Tumor-infiltrating Lymphocytes Therapy Research Articles

TIL Therapy in Lung Cancer: Current Progress and Perspectives.

Lung cancer remains the most prevalent malignant tumor worldwide and is the leading cause of cancer-related mortality. Although immune checkpoint blockade has revolutionized the treatment of advanced lung cancer, many patients still do not respond well, often due to the lack of functional T cell infiltration. Adoptive cell therapy (ACT) using expanded immune cells has emerged as an important therapeutic modality. Tumor-infiltrating lymphocytes (TIL) therapy is one form of ACT involving the administration of expanded and activated autologous T cells derived from surgically resected cancer tissues and reinfusion into patients and holds great therapeutic potential for lung cancer. In this review, TIL therapy is introduced and its suitability for lung cancer is discussed. Then its historical and clinical developments are summarized, and the methods developed up-to-date to identify tumor-recognizing TILs and optimize TIL composition. Some perspectives toward future TIL therapy for lung cancer are also provided.

Tumor-reactive T cell clonotype dynamics underlying clinical response to TIL therapy in melanoma

Adoptive cell therapy (ACT) using invitro expanded tumor-infiltrating lymphocytes (TILs) has inconsistent clinical responses. To better understand determinants of therapeutic success, we tracked TIL clonotypes from baseline tumors to ACT products and post-ACT blood and tumor samples in melanoma patients using single-cell RNA and Tcell receptor (TCR) sequencing. Patients with clinical responses had baseline tumors enriched in tumor-reactive TILs, and these were more effectively mobilized upon invitro expansion, yielding products enriched in tumor-specific CD8+ cells that preferentially infiltrated tumors post-ACT. Conversely, lack of clinical responses was associated with tumors devoid of tumor-reactive resident clonotypes and with cell products mostly composed of blood-borne clonotypes that persisted in blood but not in tumors post-ACT. Upon expansion, tumor-specific TILs lost tumor-associated transcriptional signatures, including exhaustion, and responders exhibited an intermediate exhausted effector state after TIL engraftment in the tumor, suggesting functional reinvigoration. Our findings provide insight into the nature and dynamics of tumor-specific clonotypes associated with clinical response to TIL-ACT, with implications for treatment optimization.

Advancements and Challenges in Personalized Therapy for BRAF-Mutant Melanoma: A Comprehensive Review.

Over the past several decades, advancements in the treatment of BRAF-mutant melanoma have led to the development of BRAF inhibitors, BRAF/MEK inhibitor combinations, anti-PD-1 therapy, and anti-CTLA4 therapy. Although these therapies have shown substantial efficacy in clinical trials, their sustained effectiveness is often challenged by the tumor microenvironment, which is a highly heterogeneous and complex milieu of immunosuppressive cells that affect tumor progression. The era of personalized medicine holds substantial promise for the tailoring of treatments to individual genetic profiles. However, tumor heterogeneity and immune evasion mechanisms contribute to the resistance to immunotherapy. Despite these challenges, tumor-infiltrating lymphocyte (TIL) therapy, as exemplified by lifileucel, has demonstrated notable efficacy against BRAF V600-mutant melanoma. Additionally, early response biomarkers, such as COX-2 and MMP2, along with FDG-PET imaging, offer the potential to improve personalized immunotherapy by predicting patient responses and determining the optimal treatment duration. Future efforts should focus on reducing the T-cell harvesting periods and costs associated with TIL therapy to enhance efficiency and accessibility.

Recent Advancements in Cell-Based Therapies in Melanoma.

Malignant melanoma outcomes have drastically changed in recent years due to the introduction of immune checkpoint inhibitors (ICIs). However, many patients still experience intolerable side effects, therapy resistance, and disease progression on ICI therapy. Therefore, there remains a need for novel therapeutics that address this gap in treatment options. Cell-based therapies have gained wide attention as a therapeutic option that could address this gap in treatment options for advanced melanoma. These therapies work by extracting certain cell types produced in the human body such as T-cells, modifying them based on a specific target, and transfusing them back into the patient. In the realm of cancer therapy, cell-based therapies utilize immune cells to target tumor cells while sparing healthy cells. Recently, the Food and Drug Administration (FDA) has approved the usage of lifileucel, a tumor-infiltrating lymphocyte (TIL) therapy, in advanced melanoma. This came following recent results from the C-144-01 study (NCT02360579), which demonstrated the efficacy and safety of TILs in metastatic melanoma patients who otherwise failed on standard ICI/targeted therapy. Thus, the results of this trial as well as the recent FDA approval have proven the viability of utilizing cell-based therapies to fill the gap in treatment options for patients with advanced melanoma. This review aims to provide a comprehensive overview of major cell-based therapies that have been utilized in melanoma by delineating results of the most recent multi-center phase II/ III clinical trials that evaluate the efficacy and safety of major cell-based therapies in melanoma. Additionally, we provide a summary of current limitations in each cell-based therapeutic option as well as a future direction of how to further extrapolate these cell-based therapies in advanced melanoma.

