MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY Volume 34, Number 3, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/mab.2014.0092

Production and Characterization of New Anti-HER2 Monoclonal Antibodies Manijeh Mahdavi,1 Mehrnaz Keyhanfar,2 Abbas Jafarian,1 Hassan Mohabatkar,2 and Mohammad Rabbani1

Breast cancer is a major public health problem worldwide. Although in Iran cancer is the third cause of death after coronary heart disease and accidents, mortality from cancer has been on the rise during recent decades. About 15% to 20% of patients with invasive breast cancer have abnormally high levels of HER2 protein. HER2 is a specialized protein found on breast cancer cells that controls cancer growth and spread. This study describes the generation and characterization of new anti-HER2 MAbs towards HER2 protein using a chimeric peptide immunogen containing discontinuous B-cell epitope peptide (peptide 626) and promiscuous T-helper epitope (MVF). The specificity of these MAbs was confirmed in various immunoassays, including ELISA, Western blotting, and immunofluorescence. Moreover, the MTT assay results indicated that 5H5 and 5H11 MAbs could reduce the growth of SKBR3 cells by approximately 50% ( p < 0.05). These MAbs that can reduce cancer cell proliferation would be useful for cancer therapy. Furthermore, the synthetic peptide used in the current work was able to induce the immune system to generate antibodies, especially IgG isotype. Therefore, it could be further used as a peptide cancer vaccine that targets different epitopes or structural domains of HER2 ECD.

Introduction

A

fter skin cancer, breast cancer is the most common cancer in women worldwide.(1) It represents 16% of all cancers in women.(2) This rate is twice that of colorectal cancer and cervical cancer and about three times that of lung cancer.(3) Death rates are also 25% greater than that of lung cancer in women.(4) All around the world, the incidence of this cancer shows varied rates.(2) The rates are low in lessdeveloped countries, greatest in the more-developed countries.(5) Breast cancer is related to age, with only 5% of all breast cancers occuring in women less than 40 years old.(6) In vitro studies showed that inhibition of HER2 expression induced significant apoptosis in breast cancer cells.(7,8) Thereafter, HER2 is a logical target for breast cancer therapy. Both prognostic and therapeutic values of HER2 in breast cancer have been established.(9) Most notably, the monoclonal humanized antibody against HER2 (Trastuzumab) was approved in 1998 by FDA for the treatment of HER2 positive breast cancer.(10) In addition, it has been shown in vitro that reduction of HER2 expression by antisense or siRNA resulted in growth inhibition and apoptosis in HER2 positive breast cancer cells.(8,11,12) In recent years, Pertuzumab was also approved in 2012 for the treatment of patients with advanced or late-stage and metastatic HER2 positive breast cancer.(13) All these data indicate the es-

sential role of HER2 in proliferation and antiapoptosis in HER2 positive breast cancer. Receptor tyrosine-protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent), or ERBB2 (human), is a protein that in humans is encoded by the ERBB2 gene.(14) The ERBB2 gene is also frequently called HER2 (from human epidermal growth factor receptor 2) or HER2/neu. HER2 is a 185 kDa growth factor receptor transmembrane glycoprotein and a member of the epidermal growth factor receptor (EGFR/ ERBB) family.(15) Overexpression of this oncogene has been shown to play an important role in the development of certain invasive types of breast cancer.(14) Recently, the protein has become an important target of therapy for approximately 30% of breast cancer patients. The structure of HER2 consists of three domains: N-terminal extracellular domain (ECD), a single transmembrane helix (TM), and an intracellular tyrosine kinase domain. The largest part (ECD) is divided into four subdomains (I–IV). Subdomains I and III create a binding site for the potential receptor’s ligands.(16) Furthermore, the cysteine-rich subdomains II and IV are involved in homodimerization and heterodimerization.(17,18) Current treatments for breast cancer include local/regional treatment: lumpectomy (surgical removal of tumor), mastectomy (surgical removal of the breast), with removal of axillary underam lymph nodes to determine the severity of

1 Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran. 2 Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan, Iran.

