Induction of delayed hypersensitivity to human tumor cells with a human monoclonal anti-idiotypic antibody.

There is considerable interest in the development of anti-idiotypic antibodies as vaccines in a number of diseases, including cancer. We have developed a human anti-idiotypic monoclonal antibody (105AD7) which binds at or very near to the binding site of mouse antitumor monoclonal antibody 791T/36. The 791T/36 antibody binds to a tumor-associated antigen (gp72) expressed on a number of human tumors, including colorectal and ovarian carcinomas and osteogenic sarcoma. This study shows that, in rats and mice, 105AD7 induces delayed-type hypersensitivity to human tumor cells bearing the gp72 antigen. Local transfer of delayed hypersensitivity was also demonstrated using lymphocytes from mice primed with 105AD7. These findings show that the human monoclonal anti-idiotypic antibody 105AD7 is likely to induce cellular immune responses to tumors in cancer patients.

Monoclonal antibodies reacting with normal rat liver cells as probes in hepatocarcinogenesis.

A series of four monoclonal antibodies was raised against suspensions of normal adult WAB/Not rat hepatocytes. An immunoperoxidase-staining technique was used to examine the distribution of determinants detected by these antibodies on frozen sections of fetal, neonatal, adult, and regenerating liver and on a range of 4-dimethylaminoazobenzene-induced liver lesions, including a panel of 32 primary liver carcinomas. Three of the antibodies were directed against hepatocytes, while a fourth antibody stained stromal elements within the liver. The determinants detected by the anti-hepatocyte monoclonal antibodies arose in a specific sequence during normal liver development and, when assessed in conjunction, characterized several phenotypes associated with stages in normal hepatocyte differentiation. These same antibody-defined phenotypes were expressed by the primary liver carcinomas, and the distribution of phenotypes among the tumors revealed a heterogeneity which was not evident from a conventional morphological classification. Primary liver tumors expressed a total of four antibody-defined phenotypes, whereas gamma-glutamyl transpeptidase-positive foci of hepatocytes and neoplastic nodules expressed, respectively, only one or 2 antibody-defined phenotypes. We suggest that monoclonal antibodies directed against normal liver cell components may provide a means to establish lineage relationships between cell populations involved in hepatocarcinogenesis.

Antitumor immune response and interleukin 2 production induced in colorectal cancer patients by immunization with human monoclonal anti-idiotypic antibody.

The immunogenicity of human anti-idiotypic antibody has been investigated using a human monoclonal anti-idiotypic antibody (105AD7) which interacts with the binding site of 791T/36, a mouse monoclonal antibody against gp72 antigen. This antigen is frequently expressed in gastrointestinal cancer, therefore, six patients with advanced colorectal cancer have been immunized with 105AD7 as an aluminum hydroxide gel precipitate in a phase I clinical study. Cryopreserved blood mononuclear cells were tested for in vitro proliferative responses by [3H]thymidine incorporation; plasma samples were tested by enzyme-linked immunosorbent assay for anti-anti-idiotypic and antitumor antibodies, and for interleukin 2. Proliferative responses to gp72 positive tumor cells were seen in four of five patients tested; parallel in vitro responses to 105AD7 anti-idiotypic antibody were seen in most of these patients. Interleukin 2 was detected in the plasma of four of six patients after 105AD7 immunization, with peak levels up to 7 units/ml. No toxicity related to anti-idiotype immunization and no antitumor or anti-anti-idiotype antibodies were seen. This study shows that human monoclonal anti-idiotype 105AD7 is immunogenic in cancer patients, inducing cellular antitumor responses and interleukin 2 production. This suggests that human monoclonal anti-idiotype antibodies may have considerable potential for immunotherapy of human cancer.

Flow cytometry in diagnosis and management of large fetomaternal haemorrhage.

