Construction and characterization of monoclonal antibodies against the extracellular domain of B-lymphocyte antigen CD20 using DNA immunization method

International Immunopharmacology 43 (2017) 23–32

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Construction and characterization of monoclonal antibodies against the extracellular domain of B-lymphocyte antigen CD20 using DNA immunization method Fatemeh Khademi a, Ali Mostafaie a,⁎, Shahram Parvaneh a, Farah Gholami Rad a, Pantea Mohammadi a, Gholamreza Bahrami a,b a b

Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran School of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran

a r t i c l e

i n f o

Article history: Received 23 July 2016 Received in revised form 28 October 2016 Accepted 29 November 2016 Available online xxxx Keywords: Apoptosis CD20 DNA immunization Monoclonal antibody Proliferation

a b s t r a c t To date, several new anti-CD20 monoclonal antibodies (mAbs) have been developed for potential efficacies compared with familiar mAb rituximab. Despite the recent advances in development of anti-CD20 mAbs for the treatment of B cell malignancies, the efforts should be continued to develop novel antibodies with improved properties. However, the development of mAbs against CD20 as a multi-transmembrane protein is challenging due to the difficulty of providing a lipid environment that can maintain native epitopes. To overcome this limitation, we describe a simple and efficient DNA immunization strategy for the construction of a novel anti-CD20 mAb with improved anti-tumour properties. Using a DNA immunization strategy that includes intradermal (i.d.) immunization with naked plasmid DNA encoding the CD20 gene, we generated the hybridoma cell line D4, which secretes functional mAbs against an extracellular epitope of CD20. Immunocytochemistry analysis and a cell-based enzyme-linked immunosorbent assay using a Burkitt's lymphoma cell line showed that D4 mAbs are capable of binding to native extracellular epitopes of CD20. Moreover, the binding specificity of D4 mAbs was determined by western blot analysis. Cell proliferation was examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected by the annexin V/ propidium iodide staining and dye exclusion assay. The results showed that D4 anti-CD20 mAbs produced by DNA immunization exhibit potent growth inhibitory activity and have superior direct B-cell cytotoxicity compared to rituximab. We propose that antibody-induced apoptosis is one of the mechanisms of cell growth inhibition. Taken together, the data reported here open the path to DNA-based immunization for generating pharmacologically active monoclonal antibodies against CD20. In addition, the data support future in vivo animal testing and subsequent procedures to produce a potential therapeutic mAb. © 2016 Elsevier B.V. All rights reserved.

1. Introduction CD20, a 33–35 kDa cell surface protein, is expressed on normal and most malignant B cells but not stem or plasma cells [1]. The gene for CD20, located on human chromosome 11 at position q12-q13 [2,3], encodes a protein with 4 transmembrane domains and both the C- and Nterminus in the cytoplasm [4]. CD20 is expressed at high levels on most malignant B cells, but is not internalized or shed from the plasma membrane following mAb binding [5]. These properties of CD20 allow mAbs to be remained on the cell surface for extended periods and maintain the immunological attack according to complement activation and Fcmediated effector functions. Additionally, there is the potentially important ability of CD20 to generate transmembrane signals and trigger ⁎ Corresponding author at: Medical Biology Research Center, P. O. Box: 6714869914, Sorkheh Lizheh, Kermanshah, Iran. E-mail addresses: [email protected], [email protected] (A. Mostafaie).

http://dx.doi.org/10.1016/j.intimp.2016.11.035 1567-5769/© 2016 Elsevier B.V. All rights reserved.

programmed cell death (PCD) when engaged by mAbs. These desirable features of CD20 have led to the success of anti-CD20 mAbs for treatment of B cell malignancies and autoimmune disorders [5]. However, the production of therapeutic monoclonal antibodies (mAbs) against extracellular domain of CD20, a 4-transmembrane protein, is technically challenging: the CD20 molecule is insoluble, and solubilized protein obtained from a cell lysate using a surfactant or strong alkali, and synthetic peptides frequently fail to maintain native epitopes [6]. Immunization with CD20-expressing cells has been successful in developing important therapeutic antibodies, such as rituximab and ocrelizumab [7–10]. While immunization with CD20-overexpressing cells may circumvent the problem of insolubility and loss of native epitopes, the success is often attenuated by low immunostimulatory properties and a time-consuming screening process. To overcome these limitations, we used a DNA-based immunization, avoiding laborious screening and costly antigen purification or synthesis, as an alternative and attractive method. Indeed, injection of the cDNA encoding the

