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Effective public-health measures and vaccination campaigns against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against the ectodomain and the receptor-binding domain of the spike protein as well as the nucleocapsid protein of SARS-CoV-2. We used TRABI for continuous seromonitoring of hospital patients and healthy blood donors (n=72’222) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). Seroprevalence peaked in May 2020 and rose again in November 2020 in both cohorts. Validations of results included antibody diffusional sizing and Western Blotting. Using an extended Susceptible-Exposed-Infectious-Removed model, we found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020 in the population of the canton of Zurich. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19 and up to the timepoint of survey participation. Crucially, we found no evidence for a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2- infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

CD3-based T cell engagers are highly potent therapeutic molecules which enable T cell-mediated cytotoxic activity toward cells expressing selected tumor-associated antigens. Alongside a highly potent anti-tumor activity, is the risk of on-target off-tumor side effects due to low levels of expression of the target antigen in normal tissue. We have sought to overcome this issue with the design and generation of a target-density-dependent activation mechanism. We have generated reduced-affinity antibody fragments to the tumor associated antigen mesothelin and constructed a multi-domain MATCH4 molecule encompassing bivalent mesothelin binding domains, a CD3 binding domain, and a human serum albumin (HSA)-binding domain for half-life extension. Here we report the design of the MATCH4 molecule and the preclinical activity of the molecule in vitro and in vivo. We also report the biochemical characteristics of the therapeutic candidate molecule showing its highly favorable properties for clinical development. We demonstrate that the bivalent mesothelin T cell engager has increased in vitro potency in T cell activation and tumor cell killing in the presence of high mesothelin expressing cells, when compared to a higher-affinity monovalent counterpart. We also demonstrate that the activity on low mesothelin expressing cells, such as healthy mesothelial cells, is reduced for the bivalent molecule compared to the higher affinity monovalent molecule. Due to the shedding of mesothelin from the surface of cancer cells and the high circulating levels of soluble mesothelin in patient sera, we also demonstrate that the bivalent molecule is still highly potent in cytotoxic activity in the presence of concentrations of soluble mesothelin up to 500 ng/ml. We also demonstrate dose dependent anti-tumor activity in in vivo efficacy studies in PBMC-reconstituted mice, and combination therapeutic activity with an anti-PD-L1, anti-4-1BB bispecific molecule (NM21-1480). Collectively, these data demonstrate an increased selectivity to mesothelin-overexpressing cells by this novel MATCH4 reduced affinity bivalent T cell engager. These data indicate the potential of this molecule to increase the therapeutic window by reducing safety concerns on normal tissue where mesothelin expression is low, and yet promote cytotoxicity on mesothelin over-expressing cancer cells. Citation Format: Bithi Chatterjee, Christian Hess, Daniel Snell, Tea Gunde, Stefan Warmuth, Alexandre Simonin, Matthias Brock, Fabio Spiga, Maria Johansson, Christopher Weinert, Julia Tietz, Niels Kirk, Nicole Bassler, Dana Mahler, Dania Diem, Alessandra Carrella, Noreen Giezendanner, Alessandra Alberti, Giorgio Gambino, Belinda Wickihalder, Bettina Bommer, Simone Muntwiler, Yasemin Yaman, Naomi Flueckiger, Robin Heiz, Sandro Wagen, David Urech. NM28-2746, a reduced affinity bivalent mesothelin-binding MATCH4 T cell engager, with half-life extension, increases selectivity for killing of mesothelin-overexpressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2871.

SummaryNeuropathological and experimental evidence suggests that the cell-to-cell transfer of a-synuclein has an important role in the pathogenesis of Parkinson’s disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook an siRNA, genome-wide high throughput screen to identify genes regulating the cell-to-cell transfer of a-synuclein. We transiently transfected HEK cells stably overexpressing a-synuclein with a construct encoding GFP-2a-aSynuclein-RFP. The cells expressing a-synuclein-RFP through transfection were double positive for GFP and RFP fluorescence, whereas the cells receiving it through transfer were positive only for RFP fluorescence. The amount of a-synuclein transfer was quantified by high content microscopy. A series of unbiased screens confirmed the involvement of 38 genes in the regulation of a-synuclein-RFP transfer. One of those hits was ITGA8, a candidate gene recently identified through a large PD genome wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) showed that the hits clustered in networks that included known PD Mendelian and GWAS risk genes more frequently than expected than random chance. Given the genetic overlap between a-synuclein transfer and PD, those findings provide supporting evidence for the importance of the cell-to-cell transfer of a-synuclein in the pathogenesis of PD, and expand our understanding of the mechanism of a-synuclein spread.

Valentina Discepolo1,∗, Romain Bouziat2, Jennifer Stencel3, Mine Ikizler3, Giuliana Lania4, Merlin Nanayakkara1, Alessandra Carrella1, Marialaura Cuomo1, Katia Ferrara1, Renata Auricchio1, Riccardo Troncone1, Maria Vittoria Barone1, Terence Dermody3, Luis Bruno Barreiro5, Bana Jabri2 1 Dipartimento Scienze Mediche Traslazionali, Universita degli Studi di Napoli Federico II, Napoli, Italy 2 Department of Medicine, University of Chicago, Chicago, IL, USA 3 Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN, USA 4 Dipartimento di Scienze Mediche Traslazionali, Universita degli Studi di Napoli Federico II, Napoli, Italy 5 Department of Pediatrics, University of Montreal, Montreal, Canada

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.