Autologous Tumor-infiltrating Lymphocytes Research Articles

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eye’s uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases.

Uveal melanoma (UM) is a rare melanoma originating in the eye's uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Pharmacological induction of MHC-I expression in tumor cells revitalizes T cell antitumor immunity.

Antigen presentation by major histocompatibility complex class I (MHC-I) is crucial for T cell-mediated killing, and aberrant surface MHC-I expression is tightly associated with immune evasion. To address MHC-I downregulation, we conducted a high-throughput flow cytometry screen, identifying bleomycin (BLM) as a potent inducer of cell surface MHC-I expression. BLM-induced MHC-I augmentation rendered tumor cells more susceptible to T cells in coculture assays and enhanced antitumor responses in an adoptive cellular transfer mouse model. Mechanistically, BLM remodeled the tumor immune microenvironment, inducing MHC-I expression in a manner dependent on ataxia-telangiectasia mutated/ataxia telangiectasia and Rad3-related-NF-κB. Furthermore, BLM improved T cell-dependent immunotherapeutic approaches, including bispecific antibody therapy, immune checkpoint therapy, and autologous tumor-infiltrating lymphocyte therapy. Importantly, low-dose BLM treatment in mouse models amplified the antitumor effect of immunotherapy without detectable pulmonary toxicity. In summary, our findings repurpose BLM as a potential inducer of MHC-I, enhancing its expression to improve the efficacy of T cell-based immunotherapy.

47 Generation and Characterization of Ex Vivo Expanded Tumor-Infiltrating Lymphocytes from Renal Cell Carcinoma Tumors for Adoptive Cell Therapy

Abstract Background Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance anti-tumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Methods Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Results Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. Figure: Assessment of tumor immune microenvironment (TIME) via multiplex immunofluorescence (IF). Cases that failed REP are indicated in red. (A) Representative image at 60x magnification, fluorophore colors as indicated. (B) Density of tumor-infiltrating CD8+ and FoxP3+ cells. (C) Expression of co-inhibitory exhaustion markers as a percentage of total CD8+ cells. (D) Expression of PD-1 in FoxP3+ cells. (E) Comparison of tissue TIME (dots) with TIL product post-REP (open circles) exhaustion profiles as a percentage of total CD8+ cells. Conclusions The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Lung Cancer Adoptive Cell Therapy: Inspiring TIL ACT Comes Center Stage.

Schoenfeld and colleagues report, in this issue, a measurable objective response rate in 6/28 (21.4%) of patients with advanced non-small cell lung cancer treated with lifileucel, a cell therapy product based on autologous tumor-infiltrating lymphocytes (TIL). Extending solid evidence in advanced melanoma that led to FDA approval of lifileucel, this new evidence bodes well for treating patients with other common tumor histologies, justifying important efforts by a large number of academic and biotechnology companies engaged in improving the TIL process. See related article by Schoenfeld et al., p. 1389 (1).

Generation and Characterization of Ex Vivo Expanded Tumor-infiltrating Lymphocytes From Renal Cell Carcinoma Tumors for Adoptive Cell Therapy.

Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance antitumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Lifileucel, an Autologous Tumor-Infiltrating Lymphocyte Monotherapy, in Patients with Advanced Non-Small Cell Lung Cancer Resistant to Immune Checkpoint Inhibitors.

In this phase 2 multicenter study, we evaluated the efficacy and safety of lifileucel (LN-145), an autologous tumor-infiltrating lymphocyte cell therapy, in patients with metastatic non-small cell lung cancer (mNSCLC) who had received prior immunotherapy and progressed on their most recent therapy. The median number of prior systemic therapies was 2 (range, 1-6). Lifileucel was successfully manufactured using tumor tissue from different anatomic sites, predominantly lung. The objective response rate was 21.4% (6/28). Responses occurred in tumors with profiles typically resistant to immunotherapy, such as PD-L1-negative, low tumor mutational burden, and STK11 mutation. Two responses were ongoing at the time of data cutoff, including one complete metabolic response in a PD-L1-negative tumor. Adverse events were generally as expected and manageable. Two patients died of treatment-emergent adverse events: cardiac failure and multiple organ failure. Lifileucel is a potential treatment option for patients with mNSCLC refractory to prior therapy. Significance: Autologous tumor-infiltrating lymphocyte therapy lifileucel was administered to 28 patients with heavily pretreated metastatic non-small cell lung cancer (mNSCLC). Responses were observed in patients with driver mutations, and various tumor mutational burdens and PD-L1 expression, potentially addressing an unmet medical need in patients with mNSCLC refractory to prior therapy. See related commentary by Lotze et al., p. 1366.

