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Biomedical subjects

R H Begent

Publications and source records attributed to R H Begent.

At least 37 records · Page 2Linked to original sources
PubMed1999

A novel technique, using radioluminography, for the measurement of uniformity of radiolabelled antibody distribution in a colorectal cancer xenograft model.

PURPOSE: Radioimmunotherapy of cancer employs an antitumour antibody to carry a radionuclide selectively to deposits of cancer. Conventional dose estimates, based on the Medical Internal Radiation Dose (MIRD) formulation, assume uniform distribution of radiolabelled antitumour antibody within tissue source regions. This assumption has been tested by using a statistical model to predict the pixel value distribution obtained from the digitised radioluminographs of a known radioactive source. The model uses the statistical nature of the detection of radiation where any uniform source distribution can be expected to have a detected histogram of pixel counts that is normal or Gaussian. Therefore, any test for the degree of normality in the detected distribution is also a measure of the degree of uniformity in the source. METHODS AND MATERIALS: Three statistical techniques have been used to test the normality of the histogram of pixel values produced from the antibody distribution in a tissue section. Kurtosis, skew, and Lilliefor's are tests for normality and have statistically defined critical values for a normal distribution. After administration of 125I-labelled F(ab)2 antibody to nude mice bearing the LS174T colorectal cancer xenograft, the uniformity of antibody distribution in tumour and healthy tissues is measured using the radioluminographs of formalin-fixed paraffin sections. The test statistic for kurtosis, skew, and Lilliefor's is calculated for each tissue and is compared to critical values from statistical tables. RESULTS: The radiolabelled antibody is distributed uniformly in liver, spleen, muscle, lung, and colon and, therefore, conforms to conventional use of the MIRD formulation. The study showed that the kidney cortex and medulla should be considered separately in macroscopic absorbed-dose calculations, as should bone marrow and hard bone. Antibody heterogeneity in the tumour necessitates the incorporation of a microdosimetric tumour model into a macrodosimetry model for the accurate calculation of absorbed dose in all tissues.

Animals↗
PubMed1999

In vitro assessment of Lipiodol-targeted radiotherapy for liver and colorectal cancer cell lines.

Intra-arterial Lipiodol has been used to deliver targeted therapies to primary, and some metastatic, liver cancers. Targeted radiotherapy has been used by substituting the iodine in Lipiodol with 131Iodine (131I). Early clinical results are encouraging, but the variable response may partly depend on local pharmacokinetics. This study evaluated the in vitro cytotoxic effects of 131I-Lipiodol on human hepatocellular carcinoma (Hep-G2), human colorectal metastatic cancer (SW620), human colorectal hepatic cancer (LoVo) and human umbilical vein endothelial cells (HUVEC) cell lines. The cell cultures were exposed to 131I-Lipiodol for 48 h, following which cell counts and viability were assessed by haemocytometer, S-Rhodamine uptake and radioactivity assay. The effect of exposure to control Lipiodol, 131I-Lipiodol and 131I alone was evaluated. 131I-Lipiodol was cytotoxic against all the cancer cell lines but not against the non-malignant (HUVEC) cell line. The cytotoxicity effects were very similar in all the cancer cell lines. There were no cytotoxic effects following exposure to plain 131I in any of the cell lines (malignant and non-malignant). A similar trend was seen with radioactivity counts using a gamma counter. The cytotoxic effect of 131I-Lipiodol had a graded effect with an increase in cytotoxicity following the increase in the radioactive dose. This study showed that there was a marked cytotoxic effect by 131I-Lipiodol on all the cancer cell lines. There was no difference between the controls and the 131Iodine. This suggests that effective 131I-Lipiodol targeted therapy is dependent on the uptake and retention of Lipiodol by malignant cells.

Cell Division↗
PubMed1999

Modulation of oxygen consumption rate and vascular endothelial growth factor mRNA expression in human malignant glioma cells by hypoxia.

