Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes.

CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this "difficult to-express" (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy's success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI-MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.

A Comparison of the Impact of Restrictive Diets on the Gastrointestinal Tract of Mice.

The rate of gut inflammatory diseases is growing in modern society. Previously, we showed that caloric restriction (CR) shapes gut microbiota composition and diminishes the expression of inflammatory factors along the gastrointestinal (GI) tract. The current project aimed to assess whether prominent dietary restrictive approaches, including intermittent fasting (IF), fasting-mimicking diet (FMD), and ketogenic diet (KD) have a similar effect as CR. We sought to verify which of the restrictive dietary approaches is the most potent and if the molecular pathways responsible for the impact of the diets overlap. We characterized the impact of the diets in the context of several dietary restriction-related parameters, including immune status in the GI tract; microbiota and its metabolites; bile acids (BAs); gut morphology; as well as autophagy-, mitochondria-, and energy restriction-related parameters. The effects of the various diets are very similar, particularly between CR, IF, and FMD. The occurrence of a 50 kDa truncated form of occludin, the composition of the microbiota, and BAs distinguished KD from the other diets. Based on the results, we were able to provide a comprehensive picture of the impact of restrictive diets on the gut, indicating that restrictive protocols aimed at improving gut health may be interchangeable.

CD47-targeted cancer immunogene therapy: Secreted SIRPα-Fc fusion protein eradicates tumors by macrophage and NK cell activation.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines "the best of both worlds," i.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

Peptide-Targeted Polyplexes for Aerosol-Mediated Gene Delivery to CD49f-Overexpressing Tumor Lesions in Lung.

Peptide ligands can enhance delivery of nucleic acid-loaded nanoparticles to tumors by promoting their cell binding and internalization. Lung tumor lesions accessible from the alveolar side can be transfected, in principle, using gene vectors delivered as an aerosol. The cell surface marker CD49f (Integrin α6) is frequently upregulated in metastasizing, highly aggressive tumors. In this study, we utilize a CD49f binding peptide coupled to linear polyethylenimine (LPEI) promoting gene delivery into CD49f-overexpressing tumor cells in vitro and into lung lesions in vivo. We have synthesized a molecular conjugate based on LPEI covalently attached to the CD49f binding peptide CYESIKVAVS via a polyethylene glycol (PEG) spacer. Particles formed with plasmid DNA were small (<200 nm) and could be aerosolized without causing major aggregation or particle loss. In vitro, CD49f targeting significantly improved plasmid uptake and reporter gene expression on both human and murine tumor cell lines. For evaluation in vivo, localization and morphology of 4T1 murine triple-negative breast cancer tumor lesions in the lung of syngeneic BALB/c mice were identified by MRI. Polyplexes applied via intratracheal aerosolization were well tolerated and resulted in measurable transgene activity of the reporter gene firefly luciferase in tumor areas by bioluminescence imaging (BLI). Transfectability of tumors correlated with their accessibility for the aerosol. With CD49f-targeted polyplexes, luciferase activity was considerably increased and was restricted to the tumor area.