Gordon T. Rogers

Ablation of Human Choriocarcinoma Xenografts in Nude Mice by Antibody-directed Enzyme Prodrug Therapy (ADEPT) with Three Novel Compounds

Three novel prodrugs have been designed for use as anticancer agents. Each is a bifunctional alkylating agent which has been protected to form a relatively inactive prodrug. They are designed to be activated to their corresponding alkylating agents at a tumour site by prior administration of an antitumour antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in a two-phase system called antibody-directed enzyme prodrug therapy (ADEPT). The Km and Vmax values for three different antibody-CPG2 conjugates were determined in relation to each prodrug. The Km values ranged from 4.5-12 mumol/l and the Vmax from 0.5-1.6 mumol/U/min. Athymic Nu/Nu mice with palpable transplanted human choriocarcinoma xenografts, which are resistant to conventional chemotherapy, were treated with anti-human chorionic gonadotropin antibodies conjugated to CPG2. This was followed by each of the three novel prodrugs. Significant increase in survival was obtained in three of the regimens tested using only one course of treatment. This demonstrates the potential of a tumour-localised bacterial enzyme to activate protected alkylating agents in order to eradicate an established human xenograft.

Galactosylated antibodies and antibody‐enzyme conjugates in antibody‐directed enzyme prodrug therapy

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two‐ and three‐phase system in which an antibody to a tumor‐associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic‐antigen antibody fragment A5B7‐F(ab′)2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate‐specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor.

Relationship between tumour size and uptake of radiolabelled anti-CEA in a colon tumour xenograft

The relationship between tumour size and the uptake of three radiolabelled anti-CEA localising antibodies (A5B7, 1H12 and PK2G) into a human colon tumour xenograft (MaWi) has been examined. For tumour weights greater than 100 mg (109–873 mg) there was a strong positive correlation between absolute uptake and tumour weight with mean uptakes per gram of 9.8 (r=0.92), 5.0 (r=0.93) and 5.3 (r=0.94) for A5B7, 1H12 and PK2G respectively. For tumour weights below 100 mg (17–99 mg) the percentage uptake per gram (specific uptake) increased markedly reaching 80% of the injected dose for A5B7. The above phenomena could be modelled by representing uptake by the surface area of a sphere and tumour weight by its volume. Transformation of this model produced a linear relationship suitable for regression analysis of the experimental data. The slopes of the regression lines for the three antibodies were very close to that predicted by the model suggesting that their uptake into MaWi xenografts is proportional to surface area. The main discrepancy of the actual data was shown by the intercepts which relate to the variation in uptake between different antibodies. This model provides a possible means of correcting for the effect of tumour size when investigating the uptake of antibodies into xenografts.

Application of monoclonal antibodies to purified CEA in clinical radioimmunoassay of human serum.

Double-antibody radioimmunoassay using a mouse monoclonal anti-CEA (MA/1) has been used to measure CEA in human serum. Low levels of MA/1-binding CEA have been found in serum from normal individuals and moderately raised levels are sometimes associated with certain non-malignant diseases. As with conventional anti-CEA, the MA/1 antibodies can recognize significant amounts of CEA in serum from patients with a variety of solid tumours. However they appear to recognize a different immunodeterminant and possibly a different population of CEA molecules to, or a subset of, those measured by two routine assays. Studies in which the MA/1 assay was directly compared with the results of the Charing Cross routine and Abbott EIA assays have indicated that different immunological forms of CEA may be expressed in the course of tumour progression but no prognostic value was evident in this study. Our results stress the need to resolve immunological specificities expressed by CEA-like molecules and evaluate their clinical importance.

Radioimmunoassay methods for carcinoembryonic antigen

Abstract A comparison of three radioimmunoassay methods for measuring the concentration of carcinoembryonic antigen in plasma is presented. Experimental details are outlined for a slight modification of the zirconyl phosphate gel assay and for a coated tube sandwich assay and a rabbit anti-CEA: guinea-pig anti-rabbit double antibody system. The resulting assay profiles are discussed with reference to their potential in clinical practice.

