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Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7
Biochemical and Biophysical Research Communications xxx (2016) 1e5
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7 Satita Tapaneeyakorn a, *, Warangkana Chantima b, Charin Thepthai b, Tararaj Dharakul a, b, ** a b
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2016 Accepted 11 May 2016 Available online xxx
Development of new cancer therapies based on specific recognition of molecules in cancer cells is a significant challenge, as this requires identification of such molecules (molecular targets) and subsequent development of high-affinity, selective binders (targeting molecules). While several molecular targets for cancer therapies are currently under evaluation in clinical trials, greater selectivity for cancer cells over normal cells is required to enhance efficacy. Migration-inducing gene 7 (Mig-7), a membrane protein found in various types of carcinoma cells, is a cancer-specific biomarker and a promising molecular target for targeted cancer therapies. The purpose of this study was to produce and characterize a novel monoclonal antibody (mAb) raised against an N-terminal peptide of human Mig-7 (Mig-7(1e30)). The Mig-7(1e30) peptide was conjugated with a KLH carrier protein for immunization, and the mAb specific to Mig-7 (STmAb-1) was produced using hybridoma technology. Western blot analysis showed that STmAb-1 specifically reacted with a 23-kDa Mig-7 protein expressed in cancer cell lines, and, crucially, not with primary human fibroblasts. The affinity constant (Kaff) of STmAb-1, as measured by noncompetitive enzyme immunoassay, was 1.31 109 M1, indicating high mAb affinity against Mig-7. Immunofluorescence assays demonstrated that STmAb-1 could specifically recognize Mig-7 expressed in cancer cell lines, but not in primary human fibroblasts and keratinocytes. Moreover, STmAb-1 inhibited the growth of MCF7 and HeLa cell lines in contrast to primary human fibroblasts, highlighting its potential usefulness in the development of new cancer therapeutics. © 2016 Elsevier Inc. All rights reserved.
Keywords: Biomarker Mig-7 Molecular target Monoclonal antibody Targeted cancer therapy
1. Introduction Cancer is recognized as a dominant cause of death worldwide, and therefore more effective treatments are urgently needed in order to increase survival rates. Chemotherapy is a universal treatment strategy involving the use of medicines to stop or slow tumor growth. In addition to destroying fast-proliferating cancer cells, chemotherapy is often not selective and other rapidly dividing cells, such as blood-producing cells in bone marrow, skin cells, and hair follicles are also destroyed, resulting in side-effects. Recently,
* Corresponding author. ** Corresponding author. National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand. E-mail addresses: [email protected] (S. Tapaneeyakorn), [email protected] (T. Dharakul).
cancer therapies geared towards targeting specific molecules have been introduced. These treatments are more selective for cancer cells, resulting in lower mortality rates in normal healthy cells. To date, several targeting molecules have been developed which are capable of facilitating selective drug-delivery to cancer cells through targeted binding to surface-associated marker molecules [1e4]. However, many of these molecular targets are expressed in both tumor, and normal cells so the development of targeting molecules having higher specificities and affinities against cancerspecific biomarkers will enhance the potency of such targeted therapies. Migration-inducing gene 7 (Mig-7), a cysteine-rich protein, is found in the cell membrane and cytoplasm of carcinoma cells [5]. In previous studies, it was revealed that Mig-7 mRNA levels increase in embryonic cytotrophoblast cells during placenta development and in more than 80% of tumors, but no such elevations occur in normal tissue samples or in blood from normal subjects [5e7].
