Tumor-infiltrating Lymphocytes Persistence Research Articles

Reduced dose fludarabine/cyclophosphamide lymphodepletion for tumor-infiltrating lymphocytes therapy (LN-144, lifileucel) in metastatic melanoma.

TPS9608 Background: Metastatic melanoma (MM) patients who have progressed following immune checkpoint inhibitors (ICI; anti–PD-1/CTLA-4 therapy) and targeted agents (BRAF/MEK inhibitors) have limited treatment options and dismal prognosis. An autologous tumor-infiltrating lymphocytes (TIL) therapy (LN-144, lifileucel) has demonstrated efficacy and durable responses in heavily pretreated advanced melanoma. In a phase II study of TIL therapy (lifileucel) with traditional lymphodepletion (LD), using fludarabine 25 mg/m2 for 5 days and cyclophosphamide (Cy) 60 mg/kg for 2 days, in 153 MM patients previously treated with ICI and BRAF/MEK inhibitors, an overall response rate (ORR) of 31% was noted, and the median duration of response (DOR) was not reached after 27.6 month follow up. We hypothesize that a reduced dose of Flu/Cy LD for TIL therapy will have similar TIL expansion and persistence post-infusion, resulting in similar efficacy with a reduced toxicity profile. Methods: Study NCT06151847 is a single-center, open-label phase II pilot trial evaluating the efficacy, in vivo persistence, and safety of TIL therapy after reduced dose Flu/Cy LD. Key inclusion criteria include unresectable or metastatic melanoma (stage IIIC-IV) with disease progression after one or more lines of therapy. Four of the 12 planned patients have been enrolled as of February 2, 2024. Central TIL manufacturing from at least a 1.5 cm tumor specimen involves ex vivo expansion through cell culture in the presence of the interleukin (IL)-2 and an anti-CD3 monoclonal antibody (1-150 x109 viable cells). Patients will receive an outpatient reduced-dose Flu/Cy LD regimen of Flu (30 mg/m2 on days -4, -3, -2, -1) and Cy (750 mg/m2 on days -4, -3, -2). Infusion of TIL will be performed on day 0 followed by high-dose IL-2 (600,000 IU/kg up to 6 doses). The primary objective will be to ascertain TIL persistence using T-cell receptor (TCR) sequencing at day 42 which will be compared to historical data already generated utilizing traditional Flu/Cy LD. The secondary objectives will be to evaluate the efficacy parameters including ORR, DOR, progression-free survival (PFS), and overall survival (OS), and the safety profile of TIL in combination with a reduced dose Flu/Cy LD regimen. Exploratory objectives will involvecorrelative analyses to characterize the immunome and microenvironment. If TIL persistence is roughly equivocal between the reduced and high-dose Flu/Cy LD regimens, the design of a larger-scale trial using reduced-dose Flu/Cy LD for TIL therapy may be considered. Study follow-up will continue for one year. Clinical trial information: NCT06151847 .

Exploring the safety and efficacy of GT201 as a first-in-class autologous tumor-infiltrating lymphocyte monotherapy in advanced solid tumors.

