Figure 3.

Alemtuzumab induced complement-mediated cytotoxicity of VLC. A VLCs FACS isolated from ovarian tumor tissue incubated with Alemtuzumab in the presence or absence of complement; (1) In the presence of Alemtuzumab and heat inactivated sera, the majority of VLC are viable Annexin V ^(-) and PI ^(-) cells. In contrast, in the presence of Alemtuzumab and sera (2), the majority of VLC are Annexin V⁺ and/or PI ⁺ indicating the induction of cytotoxicity (n = 3). (3) In the presence of Alemtuzumab and sera, cytotoxicity was similarly induced in control CD3+ peripheral blood T cells. B. To determine if Alemtuzumab could induce cytotoxicity of VLC in whole tumor ascites ex vivo, we incubated ascites associated cells in ascites fluid together with either heat inactivated Alemtuzumab or Alemtuzumab. (1) In the presence of heat inactivated Alemtuzumab a population of CD45⁺/VE-Cadherin+ cells was clearly detectable (box). In contrast in the presence of active Alemtuzumab there is as significant reduction of VLC. (2) Loss of CD45⁺/VE-Cadherin⁺ VLC in the presence of Alemtuzumab was associated with an appropriate increase in Annexin V/PI-labeled cells. C. Summary of Alemtuzumab anti-VLC activity from independent patient samples (n = 3) p = 0.002.