Phase 1 study of GT101 as an autologous tumor infiltrating lymphocyte (TIL) therapy in advanced solid tumors.

2548 Background: Adoptive cell therapy utilizing autologous TIL has demonstrated efficacy and durable long-term responses in patients with certain advanced solid tumors progressed after conventional therapies. We present data from 14 patients enrolled in the study of GT101, a Phase 1, open-label, single-arm, multicenter trial (NCT05430373) of autologous TIL therapy, aiming to investigate the safety profile, efficiency trend and the duration of response. Methods: The trial was designed with the primary endpoint on evaluating DLT and characterizing the safety profile of GT101 in solid tumor patients measured by the incidence of Grade ≥ 3 treatment-emergent adverse events (TEAEs). The secondary endpoints were to assess efficacy parameters including overall response rate (ORR), disease control rate (DCR), progression-free survival (PFS), duration of response (DOR), and overall survival (OS) according to RECIST v1.1. Results: As of November 10, 2023, a cohort of 14 patients received treatment with a median age of 46.9 and a median of 2.6 lines of prior therapies. Following a standard FC-based lymphodepletion, patients underwent GT101 infusion at doses ≥ 5x109 cells with median dose of 3.7x1010 cells, followed by high-dose IL-2 administration. Most of observed Grade ≥ 3 AEs were related to the FC conditioning regimen and IL-2, including decreased lymphocyte count, decreased white blood cell count, decreased neutrophil count, anemia, pyrexia and decreased platelet count. The majority of these AEs were recovered within 14 days, while a few were downgraded to ≤ Grade 2 within 4 weeks. Among 14 enrolled patients across various indications (small-cell-lung cancer, melanoma, cervical cancer), the ORR was 35.7%. Specifically, 4 pts (28.6%) had a confirmed partial response (PR), 1 pt (7.1 %) achieved complete response (CR), and 8 pts (57.1 %) had stable disease (SD) as their best response. One pt had unconfirmed disease progression (PD). Notably, among 11/14 patients with cervical cancer, the ORR was 45.5% (5/11) with 4 pts (36.4%) achieving PR and 1 pt (9.1 %) achieving CR. Disease control was observed in 10/11 pts (90.9%), and the median PFS was 4.2 months for this cohort. The CR patient underwent a long-term follow-up and the duration of CR and PFS (estimated by Kaplan-Meier) was 24 weeks and 36 weeks, respectively. Post GT101 infusion, T cell expanded robustly in the peripheral blood of all patients with median Tmax 6.83 days and average 15.9 days. Conclusions: In the GT101 Phase 1 study, no treatment-related SAEs and DLT were observed. In patients with heavily pretreated advanced or metastatic solid tumors, GT101 exhibited a manageable safety profile under the treatment protocol of lymphodepletion and high-dose IL-2 and a favorable clinical profile, particularly in cervical cancer, with an encouraging ORR and sustained response duration. Clinical trial information: NCT05430373 .

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 .

Efficacy and safety of lifileucel, an autologous tumor-infiltrating lymphocyte cell therapy, and pembrolizumab in patients with immune checkpoint inhibitor-naive unresectable or metastatic melanoma: Updated results from IOV-COM-202 cohort 1A.