2547 Background: GT201, featuring a membrane bound IL-15 stably expressed on tumor-infiltrating lymphocytes (TIL), aims to augment TIL persistence and function as well as ameliorate immune suppression within the tumor microenvironment, offering potential for durable responses in advanced solid tumor patients. We present data from 7 patients enrolled in an open-label, single-arm, multicenter clinical study (NCT05729399), investigating the safety profile and efficiency trend of GT201 therapy. Methods: The GT201 phase 1 trial is designed with the primary endpoint of DLT evaluation and the secondary endpoint of measuring preliminary efficacy parameters including overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), duration of response (DOR, and overall survival (OS) following the RECIST v1.1. Results: As of January 22, 2024, a cohort of 7 pts has been enrolled in the study, with a median age of 48 and a median of 3 prior lines of therapy. Among these patients, 1 pt has a history of bone metastases, 2 pts have a history of liver metastases, and 1 pt has a history of brain metastases. Following the standard FC lymphodepletion, pts underwent infusion with GT201 at doses ≥ 5x109 total viable cells. Five pts subsequently received IL-2 as part of the treatment protocol. Treatment-related AEs were observed in ≥ 50% of pts with no grade≥3 AEs. Grade ≥ 3 AEs related to FC lymphodepleting chemotherapy and IL-2 included decreased lymphocyte count, decreased neutrophil count, and decreased white blood cell count, pyrexia and increased heart rate. Notably, all grade ≥3 AEs were either recovered or downgraded to grade ≤ 2 within 14 days. Among the 7 response-evaluable patients across various indications, including Non-Small-Cell-Lung Cancer (NSCLC), melanoma malignant, cervical cancer, and ovarian cancer, 3 patients (42.9%) achieved a confirmed partial response (PR), and 2 patients (28.6%) demonstrated stable disease (SD) as their best response. Notably, among the NSCLC subgroup, disease control (SD ≥ 24 weeks or any PR) was observed in all 3 pts (3/3, 100 %). GT201 cells can be detected in all patients receiving treatment, indicated by both staining IL15RA protein on peripheral T cells and measuring the transgene copy number in peripheral white blood cells. GT201 cells expanded robustly in patients and persist in peripheral blood beyond at least 6 months post cell infusion. Conclusions: In patients with heavily pretreated advanced or metastatic solid tumor, GT201, when infused after FC lymphodepletion and followed by high dose IL-2, exhibits a manageable safety profile. Notably, GT201 has demonstrated a favorable clinical profile in NSCLC, with an encouraging objective response rate, response durability, and no GT201-treatment related grade≥3 AEs. Clinical trial information: NCT05729399 .

Abstract LB065: Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold tumors

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.

1687P Targeted metabolomics reveals dynamic changes and potential therapeutic targets in the serum metabolome of patients receiving adoptive cell therapy with tumor infiltrating lymphocytes

Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs) has shown clinical responses in the treatment of metastatic melanoma. Improvement in the persistence of TILs is an area of ongoing investigation. Emerging data has demonstrated that levels of circulating metabolites significantly affect T cell function and survival in multiple pre-clinical models of ACT. However few studies have interrogated the circulating metabolome in patients undergoing ACT. We aimed to investigate changes in the serum metabolome in patients with metastatic melanoma undergoing ACT with TILs.

Modulation of intracellular kinase signaling to improve TIL stemness and function for adoptive cell therapy.

Adoptive cellular therapy with tumor-infiltrating lymphocytes (TIL) has demonstrated promising clinical benefits in several solid tumors, but the efficacy of this therapy might be compromised by the "prone-to-exhaustion" phenotype of TIL and poor persistence in vivo. This calls for a robust expansion process to produce a large number of cells for clinical usage while at the same time maintaining favorable anti-tumor function and memory phenotype. Previous studies showed that the PI3K-AKT signaling pathway plays a key role in the regulation of T cell activation, differentiation and memory formation. We modulated the PI3K-AKT pathway in TIL isolated from cervical and ovarian cancer by application of AKT or PI3K inhibitors or CRISPR knockout of AKT1 and/or AKT2, and characterized their effects on TIL phenotype and effector function. Mechanistic study was further performed with RNA-seq analysis of AKT1/2 KO TIL in comparison to control TIL. The inhibition of either PI3K or AKT led to an increase in the population of effector CD8+ T cells with upregulation of activation markers, elevated CD39- CD69- memory T cells, and significantly enhanced cytotoxicity when cocultured with tumor cell lines and patient-derived tumor samples. Moreover, dual knockout of AKT1 and AKT2 largely phenocopies the functional impact of AKT or PI3K inhibition on TIL. This result was further validated by RNA-seq analysis indicating that AKT1/2 ablation primarily regulates T cell differentiation and function-related programs. Modulation of PI3K-AKT signaling represents a promising strategy to enhance TIL stemness and cytotoxicity and improve the clinical outcome of current TIL-based therapy to treat solid tumors.

