The impact of antigen density and antibody affinity on antibody-dependent cellular cytotoxicity: relevance for immunotherapy of carcinomas.

Mp Velders,E Oskam,Gj Fleuren,Sv Litvinov,Cm Van Rhijn,So Warnaar

doi:10.1038/bjc.1998.518

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism through which tumour cells, upon treatment with anti-tumour MAbs, are eliminated in vivo. However, the relative importance of various parameters that influence the efficacy of ADCC is unclear. Here we present in vitro data on the impact of MAb affinity and antigen density on ADCC, as obtained by comparison of two MAbs against the tumour-associated antigen Ep-CAM. The low-affinity MAb 17-1A (Ka = 5 x 10(7)M(-1)) currently used for therapy, and the high-affinity MAb 323/A3 (Ka = 2 x 10(9) M(-1)), were compared in ADCC experiments against murine and human tumour target cells transfected with the Ep-CAM cDNA under the control of an inducible promoter to enable regulation of the target antigen expression levels. Data obtained from these studies revealed that the high-affinity MAb, in contrast to the low-affinity MAb, could mediate killing of tumour cells with low antigen expression levels. Even at comparable MAb-binding levels, ADCC mediated by the high-affinity MAb was more effective. The kinetics of ADCC was also found to be determined by the level of antigen expression, and by the affinity and the concentration of the MAb used. The efficacy of ADCC with both low- and high-affinity MAbs further depended on adhesive interactions between effector and target cells mediated by CD18. However, at every given MAb concentration these interactions were of less importance for the high-affinity MAb than for the low-affinity MAb. As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease.

Highlights

At every given monoclonal antibody (MAb) concentration these interactions were of less importance for the high-affinity MAb than for the lowaffinity MAb
As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease
C1.5 and C1.13, obtained by transfection of L153S murine carcinoma cells with epithelial cell adhesion molecule (Ep-CAM) cDNA under the control of the inducible murine mammary tumour virus (MMTV) promotor were tested for Ep-CAM expression

Summary

Methods

For all in vitro experiments with human effector cells the chimeric human/mouse versions of the antibodies 17-1A and 323/A3 were used (Sun et al, 1987; Velders et al, 1994). The chimeric antibodies contain the same human IgG 1 and kappa-constant domains as both MAbs were chimerized at Centocor using identical methods and vectors. All antibodies were initially purified over protein ASepharose (Pharmacia, Woerden, The Netherlands), and were further purified by sequential ion-exchange chromatography on Mono S and Mono Q (Pharmacia) columns, followed by diafiltration into phosphate-buffered saline (PBS). All antibody preparations were tested using LAL assays and shown to be endotoxin negative. The anti-human CD18, clone 7E4 (Immunotech, Marseilles, France), was used in ADCC inhibition assays. To quantify the MAb binding to Ep-CAM using flow cytometry, the Fab fragments of the chimeric 323/A3 and 17-1A MAbs, directly labelled with FLUOS (Boehringer Mannheim, Mannheim, Germany), were used

Results

Discussion

Conclusion