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protein ensures in vivo expression of protein with normal posttranscriptional modifications in the membrane-bound state and thus generates antibodies against native epitopes [11]. Despite the efficiency of DNA immunization, a few antibodies against different epitopes have been generated by various researchers up to now using this approach [12– 21]. It has been shown that the efficiency of DNA immunization can be enhanced by using vectors encoding T-cell epitopes [22] or by DNA injection using electroporation [23]. Among a variety of gene delivery methods [11,24–28], direct injection of naked plasmid DNA is most attractive for DNA immunization because the delivery is simple and does not require any equipment [21]. Moreover, the route of immunization influences the magnitude of the antibody response. In demonstration of this, Boyle and et al. [29] reported that intradermal (i.d) injection induces rapid cytotoxic T cell responses and elicits higher antibody levels than intramuscular (i.m) injection after DNA immunization. Recently, it was demonstrated that hydrodynamic tail vein (HTV) DNA immunization with the pCAGGS plasmid, in conjunction with immune modulators, is a successful strategy for the production of antibodies against challenging multi-transmembrane proteins [12]. In this study, we describe an intradermal DNA immunization method with naked plasmid encoding the CD20 gene as an efficient and simple approach for generating functional mAbs against the extracellular epitopes of CD20. Anti-CD20 mAbs cause cell death through at least three different pathways, including the activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and programmed cell death (also termed direct cell death or apoptosis) [5]. Therapeutic anti-CD20 mAbs can be divided into two distinct types based on the triggered cell death pathway: type I or rituximab-like antibodies, induce CD20 translocation into lipid rafts and activate complement, but they are relatively poor at mediating programmed cell death (PCD). Type II or Bexxar-like mAbs, do not redistribute CD20 on the cell surface and tend to promote strong PCD, but they are relatively ineffective in complement activation [30–32]. Both types of mAbs are equally potent in ADCC mediated by Fcγ receptors on effector cells. The majority of anti-CD20 antibodies are type I, including rituximab [33], veltuzumab [34], ocrelizumab [35], and ofatumumab [36], whereas tositumomab (Bexxar) [37] and obinutuzumab [38] are type II. Currently available mAbs are only partially effective in lymphoid malignancies, so there is an essential need to develop new and more active antibodies [39]. Immunotherapy using rituximab has been successful, both alone and in combination with chemotherapy, in treating B-cell malignancies [40,41]. This human-mouse chimeric antibody induces apoptosis through complement fixation and antibody-dependent, cell-mediated cytotoxicity [42,43]. High response rates have been reported with rituximab in various non-Hodgkin lymphomas and chronic B-cell lymphocytic leukemia (CLL) [44–46], But a certain portion of patients are resistant to therapy with rituximab [47]. Our aim was to develop a new anti-CD20 mAb with improved in vitro anti-tumour properties using an efficient DNA immunization strategy. We examined the binding specificity of the generated D4 mAbs to the native extracellular epitope of CD20, and subsequently, the potential effects of D4 on direct B cell cytotoxicity and proliferation were compared with those of rituximab. Our findings suggest that the proposed DNA immunization strategy is a simple and efficient approach to generate functional mAbs against the extracellular epitopes of CD20.

2. Materials and methods 2.1. Mice Female BALB/c mice (Pasteur Institute, Tehran, Iran) aged 6 to 8 weeks were used in all experiments and maintained in specific pathogen-free conditions. All experiments on the animals were performed

according to Animal Care and Use Protocol of the Kermanshah University of Medical Sciences. 2.2. Cell lines The SP2/0 myeloma cell line, human Raji (Burkitt lymphoma) and Jurkat (T lymphoblastic lymphoma) cell lines were obtained from the National Cell Bank of Iran (NCBI). Cells were maintained in log-phase growth in antibiotic-free RPMI 1640 medium supplemented with 10% foetal bovine serum (FBS) and 2 mM L-glutamine (Gibco, Grand Island, NY) at 37 °C with 5% CO2. 2.3. Construction of expression vectors The complete ORF for the MS4A1 gene (GeneBank accession number NM_021950) along with a Kozak consensus sequence was synthesized by Generay (Generay Biotech. Co., Ltd., Shanghai, China). The synthetic CD20 gene with 5′ and 3′ terminal restriction sites (915 bp) was cloned into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA) under the control of a CMV promoter using the XbaI and XhoI restriction sites. The final construct was confirmed by sequencing. 2.4. DNA immunization The CD20 plasmid for injection was prepared from Escherichia coli using alkaline lysis, PEG precipitation and Triton X-114 extraction as previously described [48]. BALB/c mice were immunized intradermally (i.d.) with 100 μg of plasmid DNA encoding the cell surface marker CD20 in phosphate-buffered saline (PBS). After 2 weeks, mice were boosted with CD20 plasmid (100 μg) in PBS. The animals were bled three weeks after the second injection and tested for anti-CD20 antibody activity using a cell-based enzyme-linked immunosorbent assay (ELISA). The spleen from the mouse with the highest antibody titers was aseptically removed for hybridoma production. 2.5. Immunization with purified human CD20 BALB/c mice were also immunized with purified human CD20 from Raji cell lysates prepared in the present study. The purification was carried out in two steps using immunoprecipitation (IP) of CD20 with antiCD20 mAbs, from 1F5 hybridoma (ATCC HB-9645), and preparative gel electrophoresis. To perform immunoprecipitation of the CD20 antigen, a total of 5 × 108 Raji cells were washed twice with PBS and solubilized in lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM ethylene diamine tetra acetic acid (EDTA) and 1 mM phenyl methyl sulfonyl fluoride (PMSF) along with a cocktail of protease inhibitors) for 1 h at 4 °C. After centrifugation at 13000 ×g for 30 min, the supernatants were collected and subjected to immunoprecipitation with 1F5 antiCD20 mAbs. The purified 1F5 mAbs was added to the lysate and incubated for 1 h at 4 °C. Then Protein G Sepharose 4 Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was added and incubated for 1 h at 4 °C with gentle mixing. Beads were washed five times with lysis buffer. Bound proteins were eluted with 0.1 M glycine-HCl pH 2.7, 0.5 M NaCl, 1% Triton X-100 and neutralized with 1 M Tris-HCl, pH 8, followed by dialysis against PBS. The eluted proteins were resolved in a 12.5% sodium dodecyl sulphate (SDS) polyacrylamide gel and silver stained. Further purification of the CD20 antigen was performed by preparative gel electrophoresis as described previously [49]. Purified CD20 was identified using a sandwich ELISA. Mice were immunized three times at two-week intervals by an i.d. injection of 20 μg of purified CD20 in Complete Freund's adjuvant (CFA, Sigma-Aldrich) for the first injection, and Incomplete Freund's adjuvant (IFA, Sigma-Aldrich) for the booster injections. After completion of the immunization schedule, animals were bled and tested for antiCD20 antibody activity. The mouse with the highest antibody titers was selected for hybridoma production.

