Zenon Steplewski

Colorectal carcinoma antigens detected by hybridoma antibodies

Hybridoma cells which secrete colorectal carcinoma-specific antibodies have been produced and used to study the antigenic structure of these tumor cells. Nineteen antibodies have been studied in detail, and 15 of these are colorectal carcinoma specific. Only two antibodies reactive with carcinoembryonic antigen (CEA) have been discovered and five other antibodies that react with distinct epitopes on the cell surface have been defined. Several antigens with distinct molecular characteristics have been shown to exist by use of hybridoma antibodies. Six hybridoma antibodies have been shown to mediate antibody-dependent cell-mediated cytotoxicity (ADCC).

PHASE-I CLINICAL TRIAL OF MONOCLONAL ANTIBODY IN TREATMENT OF GASTROINTESTINAL TUMOURS

A phase-I clinical trial of a murine monoclonal antibody that specifically suppresses growth of human gastrointestinal tumours in athymic mice was conducted in four patients, who were given 15-200 mg purified antibody. The monoclonal antibody persisted in the circulation for more than a week when more than 15 mg was given. Antibodies against mouse immunoglobulin developed in three of the four patients. In one patient who received autologous mononuclear cells that had been mixed with monoclonal antibody by way of a hepatic-artery catheter, hepatic metastases became smaller and their echogenic characteristics changed, and there was heavier monocyte infiltration in the histological appearance of a resected metastasis.

Monoclonal antibody detection of a circulating tumor-associated antigen. I. Presence of antigen in sera of patients with colorectal, gastric, and pancreatic carcinoma

Hybridoma-secreted monoclonal anti-colorectal carcinoma antibodies 19-9, 52a, and C4 14 bind specifically to cells of colorectal, gastric, and pancreatic carcinoma in tissue culture. The assay for the detection of antigen in human sera is based on the inhibition of binding of monoclonal antibodies to target preparations of colorectal carcinoma cells. Binding of monoclonal antibody 52a was inhibited more than 12% by 163 of 255 sera from patients from various stages of carcinoma of colon and rectum, by 45 of 49 sera from patients with pancreatic carcinoma, and by 8 of 11 sera from patients with gastric carcinoma. By contrast, only 7 of 89 sera from patients with other malignancies and 2 of 108 sera from healthy donors inhibited binding of this monoclonal antibody by more than 12%. These studies show the potential usefulness of monoclonal antibodies in the diagnosis of human malignancy.

Malignant astrocytomas treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor receptor: A phase II trial☆

Twenty-five patients with primary presentation of malignant astrocytoma, astrocytoma with anaplastic foci, and glioblastoma multiforme were treated with surgical resection and definitive radiation therapy followed by intravenous or intra-arterial administration of Iodine-125 labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The patients presented with primary untreated disease, positive contrast enhanced computed tomography scans of the brain, and compatible clinical symptoms. In this Phase II clinical trial, the patients had surgical debulking or biopsy followed by definitively administered external beam radiation therapy and one or multiple doses (35 to 90 mCi per infusion) of radiolabeled antibody. The total cumulative doses ranged from 40 to 224 mCi. The administrations of the radiolabeled antibody were performed in most cases 4-6 weeks following completion of the primary surgery and radiation therapy. Ten patients had astrocytoma with anaplastic foci and 15 had glioblastoma multiforme. No significant life-threatening toxicities were observed during this trial. At 1 year 60% of the patients with astrocytoma with anaplastic foci or glioblastoma multiforme are alive. The median survival for both groups was 15.6 months.

Monoclonal antibodies directed against the sugar sequence of lacto-N-fucopentaose III are obtained from mice immunized with human tumors

Abstract Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III, but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues.

