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Biomedical subjects

D Snary

Publications and source records attributed to D Snary.

At least 19 recordsLinked to original sources
PubMed2005

Immune responses in advanced colorectal cancer following repeated intradermal vaccination with the anti-CEA murine monoclonal antibody, PR1A3: results of a phase I study.

BACKGROUND AND AIMS: The aim was to determine the toxicity, clinical and immune responses to the murine monoclonal anti-carcinoembryonic antigen (CEA) antibody, PR1A3, in patients with advanced colorectal cancer. MATERIALS AND METHODS: Fifteen patients with advanced colorectal cancer received either 0.5-, 1.0- or 5.0-mg doses of PR1A3 mixed with 10% w/v Alum adjuvant (Superfos Biosector, Denmark) intradermally at 4-week intervals for 3 months. Patient serum was assessed for anti-idiotypic (Ab2), anti-anti-idiotypic (Ab3) and human anti-mouse antibody (HAMA) reactivity. Peripheral blood mononuclear cell (PBMC) proliferation with phytohaemagglutinin (PHA), CEA and PR1A3, stimulated IL-2, IL-4 and IFN-gamma levels and PR1A3-stimulated IL-2 receptor expression during immunotherapy were determined. Comparisons were made with 16 age-matched controls without malignant disease. RESULTS: Hyperimmune sera from 12 of the 15 patients showed Ab2 reactivity with no detectable Ab3 responses. Strong HAMA reactivity was recorded in 7 of the 15 cases with no adverse clinical effect. Delayed-type hypersensitivity (DTH) responses developed in 12 of the 15 patients. Pre-treatment PBMC proliferation with PHA was subnormal in each patient compared with controls, becoming normal (or supranormal) in all patients during immunisation (P<0.001). PBMC proliferation with CEA and PR1A3 increased during immunotherapy (P<0.001) along with stimulated production of IL-2, IFN-gamma and IL-2 receptor expression. Progressive disease was observed in 14 of the 15 patients with minimal toxicity. CONCLUSION: PR1A3 generated limited idiotypic responses but robust DTH reactivity in most patients. In vitro PBMC proliferation with mitogens and recall antigens is greatly increased during the course of immunisation, with a shift in stimulated cytokine profile.

Aged↗
PubMed2001

Antibody-dependent cell-mediated cytotoxicity: a flow cytometry-based assay using fluorophores.

Using flow cytometry (FCM), an assay has been developed for the determination of antibody-dependent cell-mediated cytotoxicity (ADCC). Target cells were labelled with a membrane dye, PKH-26, to allow discrimination when incubated with effector cells and antibody. Post-incubation, cell death within the PKH-26+ target cell population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3). This ADCC method allows analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the assay is accurate and reproducible with the potential to be a useful tool for evaluating the therapeutic potential of antibodies and antibody-based reagents.

Animals↗
PubMed2001

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Adolescent↗
PubMed2001

Antibody targeting studies in a transgenic murine model of spontaneous colorectal tumors.

Monoclonal antibodies (mAbs) have been used to treat malignancies in humans with varying degrees of success. Progress has been hindered by the lack of suitable animal models, which would ideally consist of immunocompetent animals that are tolerant to tumor-associated antigens. Suitable models would allow the study and optimization of anti-tumor immunotherapy. We describe a murine model for the study of immunotherapy in colorectal cancers. Carcinoembryonic antigen (CEA) is a cell-surface glycoprotein that is expressed on normal human intestinal epithelium and that is overexpressed in intestinal tumors. Mice that are transgenic for the human CEA gene (CEA.Tg) were crossed with multiple intestinal neoplasia (MIN) mice. MIN mice carry a germline APC mutation and are prone to the development of intestinal adenomas. The offspring from the MIN x CEA.Tg cross developed intestinal adenomas that were shown by immunohistochemistry to overexpress CEA. Pharmacokinetic studies by using (125)I-labeled anti-CEA mAb PR1A3 showed rapid localization of antibody to tissues expressing CEA, especially the gastrointestinal tract. Macroscopic and microscopic radioautographic analysis of the gastrointestinal tracts from MIN/CEA.Tg mice indicated that PR1A3 targeted and was retained in tumors at levels higher than in areas of normal gut. These results demonstrate the utility of the MIN/CEA.Tg mouse as a model for the study of anti-CEA immunotherapy and, furthermore, demonstrate the efficiency of tumor localization by PR1A3.

Animals↗
PubMed2001

Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide.

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Animals↗
PubMed2001

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Adenoviridae↗
PubMed2000

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Animals↗
PubMed2000

The expression profile for the tumour suppressor gene PTEN and associated polymorphic markers.

