Editing Antibody Design by AI (section)

== Library Design and Contruction ==

Single framework library generation

A gene fragment encoding the germline framework combination IGHV3-23 and IGKV1-39 was synthesized by Invitrogen’s GeneArt service in Fab format and cloned into a phagemid vector serving as the base template.

IGHV3-23 and IGKV1-39 were used as they display a favorable framework combination for a phage display library and this framework combination is used in several therapeutic antibodies including trastuzumab and bevacizumab (Tiller et al., 2013). The phagemid vector consists of Ampicillin resistance, ColE1 origin, M13 origin and a bi-cistronic expression cassette under a lac promotor with OmpA - light chain followed by PhoA–heavy chain – '''Amber stop''' – truncated pIII (amino acids '''231 – 406''').

Only '''CDR-H3 '''was diversified and primers were designed to incorporate&nbsp;'''all naturally occurring amino acids excluding cysteine '''(free cysteines could form disulfide bonds), '''and asparagine''' (asparagine in conjunction with certain amino acids could undergo deamidation or become glycosylated) using trinucleotide technology (ELLA Biotech). Lengths between '''10 and 16 amino acids and 18 '''amino acids were allowed, in which the last two amino acids were kept constant with the sequence '''Asp-Tyr''' for length '''10 to 16''' and '''Asp-Val''' for length '''18'''.

The design of the final two CDR-H3 amino acids reflects human VDJ recombination. '''Short CDR-H3s''' more often use J-fragment '''IGHJ4''' with “'''DY'''” at the end of CDRH3 while '''longer '''CDR-H3s (here 18 aa) more often use&nbsp;'''IGHJ6 '''with “'''DV'''” at the end of CDR-H3.

Library inserts were generated by&nbsp;'''PCR''' using '''Phusion '''High Fidelity DNA polymerase (NEB Biolabs). The resulting CDR-H3 library inserts were ligated into the base template, transformed into E.coli&nbsp;'''TG1 DUO''' (Lucigen) with a minimal library size of '''1E+09''' transformants per CDRH3 length and phages were produced using '''M13KO7''' helper phage (NEB Biolabs) using standard previously described protocols (Proetzel and Ebersbach, 2016).