Cellular responses to hypoxia include modulation of respiration rate and up-regulation of genes which encode for angiogenesis factors. We tested whether human malignant glioma cells vary in their response to hypoxic stress over the range of oxygen concentrations which exist in tumours. In five cell lines tested, decreased oxygen availability resulted in decreased rates of oxygen utilization, however substantial differences in the magnitude of the response were observed. Northern blot analysis was used to study induction of vascular endothelial growth factor mRNA in response to hypoxia. In two cell lines, modest hypoxia increased vascular endothelial growth factor mRNA levels compared with those of aerobic controls. In two additional cell lines, vascular endothelial growth factor mRNA was constitutively expressed under aerobic conditions and was not further increased by hypoxia. These findings demonstrate that differences in the response to hypoxia exist among human malignant glioma cell lines and suggest that therapies designed to exploit tumour hypoxia may have varying effects in tumours with different hypoxic stress responses.

Blotting, Northern↗
PubMed1999

Dosimetric evaluation and radioimmunotherapy of anti-tumour multivalent Fab' fragments.

We have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab')2) which have been used previously in clinical radioimmunotherapy (RIT) studies in colorectal carcinoma. Comparative biodistribution studies of similar sized DFM and F(ab')2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model (Casey et al (1996) Br J Cancer 74: 1397-1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab')2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM.

Animals↗
PubMed1999

A comparison of image registration techniques for the correlation of radiolabelled antibody distribution with tumour morphology.

Image registration is a powerful tool for correlating functional images with images of anatomical structure. This facilitates more accurate quantitation of regional radiopharmaceutical uptake. Similarly, registration of images of radiolabelled antibody distribution, in tissue sections, with the equivalent histological images allows the comparison and measurement of radiopharmaceutical distribution with morphological structure. The images used were obtained by storage phosphor plate technology, for the radiopharmaceutical distribution, and by digitization of the stained histological sections. Here we compare four fully automatic registration techniques and one manual technique in terms of their spatial accuracy. We have found that there was no difference in accuracy between cross-correlation, minimization of variance and mutual information. These techniques were more accurate than principal axes and the manual technique. However, minimization of variance and mutual information were more time-consuming than the other methods. Consequently, cross-correlation is the method of choice for automatic registration of large numbers of these image pairs.

Adenocarcinoma↗
PubMed1998

Targeting strategy for gene delivery to carcinoembryonic antigen-producing cancer cells by retrovirus displaying a single-chain variable fragment antibody.

Cancer-specific antigens are promising targets for the specific delivery of certain drugs or genes to cancer cells in cancer therapy. Carcinoembryonic antigen (CEA) is one of the cancer-associated antigens predominantly detected in the gastrointestinal cancer of the colon and stomach. Targeting strategies for CEA-producing cancer cells have been thoroughly developed mainly by the production of monoclonal antibodies to CEA and further single-chain variable fragment (scFv) antibodies. Here, we have generated Moloney murine leukemia virus-derived retroviral vectors co-displaying an anti-CEA scFv-envelope chimeric protein and an unmodified envelope protein to deliver a gene for herpes simplex virus thymidine kinase (HSV-tk) or Escherichia coli beta-galactosidase. The harvested viruses successfully incorporated the chimeric envelope protein as well as the unmodified envelope into the viral particles, and specifically bound to and infected human CEA-producing cancer cells via recognition of CEA, depending on the CEA-producing phenotype of the target cells. These results may have significant implications for the use of scFv directed against tumor-specific antigens for targeting specific antigen-producing cancer cells, a potential step toward in vivo cancer therapy.

3T3 Cells↗
PubMed1998

Exemplifying guidelines for preparation of recombinant DNA products in phase I trials in cancer: preparation of a genetically engineered anti-CEA single chain Fv antibody.

Products of recombinant DNA technology have potential for the diagnosis or treatment of cancer. There is a need to investigate whether they function by the intended mechanism in small phase I clinical trials before their suitability for more extensive studies can be assessed. Quality and safety of these products should be assured prior to their use in humans in a way which is appropriate to the preliminary nature of the trials but not inhibitory to progress. The Cancer Research Campaign control recommendations for products derived from recombinant DNA technology (Begent RHJ and associates. Eur J Cancer 1993, 29A, 13, 1907-1910) provide guidelines for the production of new biotechnology products in academic research units within a relatively short time, while ensuring appropriate quality and safety. The practical application of the guidelines requires that solutions are found for the quality and safety issues during the production of recombinant products. We describe an approach to the relevant quality and safety issues during and after the production and purification of a genetically engineered anti-carcinoembryonic antigen (CEA) single chain Fv (scFv) antibody for a phase I trial of radioimmunoguided surgery with the intention of providing a model for other products.