Controllable regulation of CD3ζ and MyD88 signaling in T cells expands and enhances the neoantigen-reactive repertoire resulting in effective antitumor activity

Abstract Background Adoptive cell therapy (ACT) of tumor-reactive T cells including tumor infiltrating lymphocytes (TIL) represents one of the most promising forms of therapies for treating patients with solid tumors. However, durable responses are observed in a small subset of patients and in certain cancer types. Major hurdles to achieving durable responses include: 1) suboptimal TCR responses due to sparse and/or weakly immunogenic tumor antigen presentation, 2) development of dysfunctional or exhausted T cells, 3) low TIL persistence, and 4) tumor infiltration. We and others have demonstrated that activating MyD88 signaling in T cells strongly amplifies weak TCR responses and imparts T cells novel anti-tumor properties. Hypothesis TILs engineered with a novel fusion protein consisting of CD3ζ:MyD88 will activate MyD88 signals in a TCR-dependent manner resulting in enhanced activity and against a broader panel of neoantigens. Method & Results CD4 and CD8 TILs from a variety of human tumors engineered CD3ζ:MyD88 demonstrated: a) dramatically improved ability to expand in vitro resulting (generating 10–100X TILs than conventional TILs within a few weeks), b) persist for months in tissue culture, c) more polyfunctional cells, d) destruction of established melanoma tumors in vivo, e) enhanced responses against a panel of antigens with no overt autoimmunity. Conclusions CD3ζ:MyD88 represents a unique and universal approach to safely enhance T cell response to tumor antigens.

Randomized phase II trial of lymphodepletion plus adoptive cell transfer of tumor-infiltrating lymphocytes, with or without dendritic cell vaccination, in patients with metastatic melanoma

BackgroundThe adoptive transfer of tumor-infiltrating lymphocytes (TIL) has demonstrated robust efficacy in metastatic melanoma patients. Tumor antigen–loaded dendritic cells (DCs) are believed to optimally activate antigen-specific T lymphocytes. We hypothesized...

Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer.

Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39-CD69-) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39+CD69+) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39+ state. However, ACT responders retained a pool of CD39- stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity.

Abstract CT056: Durable complete responses to adoptive cell transfer using tumor infiltrating lymphocytes (TIL) in non-small cell lung cancer (NSCLC): A phase I trial

Abstract Study Objectives: A hallmark of tumor infiltrating lymphocytes (TIL) in melanoma is the potential for a complete response (CR) which can last for decades. This long-lived effect is attributed to persistence of memory T cells. However, clinical efficacy in metastatic non-small cell lung cancer (mNSCLC) has not previously been reported. We launched a phase I trial with the objective to evaluate the safety and efficacy in mNSCLC after evidence of progression on nivolumab. Methodology: Full eligibility criteria is described in trial registry (NCT03215810). Patients were required to have mNSCLC with at least 1 RECIST measurable lesion and also 1 lesion safely accessible for excisional biopsy, usually a supraclavicular lymph node or pleural nodule. This histologically confirmed metastasis was resected and TIL cultured. Autologously reactive cultures were pooled and cryopreserved. TIL was successfully expanded in 95% of patients, to a median dose of 103 billion CD3+ cells. Patients received nivolumab for 4 cycles with two serial CT scans. Patients with tumor enlargement or new lesions proceeded to lymphodepletion cyclophosphamide/fludarabine (Cy/Flu), TIL, and attenuated IL-2. After first CT scan, patients resumed nivolumab for up to 11 doses to augment TIL persistence. Results: Of 32 patients screened, 20 were eligible and enrolled. Median age 54 yrs (range 38 - 75), median PD-L1 proportion score 6%, median estimated TMB 5 mut/MB, 20% EGFR-mutant. The majority had bulky disease with mean sum of target lesion diameters of 7.0 cm. Of 20 enrolled, 13 had evidence of progression on nivolumab and received subsequent Cy/Flu/TIL/IL-2. Two more are progressing on nivolumab and set to receive TIL within next 2 months. Common non-hematologic adverse events (AEs) of the Cy/Flu/TIL/IL2 regimen included hypoalbuminemia (92%), hypophosphatemia (85%), nausea (77%), hyponatremia (69%), diarrhea (62%), the majority of these AEs resolving by Day +10 post-TIL. Initial tumor regression occurred in most patients at their first post-TIL CT scan, with the median best overall change in sum of target lesion diameters of -38% (range +20 to -100). Median time-on-trial post-TIL is 1.4 years. Two patients have achieved durable CRs which are still ongoing almost 1 year post-TIL. One is a never-smoker with EGFREx19 mutation and PD-L1 1%. Three additional patients were able to maintain a clinical remission by local ablative therapy of an isolated new lesion performed 6 to 17 months post-TIL. Persistence of the infused TCR-Vβ clonotypes at post-infusion timepoints was associated with clinical benefit. Neoantigen-specific T cells were detected in the TIL and post-TIL peripheral blood. Conclusions: TIL has manageable toxicity and capacity to achieve durable remission in mNSCLC after nivolumab treatment. TIL may be a promising option for fit mNSCLC patients. Citation Format: Ben Creelan, Chao Wang, Jamie Teer, Eric Toloza, John Mullinax, Jiqiang Yao, John Koomen, Sungjune Kim, Alberto Chiappori, James Saller, Leighann Montoya, Ana Marie Landin, Tawee Tanvetyanon, Bin Fang, Zachary Thompson, Xiaoping Yu, Andreas Saltos, Dung-Tsa Chen, Jose Conejo-Garcia, Eric Haura, Scott Antonia. Durable complete responses to adoptive cell transfer using tumor infiltrating lymphocytes (TIL) in non-small cell lung cancer (NSCLC): A phase I trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT056.