AIMS: To evaluate an indirect immunofluorescence flow cytometry technique in a series of patients with large fetomaternal haemorrhage (FMH). METHODS: Patient samples identified by Kleihauer testing in local laboratories as having FMH > 4 ml were sent for flow cytometric analysis. In a proportion of cases the mothers received anti-D immunoglobulin prophylaxis according to the flow cytometer estimate of FMH volume. RESULTS: Forty three cases of FMH were studied prospectively. The correlation between Kleihauer and flow cytometry results was poor. In 38 (88%) cases the size of FMH quantitated by flow cytometry was lower than that estimated using the Kleihauer technique. In 13 (30%) cases no Rh D immunoglobulin positive cells were detected by flow cytometry. Centralised review of the original Kleihauer films using a calibrated microscope resulted in improved, but still suboptimal correlation with flow cytometry results. In 15 cases anti-D immunoglobulin was given according to the flow cytometer estimation of FMH size, resulting in a 58% reduction in the amount of anti-D immunoglobulin given. None of the patients were immunised when tested six months later. CONCLUSIONS: Flow cytometry is helpful for the accurate quantitation and management of patients with large FMH and in cases where the presence of maternal haemoglobin F containing cells renders the Kleihauer technique inaccurate. Worthwhile reductions in the use of anti-D immunoglobulin can be achieved.

Regulation of the contact sensitivity response to urushiol with anti-urushiol monoclonal antibody ALG 991.

The objective of the studies was to demonstrate that the contact sensitivity (CS) response to poison ivy/oak could be downregulated following treatment with a monoclonal antibody (mAb) reacting with the allergen urushiol. Conjugation of urushiol and its synthetic analogue 3-n-pentadecylcatechol (PDC) to N-acetylcysteine yielded hydrosoluble derivatives which induced humoral immune responses in BALB/c mice. Hybridomas secreting monoclonal antibodies (mAbs) reacting with urushiol and PDC were generated by fusion of B lymphocytes from immunized mice with mouse myeloma P3NS0 cells. The specificity of mAb ALG 991 (IgM isotype) was defined by inhibition of antibody binding by PDC analogues. This demonstrated that mAb ALG 991 reacted with the catechol moiety of urushiol, the region of the allergen being critically important in the induction of contact dermatitis. The CS response to urushiol in BALB/c mice was suppressed by stimulation with mAb ALG 991 and the role of sensitized T cells, including suppressor T cells, has been considered. Suppression of CS was most effective with low doses (1 microg) of mAb incorporated into a vaccine with Freund's adjuvant. This treatment suppressed CS responses in BALB/c mice already sensitized to urushiol.

The role of blood services and regulatory bodies in stem cell transplantation.

Advances in stem cell research over the past few decades have coincided with large increases in haemopoietic stem cell transplantation (HSCT) using either bone marrow, peripheral blood or cord blood-derived stem cells. Alongside this growth has come an increase in the role played by regulatory bodies, both governmental and professional, to ensure that those undertaking such procedures do so in a manner so as to minimize the risk to patients. Interestingly, government legislation encompasses not only cellular therapies, but also the use of tissues and organs, as many of the processes and procurement procedures involved are similar. In this review, we analyse the trends in HSCT, describe the development and impact of legislation within Europe on this practice and outline the vital role played by the UK blood services in providing robust and high-quality HSCT services.

Evidence for the homeostatic regulation of induced beta cell mass expansion.

