Disialoganglioside GD3 Research Articles

Analysis of therapeutic and immunologic effects of R24 anti-GD3 monoclonal antibody in 37 patients with metastatic melanoma

Antitumor effects of antibodies against ganglioside antigens of melanoma have been reported, but neither optimal doses nor mechanisms have been established. This Phase IB trial of the murine immunoglobulin IgG(3) monoclonal antibody R(24) against disialoganglioside GD3 was conducted with 37 patients to define better the dose-response relation and mechanism of action of R(24) in patients with metastatic melanoma. Dose-limiting toxicity consisted of a pulmonary capillary leak syndrome in 3 of 5 patients in the 80 mg/M(2)/day dosage tier. Serial blood and tumor biopsy samples were obtained prior to therapy and on Days 5, 9, and 22 following R(24) infusion. Tumor biopsy-infiltrating lymphocytes were enumerated in peritumoral, endotumoral, and perivascular compartments: endotumoral CD4(+) and CD8(+) T cells and HLA-DR(+) T cells increased over time on R(24) antibody. Endotumoral CD4 lymphoid infiltrate activation (DR expression) and antibody-dependent cytotoxicity were the greatest in the one patient who achieved a complete response. Clinical response was associated with depression in natural killer (CD56(+) and CD56(+)DR(+)) blood cells (P = 0.03) and was associated with R(24) dosage (P = 0.01). A complete response that lasted 2 years and a partial response that lasted 2 months occurred at a dose of 1 mg/M(2)/day. The limited number of clinical responses observed in this trial hampered the correlation of antitumor and immune parameters but provided a rational foundation for the future evaluation of antiganglioside antibodies.

Tenascin-R interferes with integrin-dependent oligodendrocyte precursor cell adhesion by a ganglioside-mediated signalling mechanism.

Oligodendrocyte (OL) lineage progression is characterized by the transient expression of the disialoganglioside GD3 by OL precursor (preOL) cells followed by the sequential expression of myelin-specific lipids and proteins. Whereas GD3+ preOLs are highly motile cells, the migratory capacity of OLs committed to terminal differentiation is strongly reduced, and we have recently shown that the extracellular matrix protein tenascin-R (TN-R) promotes the stable adhesion and differentiation of O4+ OLs by a sulphatide-mediated autocrine mechanism (O4 is a monoclonal antibody recognizing sulphatides/seminolipids expressed by OLs and in myelin). Using culture conditions that allow the isolation of mouse OLs at distinct lineage stages, here we demonstrate that TN-R is antiadhesive for GD3+ preOLs and inhibits their integrin-dependent adhesion to fibronectin (FN) by a disialoganglioside-mediated signalling mechanism affecting the tyrosine phosphorylation of the focal adhesion kinase. This responsive mechanism appears to be common to various cell types expressing disialogangliosides as: (i) disialogangliosides interfered with the inhibition of cell adhesion of different neural and non-neural cells on substrata containing TN-R and FN or RGD-containing FN fragments. TN-R interacted specifically with disialoganglioside-expressing cells or immobilized gangliosides, and ganglioside treatment of TN-R substrata resulted in a delayed preOL cell detachment as a function of time. We conclude that OL response to one and the same signal in the extracellular matrix critically depends on the molecular repertoire expressed by OLs at different lineage stages and could thus define their final positioning.

