1. Expression cloning of the cDNA encoding a melanoma-associated Ag recognized by mAb HMB-45. Identification as melanocyte-specific Pmel 17 cDNA
S N Wagner, C Wagner, H Höfler, M J Atkinson, M Goos Lab Invest. 1995 Aug;73(2):229-35.
Background: mAb HMB-45 recognizes a melanocyte lineage-associated Ag present in "activated melanocytes" and malignant melanoma cells. Despite its important practical significance in diagnostic pathology and its potential role as an "activation marker" of melanocytic cells, the HMB-45-reactive Ag remained undefined. Molecular characterization of the HMB-45-reactive Ag may help in analyzing underlying mechanisms of melanocyte activation and melanoma tumor progression. Experimental design: A cDNA library constructed from the HMB-45-immunoreactive human melanoma cell line SK-MEL-28 was screened for expression of the cDNA encoding the HMB-45-reactive protein. Screening was performed by expression in COS-7 cells and subsequent immunocytochemical screening. Correlation between HMB-45 immunoreactivity and mRNA expression of the cloned cDNA was analyzed by antisense mRNA expression in HMB-45-immunoreactive SK-Mel-28 cells, by Northern blot hybridization, and by comparative immunohistochemistry and in situ hybridization. Results: A cDNA clone encoding the HMB-45-reactive Ag was isolated and shown to be homologous to the Pmel 17 cDNA. Expression of either HMB-45 or Pmel 17 cDNA in COS-7 cells induced cytoplasmic HMB-45 immunoreactivity as described for melanoma cells. Conversely, constitutional HMB-45 immunoreaction in SK-MEL-28 cells could be markedly reduced by expression of antisense Pmel 17 RNA. In several tissues, Pmel 17 mRNA content conformed to the known expression pattern of HMB-45-reactive Ag. Comparative in situ hybridization and immunohistochemistry demonstrated a consistent co-localization of Pmel 17 transcripts and HMB-45-reactive Ag at the cellular level, with strong expression in "activated" melanocytes and melanoma cells and significantly less expression in normal adult melanocytes. Conclusions: We conclude that Pmel 17 cDNA encodes the HMB-45-reactive Ag. Pmel 17 has been postulated to be involved in melanin synthesis and expression of melanoma-peptide epitopes recognized by CTLs. Differential expression of Pmel 17/HMB-45 may have considerable effects in the stepwise process of melanoma progression by aberrant pigment formation and expression of CTL-reactive epitopes.
2. Antitumour activity of a melanoma-specific immunotoxin, ME20-LysPE40
E A Wolff, I Hellström, D F Chace, K E Hellström, C B Siegall Ther Immunol. 1995 Jun;2(3):137-45.
An immunotoxin conjugate has been prepared by linking an internalizing antibody with melanoma selectivity, ME20, with a binding-defective form of Pseudomonas exotoxin A, LysPE40. ME20-LysPE40 binds to a 105,000 Da cell-surface antigen present on melanoma cells (ME20-M) within twofold of unmodified ME20 and was cytotoxic to two human melanoma cell lines, H3606 and MALME-3M, with EC50 values of 100 and 200 pM, respectively. Immunotoxin treatment, initiated 1 day following subcutaneous implantation of H3606 melanoma cells into mice, prevented outgrowth of tumour xenografts in > 50% of the mice. In contrast, only a modest inhibition in tumour growth was observed if the immunotoxin was administered 5 days after implantation of in vivo passaged H3606 tumour fragments in mice. This study shows that the internalizing monoclonal antibody ME20 IgG can be used for targeting a toxin toward melanoma cells displaying the ME20-M antigen.
3. Differential processing and secretion of the melanoma-associated ME20 antigen
G A Maresh, W C Wang, K S Beam, A R Malacko, I Hellström, K E Hellström, H Marquardt Arch Biochem Biophys. 1994 May 15;311(1):95-102. doi: 10.1006/abbi.1994.1213.
A murine monoclonal antibody, ME20, with high selectivity for melanomas, has been utilized to isolate a unique membrane-bound (designated ME20-M) and secreted (designated ME20-S) antigen from H3606 human melanoma cells. ME20-M was purified from the cell lysate and ME20-S from the conditioned medium of H3606 cells by immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weights were 105,000 and 76,000, respectively. Analyses of ME20-M and ME20-S by amino acid sequencing identified the processing sites. Signal peptide cleavage occurs at Thr-24 of pro-ME20 antigen, yielding ME20-M (25 to 661). In addition, proteolytic processing of the precursor at Val-467 yields ME20-S (25 to 467). We report the characterization of Asn-linked glycosylation sites in ME20-M and ME20-S to determine the involvement of oligosaccharides in the proteolytic processing of pro-ME20 antigen. Tryptic peptide maps of ME20-M and ME20-S were prepared and the glycosylation sites identified by sequence analyses. Oligosaccharides were enzymatically released and characterized by high-performance anion-exchange chromatography. We found high-mannose-type structures at Asn-57, Asn-82, and Asn-87 of ME20-M, whereas ME20-S contained 73% complex-type and 27% high-mannose-type oligosaccharides at the same sites. To assess the role of oligosaccharides in the processing of the ME20 antigen, we tested the effect of the oligosaccharide processing modifier deoxymannojirimycin, a compound that inhibits synthesis of hybrid- and complex-type oligosaccharides. Deoxymannojirimycin had no effect on the synthesis and relative rate of synthesis of ME20-M, but markedly reduced the synthesis of ME20-S without affecting the rate of secretion. The reported results suggest that carbohydrate maturation of the ME20 antigen may be important for processing and secretion.
