A phase I trial of antibody directed enzyme prodrug therapy (ADEPT) in patients with advanced colorectal carcinoma or other CEA producing tumours.

Antibody-directed enzyme prodrug therapy is a targeted therapy in which a prodrug is activated selectively at the tumour site by an enzyme, which has been targeted to the tumour by an antibody (antibody-enzyme conjugate). Previous clinical trials have shown evidence of tumour response, however, the activated drug had a long half-life, which resulted in dose-limiting myelosuppression. Also, the targeting system, although giving high tumour to blood ratios of antibody-enzyme conjugate (10 000 : 1) required administration of a clearing antibody in addition to the antibody-enzyme conjugate. The purpose of this current study therefore was to attempt tumour targeting of the antibody-enzyme conjugate without the clearing antibody, and to investigate a new prodrug (bis-iodo phenol mustard, ZD2767P) whose activated form is highly potent and has a short half-life. Twenty-seven patients were treated with antibody-directed enzyme prodrug therapy using A5CP antibody-enzyme conjugate and ZD2767P prodrug, in a dose-escalating phase I trial. The maximum tolerated dose of ZD2767P was reached at 15.5 mg m(-2)x three administrations with a serum carboxypeptidase G2 level of 0.05 U ml(-1). Myelosuppression limited dose escalation. Other toxicities were mild. Patients' quality of life was not adversely affected during the trial as assessed by the measures used. There were no clinical or radiological responses seen in the study, but three patients had stable disease at day 56. Human anti-mouse antibody and human anti-carboxypeptidase G2 antibody were produced in response to the antibody enzyme conjugate (A5CP). The antibody-enzyme conjugate localisation data (carboxypeptidase G2 enzyme levels by HPLC on tumour and normal tissue samples, and gamma camera analysis of I-131 radiolabelled conjugate) are consistent with inadequate tumour localisation (median tumour: normal tissue ratios of antibody-enzyme conjugate of less than 1). A clearance system is therefore desirable with this antibody-enzyme conjugate or a more efficient targeting system is required. ZD2767P was shown to clear rapidly from the circulation and activated drug was not measurable in the blood. ZD2767P has potential for use in future antibody-directed enzyme prodrug therapy systems.

The ability to accumulate deoxyuridine triphosphate and cellular response to thymidylate synthase (TS) inhibition.

Thymidylate synthase (TS) is an important enzyme catalysing the reductive methylation of dUMP to dTMP that is further metabolized to dTTP for DNA synthesis. Loss of viability following TS inhibition occurs as a consequence of depleted dTTP pools and at least in some cell lines, accumulation of dUTP and subsequent misincorporation of uracil into DNA. The expansion in dUTP pools is largely determined by the expression of the pyrophosphatase, dUTPase. Our previous work has shown that following TS inhibition the ability to accumulate dUTP was associated with an earlier growth inhibitory effect. 3 human lung tumour cell lines and HT29 human colon tumour cells transfected with dUTPase have been used to investigate the relationship between loss of viability following TS inhibition and dUTP accumulation. Cell cycle arrest typical of TS inhibition was an early event in all cell lines and occurred irrespective of the ability to accumulate dUTP or p53 function. However, a large expansion of dUTP pools was associated with mature DNA damage (4 h) and an earlier loss of viability following TS inhibition compared to cells in which dUTP pools were not expanded. In A549 cells damage to mature DNA may have been exacerbated by significantly higher activity of the excision repair enzyme, uracil-DNA glycosylase. Consistent with results using different inhibitors of TS, transfection of dUTPase into HT29 cells significantly reduced the cytotoxicity of a 24 h but not 48 h exposure to ZD9331. Although loss of viability can be mediated through dTTP deprivation alone, the uracil misincorporation pathway resulted in an earlier commitment to cell death. The relevance of this latter pathway in the clinical response to TS inhibitors deserves further investigation.

Measurement of the critical DNA lesions produced by antibody-directed enzyme prodrug therapy (ADEPT) in vitro, in vivo and in clinical material.

An antibody-directed enzyme prodrug therapy (ADEPT) system against CEA-positive tumours is currently in phase I clinical trials. It consists of a prodrug, 4-[N,N-bis(2-iodoethyl) amino] phenoxycarbonyl L -glutamic acid (ZD2767P) and a conjugate of the F(ab')(2) anti-CEA antibody A5B7 and the bacterial enzyme carboxypeptidase G2 (CPG2). ZD2767P is converted by antibody-targeted CPG2 into an active bifunctional alkylating drug (ZD2767) at the tumour site. The IC(50) value of the prodrug against the human colorectal tumour LS174T cell line was 55 +/- 9 microM following a 1 h exposure. In contrast, co-incubation of ZD2767P with CPG2 resulted in 229-fold increase in activity. Using a modified comet assay, DNA interstrand cross links (ISC) were detected within 1 h of ZD2767P + CPG2 treatment and were repaired by 24 h. A clear dose-response was seen between the level of ISC, growth inhibition and ZD2767 concentration. Administration of a therapeutic dose of ZD2767P 72 h after the F(ab')(2) A5B7 conjugate to mice bearing LS147T xenografts resulted in extensive ISC in the tumour after 1 h; repair was seen at 24 h. Tumour biopsies and peripheral lymphocytes were studied in 5 patients on the ADEPT phase I clinical trial. In 4 patients no ISC were detected. These patients also demonstrated poor localization of conjugate and no tumour response was seen. However a significant level of ISC was detected in one tumour biopsy, which also showed evidence of conjugate localization and clinical response. These studies demonstrate the application of the comet assay in the measurement of ISC in vitro and in clinical material and confirm that activation of ZD2767P results in the formation of DNA crosslinks.

Deoxyuridine triphosphatase (dUTPase) expression and sensitivity to the thymidylate synthase (TS) inhibitor ZD9331.

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC(50)3-70 nM). TS protein expression correlated with TS activity (r(2)= 0.88, P = 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 microM, 24 h) varied by approximately 2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r(2)= 0.94, P = 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10(-6)cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells.