Functional assessment in vitro of human-complement-dependent antibody-induced cytotoxicity of neoplastic cells.

The complement system is one potential cytotoxic effector mechanism that might be effective in immunotherapy of cancer using monoclonal antibodies (mAb) directed against tumor antigens. In order to evaluate the treatment outcome from trials using mAb in cancer patients, assessment of complement-dependent cytotoxicity (CDC) may therefore be of interest. Here we describe the elaboration of a CDC assay in vitro using a rat hepatoma cell line, H4-II-E, as target cells sensitised with mAb F12, directed against the tumor-associated ganglioside antigen fucosyl-GMI. Sensitised cells were incubated with various concentrations of fresh serum as complement source for 48 h and cytotoxicity was then assessed by the tetrazolium bromide (MTT) test. A large variation in CDC efficacy was observed between individual serum donors. No differences in CDC could be seen between healthy donors and cancer patients. The CDC showed a strong correlation to the serum concentrations of complement factor C4, supporting the validity of the assay. Our results suggest that there may be significant variations in complement function within and between individuals that might influence the outcome of clinical mAb therapy. The H4/F12 CDC assay described here, together with measurement of individual complement factors. such as C4, should be further validated in cancer patients at various disease stages and phases of treatment.

Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells.

Fucosyl-GM1 (Fuc-GM1) [Fucalpha1 --> 2Galbeta1 --> 3GalNAcbeta1 --> 4(NeuAcalpha2-3)Galbeta1 --> 4Glcbeta1 --> O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC.

Immunogenicity of a fucosyl-GM1-keyhole limpet hemocyanin conjugate vaccine in patients with small cell lung cancer.

Although small cell lung cancer (SCLC) is highly responsive to chemotherapy, relapses are common, and most patients die within 2 years of diagnosis. After initial therapy, standard treatment is observation alone. We have been investigating immunization against selected gangliosides as adjuvant therapy directed against residual and presumably resistant disease persisting after chemotherapy and irradiation. Previously, we reported that the presence of anti-GM2 ganglioside antibodies is associated with a prolonged disease-free survival in patients with melanoma, and that SCLC patients immunized with BEC2, an anti-idiotypic monoclonal antibody that mimics the ganglioside GD3, had a prolonged survival compared with historical controls. In the present trial, fucosyl-alpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3) Galbeta1-4Glcbeta1-1Cer (Fuc-GM1), a ganglioside expressed on the SCLC cell surface, was selected as a target for active immunotherapy. Fuc-GM1 is present on most SCLCs but on few normal tissues. SCLC patients achieving a major response to initial therapy were vaccinated s.c. on weeks 1, 2, 3, 4, 8, and 16 with Fuc-GM1 (30 microg) conjugated to the carrier protein keyhole limpet hemocyanin and mixed with the adjuvant QS-21. Ten patients received at least five vaccinations and are evaluable for response. All patients demonstrated a serological response, with induction of both IgM and IgG antibodies against Fuc-GM1, despite prior treatment with chemotherapy with or without radiation. Posttreatment flow cytometry demonstrated binding of antibodies from patients' sera to tumor cells expressing Fuc-GM1. In the majority of cases, sera were also capable of complement-mediated cytotoxicity. Mild transient erythema and induration at injection sites were the only consistent toxicities. The Fuc-GM1-KLH + QS-21 vaccine is safe and immunogenic in patients with SCLC. Continued study of this and other ganglioside vaccines is ongoing.

L3p40-50--a new lung-tumor-associated 40- to 50-kDa antigen defined by a monoclonal antibody (L3).

A monoclonal antibody (MAb), designated L3, was raised against a human-lung-adenocarcinoma-derived cell line, A549. Immunohistological studies showed reactivity of MAb L3 with 70 to 90% of lung tumors representing the 4 major histotypes. Strongest reactivity was seen with squamous-cell cancer and adenocarcinoma, with strong intracellular staining of the tumor cells. In small-cell lung cancer, staining was seen in a minority of tumor cells. Normal tissue staining was seen with thyroid follicular cells and focally with epithelial cells of the bronchi, the colon and the small intestine. Normal liver parenchyma was negative, whereas hepatocytes surrounding metastatic lesions were strongly stained. This pattern of staining was also seen for connective tissue. Western-blot analysis showed that the antigen defined by MAb L3 has an apparent molecular weight of 40 to 50 kDa, and hence is denominated L3p40-50. Our results suggest that L3p40-50 represents an undescribed tumor antigen.

