Harnessing Nanobodies for Precision Targeting of Proteoforms: Opportunities and Challenges in Therapeutics and Diagnostics

Abstract

Proteoforms are biologically distinct yet structurally similar proteins that play key roles in driving disease progression but are rarely accounted for in the development of therapeutics and diagnostics. Nanobodies (Nbs) have emerged as a therapeutic and diagnostic "silver bullet" as they possess unique structural and functional attributes that offer advantages over traditional antibodies. One of the most profound advantages of Nbs is the heightened sensitivity and ability to distinguish subtle changes in the conformation of a given protein. Thus, Nbs have significant potential as therapeutic and diagnostic agents that can identify and distinguish specific pathological proteoforms that underlie a given disease. However, there remain significant challenges in obtaining sufficient quantities and purities of specific proteoform antigens that are required for engineering proteoform-specific Nbs. Recent advancements in chemical biology tools for precision proteoform synthesis have made this task feasible for the first time. In this perspective, we discuss the advantages and challenges associated with developing proteoform-specific Nbs and how success in this endeavor will significantly advance the fields of therapeutics and diagnostics.

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(A) The structures of conventional antibodies (left) compared with a heavy-chain-only antibody (HCAb) from camelid (center) and the variable heavy chain fragment of the HCAb known as a “nanobody” (right). (B) Detailed structure of a nanobody is shown, including CDRs (colored regions) (PDB ID: 1I3V).

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Overview of human proteoform production. Human genes (DNA) are transcribed into RNA, which undergoes processing and splicing to produce alternatively spliced mRNAs (1-typical splicing, 2-mutually exclusive exons/exon skipping, 3-intron retention). The resulting variants are translated into initial proteoforms, which can then undergo post-translational modifications, producing further proteoforms.

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Comparison of traditional Nb development workflows versus proteoform-targeted strategies. The left panel illustrates the conventional process for generating nanobodies against general protein targets, while the right panel outlines a workflow optimized for developing nanobodies specific to proteoforms such as post-translationally modified proteins. (1) Target identification: traditional methods permit flexible target selection, whereas proteoform targeting requires the identification of specific modifications via mass spectrometry. (2) Antigen production: standard antigens are produced through recombinant expression or enrichment from cells, but proteoform-specific antigens necessitate precise chemical biology approaches. (3) Library selection: conventional workflows prioritize positive selection against the antigen, while proteoform workflows require both positive and negative selections to discriminate between modified and unmodified forms. (4) Nanobody validation: for traditional targets, validation emphasizes affinity and specificity; for proteoforms, validation must also assess selectivity between modified and unmodified variants.