MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 32, Number 4, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2013.0005

A Monoclonal Antibody Against GPNMB Ping Zhang,1 Jie Li,2 Xiaowen Pang,1 Xiaoying Yuan,1 Dongguang Li,1 You Li,1 Liying Guo,1 and Wei Liu1

As a melanosome-associated transmembrane glycoprotein, GPNMB plays an important role in numerous cell types, as well as in tumors. Producing a high specificity and affinity monoclonal antibody against human GPNMB provides an important tool to study the function of GPNMB protein. In this study, monoclonal antibodies to GPNMB were obtained by immunizing BALB/c mice with purified GST-GPNMB emulsified in Freund’s adjuvant. Three monoclonal antibodies with high specificity and affinity were obtained. The titers of anti-serum were 1:10,000, 1:8000, and 1:3000, respectively. Western blot and immunohistochemistry experiments were used to characterize the antibody. The anti-GPNMB antibodies G203 and F105 had high affinities (G203 around 2.7 · 10 - 8 M and F105 around 1.6 · 10 - 8 M, respectively) for the GPNMB antigen. However, M306 had a low binding activity to GPNMB. The results of Western blot and immunohistochemistry experiments showed that the antibodies could bind human GPNMB antigen. The monoclonal antibodies provided good tools for further studying functional characterization of GPNMB.



lycoprotein non-metastatic melanoma protein B (GPNMB), also known as osteoactivin, HGFIN, and DC-HIL, is a melanoma-associated transmembrane glycoprotein. GPNMB was originally observed in retinal pigment epithelial cells and melanoma as a heavily N-glycosylated type I transmembrane domain protein with a short cytoplasmic domain containing an endosomal sorting motif.(1) The GPNMB gene is a member of pmel-17/nmb family, which was initially identified in low metastatic potential human melanoma cell lines.(2) The human GPNMB gene, which is a locus involved in the human inherited disease cystoid macular edema, was localized to chromosome 7q15(3) (NCBI Unigene Cluster Hs.82226 GPNMB). GPNMB is predicted to be a 560 amino acid transmembrane protein with significant amino acid sequence homology (26% amino acid identity) to the melanosomal structural protein, pmel 17.(4,5) Cell surface transmembrane proteoglycan GPNMB is expressed in numerous cells, including macrophages, osteoclasts, and dendritic cells.(6) Moreover, a series of studies have demonstrated that GPNMB has been overexpressed in melanoma, hepatocellular carcinoma, gliomas, squamous cell lung carcinoma, soft tissue tumors, and cancers of the stomach, pancreas, and breast.(7–10) GPNMB has been found to have a strong correlation with multiple malignancies. However, the precise role of GPNMB in different types of cancer is not fully understood. Currently, immunotherapeutic methods for cancer therapy, by which monoclonal antibodies target tumor-specific antigens, have shown great potential.(11) It is

therefore believed that measuring the concentration of GPNMB in various tumors may help in diagnosing disease and that anti-GPNMB monoclonal antibodies may contribute to tumor diagnosis and therapy. In this study, we produced and characterized a murine monoclonal antibody against human GPNMB that might serve as a tool for detecting GPNMB levels in the human body. Materials and Methods Materials PrimeScript One-Step RT-PCR Kit v.2, pMD19-T vector, pyrobest DNA polymerase, EcoRI, and XhoI were purchased from Takara (Dalian, China); pGEX-4T-1 vector was purchased from Novagen (Madison, WI); TRizol LS Reagent and the cDNA Synthesis kit were purchased from Life Technologies (Gaithersburg, MD); GSTrap FF columns were purchased from GE Healthcare Biosciences (Uppsala, Sweden). Isopropyl b-D-1-thiogalactopyranoside (IPTG), HAT, HT, and RPMI 1640 medium were purchased from SigmaAldrich (Saint Louis, MO). BALB/c mice were purchased from the Wushi Animal Laboratory (Fuzhou, China). The PIG1 and SP2/0 cell lines were purchased from ATCC (Manassas, VA). BCA protein assay kit was purchased from HyClone-Pierce (South Logan, UT); the isotyping kit was purchased from Pierce (Rockford, IL); the human neurokinin 1 receptors were purchased from Neobioscience Techology Company (Shenzhen, China); and the recombinant human pmel 17-GST protein was previously prepared by our laboratory.


