Tumor-infiltrating Lymphocytes Production Research Articles

A first-in-human study of CRISPR/Cas9-engineered tumor infiltrating lymphocytes (TILs) product GT316 as monotherapy in advanced solid tumors.

2549 Background: A next-generation TIL product GT316 was engineered with CRISPR/Cas9 to genetically disrupt two key immunoregulatory targets identified from a genome-wide CRISPR screening platform and demonstrated significantly enhanced anti-tumor efficacy and persistence in pre-clinical animal studies with ameliorated TIL exhaustion profile. A first-in-human trial was initiated to evaluate the preliminary safety and efficacy of GT316 in advanced solid tumors. Methods: The first-in-class study is to enroll patients (pts) with advanced treatment-refractory solid tumors, especially gynecological tumors. Once the optimal biological dose (OBD) is identified, a monotherapy expansion phase will be initiated for pts with various solid tumors. Enrolled pts were preconditioned with a nonmyeloablative (NMA) lymphodepletion regimen and treated by infusion of the GMP-grade G316 product followed by IL-2 administration. Results: As of January 22, 2024, the dose escalation study enrolled 4 pts, all of whom were female with a median age of 58 years with 1 to 9 prior lines of systemic treatment. The median number of infused TIL cells was 1.0E+10. All pts experienced TRAEs with the majority being grade 1 or 2. Grade 3/4 TRAEs included lymphocyte count decreased, white blood cell count decreased, monocyte count decreased and neutrophil count decreased, which were related to the NMA regimen. No dose-limiting toxicities (DLTs) or TIL-related grade≥3 TRAEs were observed. Median time on study was 33.4 wks (8–35 wks). One pt with treatment-refractory cervical cancer experienced a confirmed complete response (CR) after 4 wks and the duration of response is 32 wks by now (RECIST 1.1). One ovarian cancer pt with 9 lines of previous systemic therapies experienced a confirmed partial response (PR) after 4 wks and the duration of response is 22 wks by now (RECIST 1.1). Response data will be continuously collected. The other two ovarian cancer pts experienced stable disease (SD) and progressive disease (PD), respectively. Despite varying doses of TILs (3.2E+09 to 1.90E+10) and IL-2 (up to 3.0E+05 IU/kg), all pts receiving GT316 showed robust TIL expansion post-infusion, which is positively correlated with administrated IL-2 doses. Enhanced IFN-γ secretion could be detected in the serum of the CR and PR pts compared to the SD and PD pts, indicating potential tumor antigen-specific response. Conclusions: The first-in-human study of GT316, a CRISPR/Cas9-dual KO anti-exhaustion TIL product, demonstrates good tolerability with encouraging preliminary anti-tumor efficacy as a monotherapy in pts with advanced solid cancers. The infusion of GT316 was associated with robust TIL expansion and IFN-γ secretion. Updated data with additional follow-up will be continuously collected, and OBD will be determined based on the overall data of safety and preliminary efficiency. Clinical trial information: NCT06145802 .

A pilot trial of autologous tumor infiltrating lymphocytes (lifileucel, LN-144) for patients with asymptomatic melanoma brain metastases.

TPS9603 Background: Melanoma brain metastases (MBM) are a leading cause of morbidity and mortality for patients with advanced melanoma. Modern systemic therapies are insufficient at controlling MBM, resulting in median overall survival (OS) of <1 year. Adoptive T cell therapy, using tumor infiltrating lymphocytes (TIL), has demonstrated efficacy in treating advanced melanoma. Lifileucel (LN-144), an autologous TIL product, was recently shown to be safe and effective for patients with PD-1 refractory melanoma. However, no trials have yet explored lifileucel in patients with active MBM. Methods: This single-center pilot trial (NCT05640193) aims to enroll 10 pts with asymptomatic MBM from non-uveal melanoma to receive lifileucel. Patients must have ≥1 intracranial lesion measuring 5-30mm visible on MRI to be used as a target lesion for modified (m)RECIST measurement, progression on prior anti-PD-1 therapy (with or without anti-CTLA-4), progression on targeted therapy (if BRAF V600E/K-mutated), ECOG PS ≤1, ≥1 resectable lesion(s) (≥1.5 cm), recovered from prior surgery/anticancer treatment-related AEs (grade ≤1). Patients are ineligible if they have symptomatic MBM and/or require corticosteroids of ≥10 mg/day of prednisone or equivalent. Lifileucel is generated from resected tumor in a centralized GMP process. The regimen includes nonmyeloablative lymphodepletion with cyclophosphamide 750 mg/m2 on days 1-3 and fludarabine 30 mg/kg on days 1-4 (reduced dose compared with standard lifileucel protocol), lifileucel infusion, and up to 6 doses of high-dose IL-2. The primary endpoint is feasibility, defined as ≥7/10 patients who undergo tumor resection successfully receiving lifileucel infusion. Secondary endpoints are safety, feasibility of manufacturing lifileucel in patients with MBM, and brain metastasis response rate (BMRR) per mRECIST 1.1. Exploratory endpoints include overall objective response rate by mRECIST 1.1, best extracranial response rate, intracranial progression-free survival (PFS), overall PFS, OS. Extensive correlative analyses of peripheral blood mononuclear cells, plasma, tumors, and cerebrospinal fluid are planned to better understand MBM growth, treatment resistance, and response of the central nervous system to lifileucel. Clinical trial information: NCT05640193 .

Abstract 29: A genetically modified tumor-infiltrating lymphocytes product (GT216) enhanced anti-tumor efficacy against solid tumor by fine-tuning multiple negative immune regulators

