Knowledge of tumor antigenic expression is crucial to the design of therapeutic strategy. A murine monoclonal antibody (BE4) against a human melanoma membrane antigen, was used to study the in vitro expression of this antigen. By membrane immunofluorescence, BE4 reacted against 5 of 8 melanoma lines as compared to zero of 13 other cell populations. Using flow cytometry, the antigenic M14 CEM melanoma cells consisted of 40% to 60% of the total cell population. Dual-parameter measurements of DNA content and membrane antigen demonstrated that the nonantigenic cells were predominantly in G0/G1 phase, whereas the antigenic cells were distributed throughout the cell cycle. Within one passage, the sorted and recultured nonantigenic population demonstrated a similar proportion of antigenic cells as the unsorted original population. It was concluded that the expression of human melanoma antigen was cell-cycle-dependent. Understanding factors that turn off the expression of antigen in G0/G1 phase may lead to better immunotherapeutic strategies.

A human melanoma cell line, M14 , adapted to grow in serum free synthetic media was examined for its expression and secretion of several serologically defined melanoma associated antigens (MAA) previously described in this laboratory. Melanoma associated antigen expression and secretion was identical to that of M14 cells grown in parallel in serum supplemented medium. Spent synthetic media was found to be an enriched serum free source for the initial isolation of 100 kilodalton secreted glycoprotein MAA. M14 melanoma cells grown in synthetic media were also shown to be adaptable to the double agar clonogenic assay facilitating the examination of clonal heterogeneity in functional studies of MAA in melanoma tumor biology. Recent investigations from this laboratory have focused on characterizing human melanoma associated antigens (MAA) found either as secreted or cell surface associated glycoproteins in human melanoma cell lines. In these studies, monoclonal and polyclonal antiserums to melanoma cell components have been developed to specifically identify these MAAs immunochemically and provide a means to study the structural biochemistry of these determinants. At this time we have identified two antigens on which our research efforts are targeted: 1) a 100,000 dalton secreted glycoprotein (100K) common to melanoma, sarcoma and neuroblastoma tumor cell lines, and 2) a 250,000 dalton-high molecular weight component glycoprotein-proteoglycan complex which is thus far restricted to melanoma cells. The ultimate goal of our efforts is two-fold. Initially, we hope to develop schemes to isolate these melanoma associated antigens in sufficient quantities to obtain detailed structural information on these molecules, and secondly, we wish to implicate these glycoproteins in functional aspects of the biology of metastatic human melanoma in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Sera of 8 stage II melanoma patients undergoing surgical adjunctive immunotherapy with bacille Calmette Guérin (BCG) plus melanoma cell vaccine (MCV) were assayed for humoral response by the complementdependent antibody cytotoxicity (CDAC) assay and by the microcomplement fixation (MCF) test. The patients developed high levels of cytotoxic (CTX) and complement‐fixing (CF) antibodies to the UCLA‐SO‐M14 (M14) cells, one of the three melanoma cell lines in the MCV. Significant rises in CTX and CF antibodies occurred one month post‐immunotherapy. While the level of CTX antibodies was maintained for 11 months thereafter, the titer of CF antibodies was sustained for seven months, then gradually declined. When the sera were absorbed with lymphoblastoid (ML14) cells which are autologous to the M14 cells, two residual peaks of CTX antibodies, one and four months postimmunotherapy and two peaks of CF antibodies, one and seven months postimmunotherapy, emerged. Two sera that exhibited high levels of CTX and CF antibodies one month postimmunotherapy were absorbed with ML 14 cells and human fetal brain tissue. The reactivity of one serum in both the CDAC and MCF assays was abolished, whereas the reactivity of the other serum was not significantly diminished in either assay. These data indicate that the stage II post‐surgical melanoma patients developed a humoral immune response to at least two distinct tumor antigens on the membrane of the M14 cells. One of these antigens appeared to be of fetal origin (OFA). the other M14‐associated (TAA). Both antigens, OFA and TAA, were involved in complement‐dependent antibody cytotoxic and complement fixation reactions in vitro.

