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Key Points• AMG 330 cytotoxicity against AML cells is proportional to the level of CD33 expression but is not affected by ABC transporter activity.• AMG 330 cytotoxicity is amenable to modulation and augmentation by clinically available drugs such as histone deacetylase or DNA methyltransferase I inhibitors.CD33 is a valid target for acute myeloid leukemia (AML) but has proven challenging for antibody-drug conjugates. Herein, we investigated the cellular determinants for the activity of the novel CD33/CD3-directed bispecific T-cell engager antibody, AMG 330. In the presence of T cells, AMG 330 was highly active against human AML cell lines and primary AML cells in a dose-and effector to target cell ratio-dependent manner. Using cell lines engineered to express wild-type CD33 at increased levels, we found a quantitative relationship between AMG 330 cytotoxicity and CD33 expression; in contrast, AMG 330 cytotoxicity was neither affected by common CD33 single nucleotide polymorphisms nor expression of the adenosine triphosphate-binding cassette (ABC) transporter proteins, P-glycoprotein or breast cancer resistance protein. Unlike bivalent CD33 antibodies, AMG 330 did not reduce surface CD33 expression. The epigenetic modifier drugs, panobinostat and azacitidine, increased CD33 expression in some cell lines and augmented AMG 330-induced cytotoxicity. These findings demonstrate that AMG 330 has potent CD33-dependent cytolytic activity in vitro, which can be further enhanced with other clinically available therapeutics. As it neither modulates CD33 expression nor is affected by ABC transporter activity, AMG 330 is highly promising for clinical exploration as it may overcome some limitations of previous CD33-targeted agents. (Blood. 2014;123(4):554-561) IntroductionAcute myeloid leukemia (AML) has served as a paradigm for the therapeutic use of monoclonal antibodies because of well-defined cell-surface antigens and easy tumor accessibility. The most investigated target so far is CD33, a myeloid differentiation antigen found on AML blasts in most patients and, perhaps, leukemic stem cells in some.1,2 Recent randomized phase 3 trials have demonstrated that the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO), improves survival for some patients with newly diagnosed AML when added to conventional chemotherapy, with benefit primarily seen for those with favorable-risk disease and, to a smaller extent, intermediate-risk disease. [3][4][5] Although this experience indicates that CD33 is a valid target for this disease, 1,2 it is a challenging one for toxin-loaded antibodies due to its relatively low abundance, slow internalization, and drug transporter activity in AML cells. In fact, GO given alone or in combination with other chemotherapeutics is ineffective in many patients and, as a consequence, is currently no longer commercially available in many countries. 1,2Bispecific T-cell engager (BiTE) antibodies are a novel subclass of therapeutic single-chain antibodies. [6][7][8] What distinguishes BiTE antibodies ...

In the final stages of ovarian follicular development, the mouse oocyte remains arrested in the first meiotic prophase, and cAMP-stimulated PKA plays an essential role in this arrest. After the LH surge, a decrease in cAMP and PKA activity in the oocyte initiates an irreversible maturation process that culminates in a second arrest at metaphase II prior to fertilization. A-kinase anchoring proteins (AKAPs) mediate the intracellular localization of PKA and control the specificity and kinetics of substrate phosphorylation. Several AKAPs have been identified in oocytes including one at 140 kDa that we now identify as a product of the Akap1 gene. We show that PKA interaction with AKAPs is essential for two sequential steps in the maturation process: the initial maintenance of meiotic arrest and the subsequent irreversible progression to the polar body extruded stage. A peptide inhibitor (HT31) that disrupts AKAP/PKA interactions stimulates oocyte maturation in the continued presence of high cAMP. However, during the early minutes of maturation, type II PKA moves from cytoplasmic sites to the mitochondria, where it associates with AKAP1, and this is shown to be essential for maturation to continue irreversibly.

Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.

In this article, the authors reported on four individuals with intellectual disability, severely affected speech development, behavioral problems, and missense mutations affecting the SAND domain of DEAF1. Functional studies showing a loss of function of DEAF1 and behavioral studies in a conditional knockout mouse provided additional support for causality of the DEAF1 mutations in these four reported individuals.