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M R Price

Publications and source records attributed to M R Price.

At least 73 records · Page 4Linked to original sources
PubMed1995

Targeting of bladder cancer with monoclonal antibody NCRC48--a possible approach for intravesical therapy.

OBJECTIVE: To determine the localization of the anti-MUC1 mucin monoclonal antibody (mAb) NCRC48 to bladder cancer following intravesical administration. PATIENTS AND METHODS: mAb NCRC48 (330-500 micrograms) radiolabelled with 111indium (11-17 MBq) was administered intravesically to 12 unselected patients with radiological evidence of bladder cancer. Tumour localization was assessed by gamma-camera imaging and by tissue biodistribution studies on biopsies obtained at cystoscopy at about 2 or 24 h after the procedure. After 24 h, whole blood radioactivity was measured and 3 weeks after the procedure the serum level of human anti-mouse antibodies was estimated using an ELISA method. RESULTS: Eleven patients had tumours confirmed at cystoscopy (grades 1-3, stages pTa-pT2). The mean uptake of NCRC48 by tumour and by normal urothelium (expressed as the percentage of the instilled dose/g x 10(3) +/- SD) at 2 h was 3.42 +/- 3.68 and 0.41 +/- 0.77 (P < 0.05). After 24 h, the values for tumour and normal urothelium were 1.17 +/- 1.18 and 0.17 +/- 0.11, respectively. Areas of increased activity on the scintigrams were consistent with the position of the tumours at cystoscopy. No radioactivity was detected in blood at 24 h and there was no evidence of a human anti-mouse antibody response. CONCLUSION: The MUC1 mucin may be a suitable antigen to study the potential of therapeutic strategies based on monoclonal antibody targeting of superficial bladder cancer and may allow the development of more effective agents in the treatment of this condition.

Aged↗
PubMed1994

Characterisation of a recombinant Fv fragment of anti-MUC1 antibody HMFG1.

A recombinant Fv (variable fragment) has been produced for the murine monoclonal antibody HMFG1. This antibody was raised against human milk fat globules and reacts with an epitope (PDTR) in the protein core of MUC1 mucins, which are up-regulated in human breast and other carcinomas. Binding specificity of the Fv fragment has been demonstrated through immunoaffinity purification, and by radioimmunoassay. The affinity constants for this Fv fragment and for the proteolytically produced Fab (antigen binding fragment) of the related humanised antibody HuHMFG1 were determined by monitoring the fluorescence quenching of the antibody fragments whilst adding aliquots of MUC1 related antigenic peptides KAPDTRPAPG and VTSAPDTRPAPG. Using these techniques it has been demonstrated that the products of these different methods of antibody fragmentation are comparable, and suitable for solution structure analysis using nuclear magnetic resonance (NMR) spectroscopy.

Animals↗
PubMed1994

Production of monoclonal antibodies recognising the peptide core of MUC2 intestinal mucin.

A peptide based on the tandem repeat sequence of MUC2 mucin was used to produce a series of monoclonal antibodies (MAb). The fine specificity of these antibodies and their implications for MUC2 expression are presented. Three of the MAbs, 996/1, 996/7 and 995/25, were specific to the MUC2p and failed to bind to peptides based on the MUC1,3,4 tandem repeat sequences whereas three others, 994/152, 994/91 and 996/36, cross reacted with the MUC2p and the MUC3 tandem repeat peptide but not the MUC1 and MUC4 peptides. An antigen, affinity purified from a colorectal tumour on one of the MUC2p-specific MAbs, 996/1, was shown to be a high molecular weight polydisperse, mucin-like antigen. Two of the MAbs, 996/1 and 994/152, recognised MUC2 in tissue sections, although the fine specificity varied between the two MAbs, with 994/152 strongly staining gastric, ileum and kidney epithelia, and MAb 996/1 intensely staining colon, liver and prostate tissues. These antibodies also stained a colorectal cell line, and MAb 994/152 also stained a gastric and an ovarian cell line. Six of the MAbs were used to stain colorectal tumour and adjacent 'normal' colonic mucosa sections. All six stained normal mucosa, but only two of the MAbs, 996/1 and 994/91, stained tumour tissue. The staining probably reflects exposure of cryptic epitopes due to varying levels of glycosylation in different tissues. These anti-MUC2p MAbs may help in determining the normal role of MUC2 mucin and how it is subverted in malignancy.