9505 Background: Immune checkpoint inhibitors (ICI) are front-line standard-of-care treatment (tx) for advanced (unresectable or metastatic) melanoma. Despite recent advances in front-line tx, a majority of patients (pts) do not achieve long-term benefit. Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, potentially induces durable responses in pts with advanced melanoma previously treated with ICI. This study evaluates lifileucel combined with anti-PD-1 therapy in front-line advanced melanoma. Methods: IOV-COM-202 (NCT03645928) Cohort 1A assesses the efficacy and safety of lifileucel and pembrolizumab (pembro) in pts with ICI-naive unresectable or metastatic melanoma. Pts may have received BRAF/MEK inhibitor tx if BRAF mutation-positive. Eligible pts must have ≥1 resectable lesion (≥1.5 cm diameter) for 22-day cryopreserved lifileucel manufacturing, and ≥1 measurable lesion for response assessment per RECIST v1.1. The tx regimen consists of pembro, nonmyeloablative lymphodepletion (cyclophosphamide and fludarabine), a single lifileucel infusion (1 × 109 – 150 × 109 cells), ≤6 doses of IL-2 (600,000 IU/kg IV), and continued pembro until disease progression, or unacceptable toxicity for ≤24 months. The endpoints are investigator-assessed objective response rate (ORR) and incidence of grade ≥3 treatment-emergent adverse events (TEAE). Results: As of 22-Dec-2023, 22 pts with a median (range) age of 48.5 (18–68) years received lifileucel and pembro. At baseline, the median (range) target lesion sum of diameters was 54.5 (14–355) mm; 7 (31.8%) pts had liver lesions. Metastatic staging at study entry was as follows: 4 (18.2%) had M1a, 2 (9.1%) had M1b, 10 (45.5%) had M1c, and 2 (9.1%) had M1d. Eight (36.4%) pts had V600 BRAF mutations; 3 (13.6%) pts had prior BRAF/MEK inhibitor tx. Confirmed ORR was 63.6% (14/22), including 22.7% (5/22) CR and 40.9% (9/22) PR; 6 pts (27.3%) had SD. Median time to initial response was 2.5 months. All response evaluable pts demonstrated regression of target lesions. At a median follow-up of 17.2 months, median duration of response was not reached. Responses deepened over time; 10/14 (71.4%) pts had ongoing response and 8/14 (36.4%) pts had response ≥12 months. TEAEs were consistent with the underlying disease and known safety profiles of pembro, nonmyeloablative lymphodepletion, and IL-2. Most common grade ≥3 TEAEs were thrombocytopenia (68.2%), neutropenia (50.0%), and anemia (45.5%). Conclusions: These results demonstrate encouraging efficacy and durability for the combination of lifileucel and pembro and support its further evaluation in pts with untreated advanced melanoma in the phase 3 study TILVANCE-301 (NCT05727904). Clinical trial information: NCT03645928 .

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 .

A phase 2, multicenter study of TILs treatment in advanced tumors with alterations in the SWI/SNF complex: The TILTS study.

TPS2674 Background: Tumors associated with SWI/SNF complex (SWI/SNFc) mutations, mainly SMARCA4 and SMARCB1 loss, are a heterogeneous group of malignancies with rhabdoid features that typically affect young patients (pts). These are aggressive malignancies with poor prognosis for which no standard treatment is available. Despite not having a high tumor mutation burden, tumor infiltrating lymphocytes (TILs) are usually present in high numbers. The biologic reasons for this feature are not clearly understood but might be associated to changes in gene expression due to aberrant chromatin remodeling. However, these TILs indicate that tumor antigens are recognized by lymphocytes unveiling a vulnerability that can be exploited by immunotherapy. The objective of the TILTS study is to develop a personalized adoptive cell therapy using TILs in pts affected by these tumors. Methods: Single arm, multi center, phase 2 study of TILs in pts with advanced tumors associated with SWI/SNFc mutations. Pts are treated at 3 centers in Spain: Catalan Institute of Oncology (ICO), Barcelona, Vall d'Hebron Institute of Oncology (VHIO), Barcelona, and Centro Integral Oncológico Clara Campal (HM CIOCC), Madrid. Primary endpoint is overall response rate (ORR) by RECIST 1.1. Secondary endpoints include toxicity evaluation, duration of response (DOR), progression-free survival (PFS) or overall survival (OS). After informed consent is obtained, tumor resection is performed. Total volume of tumor resected should be at least 1.5 cc to assure TIL isolation. Eligible pts enter the treatment phase. TILs are produced under GMP conditions. During the TILs manufacturing process, pts are allowed to receive standard treatment if clinically indicated. Before TIL infusion, pts receive non-myeloablative preconditioning lymphodepletion with daily intravenous (IV) cyclophosphamide (60 mg/kg; IV x2 doses) and daily fludarabine (25 mg/m2; IV x5 doses). Infusion of autologous TILs is given on Day 0 followed by administration of IL-2 at 600,000 international units (IU)/kg every 8-12 hours for a maximum of 6 doses. After 4 weeks, a new tumor biopsy is optional. First image evaluation is performed after 8 weeks from TIL infusion. Pts without progression enter the follow-up phase. An ancillary translational study will explore mechanisms of immunogenicity, antigen recognition, TILs anti-tumor activity, and changes in the stool microbiome and specific TCR population during treatment. Also, tumor immune microenvironment will be analyzed. Clinical trial information: 2023-504632-17-00 . [Table: see text]

