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Abstract
Background VHH domains, derived from the variable heavy chain of the heavy-chain-only IgG2 and IgG3 domains in camelids, represent a small, single-domain antibody fragment of ~15kDa. These small molecules are becoming increasingly popular in cancer diagnostics and treatments due to distinct advantages: 1. Size: the small size of VHHs enables efficient tissue penetration of dense tumors and transport across the blood brain barrier – a current challenge for tumor detection in the central nervous system. 2. Stability: the unique properties of VHHs provide stability across a variety of environments including temperature and pH. 3. Solubility: VHHs are quickly cleared from the blood and the kidneys decreasing toxicity and immunogenicity, reducing adverse side effects. These unique properties have made VHHs successful contenders for molecular imaging uses like PET, image-guided surgery, MRIs, and anti-cancer therapeutics like drug conjugates, bispecific therapeutic molecules, checkpoint inhibitors, and radioimmunotherapy.1 In this study we show how the AbNano™ VHH Naïve Library can be used to quickly and efficiently identify target-specific VHHs.
Methods The AbNano™ VHH Naïve Library is a fully natural, single-domain naïve library constructed from 103 naïve animals: 77 llamas and 26 alpacas. This library was derived from 1.51 x 1010 camelid PBMCs and built as multiple sublibraries to a total transformation count 1.12 x 1010 CFU. This fully natural library has higher diversity in framework regions than synthetic libraries. The AbNano™ VHH Naïve Library yielded early hit molecules that bound immune and immuno-oncology targets. These VHH binders may have functionality in flow cytometry, immunohistochemistry, western blot, and immunoprecipitation.
Results Initial screens identified VHHs with nanomolar binding affinities to key immune and therapeutic targets such as PD-L1 and EGFR. Hits identified from panning of the AbNano™ VHH Naïve Library have shown EC50 measurements from 10-9 M to 10-6 M. Sixty-eight anti-PD-L1 VHH clones were enriched, and one clone was further characterized for its use in live cell flow cytometry. This clone showed positive cell staining on live, PD-L1+ HDLM-2 cells and no staining on live, PD-L1- Ramos cells, as seen in (figure 1).
Conclusions Here, we present the construction and early validation of a large naïve library from llamas and alpacas. The data suggests that the AbNano™ VHH Naïve Library may be well-suited for rapid discovery of VHH domains binding to therapeutic targets with varying levels of affinity. Additionally, these data further support the potential to identify application-specific VHHs from the naïve library.
Reference
Cong Y, Devoogdt N, Lambin P, Dubois LJ, Yaromina A. Promising diagnostic and therapeutic approaches based on VHHs for cancer management. Cancers 2024;16(2):371. https://doi.org/10.3390/cancers16020371
PD-L1 VHH staining on positive and negative cells
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