The Role of Homophilic Binding in Anti-tumor Antibody R24 Recognition of Molecular Surfaces: DEMONSTRATION OF AN INTERMOLECULAR β-SHEET INTERACTION BETWEEN VH DOMAINS

The murine antibody R24 and mouse-human Fv-IgG1(kappa) chimeric antibody chR24 are specific for the cell-surface tumor antigen disialoganglioside GD3. X-ray diffraction and surface plasmon resonance experiments have been employed to study the mechanism of "homophilic binding," in which molecules of R24 recognize and bind to other molecules of R24 though their heavy chain variable domains. R24 exhibits strong binding to liposomes containing disialoganglioside GD3; however, the kinetics are unusual in that saturation of binding is not observed. The binding of chR24 to GD3-bearing liposomes is significantly weaker, suggesting that cooperative interactions involving antibody constant regions contribute to R24 binding of membrane-bound GD3. The crystal structures of the Fabs from R24 and chR24 reveal the mechanism for homophilic binding and confirm that the homophilic and antigen-binding idiotopes are distinct. The homophilic binding idiotope is formed largely by an anti-parallel beta-sheet dimerization between the H2 complementarity determining region (CDR) loops of two Fabs, while the antigen-binding idiotope is a pocket formed by the three CDR loops on the heavy chain. The formation of homophilic dimers requires the presence of a canonical conformation for the H2 CDR in conjunction with participation of side chains. The relative positions of the homophilic and antigen-binding sites allows for a lattice of GD3-specific antibodies to be constructed, which is stabilized by the presence of the cell membrane. This model provides for the selective recognition by R24 of cells that overexpress GD3 on the cell surface.

Variable region gene segments of nine monoclonal antibodies specific to disialogangliosides (GD2, GD3) and their O-acetylated derivatives.

Despite the weak immunogenicity of gangliosides, a limited number of highly specific murine monoclonal antibodies (MAbs) were elicited. This study investigated the reactivity and the structure of the VH and V kappa genes of nine hybridomas obtained from independent fusions producing antibodies against disialogangliosides GD2 and GD3 and their O-acetylated derivatives. These antibodies depended on four types of V kappa genes. They were also encoded by VH genes of the J558 family (5 out of 9) and occasionally by VH genes of the S107, 7183, and 3609 families, rearranged with a variety of DH and JH genes. The 8B6 and 7H2 MAbs specific for GD2-O-acetylated, respectively, used the VH gene of the S107 and 7183 families. The length of H chain CDR3 ranged from 8 to 11 amino acids. A set of S107 and 3609 germline genes closed from A/J murine fetal liver and matched with the VH segment of hybridomas 8B6 and 10B8 revealed somatic mutations. Although the relative number of sequences does not preclude any formal conclusions regarding the preferential use of V genes in the immune recognition of carbohydrate structures, our results clearly indicate that MAbs directed to very similar structures as GD2 and GD3 were encoded by different VH and V kappa genes.

Superantigen-induced lysis of melanoma cells.

Superantigens like the Staphylococcus enterotoxin A (SEA) can direct cytotoxic T lymphocytes expressing certain T cell receptor V beta regions to lyse MHC class II-positive target cells. This superantigen-dependent cellular cytotoxicity (SDCC) has been extended to MHC class II-negative tumour cells by targeting T cells via conjugates of a tumour-specific monoclonal antibody (moAb) and a superantigen. In the present study the MHC class II-negative human melanoma cell lines G361 and MaRI were tested for susceptibility to SDCC in vitro. Antibodies recognizing the disialoganglioside GD3 and the CD10 antigen were linked to SEA either by a recombinant protein A-SEA fusion protein or an anti-kappa moAb-SEA chemical conjugate. Specific lysis of melanoma cells was dose- and effector to target (E:T) cell ratio-dependent. Introduction of a point mutation into the SEA gene (producing SEAm9) in order to reduce MHC II affinity of the superantigen, which has already been shown to severely diminish superantigen-dependent binding and lysis of MHC class II-positive cells, did not influence antibody-targeted SDCC. Cytotoxicity was equal with both antibodies (anti-GD3 and anti-CD10) and independent of whether protein A-SEA, protein A-SEAm9 or anti-kappa-SEA were used.

Expression of GD3 disialoganglioside antigen on peripheral T-lymphocytes in patients with disseminated malignant melanoma.

