Melanocyte differentiation antigen RAB38/NY-MEL-1 induces frequent antibody responses exclusively in melanoma patients
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- Zippelius, A., Gati, A., Bartnick, T. et al. Cancer Immunol Immunother (2007) 56: 249. doi:10.1007/s00262-006-0177-z
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Expression pattern and immunogenicity are critical issues that define tumor antigens as diagnostic markers and potential targets for immunotherapy. The development of SEREX (serological analysis of recombinant expression libraries) has provided substantial progress in the identification of tumor antigens eliciting both cellular and humoral immune responses in cancer patients. By SEREX, we have previously identified RAB38/NY-MEL-1 as a melanocyte differentiation antigen that is highly expressed in normal melanocytes and melanoma tissues but not in other normal tissues or cancer types. In this study, we further demonstrate that RAB38/NY-MEL-1 is strongly immunogenic, leading to spontaneous antibody responses in a significant proportion of melanoma patients. The immune response occurs solely in malignant melanoma patients and was not detected in patients with other diseases, such as vitiligo, affecting melanocytes. Fine analysis of the spontaneous anti-RAB38/NY-MEL-1 antibody response reveals a polyclonal B cell recognition targeting various epitopes, although a dominant immunogenic region was preferentially recognized. Interestingly, our data indicate that this recognition is not rigid in the course of a patient’s response, as the dominant epitope changes during the disease evolution. Implications for the understanding of spontaneous humoral immune responses are discussed.
KeywordsRAB38/NY-MEL-1Tumor antigenHumoral immune responseSEREXB cell epitopeMalignant melanoma
Humoral immune responses directed against tumor antigens are frequently observed in cancer patients, reflecting the interaction between the immune system and developing tumors. The properties of self-antigens necessary to induce immune responses specific to cancer are still poorly defined. Genetic aberrations [4, 27, 29], changes in the level of gene expression [22, 30], or aberrant expression in tumor lesions [23, 26] have been shown as potential mechanisms for immunogenicity, and serological responses against these antigens have been reported in cancer patients. In the last decade, the development of novel immunological methods has greatly contributed to our understanding of serological autologous immune responses in tumor patients (for a review, see ). SEREX (serological analysis of recombinant expression libraries), in particular, an expression cloning strategy, has led to the definition of a large repertoire of immunogenic antigens in various tumor types . Ideally, this technique distinguishes antigens with a direct relevance to cancer etiology or cancer progression from antigens that represent general autoimmunogenic cellular components and provides attractive antigenic targets for immunotherapy approaches in cancer patients.
Among the SEREX-defined repertoire of tumor antigens, cancer-testis (CT) antigens and differentiation antigens have been of particular interest with respect to immunotherapy strategies in cancer . The first category of antigens (e.g. NY-ESO-1, MAGE, SSX-2, etc.) are expressed in normal adult tissues solely in testis, but are present/activated in a variety of different tumor types in a lineage-unrestricted fashion. In contrast, differentiation antigens, e.g. Melan-A/MART1, tyrosinase, and gp100, are expressed in tissues and tumors of a particular cell lineage such as melanocytes. Melanocyte differentiation antigens frequently induce specific serological and T cell responses in melanoma patients [5, 7, 12, 32].
In a previous SEREX screening of a melanoma cell line library, we identified RAB38/NY-MEL-1 (later RAB38) , a polypeptide of 211 amino acids resembling a new member of the rab family of G proteins (for a review, see ). Preliminary data indicate a highly regulated expression that is restricted to the melanocytic cell lineage . Similar to most other melanosomal proteins, a coat color defect mapping to the RAB38 genetic locus has recently been identified in the mouse system . In addition to five highly conserved regions necessary for GTP binding, the structural features are characterized by a unique COOH terminus which allows post-translational lipid modifications. Interestingly, intracellular localization of a rat homolog with 97% amino acid identity indicates that RAB38 is expressed extensively in the cytoplasm with a distribution pattern similar to the endoplasmic reticulum [18, 19].
