All Publications

  • A general platform for targeting MHC-II antigens via a single loop. bioRxiv : the preprint server for biology Du, H., Liu, J., Jude, K. M., Yang, X., Li, Y., Bell, B., Yang, H., Kassardjian, A., Mobedi, A., Parekh, U., Sperberg, R. A., Julien, J. P., Mellins, E. D., Garcia, K. C., Huang, P. S. 2024


    Class-II major histocompatibility complexes (MHC-IIs) are central to the communications between CD4+ T cells and antigen presenting cells (APCs), but intrinsic structural features associated with MHC-II make it difficult to develop a general targeting system with high affinity and antigen specificity. Here, we introduce a protein platform, Targeted Recognition of Antigen-MHC Complex Reporter for MHC-II (TRACeR-II), to enable the rapid development of peptide-specific MHC-II binders. TRACeR-II has a small helical bundle scaffold and uses an unconventional mechanism to recognize antigens via a single loop. This unique antigen-recognition mechanism renders this platform highly versatile and amenable to direct structural modeling of the interactions with the antigen. We demonstrate that TRACeR-II binders can be rapidly evolved across multiple alleles, while computational protein design can produce specific binding sequences for a SARS-CoV-2 peptide of unknown complex structure. TRACeR-II sheds light on a simple and straightforward approach to address the MHC peptide targeting challenge, without relying on combinatorial selection on complementarity determining region (CDR) loops. It presents a promising basis for further exploration in immune response modulation as well as a broad range of theragnostic applications.

    View details for DOI 10.1101/2024.01.26.577489

    View details for PubMedID 38352315

    View details for PubMedCentralID PMC10862749

  • Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins. Proceedings of the National Academy of Sciences of the United States of America Misson Mindrebo, L., Liu, H., Ozorowski, G., Tran, Q., Woehl, J., Khalek, I., Smith, J. M., Barman, S., Zhao, F., Keating, C., Limbo, O., Verma, M., Liu, J., Stanfield, R. L., Zhu, X., Turner, H. L., Sok, D., Huang, P. S., Burton, D. R., Ward, A. B., Wilson, I. A., Jardine, J. G. 2023; 120 (24): e2216612120


    Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules.

    View details for DOI 10.1073/pnas.2216612120

    View details for PubMedID 37276407

  • De Novo Design of a Highly Stable Ovoid TIM Barrel: Unlocking Pocket Shape towards Functional Design. Biodesign research Chu, A. E., Fernandez, D., Liu, J., Eguchi, R. R., Huang, P. S. 2022; 2022: 9842315


    The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels.

    View details for DOI 10.34133/2022/9842315

    View details for PubMedID 37850141

    View details for PubMedCentralID PMC10521652