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Biomedical subjects

R H Begent

Publications and source records attributed to R H Begent.

At least 55 records · Page 3Linked to original sources
PubMed1996

Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab' fragment by poly(ethylene glycol) (PEG) modification.

Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.

Adenocarcinoma↗
PubMed1996

Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab' fragments.

The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The cross-linkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy beta-emitter. We have also compared the above antibody forms conjugated to both 131I and 90Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37 degrees C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 90Y was observed. Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 10(5) Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 131I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 131I and 90Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for 131I-labelled antibodies. Because of the faster clearance, it should be possible to administer a higher total dose of 90Y-labelled TFM than IgG, which is attractive for RIT. Both A5B7 DFM and TFM, therefore, show favourable properties compared with their parent antibody forms.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library.

We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

Adult↗
PubMed1996

Technetium-99m radiolabeling using a phage-derived single-chain Fv with a C-terminal cysteine.

UNLABELLED: Single-chain Fv (scFv) antibody fragments have potential for clinical imaging because of their rapid tumor penetration and high tumor-to-tissue ratios at early time points. ScFvs clear rapidly from the circulation so radiolabels such as 99mTc which have short half-lives are desirable, but the free thiol groups necessary for labeling with 99mTc are not normally found on these molecules. METHODS: We constructed a vector which enabled a free cysteine to be linked to the C-terminus of scFvs. MFE-23, a scFv directed against carcinoembryonic antigen (CEA), was cloned into this vector and cys-tagged MFE-23 was labeled with 99mTc using a D-glucarate transfer method. RESULTS: The radiolabeled product was stable in vivo and in vitro and showed favorable tumor-to-blood ratios in vivo at early time points (4:1 at 24 hr and 8:1 at 48 hr), although high kidney levels were also detected. CONCLUSION: Our study demonstrates an effective method to enable scFvs radiolabeling with 99mTc and also shows the potential of using a 99mTc-labeled scFv for clinical imaging studies.

Adenocarcinoma↗
PubMed1996

Placental site trophoblastic tumor: molecular analysis and clinical experience.

Placental site trophoblastic tumor is a very rare variant of gestational trophoblastic disease which differs histologically and immunocytochemically from gestational choriocarcinoma. The English language literature includes only 74 reported cases. Seventeen patients have been managed at Charing Cross Hospital with this diagnosis. The median follow-up is 4.6 years, and the 5-year overall survival is 80% (95% confidence interval, 55-93%). Multivariate regression analysis identified an interval of >2 years since the preceding pregnancy as an independent adverse prognostic factor. Genotypic analysis by PCR allelotyping has confirmed the gestational origin of all 11 tumors successfully studied. More detailed molecular analysis has identified the causative pregnancy for eight tumors. Five were diploid biparental tumors following term pregnancies, and three were androgenetic tumors following monospermic complete hydatidiform moles.

Adult↗
PubMed1995

A single chain Fv derived from a filamentous phage library has distinct tumor targeting advantages over one derived from a hybridoma.

Single-chain antibodies (scFvs) can be derived from a monoclonal antibody (MAb) or produced directly using filamentous phage technology, where antibodies with desired binding and purification characteristics can be readily selected from libraries. To test the hypothesis that the latter approach is more useful, we compared 2 anti-carcinoembryonic antigen (CEA) scFvs produced by these 2 different approaches. Our study showed that, both in the purification process and in the biodistribution pattern, MFE-23, produced by filamentous phage technology, gave favourable results compared to A5-SC, which is derived from the A5B7 MAb. This indicates the value of the filamentous phage approach for obtaining tumour-targeting scFvs.

Animals↗
PubMed1995

Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography.

A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.

Amino Acid Sequence↗
PubMed1995

Galactosylated streptavidin for improved clearance of biotinylated intact and F(ab')2 fragments of an anti-tumour antibody.

Persistence of high levels of radiolabelled antibody in the circulation is a major limitation of radioimmunotherapy. Biotinylation of the radiolabelled anti-tumour antibody followed by administration of streptavidin is known to give much improved tumour to blood ratios as the radioantibody is complexed and subsequently cleared via the reticuloendothelial system, although prolonged splenic uptake is a problem. We have investigated the effect on the clearance pattern and tumour localisation of a 125I-labelled biotinylated anti-CEA antibody (A5B7) after administration of a galactosylated form of streptavidin (gal-streptavidin) in nude mice bearing a human colon carcinoma xenograft. Fifteen minutes to 1 h after gal-streptavidin administration the complexes were cleared via the liver alone (as opposed to liver and spleen after native streptavidin). Twenty-four hours after administration of gal-streptavidin, the tumour to blood ratio for biotinylated A5B7 IgG increased from 2.9 to 13.2 and for biotinylated F(ab')2 fragments an increase from 4.9 to 33.2 was achieved. The reduction in tumour accumulation of F(ab')2 24 h after injection of the clearing agent was less than that seen with intact antibody. Injection of asialofetuin inhibited clearance, confirming that removal of the gal-streptavidin-biotinylated antibody complexes from the blood was via the asialoglycoprotein receptor on liver hepatocytes. Therefore, galactosylation of the streptavidin clearing agent allows rapid removal of radiolabelled biotinylated antibodies via the liver asialoglycoprotein receptor, as opposed to the reticuloendothelial system.

