L J Old

Mouse monoclonal antibody AbR24 has a high degree of specificity for human melanoma cells when tested on viable cultured cells using the protein A mixed hemagglutinin serological assay. The antigen detected by this antibody has been isolated from melanoma cells and shown to be GD3 ganglioside by compositional and partial structural analysis and by comparison with authentic GD3 in thin layer chromatography (TLC). AbR24 reacts with authentic GD3, but not with any other ganglioside tested. Using TLC and reactivity with AbR24, a wide range of cells and tissues was examined for the presence of GD3. A new serological assay, termed glycolipid-mediated immune adherence, was devised for assaying the reactivity of AbR24 with gangliosides. Melanomas (cultured cells or tumor tissue) were shown to have GD3 and GM3 as major gangliosides. Other cells and tissues examined also contained GD3, but usually only in low amounts. Melanomas (and MOLT-4, a T cell line) were characterized by a simplified ganglioside profile with GD3 and GM3 as major components. The apparent discrepancy between the ubiquitous presence of GD3 and the serological specificity of AbR24 for melanoma cells can be explained in terms of localization and concentration of GD3 in different cells.

R24 is an IgG3 mouse monoclonal antibody that identifies GD3, a prominent ganglioside on the surface of melanoma cells and other cells of neuroectodermal origin. Twelve patients with metastatic melanoma were treated with R24 at three dose levels, 8, 80, or 240 mg/m2, over a period of 2 weeks. Peak antibody levels in the serum were dose related and ranged from less than 0.1 to 62 micrograms/ml. Inflammatory reactions (urticaria, pruritus, erythema, subcutaneous ecchymoses) were observed around tumor sites in patients treated at doses greater than or equal to 80 mg/m2. Tumor biopsies during and after treatment showed lymphocyte and mast cell infiltration, mast cell degranulation, and complement deposition. Side effects were mild and were readily controlled by antihistamines. Major tumor regression has been observed in three patients.

IFN-gamma is known to induce expression of Ia antigens on a variety of cell types. In the present study, this activity of IFN-gamma has been analyzed with a panel of 36 melanoma cell lines, normal melanocytes, and 97 cell lines representing a range of other differentiation lineages. 55% of the melanoma cell lines express Ia antigens in a constitutive manner without IFN-gamma induction. Of the 16 Ia-melanoma lines, 13 could be induced to express Ia antigens by IFN-gamma, whereas three were noninducible. Melanocytes, which do not normally express Ia antigens, are converted to Ia expression by IFN-gamma. Ia antigens expressed constitutively or after IFN-gamma induction were identified with antibodies detecting monomorphic and allomorphic products of DR and DC loci. IFN-gamma appeared to be unique in its ability to induce Ia expression on melanoma and melanocytes; 14 other agents (including IFN-alpha and IFN-beta) known to influence growth or differentiation did not have Ia-inducing activity. Equally striking is the restriction of antigenic changes following IFN-gamma induction to HLA-associated products; of the 38 systems of cell surface antigens examined, only HLA-A,B,C, beta 2m, and Ia antigens were affected. A variety of other Ia- cell types were shown to be Ia-inducible by IFN-gamma; these included established lines of breast, colon, pancreas, bladder, kidney, ovary, and brain cancers, and cultures of normal fibroblasts, kidney epithelia, and epidermal keratinocytes. In contrast, three tumor types, teratocarcinoma, choriocarcinoma, and neuroblastoma, were not inducible for Ia expression, even though IFN-gamma could induce expression of HLA-A,B,C products. The broad representation of Ia antigens on most somatic cell types expressed either constitutively or after IFN-gamma can be viewed in an immunological context (antigen presentation/immune regulatory signals) or could indicate that Ia products have functions other than those related to immune reactions.

Recent progress in the structural identification of human melanoma antigens recognized by autologous cytotoxic T cells has led to the recognition of a new melanocyte differentiation antigen, Melan-A(MART-1). To determine the properties of the Melan-A gene product, Melan-A recombinant protein was produced in Escherichia coli and used to generate mouse monoclonal antibodies (mAbs). Two prototype mAbs, A103 and A355, were selected for detailed study. Immunoblotting results with A103 showed a 20-22-kDa doublet In Melan-A mRNA positive melanoma cell lines and no reactivity with Melan-A mRNA-negative cell lines. A355, in addition to the 20-22-kDa doublet, recognized several other protein species in Melan-A mRNA-positive cell lines. Immunocytochemical assays on cultured melanoma cells showed specific and uniform cytoplasmic staining in Melan-A mRNA-positive cell lines. Immunohistochemical analysis of normal human tissues with both mAbs showed staining of adult melanocytes and no reactivity with the other normal tissues tested. Analysis of 21 melanoma specimens showed homogenous staining of tumor cell cytoplasm in 16 of 17 Melan-A mRNA-positive cases and no reactivity with the three Melan-A mRNA-negative cases.

Tyrosinase (EC 1.14.18.1), the key enzyme in melanin synthesis, has been shown to be one of the targets for cytotoxic T-cell recognition in melanoma patients. To develop serological reagents useful for immunophenotyping melanoma for tyrosinase, human tyrosinase cDNA was expressed in an Escherichia coli expression vector. The purified recombinant tyrosinase was used to generate mouse monoclonal and rabbit polyclonal antibodies. The prototype monoclonal antibody, T311, recognized a cluster of protein moieties ranging from 70 to 80 kDa in tyrosinase mRNA-positive melanoma cell lines and melanoma specimens as well as in L cells transfected with tyrosinase cDNA. Untransfected L cells and L cells transfected with tyrosinase-related protein 1, TRP-1(gp75), were nonreactive. Immunohistochemical analysis of melanomas with T311 showed tyrosinase in melanotic and amelanotic variants, and tyrosinase expression correlated with the presence of tyrosinase mRNA. Melanocytes in skin stained with T311, whereas other normal tissues tested were negative. The expression pattern of three melanosome-associated proteins--tyrosinase, TRP-1(gp75), and gp100--in melanoma was also compared. Tyrosinase and gp100 are expressed in a higher percentage of melanomas than TRP-1(gp75), and the expression of these three antigens was discordant. Tyrosinase expression within individual tumor specimen is usually homogenous, distinctly different from the commonly observed heterogeneous pattern of gp100 expression.