Clinical usefulness of serum assays of neuron-specific enolase, carcinoembryonic antigen and CA-50 antigen in the diagnosis of lung cancer.

Serum concentrations of neuron-specific enolase (NSE), carcinoembryonic antigen (CEA) and CA-50 antigen were determined in 168 consecutive patients with lung cancer. All three markers were significantly elevated compared with levels in 102 patients with non-malignant chest diseases. NSE and CEA varied significantly across histological lung cancer types, with most highly elevated serum levels in small cell lung cancer and adenocarcinomas, respectively. The overall diagnostic accuracy was 0.66 for NSE, 0.74 for CEA, and 0.62 for CA-50, implying that CEA best discriminated between lung cancer and benign chest diseases, while CA-50 was less efficient as a diagnostic marker. In multivariate analysis of the three markers combined, a positive predictive value of 95% for lung cancer could be achieved with a diagnostic sensitivity of 57%, with a cut-off level defined as 0.037.NSE + 0.052.CEA + 0.011.CA-50 > 1. In 22% of the cancer patients, the time from admission to histological or cytological lung cancer diagnosis exceeded 1 month. In 52% of these patients, the initial weighted tumour marker index was > 1, strongly implying the cancer diagnosis. The study lends support to the potential use of combined analysis of NSE, CEA and CA-50 as a complementary tool in the diagnosis of lung cancer.

Coexpression of ganglioside antigen Fuc-GM1, neural-cell adhesion molecule, carcinoembryonic antigen, and carbohydrate tumor-associated antigen CA 50 in lung cancer.

With the aid of specific monoclonal antibodies, tumor tissues from 68 patients with lung cancer were examined for their expression of two small cell lung carcinoma (SCLC) antigens, Fuc-GM1 (fucosyl GM1; IV2FucII3NeuAc GgOse4) and neural-cell adhesion molecule (NCAM), and two broader tumor antigens, carcinoembryonic antigen (CEA) and carbohydrate cancer-associated antigen CA 50. Expression of Fuc-GM1 was seen in 75% and NCAM in 78% of the SCLC specimens, but also in 12 and 20% of non-SCLC. Either or both of these antigens were expressed in more than 90% of SCLC and in 25% of non-SCLC. CEA was found in more than 80% of SCLC and non-SCLC. Expression of CA 50 was seen in 65-68% of non-SCLC and SCLC, showing preference for SCLC and lung adenocarcinoma. In SCLC, cellular expression of Fuc-GM1 was generally seen together with NCAM and CA 50, but rarely with CEA. There was considerable inter- and intratumor heterogeneity in the expression of all four antigens. The results suggest that CEA is the antigen of choice for the detection of lung cancer regardless of histotype. In combined analysis of CEA, CA 50, Fuc-GM1 and NCAM, two patterns of antigen expression were recognized that appear to discriminate between SCLC and non-SCLC tumors, respectively. A considerable fraction of SCLC and non-SCLC tumors, however, exhibited similar patterns of antigen expression. The biological and clinical significance of these observations remains to be investigated.

Tumor-cell killing by MAbs against fucosyl GM1, a ganglioside antigen associated with small-cell lung carcinoma.

Monoclonal antibodies (MAbs) reactive with the ganglioside fucosyl GM1 (Fuc-GM1), an antigen associated with small-cell carcinoma of the lung (SCLC), were tested for their ability to mediate tumor-cell killing in vitro in conjunction with humoral and cellular effectors and to inhibit tumor engraftment in nude mice in vivo. MAbs F12 and F15, both IgG3k, induced human complement-mediated cytolysis of 3 Fuc-GM1-expressing tumor cell lines: one rat hepatoma cell line, H4-II-E, and 2 human SCLC cell lines, NC1 H69 and NC1 H128. F12 and F15 also induced ADCC of these cell lines in the presence of either murine or human effector cells. Addition of sub-cytolytic amounts of fresh human serum as complement source resulted in enhanced ADCC induced by MAb F12 (IgG3). Also a Fuc-GM1-reactive MAb of IgM isotype, F9, was able to induce such complement-aided ADCC (CADCC). F12 and F15 both proved to effectively inhibit engraftment of H4-II-E tumors in nude mice. A single dose of a modest amount (40 micrograms) of MAb conferred 65 to 100% protection against development of tumors. Our results demonstrate that Fuc-GM1 can act as a target antigen on tumor cells for specific immunotherapy in vitro and in a mouse model in vivo. Complement and murine and human mononuclear effector cells were effective mediators of tumor cytolysis in vitro in the presence of murine Fuc-GM1-reactive MAbs. Our results also suggest that humoral and cellular effectors may co-operate in specific tumoricidal reactions and that these may be induced by antibodies of both IgG and IgM isotypes.

