Tumor-infiltrating Lymphocytes Therapy Research Articles

Cellular Therapy for Lung Cancer: Focusing on Chimeric Antigen Receptor T (CAR T) Cells and Tumor-Infiltrating Lymphocyte (TIL) Therapy.

Lung cancer is a leading cause of morbidity and mortality in the United States and worldwide. The introduction of immune checkpoint inhibitors has led to a marked improvement in the outcomes of lung cancer patients. Despite these advances, there is a huge unmet need for therapeutic options in patients who are not candidates for targeted or immunotherapy or those who progress after first-line treatment. With its high mutational burden, lung cancer appears to be an attractive target for novel personalized treatment approaches. In this review, we provide an overview of two adoptive cell therapy approaches-chimeric antigen receptors (CAR) T-cell therapy and Tumor-infiltrating lymphocytes (TILs) in lung cancer with an emphasis on current challenges and future perspectives. While both these therapies are still in the early phases of development in lung cancer and need more refinement, they harbor the potential to be effective treatment options for this group of patients with otherwise poor prognoses.

Successful ex vivo expansion of tumor infiltrating lymphocytes with systemic chemotherapy prior to surgical resection.

Tumor infiltrating lymphocytes (TIL) have demonstrated efficacious clinical outcomes for many patients with various types of solid cancers, including melanoma, gastrointestinal cancer, lung cancer, and head and neck cancer. Currently, the majority of clinical trials require that patients did not receive systemic therapy right before tumor tissue resection to avoid the interference of chemotherapy in the ex vivo TIL expansion. The primary disadvantage of this strategy is limiting the accessibility of TIL therapy for many eligible cancer patients. Over the past decade, substantial progress has been made for ex vivo expansion technologies in T cells. In this study, we investigated the possibility of enrolling patients who underwent chemotherapy prior to surgical resection. We collected seventeen tumor tissues from treatment naive cases, and five from cases that underwent chemotherapies. Cancer indications enrolled in this study were colorectal and lung cancers from both primary and metastatic sites, such as liver and brain. TILs from these tumors were expanded ex vivo to 2.1E8 (total viable lymphocytes counts) on average, with an overall success rate of 90.9%. Subsequently, TIL phenotypes and cytokine production were analyzed using flow cytometry and ELISA, respectively. We demonstrated functional TIL expansion from tumor tissues despite chemotherapy prior to surgical resection. We observed no significant phenotypic or functional differences between groups with and without chemotherapy. TIL expansion rate and characteristics were similar regardless of chemotherapy prior to resection, thereby providing a possibility to recruit patients with the most recent chemotherapy history in TIL therapy trials.

Tolerability and response of palliative radiotherapy in patients receiving tumor-infiltrating lymphocyte therapy.

e14527 Background: Tumor-infiltrating lymphocyte (TIL) therapy is an emerging therapeutic option for patients with metastatic tumors having progressed on multiple lines of systemic therapy. It is unknown how TIL therapy impacts the tolerability and effectiveness of palliative radiotherapy (RT). We herein present a case series of patients having received both TIL therapy and palliative RT along with the associated local responses and toxicity profiles. Methods: A single institution retrospective database review identified patients treated with TIL therapy following progression on ≥2 standard treatment(s) for metastatic disease. Patients were eligible for inclusion if they received palliative radiotherapy treatment within 1 year of TIL delivery as pre- or post-TIL therapy. Patient demographic data was collected and descriptive statistics reported. Radiographic imaging response was assigned in accordance with Response Evaluation Criteria in Solid Tumors (RECIST). Toxicities were graded in accordance with Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0. Results: Between 2017–2021, a total of 10 patients receiving TIL therapy treated across 12 courses of palliative RT were included for analysis. 8 of 10 patients (80%) had a primary diagnosis of malignant melanoma and 2 (20%) had non-small cell lung cancer. RT was administered to 7 patients pre- and 3 patients post-TIL therapy. Within the pre-TIL cohort, 2 patients received RT between TIL harvest and infusion. Patients received an average total dose of 26 Gy (range, 8-45 Gy). Median patient follow-up after RT was 6 months. Of the patients who received RT prior to TIL therapy, 6 of 9 sites demonstrated at-least stable disease at the site of RT versus 1 of 3 sites receiving post-TIL RT. Across the cohort, 2 patients experienced Grade 1 toxicities associated with RT treatment. No Grade 2+ events were reported. Conclusions: Palliative RT appears to be safe and tolerable when delivered both prior to and post-TIL therapy. RT may be an option for bridging therapy between TIL harvest and infusion. Local response rates to RT were greater in patients receiving RT prior to TIL infusion. Further investigations are warranted to determine the optimal dose, fractionation, and timing of RT when integrating it into future TIL therapy protocols. [Table: see text]

Machine learning (ML)-based quantification of tumor-infiltrating lymphocytes (TIL) and clinical outcomes of patients with melanoma treated with immune-checkpoint inhibitors (ICI).

