Tumor Biol. DOI 10.1007/s13277-014-1980-3

RESEARCH ARTICLE

Generation of a monoclonal antibody specific to a new candidate tumor suppressor, cell adhesion molecule 2 Meijin Huang & Ziyuan Xia & Yang Wang & Liang Huang & Qian Ma & Xia Chen & Hao Wang & Bin Lu & Yajun Guo

Received: 1 April 2014 / Accepted: 15 April 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Cell adhesion molecule 2 (CADM2) is an immunoglobulin (Ig)-like cell adhesion molecule, which belongs to the CADMs family. The four members of CADMs family including three Ig-like domains and a short cytoplasmic tail share high degree of identity with each other, making it difficult to specifically identify each members using western blotting or immunohistochemistry. And most of anti-CADM2 antibodies available commercially or used in published papers are rabbit polyclonal antibodies, exhibiting nonspecific recognition in studies. In this study, we developed a monoclonal antibody (mAb) specific to an epitope on the extracellular domain of CADM2 using conventional hybridoma technology. Western blot assays indicated that the established mAb, named as 2B11, was specific for CADM2 recognition without interference of other members of CADMs family. Furthermore, 2B11 was competent to detect CADM2 expression specifically on the surface membranes of several types of tumor tissues, avoiding the false results with the polyclonal antibodies due to nonspecific staining. In conclusion, the mAb 2B11 could be suitable for specific detection of CADM2 expression in tumor cells or tissues.

M. Huang : Z. Xia : Q. Ma : X. Chen : H. Wang : B. Lu (*) : Y. Guo (*) International Joint Cancer Institute, The Second Military Medical University, Shanghai 200433, China e-mail: [email protected] e-mail: [email protected] Y. Wang Department of Pathology, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China L. Huang Department of Hepatic Surgery I, Eastern Hepatobiliary Surgery Hospital, The Second Military Medical University, Shanghai 200438, China

Keywords CADM2 . Monoclonal antibody . Tumor suppressor . Immunohistochemistry

Introduction Silencing of tumor suppressor genes is a major event contributing to the development and progression of human cancers [1]. Recently, a subfamily of the immunoglobulin (Ig) superfamily, CADMs family, was reported to be tumor suppressor genes, consisting of four members (CADM1 to 4) [2–7]. Among CADMs family, cell adhesion molecule 2 (CADM2) is located on chromosome 3p12.1 and included in the candidate region harboring a possible tumor suppressor or susceptibility gene identified by several previous studies [8–12]. Previous reports showed that CADM2 gene is drastically downregulated in prostate carcinomas compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor [13]. Moreover, re-expression of CADM2 expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar, which suggests that CADM2 is critical for the regulation of prostate cancer cell proliferation [13]. The correlation between the loss of CADM2 expression and a highly malignant phenotype or a consequent poor prognosis appears to be a general event in oncology. In fact, loss of CADM2 expression has been recently shown to be also associated with increasing malignancy grade in human renal cell carcinoma [14]. As membrane proteins, CADMs are located in the cell membrane, mediating cell–cell interaction or communication [15]. All members of CADMs family consist of three extracellular Ig-like domains, one transmembrane domain and a short cytoplasmic tail containing a protein 4.1 motif and a type II PDZ interaction motif [15, 16]. And the four members share

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a high degree of amino acid identity [16]. Furthermore, CADMs are glycoproteins with multiple predicted N- or Oglycosylation sites in their extracellular Ig domains [16–18]. Meanwhile, the types and quantities of glycosylation vary between different cells and tissues [19]. Therefore, it is difficult to produce an antibody specifically identifying each members by western blotting or immunohistochemistry assays. In light of the importance of CADM2 protein in tumor progression reported above, it is urgent to prepare an effective antibody for the specific detection of CADM2 protein without the cross-reactivity among other members of CADMs family. In this study, we designed two CADM2-specific peptides to immunize mice and obtained two monoclonal antibodies specifically against CADM2, which were confirmed by enzyme-linked immunosorbent assay (ELISA), western blotting, and IHC.