Biotechnology↗
PubMed1998

Clearance of yttrium-90-labelled anti-tumour antibodies with antibodies raised against the 12N4 DOTA macrocycle.

Radioimmunotherapy (RIT) is currently limited by toxicity to normal tissues as a result of prolonged circulating radioantibody in the blood. In this study, the use of a clearing antibody was investigated (second antibody) in an attempt to reduce blood background levels of [90Y]A5B7 immunoglobulin G (IgG) activity, and, therefore, improve the therapeutic tumour-blood ratio in nude mice bearing human colorectal tumour xenografts. The second antibody was raised against the 12N4 macrocycle group used for chelation of 90Y, and is, thus, applicable to any anti-tumour antibody labelled with this methodology. Second antibody was administered 18, 24 or 48 h after radiolabelled antibody injection and produced up to a tenfold reduction in blood levels and a tenfold improvement in tumour-blood ratios. This has the advantage of reducing the risk of myelotoxicity caused by prolonged retention of activity in the blood. For all normal tissues, there was a similar or slightly lower uptake of [90Y]IgG with second antibody clearance, apart from a transient rise in liver activity due to complexes of primary and secondary antibody clearing via the liver. As a result of clearance of [90Y]IgG from the blood pool, there was an associated fall in the amount of antibody at the tumour site (up to 3.3-fold) at later time points for mice injected with second antibody. However, despite this, tumour-blood ratios remained superior to the control group at these later time points. Estimated dosimetry evaluation revealed that total dose to normal tissues, blood and tumour was lower than for the non-clearance group. Surprisingly, however, there was little improvement in total estimated tumour-blood dose ratio over the time period studied. This was probably because the majority of the dose was delivered to both the blood and tumour within the first 24 h after administration of [90Y]IgG, so that giving the clearing agent after this time did not produce a large difference in total estimated dose. The anti-macrocycle second antibody proved to be a successful clearing agent and could potentially be applied to any anti-tumour antibody coupled with the 12N4 macrocycle. In the light of the estimated dosimetry results described here, it would probably be most useful given at earlier time points (i.e. before 18 h after injection of primary antibody) to produce an improved tumour-blood ratio of total dose. Development of this strategy may allow higher levels of activity to be administered for RIT, and repeated dosing regimens.

Animals↗
PubMed1998

Management of resistant gestational trophoblastic tumors.

OBJECTIVE: To analyze the causes of therapeutic success and failure in the management of patients with high-risk gestational trophoblastic tumors (GTTs). STUDY DESIGN: Analysis of 272 consecutive high-risk patients treated at the trophoblastic disease center at the Charing Cross Hospital between 1979 and 1995. RESULTS: EMA (etoposide, methotrexate, actinomycin D)/CO (cyclophosphamide, vincristine) chemotherapy is our treatment of choice for patients with high-risk GTT. In 272 consecutive patients the cumulative five-year survival was 86.2% (95% confidence interval, 81.9-90.5%). No deaths occurred from GTT more than two years after the start of treatment. In patients whose disease became resistant to EMA/CO or relapsed after receiving EMA/CO, the majority (70%) could be salvaged with further chemotherapy (usually with the EP (etoposide, cisplatin)/EMA chemotherapy with or without surgery. Multivariate analysis identified the following adverse prognostic factors: presence of liver metastases (P < .0001), prolonged interval from antecedent pregnancy (P < .0001), presence of brain metastases (P = .0008) and term delivery of antecedent pregnancy (P = .045). Intensive chemotherapy for treating high-risk GTT carries a small risk of inducing second malignancies, and two patients developed acute myeloid leukemia, 2 cervical malignancy and 1 gastric adenocarcinoma after receiving EMA/CO chemotherapy. CONCLUSION: EMA/CO is an effective and well-tolerated regimen for high-risk GTT. Salvage chemotherapy with EP/EMA is effective in the majority of patients whose disease is resistant to EMA/CO and should be combined with surgery when the dominant site of resistant disease is known. Major adverse prognostic variables have been identified, and patients with combinations of these factors should be considered for innovative therapeutic approaches from the outset.

