Acquired resistance to cancer immunotherapy

In recent times, advances in cancer immunotherapy have yielded impressive, durable clinical responses in patients with varied subtypes of cancer. However, a significant proportion of patients who initially demonstrate encouraging tumor regression develop resistance and progress over time. The identification of novel therapeutic approaches to overcome resistance may result in significantly improved clinical outcomes and remains an area of high scientific priority. This review aims to summarize the current knowledge regarding the role of both tumor-intrinsic and tumor-extrinsic factors in the development of resistance to cancer immunotherapy and to discuss current and possible future therapeutic strategies targeting these mechanisms

ACT Up TIL Now: The Evolution of Tumor-Infiltrating Lymphocytes in Adoptive Cell Therapy for the Treatment of Solid Tumors

The past decades of cancer immunotherapy research have provided profound evidence that the immune system is capable of inducing durable tumor regression. Although many commercialized anti-cancer immunotherapies are available to patients, these treatment options only scrape the surface of the potential immune-related treatment possibilities for cancer. Additionally, many individuals are ineligible for established immunotherapies due to their cancer type. The adoptive cell transfer of autologous tumor-infiltrating lymphocytes has been used in humans for over 30 years to treat metastatic melanoma, and continued modifications are making it increasingly more effective against other types of cancer. This comprehensive review outlines this therapy from its infancy through to the present day, bringing to light modifications and optimizations to the traditional workflow, as well as highlighting the influence of new methods and technologies

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

Transcriptomic signatures of tumors undergoing T cell attack

Background: Studying tumor cell–T cell interactions in the tumor microenvironment (TME) can elucidate tumor immune escape mechanisms and help predict responses to cancer immunotherapy. Methods: We selected 14 pairs of highly tumor-reactive tumor-infiltrating lymphocytes (TILs) and autologous short-term cultured cell lines, covering four distinct tumor types, and co-cultured TILs and tumors at sub-lethal ratios in vitro to mimic the interactions occurring in the TME. We extracted gene signatures associated with a tumor-directed T cell attack based on transcriptomic data of tumor cells. Results: An autologous T cell attack induced pronounced transcriptomic changes in the attacked tumor cells, partially independent of IFN-γ signaling. Transcriptomic changes were mostly independent of the tumor histological type and allowed identifying common gene expression changes, including a shared gene set of 55 transcripts influenced by T cell recognition (Tumors undergoing T cell attack, or TuTack, focused gene set). TuTack scores, calculated from tumor biopsies, predicted the clinical outcome after anti-PD-1/anti-PD-L1 therapy in multiple tumor histologies. Notably, the TuTack scores did not correlate to the tumor mutational burden, indicating that these two biomarkers measure distinct biological phenomena. Conclusions: The TuTack scores measure the effects on tumor cells of an anti-tumor immune response and represent a comprehensive method to identify immunologically responsive tumors. Our findings suggest that TuTack may allow patient selection in immunotherapy clinical trials and warrant its application in multimodal biomarker strategies

Qualitative analysis of tumor-infiltrating lymphocytes across human tumor types reveals a higher proportion of bystander CD8+ T cells in non-melanoma cancers compared to melanoma

Background: Human intratumoral T cell infiltrates can be defined by quantitative or qualitative features, such as their ability to recognize autologous tumor antigens. In this study, we reproduced the tumor-T cell interactions of individual patients to determine and compared the qualitative characteristics of intratumoral T cell infiltrates across multiple tumor types. Methods: We employed 187 pairs of unselected tumor-infiltrating lymphocytes (TILs) and autologous tumor cells from patients with melanoma, renal-, ovarian-cancer or sarcoma, and single-cell RNA sequencing data from a pooled cohort of 93 patients with melanoma or epithelial cancers. Measures of TIL quality including the proportion of tumor-reactive CD8+ and CD4+ TILs, and TIL response polyfunctionality were determined. Results: Tumor-specific CD8+ and CD4+ TIL responses were detected in over half of the patients in vitro, and greater CD8+ TIL responses were observed in melanoma, regardless of previous anti-PD-1 treatment, compared to renal cancer, ovarian cancer and sarcoma. The proportion of tumor-reactive CD4+ TILs was on average lower and the differences less pronounced across tumor types. Overall, the proportion of tumor-reactive TILs in vitro was remarkably low, implying a high fraction of TILs to be bystanders, and highly variable within the same tumor type. In situ analyses, based on eight single-cell RNA-sequencing datasets encompassing melanoma and five epithelial cancers types, corroborated the results obtained in vitro. Strikingly, no strong correlation between the proportion of CD8+ and CD4+ tumor-reactive TILs was detected, suggesting the accumulation of these responses in the tumor microenvironment to follow non-overlapping biological pathways. Additionally, no strong correlation between TIL responses and tumor mutational burden (TMB) in melanoma was observed, indicating that TMB was not a major driving force of response. No substantial differences in polyfunctionality across tumor types were observed. Conclusions: These analyses shed light on the functional features defining the quality of TIL infiltrates in cancer. A significant proportion of TILs across tumor types, especially non-melanoma, are bystander T cells. These results highlight the need to develop strategies focused on the tumor-reactive TIL subpopulation

Rapid Identification of the Tumor-Specific Reactive TIL Repertoire via Combined Detection of CD137, TNF, and IFNγ, Following Recognition of Autologous Tumor-Antigens

Detecting the entire repertoire of tumor-specific reactive tumor-infiltrating lymphocytes (TILs) is essential for investigating their immunological functions in the tumor microenvironment. Current in vitro assays identifying tumor-specific functional activation measure the upregulation of surface molecules, de novo production of antitumor cytokines, or mobilization of cytotoxic granules following recognition of tumor-antigens, yet there is no widely adopted standard method. Here we established an enhanced, yet simple, method for identifying simultaneously CD8+ and CD4+ tumor-specific reactive TILs in vitro, using a combination of widely known and available flow cytometry assays. By combining the detection of intracellular CD137 and de novo production of TNF and IFNγ after recognition of naturally-presented tumor antigens, we demonstrate that a larger fraction of tumor-specific and reactive CD8+ TILs can be detected in vitro compared to commonly used assays. This assay revealed multiple polyfunctionality-based clusters of both CD4+ and CD8+ tumor-specific reactive TILs. In situ, the combined detection of TNFRSF9, TNF, and IFNG identified most of the tumor-specific reactive TIL repertoire. In conclusion, we describe a straightforward method for efficient identification of the tumor-specific reactive TIL repertoire in vitro, which can be rapidly adopted in most cancer immunology laboratories