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A human B-lymphoblastoid cell clone, L55-81, that produces human monoclonal antibody (MAb) to ganglioside G(M2) was established from peripheral blood B lymphocytes of a melanoma patient. L55-81 secretes IgMkappa light chain antibody in a serum-free medium. G(M2) specificity of the antibody was tested by immune adherence assay, TLC immunostaining, and ELISA. Anti-G(M2) antibody was shown to have the ability to kill the G(M2)-rich human melanoma cell line M14 in the presence of human or rabbit complement. A purified L55-81 MAb (>99.5% purity in protein concentration) was biotinylated and tested for its reactivity to various histological-type biopsied tumor and normal tissues in an avidin-biotin detection system. L55-81 MAb (20 microg/ml) reacted with several types of tumor tissues such as melanoma (7 of 10), colon carcinoma (4 of 5), ovary carcinoma (4 of 5), breast carcinoma (1 of 5), kidney carcinoma (1 of 5), and prostate carcinoma (1 of 5). None of the normal tissues derived from 24 different organs and adjacent normal tissues surrounding the cancerous tissues were stained. Production of the antibody in a serum-free medium, the cytotoxic potential with human complement, the inability to react to normal tissues, and the ability to target antigen-specific target cells make L55-81 a potential therapeutic agent for the treatment of cancers expressing ganglioside G(M2).

The human monoclonal antibody (HuMAb) L92 reacts to an M(r) 43,000 protein associated with human melanoma. To identify the gene encoding its antigenic epitope, a complementary DNA expression library constructed from the human melanoma cell line UCLASO M14 was screened with HuMAb L92. DNA sequence analysis of the isolated clone revealed that the immunoreactive peptide was composed of 10 amino acids (QDLT-MKYQIF). The peptide was expressed in Escherichia coli with beta-galactosidase as a fused protein. There is no homology between the cloned sequence and other reported DNA sequences. Western blot analysis showed that the fused protein had specific binding to HuMAb L92. An antigen-encoding peptide with 10 amino acids was synthesized and tested for its immunoreactivity in vitro. HuMAb L92 reacted specifically to the 10-amino acid peptide in both an antibody-binding inhibition to the M(r) 43,000 protein and a solid-phase enzyme-linked immunosorbent assay. Using several truncated fusion proteins, we found the minimum number of amino acids required for the antibody binding to be 4 (KYQI). These results suggest that the identified peptide sequence encodes the antigenic epitope of the M(r) 43,000 protein.

In this study we report the characterization of a human monoclonal antibody (HuMab), L612, that reacts with ganglioside GM3 and has therapeutic application for the treatment of human neoplasms, particularly melanoma. A permanent IgM-secreting Epstein-Barr virus-transformed B-cell line L612 was established. L612 HuMab bound specifically to neoplastic cell lines in culture and in tissue biopsy specimens such as melanoma, colon, breast, and lung cancer. The antibody did not bind to normal cells or biopsy tissue. HuMab L612 showed the highest reactivity to melanoma cells, particularly to those with high concentrations of GM3. Immunostaining on high-performance thin-layer chromatography plates demonstrated that L612 HuMab bound to GM3 purified from melanoma cells. Removal of the sialic acid from GM3 abolished antibody binding. HuMab L612 also reacted to GM4 purified from egg yolk, indicating that it recognizes an NeuAc alpha 2-3 galactose antigen determinant. HuMab L612 heavy and light chains were sequenced and determined to belong to the mu heavy chain variable subgroup III and kappa chain variable subgroup IV families, respectively. The studies indicate that the L612 HuMab has significant therapeutic potential for a wide variety of human cancers.

The VL and VH from a murine anti-idiotypic antibody that mimics ganglioside have been cloned, sequenced, and expressed as a chimeric mouse/human IgG1 antibody. The chimeric antibody retained a binding specificity indistinguishable from the original murine antibody. The VH was a member of Vgam 3.8 family. The sequences are discussed in terms of ways in which proteins may mimic ganglioside epitopes.

