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Biomedical subjects

M Little

Publications and source records attributed to M Little.

At least 91 records · Page 5Linked to original sources
PubMed1997

A clinical overview of WT1 gene mutations.

Mutations in the WT1 gene were anticipated to explain the genetic basis of the childhood kidney cancer, Wilms tumour (WT). Six years on, we review 100 reports of intragenic WT1 mutations and examine the accompanying clinical phenotypes. While only 5% of sporadic Wilms' tumours have intragenic WT1 mutations, > 90% of patients with the Denys-Drash syndrome (renal nephropathy, gonadal anomaly, predisposition to WT) carry constitutional intragenic WT1 mutations. WT1 mutations have also been reported in juvenile granulosa cell tumour, non-asbestos related mesothelioma, desmoplastic small round cell tumour and, most recently, acute myeloid leukemia.

Alleles↗
PubMed1997

Allelic imbalance at chromosome 1q21 in Wilms tumor.

The Wilms tumor suppressor gene 1, WT1, located on chromosome 11p13 is mutated in only a subset of Wilms tumors. Cytogenetic studies of Wilms tumors show that the most frequent structural anomalies after those affecting chromosome 11p are rearrangements of 1q, suggesting that there is a gene involved in Wilms tumor etiology in this region. The WT1 target sequence +P5 (D1S3309E), isolated using whole-genome polymerase chain reaction (PCR), binds all WT1 isoforms in vitro and has been mapped to 1q21-22. As +P5 may mark a 1q Wilms tumor gene, constitutional and tumor DNA from 33 Wilms tumor patients (36 tumors) was screened for allele imbalance using microsatellite markers from 1p21 to 1q44. Although no gross rearrangements of the +P5 region were found, this study demonstrates allele imbalance for 1q in 12% of patients (5/36 tumors), defining a smallest region of overlap at 1q21. This finding supports a role for 1q21 in Wilms tumorigenesis.

Alleles↗
PubMed1997

A TNF receptor antagonistic scFv, which is not secreted in mammalian cells, is expressed as a soluble mono- and bivalent scFv derivative in insect cells.

Single chain antibodies (scFv) are usually produced in E. coli, but generation of certain scFv derivatives, such as complex fusion proteins or glycosylated forms of scFv is restricted to eukaryotic expression systems. We investigated the production of soluble mono- and bivalent single chain antibodies (scFv) in eukaryotic cells and describe a cassette vector system for mammalian and baculovirus expression which is compatible with an established vector system for bacterial expression and phage display selection of scFvs. The applied model scFv was derived from a murine antibody (H398) against human tumor necrosis factor receptor 1 (TNFR60), known to be a potent antagonist of TNF action in its monomeric form and a potential therapeutic agent for treatment of TNF-mediated diseases. Surprisingly, the monomeric scFv form of H398 (scFv H398) is expressed but not secreted in different mammalian cells. In contrast, in insect cells using recombinant baculovirus, a monovalent scFv H398 and a bivalent scFv fusion protein with an human IgG1 Fc region were expressed and secreted with correctly processed signal sequence. Concerning the influence of valency of the model Ab and its derivatives on antigen binding affinity and neutralisation of TNF activity, we found that the mono- and bivalent form of scFv H398 possesses the same characteristics as proteolytically produced Fab H398 and original mAb H398, respectively. Furthermore, fusion of the Ig Fc protein to scFv H398 increase the in vitro half-life at 37 degrees C. We conclude that the described cassette vectors readily allow the eukaryotic expression of mono- and bivalent scFv derivatives to analyse the influence of valency of scFv molecules on antigen binding and biological activity.

Animals↗
PubMed1997

Cloning and cytotoxicity of a human pancreatic RNase immunofusion.

BACKGROUND: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins. OBJECTIVES: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines. STUDY DESIGN: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity. RESULTS: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate. CONCLUSION: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

Animals↗
PubMed1997

Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.

Amino Acid Sequence↗
PubMed1997

Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.