Antibodies, Monoclonal↗
PubMed1995

Secondary tumours following etoposide containing therapy for germ cell cancer.

BACKGROUND: Reports have implied etoposide as the cause of secondary leukaemia in patients treated for germ cell cancer. PATIENTS AND METHODS: Between 1979 and 1992, 679 male patients with germ cell cancer received etoposide containing chemotherapy. RESULTS: Six of 679 patients developed acute myeloid leukaemia (relative risk 150; CI: 55-326). None of these patients had a primary mediastinal germ cell tumour and only 1 patient received previous radiotherapy. The median interval between the onset of cytotoxic treatment and the development of leukaemia was 27 months. The FAB M4 morphology was seen in 4 of 6 cases. CONCLUSION: The benefit of etoposide containing protocols outweigh the risk of leukaemia in patients with intermediate or high risk disease, however in patients with good risk disease non-etoposide containing protocols should be explored.

Acute Disease↗
PubMed1994

Enhancement of radioimmunotherapy by drugs modifying tumour blood flow in a colonic xenograft model.

Radioimmunotherapy (RIT) is hampered clinically by poor tumour localization of antibody. In order to enhance localization we have investigated the concomitant use of RIT with 2 drugs, flavone-8-acetic acid (FAA) and its analogue 5,6-dimethylxanthenone-4-acetic acid (XAA), which both reduce tumour blood flow and induce immunomodulation. A single i.v. dose of 0.5 mCi (18.5 MBq) intact 131I anti-CEA antibody significantly delayed growth and prolonged survival over that of untreated controls, in an established LS174T colon xenograft model in nude mice. The adjuvant use of a single i.p. dose of either FAA or XAA, given 24 or 48 hr after 131I-A5B7 to allow maximum tumour levels of antibody to be attained before drug-induced blood-flow inhibition, significantly enhanced the RIT. FAA caused entrapment of antibody within the tumour in relation to the time allowed for localization before drug administration. Repeated doses of FAA prolonged tumour growth inhibition but did not enhance the therapy achieved after a single dose. Although both drugs alone induced massive tumour necrosis of all but a thin peripheral rim of viable cells, tumour regrowth was inhibited for a few days only, with no effect on survival. Drug-induced tumour necrosis, immunomodulation and retention of higher doses of 131I-A5B7 within the tumour may contribute to the enhanced RIT produced by this combined therapy.

Animals↗
PubMed1994

Production and tumour-binding characterization of a chimeric anti-CEA Fab expressed in Escherichia coli.

A recombinant chimeric Fab (rcFab), with Fv derived from the monoclonal A5B7 antibody to carcinoembryonic antigen (CEA), and with human CHI and C kappa was cloned into pUC 19 and expressed in Escherichia coli. rcFab (10 to 12 mg per litre) was produced in bacterial culture fluid, and functional purified rcFab was isolated by affinity chromatography (using antibody to human C kappa) and size-exclusion gel filtration. The rcFab did not show reduced affinity for CEA, and reacted with human colorectal tumours showing a typical anti-CEA pattern by immunocytochemistry; it was also stable after iodination. Biodistribution studies in nude mice bearing human tumour xenografts showed no toxicity and good tumour localization. Therapeutic ratios at early time points were better than those obtained with whole murine antibody. The results demonstrate that bacterially produced anti-CEA Fab is of use for tumour targeting.

Adenocarcinoma↗
PubMed1994

Phage libraries for generation of clinically useful antibodies.

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

Animals↗
PubMed1994

Radioimmunotherapy of metastatic colorectal tumours with iodine-131-labelled antibody to carcinoembryonic antigen: phase I/II study with comparative biodistribution of intact and F(ab')2 antibodies.

