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Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

Chimeric antigen receptor (CAR) protein are artificial protein created by the fusion of an extracellular domain targeting one or several cell surface antigens, a transmembrane domain and an intracellular domain responsible for signal initiation and transmission. First-generation CARs consist of an intracellular signaling domain, often a cytoplasmic tail of the CD3ζ chain. The second and third generation include the addition of one or two co-stimulation domains (e.g. cytoplasmic domain of CD28 and/or 4-1BB). NKG2D is a receptor present on the cell surface of NK cells, NK T cells and subgroups of CD8+ T cells. The receptor exists as a dimer that interacts with a co-adaptor protein called DAP10. Upon interaction of NKG2D with one of its ligand (MICA, MICB, and ULBP1-ULBP6), the DAP10 cytoplasmic tail will induce downstream signaling. DAP10 has a similar signaling motif to CD28 and similarly gets phosphorylated and recruits kinases. NKG2D ligands are expressed on a vast majority of solid and liquid tumors and are thus a very attractive target for CAR T cells. In this study, multiple NKG2D constructs were created and compared to one another. The first CAR construct tested (chNKG2D.1) consists of a fusion of full-length wild-type NKG2D and the cytoplasmic domain of CD3ζ. This construct is currently tested in clinical settings. This CAR benefits from the co-stimulatory signal provided by DAP10 and can therefore be considered as a second generation CAR. Next to this, a new chNKG2D was constructed by combining DAP10 overexpression (chNKG2D.1.DAP10). Secondly classic second-generation CARs were created by adding CD28 (chNKG2D.2.1) or 4-1BB (chNKG2D.2.2) co-stimulatory domains to the chNKG2D. Co-expression of DAP10 increased chNKG2D expression at the cell surface of both CD4 and CD8 T cells (although the increase was more pronounced in CD4 T cells). Furthermore, co-cultures of T cells expressing this construct with K562 cells led to similar IFN-γ levels irrelevant of the CD4/CD8 ratio. Other CD4 specific cytokines were highly increased when the ratio was in favor of CD4 positive T cells. Interestingly, the addition of the co-stimulatory domains to the construct (chNKG2D.2.1 and .2.2) did not lead to higher levels of IFN-γ or any of the CD4 cytokines. Next to cytokine release, cytolytic activity was also assessed which interestingly showed no difference in any of the conditions even when the CD4/CD8 cell ratio was different. In conclusion NKG2D CAR T cells only showed increased cytokine release when DAP10 was overexpressed, all other conditions did not lead to any changes when compared to the ‘NKG2D CAR T cells currently tested in the clinic. This implies that NKG2D is optimally costimulated through the DAP 10 co-signaling, and that this co-signaling is a least as potent as traditional CD28 or 4-1BB based co stimulation. Citation Format: Jennifer BOLSEE, Eytan BREMAN, Fanny HUBERTY, Benjamin VIOLLE, Jerome MARIJSSE, Céline JACQUES-HESPEL, Celine MARCHAND, Nancy RAMELOT, Thuy NGUYEN, Julien HOUSSA, David E. GILHAM, Sophie AGAUGUE. NKG2D as a chimeric antigen receptor - DAP 10 provides optimal co stimulation for NKG2D based CARs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3583.

e15040Background: The majority of CAR-T cell therapy studies use the patients’ own autologous cells which carry many potential restrictions related to manufacturing such as quantity and quality of ...

e15042Background: Most CAR T cell therapy relies on autologous cells with potential limitation in quality of the cells. Cells derived from an allogeneic healthy donor may be more consistent in qual...

