CAR affinity modulates the sensitivity of CAR-T cells to PD-1/PD-L1-mediated inhibition.

Chimeric antigen receptor (CAR)-T cell therapy for solid tumors faces significant hurdles, including T-cell inhibition mediated by the PD-1/PD-L1 axis. The effects of disrupting this pathway on T-cells are being actively explored and controversial outcomes have been reported. Here, we hypothesize that CAR-antigen affinity may be a key factor modulating T-cell susceptibility towards the PD-1/PD-L1 axis. We systematically interrogate CAR-T cells targeting HER2 with either low (LA) or high affinity (HA) in various preclinical models. Our results reveal an increased sensitivity of LA CAR-T cells to PD-L1-mediated inhibition when compared to their HA counterparts by using in vitro models of tumor cell lines and supported lipid bilayers modified to display varying PD-L1 densities. CRISPR/Cas9-mediated knockout (KO) of PD-1 enhances LA CAR-T cell cytokine secretion and polyfunctionality in vitro and antitumor effect in vivo and results in the downregulation of gene signatures related to T-cell exhaustion. By contrast, HA CAR-T cell features remain unaffected following PD-1 KO. This behavior holds true for CD28 and ICOS but not 4-1BB co-stimulated CAR-T cells, which are less sensitive to PD-L1 inhibition albeit targeting the antigen with LA. Our findings may inform CAR-T therapies involving disruption of PD-1/PD-L1 pathway tailored in particular for effective treatment of solid tumors.

Strategies for the Site-Specific Decoration of DNA Origami Nanostructures with Functionally Intact Proteins.

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry, and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as the protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to three proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral peptide nucleic acid (PNA) in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

The mannose 6-phosphate/insulin-like growth factor 2 receptor mediates plasminogen-induced efferocytosis.

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.