Nanobodies as CAR T receptors, Antivirals, and Drug-Delivery Agents
Using a unique pipeline to produce high-quality nanobodies with diversity in epitope recognition
Nanobodies have strong potential for rapid, low-cost, reliable applications in diagnostics and therapeutics. Nanobodies are high-affinity, stable antibodies of about 100 amino acids that are easily produced by bacteria as recombinant proteins. They can be modified to optimize their physiological half-life, oligomerized to increase their efficacy as viral-neutralizing agents, or combined in nanobody cocktails for synergistic improvements in activity. They may also be engineered to act as the receptors on CAR T cells.
Nanobodies originate from the unique single-chain antibodies of domesticated llamas. They are one-tenth the size of monoclonal antibodies, so they are more stable and resistant to denaturation. This makes them suitable for a variety of point-of-care diagnostic tests and therapeutics, including treatments that can be delivered topically or via inhaler.
The nanobody-generating pipeline used by the Aitchison group differs from typical methods that express a library of cDNAs in yeast or phage and competitively select target-binding nanobodies in vitro. Instead, Dr. Aitchison and colleagues immunize llamas, taking full advantage of the complex repertoire of nanobodies generated by in vivo affinity maturation. A variety of candidate nanobodies are obtained from llama serum and tested by surface plasmon resonance and in vitro assays for antigen binding and desired activities such as virus neutralization. Mass spectrometry determines the amino acid sequences of the best candidates and RNA-seq data from the donor llamas yields sequences for expression in E. coli. Within 2 weeks of obtaining blood, the Aitchison group can identify nanobodies with picomolar affinity for target proteins.
The Aitchison lab used the in vivo maturation method to generate a library of hundreds of nanobodies against the SARS-CoV-2 spike protein. Many bind to immune escape mutants including omicron variants. These nanobodies neutralize SARS-CoV-2 in vitro, ex vivo, and in animal models. These reagents are ready for further development—including for potential direct delivery in nebulized form into the lungs to treat COVID-19. This work serves as a proof-of-concept of the diverse, versatile nanobodies generated by the Aitchison group's strategy.
Dr. Aitchison is interested in partnerships to develop nanobodies into clinical resources of interest to an industry partner. Potential applications include rapid diagnosis from lateral-flow strips, including for emerging infectious diseases. Cancer treatment possibilities include selecting and optimizing nanobody sequences for use as the antigen-binding components of CAR T cells, including bivalent CAR T cells directed at cancer-specific biomarkers. Purified nanobodies could be used for targeted delivery of drugs or radionuclides. Their small size ensures that drugs or radionuclides that do not bind their target are rapidly cleared, reducing the potential for toxicity. Nanobodies are more stable than monoclonal antibodies and might have topical uses, for example in treatment of skin diseases such as psoriasis.
Stage of Development
- Preclinical in vitro
- Preclinical ex vivo
- Preclinical in vivo
- Collaborative research and development
- Sponsored research agreement
- Licensing agreement
- Consultation agreement
- Clinical trials
- Investigator-initiated clinical trials
- Contract research
- Clinical device development
- Mast FD, Fridy PC, Ketaren NE, Wang J, Jacobs EY, Olivier JP, Aitchison JD, et al. Highly synergistic combinations of nanobodies that target SARS-CoV-2 and are resistant to escape. Elife. 2021. 10:e73027. doi: 10.7554/eLife.73027.
- Fridy PC, Li Y, Keegan S, Thompson MK, Nudelman I, Scheid JF, Oeffinger M, Nussenzweig MC, Fenyö D, Chait BT, Rout MP. A robust pipeline for rapid production of versatile nanobody repertoires. Nat Methods 2014. 11(12):1253-60.