Pelareorep driven blood TIL expansion in patients with pancreatic, breast and colon cancer.

e14625 Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in cancer. One emerging cancer treatment approach to leverage the therapeutic potential of TILs is the expansion and adoptive cell transfer of autologous TILs, commonly referred to as autologous adoptive therapy (ACT). ACT clinical studies have shown encouraging results. While the majority of these trials have targeted melanoma, impressive clinical benefit has also been observed in cervical cancer, with preliminary efficacy in colorectal cancer (CRC), cholangiocarcinoma, non-small cell lung cancer, and breast cancer. Pelareorep (pela) is a live, replication competent reovirus (T3D) that selectively replicates in cancer cells and represents a new class of immunotherapy currently being explored in multiple clinical settings. To examine the effect of pela therapy on TIL expansion we applied T cell receptor sequencing of matched tumor tissue and whole blood pre- and post-treatment in a subset of breast, pancreatic, and (CRC) subjects receiving chemotherapy and atezolizumab, an anti-PD-L1 therapy, and intravenous treatment with pela. Methods: Identification of TIL clones was performed by immunosequencing of the CDR3 regions of human T-cell receptor-β (TCRβ) chains (ImmunoSEQ Assay, Adaptive Biotechnologies). DNA was isolated from tissue and blood at baseline and from blood collected post-treatment. TCRβ CDR3 regions were amplified by a multiplex, bias-controlled PCR with primers targeting the V and J genes of T cells as well as primers targeting housekeeping genes to quantitate the total nucleated cells in each sample. PCR products were sequenced on an Illumina NextSeq. Results: Pela treatment was observed to increase the expansion of pre-existing and new TIL clones in the blood in from all tumor samples after one cycle of treatment. We also observed that pre-existing TIL clonal expansion in the blood seemed to correlate with reductions in tumor volume in pancreatic cancer and to a lesser extent in CRC patients who received multiple cycles of therapy. Interestingly, unlike atezolizumab, pela’s clinical activity was lost when combined with avelumab, a PD-LI inhibitor capable of binding Fc receptors and inducing ADCC. The addition of avelumab eliminated pre-existing TIL expansion in the blood highlighting the importance of TIL expansion following pela therapy. Conclusions: These findings, while preliminary, suggest that the clinical benefits observed following pela immunotherapy may be mechanistically analogous to ACT. Accordingly, pela therapy may offer a means to directly expand TILs without the need for tumor resection, ex vivo TIL expansion, T cell ablation and IL-2 therapy. Clinical trial information: Eudra-CT: 2020-003996-16 .

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 .

Dynamics of circulating cytokines and chemokines during and after tumor-infiltrating lymphocyte cell therapy with lifileucel in advanced melanoma patients.

9594 Background: Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, has demonstrated potentially clinically meaningful activity and durable responses in patients with advanced (unresectable or metastatic) melanoma. The regimen includes nonmyeloablative lymphodepletion (NMA-LD) prior to lifileucel infusion, and a short course of interleukin-2 (IL-2) post infusion. The safety profile of the regimen is consistent with the underlying disease and the known safety profiles of NMA-LD and IL-2. Cytokine release syndrome (CRS) is an acute and severe adverse event commonly associated with chimeric antigen receptor T-cell therapy. Here, we report circulating cytokine levels during and after the lifileucel treatment to monitor immune activation, and the dynamics of cytokines and chemokines to explore mechanism of action and potential predictive clinical biomarkers. Methods: The full analysis set of C-144-01 (NCT02360579) includes 153 patients with unresectable or metastatic melanoma who have progressed on checkpoint inhibitors and, if applicable, BRAF/MEK inhibitors. After tumor resection, patients received NMA-LD (cyclophosphamide on Days −7 to −6 and fludarabine on Days −5 to −1) followed by a single lifileucel infusion (Day 0) and up to 6 doses of IL-2 (Days 0-4). Peripheral blood samples were collected at baseline (pre-NMA-LD and pre-lifileucel infusion) and post-lifileucel infusion on Days 1, 4, 14, 42, and 84. Serum cytokine and chemokine levels were measured with BioPlex and included a multiplex panel for IL-6, IL-7, IL-9, IL-10, IL-12, CCL2, CXCL10, IFN-γ, and TNF-α. Plasma samples also were tested for IL-6, IL-15, and IL-7 levels by ELISA. Results: IL-15 levels following NMA-LD peaked at Day 1 and returned to baseline by Day 42. This is consistent with prior reports that NMA-LD increases circulating IL-15 levels, which may promote TIL expansion in the absence of competing endogenous lymphocytes. Similarly, IL-7 and CCL2 also peaked at Day 1. IL-6 did not increase during or after the lifileucel regimen, consistent with absence of severe systemic inflammation including higher grade CRS after lifileucel treatment. There was no difference in cytokine and chemokine levels between responders and nonresponders in the full analysis set. Conclusions: These data support the hypothesized mechanism of action of NMA-LD in the lifileucel regimen. Cytokine levels measured during treatment are consistent with lack of severe systemic inflammation observed in patients treated with lifileucel. Cytokine and chemokine dynamics did not predict for response to lifileucel. Clinical trial information: NCT02360579 .

