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Human Antibodies 23 (2014/2015) 13–20 DOI 10.3233/HAB-140278 IOS Press
Targeted therapy of solid tumors by monoclonal antibody specific to epidermal growth factor receptor Behzad Baradarana, Jafar Majidia , Safar Farajniaa, Jaleh Bararb,c and Yadollah Omidib,c,∗ a
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran c Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b
Abstract. Tumor growth and progression depends largely on the activity of cell membrane receptors like epidermal growth factor receptor (EGFR) that plays a pivotal role in the progression and invasion of different solid tumors. As one of the most promising approaches for targeting and therapy, monoclonal antibodies (mAbs) have widely been used in the treatment of various malignancies. However, the clinical effects of mAbs appear to be dependent upon its specificity and potency. In the current investigation, a series of mAbs were produced against human EGFR using hybridoma technology. Balb/c mice were immunized against EGFR-positive A431 cancer cells and the most immune mouse was selected for fusion and generation of anti-EGFR mAbs. Isotyping of the generated mAbs was performed by ELISA method. Of various monoclones produced, IgG1 subclass (mAb BF4) displayed specific binding to the EGFR-expressing A431 cells. Flow cytometry and immunofluorescence staining revalidated its specific reactivity with EGFR and MTT assay revealed significant growth inhibition of A431 cells treated with mAb BF4 mainly through induction of apoptosis. Based on these findings, we propose the produced anti-EGFR mAb BF4 to be exploited for diagnostic and possibly treatment of various malignancies with overexpression of EGFR. Keywords: Cancer immunotherapy, epidermal growth factor receptor, hybridoma, monoclonal antibody
1. Introduction Over the past few decades, there has been considerable interest in developing new agents to improve the clinical outcome for patients with solid tumors that emerge with unique characteristics (e.g., development of tumor microenvironment, aberrant metabolism, dysregulated pH, remodeled extracellular matrix) resulting in failure of the conventional cancer therapy modalities [1,2]. In fact, the currently used cytotoxic anticancer agents are imperfectly nonspecific and unable to discriminate between normal and malignant ∗ Corresponding author: Yadollah Omidi, Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail: yomidi@tbzmed. ac.ir.
cells [3], which has led to the rational design and development of smart targeted therapies [4,5]. Such therapies are deemed to confer better clinical outcomes than the conventional anticancer therapies. The targeted therapy of cancer using cytotoxic agents requires safe drug delivery systems, however most of the lipidor polymer-based delivery systems have been shown to impose some degrees of cytotoxicity and/or genotoxicity [6–13]. Therefore, smart immunotherapies against cancer are deemed to be more effective and safer alternative to the conventional chemotherapeutics. For development of immunotherapies, it is essential to identify the anomalous biomolecules and related molecular pathways that discriminate the malignant cells from the healthy ones. Of various signaling pathways involved in tumor initiation and progression, cell membrane receptors appear to play pivotal roles in tumor growth and progression through initiation and/or
c 2014/2015 – IOS Press and the authors. All rights reserved ISSN 1093-2607/14/15/$35.00
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regulation of the intracellular signal transduction pathways involved in various functions such as cell proliferation and apoptosis, angiogenesis, adhesion, and motility [14–16]. Of these membrane receptors, epidermal growth factor receptor (EGFR) has been shown to play an important role in the growth and survival of many solid tumors. It belongs to the erb-B family consisting of four closely related cell membrane receptors (i.e., EGFR (HER1) or erbB1, erbB2 (HER2), erbB3 (HER3), and erbB4 (HER4)). Binding of a ligand to the extracellular domain activates the EGFR and results in dimerization of EGFR monomers that activates protein tyrosine kinase and the downstream signaling path favoring proliferation of cancer cells, while targeting EGFR with a specific mAb as one of the most effective strategy in cancer diagnosis and/or treatment can suppress this signaling cascade [17]. The success of this approach, however, is largely dependent upon the specificity and potency of mAbs to the designated antigens [18]. Various technologic have been exploited for optimization of the specificity and potency of antibodies (Abs), including chimerization and humanization of murine Abs, as well as reshaping and redesigning, conjugation and widening of the specificity of the mAb [19–22]. The optimized screening and selection method is believed to be one of the simple and cost-effective applicable approaches for the generation of high specific mAbs. Having capitalized on the hybridoma technology, we have recently developed several mAbs specific to EGFR in BALB/c mice using [23]. Our preliminary findings revealed that the selected mAbs display different binding affinities to EGFR. Therefore, in the current investigation, we report on the specificity of a mAb with high affinity to EGFR.
