Tumor-infiltrating Lymphocytes Production Research Articles

Effects of prophylactic high and low doses of corticosteroid on the efficacy of immune checkpoint blockade in murine hepatocellular carcinoma models.

e14596 Background: Immune-related adverse events (irAEs) are major hurdle to immune checkpoint blockade (ICB). Corticosteroid (CS) is effective in controlling the majority of irAEs. Although recent studies suggested CS use does not jeopardize the anti-tumor efficacy of ICB, prophylactic use of CS remains prohibited with the concerns of attenuating efficacy of ICB. This study aimed to investigate the effects of CS premedication on the efficacy of ICB in murine hepatocellular carcinoma models. Methods: Anti-mCTLA-4 (9D9C, BMS) and anti-mPD-1 antibodies (4H2, ONO) were intraperitoneally (ip) administered to tumor-bearing mice (subcutaneous Hepa 1-6 model and orthotopic BNL 1MEA.7R.1 model) with or without dexamethasone (DEXA) premedication (10 and 200 μg, equivalent to minimal anti-inflammatory dosage and pulse therapy in human, respectively). Efficacy of ICB was evaluated as tumor shrinkage. Tumor-infiltrating lymphocytes (TILs) were isolated for single cell RNA-sequencing and effector function analysis through flow cytometry. Results: In the subcutaneous model, all tumors treated with ICB alone (N=7) or ICB plus DEXA 10 μg (N=5) completely regressed, but 1 out of 7 tumors treated with ICB plus DEXA 200 μg escaped. However, the tumor growth was not significantly different between groups ( P-value >0.05, multiple Mann-Whitney test). In the orthotopic model (N=5/group), the mean (± standard error) tumor weights on day 21 after tumor implantation for isotype control, ICB, ICB plus DEXA 10 μg and ICB plus DEXA 200 μg were 2.45 (± 0.54), 0.73 (± 0.18), 0.98 (± 0.65), and 0.69 (± 0.17) grams, respectively ( P-value >0.05, comparing ICB plus DEXA 10 or 200 μg with ICB). At transcriptomic level, premedication with either dosage of DEXA significantly reduced the percentage of effector memory cells and increased the percentage of exhausted effector cells in the CD8 TIL population, which appeared to counterbalance the effects of ICB. However, at protein level, premedication of DEXA 10 or 200 μg did not reduce the interferon-γ or granzyme B production of CD8 TILs in both models (Table). Conclusions: CS premedication did not attenuate the efficacy of ICB. Our study provides the scientific basis to evaluate the potential of prophylactic CS in preventing ICB-induced irAEs in clinical studies.[Table: see text]

A phase I trial of T-cell receptor gene therapy targeting KK-LC-1 for gastric, breast, cervical, lung and other KK-LC-1 positive epithelial cancers.

TPS2678 Background: T cell receptor (TCR)-T cell therapy is an emerging cancer treatment strategy. Thus far, demonstration of clinical activity has been limited to a subset of solid tumors including melanoma, synovial cell sarcoma and HPV-associated cancer. It is estimated that metastatic epithelial cancers are responsible for approximately 90% of cancer deaths in the United States. The estimated 600,000 cancer deaths each year is driven largely by lung adeno- and squamous cell carcinoma and invasive breast cancer, which account for approximately 30% of all cancer-related deaths. Kita-Kyushu Lung Cancer Antigen 1 (KK-LC-1) is a cancer germline antigen with expression restricted to germ cells in adults and certain epithelial cancers including lung, breast, gastric and cervical. We identified a KK-LC-1 TCR from the tumor-infiltrating lymphocytes (TIL) of a patient with cervical cancer who had a complete tumor response to TIL therapy. The KK-LC-1 TCR was the most dominate clonotype in the infused TIL product and persisted in the peripheral blood following infusion, suggesting that it may have contributed to cancer regression in this patient. Methods: We are conducting a phase I cell dose escalation trial to test the safety and efficacy of KK-LC-1 TCR-T cell therapy in patients with metastatic KK-LC-1 positive epithelial cancer. Patients receive a lymphocyte depleting conditioning regimen followed by a one-time infusion of genetically engineered T cells expressing the KK-LC-1 TCR (KK-LC-1 TCR-T cells) and high-dose systemic aldesleukin. KK-LC-1 positivity is determined by RNAscope assay measuring the percentage of cancer cells expressing CT83 (gene encoding KK-LC-1) with a percentage of 25 or greater considered positive. Main inclusion criteria include HLA-A*01:01 allele, prior first-line therapy, ECOG of 0 or 1 and adequate organ and hematologic function. Main exclusion criteria include active major medical illness of the cardiovascular, respiratory or immune system, primary or secondary immunodeficiency and autoimmune disease. Participants will be entered in sequential dose levels and receive escalating doses of cells beginning at 1x108 and ending with 6x1010. Dose-limiting toxicities will be assessed during the first 30 days of cell infusion. The primary objective is to determine the maximally tolerated dose of KK-LC-1 TCR-T cells. Exploratory objectives include assessing the safety and efficacy of KK-LC-1 TCR-T cells and to conduct immunologic studies to understand and improve the administered treatment. Clinical trial information: NCT05035407.

