We have developed and optimized a new technology, termed Nanobody Seq/MS, which combines deep sequencing with mass spectrometry to rapidly generate large numbers of bacterially-expressed nanobodies with extremely high affinity and specificity. In our nanobody development pipeline, we first immunize llamas with the antigen target. After a strong immune response is elicited, we collect lymphocytes from bone marrow, highly enriched for active B cells. We then isolate cDNA from these cells, and perform high-throughput sequencing on the PCR-amplified variable region (VHH) of heavy-chain-only IgG variants (HCAbs). In parallel, we collect animal sera, and affinity purify HCAbs with strong affinity and specificity to each antigen. The purified HCAbs are then analyzed by MS, and by correlating peptides to the DNA database of full-length sequences, we can identify candidate nanobody clones. We also now utilize a newly-modified orthogonal and complementary approach, based on yeast surface display, to expand our nanobody repertoires.
We first demonstrated the effectiveness of this approach by generating more than 30 high affinity nanobodies against GFP and mCherry.
Since this initial proof of principle, we have applied our nanobody generation pipeline to over 30 new antigens, generating a total of more than 500 unique nanobodies. Most recently, we generated a panel of 116 nanobodies against the SARS-CoV-2 spike protein, most of which were capable of neutralizing viral infection. These neutralizing nanobodies act through a diverse range of epitopes and mechanisms, retain activity against the most recent SARS-CoV-2 variants, and can act in synergistic combinations that dramatically increase potency.
Among our current interests, we are developing new nanobodies for use as therapeutics against cancer targets and infectious agents.