Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen I (OFA-I)

Summary Oncofetal antigen I (OFA-I) has been identified by immunofluorescence and immune adherence (IA) as a membrane antigen on human tumor cells, which cross-reacts with fetal brain tissue. This antigen induces humoral antibody in patients with cancer. The present work was designed to evaluate the complement-dependent cytotoxic potential of anti-OFA-I antibody produced in melanoma patients against an OFA-I-positive melanoma cell line, UCLA SO M14 (M14). Patients' sera were chosen on the basis of anti-OFA-I activity by IA. Alloantibodies to M14 were removed by absorption of the sera with lymphoblastoid cells autologous to M14. Fourteen sera were tested, and all demonstrated cytotoxic activity in the presence of rabbit complement. Human complement was also shown to mediate cytotoxicity, although less effectively than rabbit complement. The specificity of the reaction was confirmed when the cytotoxic capability could be absorbed by fetal brain, but not by autologous fetal liver tissues. These results indicate that a patient's serum antibody to OFA-I can lyse tumor cells expressing this antigen.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

Serologic analysis of human tumor antigens

Summary The immune adherence (IA) assay was used to measure serum reactivity of patients with melanoma and osteosarcoma against paired cell lines (tumor cells and normal skin fibroblasts obtained from the same individuals) grown in tissue culture. Sera from 224 patients with various stages of melanoma were compared with sera from 100 normal age- and sex-matched donors. None of the 18 stage I sera (0%), 23 of 166 (14%) stage II sera, 3 of 40 (7%) stage III sera, and 3 of 100 (3%) normal sera were highly reactive to a standard allogeneic melanoma-fibroblast pair. None of the sera exhibited unique activity against melanoma. There was no correlation between stage of melanoma and high serum reactivity, nor was this reactivity predictive of recurrence. Sera from 39 tumor-bearing osteosarcoma patients prior to amputation were compared with sera from 50 normal age-and sex-matched donors. Eight of 39 (21%) patient sera and 1 of 50 (2%) normal sera were highly reactive to an osteosarcoma-fibroblast pair. No sera had reactivity uniquely directed against osteosarcoma. Eight osteosarcoma and two melanoma patients were tested simultaneously against their autologous cultured tumor and skin cells. Only one of these patients exhibited high reactivity towards autologous cells, and this reactivity was equal against both osteosarcoma and normal cells. None of seven highly reactive osteosarcoma or six highly reactive melanoma sera had residual tumor-specific reactivity against allogeneic osteosarcoma or melanoma after absorption with cultured fibroblasts, cultured fetal fibroblasts, or fetal calf serum.     

Functional consequence of variation in melanoma antigen expression

Summary Melanoma cells have been shown to express melanoma-associated antigens and, in many cases, the histocompatibility antigen, HLA-DR. We questioned whether the expression of these antigens was quantitatively altered during the serial passage of melanoma cells in culture. Therefore, we measured the binding of monoclonal antibodies specific for a melanoma-specific antigen and the HLA-DR antigen to melanoma cells from serial passages. Three cell lines were studied. We found that although both the melanoma-associated antigen and the HLA-DR antigen were qualitatively conserved, significant quantitative differences were seen. To study the functional consequences of these differences, we used fluorescence-activated cell sorting to create DR-enriched and DR-depleted populations from a single melanoma cell line heterogeneous for DR expression. We found that the proliferation of allogeneic T cells (measured by the ³H-TdR uptake) cultured with the DR-enriched and -depleted melanoma cell populations was directly related to the amount of the HLA-DR antigen expressed. These results indicate that in performance of experiments using melanoma cell lines quantitative assessment of antigenic expression is important, particularly if the function of a specific antigen is under examination. Further, our data clearly identify the HLA-DR antigen on melanoma cells as a participant in allogeneic lymphocyte stimulation. Abbreviations used are: FACS, fluorescence activated cell sorter; FITC, fluorescein isothocyante; ³H-TdR, tritiated thymidine     