Enriching for tumor-reactive CD8 TIL (AGX148) leads to effective tumor clearance in a patient-derived xenograph model

Abstract Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a subset of patients with melanoma; however, the efficacy of standard bulk TIL therapy can be limited. We hypothesize that the therapeutic efficacy of TIL ACT is dependent on the frequency and functionality of tumor-reactive T cells when infused back into patients. Our approach enhances the activity of the ACT product by enriching for the tumor-reactive CD8 TIL prior to ex vivo expansion by sorting on cells that co-express CD103 and CD39. The CD103/CD39 double positive (DP) CD8 TIL can be found in most human solid tumors but at varying frequencies in any given patient. Surgically resected human tumor samples were provided through a collaboration with the EACRI. Tumor samples were flow sorted and expanded ex vivo and patient-matched autologous tumor lines were generated. Tumors were then cocultured with the expanded autologous ACT product (AGX148) in vitro or used in xenograph mouse experiments. The AGX148 TIL products are effective at recognizing and killing autologous tumor cells, in vitro. Upon adoptive transfer into patient-derived tumor-bearing huIL-2 NOG mice, the transferred human T cells increased in frequency, migrated to the tumor, became activated in the tumor and recognized/destroyed established tumors. Interestingly there was little to no in vivo activity of CD8 TIL that did not express these two markers. We have generated a potent therapeutic ACT TIL product (AGX148) through the selective isolation and expansion of tumor-reactive T cells. This TIL product effectively induces durable curative responses in hosts harboring autologous tumors providing rationale for clinical investigation.

Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes

BackgroundAdoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical...

Highly efficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptive T cell therapy

Adoptive T cell therapy (ACT) with expanded tumor-infiltrating lymphocytes (TIL) can induce durable responses in cancer patients from multiple histologies, with response rates of up to 50%. Antibodies blocking the engagement of the inhibitory receptor programmed cell death protein 1 (PD-1) have been successful across a variety of cancer diagnoses. We hypothesized that these approaches could be combined by using CRISPR-Cas9 gene editing to knock out PD-1 in TILs from metastatic melanoma and head-and-neck, thyroid, and colorectal cancer. Non-viral, non-plasmid-based PD-1 knockout was carried out immediately prior to the traditional 14-day TIL-based ACT rapid-expansion protocol. A median 87.53% reduction in cell surface PD-1 expression was observed post-expansion and confirmed at the genomic level. No off-target editing was detected, and PD-1 knockout had no effect on final fold expansion. Edited cells exhibited few phenotypic differences and matched control functionality. Pre-clinical-scale results were confirmed at a clinical scale by generating a PD-1-deficient TIL product using the good manufacturing practice facilities, equipment, procedures, and starting material used for standard patient treatment. Our results demonstrate that simple, non-viral, non-plasmid-based CRISPR-Cas9 methods can be feasibly adopted into a TIL-based ACT protocol to produce treatment products deficient in molecules such as PD-1, without any evident negative effects.

385 Identification and enrichment of neoantigen-reactive T cells to optimize adoptive cell transfer with tumor-infiltrating lymphocytes (TIL)

