional B cells, precursors of mature B cells, was observed in mice with LM (23±2% and 8±4% in normal and metastatic livers respectively, p=0.02). HBC exhibited significant down-regulation of two molecules associated with T cell activation: MHCII (7.5±3.5%, down from 31±5% in normal livers, p=0.03) and CD80 (0.9±0.4%, down from 9.6±1.4% in normal livers, p=0.02). Conclusion: The ability of HBC to mount an immune response may be compromised in the setting of LM as evidenced by reduction of HBC number and diminished HBC expression of MHCII and CD80. Further research is needed to elucidate the function of hepatic B cells during liver metastasis.