Amino Acid Sequence↗
PubMed1993

Immune recognition of human colonic-tumour-associated MUC-2 mucins using an anti-peptide antibody.

In human intestinal malignancy, alterations occur in the expression of mucins defined by the MUC-2 gene. These changes include the unmasking of epitopes in the mucin protein core. In order to probe these modifications associated with mucins of the malignant phenotype, a monoclonal antibody (MAb) was developed against synthetic peptide with a sequence based upon that of the protein core of the MUC-2 mucin. The antibody (designated 996) was shown to recognize a high-molecular-weight glycoprotein from colonic carcinoma tissue. The material reacted uniformly with Concanavalin A but variably with other lectins, indicating heterogeneity in the associated oligosaccharide side chains. The protein core was accessible both to 996 antibody binding and to degradation with proteases. Immunization with the affinity-purified mucin-like material elicited antibodies reactive with both the immunogen and the synthetic peptides, confirming the immunogenic character of protein-core determinants. Epitope mapping studies, using synthetic peptides in solution and synthetic peptides tethered to the heads of plastic pins, indicated that the minimum epitope for the 996 antibody is a tetramer of T G T Q. Antibody interaction with the glutamine (Q) residue was determined to be of major importance in the antigen-antibody reaction. The findings illustrate the characterization of an anti-peptide antibody which may be used to probe alterations in MUC-2 mucin expression associated with human intestinal malignant disease.

Adsorption↗
PubMed1993

Induction of antibody responses to breast carcinoma associated mucins using synthetic peptide constructs as immunogens.

A strategy for directing and enhancing B cell immune responses against synthetic peptide determinants has been developed in order to produce antibodies specifically against protein epitopes of clinical relevance. A peptide sequence based upon the MUC-1 mucin protein core was selected for this purpose since anti-MUC-1 antibodies have proven diagnostic application and therapeutic potential in human breast and ovarian cancer. Peptide constructs were synthesised co-linearly linking the immunodominant B cell determinant region, PDTRPAP, in the protein core of the MUC-1 mucin, to sequence 111-120 of influenza haemagglutinin A/X-31, a determinant recognised by T helper cells through association with MHC class II molecules. Induction of anti-MUC-1 antibodies to the B cell determinant region by immunisation with peptide was shown to be dependent upon both the presence and the position of the T cell determinant. In addition, haplotype mismatching with respect to the T cell determinant resulted in a significant lowering of the anti-MUC-1 antibody response in peptide construct immunised mice. These findings are relevant to the design of immunogens to produce antibodies against peptide epitopes of tumour associated proteins and glycoproteins.

Amino Acid Sequence↗
PubMed1993

Development of an anti-idiotypic antibody reactive with an antibody defining the epitope RPAP in the MUC-1 epithelial mucin core.

C595 is a murine IgG3 monoclonal antibody (MAb) raised against human urinary mucin. C595 antibody binds to the protein core of the MUC-1 mucin (the polymorphic epithelial mucin, PEM) which is elevated in tissue and secretions from human breast carcinomas. The antibody defines the tetrameric epitope, RPAP. In the present study, a syngeneic anti-idiotypic MAb, 911, was raised in BALB/c mice against the C595 of C595 to its mucin core antigen. In turn, pre-blocking of C595 with a 20 amino-acid mucin core peptide specifically inhibited binding of 911 antibody to C595. Cross-reactivity of antibody 911 was only observed against the IgM MAb, 789/91, which has the same minimum antibody-binding epitope as C595, namely, RPAP. Syngeneic and xenogeneic anti-sera against 911 antibody displayed increased binding to heptameric peptide sequences containing the motif, RPA(P). The anti-idiotypic antibody 911 therefore appears to recognize a site within or close to the binding site of the C595 antibody and thus carries an internal image of the parental mucin epitope. Consequently, the 911 idiotypic network offers a promising model system to investigate the mechanism of anti-idiotype-induced tumour immunity.

Amino Acid Sequence↗
PubMed1993

Measurement of antibody binding to antigenic peptides conjugated in situ to albumin-coated microtitre plates.

