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Tissue resident memory T cells (Trm) are a subset of memory T cells mainly described in inflammation and infection settings. Their location in peripheral tissues, such as lungs, gut, or skin, makes them the earliest T cell population to respond upon antigen recognition or under inflammatory conditions. The study of Trm cells in the field of cancer, and particularly in cancer immunotherapy, has recently gained considerable momentum. Different reports have shown that the vaccination route is critical to promote Trm generation in preclinical cancer models. Cancer vaccines administered directly at the mucosa, frequently result in enhanced Trm formation in mucosal cancers compared to vaccinations via intramuscular or subcutaneous routes. Moreover, the intratumoral presence of T cells expressing the integrin CD103 has been reported to strongly correlate with a favorable prognosis for cancer patients. In spite of recent progress, the full spectrum of Trm anti-tumoral functions still needs to be fully established, particularly in cancer patients, in different clinical contexts. In this mini-review we focus on the recent vaccination strategies aimed at generating Trm cells, as well as evidence supporting their association with patient survival in different cancer types. We believe that collectively, this information provides a strong rationale to target Trm for cancer immunotherapy.

A total of 12 pediatric patients with diffuse intrinsic pontine glioma were treated with a direct infusion of an oncolytic virus, followed by radiotherapy. The tumor size was reduced in 9 patients, and disease was stable over a median follow-up of 18 months in 8 patients.

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate large amounts of tumor-specific CD8 T cells with high potential to persist . PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in and lack and expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39- Tim3- Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39- Tim3- Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and persistence of transferred cells in the context of ACT.

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions. Significance Immunotherapy of cancer with immunomodulatory agents is achieving significant efficacy in an important fraction of patients. The stimulatory inducible receptor of T and NK lymphocytes known as CD137 or 4-1BB is being stimulated with agonist antibodies to enhance antitumor immunity in clinical trials. In addition, the intracellular signaling domain of CD137 is crucial as a component of successful anti-leukemia therapies with chimeric antigen receptors transduced into adoptively transferred T lymphocytes. In this study the marked synergistic effects of adoptive T cell and agonist anti-CD137 mAb therapies are studied, providing in vivo evidence for improved, more sustained and focused tumoricidal functions of antitumor cytotoxic T lymphocytes when under the influence of CD137-targeted pharmacological stimulation with immunostimulatory monoclonal antibodies.

BackgroundGlioblastoma (GBM) is a devastating primary brain tumor with a highly immunosuppressive tumor microenvironment, and treatment with oncolytic viruses (OVs) has emerged as a promising strategy for these tumors. Our group constructed a new OV named Delta-24-ACT, which was based on the Delta-24-RGD platform armed with 4-1BB ligand (4-1BBL). In this study, we evaluated the antitumor effect of Delta-24-ACT alone or in combination with an immune checkpoint inhibitor (ICI) in preclinical models of glioma.MethodsThe in vitro effect of Delta-24-ACT was characterized through analyses of its infectivity, replication and cytotoxicity by flow cytometry, immunofluorescence (IF) and MTS assays, respectively. The antitumor effect and therapeutic mechanism were evaluated in vivo using several immunocompetent murine glioma models. The tumor microenvironment was studied by flow cytometry, immunohistochemistry and IF.ResultsDelta-24-ACT was able to infect and exert a cytotoxic effect on murine and human glioma cell lines. Moreover, Delta-24-ACT expressed functional 4-1BBL that was able to costimulate T lymphocytes in vitro and in vivo. Delta-24-ACT elicited a more potent antitumor effect in GBM murine models than Delta-24-RGD, as demonstrated by significant increases in median survival and the percentage of long-term survivors. Furthermore, Delta-24-ACT modulated the tumor microenvironment, which led to lymphocyte infiltration and alteration of their immune phenotype, as characterized by increases in the expression of Programmed Death 1 (PD-1) on T cells and Programmed Death-ligand 1 (PD-L1) on different myeloid cell populations. Because Delta-24-ACT did not induce an immune memory response in long-term survivors, as indicated by rechallenge experiments, we combined Delta-24-ACT with an anti-PD-L1 antibody. In GL261 tumor-bearing mice, this combination showed superior efficacy compared with either monotherapy. Specifically, this combination not only increased the median survival but also generated immune memory, which allowed long-term survival and thus tumor rejection on rechallenge.ConclusionsIn summary, our data demonstrated the efficacy of Delta-24-ACT combined with a PD-L1 inhibitor in murine glioma models. Moreover, the data underscore the potential to combine local immunovirotherapy with ICIs as an effective therapy for poorly infiltrated tumors.