Disialoganglioside antigens GD2 and GD3 are expressed on most melanoma cells. On melanoma surrounding T-cells in immunohistological sections, disialogangliosides can also be found, as well as in a small % of T-lymphocytes in peripheral blood from healthy persons. In order to find out if there is a difference in ganglioside expression on peripheral T-lymphocytes between melanoma patients and healthy persons, we examined the expression of CD3 as T-lymphocytic antigen and GD2 or GD3 antigens, respectively, by flow cytometry. We used peripheral mononuclear blood cells of 12 patients with advanced disseminated malignant melanoma and of 12 healthy control donors. For immunostaining, murine monoclonal antibodies Leu-4, 14G2a and MB3.6 were used, recognizing CD3, GD2 and GD3. GD2 expression was found on only a low proportion of T-lymphocytes in patients and healthy persons (pat.: mean = 1.2% +/- 0.7%, co.: mean = 0.4% +/- 0.4%). Disialoganglioside antigen GD3, however, could be demonstrated on an average of 8.4% +/- 4.6% of patients' and on 4.0% +/- 2.1% of healthy persons' T-cells. There is a statistically significant difference (P < 0.01) between the data of patients' and control group. We conclude that there is a correlation between advanced malignant melanoma and expression of GD3 antigen on patients' peripheral T-lymphocytes. The immunological relevance of our findings is discussed.

T cell activation via the disialoganglioside GD3: analysis of signal transduction.

The monoclonal antibody (mAb) R24 is a murine immunoglobulin G3 (IgG3) that reacts with the GD3 disialoganglioside present on melanoma cells as well as a subset of T cells. R24 mAb has induced antitumor responses both alone and in combination with interleukin-2 (IL-2) in clinical trials. We have reported T cell activation via GD3 as measured by the induction of tyrosine phosphorylation. In this study a more detailed analysis of signal transduction after ligation of GD3 was performed in an attempt to understand the mechanism of in vivo therapeutic benefits observed. Analysis of subsequent events indicated that GD3 engagement resulted in phospholipase C(gamma) phosphorylation and calcium flux. When ras-associated events were examined, GD3 signaling resulted in ras activation as determined by GDP/GTP conversion as well as dose-and time-dependent IP3 activation. In addition, the majority of the IP3 activation by GD3 was inhibited by herbimycin A pretreatment. Elucidation of the nature and potential role of this moiety in GD3 signal transduction should be useful. Collectively, these data suggest a novel mechanism of T cell activation via a single, non-protein, surface moiety. This novel form of T cell-mediated activation may permit the delivery and local activation of effector cells at the tumor resulting in site-specific activation of the immune system.

Anti-ganglioside antibodies in new onset type 1 diabetic patients and high risk subjects.

Insulin dependent (type 1) diabetes mellitus appears to be a genetically determined autoimmune disease. Gangliosides have been implicated in type 1 diabetes as antigenic determinants recognized by islet cell antibodies (ICA) and shown to be able to modulate autoimmune phenomena in experimental diabetes. In order to explore in type 1 diabetes the humoral immune reactivity against gangliosides, taking into account their pancreatic localization and molecular characteristics, antibodies to gangliosides GM3, GM2, GM1, GD3, GD1a, GD1b, and GT1b have been investigated in sera from new onset type 1 diabetics and relatives of type 1 diabetic patients with or without insulin (CIAA) and/or islet cell autoantibodies. Using a purposefully designed sensitive ELISA method we found that presence of antibodies directed against the pacreatic disialo-ganglioside GD3 in a significant percentage of newly diagnosed type 1 diabetics (p < 0.001 vs normal controls) but not in CIAA and/or ICA positive relatives of type 1 diabetics. These findings confirm the involvement of gangliosides in autoimmune phenomena related to type 1 diabetes and suggest disialo-ganglioside GD3 as target of a humoral immune response associated with the onset of insulin-dependent diabetes.

