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M P Deonarain

Publications and source records attributed to M P Deonarain.

At least 19 recordsLinked to original sources
PubMed2005

Characterisation and internalisation of recombinant humanised HMFG-1 antibodies against MUC1.

The humanised HMFG-1 immunoglobulin has been extensively developed as a clinical immunotherapeutic agent for MUC1 expressing tumours. We have constructed a single-chain Fv (scFv) and Fab fragment from this antibody and shown that both these species retain their specificity for MUC1. The scFv was less stable and less soluble than the Fab. Detailed analyses of the binding kinetics of the whole IgG and Fab fragment show that the affinity for MUC1 synthetic peptides is low (approximately 100 nM for the IgG and 10 muM for the Fab), with particularly low but similar dissociation rate constants (0.031-0.095 s(-1)). Binding to native antigen on the cell surface is over two orders of magnitude better. Confocal immunofluorescence microscopy shows that both the IgG and Fab are internalised rapidly (the IgG is internalised within 15 min) and colocalise to early endosomes. This work provides an appreciation of the binding, internalising and trafficking kinetics, important for the development of future therapeutics based on this antibody.

Antibodies, Monoclonal↗
PubMed2004

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme.

Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37 degrees C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies.

Amino Acid Sequence↗
PubMed2000

A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I.

A number of mammalian proteins with suitable biological activities have been considered for use in targeted tumour therapy. Deoxyribonuclease-I (DNase-I), an endonuclease that degrades double-stranded DNA, represents an attractive candidate for tumour targeting since it is normally non-toxic yet could be highly cytotoxic when redirected to the cell nucleus. Our aim was to investigate the cytotoxic potential of mammalian DNase-I and its possible use in tumour-targeting strategies for cancer therapy. A chimeric molecule comprising a scFv reactive against the human placental alkaline phosphatase (hPLAP) and bovine pancreatic DNase-I was designed and investigated. The development of a tightly controlled system for the bacterial expression of DNase-I and its chimera is described. The production and purification of active DNase-I from the soluble cell fraction and significant yields from the insoluble fraction by isolation and refolding are described. The construction, expression, purification and in vitro characterisation of an anti-PLAP scFv-DNase-I chimera is also described. This molecule was shown to possess both antigen-binding and DNA-degrading activity in in vitro assays, thus combining the specific cell-targeting properties of the scFv and the potent, highly catalytic activity of the endonuclease. Furthermore, this chimeric molecule was highly cytotoxic in vitro in cells expressing the PLAP antigen. Targeting mammalian DNase-I provides a novel therapeutic strategy for selective cell killing, with the promise of less systemic toxicity and immunogenicity than currently used immunotoxins.

Alkaline Phosphatase↗
PubMed1998

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins.

Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.

Alkaline Phosphatase↗
PubMed1997

Redesigned anti-human placental alkaline phosphatase single-chain Fv: soluble expression, characterization and in vivo tumour targeting.

Although much progress has been made in the production of recombinant antibodies and their fusions, there are still problems with solubility and folding. Useful antibodies produced from cloned hybridomas do not always result in scFvs behaving favourably. We report here further work on an scFv (H17E2) against the oncofetal antigen human placental alkaline phosphatase. The overall expression was greatly improved and the H17E2 scFv was redesigned by manipulation of the interdomain linker, resulting in much higher expression levels of the soluble scFv in its active conformation at 0.2-0.5 mg/l of bacterial culture. We show that the new soluble version of this scFv has similar characteristics to the refolded version in terms of antigen and tumour cell binding, stability and in vivo pharmacokinetics. The final tumour uptake behaviour of these scFvs is superior to that of the parental whole antibody with respect to tumour:organ ratios, but still requires further development before considering it as a suitable molecule for clinical use in ovarian or testicular cancer.

Alkaline Phosphatase↗
PubMed1995

Genetic delivery of enzymes for cancer therapy.

For many years, antibodies have been examined as means to deliver cytotoxic proteins to kill target cells (immunotoxins). More recently, there have been studies on enzymes that convert prodrugs to active drugs to kill target cells. The advances in gene therapy strategies now allow one to deliver the gene for the protein or enzyme as an alternative. This technique, although in its infancy, promises to overcome some of the problems associated with antibody-mediated delivery. Thymidine kinase and cytosine deaminase are some of the enzymes currently being exploited in this way, but more are on the horizon. However, more research is still needed to enable full exploitation of the transcriptional differences between tumour and normal cells so that more existing cancers can be treated in this way.

Animals↗
PubMed1995

DNA vaccination for cancer treatment.

The recent finding that inoculation with plasmids encoding a variety of proteins leads to T cell and antibody responses in vivo against these proteins provides a novel means of active specific immunisation by plasmid vaccination. The demonstration that both major histocompatibility complex (MHC) Class I- and Class II-mediated interactions can be elicited may make this approach suitable for development of tumour vaccines. Plasmids may prove to be an efficient way to build 'subunit' and multi-subunit vaccines based on the genetic changes that occur in carcinogenesis. Expression of DNA encoding fragments of tumour-specific proteins as neo-antigens or surrogate antigens in a novel context may be a means of breaking immunological tolerance and lead to the generation of tumour-specific immune responses.

