Tagging Nanobodies Precisely

Reflecting work in the Distefano Lab

Published here October 5, 2026

Farnesyltransferase Enables Modular Assembly of Dual-Functional Nanobody Conjugates for in Vivo Imaging of Inflammation

Sneha Venkatachalapathy, Yoon Ho Lee, Ali Salehi Farid, Heydar Moravej, Sina Djafari Rouhani, Jennifer E. Rowley, Mohammad Rashidian, Mark D. Distefano

Angew. Chem. Int. Ed. 2026, e5009703. https://doi.org/10.1002/anie.5009703

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Non-invasive PET imaging of inflammation requires probes that are target-specific, pharmacokinetically well-behaved, and chemically homogeneous. Nanobodies, the ~14 kDa single-domain antibody fragments derived from camelid heavy-chain-only antibodies, satisfy these demands in principle: their small size, high stability, and C-terminus orientation away from the antigen-binding site make them natural candidates for site-selective functionalization. The obstacle lies in the functionalization itself. Conventional chemical conjugation reacts indiscriminately with accessible residues, yielding heterogeneous mixtures in which payload position, stoichiometry, and binding capacity vary from molecule to molecule. Enzymatic alternatives such as sortase A offer more precision but require high molar excesses of peptide nucleophile and extended reaction times. Compounding the problem, unmodified nanobodies undergo rapid glomerular filtration and non-specific renal reabsorption, so any practical imaging agent must carry both a radiometal chelator and a pharmacokinetic modifier such as PEG, ideally in a single controlled workflow.

Researchers in the Distefano Group at the University of Minnesota and the Rashidian Group at the Dana-Farber Cancer Institute, published in Angewandte Chemie International Edition, report a chemoenzymatic platform built around protein farnesyltransferase, FTase, that resolves this challenge in one enzymatic step. FTase recognizes a C-terminal CaaX tetrapeptide motif appended to the nanobody and transfers an isoprenoid group from a synthetic farnesyl diphosphate analog onto the cysteine of that motif, forming a stable thioether bond. The key design advance is a trifunctional analog carrying both an azide and an aldehyde handle on the same isoprenoid scaffold. Molecular docking confirmed that this analog adopts a conformation in the FTase active site closely matching that of the natural substrate. Once the dual-handle intermediate is installed on the nanobody, strain-promoted azide-alkyne cycloaddition connects the deferoxamine chelator for 89Zr radiolabeling, while an inverse electron demand Diels-Alder reaction attaches a PEG chain of defined molecular weight through the aldehyde site. The sequential orthogonality of these reactions eliminates cross-reactivity and delivers well-defined, dual-labeled constructs at low micromolar substrate concentrations.

Applied to nanobodies targeting CD11b on myeloid cells and CD45 on all leukocytes, the strategy produced 89Zr-labeled, PEGylated probes that enabled high-contrast PET imaging of immune organs and localized inflammation in a murine complete Freund's adjuvant model. PEG size emerged as a tunable parameter governing renal clearance kinetics and imaging time windows. The platform's compatibility with any protein bearing a C-terminal CaaX motif and its near-quantitative conversions under mild aqueous conditions position it as a broadly applicable route to chemically defined nanobody-based imaging agents for inflammation and other immune-mediated diseases.

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Author

Mark Distefano is currently a Distinguished McKnight Professor of Chemistry and Medicinal Chemistry at the University of Minnesota. He received his B.A. degree in Chemistry and Biochemistry from the University of California at Berkeley, his Ph.D. degree from Massachusetts Institute of Technology and was a postdoctoral fellow at the California Institute of Technology. He has published more than 200 research articles, book chapters and reviews.

Mark is currently Editor in Chief for Bioorganic Chemistry, a member of the Executive Committee for the American Peptide Society and Chair Elect for the Biochemistry and Chemical Biology Division of the American Chemical Society. His current research is focused on understanding the role of protein prenylation in disease including Alzheimer’s disease and cancer, and in exploiting protein lipid modification for biotechnology applications.

Tagging Nanobodies Precisely

FIGURE 1 | Reaction catalyzed by farnesyltransferase and substrates and analogs used in this study. a| Schematic representation of farnesylation reaction with a protein containing a CaaX box at its C terminus. b| Structures of FPP and alternative substrates 1 and 2 . c| Docking of 1 with FTase. d| Docking of 2 with FTase, superimposed with the natural substrate, FPP, bound to FTase, from x-ray crystallographic data; PDB ID: 1JCR. FPP is shown in cyan, synthetic substrates are shown in green, C, blue, N, red, O, and orange, P. The protein structure is shown in white.

Author Spotlight

Author

Sneha Venkatachalapathy was born and raised in Thanjavur, Tamil Nadu, India. She received her B.Sc., Research, in Chemistry with a minor in Biotechnology from Shiv Nadar University, India, in 2020. As an undergraduate researcher, she worked across three synthetic chemistry laboratories on carbohydrate–drug conjugates, peptide-based sensors, and blue LED light-mediated heterocycle synthesis. She recently completed here Ph.D. in Chemistry at the University of Minnesota under the guidance of Prof. Mark D. Distefano.

Sneha's doctoral research focused on developing farnesyltransferase-mediated chemoenzymatic strategies for the site-selective modification of nanobodies. Her work integrated protein engineering, bioorthogonal chemistry, and molecular imaging to construct multifunctional nanobody conjugates and PET imaging agents for the sensitive, noninvasive detection of inflammation. She plans to start work at a biotech startup in India soon.