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.

Current status and perspectives of clinical trials for tumor-infiltrating lymphocyte therapy.

Immunotherapies, mainly immune checkpoint inhibitors (ICIs), have revolutionized cancer treatment strategies over the past decade, but their limitations have limited clinical applications. Tumor-infiltrating lymphocyte (TIL) therapy is a type of adoptive cell therapy (ACT), which collects infiltrating lymphocytes at the tumor site and expands them in vitro to obtain TIL final products cloned by various T-cell receptors, subsequently reinfused TIL into the patient, which is effective for the treatment of solid tumors. The approval of Lifileucel for commercialization marks the success of TIL therapy. This review summarizes the current status of clinical trials of TIL treatment. In addition, it is suggested that the current research trend of TIL should focus on improving the survival time of TIL in vivo, reducing drug toxicity, and searching for prognostic markers. Finally, it is expected that TIL therapy can be applied to a more wide range of clinical treatments.

Tumor-infiltrating lymphocytes: A new hope

Tumor-infiltrating lymphocytes (TILs) can be massively expanded from resected tumors and used as a cellular treatment for advanced malignancies. TILs require a preparative non-myeloablative chemotherapy followed by an abbreviated course of interleukin-2. Here, we review the historical development of TIL therapy and discuss potential solutions to ongoing roadblocks that may result in broader and improved efficacy for patients afflicted with treatment-refractory, advanced cancer.

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.

Targeted Bias: The Next Swing at IL2 Therapy.

Despite its long history of toxicity and limited efficacy, IL2 has re-entered the clinic as a companion to the recently FDA-approved tumor infiltrating lymphocyte therapy. In back-to-back articles, Moynihan and Kaptein introduce a new fusion protein that delivers a biased IL2 mutein to CD8 T cells. See related article by Moynihan et al., p. 1206 (6). See related article by Kaptein et al., p. 1226 (7).

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 .

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 .

Research advances on TIL therapy for colorectal cancer.

Colorectal cancer (CRC) is a prevalent gastrointestinal malignancy. Tumor-infiltrating lymphocyte (TIL) therapy, a form of adoptive cellular therapy (ACT), involves isolating T lymphocytes from tumor tissues, in vitro expansion, and reintroduction into the body to target and eliminate tumor cells. This article presents an overview of the development and application of TIL therapy in CRC, as well as the associated challenges.

IL-2-free tumor-infiltrating lymphocyte therapy with PD-1 blockade demonstrates potent efficacy in advanced gynecologic cancer

BackgroundTumor-infiltrating lymphocyte (TIL) therapy has been restricted by intensive lymphodepletion and high-dose intravenous interleukin-2 (IL-2) administration. To address these limitations, we conducted preclinical and clinical studies to evaluate the safety, antitumor activity, and pharmacokinetics of an innovative modified regimen in patients with advanced gynecologic cancer.MethodsPatient-derived xenografts (PDX) were established from a local recurrent cervical cancer patient. TILs were expanded ex vivo from minced tumors without feeder cells in the modified TIL therapy regimen. Patients underwent low-dose cyclophosphamide lymphodepletion followed by TIL infusion without intravenous IL-2. The primary endpoint was safety; the secondary endpoints included objective response rate, duration of response, and T cell persistence.ResultsIn matched patient-derived xenografts (PDX) models, homologous TILs efficiently reduced tumor size (p < 0.0001) and underwent IL-2 absence in vivo. In the clinical section, all enrolled patients received TIL infusion using a modified TIL therapy regimen successfully with a manageable safety profile. Five (36%, 95% CI 16.3–61.2) out of 14 evaluable patients experienced objective responses, and three complete responses were ongoing at 19.5, 15.4, and 5.2 months, respectively. Responders had longer overall survival (OS) than non-responders (p = 0.036). Infused TILs showed continuous proliferation and long-term persistence in all patients and showed greater proliferation in responders which was indicated by the Morisita overlap index (MOI) of TCR clonotypes between infused TILs and peripheral T cells on day 14 (p = 0.004) and day 30 (p = 0.004). Higher alteration of the CD8+/CD4+ ratio on day 14 indicated a longer OS (p = 0.010).ConclusionsOur modified TIL therapy regimen demonstrated manageable safety, and TILs could survive and proliferate without IL-2 intravenous administration, showing potent efficacy in patients with advanced gynecologic cancer.Trial registrationNCT04766320, Jan 04, 2021.