BackgroundAdoptive cell transfer (ACT) using tumor-infiltrating lymphocytes (TIL) has achieved an overall response rate of 39% in metastatic melanoma patients at Moffitt Cancer Center. In these trials, a substantial fraction of patients were non-responders by RECIST, but demonstrated a mixed response to therapy. These results suggest that the infused TIL product contained tumor-reactive T cells with therapeutic potential, which could be further optimized to improve ACT with TIL. We hypothesized that outcomes might be improved by identifying and enriching neoantigen-reactive TIL within bulk products. The purpose of this study is to define approaches to optimize ACT with TIL, by identifying, enriching, and analyzing neoantigen reactive TIL from the ACT infusion product of previously treated metastatic melanoma patients.MethodsPatient-derived cryopreserved tumor tissue, PBMC, and TIL from completed metastatic melanoma TIL trials were used for this study. Whole exome and RNA sequencing were performed on DNA and RNA extracted from tumor tissue and compared to DNA from autologous PBMC. Genetic sequencing and gene expression data were utilized to determine protein-modifying somatic mutations. Peptides were then predicted for their ability to be presented on MHC molecules, prioritized, and up to 192 custom 25-mers were synthesized per patient sample. Neoantigen peptides were loaded onto patient-derived dendritic cells (DC) and co-cultured with autologous TIL. These TIL were then sorted by FACS on their ability to upregulate 41BB and OX40 and expanded through the rapid expansion protocol (REP). Enriched TIL were subsequently screened for neoantigen reactivity by 41BB/OX40 upregulation, cytokine release, and degranulation.ResultsProtein-altering somatic mutations from metastatic melanoma tissues ranged from 49 to 1631 mutations (median = 389). On average, 16.2% of TIL were sorted for upregulation of 41BB/OX40 upon co-culture with DC pulsed with the neoantigen peptide pool (range: 2.7–31.1%). CD4+ TIL displayed a 3.75-fold upregulation of 41BB/OX40, while CD8+ TIL saw a 1.88-fold increase (n=6). This coincided with substantial production of IFNγ, TNFα, and granzyme B (n=6). Neoantigen-reactive (41BB+/OX40+) and non-reactive (41BB-/OX40-) TIL expanded to similar degrees in REP (average of 639-fold vs. 611-fold; n=6). Restimulation of enriched neoantigen-specific TIL resulted in superior pro-inflammatory functionality (granzyme B, IFNγ, and TNFα) when compared to non-reactive TIL.ConclusionsTIL from metastatic melanoma patient samples were successfully enriched for neoantigen-reactive TIL, which maintained increased reactivity against these predicted peptides upon restimulation when compared non-reactive TIL. These data support further investigation into the use of neoantigen-enriched TIL products to enhance efficacy of ACT.Trial RegistrationNCT01005745, NCT01659151, NCT01701674Ethics ApprovalNCT01005745 was approved by USF IRB approval number Ame5_107905.NCT01659151 was approved by Advarra IRB approval number 14.03.0083.NCT01701674 was approved by USF IRB approval number Ame13_Pro00009061.All participants gave informed consent before taking part.

Adoptive cell therapy with tumor-infiltrating lymphocytes supported by checkpoint inhibition across multiple solid cancer types

BackgroundAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown remarkable results in malignant melanoma (MM), while studies on the potential in other cancer diagnoses are sparse. Further, the prospect...

1058P Treatment outcomes with unselected autologous tumor infiltrating lymphocytes (TILs) in patients (pts) with checkpoint inhibition–refractory advanced cutaneous melanoma

TIL products made from tumor digests showed a high overall response rate (ORR; 67%) and complete response (CR) rate (19%) and a safety profile consistent with lymphodepletion and high-dose interleukin (IL)-2 in a retrospective analysis of a single-center experience of TILs for compassionate use treatment of advanced cutaneous melanoma (n=21; Hawkins, et al. AACR 2021. ePoster LB150). This subanalysis assesses outcomes for pts who received TILs after prior checkpoint inhibition, a pt subset with limited treatment options.

Abstract 1513: A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy

Abstract Immunotherapy for cancer has long been focused on the generation of CD8+ cytotoxic T lymphocyte responses, independent of their dynamic CD4+ T cell counterpart. One promising approach, adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL), has yielded response rates ranging from 28-55%. Although lasting and complete responses have been achieved, there is substantial opportunity for improvement. Investigation into the role of CD4+ TIL in this setting remains critically underexplored. CD4+ T cells recognize tumor antigen presented on MHC Class II either directly on tumor cells or indirectly through antigen presenting cells (APCs) and are able to elicit potent anti-tumor responses under the appropriate conditions. Here, we present a case study of a metastatic melanoma patient who received adoptive transfer of a predominantly (88%) CD4+ TIL product. This patient demonstrated a complete response (CR) to therapy despite a lack of detection of IFNg in the infusion product in vitro when these TIL were co-cultured with autologous tumor prior to ACT. Tumor recognition was also absent when CD8+ TIL were isolated and stimulated directly with HLA-matched tumor lines, indicating a lack of recognition of shared melanoma antigens presented on MHC Class I. Longitudinal analysis of the peripheral blood of this patient confirmed that the infused CD4+ TIL persisted after therapy for at least six weeks. Whole exome sequencing (WES) performed on the TIL surgical specimen discovered 88 non-synonymous single nucleotide variants (SNVs) as candidate neoantigens. Predicted binding of the resulting mutant peptides to autologous HLA molecules generated a predominantly MHC Class II restricted profile, with 81.8% of variants capable of MHC Class II presentation and greater than half exclusive to MHC Class II only. CD4+ TIL were screened for tumor antigen recognition by upregulation of OX40 and 41BB after stimulation with autologous APCs loaded with mutant peptides. Nearly half (49.2%) of CD4+ TIL responded to tumor-derived peptides. These CD4+ TIL were then sorted into tumor-reactive and non-reactive subsets for further clonal analysis of phenotype and transcriptional profile (scRNASeq) of these T cells in order to characterize the nature of the CD4+ TIL response to tumor antigen. Overall, thorough interrogation of this patient's case study demonstrated evidence of CD4+ TIL involvement in a complete clinical response after ACT. Ongoing studies will define the precise role of tumor-reactive CD4+ T cells in the anti-tumor immune response and provide the framework for future investigation into their function and therapeutic efficacy. Citation Format: MacLean Hall, Pat Innamarato, Amy Mackay, Holly Branthoover, Alex Alfaro, Carolyn Rich, Allison Richards, Jonathan Hensel, Matthew Beatty, John Mullinax, Amod A. Sarnaik, Shari Pilon-Thomas. A case study investigation into the role of CD4+ tumor-infiltrating lymphocytes in a metastatic melanoma patient with a complete response to adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1513.