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2.6. Enzyme-linked immunosorbent assay

2.9. Western blot analysis

A sandwich ELISA method was established to identify purified CD20. In this assay goat anti-CD20 polyclonal antibody (sc-7733, Santa Cruz Biotechnology, Santa Cruz, CA) (3.5 μg/mL) in a 0.05 M carbonate/bicarbonate buffer (pH 9.5) was coated for 2 h at room temperature. Negative controls include the wells without coating polyclonal anti-CD20 antibody. Wells were washed three times with PBS containing 0.05% Tween 20 and blocked with 250 μL of 2% BSA and 0.1% Tween 20 in PBS for 1 h at room temperature. Purified CD20 (1–10 μg/mL) or PBS were then added to the wells and incubated for 2 h at room temperature in duplicate. The plates were washed three times with PBS containing 0.05% Tween 20 and then incubated for 1 h at room temperature with 2 μg/mL mouse anti-CD20 mAbs (sc-70582, Santa Cruze Biotechnology). Then, the plate was washed three times with PBS containing 0.05% Tween 20 followed by incubation with HRP-conjugated goat antimouse IgG (Razi Biotech, Iran) at a dilution of 1/1000 in PBS with 2% BSA and 0.1% Tween 20 for 1 h at room temperature. The wells were then washed six times with PBS containing 0.05% Tween 20. A tetramethylbenzidine substrate solution (TMB, 100 μL) (Sigma -Aldrich) was added to develop the colour. The plates were incubated for 20 min in the dark. The reaction was stopped with 50 μL of 2 M sulfuric acid, and the optical density (OD) was read at a wavelength of 450 nm using a multiwell plate reader (Bio-Rad Laboratories Inc. Philadelphia, PA).

Raji or Jurkat cells were grown to confluence in 75-cm2 culture flasks and gently washed twice with PBS. The cells were solubilized in lysis buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1 mM PMSF along with a cocktail of protease inhibitors) for 1 h at 4 °C. After centrifugation at 13000 ×g for 30 min, the supernatants were resolved by 10% non-reducing SDS polyacrylamide gel electrophoresis [51]. The separated proteins were electroblotted onto polyvinylidene difluoride (PVDF) membranes over 5 h at 60 V according to standard protocols [52]. The PVDF membranes were blocked for 2 h at room temperature with 2% bovine serum albumin (BSA; Sigma-Aldrich) in PBS containing 0.1% Tween 20. The blots were incubated overnight at 4 °C with 2 μg/mL purified anti-CD20 mAbs (D4) or 1F5 in PBS containing 2% BSA and 0.1% Tween 20. The membranes were washed with PBS containing 0.05% Tween 20. The blots were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Razi Biotech, Iran) diluted 1/500 in PBS containing 1% BSA and 0.1% Tween 20. The immunoreactive bands were visualized using a diaminobenzidine and H2O2 substrate solution (Sigma-Aldrich).

2.7. Hybridoma cell production The murine myeloma cell line Sp2/0 was grown in RPMI 1640 medium containing 10% FBS. Splenocytes from immunized mice were mixed with Sp2/0 cells at a ratio of 5:1 and the cell mixture was washed twice with serum-free RPMI 1640. The fusion reaction was carried out in the presence of polyethylene glycol (PEG) 1500 (Sigma-Aldrich) in RPMI 1640 medium containing 20% FBS, 2 mM L-glutamine, and 0.01% penicillin/streptomycin (all from Gibco) and plated into 96-well plates. Hybridomas were selected by culturing in hypoxanthine, aminopterin, and thymidine (HAT medium; Gibco). The growing hybridomas were screened for their ability to produce antibodies against the native conformation of CD20 using a cell-based ELISA. Positive hybridomas were expanded and subcloned twice by the limiting dilution method to establish a hybridoma cell line that secreted mAbs. Immunoglobulin isotyping was performed using the Rapid Antibody Isotyping Kit (Pierce, Rockford, IL). 2.8. Production and purification of anti-CD20 mAbs Anti-CD20 mAbs were produced in ascitic fluid. Briefly, BALB/c mice were primed with 500 μL of 2,6,10,14-tetramethyl-pentadecane (Pristane, Sigma-Aldrich) injected intraperitoneally. One week later, 3 × 106 hybridoma cells were inoculated into the peritoneal cavity of mice, and the ascitic fluid was harvested after 7–10 days [50]. The IgG fraction of the ascitic fluid was precipitated with semi-saturated ammonium sulphate (50%). The precipitated fraction was then dialyzed against PBS, pH 7.5, and applied to a protein G Sepharose column (GE Healthcare Bio-Sciences AB) equilibrated with the same buffer. The column was washed extensively with PBS containing 0.35 M NaCl (pH 7.5). The captured antibodies were eluted from the column with a solution of 0.1 M glycine-HCl and 0.5 M NaCl (pH 2.7) and neutralized with 1 M Tris-HCl, pH 8, followed by dialysis against PBS. The concentration of the purified mAbs was determined by spectrophotometry using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The purity of the anti-CD20 mAbs was assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced and non-reduced conditions, followed by Coomassie Blue R-250 staining.

