Background
Melanoma is the most lethal form of common skin cancers. While targeted and immune-based therapies are increasingly promising, not all patients benefit, thus establishing the need to identify novel targets that can contribute to improved therapeutic strategies. Using live melanoma cell immunization and high-throughput screening (HTS) [
1], we generated a novel neutralizing monoclonal antibody (mAb) directed against a melanoma cell-surface antigen, which we subsequently identified as the functional, glycosylated protein MUC18.
MUC18, also known as MCAM (melanoma cell adhesion molecule), CD146 (cluster of differentiation 146), or METCAM/MelCAM (metastatic melanoma CAM), is expressed on the surface of metastatic melanoma and other cancer cells [
2]. Expression of MUC18 has been demonstrated to promote tumorigenesis and tumor progression, and therapeutic targeting of MUC18 can reduce bone metastasis in a prostate cancer model [
3]. Investigations outlined here demonstrated that the mAb developed was capable of specifically interacting with MUC18 on melanoma cells, initiating downstream signaling events that were associated with inhibition of melanoma cell proliferation, migration, and invasion in vitro
, and reduction in tumor growth and metastasis in vivo. Furthermore, we found that these signaling events depended on binding of the mAb to a conformational epitope on the extracellular domain of MUC18.
Materials and methods
Animal study
A/J mice (6–8 weeks old, male) (Harlan Sprague Dawley, Inc., Indianapolis, IN) and nu/nu mice (4–8 weeks old, male) (JAX, Bar Harbor, Maine) were used for this study. Mice were maintained and experiments were performed under protocols (IACUC00001239-RN00 and IACUC00000731-RN01) approved by the Institutional Animal Care and Use Committee (IACUC).
Cell lines & tissues & other resources
A375 cells (primary cell line with low metastatic capacity) [
4]; and A2058 and WM266-4 cells (highly metastatic melanoma cells), SP2/0 (mouse myeloma cells), EC-RF24 (immortalized human endothelial cells) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human peripheral blood mononuclear cells (PBMCs) were separated by using Ficoll-Plaque Plus (Ficoll, GE Healthcare Biosciences) from healthy donor blood (Gulf Coast Regional Blood Center). Other cell lines were gifts from collaborators. Cell lines were grown in serum free medium MD6 derived from Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 5% FBS and 1% penicillin-streptomycin. Formalin-fixed paraffin embedded (FFPE) tissue slides and tissue microarrays (TMAs) were prepared from a variety of tumors and normal tissues from our institutional tissue bank under institutional review board protocols (LAB09–0197 and PA11–0957). Tunicamycin (Cat. #T7765, Sigma-Aldrich, MO) and Dynabeads conjugated with Protein A (Cat. #14311D, Invitrogen, Inc., Carlsbad, CA) were also purchased.
Antigen & Antibodies
Human recombinant protein CD146 (MCAM) was purchased from OriGene Technologies (Cat. #TP308937, Rockville, MD). Commercial antibodies used in this study include anti-human MUC18 (mouse mAb, Cat. #MAB932; goat polyclonal Ab, Cat. #AF932, R&D Systems, Inc., Minneapolis, MN; mouse mAb, Cat # ab233923; AbCam, Cambridge, MA) and an irrelevant mAb (served as isotype control antibody) and goat serum (IgG). FITC, HRP and Alexa fluor 647 conjugated goat anti-mouse IgG (Cat. #115–095-071, 115–035-071, 115–606-062, Jackson ImmunoResearch Lab, West Grove, PA) served as secondary antibodies.
Live-cell immunization
Five million each of A375, A2058 and WM266-4 metastatic melanoma cells were injected subcutaneously (s.c.) into three A/J mice every 2 weeks × 3, followed by an intraperitoneal (i.p.) boost. Three days after boost, spleen cells from the immunized mice were collected and fused with myeloma SP2/0 cells to generate hybridomas [
5].
High-throughput screening (HTS) using fluorescence-activated cell sorting (FACS)
The BD LSR II Flow Cytometry System with HTS autosampler (Becton Dickinson) was used to screen for mAbs secreted from the hybridomas that bound to the mixed melanoma cell lines as outlined previously [
1]. Mean fluorescence intensity (MFI) and the percentage of the stained cell peaks from screening and counter-screening plates were determined using the BD LSR II FACS-HTS system.