Stem cell properties of GT101, a tumor infiltrating lymphocyte (TIL) product with multiple indications.

e14506 Background: Tumor infiltrating lymphocyte (TIL) is a kind of adoptive T cell therapy that has demonstrated clinical benefits in several types of solid tumors. GT101 is an autologous TIL therapy for the treatment of multiple solid tumors. Previous studies showed that in vitro expansion resulted in over 80% terminally differentiated effector T cells in the final TIL products, which may show good cytotoxicity but weak capability of persistence and proliferation in vivo. Herein we present phenotype and function characterization of our TIL product GT101 with a focus on its stemness attributes. Methods: We have produced more than 30 batches of GT101 TIL samples from 10 different cancer indications and analyzed their proliferation, cytokine production and phenotype at preREP and/or REP stage. Results: GT101 products keep a consistent T cell phenotype and good proliferation capacity, and are capable of secreting large amounts of cytokines after stimulation by anti-CD3/CD28 antibody, reflecting its good functionality. The residues in the final products, such as feeder cells and tumor cells, are below the detection limit. In contrast to equivalent TIL products previously reported across the world, we find that GT101 features a manufacturing process that preserves the stem cell characteristics of T cells. Analysis of all the 30 GT101 TIL samples demonstrates that the average percentage of central memory T cells (Tcm) and naïve T cells is 40-50% and 10-20% respectively in both CD8+ and CD4+ populations at the end of preREP stage. Meanwhile, CD8+TCF1+ cells are close to 50%, mainly in the form of PD-1-. In the phase of REP, the stemness of T cell is still well maintained—the average proportion of both Tcm and naïve T cells only decreases by about 5%, and that of CD8+TCF1+ cells down by about 10%. Conclusions: In general, GT101 demonstrates good stemness with a higher percentage of Tcm and TCF1+ than equivalent products previously reported across the world, suggesting better performance in persistence and proliferation in clinic.

A micro-organosphere potency assay for adoptive T-cell therapy.

e15027 Background: Lung cancer is the leading cause of death worldwide. Metastatic lung cancer patients often relapse or grow refractory to chemotherapy, radiotherapy, and checkpoint inhibitors. Adoptive T cell therapy (ACT) has been garnering more attention due to longitudinal complete responses. Tumor resections from patients harbor tumor-infiltrating lymphocytes (TILs) which can be cultured ex vivo and assessed for tumor reactivity before infusion. This generally follows a patient lymphodepletion regimen which allows the transferred T cells an optimal environment to proliferate and survive in the patient. TIL ACT can produce complete responses – rarely ever observed using traditional onco-therapy – in metastatic melanoma patients. However, while TILs specific to the neo-antigens expressed by tumor cells can be expanded ex vivo, this observed specificity is low in the clinic. Researchers have attempted to solve this by priming ex vivo expanded T cells with antigen-presenting cells previously pulsed with peptides representative of the neo-antigen repertoire of the matched tumor, but have rarely observed complete responses, likely due to current biomarkers for T cell activity poorly predicting anti-tumor cytotoxicity. To date there has been no acceptable potency assay for manufactured TILs, a requirement by the FDA for approval to use them in the clinic. Thus, the need to assess potency of ex vivo engineered T-cells against matched tumor cells is evident. Methods: We have developed a novel diagnostic immune-oncology (IO) pipeline, which uses a membrane-microfluidic platform to culture patient-derived tumor micro-organospheres (MOs) in extracellular matrix droplets. MOs can be rapidly established following patient tumor sample acquisition through biopsies or resection, and preserve the stromal cell populations in the original tumor microenvironment, as characterized by both flow cytometry and single-cell RNA-seq. An automated imaging assay was further established to robustly quantify the amount of immune-induced apoptosis of tumor cells in the MOs by patient-matched TILs, which is highly specific and yields minimal background. Results: We find that this method is not only amenable to high-throughput microscopy, but the larger surface-area-to-volume ratio of micro-organospheres also allows greater TIL infiltration and interaction with tumor cells. The resulting highly-sensitive assay requires far fewer input immune and tumor cells to achieve robust, clinical grade sensitivity response, making it the first clinically feasible assay for testing personalized TIL potency. Conclusions: The MO IO technology is currently being used for assessing clinical efficacy of manufactured TIL products for an upcoming ACT trial for non-small cell lung cancer patients. This technology also provides a companion to TIL ACT, CAR T therapy, and other immunotherapies, for which the ability to predict clinical potency is generally lacking.

