Differential lytic and agglutinating activity of the anti-Lewis(X) monoclonal antibody FC-2.15 on human polymorphonuclear neutrophils and MCF-7 breast tumor cells. In vitro and ex vivo studies

The Lewis(x) (Le(x)) tksaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutr ophils (PMN) and several human neoplasias, such as breast and gastrointesti nal carcinomas and chronic myeloid leukemias. FC-2.15 is an IgM murine mAb that specifically recognizes Le(x) and has been previously shown to mediate the in vitro lysis of Le(x)(+) cells by human complement. In a phase I cli nical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. Tn order to characterize FC-2.15 further and determine the physiological relevance of Le(x) binding, the reactivity of FC-2.15 on PMN was investigated under several conditions. Flow cytometr y revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scat chard analysis demonstrated an affinity constant of 5.14 x 10(9) M-1 and 1. 11 x 10(6) antigen sites/cell. In vitro, the binding of Le(x) epitopes by F C-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as s ingle cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-in cubated, FC-2.15 induced heterotypic aggregation. In Cr-51-release assays e mploying human complement, FC-2.15 lysed 93.4 +/- 7.9% of PMN and 87.8 +/- 10.7% of MCF-7 cells. However, when the effect of FC-2.15 was tested in ex vivo circulating blood, no lytic activity against PMN was detected, whereas MCF-7 cells were still lysed. Blood smears demonstrated that FC-2.15 induc ed PMN agglutination and heterotypic aggregates when MCF-7 cells were prese nt. A pretreatment of PMN with colchicine impaired PMN agglutination both i n vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating: blood. I n the latter condition, FC-2.15-lytic activity was restored, suggesting tha t PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on micr otubule integrity and that PMN agglutination hinders their lysis. Moreover, when Cr-51-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslink ing of Le(x) epitopes by FC-2.15 induces PMN to form homotypic aggregates. It is suggested that the neutropenia observed in FC-2.15-treated patients w ould be due to PMN agglutination and margination, rather than lysis. In add ition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulati on.