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JBC Papers in Press. Published on December 15, 2008 as Manuscript M806776200 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M806776200 Mesothelin and CA125/MUC16, Page 1 A BINDING DOMAIN ON MESOTHELIN FOR CA125/MUC16* Osamu Kaneko, Lucy Gong, Jingli Zhang, Johanna K. Hansen, Raffit Hassan, Byungkook Lee and Mitchell Ho From the Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Running head: Mesothelin and CA125/MUC16 *This work was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, in part by the Ovarian Cancer Research Fund Individual Investigator Award (M. Ho) and in part by the Mesothelioma Applied Research Foundation Grant in Honor of Craig Kozicki (M. Ho). Address correspondence to: Dr. Mitchell Ho, Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5002C, Bethesda, MD 20892-4264 Phone: (301) 451-8727; Fax: (301) 402-1344; E-mail: firstname.lastname@example.org Ovarian cancer and malignant new therapeutic agent for preventing or mesothelioma frequently express both treating peritoneal malignant tumors. Downloaded from www.jbc.org by guest, on August 14, 2011 mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The Ovarian cancer largely is confined to the interaction between mesothelin and CA125 may peritoneal cavity for much of its natural history facilitate the implantation and peritoneal (1). Peritoneal mesothelioma is a highly invasive spread of tumors by cell adhesion, whereas the tumor originating from the mesothelial linings of detailed nature of this interaction is still the peritoneum (2). The development of effective unknown. Here, we used truncated mutagenesis drug regimens against ovarian cancer and and alanine replacement techniques to identify mesothelioma has proven extremely difficult. a binding site on mesothelin for CA125. We Mesothelin was first identified in 1992 by the examined the molecular interaction by Western monoclonal antibody (mAb)1 K1 that was blot overlay assays and further quantitatively generated by the immunization of mice with analyzed by enzyme-linked immunosorbent human ovarian carcinoma (OVCAR-3) cells (3). assay. We also evaluated the binding on cancer The mesothelin gene encodes a 71-kDa precursor cells by flow cytometry. We identified the protein that is processed to a 40-kDa protein region (296-359) consisting of 64 amino acids at termed mesothelin, which is a glycosyl- the N terminal of cell surface mesothelin as the phosphatidylinositol (GPI)-anchored glycoprotein minimum fragment for complete binding present on the cell surface (4). Mesothelin is a activity to CA125. In addition, we found that differentiation antigen that is present on a substitution of tyrosine-318 with an alanine restricted set of normal adult tissues such as the abolished the CA125-binding. Replacement of mesothelium. In contrast, it is overexpressed in a tryptophan-321 and glutamic acid-324 with variety of cancers including mesothelioma, alanine could partially decrease binding to ovarian cancer, and pancreatic cancer (5). In CA125, while mutation of histidine-354 had no addition, mesothelin is also expressed on the effect. These results indicate that a conformation-sensitive structure of the region (296-359) is required and sufficient for the 1 binding of mesothelin to CA125. In addition, we The abbreviations used are: mAb, monoclonal have shown that a single chain monoclonal antibody; GPI, glycosyl-phosphatidylinositol; antibody (SS1) recognizes this CA125-binding FDA, Food and Drug Administration; TR, tandem domain and blocks the mesothelin-CA125 repeat; ELISA, enzyme-linked immunosorbent interaction on cancer cells. The identified assay; DMEM, Dulbecco’s modified eagle CA125-binding domain significantly inhibits medium; rFc, rabbit Fc; hnMSLN, human cancer cell adhesion and merits evaluation as a mesothelin; RT, room temperature; scFv, single chain Fv antibody; mnMSLN, mouse mesothelin Mesothelin and CA125/MUC16, Page 2 surface of non-small cell lung cancer cells (6, 7), CA125 was originally used as a biomarker in especially most lung adenocarcinomas (8). ovarian cancer due to its high expression in We and others have shown that mesothelin is ovarian carcinomas and that it is shed into the shed from tumor cells (9, 10), and antibodies serum (23). A majority (88%) of mesotheliomas specific for mesothelin are elevated in the sera of are also CA125 positive on the cell membrane patients with mesothelioma and ovarian cancer (24). It was shown that 25% of peritoneal (11). Shed serum mesothelin has been approved by mesotheliomas have high CA125 expression (25). the U.S. Food and Drug Administration (FDA) as The intensity of CA125 membranous expression is a new diagnostic biomarker in mesothelioma. In a indistinguishable between ovarian carcinomas and Phase I clinical study of an intrapleural IFN-β peritoneal mesotheliomas. Gene expression gene transfer using an adenoviral vector in patients analysis using SAGE tag database has shown that with mesotheliomas, we found that antitumor mesothelioma has the second highest co- immune responses targeting mesothelin were expression of CA125 and mesothelin after ovarian elicited in several patients (12). A recent study cancer (26). Rump and colleagues have shown that indicated that anti-mesothelin antibodies and mesothelin binds to CA125 and that this circulating mesothelin relate to the clinical state in interaction may mediate cell adhesion (26). ovarian cancer patients (13). Pastan and colleagues Scholler et al. recently showed that CA125/mesothelin-dependent cell attachment Downloaded from www.jbc.org by guest, on August 14, 2011 developed an immunotoxin (SS1P) with a Fv for mesothelin (14). Two Phase I clinical trials were could be blocked with anti-CA125 antibodies (27). completed at the National Cancer Institute (NIH, Since mesothelin is present