F Searle

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.

LIPOSOMALLY ENTRAPPED SECOND ANTIBODY IMPROVES TUMOUR IMAGING WITH RADIOLABELLED (FIRST) ANTITUMOUR ANTIBODY

The usefulness of antibodies directed against tumour products for localisation and therapy of cancer is limited by the large proportion of administered antibody which remains non-specifically in the circulation and extravascular space. Liposomally entrapped second antibody (LESA) directed against the first (antitumour) antibody has been used to accelerate clearance of non-tumour-bound first antibody without affecting its clearance from the tumour. Intravenously administered LESA binds the first antibody and LESA is cleared by the reticuloendothelial system. LESA accelerated clearance of radiolabelled antibody directed against carcinoembryonic antigen (CEA) from the circulation in four of five patients with gastrointestinal cancer, enhancing gamma-camera imaging of the tumour in three of them. These results suggest that LESA can improve the sensitivity and specificity of tumour imaging with radiolabelled antitumour antibody. This strategy may also have the potential to improve the therapeutic ratio of some toxic agents linked to antitumour antibody.

Radioimmunolocalization of Tumours by External Scintigraphy after Administration of 131I Antibody to Human Chorionic Gonadotrophin: Preliminary Communication

131Iodine (131I) labelled antibody directed against human chorionic gonadatrophin (hCG) was given on 21 occasions to 18 patients with hCG-producing neoplasms. Tumours were localized by external scintigraphy in 13 of 21 investigations. Positive results were obtained reliably when serum hCG exceeded 500 miu/ml and in some cases sensitivity was comparable to that of computerized tomography. A positive result probably implies viability in the tumour and this was of practical value in discriminating between necrotic deposits and living tumour before surgery.

Repeated antitumour antibody therapy in man with suppression of the host response by Cyclosporin A

Antibody targeted therapy of cancer results in anti-antibody production which prevents repeated treatment. Cyclosporin A (CsA) has been used to suppress this response in patients treated with a radiolabelled antibody to carcinoembryonic antigen (CEA). Patients with CEA producing tumours received a minimum of two courses consisting of an injection of radiolabelled antibody and CsA, 24 mg kg-1 day-1, for 6 days; each course was given at 2 week intervals. Two weeks after the completion of the second course the mean human antimouse antibody (HAMA) levels were 3.5 micrograms ml-1 (s.d. 2.7) in 3 patients receiving CsA and 1,998 micrograms ml-1 (s.d. 387) in 3 patients not receiving the drug. Clearance of antitumour antibody was accelerated and tumour localisation absent when HAMA levels exceeded 30 micrograms ml-1. With lower levels of HAMA in the CsA-treated patients, further antitumour antibody accumulated in the tumour after each dose. Further therapy with antitumour antibody and CsA lead to the development of HAMA, but this was less than 25% of the amount in patients not given CsA. In this preliminary study up to 4 times as many doses of antitumour antibody could be usefully given when CsA was used. This increases the potential for effective antibody targeted therapy of cancer.

Antibody distribution and dosimetry in patients receiving radiolabelled antibody therapy for colorectal cancer

The distribution of iodine-131 (131I) labelled antibody to carcinoembryonic antigen (CEA) has been studied in 16 patients with colorectal cancer. Levels of tumour and normal tissue radioactivity were measured by serial gamma-camera imaging and counting of blood and urine. Maximum concentrations were found in tumour 8 h after administration and varied up to 9-fold in different patients. Higher levels were found on average in tumour than in any other tissue. Liver, lung and blood were the other tissues in which antibody was concentrated relative to the rest of the body. Antibody cleared from all these tissues over 1 week. Second antibody directed against the antitumour (first) antibody was given 24 h after first antibody in order to accelerate clearance from the blood. This increased the tumour to blood ratio but had little effect on other tissues. Cumulative radiation dose to tumour and normal tissue was estimated. In patients with the most efficient localisation the tumour to body ratio was 20:1 and tumour to blood ratio 5:1. This may be sufficient for effective therapy of cancer in patients selected for efficient antibody localisation. The data may be used to estimate the effect of different therapeutic strategies. For instance, in the time after second antibody administration the average tumour to blood ratio of radiation dose was 11:1, suggesting that two phase systems in which the therapeutic modality is given after a good tumour to normal tissue ratio is obtained may be effective for the majority of patients.

The potential of carboxypeptidase G2-antibody conjugates as anti-tumour agents. I: Preparation of antihuman chorionic gonadotrophin-carboxypeptidase G2 and cytotoxicity of the conjugate against JAR choriocarcinoma cells in vitro

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been linked to a monoclonal antibody (W14A) raised to human chorionic gonadotrophin. The coupling efficiency and retention of antibody and enzymatic activities are compared for three separate methods of preparing 1:1 conjugates. Preliminary in vitro studies on the cytotoxicity of the free enzyme and the conjugated enzyme towards JAR choriocarcinoma cells are reported. Despite the limitations of the in vitro model, it could be demonstrated that a significant proportion of 10(6) choriocarcinoma cells lost viability when exposed to either free or conjugated enzyme for 72 hours at concentrations of carboxypeptidase G2 of 1-3 units ml-1 of medium.

The clinical value of imaging with antibody to human chorionic gonadotrophin in the detection of residual choriocarcinoma.

Choriocarcinoma can be imaged by external scintigraphy after intravenous administration of radiolabelled antibody directed against human chorionic gonadotrophin (HCG). The purpose of this study was to investigate whether antibody imaging was sufficiently sensitive and specific to improve the selection of patients for surgical resection of localised deposits of drug resistant or recurrent choriocarcinoma. Eighteen patients with raised serum HCG concentrations in whom the site of tumour was not known were investigated by antibody imaging and conventional imaging methods. When the tumour appeared localised, resection was attempted. Tumour was found at all sites in which both antibody imaging and conventional imaging methods were positive. Antibody imaging gave false positive results in 2 of 18 patients and false negatives in 5. Computerised tomography was false positive in one case and false negative in 2. In these patients, antibody imaging gave true negative and true positive results respectively. Of 8 patients with positive antibody imaging in whom resection was attempted, 5 achieved sustained complete response with up to five years follow up. It is concluded that antibody imaging is useful in selection of patients for surgery in drug resistant or recurrent choriocarcinoma.

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.

Multivariate biochemical indicators of breast cancer: An evaluation of their potential in routine practice

Abstract A vertical study has been made of the frequency of abnormalities of the levels of 3 tumour related antigens (CEA, βHCG and casein), 2 serum enzymes (gamma glutamyltranspeptidase and alkaline phosphatase), 4 acute phase reactant proteins (haptoglobin, acid glycoprotein, ceruloplasmin and antitrypsin) and a random study of C-Reactive protein in 429 women attending a breast clinic. Whilst the frequency of abnormality increases with tumour load, these parameters seem unable to discriminate high risk groups Stages II and III from benign breast disease. The women are heterogenous with respect to their individual marker profiles. Elements within this panel of potential markers could be used to design a profile for longitudinal monitoring of recurrence in certain high risk groups, but as yet they do not appear to warrant their introduction into routine medical practice until they have been proven to aid clinical decision making.

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.