Production and characterization of a recombinant anti-MUC1 scFv reactive with human carcinomas

Denton, G.; Sekowski, M.; Spencer, Daniel I. R.; Hughes, O. D. M.; Murray, Andrea; Denley, Helen; Tendler, Saul J. B.; Price, Michael R.

doi:10.1038/bjc.1997.434

Cited by 19 publications

(12 citation statements)

References 21 publications

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“…Immunohistochemistry was performed using the method described by Denton et al (1997). The biotin-labelled SC142 scFv was applied for 1 h. Optimal antibody concentrations were determined by titration.…”

Section: Immunohistochemistrymentioning

confidence: 99%

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

et al. 2002

Br J Cancer

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SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues. Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells. However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size. To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy-and variable light-chainencoding antibody genes. The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E. coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry. Analysis by DNA sequencing, SDS -PAGE and Western blotting has demonstrated the integrity of the single-chain fragments. Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody. BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent. These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent. British Journal of Cancer (2002) Mucins are high-molecular-weight glycoproteins composed by a carbohydrate moiety (mainly O-glycans) that represents 50 -80% of their total mass and peptide core; also called apomucin, it is rich in Thr and Ser (Verma and Davidson, 1994). A variety of alterations of mucins have been described in metaplastic and malignant disease of the stomach (Correa, 1988). To detect those cancer associated alterations of gastric mucin, a number of monoclonal antibodies (mAbs) were developed. Although their specificity for cancer was limited compared with that of genetic markers such as p53, APC, c-met, k-sam and c-erbB-2, immunohistological detection of antigen is much easier than molecular biological analysis of those gene abnormalities, and this is of practical importance.In the previous study, we developed monoclonal antibodies elicited to the human stomach carcinoma cell line SNU16 to detect useful markers for gastric cancer. One of these monoclonal antibodies, SC142, detects an antigen present on adenocarcinoma cells of stomach. Preliminary studies on the molecular properties of the SC142-reactive antigen suggest that the epitope is sensitive to Oglycanase and is expressed on a large, heavily glycosylated molecule indicating that the antigen is probably mucin (Hong et al, 2001).In immunohistochemical studies, SC142-reactive antigen was not detected in normal gastrointestinal epithelium, whereas it was highly expressed in 78% of gastric cancers (29 out of 37) and 87% of colon cancers (27 out of 31) indicating that SC142 antibody can be a valuable tool to detect gastrointestinal cancers.However, the use of whole antibody fo...

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Section: Immunohistochemistrymentioning

confidence: 99%

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

et al. 2002

Br J Cancer

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“…The replacement of DT by ES represents a sequence polymorphism found with a high incidence in all repeat domains of human MUC1, which were analysed on the level of genomic DNA (Engelmann et al, 2001). The C595 scFv binds to a panel of mammary and bladder carcinoma biopsies but not to benign tissues like breast ducts (Denton et al, 1997) making the scFv suitable for specific tumour cell binding.…”

Section: Discussionmentioning

confidence: 99%

“…C595 mAb has been used in immunoassays for the measurement of soluble MUC1 in breast cancer patients Dixon et al, 1993) and for in vivo immunoscintigraphy (Symonds et al, 1992;Perkins et al, 1993). The C595 mAb has been converted into a single-chain fragment of variable regions (scFv) with retained binding specificities (Denton et al, 1997). Utilising the C595 scFv, we generated an anti-MUC1-scFv-Fc-IL2 fusion protein that after binding to MUC1-positive tumour cells induces proliferation of peripheral blood T cells and mediates activation of resting NK cells to lysis of MUC1-positive tumour cells.…”

Section: Revised 15 July 2003; Accepted 17 July 2003mentioning

confidence: 99%

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An anti-MUC1-antibody–interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells

et al. 2003

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MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expession is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours.

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“…The scFv of an antibody is the smallest structural domain which retains the complete specificity and binding site capability of the parent antibody (142). One can derive scFvs from monoclonal antibodies produced by hybridomas (143). ScFvs may be isolated that bind to different domains of the IN protein.…”

Section: Scfv-nucleasementioning

confidence: 99%

Potential nuclease-based strategies for HIV gene therapy

Joshi¹

2000

Front Biosci

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Production and characterization of a recombinant anti-MUC1 scFv reactive with human carcinomas

Cited by 19 publications

References 21 publications

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas

An anti-MUC1-antibody–interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells

Potential nuclease-based strategies for HIV gene therapy

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