Abstract Adoptive cell therapy by tumor-infiltrating lymphocytes (TILs) has demonstrated promising clinical benefits for patients with solid tumors. However, the large-scale expansion during manufacture may drive hypofunction or exhaustion status of the final TIL product, leading to impaired anti-tumor efficacy and persistence post infusion. Grit Biotechnology designed a genetically engineered TIL product (GT216) to overexpress an intracellular protein that is induced by antigen-specific T cell response and mediates the degradation of multiple immunoregulatory molecules, which releases the therapeutic potential of TIL with enhanced cytotoxicity as well as persistence in vitro and in vivo. Multiple vector construct designs of GT216 were compared and the best design was selected based on stable and efficient expression of surrogate marker in TILs. GT216 could be successfully manufactured by Grit’s StemTexp® and StaViral® platforms with good memory phenotype and no signs of overactivation or exhaustion. In vitro functional analysis showed that GT216 demonstrated significantly increased cytotoxicity and cytokine production when cocultured with tumor cells and better tumor control ability during a serial killing assay than conventional TILs. Notably, GT216 TILs eradicated tumor growth with low IL-2 support in NOG mice inoculated with HLA-matched tumor cells while conventional TILs showed limited tumor control with high IL-2, corelated with their better infiltration and persistence in tumor and periphery. In summary, GT216 demonstrated enhanced anti-tumor efficacy and persistence as well as reduced IL-2 dependency in vitro and in vivo, supporting the clinical evaluation of GT216 as a next-generation TIL therapy in treatment of refractory solid tumors. Citation Format: Jingwei Sun, Yiyang Tan, Jiahui Jin, Jingman Wang, Lingyun Chen, Fengbiao Guo, Yongchao Tan, Zhao Xu, Yarong Liu. A genetically modified tumor-infiltrating lymphocytes product (GT216) enhanced anti-tumor efficacy against solid tumor by fine-tuning multiple negative immune regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 29.

Abstract 3605: Phase II study of pembrolizumab in conjunction with lymphodepletion, TIL ACT, and high- or low-dose IL-2 in patients with metastatic melanoma

Abstract Background: In metastatic melanoma (MM) patients (pts), adoptive cell transfer (ACT) using tumor-infiltrating lymphocytes (TIL) can yield lasting responses. However, TIL ACT with high-dose IL-2 has notable toxicities. Anti-PD1 (pembrolizumab) is FDA approved for MM treatment. Our study aimed to merge TIL infusion with anti-PD1 in two arms, comparing high (HD, arm 1) and low (LD, arm 2) IL-2 doses. Methods: After tumor harvest and TIL expansion, pts were lymphodepleted with cyclophosphamide and fludarabine, followed by infusion with their ex-vivo expanded TIL combined with IL-2 (arm 1: 720,000 IU/kg IV q 8 hrs up to 15 doses; arm 2: 2 million IU SC for 14 days). Pts were given 200 mg of IV anti-PD1 starting 21 days post-TIL infusion, followed by doses every 3 weeks for up to 2 years. Blood was taken before lymphodepletion and before the first two anti-PD1 doses for flow cytometry and cytokine analysis The primary endpoint of the study was determination of overall response rate by RECISTS 1.1 criteria. Enrollment aimed for 18 pts per arm, monitoring the primary ORR endpoint using a Bayesian rule. Enrollment would halt in any arm if the ORR dropped below 40%. Results: The median age was 50 years (n=14: 6 F and 8 M). Of these 14 pts, 8, 2, 2, and 2 had cutaneous, uveal, mucosal, or unknown primary melanoma. Pts had a median of 3 lines of prior therapies (range 1-6), including anti-PD-1 (n=13). In arm 1, one pt had a partial response (PR) for 10 months, 2 had stable disease (SD), 3 had progressive disease (PD), and 1 was not evaluable (NE). In arm 2, one pt had an ongoing PR for over 76 months, 1 had SD, and 5 had PD. Due to ORR <40% in each arm, enrollment was stopped after treatment of 14 pts. There was no significant different in progression-free survival or overall survival between the two arms (p=0.99 and p=0.71, respectively). In general, toxicity levels were comparable in both arms; however, pts in arm 2 had slightly lower grade 3 febrile neutropenia (57% vs. 71%) and shorter hospitalization (median 16 days vs. 18 days) than pts in Arm 1. While no correlation was observed between the phenotype of the TIL product and clinical response, both PR pts received high numbers of TIL (38.6 × 10^9 and 93.7 × 10^9; mean: 32.6 × 10^9 cells), with a high CD8+/CD4+ T cell ratio (284.7 and 2.7; median: 2.7). IL-2 dose did not affect circulating Treg frequency, phenotype, or proliferation by flow cytometry 3 weeks post TIL infusion. IL-2 dose was not associated with CD4+ non-Treg and CD8+ T cell phenotype or proliferation. Addition of anti-PD1 reduced expression of checkpoints but did not boost T cell proliferation. Conclusion: Our treatment strategy did not yield any distinct difference between low and high dose IL-2, suggesting the potential of low-dose IL-2 as an alternative. Anti-PD1 did not have any appreciable effect on immune states potentially due to the cohorts being comprised of a heavily pre-treated heterogenous pt population. Citation Format: Merve Hasanov, Simin Kiany, Marie-Andrée Forget, Roland L. Bassett, Michael A. Davies, Adi Diab, Jeffrey E. Gershenwald, Isabella C. Glitza, Jeffrey E. Lee, Anthony Lucci, Jennifer L. McQuade, Sapna P. Patel, Merrick I. Ross, Hussein A. Tawbi, Jennifer Wargo, Michael K. Wong, Chantale Bernatchez, Patrick Hwu, Cara Haymaker, Rodabe N. Amaria. Phase II study of pembrolizumab in conjunction with lymphodepletion, TIL ACT, and high- or low-dose IL-2 in patients with metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3605.

Abstract 6593: Rejuvenation of tumor-infiltrating lymphocytes (TIL): A novel strategy to revitalize TIL antitumor function for cell therapy