on peritoneal Bethesda, MD) and there was sufficient antitumor mesothelium, there may be an important role for activity of SS1P to justify a Phase II trial. A the mesothelin-CA125 interaction in the chimeric antibody containing the mouse SS1 Fv tumorigenesis of ovarian cancer and mesothelioma for mesothelin was also developed and is currently in the peritoneal cavity. The mesothelin binding examined in a Phase I clinical trial for ovarian site on CA125 may lie within the 156 amino acid cancer, mesothelioma, pancreatic cancer, and non- TR units, indicating multimeric binding of small cell lung cancer (15). mesothelin to CA125. It has been found that the Mucins are heavily glycosylated proteins extraordinarily abundant N-glycans on CA125, found in the mucus layer or at the cell surface of presumably in the TR region, are required for many epitheliums (16). There are two structurally binding to both glycosylated and non-glycosylated distinct families of mucins, secreted and mesothelin (28). membrane-bound forms. CA125 (also known as Here, we identified the binding site of CA125 MUC16) was first identified in 1981 by OC125, a on mesothelin by use of truncated mutagenesis and mAb that had been developed from mice alanine replacement approaches. We measured immunized with human ovarian cancer cells (17). binding qualitatively by Western blot overlay The first cDNA clones were reported in 2001 (18, assays and quantitatively by enzyme-linked 19). CA125 is a very large membrane-bound cell immunosorbent assay (ELISA). We also evaluated surface mucin, with an average molecular weight the interaction of CA125 and mesothelin on cancer between 2.5 and 5 million Dalton. It is also cells by flow cytometry. Furthermore, we have heavily glycosylated with both O-linked and N- shown that a single chain mAb (SS1) recognized linked oligosaccharides (20). The peptide the CA125-binding domain and blocked the backbone of CA125 is composed of the N- mesothelin-CA125 interaction on cancer cells. The terminal region, extensive Ser/Thr/Pro-rich identified CA125-binding domain-Fc fusion tandem repeats (TR) with 156 amino acids each protein also significantly inhibited cancer cell with both N- and O-glycosylations, a SEA domain adhesion. Our results suggest that conformation- with high levels of O-glycosylation and a C- sensitive structures of the region (296-359) are terminal region with a short cytoplasmic tail (19). required and sufficient for the specific binding of The SEA domain was first identified as a module mesothelin to CA125. The domain proteins or the commonly found in sea urchin sperm protein, antibodies that block the mesothelin-CA125 enterokinase and agrin (21, 22). The significance interaction merit evaluation as new therapeutic of the SEA domain in CA125 is not clear. agents in treating peritoneal malignant tumors. Mesothelin and CA125/MUC16, Page 3 phosphate buffer pH 5.0, and eluted with 100 mM EXPERIMENTAL PROCEDURES glycine-HCl pH 3.0, and neutralized in 1M Tris Cell Culture - OVCAR-3 (ovarian) cells were pH8.0. Fractions were collected using the AKTA grown in RPMI 1640 (Dulbecco) supplemented FPLC system (GE Life Sciences) and pooled and with 20% FBS, 1% penicillin-streptomycin, 1% L- concentrated. Final protein concentration was Glutamine, and 0.2% human insulin. YOU measured using Coomassie Plus Protein Assay (mesothelioma) cells were grown in RPMI 1640 Reagent (Pierce). Fractions of the dominant peak (Dulbecco) supplemented with 10% FBS, 1% were run on a SDS-PAGE gel under non-reducing penicillin-streptomycin, and 1% L-Glutamine. and reducing conditions. To verify that generated HEK 293T cells were grown in 100 mm tissue proteins were not aggregated, mesothelin and its culture dishes (Falcon) with Dulbecco’s Modified mutants were subsequently run over a TSK gel- Eagle Medium (DMEM) and supplemented with filtration size exclusion column (TOSOH 10% FBS, 1% penicillin-streptomycin, and 1% L- Bioscience LLC, Montgomeryville) at 0.5 ml/min Glutamine. in PBS pH7.5. Truncated Mutant Constructs - Full-length and Flow Cytometry - To determine binding of fragments of mesothelin were amplified by PCR mesothelin fragments to CA125 on the cell from pMH107 (GenBank Accession No. surface, OVCAR-3 or YOU cells were grown until AY743922) (29). Primers were designed to confluent, detached, and then incubated with 1 Downloaded from www.jbc.org by guest, on August 14, 2011 incorporate flanking EcoRI and NotI restriction µg/mL of mesothelin or its fragments in FACS enzyme sites to facilitate in-frame cloning into a buffer (5% BSA, 0.01% NaN3) for 1 h on ice. modified pSecTag2 vector (Invitrogen) (Table 1). Bound fragments were detected by incubating with Constructs contained an Ig-κ leader sequence a 1:200 dilution of Goat anti-Rabbit IgG-PE followed by the rabbit IgG Fc and the full-length (Biosource) secondary antibody in FACS buffer sequence of extracellular domain of mesothelin for 0.5 h on ice. Cells were analyzed using (pMH113) or its fragments, followed by a myc FACSCalibur (BD Biosciences). Each binding epitope and 6xHis tag. The rabbit IgG Fc (rFc) and experiment was repeated three to five times. mesothelin fragments were separated by a In inhibition assays, cells were incubated with thrombin cleavage site and a flexible linker. The Flag-tagged mesothelin and excess amount (10- rFc-mouse mesothelin fusion (pMH117) was fold) of mesothelin or mesothelin fragments constructed using the same strategy. without a Flag tag for 1 h on ice. Bound Flag- Alanine mutants were generated by PCR tagged mesothelin proteins were detected by mutagenesis of pMH107-derived human incubating with 1:100 dilution of an anti-Flag tag mesothelin (hnMSLN) cDNA followed by cloning mAb (Sigma) followed by PE conjugated goat into the modified pSecTag2 vector. Mutations anti-mouse IgG (Biosource). were introduced in either