Potential to Involve Multiple Effector Cells with Human Recombinant Interleukin-2 and Antiganglioside Monoclonal Antibodies in a Canine Malignant Melanoma Immunotherapy Model

Human tumors originating from neuroectodermal cells such as malignant melanoma and neuroblastoma express high levels of disialogangliosides GD2 and GD3, making these antigens ideal for targeting by monoclonal antibodies (Mabs). The purpose of this study was to investigate expression and targeting of gangliosides on canine melanoma. Using immunohistochemical methods, we analyzed the expression of disialogangliosides GD2 and GD3 on canine oral malignant melanomas with murine Mabs 14.G2a and R24 that recognize GD2 and GD3 disialogangliosides, respectively, on human tumors. We also assessed the ability of Mab 14.G2a (and its mouse-human chimera, ch 14.18) to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro against a canine malignant melanoma cell line with human recombinant interleukin-2 (IL-2) activated canine peripheral blood lymphocytes (PBL), or canine neutrophil effector cells. Our data show that Mabs 14.G2a and R24 recognized fresh frozen canine oral melanoma. Mabs 14.G2a or ch 14.18, or IL-2, potentiated lysis of the canine malignant melanoma cell line by canine PBL. The killing effect observed using the combination of either Mab with IL-2 was additive. Mab 14.G2a mediated potent ADCC of canine melanoma by canine neutrophils. These studies indicate that disialogangliosides are expressed on fresh canine melanoma cells. Mabs reactive with these antigens can target and trigger tumor killing by multiple canine effector populations and IL-2 can potentiate these effects by canine lymphocytes. Thus, canine oral malignant melanoma, a spontaneously occurring, metastatic cancer in the dog, may be a relevant animal model to investigate combination immunotherapy using antitumor Mab and IL-2.

Characterization of a hamster melanoma-associated ganglioside antigen as 7-O-acetylated disialoganglioside GD3

We previously reported a hamster animal model of melanoma in which the tumor tissue expresses gangliosides GM3, GD3, and O-acetyl GD3. This ganglioside pattern is similar to that in human melanomas (Ren, S., A. Slominski, and R. K. Yu. 1989 Cancer Res. 49: 7051). In this study, we isolated and purified these gangliosides using chloroform-methanol extraction, Folch partition, chromatographies on DEAE-Sephadex A-25, and Iatrobeads columns. The yields of gangliosides GM3, GD3, and O-acetyl GD3 were 44.1 mg, 19.6 mg, and 9 mg per 100 g of Ma melanotic melanoma tissues, respectively. The structures of these gangliosides were characterized by periodate oxidation, gas chromatographic (GC) analysis, fast-atom bombardment-mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR) studies. The structure of hamster melanoma O-acetyl GD3 is different from the 9-O-acetyl GD3 previously reported in human melanoma. The major fatty acids of this ganglioside are C16:0, C18:0, C20:0, C22:0, and C24:0 and the long-chain base is C18-sphingosine.

S4.4 Selection of human melanoma variants showing concomitantly an increase in their metastatic potential and a defect in their biosynthesis of disialogangliosides GD3 and GD2

S4.4 Selection of human melanoma variants showing concomitantly an increase in their metastatic potential and a defect in their biosynthesis of disialogangliosides GD3 and GD2

Expression of disialogangliosides GD2 and GD3 on human soft tissue sarcomas.

Fifty-six freshly frozen human sarcomas were studied for expression of disialogangliosides by avidin-biotin immunohistochemical staining with purified monoclonal antibodies (MoAb) 3F8 (antidisialoganglioside GD2) and R24 (antidisialoganglioside GD3). Ninety-three percent of the tumors tested by the immunohistochemical staining expressed GD2 and 88% expressed GD3. The intensity of expression varied among sarcomas of different histologic types. Liposarcoma, fibrosarcoma, malignant fibrous histiocytoma, leiomyosarcoma, and spindle cell sarcoma reacted strongly with 3F8 and R24. Embryonal rhabdomyosarcoma and synovial sarcoma demonstrated substantially weaker staining by either MoAb. Weakly reactive or nonreactive tumors were, in general, high-grade or metastatic sarcomas. Ganglioside extraction and thin-layer chromatography/immunothin-layer chromatography of two liposarcomas confirmed the identities of these gangliosides. GD2 and GD3 may indicate sites for MoAb-targeted imaging and therapy for sarcomas. The association of a diminished stainability by 3F8 and R24 with aggressive sarcomas may indicate prognostic significance.