The culture supernatants of hybridomas were screened for the production of Abs using enzyme-linked immunosorbent assay (ELISA) method. After screening, the clones with high absorbance were selected for subcloning by means of Limiting Dilution (L.D) method. Then, suitable monoclones, which possessed high absorbance, were selected for mass production and characterization of mAbs. 2.2. In vivo expansion For in vivo expansion, 10 days before injection with hybridoma, BALB/c female mice (5–7 weeks old) were primed through intraperitoneal injection of 0.5 mL pristane (2, 6, 10, 14-tetra methyl pentadecane) (Sigma-Aldrich Co. Louis, USA). Mice were then injected with hybridoma cells (1.0 × 106 cells/0.5 mL PBS per mouse). After successful ascites development (10–14 days), peritoneal solution was drained using 19 G needle and stored at −20◦ C for further analysis. 2.3. ELISA analysis for antibody isotyping The class and subclass of the generated mAbs were determined using an enzyme immunoassay employing a mouse hybridoma subtyping kit (ZYMED Co., California, USA). Then, the 96-well cell culture plates were coated with the human EGFR and were exposed to the supernatants of the clones. Then, Rabbit Anti-Mouse class or subclass of light and heavy chain was added. After adding the HRP conjugated Goat Anti-Rabbit IgG and specific substrate 3,3’,5,5’tetramethylbenzidine (TMB), the resultant color reaction was read by a ELISA Reader at 405 nm. 2.4. Immunoblotting
2. Materials and methods 2.1. Generation of mAbs Female BALB/c mice (5–7 weeks old) were immunized (4×) subcutaneously and intraperitoneally as 3 weeks intervals with 2 × 106 cells of human epithelial carcinoma A431 cells in Freund’s adjuvant. Complete Freund’s adjuvant (Sigma-Aldrich Co. Louis, USA) was used for the first injection, and incomplete Freund’s adjuvant was used for subsequent injections. Hybridomas were generated by cell fusion of spleen cells from A431-immunized mice and SP2/0 myeloma cells at a 5:1 ratio following our reported method [23].
The selective reactivity of the produced mAbs with EGFR-expressing cancer cells were tested by Western blot analysis using 10% Sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). Cells, at 40–50% confluency, were washed with ice cold phosphate buffered saline (PBS) (3×), then total protein were harvested using 100–200 μL lysis buffer. After boiling the samples for 5 min at 100◦ C, designated amount of protein (30 μg/lane) together with rainbow molecular weight marker were loaded onto the SDS-PAGE and run at 100 V for 2–3 h. After exposure of the nitrocellulose membrane and whatman chromatography papers to the transfer buffer, the wet nitrocellulose membrane on top of the wet whatman sheets was stacked with SDS-
B. Baradaran et al. / Targeted therapy of solid tumors by monoclonal antibody specific to EGFR
PAGE and several wet whatman sheets. The protein bands were then transferred from gel onto the nitrocellulose membrane by electroblotting at 30 V for 2 h. After blocking the free sites with 5% skim milk solution and washing (3×), the membrane was incubated with the generated mAbs over night at 4◦ C. The membranes were washed (5×) and the specific binding was detected using horseradish peroxidase-conjugated antimouse secondary antibody (1/5000 dilution) and visualizing through enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.5. Conjugation of monoclonal antibody with FITC The purified mAbs were conjugated with fluorescein isothiocyanate (FITC) through covalently coupling to the primary amines (lysines) of the immunoglobulin. Briefly, 10 mg of FITC was dissolved in 1 mL anhydrous DMSO immediately before use. The mAbs were exchanged into reaction buffer (500 mM carbonate, pH 9.5). FITC was added to give a ratio of 40–80 μg per mg of mAb and mixed immediately. The tube was wrapped with aluminum foil and incubated under rotation at room temperature for 1 h. The unreacted FITC was removed by gel filtration and the conjugated antibody was exchanged into Storage Buffer (10 mM Tris, 150 mM NaCl, 0.1% (w/v) NaN3 , pH 8.2) by dialysis. The conjugation efficiency was determined by measuring the absorbance at 280 and 495 nm. 2.6. Flow cytometry Binding activity of mAb to the surface of EGFR overexpressing A431 cells was analyzed using a flow cytometry FACSCaliburTM and analyzed with CellQuestTM Pro software (Becton Dickinson Biosciences, San Jose, CA, USA). The EGFR negative Chinese hamster ovary (CHO) cells were used as the negative control. Briefly, the grown cells (1.0 × 107 cells) washed with PBS were incubated with 10 μg/mL of FITC conjugated mouse mAb at 4◦ C for 1 h. As a negative control for the mAb, mouse immunoglobulin IgG1 mAb (Beckman-Coulter, Miami, FL) was used. After washing with PBSB, the cells analysis was carried out by flow cytometry. 2.7. Fluorescence microscopy For fluorescence microscopy, the A431 cells were cultivated in the six well plates, then, at 40–50% con-
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fluency, they were exposed to designated amount of FITC conjugated anti-EGFR mAb for 1 h. After washing with sterile PBS (3×), the cells were fixed by 10 min incubation with 3.7% formaldehyde in PBS at room temperature. The cells were washed with PBS (3×) and mounted on slides using mounting medium with DAPI (50 M, for 20 min) for nuclear staining. The prepared samples were then examined utilizing a fluorescence microscope, Nikon Eclipse 90i (Nikon, Tokyo, Japan). To optimize fluorescence excitation, a double band fluorescence mirror unit, U-DM-DA/FI2 with excitation filter at 400–420 and 480–500 nanometers, for simultaneously observation of DAPI/FITC stained samples was used. 2.8. Immunofluorescence staining for internalization Internalization of surface-bound antibody was initiated by incubation in pre-warmed (37◦ C) serum-free media. A431 and CHO cells were cultivated in the six well plates. At 40–50% confluency, they were treated with designated amount of FITC-labeled anti-EGFR mAb for 15, 30 and 60 min. The cells were washed with sterile PBS (3×) and fixed by incubation with 3.7% formaldehyde in PBS at room temperature for 10 min. After washing, the cells were permeabilized with 0.2% Triton X-100, and then washed with PBS (3×) and blocked with 1% BSA for 1 h. After washing, secondary antibody, Rabbit Anti-Mouse Alex flour 488 (Invitrogen, Carsbad, CA, USA),was added to wells. Cells were washed with PBS (3×) and incubated with Hoechst (1 μg/mL, for 10 min) for nuclear staining. Cells were subsequently analyzed with Nikon Eclipse 90i fluorescent microscope (Nikon, Tokyo, Japan) using appropriate wavelength settings. 2.9. MTT assay The MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide] assay, which involves the conversion of tetrazolium salt to coloured formazan by cells serving as an indirect measurement of cell proliferation and viability, was performed according to previously reported method [24]. The cultivated A431 cells in the 96-well plates, at 40-50% confluency, were exposed to a range of concentrations of the anti-EGFR mAbs (0.1, 0.5, 2.5 μg/100 μL) and incubated at 37◦ C. After which, cells were washed once with PBS, and then replenished with 200 μL fresh media and 50 μL MTT reagent (2.5 mg/mL in PBS). Following an incubation at 37◦ C for 4 h, medium was removed and cells
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3.2. Ability of mAb to react with cell surface-expressed EGFR
Fig. 1. Specific reactivity of mAb BF4 with A431 and CHO cell lysates. Cell lysates were run on SDS-PAGE, transferred onto the nitrocellulose membrane, incubated with the generated mAbs and the specific binding was detected using horseradish peroxidase-conjugated anti-mouse secondary antibody.
