R.A. Robins

Enhanced expression of the complement regulatory protein CD55 predicts a poor prognosis in colorectal cancer patients

This study prospectively correlated the level of expression of CD55 on tumours with 7-year survival in 136 colorectal cancer patients. Patients with tumours expressing high levels of CD55 had a significantly worse survival (24%) than patients with low CD55 levels (50%, p<0.02). A similar difference was seen for patients (Dukes B or C) with a high risk of recurrence (29% vs 58%, p<0.05). Furthermore, there was a progressive deterioration in prognosis with increasing antigen expression (p=0.01). It remains unclear if CD55 is overexpressed by tumours to protect them from complement or if it is related to the recent observation that CD55 is a ligand for the T-cell activation antigen CD97. However, it is a marker of aggression, as colorectal cancer patients whose tumours overexpress CD55 have a significantly reduced 7-year survival.

Randomized double-blind phase II survival study comparing immunization with the anti-idiotypic monoclonal antibody 105AD7 against placebo in advanced colorectal cancer

The cancer vaccine 105AD7 is an anti-idiotypic monoclonal antibody that mimics the tumour-associated antigen 791T/gp72 (CD55, Decay Accelerating Factor) on colorectal cancer cells. Phase I studies in patients with advanced disease confirmed that 105AD7 is non-toxic, and that T cell responses could be generated. A prospective, randomized, double-blind, placebo-controlled survival study in patients with advanced colorectal cancer was performed. 162 patients were enrolled between April 1994 and October 1996. Patients attended at trial entry, and at 6 and 12 weeks, where they received 105AD7 or placebo. Study groups were comparable in terms of patient demographics, and time from diagnosis of advanced colorectal cancer (277.1 v 278.6 days). Baseline disease was similar, with 50% of patients having malignancy in at least 2 anatomic sites. Compliance with treatment was poor, with only 50% of patients receiving 3 planned vaccinations. Median survival from randomization date was 124 and 184 days in 105AD7 and placebo arms respectively (P=0.38), and 456 and 486 days from the date of diagnosis of advanced disease (P=0.82). 105AD7 vaccination does not prolong survival in patients with advanced colorectal cancer. The reasons for lack of efficacy are unclear, but may reflect the high tumour burden in the patient population, and poor compliance with immunization. Further vaccine studies should concentrate on patients with minimal residual disease.

Immune complexes in cancer

ConclusionIt is clear that a wide variety of methods for measuring immune complexes are available but some may have disadvantages, such as technical difficulty, and all suffer from a lack of satisfactory standardization (WHO, 1977). However, in spite of these difficulties, the measurement of immune complexes may itself prove useful in monitoring of patients, and the complexes may provide a route to the identification and isolation of tumor related products, which would allow a more direct quantitation of tumor burden.

A Neoadjuvant/Adjuvant Randomized Trial of Colorectal Cancer Patients Vaccinated with an Anti-Idiotypic Antibody, 105AD7, Mimicking CD55

Purpose: To assess the tolerability and effectiveness of 105AD7 vaccination in colorectal cancer patients. 105AD7 is a human anti-idiotypic antibody mimicking CD55, a glycoprotein, which is more than expressed on colorectal cancer cells and protects them from attack by complement. Experimental Design: Colorectal cancer patients (n = 67) eligible for primary surgery were randomized to receive the anti-idiotypic antibody 105AD7±Bacillus Calmette-Guerin/alum or to no treatment (control group). The immunizations were given i.d./i.m. before surgery and continued for a period of 2 years. The patients were monitored in enzyme-linked immunospot (ELISPOT; γ-IFN), proliferation assay, and Luminex cytokine assays. Results: No serious adverse events were recorded. Of the 32 investigated immunized patients, 14 (44%) were considered to be responders in the ELISPOT assay. Induced proliferative responses were noted in 17 of 40 (43%) monitored patients. There was no correlation between the ELISPOT and proliferation assays. Luminex analyses revealed tumor necrosis factor-α and granulocyte macrophage colony-stimulating factor responses not only to the vaccine but also toward the native antigen CD55 in 9 of 13 (69%) patients. Conclusions: Immune responses to vaccination were induced in a majority of monitored patients measured by ELISPOT and proliferation assay. The lack of correlation between the ELISPOT and proliferation assays may reflect the fact that the two methods measure different T-cell responses and highlights the importance of multiple readouts in evaluating a potential cancer vaccine. Responses to both the anti-idiotype and the CD55 antigen were measurable, adding support to the use of CD55 as a target in cancer treatment.

Characteristics of a cell surface antigen defined by an anti-human osteogenic sarcoma monoclonal antibody.

The expression of a cell surface antigen defined by an anti-human osteogenic sarcoma monoclonal antibody was analysed by flow cytofluorometry using fluorescein-labelled antibody. Quantitative absorption tests established that the antigen was associated with plasma membranes, whereas cytosol, cellular lipids and nuclei were largely devoid of activity. Single-phase aqueous butanol solutions at non-cytolytic concentrations failed to solubilize the antigen, although treatment of cells with papain virtually abolished antigenic activity. The antigen was shown to be solubilized by the non-ionic detergent Nonidet P-40, and following lactoperoxidase-catalysed radioiodination of viable cells, extraction with detergent, immunoprecipitation of antigen with monoclonal antibody and Sepharose-Protein A, the molecular weight of antigen was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The findings indicate that this human osteogenic sarcoma antigen is a monomeric integral membrane protein with an apparent molecular weight of 72,000, which is predominantly expressed at the external face of the tumour cell plasma membrane.