2.10. Whole cell enzyme-linked immunosorbent assay We developed a cell-based ELISA assay to determine the titers of prepared antisera, screen hybridomas, detect the binding activity of purified mAbs to the extracellular domain of CD20 and compare the binding activity of prepared mAbs with that of 1F5. CD20-expressing Raji Blymphocytes were used in these experiments, and Jurkat T-lymphocytes were used as a negative control. Briefly, a total of 105 cells/well were added to 96-well plates. Cells were fixed with 4% paraformaldehyde in PBS for 20 min, air-dried, and then washed with PBS containing 0.05% Tween 20. Each well was blocked with 250 μL of 5% BSA in PBS for 2 h at room temperature. Serial dilutions of immunized mouse sera, culture filtrate, purified anti-CD20 mAbs or 1F5 was then added to each well, and the plate was incubated for 2 h at room temperature. The plates were washed with PBS containing 0.05% Tween 20 and then incubated for 1 h at room temperature with HRP-conjugated goat antimouse IgG (Razi Biotech, Iran) at a dilution of 1/1000 in PBS with 5% BSA. The wells were then washed 6 more times with PBS containing 0.05% Tween 20. A tetramethylbenzidine substrate solution (TMB, 100 μL) was added to develop the colour. The plates were incubated for 20 min in the dark. The reaction was stopped with 50 μL of 2 M hydrochloric acid, and the optical density (OD) was read at a wavelength of 450 nm using a multiwell plate reader (Bio-Rad Laboratories Inc. Philadelphia, PA). 2.11. Immunocytochemistry assay The specific binding of purified mAbs to the extracellular domain of CD20 was also determined by an indirect immunoperoxidase assay. Briefly, a total of 1 × 105 Raji (CD20+) or Jurkat (CD20−) cells were spread gently on the surface of a microscope slide. The cells were fixed with 4% paraformaldehyde in PBS for 15 min, air-dried, and then non-specific binding was blocked with 5% BSA in PBS. Subsequently, the cells were incubated with Purified D4 or 1F5 mAbs (10 μg/mL) for 2 h at 37 °C in a humidified chamber. After three washes with PBS containing 0.05% Tween 20 for 15 min, the cells were incubated for 45 min at 37 °C with HRP-conjugated goat anti-mouse IgG at a dilution of 1/500. The slides were washed four times with PBS containing 0.05% Tween 20 for 20 min. A diaminobenzidine (DAB) and H2O2 substrate solution (Sigma-Aldrich) was then added to develop the colour. The slides were incubated for 15 to 30 min at room temperature. The DAB-stained cells were visualized against controls using an inverted microscope (TS100; Nikon, Tokyo, Japan); images were captured using an appropriate camera (ELWD 0.3/OD75; Nikon).

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2.12. Cell proliferation assay The effect of generated anti-CD20 mAbs on malignant B-cell proliferation in comparison with the effect of rituximab was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) colorimetric assay. Briefly, Raji cells were seeded in 96-well flat-bottom cell culture plates at a density of 5 × 104 cells/ well. Then, 10 μL of each antibody, serially diluted, was added to each well containing 90 μL of medium. The antibody concentration in the mixture of 100 μL/well was set to 1, 0.5, 0.1 or 0.01 μg/mL. After 72 h of exposure at 37 °C, MTT was added at a final concentration of 0.5 mg/mL for 3 h. The medium with MTT was removed, and 100 μL of DMSO was added to dissolve the formazan crystals. Absorbance of each well was read at 570 nm using a microplate reader (Bio-Rad Laboratories Inc. Philadelphia, PA). The percent of cell proliferation was calculated according to the following formula: Cell proliferation (%) = [A570 (sample) / A570 (control)] × 100. The effects of the mAbs on Raji cell growth were estimated in terms of the percent proliferation and expressed as IC50, which is the antibody concentration that reduces the absorbance of treated cells by 50% with reference to the control (untreated cells). All of the antibody concentrations were tested in triplicate, and each assay was repeated at least three times. 2.13. Annexin V/PI assay Phosphatidylserine (PS) exposure and cell death was assayed with an Annexin-V-FLUS Staining Kit (Roche Applied Science, Mannheim, Germany). In general, 5 × 104 cells were seeded into 96-well plates and were left untreated (control) or treated with 2 μg/mL D4 antiCD20 mAbs or rituximab for 24 to 48 h at 37 °C. Annexin V/PI staining was performed according to the manufacturer's instructions. The stained cells were visualized under a fluorescence microscope (TS100; Nikon, Tokyo, Japan). The results are expressed as the mean percentage of total annexin V-positive, PI-negative (V+/PI−) cells and annexin V/PI double-positive cells (V+/PI+; n = 3). All experiments were repeated at least three times. 2.14. Statistical analysis Data are expressed as mean ± standard deviation (SD). One-way analysis of the variance (ANOVA) was performed, and results were considered significant when p-values b0.05. The data were analyzed using the Statistical Package for Social Sciences (SPSS) software 19.0 (SPSS, Inc., Chicago, IL). 3. Results 3.1. Production and purification of anti-CD20 mAbs We used two immunization strategies to produce antibodies against CD20 in mice, including immunization with DNA expressing CD20 and purified CD20. Immunization of mice was performed by intradermal (i.d) injection of naked plasmid DNA encoding CD20 (200 μg) or purified CD20 antigen (60 μg) as described in the Methods section. Purification of CD20 antigen from Raji cell lysates was performed using a twostep process, including immunoprecipitation and preparative gel electrophoresis. Immunoprecipitation showed a 33-kDa protein band corresponding to B-lymphocyte antigen CD20 [1,53] and two 25- and 50-kDa bands corresponding to the light and heavy chains of the antibodies (Fig. 1, Lane 2). Immunoprecipitation using Jurkat cell lysate as negative control shows no band corresponding to CD20 (Fig. 1, Lane 1). For further purification, the immune complex was then loaded onto a preparative electrophoresis gel. After electrophoresis, the separated 33-kDa CD20 band was cut and extracted from the polyacrylamide gel and used for immunization. The purified 33-kDa band was identified as CD20 using a sandwich ELISA with goat anti-CD20 polyclonal antibody,

