Biology subjects

Traxlmayr, M. W.

Publications and source records attributed to Traxlmayr, M. W..

4 recordsLinked to original sources

bioRxiv2026

Intrinsically disordered insert from SH2D2A rewires CD19 CAR signaling via Tyr290

Chimeric antigen receptor (CAR) T cells have transformed cancer immunotherapy, yet their truncated or suboptimal intracellular signaling can limit therapeutic efficacy. To enhance proximal signaling of a CD19-targeted CAR, we systematically inserted short Lck-recruiting motifs derived from Lck-adaptor proteins into the CAR intracellular tail. Six candidate sequences from four adaptor molecules (SH2D2A, SKAP1, LAT, LIME), with a sequence from CD3{varepsilon}, known to affect CAR functionality, as a positive control, were tested for expression and functional impact. Three CAR constructs (containing SH2D2A, LAT and LIME1 sequences respectively) displayed reduced surface expression, but only SH2D2A elicited a pronounced rewiring of CAR T cell phenotype following co-culture with CD19+ tumor lines. SH2D2A CAR T cells showed increased CD27 and CD56 expression and reduced expression of effector-associated mediators including granzyme B, IL-2, TNF, and IFN{gamma}. Through systematic mutagenesis and comparative phenotyping of SH2D2A CAR variants, we identified SH2D2A tyrosine 290 (Tyr290) as the critical residue mediating both the altered signaling phenotype and the low surface expression. Additionally, mutation of Tyr254 in the LIME1 CAR restored surface expression in Jurkat T cells, indicating insert- and context-dependent effects on receptor surface expression. Collectively, these results demonstrate that short, intrinsically disordered adaptor-derived sequences -- and single tyrosine residues within them -- can profoundly reprogram CAR signaling and expression.

immunology↗

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↗

bioRxiv2021

Peroxidasin protein expression and enzymatic activity in metastatic melanoma cell lines are associated with invasive potential

Peroxidasin, a heme peroxidase, has been shown to play a role in cancer progression. mRNA expression has been reported to be upregulated in metastatic melanoma cell lines and connected to the invasive phenotype, but little is known about how peroxidasin acts in cancer cells. We have analyzed peroxidasin protein expression and activity in eight metastatic melanoma cell lines using an ELISA developed with an in-house peroxidasin binding protein. RNAseq data analysis confirmed high peroxidasin mRNA expression in the five cell lines classified as invasive and low expression in the three non-invasive cell lines. Protein levels of peroxidasin were higher in the cell lines with an invasive phenotype. Active peroxidasin was secreted to the cell culture medium, where it accumulated over time, and peroxidasin protein levels in the medium were also much higher in invasive than non-invasive cell lines. The only well-established physiological role of peroxidasin is in the formation of a sulfilimine bond, which cross-links collagen IV in basement membranes via catalyzed oxidation of bromide to hypobromous acid. We found that peroxidasin secreted from melanoma cells formed sulfilimine bonds in uncross-linked collagen IV, confirming peroxidasin activity and hypobromous acid formation. Moreover, 3-bromotyrosine, a stable product of hypobromous acid reacting with tyrosine residues, was detected in invasive melanoma cells, substantiating that their expression of peroxidasin generates hypobromous acid, and showing that it does not exclusively react with collagen IV, but also with other biomolecules.

cancer biology↗