Search

Purpose: To attempt the isolation and primary culture of prostate tumor cells, to use the cultured cells for active immunotherapy, and to evaluate the safety and efficacy in a Phase I clinical trial., Materials and Methods: Tumor fragments were collected from 50 patients with prostate-specific antigen (PSA) > or = 10 ng/mL, < or = cT2 PCa who underwent radical retropubic prostatectomy (RRP) and 6 patients with metastatic PCa who underwent transurethral resection of the prostate (TURP). Cultured tumor cells were incubated with IFN-fi, irradiated, and cryopreserved. Seven vaccine inoculations were performed into > or = pT3 and/or N+ patients, and M+ patients, with the first two doses admixed with Bacille Calmette-Guerin (BCG). Follow-up was performed with measurement of delayed-type hypersensitivity (DTH) reactions, PSA and hemato-chemical tests, and bone scans., Results: No cell culture was obtained in the TURP group. Cell culture and vaccine production were obtained in 37 cases (74%) in the RRP group. Eleven > or = pT3 and/or N+ patients were vaccinated. Toxicity was generally limited to the inoculation sites. DTH reactions > or = 10 mm were observed in 2 patients and > or = 5 mm in 6 patients. Two patients had a decrease in PSA levels after vaccine administration., Conclusions: The autologous cell vaccine is safe and seems to induce a positive immune cellular response. Primary cell culture and vaccine production can be obtained for most RRP patients, but not for TURP patients using our method. There seems to be some influence of the vaccine in PSA evolution after RRP.

Gross inequities in disease burden between developed and developing countries are now the subject of intense global attention. Public and private donors have marshaled resources and created organizational structures to accelerate the development of new health products and to procure and distribute drugs and vaccines for the poor. Despite these encouraging efforts directed primarily from and funded by industrialized countries, sufficiency and sustainability remain enormous challenges because of the sheer magnitude of the problem. Here we highlight a complementary and increasingly important means to improve health equity: the growing ability of some developing countries to undertake health innovation.

The relative affinities of a panel of antibodies submitted to the ISOBM TD-3 PSA Workshop were determined by direct-binding ELISA. The Workshop antibodies were also tested for reactivity with the prostate-specific antigen alpha1-antichymotrypsin complex (PSA-ACT) and cross-reactivity with porcine pancreatic kallikrein. There was a wide range of affinities observed for the panel of antibodies. Twelve antibodies failed to react with the PSA-ACT complex, and 1 antibody was found to recognize an epitope on ACT but not on PSA. Only 2 antibodies were found to react with porcine pancreatic kallikrein, a protein with 64% sequence homology with human glandular kallikrein-2 (hK2).

Twelve research groups participated in the ISOBM TD-3 Workshop in which the reactivity and specificity of 83 antibodies against prostate-specific antigen (PSA) were investigated. Using a variety of techniques including cross-inhibition assays, Western blotting, BIAcore, immunoradiometric assays and immunohistochemistry, the antibodies were categorized into six major groups which formed the basis for mapping onto two- and three-dimensional (2-D and 3-D) models of PSA. The overall findings of the TD-3 Workshop are summarized in this report. In agreement with all participating groups, three main antigenic domains were identified: free PSA-specific epitopes located in or close to amino acids 86-91; discontinuous epitopes specific for PSA without human kallikrein (hK2) cross-reactivity located at or close to amino acids 158-163; and continuous or linear epitopes shared between PSA and hK2 located close to amino acids 3-11. In addition, several minor and partly overlapping domains were also identified. Clearly, the characterization of antibodies from this workshop and the location of their epitopes on the 3-D model of PSA illustrate the importance of selecting appropriate antibody pairs for use in immunoassays. It is hoped that these findings and the epitope nomenclature described in this TD-3 Workshop are used as a standard for future evaluation of anti-PSA antibodies.

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid-hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid-hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.

Bispecific monoclonal antibodies (bsMAbs) are unique molecules incorporating two different paratopes in a single antibody molecule. BsMAbs can be useful in different areas of research as well in clinical applications. Traditionally, bsMAbs are produced by hybrid-hybridomas that are generated by the fusion of two pre-established hybridomas. The development of such hybrid-hybridomas can be difficult and time-consuming. Here, we introduce a new technique to generate such hybrids, electro-FACS-fusion. In this procedure, before the electrofusion, one of the hybridomas is labeled with fluorescein isothiocyanate (FITC) and the other with tetramethylrhodamine isothiocyanate (TRITC). The mixture of cells is then electrofused, and cells exhibiting dual fluorescence are selected by fluorescence activated cell sorting (FACS). The fused cells are directly plated in microplates for clonal growth. Using this technique, we produced three new hybrid-hybridomas secreting bsMAb that could be used for the next generation of immunoassays.

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP-IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5-20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.

The ISOBM TD-6 Workshop is the first international workshop on monoclonal antibodies against the Sialyl Lewisa (SLea) antigen. Eight research groups participated in a blind study to characterize the epitope binding, relative affinity and performance in immunoradiometric assays, of a panel of 20 monoclonal antibodies. The antibodies were tested against a diverse panel of neoglycoconjugates, purified antigens and human serum pools from gastrointestinal malignancies. Epitope specificities were determined for the majority of antibodies in the panel. Cross-reactivity with related saccharide structures was noted in several antibodies. Overall, the results of the TD-6 Workshop show further development of SLea immunoassays may yield yet more specific assays for the detection and management of gastrointestinal and other malignancies.