forward or reverse Sandwich ELISA - Nunc MaxiSorp 96 well primers or by using a two-step overlap-extension flat-bottomed plates were incubated overnight PCR reaction (Table 2). with 5 µg/mL goat anti-rabbit IgG (Jackson Transfections and Mesothelin Mutant-Fc ImmunoResearch Laboratories) in PBS, followed Fusion Protein Production - HEK 293T cells were by an overnight block with 5% BSA, 0.01% NaN3 grown until 60% confluent on 100 mm tissue in PBS. Purified Fc mesothelin fragments were culture dishes (Falcon). Constructs encoding diluted to 1 µg/mL in ELISA buffer (0.01% mesothelin-Fc fusion proteins were transiently Tween 20, 10% Pierce SuperBlock) and incubated transfected using Lipofectamine (Invitrogen) in 6 on plate for 1 h at room temperature (RT). Plates mL serum free media. Three to 5 h later, 6 mL of were then incubated with OVCAR-3 supernatant 20% FBS DMEM was added to each dish and containing CA125 for 1 h at RT. To detect bound incubated for 48 h. Media was harvested CA125 a 1:200 dilution of anti-CA125 OC125 subsequently on a daily basis and replaced with mAb (Zymed) was incubated for an additional fresh medium. Fc fusion proteins were purified hour at RT; subsequently a 1:1500 dilution of goat from the media using columns containing Protein anti-mouse IgG HRP conjugate (Biosource) was A Sepharose (Amersham Biosciences). One mL added for 1 h at RT. The plates were washed four columns were loaded, washed with citrate- times with ELISA buffer between each coating. Mesothelin and CA125/MUC16, Page 4 Visualization was achieved with TMB detection with Dunnett's and Newman-Keuls multiple reagent (KPL) and absorbance was read at 450 nm comparison post tests. P values < 0.05 were with SpectraMax Plus plate reader (Molecular considered statistically significant. Devices). Western Blots - Purified Fc mesothelin RESULTS fragments (500 ng) were mixed with Laemmli Generation of Mesothelin Mutants - Truncated Sample Buffer (Biorad) supplemented with 5% B- mutants of mesothelin were generated to Mercaptoethanol. Samples were boiled for 2 min sequentially narrow down the binding domain to and separated on 4-20% SDS-PAGE gels CA125. As shown in Fig. 1, portions of (Invitrogen). After transfer for 4 h at 30V, the mesothelin were PCR amplified to incorporate PVDF membrane was blocked overnight at 4°C in NotI and EcoRI restriction sites and cloned into a 1% Western Blocking Reagent (Roche) in Tris modified pSecTag 2B vector containing an N- buffered saline (50 mM Tris-HCl, 150 mM NaCl). terminal rFc fragment. HEK 293T cells were The membrane was then incubated with OVCAR- transfected and Fc mesothelin proteins were 3 supernatant + 0.5% blocking solution for 1 h at collected and purified from the supernatant over RT. This was followed by incubation with 1:200 the course of 8-10 days. Constructs encoding dilution of OC125 mAb for 1 h at RT. Detection amino acid residues 296-390 (Region I), 391-486 was performed with goat anti-mouse IgG-HRP (Region II), and 487-581 (Region III) of Downloaded from www.jbc.org by guest, on August 14, 2011 conjugate (Biosource) 1:1000 for 1 h. mesothelin were initially generated and tested. Determination of Affinity Constants (KD) – As Constructs encoding smaller fragments within previously described (29), equilibrium constants Region I , Region IAB (296-359), Region IBC and Scatchard plots were determined by using the (328-405), Region IA (296-337), Region IB (328- Marquardt-Levenberg algorithm for nonlinear 369), and Region IC (360-405) were also regression with the Prism software (version 5.0, generated. It was found that secretion of the IA GraphPad Software, San Diego, CA). (296-337) fragment was prevented by protein Mammalian Cell Display – As previously aggregation; however, this problem was described (30), single chain Fv antibody (scFv) circumvented by moving the rFc to the C-terminus SS1 was cloned into an expression vector of the mesothelin fragment. (pMH112) for cell surface expression of scFv on PCR mutagenesis was used to generate a panel HEK-293 cells. of alanine mutants within region 296-359 (Region Heterotypic Cancer Cell Adhesion Assay – We IAB). As shown in Fig. 2A, both human and followed a recently developed protocol (15). mouse mesothelin (mnMSLN) proteins bind Briefly, OVCAR-3 or YOU cells (1 x 105) were human CA125. It was reasoned that those residues seeded in triplicate in microplates, incubated conserved between the two species would be more overnight at 37˚C/5% CO2 . The following day, H9 likely to be involved in the interaction. Specific (A431-Mesothelin+) (11) were harvested and amino acids were targeted based on the homology loaded with Calcein AM cell dye (Invitrogen) as between human and murine mesothelin (Fig. 2B). per the instruction manual. The OVCAR-3 or In addition, we hypothesized that these residues YOU monolayers were washed once with 200 µL are likely to be involved in the carbohydrate 10% complete RPMI and pre-incubated for 1 h at binding. In total, eight alanine mutant constructs 4oC with purified mesothelin Fc fusion proteins. were made: Y318A, W321A, E324A, F344A, The labeled H9 (2 x 105) cells were added to E347A, K353A, H354A, and K355A. Of these triplicate wells for 1 h at 4˚C. Wells were gently mutants, four were secreted at high enough levels washed five times with 200 µL PBS by inverting to purify using a Protein A column – Y318A, the plate on paper towels. Cell adherence and its W321A, E324A, and H354A. The purity and inhibition were quantitated using VICTOR3 molecular weight of each purified protein was Multilabel Counter model 1420 (Perkin Elmer). confirmed on SDS-PAGE. The other four mutants, Statistical Analysis - The data obtained was F344A, E347A, K353A and K355A, were not entered in Prism (version 5) for Windows secreted into the culture supernatants due to (GraphPad Software,) for statistical