Immunothérapie du cancer : espoirs et réalités

Au cours des deux dernières décennies, l’immunologie des tumeurs a connu un réel bouleversement et un foisonnement de découvertes fondamentales qui se sont traduites en applications cliniques. On peut notamment citer l’identification des antigènes de rejet tumoral et des fondements moléculaires et cellulaires de l’immunogénicité et de la tolérance, la connaissance fine de l’immunité innée, l’élucidation de la biologie des cellules dendritiques et une certaine compréhension des mécanismes d’échappement tumoral à l’immuno-surveillance. Ainsi, l’émergence de nouveaux concepts a fait de l’immunothérapie une quatrième modalité de traitement du cancer après les traitements conventionnels. Les acquis fondamentaux ont facilité la reconceptualisation de l’immunothérapie du cancer, qui a connu un élan majeur en devenant de plus en plus spécifique : au cours des dernières années, l’immunothérapie non spécifique, fondée sur le dopage non spécifique du système immunitaire du patient, a laissé place aux approches vaccinales antitumorales peptidiques et cellulaires. Malgré des résultats encourageants obtenus à partir des modèles expérimentaux et des résultats, certes modestes, obtenus chez les patients, plusieurs obstacles inhérents persistent, notamment celui du décryptage du conflit entre le système immunitaire et le micro-environnement tumoral.The notion that the immune system regulates cancer development is now well established. An overwhelming amount of data from animal models, together with compelling data from human patients, indicate that the immune system is instrumental in scanning and irradicating tumors. Analysis of individuals with congenital or acquired immunodeficiencies or patients undergoing immunosuppressive therapy has documented a highly elevated incidence of virally induced malignancies and cancers compared with immunocompetent individuals [1-3]. During the last decade, thanks to the breakthoughts in understanding the molecular mechanisms responsible for immune activation, the tumor antigen identification, the dendritic cell biology, the immunogenecity of tumors, the immune escape mechanisms, the host-tumor relationship, we are facing a new area of tumor immunotherapy. The basic advances were translated in therapeutical applications and have changed the view of immunotherapy from "a dream scenario" to a clinical fourth modality to cancer treatments. Multiple cancer trials using active immunization with vaccines or adoptive immunotherapy have been conducted with only very limited success. There are still a number of issues that still need to be resolved including a better understanding of immune escape mechanisms. Cancer vaccines continue to be evaluated and may lead to the emergence of clinically useful new treatments. A comprehensive approach to define the intricate molecular program initiated by tumor cells to resist to escape and the immune system of the host may help in breaking down the barriers to a more adapted cancer immunotherapy

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

International audienceBesides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis

A ras-Mutated Peptide Targeted by CTL Infiltrating a Human Melanoma Lesion 1

International audienceAgs derived from commonly mutated oncogenic proteins seem ideally suited as targets for tumor immunotherapy. Nonetheless, only a few mutated epitopes efficiently presented by human tumors have thus far been identified. We describe here an approach to identify such epitopes. This approach involves: 1) identifying tumors expressing a ras mutation and isolating the tumor-infiltrating lymphocytes (TIL); 2) transfecting COS cells to induce expression of unknown mutated peptides in the context of a patient's HLA class I molecules; and 3) screening epitope recognition by using TIL from the tumors expressing a ras mutation. By using this approach, there appeared to be a N-ras mutation (a glutamine-to-arginine exchange at residue 61 (Q61R)), detected in a melanoma lesion, which was recognized specifically by the autologous TIL in the HLA-A*0101 context. The ras peptide 55-64 Q61R was the epitope of these TIL and was regularly presented by Q61R-mutated HLA-A*0101 ؉ melanoma cell lines. This peptide and its wild-type homolog (55-64 wt) bound to HLA-A*0101 with similar affinities. However, only the mutated peptide could induce specific CTL expansion from PBL. All the CTL clones specific to the mutated peptide, failed to recognize the wild-type sequence on both COS and melanoma cells. These data thus show that oncogenic protein mutations can create shared tumor-specific CTL epitopes, efficiently presented by tumor cells, and that screening for oncogene-transfected COS cell recognition by TIL (from tumors containing mutations) is a powerful approach for the identification of these epitopes

