Intercellular nanotube-mediated mitochondrial transfer enhances T cell metabolic fitness and antitumor efficacy.

Mitochondrial loss and dysfunction drive T cell exhaustion, representing major barriers to successful T cell-based immunotherapies. Here, we describe an innovative platform to supply exogenous mitochondria to T cells, overcoming these limitations. We found that bone marrow stromal cells establish nanotubular connections with T cells and leverage these intercellular highways to transplant stromal cell mitochondria into CD8+ T cells. Optimal mitochondrial transfer required Talin 2 on both donor and recipient cells. CD8+ T cells with donated mitochondria displayed enhanced mitochondrial respiration and spare respiratory capacity. When transferred into tumor-bearing hosts, these supercharged T cells expanded more robustly, infiltrated the tumor more efficiently, and exhibited fewer signs of exhaustion compared with T cells that did not take up mitochondria. As a result, mitochondria-boosted CD8+ T cells mediated superior antitumor responses, prolonging animal survival. These findings establish intercellular mitochondrial transfer as a prototype of organelle medicine, opening avenues to next-generation cell therapies.

Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

Recent clinical researches and technological development in TIL therapy.

Tumor-infiltrating lymphocyte (TIL) therapy represents a groundbreaking advancement in the solid cancer treatment, offering new hope to patients and their families with high response rates and long overall survival. TIL therapy involves extracting immune cells from a patient's tumor tissue, expanding them ex vivo, and infusing them back into the patient to target and eliminate cancer cells. This revolutionary approach harnesses the power of the immune system to combat cancers, ushering in a new era of T cell-based therapies along with CAR-T and TCR-therapies. In this comprehensive review, we aim to elucidate the remarkable potential of TIL therapy by delving into recent advancements in basic and clinical researches. We highlight on the evolving landscape of TIL therapy as a prominent immunotherapeutic strategy, its multifaceted applications, and the promising outcomes. Additionally, we explore the future horizons of TIL therapy, next-generation TILs, and combination therapy, to overcome the limitations and improve clinical efficacy of TIL therapy.

Lung cancer immunotherapy: progress, pitfalls, and promises.

Lung cancer is the primary cause of mortality in the United States and around the globe. Therapeutic options for lung cancer treatment include surgery, radiation therapy, chemotherapy, and targeted drug therapy. Medical management is often associated with the development of treatment resistance leading to relapse. Immunotherapy is profoundly altering the approach to cancer treatment owing to its tolerable safety profile, sustained therapeutic response due to immunological memory generation, and effectiveness across a broad patient population. Different tumor-specific vaccination strategies are gaining ground in the treatment of lung cancer. Recent advances in adoptive cell therapy (CAR T, TCR, TIL), the associated clinical trials on lung cancer, and associated hurdles are discussed in this review. Recent trials on lung cancer patients (without a targetable oncogenic driver alteration) reveal significant and sustained responses when treated with programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) checkpoint blockade immunotherapies. Accumulating evidence indicates that a loss of effective anti-tumor immunity is associated with lung tumor evolution. Therapeutic cancer vaccines combined with immune checkpoint inhibitors (ICI) can achieve better therapeutic effects. To this end, the present article encompasses a detailed overview of the recent developments in the immunotherapeutic landscape in targeting small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Additionally, the review also explores the implication of nanomedicine in lung cancer immunotherapy as well as the combinatorial application of traditional therapy along with immunotherapy regimens. Finally, ongoing clinical trials, significant obstacles, and the future outlook of this treatment strategy are also highlighted to boost further research in the field.

An armed oncolytic virus enhances the efficacy of tumor-infiltrating lymphocyte therapy by converting tumors to artificial antigen-presenting cells in situ.