Tissue distribution of GD3 ganglioside detected by mouse monoclonal antibody R24.

Mouse monoclonal antibody (MoAb) R24 detects the disialoganglioside GD3, a neuroectodermal antigen expressed by normal melanocytes and melanomas. This antibody has been used to define the immunohistochemical distribution of GD3 antigen in normal and malignant human tissues. The present study shows that staining with MoAb R24 in normal organs and tissues where low concentrations of the antigen can be detected. The highest expression of GD3 was detected on the surface of melanoma cells. Therefore, MoAb R24 can be a useful reagent for studying cells of melanocytic lineage, and for imaging and therapeutic studies of melanomas.

Serological response of non-human primates to human melanoma disialoganglioside GD3.

The immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of greater than 2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer = 640) while having only marginal reactivity with GM3 (titer = 40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.

Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells.

The identification of specific cell surface glycoprotein receptors for Arg-Gly-Asp-containing extracellular matrix proteins such as fibronectin has focused attention on the role of gangliosides in this process. Is their involvement dependent or independent of the protein receptors? In attachment assays with cells from a human melanoma cell line, titration experiments with an antibody (Mel 3) with specificity for the disialogangliosides GD2 and GD3, used together with a synthetic peptide containing the cell binding sequence Arg-Gly-Asp, show that their joint effect is synergistic. Both the Mel 3 antibody and the synthetic peptide individually cause rapid detachment of melanoma cells from fibronectin substrate but, when used together, much smaller concentrations of both are required to achieve the same effect. The Mel 3 antibody was not nonspecifically reducing receptor binding to the Arg-Gly-Asp sequence since, in binding assays with radiolabeled peptide performed with cells in suspension, very little peptide is bound by the melanoma cells under these conditions but addition of Mel 3, an antibody of IgM isotype, causes a two- to threefold increase in specific binding. The simplest interpretation of these data is that the Mel 3 antibody is causing sufficient clustering of membrane gangliosides in local areas and producing a favorably charged environment to facilitate peptide binding by specific glycoprotein receptors.

Differentiation of bipotential glial precursors into oligodendrocytes is promoted by interaction with type-1 astrocytes in cerebellar cultures.

The differentiation of bipotential precursors of oligodendrocytes (OL) and type-2 astrocytes (AS) was followed in primary cultures from 8-day postnatal rat cerebellum by labeling the cells with the antibodies LB1 (which binds to the surface disialoganglioside GD3 present in glial precursors, type-2 AS, and immature OL), O4 (a marker of immature and mature OL binding to surface sulfatide), anti-galactocerebroside (GalCer, a marker of OL), and anti-glial fibrillary acidic protein (GFAP, a marker of AS). Two hours after plating, hardly any LB1+, GFAP+ cells were detectable, 40% of the O4+ cells were GalCer+, and none of the O4+ cells were GFAP+. Upon culturing cells plated at a density of 1 x 10(5) cells per cm2 in the presence of fetal calf serum, most of the LB1+ precursors differentiated into type-2 AS, even if most of them had already expressed the O4 antigen. Thus, in culture, most type-2 AS seem to derive from progenitor cells that were differentiating in vivo into OL. In higher density cultures (2.5 x 10(5) cells per cm2), however, many precursors differentiated into GalCer+ OL, rather than into AS. As a possible source of the signals responsible for the behavior of the glial precursors in high-density cultures, we focused our attention on type-1 AS, the most abundant cell type in the cultures. We found that, in low-density cultures maintained for 5-7 days in a medium conditioned by type-1 AS, the proliferation of the precursors was enhanced and their differentiation into OL or AS was prevented. In contrast, when cerebellar cells were coplated with type-1 AS dissociated from purified cultures, not only did the precursors proliferate more than in control cultures, but also a larger proportion of them differentiated into GalCer+ OL. In conclusion, type-1 AS appear to facilitate the differentiation of bipotential glial precursors into OL through direct cell-cell interactions. The influence of type-1 AS on the differentiation of the LB1+ and O4+ precursors is supported also by experiments with glial cortical cultures.