Monoclonal antibodies have been prepared against a synthetic peptide with a sequence corresponding to a repeated hydrophilic region of the protein core of the human MUC-2 gastrointestinal mucin. Peptide conjugates, prepared by glutaraldehyde cross-linking with keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), were employed as the immunogen and target antigen (for screening by ELISA), respectively. However, for the measurement of antibody binding to peptide by an ELISA procedure, an alternative strategy was developed and is described in this report: peptides were conjugated directly to BSA immobilized by physical adsorption to the surface of microtitre plate wells. This procedure permits peptides to be tested as target antigens by ELISA without prior preparation of peptide-carrier conjugates.

Amino Acid Sequence↗
PubMed1993

Discrimination of MUC1 mucins from other sialyl-Le(a)-carrying glycoproteins produced by colon carcinoma cells using a novel monoclonal antibody.

Earlier studies (Baeckström et al., J. Biol. Chem., 266: 21537-21547, 1991) have revealed that the colon carcinoma cell line COLO 205 produces two different proteins carrying the carcinoma-associated sialyl-Le(a) carbohydrate epitope. One is the MUC1 mucin apoprotein, and the other protein is smaller and has not been characterized in detail. This paper describes a comparison of COLO 205 with three other colon carcinoma cell lines, aided by the use of a novel MUC1-reactive monoclonal antibody, Ma552. Ma552 reacted with H-CanAg, the MUC1 mucin purified from COLO 205, the binding increasing greatly upon partial deglycosylation of H-CanAg with trifluoromethanesulfonic acid. This pattern of reactivity was in contrast with that of the MUC1-reactive monoclonal antibody HMFG-2, which did not recognize H-CanAg without prior deglycosylation. The nature of the epitope of Ma552 was further investigated using a synthetic peptide corresponding to the tandem-repeat sequence of the MUC1 protein. When the peptide was used as an inhibitor of antibody binding to immobilized, partially deglycosylated H-CanAg mucin, Ma552 was inhibited, as was HMFG-2. Using short, immobilized synthetic peptides identical to parts of the MUC1 tandem repeat, the reactivity of Ma552 was mapped to the hexapeptide TRPAPG. Ma552 and C50, a monoclonal antibody reactive with sialyl-Le(a), were used in immunofluorometric assays to analyze gel filtration fractions of extracts and spent media from the colorectal carcinoma cell lines COLO 205, SW1116, LoVo, and LS 174T. Using C50 in a homologous assay, all sialyl-Le(a)-carrying glycoproteins were detected. Among these, mucins based on the MUC1 apoprotein were identified using Ma552 and C50 in a combination assay. The results showed that sialyl-Le(a) was present on more than one glycoprotein not only in COLO 205 but also in SW1116 and LoVo. The Ma552/Eu-C50 assay revealed the presence of sialyl-Le(a)-carrying MUC1 in COLO 205 as expected, as well as in SW1116. The presence of MUC1 in Ma552-reactive fractions was confirmed by deglycosylation followed by assaying with the monoclonal antibody HMFG-2. Furthermore, Northern blots revealed the presence of MUC1 mRNA only in the two Ma552-positive cell lines.

Amino Acid Sequence↗
PubMed1993

Suppression of antibody responses to ricin A chain (RTA) by monoclonal anti-RTA antibodies.

Balb/c mice treated with an immunotoxin constructed by conjugation of murine monoclonal antibody 791T/36 via a disulfide linker to ricin A chain generate a pronounced antibody response to peptide epitopes on ricin A chain. Monoclonal anti-RTA antibodies which recognize peptide epitopes have been developed and these have been used to down-regulate anti-RTA antibody responses in 791T/36-RTA immunotoxin-treated Balb/c mice. Of the five MAB tests, two (608/7 and 596/134) proved most effective, inhibiting anti-RTA antibody formation by up to 73%. MAB treatment was effective when initiated up to 3 days after immunotoxin treatment. Pharmacokinetic studies with 791T/36-RTA have shown that the immunotoxin is rapidly eliminated from the circulation, with no more than 4% remaining in blood after 24 hr. It is proposed that the down-regulation of anti-RTA antibodies is effected by MAB interfering with antigen processing.

Animals↗
PubMed1993

Altered diaphragm function modifies neonatal lung growth: biologic morphometric assessment.

