Bystander Killing of Tumor-Cells by Antibody-Targeted Enzymatic Activation of a Glucuronide Prodrug

RHI-beta G-PEG, formed by linking poly(ethylene glycol)- modified beta- glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG ) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS - 30D rat hepatoma cells. Sequential treatment of cells with 10 mu g ml(-1 ) RH1-beta G-PEG and 20 mu M BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-SOD and N1S1 cells in BALB/c nu /nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-beta G-PEG in solid AS-SOD and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection , intravenous treatment with RH1-beta G-PEG and BHAMG cured seven of seven nude mice bearing solid s.c, AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo