Development of NK cell-based cancer immunotherapies through receptor engineering.

Natural killer (NK) cell-based immunotherapies are attracting increasing interest in the field of cancer treatment. Early clinical trials have shown promising outcomes, alongside satisfactory product efficacy and safety. Recent developments have greatly increased the therapeutic potential of NK cells by endowing them with enhanced recognition and cytotoxic capacities. This review focuses on surface receptor engineering in NK cell therapy and discusses its impact, challenges, and future directions.Most approaches are based on engineering with chimeric antigen receptors to allow NK cells to target specific tumor antigens independent of human leukocyte antigen restriction. This approach has increased the precision and potency of NK-mediated recognition and elimination of cancer cells. In addition, engineering NK cells with T-cell receptors also mediates the recognition of intracellular epitopes, which broadens the range of target peptides. Indirect tumor peptide recognition by NK cells has also been improved by optimizing immunoglobulin constant fragment receptor expression and signaling. Indeed, engineered NK cells have an improved ability to recognize and destroy target cells coated with specific antibodies, thereby increasing their antibody-dependent cellular cytotoxicity. The ability of NK cell receptor engineering to promote the expansion, persistence, and infiltration of transferred cells in the tumor microenvironment has also been explored. Receptor-based strategies for sustained NK cell functionality within the tumor environment have also been discussed, and these strategies providing perspectives to counteract tumor-induced immunosuppression.Overall, receptor engineering has led to significant advances in NK cell-based cancer immunotherapies. As technical challenges are addressed, these innovative treatments will likely reshape cancer immunotherapy.

Netrin-1 blockade inhibits tumor associated Myeloid-derived suppressor cells, cancer stemness and alleviates resistance to chemotherapy and immune checkpoint inhibitor.

Drug resistance and cancer relapse represent significant therapeutic challenges after chemotherapy or immunotherapy, and a major limiting factor for long-term cancer survival. Netrin-1 was initially identified as a neuronal navigation cue but has more recently emerged as an interesting target for cancer therapy, which is currently clinically investigated. We show here that netrin-1 is an independent prognostic marker for clinical progression of breast and ovary cancers. Cancer stem cells (CSCs)/Tumor initiating cells (TICs) are hypothesized to be involved in clinical progression, tumor relapse and resistance. We found a significant correlation between netrin-1 expression and cancer stem cell (CSC) markers levels. We also show in different mice models of resistance to chemotherapies that netrin-1 interference using a therapeutic netrin-1 blocking antibody alleviates resistance to chemotherapy and triggers an efficient delay in tumor relapse and this effect is associated with CSCs loss. We also demonstrate that netrin-1 interference limits tumor resistance to immune checkpoint inhibitor and provide evidence linking this enhanced anti-tumor efficacy to a decreased recruitment of a subtype of myeloid-derived suppressor cells (MDSCs) called polymorphonuclear (PMN)-MDSCs. We have functionally demonstrated that these immune cells promote CSCs features and, consequently, resistance to anti-cancer treatments. Together, these data support the view of both a direct and indirect contribution of netrin-1 to cancer stemness and we propose that this may lead to therapeutic opportunities by combining conventional chemotherapies and immunotherapies with netrin-1 interfering drugs.

TCR-engineered T cell therapy in solid tumors: State of the art and perspectives.

T cell engineering has changed the landscape of cancer immunotherapy. Chimeric antigen receptor T cells have demonstrated a remarkable efficacy in the treatment of B cell malignancies in hematology. However, their clinical impact on solid tumors has been modest so far. T cells expressing an engineered T cell receptor (TCR-T cells) represent a promising therapeutic alternative. The target repertoire is not limited to membrane proteins, and intrinsic features of TCRs such as high antigen sensitivity and near-to-physiological signaling may improve tumor cell detection and killing while improving T cell persistence. In this review, we present the clinical results obtained with TCR-T cells targeting different tumor antigen families. We detail the different methods that have been developed to identify and optimize a TCR candidate. We also discuss the challenges of TCR-T cell therapies, including toxicity assessment and resistance mechanisms. Last, we share some perspectives and highlight future directions in the field.

Radiotherapy as a means to increase the efficacy of T-cell therapy in solid tumors.