Prostate-specific antigen (PSA) is one of the most useful tumor markers for the screening and follow-up of prostate cancer. Bispecific monoclonal antibodies (bsMAbs) are unique immunoprobes that incorporate two different binding sites in the same antibody molecule. This antibody designing can bring important advantages in the development of new immunoassays. We have developed a new hybrid hybridoma that secretes bsMAb anti-PSA x anti-horseradish peroxidase. This bsMAb has shown rapid kinetics and an excellent detection limit in a sandwich single-step assay with a total incubation time of 15 min and a 5-min substrate development. This assay in a manual format has a detection limit of 0.028 microgram/L. Comparison with the Hybritech Tandem-E PSA assay yielded a regression equation with slope = 0.433 [95% confidence interval (CI) = 0.415-0.451], intercept = 0.88 (CI = 0.45-1.31), and Sy/x = 1.83 micrograms/L (r = 0.98). This new immunoprobe can be used to develop a new generation of assays for clinical laboratories and can be adapted to screening devices for physicians' offices and even home diagnostics.

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. We identified and characterized the epitopes of two anti-PSA monoclonal antibodies (mAbs) designated B80 and B87. The epitopes were initially mapped as nonoverlapping by developing a sandwich immunoassay to measure PSA with the two anti-PSA mAbs. The two antibodies do not cross-react with homologous pancreatic kallikrein, but recognize epitopes unique to PSA. B80 and B87 can recognize both free and complexed PSA and hence measure total PSA. Epitope scanning and bacteriophage peptide library affinity selection procedures were used to identify and locate an epitope on PSA. A possible epitope for B80 was identified as being located on or near PSA amino acid residues 50-58 (-GRH-SLFHP-). The epitope for B87 was likely on an exposed nonlinear conformational determinant, unique to PSA, and not masked by the binding of B80 or alpha 1-antichymotrypsin.

We evaluated the immunohistological (IH) characteristics of 22 different antibodies that were submitted for study in the frame of the TD-1 ISOBM Workshop on monoclonal antibodies against CA125. Information on relative affinities and epitope similarities was obtained from a parallel immunochemical study. Antibodies were tested at concentrations of 10 and 1 micrograms/ml on frozen and paraffin sections. Paraffin sections were stained according to the streptavidin-biotin complex protocol, and frozen sections according to a two-step immunoperoxidase technique. Aminoethylcarbazole served as the chromogen. The tissues were from normal proliferative endometrium (formalin-fixed paraffin-embedded) material and clear-cell adenocarcinoma of the ovary (formalin-fixed paraffin-embedded and frozen material). Sections were scored for staining in epithelial cells, basal, apical and diffuse cytoplasmic and in stromal components. Intensity was graded as 1, 2 or 3 for epithelial cells and as -1, -2 or -3 for stroma. The cumulative scores for each antibody expressed the discriminative properties of specific epithelial staining against background. M11 and M11-like antibodies, as well as OC125 and OC125-like antibodies, in general showed good staining results. Although there was a trend for high-affinity antibodies to show higher scores, there was no clear relationship between affinity and staining result. For nine antibodies (ZR45, MA602-1, K102, K94, K90, OV185, K97, K96, OV198), the reactions in paraffin and frozen sections were of similar intensity. Most of these were of low affinity with one exception: antibody ZR45, a rat monoclonal antibody (MAb) which had a high relative affinity. For eight antibodies (M11, K101, MA602-6, ZS33, B27.1, B43.13, K93, OC125), a loss of specific staining was observed in frozen sections. All but two of these antibodies (MA602-6 and OC125) were of high relative affinity. With four antibodies (K91, ZR38, K95, K100), the reverse situation was observed. One (K100) was of low affinity, two (K95 and K91) of high affinity and the fourth (ZR38) was a rat MAb of high affinity. Mainly due to the increased cytoplasmic staining in carcinoma, the reactivity in paraffin sections was less extensive in normal endometrium compared to ovarian carcinoma for the majority of antibodies, irrespective of their affinity or epitope group. The IH characterization of these antibodies may be of help in selecting antibodies with specific properties for further comparative studies. The reactivity of normal endometrium with all useful antibodies makes it a good candidate for standard external IH tissue control.

In a guinea-pig model of asthma, active immunization against substance P (SP) prevented the development of airways' hyperresponsiveness and reduced bronchospastic responses to SP (i.v.). The rat-mouse heterohybridoma NC1/34 secretes a specific, rat IgG1, anti-substance P antibody (alpha-SP Ab) which was isolated and purified by passing supernatant from cultures through thiophilic gel. Purity of antibody was about 50% (SDS-PAGE). The relative affinities of the alpha-SP Ab for SP, neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) were estimated by ELISA using a constant amount of SP coupled (glutaraldehyde) to bovine serum albumin (BSA) to capture the antibody, alone and in the presence of increasing concentrations of the neuropeptides. At alpha-SP Ab dilutions of 1 in 5,000 to 1 in 32,000, CGRP did not prevent antibody binding to SP-BSA conjugate bound to the plates, but both SP and NKA prevented binding. In this system, the relative affinity of the alpha-SP Ab, at dilutions of 1 in 5,000 and 1 in 10,000, was about 50 times greater for SP than NKA. Whether passive immunization with alpha-SP Ab prevented bronchospastic responses to SP and NKA (i.v.), in vivo, was determined in groups of anesthetized guinea-pigs by recording pulmonary flow resistance (RL) and dynamic pulmonary elastance (EL). Injection of alpha-SP Ab (i.v., 5:1 molar ratio: alpha-SP Ab:SP total dose) did not alter baseline values of RL and EL, but markedly inhibited increases in RL and EL induced by SP and NKA (i.v.) without affecting responses to methacholine (i.v.). A control, "irrelevant" rat IgG-type antibody at a similar concentration had no effect on responses to SP or NKA. These findings indicate that passive immunization with a monoclonal alpha-SP Ab can prevent the bronchospastic effects of exogenous SP and NKA in guinea-pigs.