longer time. Separation of reaction products are visualized by western blotting. Peptide synthesis Based on the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), we analyzed potential antigenic epitope of human CADM2 and designed two specific peptides, P1 and P2 for CADM2, which are distinguished with other members of CADMs family (Table 1). The P3 epitope peptide was designed according to published study [20], which is located in the C-terminus of the short cytoplasmic tails of CADM2 protein. All peptides were synthesized by 9-fluorenylmethoxy carbonyl-based solidphase chemistry (purity >85 % pure; Science Peptide Company, Shanghai, China) and coupled to keyhole limpet hemocyanin (KLH) for immunization. Immunization and antibody production

Material and methods Cell culture HEK293T, mouse myeloma cells NS-1, and hybridoma cells were cultured in Dulbecco’s Modified Essential Medium supplemented with 10 % fetal bovine serum (SAFC Biosciences, Lenexa, KS, USA). All cells were maintained under humidified atmosphere with 5 % CO2 at 37 °C. Plasmid construction and transfection CADM1, CADM2, CADM3, and CADM4 were cloned from human cDNA library by polymerase chain reaction (PCR) and tagged with an influenza hemagglutinin (HA) epitope at the C-terminus to facilitate the detection. Then, the right segments were inserted to the expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), and designated as pcCADM1, pcCADM2, pcCADM3, and pcCADM4, respectively. For cell transfection, HEK293T cells were transiently transfected with plasmids using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Deglycosylation assay Digestion of N-linked and O-linked glycosylation were carried out using PNGase F and O-Glycosidase, respectively (New England Biolabs, Beverly, MA, USA) according to the manufacturer’s instructions. Briefly, 30 μg of total protein from cell lysates in a 9-μL lysis buffer was denatured with 1 μL of a glycoprotein denaturing buffer at 100 °C for 10 min and then incubated with 2 μL G7 Buffer (0.5 M sodium phosphate, pH 7.5) and 2 μL 10 % NP-40 containing 1,500 U of PNGaseF and/or O-glycosidase at 37 °C for 1 h or

Eight-week-old female BALB/c mice were injected subcutaneously with 200 μL of emulsion containing 100 μg of KLHconjugated peptide and Freund’s complete adjuvant (Sigma, USA). Then, the mouse was boosted with same volume of emulsion mixed 1:1 with incomplete Freund’s adjuvant at 3 weeks and again at 6 weeks. The mouse antiserum was collected every week to assess immunity by ELISA. All the experiments were performed in accordance with the guidelines of the ethics review committee for animal experiments at Second Military Medical University. The splenocytes of immunized animals were fused with mouse myeloma cell NS-1 at a ratio of 5:1 in a 50 % polyethylene glycol (PEG 1500, Sigma, USA) solution, as described previously [21, 22]. Then, hybridoma cells were plated on 96well plates and cultured in HAT selection media (Sigma). At 10 days post-fusion, the supernatants were collected and screened by ELISA against unconjugated peptides. Positive clones were subcloned and then rescreened by ELISA and immunoblotting. The cloned hybridoma cells were injected into the peritoneal cavity of Balb/c mice to produce ascites. The mice ascites were selected and purified by affinity chromatography using protein A affinity columns (GE Health, USA). The specific immunoglobulin class of mAb was determined by using IsoQuick kit (Sigma).