Production of monoclonal antibodies in serum free medium

We have succeeded in growing several established hybridoma cell lines (1116NS-19; 116NS-33a; 1116NS-52a; 1083-17-1A; 691-19-19) in a serum free medium supplemented with physiological concentrations of insulin (5 microgram/ml) and transferrin (5 microgram/ml). The hybridoma cells replicate in this medium and will secrete monoclonal antibodies in quantities comparable to those produced in the presence of serum. Adapted hybridoma cultures have been secreting antibodies in the supplemented serum free (SSF) medium for more than 3 months. Parental mouse myeloma P3x63Ag8 cells and non-secreting subclone 653 cells cannot survive longer than 6 days under the same conditions. Both insulin and transferrin are necessary for hybridoma cells to grow in serum free medium and omission of either one will not permit cell multiplication.

Vaccination with carbohydrate peptide mimotopes promotes anti-tumor responses

Tumor-associated carbohydrate (TAC) antigens are important targets in cancer vaccine efforts. Carbohydrates are, however, frequently poor immunogens, in that they are T-cell–independent antigens. Molecular mimicry of TAC by peptides is an alternative approach to generating anti-carbohydrate immune responses. Here we demonstrate that peptide mimotopes can elicit antibody responses that cross-react with representative human TAC antigens. Primary immunization with such a multiple antigenic peptide, along with QS-21 as adjuvant, elicits cytotoxic antibodies reactive with naturally occurring forms of TAC expressed on tumor cells, and vaccination of mice with peptide mimotopes reduced tumor growth and prolonged host survival in a murine tumor model.

Monoclonal Antibody Detection of a Circulating Tumor- Associated Antigen. II, A Longitudinal Evaluation of Patients with Colorectal Cancer

An antigen identified by two monoclonal anti-colorectal cancer antibodies was studied in sera of 85 patients who had a resection of their primary colorectal cancer. Preoperative and postoperative serum samples and sera collected every 3 months for at least 1 year were included in this study. The levels of these antigens were compared to the carcinoembryonic antigen (CEA) levels. Sixty-six patients had at least one antigen elevated in the preoperative period. Malignancy recurred in 10 patients. In 8 of these the recurrence could have been predicted by the persistence or rise in antigen levels 3 to 18 months prior to the detection of the recurrence by current clinical methods. The data suggest that the assays for these antigens are valuable prognostic aids for making clinical therapeutic decisions and appropriately stratifying patients for clinical trials.

Production and characterization of monoclonal antibodies against human malignant melanoma.

The specific immunoreactivities of 31 monoclonal antibodies against human malignant melanoma were analyzed on a variety of malignant and nonmalignant human cells. Seven distinct groups were defined based on reactivity in radioimmunoassay and in mixed hemadsorption assay. The Group A antibody bound to 33% of short- and long-term cultured melanomas; Group B antibodies reacted with the majority of melanomas, astrocytomas, neuroblastomas, and fetal polygonal cells; and Group C antibodies bound to melanomas, teratocarcinomas, and to melanocytes grown in the presence of tumor-promoting phorbol esters. Antibodies of Groups D-G showed a less restricted binding pattern. In all groups, antibodies of IgG2b and IgM isotypes mediated complement-dependent lysis (CDC) and antibodies of IgG1, IgG2a, and IgG2b isotypes mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Biochemical analysis indicated that 16 different proteins with molecular weights ranging between 28,000 and 500,000 were detected by the monoclonal antimelanoma antibodies.

GD2 ganglioside biosynthesis is a distinct biochemical event in human melanoma tumor progression

Gangliosides from cell cultures established from melanocytic lesions, representing different stages of melanoma tumor progression, were analyzed by chemical and immunological means on thin‐layer chromatograms. The GD2 ganglioside and N‐acetylgalactosaminyl transferase, which catalyzes the biosynthesis of GD2 from its precursor GD3, were detected in cultures established from advanced primary and metastatic melanomas, but not in cultures of normal melanocytes. Immunohistochemical studies on tissue sections from all progression stages confirmed GD2 expression only in these advanced lesions. A distinct biochemical event thus coincides with the onset of faster growth and acquisition of metastatic competence in human melanoma tumor progression.