A human monoclonal antibody produced by in vitro sensitization of human lymphocytes with an antigen from urine of a sarcoma patient.
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Antibodies, Monoclonal↗
Biomedical subjects
R F Irie
Publications and source records attributed to R F Irie.
Gangliosides of human melanoma.
The ganglioside composition of human malignant melanoma was studied with the use of 80 melanoma specimens, including 52 surgical specimens and 28 cultured cell lines. A ganglioside fraction was isolated and purified from each of these tissues, and the amount of each component ganglioside was assessed by thin-layer chromatography (TLC) and a TLC scanner. Five gangliosides (GM3, GD3, GM2, GD2, and alkali-labile ganglioside) were most commonly expressed by these melanomas. However, the total ganglioside amount (ranging from 33 to 302 micrograms/g wet wt of tissue) as well as the distribution of each ganglioside were widely heterogeneous in both biopsied and cultured melanomas. When the ganglioside expressions of cultured and biopsied melanomas were compared, GM2 and GD2 were minor components of biopsied melanomas but often became major components of cultured melanoma cells. Conversely, alkali-labile ganglioside was expressed more strongly on biopsied melanomas. This heterogeneity suggests that it will be necessary to analyze the ganglioside composition of biopsied melanomas before using monoclonal antibodies to melanoma-associated gangliosides for melanoma diagnosis or therapy.
Biopsy↗
Gangliosides of human melanoma: GM2 and tumorigenicity.
An increased synthesis of the ganglioside GM2 has been reported on transformed murine cells, human fetal tissues, and transformed melanocytes. This study was designed to investigate whether any correlation existed between GM2 expression and the tumorigenicity of human melanoma. Ten established human melanoma cell lines, 5 rich in GM2 (group A) and 5 poor in GM2 (group B), were selected on the basis of previous ganglioside analysis of 28 melanoma cell lines. Six athymic nude mice per cell line were given an sc injection of 10(6) human melanoma cells/mouse. Tumors were measured every 2-4 days. By day 36 after the injection, 28 of 30 mice (93%) in group A developed medium to large tumors, but only 1 of 30 (3%) in group B developed a small tumor (P less than .005). The correlation of GM2 content of individual melanomas with tumor growth rate also was very high. GM2 content was expressed as nanomoles per gram wet weight of each melanoma. The area under the log values of tumor growth curves from day 0 to day 36, which represented tumor growth rate, tumor size, and latent period, was proportional to GM2 content, with a correlation coefficient of 0.927 and with P less than .001. The same log relationship when tested with other gangliosides, GM3, GD3, and GD2, was not statistically significant. These results, combined with the fact that variations in GM2 content do not affect in vitro growth of human melanoma cells, suggest that GM2 expression may be directly related to the dedifferentiation or the tumorigenicity of human melanoma.
Animals↗
Expression of heterophile, Paul-Bunnell and Hanganutziu-Deicher antigens on human melanoma cell lines.
Expression of heterophile antigens was studied on 6 human melanoma cell lines. Paul-Bunnell and Hanganutziu-Deicher (H-D) antigens, but not Forssman antigen, were demonstrated on these cell lines. H-D antigen was also demonstrated on melanoma cells isolated from primary and metastatic lesions. Evidence was also presented that HLA class I but not class II (DR and DQ) molecules are expressed on these cell lines. H-D antibodies of IgG class were detected in 10 of 23 (42%) melanoma patients suggesting the possibility that H-D antigen might have been expressed in an immunogenic form in the patients.
Antibodies, Heterophile↗
Radioimmunodetection of human melanoma tumor xenografts with human monoclonal antibodies.
We established a human IgM monoclonal antibody that defines a tumor-associated membrane antigen expressed on human melanoma cells. The antigen has been identified as the ganglioside GD2. In this paper, we describe the potential usefulness of the human monoclonal antibody for radioimaging. Nude mice bearing tumors derived from a human melanoma cell line were used as a model. Antibody activity was degradated significantly after labeling with 131I by the use of a modified chloramine-T method. After testing various concentrations, labeled antibody of a specific activity of 2.8 microCi/micrograms produced the best results. Balb/c nude mice bearing a GD2-positive M14 melanoma cell line were injected with 10-30 micrograms of labeled antibody, and its radiolocalization in different organs and in the whole body were evaluated. The best tumor image was obtained on Day 6. The labeled antibody uptake ratio between tumor and muscle was 9.2:1; the ratio between tumor and liver was 1.4:1. These studies represent the first report of experimental tumor imaging with human monoclonal antibody. Human monoclonals with probably prove to be superior reagents for tumor imaging in melanoma patients if the problem of antibody radiolysis is resolved.
