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G T Yarranton

Publications and source records attributed to G T Yarranton.

At least 19 recordsLinked to original sources
PubMed2002

Tumour targeting of humanised cross-linked divalent-Fab' antibody fragments: a clinical phase I/II study.

Antibody engineering has made it possible to design antibodies with optimal characteristics for delivery of radionuclides for tumour imaging and therapy. A humanised divalent-Fab' cross-linked with a bis-maleimide linker referred to as humanised divalent-Fab' maleimide was produced as a result of this design process. It is a humanised divalent antibody with no Fc, which can be produced in bacteria and has enhanced stability compared with F(ab')(2). Here we describe a clinical study in patients with colorectal cancer using humanised divalent-Fab' maleimide generated from the anti-carcinoembryonic antigen antibody A5B7 radiolabelled with iodine-131. Ten patients received an i.v. injection of iodine-131-labelled A5B7 humanised divalent-Fab' maleimide, and positive tumour images were obtained by gamma camera imaging in eight patients with known lesions, and one previously undetected lesion was identified. True negative results were obtained in two patients without tumour. Area under the curve analysis of serial blood gamma counting and gamma camera images showed a higher tumour to blood ratio compared to A5B7 mF(ab')(2) used previously in the clinic, implying this new molecule may be superior for radioimmunotherapy. MIRD dose calculations showed a relatively high radiation dose to the kidney, which may limit the amount of activity that could be administered in radioimmunotherapy. However the reduction in immunogenicity was also a major advantage for A5B7 humanised divalent-Fab' maleimide over murine versions of this antibody suggesting that humanised divalent-Fab' maleimide should be a useful vehicle for repeated therapies.

Area Under Curve↗
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↗
PubMed1996

The secretion of active recombinant human gastric lipase by Saccharomyces cerevisiae.

The expression of the complete human gastric lipase (HGL) gene in Saceharomyces cerevisiae grown in defined medium resulted in the secretion of active recombinant HGL (rec.HGL) to levels of up to approximately 11 mg/liter. Of the total measurable HGL activity, 90% was detected by assaying intact cells, suggesting that the majority of rec.HGL produced was secreted but stayed attached to the cell wall. The remaining 10% was present in the growth medium and from this source active rec.HGL was purified 300-fold by a combination of hydrophobic interaction and ion-exchange chromatography. Rec.HGL migrated on reduced SDS-PAGE as three bands with estimated molecular masses of 47,45, and 43 kDa. All three forms cross-reacted with an antibody raised to natural HGL and their treatment with Endo H showed them to be N-linked glycosylation variants of a single polypeptide. The 47-kDa species was isolated using lentil lectin Sepharose 4B and shown to possess a specific activity comparable to that of the natural enzyme. Rec.HGL had an acid pH activity optimum using either tributyrin or olive oil as substrate and did not lose activity if incubated in the presence of pepsin at pH 2.0. These results demonstrate that HGL secreted by Saccharomyces cerevisiae retained those properties of the natural enzyme required for its use in the treatment of pancreatic insufficiency.

Chromatography, Agarose↗
PubMed1996

Radioimmunotherapy of colorectal carcinoma xenografts in nude mice with yttrium-90 A33 IgG and Tri-Fab (TFM).

