Generation of Human Single Domain Antibody Repertoires by Kunkel Mutagenesis

Abstract

Human antibody single domains are a promising new class of antibody fragments. Here we describe methods for the cloning of human V(H) and V(L) genes into phage and phagemid vectors. Furthermore, we provide detailed protocols for the generation of single domain antibody libraries by Kunkel mutagenesis and the analysis of diversity by DNA sequencing and superantigen binding.

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies

“…For the design of site-directed mutagenesis libraries, we utilized previously reported crystal structures of antibodies developed against SARS-CoV-1 in complex with either cognate RBD (80R -PDB ID 2ghw 20 ; m396 -PDB ID 2dd8 21 ) or SARS-CoV-2 RBD in the case of the cross-specific CR3022 antibody (PDB ID 6w41 15 ). Based on the structures, we selected contact and proximal residues in CDR regions of VH and VL, which were targeted for diversification by Kunkel mutagenesis 16 (Fig. 1A-C and Supplementary Table 1; all six CDR regions were targeted for CR3014 for which no structural information has been reported).…”

“…m396, CR3022, CR3014, and 80R scFv were gene synthesized (Genscript) and cloned into the pHEN1 phagemid vector. Site-directed mutagenesis was carried out by Kunkel mutagenesis 16 . In brief, phagemid vectors were transformed into E. coli CJ236, single colonies grown in 2xYT media supplemented with 100 µg/mL ampicillin, 10 µg/mL chloramphenicol and 2% glucose until reaching an OD600nm of 0.4.…”

“…For the re-engineering strategy, we focused on two well-established in vitro methods for antibody affinity maturation: (I) site-directed mutagenesis of complementarity determining regions (CDR) of human variable domains 16 and (II) light chain shuffling 17 ( Fig. 1B-D).…”

Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies

“…Synthetic libraries were constructed essentially as described previously (25). For the generation of the Garvan I repertoire, single-stranded DNA encoding the model V H domain HEL4 in the phage vector FdMyc was isolated using a QIAprep spin M13 kit (Qiagen).…”

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies

“…16 Unlike m396, CR3014 and 80R, CR3022 displays residual binding to SARS-CoV-2 RBD; however, it does not detectably neutralize live SARS-CoV-2 virus. 16,18 For the re-engineering strategy, we focused on two wellestablished in vitro methods for antibody affinity maturation: 1) site-directed mutagenesis of complementarity-determining regions (CDRs) of human variable domains; 19 and 2) light chain shuffling 20 (Figure 1(b-d)). Library design based on the reported structures of m396, CR3022 and 80R in complex with RBD was used for the construction of site-directed mutagenesis repertoires, with antibody contact residues with antigen targeted for diversification (residues underlined in Figure 1 (b)).…”