Table 1 The sequence of peptides specific to CADM2 Name

peptide sequence

P1 P2 P3

FKNDKEIKDVKYLKEEDANRKTC KTSGSKPAADIRWFKNDKEIKC IINAEGSQVNAEEKKEYFI

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Enzyme-linked immunosorbent assay For ELISA assay, 96-well polystyrene plates (Corning, USA) were coated with 0.1 μg/well of unconjugated peptides in 0.1 M phosphate-buffered saline (PBS) overnight at 4 °C. The plates were washed and blocked with 10 % skim milk powder in PBS at 37 °C for 2 h to avoid nonspecific binding. After washing, the culture supernatant or antiserum was added and the plates were incubated at 37 °C for 1 h. After washing twice with PBS/0.1 % Tween-20, the wells were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Santa Cruz, CA, USA) and the absorbance of each well were determined at 492 nm with a microplate reader (Thermo Electron Corporation, USA). Protein extraction and western blotting analysis Cells were washed with ice-cold PBS and lysed with RIPA lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 1 % sodium deoxycholate, 1 % NP-40, and 0.1 % sodium dodecyl sulfate (SDS) supplemented with the complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The cell lysates were incubated on ice and centrifuged at 13,500 rpm for 10 min at 4 °C to remove insoluble material, and the supernatant was collected. Protein concentration in the supernatants was determined using the BCA Protein Assay Kit (Beyotime, China). The samples were fractionated in 10 % SDS-PAGE, transferred to a polyvinylidene difluoride membrane (PVDF, Millipore, Bedford, MA), and reacted with primary antibody against CADM2 or HA-tag (Cell Signaling Technology, Beverly, MA, USA). The binding of the primary antibody was detected with Chemiluminescent Substrate Kit (Thermo Scientific, Rockford, IL, USA) using a peroxidase-conjugated secondary antibody. Peptide competition assay The specificity for antibody recognition of peptide derived from CADM2 protein was confirmed by a peptide competition assay (PCA). The unconjugated peptide (0.25, 0.5, or 1 μg) and mAb 2B11 were preincubated with agitation for 30 min at room temperature. Then, the peptide-antibody solution was added to PVDF membrane followed by western blotting. A competition was observed when the addition of peptide resulted in a stepwise decrease of binding between mAb and CADM2 protein. Specimens and immunohistochemical staining The samples of normal or tumor tissues were randomly obtained from Changhai Hospital (Shanghai, China) with the informed consents. Immunohistochemically was performed as described previously [23, 24]. Tissue sections were

incubated with mAb 2B11 or rabbit polyclonal antibody R3, respectively. Then, the sections were washed and incubated with HRP-conjugated secondary antibodies (Santa Cruz). The expression and localization of CADM2 were revealed using a peroxidase–diaminobenzidine (DAB) reaction (Maxim, China). For negative controls, the primary antibody was replaced with PBS.

Result High degree of amino acid identity among CADMs family members By comparing the amino acid sequences, a high degree of identity between four members of CADMs family was found (Fig. 1). For CADM2, it shares a high degree of amino acid identity with other three members of CADMs family, ranging for the full-length proteins from 33.0 % (CADM2 vs. CADM4) to 41.5 % (CADM2 vs. CADM1 or CADM3) [16]. Furthermore, there was only a short cytoplasmic segment in the C-terminus of CADMs molecules, containing 47 amino acids (CADM1, CADM2, and CADM3) or 43 amino acids (CADM4), with higher degree of identity among CADM2 and others (e.g., 83.0 % between CADM2 and CADM1). Such high sequence homology makes it difficult to select a specific antigen epitope to discriminate different molecules of CAMDs family and generate an antibody specific to CADM2. Selection of specific antigen epitopes of CADM2 N- or C-terminal fragments of membrane proteins are commonly used to generate epitopes for antibody production. Due to the short cytoplasmic tails of CADMs family proteins and their higher degree of amino acid identity, it is difficult to select a specific epitope in the cytoplasmic domain of CADM2. On the other hand, the extracellular domains of CADMs share three Ig-like domains which contain several N- and O-linked glycosylation sites. Glycosylation have been shown to influence available epitopes and recognition of antigens. As shown in Fig. 2, CADM2 is a glycoprotein that possesses both N- and O-glycans with the assistant of IEDB analysis resource at the website (http://tools.immuneepitope. org); we chose two peptide segments within the second Ig-like domain of CAMD2, which are distinguished from other three members. Meanwhile, we also synthesized the peptide at the C-terminus of CADM2 cytoplasmic tails published in some paper [20] as a control. Generation of monoclonal antibodies against CADM2 Balb/c mice were subcutaneously immunized with three peptides, including two candidate CADM2-specific antigen

Tumor Biol. Fig. 1 Comparison of the amino acid sequences of CADMs proteins. Boxed residues (black) indicate that amino acids were highly conserved among the CADMs

epitopes and a control peptide, respectively. Three weeks after immunization, antibody responses in mice sera were revealed by ELISA every week, and when the titer reached 1:16,000, western blot analysis was performed to determine the responses of mice sera to CADM2 protein. The result showed that only the sera from mice immunized with the peptide P2 could specifically recognize CADM2 protein, but the serum from mice immunized with the peptides P1 or P3 did not recognize CADM2 protein (Fig. 3).