Detection of GD3 ganglioside in childhood acute lymphoblastic leukemia with monoclonal antibody to GD3: Restriction to immunophenotypically defined T‐cell disease

We have recently reported that the disialoganglioside GD3 is found in cellular lipid extracts of T-cell acute lymphoblastic malignancies (T-ALL) but is not detectable by resorcinol staining in extracts of non-T acute lymphoblastic leukemia blasts (non-T-ALL). We have now extended this study to assess the detectability of GD3 in T-ALL vs non-T-ALL utilizing an anti-GD3 antibody, R24. Gangliosides isolated from T-ALL and non-T-ALL blasts by two different methods were separated by thin-layer chromatography and stained with anti-GD3 and a control antibody specific for GM3 and sialosylparagloboside (SPG). Anti-GD3 reactivity was observed in extracts from T-ALL cells in all cases, whereas GD3 was not detected in any of the non-T-ALL samples. The anti-GM3/SPG antibody stained GM3 in all of the leukemic samples analyzed as well as SPG in the non-T-ALL samples. Indirect immunofluorescence was used to assess the expression of GD3 at the surface of leukemic blasts. Fluorescence-activated cell sorting analysis with R24 showed that whereas T-ALL blasts were highly reactive with this antibody, non-T-ALL blasts were totally unreactive. In an analysis of a larger number of leukemia patients by fluorescence microscopy, 20 out of 28 samples with the T-ALL phenotype were positive for R24, whereas zero out of 11 non-T-ALL samples were reactive. These results confirm our earlier finding of the specificity of GD3 to the T-ALL subclass of childhood leukemias and furthermore suggest the potential value of anti-GD3 as an immunological tool for the diagnosis and therapy of T-cell ALL.

Enzymatic basis for increased expression of GD3 on human melanoma cells derived from metastatic lesions

AbstractThe expression of disialoganglioside GD3 is examined in human melanoma cell lines derived from primary (WM 115 and WM 75) or metastatic (WM 266 and WM 373) lesions resected from the same two patients. GD3 is demonstrated to be preferentially expressed by the metastatic cell lines relative to their primary cell line counterparts as shown by thin layer chromatography and flow cytometric analysis. The enhanced expression of GD3 in the metastatic cell lines is due to the increased activity of its biosynthetic enzyme, cytidine‐5′‐monophospho‐N‐acetylneuraminic acid:GMS sialyltransferase (GD3 synthetase), as evident from assays of GD3 synthetase activity of these cell lines. The activity of this enzyme in membrane extracts of all 4 cell lines is dependent on protein concentration and time, with peak activity occurring after 30 min. GD3 synthetase in both WM 115 and WM 266 exhibits a relatively broad pH optima between 6.2 and 6.6, is stimulated by Mn2+ and Mg2+, and inhibited by EDTA and Hg2+. Subcellular fractionation indicates that GD3 synthetase activity is localized primarily within the Golgi‐enriched fraction. The enhanced expression of GD3 on the metastatic melanoma cell lines WM 266 and WM 373 relative to that of their primary cell line counterparts cannot be attributed to blocked synthesis of other gangliosides utilizing GD3 as a precursor, but is primarily due to the increased activity of GD3 synthetase.

