Differential effects of Cbl and 70Z/3 Cbl on T cell receptor-induced phospholipase Cγ-1 activity

AbstractWe demonstrate that the differential effects Cbl and oncogenic 70Z/3 Cbl have on Ca2+/Ras-sensitive NF-AT reporters is partially due to their opposing ability to regulate phospholipase Cγ1 (PLCγ1) activation as demonstrated by analysis of the activation of an NF-AT reporter construct and PLCγ1-mediated inositol phospholipid (PI) hydrolysis. Cbl over-expression resulted in reduced T cell receptor-induced PI hydrolysis, in the absence of any effect on PLCγ1 tyrosine phosphorylation. In contrast, expression of 70Z/3 Cbl led to an increase in basal and OKT3-induced PLCγ1 phosphorylation and PI hydrolysis. These data indicate that Cbl and 70Z/3 Cbl differentially regulate PLCγ1 phosphorylation and activation. The implications of these data on the mechanism of Cbl-mediated signaling regulation are discussed

C-Reactive Protein Causes Insulin Resistance in Mice Through Fcγ Receptor IIB–Mediated Inhibition of Skeletal Muscle Glucose Delivery

Elevations in C-reactive protein (CRP) are associated with an increased risk of insulin resistance. Whether CRP plays a causal role is unknown. Here we show that CRP transgenic mice and wild-type mice administered recombinant CRP are insulin resistant. Mice lacking the inhibitory Fcγ receptor IIB (FcγRIIB) are protected from CRP-induced insulin resistance, and immunohistochemistry reveals that FcγRIIB is expressed in skeletal muscle microvascular endothelium and is absent in skeletal muscle myocytes, adipocytes, and hepatocytes. The primary mechanism in glucose homeostasis disrupted by CRP is skeletal muscle glucose delivery, and CRP attenuates insulin-induced skeletal muscle blood flow. CRP does not impair skeletal muscle glucose delivery in FcγRIIB−/− mice or in endothelial nitric oxide synthase knock-in mice with phosphomimetic modification of Ser1176, which is normally phosphorylated by insulin signaling to stimulate nitric oxide–mediated skeletal muscle blood flow and glucose delivery and is dephosphorylated by CRP/FcγRIIB. Thus, CRP causes insulin resistance in mice through FcγRIIB-mediated inhibition of skeletal muscle glucose delivery

Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule.

Combination immunotherapy with antibodies directed against PD-1 and CTLA-4 shows improved clinical benefit across cancer indications compared to single agents, albeit with increased toxicity. Leveraging the observation that PD-1 and CTLA-4 are co-expressed by tumor-infiltrating lymphocytes, an investigational PD-1 x CTLA-4 bispecific DART molecule, MGD019, is engineered to maximize checkpoint blockade in the tumor microenvironment via enhanced CTLA-4 blockade in a PD-1-binding-dependent manner

Efficacy of Flotetuzumab in Combination with Cytarabine in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia

Children with acute myeloid leukemia (AML) have a poor prognosis despite the intensification of chemotherapy. Future efforts to improve outcomes should focus on more precise targeting of leukemia cells. CD123, or IL3RA, is expressed on the surface of nearly all pediatric AML samples and is a high-priority target for immunotherapy. The efficacy of an investigational dual-affinity retargeting antibody (DART) molecule (CD123 × CD3; MGD006 or flotetuzumab) was assessed in two distinct patient-derived xenograft (PDX) models of pediatric AML. MGD006 simultaneously binds to CD123 on target cells and CD3 on effector T cells, thereby activating T cells and redirecting them to induce cytotoxicity in target cells. The concurrent treatment of cytarabine and MGD006 was performed to determine the effect of cytarabine on T-cell counts and MGD006 activity. Treatment with MGD006 along with an allogeneic human T-cell infusion to act as effector cells induced durable responses in both PDX models, with CD123 positivity. This effect was sustained in mice treated with a combination of MGD006 and cytarabine in the presence of T cells. MGD006 enhanced T-cell proliferation and decreased the burden of AML blasts in the peripheral blood with or without cytarabine treatment. These data demonstrate the efficacy of MGD006 in prolonging survival in pediatric AML PDX models in the presence of effector T cells and show that the inclusion of cytarabine in the treatment regimen does not interfere with MGD006 activity

A humanized CD3ε-knock-in mouse model for pre-clinical testing of anti-human CD3 therapy.

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies