Characterisation of a humanised bispecific monoclonal antibody for cancer therapy.

A Bruynck,K Bosslet,G Seemann

doi:10.1038/bjc.1993.84

Abstract

A humanised bispecific monoclonal antibody (bsMAb) with binding specificity for carcinoembryonic antigen (CEA) on one arm and a radiolabelled chelate (DTPA-90Y) on the other arm was generated by consecutively transfecting the humanised genes of an anti-CEA MAb and the chimerised genes of an anti-chelate MAb into eucaryotic BHK cells using the calcium-phosphate coprecipitation technique. The antibodies secreted were of IgG3 isotype with a shortened hinge region (delta gamma 3) and light chains. Double transfectomas were screened for the secretion of bsMAbs using a double determinant enzyme-linked immunosorbent assay (ELISA) based on solid phase attached HSA-benzyl-DTPA and an anti-idiotypic MAb selective for the CEA-specific arm. After purification on two immunoaffinity chromatography columns, the humanised bsMAbs were characterised by SDS-PAGE and a quantitative binding assay in antigen excess. The purification procedure resulted in 95% reactive bispecific MAb. This humanised bsMAb may be employed in two phase radioimmunotherapy, binding to the tumour via the anti-CEA arm and localising a radiolabelled chelate with the other arm, without inducing a strong immune response observed sometimes with murine MAbs.

Highlights

We describe the generation of a bifunctional monoclonal antibodies (MAbs) consisting of one humanised anti-carcinoembryonic antigen (CEA) arm and a chimerised anti-DTPA-Y arm by double transfection of the corresponding genes into BHK cells, its purification using immunoaffinity chromatography and its characterisation
The generation and screening of murine MAbs directed against CEA (BW 431) and DTPA-Y (BW 2050), respectively, was described previously (Bosslet et al, 1988 and 1991)
Humanisation of an anti-CEA MAb The sequences of the heavy and light chain variable genes of MAb BW 431 were amplified and cloned as described by Orlandi et al (1989) and the complementarity-determining regions (CDRs) regions were subsequently built into the framework of human VH and VL domains according to the methodology of Jones et al (1986) and Riechmann et al (1988) (Figure 1)

Summary

Methods

Chimerisation of an anti-DTPA- Y MAb The murine variable (VH and VL) region genes of the heavy and light chain of MAb BW 2050 were recombined with the constant region genes of human IgGs (Ay3) according to the method of Boulianne et al (1984) (Figure 1). Humanisation of an anti-CEA MAb The sequences of the heavy and light chain variable genes of MAb BW 431 (anti-CEA) were amplified and cloned as described by Orlandi et al (1989) and the CDR regions were subsequently built into the framework of human VH and VL domains according to the methodology of Jones et al (1986) and Riechmann et al (1988) (Figure 1). Received 12 December 1991; and in revised form 8 October 1992

Results

Discussion

Conclusion