Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

Expression of B7 costimulatory molecules by cells infiltrating the colon in experimental colitis induced by oral dextran sulfate sodium in the mouse.

BACKGROUND AND AIM: T-cell activation, mediated by the interaction with major histocompatibility complex (MHC)-peptide complexes and B7 costimulatory molecules on antigen-presenting cells, is an essential event in the pathogenesis of inflammatory bowel disease (IBD). We investigated the expression of B7 costimulatory molecules on cells in the colon in an experimental mouse model of IBD to determine whether the B7/ligand interaction could provide a target for therapeutic intervention in IBD. METHODS: Experimental colitis was induced in mice by oral consumption of water substituted with 5% dextran sulfate sodium (DSS). Mice (n=4) were killed 1, 2, 3, 4 and 7 days after commencing DSS consumption, and colonic tissue was collected and examined immunohistochemically for T cells, B cells, macrophages and cells expressing B7-1 or B7-2. RESULTS: Compared to control mice drinking water, macrophage numbers in the colonic epithelium were elevated sevenfold by day 1 and T cells were elevated threefold by day 3 following commencement of DSS consumption. Numbers of infiltrating B7-positive (B7+) cells were not significantly elevated until day 7 when B7-1+, B7-2+ cells and macrophages were increased 20-fold compared to normal mice. CONCLUSION: These results demonstrate that an initial and rapid infiltration of the colonic epithelium by B7-negative macrophages is followed by an infiltration of T cells and subsequent upregulation of the B7 costimulatory molecules potentiating the inflammatory reaction in this disease model. These results suggest an intervention strategy based on the blockade of the B7-costimulatory axis could find application in the treatment of inflammatory bowel disease.

Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma.

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a Ka of 2.3 x 10(-9), compared with 4.2 x 10(-9) for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

A comparison of the anti-idiotypic responses generated by antibodies to a protein and a hapten: a common interspecies idiotype on antibodies against human albumin induces an idiotypic network in rabbits.

Idiotypic networks have the capacity to exert significant influences on immune responses and an understanding of the ways to manipulate these networks may lead to new modalities in immunotherapy. In order to gain further insights into the nature of the immune responses stimulated by immunoglobulin idiotypes, rabbits were immunized with a mAb (Ab1) against a large globular protein, human albumin, or a mAb against a hapten, TNP. All rabbits developed anti-idiotypic antibodies (Ab2) and the rabbits immunized with anti-human albumin concomitantly developed antibodies to human albumin (Ab3). Ab2 prepared from these rabbits blocked binding of Ab1 to antigen and the anti-human albumin Ab2 reacted with all species of anti-human albumin including sheep, rabbit, rat and goat. The anti-TNP Ab2 reacted only with the mouse anti-TNP Ab1. This TNP Ab2 bound only to intact Ab1 whereas the human albumin Ab2 reacted with the Ab1 heavy chain. To compare the relative efficiencies of anti-idiotypic antibodies and antigen in inducing antibody, mice were immunized with rabbit Ab2 or antigen. All mice immunized with Ab2 developed anti-idiotypic Ab3, but only the human albumin Ab2 preparations elicited antigen specific Ab3; the amount of antibody produced was less than 1% of that found by immunization with antigen. The type of antibody induced in the Ab2-immunized mice was compared with that found in the antigen-immunized mice and in the Ab1-immunized rabbits. The mouse anti-albumin Ab3 was comparable to mouse Ab1 in terms of affinity and specificity for proteolytic fragments of human albumin. The Ab3 which arose in Ab1-immunized rabbits had a higher affinity and broader epitope specificity and was similar to antibodies raised against antigen. These results show considerable differences in the ability of similar anti-idiotypic antibodies to induce immune responses as well as considerable differences in the nature of a response seen within an intact network compared to an artificially induced network.

Common epitope on HIV p24 and human platelets.

Mimicry of human antigen by HIV may underlie the autoreactivity seen in AIDS. A mouse monoclonal antibody (VIC8) raised against HIV p24 cross-reacted with human platelets; binding could be abolished by recombinant p24 antigen. VIC8 bound less well to platelets from patients with HIV than to those from healthy individuals. In the HIV group, binding was not related to p24 antigenaemia, disease stage, or platelet counts. This cross-reactivity is another example of antigenic mimicry by HIV and may be mechanistically important in HIV-associated autoimmune-like thrombocytopenia.