The full potential of tumor-infiltrating lymphocyte (TIL) therapy has been hampered by the inadequate activation and low persistence of TILs, as well as inefficient neoantigen presentation by tumors. We transformed tumor cells into artificial antigen-presenting cells (aAPCs) by infecting them with a herpes simplex virus 1 (HSV-1)-based oncolytic virus encoding OX40L and IL12 (OV-OX40L/IL12) to provide local signals for optimum T cell activation. The infected tumor cells displayed increased expression of antigen-presenting cell-related markers and induced enhanced T cell activation and killing in coculture with TILs. Combining OV-OX40L/IL12 and TIL therapy induced complete tumor regression in patient-derived xenograft and syngeneic mouse tumor models and elicited an antitumor immunological memory. In addition, the combination therapy produced aAPC properties in tumor cells, activated T cells, and reprogrammed macrophages to a more M1-like phenotype in the tumor microenvironment. This combination strategy unleashes the full potential of TIL therapy and warrants further evaluation in clinical studies.

Adoptive cell therapy in gynecologic cancers: A systematic review and meta-analysis.

Adoptive cell therapy (ACT) has shown promise in hematologic and solid tumors. While data supports immunogenicity of gynecologic cancers, the benefit of ACT is not yet clear. To address this question, we performed a comprehensive systematic review and meta-analysis. Eligible studies included those reporting oncologic response or toxicity data in at least one patient with any gynecologic cancer treated with ACT. Chi-square test and multivariable logistic regression were performed to identify predictors of response. We retrieved 281 articles, and 28 studies met our inclusion criteria. These comprised of 401 patients including 238 patients with gynecologic cancers (61.8% ovarian, 34.0% cervical, 2.9% endometrial, and 1.2% other). In patients with gynecologic cancers, response rates to ACT were 8.1% complete response, 18.2% partial response, and 31.4% stable disease, for an objective response rate (ORR) of 26.3%, disease control rate (DCR) of 57.6%, and median response duration of 5.5 months. Patients in studies reporting ≤1 median line of prior therapy had a higher ORR (52.9% vs. 22.6% for >1, p < 0.001), although DCR in the >1 group was still 53.2%. ORRs by ACT type were tumor infiltrating lymphocytes (TIL) 41.4%, natural killer cells 26.7%, peripheral autologous T-cells 18.4%, T-cell receptor-modified T-cells 15.4%, and chimeric antigen receptor T-cells 9.5% (p = 0.001). ORR was significantly improved with inclusion of lymphodepletion (34.8% vs. 15.4% without, p = 0.001). On multivariable analysis controlling for cancer type and lymphodepletion, TIL therapy was predictive of objective response (odds ratio 2.6, p = 0.011). The rate of grade 3 or 4 toxicity was 46.0%. All grade adverse events included fever, hypotension, dyspnea, confusion, hematologic changes, nausea/vomiting, fatigue, and diarrhea. In conclusion, ACT is a promising treatment modality in gynecologic cancer. We observed a particular benefit of TIL therapy and suggest inclusion of lymphodepletion in future trials.

Potential clinical application of tumor-infiltrating lymphocyte therapy for ovarian epithelial cancer prior or post-resistance to chemotherapy.

BACKGROUND: Immunotherapy has become a powerful treatment option for several solid tumor types. The presence of tumor-infiltrating lymphocytes (TIL) is correlated with better prognosis in ovarian cancer, pointing at the possibility to benefit from harnessing their anti-tumor activity. This preclinical study explores the feasibility of adoptive cell therapy (ACT) with TIL using an improved culture method. METHODS: TIL from high-grade serous ovarian cancer were cultured using a combination of IL-2 with agonistic antibodies targeting 4-1BB and CD3. The cells were phenotyped using flow cytometry in the fresh tissue and after expansion. Tumor reactivity was assessed against HLA-matched ovarian cancer cell lines via IFN-γ ELISPOT. RESULTS: Ovarian cancer is highly infiltrated with CD8+ TIL that are preferentially and robustly expanded with the addition of the agonistic antibodies. With a 95% success rate, the TIL are grown to ≥ 100 × 106 cells in 2-3 weeks without over differentiation. In addition, the CD8+ TIL grown with this method showed HLA-restricted tumor recognition. CONCLUSIONS: These results indicate the viability of TIL ACT for refractory ovarian cancer by allowing for the large expansion of anti-tumor TIL in a short time and consistent manner.