Oncofetal antigen-I (OFA-I) relative antigenicity on melanoma cells

Summary The oncofetal antigen-I (OFA-I) has been defined as an immunogenic antigen that is expressed on human cancer cells and is cross reactive with fetal brain tissue. Quantitative variations in the expression of OFA-I among different cultured melanoma cell lines were determined by absorption techniques based on functionally monospecific anti-OFA-I serum. Allo-antibodies were removed by absorption with lymphoblasts autologous to an OFA-I-positive target cell. Functional monospecificity toward OFA-I was confirmed by complete absorption with a specimen of fetal brain but not by liver from the same fetus.Of 14 melanoma cell lines tested, two did not express OFA-I, whereas 12 expressed the antigen to varying degrees. Five of the cell lines were highly antigenic, and serum absorbed with 5×10⁵ of any of these cell lines could reduce the anti-OFA-I titer (1 : 96) at least four-fold. OFA-I was detected on biopsied melanomas autologous to the antigenic cultured cells. The ability to select highly antigenic cell lines could be useful in further attempts to characterize OFA-I and to monitor tumor immunity in vitro. Antigenic cell lines may improve the response of patients treated in trials of immunotherapy.     

Recovery of a cell surface fetal antigen from circulating immune complexes of melanoma patients

Summary A well-characterized 69.5×10³ dalton glycoprotein fetal antigen (FA), isolated from the spent culture medium of a melanoma cell line, UCLA-SO-14 (M14), was utilized to characterize the antigen component of circulating immune complexes (CIC) from melanoma patients. Ten serum samples from five patients with stage II melanoma at 1 and 4 months prior to the clinical detection of recurrent disease were selected for study. The CIC were dissociated with low pH and ultrafiltered through a 100¢10³ dalton exclusion limit membrane. The low pH treatment resulted in an increase in antibody titer in eight of ten serum samples. The antibody activity in membrane immunofluorescence was quantitatively inhibited by the filtered antigen fraction and purified FA, suggesting the presence of anti-FA antibodies in the treated serum, which possibly were complexed with FA in the untreated sample. As determined by competitive inhibition in an enzyme-linked immunosorbent assay, the filtrate (antigen fraction) contained an antigen that was immunologically similar to FA. These results clearly demonstrate that FA, expressed on the cell surface of melanoma cells, is present in CIC of selected melanoma patients.Supported in part by NIH grants CA 30019, CA 12582, CA 09010, CA 29605 awarded by DHSS     

Circulating immune complexes as possible cause for anticomplementary activity in humans with malignant melanoma

Summary Sera from 98 melanoma patients, 20 noncancer patients with immune complex-associated diseases, and 90 normal donors were analyzed for anticomplementary (AC) activity by the complement consumption method. Some of these sera were also tested for immune complex-like materials by the Raji cell radioimmune assay. In addition, serum samples from ten melanoma patients were analyzed serially to correlate the AC activity with clinical course. Significant levels of Ac activity were found in 45% of melanoma sera, 75% of nonmalignant immune complex-associated disease sera, and 10% of normal donors' sera. In some patients, AC activity decreased and became undetectable as their disease progressed. AC-negative serum samples taken from melanoma patients late in the course of disease when the tumor burden was large became anticomplementary when mixed with autologous or allogeneic serum samples taken earlier at the time of little or no tumor burden. The early serum samples contained antibodies against autologous tumor extracts, as shown by a complement fixation test. Absorption of early serum samples with cultured allogeneic melanoma cells reduced their ability to consume complement when mixed with autologous late serum samples, suggesting the presence of free antigen in the latter. The mixed samples of early and late sera and the sera positive in the complement consumption test contained heavy nonmonomeric IgG. Therefore, the AC activity of melanoma sera could be due to tumor-associated antigen-antibody complexes.     