Fig. 2 Identification of N- and Olinked glycosylation of CADM2 protein. a Cell lysis from HEK293T cells transfected with HA-tagged CADM2 gene were treated with PNGaseF to remove the N-linked glycans for different reaction time indicated. b Cell lysis were treated with PNGaseF and/or O-Glycosidase, to remove N-linked glycans and/or O-linked glycans. Both blots were probed with anti-HA tag antibody

The splenocytes from a mouse demonstrated to have a high-titer immune response to P2 peptide and specific binding to CADM2 protein were isolated to be fused with mouse myeloma cells NS-1. Hybridomas were screened for antibodies that reacted with the peptides P2 and CADM2 protein. Finally, two different clones (2B11 and 14A5) were successfully isolated. The hybridomas clone 2B11 was inoculated into the peritoneal cavity of Balb/c mice and monoclonal antibody was purified from mouse ascites fluid using a protein A column. Immunoglobulin isotyping showed the isotype of

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Fig. 3 Characterization of antibodies from the mice sera by western blot analysis. HEK293T cells were transiently transfected with a plasmid encoding HA tagged CADM2 gene (C) or a control vector pcDNA3.1 (P). Forty-eight hours post-transfection, cells were lysed and analyzed by western blot with sera from mice immunized with P1, P2, or P3 peptides, respectively. Anti-HA tag antibody was used as a positive control

the purified anti-CADM2 mAb (2B11) as IgG3 with a Kappa (κ) light chain. In the following experiments, we would mainly use the mAb 2B11 to perform western blotting and immunohistochemistry test.

Fig. 4 Cross-activity detection of mAbs against CADM2 with other members of CAMDs family. HEK293T cells were transiently transfected with the plasmid HA-tagged CADM1, CADM2, CADM3, or CADM4, respectively. Forty-eight hours post-transfection, cells were lysed and analyzed by western blotting with mAb 2B11 and 14A5. Anti-HA tag antibody was used as a positive control

which demonstrated that mAb 2B11 could specifically bind to the peptide P2.

Specificity and cross-reactivity of anti-CADM2 monoclonal antibody To determine the specificity of mAb 2B11 and 14A5 to CADM2 protein without cross-reactivity to other members of CADMs family, we cloned the CADM1, CADM3, and CADM4 genes tagged with HA tag at their C-terminus and inserted into expression vectors pcDNA3.1 to obtain expression plasmids named pc-CADM1, pc-CADM3, and pcCADM4. Then, these plasmids together with pc-CADM2 were transfected into 293 T cells respectively and the total proteins were extracted 48 h post-transfection for western blot analysis. The results showed that mAb 2B11 and 14A5 can specifically identify CADM2 without cross-reactivity to other members of the family (Fig. 4). However, a rabbit polyclonal antibody R3 produced by immunizing with peptide P3 in rabbit could bind both CADM1 and CADM2 proteins (Fig. 5). These results indicated that we successfully obtained monoclonal antibodies specific to CADM2 without crossreactivity, which provides a useful tool to recognize CADM2 protein. A peptide competition assay was performed to demonstrate the specificity of mAb 2B11 to specific peptide P2 derived from CADM2 molecular. As shown in Fig. 6, when diluted concentrations of peptide P2 was pre-incubated with 2B11, a stepwise decrease of binding between mAb and CADM2 protein was observed in a concentration-dependent manner,

Fig. 5 Cross-reactivity detection of pAbs R3 and 2B11 between CADM2 and CAMD1. Cell lysates of HEK293T transfected with HAtagged CADM1 or CADM2 were immunoblotted to determine the crossreactivity of antibodies. A rabbit polyclonal antibody R3 purified from sera of rabbit immunized with peptide P3 or mAb 2B11 was used as primary antibody

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Fig. 6 Specificity of mAb 2B11 to peptide P2 derived from CADM2 protein. A peptide competition assay was applied to determine the specificity of antibody to peptide. MAb 2B11 were pre-incubated with diluted concentrations of unconjugated peptide (0.25, 0.5, or 1 μg) for 30 min at room temperature prior to use in western blotting assays

Characterization of anti-CADM2 monoclonal antibody by immunohistochemistry Based on the results above that anti-CADM2 mAb could recognize CADM2 without cross-reactivity to other members of CADMs family, we next explored the ability of antiCADM2 mAb to detect the expression of CADM2 on serial paraffin-embedded sections by immunohistochemical staining analysis. Consistent with its characteristics of membrane protein, the expression of CAMD2 was detected by antiCADM2 mAb 2B11 with staining mainly on the cell membrane of the normal human nasal epithelial cells (Fig. 7). And no reactivity was detected, when PBS or rabbit pAb R3 produced from rabbit immunized with peptide P3 was used in place of the primary antibody. Furthermore, we also found the staining of CADM2 in membrane of some tumor samples using anti-CADM2 mAb 2B11 (Fig. 8), though low or no expression of CADM2 was detected in most tumor tissues compared to normal tissues (data not shown). However, using rabbit pAb R3 could not detect the expression of CADM2 in adjacent serial section.