Chimeric antigen receptor (CAR)-T cells have demonstrated significant improvements in the treatment of refractory B-cell malignancies that previously showed limited survival. In contrast, early-phase clinical studies targeting solid tumors have been disappointing. This may be due to both a lack of specific and homogeneously expressed targets at the surface of tumor cells, as well as intrinsic properties of the solid tumor microenvironment that limit homing and activation of adoptive T cells. Faced with these antagonistic conditions, radiotherapy (RT) has the potential to change the overall tumor landscape, from depleting tumor cells to reshaping the tumor microenvironment. In this article, we describe the current landscape and discuss how RT may play a pivotal role for enhancing the efficacy of adoptive T-cell therapies in solid tumors. Indeed, by improving homing, expansion and activation of infused T cells while reducing tumor volume and heterogeneity, the use of RT could help the implementation of engineered T cells in the treatment of solid tumors.

Chimeric antigen receptor T-cells targeting IL-1RAP: a promising new cellular immunotherapy to treat acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) remains a very difficult disease to cure due to the persistence of leukemic stem cells (LSCs), which are resistant to different lines of chemotherapy and are the basis of refractory/relapsed (R/R) disease in 80% of patients with AML not receiving allogeneic transplantation. METHODS: In this study, we showed that the interleukin-1 receptor accessory protein (IL-1RAP) protein is overexpressed on the cell surface of LSCs in all subtypes of AML and confirmed it as an interesting and promising target in AML compared with the most common potential AML targets, since it is not expressed by the normal hematopoietic stem cell. After establishing the proof of concept for the efficacy of chimeric antigen receptor (CAR) T-cells targeting IL-1RAP in chronic myeloid leukemia, we hypothesized that third-generation IL-1RAP CAR T-cells could eliminate AML LSCs, where the medical need is not covered. RESULTS: We first demonstrated that IL-1RAP CAR T-cells can be produced from AML T-cells at the time of diagnosis and at relapse. In vitro and in vivo, we showed the effectiveness of IL-1RAP CAR T-cells against AML cell lines expressing different levels of IL-1RAP and the cytotoxicity of autologous IL-1RAP CAR T-cells against primary cells from patients with AML at diagnosis or at relapse. In patient-derived relapsed AML xenograft models, we confirmed that IL-1RAP CAR T-cells are able to circulate in peripheral blood and to migrate in the bone marrow and spleen, are cytotoxic against primary AML cells and increased overall survival. CONCLUSION: In conclusion, our preclinical results suggest that IL-1RAP CAR T-based adoptive therapy could be a promising strategy in AML treatment and it warrants the clinical investigation of this CAR T-cell therapy.

HERVs characterize normal and leukemia stem cells and represent a source of shared epitopes for cancer immunotherapy.

Human endogenous retroviruses (HERVs) represent 8% of the human genome. The expression of HERVs and their immune impact have not been extensively studied in Acute Myeloid Leukemia (AML). In this study, we used a reference of 14 968 HERV functional units to provide a thorough analysis of HERV expression in normal and AML bone marrow cells. We show that the HERV retrotranscriptome accurately characterizes normal and leukemic cell subpopulations, including leukemia stem cells, in line with different epigenetic profiles. We then show that HERV expression delineates AML subtypes with different prognoses. We finally propose a method to select and prioritize CD8+ T cell epitopes derived from AML-specific HERVs and we show that lymphocytes infiltrating patient bone marrow at diagnosis contain naturally occurring CD8+ T cells against these HERV epitopes. We also provide in vitro data supporting the functionality of HERV-specific CD8+ T-cells against AML cells. These results show that HERVs represent an important source of genetic information that can help enhancing disease stratification or biomarker identification and an important reservoir of alternative tumor-specific T cell epitopes relevant for cancer immunotherapy.

Tumour burden and antigen-specific T cell magnitude represent major parameters for clinical response to cancer vaccine and TCR-engineered T cell therapy.

BACKGROUND: Cancer vaccines and T-cell receptor (TCR) engineered T cells (Tg-T cell) represent two different therapeutic strategies that can target the same tumour epitopes. The first approach requires the induction of a specific immune response in patients, while the second relies on the efficacy of adoptively transferred T cells. Because the ratio of antigen-specific T cells to tumour cells engaged by these strategies may influence the clinical outcome, we evaluated the efficacy of these two therapeutic approaches in solid tumours according to the tumour burden. METHODS: We performed a meta-analysis restricted to the therapeutic vaccine and Tg-T cell trials, presenting annotated individual clinical data. We adapted a previously published mathematical model for tumour immune dynamics to estimate the clinical impact of the number of specific T cells in regard to the tumour burden. RESULTS: A focused analysis of Tg-T cell studies revealed that clinical responses were mostly observed with the highest doses of infused T cells, suggesting that exceeding a threshold of effector T cells may be required for clinical efficacy. In silico modelling of cancer vaccine and Tg-T cell therapies starting at different tumour burdens showed that therapeutic vaccines control low or moderate tumour burdens, whereas increasing the amount of infused Tg-T cells succeeds in controlling high tumour masses. CONCLUSION: We propose that therapeutic vaccines should be considered in the context of low or moderate tumour burden, whereas Tg-T cell strategies may be more adapted for the treatment of advanced metastatic diseases.