Applied Cardio-Oncology in Hematological Malignancies: A Narrative Review.

Applied cardio-oncology in hematological malignancies refers to the integration of cardiovascular care and management for patients with blood cancer, particularly leukemia, lymphoma, and multiple myeloma. Hematological cancer therapy-related cardiotoxicity deals with the most common cardiovascular complications of conventional chemotherapy, targeted therapy, immunotherapy, chimeric antigen receptor T (CAR-T) cell and tumor-infiltrating lymphocyte therapies, bispecific antibodies, and hematopoietic stem cell transplantation. This narrative review focuses on hematological cancer-therapy-related cardiotoxicity's definition, risk stratification, multimodality imaging, and use of cardiac biomarkers to detect clinical and/or subclinical myocardial dysfunction and electrical instability. Moreover, the most common cardiotoxic profiles of the main drugs and/or therapeutic interventions in patients with hematological malignancies are described thoroughly.

Abstract 20: KSQ-001EX: An engineered TIL therapy manufactured from a clinical-scale, feeder-free process for the treatment of solid tumor indications

Abstract Tumor Infiltrating Lymphocyte (TIL) therapy is an autologous adoptive cell therapy involving the isolation, ex vivo expansion and infusion of tumor infiltrating lymphocytes (TIL) into late-stage cancer patients for therapeutic benefit. While clinical responses to TIL therapy have been observed in metastatic melanoma, efficacy in other tumor types including non-small cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC) [SM1] is limited by the intrinsic functionality and persistence of transferred T cells and the immunosuppressive tumor microenvironment. To improve the clinical outcome of TIL therapy, we have developed KSQ-001EX, an engineered TIL therapy using CRISPR/Cas9 technology to knockout the SOCS1 gene, for the treatment of solid tumors. SOCS1 is a negative regulator of cytokine signaling in T cells that we previously identified as a top target restraining T cell anti-tumor function and persistence using genome-wide in vivo CRISPR screens. To streamline and shorten TIL manufacture, we established a next-generation manufacturing process with resected tumors or core biopsies as starting materials that eliminates the use of feeder cells and REP, shortening the length of manufacture to 22 days. We demonstrated robust manufacture of cryopreserved KSQ-001EX at clinical scale from NSCLC, HNSCC, checkpoint refractory melanoma, and heavily pre-treated colorectal carcinoma (CRC). KSQ-001EX manufactured from clinical-scale runs were highly viable (&amp;gt;90%) following cryopreservation and thaw, with &amp;gt; 90% SOCS1 on-target editing and complete knockdown of SOCS1 at the protein level. Importantly, KSQ-001EX across all indications assessed showed high frequency of CD8 (median ~80%), an attribute associated with TIL clinical responses. KSQ-001EX also retained high diversity of TCR repertoire. Consistent with the biology of SOCS1 editing, KSQ-001EX exhibited greater cytotoxicity and higher IFNγ production against tumor spheroids in comparison to TIL and enhanced induction of pSTAT4 upon IL-12 stimulation, demonstrating heightened sensitivity of KSQ-001EX to cytokines. In conclusion, KSQ-001EX, an engineered TIL therapy with CRISPR/Cas9 mediated inactivation of SOCS1, can be manufactured at clinical doses from surgical resections or core biopsies within 22 days using a simplified feeder-free manufacturing process from multiple indications. KSQ-001EX exhibits heightened anti-tumor activity in preclinical models, with therapeutic benefit to be evaluated in a planned clinical trial in patients with treatment-refractory melanoma, NSCLC, and HNSCC. [SM1]Spell out these at first instance? Citation Format: Sharon Lin, Gustavo Martinez, Marie-Andrée Forget, Katrina Adlerz, Mitali Ghose, Leila Williams, Paul Dunbar, Frank Thompson, Fiona Sharp, Hugh Gannon, Conor Calnan, Ashish Yeri, Dipen Sangurdekar, Donald Sakellariou-Thompson, Samantha Fix, Tamara Griffith, Ellie Jafari, Jane Zulovich, Priya Balasubramanian, Erin Willert, MIcah Benson, Chantale Bernatchez, Karrie Ka Wai Wong. KSQ-001EX: An engineered TIL therapy manufactured from a clinical-scale, feeder-free process for the treatment of solid tumor indications [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 20.