Adult↗
PubMed1998

The management of high-risk gestational trophoblastic tumours (GTT).

Between 1979 and 1995 we have treated 272 consecutive women with high-risk (GTT including 121 previously treated patients who were treated with the weekly EMA/CO (etoposide, methotrexate, actinomycin D alternating with cyclophosphamide and vincristine). The median follow-up is 4.5 years (range 1-16 years). The cumulative 5 year survival is 86.2% (95% confidence interval 81.9-90.5%). No deaths from GTT occurred later than 2 years after starting EMA/CO. In a multivariate analysis, adverse prognostic factors were the presence of liver metastases (p < 0.0001), interval from antecedent pregnancy > 24 months (p < 0.0001), brain metastases (p=0.0008) and term delivery of antecedent pregnancy (p=0.045). There were 11 (4%) early deaths while 213 (78%) achieved complete remission. 47 (17%) developed drug resistance to EMA/CO of whom 33 (70%) were salvaged by further cisplatinum based chemotherapy and surgery. 2 women developed acute myeloid leukaemia after treatment with EMA/CO. 56% of women who have been in remission for at least 2 years and had fertility conserving surgery have achieved pregnancy since completing EMA/CO and there have been 112 live births including 3 babies with congenital abnormalities. EMA/CO is an effective, easy to administer and well tolerated regimen for treating patient with high-risk GTT. More than half of these women will retain their fertility. However, there is a small but significant increase in second malignancies.

Adolescent↗
PubMed1997

Antibody-directed enzyme prodrug therapy: pharmacokinetics and plasma levels of prodrug and drug in a phase I clinical trial.

Antibody-directed enzyme prodrug therapy (ADEPT) was administered to ten patients in a phase I clinical trial. The aim was to measure plasma levels of the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl) amino] benzoyl-L-glutamic acid (CMDA) and the bifunctional alkylating drug (CJS11) released from it by the action of tumour-localised carboxypeptidase G2 (CPG2) enzyme. New techniques were developed to extract the prodrug and drug from plasma by solid-phase absorption and elution and to measure CPG2 activity in plasma and tissue. All extracts were analysed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). CPG2 activity was found in metastatic tumour biopsies but not in normal tissue, indicating that localisation had been successful. The clearing agent SB43-gal, given at 46.5 mg/m2, achieved the aim of clearing non-tumour-localised enzyme in the circulation, indicating that conversion of prodrug to drug could take place only at the site of localised conjugate. Plasma prodrug did not always remain above its required threshold of 3 microM for the "therapeutic window" of 120 min after dosing, but the presence of residual prodrug after the first administration of each day indicated that this could be achieved during the remaining four doses over the following 8 h. Despite considerable inter-patient prodrug plasma concentration variability, the elimination half-life of the prodrug was remarkably reproducible at 18 +/- 8 min. Rapid appearance of the drug in plasma indicated that successful conversion from the prodrug had taken place, but also undesirable leakback from the site of localisation into the bloodstream. However, drug plasma levels fell rapidly by at least 50% at between 10 and 60 min with a half-life of 36 +/- 14 min. Analysis of the plasma extracts by LC/MS indicated that this technique might be used to confirm qualitatively the presence of prodrug, drug and their metabolites.

Antibodies, Monoclonal↗
PubMed1997

Treatment of men with metastatic non-seminomatous germ cell tumours with cyclical POMB/ACE chemotherapy.

