Biology subjects

Salzer, B.

Publications and source records attributed to Salzer, B..

4 recordsLinked to original sources

bioRxiv2025

MiniCARbids: Minimalistic human binding domains specifically tailored to CAR T applications

Traditionally, chimeric antigen receptor (CAR) T cells employ single-chain variable fragments (scFvs) as binding entities. While scFvs represent a convenient option due to their broad availability, they also come with drawbacks, in particular their tendency to cluster and their relatively large size. Moreover, most scFvs used in the CAR field are of non-human origin, potentially causing immunogenicity. Therefore, we established an engineering platform for minimalistic CAR binding domains (miniCARbids), which combine several critical advantages: (i) human origin, (ii) small size, (iii) efficient expression in T cells and (iv) single-domain architecture, among others. We demonstrate that miniCARbids can be engineered to recognize various antigens with antibody-like affinities, while being stable and aggregation-resistant. When miniCARbids are incorporated into CARs, they induce high anti-tumor potency in both adapter and conventional CAR formats. Remarkably, CD22-directed miniCARbid-based CARs showed similar or even more efficient tumor clearance in leukemia-bearing mice when compared with a CAR comprising the clinically tested m971-1xG4S scFv. Together, we introduce the miniCARbid engineering platform, enabling the generation of small, human antigen-binding domains with high potency in CAR T cells against virtually any target antigen.

synthetic biology↗

bioRxiv2025

Caffeine-regulated molecular switches for functional control of CAR T cells in vivo

The limited controllability of CAR T cells in patients represents a key challenge of this highly potent immunotherapy. A molecular ON-switch, which can be regulated with a non-toxic and readily available small molecule drug, would represent a major advance towards controllable CAR T therapeutics. For that purpose, we engineered caffeine-responsive heterodimeric ON-switches (CaffSwitches) and demonstrate their high caffeine-dependency and virtually absent leakiness. When incorporating these CaffSwitches into CARs, the resulting CaffCARs were efficiently activated by caffeine concentrations achieved in human plasma after drinking one cup of coffee. Moreover, CaffCAR T cells also showed efficient tumor clearance in an in vivo mouse model, which was completely abolished in the absence of caffeine. This tight control was even observed with c-Jun overexpressing CaffCAR T cells, despite their massive expansion. Together, we anticipate that these novel CaffSwitches will be valuable tools for the development of safe and efficient next generation CAR T cells.

synthetic biology↗

bioRxiv2023

TCR/CD3-based synthetic antigen receptors (TCC) convey superior antigen sensitivity combined with high fidelity of activation

Low antigen sensitivity and a gradual loss of effector functions limit the clinical applicability of chimeric antigen receptor (CAR)-modified T-cells and call for alternative antigen receptor designs for effective T-cell-based cancer immunotherapy. Here we applied advanced microscopy to demonstrate that TCR/CD3-based synthetic constructs (TCC) outperform second-generation CAR formats with regard to conveyed antigen sensitivities by up to a thousand-fold. TCC-based antigen recognition occurred without adverse non-specific signaling, which is typically observed in CAR-T-cells, and did not depend - unlike sensitized peptide/MHC detection by conventional T-cells - on CD4- or CD8- coreceptor engagement. TCC-endowed signaling properties may prove critical when targeting antigens in low abundance and aiming for a durable anti-cancer response.

immunology↗

bioRxiv2021

Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

Chimeric antigen receptors (CARs) can re-direct T cells to target abnormal cells but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here we show that, while CARs have a >100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting-cells (APCs), they have nearly identical sensitivity when antigen is presented as purified protein on artificial surfaces. We next measured the impact of engaging accessory receptors (CD2, LFA-1, CD28, CD27, 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved TCR antigen sensitivity by 125 and 22-fold, respectively, but only improved CAR sensitivity by <5-fold. This differential effect of CD2 and LFA-1 engagement on TCR versus CAR sensitivity was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3{varepsilon} subunit (also known as a TRuC), and fully restored by exchanging the CAR variable domains with the TCR{beta} variable domains (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors, and suggest approaches to increase sensitivity.

immunology↗