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Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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Furthermore, in vitro studies have demonstrated that expression of Mig-7 protein contributes to cancer cell invasion and vascular cell mimicry [7e10]. The molecular mechanisms underlying the inhibition of cancer invasion and metastasis by Mig-7 remain largely unexamined. It has been reported that silencing Mig-7 expression inhibited early tumor growth in vivo presumably, in part, by deactivation of MT1-MMP, ERK1/2, Akt, and S6 kinase [11]. Recently, Mig-7 was also proposed as a potential therapeutic target for Akt/ GSK-3b-, and COX-2/PGE2-mediated cancer metastasis [12e14]. In this study, we report the successful production, characterization, and in vitro cytotoxic activity of a novel anti-Mig-7 monoclonal antibody having potential applications in cancer diagnosis and therapy. 2. Materials and methods 2.1. Cell culture Cancer cell lines (MCF7 breast adenocarcinoma, HeLa cervical adenocarcinoma, HL60 promyelocytic leukemia), normal human fibroblasts, and normal human keratinocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and were cultured at 37 C in a humidified atmosphere of 5% CO2 and 95% air. MCF7, HeLa, and normal human fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM), and HL60 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Normal human keratinocytes were cultured in DMEM supplemented with 1% human keratinocyte growth supplement (HKGS) and 10% heat-inactivated FBS. All media and supplements for culture were purchased from Gibco, Thermo Fisher Scientific Inc. (Waltham, MA). 2.2. Preparation of Mig-7 peptide conjugates The N-terminal amino acids 1e30 of the Mig-7 protein sequence (Mig-7(1e30)) (Accession DQ080207), a putative extracellular domain of Mig-7, was chosen as the immunogen for monoclonal antibody (mAb) production. The carboxyl group of the Mig-7(1e30) peptide was conjugated with Keyhole Limpet Hemocyanin (KLH) using an Imject® Immunogen EDC Conjugation Kit (Thermo Fisher Scientific Inc.). Conjugation of Mig-7(1e30) to ovalbumin (OVA) utilized an identical protocol as used for KLH, with the conjugate being subsequently used for screening of Mig-7 specific monoclonal antibodies. All peptides used in the study were purchased from GenScript (Piscataway, NJ). 2.3. Production of the anti-Mig-7 monoclonal antibody Four BALB/c mice were each immunized by injecting intraperitoneally at monthly intervals for 4 months with 50 mg of KLHconjugated Mig-7(1e30) emulsified in Freund’s complete adjuvant. After each immunization, the mice were bled to monitor levels of Mig-7 specific antibodies in serum using OVA-conjugated Mig-7(1e30). The mice showing the highest titers as determined by ELISA were boosted intraperitoneally with 50 mg of KLH-conjugated Mig-7(1e30) without adjuvant 3 days before cell fusion. After sacrificing, the spleens were removed and the splenocytes were fused with the X63-Ag8.653 mouse myeloma cell line. Culture supernatants from individual hybridoma clones were collected after fusion and initially screened against OVA-conjugated Mig-7(1e30) and KLH by ELISA, then against the MCF7 cancer cell line by dotblot assay. An antibody isotype was identified with rabbit antimouse isotype-specific antibodies, and the mAb was purified by precipitation with 50% (v/v) saturated ammonium sulfate followed
by affinity chromatography based on its isotype. The concentration of mAb was determined using the Bradford protein assay (Bio-Rad, Philadelphia, PA). 2.4. Antiserum titer determination by ELISA Immunoplates (Thermo Fisher Scientific Inc.) were coated with 2 mg/ml of OVA-conjugated Mig-7(1e30) or KLH and kept overnight at 4 C. After washing five times with PBS containing 0.05% tween (PBST), and blocking with 5% skim-milk in PBST for 1 h at 37 C, plates were incubated with either serum (1:500 to 1:50,000 dilution with 5% skim-milk in PBST) or individual culture supernatant (1:5 dilution with 5% skim-milk in PBST) for 30 min at 37 C. Plates were then washed 5 times with PBST and subsequently incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgM (Thermo Fisher Scientific Inc.; 1:10,000) for 1 h at 37 C. Peroxidase activity was detected using the TMB substrate (KPL, Inc., Gaithersburg, MD). Plates were read at an absorbance of 450 nm using a microplate reader (Bio-tek instrument, USA). Skim-milk was used as a negative control. All experiments were done in triplicate, and the data are presented as mean ± standard deviations. 2.5. Affinity constant determination A non-competitive enzyme immunoassay [15] was used to measure the affinity constant (Kaff) of anti-Mig-7(1e30) mAb serially diluted to concentrations of 21, 18, 9, 6, 3, 1, 0.781, 0.390, 0.195, 0.0195, and 0.00195 mg/mL. The sigmoid curve was plotted to depict the relationship of OD450 versus mAb concentration. Accordingly, Kaff could be calculated using the following equation: Kaff ¼ (n 1)/ 2(n[Ab0 ]t [Ab]t), where n ¼ [Ag]t/[Ag0 ]t. [Ag]t and [Ag0 ]t represent antigen concentrations, with [Ab0 ]t and [Ab]t corresponding to antibody concentrations at the half maximum OD (OD-50) for plates coated with [Ag0 ]t and [Ag]t, respectively. 