Biology subjects

Spiller, B. W.

Publications and source records attributed to Spiller, B. W..

3 recordsLinked to original sources

bioRxiv2026

Novel COX-2 Targeted Nanobodies for Molecular Endoscopic Imaging of Colorectal Adenomas

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality in men and women. Timely detection and diagnosis are key to management of CRC, which is under-diagnosed because colorectal aberrant crypt foci, hyperplastic polyps, and microadenomas are often missed with conventional colonoscopy. The enzyme cyclooxygenase-2 (COX-2) is overexpressed in early stages of colorectal carcinogenesis and plays an important regulatory role in the process, suggesting that it could be a valuable target for enhanced imaging of nascent disease. Thus, we have generated an alpaca-derived library of 73 COX-2-specific nanobody clones. Here, we describe one such nanobody, F9-K45Q-K77Q-ROX, in which two native lysine residues have been mutated followed by conjugation to a fluorophore at the N-terminus with retention of COX-2-selective binding. The site of fluorophore conjugation and COX-2 binding affinity of F9-K45Q-K77Q-ROX were determined by proteomic and microscale thermophoretic analyses, respectively. In cell culture studies using 1483 human head and neck squamous cell carcinoma cells, F9-K45Q-K77Q-ROX accumulated inside cells and bound to intracellular COX-2, as visualized by fluorescence microscopy. In vivo pharmacokinetic, and toxicological analyses revealed that F9-K45Q-K77Q-ROX is detectable in circulation with a plasma half-life of 17.9 min and there is no short-term toxicity associated with single injections of 10 mg/kg, 20 mg/kg, or 40 mg/kg doses at 24 h post-administration. Noninvasive in vivo fluorescence endoscopic imaging validated tumor-specific accumulation of F9-K45Q-K77Q-ROX in azoxymethane/dextran sodium sulfate-induced colorectal adenomas in mice. This work demonstrates the first COX-2-targeted nanobodies including a fluorescent derivative that offers significant promise for targeted endoscopic imaging of COX-2-expressing neoplasms. Significance StatementCurrent colorectal cancer screening procedures, such as white-light colonoscopy, chromoendoscopy, and narrow-band imaging aim to detect solid colon tumors and precursor lesions. However, these methods tend to detect only raised solid tumors and mature cancers, whereas precursor lesions, such as aberrant crypt foci, hyperplastic polyps, and small adenomas are frequently missed. To address the need for better visualization of early lesions, we developed a library of alpaca-derived nanobodies targeted to cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in colorectal adenomas. COX-2-targeted nanobodies bearing a fluorescent tag accumulate and are retained in colonic adenomas, facilitating their endoscopic visualization. This novel COX-2-targeted nanobody platform may also be valuable for early detection of other neoplastic diseases in which COX-2 overexpression occurs. (Word counts 119, limit 120)

bioengineering↗

bioRxiv2025

Development and application of nbLIBRA-seq for high-throughput discovery of antigen-specific nanobodies

Nanobodies are of high interest in many fields of medicine and biotechnology due to their high stability, tissue penetration, and engineering adaptability compared to monoclonal antibodies. However, nanobody discovery has been limited by technologies that rely on laborious library generation, panning, and clone screening techniques. Here, we demonstrate the successful adaptation of Linking B-Cell Receptor to Antigen Specificity through Sequencing (LIBRA-seq) to immunized alpacas for the rapid identification of antigen-specific nanobodies, derived from heavy-chain antibodies. We validated for nanobody discovery (nbLIBRA-seq) in two different disease settings. First, we identified over 300 antigen-specific heavy chain antibodies against human Transferrin Receptor (TfR1), also known as CD71, from a single alpaca blood sample. Experimental validation showed nbLIBRA-seq was able to identify nanobodies that exhibit specific binding to CD71, with two nanobodies also showing receptor internalization on human T cells. In a separate experiment, we tested the ability of nbLIBRA-seq to perform nanobody discovery with multiple antigens in the antigen screening library. Using fusion glycoproteins from the related respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), 1,125 antigen-specific heavy-chain expressing B cells were recovered via nbLIBRA-seq. A subset of these nanobodies was validated experimentally to possess the target antigen specificity. Together, our results illustrate the potential of nbLIBRA-seq to rapidly identify antigen-specific heavy chain antibodies for a range of diverse targets, a capability that will be of critical significance for the effective and efficient development of novel nanobody-based therapeutics against targets of biomedical significance.

immunology↗

bioRxiv2023

Nanobodies against C. difficile TcdA and TcdB reveal unexpected neutralizing epitopes and provide a toolkit for toxin quantitation in vivo

Clostridioides difficile is a leading cause of antibiotic-associated diarrhea and nosocomial infection in the United States. The symptoms of C. difficile infection (CDI) are associated with the production of two homologous protein toxins, TcdA and TcdB. The toxins are considered bona fide targets for clinical diagnosis as well as the development of novel prevention and therapeutic strategies. While there are extensive studies that document these efforts, there are several gaps in knowledge that could benefit from the creation of new research tools. First, we now appreciate that while TcdA sequences are conserved, TcdB sequences can vary across the span of circulating clinical isolates. An understanding of the TcdA and TcdB epitopes that drive broadly neutralizing antibody responses could advance the effort to identify safe and effective toxin-protein chimeras and fragments for vaccine development. Further, an understanding of TcdA and TcdB concentration changes in vivo can guide research into how host and microbiome-focused interventions affect the virulence potential of C. difficile. We have developed a panel of alpaca-derived nanobodies that bind specific structural and functional domains of TcdA and TcdB. We note that many of the potent neutralizers of TcdA bind epitopes within the delivery domain, a finding that could reflect roles of the delivery domain in receptor binding and/or the conserved role of pore-formation in the delivery of the toxin enzyme domains to the cytosol. In contrast, neutralizing epitopes for TcdB were found in multiple domains. The nanobodies were also used for the creation of sandwich ELISA assays that allow for quantitation of TcdA and/or TcdB in vitro and in the cecal and fecal contents of infected mice. We anticipate these reagents and assays will allow researchers to monitor the dynamics of TcdA and TcdB production over time, and the impact of various experimental interventions on toxin production in vivo. Author SummaryC. difficile (C. diff) is a leading cause of diarrhea and is recognized as an urgent threat by the Centers for Disease Control. Disease symptoms are caused by two large, similar, protein toxins, TcdA and TcdB. These toxins are drug targets and are also important for diagnosis. Despite their clear importance, the understanding of how to neutralize toxin activity is incomplete, and there are no freely available tools to quantify toxin concentration in research studies. To address these issues, we have developed nanobodies that bind and neutralize TcdA and TcdB and have also used these nanobodies to develop quantitative assays for TcdA and TcdB detection. Neutralization studies led us to discover that many of the potent neutralizers of TcdA bind epitopes within the delivery domain. This finding suggests either a role for the delivery domain in receptor binding or that the nanobodies block pore-formation and thereby inhibit delivery of the toxin enzyme domains to the cytosol. The availability of nanobody assays that can differentiate the quantities of TcdA from TcdB should permit a better understanding of toxin-specific effects and how toxin levels change over the course of infection.

microbiology↗