Fig. 5

Factors affecting immunotherapeutic efficacy of AVEC: anti-HER-2 × HBsAg. (a–d) Blood of the HBV vaccinated patients was depleted of erythrocytes and analyzed. Three main populations of cells were revealed by forward and side scattering (a). Fractions of cells were determined through cell counts (b). Various fractions of white blood cells (WBC) were sorted based upon clusters of differentiations’ display, so that the number of cells and inter-fractions’ ratios could be adjusted in the forthcoming experiments. The OV90 cells were labeled with anti-HER-2 × HBsAg followed by anti-Fc-R-BD and anti-HBsAg (d). These assays were repeated four times. The data presented are representative for all performed. (e) OV90 cells were treated at 37 °C with trastuzumab, biosimilar anti-HER-2, anti-HBV, and anti-HER-2 × HBsAg in erythrocyte-free blood from the HBV vaccinated patients, while concentrations of the complement system were adjusted for C1q and C3 as indicated on the diagram. Increasing concentrations of complement system components resulted in increased efficacy of the ovarian cancer cells killing as complement dependent cytotoxicity (CDC). Importantly, treatment with AVEC at nearly three times lower concentrations of C1q and C3 resulted in nearly the same therapeutic efficacy as naked anti-HER-2 antibodies at three times higher concentrations. This efficacy was at significantly higher statistical rate than with the relevant isotype antibodies. This impact onto cancer cells was also of statistical significance difference over that onto HOSE and HAE cells. These assays were repeated four times. The data presented are representative for all performed. I isotype antibodies. (f) OV90 cells were treated at 37 °C with trastuzumab, biosimilar anti-HER-2, anti-HBV, and anti-HER-2 × HBsAg in erythrocyte-free blood from the HBV vaccinated patients, while the ratios between cytotoxic effector cells and the ovarian cancer cells were adjusted as indicated on the diagram. Increasing the ratio of the effector cytotoxic cells to ovarian cancer cells clearly increased efficacy of killing cancer cells by antibody dependent cytotoxic cells (ADCC). Importantly, ratios of effector to cancer cells, when AVEC were administered, resulted in the same immunotherapeutic efficacy as compared to higher ratios when naked antibodies were administered. In other words less cytotoxic cells were needed for AVEC to deliver the same therapeutic effect as more cells when naked antibodies were administered. This feature is critically important, when the patients are immunocompromised after the rounds of systemic therapy and the patients’ ability grow the immune cells is annihilated by intended to suppress proliferation of cancer cells, but as side effects universally suppressing proliferation of all patients’ cells. The AVEC’s efficacy was at statistically significant advantage over the relevant isotype antibodies. The AVEC’s impact onto cancer cells was also of statistically significant difference over that onto HOSE and HAE cells. These assays were repeated four times. The data presented are representative for all performed. I isotype antibodies