T cells expanded from renal cell carcinoma display tumor-specific CD137 expression but lack significant IFN-γ, TNF-α or IL-2 production

ABSTRACT Metastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies. As only a subset of RCC patients respond, alternative strategies should be explored. Patients refractory to anti-PD-1 therapy may benefit from autologous tumor-infiltrating lymphocyte (TIL) therapy. Even though efficient TIL expansion was reported from RCC lesions, it is not well established how many RCC TIL products are tumor-reactive, how well they produce pro-inflammatory cytokines in response to autologous tumors, and whether their response correlates with the presence of specific immune cells in the tumor lesions. We here compared the immune infiltrate composition of RCC lesions with that of autologous kidney tissue of 18 RCC patients. Tcell infiltrates were increased in the tumor lesions, and CD8+ Tcell infiltrates were primarily of effector memory phenotype. Nine out of 16 (56%) tested TIL products we generated were tumor-reactive, as defined by CD137 upregulation after exposure to autologous tumor digest. Tumor reactivity was found in particular in TIL products originating from tumors with ahigh percentage of infiltrated Tcells compared to autologous kidney, and increased CD25 expression on CD8+ Tcells. Importantly, although TIL products had the capacity to produce the key effector cytokines IFN-γ, TNF-α or IL-2, they failed to produce significant amounts in response to autologous tumor digests. In conclusion, TIL products from RCC lesions contain tumor-reactive Tcells. Their restricted tumor-specific cytokine production requires further investigation of immunosuppressive factors in RCC and subsequent optimization of RCC-derived TIL culture conditions.

Development of an optimized closed and semi-automatic protocol for Good Manufacturing Practice manufacturing of tumor-infiltrating lymphocytes in a hospital environment

Background aimsSeveral studies report on Good Manufacturing Process (GMP)-compliant manufacturing protocols for the ex vivo expansion of tumor-infiltrating lymphocytes (TILs) for the treatment of patients with refractory melanoma and other solid malignancies. Further opportunities for improvements in terms of ergonomy and operating time have been identified. MethodsTo enable GMP-compliant TILs production for adoptive cell therapy needs, a simple automated and reproducible protocol for TILs manufacturing with the use of a closed system was developed and implemented at the authors’ institution. ResultsThis protocol enabled significant operating time reduction during TILs expansion while allowing the generation of high-quality TILs products. ConclusionsA simplified and efficient method of TILs expansion will enable the broadening of individualized tumor therapy and will increase patients’ access to state-of-the-art TILs adoptive cell therapy treatment.

Abstract 875: Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products

Abstract Adoptive transfer of tumor infiltrating lymphocytes (TIL) has generated objective clinical responses in patients with advanced metastatic cancers. Therapeutic exploitation of neoantigens as targets can potentially lead to safer and more effective treatment modalities with reduced toxicities. The Achilles Therapeutics trial NCT03517917 enabled the acquisition of matched tumor specimens and peripheral blood samples from patients undergoing routine surgery and facilitated the development of the proprietary VELOSTM manufacturing process, generating a personalized clonal neoantigen specific T cell product. An in-depth characterization of T cells expanded with the VELOSTM process was performed and compared to a standard TIL product. Samples were obtained from patients with primary NSCLC or metastatic melanoma. TIL were expanded from tumor fragments after dissection in the presence of IL-2. Peptide pools corresponding to the clonal mutations that were identified using the PELEUSTM bioinformatics platform were used to pulse dendritic cells (DC) generated from peripheral blood monocytes from each patient. Clonal neoantigen specific T cells (cNeT) were expanded using the VELOSTM process by co-culture of TIL with the peptide-pulsed autologous DC. As a comparison, TIL were expanded with a rapid expansion protocol (REP-TIL) in the presence of allogeneic feeders, anti-CD3 antibody and high-dose IL-2. Intracellular cytokine staining was performed following rechallenge with individual peptide pools encoding the clonal mutations. Single peptide reactivities were identified using ELISPOT and extended flow cytometric analysis of markers associated with T cell fitness or dysfunction was performed to phenotypically characterize the cNeT, TIL and REP-TIL. Analysis of the immune cell composition showed that cNeT, TIL and REP-TIL have similar CD3+ T cell content (median cNeT 90.2%, TIL 87.3%, REP-TIL 95%, n=6) and are composed of CD4+ and CD8+ T cells (median CD4:CD8 ratio- cNeT 11.1, TIL 2.03 and REP-TIL 4.7, n=6). cNeT showed superior clonal neoantigen specificity compared to TIL or REP-TIL. The proportion of CD3+ T cells responding to clonal neoantigen rechallenge was increased in cNeT (median 24.3%) compared to TIL (median 0.6%) and REP-TIL (median 1.8%) (n=5). The VELOSTM process incorporating the PELEUSTM platform for prediction of clonal neoantigens generates T cell products enriched for clonal neoantigen reactivities and superior phenotypic characteristics compared to conventional TIL. The VELOSTM process is currently being used to manufacture cNeT for two first-in-human studies including NSCLC and melanoma patients (NCT04032847, NCT03997474). Ethical approval: The samples for the study were collected under an ethically approved protocol (NCT03517917). Citation Format: Eleni Kotsiou, Tie Zheng Hou, Joseph Robinson, Sonal Varsani, Theres Oakes, Pablo D. Becker, Shreenal Patel, Jennine Mootien, Andrew Craig, Jane Robertson, Edward Samuel, James Reading, Lyra Del Rosario, Andrew Haynes, Samra Turajlic, Farah Islam, David Lawrence, Mariam Jamal-Hanjani, Martin Foster, Sergio A. Quezada, Katy Newton. Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products [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 875.

Tumor infiltrating lymphocytes (TIL) therapy in metastatic melanoma: boosting of neoantigen-specific T cell reactivity and long-term follow-up

Treatment of metastatic melanoma with autologous tumor infiltrating lymphocytes (TILs) is currently applied in several centers. Robust and remarkably consistent overall response rates, of around 50% of treated patients, have...

Evaluating different adoption scenarios for TIL-therapy and the influence on its (early) cost-effectiveness

BackgroundTreatment with tumor-Infiltrating Lymphocytes (TIL) is an innovative therapy for advanced melanoma with promising clinical phase I/II study results and likely beneficial cost-effectiveness. As a randomized controlled trial on the effectiveness of TIL therapy in advanced melanoma compared to ipilimumab is still ongoing, adoption of TIL therapy by the field is confronted with uncertainty. To deal with this, scenario drafting can be used to identify potential barriers and enables the subsequent anticipation on these barriers. This study aims to inform adoption decisions of TIL by evaluating various scenarios and evaluate their effect on the cost-effectiveness.MethodsFirst, 14 adoption scenarios for TIL-therapy were drafted using a Delphi approach with a group of involved experts. Second, the likelihood of the scenarios taking place within 5 years was surveyed among international experts using a web-based questionnaire. Third, based on the questionnaire results and recent literature, scenarios were labeled as being either “likely” or “-unlikely”. Finally, the cost-effectiveness of TIL treatment involving the “likely” scored scenarios was calculated.ResultsTwenty-nine experts from 12 countries completed the questionnaire. The scenarios showed an average likelihood ranging from 29 to 58%, indicating that future developments of TIL-therapy were surrounded with quite some uncertainty. Eight of the 14 scenarios were labeled as “likely”. The net monetary benefit per patient is presented as a measure of cost-effectiveness, where a positive value means that a scenario is cost-effective. For six of these scenarios the cost-effectiveness was calculated: “Commercialization of TIL production” (the price was assumed to be 3 times the manufacturing costs in the academic setting) (−€51,550), “Pharmaceutical companies lowering the prices of ipilimumab” (€11,420), “Using TIL-therapy combined with ipilimumab” (−€10,840), “Automatic TIL production” (€22,670), “TIL more effective” (€23,270), “Less Interleukin-2” (€20,370).ConclusionsIncorporating possible future developments, TIL-therapy was calculated to be cost-effective compared to ipilimumab in the majority of “likely” scenarios. These scenarios could function as facilitators for adoption. Contrary, TIL therapy was expected to not be cost-effective when sold at commercial prices, or when combined with ipilimumab. These scenarios should be considered in the adoption decision as these may act as crucial barriers.

Long-term follow up of lifileucel (LN-144) cryopreserved autologous tumor infiltrating lymphocyte therapy in patients with advanced melanoma progressed on multiple prior therapies.