Immune cytokines have been shown to play important roles in regulating immune cell functions as well as neoplastic cells. Interleukin-4 (IL4), primarily known as a B-cell growth factor, can also activate and differentiate other immune cells. This cytokine has recently been shown to have immunotherapeutic benefit in tumor-bearing hosts. The present study assessed the effect on human renal cell carcinoma cell lines of recombinant IL4 alone and in combination with recombinant gamma-interferon (IFN) or recombinant alpha-tumor necrosis factor (TNF). IL4 inhibited cell growth of all lines at 250-500 units/ml in a differential manner. Expression of IL4 receptors was demonstrated on renal cell carcinomas. Overall, IFN (500 units/ml) alone inhibited cell growth; however, TNF (500 units/ml) was not as strong an inhibitor. When IL4 was combined with IFN or TNF there was a significant augmentation of cell growth inhibition and modulation of cell morphology of the cell lines. Tumor-associated ganglioside antigens (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, GalNAc beta 1-4 (NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer, and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer) HLA class I, HLA-DR, and beta 2-microglobulin on the cell surface of renal cancer lines were assessed by flow cytometry and radiometric binding assay. IL4 alone or in combination with other cytokines modulated HLA class I and HLA-DR expression. IL4 and IFN consistently enhanced NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer expression on individual cell lines. The study demonstrated that IL4 alone or in combination with other cytokines can significantly inhibit growth, and modulate the expression of surface major histocompatibility and tumor-associated antigens of renal cell carcinomas.

Immune cytokines have been shown to play important roles in regulating immune cell functions. Interleukin-4 (IL4), originally described as a B-cell growth factor, is known to activate and differentiate other immune cells. IL4 has been given as an immunotherapeutic to tumor-bearing hosts. In this report, we set out to determine whether IL4 can directly modulate growth and expression of surface antigens on human melanoma cells. The effect of recombinant IL4 alone and in combination with recombinant gamma-interferon (IFN) or recombinant alpha-tumor necrosis factor (TNF) on melanoma cell lines was examined. IL4 significantly inhibited cell growth of all cell lines examined at 100-500 units/ml; but a dose-dependent differential response to individual cell lines occurred. The effect of IL4 was augmented by combination with IFN or TNF. Melanoma-associated ganglioside antigens (GM3, GD3, GM2, GD2) and human leukocyte antigen class I and DR on the cell surface of melanoma cells were assessed by flow cytometry and/or a radiometric binding assay. IL4, IFN, or TNF alone enhanced human leukocyte antigen class I, DR, and beta 2-microglobulin antigen expression. IL4 alone and in combination with IFN or TNF increased the GM3/GD3 ratio expression. GD2 was enhanced significantly by IL4, IFN, and TNF. Pretreatment of melanoma with IL4 or with other cytokines prior to stimulation with peripheral blood lymphocytes significantly enhanced mixed lymphocyte tumor reaction activity as compared with non-treated melanoma used as stimulators. These studies demonstrate that IL4 alone or in combination with IFN and TNF can modulate melanoma growth activity and surface antigen expression to a more differentiated and immunogenic phenotype.

The mixed hemadsorption (MHA) techniques demonstrated antibody and complement fixed in vivo to the surface of human cancer cells. Tumors from 12 cancer patients and normal tissues from 5 cancer patients and 8 patients with cerebrovascular or cardiac diseases were collected from biopsy and autopsy for in vitro testing. Antiserum to human whole immunoglobulins and antiserum to human C3 were used in the MHA techniques. Positive MHA patterns were demonstrated on the surface of cancer cells by both methods. Positive reactions ranged from 12 to 32% in mixed hemadsorption for anitbody detection and from 10 to 34% in mixed hemadsorption for complement component 3 detection. Normal tissues obtained from cancer patients or from patients who died of causes other than cancer rarely exhibited distinct MHA reactivity. Collectively, the data suggest that most human cancers are antigenic in the autologous host and that tumor-associated antigens of cancer cells react in vivo with their humoral antibody to fix complement.

