A humanized monoclonal antibody specific for invariant Natural Killer T (iNKT) cells for in vivo depletion.

Invariant Natural Killer T (iNKT) cells are a subset of T cells recognizing glycolipid antigens presented by CD1d. Human iNKT cells express a conserved T cell receptor (TCR)-α chain (Vα24-Jα18) paired with a specific beta chain, Vβ11. The cells are both innate-like, with rapid cytokine release, and adaptive-like, including thymic positive selection. Over activation of iNKT cells can mediate tissue injury and inflammation in multiple organ systems and play a role in mediating the pathology associated with clinically important inflammatory diseases. At the same time, iNKT cell activation can play a role in protecting against infectious disease and cancer or modulate certain autoimmune diseases through its impact on both the innate and adaptive immune system. This suggests that approaches to cause iNKT cell reduction and/or depletion could treat inflammatory diseases while approaches to promote activation may have therapeutic potential in certain infections, cancer or autoimmune disease. This report summarizes the characterization of a humanized monoclonal depleting antibody (NKTT120) in the cynomolgus macaque. NKTT120 is being developed to treat iNKT mediated inflammation that is associated with chronic inflammatory conditions like sickle cell disease and asthma. NKTT120 binds to human iTCRs and to FCγRI and FCγRIII and has been shown to kill target cells in an ADCC assay at low concentrations consistent with the FCγR binding. iNKT cells were depleted within 24 hours in cynomolgus macaques, but T cell, B cell, and NK cell frequencies were unchanged. iNKT cell recovery was dose and time dependent. T cell dependent antigen responses were not impaired by NKTT120 mediated iNKT depletion as measured by response to KLH challenge. NKTT120 administration did not induce an inflammatory cytokine release at doses up to 10 mg/kg. These data support the use of NKTT120 as an intervention in inflammatory diseases where iNKT reduction or depletion could be beneficial

: PD-1 / PD-L1 - PD-L2 interactions

International audienceThe programmed death-1 (PD-1) molecule is involved in peripheral tolerance and in the immune escape mechanisms during chronic viral infections and cancer. PD-1 interacts with two ligands, PD-L1 and PD-L2. We have investigated the molecular mechanisms of PD-1 interactions with its ligands by surface plasmon resonance and cell surface binding as well as the ability of the two ligands to compete for PD-1 binding. PD-L1 and PD-L2 bound PD-1 with comparable affinities, but striking differences were observed at the level of the association and dissociation characteristics. PD-L1, but not PD-L2, had a delayed interaction reminiscent of a phenomenon of conformational transition. These mechanisms were confirmed by using PD-L1 mAbs that delayed the dissociation of PD-L1 from PD-1. This mechanism was not restricted to PD-1 binding since PD-L1 behaved in a similar manner with its second ligand, CD80. Finally, we could demonstrate that PD-L1 and PD-L2 competed for PD-1 binding and conversely, an antagonist PD-1 mAb blocked both PD-L1 and PD-L2 binding to PD-1 and strongly enhanced T-cell proliferation. These data further emphasize the differential molecular mechanisms of interaction of PD-L1 and PD-L2 with PD-1, and suggest possible new approach for the therapy of chronic infection, cancer and transplantation

Cynomolgus macaques (n=3 per dose group) were dosed with a single dose of 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg and 0.3 mg/kg of NKTT120, respectively and subsequently bled at indicated time points.

<p>Whole blood samples were stained with antibodies against CD3 (SP34.2), CD20 (2H7), TCR Vα24 (C15), CD159a (Z199) and invariant TCR (6B11). Subsequently red blood cells lysed, cells washed and analyzed by flow cytometry. T cells were identified as CD20-, CD3+ and 6B11- lymphocytes, B cells were identified as CD3-, CD20+ lymphocytes and NK cells were identified as CD3-CD159a+ lymphocytes. T, B and NK cells are reported as % of lymphocytes One way ANOVA with multiple comparison followed by a non-parametric T test was performed using PRISM software on all the subsets. * indicates significant difference to pre-bleed values (P=0.01).</p

Cynomolgus macaques in a dose-range finding study (n=1 per dose) were dosed with a single dose of 0.01 mg/kg and 0.1 mg/kg NKTT320, respectively and subsequently bled at indicated timepoints.

<p>Whole blood samples were stained with antibodies against CD3 (SP34.2), CD20 (2H7), TCR Vα24 (C15), CD159a (Z199) and invariant TCR (6B11). Red blood cells were subsequenltly lysed, cells washed and analyzed by flow cytometry. iNKT cells were identified as CD3+, Vα24+ and 6B11+ lymphocytes and reported as % of CD3+ cells.</p

Cynomolgus macaques (n=3 per dose group) were dosed with a single dose of 0.01 mg/kg (filled circles), 0.03 mg/kg (filled squares), 0.1 mg/kg (filled diamonds) and 0.3 mg/kg (open squares) of NKTT120, respectively and subsequently bled at indicated time points.

<p>NKTT120 plasma concentrations were measured by ELISA. Shown are means within groups with standard error.</p