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.

Abstract

The Lewis(x) (Le(x)) trisaccharide (CD15) linked to proteins and glycolipids is highly expressed on the surface of normal human polymorphonuclear neutrophils (PMN) and several human neoplasias, such as breast and gastrointestinal 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 clinical trial of FC-2.15, a temporary neutropenia was the main toxicity, and antitumor responses were observed. In 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 cytometry revealed a strong reactivity of FC-2.15 with almost 100% of PMN, and Scatchard 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 FC-2.15 induced PMN homotypic aggregation, only 28.4 +/- 4.1% remaining as single cells. When PMN and the Le(x)(+) MCF-7 breast cancer cells were co-incubated, FC-2.15 induced heterotypic aggregation. In 51Cr-release assays employing 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 induced PMN agglutination and heterotypic aggregates when MCF-7 cells were present. A pretreatment of PMN with colchicine impaired PMN agglutination both in vitro (single PMN = 81.15 +/- 4.35%) and in ex vivo circulating blood. In the latter condition, FC-2.15-lytic activity was restored, suggesting that PMN homotypic aggregation by FC-2.15, but not lysis, is dependent on microtubule integrity and that PMN agglutination hinders their lysis. Moreover, when 51Cr-release assays were performed following agglutination, FC-2.15 cytotoxicity was restricted to isolated PMN. It is suggested that crosslinking 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 would be due to PMN agglutination and margination, rather than lysis. In addition, FC-2.15 appears to be able to lyse Le(x)(+) tumor cells in circulation.