Adenovirus Coding for Interleukin-2 and Tumor Necrosis Factor Alpha Replaces Lymphodepleting Chemotherapy in Adoptive T Cell Therapy.

Lymphodepleting preconditioning with high-dose chemotherapy is commonly used to increase the clinical efficacy of adoptive T cell therapy (ACT) strategies, however, with severe toxicity for patients. Conversely, oncolytic adenoviruses are safe and, when engineered to express interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α), they can achieve antitumor immunomodulatory effects similar to lymphodepletion. Therefore, we compare the safety and efficacy of such adenoviruses with a cyclophosphamide- and fludarabine-containing lymphodepleting regimen in the setting of ACT. Human adenovirus (Ad5/3-E2F-D24-hTNF-α-IRES-hIL-2; TILT-123) replication was studied using a Syrian hamster pancreatic tumor model (HapT1) infused with tumor-infiltrating lymphocytes (TILs). Using the oncolytic virus instead of lymphodepletion resulted in superior efficacy and survival. Immune cells responsive to TNF-α IL-2 were studied using an immunocompetent mouse melanoma model (B16.OVA) infused with ovalbumin-specific T (OT-I) cells. Here, the adenovirus approach improved tumor control together with increased intratumoral Th1 cytokine levels and infiltration of CD8+ T cells and CD86+ dendritic cells. Similar to humans, lymphodepleting preconditioning caused severe cytopenias, systemic inflammation, and damage to vital organs. Toxicity was minimal in adenovirus- and OT-I-treated mice. These findings demonstrate that ACT can be effectively facilitated by cytokine-coding adenovirus without requiring lymphodepletion, a rationale being clinically investigated.

TIL 2.0: More effective and predictive T-cell products by enrichment for defined antigen specificities.

Adoptive transfer of in vitro-expanded T cells derived from tumor-infiltrating lymphocytes (TILs) in melanoma patients started the era of tumor immunotherapy three decades ago. The approach has demonstrated remarkable clinical responses in several studies since. Reinfusion of TIL-derived T cells represents a highly personalized form of immunotherapy, taking into account the enormous interindividual tumor heterogeneity. However, despite its successes, TIL therapy does not lead to objective clinical responses in all cases. It is thus crucial to find out which tumor antigens are particularly valuable targets and to develop strategies to enhance the reactivity of T-cell products toward them. In this issue of the European Journal of Immunology, Kelderman et al. [Eur. J. Immunol. 2016. 46: 1351-1360] present a platform for the generation of antigen-specific TIL therapy. Combining recently developed technologies for clinical identification and enrichment of antigen-specific CD8(+) T cells, such as MHC Streptamers and UV-mediated peptide exchange, the authors could enrich T-cell populations with defined antigen specificities from melanoma-derived TILs. This T-cell product showed higher reactivity against autologous tumor cell lines than bulk TIL-derived T cells. The novel platform might enable the generation of more effective and predictable TIL-derived T-cell products for future clinical applications.

Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy.

Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma-associated epitopes. Because of this, the development of a technology to create T-cell products that are enriched for reactivity against defined melanoma-associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer-based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high-frequency (≥1%) and low-frequency (<1%) tumor-specific CD8(+) T-cell populations, and thereby created T-cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen-specific T-cell populations can be used to create defined T-cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy.

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.

A combination of protein phosphatase 2A inhibition and checkpoint immunotherapy: a perfect storm.

Immune checkpoint blockade has emerged as a potent new tool in the war on cancer. However, only a subset of cancer patients benefit from this therapeutic modality, sparking a search for combination therapies to increase the fraction of responding patients. We argue here that inhibition of protein phosphatase 2A (PP2A) is a promising approach to increase responses to immune checkpoint blockade and other therapies that rely on the presence of tumor-reactive T cells. Inhibition of PP2A increases neoantigen expression on tumor cells, activates the cGAS/STING pathway, suppresses regulatory T cells, and increases cytotoxic T cell activation. In preclinical models, inhibition of PP2A synergizes with immune checkpoint blockade and emerging evidence indicates that patients who have tumors with mutations in PP2A respond better to immune checkpoint blockade. Therefore, inhibition of PP2A activity may be an effective way to sensitize cancer cells to immune checkpoint blockade and cell-based therapies using tumor-reactive T cells.