Recombinant antibody fragments directed against cell surface antigens have facilitated the development of novel therapeutic agents. As a first step in the creation of cytotoxic immunoconjugates, we constructed a single-chain Fv fragment derived from the murine hybridoma OKT3, that recognizes an epitope on the epsilon-subunit of the human CD3 complex. Two amino acid residues were identified that are critical for the high level production of this scFv in Escherichia coli. First, the substitution of glutamic acid encoded by a PCR primer at position 6 of VH framework 1 by glutamine led to a more than a 30-fold increase in the production of soluble scFv. Second, the substitution of cysteine by a serine in the middle of CDR-H3 additionally doubled the yield of soluble antibody fragment without any adverse effect on its affinity for the CD3 antigen. The double mutant scFv (Q,S) proved to be very stable in vitro: no loss of activity was observed after storage for 1 month at 4 degrees C, while the activity of scFv containing a cysteine residue in CDR-H3 decreased by more than half. The results of production yield, affinity, stability measurements and analysis of three-dimensional models of the structure suggest that the sixth amino acid influences the correct folding of the VH domain, presumably by affecting a folding intermediate, but has no effect on antigen binding.

Amino Acid Sequence↗
PubMed1996

Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry.

Cloning the correct genes coding for antibody variable domains (especially VL kappa) from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from the myeloma cell line. Indeed, four different VL genes were obtained after the amplification of immunoglobulin genes by PCR from the hybridoma HD37, which produces an antibody against the human CD19 B cell differentiation antigen. Most of the variants (eight out of 15) were derived from the kappa chain of the myeloma MOPC-21. For the rapid functional evaluation of recombinant antibody fragments against cell surface antigens, we established an efficient expression and detection system. First, deleted and mutated genes were eliminated by a colony screening procedure. Bacteria from picked colonies were then induced and grown in the presence of 0.4 M sucrose to increase the accumulation of soluble scFv in the periplasm (5-10 micrograms per ml of bacterial shake-tube culture). Finally, the cell-specific binding of scFv in crude periplasmic extracts was detected by flow cytometry. This procedure facilitated the efficient cloning of a functional anti-CD19 VH/VL combination from the hybridoma cDNA.

Amino Acid Sequence↗
PubMed1996

RNA binding by the Wilms tumor suppressor zinc finger proteins.

The Wilms tumor suppressor gene WT1 is implicated in the ontogeny of genito-urinary abnormalities, including Denys-Drash syndrome and Wilms tumor of the kidney. WT1 encodes Kruppel-type zinc finger proteins that can regulate the expression of several growth-related genes, apparently by binding to specific DNA sites located within 5' untranslated leader regions as well as 5' promoter sequences. Both WT1 and a closely related early growth response factor, EGR1, can bind the same DNA sequences from the mouse gene encoding insulin-like growth factor 2 (Igf-2). We report that WT1, but not EGR1, can bind specific Igf-2 exonic RNA sequences, and that the zinc fingers are required for this interaction. WT1 zinc finger 1, which is not represented in EGR1, plays a more significant role in RNA binding than zinc finger 4, which does have a counterpart in EGR1. Furthermore, the normal subnuclear localization of WT1 proteins is shown to be RNase, but not DNase, sensitive. Therefore, WT1 might, like the Kruppel-type zinc finger protein TFIIIA, regulate gene expression by both transcriptional and posttranscriptional mechanisms.

Animals↗
PubMed1996

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.

BACKGROUND: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). OBJECTIVES: pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. STUDY DESIGN AND RESULTS: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. CONCLUSIONS: Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.

Amino Acid Sequence↗
PubMed1996

An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling.