Abstract CT285: Trial in progress: A phase 1/2 study to investigate the safety and efficacy of OBX-115 engineered tumor-infiltrating lymphocyte (TIL) cell therapy in patients (pts) with immune checkpoint inhibitor (ICI)-resistant advanced or metastatic melanoma

Abstract Introduction ICI have improved outcomes for pts with melanoma; however, most pts (~60%) do not achieve long-term survival. Unengineered bulk TIL cell therapy has an objective response rate (ORR) of 31-49% in pts with unresectable or metastatic melanoma (Rohaan NEJM 2022, Chesney JITC 2022), but requires co-administration of systemic high-dose IL2, which is associated with safety risks and limits pt eligibility. OBX-115 TIL are engineered to express regulatable membrane-bound IL15 (mbIL15) under dose-dependent regulation using acetazolamide (ACZ), an FDA-approved small-molecule diuretic, avoiding the need for high-dose IL2. A first-in-human single-institution study (NCT05470283) evaluating the safety of OBX-115 in metastatic melanoma is currently enrolling. The current study (NCT06060613) is enrolling at multiple US sites using centralized manufacturing. Methods This phase 1/2, single-arm, open-label, nonrandomized, multicenter study will assess the safety, tolerability, and efficacy of the OBX-115 engineered autologous TIL cell therapy regimen in pts with unresectable or metastatic melanoma resistant to ICI (Table). Primary objectives of Phase 1 are to characterize safety and tolerability and identify a recommended Phase 2 dose of the OBX-115 regimen; Phase 2 will evaluate efficacy (ORR using RECIST v1.1). Cryopreserved OBX-115 is generated from the pt’s own tumor tissue procured by surgical excision or core biopsy, and is infused after standard- or low-dose (based on clinical eligibility) lymphodepletion (cyclophosphamide and fludarabine). ACZ is administered at cohort-defined doses once daily for up to 10 days, with ACZ redosing at Week 6-8 for 10 days if the initial observed tumor response is less than partial response. No systemic high-dose IL2 is administered. Two sites are open and recruiting pts. TABLE 1. NAND Key Eligibility Criteria Key Inclusion Criteria Key Exclusion Criteria Age ≥18 years Uveal melanoma Histologically confirmed diagnosis of unresectable Stage IIIC, IIID, or Stage IV metastatic melanoma Brain metastasis or leptomeningeal disease Documented radiographic disease progression after systemic therapy containing a PD-1- or PD-L1-blocking antibody (if adjuvant setting, progression during or within 12 weeks after the last dose) Active autoimmune disease, including active uveitis or any other medical illness that would pose increased risks for study participation Received a BRAF inhibitor ± MEK inhibitor if BRAF V600 mutation-positive Prior allogeneic organ transplant, allogeneic cell therapy, or genetically engineered cell therapy (not including autologous stem cell or unengineered TIL cell therapy) ≥1 lesion suitable for OBX-115 generation with expected minimum of 1.5-cm diameter; minimally invasive tumor tissue procurement (core biopsy) may be considered on a case-by-case basis after discussion with the Medical Monitor Systemic steroid therapy >10 mg/day of prednisone or equivalent ≥1 RECIST 1.1-measurable lesion remaining after tumor tissue procurement ECOG performance status 0 or 1 Estimated life expectancy >6 months Citation Format: Sajeve S. Thomas, Jason A. Chesney, Omid Hamid, Gino K. In, Alexander N. Shoushtari, Yazan Samhouri, Parameswaran Hari, Giridharan Ramsingh, Prakash Prabhakar, Lauren Mclaughlin, Allison Betof Warner. Trial in progress: A phase 1/2 study to investigate the safety and efficacy of OBX-115 engineered tumor-infiltrating lymphocyte (TIL) cell therapy in patients (pts) with immune checkpoint inhibitor (ICI)-resistant advanced or metastatic melanoma [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 CT285.