Advancements in tumor-infiltrating lymphocytes: Historical insights, contemporary milestones, and future directions in oncology therapy.

Tumor-infiltrating lymphocytes (TILs) are a subtype of immune cells that infiltrate and accumulate within tumors. Studies proved that TILs can be used as prognostic and predictive markers for cancer patients' responses to immunotherapy. This review explores the modern knowledge of TILs, the challenges and opportunities for utilizing TILs in cancer treatment, such as the rise of therapies under TIL circumstances, the identification of biomarkers for TIL activity, and methods used to isolate and expand TILs for therapeutic use. Ongoing clinical trials and promising results in different cancer types are highlighted, including melanoma, ovarian, and colorectal cancer. This also focuses on ongoing efforts to improve TIL-based therapies by identifying the specific subsets of TILs that are most effective in treating cancer and developing methods to increase the functionality and persistence of TILs in the tumor microenvironment. The article recapitulates the present state TILs therapy, ongoing research, and improvements to its potency.

Tertiary lymphoid structure-related immune infiltrates in NSCLC tumor lesions correlate with low tumor-reactivity of TIL products.

Adoptive transfer of tumor infiltrating lymphocytes (TIL therapy) has proven highly effective for treating solid cancers, including non-small cell lung cancer (NSCLC). However, not all patients benefit from this therapy for yet unknown reasons. Defining markers that correlate with high tumor-reactivity of the autologous TIL products is thus key for achieving better tailored immunotherapies. We questioned whether the composition of immune cell infiltrates correlated with the tumor-reactivity of expanded TIL products. Unbiased flow cytometry analysis of immune cell infiltrates of 26 early-stage and 20 late-stage NSCLC tumor lesions was used for correlations with the T cell differentiation and activation status, and with the expansion rate and anti-tumor response of generated TIL products. The composition of tumor immune infiltrates was highly variable between patients. Spearman's Rank Correlation revealed that high B cell infiltration negatively correlated with the tumor-reactivity of the patient's expanded TIL products, as defined by cytokine production upon exposure to autologous tumor digest. In-depth analysis revealed that tumor lesions with high B cell infiltrates contained tertiary lymphoid structure (TLS)-related immune infiltrates, including BCL6+ antibody-secreting B cells, IgD+BCL6+ B cells and CXCR5+BLC6+ CD4+ T cells, and higher percentages of naïve CD8+ T cells. In conclusion, the composition of immune cell infiltrates in NSCLC tumors associates with the functionality of the expanded TIL product. Our findings may thus help improve patient selection for TIL therapy.

Adoptive T cell therapy for solid tumors: current landscape and future challenges.

Adoptive cell therapy (ACT) comprises different strategies to enhance the activity of T lymphocytes and other effector cells that orchestrate the antitumor immune response, including chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR) gene-modified T cells, and therapy with tumor-infiltrating lymphocytes (TILs). The outstanding results of CAR-T cells in some hematologic malignancies have launched the investigation of ACT in patients with refractory solid malignancies. However, certain characteristics of solid tumors, such as their antigenic heterogeneity and immunosuppressive microenvironment, hamper the efficacy of antigen-targeted treatments. Other ACT modalities, such as TIL therapy, have emerged as promising new strategies. TIL therapy has shown safety and promising activity in certain immunogenic cancers, mainly advanced melanoma, with an exciting rationale for its combination with immune checkpoint inhibitors. However, the implementation of TIL therapy in clinical practice is hindered by several biological, logistic, and economic challenges. In this review, we aim to summarize the current knowledge, available clinical results, and potential areas of future research regarding the use of T cell therapy in patients with solid tumors.

Targeting tumor microenvironment using tumor-infiltrating lymphocytes as therapeutics against tumorigenesis.