For further screening of the selected mAb ability to react with cell surface-expressed EGFR, the cell lysates of A431 cells and CHO cells were prepared. While mAb BF4 exhibited significant binding to the cell lysates of A431 cells, it did not show any reaction with the cells lysate of CHO cells known as EGFRnegative cells (Fig. 1). 3.3. Flow cytometry analysis of mAb BF4 interaction with EGFR-positive A431cells
were exposed to 200 μL DMSO and 50 mL of Sorenson buffer (pH 7.4). They were incubated at 37◦ C for 30 min, and then the absorbance was measured at 570 nm using a spectrophotometric plate reader. The inhibition of proliferation was shown as percentage of cell growth induced by the produced anti-EGFR mAbs. The untreated cells with maximum cell growth point were considered as control.
We carried out flow cytometry to determine whether mAb BF4 is able to bind to EGFR expressed on the surface of the EGFR-positive A431 tumor cells in comparison with the EGFR-negative CHO cells. The mAb BF4 failed to react with CHO cells, however it significantly bound to the EGFR-positive A431 cells (Fig. 2).
2.10. Statistical analysis
Molecular interaction analysis of the FITC conjugated mAb BF4 with EGFR-positive A431 cells and EGFR-negative CHO cells revealed that the anti– EGFR BF4 antibody was able to strongly bind to the EGFR-positive A431 cells (Fig. 3), but not the EGFR-negative CHO cells. An isotype-matched IgG1 antibody conjugated to FITC was used as a negative control while neither antibody showed any interactions with the EGFR-negative control CHO (data not shown).
The in vitro viability data were evaluated by the unpaired t-test and a p value less than 0.05 was considered for statistical differences.
3. Results 3.1. Generation of anti-EGFR mAb Hybridomas were established from mice immunized with A431 cells that overexpress EGFR. Based on the amount of titer of supernatants with EGFR protein, four clones were initially selected for further characterization, including: BF4 (IgG1), BF8 (IgA), BE10 (IgA) and BG5 (IgA). Ten days after injection of hybridoma cells, ascitic fluid was collected from each mouse in two time periods and then precipitated with 45% ammonium sulfate for purification with ion exchange chromatography (IEC). The purified proteins analyzed by SDS-PAGE and PAGE showed high quality (data not showed). Isotyping of the generated mAbs from the hybridoma by ELISA method revealed that the subclass of mAb BF4 was IgG1. Thus, the BF4 clones were selected for production of the anti-EGFR mAbs.
3.4. Fluorescence microscopy of A431 cells treated with FITC-conjugated mAb BF4
3.5. Internalization of mAb BF4 MAb BF4 internalization, after binding to EGFR and recognition by the secondary antibody in A431 and CHO cells was assessed using fluorescence microscopy. BF4 mAb showed no interaction with the EGFR-negative CHO cells (Fig. 4A). To show the interaction of BF4 antibody with EGFR expressing A431 cells, the cells after being exposed to BF4 mAb were incubated at 4◦ C (Fig. 4B). For internalization, cells were incubated at 37◦ C for a designated time points. We witnessed fast internalization of mAb BF4 within 15–60 min, which was indicated by the appearance of small intracellular punctate structures (Fig. 2C), which were increased over the time (Figs 4D and E). Quantification of the mean fluorescence intensity ratio be-
B. Baradaran et al. / Targeted therapy of solid tumors by monoclonal antibody specific to EGFR
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Fig. 2. Interaction of anti-EGFR mAb BF4 with EGFR-positive A431 cells (A) and EGFR-negative CHO cells (B).
colorimetric assay (Fig. 5). While mAb BF4 showed significant decrease in cell proliferation in EGFR overexpressing A431 cancer cells, it had no effects on the EGFR negative CHO cells (data not shown).
