Circulating immune profile in granulomatosis with polyangiitis reveals distinct patterns related to disease activity.

Granulomatosis with polyangiitis (GPA) is an autoimmune disorder characterized by recurrent relapses that can cause severe tissue damage and life-threatening organ dysfunction. Multiple immune cells and cytokines/chemokines are involved in the different stages of the disease. Immune profiling of patients may be useful for tracking disease activity, however, reliable immune signatures for GPA activity are lacking. In this study, we examined circulating immune profiles in GPA patients during active and remission disease states to identify potential immune patterns associated with disease activity. The distribution and phenotypic characteristics of major circulating immune cells, and the profiles of circulating cytokines/chemokines, were studied on cryopreserved peripheral blood mononuclear cells from GPA patients (active, n = 20; remission, n = 20) and healthy controls (n = 20) leveraging a 40-color optimized multicolor immunofluorescence panel (OMIP-69) and in serum using a 46-plex Luminex multiplex assay, respectively. Deep phenotyping uncovered a distinct composition of major circulating immune cells in active GPA and GPA in remission, with the most significant findings emerging within the monocyte compartment. Our detailed analysis revealed circulating monocyte diversity beyond the conventional monocyte subsets. We identified eight classical monocyte populations, two intermediate monocyte populations, and one non-classical monocyte population. Notably, active GPA had a higher frequency of CD45RA+CCR5+CCR6-CCR7+/lowCD127-HLA-DR+CD2- classical monocytes and a lower frequency of CD45RA-CCR5-/lowCCR6-CCR7-CD127-HLA-DR+CD2+/- classical monocytes, which both strongly correlated with disease activity. Furthermore, serum levels of CXCL1, CXCL2, and CCL20, all linked to monocyte biology, were elevated in active GPA and correlated strongly with disease activity. These findings shed light on the circulating immune profile of GPA and may lead to immune signature profiles for assessing disease activity. Monocytes in particular may be studied further as potential markers for monitoring GPA.

A novel AML-selective TRAIL fusion protein that is superior to Gemtuzumab Ozogamicin in terms of in vitro selectivity, activity and stability.

Gemtuzumab ozogamicin (GO, Mylotarg) is a targeted therapeutic agent in which an anti-CD33 antibody is chemically coupled to a highly cytotoxic calicheamicin derivative through a hydrolysable linker. GO has improved the treatment outcome for a subgroup of acute myeloid leukemia (AML) patients, but its use is associated with severe myelosuppression and hepatotoxicity. Here, we report on a novel anti-leukemia agent, designated scFvCD33:sTRAIL, in which an anti-CD33 single chain fragment of variable regions (scFv) antibody fragment is genetically linked to soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). Normal CD33-positive monocytes were fully resistant to prolonged treatment with scFvCD33:sTRAIL, whereas treatment with GO resulted in substantial cytotoxicity. The activity of scFvCD33:sTRAIL towards AML cells was up to 30-fold higher than GO. The CD33-restricted anti-leukemia activity of scFvCD33:sTRAIL remained stable during prolonged storage at 37 degrees C, whereas GO showed a rapid increase in CD33-independent cytotoxicity. Moreover, scFvCD33:sTRAIL showed potent anti-leukemia activity towards CD33+ CML cells when treatment was combined with the Bcr-Abl tyrosine kinase inhibitor, Gleevec. Importantly, ex vivo treatment of patient-derived CD33+ AML tumor cells with scFvCD33:sTRAIL resulted in potent apoptosis induction that was enhanced by valproic acid, mitoxantrone and 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG). Taken together, scFvCD33:sTRAIL is superior to GO in terms of tumor selectivity, activity and stability, warranting its further development for the treatment of CD33-positive leukemias.

The histone deacetylase inhibitor valproic acid potently augments gemtuzumab ozogamicin-induced apoptosis in acute myeloid leukemic cells.

Gemtuzumab ozogamicin (GO) is a calicheamicin-conjugated antibody directed against CD33, an antigen highly expressed on acute myeloid leukemic (AML) cells. CD33-specific binding triggers internalization of GO and subsequent hydrolytic release of calicheamicin. Calicheamicin then translocates to the nucleus, intercalates in the DNA structure and subsequently induces double-strand DNA breaks. GO is part of clinical practice for AML, but is frequently associated with severe side effects. Therefore, combination of GO with other therapeutics is warranted to reduce toxicity, while maximizing therapeutic selectivity. We hypothesized that the histone deacetylase inhibitor valproic acid (VPA) sensitizes AML cells to GO. VPA-induced histone hyperacetylation opens the chromatin structure, whereby the DNA intercalation of calicheamicin should be augmented. We found that clinically relevant concentrations of VPA potently augmented the tumoricidal activity of GO towards AML cell lines and primary AML blasts. Moreover, VPA treatment indeed augmented the DNA intercalation of calicheamicin and enhanced DNA degradation. Importantly, synergy was restricted to CD33-positive AML cells and did not require caspase activation. In conclusion, the synergistic proapoptotic activity of cotreatment of AML cells with VPA and GO indicates the potential value of this strategy for AML.