10006 Background: Treatment options are limited for patients with advanced melanoma who have progressed on checkpoint inhibitors and targeted therapies. Adoptive cell therapy using tumor-infiltrating lymphocytes (TIL) leverages and enhances the body’s natural defense against cancer and has shown durable responses in heavily pretreated melanoma patients. Methods: C-144-01 is a global Phase 2 open-label, multicenter study of efficacy & safety of lifileucel in patients with unresectable metastatic melanoma who have progressed on checkpoint inhibitors and BRAF/MEK inhibitors, if BRAFv600 mutant. We report on Cohort 2 (N = 66) patients who have received TIL. Tumors were resected at local institutions, processed in central GMP facilities for TIL production, manufactured, cryopreserved & shipped back to sites in a 22-day process. Therapy consisted of one week of lymphodepletion, a single lifileucel infusion, and up to 6 IL-2 doses. ORR was based on RECIST v1.1 by investigator assessment. Data cutoff was Feb 2, 2020. Results: Baseline characteristics: 3.3 mean prior therapies (anti-PD1 100%; anti-CTLA-4 80%; BRAF/MEK inhibitor 23%), high baseline tumor burden (106 mm mean target lesion sum of diameters), 44% liver/brain lesions, 40.9% LDH > ULN. ORR by investigator was 36.4% (2 CR, 22 PR) and DCR was 80.3%. Mean time to response was 1.9 months (range: 1.3-5.6). After a median study follow-up of 17.0 months, median DOR (mDOR) was still not reached. Six responders have progressed, 2 have died and 2 started other anti-cancer therapy without progression. The adverse event profile was consistent with the underlying advanced disease and the lymphodepletion and IL-2 regimens. Additional follow-up data will be available for presentation. Conclusions: Lifileucel treatment results in a 36.4% ORR and mDOR was not reached at 17.0 months of median study follow up in a heavily pretreated metastatic melanoma patients with high baseline disease burden who progressed on multiple prior therapies, including anti-PD1 and BRAF/MEK inhibitors, if BRAFv600 mutant. Clinical trial information: NCT02360579.

TUMOR INFLATING LYMPHOCYTES. PURIFICATION, EXPANDING AND CYTOTOXICITY ANALISYS ON PRIMARY TUMOR CULTURES

Tumor Infiltrating Lymphocytes (TILs) is one of the most promising sources of autologous cytotoxic T-cells for adoptive immunotherapy, which has already shown high efficiency in the treatment of metastatic melanoma. In this study, we isolated TILs from surgical material obtained by resection of solid tumors (primary and metastatic adenocarcinomas of various localization, melanoma, glioblastoma), studied their population composition and developed protocols for the purification expanding, and activation of CD4 +, CD8 + cytotoxic antitumor lymphocytes. Cells were obtained from samples of resected tumors in 15 patients; in each case we obtain an autologous pair: the primary tumor culture and the TILs culture. We could isolate viable lymphocytes in 100% of cases. Isolated TILs were successfully expanded in our specialized medium using various combinations of IL-2 / IL-15 / IL-21 // IL-7, anti-CD3 and anti-CD28. Immunophenotyping showed that the obtained TILs are a heterogeneous mixture of CD4 +, CD8 + cells containing populations of CD3 +, CD4, CD8 cells, CD4 + CD25 + CD127 T-regulatory cells, CD45RA, CCR7 + memory T-cells, CD56 + CD16- NK cells. The initial cultures of TILs were also characterized by a high level of PD1 expression, indicating their low antitumor cytotoxicity. Using different protocols of isolation, expansion, and activation, we obtained a cell preparation containing 80% of CD8+ PD1 activated TILs in an amount sufficient for adoptive therapy (500106 or more). An in vitro study of the cytotoxicity of obtained TILs in primary cultures of homologous tumors using RTCA Icelligence showed high cytotoxicity, providing almost 100% tumor cell death. Our developed protocol for the production and activation of TILs can be recommended for the phase I-II clinical trials of adoptive immunotherapy of recurrent, highly metastatic solid tumors.