Fig. 1. SDS-PAGE of immunoadsorbed Raji cell extract with a mouse anti-CD20 antibody (1F5) in 12.5% acrylamide separating gel under reducing conditions, followed by the silver staining. Lane 1, negative control, immunoprecipitation using Jurkat cell lysate shows no band corresponding to CD20; Lane 2, immunoprecipitation using Raji cell lysates shows a 33-kDa protein band corresponding to CD20 and two 25- and 50-kDa bands corresponding to the light and heavy chains of the antibodies; Lane M, protein standard markers.

sc-7733, as capture antibody and mouse anti-CD20 monoclonal antibody, sc-70,582, as detecting antibody. The initial assessment for anti-CD20 antibodies was performed by measuring the antibody levels in individual sample bleeds of all mice. Three weeks after the final immunization, only animals immunized with CD20 plasmid demonstrated a marked elevation in anti-CD20 antibody level. According to the cell-based ELISA, The anti-CD20 antibody titers of 1000–2000 were obtained after DNA immunization. Titers were defined as the highest dilution of antiserum to reach an OD of 1 at 450 nm. Thus, it was found that DNA immunization is more efficient for production of anti-CD20 antibodies than protein immunization, as reflected by higher titers of antibodies in mouse sera (p b 0.01, Fig. 2). Mice receiving empty vector (pcDNA 3.1) or PBS did not elevate a CD20-specific antibody response. Hybridization of the splenocytes from DNA-immunized mice with SP2/0 myeloma cells produced a total of 155 hybridoma clones. The culture supernatants of the growing hybridomas (100 μL) were collected from 70 to 80% confluent wells and tested for anti-CD20 antibody activity. Of these hybridoma clones, 5 stable positive hybridomas were established and then subcloned twice by a limiting dilution. Finally, one stable hybridoma clone, designated D4, was generated, and then, corresponding mAbs were obtained and further characterized. Isotyping of mAbs revealed that the D4 clone had the IgG isotype. However, fusions of SP2/0 myeloma cells with spleen cells isolated from mouse immunized with purified CD20 failed to produce any suitable and stable clones. This is because the purified CD20 which was solubilized using detergent during purification steps fails to maintain native epitopes. The CD20 molecule as a multi-transmembrane protein is insoluble, and we need to use detergent in all steps of purification in order to solubilize the protein. In such conditions, the purified CD20 fails to maintain native epitopes which is the main reason why no suitable clones have been generated from animals immunized with purified proteins. D4 anti-CD20 mAbs were then produced in ascitic fluid, and approximately 5 mL of ascitic fluid was harvested from each mouse. To estimate the biological properties of D4, we purified antibodies from ascitic fluid using ammonium sulphate (50% v/v) fractionation and protein G affinity chromatography. The purity of the mAbs was confirmed

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Fig. 2. The CD20-specific IgG titers were determined by cell-based ELISA. BALB/c mice were immunized i.d with naked plasmid DNA encoding CD20 or purified CD20 antigen in PBS. Sera were obtained three weeks after the final immunization. Titers were defined as the highest dilution of antiserum to reach an OD of 1 at 450 nm. Results are expressed as the mean of the log10 IgG titer ± SD from three mice in each group. pvalues were determined using one-way ANOVA (*p b 0.01 compared with protein immunization).

by SDS-PAGE and Comassie Blue staining, which showed two bands at the expected positions for the antibody light and heavy chains under reduced conditions and a band at the molecular weight (MW) of 150– 155 kDa with high purity under non-reduced conditions (Fig. 3).

3.2. DNA immunization generates antibodies that specifically bind to native human CD20 The CD20 binding specificity of the generated D4 anti-CD20 mAbs was investigated using immunocytochemistry (ICC), western blot, and cell-based ELISA. ICC was performed by adding D4 or 1F5 mAbs to CD20-expressing Raji cells on the surface of a microscope slide, followed by staining with an HRP-conjugated goat anti-mouse IgG. Microscopic analysis revealed a brown colour on the surface of Raji cells, but not on the surface of control Jurkat cells (CD20−), which indicated that D4 mAbs bind specifically to extracellular epitopes of CD20. The binding activity of D4 mAbs was further compared with that of 1F5 mAbs using ICC. Interestingly, we found that D4 mAbs binding resulted in a more intense brown colour on the surface of Raji cells than that of 1F5 mAbs, indicating the higher binding activity of D4 (Fig. 4). 1F5 is one of a panel of monoclonal antibodies that have been produced recognizing the CD20 pan B cell antigen, and it was the first anti-CD20 monoclonal antibody used in serotherapy of human B cell lymphoma [54]. Additionally, we developed a cell-based ELISA assay for the detection and comparison of the relative binding activities of D4 and 1F5, and the results from the cell-based ELISA were compared with those from ICC. The cell-based ELISA was performed by adding 100 μL of various concentrations (0.5–20 μg/mL) of D4 or 1F5 mAbs to 105 Raji cells, followed by the addition of a secondary antibody. It was found that the generated D4 mAbs can bind specifically to native human CD20 on Raji cells because nonspecific binding of D4 was not found with Jurkat cells. Comparison of the binding activity of D4 mAbs with 1F5 anti-CD20 mAbs by ELISA indicated that the binding activity of the generated D4 mAbs was higher than that of the murine 1F5 mAbs (P b 0.05) (Fig. 5). This