4. Discussion
Fig. 3. Specific binding of mAb BF4 to EGFR in A431 cells. (Colours are visible in the online version of the article; http://dx.doi. org/10.3233/HAB-140278)
tween the intracellular region versus the cell surface and vice versa, over the time course of 60 min was calculated. This ratio steadily increased over time, highlighting the gradual accumulation of internalized antibody within intracellular compartments. Concurrently, the ratio of membrane to intracellular fluorescence was seen to decrease over the time, consistent with the progressive removal of antibody from the cell surface and its concomitant intracellular accumulation. Isotype control antibody tagged with Alex Flour did not bind to cells and was not internalized. 3.6. Anti-proliferative effects of anti-EGFR mAb The incubation of A431 tumor cells with the antiEGFR mAb BF4 (0.1 μg/dL) significantly (unpaired ttest, p < 0.05) decreased the number of viable cells when compared with control sera measured by MTT
Targeted immunotherapy of cancer continues to grow as one of the most promising treatment modalities against various malignancies [3]. The proof-ofconcept of this powerful approach includes a number of successfully clinical applications such as targeting CD20 antigen in B cell lymphomas and leukemias (using rituximab, ibritumomab tiuxetan, and tositumomab) [25], and HER2/neu in breast cancer (using trastuzumab and pertuzumab) [26–31]. To date, a number of clinical studies have been capitalized on anti-EGFR Abs (e.g., cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab), which have resulted in profound clinical impacts in particular when used with other treatment modalities such as radiation or chemotherapy [32,33]. Given the fact that the specificity of mAbs are the main factor for successful immunotherapy, in the current study, we aimed to develop mAbs with high specificity to EGFR using hybridoma technology. To this end, the EGFRoverexpressing A431 cell line was exploited as the source of immunogen EGFR, and mAbs were chosen based on the intensity of selective reactivity with the cell-surface EGFR overexpressed on A431 cells. A panel of anti-EGFR mAbs has been developed by immunizing BALB/c female mice with human EGFRoverexpressing tumor cells, selecting mAbs that bind specifically to the soluble (sEGFR), the mAb BF4 (Figs 1 and 2).
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Fig. 4. Fluorescent microscopy of antibody internalization in A431 cells. (A) Cells were preincubated with mAb BF4 at 4◦ C (0 minute). Internalized antibody is indicated in sequential images representing 15, 30, and 60 minutes of incubation at 37◦ C. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/HAB-140278)
Fig. 5. Cell viability analysis by MTT colorimetric assay in A431 cells treated with mAb BF4.
Previously, the chimeric mAb cetuximab derived from murine mAb 225 was raised by immunizing mice with A431 cells [34], which was shown to bind to a site on the domain III of EGFR that overlaps with EGF binding site and hence can inhibit the attachment of the EGFR-activating ligands (EGF and TGF-αp to EGFR. Such reactivity results in prevention of EGFR dimer-
ization and its downstream signaling pathway [35,36]. In our study, the mAb BF4 showed strong binding to a variety of human tumor cells expressing EGFR such as PC3 [37] and A431 cells, but not to the EGFR-negative CHO cells (Figs 3 and 4). Binding of a designated mAb to its cognate receptor is able to potentially block the receptor resulting in
B. Baradaran et al. / Targeted therapy of solid tumors by monoclonal antibody specific to EGFR
cytotoxic effects attributable to the toxin or radioisotope. The interaction of mAb with its target receptor is critical to its function, and therefore the greater the binding affinity to the receptor, the higher the impact of the mAb. Further, the internalization and intracellular retention of a mAb are pivotal factors in terms of its therapeutic function. To evaluate the internalization of mAb BF4, we capitalized on the wellcharacterized A431 cell line that overexpresses EGFR. Using fluorescent microscopy, we found that mAb BF4 undergoes rapid internalization following binding to EGFR overexpressed on A431 cells (Fig. 4). We speculate that, following internalization, the mAb BF4 is trafficked into the early endocytic compartments and then into the lysosomal compartments after 30 to 60 min. Since the fast internalization and accumulation of the mAb within the target cells is crucial for a selected therapeutic mAb, we envision that the retention of mAb BF4 within the target tumor cells makes it an ideal candidate for targeted delivery of cytotoxic agents (e.g. radioisotopes or toxins) into EGFR-expressing cells. Further, despite the high binding of the mAb BF4 to the EGFR-positive A431 cells indicative of specific association of the mAb with its cognate receptor EGFR, it was not able to specifically bind to the HER-2 receptors that have high homology in comparison with compare to EGFR (our unpublished data). The cultivated cells treated with various doses of the anti-EGFR mAb BF4 resulted in significant inhibition of cell growth in A431 cells, whose maximum inhibition appeared to be concentration dependent (Fig. 5). The growth inhibition rate of A431 cells appeared to enhance with increasing incubation time of anti-EGFR mAb. Given the fact that the expression of EGFR in PC3 cells (30000/cell) is less than A431 cells (1200000/cell) [38], studies carried with the anti-EGFR mAb on PC3 resulted in 20–30% decrease in cell growth [37]. This clearly indicates that the mAb effects are largely dependent upon the level of EGFR expression on the cell surface. Similarly, it has been reported that cetuximab inhibitory effect on tumor cells can be in an intimate association with the number of EGFR on the cell surface and concentration of the mAb used [39]. While the inhibitory effects of cetuximab on the growth of EGFR-expressing cancer cells have well been proven [40], the cetuximab impact on lung cancer cell lines has been shown to be concentrationdependent correlating with the number of cell surface EGFR [41]. Besides, we also believe that mutations in genes encoding EGFR antigens may also influence the final goal of immunotherapy [42].