2.6. Western blot analysis Cell pellets were lysed with M-PER lysis buffer (Thermo Fisher Scientific Inc.) and a mixture of protease inhibitor cocktail (SigmaAldrich, St. Louis, MO) for 10 min while standing on ice. The lysate supernatants were resolved in SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. After blocking with 5% skim-milk in PBST for 1 h, the membrane was incubated with the monoclonal antibody (1:750) and mouse anti-b-actin mAb (Santa Cruz Biotechnology, Inc., Dallas, TX; 1:200; loading control), and thereafter rinsed three times with PBST. The membrane was then incubated with HRP-conjugated goat anti-mouse IgM and HRPconjugated goat anti-mouse IgG (H þ L) (Thermo Fisher Scientific Inc.; 1:10,000) prior to rinsing three times with PBST. The blot was developed using the TMB membrane peroxidase substrate (KPL, Inc.). Normal human fibroblasts served as a negative control. 2.7. Immunofluorescence Cell pellets were diluted with PBS and smeared onto clean microscope slides. After air-drying, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% TritonX-100/PBS. Permeabilized cells were then treated with 10% normal goat serum in PBS to block nonspecific binding sites, followed by incubation with the mAb (1:750). After washing with PBS cells were incubated with FITC-conjugated anti-mouse IgM (1:50). Nuclei were visualized after staining with Hoechst 33,342 dye (Thermo Fisher Scientific Inc.). Images of the stained cells were acquired using a Fluoview FVi10 confocal microscope (Olympus, Japan). Cells
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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without primary or secondary antibodies were utilized as controls. 2.8. MTT assay Cytotoxic activities of the monoclonal antibody on MCF7, HeLa and normal fibroblast cells was determined using the 3-(4,5dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT, Sigma-Aldrich) assay [16]. Initially, cells were seeded on a 96 well microplate and pre-cultured overnight. Following this 100 ml of the mAb (concentration range 0e50 mM) was added to each well, then microplates were incubated for 72 h. An aliquot (20 mL) of MTT solution (5 mg/mL) was then added to each well prior to further incubation (3 h). The colored formazan crystal produced in each well was then dissolved through addition of 100 mL of dimethyl sulfoxide (DMSO), with the optical density (OD) value of each solution being measured at 540 nm using a BioTek microplate reader. Data obtained represent a mean value ± SD and are derived from the results of at least two independent experiments, conducted in triplicate. 3. Results 3.1. Production of a monoclonal antibody (mAb) against Mig7(1e30) To generate anti-Mig-7 mAbs, BALB/c mice were immunized with the N-terminus region of the putative extracellular domain of Mig-7. Blood was collected from mice after multiple injections. Antibody tiers were determined by ELISA on plates coated with OVA-conjugated Mig-7(1e30) (data not shown). The two mice showing the highest titers were sacrificed, and the mouse splenocytes were fused with myeloma cells for hybridoma production. Supernatant screening from the growing hybridoma clones was initially done with ELISA in 96-well plates with wells coated with OVA-conjugated Mig-7(1e30) and KLH, followed by a dot-blot assay against the MCF7 cancer cell line. From these a hybridoma clone (STmAb-1; mouse IgM, kappa) against Mig-7 was established which was shown to react selectively with Mig-7(1e30) over KLH (data not shown).
Fig. 1. Western blot analysis of anti-Mig-7 monoclonal antibodies (STmAb-1) against fibroblast, MCF7 and HeLa cells (A). The b-actin probe was used as a loading control. Densitometry of immunoblots in A (B).
3.4. Immunofluorescence studies by STmAb-1 against cancer cell lines Based on the specificity profiles identified by Western blot analysis, immunofluorescence studies were used to probe STmAb-1 binding specificity, and cellular localization of Mig-7 protein. As shown in Fig. 2, STmAb-1 specifically recognized cytoplasmic and cell-surface Mig-7 in MCF7 and HeLa carcinoma cell lines, as evident by strong signal intensities. On the other hand, STmAb-1 showed no binding to normal fibroblasts and keratinocytes, illustrating the high specificity of STmAb-1 against cancer cell lines. Furthermore, only weak expression of Mig-7 was evident in the HL60 cell line. These results highlight the utility of immunofluorescence to probe the specificity of mAb for detection of Mig-7 protein in cancer cell lines, and its extracellular and intracellular localization.
3.2. Confirmation of specificity against cancer cell lines
3.5. Evaluation of in vitro cytotoxicity activity of STmAb-1
To determine the specificity of STmAb-1 against cancer cell lines, MCF7 and HeLa carcinoma cell lines were used to perform Western blot analysis, with normal human fibroblasts as a negative control. STmAb-1 showed reactivity with an approximately 23-kDa protein band corresponding to the Mig-7 protein in all cancer cell lines, but not in normal human fibroblasts (Fig. 1). This indicated that STmAb-1 reacted with cancer cell lines specifically, with no cross-reactivity with normal cells. Densitometry measurements of these immunoblots contrasting the intensities of Mig-7 with bactin bands indicated that levels of the Mig-7 band in MCF7 cells were significantly (50%) higher than in HeLa cells, although protein loadings were equal as determined by b-actin antibody levels.