Abstract TIL therapy is a promising approach for the treatment of metastatic solid tumors, but its efficacy is limited by T-cell exhaustion and terminal differentiation. In addition, recent studies have underscored the adverse effects of aging on T cells. Reprogramming T cells into induced pluripotent stem cells (iPSC) and differentiating them back to the T-cell lineage has proven to be complex and time-consuming. In particular for TIL applications, de novo T cell production via iPSCs requires each TCR in the final product to be derived from an independently established iPSC clone. To overcome these barriers, we developed a novel technology called Rejuvenation, which bypasses the need for reprogramming of T cells into iPSCs. Rejuvenated TIL products retain their TCR diversity while offering several significant advantages, including reduced T-cell epigenetic age, improved expansion capacity, stem cell-like phenotype, and enhanced secretion of cytokines upon activation by target antigens. In this study, we used TIL derived from patients with melanoma and NSCLC. Rejuvenated TIL were generated by the transient expression of transcription factors in a process called ‘Partial Reprogramming’ and returned back to a T-cell lineage phenotype by a process called ‘Redirection’. Partially reprogrammed TIL showed an altered morphology resembling adherent stromal cells and down-regulated conventional T-cell markers including CD3, CD4, and CD8. Redirected T cells reacquired a conventional CD4/CD8 phenotype including morphology, surface markers and function, and demonstrated decreased epigenetic age (>7 years younger, N=3). We also observed a higher proliferative capacity, improved metabolic state, and increased expression of biomarkers associated with T-cell stemness, including CCR7 and CD62L, when compared with untreated TIL. Rejuvenated TIL demonstrated improved cytokine secretion, including IFNγ, TNFα and IL-2 in functional assays. Immune repertoire analysis revealed a significant polyclonal population of putative tumor-reactive TCRs. In vivo functionality was also evaluated using a xenograft model of engineered A375 melanoma cell line that agonizes T-cells in hIL2-NOG mice. Adoptive transfer of rejuvenated TIL resulted in significantly improved suppression of tumor growth and prolonged survival compared with TIL expanded by conventional methods. These results were also confirmed in other surrogate models, including rejuvenated PBMC, TCR and CAR T cells, demonstrating the broad applicability of Rejuvenation technology. Our results indicate TIL Rejuvenation enhances the functionality and antitumor characteristics of T cells, while preserving a broad TCR repertoire. This technology holds substantial promise and may pave the way for the development of the first rejuvenated autologous polyclonal TIL therapy for treatment of advanced solid tumors. Citation Format: Raul Vizcardo, Yin Huang, Jessica Fioravanti, Takuya Maeda, Naritaka Tamaoki, Yasuhiro Yamazaki, Burak Kutlu, Kriti Bahl, Biao Wang, Zheng Zhong, Shobha Potluri, Nicholas P. Restifo, Gary Lee. Rejuvenation of tumor-infiltrating lymphocytes (TIL): A novel strategy to revitalize TIL antitumor function for cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6593.

CTIM-16. DEVELOPING PATIENT SPECIFIC TUMOR INFILTRATING LYMPHOCYTES (TILS) WITH INCREASED TUMOR SPECIFICITY AS A THERAPY FOR GLIOBLASTOMA

Abstract Immunotherapy failure in glioblastoma results from three main hurdles; limited numbers of T cells within the tumor, T cell exhaustion and significant intra-tumoral and intra-patient heterogeneity leading to lack of T cell tumor recognition. Our group has developed an expansion protocol to isolate and expand less exhausted tumor infiltrating lymphocytes (TILs) from the tumor periphery, selecting for TILs that recognize tumor antigen ex vivo. This process will increase the number of T-cells specific for an individual patient’s diverse tumor neoantigens. We have successfully expanded glioblastoma associated TILs from 66% of the novel Lukens trap substrate, which collects TILs and tumor from the tumor periphery. Cells are expanded in two stages, preREP and REP, through novel methods based on a protocol with clinical success in lung cancer and modified to increase the percent of tumor specific T cells. Through co-culture with patient matched glioma neurospheres we have identified activated T cells from each stage of culture (initial product, preREP, REP) based on CD137+ expression and tumor specific cytokine release (IFNy+TNFa+IL2+IL17a+) by flow cytometry. Through 5’scRNA sequencing paired with TCR repertoire analysis we can identify activation specific TCRs. Overrepresented TCR alpha and beta chains will be linked by a P2A site, cloned into a lentiviral vector, and transduced into TCR negative Jurkat cells to confirm tumor reactivity. This strategy will allow us to enrich and assess tumor specific TCR activity compared to bystander activation in our TIL product. Following additional preclinical assessment of cytotoxicity against autologous tumor, we will evaluate safety and feasibility of by administering our novel TIL product intrathecally in patients with glioblastoma.

Neoantigen-specific CD4+ tumor-infiltrating lymphocytes are potent effectors identified within adoptive cell therapy products for metastatic melanoma patients

BackgroundAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a promising immunotherapeutic approach for patients with advanced solid tumors. While numerous advances have been made, the contribution of neoantigen-specific CD4+T...

Abstract B023: Using hypoxia to improve tumor infiltrating lymphocytes therapy in different subtypes of renal cell carcinoma

Abstract BACKGROUND: Improving the long-term efficacy for advanced renal cell carcinoma (RCC) remains challenging after progression on first line therapy. Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) is a promising personalized immunotherapeutic approach to treating solid tumors. Previously, TIL therapy for clear cell RCC patients showed moderate success but did not account for the distinct immune microenvironments in different subtypes of RCC such as clear cell (ccRCC), papillary (pRCC) or unclassified (uRCC) known to have different response to immunotherapy. In addition, hypoxia plays a critical role in tumor progression and therapy resistance in RCC. Therefore, the aim of this study was to evaluate TIL expansion from different subtypes of RCC under varying oxygen concentrations. METHODS: Tumor types collected from 48 patients included ccRCC (81.25%), pRCC (16.7%) and uRCC (8.3%). Tumors were minced into fragments and cultured for 4 weeks in media supplemented with high dose IL-2 (6000 IU/mL). At the end of the 4 weeks, TIL were further expanded using a rapid expansion protocol (REP) in either 20% or 5% oxygen. TIL expansion, reactivity to autologous tumor, and phenotype were evaluated in pre-REP and post-REP samples. RESULTS: Among the ccRCC, 91.7% of the samples expanded, while 100% of the pRCC and uRCC expanded. pRCC expanded more TIL than ccRCC (1.05e8 vs 6.7e7 total TIL expanded, respectively) while uRCC expanded less than the other subtypes (2.03e7). Pre-REP reactivity against autologous tumors was high for all the subtypes (88.3%, 75% and 100% for ccRCC, pRCC and uRCC respectively). The T-cell phenotype across subtypes showed a tendency of more CD4+ T-cells and less CD8+ T-cells for pRCC and uRCC, compared to ccRCC. All subtypes that underwent REP were able to expand in both 20% and 5% O2 (hypoxia) conditions. TIL expanded in hypoxia had a significantly higher percentage of tissue resident memory T-cells (CD69+CD103+; 15.1%, p<0.0001) when compared to the pre-REP TIL population (2%) or TIL expanded at 20% O2 (3.1%). CONCLUSION: These results demonstrate the feasibility of expanding tumor-reactive TIL from different subtypes of RCC for the first time, and highlight the advantage of exposing TIL to hypoxic conditions to enhance the differentiation of resident memory T-cells in TIL products. Citation Format: Marine Potez, Mohammed Alkhouli, Johannes Ali, Michael Carter, Matthew Beatty, Shari Pilon-Thomas, Jad Chahoud. Using hypoxia to improve tumor infiltrating lymphocytes therapy in different subtypes of renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B023.

