LUE Développement de cellules T anti-virus modifiées par un récepteur d'antigène chimérique anti-GD2 : migration et persistance dans un modèle allogénique de tumeur solide

école doctorale

équipe

Equipe 6 : Ingénierie Cellulaire, Immunothérapie Cellulaire et Approches Translationnelles

contexte

Chimeric antigen receptor T cells, known as CAR-T cells, have generated extraordinary results in phase I/II clinical trials in the treatment of CD19+ B-cell hematological malignancies. The principle is based on the genetic modification of the patient's immune T cells by transferring a transgene coding for a chimeric receptor. This receptor recognizes antigens present on the surface of targeted tumor cells, regardless of any major histocompatibility complex (MHC) restriction, leading to their destruction. CARs are usually composed of an extracellular domain—a single-chain variable fragment (scFv) of a monoclonal antibody implicated in the recognition of the target cell antigen—and an intracellular domain responsible for the activation and the function of T cells [1]. Different generations of CAR-T cells have been raised, depending on the composition of the intra-cellular domain: (i) first generation, CD3z chain [2]; (ii) second generation, CD3z chain and a costimulation domain such as CD28, 4-1BB or OX40 [3,4]; and (iii) third generation, CD3zchain and two co-stimulation domains. A fourth generation called TRUCKS (T cells Redirected for antigen-Unrestricted Cytokine-initiated Killing) has been recently developed, where the transgene coding for a second-generation CAR-T is completed with a gene coding for a cytokine such as IL-12 or IL-15, for example [5]. Clinical trials for CD19 CAR-T cells have shown a high level of complete or partial remissions in patients with poor prognoses. For example, the results from the ELIANA and ENSIGN studies [6] showed a complete remission of 67% at 3 months in patients with acute lymphoblastic leukemia (ALL), which was maintained in almost 40% of patients after a median follow-up of 9 months. Nevertheless, despite these results and an effective management of adverse effects, more than half of patients will experience a relapse. In fact, according to data from follow-up studies, between 30-50% of patients who have been in remission are found to relapse within one year of the infusion of anti-CD19 CAR-T cells [7]. Different reasons were highlighted: the absence of persistence of CAR T cells, the absence or loss by the tumor cells of the antigen targeted by CAR T cells. Moreover, it is still difficult to transpose this therapy to solid tumors. This is due to the tumor microenvironment (TME) and involves numerous barriers of physical (access to the tumor), immunological (immunosuppressive environment induced by the tumor) and tumor targeting specificities, with an “off target” effect observed on healthy tissues. The development of this therapy in solid cancers is a major challenge for academic research teams and the pharmaceutical industry. To address these drawbacks, different groups, including ours, investigated different strategies to improve CAR-T cell efficiency and persistence. The first strategy is the engineering of the CAR construct: as an example, we can mention the work performed by Dr Loïc Reppel during a mobility in the Department of Immunology and Microbiology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina (USA). He optimized the GD2-CAR-T cells by incorporating IL-15 gene in the construct to promote CAR-T cell expansion and persistence. They studied the antitumor activity in vitro and in vivo in a solid tumor model of lung cancers [8]. Although effective, the transgenic expression of cytokines can be associated with systemic toxicities [9], and genetically modified T cells might not express cognate cytokine receptors as they undergo activation and differentiation. Although this cytokine expression brings improvement in CAR T cells efficacy, it does not seem enough to modulate the TME. The second strategy relies on the raw cellular material used for transduction: instead of using T cells or mononuclear cells (PBMCs), it seems of interest to focus on a subpopulation of T cells known to present a low alloreactivity with a native TcR that can be challenged in vivo to maintain proliferation, efficacy and persistence of CAR T cells [10]. The idea of preparing CAR T cells from a defined population of T lymphocytes with anti-viral specificity (VSTs) could make it possible to maintain the expansion of CAR T cells, while controlling viral infections, given the frequent viral reactivations in these patients. This dual-potential drug would exert an anti-viral effect related to VSTs to control the infection and promote the in vivo expansion and persistence of CAR-VSTs thanks to a viral rechallenge, and finally an anti-tumor effect related to the CAR. Considering the experience of our team 6 of IMoPA and of the Nancy university hospital cell therapy department (UTCT) on VSTs in the treatment of viral infections in immunocompromised patients [11, 12], we decided to investigate, during the PhD of Valentine Wang, the generation of CD19 CAR-VSTs, as a proof of concept, using VSTs isolated by an immunomagnetic method based on IFN secretion after viral antigen stimulation. We optimized the protocol to generate those CD19 CAR-VSTs and could observe a high anti-tumoral and anti-viral dual effect. During the coming months of the PhD of Valentine Wang, we will check, both in vitro and in vivo, that CAR-VSTs are able to reinitiate an anti-tumor effect after a viral antigen restimulation. Moreover, during her second year of PhD, Valentine Wang, in the laboratory of G Dotti, implemented gene editing using CRISPR/Cas9 technology to disrupt the expression of molecules involved in graft rejection. In the end, while combining CAR-VSTs including the knock-out of HLA class I molecules, the PhD of Valentine Wang aims at generating allogeneic CAR cells. At that time, good results have been obtained on the two parts of the subject and efforts have to be maintained to combine the two strategies to develop allogeneic CD19-CAR-VST-HLA cl I KO. The aim of this project is to transpose this CAR-VST strategy to a solid tumor model, previously explored by Loïc Reppel during his post-doctorat. We aim at generating GD2-CAR-VST using a CAR that will be improved compared to the one that has been used in the work of Loïc Reppel [8]. As mentioned before, one of the most important challenge in solid tumor is the TME impairing CAR T cells to reach the tumor and to maintain their capacity to kill tumor cells. Incorporation of the IL-15 cytokine within the CAR construct produces an enrichment of cells with ‘memory‘ and ‘stem-cell' like phenotypes, and provides enhanced antigen-independent expansion and persistence in tumor sites and improved antitumor activity. However, as previously mentioned, as the transgenic expression of soluble cytokines is sometimes associated with systemic toxicities, the strategy needs to be improved. Moreover, CAR T cells efficacy is dependent on a reduction of TME blockade. In collaboration with Pr G Dotti, we propose to generate a CAR construct containing a gene coding for a membrane-bound form of IL12. This construct has been developed by Pr G Dotti's lab and it was shown that engineering a membrane-bound form of IL12 enhanced CAR T-cell and anti-tumor activity.

spécialité

Sciences de la Vie et de la Santé - BioSE

laboratoire

IMoPA - Ingénierie Moléculaire et Physiopathologie Articulaire