Antibody cytotoxicity of STmAb-1 against MCF7, HeLa and normal fibroblast cells was assessed by MTT assay 72 h posttreatment. As shown in Fig. 3, STmAb-1 shows cytotoxicity to MCF7 and HeLa cancer cells, but not towards normal fibroblasts. Cell viability decreased with increasing antibody concentration; IC50 values of 20.5 and > 50 mg/mL were obtained for STmAb-1 with MCF7 and HeLa cells, respectively. These results demonstrate the significant cytotoxic effect of STmAb-1 in cancer cells when compared with the anti-p16 mAb control, which is non-cytotoxic to either cancer cell lines or normal fibroblasts.
3.3. Affinity constant of anti-Mig-7(1e30) mAb
Targeting of cancer cell-specific proteins could result in more effective cancer treatments with less side effects. Mig-7 protein, a promising biomarker for cancer targeted therapeutics, is found in cell membranes and the cytoplasm in solid carcinoma cells. Recently, Mig-7 specific shRNA and anti-Mig-7(1e9) polyclonal antibody were produced and it was reported that targeting Mig-7 protein with these reduced HEC1A endometrial carcinoma cell chemoinvasion and MT1-MMP activation [11]. However, anti-Mig-7
The affinity constant (Kaff) of mAb binding to Mig-7(1e30) was measured by the non-competitive enzyme immunoassay, using serial dilutions of mAb and three concentrations of KLH-Mig7(1e30) for immunoplate coating (500, 250, 125 ng/mL). The results suggested high affinity of STmAb-1 for KLH-Mig-7(1e30) (mean Kaff 1.31 109 M1, Table 1).
4. Discussion
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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Table 1 Affinity constants for anti-Mig-7 monoclonal antibody (STmAb-1) at different [Ag] as determined by non-competitive ELISA. mAb
[Ag] (ng/mL)
OD-50a
[Ab] at OD-50 (ng/mL)
Kaff (M1)
Average Kaff (M1)
STmAb-1
500 250 125
0.15 0.125 0.1085
3.158 2.453 2.379
1.53E09 1.16E09 1.26E09
1.31E09
a
OD-50 represents the half maximum optical density obtained for a given concentration of KLH-Mig-7(1-30) ([Ag]) and the corresponding mAb ([Ab]).
Fig. 2. Immunofluorescence analyses using anti-Mig-7 monoclonal antibody (STmAb-1). Cancer cell lines MCF7, HeLa, and HL60 (top), and normal human fibroblast and keratinocyte cells (bottom) were treated with STmAb-1 followed by FITC-conjugated anti-mouse IgM (green). Hoechst 33,342 (blue) was used to achieve nuclear staining. All images were acquired under identical conditions and are displayed at the same scale. Magnification: 60. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
monoclonal antibodies (mAb) have not been developed for therapeutic use. Here, we demonstrate the successful production and characterization of a novel mAb, STmAb-1, that recognizes the N-terminal amino acids 1e30 of the Mig-7 protein sequence. The high affinity STmAb-1 specifically reacted with cancer cell lines, and exhibited no cross-reaction with normal cells. The level of Mig-7 expression in MCF7 cells was 50% higher than in HeLa cells as shown in
immunoblotting studies, in agreement with the previous reports documenting a higher expression level of Mig-7 in breast cancer cells in comparison to cervical cancer cells [6]. Immunofluorescence experiments resulted in strong signal intensities being obtained for STmAb-1 interactions with Mig-7 in MCF7 and HeLa cells, but only very weak signals in HL60 cells. These results are consistent with greater levels of Mig-7 protein expression in solid tumor cancer cell lines (e.g. MCF7 and HeLa) over lymphoma cells (e.g. HL60) [6]. Moreover, STmAb-1 inhibited growth of MCF7 and HeLa cells, but did not retard normal fibroblast growth. Our results suggest that STmAb-1 may have potential applications in the development of cancer therapeutics. Combination of this mAb with other therapeutic molecules could decrease migration and invasion of cancer cells. Humanization of STmAb-1 for therapeutic purposes is currently underway and in vivo studies will be further investigated.
Acknowledgments
Fig. 3. Cytotoxicity profiles of anti-Mig-7 monoclonal antibody (STmAb-1) against MCF7, HeLa, and normal fibroblast cells with anti-p16 monoclonal antibody used as a control.
We acknowledge the Thailand Research Fund (TRG5580019) and the National Nanotechnology Center (NANOTEC) for funding to ST. We also thank the Olympus Bioimaging Center, Faculty of Science, Mahidol University for use of the confocal microscopy facility and Miss Chayachon Apiwat and Miss Suchintana Chumseng for technical assistance.
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.062.
