Graeme Denton

External quality assurance of circulating tumor cell enumeration using the CellSearch(®) system: a feasibility study.

Circulating tumor cells (CTCs) are cells that have detached from solid tumors and entered the blood. CTCs can be detected, among others, by semi‐automated immunomagnetic enrichment and image cytometry using CellSearch® (Veridex, Raritan, NJ). We studied the feasibility of external quality assurance (EQA) of the entire CellSearch procedure from blood draw to interpretation of results in multiple laboratories.

Purification of monoclonal antibodies by epitope and mimotope affinity chromatography.

Murine monoclonal antibodies raised against the carcinoma-associated MUC1 mucin have applications in the diagnosis and therapy of human cancer. Many of these antibodies define linear epitopes of three, four or five amino acids within an immunodominant region of the MUC1 protein core. Various synthetic peptides which incorporated this region were prepared and covalently linked to agarose beads for use as affinity matrices. An unrelated peptide was identified as a mimotope for one of the anti-MUC1 antibodies using phage display technologies and this was also evaluated as a potential ligand in an affinity matrix. Epitope affinity chromatographic purification of an anti-MUC1 antibody was performed using hybridoma tissue culture supernatants as sample. Following sample application and column washing, antibody was desorbed from the matrix by gradient elution with increasing concentrations of NaSCN. The procedure has proved efficient for the purification of anti-MUC1 antibodies and the concentration of NaSCN required for antibody desorption gives a measure of the relative binding affinity of the antibody for the peptide epitope matrix so that separation strategies may be optimised.

Preclinical evaluation of copper-67 labelled anti-MUC1 mucin antibody C595 for therapeutic use in bladder cancer

Transitional cell carcinoma of the bladder is the fifth commonest cause of death from cancer in men in the United Kingdom. Most patients present early with superficial disease, though with current treatment up to 20% progress to invasive disease, which has a poor prognosis. Better local treatments are required to limit this tumour progression. The ease of access to the bladder via a catheter provides the ideal opportunity for antibody (Ab) targeted therapy. We have previously shown that indium-111 labelled anti-MUCI mucin Ab C595 selectively localises to bladder tumours after intravesical administration. We have selected copper-67 as an alternative radiolabel with suitable physical characteristics for radioimmunotherapy. This communication demonstrates that C595 can be reproducibly labelled with67Cu and that the radioimmunoconjugate is both stable and maintains high immunoreactivity. Pilot studies on cystectomy specimens in a novel ex vivo system and in one patient confirmed the ability of this conjugate to localise to tumour after intravesical administration. On the basis of these studies we are now in a position to study the intravesical administration of67Cu-labelled C595 in patients with bladder cancer with a view to a therapeutic trial.

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.

Direct isolation of monoclonal antibodies from tissue culture supernatant using the cation-exchange cellulose Express-Ion S

The chromatography of the murine hybridoma cell C595/102 culture supernatant expressing the therapeutic monoclonal antibody C595, on the cation-exchange cellulose Whatman Express-Ion Exchanger S has been investigated. Initial method scouting studies using purified C595 in 1-ml mini columns demonstrated that binding capacity and binding efficiency were dependent not only on decreasing pH but also on the buffer salts used to prepare the mobile phase. Under optimised conditions of 0.1 M sodium acetate buffer, pH 5.0, we were able to separate purified C595 from BSA, the major contaminant in tissue culture fluid. Under these conditions immunoreactive C595 could be isolated directly from tissue culture supernatant. A scale-down study was carried out using a 25-ml column operated at a flow-rate of 150 cm/h which also yielded purified immunoreactive antibody. This procedure should now be suitable for scale-up.

Production and characterization of an anti-(MUC1 mucin) recombinant diabody.

Abstract A recombinant diabody fragment based on the anti-MUC1 monoclonal antibody, C595 has been produced in a bacterial expression system. Substitution of a 7-amino-acid linker sequence (Gly6Ser) for the original single-chain (sc)Fv 15-amino-acid linker (Gly4Ser)3, using polymerase-chain-reaction-based strategies, forces variable heavy (VH) and light (VL) domains to pair with complementary domains on neighbouring scFv molecules, forming a scFv dimer (diabody). This recombinant protein shows similar binding characteristics to the parental C595 monoclonal antibody. The ability to bind to MUC1 mucin on carcinoma cell surfaces will allow its potential as a diagnostic and therapeutic reagent of clinical utility to be investigated.

