Abstract Background: Metastatic breast cancer is the second leading cause of cancer-related deaths of women in the United States. First line treatment for Her2-positive metastatic breast cancer patient is combination therapy involving pertuzumab, trastuzumab, and docetaxel. Both pertuzumab and trastuzumab targets Her2, but works in complementary ways in increasing the cytotoxicity. Indeed, combination of these two antibody drugs for treatment has shown excellent survival benefits. We hypothesized that by covalently attaching these two antibodies to a single nanoparticle would accelerate the cancer cell death in lower concentrations of antibodies. In this study, we covalently conjugated trastuzumab and pertuzumab to gold nanoparticle (AuNP) and investigated the cytotoxicity in metastatic breast cancer cells. Methods: Gold nanoparticles of core size 16 nm were prepared and modified using heterofunctional end terminal carboxylic polyethylene glycol (PEG) linker. The carboxyl terminals were activated by conventional EDC/NHS procedure followed by simultaneous addition of antibodies. The dual antibody gold nanoconjugates (AuNP-Dual Ab) was purified by HPLC using size exclusion column and characterized in detail by routine analytical techniques. Detailed proteomic analysis was performed on the dual antibody gold nanoconjugate. The binding affinity of AuNP-Dual Ab was investigated on HER2+ve SkBR3 cells using dark field microscopy. In vitro cytotoxicity evaluations of AuNP-Dual Ab were performed on HER2 expressing SKBR3 and BT474 cell lines. The effect of AuNP-Dual Ab on signal transduction and phosphorylation status mediated by HER family proteins were evaluated by monitoring the HER2, HER3, AKT, ERK, MEK protein expression through western blot analysis. In vivo studies have been performed in metastatic breast tumor mice models to evaluate the therapeutic efficacy of the nanoconjugate. Results: AuNP-Dual Ab was isolated from the free antibodies by HPLC. We confirmed both the presence and the structural identity of antibodies covalently attached to AuNP using proteomics. TEM immunohistochemistry and dark field microscopy showed AuNP-Dual-Ab possess high receptor binding affinity with Her2+ve breast cancer cells. We investigated the cytotoxicity of AuNP-Dual-Ab in SKBR-3 and BT474 human breast cancer cells. Our studies reveal that AuNP-Dual-Ab is 60 fold higher cytotoxic than non-antibody coated AuNPs, antibodies, and combination antibodies. Western blot analysis showed down regulation of AkT and phospho-AkT proteins while expression of MEK and phospho-MEK proteins was un-altered. We believe the increase in cytotoxicity is possibly due to effective transport of antibodies to cancer cells. Conclusions: The in vitro studies demonstrate that AuNP-Dual Ab is promising and can potentially overcome the problem of resistance. In vivo studies are ongoing on metastatic breast tumor mice models for therapy evaluation. Citation Format: Ajit Zambre, Anandhi Upendran, Matthew Leevy, Sarah Chapman, Raghuraman Kannan. Dual antibody gold nanoconjugate significantly enhances cytotoxicity in metastatic breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1332.