J Boden

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.

Polyethylene glycol modification of a galactosylated streptavidin clearing agent: effects on immunogenicity and clearance of a biotinylated anti-tumour antibody.

Effective radioimmunotherapy is limited by slow antibody clearance from the circulation, which results in low tumour to blood ratios and restricts the dose of radiolabelled anti-tumour antibody that can be safely administrated. Avidin and streptavidin clearing agents have been shown to effectively complex and clear radioactive biotinylated antibodies from the circulation, but their immunogenicity may limit their repeated use. We have investigated whether polyethylene glycol (PEG) modification can reduce the immunogenicity of our galactosylated streptavidin (gal-streptavidin) clearing agent without altering its effectiveness as a clearing agent. The immune response evoked in mice after intraperitoneal infection of 30 micrograms of gal-streptavidin was decreased after PEG modification, as shown by lower antibody titres and a reduction in the number of mice that elicited an anti-gal-streptavidin response. The effect of PEG-modified gal-streptavidin on the blood clearance and tumour localisation of a 125I-labelled biotinylated anti-CEA was investigated in the LS174T human colon carcinoma xenograft in nude mice. Although PEG modified gal-streptavidin bound the [125I]biotinylated antibody in vivo, effective clearance from the circulation was inhibited, resulting in very little reduction in the levels of circulation radioactivity, together with a decrease in the antibody localised to the tumour.

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.

The effect of liposome (phospholipid vesicle) entrapment of actinomycin D and methotrexate on the in vivo treatment of sensitive and resistant solid murine tumours

Abstract When tested in mice bearing the Ridgway osteosarcoma (ROS) the activity of methotrexate entrapped in small cationic liposomes was increased, both in terms of tumour growth inhibition and toxicity for normal tissues. Conversely, actinomycin D entrapped in small cationic liposomes lost its cytotoxic activity, both against the tumor and normal tissues. In addition, liposome-entrapped actinomycin D proved ineffective therapeutically against a new ROS tumour subline in which resistance to free drug had been derived in vivo ; moreover, this resistance appeared to have resulted from failure to retain drug rather than through impaired drug uptake. These results, and those obtained from tissue distribution studies using free and liposome-entrapped actinomycin D, suggested that (a) liposome entrapment modifies the pharmacokinetics and hence activity of these drugs by acting as a slow release system rather than by providing a means of selective delivery to tumours, and (b) the use of liposome-entrapped actinomycin D to overcome drug resistance acquired in vivo may be inappropriate.

Clearance of circulating radio-antibodies using streptavidin or second antibodies in a xenograft model.

The improved tumour to non-tumour ratios needed for effective tumour targeting with antibodies requires that blood background radioactivity is reduced. We investigated the effect of streptavidin as a clearing agent for 125I-labelled biotinylated anti-CEA antibodies in a human colon carcinoma xenograft model. By comparing the biodistribution of the monoclonal antibody A5B7 with four, nine or 22 biotins per antibody molecule, we investigated how the degree of biotinylation of the primary radiolabelled antibody affects its clearance with streptavidin. Limiting the degree of biotinylation limited blood clearance, whereas nine or 22 biotins per antibody molecule resulted in a 13- to 14-fold reduction in blood radioactivity, the streptavidin-biotinylated antibody complexes clearing rapidly via the liver and spleen. Although a reduction in tumour activity was also seen, a 6.6-fold improvement in the tumour to blood ratio was achieved. A comparative study of streptavidin versus second antibody clearance was carried out using the polyclonal antibody PK4S biotinylated with 12 biotins per antibody molecule. This study indicated that second antibody was superior for clearance of the polyclonal antibody, resulting in a larger and faster reduction in blood radioactivity and improved tumour to blood ratios. In this case the primary antibody was polyclonal, and therefore non-uniformity of biotinylation may affect complexation with streptavidin. Therefore, the degree of biotinylation and type of antibody must be carefully considered before the use of streptavidin clearance.

A human choriocarcinoma xenograft in nude mice; a model for the study of antibody localization.

The successful development of the concept of linking cell-killing agents to tumour-specific antibodies will be largely determined by the extent to which the antibodies are preferentially localized in the malignant tissue. A xenograft of human choriocarcinoma (CC3) has been established in nude mice, and the relative distribution of affinity-purified specific antibodies to human chorionic gonadotrophin has been compared with that of nonspecific antibodies from the same species. Treatment of the nonspecific antibodies with ammonium thiocyanate appeared to be important to ensure that the distributions in normal nude mice were equivalent. Specificity indices, derived from the comparative distributions of isotope activity in the tumour and lung of labelled specific and nonspecific antibodies, ranged between 1.3 and 2.0.

Antibody-directed enzyme prodrug therapy (ADEPT): A three-phase study in ovarian tumor xenografts

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with125I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab′)2 fragment showed localization in these xenografts. The AB57-F(ab′)2 fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with125iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon).A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab′)2-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress.

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.