Abstract CT286: TILVANCE-301, a phase 3 study of lifileucel tumor-infiltrating lymphocyte (TIL) cell therapy in combination with pembrolizumab vs pembrolizumab alone in treatment-naive unresectable or metastatic melanoma

Abstract Introduction: Novel early-line therapies for advanced (unresectable or metastatic) melanoma are needed to improve the rate of deep and durable responses and increase the proportion of patients with long-term benefit. TILVANCE-301 will evaluate the efficacy and safety of lifileucel autologous TIL cell therapy plus pembrolizumab in patients with untreated advanced melanoma. Methods: TILVANCE-301 (NCT05727904) is a phase 3, multicenter, randomized, open-label, parallel group study that will randomize ~670 patients (1:1; Day 0) to Arm A: lifileucel plus pembrolizumab (Day 3: tumor tissue resection for TIL manufacturing; Day 5: pembrolizumab 200 mg; Day 26: pembrolizumab 400 mg; Day 28-29: cyclophosphamide 60 mg/kg; Day 28-32: fludarabine 25 mg/m2; Day 33: lifileucel; Day 34-37: ≤6 doses of high-dose IL-2; Week 10: pembrolizumab 400 mg Q6W) or Arm B: pembrolizumab alone (same pembrolizumab dosing as Arm A). Patients in Arm B with confirmed progressive disease verified by blinded independent review committee (BIRC) have the option to receive lifileucel monotherapy as immediate next treatment and may continue pembrolizumab until start of nonmyeloablative lymphodepletion. Eligible adults have histologically confirmed advanced melanoma, Eastern Cooperative Oncology Group performance status of 0-1, estimated life expectancy >6 months, ≥1 resectable lesion to generate lifileucel, and ≥1 remaining measurable lesion. Prior neoadjuvant or adjuvant treatment including immune checkpoint inhibitors may be allowed. Prior therapy for metastatic disease, symptomatic untreated brain metastases, organ allograft or prior cell therapy, uveal/ocular melanoma, and chronic systemic steroid therapy are not permitted. The dual primary efficacy endpoints are BIRC-assessed (RECIST v1.1) objective response rate (ORR) and progression-free survival (PFS). Key secondary efficacy endpoint is overall survival. Additional secondary efficacy endpoints include BIRC-assessed complete response (CR) rate, duration of response (DOR), and event-free survival (EFS); investigator-assessed ORR, PFS, CR rate, DOR, EFS, and PFS2; and safety as characterized by severity and seriousness of treatment-emergent adverse events and relationship to study drug. Exploratory endpoints include in vivo T-cell persistence (unique CDR3 sequences in peripheral blood mononuclear cell [PBMC] over time) and correlative biomarkers (eg, lifileucel phenotypic and functional characteristics; lifileucel, tumor, and PBMC gene expression profiles; tumor mutational landscape). The study will enroll globally, with initial sites in Europe, North America, and Australia. Trial registration: NCT05727904 Citation Format: Sajeve Thomas, Young Ki Hong, Yazan Samhouri, James Larkin, David J. Olson, Gino K. In, Victoria Atkinson, Philip Lammers, Andrew J. Furness, Juan Martin-Liberal, Patrick Terheyden, Andrew S. Poklepovic, Ryan H. Nguyen, Idit Peretz, Marcus Butler, Adnan Khattak, Lavinia Spain, E.M. Gaughan, Melissa Wilson, John W. Dubay, Anja Williams, Stephanie Goff, Gary C. Doolittle, Jason Chesney, Friedrich Graf Finckenstein, Jeffrey Chou, Xiao Wu, Giri Sulur, Wen Shi, John Haanen. TILVANCE-301, a phase 3 study of lifileucel tumor-infiltrating lymphocyte (TIL) cell therapy in combination with pembrolizumab vs pembrolizumab alone in treatment-naive unresectable or metastatic melanoma [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 CT286.