Reactive Myelopoiesis Triggered by Lymphodepleting Chemotherapy Limits the Efficacy of Adoptive T Cell Therapy

Adoptive Tcell therapy (ACT) in combination with lymphodepleting chemotherapy is an effective strategy to induce the eradication of tumors, providing long-term regression in cancer patients. Despite that lymphodepleting regimens condition the host for optimal engraftment and expansion of adoptively transferred Tcells, lymphodepletion concomitantly promotes immunosuppression during the course of endogenous immune recovery. In this study, we have identified that lymphodepleting chemotherapy initiates the mobilization of hematopoietic progenitor cells that differentiate to immunosuppressive myeloid cells, leading to a dramatic increase of peripheral myeloid-derived suppressor cells (MDSCs). In melanoma and lung cancer patients, MDSCs rapidly expanded in the periphery within 1week after completion of a lymphodepleting regimen and infusion of autologous tumor-infiltrating lymphocytes (TILs). This expansion was associated with disease progression, poor survival, and reduced TIL persistence in melanoma patients. We demonstrated that the interleukin 6 (IL-6)-driven differentiation of mobilized hematopoietic progenitor cells promoted the survival and immunosuppressive capacity of post-lymphodepletion MDSCs. Furthermore, the genetic abrogation or therapeutic inhibition of IL-6 in mouse models enhanced host survival and reduced tumor growth in mice that received ACT. Thus, the expansion of MDSCs in response to lymphodepleting chemotherapy may contribute to ACT failure, and targeting myeloid-mediated immunosuppression may support anti-tumor immune responses.

Bhlhe40 Keeps Resident T Cells Too Fit to Quit

Factors regulating the differentiation of tissue-resident memory T (TRM) cells and tumor-infiltrating lymphocytes (TILs) are incompletely understood. In this issue of Immunity, Li etal. identify Bhlhe40 as a transcriptional regulator of CD8+ TRM cell and TIL persistence and activity by orchestrating metabolic and epigenetic programming.

Prospective Analysis of Adoptive TIL Therapy in Patients with Metastatic Melanoma: Response, Impact of Anti-CTLA4, and Biomarkers to Predict Clinical Outcome.

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence.Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated.Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome.Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416-28. ©2018 AACR.

Efficacy of single administration of tumor-infiltrating lymphocytes (TIL) in heavily pretreated patients with metastatic melanoma following checkpoint therapy.

3045 Background: Adoptive cell therapy with TIL involves collection of autologous lymphocytes from the tumor via surgical resection, ex vivo expansion of TIL, lymphodepletion of the patient prior to infusion of TIL using Fludarabine and Cyclophosphamide, followed by infusion of TIL. Up to 6 doses of IL-2 (600,000 IU/kg) is administered to support multiplication of TIL and engraftment. Here, we present the preliminary results from an ongoing, multi-site Phase 2 study of TIL for advanced metastatic melanoma. Methods: Patients with advanced metastatic melanoma who have failed at least one prior systemic therapy were enrolled. Primary objective of the study was to characterize safety profile of LN-144. At baseline, patients had a median age of 56 (41-72) years; 44% were ≥ 60 years old. Median sum of tumor diameters for the target lesions was 10.4 cm, and median of 3 prior therapies. All enlisted patients had prior anti-PD1 as well as anti-CTLA4 and 67% had received ≥ 3 prior therapies. Responses were assessed by RECIST 1.1. TIL products were centrally manufactured. No complications arose from shipment of tumors or TIL. Results: Results are presented through 31 Jan 2017 for the first 9 infused patients evaluable by two assessments. Eight of 9 patients received all 6 doses of IL-2 per protocol. The most common (≥3 patients) non-hematologic grade 3-4 TEAE was hypophosphatemia. No neurotoxicity of grade ≥ 3 was reported. There were no deaths or discontinuations due to SAEs related to study treatment. ORR was 33% (CR = 11%, PR = 22%, SD = 22%, PD = 33%, NE = 11%). Mean time to best response was 3.0 months and median duration of follow up was 3.6 months (1.1+, 12.1). Responses were observed in patients with tumors carrying wild type or BRAF mutations. All patients demonstrated persistence of TIL on day 14 post-infusion. Conclusions: Cell therapy with TIL is an effective treatment with acceptable safety profile for advanced metastatic melanoma patients who are refractory to anti-PD1. TIL products can be centrally manufactured for broad clinical application. This study will be expanded to enroll patients with a shorter manufacturing process as well as offering retreatment for study patients. Clinical trial information: NCT02360579.