Animals↗
Regression of cutaneous metastatic melanoma by intralesional injection with human monoclonal antibody to ganglioside GD2.
In this study we used human monoclonal antibody (Hu-mAb) L72 as an intratumoral injection of cutaneous metastasis of melanoma to study its anti-tumor effects in human patients. Hu-mAb L72 was developed by transforming peripheral blood lymphocytes from a melanoma patient in vitro with the Epstein-Barr virus, forming a human lymphoblastoid cell line that produces 2-5 micrograms of IgM per ml. This IgM Hu-mAb was shown to react specifically with ganglioside GD2 and have a strong cytotoxic effect on human melanoma cells in the presence of complement. Patients with cutaneous metastatic melanoma were given intralesional injections on a daily or weekly injection schedule. Regression was seen in all tumors except in those of two patients whose tumors were shown to have low antigenicity. Histopathological data showed tumor degeneration, fibrosis, free melanin, and some degree of lymphocyte or macrophage infiltration. One patient with melanoma satellitosis treated with Hu-mAb showed complete regression with no sign of recurrence 20 months after the initial treatment. With the exception of mild erythema, no side effects were observed in any patient.
Adult↗
Human IgM monoclonal anti-GD2 antibody: reactivity to a human melanoma xenograft.
The cell membranes of human melanoma express a tumor-associated ganglioside, GD2. We previously established a human melanoma cell line, M14-A, that metastasizes to the lung, liver, skin, lymph nodes, and abdominal organs of nude mice in addition to forming ascites and pleural effusions. We also reported the successful in vitro production of human IgM monoclonal antibody to GD2. In the present study, we evaluated the GD2 expression of human melanoma cells at the primary and metastatic sites and their reactivity to human monoclonal anti-GD2 antibody in vivo. GD2 was expressed strongly on the melanoma cells from both primary and metastatic sites, except for cells from pleural effusions and ascites. When M14-A-bearing nude mice received systemic injections of the human monoclonal antibody, the anti-GD2 titer in the sera was reduced markedly at 2 hours, whereas the reduction was minimal in sera from tumor-free mice and mice bearing GD2-negative human M24 cells. The immune adherence test confirmed that antibody was fixed on cells of primary subcutaneous M14-A tumors and on their metastases to liver, lung, abdominal organs and skin. These results suggest that this large molecule protein can penetrate the blood-tumor barrier and bind immunologically to antigen-positive melanoma cells in vivo.
Animals↗
[Cancer therapy with monoclonal antibody].
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Animals↗
Immunogenicity of melanoma-associated gangliosides in cancer patients.
The immunogenicity of gangliosides found on human melanoma cells was determined from sera of 26 melanoma patients who were immunized every 1-4 weeks for 4 months with tumor-cell vaccine (TCV) prepared from cultured melanoma cells. Total lipid-bound sialic acid in the gangliosides isolated from TCV was 0.38 mumol/10(8) cells, and was distributed as follows: 44.8% to GM3, 44.2% to GD3, 5.6% to GM2, and 4.6% to GD2. Sera were tested at monthly intervals for antibodies to each ganglioside by ELISA with purified gangliosides as the antigen source. The immunologic specificity of the antibody was confirmed by absorption tests. None of the 26 patients had detectable anti-GM3, anti-GD3, or anti-GD2 antibodies before immunization, although anti-GM2 antibody was detected in 3 patients. After immunization, 2 patients developed IgM anti-GD2, 10 developed IgM anti-GM2, and 2 developed IgG anti-GM2 antibodies. No patient developed detectable anti-GM3 or anti-GD3 antibodies. These results indicate that both GD2 and GM2 expressed on human melanoma cells are immunogenic in humans, although GM2 appears to be more immunogenic. The other two gangliosides, GM3 and GD3, are present in human sera and in human normal tissues, and thus immunologic tolerance may have been established against these gangliosides. Alternatively, circulating GM3 and GD3 may have neutralized anti-GM3 and anti-GD3 antibodies, if any were induced by TCV immunization.
Antibodies↗
Establishment of an ascitic human melanoma cell line that metastasizes to lung and liver in nude mice.