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Fig. 3. SDS-PAGE analysis of the purified D4 anti-CD20 mAbs. The antibodies were purified by ammonium sulphate (50% v/v) fractionation and chromatography on a protein GSepharose column. SDS-PAGE was performed under reduced and non-reduced conditions, followed by Coomassie Blue R250 staining. (A) Lane 1, SDS-PAGE pattern of purified D4 mAbs using 12.5% acrylamide separating gel under reduced conditions. The two bands at the MWs of 25–27 and 50–52 kDa respectively correspond to the antibody light and heavy chains (B) SDS-PAGE pattern of purified D4 mAbs using 10% acrylamide separating gel under non-reduced conditions; Lane 1, SDS-PAGE pattern of 1F5 mAbs include a band at the MW of 150–155 kDa; Lane 2, SDS-PAGE pattern of purified D4 mAbs also include a band at the MW of 150–155 kDa with high purity; Lane M, protein standard markers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

result is in agreement with the results obtained from ICC, which are shown in Fig. 4. Immunocytochemistry analysis and cell-based ELISA demonstrated that D4 mAbs are capable of binding to native CD20 molecules on B lymphocytes. The binding specificity of D4 mAbs was also detected by nonreducing western blot using the Raji cell lysate. The results presented in Fig. 6 show that the D4 or 1F5 mAbs specifically recognized a 33-kDa band corresponding to CD20. It appears probable that native CD20 epitopes can be recognized by the D4 or 1F5 mAbs after the antigen has been separated by SDS-PAGE under denaturing, non-reducing conditions. The mechanism permitting the recognition of native epitopes on western blots following denaturation is not known. However, it is more probable that the denatured polypeptides in the gel might renature enough to reform epitopes during or after transfer to the membranes [55]. As the transfer buffer is often used without SDS, the protein-bound SDS gradually decreases during the transfer, so, after the transfer, the proteins on the membrane are no longer under denaturing conditions [56]. In this regards, previous studies found that western blot-reactive mAbs also recognized native conformation of epitopes [57–59]. 3.3. Effect of D4 anti-CD20 mAbs on proliferation of Raji cells We next investigated whether D4 anti-CD20 mAbs also had antiproliferative activity on the Burkitt's lymphoma cell line. For this purpose, we measured proliferation by the MTT assay in the presence or absence of antibodies. The proliferation of Raji cells was progressively inhibited as the concentration of D4 mAbs increased, with maximum inhibition at concentrations of ≥0.01 μg/mL (Fig. 7). The percent cell proliferation after treatment with various concentrations (0.01–1 μg/mL) of D4 mAbs was dramatically lower than that of the untreated control (p b 0.001). At antibody concentrations of lower than 1 μg/mL, the

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Fig. 4. Binding specificity of the generated D4 anti-CD20 mAbs to native CD20 was detected by immunocytochemistry (ICC). The results were compared with those of 1F5 anti-CD20 mAbs. ICC was performed by adding D4 or 1F5 mAbs to CD20-expressing Raji cells followed by incubating with an HRP-conjugated goat anti-mouse IgG. (A) ICC using 1F5 mAbs. Under an inverted microscope, brown staining can be clearly observed on the surface of Raji cells, indicating the binding of 1F5 mAbs to the CD20 cell surface antigen. (B) ICC using D4 mAbs. D4 anti-CD20 mAbs resulted in a more intense brown colour on the surface of Raji cells than that of 1F5 mAbs. (C) ICC on negative control Jurkat cells. There is no colour on the surface of Jurkat cells. (D) ICC on negative control using Raji cells without D4 or 1F5 primary Abs. There is no colour on the surface of Raji cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

percent cell proliferation in D4-treated cells was significantly lower than that in rituximab-treated cells (p b 0.001) (Fig. 7). The IC50 of D4 on Raji cell proliferation was estimated to be 0.01 μg/mL. The cell

growth inhibitory activity of D4 mAbs (IC50 = 0.01 μg/mL) was substantially higher than that of rituximab (IC50 = 0.5 μg/mL), showing the strong ability of D4 anti-CD20 mAbs to inhibit cell proliferation.

3.4. Superior ability of D4 mAbs in inducing direct cell death in Raji cells

Fig. 5. Binding specificity of the generated D4 anti-CD20 mAbs to native CD20 was detected by whole-cell ELISA. The results were compared with those of 1F5 anti-CD20 mAbs. The cell-based ELISA was performed by adding various concentrations (0.5– 20 μg/mL) of D4 or 1F5 mAbs to 105 Raji cells, followed by the addition of a secondary antibody. These results confirmed the results obtained from ICC, as both indicate the higher binding activity of D4 compared with 1F5 (p b 0.05). Controls include wells with Jurkat cells or wells incubated without D4 or 1F5 primary Abs. Represented data are mean ± SD of three concurrent experiments. p-values were determined using one-way ANOVA (*p b 0.05 compared with 1F5).