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In short, given that the selection of mAbs against EGFR with high specificity and anti-tumor activity, yet with low cost, is a challenging issue, we envision that this approach may be considered as an attractive alternative strategy to the one that use other technologies such as phage display for the development of mAbs [22,43–46]. Based on our findings, the isolated mAb BF4 showed high specificity to the EGFRpositive A431 cells, but not the EGFR-negative CHO cells. Given the fact that the mAb BF4 represents great targeting potential against EGFR with fast internalization potential, it is proposed to be translated into relevant clinical applications. References [1] [2]
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Human Antibodies 23 (2014/2015) 13–20 DOI 10.3233/HAB-140278 IOS Press
Targeted therapy of solid tumors by monoclonal antibody specific to epidermal growth factor receptor Behzad Baradarana, Jafar Majidia , Safar Farajniaa, Jaleh Bararb,c and Yadollah Omidib,c,∗ a
Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran c Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b
Abstract. Tumor growth and progression depends largely on the activity of cell membrane receptors like epidermal growth factor receptor (EGFR) that plays a pivotal role in the progression and invasion of different solid tumors. As one of the most promising approaches for targeting and therapy, monoclonal antibodies (mAbs) have widely been used in the treatment of various malignancies. However, the clinical effects of mAbs appear to be dependent upon its specificity and potency. In the current investigation, a series of mAbs were produced against human EGFR using hybridoma technology. Balb/c mice were immunized against EGFR-positive A431 cancer cells and the most immune mouse was selected for fusion and generation of anti-EGFR mAbs. Isotyping of the generated mAbs was performed by ELISA method. Of various monoclones produced, IgG1 subclass (mAb BF4) displayed specific binding to the EGFR-expressing A431 cells. Flow cytometry and immunofluorescence staining revalidated its specific reactivity with EGFR and MTT assay revealed significant growth inhibition of A431 cells treated with mAb BF4 mainly through induction of apoptosis. Based on these findings, we propose the produced anti-EGFR mAb BF4 to be exploited for diagnostic and possibly treatment of various malignancies with overexpression of EGFR. Keywords: Cancer immunotherapy, epidermal growth factor receptor, hybridoma, monoclonal antibody
1. Introduction Over the past few decades, there has been considerable interest in developing new agents to improve the clinical outcome for patients with solid tumors that emerge with unique characteristics (e.g., development of tumor microenvironment, aberrant metabolism, dysregulated pH, remodeled extracellular matrix) resulting in failure of the conventional cancer therapy modalities [1,2]. In fact, the currently used cytotoxic anticancer agents are imperfectly nonspecific and unable to discriminate between normal and malignant ∗ Corresponding author: Yadollah Omidi, Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. E-mail: yomidi@tbzmed. ac.ir.
cells [3], which has led to the rational design and development of smart targeted therapies [4,5]. Such therapies are deemed to confer better clinical outcomes than the conventional anticancer therapies. The targeted therapy of cancer using cytotoxic agents requires safe drug delivery systems, however most of the lipidor polymer-based delivery systems have been shown to impose some degrees of cytotoxicity and/or genotoxicity [6–13]. Therefore, smart immunotherapies against cancer are deemed to be more effective and safer alternative to the conventional chemotherapeutics. For development of immunotherapies, it is essential to identify the anomalous biomolecules and related molecular pathways that discriminate the malignant cells from the healthy ones. Of various signaling pathways involved in tumor initiation and progression, cell membrane receptors appear to play pivotal roles in tumor growth and progression through initiation and/or
c 2014/2015 – IOS Press and the authors. All rights reserved ISSN 1093-2607/14/15/$35.00
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regulation of the intracellular signal transduction pathways involved in various functions such as cell proliferation and apoptosis, angiogenesis, adhesion, and motility [14–16]. Of these membrane receptors, epidermal growth factor receptor (EGFR) has been shown to play an important role in the growth and survival of many solid tumors. It belongs to the erb-B family consisting of four closely related cell membrane receptors (i.e., EGFR (HER1) or erbB1, erbB2 (HER2), erbB3 (HER3), and erbB4 (HER4)). Binding of a ligand to the extracellular domain activates the EGFR and results in dimerization of EGFR monomers that activates protein tyrosine kinase and the downstream signaling path favoring proliferation of cancer cells, while targeting EGFR with a specific mAb as one of the most effective strategy in cancer diagnosis and/or treatment can suppress this signaling cascade [17]. The success of this approach, however, is largely dependent upon the specificity and potency of mAbs to the designated antigens [18]. Various technologic have been exploited for optimization of the specificity and potency of antibodies (Abs), including chimerization and humanization of murine Abs, as well as reshaping and redesigning, conjugation and widening of the specificity of the mAb [19–22]. The optimized screening and selection method is believed to be one of the simple and cost-effective applicable approaches for the generation of high specific mAbs. Having capitalized on the hybridoma technology, we have recently developed several mAbs specific to EGFR in BALB/c mice using [23]. Our preliminary findings revealed that the selected mAbs display different binding affinities to EGFR. Therefore, in the current investigation, we report on the specificity of a mAb with high affinity to EGFR.
The culture supernatants of hybridomas were screened for the production of Abs using enzyme-linked immunosorbent assay (ELISA) method. After screening, the clones with high absorbance were selected for subcloning by means of Limiting Dilution (L.D) method. Then, suitable monoclones, which possessed high absorbance, were selected for mass production and characterization of mAbs. 2.2. In vivo expansion For in vivo expansion, 10 days before injection with hybridoma, BALB/c female mice (5–7 weeks old) were primed through intraperitoneal injection of 0.5 mL pristane (2, 6, 10, 14-tetra methyl pentadecane) (Sigma-Aldrich Co. Louis, USA). Mice were then injected with hybridoma cells (1.0 × 106 cells/0.5 mL PBS per mouse). After successful ascites development (10–14 days), peritoneal solution was drained using 19 G needle and stored at −20◦ C for further analysis. 2.3. ELISA analysis for antibody isotyping The class and subclass of the generated mAbs were determined using an enzyme immunoassay employing a mouse hybridoma subtyping kit (ZYMED Co., California, USA). Then, the 96-well cell culture plates were coated with the human EGFR and were exposed to the supernatants of the clones. Then, Rabbit Anti-Mouse class or subclass of light and heavy chain was added. After adding the HRP conjugated Goat Anti-Rabbit IgG and specific substrate 3,3’,5,5’tetramethylbenzidine (TMB), the resultant color reaction was read by a ELISA Reader at 405 nm. 2.4. Immunoblotting
2. Materials and methods 2.1. Generation of mAbs Female BALB/c mice (5–7 weeks old) were immunized (4×) subcutaneously and intraperitoneally as 3 weeks intervals with 2 × 106 cells of human epithelial carcinoma A431 cells in Freund’s adjuvant. Complete Freund’s adjuvant (Sigma-Aldrich Co. Louis, USA) was used for the first injection, and incomplete Freund’s adjuvant was used for subsequent injections. Hybridomas were generated by cell fusion of spleen cells from A431-immunized mice and SP2/0 myeloma cells at a 5:1 ratio following our reported method [23].