9560 Background: TIL quantification has shown promising prognostic and predictive impact in various tumors treated with ICI. More recently, TIL therapy has become an emerging treatment agent for ICI-refractory melanoma. In this work, we studied the effect of intrinsic TILs on clinical outcomes of patients with melanoma treated with ICI and quantified its utility as a biomarker in combination with tumor mutational burden (TMB). Methods: We applied a previously developed ML model to process digital whole-slide images of hematoxylin and eosin slides to quantify TIL density (TIL/mm2) in 182 metastatic melanoma samples treated with ICI at the Dana-Farber Cancer Institute. All samples underwent next-generation targeted-panel sequencing and had available TMB data (mutations/megabase). Overall survival (OS) was calculated from the data of ICI initiation to the date of death. TTF was calculated from the data of ICI initiation to the date of next line treatment or death. Alive patients were censored at the date of last follow-up. Multivariable cox regression was used to estimate adjusted hazard ratios (adj-HR) and p-values (adj-p), with TMB, prior lines of treatment and regimen type (single vs. combination ICI) used as covariates. TMB-high (TMB-H) group was defined as samples with TMB≥10. TIL-high (TIL-H) group was defined as samples with TIL density higher than the median. Results: Of 182 patients, 60% (110/182) were male and the median age at ICI start was 65.5 years. 63% (115/182) of patients received single-agent ICI (anti-PD1 or anti-CTLA4). Of 182 tumor specimens assessed for TILs, 47 (25.8%) were obtained from the primary site. There was no difference in TIL density (medians 583 vs. 817 TILs/mm2, p=0.57; 709 TIL/mm2 for full cohort) or TMB (medians 11.4 vs 12.2 mutations/megabase) between primary and metastatic samples, respectively. Moreover, there was no correlation between TIL density and TMB (pearson’s correlation coefficient = 0.92, p=1) across all samples. Compared to patients in the TIL-low (TIL-L) category, patients in TIL-H had significantly longer median TTF (33.9 [15.9-NR] versus 13.0 [7.8-18.6] months, adj-HR: 0.53 (95% CI: 0.36-0.78); adj-P=0.0012), while there was no difference in OS (P=0.57). In fact, patients in the TMB-H/TIL-H group had the longest TTF, followed by TMB-H/TIL-L, TMB-L/TIL-H then TMB-L/TIL-L (table). Conclusions: Our results support the potential use of ML-based TIL scoring as a novel and independent biomarker to predict time to failure on ICI. There is ongoing effort to validate our findings in an external dataset. [Table: see text]

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

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

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Tumor-infiltrating lymphocyte therapy: Clinical aspects and future developments in this breakthrough cancer treatment.

Tumor-infiltrating lymphocyte (TIL) therapy is a promising approach for treating refractory or advanced solid cancers by using autologous TILs harvested from cancer tissues. Despite the heterogeneity of cancer, TIL therapy can potentially produce a positive therapeutic response, including complete remission. After decades of research on lymphocyte functions, culture/expansion methods, therapeutic protocols, and multiple clinical trials, TIL therapy has finally reached a stage where it can be formally approved for clinical use. TIL therapy is expected to hold a unique position among anti-cancer therapeutic options as a standard intervention. To successfully introduce TIL therapy into clinical settings, there is a need to expand therapeutic indications and set up the best protocols for cancer tissue sampling and manufacturing, and related clinical trials. Moreover, studies on next-generation TIL therapy have already begun, and post-approval real-world data will promote and support further research.

Bispecific antibodies redirect synthetic agonistic receptor modified T cells against melanoma

BackgroundMelanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating...