Department of Dermatology, The General Hospital of the Air Force, Beijing, China. Department of Gynaecology and Obstetrics, The General Hospital of PLA, Beijing, China.



266 Cell culture The human melanocyte cell line PIG1 was maintained in 254 medium, supplemented with 5% fetal bovine serum (FCS) and human melanocyte growth supplement (S-002-5). SP2/0 cells were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum. All cells were grown at 37C in a high humidity 5% CO2–95% air incubator. Total RNA extraction and cDNA cloning Total RNA was extracted from cultured PIG1 cells using TRizol LS Reagent according to the manufacturer’s instructions. The purified total RNA (*4.5 mg) was reverse-transcribed to synthesize the cDNA with the oligo (dT)18 primer, according to the instructions provided with the cDNA Synthesis kit. A pair of specific clone primers—GPNMB (sense primer 1) 5¢-AACCTT GAGTGCCTGCGTC-3¢, GPNMB (anti-sense primer 1) 5¢ACTTCCCCAAACCACAATATCTA-3¢—was designed according to the deduced sequence. The PCR was carried out in a final volume of 25 mL. The reverse transcription reaction was performed at 37C for 1 h, and 15 mL of the reaction mixture was used for PCR amplification under the following conditions: 95C for 3 min; followed by 30 cycles of amplification (95C for 1 min, 56C for 1 min, 72C for 3 min, and a final extension at 72C for 10 min). The PCR products were purified and ligated into pMD19-T vector according to the manufacturer’s instructions. The recombinant plasmid was transformed into Escherichia coli DH5a; the positive clones were sequenced with vector-specific primers. Construction of expression vector pGEX-4T-1-GPNMB The cDNA encoding GPNMB was generated by PCR using a pair of gene-specific primers (GPNMB sense primer 2: 5¢CCGGAATTCGAATGTCTCTC-TATTTCCTGG-3¢, containing an EcoRI restriction enzyme site; GPNMB anti-sense primer 2: 5¢-CCGCTCGAGTTAAGAAACTCCTTTAAATTCTTG-3¢, containing an XhoI restriction enzyme site). Then, it was ligated into a pMD19-T vector. The recombinant plasmid was digested with two restriction enzymes (EcoRI and XhoI), and the full-length GPNMB gene was sub-cloned into the pGEX-4T-1 vector, which had been digested by EcoRI and XhoI. The obtained positive recombinant plasmid (pGEX-4T-1-GPNMB) was then transformed into E. coli BL21 (DE3) pLysS for expression. Expression and purification of recombinant protein from E. coli Expression of the fusion protein was performed according to methods described previously with minor modifications.(12) The induction of expression was performed with 1 mM isopropyl-1-thi-D-galactopyranside (IPTG) at 28C and 250 rpm for 6 h. The bacteria were then harvested. GSTGPNMB fusion protein was purified with a GSTrap FF column.(13) Expression and purification of GPNMB were analyzed by SDS-PAGE. The concentration of purified fusion proteins was detected by a BCA protein assay kit. Immunization of animals Female, 6-week-old BALB/c mice were immunized with 20 mg purified recombinant GPNMB protein emulsified in