The selective reactivity of the produced mAbs with EGFR-expressing cancer cells were tested by Western blot analysis using 10% Sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE). Cells, at 40–50% confluency, were washed with ice cold phosphate buffered saline (PBS) (3×), then total protein were harvested using 100–200 μL lysis buffer. After boiling the samples for 5 min at 100◦ C, designated amount of protein (30 μg/lane) together with rainbow molecular weight marker were loaded onto the SDS-PAGE and run at 100 V for 2–3 h. After exposure of the nitrocellulose membrane and whatman chromatography papers to the transfer buffer, the wet nitrocellulose membrane on top of the wet whatman sheets was stacked with SDS-
B. Baradaran et al. / Targeted therapy of solid tumors by monoclonal antibody specific to EGFR
PAGE and several wet whatman sheets. The protein bands were then transferred from gel onto the nitrocellulose membrane by electroblotting at 30 V for 2 h. After blocking the free sites with 5% skim milk solution and washing (3×), the membrane was incubated with the generated mAbs over night at 4◦ C. The membranes were washed (5×) and the specific binding was detected using horseradish peroxidase-conjugated antimouse secondary antibody (1/5000 dilution) and visualizing through enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.5. Conjugation of monoclonal antibody with FITC The purified mAbs were conjugated with fluorescein isothiocyanate (FITC) through covalently coupling to the primary amines (lysines) of the immunoglobulin. Briefly, 10 mg of FITC was dissolved in 1 mL anhydrous DMSO immediately before use. The mAbs were exchanged into reaction buffer (500 mM carbonate, pH 9.5). FITC was added to give a ratio of 40–80 μg per mg of mAb and mixed immediately. The tube was wrapped with aluminum foil and incubated under rotation at room temperature for 1 h. The unreacted FITC was removed by gel filtration and the conjugated antibody was exchanged into Storage Buffer (10 mM Tris, 150 mM NaCl, 0.1% (w/v) NaN3 , pH 8.2) by dialysis. The conjugation efficiency was determined by measuring the absorbance at 280 and 495 nm. 2.6. Flow cytometry Binding activity of mAb to the surface of EGFR overexpressing A431 cells was analyzed using a flow cytometry FACSCaliburTM and analyzed with CellQuestTM Pro software (Becton Dickinson Biosciences, San Jose, CA, USA). The EGFR negative Chinese hamster ovary (CHO) cells were used as the negative control. Briefly, the grown cells (1.0 × 107 cells) washed with PBS were incubated with 10 μg/mL of FITC conjugated mouse mAb at 4◦ C for 1 h. As a negative control for the mAb, mouse immunoglobulin IgG1 mAb (Beckman-Coulter, Miami, FL) was used. After washing with PBSB, the cells analysis was carried out by flow cytometry. 2.7. Fluorescence microscopy For fluorescence microscopy, the A431 cells were cultivated in the six well plates, then, at 40–50% con-
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fluency, they were exposed to designated amount of FITC conjugated anti-EGFR mAb for 1 h. After washing with sterile PBS (3×), the cells were fixed by 10 min incubation with 3.7% formaldehyde in PBS at room temperature. The cells were washed with PBS (3×) and mounted on slides using mounting medium with DAPI (50 M, for 20 min) for nuclear staining. The prepared samples were then examined utilizing a fluorescence microscope, Nikon Eclipse 90i (Nikon, Tokyo, Japan). To optimize fluorescence excitation, a double band fluorescence mirror unit, U-DM-DA/FI2 with excitation filter at 400–420 and 480–500 nanometers, for simultaneously observation of DAPI/FITC stained samples was used. 2.8. Immunofluorescence staining for internalization Internalization of surface-bound antibody was initiated by incubation in pre-warmed (37◦ C) serum-free media. A431 and CHO cells were cultivated in the six well plates. At 40–50% confluency, they were treated with designated amount of FITC-labeled anti-EGFR mAb for 15, 30 and 60 min. The cells were washed with sterile PBS (3×) and fixed by incubation with 3.7% formaldehyde in PBS at room temperature for 10 min. After washing, the cells were permeabilized with 0.2% Triton X-100, and then washed with PBS (3×) and blocked with 1% BSA for 1 h. After washing, secondary antibody, Rabbit Anti-Mouse Alex flour 488 (Invitrogen, Carsbad, CA, USA),was added to wells. Cells were washed with PBS (3×) and incubated with Hoechst (1 μg/mL, for 10 min) for nuclear staining. Cells were subsequently analyzed with Nikon Eclipse 90i fluorescent microscope (Nikon, Tokyo, Japan) using appropriate wavelength settings. 2.9. MTT assay The MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide] assay, which involves the conversion of tetrazolium salt to coloured formazan by cells serving as an indirect measurement of cell proliferation and viability, was performed according to previously reported method [24]. The cultivated A431 cells in the 96-well plates, at 40-50% confluency, were exposed to a range of concentrations of the anti-EGFR mAbs (0.1, 0.5, 2.5 μg/100 μL) and incubated at 37◦ C. After which, cells were washed once with PBS, and then replenished with 200 μL fresh media and 50 μL MTT reagent (2.5 mg/mL in PBS). Following an incubation at 37◦ C for 4 h, medium was removed and cells
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3.2. Ability of mAb to react with cell surface-expressed EGFR
Fig. 1. Specific reactivity of mAb BF4 with A431 and CHO cell lysates. Cell lysates were run on SDS-PAGE, transferred onto the nitrocellulose membrane, incubated with the generated mAbs and the specific binding was detected using horseradish peroxidase-conjugated anti-mouse secondary antibody.