Mononuclear cells in normal colon and colonic carcinoma express the same T cell activation markers.

The proportion of mononuclear cells (MC) expressing some of the T cell activation markers in normal colon and colonic carcinomas was determined by immunoperoxidase staining and computer-assisted video image analysis (VIA). Tissue sections were stained with monoclonal antibodies against the T cell activation markers which included the MHC class II antigens DR, DP and DQ, interleukin 2 receptor (CD25) and the p180 (CD45RO) form of the leucocyte common antigen. The mean values for the proportion of leucocytes expressing these activation markers were 92% for DR, 61% for DP, 68% for DQ and 60% for CD45RO in tumours and 90, 62, 70 and 55% in normal tissue respectively. Few cells were stained for the IL2 receptor (mean values 7% for tumours and 5% for normal tissue) or the p220 form (CD45RA) of the leucocyte common antigen (mean values 6% for tumours and 4% for normal tissue). The proportion of MC bearing any of these markers in the normal colon was not significantly different from the matched colonic carcinomas.

Influence of recombinant interferon-gamma QN the expression of MHC class I and class II antigens on four human colonic carcinoma cell lines.

The occurrence of MHC class I and class II antigens on four human colonic carcinoma cell lines and the effect of recombinant interferon-gamma (rIFNg) on the expression of these antigens was investigated by immunofluorescent flow cytometry. The concentration of rIFNg which resulted in the largest increase in expression of class I and class II antigens was determined. Changes in the amount of MHC antigen on the membrane were indicated by a shift in the mean fluorescence intensity (MFI) of the cell population. Without addition of rIFNg, the COLO 206, COLO 320F and COLO 397 cell lines were class I positive although the COLO 206 cell line expressed less class I antigen than the other two lines. The HT-29 cell line expressed only a minimal level of class I antigen. Treatment with rIFNg increased the amount of class I antigen on these cell lines 5, 1.4, 2.5 and 20 times respectively. Maximum levels of class I antigen were found two days after treatment. Class I antigen expression returned to pre-treatment levels by day 8 in all but the HT-29 cell line, which maintained its increased level following a single dose of rIFNg. All four cell lines had little or no class II antigens. Following treatment with rIFNg, DR antigen appeared on all four lines whereas DP and DQ antigens could be induced only on the 320F and 397 lines. The amount of class II antigen reached its peak two days after treatment and gradually decreased over the next 6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of computer-assisted video image analysis in the enumeration of immuno-stained cells in tissue sections.

A novel method of computer-assisted video image analysis (VIA) was used to determine the number of immunostained cells in tissue sections. This method permitted an accurate and objective quantification of cells of a particular phenotype. This enumeration was achieved by measuring the area stained by a test monoclonal antibody (such as the T cell marker, CD3) and comparing it with the area stained by a leukocyte common (LCA) monoclonal antibody (CD45). The proportion of T cells within the total leukocyte population in a particular tissue was then calculated. The differentiation of positive (stained) and negative (unstained) cells was uniformly maintained by setting the computer to detect a threshold for staining intensity. This enabled consistency to be maintained within a tissue section as well as between sections stained with the same antibody. In the present study, we determined the phenotype of leukocytes in colonic carcinomas by VIA and compared this with results obtained by normal visual analysis. The VIA method showed distinct advantages over normal visual analysis especially in sections which contained moderate numbers of stained cells.

Density and phenotype of tumour-associated mononuclear cells in colonic carcinomas determined by computer-assisted video image analysis.