Abstract A15: Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy

Abstract The natural occurrence of immune cells in the tumor microenvironment of ovarian cancer (OC) facilitates the ex vivo generation and expansion of tumor-infiltrating lymphocytes (TIL) for infusion in the context of adoptive T-cell transfer. Yet, the lack of tumor-reactive TILs and the suppressive microenvironment have hindered the translation of TIL therapy into meaningful clinical responses in OC patients. Therefore, generating a proinflammatory tumor microenvironment with oncolytic adenoviruses, in order to induce a steady and potent antitumor activity, may prove an effective add-on to TIL therapy for OC. Besides their well-established efficacy in OC, oncolytic adenoviruses genetically modified to carry the interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFa) transgenes may further support and promote T-cell mediated antitumor immunity. In this study, we assessed the capability of an oncolytic virus coding for IL-2 and TNFa (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) to enhance the reactivity of TILs towards OC in a suppressive microenvironmental context. Fresh tumor tissue from regional metastatic or primary sites was obtained from stage III-IV OC patients. Part of the samples were diced in small fragments for the generation and rapid expansion of TILs under clinically relevant in vitro conditions. Phenotypic characterization of TILs was performed by flow cytometry after rapid expansion. Following TIL production and characterization, TILs were co-cultured with T cell-depleted autologous OC single-cell suspensions in the presence or absence of oncolytic virus. TIL reactivity was determined by quantifying Interferon (IFN)-gamma in culture supernatants through ELISA. The remainder of the tumor sample was dissociated overnight and cultured in the presence or absence of oncolytic virus. Supernatants were harvested at different days post-infection for detection of relevant cytokines. Following a 14-day rapid expansion period, the final TIL product was composed mostly of CD8+ T cells (56%), CD4+ T cells (27%) and a small fraction of CD3+CD56+ NK-like T cells (2%). When TILs were cocultured with T-cell depleted OC single-cell suspensions, treatment with oncolytic virus coding for IL-2 and TNFa caused a significant increase in the release of IFNg by TILs as compared to conditions where no virus or the backbone oncolytic virus (without cytokine transgenes) was added (p<0.05). Microenvironmental changes by the cytokine-encoding oncolytic adenovirus were evidenced by increased release of proinflammatory cytokines such as, TNFa, IL-2 and IFNg in 7-day cell cultures of ex vivo single-cell suspensions derived from OC patients. In summary, tailoring the tumor microenvironment with IL-2 and TNFa using oncolytic viruses sharpens the TIL reactivity in an immune-suppressive OC microenvironmental context. Hence, this approach may enable effective responses in OC patients treated with TIL therapy. This evidence supports further clinical validation. Citation Format: João Manuel Santos, Camilla Hëinio, Victor Cervera-Carrascon, Mikko Siurala, Dafne Quixabeira, Tanja De Gruijl, Heini Lassus, Anna Kanerva, Akseli Hemminki. Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A15.

Abstract LB-069: Persistence of cryopreserved tumor-infiltrating lymphocyte product lifileucel (LN-144) in C-144-01 study of advanced metastatic melanoma

Abstract Adoptive cell transfer utilizing tumor-infiltrating lymphocytes (TIL) is recognized as an effective treatment in metastatic melanoma and other solid tumors eliciting durable and complete responses in heavily pretreated patients, presumably by targeting somatic mutations specific to each tumor1. We have treated patients with anti-PD-1-refractory advanced melanoma with lifileucel and reported an overall response rate of 38%2. Here, we analyzed the composition of the initial TIL products and the T cells circulating 42 days post-infusion (D42) to uncover a potential link between clonal diversity, TIL in vivo persistence, and anti-tumor activity. Since TIL products are preparations of polyclonal autologous T cells, each T cell clone expresses a unique T cell receptor (TCR) that can be identified by its complementary determining region 3 (CDR3). CDR3 of TIL products and corresponding post-infusion peripheral blood samples were subjected to RNA-seq, using iRepertoire technology (Huntsville, AL). The average number of unique TCR CDR3 sequences (uCDR3) was 17511 [3574-110797] across TIL products, with Shannon diversity indexes varying from 2.7 to 10.8. Correlation analyses revealed no association between either parameter and clinical response, suggesting that tumor-reactive T cells may be present in low and high diversity bulk TIL products. At D42, TIL clones could be detected in the circulation of all treated patients. Numbers of Shared uCDR3 ranged from 28 to 6964 unique clonotypes and represented highly variable fractions of both the TIL product and the D42 circulating T cells. A link between Shared T cell clones and clinical response was previously hypothesized3. In this study, comparable percentages of Shared uCDR3 were detected in responders and non-responders. Importantly, the majority of Shared uCDR3 were not detected in the patients’ peripheral blood at the time of enrollment, indicating that they represented intratumoral clonotypes that persisted after TIL administration. In addition, Shared uCDR3 clones represented at either high or low frequencies in the TIL product could persist for at least 6 weeks post-infusion. Finally, more than 97% of persisting clones were uniquely present in individual responding and non-responding patients, indicating a unique repertoire in each TIL preparation. Overall, the data demonstrate that a fraction of TIL clones persisted in all patients. The uniqueness of the clonal profiles associated with response highlights the challenge of identifying a single TCR as mediator of activity and supports using a polyclonal product such as bulk TILs to treat solid tumors with their associated unique, patient-specific, mutational and neoantigen spectra. 1 Rosenberg et al. CCR 2011; 2 SITC Nov2018; 3 Robbins et al. J Immunol 2004 Citation Format: Viktoria Gontcharova, Sam Suzuki, Michelle R. Simpson-Abelson, Michelle Blaskovich, Cécile Chartier. Persistence of cryopreserved tumor-infiltrating lymphocyte product lifileucel (LN-144) in C-144-01 study of advanced metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-069.