Biology subjects

Rivera-de-Torre, E.

Publications and source records attributed to Rivera-de-Torre, E..

6 recordsLinked to original sources

bioRxiv2026

Accelerating multi-objective VHH discovery via integrated high-throughput selection and AlphaFold3-guided structure prediction

Discovering therapeutic antibodies that bind multiple related targets with high affinity and favourable biophysical properties remains challenging and resource intensive. For snakebite antivenoms, this challenge is critical as treatments must neutralise toxins across multiple snake species. We developed a pipeline combining high-throughput yeast screening, deep sequencing, and AlphaFold3 structure prediction to rapidly identify poly-specific variable domains of heavy-chain-only antibodies (VHHs) against long-chain -neurotoxins. Multiplexed yeast display screening generated a dataset of diverse candidates with varying binding specificities. AlphaFold3-generated VHH-toxin complex predictions enabled structure-based prioritisation that accurately predicted poly-specific binders targeting conserved epitopes across multiple toxins. These structural insights enabled computational optimisation of both affinity and solubility without disrupting target recognition. Experimental validation confirmed improved variants maintained broad specificity across toxins. This integrated approach accelerates multi-objective antibody discovery by predicting which candidates will bind multiple targets before extensive laboratory testing, providing a generalisable strategy applicable beyond antivenoms to any therapeutic requiring broad target coverage.

bioengineering↗

bioRxiv2025

De novo designed protein enables precise epitope-level control of Gremlin-1 antagonism

Growth-factor antagonists regulate key developmental and pathological processes, yet the molecular principles governing their control remain poorly understood. Gremlin-1 (GREM1) is a secreted antagonist of bone morphogenetic proteins (BMPs) whose dysregulation is implicated in fibrosis and cancer. Here, we report a de novo designed protein that binds GREM1 with sub-nanomolar affinity and selectively releases BMPs for downstream signalling. By integrating generative deep-learning-based protein design with molecular-dynamics-derived flexibility descriptors, we identify a predictive relationship between interface rigidity, desolvation energy, and binding success. The resulting binder, RF1-2, reproduces the native BMP-binding epitope on GREM1 at near-atomic precision, as confirmed by cryo-electron microscopy, and competitively blocks BMP-2 and BMP-4 association. These results establish interface rigidity as a key physical determinant of antagonist inhibition and demonstrate how AI-guided protein design can uncover molecular principles underlying extracellular signalling control.

biochemistry↗

bioRxiv2024

De novo designed pMHC binders facilitate T cell induced killing of cancer cells

The recognition of intracellular antigens by CD8+ T cells through T-cell receptors (TCRs) is central to adaptive immunity, enabling responses against infections and cancer. The recent approval of TCR-gene-edited T cells for cancer therapy demonstrates the therapeutic advantage of using pMHC recognition to eliminate cancer. However, identification and selection of TCRs from patient material is complex and influenced by the TCR repertoire of the donors used. To overcome these limitations, we here present a rapid and robust de novo binder design platform leveraging state-of-the-art generative models, including RFdiffusion, ProteinMPNN, and AlphaFold2, to engineer minibinders (miBds) targeting the cancer-associated pMHC complex, NY-ESO-1(157-165)/HLA-A*02:01. By incorporating in silico cross-panning and molecular dynamics simulations, we enhanced specificity screening to minimise off-target interactions. We identified a miBd that exhibited high specificity for the NY-ESO-1-derived peptide SLLMWITQC in complex with HLA-A*02:01 and minimal cross-reactivity in mammalian display assays. We further demonstrate the therapeutic potential of this miBd by integrating it into a chimeric antigen receptor, as de novo Binders for Immune-mediated Killing Engagers (BIKEs). BIKE-transduced T cells selectively and effectively killed NY-ESO-1+ melanoma cells compared to non-transduced controls, demonstrating the promise of this approach in precision cancer immunotherapy. Our findings underscore the transformative potential of generative protein design for accelerating the discovery of high-specificity pMHC-targeting therapeutics. Beyond CAR-T applications, our workflow establishes a foundation for developing miBds as versatile tools, heralding a new era of precision immunotherapy.