For further screening of the selected mAb ability to react with cell surface-expressed EGFR, the cell lysates of A431 cells and CHO cells were prepared. While mAb BF4 exhibited significant binding to the cell lysates of A431 cells, it did not show any reaction with the cells lysate of CHO cells known as EGFRnegative cells (Fig. 1). 3.3. Flow cytometry analysis of mAb BF4 interaction with EGFR-positive A431cells
were exposed to 200 μL DMSO and 50 mL of Sorenson buffer (pH 7.4). They were incubated at 37◦ C for 30 min, and then the absorbance was measured at 570 nm using a spectrophotometric plate reader. The inhibition of proliferation was shown as percentage of cell growth induced by the produced anti-EGFR mAbs. The untreated cells with maximum cell growth point were considered as control.
We carried out flow cytometry to determine whether mAb BF4 is able to bind to EGFR expressed on the surface of the EGFR-positive A431 tumor cells in comparison with the EGFR-negative CHO cells. The mAb BF4 failed to react with CHO cells, however it significantly bound to the EGFR-positive A431 cells (Fig. 2).
2.10. Statistical analysis
Molecular interaction analysis of the FITC conjugated mAb BF4 with EGFR-positive A431 cells and EGFR-negative CHO cells revealed that the anti– EGFR BF4 antibody was able to strongly bind to the EGFR-positive A431 cells (Fig. 3), but not the EGFR-negative CHO cells. An isotype-matched IgG1 antibody conjugated to FITC was used as a negative control while neither antibody showed any interactions with the EGFR-negative control CHO (data not shown).
The in vitro viability data were evaluated by the unpaired t-test and a p value less than 0.05 was considered for statistical differences.
3. Results 3.1. Generation of anti-EGFR mAb Hybridomas were established from mice immunized with A431 cells that overexpress EGFR. Based on the amount of titer of supernatants with EGFR protein, four clones were initially selected for further characterization, including: BF4 (IgG1), BF8 (IgA), BE10 (IgA) and BG5 (IgA). Ten days after injection of hybridoma cells, ascitic fluid was collected from each mouse in two time periods and then precipitated with 45% ammonium sulfate for purification with ion exchange chromatography (IEC). The purified proteins analyzed by SDS-PAGE and PAGE showed high quality (data not showed). Isotyping of the generated mAbs from the hybridoma by ELISA method revealed that the subclass of mAb BF4 was IgG1. Thus, the BF4 clones were selected for production of the anti-EGFR mAbs.
3.4. Fluorescence microscopy of A431 cells treated with FITC-conjugated mAb BF4
3.5. Internalization of mAb BF4 MAb BF4 internalization, after binding to EGFR and recognition by the secondary antibody in A431 and CHO cells was assessed using fluorescence microscopy. BF4 mAb showed no interaction with the EGFR-negative CHO cells (Fig. 4A). To show the interaction of BF4 antibody with EGFR expressing A431 cells, the cells after being exposed to BF4 mAb were incubated at 4◦ C (Fig. 4B). For internalization, cells were incubated at 37◦ C for a designated time points. We witnessed fast internalization of mAb BF4 within 15–60 min, which was indicated by the appearance of small intracellular punctate structures (Fig. 2C), which were increased over the time (Figs 4D and E). Quantification of the mean fluorescence intensity ratio be-
B. Baradaran et al. / Targeted therapy of solid tumors by monoclonal antibody specific to EGFR
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Fig. 2. Interaction of anti-EGFR mAb BF4 with EGFR-positive A431 cells (A) and EGFR-negative CHO cells (B).
colorimetric assay (Fig. 5). While mAb BF4 showed significant decrease in cell proliferation in EGFR overexpressing A431 cancer cells, it had no effects on the EGFR negative CHO cells (data not shown).