Identification of shared tumor epitopes from endogenous retroviruses inducing high-avidity cytotoxic T cells for cancer immunotherapy.

Human endogenous retroviruses (HERVs) represent 8% of the human genome. HERV products may represent tumor antigens relevant for cancer immunotherapy. We developed a bioinformatic approach to identify shared CD8+ T cell epitopes derived from cancer-associated HERVs in solid tumors. Six candidates among the most commonly shared HLA-A2 epitopes with evidence of translation were selected for immunological evaluation. In vitro priming assays confirmed the immunogenicity of these epitopes, which induced high-avidity CD8+ T cell clones. These T cells specifically recognize and kill HLA-A2+ tumor cells presenting HERV epitopes on HLA molecules, as demonstrated by mass spectrometry. Furthermore, epitope-specific CD8+ T cells were identified by dextramer staining among tumor-infiltrating lymphocytes from HLA-A2+ patients with breast cancer. Last, we showed that HERV-specific T cells lyse patient-derived organoids. These shared virus-like epitopes are of major interest for the development of cancer vaccines or T cell-based immunotherapies, especially in tumors with low/intermediate mutational burden.

[Development of allogeneic CAR T-cells]. / Développement des CAR-T allogéniques.

Chimeric antigen receptor (CAR) T-cell therapy represents a major breakthrough in the field of hematology. "Off-the-shelf" allogeneic CAR T-cells from donors have many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches, possible standardization of the cell product, time for multiple cell modifications, redosing and decreased cost. However, allogeneic T-cells possess foreign immunological identities that can lead to graft-versus-host disease (GvHD) and their rejection by the host immune system. In this review, we describe the different approaches to produce allogeneic CAR T-cells with limited potential for GvHD and that can persist in the recipient. The preliminary clinical results obtained with the first generation of allogeneic CAR T-cells are presented as well as the perspectives in hematological malignancies and solid tumors.

[T cell-based immunotherapies in solid tumors]. / Approches d'immunothérapie cellulaire T dans les tumeurs solides.

In solid tumors, adoptive T cell therapies based on ex vivo amplification of antitumor T cell are represented by three main complementary approaches : (i) tumor infiltrating lymphocytes (TILs) which are amplified in vitro before reinjection to the patient, (ii) chimeric antigen receptor (CAR) engineered T cells and (iii) T cell receptor (TCR) engineered T cells. Despite encouraging results, some obstacles remain, such as optimal target selection and tumor microenvironment. In this Review, we discuss pros and cons of these different therapeutic strategies that may open new perspectives in the treatment of solid tumors.

[CD4+ T Lymphocytes: major players in antitumor immune response]. / Les lymphocytes T CD4+ jouent un rôle majeur dans la réponse immunitaire antitumorale.

TITLE: Les lymphocytes T CD4+ jouent un rôle majeur dans la réponse immunitaire antitumorale. ABSTRACT: Cette année encore, dans le cadre d'un partenariat avec médecine/sciences, les étudiants de l'unité d'enseignement « Immunologie, virologie et cancer ¼, dirigée par le Dr Julien Marie, au sein du Master Cancer (Université Lyon 1/VetAgroSup), présentent une analyse d'articles scientifiques récents faisant état d'observations innovantes et importantes. Ce travail de M1 a été encadré par des chercheurs immunologistes et virologistes du Centre de Recherche en Cancérologie de Lyon. Le master de cancérologie de Lyon est une formation dite d'excellence, qui accueille chaque année 30 à 40 étudiants en M1 et en M2. Ce master assure aux étudiants de M1 une formation à la cancérologie reposant sur un socle de base commun. En M2, les étudiants peuvent choisir l'une des trois spécialités suivantes : « Recherche en cancérologie ¼, « Technologie haut débit en cancérologie ¼ ou « Innovations thérapeutiques en cancérologie ¼. Créé en 2013, le Master de cancérologie de Lyon repose sur une forte implication des chercheurs et enseignants-chercheurs du laboratoire d'excellence (LabEx DEV2CAN), ainsi que sur un partenariat solide avec des laboratoires académiques situés à Oxford, Bruxelles, Tokyo, Boston, New York, San Diego, etc.