A pilot trial of autologous tumor infiltrating lymphocytes (lifileucel, LN-144) for patients with asymptomatic melanoma brain metastases.

TPS9606 Background: Melanoma brain metastases (MBM) are a leading cause of morbidity and mortality for patients with advanced melanoma. Modern systemic therapies are insufficient at controlling MBM, and median overall survival (OS) for pts with MBM is < 1 year. Adoptive T cell therapy using tumor infiltrating lymphocytes (TIL) has demonstrated efficacy in advanced melanoma. Lifileucel (LN-144), an autologous TIL product, was recently shown to be safe and effective for patient with PD-1 refractory melanoma. Patients with active MBM were excluded from lifileucel trials to date. Methods: This single-center pilot trial (NCT05640193) is enrolling up to 10 pts with asymptomatic MBM from non-uveal melanoma to receive lifileucel. Patients must have ≥1 intracranial lesion measuring 5-30mm visible on MRI to be used as a target lesion for modified (m)RECIST measurement, progression on prior anti-PD-1 therapy (with or without anti-CTLA-4), progression on targeted therapy if BRAF V600E/K-mutated), ECOG PS ≤1, ≥1 resectable lesion(s) (≥1.5 cm), recovered from prior surgery/anticancer treatment-related AEs (grade ≤1). Patients are not eligible if they have symptomatic MBM and/or require corticosteroids of ≥10 mg/day of prednisone or equivalent. Lifileucel is generated from resected tumor in a centralized GMP process. The regimen includes nonmyeloablative lymphodepletion, lifileucel infusion, and a short course of high-dose IL-2. The primary endpoint is feasibility, defined as ≥7/10 patients who undergo tumor harvest receiving lifileucel infusion. Secondary endpoints are safety, feasibility of manufacturing lifileucel in patients with MBM, and brain metastasis response rate (BMRR) per mRECIST 1.1. Exploratory endpoints include overall objective response rate by mRECIST 1.1, best extracranial response rate, intracranial progression-free survival (PFS), overall PFS, OS. Extensive correlative analyses of peripheral blood mononuclear cells, plasma, tumors, and cerebrospinal fluid are planned to better understand MBM growth, treatment resistance, and response of the central nervous system to lifileucel. Clinical trial information: NCT05640193 .

Tumor infiltrating lymphocyte (TIL) harvest and ex vivo expansion from primary and metastatic (met) uveal melanoma (UM) tumors.

9513 Background: Adoptively transferred autologous cell therapy using tumor-infiltrating lymphocytes (TIL) is an emerging treatment for met melanoma. Responses have been seen in patients (pts) with checkpoint inhibitor-naïve and -refractory cutaneous melanoma. UM represents a disease subtype that is traditionally less responsive to immune checkpoint blockade (ICB). Preliminary studies have shown clinical utility of TIL in UM pts. However, data describing the success rates of TIL harvest and ex vivo expansion of cells from primary and met UM tumors, and of the treatment of pts with met UM, is limited. Methods: Between 2004 and 2019, we conducted a single-center IRB-approved trial (NCT NCT00338377) for TIL harvest in pts age 18 and older with documented met melanoma, including UM pts. The protocol was amended to allow harvest of primary UM tumors in 2015 if the pt was dispositioned for enucleation as primary treatment. Results: A total of 96 UM pts underwent TIL harvest; 9 underwent treatment with TIL. Median age was 54 years (range: 28 – 86); 56% of the pts were male; 78% were Caucasian, 9% Hispanic, and 13% unknown ethnicity. Overall successful ex vivo expansion of TIL occurred in 34.8% (32/92) of UM tumors in typical tumor fragment culture with IL-2 (TIL 1.0), with higher rates observed with UM mets (29/65; 44.6%) than UM primary tumors (3/27; 11.1%). Better results were observed with a culture method (TIL 3.0) that includes agonistic stimulation of CD3 and 41BB, with 96.8% (30/31 tumors). Expansion using this new method was successful in 9/10 primary UM tumors and 21/21 UM mets. Average number of days in culture for successful initial ex vivo expansion was 33.0 days for TIL 1.0 and 17.9 days for TIL 3.0 ( p-value<0.0001). Overall, the average number of TIL expanded from culture and cryopreserved for clinical use was 130.4 million for TIL 1.0 (range: 34-460) and 326.2 million for TIL 3.0 (range 102-760) ( p-value<0.0001). Nine met UM pts received treatment with up to 150 x 109 post-Rapid Expansion Protocol (REP) TIL (5 TIL 1.0, 4 TIL 3.0). Median age was 53, 44% were male, and all had active liver mets with a median of 3 lines of prior treatment for advanced disease. 8 of the TIL products were from UM mets (4 soft tissue, 3 liver, 1 combination of soft tissue and liver tumors), and 1 was from primary UM tumor, for which TIL could only be expanded with TIL 3.0. Best overall response rate per immune-related response criteria was 22% partial response (PR, duration of response was 22.1 months and 16.5 months), 44% stable disease (SD), and 33% progression of disease (PD) for a disease control rate of 66% (PR + SD). Both responding pts had TIL harvests from mets and had previously progressed on ICB, one was treated with TIL 3.0. Conclusions: TIL harvest and successful ex vivo expansion of cells is possible from both primary and met UM with TIL 3.0 culture method and can be therapeutically effective. Clinical trial information: NCT00338377 .

Investigator-initiated phase 1 clinical trial of synthetic TIL: Autologous peripheral T cells engineered with polyclonal TIL-derived TCRs.