analysis. Flow aggregation inside cells according to cytometry raw data were analyzed by ANOVA immunoblotting of whole cell lysates (data not Mesothelin and CA125/MUC16, Page 5 shown), indicating that mutations of these residues for the mesothelin-CA125 interaction is consistent may cause misfolding of mesothelin. For all with the value (~5 nM) previously obtained on proteins purified, a distinct peak was found on a OVCAR-3 cells by flow cytometry (28). Three TSK size exclusion column (Fig. 3). The rFc – smaller fragments (296-337, 328-369, 360-405) extracellular portion of mesothelin 296-581 fusion within Region I showed no binding to CA125 in protein (full-length) was estimated to be ELISA (data not shown), indicating the first 64 approximately 75-kDa, while truncated mutants residues at the N-terminus of cell surface were relatively smaller in molecular weight (~50- mesothelin is the irreducible binding domain on kDa for Regions I, II and III and ~40-kDa for IAB, mesothelin for the CA125 protein. It is striking IBC and alanine mutants of IAB). that substitution of the tyrosine at position 318 Binding of CA125 to Mesothelin and its with an alanine (Y318A) completely disrupted the Mutants - To examine the interaction of CA125 interaction with CA125. Alanine mutations at Glu- and mesothelin mutants, we used Western blot 324 (E324A; KD = 42.4 nM) and Trp-321 overlay analysis. We ran equal amounts (500 ng) (W321A; KD = 19.5 nM) partially reduced the of each protein an SDS-PAGE gel and transferred binding of mesothelin to CA125. The alanine to a PVDF membrane. Membrane was blotted with mutation at His-354 (H354A) did not change the OVCAR-3 supernatant containing CA125 mesothelin-CA125 interaction (KD = 2.71 nM). followed by OC125, an anti-CA125 mAb. As To verify that fragments were specifically Downloaded from www.jbc.org by guest, on August 14, 2011 shown in Fig. 4, full-length extracellular domain binding to CA125, an ELISA assay was employed of mesothelin (296-581), Region I (296-390) and wherein captured mesothelin fragments were Region IAB (296-359) bound CA125. A 64 amino incubated with either OVCAR-3 supernatant acid fragment (IAB) at the N-terminus of containing CA125 or supernatant from the mesothelin (296-359) retained 100% binding OVCAR-3 with knockdown of CA125 expression. capability to CA125. However, three smaller No signal was detected from those fragments fragments, Region IA (296-337), Region IB (328- incubated with the supernatant from CA125- 369), and Region IC (360-405), consisting of knockdown cells (data not shown), indicating that approximately 42 amino acids covering all the the binding between mesothelin and CA125 in residues within Region I showed no binding to ELISA was specific. CA125 in Western blot. The alanine mutation at Cell Binding Assays by Flow Cytometry - To His-354 did not change the mesothelin-CA125 assess binding to CA125 on cancer cells, wild-type interaction. Interestingly, alanine mutations at Tyr- mesothelin and its mutants were incubated with 318 (Y318A) and Glu-324 (E324A) abolished the cells. As shown in Fig. 6, Region I (a 95 amino binding of mesothelin to CA125. Alanine mutation acid fragment consisting of residues 296-390 at at Trp-321 (W321A) partially reduced the binding the N-terminus) of mesothelin was found to bind of mesothelin to CA125. to OVCAR-3 cells, while fragments Region II Kinetic Studies by ELISA - To precisely (391-486) and Region III (487-581) showed no characterize the interaction between CA125 and binding. The smallest fragment that still contained mesothelin and to determine a binding affinity most (~90%) of binding activity to CA125 was (KD) for each mesothelin fragment, we used a Region IAB. Three smaller fragments within quantitative ELISA-based assay. ELISA plates Region I, Region IA (296-337), IB (328-369), and were coated with a goat anti-rabbit IgG antibody IC (360-405), were also tested. Only Region IB overnight for capturing the Fc mesothelin mutant had modest (~10%) CA125-binding activity. fusion proteins. OVCAR-3 supernatant containing These data indicate that IAB, the first 64 residues soluble CA125 was then added, followed by the at the N-terminus of cell surface mesothelin, is the OC125 mAb. Overall results from the ELISA were minimum region which retains the most binding consistent with those seen in the Western blot activity to CA125. The IAB binding domain was overlay assays except for the E324A mutant of found to bind with comparable affinity, when mesothelin (Fig. 5). The average KD for the compared to the full-length mesothelin (FULL). binding of the Fc fusion protein of wild-type This suggests that it is primarily the N-terminus of mature mesothelin (296-598), Region I (296-390) cell surface mesothelin that is involved in the and IAB (296-359) to CA125 was ~3 nM. The KD interaction with CA125 and the minimum Mesothelin and CA125/MUC16, Page 6 sequence for CA125 binding activity is Region CA125 interaction but not induce cell adhesion IAB (328-405). and signaling. To this end, we examined if any of Four alanine mutants (Y318A, W321A, the mesothelin truncated or alanine mutants E324A, and H354A) generated within the region reported here can compete with the binding of 296-359 were similarly assessed for their ability to wild-type mesothelin to CA125. We co-incubated bind to CA125 on the surface of OVCAR-3 cells. a Flag-tagged wild-type mesothelin with a panel of It was found that the substitution of alanine for our mesothelin mutants (Fig. 8). Region IAB can tyrosine at residue 318 completely ablated binding effectively inhibit the mesothelin-CA125 (Fig. 6). The H354A mutant conversely showed interaction. Other constructs such as Region I and comparable binding to CA125 as the wild-type H354A can also inhibit the mesothelin-CA125 region 296-359. Alanine mutants of the tryptophan interaction while