[7]
[8]
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Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7 Satita Tapaneeyakorn a, *, Warangkana Chantima b, Charin Thepthai b, Tararaj Dharakul a, b, ** a b
National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 May 2016 Accepted 11 May 2016 Available online xxx
Development of new cancer therapies based on specific recognition of molecules in cancer cells is a significant challenge, as this requires identification of such molecules (molecular targets) and subsequent development of high-affinity, selective binders (targeting molecules). While several molecular targets for cancer therapies are currently under evaluation in clinical trials, greater selectivity for cancer cells over normal cells is required to enhance efficacy. Migration-inducing gene 7 (Mig-7), a membrane protein found in various types of carcinoma cells, is a cancer-specific biomarker and a promising molecular target for targeted cancer therapies. The purpose of this study was to produce and characterize a novel monoclonal antibody (mAb) raised against an N-terminal peptide of human Mig-7 (Mig-7(1e30)). The Mig-7(1e30) peptide was conjugated with a KLH carrier protein for immunization, and the mAb specific to Mig-7 (STmAb-1) was produced using hybridoma technology. Western blot analysis showed that STmAb-1 specifically reacted with a 23-kDa Mig-7 protein expressed in cancer cell lines, and, crucially, not with primary human fibroblasts. The affinity constant (Kaff) of STmAb-1, as measured by noncompetitive enzyme immunoassay, was 1.31 109 M1, indicating high mAb affinity against Mig-7. Immunofluorescence assays demonstrated that STmAb-1 could specifically recognize Mig-7 expressed in cancer cell lines, but not in primary human fibroblasts and keratinocytes. Moreover, STmAb-1 inhibited the growth of MCF7 and HeLa cell lines in contrast to primary human fibroblasts, highlighting its potential usefulness in the development of new cancer therapeutics. © 2016 Elsevier Inc. All rights reserved.
Keywords: Biomarker Mig-7 Molecular target Monoclonal antibody Targeted cancer therapy
1. Introduction Cancer is recognized as a dominant cause of death worldwide, and therefore more effective treatments are urgently needed in order to increase survival rates. Chemotherapy is a universal treatment strategy involving the use of medicines to stop or slow tumor growth. In addition to destroying fast-proliferating cancer cells, chemotherapy is often not selective and other rapidly dividing cells, such as blood-producing cells in bone marrow, skin cells, and hair follicles are also destroyed, resulting in side-effects. Recently,
* Corresponding author. ** Corresponding author. National Nanotechnology Center (NANOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani 12120, Thailand. E-mail addresses: [email protected] (S. Tapaneeyakorn), [email protected] (T. Dharakul).
cancer therapies geared towards targeting specific molecules have been introduced. These treatments are more selective for cancer cells, resulting in lower mortality rates in normal healthy cells. To date, several targeting molecules have been developed which are capable of facilitating selective drug-delivery to cancer cells through targeted binding to surface-associated marker molecules [1e4]. However, many of these molecular targets are expressed in both tumor, and normal cells so the development of targeting molecules having higher specificities and affinities against cancerspecific biomarkers will enhance the potency of such targeted therapies. Migration-inducing gene 7 (Mig-7), a cysteine-rich protein, is found in the cell membrane and cytoplasm of carcinoma cells [5]. In previous studies, it was revealed that Mig-7 mRNA levels increase in embryonic cytotrophoblast cells during placenta development and in more than 80% of tumors, but no such elevations occur in normal tissue samples or in blood from normal subjects [5e7].