The Wilms' tumour suppressor gene 1 (WT1) (1,2) encodes four C2H2 zinc finger-containing proteins (3) critical for normal mammalian urogenital development (4). Mutations in this gene are observed in the childhood kidney cancer, Wilms' tumour (WT) (5). WT1 can bind specific DNA targets within the promoters of many genes (6-9) and both transcriptional repression and activation domains have been identified (10). On this basis, it has been assumed that regulation of transcription is the basis of WT1 tumour suppressor activity. However, subnuclear localization studies have revealed an association between WT1 proteins and 'speckled bodies' within the nucleus. Degradation of nuclear RNA in cells expressing WT1 abolishes this speckled localization and WT1 co-immunoprecipitates with a number of spliceosomal proteins, suggesting that it may also bind to RNA (11). Using structural rather than sequence comparison, we have now identified an evolutionarily conserved N-terminal RNA recognition motif (RRM) in all known WT1 isoforms similar to that in the constitutive splicing factor U1A. Given the association between WT1 mutations and Wilms' tumours, this study, together with other recent findings, may suggest a novel tumour suppression mechanism.

Amino Acid Sequence↗
PubMed1996

Affinity enhancement of a recombinant antibody: formation of complexes with multiple valency by a single-chain Fv fragment-core streptavidin fusion.

In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites. To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin. The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies. After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed. The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C). Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable. The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers. This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies. We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium. These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands.

Antibodies↗
PubMed1996

Cell surface display of a single-chain antibody for attaching polypeptides.

To provide an efficient means of coupling proteins, peptides and other suitable moieties to cells, we have constructed a retroviral expression vector for cell surface display of a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-oxazo-line-5-one (phOx). The hapten phOx can be easily conjugated to primary amino and sulfhydryl groups, thus providing points of attachment for the cell surface-bound anti-phOx scFv. This universal cell coupling system could prove to be particularly useful for anchoring monoclonal antibodies for tumor targeting and to present co-stimulatory molecules and other ligands (even mixtures) at the cell surface for gene therapy.

Animals↗
PubMed1996

The enemy within: diarrheal rates among British and Australian troops in Iraq.

British and Australian medical teams working in Northern Iraq in 1991 providing primary care to refugees and the war wounded were subjected to a descriptive retrospective survey, 5 weeks after arriving in Iraq. The aim was to document different rates of diarrhea in British and Australian troops. The British, who were not taking daily doxycycline and did not enforce a plate- and hand-washing routine, experienced higher rates of diarrhea (69% of British troops compared with 36% of Australian troops), which was more severe and of a longer duration (p < 0.001) and resulted in twice as many days being lost (p < 0.001) in spite of the British team being half the size of the Australian contingent, and the region having enteropathogens with a high rate of antibiotic resistance. Vigorous hand- and plate-washing routines along with doxycycline prophylaxis appear to significantly reduce incapacitation from diarrhea in this military setting and have an important implication for operational effectiveness.

Anti-Bacterial Agents↗
PubMed1995

Human antibody libraries in Escherichia coli.

A potentially vast pool of human antibodies with novel specificities for diagnostic and therapeutic purposes can be generated in Escherichia coli. Antibodies to infectious agents have already been isolated by amplifying the heavy and light chain repertoires of donor lymphocytes and they have even been rescued many years after the initial infection from memory cells cultivated in SCID mice. Eventually, however, the creation of extremely large and diverse libraries from the naive antibody repertoire of unactivated B lymphocytes or by gene synthesis using random oligonucleotides for the hypervariable regions could provide a rapid means of obtaining human antibodies to any particular antigen. An important breakthrough for exploiting the potential size and diversity of these libraries has been the development of systems for the surface display of antibodies that are physically linked to their own genes. This allows large numbers of clones to be screened simultaneously and antibodies with affinities of up to 10(8) M-1 have already been obtained using these vectors. It seems quite feasible, therefore, that antibodies with affinities approaching those obtained in the secondary immune response can be obtained by systematically optimizing the strategies for making antibody libraries. Furthermore, it might be possible to establish extremely large antibody repertoires in E. coli by the in vivo recombination of phage and plasmid antibody libraries. The affinity of the selected antibodies could be increased by chain shuffling or random mutagenesis followed by several rounds of selection under increasingly stringent conditions.

Animals↗