Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition (IMAGE)
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Figure 1: 3E10 humanization and heavy and light chain variant screening. (A) Diagram of the 3E10 antibody engineering process. The original murine wild-type 3E10 was modified to contain a human IgG1 Fc region. This chimera was subsequently engineered with a D31N mutation in heavy chain CDR1. CDR grafting was performed to produce a fully humanized IgG1 framework; 22 variants were created by introducing point mutations into the VH and VL regions outside of the CDRs. (B) Nucleic acid affinity screening of humanized 3E10 variants. The 22 full-length antibodies were screened for their affinity to a 20-mer poly(dT) DNA oligo by ELISA for EC50 determination. All EC50s presented in the heat map are normalized to a chimeric 3E10 D31N positive control. (C) Representative ELISA assay data for poly(dT) binding by humanized V66, V13, and V31, and chimeric D31N. One biological replicate was performed. Humanized variant EC50s are 5.933, 44.34, and 685.2 nM, respectively. (D) Variable heavy chain (top) and variable light chain (bottom) sequence alignments, with deviations from WT sequence highlighted in red if substitution is increasingly anionic, blue if substitution is increasingly cationic, and grey if no significant change in formal charge at physiological pH.
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2024 Rackear et al.
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