UCLA-SO-M14, a human melanoma cell line, was cultured for ten passages in vitro. The line was converted to an ascitic form, M14-A, by transplanting M14 into CD-1 nude mice and back into tissue culture. The minimum number of M14-A cells that formed ascites in all mice (within 10-21 days) was 5 X 10(5), and over 80% of such mice developed macroscopic liver metastases. M14-A inoculated subcutaneously formed tumor at the site of injection, but rarely led to the development of metastases. However, when M14-A was inoculated subcutaneously in young mice (1-14 days old), more than 50% developed lung (but not liver) metastases. The reproducibility of the formation of ascites and metastases was confirmed by testing M14-A at various passages. M14-A may be useful as a model for the metastatic process in human melanoma.
Animals↗
Human monoclonal antibody to tumor-associated ganglioside GD2: suppressed growth of human melanoma in nude mice.
Human IgM kappa monoclonal antibody (anti-GD2) to a tumor antigen, ganglioside GD2, has been produced by Epstein-Barr virus-transformed human B lymphoblasts. In the present study, we demonstrated the anti-tumor effects of passively administered anti-GD2 against an ascites-form human melanoma cell line, M14-A, transplanted into athymic nude mice. M14-A expresses GD2 in vivo. Cells (5 X 10(5) were inoculated intraperitoneally (i.p.) or subcutaneously (s.c.) into CD-1 nude mice that had received i.p. injections of 200 micrograms anti-GD2 and rabbit complement. Significant tumor-free intervals were observed in the treated mice (P less than 0.005) for i.p. tumors and P less than 0.025 for s.c. tumors). M14-A formed well-vascularized s.c. tumors if injected into young nude mice. Three-wk-old CD-1 nude mice bearing 2-3 mm M14-A s.c. tumor nodules were treated i.p. with anti-GD2 and rabbit complement. Tumor growth was delayed for 25 days. On day 15, treated tumors were 20% the size of control tumors. Because most biopsied or autopsied melanomas express GD2, and because patients with melanoma produce autoantibodies to GD2, the results in this study may provide important information for future passive immunization with human monoclonal antibody and for active specific immunization with GD2 antigen.
Animals↗
Human monoclonal antibody to ganglioside GD2-inhibited human melanoma xenograft.
A human IgM monoclonal antibody was produced in vitro against OFA-I-2, a human tumor membrane antigen. This antigen is expressed on tumors of neuroectodermal origin, and has been identified as the ganglioside GD2. This study examines the anti-tumor effect of the monoclonal antibody against a GD2-positive human melanoma cell line, M14, inoculated subcutaneously into athymic CD-1 nude mice. Tumor-free survival was prolonged markedly when the monoclonal antibody and M14 cells were inoculated simultaneously. When antibody and complement were also injected into established tumor nodules, M14 tumor growth was suppressed. However, intraperitoneal injection of the antibody did not alter the growth of the subcutaneously inoculated M14 cells. The antibody has no effect on the growth of a GD2-negative melanoma cell line, M24. These results indicate that the human monoclonal antibody to GD2 may be useful for the suppression of GD2-positive tumor cells in cancer patients if the tumor can be directly exposed to the antibody and complement.
Animals↗
Human monoclonal antibody to tumor-associated ganglioside GD2.
Human IgM kappa antibody to a membrane antigen of human tumors of neuroectodermal origin (melanoma, glioma and neuroblastoma) has been detected in the spent culture fluid of an Epstein-Barr virus (EBV)-transformed human B-lymphoblastoid cell line, L72. The chemical nature of the antigen was identified as ganglioside GD2. The antibody was purified by precipitation of L72 culture fluid with ammonia sulfate and hypotonic buffer followed by ultracentrifugation and Sephacryl S-300 super gel filtration. Approximately 27 mg of pure human IgM was obtained from 101 of spent medium. Total IgM and antibody activity recovery efficiency was 60% and 75%, respectively. The monoclonal character of the immunoglobulin produced by the L72 cell line was determined by agarose isoelectric focusing and immunofixation techniques. 1 mg of the purified IgM possessed an antibody titer endpoint to a GD2-positive melanoma cell line of 1:10,000 as assayed by immune adherence and 1:100 titer by complement-dependent cytotoxicity in vitro. The effect of pure anti-GD2 on suppression of melanoma growth in vivo was tested using a nude mouse model. Three-week-old CD-1 nude mice bearing 2-3 mm M14-A subcutaneous melanoma nodules were treated intraperitoneally with anti-GD2 and rabbit complement. Tumor growth was retarded for 25 days when compared to that of control mice receiving non-specific human IgM and complement. On Day 15, treated tumors were 80% smaller than control tumors. These result indicated that the pure human monoclonal antibody to GD2 may have potential for cancer therapy.
Animals↗
Human monoclonal antibody against ganglioside GD2: use in development of enzyme-linked immunosorbent assay for the monitoring of anti-GD2 in cancer patients.