The monoclonal antibody A33 recognises a tumour-associated antigen on human colorectal carcinoma, and has undergone preliminary evaluation in the clinic where selective localisation to hepatic metastases has been demonstrated [Welt et al. (1994) J. Clin. Oncol. 12, 1561-1571]. A33 and an A33 tri-fab fragment (TFM) were labelled with 90Y via a stable macrocyclic ligand for biodistribution and therapy studies in nude mice bearing SW1222 colon carcinoma xenografts. Biodistribution studies demonstrated tumour localisation for both A33 IgG and TFM with low bone, liver and kidney levels. Clearance of TFM from the blood was much faster than IgG and this led to lower tumour accumulation for TFM but superior tumour-blood ratios. The maximum per cent injected dose per g localised to tumour was 35.9% +/- 5.3% for A33 IgG and 12.9% +/- 4.6% for A33 TFM with tumour-blood ratios at 48 h after administration of 5.6 +/- 1.8 and 29.2 +/- 9.8 respectively. Autoradiography studies with 125I-labelled A33 IgG and TFM demonstrated a homogeneous distribution within tumour tissue which was not observed with other anti-colorectal tumour antibodies. TFM penetrated into the tumour tissue more rapidly than IgG. In therapy studies, a single dose of 90Y-A33 IgG (250 microCi per mouse) or 90Y-A33 TFM (300 microCi per mouse) led to complete regression of 2-week-old tumour xenografts with long-term tumour-free survivors. A transient drop in white blood cell count was observed with both IgG and TFM but was significantly more pronounced with IgG. The cell count fell to 8.4% of control for IgG, whereas with TFM cell counts fell to 51% of control before recovery. These results indicate that the more rapid blood clearance of 90Y-TFM confers reduced toxicity compared with 90Y-IgG although similar therapeutic effects are achieved. When the dose of 90Y-IgG was adjusted to give the same dose to tumour achieved with 300 microCi 90Y-TFM, a lesser therapeutic effect was observed. This may be owing to more rapid tumour penetration achieved with TFM. Both A33 IgG and TFM demonstrated potent anti-tumour effects against human tumour xenografts in this mouse model system. The stability of these 90Y-labelled conjugates and their effective tumour penetration are promising for the development of humanised reagents for clinical studies.

Animals↗
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↗
PubMed1993

Expression, purification and characterization of B72.3 Fv fragments.

The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.

Animals↗
PubMed1992

Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab' fragment.

B72.3 is a mouse monoclonal antibody against a tumour-associated antigen, TAG72, which recognizes breast, ovarian and colorectal tumour tissue. A mouse-human chimeric version of B72.3 has been expressed in Chinese-hamster ovary cells. This molecule has the binding specificity of B72.3 and constant regions from human IgG4. The chimeric B72.3 assembles to intact IgG and recognizes TAG72 as well as B72.3 in competitive binding assays. A proportion of the chimeric B72.3 (approx. 10%) does not form inter-heavy-chain disulphide bonds but still assembles into the IgG tetramer. This appears to be a general property of human IgG4 molecules. Co-expression of the chimeric light chain with a chimeric Fd' gene resulted in the expression of functional Fab'. Very little F(ab')2 is produced, although the Fab' can be oxidized to the dimeric F(ab')2 in vitro. The production of Fab' and F(ab')2 by this method is an attractive alternative to proteolytic digestion of IgG. The ability to produce these molecules in large quantities will allow the production and testing of a range of anti-tumour antibody and antibody fragment conjugates.

Animals↗
PubMed1992

Inducible vectors for expression in mammalian cells.

The most recent developments in mammalian cell inducible expression systems have involved the use of bacterial gene control elements and viral transactivator proteins. The combination of hybrid viral transactivator and bacterial repressor proteins, and simple chemical inducers can provide induction ratios of over 1000-fold. These developments will have applications in both cell-based research and the generation of transgenic animals.

Animals↗
PubMed1992

High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker.

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.

Animals↗
PubMed1991

The use of engineered E1A genes to transactivate the hCMV-MIE promoter in permanent CHO cell lines.

Vectors expressing adenovirus 5 E1A or a domain 2 mutant E1A were introduced into CHO-K1 cells in order to transactivate the hCMV-MIE promoter in transient and stable transfections. Expression from the hCMV promoter was efficiently activated by both wild-type and mutant E1A in contrast to other viral promoters such as the SV40 early promoter which are repressed by E1A. E1A genes expressed from a strong promoter were inhibitory to the growth of CHO cells. Nevertheless, by the use of a weaker promoter, it was possible to isolate stably transfected cell lines containing a level of E1A compatible with both continued cell growth and significant transactivation of the hCMV promoter. By this means we have generated cell lines secreting tissue inhibitor of metalloproteinases (TIMP) at levels approaching those previously attained using gene amplification. CHO cell lines constitutively expressing wild-type and mutant E1A genes have been derived which can serve as new host cell lines for transient expression and efficient stable expression without gene amplification.

