Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library

Picloram-specific variable fragments (VHHs) of heavy chain antibodies (HCAbs) were selected from a naı̈ve-llama library using ribosome display technology. A cDNA library of VHHs was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different VHHs all belonging to the VHH subfamily 1 were isolated. Following another three cycles of selection, only two of the seven VHHs persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured KDs were 3 and 254 μM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naı̈ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a VHH naı̈ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.