Discussion CADM2 (alternatively named as TSLC2, IGSF4D, and SynCAM2), belongs to CADMs family and was reported to be a new candidate tumor suppressor. Down-regulation of CADM2 in tumor tissues and its suppressor function upon cell proliferation reported previously suggested that silencing

of CADM2 gene is related to the development and progression of some tumors. However, the expression and function of CADM2 in tumor cell have not yet been fully characterized. To evaluate the protein expression levels and localization of CADM2 in tumor tissues, a specific antibody without cross-reactivity to other proteins was needed. However, most of anti-CADM2 antibodies available commercially or used in published papers are rabbit polyclonal antibodies, exhibiting nonspecific recognition in some studies. In this study, we have designed two CADM2-specific peptides by IEDB-AR, as well as a published peptide located in the Cterminus of the short cytoplasmic tails of CADM2 protein [20]. The synthesized and KLH-conjugated peptides were applied to immunize the mice. Finally, we obtained two mouse monoclonal antibodies (2B11 and 14A5) specifically against CADM2 by hybridoma technology, which were confirmed by ELISA, western blotting, peptide competition assay, and IHC. To address the specificity and cross-reactivity of this monoclonal antibody, we performed peptide competition assay and demonstrated that mAb 2B11 could specifically bind to the peptide P2 derived from CADM2 molecule. Furthermore, the cross-reactivity among four members of CADMs family was detected by western blotting, and mAb 2B11 and 14A5 could only recognize CADM2 protein, but not the other three members. However, a rabbit polyclonal antibody R3 produced by immunizing rabbit with peptide P3 could bind both CADM1 and CADM2 proteins, which may be due to the high degree identity of the C-terminus between CADM1 and CAMD2. Finally, we examined the specificity of mAb 2B11 to detection of CADM2 expression on serial paraffinembedded sections by immunohistochemistry. In the normal human nasal mucosa, CADM2 was expressed and located on the cell membrane of nasal epithelial cells using mAb 2B11, while rabbit pAb R3 or PBS could not detect the reactivity. Furthermore, in a panel of tumor tissue specimens, the expression of CADM2 was down-regulated in most hepatic carcinoma, bladder cancer, and kidney cancer. We also found the staining of CADM2 in the membrane of some tumor samples using anti-CADM2 mAb 2B11, but not rabbit pAb R3. Our

Fig. 7 Representative immunohistochemical staining of normal human nasal mucosa. Immunohistochemistry on serial sections shows the expression of CADM2 in normal human nasal epithelial cells. a Primary antibody: mAb 2B11; b primary antibody: pAb R3; c PBS as a negative control

Tumor Biol. Fig. 8 Representative immunohistochemical staining for CADM2 in serial sections of hepatic carcinoma, bladder cancer, and kidney cancer. Left panel, mAb 2B11; right panel, pAb R3. Magnification, ×200

results suggest that mAb 2B11 could be applied in the specific detection of CADM2 using western blotting and immunohistochemistry.

Conflicts of interest None

References Conclusion In this study, we designed two CADM2-specific peptides to immunize mice and obtained two monoclonal antibodies specifically against CADM2, without cross-reactivity to other members of CADMs family. It would be appealing to evaluate the expression and localization of CADM2 protein in tumor cell or tissues for future studies. Acknowledgments This work was supported by grants from National Natural Science Foundation of China, Ministry of Science and Technology of China (973 and 863 project), Shanghai Commission of Science and Technology, and National Special Projects for New Drug Development and Infectious Diseases.

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Generation of a monoclonal antibody specific to a new candidate tumor suppressor, cell adhesion molecule 2.

Cell adhesion molecule 2 (CADM2) is an immunoglobulin (Ig)-like cell adhesion molecule, which belongs to the CADMs family. The four members of CADMs f...
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