Abstract Background: Improving drug delivery in oncology by developing antibodies targeted nanoparticles loaded with cytotoxics is a rising strategy in oncology. Determination of the optimal density of antibodies on nanoparticles surface remains an open question, mainly due to the difficulty in measuring accurately the actual level of coating. We have developed a stealth immunoliposome encapsulating docetaxel and harboring anti-Her2 trastuzumab. To better prototyping the nanoparticles, we have developed a method for the absolute quantitation of trastuzumab, so as to compare the performance of the immunoliposomes in terms of efficacy in breast cancer depending on the number of antibodies. Methods: Using flow cytometry we have developed a quick and quantitative assay to measure the amount of trastuzumab coated per docetaxel immunoliposome. Three batches of immunoliposomes exhibiting different densities of trastuzumab were synthesized using the standard thin-film method and a maleimide linker. Quality control was operated regarding size, docetaxel encapsulation and trastuzumab coating levels, including stability studies. In vitro and in vivo efficacy studies were performed on MDA-MB-453 used as a canonical model of Her-2+ human breast cancer cells. Results: The three batches of Immunoliposomes exhibited 330 ± 30 (batch1), 480 ± 110 (batch2) and 690 ± 80 (batch3) trastuzumab IgG molecules per liposome, respectively (p<0.01, One-Way Anova). In 2D apoptosis induction experiments, no difference was observed between free drugs and immunoliposomes, regardless of trastuzumab density. When shifting to 3D spheroids, higher antiproliferative efficacy was observed with batch2 as compared with other immunoliposomes or free drugs (+76%) or reference T-DM1 (+85%). Next, batch2 was further tested in mice bearing MDA-MB-453 xenografts. Immunoliposomes achieved a tumor reduction of 89 % when compared to free drugs and 66% as compared with T-DM1 (p<0.05, One-Way Anova). Conclusion: Our assay based on flow cytometry of submicron particles can accurately quantify the number of coated antibodies on single nanoparticles. In vitro studies demonstrated that maximal density of targeting agent on nanoparticles was not a prerequisite for maximal therapeutic effect. In vitro spheroids and in vivo studies in mice demonstrated higher efficacy of our immunoliposomes when compared to free trastuzumab docetaxel used in combo and antibody-drug conjugate T-DM1. Beyond the current project, this new quantitation method could be valuable when prototyping nanoparticles or conjugated drugs using monoclonal antibodies as targeting agent. Citation Format: Anne Rodallec, Corentin Franco, Stéphane Robert, Guillaume Sicard, Sarah Giacometti, Ariel Savina, Fanny Bouquet, Bruno Lacarelle, Joseph Ciccolini, Philippe Poncelet, Raphaelle Fanciullino. Impact of trastuzumab coating when prototyping immunoliposomes in breast cancer models: The more the merrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3000.