Animals↗
PubMed1994

Reductive and oxidative half-reactions of glutathione reductase from Escherichia coli.

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH and has a redox active disulfide and an FAD cofactor in each monomer. In the reductive half-reaction, FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox active disulfide. The oxidative half-reaction is dithiol-disulfide interchange between the enzyme dithiol and glutathione disulfide. We have investigated the reductive and oxidative half-reactions using wild-type glutathione reductase from Escherichia coli and in an altered form of the enzyme in which the active site acid-base catalyst, His439, has been changed to an alanine residue (H439A). H439A has 0.3% activity in the NADPH/GSSG assay. The replacement affects both the oxidative half-reaction, as expected, and the reductive half-reaction--specifically, the passage of electrons from reduced flavin to the disulfide. Reduction of H439A by NADPH allows direct observation of flavin reduction. The NADPH-FAD charge transfer complex is formed in the dead time. Reduction of FAD, at a limiting rate of 250 s-1, is observed as a decrease at 460 nm and an increase at 670 nm (FADH(-)-NADP+ charge transfer). Subsequent passage of electrons from FADH- to the disulfide (increase at 460 nm and a decrease at 670 nm) is very slow (6-7 s-1) and concentration independent in H439A. The monophasic oxidative half-reaction is very slow, as expected for reduced H439A.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1994

Deoxyribonuclease I (DNAse I). A novel approach for targeted cancer therapy.

A number of phosphodiesterases, some of which possess additional biological activities (e.g., antitumor, immunosuppressive, and so on), have been considered for use in targeted tumor therapy. We propose Deoxyribonuclease I (DNase I), a compact, monomeric enzyme, as a very attractive candidate for targeting to tumor cells. Only a small amount of enzyme targeted to a cell needs to enter the nucleus in order to degrade the chromosomal DNA, making a cell incapable of further replication. We describe preliminary data on the construction of a potent single-chain antibody (scFv) immunotoxin based on bovine pancreatic DNAse I. The use of a mammalian enzyme should be much less toxic and less immunogenic than current immunotoxins and may expand the current limits of immunotoxin therapy.

Amino Acid Sequence↗
PubMed1994

Targeting phosphodiesterases as a strategy for killing tumor cells.

Ribonucleases (RNases) are being employed as alternative cytotoxic proteins to the conventionally used ones such as ricin and Pseudomonas exotoxin. Mammalian RNases are attractive enzymes because of their comparable cytotoxicity when suitably directed and the likelihood of lower immunogenicity compared to plant and bacterial toxins. Bovine seminal RNase (BSRNase) is a member of the RNase superfamily, but differs in many interesting ways. Unlike the rest of the family it is dimeric, and possesses antitumor and immunosuppressive properties. These features make it a choice candidate for a single-chain antibody (scFv) based immunotoxin. This work describes preliminary data on the construction, expression in Escherichia coli and characterization of a tumor-specific scFv (directed against human placental alkaline phosphatase)-BSRNase chimeric molecule. It is shown that the created molecule has RNA degrading activity and antigen-binding activity when refolded from bacterial inclusion bodies.

Animals↗
PubMed1994

Genetically engineered antibodies for diagnostic pathology.

Antibody genes can be cloned, genetically manipulated, and expressed in both homologous and heterologous expression systems to produce viable antigen-binding proteins complete with natural effector functions. Manipulation of antibody genes permits the expression of fusion proteins or truncated proteins that retain antigen-binding activity. The new antibody technologies are becoming increasingly sophisticated, permitting the alteration of antigen-binding responses, the transfer of antigen specificity between antibodies, and the expression of minimal-size antigen-binding protein domains. These new molecules have been made mostly for studies on function or to provide molecules suited for in vivo diagnosis and therapy; very few have been specifically designed for, or used for, diagnostic histopathology. We describe here the adaptation of small antibody derivatives for use in immunohistochemistry. Molecules suitable for this purpose need only to possess specific antigen-binding ability and some means of detection of antigen-bound material. Detection could be by recognition of a genetically fused flag or tag epitope, by the fusion of an enzyme whose activity can be assayed, or by fusion with a protein that can interact with pre-existing histopathological reagents.

Animals↗
PubMed1994

Targeting enzymes for cancer therapy: old enzymes in new roles.

Enzymes which traditionally have played no role in cell-directed cytotoxicity are finding their way into schemes for prodrug activation and immunotoxins owing to such useful enzymatic activity. Alkaline phosphatase, carboxypeptidases, beta-glucosidases and beta-lactamases among many others are being utilised to regenerate potent anti-cancer drugs or toxic small molecules from precursors in a bid to enhance their activity in tumours. These prodrug activation systems require the pretargeting of the enzyme to the surface of a tumour cell, usually by an antibody or its immunoreactive fragment. A recent novel approach proposes the intracellular delivery of appropriate enzymes, such as phosphodiesterases, to particular cellular compartments. There, enzyme activity can cause substantive damage resulting in cell death. Cell targeting of mammalian phosphodiesterase promises to improve upon conventional immunotoxins because of their increased cytotoxicity when targeted to the appropriate compartment and their expected lack of, or lower, immunogenicity in clinical use.