In immunotherapy of cancer, different vaccination strategies have been developed and evaluated in clinical trials, e.g. monoclonal antibodies, injection of DNA, peptides, proteins, autologous cells (tumoral cells, dendritic cells (DC) or T cells) processed in vitro or even direct injection of modified viral vectors.THERAVECTYS is a Paris-based, privately-owned, fully integrated discovery & clinical development biotech company, developping lentiviral vectors for human vaccination and immunotherapy applications. THERAVECTYS’ vaccine candidates based on a proprietary lentiviral vector technology enable a direct vaccination mechanism in patients by inducing a broad, intense and long-lasting T cell mediated response. Theravectys’ technology presents several advantages compared to these various vaccination approaches: -Our lentivectors encompasse up to 15kb DNA, the resulting polypeptide hence targeting a combination of different antigens which mobilizes different actors of the anti-tumor immune system-No selection of patients is needed according to HLA haplotype since epitopes are physiologically processed in vivo.-Unlike all other vaccination approaches, this technology allows the transduction of non-dividing cells such as DC, the most potent antigen-presenting cells (APC) of the immune system.-Break of immunotolerance: lentivectors not only provide sufficient amounts of antigens to DCs but also ensure an efficient processing and prolonged presentation of the antigens, an efficient maturation of DCs that possess all the stimulating abilities to induce an efficient activation of T cells-Non replicative and non persistent lentivectors-antigen expressing cells and transduced cells are eliminated by the immune response elicited after injection.-Direct injections in patients, without ex vivo process of autologous cells.-Different pseudotypes of lentivectors are developed to avoid specific immune reaction against the vector during boost injection(s).-Vaccination with lentiviral vectors are more potent and efficient than DNA or peptide or protein vaccination.The phase I/II clinical trial with our first vaccine candidate has been completed in HIV-infected patients. The vaccine is safe, as demonstrated by the clinical trial ongoing in 38 patients (no Serious Adverse Events have been reported during the trial). The interim analysis of the immunological data from the first two cohorts of patients performed by THERAVECTYS demonstrates the ability of the vaccine candidate to elicit multi-specific and poly-functional CD4+ and CD8+ cellular immune responses in vaccinated patients, even at the lowest dose.The interim analysis of this trial supports the potential of the lentiviral vector platform developed by THERAVECTYS for the future development of therapeutic vaccines and immunotherapies in oncology and infectious diseases. As a second indication, THERAVECTYS is developing an anti-HTLV-1 vaccine candidate to treat patients who have developed a virally-induced adult T cell leukemia. This vaccine candidate should enter into the clinics in mid-2015. Other indications in the pipeline are urogenital cancers, triple-negative breast cancer, EBV-induced nasopharyngeal cancers, HPV-induced cancers (cervix, oropharyngeal and anal cancers) and multiple myeloma.

Chimeric antigen receptors (CAR) modified T cells have shown very promising clinical results in hematological malignancies. CAR are usually composed of a scFv antigen binding domain linked to a spacer and intracellular signaling domains. CAR introduction into T cells leads to cells with optimal effector functions and with a new antigen binding ability. This strategy is well suited to skirt immune escape mechanisms.Despite their efficacy, CAR-T cells still show toxicity due to a cytokine storm, even if it is manageable. Improving the safety while maintaining or even improving the anti-tumor efficacy of CAR-T cells is therefore a crucial therapeutic challenge.Based on our proprietary lentiviral vector technology, we are developing a CAR-T cell platform aiming at producing innovative CAR-T cells. The development conducted by Theravectys are: -Development of proprietary lentiviral vectors coding for CAR of the second (containing the CD3ζ and the 4-1BB cosignaling domains) and third generations (containing the CD3ζ, the CD28 and the 4-1BB domains) directed against CD19 (for CD19+ leukemias and lymphomas), LMP-1 and -2 (for EBV-induced leukemias). Our lentiviral vectors will allow optimal expression of CAR therefore leading to a better efficacy of CAR-T cells. Hematological malignancies will be used as a benchmark.-Development of an inducible RUSH (Retention Using Selective Hook) system which is based on the streptavidin anchored to the membrane of the endoplasmic reticulum (ER) through a hook, and on the SBP (streptavidin binding protein) introduced into the CAR structure. The interaction between the streptavidin-hook and the SBP-CAR allows the CAR retention inside the ER. The addition of biotin displaces the equilibrium of binding of streptavidin towards biotin instead of SBP, thus leading to the release of the CAR from the ER and its expression to the cytoplasmic membrane. View Large Image | Download PowerPoint SlideThe release of the CAR will stop with drug exhaustion (or antagonist) and remaining cells can be easily reactivated through reintroduction of biotin. This system will increase safety of CAR-T cells. A CD19-CAR RUSH will be used as a proof-of-concept of the RUSH system.Most of the scFv used to date are of murine origin. Neutralizing antibodies against these murine scFv can limit the efficacy of CAR. As an alternative, we are screening and engineering specific humanized camelid nanobodies to be used as binding domains since they are highly homologous to the human VH domain of antibodies and they display high antigen binding capacities. The proof-of-concept will be made with a second generation CAR containing a nanobody directed against Her2 as a binding domain.Theravectys’ technological platforms allows flexibility and reactivity in the CAR design, production and evaluation, thus leading to the fast generation of safe and optimized CAR-T cells.