Glycosylation and Lectin binding analyses
FACS analysis was used to detect the carbohydrate group of the MUC18 glycoprotein from cells (1 × 105) individually seeded in flasks with or without conditioned medium containing 3.0 μg/mL tunicamycin in DMSO (Dimethyl sulfoxide) and cultured for 24 h. Cells were then harvested and stained with JM1-24-3 or an irrelevant mAb for quantification of MFI. Cell lysates were probed with JM1-24-3 and β-actin mAb in WB (western blot). FLISA (Fluorescence-linked immunosorbent assay) was used for SNL (Sambucus nigra lectin) binding analysis, in which individual cell lysates were coated on plates and incubated with SNL-FITC (Fluorescein isothiocyanate) which was then competed by serial dilution of JM1-24-3, starting with 20 μg/mL, with or without SNL. Similar FLISA assays were conducted for other lectins [DSA (Datura stramonium agglutinin) lectin and DSA-FITC; LCA (Lens culinaris agglutinin) lectin and LCA-FITC; WGA (Wheat germ agglutinin) lectin and WGA-FITC].
Epitope mapping
The biochip was spotted in triplicate with MUC18 8-mer and 6-mer oligopeptides at one amino acid resolution (acetyl capped at the N-termini on the ChipMDA_130046) and then incubated with JM1-24-3 (1.0 μg/mL) at 4 °C for 2 h, followed by washing and incubation with goat anti-mouse IgGFc Alexa 647 conjugated (0.01 μg/mL) at 4 °C for 2 h, and then washed again. The image of the biochip was scanned in Cy5 channel and data analyzed.
Immunohistochemistry (IHC) analysis
The expression of MUC18 on melanoma patient tissues and on TMA containing several normal tissues and cancers was detected by IHC using JM1-24-3 mAb (1:1000) as described previously [
1].
Immunoprecipitation (IP) and mass spectrometry (MS)
IP was conducted on cell lysates (100 μg) using Protein A beads (0.2 mL) and JM1-24-3 (10 μg) or irrelevant control antibodies as described previously [
1]. Excised bands were analyzed with a ProteinChip system in Series 4000 Mass Spectrometry (Bio-Rad) as described previously [
1].
Reverse phase protein Array (RPPA)
WM266-4 cells were treated with JM1-24-3 or its F (ab’)
2 fragment for 1 h or 6 h and RPPA conducted [
6]. Heat map results were analyzed for changes in protein expression using Ingenuity Pathway Analysis (IPA) [
7] software (Qiagen Bioinformatics) to identify down-stream signaling pathways.
Structural modeling
The homology model of the extracellular domain (residues 5–559) of MUC18 was generated using the Swiss Model web interface [
8], using the functional motif of “search for templates”. The first approach modes were used to generate an initial homology model and accompanying sequence alignment, which was then manually modified using Coot [
9]. A final correction of the alignment was then performed in Swiss Pdb Viewer. This model and the accompanying corrected sequence alignment were then input into the Optimization Mode of Swiss Model. The final pdb files were used to display the overall conformation and the antibody epitope locations.
The homology model of JM1-24-3 variable domain was generated using BioLuminate v.1.1 (Schrödinger, New York, NY). Template coordinates for heavy chain and light chain were chosen from PDB structure 4BKL and 6I1O respectively based on the sequence homology search. CDRs were modeled using the BioLuminate Basic Loop Model function. Experimental data were incorporated into the program for interaction. All antibody structure images were generated using Maestro and PyMol (Schrödinger, New York, NY).
In vitro cell proliferation, migration and invasion studies
These studies were conducted on incubating cells with and without JM1-24-3 or irrelevant mAb (150 μg/mL) for 1 week as described previously [
1]. For migration assays, 3 × 10
4 cells in serum-free medium (300 μL) were incubated with and without JM1-24-3 (150 μg/mL) or irrelevant mAb for 24 h. Invasion assays were carried out by a similar protocol using QCM ECMatrix cell Invasion Assay Kit (EMD Millipore).
In vivo tumor Xenograft studies of melanoma growth and metastasis
Athymic nude (nu/nu) mice were injected subcutaneously with WM266-4 cells (1 million/0.1 mL). After 5 days mice were randomly divided into two groups and treated with either JM1-24-3 (6 mg/kg body weight/i.p./twice a week) (n = 11) or with the same dose of irrelevant mAb (n = 8) for 45 days. Tumor volume was measured with calipers every 4 days. At the end of the experiment, tumors were excised and weighed. Mice body weight was measured every 4 days and any reduction > 10% from initial weight was considered as toxicity. For metastasis studies, mice were pre-treated 1 day before with JM1-24-3 (n = 5) or with irrelevant mAb (n = 7), following which all received a tail vein injection with WM266-4 cells (1 million); mAb treatment was administered every 4 days. All mice were sacrificed at day 45 and lungs were harvested for H&E staining.