Thoracic volume relationships supported by the diaphragm are important mechanical components of neonatal lung growth. Alterations modify regional lung growth. We used stereologic morphometry to study effects of altered diaphragm function on alveolar growth in neonatal pigs. Nine piglets (1 mo) were divided into three groups: unilateral phrenectomy, noncompliant patch replacement of diaphragm, and sham. Seven days later lungs were fixed in situ with 2.5% glutaraldehyde by airway installation at 20 cm H2O. Five 1-mm cubes were cut from lung corresponding to apex/RUQ(A), base/RLQ(B), apex/LUQ(C), and base/LLQ(D) and prepared for light microscopy (n = 25/quad/animal). Stereologic morphometry involved point counting for air volume density and point intersect to determine the surface area density of the alveolar spaces. Results were analyzed for variance and by Tukey range testing. Variance of air volume % between groups-quadrants B, C, D had decreased air volume % in phrenectomy group compared with patch and sham group (P < .05). Variance of air volume % within groups, for phrenectomy group A (81.5%), is different from all others (B = 70.6%, C = 75.5%, D = 66.7%); C is different from D (P < .05). For patch and sham group, D (P = 74.8%, S = 80.7%) is different from A (P = 84.1%, S = 86.6%) and C (P = 86.2%, S = 84.4%). Variance of surface area density between groups--quadrant D had increased surface area density % in phrenectomy group compared to sham group (P < .05). Variance of surface area density % within groups, for phrenectomy group, A (37.9 mm-1) is different from D (55.7 mm-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Fine specificity of antibody recognition of carcinoma-associated epithelial mucins: antibody binding to synthetic peptide epitopes.

The protein core of polymorphic epithelial mucins consists predominantly of a repeating 20 amino acid peptide motif. Many monoclonal antibodies reactive with breast carcinomas recognise determinants located within the mucin protein core, and epitope mapping techniques have demonstrated that these antibodies bind to epitopes of three, four or five amino acids within the hydrophilic sequence, P D T R P A P. Each of these mucin core-reactive antibodies map to epitopes containing the central arginine residue. The fine specificity of a panel of anti-mucin antibodies binding to the tetrameric peptides P D T R or R P A P (synthesised on the heads of polyethylene pins) was examined by systematically replacing each amino acid in turn with all other 19 natural amino acids, and then testing these analogues for antibody binding. We have (i) identified those amino acids in epitopes which are essential for antibody binding, (ii) shown that for each epitope there is a hierarchy of residues required for immune recognition--certain amino acids may be replaced with little or no loss of antibody binding, while the presence of others is essential, and (iii) concluded that antibody specificity is further regulated by the residue(s) flanking an epitope motif which may impose conformational constraints upon the presentation of the epitope to an antibody.

Amino Acid Sequence↗
PubMed1993

Epithelial mucin core antigen (EMCA) in assessing therapeutic response in advanced breast cancer--a comparison with CA15.3.

We report a comparative study of CA 15.3 and EMCA (epithelial mucin core antigen) in 77 consecutive women with newly diagnosed UICC assessable metastatic breast cancer, 59 patients received hormones and 18 chemotherapy. Assessments of response were made prior to commencing therapy and repeated 2 monthly. Sites of metastatic disease included bone (34), pulmonary (8), bone and pulmonary (14) and visceral (21). Using a cut-off of 33 U ml-1 changes in EMCA at 2, 4 and 6 months showed a highly significant correlation (P < 0.001) with UICC assessed response at 6 months; selectivity 70%, sensitivity 80%, specificity 91%, positive predictive value 84%; negative predictive value 89% at 2 months. Corresponding values for CA 15.3: selectivity 89%, sensitivity 85%, specificity 91%, PPV 92% and NPV 91%. Four of eight patients unassessable by CA 15.3 were assessable by EMCA; four patients expressed neither marker. EMCA appears to reflect tumour bulk and may be useful in monitoring therapy in patients with advanced breast cancer. With an easier and more robust assay format than CA 15.3, EMCA is potentially a more useful marker.

Adult↗
PubMed1993

Development of second generation monoclonal antibodies recognising Lewisy/b antigen by anti-idiotypic immunisation.

Five new monoclonal antibodies (Mabs) recognising the Lewisy/b hapten of the IgG isotype have been produced following immunisation with rat anti-idiotypic antiserum to C14, an IgM Lewisy/b hapten Mab and boosting with C14gp200 antigen. They have the same fine specificity as the original Mab binding to a cell surface and secreted antigen preferentially expressed by colorectal tumour cells.

