pMel17 is recognised by monoclonal antibodies NKI-beteb, HMB-45 and HMB-50 and by anti-melanoma CTL.

Recently, we cloned the cDNA encoding the melanocyte lineage-specific antigen gp100 and demonstrated that gp100 is recognised by three different monoclonal antibodies (MAbs) used to diagnose malignant melanoma. In addition, we showed that tumour-infiltrating lymphocytes (TIL 1200) from a melanoma patient reacted specifically with cells transfected with the gp100 cDNA. Molecular characterisation of the gp100 cDNA revealed that the gp100 antigen is highly homologous, but not identical, to another melanocyte-specific protein, pMel17. Here, we report that cells transfected with pMel17 cDNA also react with all three MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-45 and HMB-50. Moreover, pMel17 transfectants are specifically lysed by TIL1200. These data demonstrate that antigenic processing of both gp100 and pMel17 give rise to peptides seen by anti-melanoma cytotoxic T lymphocytes (CTL) and are therefore potential targets for immunotherapy of malignant melanoma.

Melanoma is a neoplasm that originates from melanocytes, pigment-producing cells in the skin. Melanoma is a relatively immunogenic tumour, as demonstrated by the presence of both cytotoxic T lymphocytes (CTLs) and antibodies (Mattes et al., 1983;Knuth et al., 1992) in melanoma patients that react with melanoma tumour cells. The availability of antibodies and CTLs with anti-melanoma reactivity allowed the identification of several tumour-associated antigens. These include tumour-specific antigens and melanocyte differentiation antigens that are expressed by melanoma tumour cells as well as by normal melanocytes and retina (van der Bruggen et al., 1991;Brichard et al., 1993;Vijayasaradhi et al., 1990;Bakker et al., 1994;Kawakami et al., 1994a,b;Coulie et al., 1994;Gaugler et al., 1994 andWang et al., 1995). Identification of the antigens recognised by anti-tumour CTL is important for understanding the molecular basis of tumour recognition by T cells and may lead to the development of new immunotherapeutical strategies to treat cancer patients.
Recently, we cloned the cDNA encoding the melanocyte differentiation antigen gplOO and demonstrated that it is recognised by three different MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-50 andHMB-45 (Adema et al., 1993, 1994). In addition, we demonstrated that gplOO is recognised by a tumour-infiltrating T-cell line (TIL 1200), isolated from a melanoma patient (Bakker et al., 1994). Molecular characterisation of the cDNA encoding gplOO revealed that it is a type I transmembrane glycoprotein of 641 amino acids highly homologous to another melanocyte-specific protein, pMell7 Kwon et al., 1991). Nucleotide sequence analysis of genomic DNA indicated that the transcripts corresponding to gplOO and pMell7 cDNAs originate from a single gene via alternative splicing. The difference between gplOO and pMell7 consists of a stretch of seven amino acids in the carboxy terminal part of pMell7 (position 567;Adema et al., 1994) that is absent in gplOO. In all normal and malignant melanocytic cells expressing the gplOO/pMell7 gene, gplOO and pMell7 mRNAs are expressed simultaneously.
Here, we demonstrate that pMell7, like gplOO, is recognised by all three MAbs used to diagnose malignant melanoma and is properly processed and presented to antimelanoma tumour-infiltrating lymphocytes.
Molecular cloning and nucleotide sequence analysis Using a reverse transcriptase-polymerase chain reaction (PCR) (GeneAmp kit, Perkin elmer, the Netherlands) approach with 5'-ctgcatggagatcttcatcg-3' as the 5' primer and 5'-ttctgtgagctccaggaaaatcacagcat-3' as the 3' primer we isolated the 3' part of pMell7 cDNA from total RNA isolated from the melanoma cell line MEWO. The PCR product was used to replace the 3' part of the gplOO cDNA as present in pSVLgp OO+  using BglII and the newly created Sacd site in the 3' primer (underlined). The resulting construct, pSVLpMell7, was sequenced by the dideoxy-nucleotide sequencing method using T7 DNA polymerase (Pharmacia, Woerden, The Netherlands). pCMVneopMell7 was constructed by cloning the complete pMell7 cDNA from pSVLpMell7 as a blunt-ended XbaI-SacI fragment in the blunt-ended BamHI site of PCMVneo (Bakker et al., 1994).
Transfections and immunostaining Transient expression of DNA constructs in COS-7 cells was performed using 40 Mg ml-' lipofectin reagent (BRL, Gaithersburg, MD, USA) and 7.5 ,ug of DNA. BLM cells were transfected with 20 jug of pCMVneopMell7 DNA using calcium phosphate transfection systems (BRL, Gaithersburg, MD, USA) and stable clones were isolated by G418 selection (1 mg ml-') as previously described (Bakker et al., 1994).
Chromium-release assay Chromium release assays were performed as described previously (Bakker et al., 1994 The gplOO/pMell7 gene encodes both gplOO and pMell7 mRNA as a consequence of alternative RNA processing. The pMell7 mRNA encodes a stretch of seven amino acids not encoded by the gplOO mRNA ( Figure 1). To investigate the immunological properties of pMell7 we constructed a pMell7 cDNA. Using an RT-PCR approach we first cloned the 3' part of the pMell7 cDNA encoding the carboxy terminal part of pMell7, including the additional seven amino acids absent in gplOO. Nucleotide sequence analysis of the 3' part confirmed the presence of the nucleotide sequence pMell7 is recognised by anti-melanoma CTL and MAbs GJ Adema et al 1046 encoding the pMell7-specific amino acids. In addition, we found pMell7 cDNAs containing either a thymidine (as in the gplOO cDNA; Adema et al., 1994) or a cytosine at position 1998. This nucleotide change does not result in an amino acid substitution. The finding that the same nucleotide difference was found in a gplOO cDNA clone isolated from the same cell line, indicates that this particular cell line contains two different alleles of the gplOO/pMell7 gene. Subsequently, we created a full length pMell7 cDNA by exchanging the 3' part of the gplOO cDNA with the 3' part of pMell7 cDNA. To investigate whether the difference between gplOO and pMell7 affects recognition by the anti-gplOO MAbs NKIbeteb, HMB-45 and HMB-50, we transfected the cDNA encoding pMell7 into the gplOO/pMell7 negative melanoma cell line BLM. As shown in Figure 2, expression of the pMell7 cDNA resulted in immunoreactivity with all three MAbs. The typical speckled staining pattern of the pMell7 transfectants was identical to that previously observed for gplOO, suggesting that pMell7, like gplOO, localises in melanosomes. Immunoreactivity with all three MAbs was also observed when pMell7 cDNA was transiently expressed in non-melanocytic COS-7 cells (data not shown). We also analysed the pMell7 protein detected by MAbs NKI-beteb or HMB-50 in COS-7 cells transfected with the pMell7 cDNA using immunoprecipitations reactions. As shown in Figure  NKI-beteb and HMB-50 both specifically detect proteins of approximately 100 kDa (95-110 kDa) in extracts of metabolically labelled COS-7 cells transfected with the pMell7 cDNA. The pMell7 protein co-migrates with the gplOO protein immunoprecipitated from COS-7 cells transfected with gplOO cDNA as well as with the proteins immunoprecipitated from MEWO melanoma cells. The slight difference in mobility between transfected COS-7 cells and MEWO melanoma cells, which express both gplOO and pMell7 endogenously, has previously been shown to be due to differential glycosylation . Collectively, these data demonstrate that the difference between gplOO and pMell7 does not affect recognition by either of the MAbs used to diagnose malignant melanoma, NKI-beteb, HMB-50 and HMB-45. The data describing the specificity of these MAbs for cells of the melanocytic lineage can therefore be extrapolated to the expression of pMell7.
Previously, we showed that gplOO is recognised by tumour-infiltrating lymphocytes, (TIL)1200, isolated from a melanoma patient in an HLA-A2.1 restricted manner (Bakker et al., 1994). To investigate whether the pMell7 antigen also gives rise to peptide epitopes that gain access to the MHC class I antigen presentation pathway, we determined the cytolytic activity of TIL 1200 against HLA-A2.1 + BLM cells transfected with the pMell7 cDNA. As demonstrated in Figure 4, the pMel 17 transfectants were efficiently lysed by TIL 1200. No specific lysis was observed using the parental, untransfected BLM cells or BLM cells transfected with the expression vector without an insert (not shown). These data demonstrate that peptide epitope(s) recognised by TIL 1200 are properly processed from the pMell7 protein and presented in the context of HLA-A2.1. Two gplOO-derived peptides (corresponding to the amino acids at positions 154-162 and 457-466) have been identified that are recognised by TIL 1200 (Bakker et al., 1995;Kawakami et al., 1995). These peptides are located in the common part between gplOO and pMell7. Since TIL 1200 is an oligoclonal, CD8 + T-cell line expressing a restricted number of T-cell receptors (Shilyanski et al., 1994), it is most likely that either one or both the aforementioned immunogenic peptides are responsible for the observed lysis of the pMell7 and gplOO transfectants. ; the amino acids uniquely present in pMell7 are in italic capitals.