Soluble HLA-I/Peptide Monomers Mediate Antigen-Specific CD8 T Cell Activation through Passive Peptide Exchange with Cell-Bound HLA-I Molecules

International audienceAccumulating evidence that serum levels of soluble class I HLA molecules (sHLA-I) can, under various pathological conditions, correlate with disease stage and/or patient survival, has stimulated interest in defining whether sHLA-I can exert immunological functions. However, despite a mounting number of publications suggesting the ability of sHLA-I to affect immune effectors in vitro, the precise underlying mechanism still remains controversial. In this article, we address potential functions of both classical and nonclassical sHLA-I, using soluble recombinant HLA-I/peptide monomers, and clearly demonstrate their ability to trigger Ag-specific activation of CD8 T cells in vitro. Furthermore, we provide strong evidence that this behavior results from the passive transfer of peptides from monomers to T cell-bound HLA-I molecules, allowing for fratricide representation and activation. Hence, we proposed a unifying model of T cell activation by HLA-I/peptide monomers, reappraising the potential involvement of sHLA-I molecules in the immune response

Infusion of Melan-A/Mart-1 speciWc tumor-inWltrating lymphocytes enhanced relapse-free survival of melanoma patients

International audienceAdoptive therapy of cancer has been mostly tested in advanced cancer patients using tumor-inWl-trating lymphocytes (TIL). Following discouraging results likely due to poor tumor-speciWcity of TIL and/ or high tumor burden, recent studies reiterate the enormous potential of this therapy, particularly in mel-anoma. We had performed a phase II/III randomised trial on 88 stage III melanoma patients, who received autologous TIL plus IL-2 or IL-2 alone, after complete tumour resection. We reported previously clinical and immunological results supporting the ability of tumor reactive TIL infusion to prevent further development of the melanoma disease and to increase overall survival of patients bearing only one tumor invaded lymph node. The absence of correlation between overall and disease-free survival and the amount of infused tumor-speciWc TIL suggested that therapeutic eYciency might depend on other parameters such as antigen speciWcity, function or persistence of TIL. Here we studied the recognition of a panel of 38 shared tumor-associated antigens (TAA) by TIL infused to the patients included in this assay, in order to determine if treatment outcome could correlate with particular antigen speciWcities of infused TIL. Results show that the infusion of Melan-A/MART-1 reactive TIL appears to be associated with a longer relapse-free survival for HLA-A2 patients. These results further support the relevance of Melan-A/MART-1 antigen as a prime target for immunotherapy protocols in melanoma

Identification of Five New HLA-B*3501-Restricted Epitopes Derived from Common Melanoma-Associated Antigens, Spontaneously Recognized by Tumor-Infiltrating Lymphocytes 1

International audienceWe previously described HLA-B35-restricted melanoma tumor-infiltrating lymphocyte responses to frequently expressed mela-noma-associated Ags: tyrosinase, Melan-A/MART-1, gp100, MAGE-A3/MAGE-A6, and NY-ESO-1. Using clones derived from these TIL, we identified in this study the corresponding epitopes. We show that five of these epitopes are new and that melanoma cells naturally present all the six epitopes. Interestingly, five of these epitopes correspond to or encompass melanoma-associated Ag epitopes presented in other HLA contexts, such as A2, A1, B51, and Cw3. In particular, the HLA-B35-restricted Melan-A epitope is mimicked by the peptide 26-35, already known as the most immunodominant melanoma epitope in the HLA-A*0201 context. Because this peptide lacked adequate anchor amino acid residues for efficient binding to HLA-B35, modified peptides were designed. Two of these analogues were found to induce higher PBL-and tumor-infiltrating lymphocyte-specific responses than the parental peptide, suggesting that they could be more immunogenic in HLA-B*3501 melanoma patients. These data have important implications for the formulation of polypeptide-based vaccines as well as for the monitoring of melanoma-specific CTL response in HLA-B*3501 melanoma patients