immunology↗

bioRxiv2024

The action mechanism of actinoporins revealed through the structure of pore-forming intermediates

Pore-forming proteins exemplify the transformative potential of biological molecules. Initially produced in a monomeric, water-soluble form, they spontaneously assemble into multimeric integral membrane proteins in the presence of suitable target lipids. Their functions include roles in apoptosis, cell signaling, immunity, as well as attack and defense systems between different organisms. This latter group encompasses actinoporins, a family of pore-forming toxins from sea anemones that kill target cells by perforating their plasma membrane. Here, we have determined the structures of two such toxins, fragaceatoxin C and sticholysin II, in a membrane environment using cryogenic electron microscopy. The structures reveal how dozens of lipid molecules interact in an orderly manner, forming an intrinsic part of the pore. We have also isolated different pore-forming intermediates, where only a fraction of the constituent monomers is incorporated, exhibiting non-closed, arc-shaped structures. Based on these structures we propose a mechanism of action where the sequential assembly of toxin monomers onto the membrane, accompanied by conformational changes, triggers pore formation and membrane perforation. Our results contribute to a better understanding of the transforming capacity of these pore-forming proteins, which are becoming increasingly important for their diverse biotechnological applications.

biochemistry↗

bioRxiv2024

mRNA-delivered consensus allergens induce a neutralizing IgG response against food and pollen allergens

Pollen-food allergy syndrome (PFAS) affects a significant proportion of the global population with a major health and socioeconomic impact. Patients are generally treated against the major sensitized allergen which does not warrant protection against cross-reactive allergens, leading to long and ineffective treatment regimens. For food allergies, patient guidelines rely on source avoidance, leading to dietary restrictions and reduced quality of life - in particular for those suffering from PFAS. To overcome these limitations, we introduce a novel allergy immunotherapy (AIT) approach utilizing consensus allergens and mRNA technology to achieve broader, safer, and faster desensitization in PFAS patients. We first designed a consensus allergen of orthologs of non-specific Lipid Transfer Proteins (cnsLTP-1) representing a broad spectrum of nsLTP allergens prevalent in food and pollen sources. CnsLTP-1 was delivered to naive BALB/c mice using mRNA-lipid nanoparticles (mRNA-LNP) as vehicle, or by a traditional protein formulation, to assess if it elicits broad protection against allergens from different sources. Immunization with both mRNA-LNP and protein formulations demonstrated that cnsLTP-1-specific IgGs could be induced, whilst the mRNA-LNP formulation notably avoided the induction of allergen-specific IgEs. The induced antibodies were capable of recognizing and binding to a variety of nsLTPs, and effectively blocked the binding of allergens by allergic patient serum IgEs. This study thus demonstrates that the presented AIT strategy, based on mRNA-LNP technology and consensus allergens, could find clinical utility by addressing the limitations of current AIT. Further development of this technology platform could pave the way for more effective and patient-friendly treatments for PFAS and other cross-reactive allergies.

bioengineering↗

bioRxiv2023

Discovery of broadly-neutralizing antibodies against brown recluse spider and Gadim scorpion sphingomyelinases using consensus toxins as antigens

Broadly-neutralizing monoclonal antibodies are becoming increasingly important tools for treating infectious diseases and animal envenomings. However, designing and developing broadly-neutralizing antibodies can be cumbersome using traditional low-throughput iterative protein engineering methods. Here, we present a new high-throughput approach for the standardized discovery of broadly-neutralizing monoclonal antibodies relying on phage display technology and consensus antigens representing an average sequence of related proteins. We showcase the utility of this approach by applying it to toxic sphingomyelinases from the venoms of very distant orders of the animal kingdom, the recluse spider and Gadim scorpion. First, we designed a consensus sphingomyelinase and performed three rounds of phage display selection, followed by DELFIA-based screening and ranking, and benchmarked this to a similar campaign involving cross-panning against recombinant versions of the native toxins. Second, we identified two scFvs that not only bind the consensus toxins, but which can also neutralize sphingomyelinase activity in vitro. Finally, we conclude that the phage display campaign involving the use of the consensus toxin was more successful in yielding cross-neutralizing scFvs than the phage display campaign involving cross-panning.

bioengineering↗