e14525 Background: The efficacy of tumor-infiltrating lymphocyte (TIL) therapy is limited by the rarity of anti-tumor TCRs and exhausted status of the product. Its improvement should focus on a high frequency of diverse anti-tumor TCRs with stem-like phenotype, which can be achieved synthetically by identifying multiple anti-tumor TCRs from the patient's intra-tumor TCR repertoire and subsequently engineering them into the patient’s peripheral T cells. Such a ‘synthetic TIL’ is cost-prohibitive using existing technologies. To enable a synthetic TIL product, we developed ultra-high throughput TCR gene synthesis and screening methods to synthesize and screen > 50,000 TCRs from scRNA-Seq data from > 30 patients and identified > 100 tumor/NeoAg-reactive TCRs. The gene expression patterns of the T cells carrying such experimentally validated tumor/NeoAg-reactive TCRs gave rise to an algorithm called Interpreter to predict potentially anti-tumor TCRs from scRNA-Seq data. Methods: To translate synthetic TIL to the clinic, we developed a method to generate up to 100 sequence-verified TCR-encoding plasmids (selected by Interpreter) from fresh tumor biopsy in just 3 weeks. Our GMP-grade production uses a collective knock-in approach, where a mixture of up to 100 plasmids (each encoding a distinct TCR) at equal proportion are packaged into AAV which serve as template to precisely insert the TCR genes into a primarily monoallelic locus and simultaneously inhibit the endogenous TCR. This gene editing is characterized by a modified scRNA-Seq method called EntrySeq. In first-in-human, N-of-1 trials, 109 to 6x109 synthetic TIL cells were administered to solid tumor patients with or without lymphodepletion and IL2. An investigator-initiated Phase 1 trial was then opened to evaluate safety, preliminary efficacy, and cellular activity of synthetic TIL and to determine RP2D and MTD. Subjects with relapsed, unresectable locally advanced solid tumors who received at least 1 standard systemic therapy are allowed to enroll in this study. Those patients with no standard treatment regimens available, or those who are unable to obtain effective treatment due to economic conditions are also permitted. Results: EntrySeq confirmed that > 80% of the T cells express only 1 TCR, avoiding mispairing of different TCRs. The frequencies of different TCRs were largely uniform and were stable through plasmid, AAV and T-cell mixtures. Up to 1.5x1010 synthetic TIL cells were produced from a patient’s leukopak. 40 to 60% of the T cells in the product for infusion express an exogenous TCR. In first-in-human trials, the administered T cells showed favorable persistence and safety profile. The Phase 1 trial is ongoing. Conclusions: We established a fast, affordable, and GMP-compliant process to manufacture synthetic TIL products, enabling a Phase 1 trial.

Effect of a novel expansion process on tumor-infiltrating lymphocyte (TIL) polyfunctionality, cytotoxicity, and expansion, while preserving cells in a less differentiated and more stem-like phenotype.

2542 Background: Adoptive cell therapy using autologous TIL has shown durable responses in patients with metastatic melanoma and some epithelial tumors (Chesney JITC 2022; Schoenfeld SITC 2021; O’Malley SITC 2021). Data suggest that maintaining a higher proportion of less differentiated and more stem-like TIL can associate with persistence and response in patients with metastatic melanoma (Krishna Science 2020; Rosenberg Clin Cancer Res 2011). Here, we describe a novel TIL expansion process that increases TIL expansion and preserves cells in a less-differentiated and more stem-like phenotype with enhanced functional output. Methods: Tumors of various histologies, including lung, breast, and renal cancer, were fragmented and expanded using either a standard process or a newly developed process that uses a different combination of cytokines during the pre-rapid expansion protocol (pre-REP) stage and another combination of cytokines and a pathway inhibitor during the REP stage to control T-cell activation and differentiation. The expansion potential, viability, and phenotypic and functional attributes of the final TIL products were evaluated by a variety of assays. Results: When compared with standard TIL expansion, the novel process increased yield and viability while preserving TIL in a less differentiated and more functional state, as evidenced by increased expression of the memory-associated markers CD27, CD28, CD62L, and IL-7R, with reduced expression of the activation markers CD38, CD39, and CD69, and lower levels of the inhibitory markers LAG3, TIM3, TIGIT, and TOX. Importantly, the novel process led to a pronounced increase in the tumor-homing marker CXCR3 as well as TIL polyfunctionality as evidenced by increased co-expression of IFNγ, TNFα, and IL-2 while showing an enrichment in genes associated with stem-like cells and a reduction in exhaustion-associated genes. Pseudotime trajectory analysis also demonstrated that the new expansion process maintained TIL in a less differentiated and more stem-like state. Together, these phenotypic and functional characteristics translated into increased cytotoxicity even after repeated stimulation. Conclusions: Our novel TIL expansion process improves multiple metrics that correlate with both TIL persistence and response, including enhanced polyfunctionality, reduced inhibitory receptor expression and a less differentiated and more stem-like phenotype, while increasing yield. These effects may translate into a more vigorous and less differentiated TIL infusion product with improved cytotoxicity and persistence.

Abstract 4062: The discovery and development of a CRISPR/Cas9-engineered tumor-infiltrating lymphocytes product (GT316) as a next-generation TIL therapy

Abstract Adoptive cell therapy by tumor-infiltrating lymphocytes (TILs) has demonstrated promising therapeutic effects in multiple types of solid tumors and significantly prolonged the survival of late-stage patients. However, the “prone-to-exhaustion” phenotype of the final TIL product after rapid expansion during manufacture and the presence of various immunosuppressive mechanisms in tumor microenvironment (TME) compromises the persistence and anti-tumor efficacy of TIL post infusion. To discover potential immunoregulatory targets that could maximize the function of TIL, we established a genome-wide CRISPR/Cas9 screening platform, ImmuT Finder®, to unbiasedly identify functional gene targets in primary T cells. Notably, this platform employed T cells bearing a tumor-antigen specific TCR-T that were repeatedly stimulated by tumor cells with cognate tumor antigen and demonstrated similar exhaustion profile as TIL. We selected top 30 targets after several rounds of screening and quickly validated their function in a 96-well array. Then, the top targets that showed significantly enhancement in proliferation and function of TCR-T were further selected, and their combinational effects on TIL were tested both in vitro and in vivo. Finally, double KO of GT304 and GT312 (GT316) stands out as the most potent combination, which significantly improves TIL proliferation and cytokine release, eradicates tumor growth in both CDX and PDX mouse models and promotes long-term in vivo persistence with low-dependence on IL-2. Specific and potent sgRNAs targeting both targets have been selected, and manufacturing process of GT316 TIL product has been developed (KOReTIL®). In summary, these data demonstrated that our ImmuT Finder® platform enables unbiased discovery of potent and novel targets for the development of T cell therapy and support the clinical assessment of GT316 as a next-generation TIL therapy. Currently, an investigator-initiated trial of GT316 is ongoing in China. Citation Format: Yarong Liu, Jingwei Sun, Yao Sheng, Jingman Wang, Jiahui Jin, Fei Li, Lingyun Chen, Di Zhou, Yongchao Tan, Quanwei Wang, Zhao Xu, Haibao Li, Pin Wang, Jun Cui. The discovery and development of a CRISPR/Cas9-engineered tumor-infiltrating lymphocytes product (GT316) as a next-generation TIL therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4062.