Ganglioside GD2 is expressed on membranes of human melanoma cells and induces antibody responses in patients with melanoma. In this study a new enzyme-linked immunosorbent assay was developed for detection of human antibody against GD2. A human IgM monoclonal anti-GD2 antibody was used for development of the assay. The antigen, GD2, was prepared from human brain. Four micrograms of GD2 was required to coat a well in a polystyrene microtiter plate. As little as 10 ng of antibody could be detected. The applicability of the assay for detection of serum anti-GD2 antibody was demonstrated with sera from patients immunized with GD2-positive melanoma cell vaccine.
Antibodies, Monoclonal↗
Ganglioside GM2 as a human tumor antigen (OFA-I-1).
A monospecific antibody produced in vitro by a B-lymphoblastoid cell line transformed with Epstein-Barr virus has been shown to recognize a membrane antigen (OFA-I-1) on human tumors and fetal brain. This study identifies the chemical nature of OFA-I-1. The glycolipid fraction of antigen-rich spent medium of an OFA-I-1-positive melanoma cell line, M14, was extracted by chloroform/methanol/water, 4:8:3 (vol/vol), and was separated into fractions of neutral glycolipids and gangliosides by DEAE-Sephadex followed by base treatment and Bio-sil A column elution. OFA-I-1 antigens were found exclusively in the ganglioside fraction when assayed with monospecific anti-OFA-I-1 by an immune adherence inhibition test. The results obtained from thin-layer chromatography of the antigenic M14 ganglioside and sequential glycosidase digestion suggested that the antigen was a ganglioside GM2, GalNAc beta 1 leads to 4(NeuAc alpha 2 leads to 3)Gal beta 1 leads to 4Glc leads to Cer. These results were further supported by testing various authentic gangliosides and neutral glycolipids for OFA-I-1 antigenicity. Only GM2 showed positive reactivity.
Adult↗
Human monoclonal antibody to a neuroectodermal tumor antigen (OFA-I-2).
Human IgM kappa monoclonal antibody to human tumors of neuroectodermal origin was produced in the spent medium of an Epstein-Barr virus-transformed B-lymphoblastoid cell line, L72. Chemically, the antigen was identified as ganglioside GD2 [Gal NAc beta 1----4 (Neu Ac alpha 2----8 Neu Ac alpha 2---3) Gal beta 1----4 Glc----ceramide]. Twenty-seven mg of pure human IgM were obtained from 10 liters of L72 spent medium using salt and hypotonic precipitation, ultracentrifugation, and Sephacryl-S 300 superfine gel filtration. The monoclonal origin of the antibody was determined by agarose isoelectrofocusing. This human monoclonal antibody may be a particularly useful reagent for immunotherapy trials in cancer patients.
Antibodies, Monoclonal↗
Comparison of the techniques used for monitoring humoral immunity in cancer patients.
The humoral immune response to tumor-associated antigens (TAA) in cancer patients undergoing immunotherapy can vary considerably. This variability is inherent from patient to patient and is also dependent on the type of assay used to measure patient antibody response to tumors. In this investigation four serological techniques, complement-dependent antibody-mediated cytotoxicity, immune adherence, complement fixation, and indirect membrane immunofluorescence, were used to assess the humoral immune response in melanoma patients receiving specific immunization with cultured allogeneic melanoma cell vaccine. Two different patterns of antibody response were elicited. One was directed against TAA and oncofetal antigens (OFA) and the other was against HLA antigens. The sensitivity and reactivity varied among the different techniques suggesting that one test or technique may not be sufficient to evaluate critically a patient's response to immunotherapy.
Antibody Formation↗
Human antibody to OFA-I, a tumor antigen, produced in vitro by Epstein-Barr virus-transformed human B-lymphoid cell lines.
We established two long-term human B-lymphoblastoid cell lines (L55 and L72) transformed by Epstein-Barr virus that produced IgM kappa antibodies to the human tumor antigen, OFA-I. Periphral blood lymphocytes obtained from melanoma patients were used as the source of the B lymphocytes. Antibody specificity was determined by the immune adherence assay using various human cancer and noncancer tissues as targets. L55 antibody (designated anti-OFA-I-1) reacted with a variety of human tumor types whereas L72 antibody (designated anti-OFA-I-2) reacted only with tumor cells of neuroectodermal origin (melanoma, glioma, and neuroblastoma). The levels of IgM detected in the spent medium of 1 X 10(6) L55 and L72 cells were 4 and 9 micrograms/ml, respectively, by radioimmunoassay.
Antibody Formation↗