How to overcome the disturbing effects of human anti-mouse antibodies (HAMA) on in vitro assays.

The development of human anti-mouse antibodies (HAMA) is a common immune response in patients with ovarian carcinoma after repeated injections of murine anti-CA 125 monoclonal antibodies for immunoscintigraphy. As a tumor marker with significant diagnostic value CA 125 is routinely measured in the follow-up of tumor patients by immunoradiometric assays (IRMA) based on murine anti-CA 125 monoclonal antibodies. HAMA may cause false-positive findings in a CA 125-IRMA. In this report our group demonstrates a simple way of eliminating HAMA by protein A/G affinity chromatography allowing the reliable detection of the tumor marker CA 125 in the serum of patients with ovarian carcinoma.

Reduced glutathione and S-acetylglutathione as selective apoptosis-inducing agents in cancer therapy.

The effect of reduced glutathione (GSH) and S-acetylglutathione (S-acglu) treatment on several tumor cell lines and normal cells in vitro was investigated. GSH and S-acglu applied at concentrations of 1 mM and 2 mM induced apoptosis in malignant cells as shown by DNA-fragmentation and staining of apoptotic cells with 7-amino-actinomycin D while viability and growth of normal cells were not significantly influenced by this treatment. The results demonstrated that GSH and S-acglu may be selective inducers of apoptosis in malignant cells.

Generation of human monoclonal anti-idiotypic antibodies with specificity to the murine monoclonal anti-CA 125 antibody B43.13.

Two human monoclonal antibodies, HID-7E7 and ROB-6F2, were produced by EBV transformation of peripheral blood lymphocytes (PBL). PBL were obtained from a patient with ovarian cancer who had been exposed several times to a Tc-99m labeled murine monoclonal anti-CA 125 antibody (B43.13, Biomira, Edmonton) for immunoscintigraphy. The HID-7E7 and ROB-6F2 producing B-cells were cloned with a limiting dilution technique and have shown stable immunoglobulin secretion within a period of three years. The human monoclonal antibodies HID-7E7 and ROB-6F2 are of the IgG isotype, and bind with significant affinity to the murine monoclonal antibody B43.13, which was used for immunoscintigraphy. Binding affinity of ROB-6F2 to other murine antibodies could not be detected. Cross reactivity of HID-7E7 to a murine anti-CEA monoclonal antibody was observed. In order to verify the anti-idiotypic character of the generated human antibodies, the ability of HID-7E7 and ROB-6F2, respectively, to inhibit the formation of the CA125/B43.13 complex is demonstrated via an enzyme-linked immunosorbent assay. These human anti-idiotypic antibodies are possible candidates for immunotherapy of ovarian cancer in patients with a small tumor burden following surgery and/or chemotherapy.

Immunological profile of patients with ovarian-cancer under immunostimulation with murine monoclonal-antibodies.

The longer survival of ovarian cancer patients after immunostimulation has been connected with the induction of an anti-tumor activity triggered by cellular and humoral immune responses. Our interest was to study the long-term influence on the immune system in relation to the various levels of the HAMA response and the disease stage. The immunological profile was evaluated by regularly performing phenotyping and functional analysis of peripheral blood lymphocytes (PBL). We report the statistical analysis of immunological data obtained in a study with 77 ovarian cancer patients examined over a period of up to 28 months. The results demonstrate that these immunological data are important for monitoring cancer patients under immunotherapy whereas they provide no prognostic significance.

A preliminary study on the functional analysis of peripheral blood lymphocytes from ovarian cancer patients developing HAMA after immunoscintigraphy.

In the follow-up of ovarian cancer patients, rising levels of the tumor-associated antigen CA 125 are an indication for immunoscintigraphy. Human anti-mouse antibodies (HAMA) are frequently found after immunoscintigraphy with murine MAb directed against CA 125. Since we observed that patients developing high HAMA-levels in serum remained free of tumor or had stable disease, we examined the cytotoxic activity of peripheral blood lymphocytes (PBL) by a fluorescence-based assay. Our results demonstrate that PBLs of patients with high anti-idiotypic antibodies show an increased cytotoxic activity (by a factor of 4) compared to those of patients with low HAMA levels. The clinical course of the patients after the first injection of murine monoclonal antibody was observed over a period of 1 to 3 years. Improvement or deterioration of patients' clinical condition corresponded with the results obtained by functional analysis. Further investigations concerning the course of cytotoxic activity in HAMA-positive patients will have to clarify HAMA's role in the immune response.

Activating anti-idiotypic human anti-mouse antibodies for immunotherapy of ovarian carcinoma.

