We previously identified Sys1p as a high copy number suppressor of Ypt6 GTPase-deficient yeast mutants that are defective in endosome-to-Golgi transport. Here, we show that Sys1p is an integral membrane protein that resides on a post-endoplasmic reticulum (ER) organelle(s). Affinity studies with detergent- solubilized yeast proteins showed that the C-terminal 53 amino acid tail of Sys1p binds effectively to the cytoplasmic Sec23p-Sec24p COPII subcomplex. This binding required a di-acidic Asp-Leu-Glu (DXE) motif, previously shown to mediate efficient ER export of the vesicular stomatitis virus glycoprotein in mammalian cells. In Sys1p, a Glu-Leu-Glu (EXE) sequence could not substitute for the (DXE) motif. Mutations of the (DXE) sequence resulted in ER retention of approximately 30% of the protein at steady state, whereas addition of the Sys1p tail to an ER-resident membrane protein led to an intracellular redistribution of the chimeric protein. Our study demonstrates for the first time that, in yeast, a di-acidic sequence motif can act as a sorting signal for cargo selection during the formation of transport vesicles at the ER by direct binding to COPII component(s).

Background: Therapeutic antibodies such as adalimumab can elicit anti-drug antibodies. Results: Monoclonal patient-derived antibodies were generated and found to compete for binding to adalimumab and are neutralizing, but they show markedly different fine-specificity. Conclusion: Anti-adalimumab antibodies bind to overlapping but distinct epitopes on adalimumab. Significance: Even for a fully human therapeutic antibody, there may be multiple determinants that contribute to immunogenicity.

In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.

TWEAK receptor (TWEAKR, FN14) is a member of the tumor necrosis factor receptor superfamiliy and is highly expressed in a variety of human solid tumor types, and its overexpression is associated with poor prognosis and metastasis. To explore targeting of TWEAKR for cancer therapy we have generated the novel, anti-TWEAKR antibody BAY-356. Its potent agonistic activity leads to TWEAKR hyperactivation and subsequent induction of cell death in vitro and tumor growth inhibition in vivo. BAY-356 is a fully human aglycosylated antibody (Kd ∼ 10nM) that binds to a novel epitope within the TWEAKR ectodomain of various species as determined by BiaCore. In vitro, BAY-356 showed strong agonistic activity on TWEAKR-positive tumor cells, including activation of NFκB- and STAT1 pathways, increase of TWEAKR protein expression, increased IL-8 secretion, caspase 3/7 activation, and proliferation inhibition in a dose-dependent manner. BAY-356 inhibited tumor growth in several TWEAKR-positive tumor models (NCI-H1975, WiDr, ScaBER, and HN10321) with growth inhibition rates of 49-71% when treated with 3-10 mg/kg BAY-356 twice weekly for up to 3 weeks. The activity of BAY-356 was independent of ADCC activation. In a preventative syngeneic CT26-tumor model in Balb/c mice, BAY-356 induced complete responses. Anti-tumor activity of BAY-356 was associated with high tumor levels of TNF alpha protein. To investigate the toxicity of BAY-356, a repeated dose-toxicity study was performed in Cynomolgus monkeys. Animals were dosed with 10, 20, and 40 mg/kg by weekly intravenous injection for 4 weeks. Compound-related clinical findings consisted of an increase of the serum markers amylase and lipase from 10 mg/kg onwards, urea and creatinine from 20 mg/kg onwards and the transaminases ALT and GDPH at 40 mg/kg. Histopathological evaluation revealed focal ductular epithelial hyperplasia with periductular fibrosis in the exocrine pancreas (at 10 & 20 mg/kg), renal tubular hyperplasia and degeneration, Bowman capsule hyperplasia, and glomerulosclerosis in the kidney starting at 10 mg/kg and bile duct hyperplasia in liver at 20 mg/kg and higher. The HNSTD was set as the highest tested dose of 40 mg/kg. Immunohistochemical analysis of TWEAKR expression in these organs demonstrated a dose dependent induction and increase when compared to untreated controls which correlated with the histopathological findings. From these data it can be concluded that hyperactivation of TWEAKR signaling by BAY-356 leading to strong anti-tumor efficacy in various mouse models is invariably accompanied by target-mediated side-effects originating from enhanced TWEAKR induction in in particular in kidneys, pancreas, and liver of sensitive species such as Cynomolgus monkeys. Citation Format: Sandra Berndt, Christian Votsmeier, Ruprecht Zierz, Jakob Walter, Anna-Lena Frisk, Stefanie Hammer, Heiner Apeler, Bertolt Kreft. Preclinical pharmacology and repeated dose toxicity of the novel agonistic TWEAK receptor binding antibody BAY-356. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1210.

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