Valentine Péri

Gamma/delta T cells (Vgamma9delta2) contribute to innate immunity and exert natural cytotoxicity against a variety of tumors. Using a synthetic phosphoantigen (Bromohydrin Pyrophosphate, BrHPP), we amplified Vgamma9delta2 T cells in vitro from neuroblastoma patients. In the presence of BrHPP and low doses of IL-2, robust proliferation of Vgamma9delta2 T cells was obtained from peripheral blood mononuclear cells (PBMC) harvested at diagnosis. Moderate proliferation was observed from PBMC harvested after stem cell transplantation, whereas modest levels of Vgamma9delta2 T cells were obtained from PBMC harvested after induction therapy. Proliferation was observed after a single in vitro stimulation with BrHPP. After 21 days in culture, Vgamma9delta2 T cells represented more than 80% of cultured cells (a 50-fold expansion from baseline). Moreover, BrHPP-amplified Vgamma9delta2 T cells from patients-expressed activation markers and were able to lyse allogeneic and autologous neuroblasts. This cytotoxic activity was gammadelta T-cell receptor-dependent. Clinical trials using BrHPP are warranted in patients with poor-prognosis neuroblastoma, either to expand patient-derived Vgamma9delta2 T cells ex vivo or by direct administration to in vivo to boost the pool of resident Vgamma9delta2 T cells in vivo.

LIGHT (TNFSF14) is a newly identified tumor necrosis factor superfamily member involved in the regulation of immune responses by control of activation, maturation, and survival of immune effector cells. Despite the immunological relevance of the LIGHT protein, little knowledge is available as to how light gene expression is regulated. In T-lymphocytes, most LIGHT surface expression and transcript accumulation occurs after T cell activation. In this study, we have shown that these events are blocked at the transcriptional level by cyclosporin A, an immuno-suppressive drug. Besides, we identified a role for Ca2+ -signaling pathways and NFAT transcription factors in T cell activation-induced LIGHT expression. To further investigate this process, we have identified, cloned, and characterized a 2.1-kilobase 5'-flanking DNA genomic fragment from the human light gene. We have shown the transcriptional activity of the herein-identified minimal 5' regulatory region of human light gene parallels the endogenous expression of light in T cells. Moreover, we demonstrated that induced LIGHT promoter activity can be equally blocked by cyclosporin A treatment or dominant negative NFAT overexpression and further identified by site-directed mutagenesis and electrophoretic mobility supershift analysis of a NFAT transcription factor binding site within the human light minimal promoter. Finally, Sp1 and Ets1 binding sites were identified and shown to regulate light basal promoter activity. Thus, the present study establishes a molecular basis to further understand the mechanisms governing human light gene expression and, consequently, could potentially lead to novel therapeutic manipulations that control the signaling cascade, resulting in LIGHT production in conditions characterized by immunopathologic activation of T cells.

Meeting abstracts MICA and MICB, along with ULPBs, are ligands for the activating receptor NKG2D expressed on NK cells and subsets of T cells in Human. NKG2D ligands are induced by cellular stress and pathogen infections. Their expression is tightly regulated by complex mechanisms both at the mRNA

Introduction Cutaneous T-cell lymphoma (CTCL) is a form of non-Hodgkin lymphoma, which includes Sézary Syndrome (SS), a rare, aggressive and advanced type of CTCL characterized by erythroderma, significant blood involvement, and lymphadenopathy. SS is associated with poor prognosis with a median patient survival of approximately 5 years. KIR3DL2, is expressed on circulating tumor cells (CTCs) of all SS patients and in the skin of more than 85% of SS patient. Lacutamab is a first-in-class monoclonal antibody designed to deplete KIR3DL2-expressing cells via antibody-dependent cell-cytotoxicity (ADCC) and phagocytosis (ADCP). TELLOMAK is an international, open-label, multi-cohort Phase 2 trial investigating the safety and efficacy of single agent lacutamab in patients with relapsed/refractory (R/R) CTCL (NCT03902184). Here we report the results of translational analysis of the SS cohort exploring the association between biomarkers and clinical outcomes. Methods Blood and skin samples were collected at baseline and on treatment to evaluate biomarkers association with clinical outcomes and to inform lacutamab mechanism of action through several exploratory objectives. Association between baseline and longitudinal frequency and counts of KIR3DL2-expressing cells or other biomarkers and clinical activity, as well as impact of treatment on KIR3DL2-expressing cells and on other biomarkers were explored using flow cytometry (blood) and multiplex imaging (skin), and are presented here. Additional translational data may be presented. Results At DCO of May 1, 2023, 56 SS pts were enrolled, treated and evaluated. Analysis of blood samples by flow cytometry revealed that all patients expressed KIR3DL2 on their CTCs as measured by flow-cytometry, with a median of 92.2% [0.6-99.6] KIR3DL2-expressing CTCs. Among 55 patients evaluable for translational analysis, lacutamab treatment resulted in depletion of KIR3DL2-expressing CTCs in 48/55 pts, with 15/55 patients reaching a depletion ≥99%. Importantly, depletion of KIR3DL2-expressing CTCs occurred as early as week 5 of treatment. Response to lacutamab in blood was observed regardless of the baseline CTC burden, as blood response was observed for patients with CTCs ranging from 311 to 74634 cells /µl. In the skin, using a threshold of ≥1% among mononucleated cells, KIR3DL2 expression was observed in 87.5% patients, consistent with the literature. Skin responses were observed irrespective of KIR3DL2 expression in baseline biopsies. A decrease of KIR3DL2 expressing cells in skin is observed as early as week 5, prior to the clinical skin response (median time to response in skin of 2.8 months; range 1-10 months) determined by modified Severity-Weighted Assessment Tool (mSWAT). Imaging analysis of skin biopsies at baseline revealed a higher infiltration by CD68+ macrophages than NK cells. Importantly, a higher baseline skin density of cells expressing the Fc receptor CD16, especially CD68+ macrophages were associated with better skin responses to lacutamab (p=0.027 and p=0.007, respectively). Conclusion Lacutamab monotherapy shows promising clinical activity in a R/R SS population previously treated with 2 or more prior systemic therapies including mogamulizumab. The exploratory translational data from the Tellomak SS cohort demonstrate rapid and deep depletion of KIR3DL2-expressing CTCs, irrespective of the baseline blood tumor burden. In skin, while no association was observed between response to lacutamab and KIR3DL2-expression in biopsies or KIR3DL2+ cell density at baseline, higher baseline infiltration by CD16-expressing cells, in particular CD16-expressing CD68+ macrophages was associated with better response in this compartment, consistent with lacutamab mechanism of action. These data confirm at a translational level the activity of lacutamab in the clinical trial setting, and its potential as a compelling future treatment option for CTCL patients with unmet need.