Kulaksizoglu, E. | Search bioRxiv

bioRxiv2025

Deep mapping of the TCR-antigen interface using pMHC-pseudotyped viruses and yeast display

T cell receptor (TCR) specificity is central to the efficacy of T cell therapies, yet scalable methods to map how TCR sequences shape antigen recognition remain limited. To address this, we introduce VelociRAPTR, a library-on-library approach that combines yeast-displayed TCR libraries with pMHC-displaying virus-like particles (pMHC-VLPs) to rapidly screen millions of TCR-antigen interactions. We show that pMHC-VLPs efficiently bind TCRs on yeast and generate equivalent data to recombinantly produced pMHC protein. We then apply VelociRAPTR to screen 47 million variants of the A6 and 868 TCRs against 92 pMHCs simultaneously, mutating both the CDR3 loops and cognate peptides. The resulting CDR3-pMHC maps reveal biased recognition patterns, where mutations to CDR3 loops can selectively constrain or broaden specificity to peptide analogs. These insights provide a foundation for engineering TCRs with defined pMHC binding profiles and improving models that predict TCR-antigen interactions, including the prediction of off-target recognition. By coupling the scale of yeast display with the modularity of VLPs, VelociRAPTR offers a generalizable strategy for generating deep, high-throughput protein- protein interaction data.

immunology↗

bioRxiv2025

Closed-loop sonothermogenetic control of CAR T cells for metronomic brain cancer therapy

Achieving durable CAR T cell responses against primary brain tumors and metastases requires strategies that enable intracranial control of therapy to overcome the barriers of solid tumor treatment without compromising safety. Here, we show that closed-loop sonothermogenetics enables remote regulation of CAR T cell therapeutic activity through the intact skull. Using MR-guided focused ultrasound with closed-loop temperature feedback, we modulate CAR T cells engineered with a genetically encoded thermal bioswitch to achieve metronomic activation in the brain without lasting adverse effects on healthy brain tissue. In murine models of brain cancer, metronomic production of NKG2D T cell engagers by intratumoral CAR T cells overcomes antigen heterogeneity in breast cancer brain metastasis and myeloid-derived immunosuppression in glioblastoma to drive antitumor responses. Our findings support the use of closed-loop sonothermogenetics for spatial and temporal control of CAR T cell therapies targeting solid brain tumors.

bioengineering↗