Biology subjects

Munoz-Lopez, A.

Publications and source records attributed to Munoz-Lopez, A..

4 recordsLinked to original sources

bioRxiv2025

Engineering human protein switches for functional control of CARs and transcription factors via oral drug administration

While CAR T cells have revolutionized the treatment of certain hematologic malignancies, they can cause severe toxicities, which are expected to be exacerbated with next-generation CAR Ts engineered for improved proliferation, persistence, and efficacy. Therefore, regulatory systems are urgently needed to be able to control these living drugs directly in patients. Here, we engineered a molecular switch, in which the interaction of two human proteins is efficiently induced with the orally available and non-toxic drug A1120. We demonstrate the versatility of this switch by regulating CAR signaling and transcriptional activity in human T cells in vitro and in vivo. Both systems were tightly controlled in the absence of the drug but strongly activated upon administration of the small molecule. Since this switch enables the regulation of diverse systems including CARs and transcription factors, we anticipate that it represents an important step towards next-generation cellular therapies with improved safety and efficacy.

synthetic biology↗

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

Long noncoding RNA-mediated epigenetic regulation of auxin-related genes controlling shade avoidance syndrome in Arabidopsis thaliana

The long noncoding RNA (lncRNA) AUXIN-REGULATED PROMOTER LOOP (APOLO) recognizes a subset of target loci across the Arabidopsis thaliana genome by forming RNA-DNA hybrids (R-loop) and modulating local three-dimensional chromatin conformation. Here we show that APOLO is involved in regulating the shade avoidance syndrome (SAS) by dynamically modulating the expression of key factors. In response to far-red (FR) light, the expression of APOLO anticorrelates with its target BRANCHED1 (BRC1), a master regulator of shoot branching in Arabidopsis thaliana. APOLO deregulation results in BRC1 transcriptional repression and an increase in the number of branches. APOLO transcriptional accumulation fine-tunes the formation of a repressive chromatin loop encompassing the BRC1 promoter, which normally occurs only in leaves as well as in a late response to FR treatment in axillary buds. In addition, our data reveal that APOLO participates in leaf hyponasty, in agreement with its previously reported role in the control of auxin homeostasis through direct modulation of YUCCA2 (auxin synthesis), PID and WAG2 (auxin efflux). We found that direct application of APOLO RNA to leaves results in a rapid increase in auxin accumulation that is associated with changes in the response of the plants to FR light. Collectively, our data support the view that lncRNAs coordinate the shade avoidance syndrome in Arabidopsis thaliana and shed light on the potential of lncRNAs as bioactive exogenous molecules. Deploying exogenous RNAs that modulate plant-environment interactions are important new tools for sustainable agriculture.

plant biology↗