ZHANG ET AL. complete Freund’s adjuvant by intraperitoneal injections as the first immunization. Booster immunizations were carried out every 2 weeks in the same manner, but using incomplete Freund’s adjuvant. After the sixth injection, the titer of the anti-serum was determined by enzyme-linked immunosorbent assay (ELISA). The last injection was performed 3 days before cell fusion. Cell fusion and purification of anti-GPNMB antibody Three days after the final immunization, the dissociated splenocytes (6.5 · 107) were fused with SP2/0 myeloma cells (4.8 · 107) in the presence of 50% polyethylene glycol (PEG) 4000, which was used as a fusion reagent. After fusion, the cells were centrifuged for 5 min at 500 g and the fusion product was resuspended in RPMI 1640 medium containing 10% FCS and HAT. The cells were cultivated in 96-well plastic plates and incubated overnight at 37C in a high humidity 5% CO2–95% air incubator. Subsequently, HAT selection medium was added to the cells for hybrid selection. The hybridoma clones were screened by indirect non-competitive ELISA. Among the positive wells, three (G203, F105, and M306) were selected and the hybrids were cloned according to the method described previously.(14,15) The anti-GPNMB monoclonal antibodies were purified by the FPLC method. Determination of isotype, titer, and affinity of monoclonal antibodies The isotype of the antibody was determined using a commercially available isotyping kit following the manufacturer’s protocol. Antibody titers were measured according to the method described previously.(16) The affinity constant of the MAb was measured according to the competitive ELISA methods described previously with minor modifications.(12,17) Briefly, serially diluted GST-GPNMB was mixed with a constant quantity of the anti-GPNMB in PBS, and incubated at 4C overnight; the mixture was added to a GST-GPNMB– coated 96-well plate. The affinity constant was calculated according to the formula: A0/(A0 – A) = 1 + K/a0. Cross-reactivity assay To detect the specificity of the anti-GPNMB hybridoma clones, Western blot analysis was performed.(18) Briefly, pmel 17, neurokinin 1 receptor (NK-1), and GPNMB were dissolved in 2 · SDS sample buffer, respectively. After electrophoresis, the gel-containing bands were transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked in blocking buffer. After washing three times, the membranes were incubated with anti-GPNMB antibodies at 37C for 30 min. The second antibody of the goat anti-mouse IgG with HRP label was added onto the membrane at 37C for 2 h. The membrane was washed three times, and then the ABTS was added for coloration. Immunohistochemistry Immunohistochemistry studies were performed as previously described with minor modifications.(12) Paraffin sections of human seborrheic keratosis tissue were obtained from the Department of Dermatology, General Hospital of the Air Force (Beijing, China), and used for immunohistochemistry. The sections were deparaffinized with xylene and rehydrated



in ethanol. Following this, 0.3% hydrogen peroxide in methanol was added to the mixture to quench the activity of endogenous peroxidase. After blocking with 10% bovine serum, sections were subsequently incubated with purified antiGPNMB G203 overnight at 4C. Isotype control antibody (isotype is IgG1/k) was used as a negative control. After being washed four times, they were treated with biotin-conjugated goat anti-mouse IgG. The staining of reaction products was achieved by the ABC horseradish peroxidase method, and the sections were then dehydrated and mounted. Results Cloning of GPNMB cDNA The purified total RNA was reverse-transcribed to synthesize the cDNA. According to the predicted sequence, we designed a pair of specific primers and amplified the fulllength DNA fragment. The PCR amplification product was obtained. Electrophoresis results showed that the PCR yielded an 1836 bp DNA fragment (Fig. 1). DNA sequencing of the GPNMB cDNA confirmed that the sequence of the gene was identical to the reported sequence in GenBank under the accession no. NM_002510. Expression and purification of GST-GPNMB The confirmed construct pGEX-4T-1-GPNMB was transformed into E. coli BL21 (DE3) pLysS cells. The expression of the GST-GPNMB fusion protein was induced by IPTG at 32C for 2 h. Cell supernatant and the thallus lysate were analyzed by SDS-PAGE and Western blot, respectively. After IPTG

FIG. 2. Expression of recombination GPNMB-6 · His. Lanes A–C were expressed GPNMB protein of SDS-PAGE. Lane A, protein marker (kDa); lane B, without induction with IPTG; lane C, induced with IPTG; lane D, Western blot with anti-GST MAb.

induction, the expression of a GST-GPNMB protein in band at about 70 kDa was found in the supernatant of the lysate (Fig. 2), but not in the cell supernatant. Moreover, the corresponding band was not observed without IPTG induction. The GST-GPNMB recombinant protein was purified with GSTrap FF column.(13)

FIG. 1. Agarose gel electrophoresis of PCR production. Lane 1, DNA marker; lane 2, PCR production (GPNMB).

FIG. 3. Determination of the specificity of monoclonal antibodies by Western blot analysis. The MAb G203 reacted only with GST-GPNMB; M306 reacted with both GSTGPNMB and NK-1. F105 reacted with both GST-GPNMB and pmel 17.