Effect of dose escalation of a monoclonal anti-CEA IgG on tumour localisation and tissue distribution in nude mice xenografted with human colon carcinoma

SummaryA monoclonal anti-CEA antibody (1H12) has been examined for the effect of dosage on tumour localisation in immunodeprived mice xenografted with human colon carcinoma. Increased doses produced a linear rise in the absolute concentration found in the tumour but this was found to depend on tumour size, with the smaller tumours (mean weight 44 mg) accumulating significantly more antibody compared to larger tumours (mean weight 146 mg). With the smallest tumour (18 mg), in which saturation was achieved, a 500 μg dose produced a concentration in tumour of 60 μg/g. In the larger tumours a dose of 256 μg produced a mean concentration of 5.2 μg/g. Prolonged retention of 1H12 by tumour up to 8 days, observed at doses of 4, 128 and 256 μg, indicated that the dynamics of localisation is unaffected by dosage.Increased doses of 125I-1H12 caused an increase in the levels of radioactivity associated with all normal tissues studied. Thus at 8 days after injection an increase from 4 to 128 μg produced 50% and 42% declines in the tumour to blood and liver ratios, respectively. Cumulative localisation of 1H12 in tumour, from 4 h to 8 days, relative to normal tissue clearance was diminished on increasing dosage. This study shows that attempted therapy with escalated amounts of intact antibody is likely to be limited by a protracted excretory process and measures aimed at accelerating circulatory clearance are necessary.

Plasma clearance of an antibody-enzyme conjugate in ADEPT by monoclonal anti-enzyme : its effect on prodrug activation in vivo

The effect of anti-enzyme antibody clearance on prodrug turnover in antibody-directed enzyme prodrug therapy (ADEPT) has been studied. Mice bearing LS174T xenografts were given localising carboxypeptidase G2 (CPG)2 conjugate (AEC) and 19 h later galactosylated anti-CPG2 antibody (SB43-GAL). In regimen I prodrug was injected 5 h after SB43-GAL as previously described. In regimen 2 and 3 a shortened and extended clearance time was used in which prodrug was administered 0.5 h or 53 h after SB43-GAL respectively. Regimen 1 resulted in similar tumour and normal tissue levels of active drug to those of the control in which prodrug was given 72 h after AEC. SB43-GAL therefore accelerated clearance of enzyme allowing early administration of prodrug. In regimen 2, very high active drug levels were found in the liver, showing removal of AEC from the blood followed by reactivation of enzyme and extensive and rapid prodrug turnover. Active drug levels in tumour and blood reached similar peak levels to those of the control. Regimen 3 resulted in lower active drug levels in tissues, consistent with degradation and excretion of enzyme. Regimen 3 also produced the best tumour to normal ratios for active drug. Residual prodrug in tumour was unaffected by SB43-GAL, showing the advantage of galactosylation in minimising inactivation of CPG2 in tumour. By contrast, residual prodrug in blood persisted for longer when SB43-GAL was used. Circulatory clearance of enzyme with SB43-GAL allows prodrug to be administered expediently with reduced toxicity and with the prospect of increasing the dosage.

Carcinoembryonic antigen: isolation of a sub-fraction with high specific activity.

Four sub-fractions of carcinoembryonic antigen have been obtained by chromatography of conventional CEA on Con A-Sepharose and their immunoreactive contents have been determined. Comparative studies have shown that a fraction eluted with 2% methyl glucoside (CEA 2B) had the highest activity, with a potency (60 u/mug) twice that of unfractionated CEA although appreciable activity was also found in the other fractions. The amino acid composition of CEA 2B is similar to that reported for conventional CEA but there is a lower content of neutral hexoses and a comparatively high content of N-acetylglucosamine. Experiments with a pool of sera from 11 patients with colonic cancer, which had been fractionated on Con A-Sepharose, have shown that nearly all the CEA activity was contained in a fraction eluted with 2% methyl glucoside. A convenient method of isolating CEA with high specific activity directly from perchloric acid extracts of tumour tissue is also described.

Prolonged localisation of a monoclonal antibody against CEA in a human colon tumour xenograft

Prolonged localisation of a monoclonal antibody against CEA in a human colon tumour xenograft

A monoclonal antibody against a CEA-related antigen expressed on HT29 colon tumour cells

A new monoclonal antibody, that binds to CEA and with low cross-reactivity with NCA, has been raised to an antigen expressed on HT29 colon tumour cells. This antibody (H58) reacts strongly with high-molecular-weight protein (50 X 10(4)) isolated from a crude plasma membrane preparation of HT29 cells as well as binding to purified CEA of molecular size (20 X 10(4)) isolated both from those cells and liver metastases of colon tumour. H58 binds to an epitope sterically unrelated to the binding site of the previously described anti-CEA monoclonal antibody MA/1 and our routine anti-CEA polyclonal serum PKIG. Augmented binding of antibody to either the cell membrane preparation or conventional CEA can be achieved using a mixture comprising equal weights of specific immunoglobulin from H58 and MA/1. The value of solid-phase binding assays using microtitre plates for selecting potentially useful antibodies is discussed.