In this study, we further analyzed the expression pattern and immunogenicity of RAB38. As anticipated, RAB38 exhibits the characteristics of a melanocyte differentiation antigen as it is abundantly expressed in normal melanocytes and melanoma and not in any other normal tissues or non-melanocytic tumor. Spontaneous humoral immune responses against RAB38 are characterized by (i) their presence in a large proportion of melanoma patients, (ii) their restriction to patients with melanoma but not other tumor types, and (iii) polyclonal responses targeting various epitopes with a dominant immunogenic region identified. The restricted expression pattern and the immunogenicity of this antigen suggest that RAB38 may be used as a marker protein and as an appropriate target for antigen-specific immunotherapy in patients with malignant melanoma.
Materials and methods
Sera, tumor samples, and melanoma cell lines
Twelve metastatic melanoma lesions derived from 10 melanoma patients, and serum samples from 52 melanoma patients, 15 non-melanoma cancer patients, 13 vitiligo patients, and 15 healthy individuals were obtained at the University Hospital, Zurich, Switzerland, and Krankenhaus Nordwest Frankfurt, Germany, under consideration of local legal regulations. Melanoma cell lines were derived from the repository of the Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center.
RNA extraction and real-time RT-PCR
Total RNA was extracted from melanoma lesions by the Qiagen RNA Extraction Kit (Qiagen, Hombrechtikon, Switzerland) and quantified by spectroscopy (SmartSpec 3000 Spectrophotometer, BioRad). In addition, RNA was obtained from 16 different normal tissues (Clontech, Basel, Switzerland) and cultured human melanocytes (Gentaur). RNA was reverse-transcribed into cDNA using the TaqMan EZ RT-PCR kit (Applied Biosystems, Rotkreuz, Switzerland). Gene-specific TaqMan probes with a predetermined, optimum concentration of gene-specific forward and reverse primers (300–900 nM) were purchased from Applied Biosystems (TaqMan Gene Expression Assay on Demand). For endogenous control, 18S RNA-specific primers/probes were purchased from Applied Biosystems. The PCR consisted of 40 cycles of 95°C denaturation (15 s) and of 60°C annealing/extension (60 s). A total of 20 ng/μl were reverse-transcribed and 2.5 μl of cDNA was diluted in TaqMan Universal PCR Master Mix supplemented with (Fam)-labeled gene-specific TaqMan probe, and an optimal concentration of the gene-specific forward and reverse primers (300–900 nM) were used for PCR. All PCR reactions were run as triplicates, and thermal cycling and fluorescent monitoring were performed using an ABI7000 thermal cycler (Applied Biosystems).
Production of the recombinant RAB38 protein
The entire coding region of RAB38 (636 bp) was amplified with primers including specific cleavage sites for BamH1 (Bam-5′: CACACAGGATCCATGCAGGCCCCGCACAAGGAG) and HindIII (Hind-3′: CACACAAAGCTTCTAGGATTTGGCACAGCCAGAG). After digestion with BamH1 and HindIII, the PCR product was cloned into the pQE30 expression vector (Qiagen). Competent M15 (pREP4) Escherichia coli were transformed and the recombinant protein was produced and purified under denaturing condition following the manufacturer’s protocol. The purified recombinant His-tag protein was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by silver staining.
Detection of RAB38-specific antibody responses
For Western blot assays, the purified recombinant protein and lysate of a RAB38-expressing melanoma cell line (SK-MEL-37) resuspended in 1:2 in Laemmli sample buffer and denaturated for 5 min at 95°C were used. 1D SDS–PAGE was performed on 15% acrylamide gel. The protein was then transferred onto Protan Nitrocellulose Membranes (Schleicher and Schuell, BioScience, Dassel, Germany) in a Mini Trans-Blot cell (BioRad). The efficiency of the electrotransfer was assessed by Ponceau Red staining of the membranes. After incubation with patients’ sera diluted at 1:250 for 60 min, reactivity was determined using an AP-conjugated goat-anti-human IgG-specific antibody (Jackson ImmunoResearch Laboratories, LaRoche, Switzerland) diluted 1:5,000. Immunoreactive proteins were visualized with BCIP and NBT (Roche, Rotkreuz, Switzerland). All steps were performed at room temperature.