Nature of adult T-cell leukemia-associated membrane antigen on the surface of adult T-cell leukemia virus-carrying cells as revealed by membrane immunofluorescence

Adult T-cell leukemia-associated membrane antigen (ATLMA) expressed on the surface of living ATL virus (ATLV)-carrying cells was investigated by an indirect membrane immunofluorescence method using natural antibodies to ATLV in human sera. All the ATLV-positive cell lines tested that had cytoplasmic ATL-associated antigen (ATLA) detectable in acetone-fixed cell smears were also positive for ATLMA, but ATLMA was not detected in any ATLV-negative cell lines. The frequencies of ATLA- and ATLMA-bearing cells in seven cell lines tested were roughly parallel. The frequency of expression of both ATLMA and ATLA in cultures of MT-1 cells increased in the presence of 5-iodo-2'-deoxyuridine. All human sera having ATLA antibody had ATLMA antibody and the titers of the two were similar in most of the sera. The anti-ATLMA titers of human sera determined by using an ATLV-bound non-ATL T-cell line as antigen were also similar to the anti-ATLA titers. Absorption of anti-ATLMA-positive sera with living MT-2 cells, in which almost 100% of the cells express ATLA and ATLMA, caused parallel decreases in the anti-ATLA and anti-ATLMA titers. Analysis of the 125I-labeled surface of MT-2 cells by immunoprecipitation with anti-ATLMA-positive human serum followed by gel electrophoresis revealed that p19, p24, p28, and p46 polypeptides were specifically precipitated. These data suggest that ATLMA on the cell surface is not distinguishable from ATLA in the cytoplasm.     

Tissue Distribution of an Immunogenic Melanoma Associated Antigen,D-1

A human melanoma-associated antigen immunogenic in patients was recently identified by screening an expression cDNA library constructed from cultured human melanoma cell line with sera from patients with melanoma. The nucleic acid sequence of the cloned D-1 cDNA has no significant homology with previously reported mammalian genes. The cDNA D-1 encodes a peptide of about 37 kDa, which showed fivefold higher reactivity with sera from patients with melanoma than with sera from normal donors. In order to detect D-1 gene expression in vivo, in-situ hybridization and immunostaining with cRNA probe and murine anti-D-1 sera were carried out on surgically removed tissues. Digoxigenin-labeled cRNA D-1 was exclusively hybridized with mRNA in the cytoplasm of melanoma cells but not with keratinocytes and fibroblasts adjacent to melanoma nests. Polyclonal anti-D-1 antibodies were obtained by immunization of Balb/c mice with recombinant D-1 peptide and clearly reacted with melanoma cells but not with keratinocytes and fibroblasts, similar to the results of in-situ hybridization. The above information will help to assess the suitability of recombinant D-1 peptide to implement active specific immunotherapy in patients with melanoma.     

Complement-dependent lysis of tumor cells by a baboon IgM antibody to a tumor-associated antigen

Summary We developed a high-titer polyclonal antiserum to a glycoprotein tumor-associated antigen (TAA) by immunization of a baboon with the purified glycoprotein antigen. The baboon serum was fractionated into IgG and IgM components by DEAE Affi-Gel blue chromatography. The ability of the baboon IgM anti-TAA antibody to effect tumor cell lysis in the presence of complement was tested using a chromium-release assay. The baboon antibody was able to lyse melanoma target cells (20.8%–71.4% cytolysis), breast carcinoma cells (36.5%–38.9% cytolysis), and a neuroblastoma cell line (35.5% cytolysis) in the presence of complement but did not effect significant lysis of autologous lymphoblastoid cell lines (4.9% cytolysis) or peripheral blood lymphocytes from healthy volunteers (12.6% cytolysis). Cytolysis of melanoma target cells was completely inhibited by preabsorption of the IgM anti-TAA antibody with UCLA-SO-M14 (M14) cells and partially inhibited by preabsorption with several other melanoma cell lines. There was no significant inhibition of tumor cell lysis after preabsorption of the antibody with lymphoblastoid cell lines. Complement-dependent lysis of M14 targets could be blocked by addition of the purified antigen to the antibody prior to incubation with the tumor cells. Our results suggest that the glycoprotein TAA resides on the tumor cell surface and that the baboon IgM anti-TAA antibody recognizes the antigen on the cell surface and is able to fix complement and effect the lysis of the tumor cells.     