FIG. 4. Immunohistochemistry staining for GPNMB using anti-GPNMB monoclonal antibody G203 in paraffin-embedded tissues (original magnification, · 400). (A) Human seborrheic keratosis tissue reacted with anti-GPNMB antibody G203. (B) The isotype control of human seborrheic keratosis tissue. Characterization of hybridomas secreting anti-GPNMB antibody After immunization, the titers of the anti-GPNMB in mouse serum were determined by ELISA and the titers reached 1:10,000, 1:8000, and 1:3000, respectively. Purified GSTGPNMB fusion protein was used for ELISA screening of the hybridomas. Three hybridomas secreting anti-GPNMB (G203, F105, and M306) were selected and their isotypes were determined by the commercially available isotyping kit. G 203 is IgG1/k, F 105/k is IgG2b, and M 306 is IgM/k. The affinities of three anti-GPNMB antibodies were determined by competition ELISA. The affinities of G203 and F105 were 2.7 · 10 - 8 M and 1.6 · 10 - 8 M, respectively. However, the affinities of M306 could not be detected by the method, because the M306 clone had low binding activities to GPNMB. Cross-reactivity assay GPNMB shares broad homology to pmel 17 and shares similarities with NK-1; thus, we choose these proteins to determine the specificity of the monoclonal antibodies. Western blotting was used to detect the antibodies’ specificity (Fig. 3). The results showed that G203 reacted with the GPNMB protein, whereas M306 reacted with the GPNMB and NK-1, and F105 could react with both GPNMB and pmel 17. Based on these results, it was concluded that the G203 hybridoma clone was a specific monoclonal antibody against GPNMB. Immunohistochemistry The results of immunohistochemistry indicated that the monoclonal antibody we prepared could react with human GPNMB. The skin tissue of human seborrheic keratosis was strongly stained with anti-GPNMB monoclonal antibody, and the isotype control antibody could not react with the GPNMB antigen in the skin tissue of seborrheic keratosis (Fig. 4).

melanosomal proteins, such as pmel 17.(19) GPNMB cDNA was first identified as preferentially expressed in human low metastatic melanoma cell lines.(20) Increasing expressions of GPNMB protein were observed in melanoma, hepatocellular carcinoma, gliomas, and so on. However, their subcellular localization and function in these cells were not clear.(21) In this article, we reported the preparation of an anti-GPNMB monoclonal antibody that reacts with human GPNMB protein. Three hybridoma clones that secreted monoclonal antibody against GPNMB antigen were obtained according to the cell fusion technique described previously.(15) It is possible to select cell clones that produce antibodies of desired specificity and affinity by this method. Ultimately, we obtained three monoclonal antibodies—G203, F105, and M306. The preparation of anti-GPNMB antibodies may be a much more helpful tool in the future and greatly advance our understanding of the specific roles of GPNMB. These monoclonal antibodies can bind human GPNMB antigen since it can be used in Western blot analysis. By Western blotting, we confirmed that the G203 antibodies reacted only with the GPNMB, but not with NK-1 and pmel 17. However, the F105 antibody could react with both GPNMB and pmel 17 and the M306 antibody could react with both GPNMB and NK-1. In addition, the titers of the hybridoma clones were determined by ELISA, and the G203 clone reached 1:10,000; the F105 clone reached 1:8000, whereas the titer of M306 clone was just 1:3000. Because the M306 has a low titer and the F105 and M306 have low specificity, the G203 antibody was used in immunohistochemistry. The results showed that the G203 antibody can bind human GPNMB antigen. Based on these data, we concluded that the G203 hybridoma clone produced a specific monoclonal antibody against the GPNMB antigen. The relative affinity constant of G203 was much higher than the other antibodies. Because of its high specificity, stability, and affinity, this new GPNMB monoclonal antibody (G203) will be a valuable tool for studying the exact role of the GPNMB protein in multiple cells.

Discussion GPNMB is a highly glycosylated type I transmembrane protein that shares significant sequence homology to several

Author Disclosure Statement The authors have no financial interests to disclose.

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Address correspondence to: Dr. Wei Liu Department of Dermatology The Air Force General Hospital of PLA No. 30, Fucheng Road Beijing 100142 China E-mail: [email protected] Received: January 18, 2013 Accepted: April 2, 2013

A monoclonal antibody against GPNMB.

As a melanosome-associated transmembrane glycoprotein, GPNMB plays an important role in numerous cell types, as well as in tumors. Producing a high sp...
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