For ELISA assays, 96-well plates (Corning, Wohlen, Switzerland) were coated with 50 ng of recombinant RAB38 protein, tetanus toxoid (Berna), or overlapping peptides of 18 aa length derived from the RAB38 protein sequence (BioSynthesis, Lewisville, TX, USA). All peptides were diluted in PBST (PBS/0.5% Tween-20). Plates were incubated overnight at 4°C and consecutively blocked with PBST/5% fetal calf serum (FCS) for 2 h at 4°C and then washed twice. Serum samples diluted 1:250 in PBST/5% FCS were incubated for 2 h at room temperature, followed by incubation for 30 min with a secondary, peroxidase-conjugated, goat-anti-human IgG antibody (Sigma, Darmstadt, Germany), diluted 1:20,000 in PBST/5% FCS. The plates were subsequently developed at room temperature for 30 min with 150 μl/well tetramethylbenzidine (Sigma, Darmstadt, Germany) and analyzed using an ELISA reader (Labexim LMR1) at λ = 450 nm.
Tissues were mounted in OCT, snap-frozen in isopentane pre-cooled liquid nitrogen upon surgical removal. Five micrometers thick frozen tissue sections were placed on glass slides, air-dried, fixed with cold acetone for 10 min, and stored at −70°C. As a positive control, melanoma cell line SK-MEL-37 grown in slide chambers was used. After removal of media, cells were air-dried and fixed with cold acetone (data not shown). Slides were incubated with affinity-purified polyclonal rabbit anti-RAB38 antibodies [18, 19], diluted 1:200 in TBS for 30 min at room temperature. Primary antibodies were detected with a goat anti-rabbit secondary, followed by alkaline phosphatase labeled donkey antibodies to goat immunoglobulins (Jackson ImmunoResearch Laboratories). Dilutions of secondary reagents were made in TBS containing 1% FCS. Alkaline phosphatase was then detected by a red color reaction using naphthol AS-BI phosphate as a substrate and new fuchsin as a chromogen. Endogenous alkaline phosphatase was blocked by levamisole. Slides were counterstained with Meyer’s hematoxylin for 2 min.
Prediction of the protein structure and B cell epitopes
Hydrophilicity was calculated based on the Kyte–Doolittle  method with a window size of 17 amino acid residues. Surface accessibility and buried residues were calculated using the ExpASy (Expert Protein Analysis System) with a window of 17 amino acid residues . To visualize potential binding sites depending on the 3D structure, the protein sequence was blasted against the pdb database . 3D graphs were designed using the RasMol software Version 2.6.
RAB38 mRNA expression in normal tissues and melanomas
RAB38 protein expression in malignant melanoma
Melanoma patients frequently develop humoral immune responses against RAB38
Identification of RAB38 epitopes recognized by antibodies present in melanoma sera
The recognition of tumor cells by the immune system is reflected by spontaneous cellular and humoral immune responses. The immune system can target and destroy tumor cells via the recognition of specific tumor antigens . Promising target antigens for immunotherapeutic approaches are tumor-restricted immunogenic proteins that are expressed at high levels in malignant cells. A growing number of tumor antigens have been identified by methods based on autologous T cell responses and serological responses in cancer patients. To date, only few of these antigens, e.g. NY-ESO-1, have been reported to frequently elicit spontaneous cellular as well as humoral immune responses. Like RAB38, the CT antigen NY-ESO-1 was initially identified based on a spontaneous antibody response employing the SEREX method . Interestingly, most NY-ESO-1 seropositive patients have simultaneous NY-ESO-1-specific CD8 and CD4 T cell responses [6, 11]. This observation supports the strategy of exploiting serological responses in order to identify relevant T cell epitopes that can be used as targets for vaccine-based immunotherapy. Ideal target antigens should exhibit a frequent and tumor-restricted expression as well as a high immunogenicity, as reflected by frequent spontaneous immune responses in patients with antigen-positive tumors.