Immunopurification, characterization, and nature of membrane association of human melanoma-associated oncofetal antigen gp87 defined by monoclonal antibody 140.240

A melanoma-associated oncofetal antigen, gp87 (a p97-like molecule), defined by the monoclonal antibody (MoAb) 140.240 has been purified to homogeneity from the spent medium of cultured melanoma cells by a two-step immunoadsorbent procedure. The first immunoadsorbent step using glutaraldehyde-insolubilized MoAb 140.240 (ascites fluid) resulted in a 13-fold enrichment with 93% recovery in the bound material. In the second immunoadsorbent step constructed by the purified IgG2a of MoAb 140.240 (culture fluid) coupled to CNBr-activated Sepharose 4B the bound material from the first step was further purified resulting in a 330-fold purification with 90% recovery. SDS-polyacrylamide gel electrophoretic analysis of the final purified material revealed a single band migrating as a polypeptide with an approximate molecular weight of 87 Kd, consistent with the size of the molecule immunoprecipitated by MoAb 140.240 from lysates of radiolabelled melanoma cells. Preliminary amino acid analysis indicates a particularly high proportion of phenylalanine in gp87. We have also compared gp87 with two well defined antigens, HLA-A,B,C (integral membrane protein) and "94K" melanoma/carcinoma-associated antigen (peripheral membrane protein) with respect to antigen extractability from melanoma cells using phosphate-buffered saline, 0.1 M urea, 3 M NaCl, or nonionic detergent (NP-40). The results showed that whereas 94K antigen was extractable by each of the four different solutions, gp87, similar to HLA-A,B,C antigens, could only be extracted with NP-40, strongly suggesting that gp87 is an integral melanoma cell component.     

Circulating melanoma-associated antigen detected by monoclonal antibody NKI/C-3

Summary NKI/C-3 and NKI/black-13 are monoclonal antibodies recognizing different epitopes on a melanoma-associated antigen that is preserved after fixation in formalin and embedding in paraffin in virtually all melanoma tissues. The antigen, a predominantly cytoplasmic vesicle membrane-bound heterogeneous glycoprotein of 25–110×10³ daltons, was shown to be a single 25×10³ dalton polypeptide when incorporation of N-linked carbohydrates was inhibited by tunicamycin. The antigen was measured in a double determinant enzyme immunoassay (DDEIA) using NKI/C-3 as catcher antibody. Results from in vitro experiments indicated that the antigen is actively shed from living cells. In sera from melanoma patients with a small tumor burden, the antigen concentrations were in the range of those of controls (0–22 U/ml). Significantly increased values (33–600 U/ml) were found in sera from patients with a moderate or large tumor burden. The antigen concentrations in sera from patients with multiple metastases of other tumors were within the range of controls. Several sera from patients with multiple metastases of colon, pancreatic, and stomach carcinoma, however, contained increased antigen concentrations (45–80 U/ml). These results correspond with the reactions of NKI/C-3 in tissue sections of some malignancies other than melanoma.During the follow-up of melanoma patients the concentrations of circulating antigen correlated with tumor progression. The predictive value of the NKI/C-3 assay was no better than determination of serum lactate dehydrogenase, alkaline phosphatase or gamma glutamyl transferase activity.     

