Structure-Function Studies of a Cell Penetrating Antibody that Inhibits DNA Repair

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In this multi-PI R21 application, we propose to advance the anti-cancer efficacy of a tumor targeting and cell-penetrating antibody (3E10) that directly inhibits the DNA repair factor, RAD51, and thereby suppresses homology-directed DNA repair (HDR), sensitizes cancer cells to radiation, and is synthetically lethal to BRCA2- and PTEN-deficient cells. We will do so by a combination of structural, biochemical, cell-based and animal tumor studies. This proposal will leverage complementary expertise of the two PI’s in structural biology of DNA binding proteins and macromolecular machines (Bleichert) and in DNA repair, tumor biology and pre-clinical development of cancer therapeutics (Glazer). 3E10 was derived from a mouse model of systemic lupus erythematosus and was initially characterized as an anti-DNA antibody that could penetrate cells and localize in the nucleus. Our subsequent work further defined 3E10 as an inhibitor of the RAD51 recombinase via direct binding, providing a mechanistic explanation for its effect on DNA repair. Importantly, we have also established that 3E10 targets solid tumors in vivo in mice with extraordinarily high specificity compared to healthy tissues. This targeting specificity is due to the mechanism of cell penetration, which depends on engagement with the ENT2 transporter, which is highly over-expressed in human cancers compared to healthy tissues, and the presence of extracellular DNA in the tumor microenvironment, which promotes 3E10 interaction with ENT2. The ability of 3E10 to target tumors and directly penetrate cells and nuclei distinguishes it from all other antibodies currently approved for cancer therapy; as such, 3E10 potentially represents a totally new class of cancer therapy agents. In recent work studying variants of 3E10 with specific amino acid substitutions, we have discovered that the DNA binding, cell penetration, and tumor targeting properties of 3E10 are separable from RAD51 binding. This has led us to hypothesize that it should be possible to generate variants of 3E10 that have increased RAD51 binding affinity while retaining the cell penetration and tumor targeting properties. We further hypothesize that such a 3E10 variant would be an even more potent RAD51 inhibitor and a superior anti-cancer agent for DNA repair inhibition, radiation sensitization and synthetic lethal approaches. With increased synthetic lethality against BRCA2- and PTEN-deficient cancers, such a second-generation antibody would be particularly useful against the wide range of human cancers in which these factors are deficient. In this application, we propose to conduct structural studies of the 3E10 antibody to gain insight into the molecular basis of its DNA binding and RAD51 inhibition. This work will guide the design of novel 3E10 variants that will be tested for DNA binding, cell penetration, RAD51 inhibition, synthetic lethality, and tumor targeting, with the goal of identifying an optimized second-generation antibody to advance for clinical development. Consequently, the proposed work has a very high potential for translation into the clinic and could have a direct and substantial impact on human health.