PTEN, a putative tumour suppressor gene associated with prostate and other cancers, is known to be located within the chromosomal region 10q23.3. Transcription of the PTEN gives rise to multiple mRNA species. Analyses by Northern blots, using cell lines which express PTEN together with cell lines which have lost the PTEN or carry a truncated version of the gene, has allowed us to demonstrate that the pseudogene is not transcribed. In addition, 3' RACE studies confirmed that the multiple mRNA species arising from the gene probably result from the use of alternative polyadenylation sites. No evidence for tissue- or cell-specific patterns of transcription was found. Analysis by 5' RACE placed the putative site for the start of transcription around 830 bp upstream of the start codon. A map of the location of the PTEN gene with a series of overlapping YAC, BAC and PACs has been constructed and the relative position of eight microsatellite markers sited. Two known and one novel marker have been positioned within the gene, the others are in flanking regions. The more accurate location of these markers should help in future studies of the extent of gene loss. Several polymorphisms were also identified, all were within introns. Four of the common polymorphisms appear to be linked. In blood, DNA from 200 individuals, including normal, BPH and prostate cancer patients, confirmed this link. Only two samples of 200 did not carry the linked haplotype, both were patients with advanced prostate cancer. It is possible that such rearrangements within PTEN could be evidence of predisposition to prostate cancer in this small number of cases.

Alternative Splicing↗
PubMed2000

A transgenic mouse model for tumour immunotherapy: induction of an anti-idiotype response to human MUC1.

MUC1 is a membrane bound, polymorphic epithelial mucin expressed at the luminal surface of glandular epithelium. It is highly expressed in an underglycosylated form on carcinomas and metastatic lesions and is, therefore, a potential target for immunotherapy of cancer. The monoclonal antibody HMFG1 binds the linear core protein sequence, PDTR, contained within the immunodominant domain of the tandem repeat of MUC1. The efficacy of murine and humanized HMFG1 (Ab1) used as an anti-idiotypic vaccine was examined in mice transgenic for human MUC1 (MUC1.Tg) challenged with murine epithelial tumour cells transfected with human MUC1. Humoral idiotypic cascade through Ab2 and Ab3 antibodies was observed in MUC1.Tg mice following multiple antibody inoculations in the presence of adjuvant. Impaired tumour growth at day 35 and highest Ab3 levels were found in mice that had received mHMFG1 with RAS adjuvant. However, comparison of Ab3 levels in individual mice with tumour size in all treatment groups did not show a correlation between smaller tumours and increased levels of anti-idiotype antibody. This suggests that the anti-tumour effects of anti-idiotype vaccination are not solely related to the induction of idiotypic antibody cascades and probably involve other mechanisms.

Adjuvants, Immunologic↗
PubMed1999

Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen.

Carcinoembryonic antigen (CEA) is highly expressed by most tumours of gastrointestinal origin, but its use as a target for tumour therapy is complicated by the high levels of soluble CEA that are found circulating in the blood of cancer patients. A monoclonal antibody PR1A3 has been prepared, which binds preferentially to cell-surface rather than soluble CEA, this cell selectivity should make PR1A3 an ideal candidate for antibody-targeted tumour therapy. PR1A3 has been humanised and shown to retain its cell-surface specificity and affinity. Stable expression of the humanised antibody from chinese hamster ovary (CHO) cells has been achieved after transfection and amplification. Since PR1A3 binds preferentially to cell-associated CEA, a cell-free enzyme-linked immunosorbent assay (ELISA) has been developed to allow characterisation and routine assay of the antibody. This assay was developed using a recombinant chimeric protein constructed by cloning the domain of CEA that is bound by PR1A3 (the B3 domain) into a hybrid gene containing the Fc portion of IgG and three domains of biliary glycoprotein. Stable expression of this hybrid protein has been achieved from CHO cells. In ELISA both humanised and murine PR1A3 bound strongly to this antigen but only at a minimal level to soluble CEA. Two binding sites for the antibody were found on the gastric carcinoma cell line MKN45, one of higher affinity (1 nM) and the other at lower affinity (60 nM). Similar affinities were found for both murine and humanised antibodies. The data presented make it unlikely that the differential binding to cell-surface as distinct from soluble CEA can be accounted for by low affinity of PR1A3 for CEA, and provides further support for the hypothesis that some conformational change takes place on CEA release from cells and that it is this change that blocks PR1A3 binding to its epitope.

Amino Acid Sequence↗
PubMed1998

Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition.

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.

Amino Acid Sequence↗
PubMed1998

The detection of K-ras mutations in colorectal cancer using the amplification-refractory mutation system.

A total of 301 colorectal carcinoma (CRC) archival samples were analysed using the amplification-refractory mutation system (ARMS). Each sample was examined to determine the mutation status of codons 12 and 13 of the K-ras oncogene. The results from direct DNA sequence analysis carried out on 30 of the samples differed from the ARMS result in almost 50% of the cases as a result of the relative excess of wild-type to mutated DNA sequences. To assess the validity of the ARMS data, the polymerase chain reaction (PCR) was used to generate an amplicon from K-ras exon 1 from 23 of the samples. The PCR amplicons were cloned and sequenced, and the DNA sequence analysis of the cloned material was in agreement with the ARMS results in all but one case. This case represented a tumour that exhibited a five-nucleotide reversed inversion. The cloned sequence data confirm the sensitivity and specificity of the individual ARMS reactions and that it is possible in certain cases to detect additional, more complex, sequence variations.