Animals↗
PubMed1990

High level expression of tissue inhibitor of metalloproteinases in Chinese hamster ovary cells using glutamine synthetase gene amplification.

We have used a glutamine synthetase (GS) gene as an amplifiable marker in Chinese hamster ovary (CHO) cells. GS was combined with an efficient transcription unit to produce tissue inhibitor of metalloproteinases (TIMP). Initial transfectant cell-lines selected using a GS gene secreted up to 9 micrograms TIMP/10(6) cells/24h. After one round of GS gene amplification expression levels of 110 micrograms TIMP/10(6) cells/24h were achieved. These GS gene amplified CHO cells, when adapted to grow in suspension, accumulated 180mg/l in shake flask culture. This system therefore provides a rapid method of achieving high level gene expression in mammalian cells.

Animals↗
PubMed1990

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity.

The expression of immunoglobulin heavy and light chain variable regions in the cytoplasm of Escherichia coli and formation of a functional heterodimer has been demonstrated. Variable domain sequences were taken from the heavy and light chain cDNAs of the monoclonal antibody Gloop 2 and engineered for expression in a dual origin expression vector. The engineered genes vhg2 and vlg2 were separately subcloned into the vector, creating two expression plasmids. Expression of the heavy and light chain variable region genes (encoding 116 and 109 amino acids respectively) was investigated in eight E. coli strains; the polypeptides were rapidly degraded in a host strain optimized for expression and in E. coli strains deficient in the major protease La (lon-). Accumulation was permitted in severely protease-deficient E. coli having a defective heat-shock response. A lon- mutation in this genetic background permitted even higher accumulation. Expression levels were 7 and 1% of total bacterial protein for light and heavy chain variable regions respectively. Expression of the heavy chain variable region gene was increased by including a longer Shine-Dalgarno sequence. Similar constructions in the light chain vector had no effect on expression levels. The insoluble variable region polypeptides were reconstituted into a heterodimer possessing the full antigen binding characteristics of both the parent monoclonal antibody and its Fab fragment.

Animals↗
PubMed1990

Site-specific attachment to recombinant antibodies via introduced surface cysteine residues.

Many diagnostic and therapeutic applications of monoclonal antibodies require the covalent linking of effector or reporter molecules to the immunoglobulin polypeptides. Existing methods generally involve the non-selective modification of amino acid side chains, producing one or more randomly distributed attachment sites. This results in heterogeneous labelling of the antibody molecules and often to a decrease in antigen-binding due to the modification of residues close to the antigen-binding site. We report a novel strategy for site-specifically labelling antibodies through surface cysteine residues. Examination of molecular structures was used to identify amino acids of the CH1 domain of the IgG heavy chain that were accessible to solvent but not to larger molecules. Site-directed mutagenesis was used to substitute cysteine residues at these positions in the heavy chain of a mouse/human chimaeric version of the tumour-binding monoclonal antibody, B72.3. Expression of the modified antibody genes in mammalian cells yielded correctly assembled proteins that had thiol groups in pre-determined positions and showed no loss of antigen-binding activity. One of the mutants was used to demonstrate the site-specific attachment of a radio-iodinated ligand to the chimaeric B72.3 antibody.

Algorithms↗
PubMed1987

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast.

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Amino Acid Sequence↗
PubMed1986

Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes.

Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the lambda pR promoter, the cI857 temperature-sensitive repressor gene and the pSC101 origin of replication and its associated par sequence, were constructed. Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction. Cloned gene expression increased with copy number, and accumulation values of greater than 20% total cellular protein were detected. These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.

Acetyltransferases↗