Polyfunctional incompletely differentiated CD8+ T cells accumulates after adoptive transfer of tumor-infiltrating lymphocytes and establish immunological memory in patients with metastatic melanoma

Infusion of highly heterogeneous populations of autologous tumor-infiltrating lymphocytes (TILs) can result in tumor regression of exceptional duration. Initial tumor regression has been associated to persistence of TILs shortly after infusion, but it is currently not clear whether a single infusion of TILs can establish long term memory antitumor responses.

Akt inhibition enhances expansion of potent tumor-specific lymphocytes with memory cell characteristics.

Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer.

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8+ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8+ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8+ T cells, on the yield, phenotype, and functional activity of expanded CD8+ T cells during the REP. We found that CD8+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8+ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.

Persistence of tumor-infiltrating CD8 T cells is tumor-dependent but antigen-independent.

How tumor-infiltrating lymphocytes (TILs) that are tumor-specific but functionally tolerant persist in the antigen-expressing tumor tissue is largely unknown. We have previously developed a modified TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model where prostate cancer cells express the T-cell epitope SIYRYYGL (SIY) recognized by CD8 T cells expressing the 2C T-cell receptor (TCR) (referred to as TRP-SIY mice). In TRP-SIY mice, activated 2C T cells rapidly become tolerant following infiltration into the prostate tumor. In this study, we show that tolerant 2C T cells persist in the prostate tumor of TRP-SIY mice by proliferating slowly in a tumor-dependent, but antigen-, interleukin (IL)-7- and IL-15-independent manner. We also show that disappearance of 2C T cells from the lymphoid organs of TRP-SIY mice are due to antigen-induced T-cell contraction rather than altered trafficking or generalized T-cell depletion in the mice. Finally, we show that clonal T cells unreactive to SIY are equally capable of persisting in the prostate tumor. These findings suggest that while functional tolerance of TILs is induced by antigen, persistence of tolerant TILs in the tumor tissue is mediated by a novel mechanism: slow proliferation independent of antigen and homeostatic cytokines. These results also allow CD8 T-cell survival in the tumor environment to be compared with T-cell survival in chronic infection.

Costimulation through 4-1BB/CD137 improves the expansion and function of CD8+ melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy (66.21)

Abstract Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) is a powerful therapy for Stage IV melanoma patients who have failed first and second line therapies. TIL for adoptive transfer are expanded from melanoma surgical specimens with IL-2 followed by a “rapid expansion protocol” (REP), where the cells are activated with anti-CD3 antibody, PBMC feeder cells and IL-2 for two weeks, generating a final infusion product. The REP yields billions of highly-differentiated cytolytic CD8+ T cells that lose costimulatory molecules CD28 and CD27, and are hyporesponsive to further proliferative signals. We found “post-REP” CD8+ TIL express an alternative costimulatory molecule of the TNF-R family, 4-1BB. Our study tests the effects of an agonistic anti-4-1BB antibody added at the time of initiating the REP. We found adding an agonistic anti-4-1BB antibody increased CD8+ T-cell recovery, antigen specificity, BCL-2 gene expression and increased the frequency of CD28+ cells in the recovered CD8+ post-REP TIL. Adding anti-4-1BB improved post-REP TIL survival capacity in the absence of cytokine, with TIL continuing to divide even without added IL-2. Our findings suggest adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.