Statistical analysis
Experiments were repeated at least in replicate and data expressed as mean ± standard deviation (SD). Representative figures are presented for FACS, ELISA, FLISA and MS analysis. Differences were analyzed with a two-tailed Student’s t-test or Wilcoxon rank-sum test and paired t-test for serial dilution studies. P values < 0.05 were considered as statistically significant.
Discussion
In the current study, a novel strategy of live cell immunization and live cell high-throughput screening by FACS analysis was used for the development and screening of antibodies directed against antigens on the surface of metastatic melanoma tumor cells. Multiple protein-, peptide- and cell-based assays were used to define JM1-24-3 as a specific antibody binding to an identified conformational epitope on its target on cancer cells. Mass spectrometry analysis suggested the target as MUC18 (CD146).
Circulating MUC18 is a promising melanoma biomarker; circulating levels are significantly associated with poor prognosis and death [
12,
13]. MUC18 interacts with several receptor proteins, including Calprotectin (S100 calcium-binding protein A8/A9, S100A8/A9), heparin sulfate, Toll Like Receptor 4 (TLR4) and Receptor for Advanced Glycation End products (RAGE) [
14]. Thus, MUC18 may play role in multiple processes including inflammation, cell differentiation, adhesion, tumorigenesis, migration, invasion, angiogenesis, and metastasis [
15]. MUC18 has been shown to induce translational initiation and transcriptional activation of c-Jun/c-Fos in hepatocellular carcinoma [
16] and has been suggested as a potential therapeutic target in malignant rhabdoid tumors through induction of apoptosis by inactivating protein kinase B (PKB) or the serine/threonine-specific kinase (AKT) signaling pathway [
17].
Evaluation of glycosylation of MUC18 determined that sugar residues were involved in the structure of the molecule, and that functional interactions between JM1-24-3 and the conformational epitope of MUC18 were dependent on glycosylation. Recent literature suggests that MUC18 can be glycosylated via N-acetyl-glucosaminyltransferease III and V without a role in migration [
18] or could be glycosylated and stabilized by β-1,3-galactosyl-O-glycosyl-glycoprotein β-1,6-N-acetylglucosaminyltransferase 3 (GCNT3) playing a major role in melanoma migration and invasion [
19]. Thus, involvement of glycosylated conformational epitope in the function of MUC18 could therefore have implications in melanoma progression or metastasis.
Importantly, JM1-24-3 was capable of binding to MUC18 expressed on the melanoma cell surface, subsequently inducing downstream signaling pathways and further inhibition of cell growth and metastasis. Mechanistic investigations demonstrated induction of multiple changes in downstream signaling pathways following binding of JM1-24-3 to MUC18. Furthermore, JM1-24-3 demonstrated only weak, patchy binding to smooth muscle cells in small vessels of the kidney, lymph nodes and skin, and was otherwise unreactive across a cross-section of normal tissues. Hence, this preliminary data suggests that targeting MUC18 via the conformational epitope identified by JM1-24-3 may have limited toxicity. Finally, in addition to confirming that MUC18 was highly expressed on melanoma tumors, especially metastatic tumors, we identified significant MUC18 expression in several other cancers, including those of the gastrointestinal tract and TNBC.
Although a small number of potentially therapeutic antibodies have been recently developed against MUC18 [
20], the specific advantage of JM1-24-3 is that our studies document that it recognizes a conformational epitope of MUC18 on the cancer cell surface, and in doing so alters downstream signaling pathways, resulting in reduction of associated tumor-promoting functions. While we cannot completely exclude the possibility that JM1-24-3 could have additional, functionally relevant melanoma targets, we submit that the data provided are strong evidence that JM1-24-3 recognizes a specific conformational epitope on MUC18, and in binding to that epitope induces downstream signaling events that mediate the metastatic phenotype, thus identifying the MUC18 conformational epitope as a promising therapeutic target potentially amenable to mAb treatment. Also, since MUC18 is expressed on the surface of other cancers, JM1-24-3 could be a useful therapeutic agent for therapy of other cancers, either alone or in combination with other agents.
Conclusion
A novel mouse monoclonal antibody JM1-24-3, developed by melanoma live cells immunization, was identified to be directed against the conformational epitope of the cell surface antigen MUC18. JM1-24-3 blocked MUC18 downstream signaling pathways and cellular functions in vitro and in vivo. Thus, the MUC18 conformational epitope identified represents a promising therapeutic target, and the JM1-24-3 mAb might serve as the basis for a potential therapeutic agent.
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