Quantitation of MHC antigen expression on colorectal tumours and its association with tumour progression.

A flow cytometric technique has been established for accurately quantitating the cell surface density of MHC antigens and the percentage of cells expressing MHC antigens in 38 colorectal tumours. Thirty-four percent of tumours were partially or completely negative for HLA-ABC antigen expression. Although the quantity of HLA-ABC antigens varied widely, there was no correlation between the density of HLA-ABC antigens, or the percentage of cells expressing these antigens and clinicopathological stage. Fifty percent of the colorectal tumours expressed HLA-DR with varying antigen densities. All of the poorly differentiated tumours expressed HLA-DR but there was no correlation between expression of HLA-DR and clinicopathological stage. The aneuploid tumours expressed more HLA-ABC and HLA-DR antigens on a higher percentage of cells than the diploid tumours. Abnormal expression of the tumour associated antigens CEA, Y haptenic blood group and 791T p72 also correlated with expression of HLA-ABC and HLA-DR antigens on colorectal tumours. The majority of early derived in vitro dividing cells failed to express both HLA-ABC and HLA-DR antigens although they expressed high levels of tumour associated antigens. If there is a correlation between in vitro and in vivo growth perhaps tumours are maintained and seeded by MHC antigen negative cells.

Measurement of tumour reactive antibody and antibody conjugate by competition, quantitated by flow cytofluorimetry

Binding of unlabelled monoclonal antibody preparations has been assessed by competition at saturation with fluorochrome labelled homologous antibody for binding to antigen bearing target cells. The extent of competition was measured by quantitative flow cytofluorimetry, and simple mathematical procedures have been developed to allow the interpretation of competition data in terms of antibody binding activity. In the system studied, non-specific (non-competitive) fluorescence was minimal, but an iterative method to calculate its contribution to the measured signal is given. This approach has the advantage that the antibody preparation to be tested does not need to be labelled or modified; this is particularly important when evaluating the binding activity of therapeutic antibody conjugates. Comparison with a well characterized standard antibody preparation provides a rapid, sensitive and accurate quality control procedure. This test is also simple to perform, requiring only the mixing of labelled and unlabelled antibodies with target cells, a single incubation, followed by analysis without washing of the target cells.

Reactivity of an anti-(human gastric carcinoma) monoclonal antibody with core-related peptides of gastrointestinal mucin

SummaryA murine anti-(human gastric carcinoma) monoclonal antibody, GL-013 (IgG1), which reacts with a high-molecular-mass glycoprotein from colorectal tumour tissue [Yang and Price (1989) Anticancer Res 9: 1707], was examined for reactivity against a panel of purified and partially purified antigens associated with tumours of the gastrointestinal tract. These included carcinoembryonic antigen (CEA), normal cross-reacting antigen, Y-hapten glycoproteins, and perchloric acid extracts and glycolipid preparations from colorectal tumours. While the GL-013 antibody failed to bind to these antigens, it was found to react strongly with synthetic peptides with sequences based upon that reported for the protein core of a human gastrointestinal mucin [Barnd et al. (1989) Proc Natl Acad Sci USA 86: 7159; Gum et al. (1989) J Biol Chem 264: 6480]. In control tests, a series of other anti-(colorectal tumour) antibodies (IgG1 and IgG3), with broad reactivity towards gastrointestinal carcinomas, as well as an anti-CEA antibody, (IgG1) failed to react with the synthetic peptides. It is concluded that the anti-(gastric carcinoma) monoclonal antibody GL-013 binds to a threonine-rich peptide epitope expressed within the protein core of gastrointestinal mucins.

Recombinant ricin toxin A chain cytotoxicity against carcinoembryonic antigen expressing tumour cells mediated by a bispecific monoclonal antibody and its potentiation by ricin toxin B chain.

A bispecific monoclonal antibody recognising both carcinoembryonic antigen (CEA) and ricin toxin A chain (RTA) was tested for its ability to target recombinant RTA (r-RTA) to CEA-expressing tumour cells, alone and in combination with ricin B chain (RTB). The antibody, 636 (Robins et al., 1990), induced significant RTA cytotoxicity against MKN45 gastric carcinoma cells which express high levels of CEA, using the r-RTA at a concentration below that known to be intrinsically cytotoxic. The addition of ricin toxin B chain (RTB) also potentiated cytotoxicity of r-RTA, and there was an additive increase in potentiation against CEA-positive cells when both RTB and 636 were included. The bispecific antibody restored potentiation by RTB after blocking of its binding site with excess galactose, and also the cytotoxic activity of whole ricin which had been blocked with galactose. It was concluded that the 636 bispecific antibody was highly effective in targeting the toxic moiety of the molecule to CEA-expressing cells, and allowed exploitation of the additional ability of the B chain to facilitate cellular incorporation. The facilitating function of the B chain was equally effective whether or not its lectin site was active.

Induction of immunity to chemically-induced rat tumours by cellular or soluble antigens

SummaryRadiation-attenuated, aminoazo dye-induced rat hepatoma cells were employed as a standard immunogen in tests evaluating the optimal conditions for the demonstration of resistance to subcutaneous challenge with viable tumour cells. These tests revealed that maximal sensitivity for the detection of the immunogenicity of γ-irradiated cells was obtained using multiple immunisations via the intraperitoneal route rather than the subcutaneous route. When 3 M KCl extracts of tumours (as soluble tumour antigens) were evaluated for the induction of specific immunoprotection under comparable conditions, weak resistance to tumour was demonstrable, but only within a restricted dose range of the antigen preparation. No significant effects upon tumour growth were observed in rats pretreated with fractions of tumour-bearer serum.