Human anti-mouse antibodies (HAMA) are observed frequently after immunoscintigraphy with monoclonal antibodies (MoAb) directed against CA-125. As the authors have shown previously, HAMA can cause false-positive CA-125 values in routine CA-125 immunoradiometric assay (IRMA) tumor-marker assays (in one case, up to 900 days after immunoscintigraphy). In 32 patients, the authors found a HAMA frequency of 34% (11/32: 3/7 after the first administration, 6/13 after the second, and 2/2 after the third). Ten patients developed extremely high CA-125 levels after undergoing the CIS IRMA assay (up to 80,000 U/ml) in parallel to a significant HAMA increase. The use of different assays, or HAMA removal before in vitro testing, can solve this problem. After a new CA-125 assay containing antibodies that recognize different epitopes on the CA-125 antigen (Biomira Tru-Quant OV) was applied, only mildly increased assay results or normal levels were measured. Most of HAMA-positive patients demonstrated a predominantly anti-idiotypic response, determined with two different HAMA assays. Seven patients with anti-idiotypic HAMA responses after OC-125 immunoscintigraphy remained free of tumor or had stable disease (2-42 or more months), contrary to their poor prognoses that had been made based on the underlying stages of their tumors. All of these patients are currently doing well (Karnofsky Index > 70%) and show no significant tumor progression. In light of their extremely poor prognoses (5-year survival rates of 3-5% in recurrent International Federation of Gynecology and Obstetrics III/IV stages), without further chemotherapy, these courses are extremely unusual. Preliminary in vitro experiments lead to the postulation that anti-idiotypic HAMA may trigger an antitumor effect either by suppressing the growth of CA-125-expressing cancer cells directly, or by activating the patient's immune response via induction of Ab3. Similar results are observed after immunoscintigraphy with a technetium-99m-labeled anti-CA-125 monoclonal antibody (B43.13), which the authors now also use for immunotherapy of ovarian cancer patients by repeated injections, hoping that induction of anti-idiotypic HAMA will be beneficial for prolonged survival of patients with ovarian carcinoma.

Interactions between tumor cells and tumor infiltrating lymphocytes in human ovarian carcinoma.

The aim of our study was to investigate the lymphocytic infiltration rate of ovarian tumors and the possible use of tumor-infiltrating lymphocytes (TIL) as a therapeutic tool in gynecologic oncology. Twelve tumors were treated with digesting enzymes in order to isolate tumor-infiltrating lymphocytes as well as tumor cells, TIL were expanded by culture in the presence of human interleukin-2 (IL-2). Freshly prepared tumor cells were allowed to grow in culture medium for several days before the first investigations were performed. TIL could only be isolated from 50% of the investigated tumors. In contrast to this the isolated tumor cells could be largely expanded in 73% of the cases. The expression of the CA125 antigen in the culture supernatants served as control and could still be evaluated up to three months after isolation. In parallel the antigen expression on the cellular surface was estimated by immunocytochemistry. Evaluating the phenotypes of TIL showed predominantly CD3+, their expansion rate was only poor. Tumor cells were isolated and expanded in order to test the tumor-directed cytotoxic efficacy of TIL and for further use in transplantation to nude mice.

Relevance of immunological parameters in progression of colorectal-carcinoma.

The long-term influence on the immunological stage from surgery and/or adjuvant or palliative therapy of 23 patients with metastatic colorectal carcinoma was investigated by performing regular phenotyping and functional analysis of peripheral blood lymphocytes (PBL). The following groups were chosen: A (n=6); patients after resection of primary tumor and liver-metastases without chemotherapy. B (n=3); patients with catheter implantation after resection of primary tumor and liver metastases receiving an adjuvant arterial chemotherapy with 5-fluorouracil (5-FU) and folinic acid (5-formyltetrahydrofolic acid, FA). C (n=7); patients with non-resectable liver-metastases, receiving arterial or systemic chemotherapy after catheter implantation. D (n=7); patients with extrahepatic filiae receiving systemic palliative chemotherapy. Lymphocytes of 10 healthy volunteers served as controls. Furthermore, we were able to show effects of 5-FU and FA on the immune system.

Functional analysis of tumor-associated lymphocytes from gynecological tumors.

Tumor-Associated Lymphocytes (TAL) were isolated from peritoneal fluids of six ovarian cancer patients and pleural effusion from eight breast cancer patients, respectively. In one case we obtained ascitic fluid as well as pleural effusion because of intraabdominal metastatic breast carcinoma. The collected cells were cultured in a complete medium and supplemented with human interleukin-2 (nIL-2) in a concentration of 1000 Units/ml. Phenotyping was not always possible due to rapid decay of the cells. Cytotoxicity was determined with a fluorescence-based assay, in some cases at different stages of cell growth. In two cases TAL from ascitic fluids showed increased cytotoxic activity after a longer cultivation period. TAL from pleural effusions showed cytotoxic activity against the target cell lines in two cases only. Some of these TAL did not proliferate any more but died within 24 h. With the functional analysis we wanted to investigate the cytotoxic potential against natural killer (NK)-sensitive and NK-resistant (Raji) cell lines. The results demonstrate the ability of some of the TAL populations to destroy tumor cells.

Functional analysis of peripheral blood lymphocytes from healthy volunteers.

Functional analysis of peripheral blood lymphocytes (PBL) from 46 healthy donors is described. For quantitation of cytotoxic activity a fluorescence-based assay is performed with commercially available human tumor cell lines as targets. For evaluation of natural-killer cell activity, the cell line K562 (NK-cell sensitive) and for T-cell activity Raji cells (NK-cell resistant) are used as targets. Since we are not using autologous cells as targets we are measuring the function of non MHC-restricted cytotoxic T-lymphocytes. The results show a median NK-cell activity of 77.4% (given in % lysis) and a median T-cell activity of 21.4% in healthy individuals at an effector-/target-cell ratio (E/T ratio) of 40:1. For correlating immunological parameters such as functionality of PBL from tumor patients with the progression of tumor growth, normal values are of great importance.