BACKGROUND: An alternating combination chemotherapy schedule for advanced nonseminomatous germ cell tumours (NSGCT) has been in use since 1977. PATIENTS AND METHODS: Three hundred thirty-nine men with metastatic NSGCT were treated with POMB/ACE (cisplatin, vincristine, methotrexate, bleomycin, actinomycin D, cyclophosphamide and etoposide), including 42 who had received previous chemotherapy or radiotherapy. Previously untreated patients were classified according to the International Germ Cell Cancer Collaborative Group (IGCCCG) model, and 31% were in the worst prognostic group. RESULTS: The median follow-up is eight years. The overall survival at five years is 82% (95% confidence interval (CI); 78%-85%). The survival of untreated patients exceeded that for previously treated patients (log-rank P = 0.04) and of testicular tumours exceeded that for primary extragonadal tumours (log-rank P < 0.0001). The survival of men with IGCCCG poor prognosis disease at three years is 75% (95% CI: 65%-84%) compared to 50% in the large cohort which was used to derive the model. There were five early treatment related deaths. In addition, five patients developed acute leukaemia, one developed a second primary lung adenocarcinoma, one man died of pulmonary fibrosis and three men died of cerebrovascular or cardiovascular disease. CONCLUSIONS: The POMB/ACE schedule has been employed in a large series of men with metastatic NSGCT over two decades. The fatal toxicity is equivalent to that described for simpler regimens. It yields equivalent response rates and survival in men with good prognosis disease and appears to achieve better survival in patients with poor prognosis disease.

Adolescent↗
PubMed1997

The importance of measuring quality of life in phase I/II trials of cancer therapy--the effects of antibody targeted therapy: Part I.

Phase I/II trials of new cancer therapies are designed to determine safety, efficacy and the optimal regimen for treatment. In order to uphold the principle that 'the interests of the subject must prevail over the interests of science and society' as laid down in the Declaration of Helsinki the effects of these trials on quality of life must be determined. Antibody-targeted therapies are a new form of cancer therapy designed to selectively deliver cytotoxic agents to cancer cells. Twenty-four patients with advanced colorectal cancer were enrolled into trials of three different treatment modalities based on this theme. Quality of life was measured using the Hospital Anxiety and Depression Scale and the Rotterdam Symptom Checklist prior to and 4 weeks after treatment. The Common Toxicity Criteria of the NCI were used to assess treatment related toxicity. There was no significant difference between pre- and post-treatment scores for all three treatment modalities. There was also no significant correlations between toxicity and physical or psychological morbidity. We suggest that the quality of life of patients undergoing phase I/II clinical trials with antibody targeted therapies for metastatic colorectal carcinoma was not adversely affected. We feel that in part this can be attributed to the high level and the intensity of support provided for the patients undergoing these treatments.

Adult↗
PubMed1997

EMA/CO for high-risk gestational trophoblastic tumors: results from a cohort of 272 patients.

PURPOSE: To evaluate the results of etoposide, methotrexate, and dactinomycin alternating with cyclophosphamide and vincristine (EMA/CO) chemotherapy in women with high-risk gestational trophoblastic tumors (GTT) and to document the middle- and long-term toxicity of the regimen. PATIENTS AND METHODS: A total of 272 consecutive women with high-risk GTT, including 121 previously treated patients, were treated with weekly EMA/CO. The median follow-up duration is 4.5 years (range, 1 to 16). RESULTS: The cumulative 5-year survival rate is 86.2% (95% confidence interval, 81.9% to 90.5%). No deaths from GTT have occurred later than 2 years after the end [corrected] of EMA/CO. In a multivariate model, adverse prognostic factors were the presence of liver metastases (P < .0001), interval from antecedent pregnancy (P < .0001), brain metastases (P = .0008), and term delivery of antecedent pregnancy (P = .045). There were 11 (4%) early deaths, while 213 patients (78%) achieved a complete remission. Forty-seven (17%) developed drug resistance to EMA/CO, of whom 33 (70%) were salvaged by further cisplatin-based chemotherapy and surgery. Two women developed acute myeloid leukemia, two cervical malignancy, and one gastric adenocarcinoma after EMA/CO. More than half (56%) of the women who had fertility-conserving surgery and who have been in remission at least 2 years have become pregnant since the completion of EMA/CO, with 112 live births, including three infants with congenital abnormalities. CONCLUSION: EMA/CO is an effective and well-tolerated regimen for high-risk GTT. More than half of the women will retain their fertility; however, there is a small but significant risk of second malignancy.