We further tested whether the D4 anti-CD20 mAbs are capable of inducing B cell death by apoptosis after inhibiting cell growth. Raji cells were cultured in the presence or absence of 2 μg/mL D4 mAbs or rituximab for 24 to 48 h. The number of apoptotic and necrotic cells was measured by annexin V binding and propidium iodide staining. As shown in Fig. 8A, D4 mAbs did induce a clear increase in early apoptotic cells at 24 h. As expected, these cells became necrotic after 48 h. On the other hand, rituximab did induce a small increase (10%) in the number of dead cells detected at 48 h. The number of dead cells did not increase further after 48 h (data not shown). Fig. 8C shows the diagram of the annexin V/PI assay for D4 mAbs and for rituximab. Altogether, PS exposure and cell death in D4-treated was dramatically higher (80 ± 4.5%) than that in rituximab-treated (10 ± 1.9%, p b 0.001). As previously reported [60], rituximab alone does not induce significant apoptosis on malignant B cells. We also directly counted the number of live and dead cells (after treatment with D4 or rituximab) by trypan blue exclusion after 48 h, and similar results were obtained (Fig. 8B). We conclude that D4 mAbs generated by DNA immunization have a superior growth inhibitory activity and a superior ability to induce direct cell death and PS exposure in a B cell lymphoma cell line, as shown by tests in Raji cells.

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Fig. 6. Binding specificity of D4 mAbs detected by western blotting. SDS-PAGE of the Raji or Jurkat cell lysate was performed under denaturing, non-reducing conditions. After transferring the resolved proteins onto a PVDF membrane, the membrane was incubated with purified D4 or 1F5 mAbs, followed by incubation with a peroxidaseconjugated anti-mouse IgG. The CD20 antigen was developed by DAB substrate solution. Lane 1, control negative Jurkat cells; Lane 2, D4 mAbs recognized CD20 at approximately 33 kDa; Lane 3, 1F5 mAbs also recognized CD20; Lane M, protein standard markers.

4. Discussion To date, rituximab combined with chemotherapy is the gold standard for the treatment of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), but patients often relapse. Over the last few years, several new anti-CD20 mAbs have been developed using mAbs engineering techniques, which are being evaluated in clinical trials [39,61]. Despite the recent advances in development of anti-CD20 mAbs, the efforts should be continued to develop novel antibodies with improved properties by antibody engineering or other strategies.

Fig. 7. Comparison of the effects of D4 anti-CD20 mAbs and rituximab on the proliferation of Raji cells, as measured by the MTT assay; 5 × 104 Raji cells/well were incubated continuously with 0.01, 0.1, 0.5, 1 μg/mL D4 or rituximab at 37 °C for 72 h, followed by addition of 20 μL MTT (5 μg/mL) and incubating for another 4 h. After dissolving the formazan crystals with DMSO, the OD was measured at 570 nm. Cells incubated in medium without D4 or Rituximab were used as control. The represented data are the means of three independent experiments ± SD. p-values were determined using oneway ANOVA (*p b 0.001 compared with rituximab).

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The production of potential therapeutic mAbs against CD20 as a multi-transmembrane protein is challenging due to the insolubility of this protein and the difficulty of producing a soluble protein that maintains its native conformation. Although production of mAbs against multi-transmembrane proteins is possible using classic immunization approaches with intact cells, membrane lysates, solubilized protein, or linear peptides, these approaches are not always successful at generating antibodies that have the ability to recognize the native extracellular epitopes or require laborious screening process [12]. While immunization with CD20-overexpressing cells may circumvent the problem of insolubility and loss of native epitopes, the success is often attenuated by low immunostimulatory properties and a time-consuming screening process. Moreover, the biological effects of anti-CD20 mAbs are not only determined by their epitope sequences but also depend on other factors, such as the orientation of the antibodies with their respective CD20 epitopes [62]. For example, antibodies may show similar biological functions but recognize different CD20 epitopes (e.g., ofatumumab and rituximab). Conversely, tositumomab and rituximab target a similar epitope, but show different biological properties, so subtle differences in the interaction of anti-CD20 antibodies with their target can greatly change the functional activities [62]. Because the orientation of the bound antibodies is a critical factor for these differences, generating anti-CD20 mAbs using the native conformation of CD20 epitopes can influence the clinical efficacy of anti-CD20 antibodies. To date, however, classic immunization approaches have been used to produce parental mouse mAbs against B-lymphocyte antigen CD20. This is the first report describing a robust DNA immunization strategy for generating mAbs against native extracellular epitopes of CD20 and demonstrating some of the effector functions of the produced mAbs. The results show that the described DNA immunization strategy would be useful for the production of tumour-targeting anti-CD20 mAbs with improved direct Bcell cytotoxicity. As has been previously shown [12,63], immunization with DNA induces a powerful immune response against the native epitopes of proteins. Thus, DNA-based immunization creates an opportunity for the development of mAbs against the native extracellular domain of CD20 with new desirable characteristics. A few mAbs against structurally complex target antigen have been generated using DNA immunization which might be used as potentially therapeutic monoclonal antibodies [12,17,64]. The intramuscular (i.m.) injection of plasmid DNA mediated by electroporation has been shown by many groups to be an effective method for the production of mAbs [65]. However, myocytes are unlikely candidates for the induction of antibody or cytotoxic T cell responses after i.m. DNA immunization. This is because myocytes express very low or undetectable levels of major histocompatibility complex class I or II molecules and their ability is limited to processing and presenting antigenic peptides [66]. On the contrary, as mentioned earlier, i.d. immunization with DNA may directly transfect antigen presenting cells (APCs), followed by an early cytotoxic T cell response [29,67,68]. In the present study, genetic immunization involving the intradermal administration of naked plasmid DNA via needle injection appears to be a simple and robust technique for generating pharmacologically active mAbs against CD20. We tested two immunization procedures, using either plasmid DNA or purified protein, for the induction of antibody responses. It was found that the DNA immunization strategy described elicited higher antibody titers, as detected by cell-based ELISA, than immunization with protein in mice (p b 0.01). Despite the great potential of genetic immunization for development of mAbs, the uptake of DNA plasmids by cells upon injection is inefficient [69]. To increase the potency of DNA immunization, our strategy involving intradermal administration of naked plasmid DNA that may directly transfect antigen presenting cells was applied to achieve higher levels of antigen and antibody production. It is interesting that while this approach was effective for the induction of an immune response to native CD20, it is simple and does not need any modulator or equipment. Using DNA immunization, 5