Discussion
The data described in this report demonstrate that pMell7, like gplOO, is recognised by three MAbs frequently used to diagnose melanoma. In addition, we demonstrate that cells transfected with pMell7 cDNA are effectively lysed by antimelanoma T cells.
Because of the exclusive reactivity of MAbs NKI-beteb, HMB-45 and HMB-50 with cells of the melanocyte lineage, they are frequently used to diagnose malignant melanoma (Ruiter, 1990). The finding that not only gplOO but also pMell7 reacts with these antibodies emphasises the specific expression of both gplOO and pMell7 in cells of the melanocyte lineage. The identical staining pattern observed with the MAbs in gplOO and pMell7 transfectants further indicates that, like gplOO, pMell7 is also present in melanosomes, which is in line with their proposed role in the process of pigmentation (Kwon et al., 1991). Whether there exists a functional difference between gplOO and pMell7 remains to be determined.
The relative immunogenicity of melanoma tumours has long been recognised. Both cytotoxic T cells and MAbs have been identified that specifically recognise melanoma tumour cells. So far, a number of the antigens recognised have been characterised in detail. They include the tumour-specific proteins, MAGE-1 (van der Bruggen et al., 1991) and MAGE-3 (Gaugler et al., 1994), which are expressed in different types of tumour cells and in testis. In addition, the melanocyte differentiation antigens tyrosinase, gplOO, Melan-A/MART-1 and gp75 (Brichard et al., 1993;Bakker et al., 1994;Kawakami et al., 1994a,b;Coulie et al., 1994;Wang et al., 1995) that are expressed in normal and malignant melanocytes as well as in retina have been identified as targets for anti-melanoma CTLs. Potentially, these antigens are targets for specific immunotherapy. TIL1200 was isolated from a melanoma metastasis and was shown to recognise the melanocyte differentiation antigen gplOO. Interestingly, reinfusion of in vitro expanded TIL1200 together with IL-2 in the autologous patient resulted in objective tumour regression (Kawakami et al., 1994b. Here we demonstrate that TIL1200 not only recognises the gplOO antigen, but also the pMell7 antigen that is encoded by an mRNA species derived from the same gene via alternative splicing. Since we have previously shown that melanoma cells express gplOO and pMell7 mRNA simultaneously, both proteins contribute to the total amount of immunogenic peptides presented in the context of HLA-A2.1 that are recognised by TIL1200. This finding is also consistent with the recent mapping of two peptide epitopes (corresponding to the amino acids at positions, 154-162 and 457-466) recognised by TIL1200 in the common part of gplOO and pMell7 (Bakker et al., 1995;Kawakami et al., 1995 and Figure 5). Although it has been observed that sequence context can affect processing and/or presentation of T-cell epitopes (Eisenlohr et al., 1992;Del Val et al., 1991), this does not seem to be the case for pMell7 and gplOO. The fact that TIL1200 is an oligoclonal, CD8+ T-cell line expressing a restricted number of T-cell receptors (Shilyanski et al., 1994), implies that either one or both these epitopes are properly processed from the pMell7 antigen and presented by HLA-A2.1. Attempts to further investigate the recognition of the pMell7 epitopes using cloned TIL have not been successful.
Besides TIL1200, other CTL-recognising distinct epitopes encoded by the gplOO/pMell7 have recently been characterised (Cox et al., 1994;Kawakami et al., 1995). A total of five distinct peptides have now been identified ( Figure 5), all of which are present in both gplOO and pMell7, and are presented by the same restriction element, HLA-A2. 1. Examination of the additional amino acid sequence present pMI17 is recognised by meanona CTL aid A __ kGJ Adema et al 1048 in pMell 7 revealed that six peptides (including 9-and 10mers) bearing the HLA-A2.1 binding motif are uniquely present in pMell7, whereas three peptides are specifically present in gplOO (Figure 1). HLA-A2.1 stabilisation experiments revealed that two of the pMell7-specific peptides listed in Figure 1 bind to HLA-A2.1 (ABHB and GJA unpublished observation). When analysing immunoreactivity against the products of the gplOO/pMell7 gene, one should therefore include both gplOO and pMell7.
In conclusion, the data presented in this report demon-strate that pMell 7 is recognised by three different MAbs used to diagnose melanoma and functions as a target for antimelanoma CTLs.'