Membrane associated antigens of human malignant melanoma V: Serological typing of cell lines using antisera from nonhuman primates

Summary Antisera against various melanoma cell lines were raised in nonhuman primates (Cercopithecus aethiop.). After exhaustive absorption with AB Rh + red blood cells and pooled platelets from about 200 donors the sera were still reactive to various degrees in the microimmune adherence test with other melanoma lines, with embryonic fibroblasts, and with non-melanoma lines. As proven by absorption experiments, the main-specificity of the antisera was not directed against components of the fetal calf serum used for cell culture or against mycoplasma grown from commercial fetal calf serum. In addition, no cross-reactivity was observed with Bacillus Calmette-Gérin, and in blocking experiments no reactivity against extracts of common bacterial antigens or mixed molds was detected. Absorption with embryonic fibroblasts or embryonic tissue showed that the reactivity of most antisera was directed against melanoma-associated antigens expressed also on fetal tissue. It was not possible to determine whether the remaining reactivity on some cell lines was melanoma-specific or directed against fetal antigens not contained in the fetal material used for absorption. Cross-absorption of antisera with other melanoma cells revealed that various cell lines express different patterns of tumor-associated antigens with no, or only partial, overlap. The cross-absorption experiments made it possible to type the cell lines according to their surface antigens and arrange the cell lines in order according to the degree of mutual antigenic relationship.     

Membrane-associated antigens of human malignant melanoma IV: Changes in expression of antigens on cultured melanoma cells

Summary Established melanoma cell lines were cultured for one passage (approximately 1 week) in different lots of fetal calf and new born calf sera and then tested against a panel of previously positively reacting sera from melanoma patients and polyspecific HL-A alloantisera. Using indirect immunofluorescence the cells showed varying degrees of reactivity ranging from positive to negative reactions depending on the supplementing serum in the culture medium. When standardized culture conditions were used and the cells were tested by immune adherence at several weeks intervals against panels of sera from melanoma patients, from tumor patients other than melanoma, from pregnant women, and from normal donors, most of the sera reacted identical, but some sera not only had changed quantitatively but also qualitatively from a negative to a positive reaction and vice versa indicating a shift in the spectrum of expressed antigens. When single cell clones from a cell line were isolated and tested against a panel of antisera, striking differences in reactivity were observed suggesting that the shift in the spectrum of expressed antigens was due to the outgrowth of dominating subclones with antigen patterns different from the previously dominating subclones. This conclusion was further supported by experiments in which a weakly positive reacting serum was employed to separate a cell line into positively and negatively reacting sublines. Unit gravity sedimentation and density gradient sedimentation were used in order to separate rosetted from non-rosetted tumor cells which had been prepared by immune adherence. It is concluded that cultured cell lines are in a dynamic state and that differentiation is one of the major mechanisms accounting for a change in antigen expression.     

Phase II study of vaccinia melanoma cell lysates (VMCL) as adjuvant to surgical treatment of stage II melanoma

Summary Patients with stage II melanoma were vaccinated with vaccinia virus-induced melanoma cell lysates (VMCL). The vaccine contained viable vaccinia virus, membranous fragments and no intact nuclei. A number of antigens defined by monoclonal antibodies were detected in the vaccine including the ganglioside GD3 and DR antigens. Administration of the vaccine was associated with depression of natural killer cell activity against melanoma and K562 target cells in the first 3–6 months of treatment. Leucocyte dependent antibody (LDA) activity against melanoma cells was induced or increased in titre in approximately half of the patients studied. Continued vaccination was associated in a number of patients with a decrease in LDA titres. Studies on a small sample of patients revealed that this was associated with the development of serum factors which inhibited LDA activity. LDA activity appeared directed to non-MHC antigens on melanoma cells which were of at least two specificities. One specificity which was shared with antigens on a number of non-melanoma carcinoma cells was removed by absorption on fetal brain and may be similar to oncofetal antigens described by other workers. Reactivity against melanocytes was induced in some patients and may underlie the development of vitiligo in several patients. These results suggest that vaccines prepared from VMCL may be a favourable method for increasing immune responses against melanoma.     