Author Correction: Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Human Endogenous Retroviruses (HERVs): Shaping the Innate Immune Response in Cancers.

Human Endogenous Retroviruses (HERVs) are accounting for 8% of the human genome. These sequences are remnants from ancient germline infections by exogenous retroviruses. After million years of evolution and multiple integrations, HERVs have acquired many damages rendering them defective. At steady state, HERVs are mostly localized in the heterochromatin and silenced by methylation. Multiple conditions have been described to induce their reactivation, including auto-immune diseases and cancers. HERVs re-expression leads to RNA (simple and double-stranded) and DNA production (by reverse transcription), modulating the innate immune response. Some studies also argue for a role of HERVs in shaping the evolution of innate immunity, notably in the development of the interferon response. However, their exact role in the innate immune response, particularly in cancer, remains to be defined. In this review, we see how HERVs could be key-players in mounting an antitumor immune response. After a brief introduction on HERVs characteristics and biology, we review the different mechanisms by which HERVs can interact with the immune system, with a focus on the innate response. We then discuss the potential impact of HERVs expression on the innate immune response in cancer.

Repurposing endogenous immune pathways to tailor and control chimeric antigen receptor T cell functionality.

Endowing chimeric antigen receptor (CAR) T cells with additional potent functionalities holds strong potential for improving their antitumor activity. However, because potency could be deleterious without control, these additional features need to be tightly regulated. Immune pathways offer a wide array of tightly regulated genes that can be repurposed to express potent functionalities in a highly controlled manner. Here, we explore this concept by repurposing TCR, CD25 and PD1, three major players of the T cell activation pathway. We insert the CAR into the TCRα gene (TRACCAR), and IL-12P70 into either IL2Rα or PDCD1 genes. This process results in transient, antigen concentration-dependent IL-12P70 secretion, increases TRACCAR T cell cytotoxicity and extends survival of tumor-bearing mice. This gene network repurposing strategy can be extended to other cellular pathways, thus paving the way for generating smart CAR T cells able to integrate biological inputs and to translate them into therapeutic outputs in a highly regulated manner.

Repurposing rotavirus vaccines for intratumoral immunotherapy can overcome resistance to immune checkpoint blockade.

Although immune checkpoint-targeted therapies are currently revolutionizing cancer care, only a minority of patients develop durable objective responses to anti-PD-1, PD-L1, and CTLA-4 therapy. Therefore, new therapeutic interventions are needed to increase the immunogenicity of tumors and overcome the resistance to these immunotherapies. Oncolytic properties of common viruses can be exploited for the priming of antitumor immunity, and such oncolytic viruses are currently in active clinical development in combination with immune checkpoint-targeted therapies. However, the routine implementation of these therapies is limited by their manufacturing constraints, the risk of exposure of clinical staff, and the ongoing regulations on genetically modified organisms. We sought to determine whether anti-infectious disease vaccines could be used as a commercially available source of immunostimulatory agents for cancer immunotherapy. We found that rotavirus vaccines have both immunostimulatory and oncolytic properties. In vitro, they can directly kill cancer cells with features of immunogenic cell death. In vivo, intratumoral rotavirus therapy has antitumor effects that are dependent on the immune system. In several immunocompetent murine tumor models, intratumoral rotavirus overcomes resistance to and synergizes with immune checkpoint-targeted therapy. Heat- and UV-inactivated rotavirus lost their oncolytic activity but kept their synergy with immune checkpoint-targeted antibodies through the up-regulation of the double-stranded RNA receptor retinoic acid-induced gene 1 (RIG-I). Rotavirus vaccines are clinical-grade products used in pediatric and adult populations. Therefore, in situ immunization strategies with intratumoral-attenuated rotavirus could be implemented quickly in the clinic.

Modulation of chimeric antigen receptor surface expression by a small molecule switch.

BACKGROUND: Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells. Therefore, the development of strategies to control CAR expression at the surface of T-cells and their functions is under intense investigations. RESULTS: Here, we report the development and evaluation of an off-switch directly embedded within a CAR construct (SWIFF-CAR). The incorporation of a self-cleaving degradation moiety controlled by a protease/protease inhibitor pair allowed the ex vivo tight and reversible control of the CAR surface presentation and the subsequent CAR-induced signaling and cytolytic functions of the engineered T-cells using the cell permeable Asunaprevir (ASN) small molecule. CONCLUSIONS: The strategy described in this study could, in principle, be broadly adapted to CAR T-cells development to circumvent some of the possible hurdle of CAR T-cell manufacturing. This system essentially creates a CAR T-cell with an integrated functional rheostat.

