image: 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.
Credit: 2024 Rackear et al.
“Overall, the data presented in this study affirm that humanizing 3E10 preserves its crucial biological properties essential for therapeutic efficacy.”
BUFFALO, NY- October 21, 2024 – A new research paper was published in Oncotarget's Volume 15 on October 1, 2024, entitled, “Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition.”
As highlighted in the abstract, monoclonal antibody therapies for cancer have shown extraordinary clinical success in recent years. However, these strategies are primarily limited to targeting specific cell surface antigens, despite many disease targets being located intracellularly.
In their paper, researchers Madison Rackear, Elias Quijano, Zaira Ianniello, Daniel A. Colón-Ríos, Adam Krysztofiak, Rashed Abdullah, Yanfeng Liu, Faye A. Rogers, Dale L. Ludwig, Rohini Dwivedi, Franziska Bleichert, and Peter M. Glazer from the Departments of Therapeutic Radiology and Genetics at Yale University School of Medicine, Gennao Bio, and the Department of Molecular Biophysics and Biochemistry at Yale University report on the humanization of the full-length, nucleic acid-binding monoclonal lupus-derived autoantibody 3E10, which exhibits a novel mechanism for cell penetration and tumor-specific targeting.
The authors compare humanized variants of 3E10 and demonstrate that cell uptake relies on the nucleoside transporter ENT2. They also find that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding.
“We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro.”
Continue reading: DOI: https://doi.org/10.18632/oncotarget.28651
Correspondence to: Peter M. Glazer - peter.glazer@yale.edu
Keywords: cancer, 3E10, cell penetration, nucleic acid binding, nucleic acid delivery, RAD51
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About Oncotarget:
Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science.
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Journal
Oncotarget
Method of Research
News article
Subject of Research
Cells
Article Title
Next-generation cell-penetrating antibodies for tumor targeting and RAD51 inhibition
Article Publication Date
1-Oct-2024
COI Statement
E.Q. and P.M.G. are inventors on patent applications assigned to Yale University related to the work and are co-founders, hold equity in, and consult for Gennao Bio, which has licensed related intellectual property from Yale. R.A. and D.L.L. are employees of and hold equity in Gennao Bio. P.M.G. also holds equity in Patrys, Ltd., is a founder of and consultant for Cybrexa Therapeutics, and is a consultant for pHLIP Inc., none of which have a direct connection to this manuscript.