Pediatric diffuse midline gliomas (DMG) with altered H3-K27M are aggressive brain tumors that arise during childhood. Despite advances in genomic knowledge and the significant number of clinical trials testing new targeted therapies, patient outcomes are still poor. Immune checkpoint blockades with small molecules, such as aptamers, are opening new therapeutic options that represent hope for this orphan disease. Here, we demonstrated that a TIM-3 aptamer (TIM-3 Apt) as monotherapy increased the immune infiltration and elicited a strong specific immune response with a tendency to improve the overall survival of treated DMG-bearing mice. Importantly, combining TIM-3 Apt with radiotherapy increased the overall median survival and led to long-term survivor mice in 2 pediatric DMG orthotopic murine models. Interestingly, TIM-3 Apt administration increased the number of myeloid populations and the proinflammatory CD8-to-Tregs ratios in the tumor microenvironment as compared with nontreated groups after radiotherapy. Importantly, the depletion of T cells led to a major loss of the therapeutic effect achieved by the combination. This work uncovers TIM-3 targeting as an immunotherapy approach to improve the radiotherapy outcome in DMGs and offers a strong foundation for propelling a phase I clinical trial using radiotherapy and TIM-3 blockade combination as a treatment for these tumors.

BackgroundMore efficient therapeutic options for non-small cell lung cancer (NSCLC) are needed as the survival at 5 years of metastatic disease is near zero. In this regard, we used a preclinical model of metastatic lung adenocarcinoma (SV2-OVA) to assess the safety and efficacy of novel radio-immunotherapy combining hypofractionated radiotherapy (HRT) with muPD1-IL2v immunocytokine and muFAP-CD40 bispecific antibody.MethodsWe evaluated the changes in the lung immune microenvironment at multiple timepoints following combination therapies and investigated their underlying antitumor mechanisms. Additionally, we analyzed the tumor clonal heterogeneity upon the combination treatments to explore potential mechanisms associated with the lack of complete response.ResultsThe combination of HRT with muPD1-IL2v had a potent antitumor effect and increased survival in the SV2-OVA lung cancer model. Importantly, this combination therapy was devoid of measurable toxicity. It induced remodeling of the immune contexture through the increase of CD8+ T and natural killer (NK) cells. The addition of muFAP-CD40 to the combination treatment further increased infiltrating CD8+ T cells, expressing high levels of effector molecules, both in the periphery and core tumor regions. An accumulation of CD8+ PD-1+ TOX+ (exhausted) T cells, already at the ‘early’ timepoint, is consistent with the limited clinical benefits provided by the various combination treatments in this model. The study of the clonal dynamics of tumor cells during disease progression and therapy highlighted a clonal selection upon HRT+muPD1-IL2v therapy.ConclusionsWe demonstrated that HRT+muPD1-IL2v combination is a potent therapeutic strategy to delay tumor growth and increase survival in a metastatic lung cancer model, but additional studies are required to completely understand the resistance mechanisms associated with the lack of complete response in this model.