Serum antibodies to human fetal antigens in patients with systemic lupus erythematosus (SLE)

Serum antibodies to human fetal antigens were measured by a radiolabeled anti-immunoglobulin binding assay by using human fetal fibroblasts (Flow cell line No. 1000) as target cells. High titers of IgG antibody to the fetal cells were found in sera of patients with systemic lupus erythematosus (SLE). The antibody reacted with surface membrane antigens shared by various fetal tissues of human and murine origin but not by adult tissues. The reaction of the SLE antibody to the fetal cells was inhibited by heterologous antiserum to the Flow 1000 cells and antiserum to murine embryonic fibroblasts, but not by antiserum to human alpha-fetoprotein or human fibronectin. Absorption of SLE serum with isolated nuclei did not abolish the reaction indicating that these were not anti-nuclear antibodies. The antibody activity was found to reside in the F(ab')2 fragment. The serum titer of the anti-fetal antibody was higher in SLE patients with active disease than those in clinical remission.     

Expression of Thy 1 thymocyte differentiation antigen in mouse mammary cancer cells in vitro

Allogeneic AKR-anti C3H Thy 1.2 antigen serum and monoclonal anti-Thy 1.2 and anti-Thy 1.1 antigen antibodies were used to study the expression of lymphocyte differentiation antigen in a clonal mammary carcinoma cell line originated from a GR/mt stable cell line. Both allogeneic antiserum and monoclonal anti-Thy 1.2 (but not Thy 1.1) antibody were active with the hormone-treated fixed cells in an indirect immunofluorescence test. However, antigen on the cellular membranes could be detected only with the use of allogeneic (but not with monoclonal antibody) anti-Thy 1.2 serum.     

Natural antibody to a human bladder carcinoma cell line

Summary Sera from 144 healthy blood donors were screened for the presence of antibody against the bladder carcinoma cell line RT4 by means of an immune adherence assay. Only one serum (9241) demonstrated high activity with a titer of 1/124. Qualitative and quantitative absorption analyses were carried out with a wide variety of cells to characterize the specificity of the reaction. Cells used for absorption were both cultured and non-cultured and normal and malignant. The reactivity was absorbed by the bladder cancer cell lines RT4 and 5637 and the lung cancer cell line SK-LC-LL. In addition, surgically obtained urothelium from a patient with transitional cell carcinoma abrogated reactivity. No other absorbing cells removed the reactivity of serum 9241 for RT4. The antigen defined by this serologic reaction is heat-stable and trypsin-resistant, as demonstrated by using heated or trypsinized RT4 cells for absorption.This study shows that naturally occurring antibody directed toward cell surface antigens of tumor cells is present in some normal individuals. This finding is important in the evaluation of a possible causal relationship between the presence of antibody specific for tumor cell antigens and the development of neoplasia.     

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

Summary 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 >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.Supported in part by grant CA32672 from the National Cancer Institute, Veterans Administration Program 821 and by the Yerkes Regional Primate Center, Atlanta, Georgia. The Yerkes Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care     

Higher cytolytic efficiency of an IgG2 alpha than of an IgG1 monoclonal antibody reacting with the same (or spatially close) determinant on a human high-molecular-weight melanoma-associated antigen

Serological and immunochemical assays showed that the monoclonal antibody (MoAb) 225.28S, an IgG_2α, and the MoAb 653.40S, an IgG₁, react with the same (or spatially close) antigenic determinant expressed on a set of molecules carrying a high-molecular-weight human melanoma-associated antigen. Neither monoclonal antibody mediates complement-dependent lysis of cultured melanoma cells, but both of them specifically mediate lysis of target cells in an antiglobulin cytotoxic assay and in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In the latter two assays the IgG_2α displays a higher lytic activity than the IgG₁. The differential lytic activity of the IgG_2α and IgG₁ monoclonal antibodies was detected also when the sensitivity of the ADCC assay was increased either by boosting the cytolytic activity of the effector cells or by enhancing the susceptibility to lysis of target cells.