Regions II, III and IBC, mutants at 321 and glutamic acid at 324 also demonstrated Y318A, E324A and W321A cannot. We have decrease in binding (10-20%) to CA125 on the demonstrated that full-length mesothelin (FULL), surface of OVCAR-3 cells. Region I, IAB and the H354A mutant significantly Using ANOVA with Dunnett's and Newman- inhibited the binding of mesothelin with a Flag tag Keuls multiple comparison post tests, we have to CA125 on OVCAR-3 cells, as compared to demonstrated that full-length mesothelin (FULL), Regions II, III, IBC and the Y318A, W321A and Region I, IAB and the H354A mutant significantly E324A mutants (p < 0.05). As compared to Region Downloaded from www.jbc.org by guest, on August 14, 2011 bound to CA125 on OVCAR-3 cells, as compared IAB, the inhibition of the methelin-CA125 to Regions II, III, IBC, IA, IB, IC and the Y318A, interaction by full-length mesothelin (FULL) and W321A and E324A mutants (p < 0.05). As Region I was not significant (p > 0.05), clearly compared to Region IAB, the binding by full- indicating that IAB is the minimal sequence (296- length mesothelin (FULL), the H354A mutant and 359) of CA125-binding domain. Region I was not significant (p > 0.05), indicating Epitope Mapping of scFv SS1 by Mammalian that IAB is the minimal sequence of CA125- Cell Display – Since Fv SS1-derived antibody binding domain. drugs are currently in several multi-center clinical Since surface protein expression of mesothelin trials to treat mesothelioma and ovarian cancer, we and CA125 are also found frequently in malignant decided to examine its epitope using the mesothelioma, we then examined the binding of mesothelin fragments we have made. To this end, mesothelin and its mutants to the YOU we used a new method (called “mammalian cell mesothelioma cell line (Fig. 7). The binding display”) recently developed in the laboratory (30) patterns were similar to those seen in OVCAR-3 to express the SS1 scFv on HEK-293 cells. We cells except for Region I. As shown in Fig. 8, incubated the HEK-293 cells expressing scFv SS1 wild-type mesothelin (FULL), Regions I and IAB with the mesothelin and its fragments or mutants. bound CA125 on YOU cells significantly stronger As shown in Fig. 9, the epitope of scFv SS1 than Regions II, III and IBC (p < 0.05). Unlike overlaps the CA125-binding site on mesothelin. what we saw in OVCAR-3 cells, Region I retained Inhibition of the Mesothelin-CA125 about 40% of the CA125 binding activity on YOU Interaction by the SS1 scFv – We investigated cells. Nevertheless, the binding of Region IAB to whether or not the SS1 scFv could block the CA125 on YOU cells is comparable to the full- mesothelin-CA125 interaction. We made the SS1 length mesothelin (FULL) (p > 0.05). We also in the format of a scFv SS1-PE38 fusion protein found that Region IB had modest (~10%) binding (8). We co-incubated the SS1 or HA22, a scFv- activity. The alanine replacements at positions PE38 specific for CD22 (29) with mesothelin and 318, 321 and 324 showed significant decrease of OVCAR-3 or YOU cells. As shown in Fig. 10, the CA125 binding activities (10-30%) on YOU cells. SS1 scFv remarkably inhibited the mesothelin- Inhibition of the Mesothelin-CA125 CA125 interaction while the HA22 scFv did not. Interaction by the CA125-binidng Domain - The Inhibition of Cancer Cell Adhesion by the molecular mechanisms underlying the cell CA125-binding Domain – Finally we explored the adhesion and signaling induced by the mesothelin- possibility that the CA125-binding domain can CA125 interaction are not clear. An ideal functionally block cancer cell adhesion. We used antagonist drug would disrupt the mesothelin- the assay system recently established by Hassan et Mesothelin and CA125/MUC16, Page 7 al. (15). We measured adhesion of fluorescently Here we have shown that it is primarily the N- labeled mesothelin-expressing H9 cells (11) onto terminus of the extracellular domain of mesothelin CA125 positive OVCAR-3 or YOU cancer cells. (residues 296-359) that is sufficient and necessary Fig. 11 shows the results of this study. Significant for binding to CA125. A fragment (IAB) heterotypic cell binding was seen on a monolayer consisting of the first 64 amino acids displayed of OVCAR-3 or YOU cells with the control binding to CA125 on ovarian cancer and CD22-Fc fusion protein or a control fragment mesothelioma cells as detected by flow cytometry (Region IBC). The CA125-binding domain (IAB)- as well as in ELISA and Western blot assays. Fc fusion protein completely abolished H9 cell Other regions of mesothelin (391-486; 487-581) adhesion onto OVCAR-3 or YOU cells. A showed no affinity for CA125. Efforts to further statistically significant inhibition with the CA125- narrow down the binding domain with the domain was detected with concentrations as low as truncated mutant approach, however, showed that 1 µg/mL (Fig. 11). this fragment was irreducible; smaller fragments Structure of CA125-binding Domain - Since a 296-337, 328-369, and 360-405 showed no or only three-dimensional structure of mesothelin is modest binding to CA125. This may be due to a currently not available, the secondary structure specific conformation that is required for binding was evaluated by the algorithms PROF that is lost when mesothelin is truncated smaller (http://www.predictprotein.org) and APSSP2 than the 64 amino acid length. Downloaded from www.jbc.org by guest, on August 14, 2011 (http://www.imtech.res.in/raghava/apssp2). The Our efforts to further find specific amino acids CA125-binding domain is primarily composed of within CA125-binding domain (296-359) of helix-turn-helix repeats (Fig. 12). Interestingly, mesothelin that are involved in the interaction with Tyr-318 whose alanine replacement significantly CA125 led us to mutate several suspected amino reduces the CA125 binding is located at the coil acid