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Fig. 8. Greater PS exposure and cell death were induced by D4 mAbs compared than by rituximab, as measured in the annexin V/PI and trypan blue exclusion assay. Raji cells were cultured in the presence or absence of 2 μg/mL D4 or rituximab for 24 to 48 h. (A) Annexin V binding and PI staining. After 24 h, D4 mAbs induce a clear increase in early apoptotic cells, and the cells became necrotic after 48 h. Rituximab induces a small increase in the number of dead cells after 48 h. (B) After 48 h, the number of live and dead cells was directly counted after treatment with D4 or rituximab by trypan blue exclusion. (C) The graph indicates the mean percentage of total annexin V-positive, PI-negative (V+/PI−) cells and annexin V/PI double-positive cells (V+/PI+; n = 3). D4 induces superior PS exposure and Cell death induction compared with rituximab in the annexin V/PI assay. Data are represented as the means of three independent experiments ± SD. p-values were determined using one-way ANOVA (*p b 0.001 compared with rituximab). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

stable hybridoma clones were established, and finally, 1 stable hybridoma cell line (D4) producing anti-CD20 mAbs was generated by the limiting dilution method. However, immunization using purified CD20 failed to produce suitable titers of antibodies or any stable clones. The insolubility of purified CD20 probably prevented immunized mice from mounting a strong immune response. In summary, using an efficient DNA immunization strategy, we developed a new anti-CD20 antibody that is capable of binding to the native extracellular domain of CD20 with in vitro efficacy superior to that of rituximab. The binding specificity of the generated D4 anti-CD20 mAbs was confirmed by cell-based ELISA, immunocytochemistry, and western blot. Immunocytochemistry analysis and cell-based ELISA demonstrated that D4 mAbs are capable of binding to native CD20 molecules on B lymphocytes. Western blot analysis showed that the D4 mAbs specifically recognize CD20 molecules. Considering that the renaturation of native epitopes on Western blots after denaturing SDS-PAGE apparently is not a rare event [56], it is probable that native CD20 epitopes could be recognized by the D4 mAbs under denaturing, non-reducing western blot. Several properties of D4 may explain its higher efficacy. First, D4 exhibits superior cell growth inhibitor activity (IC50 = 0.01 μg/mL) relative to rituximab (IC50 = 0.5 μg/mL). The percent cell proliferation in D4treated cells was significantly lower than that in rituximab-treated cells (p b 0.001) at antibody concentrations of lower than 1 μg/mL. Moreover, D4 induces significantly stronger direct cell death (80 ±

4.5%), without effector cells, than rituximab (10 ± 1.9%, P b 0.001). Although similar observations have been reported for B1 and GA101 anti-CD20 antibodies, D4 exhibits more induction of apoptosis in Raji cells, which modestly express CD20, compared to GA101 as previously reported [38]. GA101 (obinutuzumab) is a humanized, type II, anti-CD20 mAb derived from humanization of the parental Bly-1 mouse antibody [38]. GA101 was engineered for enhanced direct cell cytotoxicity and increased ADCC in comparison with rituximab. Altogether, the results suggesting that antibody-induced apoptosis is one of the mechanisms responsible for the inhibition of cell growth by D4 mAbs. However, the molecular mechanism of direct cell death after binding of D4 to CD20 remains to be clarified. In conclusion, the data reported here open the way to DNA-based immunization for generating pharmacologically active monoclonal antibodies against CD20. The proposed DNA immunization strategy, including intradermal injection of naked DNA plasmid encoding the CD20 gene, has the potential to provide a strong and rapid immune response to the extracellular epitope of CD20. The generated D4 mAbs could specifically recognize the native CD20 molecules on B lymphocytes. In addition, we show that D4 mAbs induced superior apoptosis on a Burkitt's lymphoma cell line and propose that antibody-induced apoptosis is one of the mechanisms of cell growth inhibition. However, further studies are needed to determine functional activity and produce chimeric or immunoconjugated D4 mAbs that can be used for treatment of lymphoid malignancies.

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Conflict of interest The authors report no conflicts of interest. Acknowledgments We would like to express our gratitude to all members of the Medical Biology Research Center for valuable discussion and expert technical support. We are also particularly indebted to Dr. Reza Khodarahmi, Dr. Kamran Mansouri and Bijan Soleimani, and Ahmad Bagheri for helpful discussion. This work was supported by the Vice Chancellor for Research and Technology, Kermanshah University of Medical Sciences (91398).

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