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

Recombinant single-chain fragments (scFv) of the murine anti-MUC1 monoclonal antibody C595 have been produced using the original hybridoma cells as a source of variable heavy (V(H))- and variable light (V(L))-chain-encoding antibody genes. The use of the polymerase chain reaction (PCR), bacteriophage (phage) display technology and gene expression systems in E. coli has led to the production of soluble C595 scFv. The scFv has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C595 scFv, which was similar in activity to the C595 parent antibody. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the scFv, while ELISA, FACScan analysis, fluorescence quenching, quantitative immunoreactivity experiments and immunohistochemistry confirm that the activity of the scFv compares favourably with that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens illustrates the potential application of this novel product as an immunodiagnostic and immunotherapeutic reagent.

Imaging for staging bladder cancer: a clinical study of intravenous 111indium-labelled anti-MUC1 mucin monoclonal antibody C595.

Objective To investigate the clinical application of an 111In‐labelled anti‐MUC1 mucin monoclonal antibody (mAb) imaging for staging invasive bladder cancer.

Production and characterisation of a C595 antibody-99mTc conjugate for immunoscintigraphy of bladder cancer.

Abstract Current radiological techniques for staging bladder cancer are inaccurate, especially in the identification of pelvic lymph node metastases. Immunoscintigraphy has the potential to offer improved staging for bladder cancer. The aim of this study was to label the anti-MUC1 monoclonal antibody C595 with 99mtechnetium (Tc), the most widely used diagnostic radionuclide, and assess the potential of the resultant conjugate for intravenous immunoscintigraphy of bladder cancer. A direct, reduction-mediated technique was used to label the antibody. The resultant conjugate was shown to be highly immunoreactive, stable and bound specifically to MUC1. The ability of the conjugate to bind to bladder tumours was demonstrated in an ex vivo model where the mean tumour:normal urothelial uptake was 5.7:1 and by intravesical administration in patients with bladder cancer where the mean tumour:normal urothelial uptake was 20.4:1. The ability of the conjugate to localise MUC1-expressing tumours was demonstrated in a nude mouse xenograft model. A conjugate of 99mTc-C595 has been produced and characterised, and it may be suitable for intravenous immunoscintigraphy, a potential novel staging tool for bladder cancer.

Immune responses to the MUC1 mucin

MUC1 mucins are highly glycosylated glycoproteins expressed on the luminal surfaces of glandular epithelia. In breast and ovarian carcinomas, their expression is frequently upregulated and they may be secreted into the circulation of cancer patients. Early studies aimed at the production of anti-MUC1 monoclonal antibodies revealed that MUC1 was a potent immunogen in mice with many monoclonal antibodies raised defining epitopes within the protein core of MUC1. The immunogenicity of MUC1 has now been extended to human studies and it is apparent that patients with breast and ovarian malignant disease are able to mount immune responses against MUC1. These findings provide information on the mechanisms involved in the recognition of MUC1 expressing tumours. The utilisation of MUC1 related immunogens to stimulate immune responses to tumours could lead to the improved management of patients and the development of new immunotherapeutic strategies aimed at the eradication of MUC1 mucin expressing cancers.MUC1 mucins are highly glycosylated glycoproteins expressed on the luminal surfaces of glandular epithelia. In breast and ovarian carcinomas, their expression is frequently upregulated and they may be secreted into the circulation of cancer patients. Early studies aimed at the production of anti-MUC1 monoclonal antibodies revealed that MUC1 was a potent immunogen in mice with many monoclonal antibodies raised defining epitopes within the protein core of MUC1. The immunogenicity of MUC1 has now been extended to human studies and it is apparent that patients with breast and ovarian malignant disease are able to mount immune responses against MUC1. These findings provide information on the mechanisms involved in the recognition of MUC1 expressing tumours. The utilisation of MUC1 related immunogens to stimulate immune responses to tumours could lead to the improved management of patients and the development of new immunotherapeutic strategies aimed at the eradication of MUC1 mucin expressing cancers.