AIMS/HYPOTHESIS: Diabetes results from an insufficient insulin-secreting beta cell mass. Restoration of beta cell mass through pharmaceutically induced endogenous beta cell mass expansion may revolutionise diabetes therapy. However, it remains to be determined whether the induced beta cell mass expansion is under homeostatic regulation. METHODS: Beta cell mass expansion rates were derived from three separate studies of continuous stimulation of islet neogenesis, including the partial duct obstruction of euglycaemic Syrian hamsters, administration of a pentadecapeptide with the same amino acid sequence as residues 104-118 of islet neogenesis-associated protein (INGAP(104-118)) to euglycaemic Syrian hamsters, as well as to euglycaemic CD-1 mice. The incidence of islet neogenesis, average beta cell size, and beta cell replication and apoptotic rates were determined. RESULTS: Partial duct obstruction led to a approximately 2.5-fold increase in endocrine tissue at day 56 (p<0.05). From day 0 to day 7 the average rate of change of islet area was 12.7% per day, and this rate decreased to 5.3% per day from day 7 to day 42, and to 2.8% per day from day 42 to day 56. Administration of INGAP(104-118) to adult hamsters led to a 31% increase in total beta cell mass at day 30 (p=0.031). From day 0 to day 10 the average rate of beta cell mass expansion was 148 mug/day, whereas from day 10 to day 30 it decreased to 45 mug/day. INGAP(104-118) administration to adult CD-1 mice resulted in an approximately twofold increase in beta cell mass after 31 days (p=0.021). However, at day 90, there was no significant difference vs age-matched control mice (p=0.30), even though the neogenic beta cell mass was approximately fourfold greater (p=0.026). Beta cell replication was decreased by 56% (p<0.048), whereas beta cell apoptosis was fourfold greater (p<0.003) in 90-day INGAP(104-118)-treated mice compared with age-matched control mice. CONCLUSIONS/INTERPRETATION: These data indicate that in the presence of ongoing islet neogenesis, homeostatic regulatory mechanisms intervene to regulate beta cell mass according to the prevailing metabolic requirements.

Anti-D quantification by flow cytometry:a comparison of five methods.

UNLABELLED: BACKGROUND Three flow cytometric methods for anti-D quantification have been published. All use different cell sensitization and antibody detection conditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely. STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in-house modifications were compared. Ten serum samples containing anti-D at levels between 11.6 and 915 IU per mL were selected for analysis, and each was tested a minimum of three times. Anti-D bound to cells was detected with fluorescence-labeled anti-human IgG reagents. RESULTS The interassay CV of the standard curves for each of the five methods was less than 10 percent. The intra-assay CV was consistently <10 percent with four out of the five methods, but, by the fifth method, it was >20 percent in more than one-third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the mean anti-D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods. CONCLUSION: Anti-D quantification by flow cytometry is influenced by the serum antibody characteristics and the method used. The differences between the flow cytometric and AutoAnalyzer techniques indicate that further validation of the flow cytometric method is required before routine use.

Anti-D quantification by flow cytometry: an alternative to the AutoAnalyser?

A flow cytometry method for quantifying levels of anti-D is described. The method is based on the indirect antiglobulin test and uses an FITC Fab anti-human IgG reagent for detection of bound anti-D. A standard curve is generated from the fluorescence measured by flow cytometry using the British Standard anti-D preparation 73/515. The fluorescence obtained from test samples by flow cytometry is converted into IU/mL anti-D, using the standard curve. In a limited study with 42 serum samples the assay showed good correlation with results obtained using the AutoAnalyser for sera with anti-D levels less than 100 IU/mL. The method is simple to perform and has potential as a replacement for the AutoAnalyser.

Comparative studies of the metastatic potential of three transplantable rat mammary carcinomas of spontaneous origin.

The metastatic potential of 3 spontaneously arising mammary carcinomas (Sp4, Sp15 and Sp22) has been examined when transplanted in the form of a viable cell suspension into the tissue of origin. Primary tumours were excised at different times after implantation and it was found that the metastatic potential of the immunogenic tumour Sp4 was directly correlated with the size of the primary tumour when excised. By contrast, the incidence of metastases from the non-immunogenic tumours Sp15 and Sp22 was similar irrespective of the size of the primary tumour on excision. The pattern of metastasis also differed between the tumours, although here there was no relation to immunogenicity. Thus, resection en bloc of large primary Sp4 or Sp15 tumours plus regional lymph nodes could be completely curative, signifying initial spread of tumours via the lymphatics and only subsequently via the blood stream. On the other hand, resection en bloc of primary Sp22 tumours plus regional lymph nodes at a similar stage of primary tumour development was never curative, signifying early spread via the blood stream. Other studies showed that the metastatic potential of mammary carcinoma Sp4 was an innate characteristic of the tumour and not related to the tissue of implantation since in addition to metastasizing from the mammary pad it metastasized when implanted either s.c. or intradermally in a region devoid of mammary tissue. Furthermore, a rat sarcoma Mc7 showed a negligible tendency to metastasize when implanted either in the mammary pad or in the s.c. tissue, where it had been induced with methylcholanthrene.