http://dx.doi.org/10.1016/j.bbrc.2016.05.062 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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S. Tapaneeyakorn et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e5
Furthermore, in vitro studies have demonstrated that expression of Mig-7 protein contributes to cancer cell invasion and vascular cell mimicry [7e10]. The molecular mechanisms underlying the inhibition of cancer invasion and metastasis by Mig-7 remain largely unexamined. It has been reported that silencing Mig-7 expression inhibited early tumor growth in vivo presumably, in part, by deactivation of MT1-MMP, ERK1/2, Akt, and S6 kinase [11]. Recently, Mig-7 was also proposed as a potential therapeutic target for Akt/ GSK-3b-, and COX-2/PGE2-mediated cancer metastasis [12e14]. In this study, we report the successful production, characterization, and in vitro cytotoxic activity of a novel anti-Mig-7 monoclonal antibody having potential applications in cancer diagnosis and therapy. 2. Materials and methods 2.1. Cell culture Cancer cell lines (MCF7 breast adenocarcinoma, HeLa cervical adenocarcinoma, HL60 promyelocytic leukemia), normal human fibroblasts, and normal human keratinocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and were cultured at 37 C in a humidified atmosphere of 5% CO2 and 95% air. MCF7, HeLa, and normal human fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM), and HL60 was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). Normal human keratinocytes were cultured in DMEM supplemented with 1% human keratinocyte growth supplement (HKGS) and 10% heat-inactivated FBS. All media and supplements for culture were purchased from Gibco, Thermo Fisher Scientific Inc. (Waltham, MA). 2.2. Preparation of Mig-7 peptide conjugates The N-terminal amino acids 1e30 of the Mig-7 protein sequence (Mig-7(1e30)) (Accession DQ080207), a putative extracellular domain of Mig-7, was chosen as the immunogen for monoclonal antibody (mAb) production. The carboxyl group of the Mig-7(1e30) peptide was conjugated with Keyhole Limpet Hemocyanin (KLH) using an Imject® Immunogen EDC Conjugation Kit (Thermo Fisher Scientific Inc.). Conjugation of Mig-7(1e30) to ovalbumin (OVA) utilized an identical protocol as used for KLH, with the conjugate being subsequently used for screening of Mig-7 specific monoclonal antibodies. All peptides used in the study were purchased from GenScript (Piscataway, NJ). 2.3. Production of the anti-Mig-7 monoclonal antibody Four BALB/c mice were each immunized by injecting intraperitoneally at monthly intervals for 4 months with 50 mg of KLHconjugated Mig-7(1e30) emulsified in Freund’s complete adjuvant. After each immunization, the mice were bled to monitor levels of Mig-7 specific antibodies in serum using OVA-conjugated Mig-7(1e30). The mice showing the highest titers as determined by ELISA were boosted intraperitoneally with 50 mg of KLH-conjugated Mig-7(1e30) without adjuvant 3 days before cell fusion. After sacrificing, the spleens were removed and the splenocytes were fused with the X63-Ag8.653 mouse myeloma cell line. Culture supernatants from individual hybridoma clones were collected after fusion and initially screened against OVA-conjugated Mig-7(1e30) and KLH by ELISA, then against the MCF7 cancer cell line by dotblot assay. An antibody isotype was identified with rabbit antimouse isotype-specific antibodies, and the mAb was purified by precipitation with 50% (v/v) saturated ammonium sulfate followed
by affinity chromatography based on its isotype. The concentration of mAb was determined using the Bradford protein assay (Bio-Rad, Philadelphia, PA). 2.4. Antiserum titer determination by ELISA Immunoplates (Thermo Fisher Scientific Inc.) were coated with 2 mg/ml of OVA-conjugated Mig-7(1e30) or KLH and kept overnight at 4 C. After washing five times with PBS containing 0.05% tween (PBST), and blocking with 5% skim-milk in PBST for 1 h at 37 C, plates were incubated with either serum (1:500 to 1:50,000 dilution with 5% skim-milk in PBST) or individual culture supernatant (1:5 dilution with 5% skim-milk in PBST) for 30 min at 37 C. Plates were then washed 5 times with PBST and subsequently incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgM (Thermo Fisher Scientific Inc.; 1:10,000) for 1 h at 37 C. Peroxidase activity was detected using the TMB substrate (KPL, Inc., Gaithersburg, MD). Plates were read at an absorbance of 450 nm using a microplate reader (Bio-tek instrument, USA). Skim-milk was used as a negative control. All experiments were done in triplicate, and the data are presented as mean ± standard deviations. 2.5. Affinity constant determination A non-competitive enzyme immunoassay [15] was used to measure the affinity constant (Kaff) of anti-Mig-7(1e30) mAb serially diluted to concentrations of 21, 18, 9, 6, 3, 1, 0.781, 0.390, 0.195, 0.0195, and 0.00195 mg/mL. The sigmoid curve was plotted to depict the relationship of OD450 versus mAb concentration. Accordingly, Kaff could be calculated using the following equation: Kaff ¼ (n 1)/ 2(n[Ab0 ]t [Ab]t), where n ¼ [Ag]t/[Ag0 ]t. [Ag]t and [Ag0 ]t represent antigen concentrations, with [Ab0 ]t and [Ab]t corresponding to antibody concentrations at the half maximum OD (OD-50) for plates coated with [Ag0 ]t and [Ag]t, respectively. 