BackgroundOsteosarcoma is an aggressive bone tumor, primarily arising in the pediatric age. Despite years of intensive research, outcome for metastatic and non-responder patients is very poor and has not improved in the last 30 years. These tumors harbor a highly immunosuppressive environment, making the existing immunotherapies ineffective. Inhibition of galectin-3 (Gal3), a protein involved in immunosuppression,1–3 adhesion of tumor cells,4 5 and metastases,6–9 has been demonstrated to reduce tumor progression in different tumor models, including osteosarcoma.10–12 On the other hand, virotherapy based on recombinant Semliki Forest Virus (SFV), a self-replicating RNA virus, has shown therapeutic effect in orthotopic osteosarcoma mouse models.13 MethodsWe generated SFV vectors expressing truncated forms of Gal3, including its carboxy-terminal domain (SFV-Gal3-C) and its amino-terminal domain alone (SFV-Gal3-N) or fused to the Gal3 inhibitor peptide C12 (SFV-Gal3-N-C12). An additional construct expressed the C12 peptide14 15 (SFV-C12). We analyzed Gal3 expression in different murine and human osteosarcoma cell lines. Orthotopic osteosarcoma tumors, induced by intratibial injection of K7M2 murine cells, which showed high expression of Gal3, were treated with SFV vectors expressing Gal3 inhibitors or luciferase or with PBS (control). Animals were maintained under standard conditions, and all procedures were approved by the Institutional Ethical Committee (CEEA) in accordance with the guidelines of the University of Navarra, approval number 044-21.ResultsTreatment with the SFV-Gal3-N-C12 vector showed the highest antitumor activity, significantly reducing tumor growth compared to control mice that received PBS. In fact, this vector prolonged animal survival, leading to 47% of complete regressions. Among the other vectors, SFV-Gal3-N and SFV-C12 were also able to transiently decrease tumor growth, although they had no impact on animal survival. Moreover, the number of spontaneous lung metastasis were reduced in mice treated with SFV vectors expressing Gal3 inhibitors. Preliminary mechanistic studies showed an increase of CD3 cells infiltration in tumors treated with SFV-Gal3-N-C12 and SFV-Gal3-N vectors. Despite the antitumor effect observed with SFV-Gal3-N-C12, no protection against tumor rechallenge was observed in cured mice, indicating the lack or insufficient memory immune response generation. These data suggested that this therapeutic approach might benefit from combination with other immunodulatory strategies. We are currently characterizing the underpinnings of the mechanisms underlying this strategy.ConclusionsIn summary, we believe that inhibition of Gal3 using SFV vectors could constitute a potential approach to explore as therapy for pediatric osteosarcoma.ReferencesFarhad M, Rolig AS, Redmond WL. The role of Galectin-3 in modulating tumor growth and immunosuppression within the tumor microenvironment. Oncoimmunology 2018;7(6):p.e1434467.Ruvolo PP. Galectin 3 as a guardian of the tumor microenvironment. Biochim Biophys Acta 2016;1863(3):427–437.Guo Y, et al. Roles of galectin3 in the tumor microenvironment and tumor metabolism (Review). Oncol Rep 2020;44(5):1799–1809.Cao Z, et al. Endogenous and exogenous galectin-3 promote the adhesion of tumor cells with low expression of MUC1 to HUVECs through upregulation of N-cadherin and CD44. Lab Invest 2018;98(12):1642–1656.Kim SJ, Chun KH. Non-classical role of Galectin-3 in cancer progression: translocation to nucleus by carbohydrate-recognition independent manner. BMB Rep 2020;53(4):173–180.Pereira JX, et al. Galectin-3 regulates the expression of tumor glycosaminoglycans and increases the metastatic potential of breast cancer. J Oncol 2019;2019:9827147.Song M, et al. Galectin-3 favours tumour metastasis via the activation of beta-catenin signalling in hepatocellular carcinoma. Br J Cancer 2020;123(10):1521–1534.Fortuna-Costa A, et al. Extracellular galectin-3 in tumor progression and metastasis. Front Oncol 2014;4:138.Nakajima K, et al. Galectin-3 in bone tumor microenvironment: a beacon for individual skeletal metastasis management. Cancer Metastasis Rev 2016;35(2):333–46.Nakajima K, Balan V, Raz A. Galectin-3: an immune checkpoint target for musculoskeletal tumor patients. Cancer Metastasis Rev 2021;40(1):297–302.Lei P, et al. Small interfering RNA-induced silencing of galectin-3 inhibits the malignant phenotypes of osteosarcoma in vitro. Mol Med Rep 2015;12(4):6316–22.Park GB, et al. Silencing of galectin-3 represses osteosarcoma cell migration and invasion through inhibition of FAK/Src/Lyn activation and beta-catenin expression and increases susceptibility to chemotherapeutic agents. Int J Oncol 2015;46(1):185–94.Ketola A, et al. Oncolytic Semliki forest virus vector as a novel candidate against unresectable osteosarcoma. Cancer Res 2008;68(20):8342–50.Sun W, et al. G3-C12 peptide reverses galectin-3 from Foe to friend for active targeting cancer treatment. Mol Pharm 2015;12(11):4124–36.Sun W, et al. Two birds, one stone: dual targeting of the cancer cell surface and subcellular mitochondria by the galectin-3-binding peptide G3-C12. Acta Pharmacol Sin 2017;38(6):806–822.Ethics ApprovalAnimals were maintained under standard conditions, and all procedures were approved by the Institutional Ethical Committee (CEEA) in accordance with the guidelines of the University of Navarra, approval number 044-21.

BackgroundNovel efficient therapeutic options for lung carcinoma are needed as the survival at 5 years of metastatic disease treated by the standard of care is near zero.MethodsIn this regard, we used a novel preclinical model of lung adenocarcinoma to assess the safety and efficacy of radio-immunotherapy combining local hypofractionated radiotherapy with tumor-stroma targeted FAP-CD40 agonistic bispecific antibody and PD1-IL2v immunocytokine.ResultsIn this model, combination treatments using hypofractionated RT + anti-FAP-CD40 and/or anti-PD1-IL2v mediate tumor growth retardation and extend mouse survival compared with radiotherapy alone or immunotherapy used as a single agent. Importantly, they were devoid of measurable toxicity. The best anti-tumor activity was obtained with the RT + anti-PD1-IL2v and RT + anti-FAP-CD40 + anti-PD1-IL2v combinations. In these treatment groups, survival of mice was extended more than 30 days (60 days post tumor engraftment) compared to the control cohorts. Immuno pharmacodynamic analysis and immunofluorescence data in these groups show that treatment with radiotherapy and anti-PD1-IL2v + anti-FAP-CD40 associates with increases in the endogenous CD8 T cells and transferred OT-1 infiltrating the lungs. The anti-tumor activity of the anti-PD1-IL2v monotherapy required CD8 T cells as suggested by immune cell depletion experiments. Additionally, the SV2 lung adenocarcinoma cell line has been transduced with a library of barcodes to study clonal dynamics of tumor cells during disease progression and therapy.ConclusionsThe results of our preclinical study warrant and inform the translation and clinical testing of these novel immunotherapeutic agents in patients with metastatic lung adenocarcinoma.Ethics ApprovalAll animal procedures were carried out under the license VD3173.1c which was approved by the Veterinary Authority of the Swiss Canton of Vaud.