Background: T-cells engineered to express a chimeric antigen receptor (CAR-T-cells) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse survival in patients with B cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening, side effect often associated with CAR-T cells therapy. The Janus kinase (JAK) tyrosine kinase family is pivotal for the downstream signaling of inflammatory cytokines, including interleukins (ILs), interferons (IFNs), and multiple growth factors. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFN-g and IL-6. Itacitinib is a potent, selective JAK1 inhibitor which is being clinically evaluated in several inflammatory diseases. Aims: To evaluate in vitro and in vivo the potential of itacitinib to modulate CRS without impairing CAR-T cell anti-tumor activity. Materials and Methods: In vitro proliferation and cytotoxic activity of T cells and CAR-T cells was measured in the presence of increasing concentrations of itacitinib or tocilizumab (anti-IL-6R). To evaluate itacitinib effects in vivo, we conducted experiments involving adoptive transfer of human CD19-CAR-T-cells in immunodeficient animals (NSG) bearing CD19 expressing NAMALWA human lymphoma cells. The effect of itacitinib on cytokine production was studied on CD19-CAR-T-cells expanded in the presence of itacitinib or tocilizumab. Finally, to study whether itacitinib was able to reduce CRS symptoms in an in vivo setting, naïve mice were stimulated with Concanavalin-A (ConA), a potent T-cell mitogen capable of inducing broad inflammatory cytokine releases and proliferation. Results: In vitro, itacitinib at IC50 relevant concentrations did not significantly inhibit proliferation or anti-tumor killing capacity of human CAR-T-cells. Itacitinib and tocilizumab (anti-IL-6R) demonstrated a similar effect on CAR T-cell cytotoxic activity profile. In vivo, CD19-CAR-T-cells adoptively transferred into CD19+ tumor bearing immunodeficient animals were unaffected by oral itacitinib treatment. In an in vitro model, itacitinib was more effective than tocilizumab in reducing CRS-related cytokines produced by CD19-CAR-T-cells. Furthermore, in the in vivo immune hyperactivity (ConA) model, itacitinib reduced serum levels of CRS-related cytokines in a dose-dependent manner. Conclusion: Itacitinib at IC50 and clinically relevant concentrations did not adversely impair the in vitro or in vivo anti-tumor activity of CAR-T cells. Using CAR-T and T cell in vitro and in vivo systems, we demonstrate that itacitinib significantly reduces CRS-associated cytokines in a dose dependent manner. Together, the data suggest that itacitinib may have potential as a prophylactic agent for the prevention of CAR-T cell induced CRS. Disclosures Huarte: Incyte corporation: Employment, Equity Ownership. Parker:Incyte corporation: Employment, Equity Ownership. Huang:Incyte corporation: Employment, Equity Ownership. Milone:Novartis: Patents & Royalties: patents related to tisagenlecleucel (CTL019) and CART-BCMA; Novartis: Research Funding. Smith:Incyte corporation: Employment, Equity Ownership.
C-185cellular signals into intracellular responses. Upon nucleotide exchange catalyzed by activated receptors, heterotrimers dissociate into Ga-GTP subunits and Gsv dimers. either of which can modulate numerous downstream effectors. The crystal structures of the trimeric (Gra.py). dimeric CGtpy). and three monomeric forms (Gw:·GDP, Gw·GTPyS. Gw·GDP·AIF...-) of transducin, the heterotrimeric G protein involved in the visual pathway, have been solved and refined to high resolution.The heterotrimeric form of transducin, Gmi'Y• reveals the mechanism of the nucleotide dependent engagement/disengagement between the a and ~y subunits that regulates their interaction with receptor and effector molecules. The interaction involves two distinct interfaces and dramatically alters the conformation of the a but not the ~y subunits. The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation for the heterotrimer with respect to both the membrane smface and the activated heptahelical receptor.Multiwavelength anomalous diffraction data were used to solve the crystal structure of Gtpy . The ~ subunit is primarily a seven-bladed ~-propeller that is partially encircled by an extended y subunit. The ~-propeller, which contains seven structurally similar WD-repeats, defines the stereochemistry of the WD-repeat and the probable architecture of all \VD-repeat containing domains. The structure details interactions between G protein ~ andy subunits and highlights regions implicated in effector modulation for the conserved family of G protein ~y dimers. Like most of the members of the receptor-like protein tyrosine phosphatase (RPTP) family, the protein tyrosine phosphatase alpha (PTPa) is involved in key cellular functions such as cell growth division and differentiation. All exhibit common features: an extracellular N-terminal domain, a membrane-spanning helix and one or two intracellular domains (D l and D2) displaying tyrosine phosphatase activity at different levels. Extracellular domains vary drastically in structure and are likely to bind ligands. Here we describe the X-ray structure of PTPa's domain Dl. An unexpected tight. symmetric and apparently inactive dimer was present in the crystal. The amino-terminal segment of one monomer which adopts a helix-loop-helix conformation is wedged into the other monomer's active site preventing the swing of its catalytically essential loop. This amino-terminal motif resembles an elbow and the encompassed sequence is conserved amongst D 1 domains of all the receptor-like PTPs (except PTPx) and not in D2 domains or non-receptor PTPs. We propose that dimerization of D l domains is a physiological event inducing a reversible inactive state of the enzyme as a consequence of colocalization of the intracellular domains by extracellular mediated events. In efforts to understand intermolecular interactions that control signal transduction, we have studied the structure and function of pleckstrin homology (PH) domains. These domains of= 120 ...
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