The ganglioside composition of human melanoma was analyzed in five sets of tumor specimens obtained directly from surgery, from the autologous tissue culture cell lines, and from the autologous cell lines grown in athymic nude mice. Total gangliosides of these 15 melanoma specimens were isolated and purified, and the amount of each component ganglioside was analyzed by thin-layer chromatography and a thin-layer chromatography scanner. The ganglioside composition of the five surgical melanoma specimens clearly exhibited different patterns from each other. Moreover, none of the autologous cultured melanomas possessed the same ganglioside composition as their original biopsied tumors. However, when these melanoma cell lines were transplanted into nude mice, the ganglioside composition was converted back to the same ganglioside pattern as in the original surgical specimens. The results support the view that changes in the ganglioside composition of melanoma during in vitro growth are caused by the culture environment rather than by selection of melanoma cells with a particular genotype. Reestablishment of the original ganglioside patterns after passage in nude mice provides clear evidence that in vivo expression of gangliosides is a conserved and stable function specified by the human melanoma cells.

Membrane antigens of a cultured human melanoma line, UCLASO-M14, were studied using immune adherence techniques. Allogeneic sera from melanoma patients that were reactive with the M14 but nonreactive with lymphoid cells of the M14 donor were used as antibodies. The antigen responsible for the reaction between M14 and the antibodies was searched for in other cancer, normal, and fetal tissues using antibody absorption techniques. The antigen was found in a variety of different histological types of biopsied and cultured cancer cells as well as in melanomas. The antigen did not exist in biopsied normal tissues, but it appeared in cultured normal skin and muscle. Neither normal lymphocytes nor cultured lymphoid cells showed any antigenicity. The antigen was present in human fetal tissues and was the strongest in fetal brain tissues at 22 weeks of development. Liver, spleen, thymus, and small intestine from the same fetus were negative for antigen.

GD3 is a major ganglioside of human melanoma and was shown to be an effective target for passive immunotherapy with murine monoclonal antibodies. It was noted earlier that GD3 neither purified nor in melanoma cell vaccine (MCV), could elicit an antibody response in melanoma patients. In this study, we demonstrate that melanoma patients who received MCV had autoantibodies against a derivative of GD3, O-acetylated GD3 (O-AcGD3), a minor ganglioside expressed on human melanoma cells, and that the antibodies cross-reacted with GD3. Thin layer chromatographic immunostaining revealed that all of the sera containing antibodies against O-AcGD3 also reacted to GD3. None of the other sera responded only to GD3, although the MCV contained 7- to 12-fold higher GD3 than O-AcGD3. Furthermore, the antibody activity was completely abolished by absorption with animal erythrocytes expressing either O-acetyl disialogangliosides or GD3, indicating that the antibodies recognize an epitope commonly shared by GD3 and O-AcGD3. The antibodies bound only to the sialyloligosaccharide moiety but not to the ceramide portion of GD3 after endoglycosylceramidase treatment. The antibodies failed to bind to GD3 after neuraminidase treatment. These results indicate that the sialyloligosaccharides of the gangliosides are important components of the epitope. Periodate oxidation abolished reactivity of the antibodies to GD3 but not that to O-AcGD3, revealing that the glycerol side chain of the sialic acids in both GD3s was an important structure of the epitope. The binding of the antibodies to melanoma cell surface gangliosides was confirmed by an absorption with a GD3- and O-AcGD3-positive melanoma cell line. These results in the light of previous reports on the inability of GD3 to elicit immune response in humans suggest that anti-GD3 antibodies found in the melanoma patients were induced by immunization with O-AcGD3 and O-AcGD3 present in the MCV would serve as an antigen source for GD3-targeted active specific immunotherapy of melanoma.