Abstract 4052: Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells

Abstract Background: The autologous cell therapy field has investigated numerous novel strategies and processes to improve response rates and expand the use of adoptive cell therapies (ACT) to patient suffering from a broad range of cancers. Tumor infiltrating lymphocytes (TIL) can be harvested from tumors, expanded in vitro, and infused to patients leading to substantial clinical benefits in some patient populations. Tumor reactivity of TIL can vary greatly, representing a key limiting factor of bulk TIL therapies. The ability to generate an autologous cell product enriched in tumor reactive cells while limiting the presence of potentially detrimental and/or competitive non-reactive cells is highly desirable for next generation TIL products. Here we demonstrate that direct selection of antigen experienced TIL from tumor dissociates is feasible and allows to obtain functional cells enriched for tumor reactivity from various tumor indications. Methods: Patient-derived tumor material from various indications, including ovarian, kidney, colorectal cancer and lung tumors were first processed into single cell suspensions, maximizing TIL recovery and viability. Antigen experienced TIL were immediately selected based on the co-expression of PD-1 and CD39, using fluorescence-activated single cell sorting (FACS). The sorted cells or the unselected counterparts were expanded in a rapid expansion protocol (REP), then analyzed for phenotypic and functional characteristics and reactivity against autologous tumor cells. Results: The PD-1+ CD39+ selection strategy consistently allowed sorting of a population of viable TIL that was amenable to in vitro culture from tumors of various cancer indications. The selected TIL successfully expanded in a rapid expansion protocol. TCR sequencing analysis revealed that the PD-1+ CD39+ sorted populations were enriched in unique clones compared to the unselected populations. Expanded PD-1+ CD39+ selected cells demonstrated the ability to produce key effector cytokines upon restimulation in polyclonal assays. Importantly, the PD-1+ CD39+ expanded TIL were enriched for tumor reactive T cells and showed improved cytotoxic activity against autologous tumors. Conclusions: PD-1+ CD39+ selected TIL can be successfully isolated and expanded in vitro, generating a TIL product of superior reactivity in multiple cancer indications. PD-1+ CD39+ selected TIL showed increased cytokine secretion and cytotoxic activity against autologous material, indicating that this selection strategy enriches for functional tumor-reactive lymphocytes, which is likely to be a key feature of successful ACT. Citation Format: Christophe Pedros, Larissa Pikor, Anna Fritzsche, Zachary K. Jilesen, Bethany Macleod, Niloufar Khojandi, Bryant Thompson, Matthew Thayer, Nikolas Bryan, Emily Carron, Sowbarnika S Ratliff, April Fraley, Jake Nikota, Robert Fisher, Sebastien Delpeut, Anna Kluew, Madysson Scott, Christian Laing, Leo He, Antoine Bernard, Nathalie Brassard, Simon Turcotte, Timothy J Langer, David Stojdl, Stewart Abbot, Barbara Sennino. Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4052.

Abstract 4055: CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) is a feasible and effective therapy for melanoma and lung cancer[1,2]. Multiple factors may determine the quality of the TIL product including components of the tumor microenvironment. In this work, we analyzed the role of melanoma infiltrating B cells in the context of TIL expansion based on their documented association with response to other types of immunotherapies[3]. We stimulated melanoma infiltrating B cells using human recombinant CD40L on the first day of ex-vivo TIL expansion. Samples were expanded from cryopreserved melanoma tumor single cell suspensions, in high dose IL-2 alone (standard protocol), or in high dose IL-2 plus CD40L. After 48h, analysis of activation markers on the CD40-expressing cells by flow cytometry was performed. For further investigation of the changes induced by CD40L stimulation, TIL expansion cultures (+/- CD40L) were analyzed using scRNA-seq (10X Genomics Chromium NextGEM Single Cell 5’ v2 and V(D)J Reagent kits; Illumina NovaSeq 6000 instrument with S4 sequencing flow cell) at 48h of culture (n=7 patients). The TIL expansion success rate was 68% with the CD40L treatment condition compared to 36% with the standard protocol. TILs cultured in the presence of CD40L expanded to on average three times more than with the standard protocol (P ≤ 0.01). Treatment with CD40L increased the percentage of CD39- CD69- T cells (P ≤ 0.05). Within the tumor digests, a higher percentage of B cells, including switched memory B cells (CD27+ IgD− ), was associated with successful TIL expansion (P=0.04). scRNA-seq analysis demonstrated different clustering patterns within the B cell compartment based on culture conditions. No clear partition was observed for other cell types, including the myeloid compartment. B cells displayed 126 DEGs associated to CD40L addition, CCL22, CD83, EBI3 and CD58 were among the upregulated genes in the CD40L-treated B cells. Other cell types experienced minimal to no change in transcriptomic profiles. B cell clusters were sub-classified based on CD27 and IgD expression[4], showing a predominance of naïve and switched memory B cells. Our results show that higher presence of B cells within tumors is associated with better TIL expansion, suggesting an interplay between T and B cells, and providing rationale for the design of improved TIL expansion protocols based on B cell stimulation with CD40L. This work has been supported in part by the Flow Cytometry, Genomics and Biostatistics and Bioinformatics Core Facilities at Moffitt Cancer Center, an NCI designated Comprehensive Cancer Center (P30-CA076292). We acknowledge Moffitt’s Melanoma Center of Excellence and the Mark Foundation for the financial support. [1] Sarnaik, A.A. et al. JCO 39, 2656-2666 (2021). [2] Creelan, B. et al. Nat Med 27, 1410-1418 (2021). [3] Cabrita, R. et al. Nature 577, 561-565 (2020). [4] Sanz, I. et al. Front Immunol 10, 2458 (2019). Citation Format: Renata Ariza Marques Rossetti, Leticia Tordesillas, Matthew Beatty, Dongliang Du, Yian Ann Chen, Amod Sarnaik, Shari Pilon-Thomas, Daniel Abate-Daga. CD40L stimulates melanoma infiltrating B cells and enhances ex vivo TIL expansion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4055.