residues to alanine to see if it would affect between two helical secondary structures. The binding affinity. We chose residues that were tyrosine seems a critical residue which either homologous between mouse and human and those directly binds the N-glycan on CA125 or indirectly likely to be involved in the carbohydrate binding. plays an important role by maintaining a The replacement of tyrosine with alanine in the conformation required for CA125 binding. The IAB fragment resulted in no binding to CA125 on partial loss of CA125 binding activities of the ELISA and Western blot and significantly reduced W321A and E324A mutants may indicate that most CA125-binding activity on cancer cells by other residues near 318 are also involved. The fact flow cytometry indicating the tyrosine at position that Region IB alone had only a modest CA125- 318 is critical for either direct binding to CA125 binding activity (~10%) indicates that the CA125- or maintaining the conformation of mesothelin to binding domain requires Region IA for its full allow binding to occur. Alanine mutants of W321 activity. and E324 in the vicinity of the tyrosine at 318 also showed decreased binding to CA125 in all assays DISCUSSION but were not nearly as dramatic. Mutating the Cancer cells commonly spread within the histidine at 354 had no effect on the mesothelin- peritoneal cavity via seeding to mesothelium-lined CA125 interaction. structures. The interaction between CA125, a During the preparation of SDS-PAGE, mucin present on a majority of ovarian cancer and proteins are typically reduced and denatured by mesothelioma cells, and mesothelin, a GPI- treatment with the Laemmli sample buffer. Many anchored glycoprotein present on the mesothelial protein functions depend on protein secondary and cells lining along the peritoneal cavity, has been tertiary structures, which could be disrupted under suggested to facilitate implantation and metastasis reducing and denaturing conditions. The fact that of tumors (26, 28). Blocking the mesothelin- CA125 bound to mesothelin (296-581), Region I CA125 interaction may prevent or reverse (296-390) and IAB (296-359) in Western blot metastasis and lead to overall improved survival in overlay assays may indicate that the mesothelin cancer patients. A better understanding of how proteins were renatured rapidly during the PVDF these two proteins interact may eventually aid in membrane transfer. The renaturing may allow developing such a therapy. Mesothelin and CA125/MUC16, Page 8 mesothelin to recover their secondary and tertiary Furthermore, we have shown that a single structures specific for CA125 binding. chain mAb (SS1) recognizes the CA125-binding Mesothelin is predicted to be primarily domain and blocks the mesothelin-CA125 composed of helix-turn-helix repeats. interaction on cancer cells. Most interestingly, the Interestingly, Tyr-318 whose alanine replacement CA125 binding domain-Fc fusion significantly abolishes the CA125 binding of IAB is located at inhibits heterotypic cancer cell adhesion. The SS1 the coil between two helical secondary structures Fv-derived immunotoxin and a chimeric (the SS1 (Fig. 12), indicating that binding could be mouse Fv/human Fc) antibody are currently in dependent on the conformation of this region. clinical trials for mesothelioma, ovarian cancer, We further showed that the CA125-binding pancreatic cancer and non-small cell lung cancer. domain (IAB) or its alanine mutant could Here, we have shown that mesothelin binds to effectively block the binding of wild-type CA125 primarily with the N-terminal portion of mesothelin to CA125, indicating the CA125 cell surface mesothelin. The CA125-binding binding domain Fc-fusion protein is a good domain of 64 residues, likely a helical structure, is candidate to be used as a potential antagonist to irreducible. We have further shown that a tyrosine inhibit the mesothelin-CA125 interaction. at position 318 is critical for binding to CA125. Moreover, the Fc portion of the molecule with The CA125-binding domain significantly inhibits antibody dependent cell mediated cytotoxicity or cancer cell adhesion and merits evaluation as a Downloaded from www.jbc.org by guest, on August 14, 2011 complement dependent cytotoxicity could recruit new therapeutic agent in preventing or treating NK cells or complements to kill the CA125- peritoneal malignant tumors. positive tumor cells. Since Enbel (TNFR-Fc), an FDA-approved Fc fusion drug, many peptide-Fc ACKNOWLEDGMENTS fusion proteins are currently in clinical trials (32). We thank Ira Pastan for helpful discussions, and Anna Mazzuca for editorial assistance. REFERENCES 1. Runowicz, C. D. (2008) Cancer J. 14, 7-9 2. Palumbo, C., Bei, R., Procopio, A., and Modesti, A. (2008) Curr. Med. Chem. 15, 855-867 3. Chang, K., Pastan, I., and Willingham, M. C. (1992) Int. J. Cancer 50, 373-381 4. Chang, K., and Pastan, I. (1996) Proc. Natl. Acad. Sci. USA 93, 136-140 5. Hassan, R., and Ho, M. 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Pharmacol. 