Chronic neuroinflammation driven by microglia is a characteristic feature associated with numerous neurodegenerative diseases. While acute inflammation can assist with recovery and repair, prolonged microglial pro-inflammatory responses are known to exacerbate neurodegenerative processes. Yet, detrimental outcomes of extended microglial activation are counterbalanced by beneficial outcomes including phagocytosis and release of trophic factors promoting neuronal viability. Our past work has shown that the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is a key signaling hub driving pro-inflammatory microglia responses, but the signaling pathway maintaining PARP-1 activation remains elusive. While best understood for its role in promoting DNA repair, our group has shown that PARP-1 activity can be stimulated via Ca 2+ influx-dependent ERK1/2-mediated phosphorylation. However, to date, the route of Ca 2+ entry responsible for stimulating PARP-1 has not been identified. A likely candidate is via Ca 2+ -permeable transient receptor potential melastatin 2 (TRPM2) channels activated downstream of PARP-1 in a cascade that involves ADP-ribose (ADPR) production by poly(ADP-ribose) glycohydrolase (PARG). Here we demonstrate that NMDA receptor (NMDAR) stimulation in primary cultured microglia induces their proliferation, morphological activation and release of pro-inflammatory mediators. These responses were contingent on the recruitment of PARP-1, PARG and Ca 2+ permeable TRPM2 channels. Furthermore, we show that Ca 2+ influx is necessary to activate PARP-1/TRPM2 signaling, in an ERK1/2-dependent, but DNA damage independent, manner. Our findings, showing that PARP-1/TRPM2 mediate the pro-inflammatory effects of NMDAR stimulation, provides a unifying mechanism linking elevated glutamate levels to chronic neuroinflammation.

In the present study, an attempt to evaluate the antimicrobial and antioxidant activity of fungal endophytes inhabiting Emblica officinalis has been made keeping in view the medicinal importance of the selected host plant in Indian traditional practices. A total of four endophytic fungi belonging to Phylum Ascomycetes were isolated from different parts of the plant which were characterized morphologically and by using rDNA-internal transcribed spacer. The most frequently isolated endophyte was Phomopsis sp. The antioxidant activity by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and reducing power assay, and total phenol were evaluated using ethanolic extract of endophytic fungi. DPPH activities in all the ethanolic extract increased with the increase in concentrations. Endophytes, Phomopsis sp. and Xylaria sp. showed highest antioxidant activity and also had the higher levels of phenolics. Antimicrobial activity of fungal extract were tested against four bacteria namely, Escherichia coli MTCC730, Enteroccocus faecalis MTCC2729, Salmonella enterica ser. paratyphi MTCC735 and Streptococcus pyogenes MTCC1925, and the fungus Candida albicans MTCC183. In general, the fungal extracts inhibited the growth of test organisms except E. coli.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Many T-cell engagers (TCE) and immuno-oncology biologics have limited efficacy in the clinic due to narrow therapeutic windows, checkpoint upregulation, and emergence of resistance mechanisms over time. The PROTECTTM (PROgrammed Tumor Engagement & Checkpoint/Costimulation Targeting) platform is designed to tackle these challenges by combining a masking domain that, when cleaved, provides additional immune-modulatory properties. The PROTECTTM mask consists of a PD1-PDL1 protein pair that sterically hinders CD3 binding in the periphery. The PD-L1 moiety is fused to the anti-CD3 antibody via a linker sequence containing a protease cleavage site. Once cleaved, the resultant molecule is a trispecific antibody providing TCE activity, checkpoint inhibition, and additional differentiated functionality. Previous studies employing pan T cell cytotoxicity assays showed the PROTECTTM mask increased the therapeutic window by 400-fold. We have now expanded our in vitro evaluation to include more relevant PBMC systems showing a greater expansion of the therapeutic window. In vivo treatment of established tumours with the unmasked trispecific results in complete and durable anti-tumour responses. Here we extend those in vivo studies (in context of established tumour models) to evaluate unmasking and pharmacokinetic properties. We further evaluated the unique and differentiated mechanism of action of the cleaved trispecific to not only enhance engagement of effector T-cells with tumor cells through avidity driven cis-engagement of a tumor associated antigen (TAA) and PD-L1 co-expressed on tumor cells, but to also enhance co-engagement of a TAA with effector T cells co-expressing PD-L1. In addition, the ability of the cleaved trispecific to bridge autologous T cells with DCs results in enhanced T cell activation and proliferation. Finally, TCR dependent signaling assays confirmed the ability of the cleaved trispecific to overcome PD1/PDL1 checkpoint activity. Taken together, the PROTECTTM platform integrates a masking and immune-modulatory technology that has the potential to widen the therapeutic window of CD3 engagers. Citation Format: Anna von Rossum, Genevieve Desjardins, Nichole Escalante, Wingkie Wong, Bryant Harbourne, Janessa Li, Begonia Silva Moreno, Prajwal Raghunatha, Richard Kunze, Madeline Fung, Florian Heinkel, Harsh Pratap, Kevin Haworth, Eric Escobar-Cabrera, Brandon Clavette, Surjit Dixit, Nina Weisser, Thomas Spreter von Kreudenstein. PROTECTTM, a novel trispecific antibody masking platform with integrated immune modulation displays unique activity and differentiated modes of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2926.