Abstract CT176: OBX-115 engineered tumor-infiltrating lymphocyte (TIL) cell therapy induced deepening and durable responses without interleukin 2 (IL2) in patients (pts) with immune checkpoint inhibitor (ICI)-resistant unresectable or metastatic melanoma

Abstract Introduction: Treatment options are limited for pts with ICI-resistant metastatic melanoma. Investigational unengineered TIL cell therapy has shown promising activity in this setting, but co-administration of systemic high-dose IL2 limits pt eligibility and is associated with safety risks. OBX-115 is an investigational, autologous TIL cell therapy engineered to express membrane-bound IL15 (mbIL15) under pharmacologic regulation by the FDA-approved small-molecule drug acetazolamide (ACZ) using cytoDRiVE® technology, to induce cytokine-driven TIL expansion and persistence without co-administration of IL2. Methods: This Phase 1 first-in-human study (NCT05470283) is assessing the safety, tolerability, and efficacy of the OBX-115 regimen in pts with unresectable or metastatic melanoma progressing after ICI. Initial pts are enrolled into dose-escalation cohorts (n=3 each; maximum doses: Cohort 1 [C1], 30 × 109 cells + ACZ 125 mg; Cohort 2b [C2b], 100 × 109 cells + ACZ 125 mg). OBX-115 is manufactured using the pt’s tumor tissue (procured by surgical excision or core needle biopsy) and infused after completion of cyclophosphamide and fludarabine lymphodepletion. ACZ is administered orally once daily for up to 7 days, with redosing (Week 6, up to 7 days) permitted for pts without radiographic response at Week 6. No systemic IL2 is administered. The primary objective is to evaluate safety (dose-limiting toxicities [DLTs] and treatment-emergent adverse events [TEAEs]). Results: As of 1 December 2023, 6 pts (age, 28-64 y; C1, n=3; C2b, n=3) with ICI-resistant metastatic melanoma completed the DLT-evaluation period. Median lines of prior therapy was 2.5 (range, 1-5). OBX-115 was successfully manufactured for all 6 pts, including from core needle biopsy tumor tissue (n=5). At 20 weeks median study follow-up (range, 11-51 weeks), no DLTs were observed; 3 pts experienced Gr 3 nonhematologic TEAEs (abdominal pain, ALT elevation, hypokalemia, hyponatremia, syncope) and no Gr 4 nonhematologic TEAEs were reported. Four pts received and tolerated ACZ redosing. ORR per RECIST 1.1 was 50% (2 CR, 1 PR, 3 SD); responses were established between Week 6-18 and ongoing at the time of data cut. All pts had consistent reduction of lesions at each assessment through Week 18. One pt developed new metastatic disease (liver) and progressed at Week 24, despite continued target lesion reduction. Updated data will be presented. Conclusions: OBX-115, a one-time, IL2-free, regulatable, engineered TIL cell therapy, was well-tolerated and induced consistently deepening and durable responses, with the potential to fulfill the unmet need in ICI-resistant advanced melanoma. These early results support continued investigation of the OBX-115 regimen in this and an ongoing Phase 1/2 multicenter study (NCT06060613). Citation Format: Rodabe N. Amaria, Jennifer L. McQuade, Michael A. Davies, Isabella C. Glitza Oliva, Steffy Jose, Erik N. Cressman, Ashlynd L. Clausell, Roland Bassett, Sapna Patel, Adi Diab, Hussein A. Tawbi, Michael K. Wong, Alexandra P. Ikeguchi, Madan Jagasia, Giridharan Ramsingh, Prakash Prabhakar, Raina Duan, Parameswaran Hari. OBX-115 engineered tumor-infiltrating lymphocyte (TIL) cell therapy induced deepening and durable responses without interleukin 2 (IL2) in patients (pts) with immune checkpoint inhibitor (ICI)-resistant unresectable or metastatic melanoma [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 CT176.