Cancer-Associated Fibroblasts Produce Netrin-1 to Control Cancer Cell Plasticity.

Netrin-1 is upregulated in a large fraction of human neoplasms. In multiple animal models, interference with netrin-1 is associated with inhibition of tumor growth and metastasis. Although netrin-1 upregulation was initially described in cancer cells, we report here that in the human colorectal cancer database, the expression of netrin-1 and its receptor UNC5B correlates with a cancer-associated fibroblasts (CAF) signature. Both colon and lung CAF secreted netrin-1 when cocultured with respective cancer cells, and netrin-1 upregulation in CAF was associated with increased cancer cell stemness. Pharmacologic inhibition of netrin-1 with a netrin-1-mAb (Net1-mAb) abrogated the CAF-mediated increase of cancer stemness both in coculture experiments and in mice. Net-1-mAb inhibited intercellular signaling between CAF and cancer cells by modulating CAF-mediated expression of cytokines such as IL6. Together these data demonstrate that netrin-1 is upregulated not only in cancer cells but also in cancer-associated stromal cells. In addition to its direct activity on cancer cells, inhibition of netrin-1 may reduce proneoplastic CAF-cancer cell cross-talk, thus inhibiting cancer plasticity. SIGNIFICANCE: Netrin-1, a navigation cue during embryonic development, is upregulated in cancer-associated fibroblasts and regulates cancer cell stemness.

Granulocyte-macrophage colony-stimulating factor inactivation in CAR T-cells prevents monocyte-dependent release of key cytokine release syndrome mediators.

Chimeric antigen receptor T-cell (CAR T-cell) therapy has been shown to be clinically effective for managing a variety of hematological cancers. However, CAR T-cell therapy is associated with multiple adverse effects, including neurotoxicity and cytokine release syndrome (CRS). CRS arises from massive cytokine secretion and can be life-threatening, but it is typically managed with an anti-IL-6Ra mAb or glucocorticoid administration. However, these treatments add to a patient's medication burden and address only the CRS symptoms. Therefore, alternative strategies that can prevent CRS and neurotoxicity associated with CAR T-cell treatment are urgently needed. Here, we explored a therapeutic route aimed at preventing CRS rather than limiting its consequences. Using a cytokine-profiling assay, we show that granulocyte-macrophage colony-stimulating factor (GMCSF) is a key CRS-promoting protein. Through a combination of in vitro experiments and gene-editing technology, we further demonstrate that antibody-mediated neutralization or TALEN-mediated genetic inactivation of GMCSF in CAR T-cells drastically decreases available GMCSF and abolishes macrophage-dependent secretion of CRS biomarkers, including monocyte chemoattractant protein 1 (MCP-1), interleukin (IL) 6, and IL-8. Of note, we also found that the genetic inactivation of GMCSF does not impair the antitumor function or proliferative capacity of CAR T-cells in vitro We conclude that it is possible to prevent CRS by using "all-in-one" GMCSF-knockout CAR T-cells. This approach may eliminate the need for anti-CRS treatment and may improve the overall safety of CAR T-cell therapies for cancer patients.

Cold Tumors: A Therapeutic Challenge for Immunotherapy.

Therapeutic monoclonal antibodies targeting immune checkpoints (ICPs) have changed the treatment landscape of many tumors. However, response rate remains relatively low in most cases. A major factor involved in initial resistance to ICP inhibitors is the lack or paucity of tumor T cell infiltration, characterizing the so-called "cold tumors." In this review, we describe the main mechanisms involved in the absence of T cell infiltration, including lack of tumor antigens, defect in antigen presentation, absence of T cell activation and deficit of homing into the tumor bed. We discuss then the different therapeutic approaches that could turn cold into hot tumors. In this way, specific therapies are proposed according to their mechanism of action. In addition, ''supra-physiological'' therapies, such as T cell recruiting bispecific antibodies and Chimeric Antigen Receptor (CAR) T cells, may be active regardless of the mechanism involved, especially in MHC class I negative tumors. The determination of the main factors implicated in the lack of preexisting tumor T cell infiltration is crucial for the development of adapted algorithms of treatments for cold tumors.