4. Discussion
Fig. 3. Specific binding of mAb BF4 to EGFR in A431 cells. (Colours are visible in the online version of the article; http://dx.doi. org/10.3233/HAB-140278)
tween the intracellular region versus the cell surface and vice versa, over the time course of 60 min was calculated. This ratio steadily increased over time, highlighting the gradual accumulation of internalized antibody within intracellular compartments. Concurrently, the ratio of membrane to intracellular fluorescence was seen to decrease over the time, consistent with the progressive removal of antibody from the cell surface and its concomitant intracellular accumulation. Isotype control antibody tagged with Alex Flour did not bind to cells and was not internalized. 3.6. Anti-proliferative effects of anti-EGFR mAb The incubation of A431 tumor cells with the antiEGFR mAb BF4 (0.1 μg/dL) significantly (unpaired ttest, p < 0.05) decreased the number of viable cells when compared with control sera measured by MTT
Targeted immunotherapy of cancer continues to grow as one of the most promising treatment modalities against various malignancies [3]. The proof-ofconcept of this powerful approach includes a number of successfully clinical applications such as targeting CD20 antigen in B cell lymphomas and leukemias (using rituximab, ibritumomab tiuxetan, and tositumomab) [25], and HER2/neu in breast cancer (using trastuzumab and pertuzumab) [26–31]. To date, a number of clinical studies have been capitalized on anti-EGFR Abs (e.g., cetuximab, panitumumab, zalutumumab, nimotuzumab, and matuzumab), which have resulted in profound clinical impacts in particular when used with other treatment modalities such as radiation or chemotherapy [32,33]. Given the fact that the specificity of mAbs are the main factor for successful immunotherapy, in the current study, we aimed to develop mAbs with high specificity to EGFR using hybridoma technology. To this end, the EGFRoverexpressing A431 cell line was exploited as the source of immunogen EGFR, and mAbs were chosen based on the intensity of selective reactivity with the cell-surface EGFR overexpressed on A431 cells. A panel of anti-EGFR mAbs has been developed by immunizing BALB/c female mice with human EGFRoverexpressing tumor cells, selecting mAbs that bind specifically to the soluble (sEGFR), the mAb BF4 (Figs 1 and 2).
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Fig. 4. Fluorescent microscopy of antibody internalization in A431 cells. (A) Cells were preincubated with mAb BF4 at 4◦ C (0 minute). Internalized antibody is indicated in sequential images representing 15, 30, and 60 minutes of incubation at 37◦ C. (Colours are visible in the online version of the article; http://dx.doi.org/10.3233/HAB-140278)
Fig. 5. Cell viability analysis by MTT colorimetric assay in A431 cells treated with mAb BF4.
Previously, the chimeric mAb cetuximab derived from murine mAb 225 was raised by immunizing mice with A431 cells [34], which was shown to bind to a site on the domain III of EGFR that overlaps with EGF binding site and hence can inhibit the attachment of the EGFR-activating ligands (EGF and TGF-αp to EGFR. Such reactivity results in prevention of EGFR dimer-
ization and its downstream signaling pathway [35,36]. In our study, the mAb BF4 showed strong binding to a variety of human tumor cells expressing EGFR such as PC3 [37] and A431 cells, but not to the EGFR-negative CHO cells (Figs 3 and 4). Binding of a designated mAb to its cognate receptor is able to potentially block the receptor resulting in
B. Baradaran et al. / Targeted therapy of solid tumors by monoclonal antibody specific to EGFR
cytotoxic effects attributable to the toxin or radioisotope. The interaction of mAb with its target receptor is critical to its function, and therefore the greater the binding affinity to the receptor, the higher the impact of the mAb. Further, the internalization and intracellular retention of a mAb are pivotal factors in terms of its therapeutic function. To evaluate the internalization of mAb BF4, we capitalized on the wellcharacterized A431 cell line that overexpresses EGFR. Using fluorescent microscopy, we found that mAb BF4 undergoes rapid internalization following binding to EGFR overexpressed on A431 cells (Fig. 4). We speculate that, following internalization, the mAb BF4 is trafficked into the early endocytic compartments and then into the lysosomal compartments after 30 to 60 min. Since the fast internalization and accumulation of the mAb within the target cells is crucial for a selected therapeutic mAb, we envision that the retention of mAb BF4 within the target tumor cells makes it an ideal candidate for targeted delivery of cytotoxic agents (e.g. radioisotopes or toxins) into EGFR-expressing cells. Further, despite the high binding of the mAb BF4 to the EGFR-positive A431 cells indicative of specific association of the mAb with its cognate receptor EGFR, it was not able to specifically bind to the HER-2 receptors that have high homology in comparison with compare to EGFR (our unpublished data). The cultivated cells treated with various doses of the anti-EGFR mAb BF4 resulted in significant inhibition of cell growth in A431 cells, whose maximum inhibition appeared to be concentration dependent (Fig. 5). The growth inhibition rate of A431 cells appeared to enhance with increasing incubation time of anti-EGFR mAb. Given the fact that the expression of EGFR in PC3 cells (30000/cell) is less than A431 cells (1200000/cell) [38], studies carried with the anti-EGFR mAb on PC3 resulted in 20–30% decrease in cell growth [37]. This clearly indicates that the mAb effects are largely dependent upon the level of EGFR expression on the cell surface. Similarly, it has been reported that cetuximab inhibitory effect on tumor cells can be in an intimate association with the number of EGFR on the cell surface and concentration of the mAb used [39]. While the inhibitory effects of cetuximab on the growth of EGFR-expressing cancer cells have well been proven [40], the cetuximab impact on lung cancer cell lines has been shown to be concentrationdependent correlating with the number of cell surface EGFR [41]. Besides, we also believe that mutations in genes encoding EGFR antigens may also influence the final goal of immunotherapy [42].
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In short, given that the selection of mAbs against EGFR with high specificity and anti-tumor activity, yet with low cost, is a challenging issue, we envision that this approach may be considered as an attractive alternative strategy to the one that use other technologies such as phage display for the development of mAbs [22,43–46]. Based on our findings, the isolated mAb BF4 showed high specificity to the EGFRpositive A431 cells, but not the EGFR-negative CHO cells. Given the fact that the mAb BF4 represents great targeting potential against EGFR with fast internalization potential, it is proposed to be translated into relevant clinical applications. References [1] [2]
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