8, 616-619 Mesothelin and CA125/MUC16, Page 10 TABLE 1 Primers used to construct truncated mutants of mesothelin. The restriction enzyme sites are underlined. Fragments Primers (5’ -> 3’) Region I Forward: (296-390) AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: CTCTTCTTCTGCGGCCGCCGTCACATTCCACTTGCGAAT Region II Forward: (391-486) AGAAGAAGAGAATTCTCCCTGGAGACCCTGAAGGCT Reverse: CTCTTCTTCTGCGGCCGCCTGGAAAGCAAGGCGGGCCTT Region III Forward: (487-581) AGAAGAAGAGAATTCAACATGAACGGGTCCGAATAC Reverse: CTCTTCTTCTGCGGCCGCGCCCTGTAGCCCCAGCCCCAG Downloaded from www.jbc.org by guest, on August 14, 2011 Region IAB Forward: (296-359) AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: CTCTTCTTCTGCGGCCGCGAGCTCATCCAGTTTATGCTT Region IBC Forward: (328-405) AGAAGAAGAGAATTCGATGCGGCCCTGCTGGCCACC Reverse: CTCTTCTTCTGCGGCCGCGTGCCCTTTGTTGACTTCAAG Region IA Forward: (296-337) AGAAGAAGAAAGCTTGAAGTGGAGAAGACAGCCTGT Reverse: TCTTCTTCTGGATCCGTCCATCTGGGTGGCCAGCAG Region IB Forward: (328-369) AGAAGAAGAGAATTCGATGCGGCCCTGCTGGCCACC Reverse: CTCTTCTTCTGCGGCCGCGATCACAGACTCGGGGTAACC Region IC Forward: (360-405) AGAAGAAGAGAATTCTACCCACAAGGTTACCCCGAG Reverse: CTCTTCTTCTGCGGCCGCGTGCCCTTTGTTGACTTCAAG Mesothelin and CA125/MUC16, Page 11 TABLE 2 Primers used to generate alanine replacement mutants of mesothelin. The restriction enzyme sites are underlined. Mutants Primers (5’ -> 3’) Y318A Forward: AGAAGAGAATTCGAAGTGGAGAAGACAGCCTGTCCTTCAGGCAAGAAGG CCCGCGAGATAGACGAGAGCCTCATCTTCGCCAAGAAGTGGGAG Reverse: CTCTTCTTCTGCGGCCGCGAGCTCATCCAGTTTATGCTT W321A Forward: TCATCTTCTACAAGAAGGCGGAGCTGGAAGCCTGCGTGG Reverse: CCACGCAGGCTTCCAGCTCCGCCTTCTTGTAGAAGATGA E324A Forward: AGAAGAGAATTCGAAGTGGAGAAGACAGCCTGTCCTTCAGGCAAGAAGG CCCGCGAGATAGACGAGAGCCTCATCTTCTACAAGAAGTGGGAGCTGGCA Downloaded from www.jbc.org by guest, on August 14, 2011 GCCTGCGTG Reverse: CTCTTCTTCTGCGGCCGCGAGCTCATCCAGTTTATGCTT F344A Forward: AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: TTCTTCTGCGGCCGCGAGCTCATCCAGTTTATGCTTTAGGACGTCCAGCTG CTCGTAGGT GGCGGGGATGGCGTT E347A Forward: AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: TTCTTCTGCGGCCGCGAGCTCATCCAGTTTATGCTTTAGGACG TCCAGCTGCGCGTAGGTGAAGGG K353A Forward: AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: TTCTTCTGCGGCCGCGAGCTCATCCAGTTTATGCGCTAGGACGTCCAG H354A Forward: AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: CTCTTCTTCTGCGGCCGCGAGCTCATCCAGTTTAGCCTTTAGGACGTC K355A Forward: AGAAGAAGAGAATTCGAAGTGGAGAAGACAGCCTGT Reverse: CTCTTCTTCTGCGGCCGCGAGCTCATCCAGTGCATGCTTTAGGACGTC Mesothelin and CA125/MUC16, Page 12 FIGURE LEGENDS FIGURE 1. Generation of truncated and alanine replacement mutants of mesothelin. A. The mesothelin (MSLN) gene encodes a precursor protein of 622 amino acids. On translocation into the endoplasmic reticulum the N-terminal signal peptide (red; residues 1–33) and the C-terminal GPI anchor addition signal (blue; a predicted cleavage site: Ser598) are removed and the latter replaced with a GPI anchor. The MSLN precursor (71-kDa) is cleaved into two products, the 30-kDa megakaryocyte potentiating factor (MPF; residues Ser34–Arg286) (31) and the 41-kDa GPI-anchored membrane-bound mature MSLN (orange) starting from Glu296. The proteolytic cleavage region (green) contains a furin cleavage site at Arg295, and other protease cleavage sites including a trypsin cleavage site at Arg286. The four predicted N-linked glycans (black lollipops; Asn57, Asn388, Asn488 and Asn515) on mesothelin are indicated. Truncated mutants (Regions I, II, III, IAB, IBC, IA, IB and IC) were generated as rabbit Fc fusion proteins to sequentially narrow down the CA125 binding domain of mesothelin. B. Mesothelin and truncated mutants were generated using a modified pSecTag2B vector that when transfected into HEK 293T cells created secreted rabbit Fc-fusion proteins. A CMV promoter (PCMV) drove the expression of an Ig-κ signal, followed by a rabbit Fc fragment, a thrombin cleavage site and the desired portion of mesothelin. A 6xHis tag was added at the C-terminal of the construct. Downloaded from www.jbc.org by guest, on August 14, 2011 FIGURE 2. Key residues proposed for alanine mutations. A. OVCAR-3 cells were incubated with 1 µg/mL of human mesothelin (hnMSLN) or mouse mesothelin (mnMSLN) rFc fusion proteins. The mesothelin binding on OVCAR-3 cells was detected by a goat anti-rabbit IgG Fc PE conjugate. B. Eight residues identical between hnMSLN and mnMSLN in Region IAB were selected for alanine replacement. Region IAB starts at E296 and ends at L359 (*) in hnMSLN. Alanine mutants within Region IAB (296- 359) were expressed. Alanine mutants Y318A, W321A, E324A and H354A (arrows) were secreted and purified for analysis. The other four mutants, F344A, E347A, K353A and K355A, were not secreted and found aggregated inside transfected HEK 293T cells. FIGURE 3. Chromatograms from TSK size exclusion columns. Purified mesothelin (A) and truncated mutants of mesothelin (B-I) and alanine mutants within Region IAB (J-K) were run over a TSK size exclusion column to verify that the generated proteins were not aggregated. Truncated mutants and alanine mutants had a dominant peak that matched with the wild-type fragment, indicating that there was minimal aggregation. A. Full-length mature MSLN 296-581; B. Region I 296-390; C. Region II 391-486; D. Region III 487-581; E. Region IAB 296-359; F. Region IBC 328-405; G. Region IA 296-337; H. Region IB 328-369; I. Region IC 360-405; J. Y318A; K. W321A; L. E324A; and M. H354A. FIGURE 4. Western blots of mesothelin fragments and alanine mutants. Each protein (500 ng) was run on an SDS-PAGE gel and transferred to a PVDF membrane. Membrane was blotted with OVCAR-3 supernatant containing CA125 followed by OC125 (anti-CA125 mAb). A. Full-length extracellular domain of mesothelin (FULL, 296-581), Region I (296-390) and Region IAB (296-359) bind CA125. Regions II (391-486), III (487-581), IBC (327-390), IA (296-337), IB (328-369) and IC (360-405) do not bind CA125. B. Alanine mutants within Region IAB (296-359) show differential binding. Alanine mutations at Tyr-318 (Y318A) and Glu-324 (E324A) abolish the binding of mesothelin to CA125. Alanine mutation at Trp-321 (W321A) partially reduce the binding of mesothelin to CA125. The alanine mutation at His-354 does not change the mesothelin-CA125 interaction. Mesothelin and CA125/MUC16, Page 13 FIGURE 5. Biniding kinetics of mesothelin mutants and CA125. Scatchard plots (top left corner) were made. ELISA plates captured the Fc mesothelin mutant fusion proteins at various concentrations (x axis; see Experimental Procedures). OVCAR-3 supernatant containing soluble CA125 was then added, followed by the OC125 mAb and a goat anti-mouse IgG HRP. Visualization was achieved