A monoclonal antibody, F11, was produced against a tumor-associated antigen from the spent medium of the M14 human malignant melanoma cell line which was grown continuously in serum-free medium. Ouchterlony double-diffusion study revealed that the F11 monoclonal antibody is an immunoglobulin G1. The F11 monoclonal antibody reacted positively with seven of eight (88%) melanoma, five of five (100%) carcinoma, zero to five normal, and zero of two lymphoblastoid cell lines by indirect immunofluorescence test. Also, by indirect immunofluorescence test, F11 monoclonal antibody reacted with cryostat sections from four of five (80%) melanomas, six of seven (86%) carcinomas, zero of one benign nevus, and zero of two benign breast diseases. By the indirect avidin:biotin:peroxidase complex immunoperoxidase method, the F11 monoclonal antibody reacted positively with cryostat sections from five of five (100%) melanomas, five of five (100%) breast cancers, two of two (100%) colon cancers, zero of one benign nevus, and zero of one Hodgkin's disease spleen. Thus, the tumor-associated antigen that the F11 monoclonal antibody recognizes appears to be expressed by melanomas and carcinomas, hence the designation melanoma-carcinoma-associated antigen. Microscopic observations disclosed that the melanoma-carcinoma-associated antigen is present in the cytoplasm, on the membrane of melanoma and carcinoma cells, and in the lumen of glandular structures of breast and colon carcinomas. The molecular weight of the melanoma-carcinoma-associated antigen in spent medium from the M14 CEM cell line is 100,000 as determined by sodium dodecyl sulfate:polyacrylamide gel electrophoretic analysis of indirect immunoprecipitates obtained with the F11 monoclonal antibody.

Immunoprecipitation studies with application of monoclonal antibody F11 originally made to a partially purified spent medium antigen of melanoma cells, made it possible to delineate the molecular profiles of both the cell associated and spent medium antigens recognized on melanoma cells intrinsically labeled with glycoprotein precursors. F11 distinguishes a glycoprotein of Mr 100,000 (100 K) in the spent media of melanoma cells while a parallel analysis of detergent lysates of cells reveals a pattern of three glycoproteins of Mr 75, 77, and 100 K. Pulse-chase analysis of the biosynthesis of these antigens indicated that F11 first recognizes the 75 and 77 K antigens in the absence of a 100K component suggesting strongly that these molecules contain an antigenic site recognized by F11. The 100 K antigen appears later in the pulse-chase analysis with kinetics that suggest some of the 75 and 77 K antigens are biosynthetic precursors of the 100 K antigen. This molecule is ultimately secreted into the extracellular media and appears to be a sialoglycoprotein judging from its sensitivity to neuraminidase. A cross-reactive species with an approximate Mr 90 K is also recognized by F11 in indirect immunoprecipitation analysis of spent media from a neuroblastoma cell line indicating that a common antigenic site exists on this secreted but structurally different neuroblastoma antigen. Thus, a combination of immunochemical and biosynthetic analyses of cell-associated and secreted antigens recognized by monoclonal antibody F11 demonstrate such molecules can differ structurally when isolated from the same or different tumor cells. These findings indicate the necessity to establish molecular profiles of melanoma-associated glycoprotein antigens recognized by monoclonal antibodies to characterize and define their potential biological functions within tumor cells.

The human colon carcinoma cell line HT‐29 was adapted to grow in chemically defined medium (CDM). The spent CDM (S‐CDM) was concentrated by Amicon filtration and the crude HT‐29 S‐CDM purified by 40% saturated(NH 4 ) 2 SO 4 precipitation. The purified antigen was tested by a micro‐complement fixation (MCF) assay against the sera of cancer patients of various histologic types and against the sera of normal donors. Fifteen of 20 (75%) colon cancer, 16/20 (80%) breast cancer sera, 14/19 (74%) lung cancer sera, and 13/20 (65%) miscellaneous carcinoma sera were positive in the MCF. By contrast, 2/21 (10%) melanoma sera, 7/20 (35%) sarcoma sera, and 2/19 (11 %) normal sera were positive. These data suggest the presence of a carcinoma‐associated antigen in the spent CDM of the HT‐29 colon carcinoma cell line adapted to grow in CDM.