Abstract 1: Biomarkers of response and acquired resistance to TIL-ACT in solid tumors

Abstract Background: Cancer immunotherapy has revolutionized cancer treatment in the last decade. However, most patients either do not respond to immunotherapy or eventually relapse so there is a need to identify biomarkers of response and understand mechanisms of resistance. Most biomarker studies have analyzed patients treated with immune checkpoint inhibitors (CPI) therapy while the few studies of adoptive cell transfer of tumor infiltrating lymphocytes (TIL-ACT) have analyzed relatively small patient cohorts. Methods: To systematically identify biomarkers of response and resistance to TIL-ACT, we collected published and unpublished whole-exome and transcriptomic data for tumors from 400 patients treated with TIL-ACT from five tumor types across four institutions and processed these datasets using a standardized bioinformatic pipeline. Results: To identify biomarkers of response in pre-treatment tumors, we analyzed previously reported biomarkers and performed biomarker discovery in our cohorts. For the subset of 200 patients with T cell reactivities identified from the infusion products and fresh tumor, we refined our biomarkers to include neo-antigen reactivities in addition to identifying both mutational processes and transcriptional signatures associated with neo-antigens reactivity. To identify mechanisms of acquired resistance to TIL-ACT, we analyzed 15 patients with paired pre- and post-treatment tumors and found multiple mechanisms of immune evasion specific to infused reactive T cells including loss of heterozygosity of the HLA allele presenting the neo-antigen, genomic loss of the neo-antigen and inactivation of interferon-gamma signaling pathway. Conclusions: In summary, we will present the biomarkers landscape of TIL-ACT therapy including differences between histology and across treatment modalities to improve our understanding of the mechanisms of treatment response. Citation Format: Welles Robinson, Andrea Castro, Maise Al-Bakir, Jared J. Gartner, Christopher Aled Chamberlain, Anders H. Kverneland, Troels H. Borch, Inge Marie Svane, Marco Donia, Paul Robbins, Kevin Litchfield. Biomarkers of response and acquired resistance to TIL-ACT in solid tumors [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 1.

Abstract 25: Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features

Abstract Introduction: Autologous TIL cell therapies such as lifileucel have demonstrated durable responses in patients (pts) with various solid tumors including melanoma and lung cancer. The TIL drug product (DP) phenotype was previously assessed and extensively characterized based on the bulk population. The current research further characterizes the TIL DP at a single-cell level using high-dimension multimodal sequencing and explores features that have a potential to inform future development of TIL therapies. Methods: Manufactured lifileucel TIL DP from 20 pts (best overall response [BOR]: 6 complete response [CR], 4 partial response, 5 stable disease, 5 progressive disease [PD]) from the registrational melanoma C144-1 trial were analyzed using single-cell RNA and T-cell receptor (TCR) sequencing, resulting in 153K single-cell transcriptomes. To aid with cell-type annotation, 130 surface proteins on TIL DP samples from a subset of 4 pts (BOR: 2 CR, 2 PD) were detected via Cellular Indexing of Transcriptomes and Epitopes by sequencing. Multimodal weighted nearest-neighbor analysis combined with manual cluster annotation (via known marker genes and proteins), cell-type-specific gene signatures, and automated annotation were used to develop a single-cell TIL (scTIL) reference map, resulting in high-resolution subsets of cells. TCR clonotypes from lifileucel TIL DP were compared to those from respective pretreatment tumors previously identified by bulk TCR sequencing. Results: Mapping of the full dataset to the novel scTIL reference revealed a CD8+ TIL-dominant composition of responder TIL DP. TIL from pts with BOR PD exhibited reduced CD8+ effector memory (Tem)-like and resident memory (Trm)-like T cells. Differential expression analysis showed that responder CD8+ TIL had increased expression of TCR signaling genes while responder CD4+ TIL exhibited gene expression consistent with a more cytotoxic-like phenotype. Relative to responders, CD8+ TIL of pts with BOR PD had lower expression of the CD39−CD69− (stem-like) gene signature and higher expression of the CD39+CD69+ (terminal differentiation) gene signature. Pseudotime trajectory analysis found that both CD8+ and CD4+ TIL of pts with BOR PD were more terminally differentiated. Single-cell TCR sequencing revealed that CD8+ T-cell clusters had greater clonal overlap than CD4+ T-cell clusters. Expanded clonotypes in pretreatment tumor tissues were more prevalent in TIL TCR repertoires of responders and were more likely to be expressed by CD8+ (specifically CD8+ Tem- and Trm-like TIL) than CD4+ T cells. Conclusions: Collectively, these results shed additional light on the cellular and molecular characteristics of ex vivo expanded lifileucel TIL DP and signal additional features with a potential to guide future TIL product development. Citation Format: Joe Dean, Joe Yglesias, Hequn Yin, Rongsu Qi. Multimodal single-cell analysis of lifileucel tumor-infiltrating lymphocyte (TIL) product identifies additional high-resolution potential features [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 25.

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 (&amp;gt;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.