Abstract OT2-10-04: Treatment of advanced or metastatic triple-negative breast cancer with adoptive therapy of PD1+ tumor-infiltrating lymphocytes (TILS001 trial)

Abstract Background Metastatic triple-negative breast cancer (mTNBC) exhibits a particularly poor clinical outcome, generally with rapid progression and worse overall survival (OS) than other BC subtypes. Among the few therapeutic options, chemotherapy-based combinations are associated with increased toxicity and limited survival benefit, being treatment with sequential single agents, such as paclitaxel considered an appropriate first-line regimen for the metastatic setting for PDL-1 negative patients. Herein, there is an urgent need for clinically active agents for the mTNBC. Adoptive cell transfer (ACT)-based immunotherapy using ex vivo activated and expanded tumor-infiltrating lymphocytes (TILs) has shown promising therapeutic outcomes in some patients with metastatic tumors. The identification, selection, and enrichment of tumor-reactive lymphocytes at the early stages of the ACT generation could enhance their clinical activity. Thus, the selection of reactive T cells, such as PD1-positive (PD1+) TILs, could improve the responses achieved in those settings. TILS001 trial aims to explore the safety, tolerability and efficacy of selected PD1+ T-cell infusion with a previous pre-selection of mRNA PD1-high expression in patients with mTNBC. Study design: TILs001 trial is an open-label, single-arm, multicenter phase I/II prospective study with a two-stage design evaluating treatment with PD1+ TILs infusion in advanced or mTNBC, defined as HER2 negative and Hormonal Receptor < 10%. The study involves three different parts before PD1+TILs treatment. Tumor samples evaluable for PD1 mRNA expression and life expectancy ≥6 months are mandatory for part 1. Patients with high levels of PD1 mRNA, defined by the pre-specified cutoff, candidates for receiving a first-line taxane-based containing regimen and with at least 1 accessible target lesion to generate TILs are eligible for part 2. Finally, once the complete expansion of PD1+TILs is reached, patients will receive the non-myeloablative lymphodepleting chemotherapy regimen followed by PD1+TIL infusion and IL-2 treatment. Allogeneic hematopoietic stem cell transplantation, immune system-related disease or clinically active cerebral metastasis are not allowed. The primary objectives are to evaluate the safety and tolerability of the PD1+ TIL product, as per incidence of grade 3-5 adverse events (AE) or any grade AE that leads to treatment discontinuation and to assess the efficacy of ACT therapy with selected PD1+ TILs in mTNBC in terms of progression-free survival (PFS) at 6 months. The secondary endpoints are clinical benefit rate at 6 months (CBR6), overall response rate, duration of response (DoR), PFS and OS. Further translational research including immunophenotyping, TCR sequencing and mutational analysis will also be performed. The first 3 patients will be included in a safety run-in phase where safety will be evaluated 24h after PD1+/TILs infusion (before IL-2 treatment) and a phase II stage where efficacy will be evaluated, which will include up to 20 patients. Patients will be enrolled in 4 sites in Spain. Recruitment is expected to start by July 2022 and to be completed within 24 months. This study is financially supported by the Asociación Española Contra el Cáncer (GCAEC19010PRAT). NCT05451784 Citation Format: Nuria Chic, Eva Ciruelos, Cristina Saura, Europa-Azucena Gonzalez, Luís Álvarez-Vallina, Juan José Lasarte, Alena Gros, Lorea Villanueva, Jordi Canes, Laura Angelats, Aleix Prat, Tomás Pascual, Marta Santisteban, Manel Juan. Treatment of advanced or metastatic triple-negative breast cancer with adoptive therapy of PD1+ tumor-infiltrating lymphocytes (TILS001 trial). [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT2-10-04.

Expansion of KRAS hotspot mutations reactive T cells from human pancreatic tumors using autologous T cells as the antigen-presenting cells.

Adoptive cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) or TCR gene-modified T cells (TCR-T) that recognize mutant KRAS neo-antigens can mediate tumor regression in patients with advanced pancreatic ductal adenocarcinoma (PDAC) (Tran et al in N Engl J Med, 375:2255-2262, 2016; Leidner et al in N Engl J Med, 386:2112-2119, 2022). The mutant KRAS-targeted ACT holds great potential to achieve durable clinical responses for PDAC, which has had no meaningful improvement over 40years. However, the wide application of mutant KRAS-centric ACT is currently limited by the rarity of TIL that recognize the mutant KRAS. In addition, PDAC is generally recognized as a poorly immunogenic tumor, and TILs in PDAC are less abundant than in immunogenic tumors such as melanoma. To increase the success rate of TIL production, we adopted a well-utilized K562-based artificial APC (aAPC) that expresses 4-1BBL as the costimulatory molecules to enhance the TIL production from PDCA. However, stimulation with K562-based aAPC led to a rapid loss of specificity to mutant KRAS. To selectively expand neo-antigen-specific T cells, particularly mKRAS, from the TILs, we used tandem mini gene-modified autologous T cells (TMG-T) as the novel aAPC. Using this modified IVS protocol, we successfully generated TIL cultures specifically reactive to mKRAS (G12V). We believe that autologous TMG-T cells provide a reliable source of autologous APC to expand a rare population of neoantigen-specific T cells in TILs.

Abstract 5581: A genetically modified tumor-infiltrating lymphocytes product (GT202) exhibits enhanced persistence and anti-tumor efficacy against solid tumor in vivo

Abstract Adoptive cell therapy by tumor-infiltrating lymphocytes (TILs) has demonstrated significant clinical benefits for patients with solid tumors. However, the toxicities related to high-dose IL-2 that is required to support TIL survival and the exhaustion status of the final TIL product after rapid expansion in culture largely limits the clinical application and long-term efficacy of TIL therapy. Grit Biotechnology designed a genetically engineered TIL product (GT202) to express a membrane-bound cytokine that is critical for T cell activation and effector function, leading to enhanced cytotoxicity as well as persistence in vitro and in vivo. Multiple designs of the transmembrane domain of GT202 were compared and the best design was selected based on stable and efficient expression of GT202 on the membrane of TILs. GT202 demonstrated significantly increased cytotoxicity and cytokine production when cocultured with tumor cells in vitro than conventional TILs and better tumor control ability during a serial killing assay. In the meanwhile, GT202 TILs showed enhanced long-term survival and better preservation of TCR clonality without IL-2 in vitro, as well as persisted longer in multiple tissues without IL-2 over conventional TILs + IL-2 in NGS mice. Notably, GT202 TILs achieved better anti-tumor efficacy with low IL-2 in NSG mice inoculated with HLA-matched tumor cells than conventional TILs, corelated with their better infiltration and proliferation in spleen and tumor. In summary, GT202 demonstrated enhanced anti-tumor efficacy and persistence as well as decreased IL-2 dependency in vitro and in vivo. Currently, an investigator-initiated trial of GT202 is ongoing in China. Citation Format: Jingwei Sun, Jiahui Jin, Yongchao Tan, Lingyun Chen, Quanwei Wang, Yarong Liu. A genetically modified tumor-infiltrating lymphocytes product (GT202) exhibits enhanced persistence and anti-tumor efficacy against solid tumor in vivo [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 5581.