IL-21 or synthetic IL-2 treatment drives T cell IFNγ production or expansion, respectively, during adoptive cell therapy of melanoma

Abstract Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) can generate long-term durable responses in patients with melanoma and can even successfully treat patients that are refractory to anti-PD-1 checkpoint blockade. IL-2 is often used to expand TILs, however the administration of this cytokine can induce systemic toxicities in patients. Synthetic cytokine mimetics of IL-2 have been developed, including neoleukin-2 (Neo2), which has been shown to have enhanced therapeutic efficacy over IL-2, with reduced toxicity. IL-21 has also been used in TIL therapy to promote T cell expansion. Here, we tested the efficacy of Neo2 or IL-21 in treating mice bearing B16 tumors that received ACT with melanoma-specific T cells (TRP1 Highand TRP1 Low; TCRs recognizing antigen with high or low affinity). Both Neo2 and IL-21 could slow growth of B16 tumors, curing several mice. We performed single-cell RNA-sequencing on these tumors to analyze cytokine therapy-induced changes in the microenvironment. Neo2-treated tumors had a signature primarily of T cell expansion, with few other associated transcriptional changes. IL-21 therapy generated a profound IFNγ signature, with an increase in T cells production of IFNγ and many cell types, including T cells, macrophages, and tumor cells responding with interferon-stimulated gene expression. Interestingly, IL-21 therapy did not induce profound T cell expansion as Neo2 did, but importantly both IL-21 and Neo2 therapy reduced levels of Tregs within the tumor. IL-21 also improved T cell production of granzyme B. Thus, IL-2 primarily expands T cells and IL-21 generates higher quality anti-tumor T cells. Therefore, IL-21 is a promising cytokine for improving ACT and other immunotherapies.

Adoptive Cell Therapy for Nonhematologic Solid Tumors.

The long-term benefits demonstrated by immunotherapy in select tumors have failed to generalize to most nonhematologic solid tumors. Adoptive cell therapy (ACT)-a treatment on the basis of the isolation and engineering of living T cells and other immune cells-has shown early clinical advances. ACT, through tumor-infiltrating lymphocyte therapy, has shown activity in traditionally immunogenic tumors such as melanoma and cervical cancers, and has the potential to improve immune reactivity in these tumor types where traditional therapies have failed. Engineered T-cell receptor and chimeric antigen receptor T-cell therapies have also shown activity in select nonhematologic solid tumors. Through receptor engineering, and improved understanding of tumor antigens, these therapies have the potential to target poorly immunogenic tumors to deliver long-lasting responses. Additionally, non-T-cell therapies such as natural killer-cell therapy may allow for allogeneic forms of ACT. Each form of ACT has trade-offs that will likely limit their application to specific clinical settings. Key challenges with ACT include the logistical challenges of manufacturing, accurate antigen identification, and the risk of on-target, off-tumor toxicity. The successes of ACT are built on decades of advances in cancer immunology, antigen identification, and cell engineering. With continued refinements in these processes, ACT may extend the benefits of immunotherapy to more patients with advanced nonhematologic solid tumors. Herein, we review the major forms of ACT, their successes, and strategies to overcome the trade-offs of current ACTs.

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

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

Tumor-infiltrating lymphocytes – from theory to real practice

Adoptive therapy with tumor-infiltrating lymphocytes (TIL) has been studied since 1980s. Although some trials showed very high efficacy in melanoma and other tumors no enough data were available for approval of the method perhaps due to the cost, complexity and reproducibility limited to academic centers. Last year the results of prospective phase III trial comparing TIL therapy to standard immunotherapy for melanoma were reported which proved the efficacy and even superiority of the method. This paper reviews the principles of adoptive TIL therapy and the perspectives for further developement.

Therapeutic implication of chimeric antigen receptor T-cell therapy for head and neck squamous cell carcinoma.

Therapeutic implication of chimeric antigen receptor T-cell therapy for head and neck squamous cell carcinoma.