Immunohistochemical detection of two small cell lung carcinoma-associated antigens defined by MAbs F12 and 123C3 in bronchoscopy biopsy tissues.

We have studied MAbs* for their ability to detect SCLC and differentiate this tumor type from the other lung tumor histotypes in cryostat sections of biopsy specimens taken at bronchoscopy from patients with suspected primary lung tumor disease. MAb F12, specific for the ganglioside fucosyl-GM1, reacted with 58% of the cases with SCLC (n = 19) and with less than 3% of those with non-SCLC (n = 38). MAb 123C3, specifically reactive with NCAM, reacted with 78% of the SCLC cases (n = 23). With this MAb no positive staining was seen in the non-SCLC cases (n = 41). None of the two MAbs reacted with tissue sections without tumor. In combined analysis with MAbs F12 and 123C3, all SCLC cases (n = 15) were positive with either and 47% with both of the MAbs. Our results show that both MAbs F12 and 123C3 are highly specific for SCLC in bronchoscopic biopsy tissue specimens, whereas the sensitivity for this histotype tends to be higher with MAb 123C3 than with F12 (P = 0.14). When used in combination, all SCLC cases could be identified. These MAbs may therefore be valuable as complements to current histopathologic characterization and differentiation of lung cancer.

Immunohistological detection of fucosyl-GM1 ganglioside in human lung cancer and normal tissues with monoclonal antibodies.

With the aid of a highly specific murine monoclonal antibody, F12, an immunofluorescence method was elaborated that allowed sensitive and specific detection of the ganglioside antigen fucosyl-GM1 (IV2FucII3NeuAcGgOse4Cer) in different types of human lung cancer and normal tissues. Nineteen of 21 cases of small cell lung cancer were positive with the F12 immunofluorescence method as compared to 2 of 10 squamous epithelial cell lung cancers and 1 of 5 large cell lung cancer specimens. Specimens of lung adenocarcinoma (8 cases) and bronchial carcinoid (3 cases) were all negative, as were 2 examined cases of neuroblastoma. No fucosyl-GM1 could be detected in normal lung and bronchus. However, in thymus, spleen, and lamina propria of the small intestine sparsely distributed clusters of small round cells were stained as well as intramural ganglionic cells of the small intestine and islet cells of the pancreas. All other normal tissues tested were negative. Results obtained with immunofluorescence closely agreed with immunochemical determination of fucosyl-GM1 in lipid extracts of tissues. Our findings suggest that fucosyl-GM1 is strongly associated with small cell cancer of the lung and demonstrate that this tumor-associated antigen can be detected with high sensitivity and specificity with an immunofluorescence method based on the use of the F12 monoclonal antibody.

Detection of a ganglioside antigen associated with small cell lung carcinomas using monoclonal antibodies directed against fucosyl-GM1.

Monoclonal antibodies with an apparent specificity for fucosyl-GM1 (Fuc-GM1) were produced by the immunization of mice with Fuc-GM1 adsorbed to Salmonella minnesota bacteria and fusion of the spleen cells with the myeloma cell line Sp 2/0. The antibodies detected Fuc-GM1 with a unique ceramide composition containing 2-hydroxy fatty acids in 11 of 12 cases of small cell carcinoma of the lung. Trace amounts of Fuc-GM1 were detected in 1 of 11 squamous epithelial cell lung carcinomas. Fuc-GM1 was also detected in 1 of 7 pancreas carcinomas but was not detected in any of the other cancers analyzed. Small amounts of Fuc-GM1 without 2-hydroxy fatty acids were detected in normal adult pancreas, spleen, and brain but could not be detected in normal lung tissue. Fuc-GM1 with 2-hydroxy fatty acids is suggested to be a specific ganglioside associated with small cell lung carcinomas. The monoclonal antibodies directed against Fuc-GM1 may be useful for specific immunodiagnosis of small cell lung carcinomas and might also be useful for specific immunotherapy of these malignant tumors.