2.6. Western blot analysis Cell pellets were lysed with M-PER lysis buffer (Thermo Fisher Scientific Inc.) and a mixture of protease inhibitor cocktail (SigmaAldrich, St. Louis, MO) for 10 min while standing on ice. The lysate supernatants were resolved in SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. After blocking with 5% skim-milk in PBST for 1 h, the membrane was incubated with the monoclonal antibody (1:750) and mouse anti-b-actin mAb (Santa Cruz Biotechnology, Inc., Dallas, TX; 1:200; loading control), and thereafter rinsed three times with PBST. The membrane was then incubated with HRP-conjugated goat anti-mouse IgM and HRPconjugated goat anti-mouse IgG (H þ L) (Thermo Fisher Scientific Inc.; 1:10,000) prior to rinsing three times with PBST. The blot was developed using the TMB membrane peroxidase substrate (KPL, Inc.). Normal human fibroblasts served as a negative control. 2.7. Immunofluorescence Cell pellets were diluted with PBS and smeared onto clean microscope slides. After air-drying, cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% TritonX-100/PBS. Permeabilized cells were then treated with 10% normal goat serum in PBS to block nonspecific binding sites, followed by incubation with the mAb (1:750). After washing with PBS cells were incubated with FITC-conjugated anti-mouse IgM (1:50). Nuclei were visualized after staining with Hoechst 33,342 dye (Thermo Fisher Scientific Inc.). Images of the stained cells were acquired using a Fluoview FVi10 confocal microscope (Olympus, Japan). Cells
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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without primary or secondary antibodies were utilized as controls. 2.8. MTT assay Cytotoxic activities of the monoclonal antibody on MCF7, HeLa and normal fibroblast cells was determined using the 3-(4,5dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT, Sigma-Aldrich) assay [16]. Initially, cells were seeded on a 96 well microplate and pre-cultured overnight. Following this 100 ml of the mAb (concentration range 0e50 mM) was added to each well, then microplates were incubated for 72 h. An aliquot (20 mL) of MTT solution (5 mg/mL) was then added to each well prior to further incubation (3 h). The colored formazan crystal produced in each well was then dissolved through addition of 100 mL of dimethyl sulfoxide (DMSO), with the optical density (OD) value of each solution being measured at 540 nm using a BioTek microplate reader. Data obtained represent a mean value ± SD and are derived from the results of at least two independent experiments, conducted in triplicate. 3. Results 3.1. Production of a monoclonal antibody (mAb) against Mig7(1e30) To generate anti-Mig-7 mAbs, BALB/c mice were immunized with the N-terminus region of the putative extracellular domain of Mig-7. Blood was collected from mice after multiple injections. Antibody tiers were determined by ELISA on plates coated with OVA-conjugated Mig-7(1e30) (data not shown). The two mice showing the highest titers were sacrificed, and the mouse splenocytes were fused with myeloma cells for hybridoma production. Supernatant screening from the growing hybridoma clones was initially done with ELISA in 96-well plates with wells coated with OVA-conjugated Mig-7(1e30) and KLH, followed by a dot-blot assay against the MCF7 cancer cell line. From these a hybridoma clone (STmAb-1; mouse IgM, kappa) against Mig-7 was established which was shown to react selectively with Mig-7(1e30) over KLH (data not shown).
Fig. 1. Western blot analysis of anti-Mig-7 monoclonal antibodies (STmAb-1) against fibroblast, MCF7 and HeLa cells (A). The b-actin probe was used as a loading control. Densitometry of immunoblots in A (B).
3.4. Immunofluorescence studies by STmAb-1 against cancer cell lines Based on the specificity profiles identified by Western blot analysis, immunofluorescence studies were used to probe STmAb-1 binding specificity, and cellular localization of Mig-7 protein. As shown in Fig. 2, STmAb-1 specifically recognized cytoplasmic and cell-surface Mig-7 in MCF7 and HeLa carcinoma cell lines, as evident by strong signal intensities. On the other hand, STmAb-1 showed no binding to normal fibroblasts and keratinocytes, illustrating the high specificity of STmAb-1 against cancer cell lines. Furthermore, only weak expression of Mig-7 was evident in the HL60 cell line. These results highlight the utility of immunofluorescence to probe the specificity of mAb for detection of Mig-7 protein in cancer cell lines, and its extracellular and intracellular localization.
3.2. Confirmation of specificity against cancer cell lines
3.5. Evaluation of in vitro cytotoxicity activity of STmAb-1
To determine the specificity of STmAb-1 against cancer cell lines, MCF7 and HeLa carcinoma cell lines were used to perform Western blot analysis, with normal human fibroblasts as a negative control. STmAb-1 showed reactivity with an approximately 23-kDa protein band corresponding to the Mig-7 protein in all cancer cell lines, but not in normal human fibroblasts (Fig. 1). This indicated that STmAb-1 reacted with cancer cell lines specifically, with no cross-reactivity with normal cells. Densitometry measurements of these immunoblots contrasting the intensities of Mig-7 with bactin bands indicated that levels of the Mig-7 band in MCF7 cells were significantly (50%) higher than in HeLa cells, although protein loadings were equal as determined by b-actin antibody levels.