The immune system plays a vital role in suppressing tumor cell progression. The tumor microenvironment augmented with significant levels of tumor-infiltrating lymphocytes has been widely investigated and it is suggested that tumor-infiltrating lymphocytes have shown a significant role in the prognosis of cancer patients. Compared to ordinary non-infiltrating lymphocytes, tumor-infiltrating lymphocytes (TILs) are a significant population of lymphocytes that infiltrate tumor tissue and have a higher level of specific immunological reactivity against tumor cells. They serve as an effective immunological defense against various malignancies. TILs are a diverse group of immune cells that are divided into immune subsets based on the pathological and physiological impact they have on the immune system. TILs mainly consist of B-cells, T-cells, or natural killer cells with diverse phenotypic and functional properties. TILs are known to be superior to other immune cells in that they can recognize a wide range of heterogeneous tumor antigens by producing many clones of T cell receptors (TCRs), outperforming treatments like TCR-T cell and CAR-T therapy. With the introduction of genetic engineering technologies, tumor-infiltrating lymphocytes (TILs) have become a ground-breaking therapeutic option for malignancies, but because of the hindrances opposed by the immune microenvironment and the mutation of antigens, the development of TILs as therapeutic has been hindered. By giving some insight into the many variables, such as the various barriers inhibiting its usage as a potential therapeutic agent, we have examined various aspects of TILs in this work.

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.

Tumor-infiltrating lymphocytes for treatment of solid tumors: It takes two to tango?

Tumor-infiltrating lymphocytes (TILs), frontline soldiers of the adaptive immune system, are recruited into the tumor site to fight against tumors. However, their small number and reduced activity limit their ability to overcome the tumor. Enhancement of TILs number and activity against tumors has been of interest for a long time. A lack of knowledge about the tumor microenvironment (TME) has limited success in primary TIL therapies. Although the advent of engineered T cells has revolutionized the immunotherapy methods of hematologic cancers, the heterogeneity of solid tumors warrants the application of TILs with a wide range of specificity. Recent advances in understanding TME, immune exhaustion, and immune checkpoints have paved the way for TIL therapy regimens. Nowadays, TIL therapy has regained attention as a safe personalized immunotherapy, and currently, several clinical trials are evaluating the efficacy of TIL therapy in patients who have failed conventional immunotherapies. Gaining favorable outcomes following TIL therapy of patients with metastatic melanoma, cervical cancer, ovarian cancer, and breast cancer has raised hope in patients with refractory solid tumors, too. Nevertheless, TIL therapy procedures face several challenges, such as high cost, timely expansion, and technical challenges in selecting and activating the cells. Herein, we reviewed the recent advances in the TIL therapy of solid tumors and discussed the challenges and perspectives.

Effective generation of tumor-infiltrating lymphocyte products from metastatic non-small-cell lung cancer (NSCLC) lesions irrespective of location and previous treatments.

Background: Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Because current treatment regimens show limited success rates, alternative therapeutic approaches are needed. We recently showed that treatment-naïve, stage I/II primary NSCLC tumors contain a high percentage of tumor-reactive T cells, and that these tumor-reactive T cells can be effectively expanded and used for the generation of autologous tumor-infiltrating T cell (TIL) therapy. Whether these promising findings also hold true for metastatic lesions is unknown yet critical for translation into the clinic. Materials and methods: We studied the lymphocyte composition using flow cytometry from 27 metastatic NSCLC lesions obtained from different locations and from patients with different histories of treatment regimens. We determined the expansion capacity of TILs with the clinically approved protocol, and measured their capacity to produce the key pro-inflammatory cytokines interferon-γ, tumor necrosis factor and interleukin 2 and to express CD137 upon co-culture of expanded TILs with the autologous tumor digest. Results: The overall number and composition of lymphocyte infiltrates from the various metastatic lesions was by and large comparable to that of early-stage primary NSCLC tumors. We effectively expanded TILs from all metastatic NSCLC lesions to numbers that were compatible with TIL transfusion, irrespective of the location of the metastasis and of the previous treatment. Importantly, 16 of 21 (76%) tested TIL products displayed antitumoral activity, and several contained polyfunctional T cells. Conclusions: Metastatic NSCLC lesions constitute a viable source for the generation of tumor-reactive TIL products for therapeutic purposes irrespective of their location and the pre-treatment regimens.

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.