Adolescent↗
PubMed1996

Ablation of colorectal xenografts with combined radioimmunotherapy and tumor blood flow-modifying agents.

Radioimmunotherapy (RIT) does not readily eradicate common solid tumors and therefore requires augmentation by complementary therapies that do not increase normal tissue damage. We have examined the efficacy of RIT combined with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a drug which induces immunomodulation and cytokine production and preferentially reduces tumor blood flow, using a colorectal xenograft model in nude mice. Although an optimal i.p. dose (27.5 mg/kg) of drug alone induced massive hemorrhagic necrosis of all but a thin peripheral rim of viable tumor cells, survival was unaffected. However, when combined with i.v. 18.5 MBq 131I-labeled anti-carcinoembryonic antigen IgG, DMXAA significantly potentiated the RIT without increased toxicity, with five of six mice showing complete cures. Scheduling was critical because the antibody must be allowed to reach maximum tumor accumulation before initiation of drug-induced blood flow inhibition. Subsequently, the antibody was retained preferentially in the tumor, reaching approximately twice control levels by 5 days after drug delivery. In combined studies, the drug had a narrow therapeutic window, 30 mg/kg being toxic to two of six mice, whereas 20 mg/kg were ineffective. However, the addition of a second vasoactive agent, serotonin, to RIT plus 20 mg/kg DMXAA enhanced therapy without increasing systemic toxicity. Tumor histology and phosphor image plate analysis reflected these results. When given without RIT, the two drugs combined, although not alone, also significantly inhibited tumor growth. Drug-induced tumor necrosis and tumor retention of radioantibody may both contribute to the enhanced RIT produced by this combined complementary therapy.

Animals↗
PubMed1996

Extended glycoprotein structure of the seven domains in human carcinoembryonic antigen by X-ray and neutron solution scattering and an automated curve fitting procedure: implications for cellular adhesion.

Carcinoembryonic antigen (CEA) is one of the most widely used cell-surface tumour markers for tumour monitoring and for targeting by antibodies. It is heavily glycosylated (50% carbohydrate) and a monomer is constructed from one V-type and six C2-type fold domains of the immunoglobulin superfamily. The solution arrangement at low resolution of the seven domains in CEA cleaved from its membrane anchor was determined by X-ray and neutron scattering. Guinier analyses showed that the X-ray radius of gyration RG of CEA was 8.0 nm. The length of CEA was 27 to 33 nm, and is consistent with an extended arrangement of seven domains. The X-ray cross-sectional radius of gyration RXS was 2.1 nm, and is consistent with extended carbohydrate structures in CEA. The neutron data gave CEA a relative molecular mass of 150,000, in agreement with a value of 152,500 from composition data, and validated the X-ray analyses. The CEA scattering curves were analysed using an automated computer modelling procedure based on the crystal structure of CD2. The V-type and C2-type domains in CD2 were separated, and the C2-type domain was duplicated five times to create a linear seven-domain starting model for CEA. A total of 28 complex-type oligosaccharide chains in extended conformations were added to this model. By fixing the six interdomain orientations to be the same, three-parameter searches of the rotational orientations between the seven domains gave 4056 possible CEA models. The best curve fits from these corresponded to a family of zig-zag models. The long axis of each domain was set at 160(+/-25) degrees relative to its neighbour, and the two perpendicular axes were orientated at 10(+/-30) degrees and -5(+/-35) degrees. Interestingly, the curve fit from this model is within error of that calculated from a CEA model generated directly from the CD2 crystal structure by the superposition of adjacent domains. Zig-zag models of this type imply that the protein face of the GFCC' beta-sheet in neighbouring CEA domains lie on alternate sides of the CEA structure. Such a model has implications for the adhesion interactions between CEA molecules on adjacent cells or for the antibody targeting of CEA.

Amino Acid Sequence↗
PubMed1996

In vitro and in vivo characterisation of a recombinant carboxypeptidase G2::anti-CEA scFv fusion protein.

BACKGROUND: There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site. OBJECTIVES: To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins. STUDY DESIGN: To introduce unique cloning restriction sites into the 5'-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3'-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide. RESULTS: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-CEA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7-1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios. CONCLUSION: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.

Animals↗