Antibody cytotoxicity of STmAb-1 against MCF7, HeLa and normal fibroblast cells was assessed by MTT assay 72 h posttreatment. As shown in Fig. 3, STmAb-1 shows cytotoxicity to MCF7 and HeLa cancer cells, but not towards normal fibroblasts. Cell viability decreased with increasing antibody concentration; IC50 values of 20.5 and > 50 mg/mL were obtained for STmAb-1 with MCF7 and HeLa cells, respectively. These results demonstrate the significant cytotoxic effect of STmAb-1 in cancer cells when compared with the anti-p16 mAb control, which is non-cytotoxic to either cancer cell lines or normal fibroblasts.
3.3. Affinity constant of anti-Mig-7(1e30) mAb
Targeting of cancer cell-specific proteins could result in more effective cancer treatments with less side effects. Mig-7 protein, a promising biomarker for cancer targeted therapeutics, is found in cell membranes and the cytoplasm in solid carcinoma cells. Recently, Mig-7 specific shRNA and anti-Mig-7(1e9) polyclonal antibody were produced and it was reported that targeting Mig-7 protein with these reduced HEC1A endometrial carcinoma cell chemoinvasion and MT1-MMP activation [11]. However, anti-Mig-7
The affinity constant (Kaff) of mAb binding to Mig-7(1e30) was measured by the non-competitive enzyme immunoassay, using serial dilutions of mAb and three concentrations of KLH-Mig7(1e30) for immunoplate coating (500, 250, 125 ng/mL). The results suggested high affinity of STmAb-1 for KLH-Mig-7(1e30) (mean Kaff 1.31 109 M1, Table 1).
4. Discussion
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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Table 1 Affinity constants for anti-Mig-7 monoclonal antibody (STmAb-1) at different [Ag] as determined by non-competitive ELISA. mAb
[Ag] (ng/mL)
OD-50a
[Ab] at OD-50 (ng/mL)
Kaff (M1)
Average Kaff (M1)
STmAb-1
500 250 125
0.15 0.125 0.1085
3.158 2.453 2.379
1.53E09 1.16E09 1.26E09
1.31E09
a
OD-50 represents the half maximum optical density obtained for a given concentration of KLH-Mig-7(1-30) ([Ag]) and the corresponding mAb ([Ab]).
Fig. 2. Immunofluorescence analyses using anti-Mig-7 monoclonal antibody (STmAb-1). Cancer cell lines MCF7, HeLa, and HL60 (top), and normal human fibroblast and keratinocyte cells (bottom) were treated with STmAb-1 followed by FITC-conjugated anti-mouse IgM (green). Hoechst 33,342 (blue) was used to achieve nuclear staining. All images were acquired under identical conditions and are displayed at the same scale. Magnification: 60. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
monoclonal antibodies (mAb) have not been developed for therapeutic use. Here, we demonstrate the successful production and characterization of a novel mAb, STmAb-1, that recognizes the N-terminal amino acids 1e30 of the Mig-7 protein sequence. The high affinity STmAb-1 specifically reacted with cancer cell lines, and exhibited no cross-reaction with normal cells. The level of Mig-7 expression in MCF7 cells was 50% higher than in HeLa cells as shown in
immunoblotting studies, in agreement with the previous reports documenting a higher expression level of Mig-7 in breast cancer cells in comparison to cervical cancer cells [6]. Immunofluorescence experiments resulted in strong signal intensities being obtained for STmAb-1 interactions with Mig-7 in MCF7 and HeLa cells, but only very weak signals in HL60 cells. These results are consistent with greater levels of Mig-7 protein expression in solid tumor cancer cell lines (e.g. MCF7 and HeLa) over lymphoma cells (e.g. HL60) [6]. Moreover, STmAb-1 inhibited growth of MCF7 and HeLa cells, but did not retard normal fibroblast growth. Our results suggest that STmAb-1 may have potential applications in the development of cancer therapeutics. Combination of this mAb with other therapeutic molecules could decrease migration and invasion of cancer cells. Humanization of STmAb-1 for therapeutic purposes is currently underway and in vivo studies will be further investigated.
Acknowledgments
Fig. 3. Cytotoxicity profiles of anti-Mig-7 monoclonal antibody (STmAb-1) against MCF7, HeLa, and normal fibroblast cells with anti-p16 monoclonal antibody used as a control.
We acknowledge the Thailand Research Fund (TRG5580019) and the National Nanotechnology Center (NANOTEC) for funding to ST. We also thank the Olympus Bioimaging Center, Faculty of Science, Mahidol University for use of the confocal microscopy facility and Miss Chayachon Apiwat and Miss Suchintana Chumseng for technical assistance.
Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062
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Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.062.
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Please cite this article in press as: S. Tapaneeyakorn, et al., Production, characterization, and in vitro effects of a novel monoclonal antibody against Mig-7, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.05.062