Semisynthetic Phage Display Library Construction: Generation of Single <i>-</i> Chain Variable Fragment Secondary Libraries

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.