Animals↗
PubMed1993

Immunoscintigraphy of ovarian carcinoma using a monoclonal antibody (111In-NCRC48) defining a polymorphic epithelial mucin (PEM) epitope.

An anti-polymorphic epithelial mucin (PEM) monoclonal antibody NCRC48 (IgG3) has been tested for its capacity to localize in tumours according to accepted guidelines for human administration. Following radiolabelling with 111In, 1 mg antibody was administered to 19 patients with a clinical suspicion of ovarian malignancy. Initial imaging and biodistribution studies confirm the safety of this conjugate although six out of 11 patients tested developed an antibody response to the monoclonal antibody. Immunoscintigraphy with this antibody was compared with magnetic resonance imaging and ultrasound in relation to the final tumour histology, the final accuracies being 79, 79 and 64% respectively. Positive localization of antibody was confirmed in malignant tissue with little evidence of uptake in benign tissue.

Adenocarcinoma↗
PubMed1993

Construction of a reshaped HMFG1 antibody and comparison of its fine specificity with that of the parent mouse antibody.

A human antibody with milk mucin specificity was obtained by transferring the complementarity determining regions (CDR) of the mouse antibody HMFG1 onto carefully selected human framework regions. The resulting reshaped human antibody, HuHMFG1, showed no difference in relative affinity for its antigen compared with the parent mouse HMFG1. The minimum epitope recognized by both the mouse and reshaped antibodies was demonstrated by epitope mapping to be identical, and consists of the tetramer PDTR. In a replacement net analysis, in which each of the amino acids was replaced in turn with the 19 other residues, it was determined that mouse HMFG1 and HuHMFG1 reacted with this series of synthetic peptides in an equivalent manner, indicating retention of identical fine specificity in the HuHMFG1 antibody. In contrast to other published reports, this was achieved without involvement of any framework residues in the binding site transfer. These data demonstrate that if well-matching human framework regions are employed grafting the CDR only can be sufficient to confer desired specificities to human antibodies and can, indeed, provide human analogues of mouse antibodies with virtually indistinguishable affinities and fine specificities relative to the mouse parent antibodies.

Amino Acid Sequence↗
PubMed1992

Structural and computational investigations of the conformation of antigenic peptide fragments of human polymorphic epithelial mucin.

Human polymorphic epithelial mucins (PEM) are complex glycoproteins that are associated with breast and ovarian carcinomas. The PEM core protein consists of variable numbers of a tandem repeat sequence which contains a short antigenic hydrophilic region (Pro1-Asp-Thr-Arg-Pro-Ala-Pro7). High-field n.m.r. studies undertaken on antigenic 20- and 11-amino acid fragments of the PEM core protein in dimethyl sulphoxide have identified a type-I beta-turn to be present in the region Pro1-Asp-Thr-Arg4. This region includes and overlaps the identified type-I (Asp2-Thr-Arg4) and type-II (Arg4-Pro-Ala6) epitopes of anti-PEM monoclonal antibodies. The studies indicate that the beta-turn is stabilized by the presence of a salt-bridge interaction between Asp-2 and Arg-4. In order to probe the conformations accessible to the PEM peptides a computational study was undertaken independently on the peptide Pro-Asp-Thr-Arg-Pro using a modified Metropolis Monte Carlo algorithm. This study identified the n.m.r.-observed salt-bridge type-I beta-turn as the major low-energy conformer. These results suggest that this structural motif may be involved in the immune recognition of PEM.

Amino Acid Sequence↗
PubMed1992

Peptide epitope binding and fluorescence quenching with the anti-mucin antibody C595.

The Fab fragment of the anti-polymorphic epithelial mucin antibody C595 (IgG3, kappa) has been produced by digestion with papain. The purified antibody fragment retained its binding activity for the mucin in an indirect radioisotopic antiglobulin assay. The binding constant of the Fab fragment for the synthetic peptide (PDTRPAPGSTAPPAHGVTSA) corresponding to the 20-amino acid tandem repeat sequence found in the mucin protein core was determined to be 0.3 x 10(6) M-1 by measuring the capacity of the peptide to quench the fluorescence of the Fab fragment.

Amino Acid Sequence↗