Animals↗
PubMed1992

Cooperativity induced by a single mutation at the subunit interface of a dimeric enzyme: glutathione reductase.

When glycine418 of Escherichia coli glutathione reductase, which is in a closely packed region of the dimer interface, is replaced with a bulky tryptophan residue, the enzyme becomes highly cooperative (Hill coefficient 1.76) for glutathione binding. The cooperativity is lost when the mutant subunit is hybridized with a wild-type subunit to create a heterodimer. The mutation appears to disrupt atomic packing at the dimer interface, which induces a change of kinetic mechanism. A single mutation in a region of the protein remote from the active site can thus act as a molecular switch to confer cooperativity on an enzyme.

Base Sequence↗
PubMed1992

Engineering surface charge. 1. A method for detecting subunit exchange in Escherichia coli glutathione reductase.

The gene gor encoding Escherichia coli glutathione reductase was mutated to create a positively charged N-terminal extension consisting of five arginine residues followed by a factor Xa cleavage site to the enzyme polypeptide chain. The modified protein assembled in vivo to yield a dimeric enzyme with kinetic parameters indistinguishable from those of wild-type glutathione reductase. The N-terminal extension could not be released by treatment with factor Xa but could be removed by exposure to trypsin, again without effect on the enzyme activity. The modified enzyme was readily separated from the wild-type enzyme by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis. Incubation of the modified and wild-type enzymes, separately or as a mixture, with NADH led to their partial inactivation, and activity was restored by exposure to 1 mM reduced glutathione. No hybrid dimer was formed in the mixture of modified and wild-type enzymes, as judged by polyacrylamide gel electrophoresis, strongly suggesting that the inactivation induced by NADH was not due to dissociation of the parental dimers. The addition of otherwise benign positively or negatively charged extensions to the N- or C-terminal regions of the constituent polypeptide chains of oligomeric enzymes offers a simple route to detecting hybrid formation and the causative subunit dissociation and exchange.

Amino Acid Sequence↗
PubMed1992

Engineering surface charge. 2. A method for purifying heterodimers of Escherichia coli glutathione reductase.

Two gor genes encoding different mutants of Escherichia coli glutathione reductase have been expressed in the same E. coli cell, leading to the creation of a hybrid form of the enzyme dimer. One of the gor genes carried, in addition to various directed mutations, a 5' extension that encodes a benign penta-arginine "arm" added to the N-terminus of the glutathione reductase polypeptide chain [Deonarain, M.P., Scrutton, N.S., & Perham, R.N. (1992) Biochemistry (preceding paper in this issue)]. This made possible, by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis, the facile separation of the hybrid enzyme from the two parental forms. Moreover, the two subunits in the hybrid enzyme could be made to carry different mutations. In this way, glutathione reductases with only one active site per dimer were generated: the effects of replacing tyrosine-177 with glycine in the NADPH-binding site, which greatly diminishes the Km for glutathione and switches the kinetic mechanism from ping-pong to ordered sequential, and of replacing His-439 with glutamine in the glutathione-binding site, which greatly diminishes the Km for NADPH, were both found to be restricted to the one active site carrying the mutations. This system of generating separable enzyme hybrids is generally applicable and should make it possible now to undertake a more systematic study of catalytic mechanism and assembly for the many enzymes with quaternary structure.

Bacterial Proteins↗
PubMed1990

Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo.

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the dimeric flavoprotein glutathione reductase, Cys-47 (a cysteine residue forming an essential charge-transfer complex with enzyme-bound FAD) was converted to serine (C47S) and His-439 (required to facilitate protonation of the reduced glutathione) was converted to glutamine (H439Q). Both mutant genes were placed in the same plasmid, pHD, where each of them came under the control of a strong tac promoter. This was designed to achieve equal over-expression of both genes in the same E. coli cell. The parental homo-dimers show no (C47S) or very little (H439Q) activity as glutathione reductases. The formation in vivo of heterodimers, carrying one crippled and one fully functional active site, was detected by absorbance spectroscopy and fluorescence emission spectrometry of enzyme-bound FAD and by active site complementation. The fractional distribution of homo- and hetero-dimers was in accord with that expected for a random association of enzyme subunits. In a homo-dimer, the H439Q mutation leads to a big fall in the value of Km for NADPH which binds some 1.8 nm from the point of mutation (Berry, A., Scrutton, N.S. & Perham, R. N. Biochemistry 28, 1264-1269 (1989)). However, the one active site in the H439Q/C47S hetero-dimer exhibited kinetic parameters similar to those of the wild-type enzyme. Thus, the effect of the H439Q mutation must be retained within the active site that accommodates it and is not transmitted through the protein to the second active site across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli.

Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned.

Alanine↗