Immune and nonimmune effector functions of IgG3 mouse monoclonal antibody R24 detecting the disialoganglioside GD3 on the surface of melanoma cells

R24, an IgG3 mouse monoclonal antibody reactive with the disialoganglioside GD3, was found to be a potent mediator of human complement cytotoxicity and human effector cell cytotoxicity. Cytotoxicity correlated with the degree of antibody binding (GD3 cell surface expression) for each of the melanoma cell lines and melanocyte cell cultures tested. Melanoma cell lines binding low amounts of R24 (low GD3 cell surface expressors) were not lysed in R24-directed immune reactions, suggesting that a threshold number of R24 molecules bound per cell is necessary to initiate these cytotoxic mechanisms. Since both complement- and cell-mediated reactions lysed the same subpopulations of cells in each cell line, both mechanisms appeared to depend on similar threshold quantities of bound R24 molecules. However, due to the heterogeneity of R24 binding in each cell line, the numerical value for this threshold could not be determined. Only in cell lines binding >10 7 R24 molecules per cell were >90% of the cells lysed. Normal melanocytes in culture were not lysed by R24-directed immune mechanisms, due to their low GD3 expression, indicating that monoclonal antibodies such as R24 may show tumor specificity with regard to effector functions even though normal cells express the relevant antigen. In contrast to the potent in vitro activity of R24, treatment of nu nu mice bearing human melanoma grafts resulted in tumor inhibition only when started within 3 days of tumor cell inoculation. No effect was seen on established tumors. Thus, this in vivo mouse model failed to predict the clinical and pathological findings observed in treatment trials of R24 in human melanoma patients—urticaria involving skin metastases, cellular infiltration of tumor tissue, and tumor regression. In addition to activating immunologic effector functions, R24 had direct effects on melanoma cells, blocking their ability to attach to surfaces and causing tumor cell aggregation. These effects were again related to the number of R24 molecules bound to the cell surface; no aggregation was seen with cell lines binding less than 4 × 10 5 molecules per cell. Both immune and nonimmune effector functions may be involved in the tumor inhibitory activity of R24 in humans.

An Arg-Gly-Asp-directed receptor on the surface of human melanoma cells exists in an divalent cation-dependent functional complex with the disialoganglioside GD2.

The disialogangliosides GD2 and GD3 play a major role in the ability of human melanoma cells to attach to Arg-Gly-Asp-containing substrates such as fibronectin and vitronectin, since pretreatment of these cells with monoclonal antibodies to the oligosaccharide of GD2 and GD3 can inhibit their attachment and spreading on such adhesive proteins. This report demonstrates that human melanoma cells (M21) synthesize and express a glycoprotein receptor that shares antigenic epitopes with the vitronectin receptor on human fibroblasts and is capable of specifically recognizing the Gly-Arg-Gly-Asp-Ser-Pro sequence. In the presence of calcium, GD2, the major ganglioside of M21 cells, colocalized with this receptor on the surface of human melanoma cells and their focal adhesion plaques as demonstrated by double-label transmission immunoelectron microscopy and indirect immunofluorescence. Biochemical evidence is presented indicating that the vitronectin receptor on M21 human melanoma cells contains associated calcium and GD2. This ganglioside copurified with the glycoprotein receptor for vitronectin on affinity columns containing either an Arg-Gly-Asp-containing peptide, concanavalin A, or lentil lectin. This major Arg-Gly-Asp-directed receptor on M21 cells could be metabolically labeled with 45Ca2+. Chelation of this ion with EDTA caused the dissociation of GD2 from the receptor and rendered the remaining glycoprotein incapable of binding to an Arg-Gly-Asp-containing peptide. Reconstitution experiments demonstrated a requirement for calcium, and not magnesium, for receptor binding to Arg-Gly-Asp and indicated that addition of ganglioside can enhance this interaction.