Development of a novel bi-specific monoclonal antibody approach for tumour targeting

Abstract

SummaryTo overcome the disadvantages of bi-specific antibody methodologies in vivo, a novel antibody approach has been designed to improve tumour targeting and effector to target ratio. The technique involves biotinylated anti-CD3 Fab fragments and streptavidinylated anti-tumour monoclonal antibodies (mAbs) that can spontaneously form cross-links. We describe here a method for the direct cross-linking of sulphydryl-conjugated HMFG1 (anti-MUC1 mucin mAb) to streptavidin by sulphosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate. Fab fragments generated by papain digestion of the 1452C11 antibody (anti-CD3 mAb without Fc to avoid peripheral activation of T-cells) were biotinylated with NHS-Iminobiotin. MUC1-transfected BALB/c breast cancer cell lines 413BCR and 425CCR and the parental cell line (410.4) were labelled with streptavidinylated mouse anti-MUC1 mucin mAb. BALB/c effector T-cells were separately labelled with biotinylated anti-CD3 Fab fragments (1452C11) and mixed with tumour cells in different effector to target ratios. Percentage of killing was assessed using the 51 Cr cytotoxicity assay. Seventy per cent lysis was measured in the case of 413BCR (high MUC1 mucin expressor) and 40% in the case of 425CCR (low expressor) cell line. No lysis was apparent in the MUC1 negative cell line. These results demonstrate that the novel T-cell redirecting approach we have developed can produce effective immune lysis of target cells in vitro. © 1999 Cancer Research Campaign Keywords: bi-specific monoclonal antibody; tumour immunotherapy; directly streptavidinylated antibody; biotinylated anti-CD3 Fab fragments; MUC1 mucin 431British Journal of Cancer (1999) 81(3), 431-439 © 1999 Cancer Research Campaign Article no. bjoc.1999 Received 3 December 1998 Revised 22 April 1999 Accepted 22 April 1999 Correspondence to: GWH Stamp increase in the tumour:background ratio with the SA-biotin approach as compared with the BiAb method (Zhu et al, 1998). Furthermore, the latter study showed that tumour antigen expression and shedding had little effect on the isotope delivery to the tumour. Despite the potential benefits of this approach, it has yet to be investigated in the therapeutic context.The aim of this study has been to develop the SA-biotin approach to improve T-cell redirection at the tumour site for immunotherapeutic applications. In order to achieve this we have directly streptavidinylated an anti-tumour antibody and biotinylated monovalent antibody Fab fragments (without Fc region to avoid T-cell activation/sequestration by cells expressing membrane receptors for the Fc) raised against the CD3 receptor on T-cells. In theory, tumour cells targeted by the antibody conjugated with SA will bind the biotinylated anti-CD3 Fab causing its aggregation (i.e. making an artificially bivalent or polyvalent antibody construct), thus rendering it able to deliver an efficient activation signal for the T-cells (Figure 1). We selected MUC1 mucin, a well-documented antigen of human adenocarcinomas (Taylor et al, 1994) as ...

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