Studies in animal tumour models of colorectal cancer suggest that F(ab')2 antibody fragments to carcinoembryonic antigen (CEA) labelled with iodine-131 give superior therapy compared with intact anti-CEA antibody. The purpose of this study was to investigate this hypothesis in patients. Ten patients received intact A5B7 IgG1 mouse monoclonal antibody (MAb) to CEA and nine patients received the F(ab')2 fragment of the same antibody. The biodistribution for each molecule was compared using quantitative single-photon emission computerised tomographic (SPECT) gamma-camera imaging. Tumour responses were seen in both groups and myelosuppression was the limiting toxicity. F(ab')2 localised more rapidly than intact antibody in tumour, giving a mean percentage injected activity per kg at 4.25 h after injection of 8.2% for F(ab')2 compared with 4.4% for intact antibody (P < 0.05). No significant difference in antibody clearance from, or cumulative dose per unit administered activity (cGy MBq-1) to, tumour was seen. Distribution in blood was similar for both the intact and fragment antibody. These findings are consistent with more rapid penetration of the smaller F(ab')2 into tumour masses. More efficient early uptake will give higher maximum dose rates to the tumour which is valuable for radioimmunotherapy (RIT) when low dose rates may limit effectiveness of treatment. F(ab')2 fragments may provide a substantially enhanced method of delivering RIT.

Adult↗
PubMed1994

The potential for enhanced tumour localisation by poly(ethylene glycol) modification of anti-CEA antibody.

Attachment of poly(ethylene glycol) (PEG) to proteins can greatly alter their pharmacological properties, including extending the plasma half-life and reducing immunogenicity, both of which are potentially beneficial to tumour targeting. IgG, F(ab')2 and Fab' fragments of the anti-CEA antibody A5B7 were chemically modified with PEG (M(r) 5,000), labelled with 125I and their pharmacokinetics compared with the unmodified forms in the LS174T colonic xenograft in nude mice. PEG modification of the intact antibody had little effect on biodistribution, although tumour localisation was slightly reduced. In contrast, similar modification of F(ab')2 and Fab'A5B7 significantly prolonged plasma half-life and increased radioantibody accumulation in the tumour and to a lesser extent in normal tissues, but reduced tissue to blood ratios. Prior to modification, Fab' A5B7 (M(r) 50,000) cleared more rapidly from the circulation than F(ab')2 (M(r) 100,000), but after PEG attachment their biodistributions converged, while the tumour to blood ratios were reduced and resembled that of the intact antibody. The enhanced tumour accumulation, reduced normal tissue to blood ratios and potentially reduced immunogenicity of fragments after PEG attachment may therefore prove superior to either unmodified fragments or intact antibody for antibody-targeted therapy, although the increased plasma half-life may necessitate the use of a clearance mechanism.

Adenocarcinoma↗
PubMed1994

Localisation of monoclonal antibodies reacting with different epitopes on carcinoembryonic antigen (CEA)--implications for targeted therapy.

Antibody targeting has potential for selective delivery of cancer therapy. However, there is a wide variation in the degree of antibody localisation in individual patients with colorectal adenocarcinoma. Colorectal adenocarcinomas are composed of glandular structures separated from fibrovascular stroma by a basal lamina which may represent a significant barrier to extravasated antibody. Basement membrane-associated CEA epitopes may be more accessible to antibodies than those which are cytoplasmic or lumenal. We have investigated by immunohistochemistry and in vivo localisation, the extent to which distribution of antigen epitopes influences targeting. Two monoclonal antibodies (A5B7 and EA77) recognising non-overlapping CEA epitopes were reacted immunohistochemically with samples of 39 tumours. Intensity and site of reaction were assessed for basement membrane, cytoplasmic or lumenal surface association. 125I-labelled antibodies were injected into nude mice bearing LS174T tumour. Per cent injected activity per gram was measured in tumour and normal tissues, 24, 72 and 168 h later. Tissues reacted immunohistochemically for CEA were autoradiographed to assess the relationship of injected antibody to target antigen. Immunohistochemistry showed that A5B7 antibody favours basement membrane aspects of malignant glands; in contrast, EA77 concentrated generally on lumenal surfaces. In vivo localisation showed that per cent inj.act g-1 in tumour for A5B7 reached 36.5% at 24 h. EA77 localised to a lesser extent (9.1% at 24 h), despite a longer circulatory half-life. Autoradiography combined with immunohistochemistry showed A5B7 reacting with antigen close to vasculature after 24 h, slowly penetrating deeper parts of the tumour by 72 h. In contrast, EA77 was confined mainly to fibrovascular stroma, showing little labelling of antigen-positive tumour cells. Localisation differences between A5B7 and EA77 may partly be due to accessibility of epitopes on tumour cells.

Adenocarcinoma↗
PubMed1994

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.

Animals↗