Adenoma↗
PubMed1998

Mutation and expression analysis of the putative prostate tumour-suppressor gene PTEN.

The chromosomal region 10q23-24 is frequently deleted in a number of tumour types, including prostate adenocarcinoma and glioma. A candidate tumour-suppressor gene at 10q23.3, designated PTENor MMAC1, with putative actin-binding and tyrosine phosphatase domains has recently been described. Mutations in PTEN have been identified in cell lines derived from gliomas, melanomas and prostate tumours and from a number of tumour specimens derived from glial, breast, endometrial and kidney tissue. Germline mutations in PTEN appear to be responsible for Cowden disease. We identified five PTEN mutations in 37 primary prostatic tumours analysed and found that 70% of tumours showed loss or alteration of at least one PTEN allele, supporting the evidence for PTEN involvement in prostate tumour progression. We raised antisera to a peptide from PTEN and showed that reactivity occurs in numerous small cytoplasmic organelles and that the protein is commonly expressed in a variety of cell types. Northern blot analysis revealed multiple RNA species; some arise as a result of alternative polyadenylation sites, but others may be due to alternative splicing.

Amino Acid Sequence↗
PubMed1997

Crystallization of an antitumour antibody SM3 complexed with a peptide epitope.

SM3 antibody binds to a tumour-associated epitope on polymorphic epithelial mucin (PEM). Crystals of the Fab fragment of SM3 in complex with a peptide antigen were obtained by vapour diffusion against mother liquor containing acetate buffer, pH 6.5, cadmium chloride and polyethylene glycol (PEG) 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 83.9, c = 64.5 A and beta = 93.4 degrees. One Fab-antigen complex is present in the asymmetric unit. Diffracted intensities up to 1.95 A resolution have been measured from a frozen crystal using synchrotron radiation.

Journal Article↗
PubMed1997

Crystallization of the Fab fragment of the tumour-specific antibody PR1A3.

PR1A3 antibody binds specifically to the tumour-associated cell-surface antigen, carcinoembryonic antigen. Crystals of the Fab fragment of the PR1A3 antibody were obtained by vapour diffusion against mother liquor containing Tris-HC1 buffer, pH 8.6, magnesium chloride and polyethylene glycol 4000 as precipitating agent. Crystals belong to the monoclinic space group P2(1) with cell dimensions a = 42.2, b = 216.7, c = 45.9 A and beta = 95.6 degrees. Two Fab fragments are proesent in the asymmetric unit. Diffracted intensities up to 2.9 A resolution have been measured from frozen crystals.

Journal Article↗
PubMed1994

An epitope on carcinoembryonic antigen defined by the clinically relevant antibody PR1A3.

The monoclonal antibody PR1A3 has been used successfully for in vivo imaging of colorectal cancers, and several properties associated with this antibody, including minimal reactions of the antibody with circulating antigen in patients' sera, differentiate it from anti-carcinoembryonic antigen (CEA) antibodies used in similar studies. However, the antigen bound by PR1A3 was identified as CEA by analysis of somatic cell hybrids and by antigen expression from yeast artificial chromosomes, cosmids, and cDNA clones. The molecular weight, presence of a glycosyl-phosphatidylinositol anchor, elevation of surface expression by gamma-interferon, and N-terminal amino acid sequence all confirmed the antigen identification as CEA. A series of biliary glycoprotein-CEA hybrid proteins was produced which demonstrated that the epitope bound by the antibody was at the site of membrane attachment and involved parts of the glycosyl-phosphatidylinositol anchor and the B3 domain of CEA to form a conformational epitope. Access to this epitope, although possible when the antigen was on the cell surface, appeared to be blocked when CEA was released from the cell. The nature and location of the epitope on CEA are proposed to be responsible for the unique properties of the antibody.

Animals↗
PubMed1989

Cloning and expression of a trypomastigote-specific 85-kilodalton surface antigen gene from Trypanosoma cruzi.

An 85-kDa trypomastigote-specific surface antigen gene from Trypanosoma cruzi has been identified by screening a genomic library in lambda gt10 with trypomastigote and epimastigote cDNA. The 1.3-kb genomic clone (pTt34) hybridizes to a single trypomastigote mRNA of 3.7 kb and to multiple bands in genomic Southern blots. Dot-blot experiments show that there are 5-10 copies of this sequence per haploid genome, and these are arranged in a non-tandem manner. pTt34 has been expressed as an anthranilate synthetase fusion protein in Escherichia coli, and inclusion bodies have been used to raise antiserum in rabbits. This antiserum immunoprecipitates a cell surface trypomastigote-specific protein of 85 kDa. The DNA and predicted amino acid sequences of pTt34 are given. Four further clones obtained from a PvuII/HpaI partial genomic library in pUC13 have extended the sequence of the 3' end of pTt34; each of these clones has regions of sequence divergence and each could represent a different member of the gene family.

Amino Acid Sequence↗