Conduit CAR: Redirecting CAR T-Cell Specificity with A Universal and Adaptable Bispecific Antibody Platform.

The success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies has altered the treatment paradigm for patients with these diseases. Nevertheless, the occurrence of relapse due to antigen escape or heterogeneous antigen expression on tumors remains a challenge for first-generation CAR T-cell therapies as only a single tumor antigen can be targeted. To address this limitation and to add a further level of tunability and control to CAR T-cell therapies, adapter or universal CAR T-cell approaches use a soluble mediator to bridge CAR T cells with tumor cells. Adapter CARs allow simultaneous or sequential targeting of multiple tumor antigens, control of immune synapse geometry, dose control, and the potential for improved safety. Herein, we described a novel CAR T-cell adapter platform that relies on a bispecific antibody (BsAb) targeting both a tumor antigen and the GGGGS (G4S) linker commonly used in single-chain Fv (ScFv) domains expressed on CAR T-cell surfaces. We demonstrated that the BsAb can bridge CAR T cells to tumor cells and potentiate CAR T-cell activation, proliferation, and tumor cell cytolysis. The cytolytic activity of CAR T-cells was redirected to different tumor antigens by changing the BsAb in a dose-dependent manner. This study highlights the potential of G4S-displaying CAR T cells to be redirected to engage alternative tumor-associated antigens (TAA). Significance: New approaches are needed to address relapsed/refractory disease and manage potential toxicities associated with CAR T-cell therapy. We describe an adapter CAR approach to redirect CAR T cells to engage novel TAA-expressing cells via a BsAb targeting a linker present on many clinical CAR T-cell therapeutics. We anticipate the use of such adapters could increase CAR T-cell efficacy and reduce potential CAR-associated toxicities.

Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment.

The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.

The structure of ferricytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H.

Approximately 40% of all proteins are metalloproteins, and approximately 80% of Earth's ecosystems are at temperatures ≤5 °C, including 90% of the global ocean. Thus, an essential aspect of marine metallobiochemistry is an understanding of the structure, dynamics, and mechanisms of cold adaptation of metalloproteins from marine microorganisms. Here, the molecular structure of the electron-transfer protein cytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H has been determined by X-ray crystallography (PDB: ). The structure is highly superimposable with that of the homologous cytochrome from the mesophile Marinobacter hydrocarbonoclasticus. Based on structural analysis and comparison of psychrophilic, psychrotolerant, and mesophilic sequences, a methionine-based ligand-substitution mechanism for psychrophilic protein stabilization is proposed.

Overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism.

The psychrophilic, hydrocarbonoclastic microorganism Colwellia psychrerythraea is important in global nutrient cycling and bioremediation. In order to investigate how this organism can live so efficiently at low temperatures (~4°C), thermal denaturation studies of a small electron transfer protein from Colwellia were performed. Colwellia cytochrome c552 was overexpressed in Escherichia coli, isolated, purified, and characterized by UV-visible absorption spectroscopy. The melting temperature (Tm) and the van't Hoff enthalpy (ΔHvH) were determined. These values suggest an unexpectedly high stability for this psychrophilic cytochrome.