with TMB detection reagent and absorbance was read at 450 nm (y axis). The full-length mature form of mesothelin (FULL) bound to CA125 with an approximate affinity of 1.54 nM. Region IAB wild-type (wt) (296-359) had a KD of 3.35 nM. Substitution of the tyrosine at position 318 with an alanine (Y318A) completely disrupted the interaction with CA125. Alanine mutation at Glu-324 (E324A; KD = 42.4 nM) and Trp-321 (W321A; KD = 19.5 nM) reduce the binding of mesothelin to CA125. The alanine mutation at His-354 (H354A) does not change the mesothelin-CA125 interaction (KD = 2.71 nM). FIGURE 6. Binding of truncated mutants and alanine mutants to CA125 on the cell surface of OVCAR-3 cells. A. OVCAR-3 ovarian cancer cells were incubated with full-length extracellular domain of mesothelin (FULL), Region I, IAB or IBC. The binding was visualized with a goat anti-rabbit IgG PE- conjugated secondary antibody by flow cytometry (gray line). Light gray shaded plot: secondary antibody only. B. A fluorescence intensity (geometrical mean) was used to quantitatively measure the CA125 binding. In each experiment, the binding of the full-length mature form of mesothelin (FULL) to CA125 Downloaded from www.jbc.org by guest, on August 14, 2011 was determined as 100% of binding. The secondary antibody only was used as a negative (0%) control. Full-length extracellular domain of mesothelin (296-581), Region I (296-390), Region IAB (296-359) and the H354A mutant of IAB bound to CA125 on OVCAR-3 cells significantly stronger than any other fragments or mutants on the figure (* p < 0.05). FIGURE 7. Binding of truncated mutants and alanine mutants to CA125 on the cell surface of YOU cells. A. YOU mesothelioma cells were incubated with full-length extracellular domain of mesothelin (FULL) or a mutant of mesothelin. The binding was visualized with a goat anti-rabbit IgG PE-conjugated secondary antibody by flow cytometry (gray line). Light gray shaded plot: secondary antibody only. B. A mean fluorescence intensity (geometrical mean) was used to quantitatively measure the CA125 binding on YOU cells. In each experiment, the binding of the full-length mesothelin (FULL) to CA125 was determined as 100% of binding. The secondary antibody only was used as a negative (0%) control. Full- length extracellular domain of mesothelin (296-581), Region I (296-390), Region IAB (296-359) and the H354A mutant of IAB bound to CA125 on YOU cells significantly stronger than any other fragments or mutants on the figure (* p < 0.05). FIGURE 8. Inhibition of the mesothelin-CA125 interaction by mesothelin mutants. OVCAR-3 cells were incubated with mesothelin or each mutant and the Flag-tagged mesothelin (see Experimental Procedures). The binding of Flag-mesothelin (Flag-MSLN) to CA125 was detected by an anti-Flag mAb. Full-length extracellular domain of mesothelin (296-581), Region I (296-390), CA125 binding domain or Region IAB (296-359) and the H354A mutant of IAB (dark gray shading) can significantly block the binding of Flag-tagged MSLN to CA125 on OVCAR-3 cells (* p < 0.05). FIGURE 9. Epitope mapping of single chain SS1 monoclonal antibody by mammalian cell display. HEK-293 cells expressing the anti-mesothelin SS1 single chain Fv were incubated with 5 µg/mL of with full-length extracullular domain of human mesothelin (100% binding), mouse mesothelin (mnMSLN) or a mutant of human mesothelin. The binding was visualized with a goat anti-rabbit IgG PE-conjugated secondary antibody by flow cytometry. SS1 recognized the CA125 binding domain in mesothelin. Mesothelin and CA125/MUC16, Page 14 FIGURE 10. Single chain Fv (scFv) SS1 blocks the mesothelin-CA125 interaction. OVCAR-3 (A, B) or YOU (C, D) were incubated with an equimolar concentration of mesothelin and a scFv-PE38 fusion protein (HA22 or SS1P). HA22 contains Fv specific for CD22. SS1P contains the SS1 Fv specific for mesothelin. SS1P significantly blocked the binding of mesothelin to CA125 on OVCAR-3 cells (B) or YOU cells (D) while HA22 did not (A and C). FIGURE 11. The CA125-binding domain blocks the mesothelin/CA125-mediated cancer cell adhesion. OVCAR-3 (A, C) or YOU cancer cells (B, D) formed monolayers. The OVCAR3 or YOU cancer cell monolayer was pre-incubated with full-length mesothelin, the CA125-binding domain (IAB), IBC or CD22 Fc fusion proteins before fluorescently labeled mesothelin-expressing H9 cells were added. Mesothelin or IAB significantly blocked cancer cell adhesion with concentrations as low as 10 µg/mL on OVCAR3 cells (A, C) or 1 µg/mL on YOU cells (B, D) as compared to the control protein (CD22 or IBC) (* p < 0.05). FIGURE 12. Interaction of CA125 and mesothelin. A. CA125 is heavily glycosylated with both O- linked and N-linked oligosaccharides. The peptide backbone of CA125 is composed of the N-terminal region, extensive Ser/Thr/Pro-rich tandem repeat (TR) containing 165 amino acids each with both N- and Downloaded from www.jbc.org by guest, on August 14, 2011 O-glycosylations, a SEA domain with high levels of O-glycosylation and a C-terminal region with a short cytoplasmic tail. Previous studies have shown that the N-glycan (28) in the TR region (27) is required for the binding of CA125 to mesothelin. B. The secondary structure of mesothelin was evaluated by the algorithms PROF and APSSP2. The N-glycan moiety of CA125 repeat units binds to CA125-binding domain (Region IAB), likely the helical structures around Tyr-318 at the N terminal of cell surface mesothelin. Line: coil. Tube: helix. Downloaded from www.jbc.org by guest, on August 14, 2011 A 1.50 B 1.25 Cell adhesion (x 100%) 1.00 0.75 0.50 0.25 Mesothelin * CD22 * 0.00 0.001 0.01 0.1 1 10 100 Fc fusion (µg/ml) C1.25 D 1.00 Cell adhesion (x 100%) 0.75 * 0.50 0.25 IAB * IBC 0.00 0.001 0.01 0.1 1 10 100 Fc fusion ( µ g/ml) Figure 11. Kaneko et al.
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