Abstract 575: Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors

Abstract In this study, we evaluated the feasibility of expanding tumor infiltrating lymphocytes (TIL) from surgically resected renal cell carcinoma (RCC) tumors. Tumors were collected from 43 patients undergoing surgery to remove primary kidney tumors. Tumor types included clear cell RCC (86.0%), papillary RCC (11.6%) as well as chromophobe RCC (2.3%). Tumors were minced into fragments, placed in individual wells of a 24-well plate containing media with high dose IL-2 (6000 IU/mL), and cultured for four weeks. Successful expansion was considered when at least one fragment expanded to a minimum of 2 wells. Successfully expanded TIL were then evaluated for T-cell phenotypes as well as reactivity to the autologous tumor. Of all RCC tumors collected, TIL were successfully grown for 79% of the samples (34/43). The T-cell phenotype of these TIL was highly variable across samples, although there was a higher percentage of CD4+ T-cells relative to CD8+ T-cells. The reactivity of expanded TIL was then assessed by IFNγ release. We found that TIL secreted IFNγ in response to autologous tumor in 71% of the samples. Together, these results demonstrated the feasibility of expanding functional tumor reactive TIL from RCC. Given that hypoxia plays an important role in the development of RCC tumors and is associated with poor prognosis, we then evaluated the ability to expand TIL under hypoxic conditions. To this end, primary TIL were cultured at various O2 levels (20% O2, 5% O2 or 1% O2) and the cell yield as well as the T-cell memory phenotypes were evaluated. We found that the yield of TIL grown in hypoxic conditions was lower when compared to normoxic conditions (20% O2), suggesting a reduction of TIL proliferation in hypoxia. TIL grown in hypoxic conditions displayed increased percentage of central memory T-cells compared to TIL cultured at 20% O2, as evaluated by CD45RA and CCR7 expression (CD45RA-CCR7+). Primary TIL were then expanded to larger numbers using a rapid expansion protocol (REP) by stimulation with anti-CD3 antibody in the presence of allogenic feeder cells. REP of TIL was carried out at either atmospheric O2 (20% O2) or at a hypoxic O2 level (5% O2), and the T-cell phenotype as well as reactivity to the autologous tumor were assessed. Unlike pre-REP TIL, TIL that underwent REP were able to expand in hypoxic conditions (5% O2) and displayed increased percentage of tissue resident memory T-cells (CD69+CD103+; 19%) when compared to the starting TIL population (0.11%) or TIL expanded at atmospheric O2 (0.17%). When co-cultured with the autologous tumor, hypoxic TILs displayed increased IFNγ, TNFα as well as increased Granzyme B release (p<0.001) when compared to TIL expanded in normoxia (20% O2). Collectively, these results support the advantage of adapting TIL to hypoxic conditions in the production of tumor-reactive TIL. Citation Format: Mohammed Alkhouli, Veronica Martinez-Brockhus, Matthew S. Beatty, Robert J. Gillies, Shari Pilon-Thomas, Jad Chahoud. Ex vivo expansion of tumor infiltrating lymphocytes (TIL) from human renal cell carcinoma (RCC) tumors [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 575.

Abstract 3542: Oncolytic adenovirus encoding transgenes for IFN-beta and recombinant CD40 ligand promotes conventional dendritic cell activation and trafficking with enhanced tumor infiltrating lymphocytes for effective cancer immunotherapy

Abstract Clinical studies have shown that pre-existing tumor-reactive T cells are critically important for effective cancer immunotherapy. Methods to augment the number and functionality of tumor-reactive T cells are therefore vital. Given that cytotoxic CD8 T cell priming requires conventional dendritic cells (cDC), we hypothesized that strategies to enhance cDC functionality in tumors would augment anti-tumor CD8 T cell responses. CD40 and type 1 IFN signaling pathways each promote the cross-presentation function of cDCs to activate CD8 T cells. However, it is unclear whether these pathways are functionally independent and whether their combined activation in tumor cDC leads to higher levels of T cell priming. Using adenovirus vectors, we tested the impact of expression of IFNβ and a chimeric membrane-stable CD40 ligand (MEM40) in syngeneic mouse tumor models. Compared to intratumoral administration of each individual transgene-encoding adenovirus, combined MEM40 + IFNβ expression resulted in the best tumor growth control, highest levels of tumor infiltrating lymphocytes (TILs), and systemic CD8 T cell responses. MEM40 and IFNβ were independently capable of enhancing T cell responses as we found IFNβ in CD40-/- mice and MEM40 in type 1 IFN receptor IFNAR1-/- mice induced a strong T cell response. These results were consistent with a cDC-mediated priming mechanism. Studies with cDC1 deficient BATF3-/- mice revealed a crucial role of this cDC subset in MEM40 + IFNβ induced T cell responses. IFNβ alone and MEM40 + IFNβ triggered increase in lymph node (LN) homing receptor CCR7 expression in cDC1 concurrent with a reduction in cDC1 in tumors and gain in tumor draining LNs. Simultaneously, combined MEM40 + IFNβ combination led to the highest levels of expression of CD80 and CD86 co-stimulatory molecules in both tumor and LN cDC1. Single cell (sc)-RNA sequencing of tumor DCs further showed that MEM40 + IFNβ led to the highest proportion of a recently defined mature/regulatory cDC subset characterized by high expression of CCR7, CD40 and IL12b. Applying these results towards an effective therapeutic modality, we developed a dual-transgene-armed MEM40 + IFNβ expressing conditionally replicative adenovirus (MEM-288) that simultaneously confers tumor-specific oncolysis and promotes tumor antigen release. MEM-288 drives MEM40 + IFNβ expression in freshly resected human lung tumors and generates potent anti-tumor responses as a monotherapy and in combination with immune checkpoint inhibitors. Furthermore, MEM-288 induced enhancement of tumor-reactive TILs and provides a rationale for studies to determine whether MEM-288 pre-treatment generates a superior TIL product for more effective adoptive T cell therapy. Citation Format: Hong Zheng, Scott Antonia, Mark J. Cantwell, Xiaoqing Yu, Amer A. Beg. Oncolytic adenovirus encoding transgenes for IFN-beta and recombinant CD40 ligand promotes conventional dendritic cell activation and trafficking with enhanced tumor infiltrating lymphocytes for effective cancer immunotherapy [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 3542.