Tumor-infiltrating lymphocyte therapy: an overview

Tumor-infiltrating lymphocyte therapy: an overview

Towards next‐generation TIL therapy: TILs enriched in neoepitope‐specific T cells

Towards next‐generation TIL therapy: TILs enriched in neoepitope‐specific T cells

Cancer immunotherapeutics: transforming T-cells to superior cancer killers

The evolution of cancer treatment has marked a significant shift from traditional therapies such as surgery, chemotherapy, and radiation to more targeted T-cell-based therapies. The emergence of immune checkpoint inhibitors (ICIs) and gene-editing techniques like CRISPR/Cas9 has revolutionized cancer immunotherapy. Currently, adoptive cell transfer (ACT) therapies, such as tumor-infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor (CAR) T-cell therapy, are giving promising results in melanoma and leukemia, respectively. Challenges occur to infiltrating solid tumors due to their dense stromal network, and abnormal vasculature has become a barrier. Hence, focusing on these strategies to modify cancer vasculature, immune suppression, and chemokines are major goals of current ACTs. The present review provides insight into the emerging cancer immune therapeutics, their advantages, and limitations. Emphasis is given to the recently discovered “superior” T-cells. These cells exhibit a unique ability to recognize multiple cancer-associated proteins effectively, making TIL therapy the future of cancer management.

Personalized Immunotherapy of Patients: Defining by Single-cell RNA-seq with Artificial Intelligence.

Immunotherapy, including immune cell therapy and targeted therapy, is gradually developed through the ongoing discovery of molecular compounds or immune cells. Choosing the best one or the best combination of target compounds and immune-cell therapy is a challenge for clinical scientists and clinicians. We have found variable efficacy individually after tumor-infiltrating lymphocyte (TIL) therapy, and now TILs have been discovered in a group of heterogeneous immune cells. To select the best immunotherapy for each patient, we started to study TIL genomics, including single-cell mRNA differential display from TIL published in 2007 and single-cell RNA-seq from TIL published in 2013, set up TIL quantitative network in 2015, researched machine-learning model for immune therapy in 2022. These manual reports single-cell RNA-seq data combined with machine learning to evaluate the optimal compounds and immune cells for individual patients. The machine-learning model, one of artificial intelligence, can estimate targeting genomic variance from single-cell RNA-seq so that they can cover thirteen kinds of immune cell therapies and ongoing FDA-approved targeted therapies such as PD1 inhibitors, PDL1 inhibitors, and CTLA4 inhibitors, as well as other different treatments such as HDACI or DNMT1 inhibitors, FDA-approved drugs. Moreover, also cover Phase-1, Phase-2, Phase-3, and Phase-4 of clinical trials, such as TIL, CAR T-cells, TCR T-cells. Single-cell RNA-seq with an Artificial intelligence estimation system is much better than our published models from microarrays or just cell therapy. The medical goal is to address three issues in clinical immunotherapy: the increase of efficacy; the decrease of adverse effects and the decrease of the cost in clinical applications.

Current Situation and Prospect of Adoptive Cellular Immunotherapy for Malignancies.

Adoptive cell immunotherapy (ACT) is an innovative promising treatment for tumors. ACT is characterized by the infusion of active anti-tumor immune cells (specific and non-specific) into patients to kill tumor cells either directly or indirectly by stimulating the body's immune system. The patient's (autologous) or a donor's (allogeneic) immune cells are used to improve immune function. Chimeric antigen receptor (CAR) T cells (CAR-T) is a type of ACT that has gained attention. T cells from the peripheral blood are genetically engineered to express CARs that rapidly proliferate and specifically recognize target antigens to exert its anti-tumor effects. Clinical application of CAR-T therapy for hematological tumors has shown good results, but adverse reactions and recurrence limit its applicability. Tumor infiltrating lymphocyte (TIL) therapy is effective for solid tumors. TIL therapy exhibits T cell receptor (TCR) clonality, superior tumor homing ability, and low targeted toxicity, but its successful application is limited to a number of tumors. Regardless, TIL and CAR-T therapies are effective for treating cancer. Additionally, CAR-natural killer (NK), CAR-macrophages (M), and TCR-T therapies are currently being researched. In this review, we highlight the current developments and limitations of several types of ACT.

Genetics and Therapeutic Responses to Tumor-infiltrating Lymphocyte Therapy of Pancreatic Cancer Patient-derived Xenograft Models

Genetics and Therapeutic Responses to Tumor-infiltrating Lymphocyte Therapy of Pancreatic Cancer Patient-derived Xenograft Models

Tumor-Infiltrating Lymphocyte Therapy for Advanced Melanoma: Ready for Prime Time?

Tumor-Infiltrating Lymphocyte Therapy for Advanced Melanoma: Ready for Prime Time?