An anti-idiotopic antibody-based enzyme-linked immunosorbent assay for the quantification of the monoclonal anti-D BRAD-5.

OBJECTIVES: We aimed to develop an anti-idiotopic antibody-based enzyme-linked immunosorbent assay (ELISA) to quantify human monoclonal anti-D using BRAD-5 as a model system. MATERIALS AND METHODS: One of the anti-idiotopic antibodies 2E6 was used to capture BRAD-5 with the other anti-idiotopic antibody 3B1 biotinylated for detection. RESULTS: The assay developed can detect BRAD-5 at < 2 ng/ml. Assay interference caused by heterophilic antibodies in some human sera was removed by preincubation with bovine serum. The assay is reproducible with intra- and inter-assay coefficient of variation (CV) < 10%. CONCLUSION: This ELISA should prove of benefit in developing a monoclonal anti-D for prophylactic use.

Murine monoclonal antibodies reactive with a human monoclonal anti-RhD antibody (BRAD-5).

BRAD-3 and BRAD-5 are human monoclonal antibodies that recognize the RhD antigen on red blood cells. Both antibodies are currently in clinical trials for use as a replacement for polyclonal anti-RhD in the prophylactic treatment of haemolytic disease of the newborn. We have produced three murine IgG1 antibodies that cause agglutination of cells sensitized with BRAD-5 and also block binding of BRAD-5 to its target antigen. Using a haemagglutination assay, these antibodies, 1D7, 2E6 and 3B1, have shown specificity for BRAD-5 as they did not bind to other monoclonal anti-RhD antibodies of differing specificity or derived from other donors. This assay has also been used to show a lack of reactivity with anti-RhD antibodies present in 198 human serum samples from 44 anti-RhD immune individuals. The three anti-BRAD-5 antibodies have been shown to recognize different epitopes on the BRAD-5 molecule using a blocking ELISA. These antibodies appear to recognize private idiotopes on BRAD-5 that were not detectable in RhD immune sera, and therefore they will be of use for monitoring BRAD-5 in clinical trials.

Induction of cellular immune responses by a murine monoclonal anti-idiotypic antibody recognizing the 791Tgp72 antigen expressed on colorectal, gastric and ovarian human tumours.

There is accumulating evidence that cellular rather than antibody responses are more effective for tumour rejection. It is therefore important to screen anti-idiotypic (anti-id) antibodies for their ability to stimulate anti-tumour T-cell responses. The human anti-id monoclonal antibody (MAb) 105AD7 stimulated both delayed-type hypersensitivity (DTH) responses in animals and antigen-specific blastogenesis and IL-2 induction in advanced cancer patients. It may not be necessary to use human anti-id antibodies as murine anti-id antibodies, which elicit DTH responses against immunodominant human T-cell epitopes and may be just as useful in the clinic. We have therefore produced a murine anti-id antibody to the same MAb as was used to generate the human anti-id antibody and screened it for its ability to generate cellular anti-tumour immune responses. Low-dose immunization with the murine anti-id MAb NCRC60, which recognises the paratope of the anti-791Tgp72 MAb 791T/36, induced DTH responses to 791Tgp72-expressing tumour cells but not to antigen-negative cells. DTH responses with no detectable antibody responses were induced with 5 micrograms of anti-id NCRC60 without adjuvant. Addition of either complete Freund's adjuvant or Quil A did not enhance DTH responses. However, when the anti-id NCRC60 was linked to KLH and injected in the presence of Freund's adjuvant anti-anti-id antibodies and anti-791Tgp72 antibodies were induced. NCRC60 anti-id was also capable in vitro of priming human T cells from cancer patients to proliferate in response to secondary stimulation with 791Tgp72-expressing tumour cells, suggesting that it may have therapeutic potential in cancer patients.

Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry.

The use of flow cytometry for quantifying fetomaternal haemorrhage is increasing, and has been shown to be more accurate than the Kleihauer-Betke test for evaluating larger bleeds of over 4 mL in volume. Red cells are stained with fluorescently labelled monoclonal anti-D. Cells for analysis are normally gated manually on the basis of forward and side scatter. We investigated whether the use of an antiglycophorin A monoclonal antibody conjugate (red cell specific) in a dual labelling technique would improve the gating of RBC and FMH quantification. Mixes of adult rr and cord R1r RBC were prepared to simulate 1, 0.5, 0.25, 0.12 and 0.06% fetal bleeds. Phycoerythrin-conjugated BRIC 256 (mouse monoclonal antiglycophorin A) was used to label all RBC, and FITC-BRAD-3 monoclonal anti-D was used to determine the proportion of D-positive cells. Results from the dual labelling experiments were compared to those from single labelling of the same mixtures with FITC-BRAD-3 alone, using gated and ungated data. The results showed that single labelling with manual gating gave falsely low FMH estimates. We conclude that use of a fluorescently labelled antiglycophorin A antibody improves the accuracy of the FMH measurement by flow cytometry, as manual subjective gating of RBC excludes a higher proportion of fetal than of adult RBC.

Flow cytometric analysis of proflavine uptake into normal rat hepatocytes and cells arising during AAF-induced hepatocarcinogenesis.

The uptake of the fluorescent drug proflavine was measured in suspensions of hepatocytes from normal and carcinogen (2-acetylaminofluorine, AAF)-fed rats by flow cytometry. Drug uptake into hepatocytes from carcinogen-fed animals was consistently lower than that into hepatocytes from normal animals. Isolated nuclei, prepared from the livers of normal and AAF-fed rats showed similar proflavine uptake. Drug uptake into hepatocytes from AAF-fed animals, however, was increased by prior exposure to a metabolic inhibitor. Thus, differences in drug uptake may reflect changes in the cell membrane, together with an alteration in the metabolic integrity of the cells. The uptake of drug in hepatocytes from AAF-fed rats was uniformly low within each cell preparation. However, drug uptake varied not only between tumours arising in the livers of these animals but also within each tumour cell preparation. This study indicates that flow cytometry can provide an effective means for analysing drug uptake into cell populations arising during hepatocarcinogenesis.

Expression of a monoclonal antibody-defined liver-associated antigen in normal rat hepatocytes and hepatocellular carcinoma cells.

In order to examine changes in the expression of normal cellular antigens during hepatocarcinogenesis, we have developed and characterized a monoclonal antibody detecting an antigen which is particularly associated with adult rat hepatocytes. The antibody showed no reactivity with a range of freshly-prepared normal syngeneic adult rat cell types, but showed strong reactivity with hepatocytes derived from several rat strains in antibody binding tests. Immunoperoxidase staining of sections of normal rat liver showed that the antigen was distributed throughout hepatocytes within the liver, but did not stain cells other than hepatocytes. The antigen detected by this monoclonal antibody was not detectable on a number of transplanted and primary dimethylaminoazobenzene-induced hepatomas. Preliminary data indicated that this monoclonal antibody may be suitable for use in flow cytofluorimetric sorting of mixed populations of normal and tumour cells, allowing the investigation of cell phenotypes during hepatocarcinogenesis.

IgG anti-Jka/Jkb antibodies are unlikely to fix complement.