Acute myeloid leukemia (AML) is a heterogeneous group of malignancies sensitive to natural killer (NK) cell anti-tumor responses. However, NK cells are frequently defective in AML. Here, we found in an exploratory cohort (n = 46) that a different expression of activating receptors (NCR) on NK-cells identified a subgroup of patients with reduced expression of DNAM-1, NKG2D, NCRs and perforin especially in CD56bright NK-cells. Patients with a deficient NK-cell profile had a significantly higher risk of relapse (P = 0.03) independently of cytogenetic classification in multivariate analysis. We hypothesized that the transcription patterns in AML cells could give a clue on the NK/AML crosstalk. Analysis of the gene expression in AML blasts following classification according to their NK-cell status showed extensive differences in immune-related and oncogenic pathways. A favorable NK status was associated with pro-inflammatory AML transcription patterns whereas patients with defective NK cells showed a profound gene expression decrease in AML blasts for cytokine and chemokine signaling, antigen processing and adhesion molecule pathways. A set of 388 leukemic classifier genes defined in the exploratory cohort was independently validated in a multicentric cohort of 194 AML patients (ALFA-0701). In total, these data evidenced the prognostic value of NK-cell responses at diagnosis of AML and the relationships between NK-cells and AML independently of known cytogenetic status.

International audience; Macrophages play an important role in tissue homeostasis, tissue remodeling, immune response, and progression of cancer. Consequently, macrophages exhibit significant plasticity and change their transcriptional profile and function in response to environmental, tissue, and inflammatory stimuli resulting in pro- and anti-tumor effects. Furthermore, the categorization of tissue macrophages in inflammatory situations remains difficult; however, there is an agreement that macrophages are predominantly polarized into two different subtypes with pro- and anti-inflammatory properties, the so-called M1-like and M2-like macrophages, respectively. These two macrophage classes can be considered as the extreme borders of a continuum of many intermediate subsets. On one end, M1 are pro-inflammatory macrophages that initiate an immunological response, damage tissue integrity, and dampen tumor progression by fostering robust T and natural killer (NK) cell anti-tumoral responses. On the other end, M2 are anti-inflammatory macrophages involved in tissue remodeling and tumor growth, that promote cancer cell proliferation, invasion, tumor metastasis, angiogenesis and that participate to immune suppression. These decisive roles in tumor progression occur through the secretion of cytokines, chemokines, growth factors, and matrix metalloproteases, as well as by the expression of immune checkpoint receptors in the case of M2 macrophages. Moreover, macrophage plasticity is supported by stimuli from the Tumor Microenvironment (TME) that are relayed to the nucleus through membrane receptors and signaling pathways that result in gene expression reprogramming in macrophages, thus giving rise to different macrophage polarization outcomes. In this review, we will focus on the main signaling pathways involved in macrophage polarization that are activated upon ligand-receptor recognition and in the presence of other immunomodulatory molecules in cancer.