Abstract 4285: Tumor-on-chip co-culture system: A comprehensive tool in personalized medicine to study cancer progression

Abstract As a part of the rapidly advancing organ-on-chip field of research, tumor-on-chip technology (TOC) has emerged as a promising co-culture system to mimic the mechanical and biochemical properties of the tumor microenvironment. This includes factors such as oxygen and nutrient gradients, extracellular matrix stiffness, and cell-cell interactions. Depending on the complexity of system, TOCs are miniature tumor models in a microfluidic chip allowing researchers to gain a better understanding and controlled cancer environment to effectively test potential drug treatments and study immune cell co-cultures. Champions’TumorGraft3D (CTG3D), established from patient-derived xenografts, is the essential component used to recapitulate tumor microenvironment in the TOC platform which provides an even more powerful tool to gain more insight on the efficacy of various therapeutics. Here, we demonstrate the value in combining microfluidic devices with well characterized CTG3D models at Champions Oncology focused on indications including colorectal, breast, gastric and lung cancer. In brief, we optimized cell seeding conditions in both top (seeded with CTG3Ds) and bottom (seeded with endothelial cells) channels to develop TOCs interfacing epithelial-endothelial cells. These established systems may be applied as a baseline for introducing additional cell types such as autologous tumor-infiltrating lymphocytes (TILs) and studying their effect in the presence and absence of additional treatments. With an initial focus on colorectal cancer, we successfully developed TOC epithelial-endothelial interface using CTG3Ds originated from patients with various clinical backgrounds. We measured the permeability as an initial read-out to prove that although tumor cells do not inherently form a tight barrier, they can still form a non-leaky barrier with distinct organoid-like structures. Next, we extended our study to additional indications featuring unique characteristics and proved the feasibility of applying TOCs as a preclinical tool regardless of the tissue-of-origin. In all cases, the endothelial cells formed mature microvascular structures and to confirm the consistency of formed epithelial-endothelial interface among the replicates of each experimental group, we did Luminex studies to prove high consistency in secreted proteins. Regarding the functionality of TOCs, we investigated the effect of relevant test agents to each indication and showed that that the combination of DRAQ7 dye and high-content imaging may be a reliable read-out to detect the response and viability of cancer cells pre- and post-treatment. In summary, our established tumor-on-chip system proves to be a robust and powerful tool for studying the effect of microenvironmental factors in drug testing and immune cells in cancer progression. Citation Format: Samaneh Kamali, Mara Gilardi, Brandon Walling, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. Tumor-on-chip co-culture system: A comprehensive tool in personalized medicine to study cancer progression [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 4285.

Abstract 5342: Evaluation of autologous and allogeneic T cell-based therapies in ex-vivo and in-vivo xenograft tumor models

Abstract The field of adoptive T cell-based immunotherapy, including tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR) & Chimeric antigen receptor (CAR) - T cells, has undergone unprecedented growth, specifically later in delivering effective and durable clinical response. Notable success with CAR-T cells is limited to hematological malignancies while clinical gains with solid tumors is still a challenge. Solid tumors’ heterogenous target expression and loss, T cell status and metabolic fitness, T cell trafficking into the tumor, and the hostile immunosuppressive tumor microenvironment (TME) have hampered T cell efficacy in the patients. There is a lack of clinically relevant solid tumor models for the translation of T cell-based therapies into the clinic. We report the development of translational ex-vivo and in-vivo platforms for evaluating cell line-derived and patient-derived xenografts (CDX and PDX) models (BarneyOI™️ model), validated with hCD3 T-cells, autologous TILs and/or allogeneic 3rd generation CAR-T cells (scFv(HER2)-CD28-4-1BB-CD3zeta CAR-T). Anti-hPDL1 but not anti-hPD1 significantly enhanced hCD3 T-cell and autologous TIL-mediated killing of tumor cells in an ex-vivo co-culture assay using Incucyte-based live cell imaging. Also, a dose-dependent killing of HER-2 expressing CDX (A549 & Colo205) and PDX was observed with HER-2 targeting CAR-T cells in an ex-vivo co-culture assay. This was in concordance with the activation, effector function, and differentiation status of the CAR-T cells, immune phenotype by Cytek aurora spectral flow cytometry. Further, to imitate the patient population and the effect of solid TME, HER-2 targeting CAR-T cells were assessed for their in-vivo efficacy and immune response differences in the subcutaneous and orthotopically implanted HER-2 expressing PDX in immunodeficient NOG mice. Our findings highlight the importance of testing T cell-based immune therapies in the appropriate translational tumor models with better certainty of clinical outcome. Citation Format: Sherry Liu, Jasmine Borroel, Carmen Sunico, Emily Eastwood, Bridget Corcoran, Bianca Carapia, Jantzen Sperry, Jonathan Nakashima, Rajeev Shrimali. Evaluation of autologous and allogeneic T cell-based therapies in ex-vivo and in-vivo xenograft tumor models [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 5342.