Specific targeting of MR1-antigen complexes using nanobodies

Hussain, M. H. S.; Redmond, S. J.; Awad, W.; Xu, C.; Soliman, C.; Ciacchi, L.; Gonzalez, A. P.; Mak, J. Y.; Fairlie, D. P.; McCluskey, J.; Uldrich, A. P.; Rossjohn, J.; Godfrey, D. I.; Koay, H.-F.; Gherardin, N. A.

doi:10.64898/2026.03.22.713551

bioRxiv · 10.64898/2026.03.22.713551

Specific targeting of MR1-antigen complexes using nanobodies

Hussain, M. H. S.·Redmond, S. J.·Awad, W.·Xu, C.·Soliman, C.·Ciacchi, L.·Gonzalez, A. P.·Mak, J. Y.·Fairlie, D. P.·McCluskey, J.·Uldrich, A. P.·Rossjohn, J.·Godfrey, D. I.·Koay, H.-F.·Gherardin, N. A.

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Abstract

T cell receptor mimic (TCRm) antibodies and nanobodies that specifically bind peptide-HLA complexes have great therapeutic potential, as they can target polymorphic HLA on tumour cells furnishing peptides derived from tumour-associated antigens. MR1 is an MHC class-I-like molecule that exhibits limited polymorphism that binds and presents conserved metabolites, such as 5-OP-RU, derived from microbial riboflavin biosynthesis. Whether antibodies targeting such MR1-5-OP-RU complexes can be generated remains unclear. Using yeast display technology and in vitro affinity maturation, a nanobody with high affinity and fine specificity toward MR1-5-OP-RU complex was generated. These nanobodies bind both mouse and human MR1-5-OP-RU and inhibited MAIT cell responses to 5-OP-RU in vitro and in vivo demonstrating therapeutic potential. Moreover, we provide a molecular basis underpinning the fine specificity of these nanobodies, solving the crystal structures of MR1 in complex with either 5-OP-RU or Ac-6-FP. Here, the nanobody co-bound MR1 and 5-OP-RU, akin to a TCRm antibody. Moreover, we engineer bispecific antibodies targeting both MR1-5-OP-RU and CD3, that drive broad T cell killing of bacterially-infected cells as well as tumour cells treated with 5-OP-RU, thereby providing proof-of-principle for targeting the MR1 molecule with with TCRm-based nanobodies. One Sentence SummaryWe report the development of a nanobody targeting MR1-5-OP-RU complex and demonstrate its utility to modulate MAIT cells responses, and as a bispecific engager.

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Hussain, M. H. S., Redmond, S. J., Awad, W., Xu, C., Soliman, C., Ciacchi, L., Gonzalez, A. P., Mak, J. Y., Fairlie, D. P., McCluskey, J., Uldrich, A. P., Rossjohn, J., Godfrey, D. I., Koay, H.-F., Gherardin, N. A.. 2026-03-25. Specific targeting of MR1-antigen complexes using nanobodies. https://doi.org/10.64898/2026.03.22.713551

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