The antiangiogenic activtivity of IL-12 is increased in iNOS-/- mice and involves NK cells

International audienceWe have previously reported that the in vivo transfer of murine interleukin-12 (IL-12) gene using a Semliki Forest virus vector induced tumor regression through inhibition of tumor blood vessel formation. To examine whether IL-12 anti-angiogenic activity interferes with the NO pathway, we used inducible nitric oxide synthase-deficient mice (iNOS−/−) and demonstrated that the anti-tumor effect of IL-12 is more pronounced in these mice. In addition, despite the increased level of intratumoral VEGF in iNOS−/− mice, IL-12 induced a stronger inhibition of blood vessel formation. Histological analysis of SFV-IL-12-treated tumors showed an increase in natural killer (NK) perivascular infiltration in iNOS−/− as compared to control mice. In vitro IL-12-stimulated murine splenic NK cells displayed significant killing activity towards established murine endothelial cells used as targets. These studies indicate that the anti-angiogenic activity of IL-12 interferes with iNOS pathway and involves NK cell recruitment

Merkel cell carcinoma and cellular cytotoxicity: sensitivity to cellular lysis and screening for potential target antigens suitable for antibodydependent cellular cytotoxicity

The online version of this article ( https://doi.org/10.1007/s00262-018-2176-2) contains supplementary material, which is available to authorized users.International audienceThe recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpointinhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further

Hypoxia-dependent inhibition of tumor cell susceptibility to CTL-mediated lysis involves NANOG induction in target cells.

International audienceHypoxia is a major feature of the solid tumor microenvironment and is known to be associated with tumor progression and poor clinical outcome. Recently, we reported that hypoxia protects human non-small cell lung tumor cells from specific lysis by stabilizing hypoxia-inducible factor-1α and inducing STAT3 phosphorylation. In this study, we show that NANOG, a transcription factor associated with stem cell self renewal, is a new mediator of hypoxia-induced resistance to specific lysis. Our data indicate that under hypoxic conditions, NANOG is induced at both transcriptional and translational levels. Knockdown of the NANOG gene in hypoxic tumor cells is able to significantly attenuate hypoxia-induced tumor resistance to CTL-dependent killing. Such knockdown correlates with an increase of target cell death and an inhibition of hypoxia-induced delay of DNA replication in these cells. Interestingly, NANOG depletion results in inhibition of STAT3 phosphorylation and nuclear translocation. To our knowledge, this study is the first to show that hypoxia-induced NANOG plays a critical role in tumor cell response to hypoxia and promotes tumor cell resistance to Ag-specific lysis

Engineering a Human Plasmacytoid Dendritic Cell-Based Vaccine to Prime and Expand Multispecific Viral and Tumor Antigen-Specific T-Cells.

Because dendritic cells are crucial to prime and expand antigen-specific CD8 T-cells, several strategies are designed to use them in therapeutic vaccines against infectious diseases or cancer. In this context, off-the-shelf allogeneic dendritic cell-based platforms are more attractive than individualized autologous vaccines tailored to each patient. In the present study, a unique dendritic cell line (PDC*line) platform of plasmacytoid origin, already used to prime and expand antitumor immunity in melanoma patients, was improved thanks to retroviral engineering. We demonstrated that the clinical-grade PDC*line, transduced with genes encoding viral or tumoral whole proteins, efficiently processed and stably presented the transduced antigens in different human leukocyte antigen (HLA) class I contexts. Moreover, the use of polyepitope constructs allowed the presentation of immunogenic peptides and the expansion of specific cytotoxic effectors. We also demonstrated that the addition of the Lysosome-associated membrane protein-1 (LAMP-1) sequence greatly improved the presentation of some peptides. Lastly, thanks to transduction of new HLA molecules, the PDC platform can benefit many patients through the easy addition of matched HLA-I molecules. The demonstration of the effective retroviral transduction of PDC*line cells strengthens and broadens the scope of the PDC*line platform, which can be used in adoptive or active immunotherapy for the treatment of infectious diseases or cancer