The density and phenotypes of tumour-associated mononuclear cells (TAMC) in tissue sections of colonic carcinomas was determined by the technique of video image analysis (VIA). This technique allowed an accurate and objective enumeration of both total mononuclear cells (MC) in H&E stained sections and individual types of cells as revealed by immunoperoxidase staining with monoclonal antibodies in frozen sections. This enumeration allowed reliable statistical analysis of the differences between sample groups. Using this technique it was found that the density of MC in histiologically normal tissue was significantly higher than in tumour tissue. Tumours from patients with the best prognosis (stage A) had significantly higher numbers of TAMC than stage B (P less than 0.02), C (P less than 0.002) and D (P less than 0.002) tumours. The differences in the density of TAMC between tumours obtained from stage B and C and that between C and D were not significant, whereas stage B had a significantly higher TAMC density than stage D tumours (P less than 0.05). Comparing tumour differentiation, well differentiated adenocarcinomas had a significantly higher (P less than 0.05) TAMC density than poorly differentiated tumours but not moderately differentiated tumours. Moderately and poorly differentiated adenocarcinomas did not differ significantly in the density of TAMC. In examining the phenotype of these cells, it was found that T lymphocytes formed the majority of the TAMC with the CD4+ subset predominating in 28 of 29 cases. Similarly, all sections of normal colon (taken at least 4 cm away from the tumour) had more CD4+ than CD8+ cells. The proportion of the total leucocyte population that was CD3+ was comparable in normal and tumour tissue. Generally, few macrophages were present in either tumour or normal tissues. B cells (CD21%) and subset of NK cells (CD57+) were not detected in the tumours. There were no significant differences in the proportion of leucocytes which were CD4+, CD8+ and CD14+ (macrophages) between the normal colon and the tumour tissues. The types of cells in the TAMC population did not differ with tumour stage or differentiation or with the density of the TAMC itself.

Expression of MHC class I and class II antigens in colonic carcinomas.

Malignant and non-malignant ('normal') colonic tissues from patients with colonic carcinoma were examined for the expression of MHC class I and class II antigens by immunoenzymatic staining using monoclonal antibodies. The amount of class I antigen as detected by 2 monoclonal antibodies, FMC 16 or W6/32 was clearly diminished in 11 of 14 tumours when compared to the amount present on 'normal' colonic tissue from the same individual. The loss of class I antigen did not correlate with tumour stage or differentiation. The reactivities of FMC 16 and W6/32 with these tissues were not identical, which indicates that the 2 monoclonal antibodies may recognize different epitopes on the HLA class I molecule. Class II antigens were absent from 'normal' colonic epithelium but were present on 20 of 28 tumours, with DR being detected more often than DP, and DQ found only on 4 of 28 tumours. When present, staining for class II antigens was heterogeneous within the tumour, in that all tumour cells did not stain equally. DR and DP antigens were found more often on moderately or poorly differentiated adenocarcinomas and on stage B, C and D tumours in that order of frequency. Thus tumours with a better prognosis were less likely to express DR and DP. The expression of DQ was unrelated to staging or differentiation.

IgG antibodies to Aspergillus fumigatus in cystic fibrosis: a laboratory correlate of disease activity.

Serum was collected from 50 patients with cystic fibrosis, and IgG antibodies to Aspergillus fumigatus were measured by enzyme linked immunosorbent assay (ELISA). In addition, total IgE and Aspergillus specific IgE antibodies were measured in 41 of the 50. A close association was found between pulmonary function and clinical state, and IgG antibodies to Aspergillus. There was no association between pulmonary function or clinical state and IgE antibodies. It is postulated that in patients with cystic fibrosis, Aspergillus fumigatus may contribute to deterioration in pulmonary function by local pathogenicity, or by hypersensitivity mechanisms mediated by IgG.

Production of a polyspecific human monoclonal antibody reacting with an epidermal antigen.

A clone of cells secreting an antibody to an epidermal antigen was generated from a patient with a blistering skin lesion. Although produced by fusion of human lymphocytes to a HAT-sensitive myeloma, this clone of cells did not have characteristics of a hybridoma. A true hybridoma was produced by fusion of this clone to a HATr/ouabain(r) myeloma line. The IgM antibody secreted by this clone reacted with the intercellular region of the epidermis of normal human skin in a manner similar to pemphigus autoantibodies. In addition, in normal human kidney the antibody bound to glomeruli and tubules. It also reacted with an antigen present in the cytoplasm of a wide variety of cell lines including epithelial, lymphoid and myeloid types. No reaction was found with the surface of any of the cell lines, nor with DNA or phospholipid antigens. This monoclonal antibody may define an autoantibody specificity which mediates some autoimmune skin lesions. Its polyspecificity is reminiscent of some other human hybridoma autoantibodies, and its reaction with components of the kidney suggests an alternative pathology for renal disease in such patients.

Intestinal and serum antibody in coeliac disease: a comparison using ELISA.

