NK cell anti-tumor cytotoxicity against breast cancer targets is mediated by several mechanisms which are further augmented through ADCC by an antibody that recognizes LFA-3

Treatment of advanced breast cancer with autologous stem cell transplantation is limited by a high probability of disease relapse and we have been testing whether IL-2-based immunotherapy may mediate an antitumor response. In clinical trials, IL-2 alone can expand NK cells in vivo and increase their cytotoxic activity against breast cancer cell lines but this increase is modest. We hypothesized that better understanding of the mechanisms that mediate ANK killing may lead to improvements of current immunotherapy strategies. Activated NK (ANK) populations exhibit significant but variable cytotoxicity against five breast cancer cell lines in the following order from most to least susceptible to ANK mediated killing (MCF-7, T47D, MDA-MB-231, BT-20, SKBR-3). Cytotoxicity in the absence or presence of antibodies against ICAM-1, ICAM-2, ICAM-3, CD18, CD2, and LFA-3 used alone or in combination was then tested. Control antibodies, ICAM-2 and ICAM-3 antibodies did not alter killing compared to controls without antibody. In contrast, ICAM-1, CD18 and CD2 variably decreased target cell lysis and the combination of all antibodies were additive suggesting independent roles of recognition by ANK through β2 integrins and CD2. However, contrary to our expectations, an anlibody against LFA-3 (the ligand for CD2), failed to block killing but instead, significantly increased cytotoxicily which correlated with target expression of LFA-3 (all but SKBR-3). Resting NK cells, which exhibit little lytic activity, could kill LFA-3 positive targets suggesting that the LFA-3 antibody was mediating ADCC. The LFA-3 induced target lysis did not occur when NK cells devoid of CD16 or CD2 (derived from CD34+/CD38- marrow progenitors in long-term culture) were tested. Further blocking studies showed that the LFA-3 augmented killing was blocked by treatment of NK with anti-CD2 but not antt-CD18. The LFA-3 antibody augmented killing was also prominent when lymphocytes were activated in patients after autologous stem cell transplant with subcutaneous IL-2. These studies show that multiple mechanisms are involved in NK killing of breast cancer targets, none of the targets were inherently resistant to NK killing and the LFA-3 mediated ADCC can target immunotherapy to breast cancer cells. This or other methods of targeting cytotoxic lymphocytes may be useful to prevent relapse after autologous stem cell transplantation for breast cancer and other malignant diseases.