A Novel Cell-Penetrating Antibody Fragment Inhibits the DNA Repair Protein RAD51

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• DNA damaging chemotherapies are vital cancer treatments.
• Cancer cells can up-regulate DNA repair mechanisms, reducing chemotherapy efficacy.
• Homologous recombination repair, mediated by RAD51, is a key DNA repair pathway.

Purpose of the Study:

• To inhibit RAD51 function using a novel antibody fragment.
• To restore the effectiveness of DNA damaging chemotherapy by blocking DNA repair.
• To develop a cell-penetrating antibody fragment for intracellular targeting.

Main Methods:

• Phage-display technology was used to generate a synthetic antibody fragment against human RAD51.
• The antibody fragment's affinity and inhibition of RAD51 ssDNA binding were assessed in vitro.
• An intrabody fragment was created for intracellular targeting, and a cell-penetrating peptide (iPTD) was fused to generate a therapeutic candidate.

Main Results:

• A high-affinity antibody fragment (K_D = 8.1 nM) targeting human RAD51 was successfully generated.
• The intrabody fragment induced significant growth inhibition in human cells.
• The iPTD-fused antibody fragment effectively entered living cells and potentiated the cytotoxic effects of a DNA alkylating agent.

Conclusions:

• Inhibiting RAD51 via a novel antibody fragment can overcome chemotherapy resistance.
• The iPTD peptide facilitates intracellular delivery of antibody fragments, enabling targeting of previously inaccessible intracellular targets.
• This strategy holds promise for enhancing cancer therapy and developing new treatments for intracellular targets.