Here we report on RAB38, a novel melanocyte differentiation antigen, that frequently induces humoral immune responses in melanoma patients. Extending the initial expression analysis by northern blot , we performed real-time RT-PCR in normal tissues and melanoma lesions, and confirmed the tissue-restricted expression of RAB38 with predominant expression in melanocytes and in melanoma tissues. The low-level mRNA expression in adrenal gland needs to be confirmed in a higher number of cases and especially on a protein level. However, like melanocytes, adrenal medulla is a neuroectodermal tissue and some relation between both tissues may exist. Among the melanomas of our series, the only negative cases were specimens of an amelanotic melanoma and uveal melanoma. Amelanotic melanoma is characterized by the low number of melanosomes which may explain the low expression of some melanocyte differentiation antigens. However, on a protein level, melanocyte differentiation antigens such as Melan-A, gp100, and tyrosinase did not show a lower expression in amelanotic melanoma. The biology of uveal melanoma is poorly understood and it has been reported that the expression of melanocyte lineage markers differs from cutaneous melanoma  (E. Jaeger, personal communication). RAB38 mRNA expression data obtained by real-time RT-PCR correlated to RAB38 protein expression analyzed by immunohistochemistry using a polyclonal rabbit anti-rat RAB38 antibody. RAB38-positive cells showed cytoplasmatic staining which is in line with previous reports localizing murine RAB38 to the melanosomal compartment in mice . We are currently generating mAbs to human RAB38 in order to analyze its protein expression in normal tissues and tumors.
In addition to its tumor-restricted expression, we demonstrated that RAB38 is highly immunogenic in melanoma patients. Spontaneous RAB38-specific antibody responses were present in 23% of melanoma patients (12 out of 52). As not all melanomas express RAB38, the frequency of anti-RAB38 response in patients with RAB38-positive melanoma is likely to be higher. The immune response was present solely in melanoma patients and none of the 13 vitiligo patients had detectable RAB38-specific antibodies. This suggests that serological responses to RAB38 likely result from ongoing interactions of the immune system with evolving and progressing tumors, rather than a tumor-unrestricted process following cell necrosis and release of intracellular proteins. However, this does not exclude the possibility of side effects such as vitiligo as a result of immune responses to RAB38 vaccines as it has been observed in clinical trials using vaccines to other melanocyte differentiation antigens such as Melan-A and tyrosinase [13, 17].
To identify antibody epitopes within the RAB38 sequence, we tested RAB38 antibody-positive sera against RAB38-derived peptides in ELISA assays and correlated the serological findings with the results of computer algorithms predicting potential immunogenic sites in the protein structure [15, 20]. The results of both methods concurred indicating an immunodominant region encompassing position aa 153–178. Notably, this dominant B cell epitope may by used to measure antibody responses, as previously reported for NY-ESO-1 . This would bypass the requirement for purification of the recombinant protein which is often difficult to achieve. However, as demonstrated here, a potential caveat of the peptide-based approach for measuring antibodies is the possibility of changing the epitope during the course of the disease (Fig. 6c). Consequently, changing epitopes may simply be missed in epitope-restricted assays. Moreover, the present approach is not suitable for the identification of conformational epitopes.
In conclusion, the present data support our initial analyses and demonstrate that RAB38 is a true melanocyte differentiation antigen which may be useful for diagnostic purposes or may serve as a target for immunotherapy of melanoma. Further studies about the protein expression and potential T cell responses are necessary and underway to assess the role of RAB38 as a cancer target and diagnostic marker.
This work was supported in part by a Swiss National Science Foundation special program, and a grant from the Cancer Research Institute/Ludwig Institute for Cancer Research Cancer Vaccine Collaborative, the Terry-Fox, Hanne-Liebermann and Claudia-von-Schilling Foundation, and the UBS Wealth Management. A.Z., A.G., and S.W. were supported in part by the Emmy-Noether Program (Zi685-2/3) of the Deutsche Forschungsgemeinschaft. The excellent technical assistance of Claudia Frei and Julia Karbach is greatly appreciated.