Intestinal and serum antibody to antigens derived from gluten and other food proteins in 16 children with coeliac disease and 15 control subjects was measured using an enzyme-linked immunosorbent assay (ELISA). High concentrations of antibody to gluten antigens were found in children with coeliac disease who were on a diet which contained gluten. This antibody was predominantly in the IgA and IgM classes in intestinal fluid, and in the IgG and IgA classes in serum. When coeliac children transferred to a gluten-free diet for 6 months or more, anti-gluten antibody fell much more rapidly in serum than in intestinal fluid. Although no single measure of antibody, in any immunoglobulin class, to a gluten-derived antigen proved sufficiently discriminating to be suggested as a diagnostic test for coeliac disease, serum antibody, particularly in the IgA class, may be of value in following the progress of patients and in assessing their adherence to a gluten-free diet.

Monoclonal antibodies to baculovirus structural proteins: determination of specificities by Western blot analysis.

Conventional mouse hybridoma technology was utilized to produce a panel of monoclonal antibodies which reacted with baculovirus proteins. Using an enzyme-linked immunosorbent assay (ELISA), the hybridomas which were raised against polyhedrin from Autographa californica nuclear polyhedrosis virus (AcNPV) and Choristoeura fumiferana nuclear polyhedrosis virus (CfNPV) were found to cross-react differentially with polyhedrins and granulins from several species of baculoviruses. Hybridoma antibodies which reacted against the nonoccluded form (NOV) of AcNPV in an ELISA test expressed different specificities for the occluded form of the virus (OV), a mutant strain of AcNPV, and CfNPV. Four hybridoma clones produced antibody which neutralized the infectivity of AcNPV NOV. One hybridoma antibody reacted strongly with the uninfected Spodoptera frugiperda host cell line. Using Western blot analysis, it was shown that hybridoma antibodies against polyhedrin reacted differentially with the complete polypeptide and protease-generated fragments of polyhedrin. The polypeptide specificity of 19 of 28 hybridoma antibodies which reacted with OV and NOV of AcNPV was assigned using Western blot analysis.

Intestinal colonization and virulence of Salmonella in mice.

Within 3 h after oral challenge of mice with Salmonella typhimurium, foci of infection developed in the Peyer's patches of the small intestine. The numbers of organisms in the cecum, although in excess of those found in the small intestine, were not firmly associated with the cecal wall but were present largely in the cecum's contents. The Peyer's patches at first were remarkably incapable of eliminating even small numbers of Salmonella, but at about 7 days after infection developed the ability to eliminate a less virulent strain of S. typhimurium. Selected strains of Salmonella of varied virulence, and hybrid Escherichia coli/Salmonella typhimurium with varied O-antigens, revealed that those of low virulence could multiply within the intestinal Peyer's patches at nearly the same rate as a virulent strain, and the ability to multiply within the Peyer's patches was not dependent upon O-antigen type or smooth lipopolysaccharide. The ability of these strains to adhere to intestinal mucosa in vitro did not reflect on their ability to colonize the Peyer's patches, although strains of high in vitro adhesive ability appeared in greater numbers initially after oral challenge. Anti-O serum, ineffective in reducing the in vitro adhesive ability of virulent S. typhimurium, when given with the oral challenge prevented Peyer's patch colonization but was unable to prevent the appearance of a systemic infection. Anti-H serum, although effective in vitro in preventing adherence, had no effect in vivo. These experiments suggest that adhesiveness is neither essential nor sufficient for the virulence of Salmonella and that the usual development of a systemic infection after colonization of the small intestinal Peyer's patches may be subverted by the presence of O-antibody.

Immunity to Escherichia coli in pigs: adhesion of enteropathogenic Escherichia coli to isolated intestinal epithelial cells.

A method was developed to test for the ability of Escherichia coli to adhere to isolated intestinal epithelial cells. Of the E. coli tested, those having either K88ac or K88ab antigens adhered to the cells, and those which did not have these antigens did not. Since some enteropathogenic E. coli did not have the ability to adhere, it is assumed that adherence is not an essential factor of pathogenesis but rather should be considered an enhancement to the pathogenicity of some E. coli. None of the E. coli enteropathogens of cattle tested adhered to either pig or cattle cells. Similarly, human strains did not adhere to pig cells. Although the test system may not have been ideal for human or bovine E. coli, the results reported here suggest that adhesiveness is a property limited to porcine enteropathogenic E. coli carrying one of the K88 antigens. Adhesiveness is associated with the K88c or K88b antigens, and their adhesive ability is only neutralizable by the homologous antisera.