Antibodies in the Kidd blood group system show a great deal of serological variability, are notoriously elusive and hence evoke difficulties in detection. However, they have been regularly reported as causing severe immediate or delayed haemolytic transfusion reactions and this clinical potential has been largely attributed to their complement binding ability. In initial investigations on 43 anti-Jka/Jkb sera with a range of titres of IgG antibody only a few seemed to fix complement, though following repeated tests on 20 of these sera a further five were shown to bind complement, making a total of 12 (27.9%) showing evidence of complement binding. Twenty-three sera were unavailable for re-testing. Subsequent tests revealed that only those sera which showed direct agglutination or were positive with an anti-IgM reagent in an indirect antiglobulin test (IAT) fixed complement. Evaluations on the IgG fractions of six selected potent anti-Jka sera failed to reveal any complement-fixing ability although all the original sera bound complement avidly and contained variable amounts of IgM antibody, some at very low subagglutinating levels. These findings challenge past perceptions and give cause for reflection on the changing methodologies and strategies which could unduly compromise the detection of these potentially clinically damaging antibodies.

Patterns of reactivity of the monoclonal antibody 791T/36 with different tumour metastases in the liver.

Reactivity of the monoclonal antibody 791T/36 with secondary malignant deposits has been investigated in frozen sections of 74 human liver biopsy specimens. There was no reactivity with hepatocytes but in some instances binding to fibrous tissues and in one case to portal tract lymphocytes was observed. Sections from 9 biopsy specimens contained malignant deposits. In seven of these 791T/36 bound either to malignant cells or to pseudoacinar contents (3 colorectal adenocarcinomas; 1 probable pancreatic adenocarcinoma; 1 medullary cell carcinoma of thyroid; 1 oat cell carcinoma of bronchus and 1 deposit of nodular sclerosing Hodgkins Disease). Two undifferentiated tumours (1 gastric adenocarcinoma and 1 oat cell bronchial carcinoma) showed no antibody binding. The histological pattern of reactivity previously reported with primary tumours appears to be similar in secondary deposits. A wider range of tumours than recognised hitherto binds 791T/36. Whether the binding to fibrous tissue seen in some instances is sufficient to cause diagnostic uncertainty when 791T/36 is used for scanning requires further investigation.

Comparison of flow cytometric assays with isotopic assays of (51)chromium-labeled cells for estimation of red cell clearance or survival in vivo.

BACKGROUND: A comparison was made between flow cytometric and conventional radioisotopic assays in the determination of the clearance or survival of small volumes of (51)chromium-labeled D+ red cells after injection into volunteers. STUDY DESIGN AND METHODS: Four clearance studies were performed using 4 mL of autologous D+ cells coated with anti-D at two concentrations (5 or 10 microg anti-D/mL red cells) transfused to two subjects at separate times. Five survival studies were carried out using 5 mL of frozen-thawed D+ cells transfused to five D- subjects with no detectable anti-D. Sequential blood samples were taken for gamma counting and flow cytometry. Several methods were used to stain the transfused red cells, and the data were analyzed by using three flow cytometers. RESULTS: The determination of red cell clearance or survival by radioactivity measurements gave results consistent with published data. However, none of the flow cytometric assays exhibited the necessary sensitivity or accuracy in quantitation of the rare events to provide reliable data for the calculation of the initial clearance rate, the red cell half-life, or the mean cell lifespan, although rough estimates of red cell clearance were obtained in some subjects. This inability to accurately enumerate rare fluorescence-labeled cells was due mainly to the presence of "background" events, which were a considerable problem in some samples, when the coating level of anti-D was less than 3000 molecules of IgG per cell. CONCLUSION: Flow cytometry may enable the crude estimation of the percentage of small volumes (<5 mL) of transfused D+ red cells, but in this study it was found that this method was not sufficiently accurate to determine the initial clearance rate, red cell half-life, or mean cell lifespan. If the proportion of transfused cells in the recipient is about 0.2 percent or less, the use of radioisotopes for labeling cells for quantitative in vivo red cell clearance or survival data should remain the method of choice.