Strategy for capturing CDRH3 diversity into a human antibody library.

<p>(A) Natural diversity was incorporated from naive human donors and BalbC mice either kept naive or immunized with hIFNγ or hCCL5. (B) Their CDRH3 repertoires were extracted and amplified by PCR. During this step, recognition sites for the type IIS enzyme FokI were added. CDR3 are represented in dark grey, CDR1 and 2 in light grey. (C) Following digestion by FokI, repertoires of CDRH3 were cloned into an acceptor library itself digested with BsmBI. This second type IIS enzyme allowed the removal of a non diversified stuffer sequence (“S”) at the location of CDRH3 and the generation of compatible cohesive ends for the incorporation of CDRH3. The acceptor library combines 7 human VH and 7 human VL in a format of scFv as well as additional synthetic diversity in the CDRL3 (“L3” in the picture). Tags allow for purification while gIII allows for display on phage. After cloning of CDRH3, resulting libraries were analyzed by NGS covering the location of the CDRH3 and part of VH at the 5′ border (102 bp covered). (D) Similarly, sequences from a library of synthetic CDRH3, SnA, were explored in the context of the human acceptor library. Prior to the amplification, the repertoire of SnA was biased in vitro towards hIFNγ by performing three rounds of selection by phage display.