Development of an in vivo antibody-mediated killing (IVAK) model, a flow cytometric method to rapidly evaluate therapeutic antibodies.

The efficacy and mechanism of action of therapeutic antibodies that target cancer cells have typically been evaluated using in vitro assays and long-term in vivo tumor models. To allow for a more efficient assessment of the function of candidate therapeutic antibodies, we have developed a flow cytometric-based method that rapidly and directly quantifies antibody-mediated killing in a short term in vivo assay. Target cells that express human CD52, including huCD52(+) splenocytes from huCD52 transgenic mice and Ramos cells, a CD52(+) human B cell lymphoma line, and CD52(-) reference cells were differentially labeled by using two fluorescent dyes to distinguish target and reference cell populations. Labeled cells were injected into mice with or without Campath-1H (Alemtuzumab) and then recovered for flow cytometric analysis 5 h later. We found that huCD52(+) transgenic splenocytes and Ramos cells were selectively depleted in Campath-treated animals but not in animals treated with a negative control antibody. Furthermore, it is likely that the cells were depleted in vivo by a complement-dependent mechanism since target cell depletion was significantly reversed after complement inactivation using cobra venom factor. This report demonstrates the feasibility and utility of a powerful method for the rapid evaluation in vivo of therapeutic antibody candidates for cancer.