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disease (if cancer has spread to lymph nodes; node-positive or node-negative status), and irradiation. It can also entail adjuvant systemic therapy including DNA-targeted chemotherapy (doxorubicin, cyclophosphamide, and paclitaxel). Additionally, if an individual’s tumor specimen is estrogen receptor (ER) positive, hormone therapy (tamoxifen, aromatase inhibitors) is a possible treatment. Monoclonal antibody therapy with Trastuzumab and Pertuzumab is also a possible treatment if a patient’s tumor specimen is HER2 positive.(19,20) Although the MAbs have shown promising results in clinical settings, some concerns, such as repeated therapies and high costs, limited period of effective therapy, undesired immunogenicity, and possible tolerance, must be considered.(19,21) Trastuzumab is a humanized monoclonal antibody by binding to the ECD of HER2.(10,22) It has therapeutic efficacy in early stage breast cancers and HER2 over-expressing metastatic cancers.(23) Furthermore, Pertuzumab as a HER dimerization inhibitor, blocks HER2 dimerization with other HER receptors by binding to the ECD of HER2.(24,25) It has been reported that Trastuzumab and Pertuzumab synergistically can prevent the growth of overexpressing HER2 breast cancer cells.(26) Pertuzumab and Trastuzumab bind to the ECD of HER2 at different sites, corresponding to subdomain II and IV, respectively.(27) Moreover, anti-HER2 MAbs muA21 and CH401 were developed by Zhang and associates and Ishida and colleagues, respectively.(28,29) MAb muA21 specifically inhibited the growth of the human breast cancer SK-BR-3 cells and MAb CH401 had cytotoxity effect on HER2 overexpressing tumor cells. These MAbs targeted the subdomain I of HER2 ECD. Since the MAbs that target different epitopes have distinct biological functions on cancer cells, finding further new epitopes of HER2 ECD is needed.(30) As subdomain III of HER2 ECD plays an important role in the formation of ligand binding site of the receptor and there is no MAb against it,(31) we decided to produce new MAbs from subdomain III of HER2 ECD. Methods Bioinformatics analyses and peptide synthesis

In our previous studies, the selection of candidate peptide within subdomain III of the hHER2 ECD was performed by bioinformatics analyses.(31,32) Integrated strategy, SUPERFICIAL software, and PEPOP tool were used for linear and discontinuous B-cell epitope prediction.(31,33,34) In this strategy, a combination of linear B-cell epitope prediction web servers, such as ABCpred, BCPREDs, Bepired, Bcepred, Elliprro, Discotope, and CBtope, were used for prediction of B-cell epitope peptides.(31) In addition, PEPOP, as a newly developed immunoinformatics tool, was used for prediction of discontinuous B-cell epitope peptides.(32) Also SUPERFICIAL software predicted new conformational peptides that were linked by the linkers. Furthermore, SYFPEITHI, MHCPred, Propred, SVMHC, and HLA peptide motif search for prediction of T-cell epitope peptides were employed.(32) Both B- and T-cell epitope predictions were essential for effective induction of immune responses. Thus, in the present study the results of B-cell epitope prediction were linked to the T-cell epitope prediction results. In this way, as previously described, the selected discontinuous peptide containing both B- and T-cell epitopes was synthesized colinearly with the

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T-helper epitope measles virus fusion (MVF) using a four residue linker (GPSL). The candidate peptide antigen was synthesized as a single peptide at purity >95% by Peptron (Daejeon, South Korea).(35) Generation of hybridomas to produce MAbs

Immunization was performed as previously described.(35) Female BALB/c mice (8 weeks old) were immunized with 100 mg/mL of the synthetic peptide antigen by intraperitoneal injection. The antigen was prepared in phosphate-buffered saline (PBS, pH 7.2) and then mixed with Freund’s complete adjuvant (Sigma, St. Louis, MO) in a ratio of 2:1 in favor of the antigen.(36) In four weekly intervals, the booster injections were performed by Freund’s incomplete adjuvant. In the next step, the mice were bled through the microhematocrit tube, 10–14 days after the primary immunization dose and each of the next booster doses.(37) Then, the separated sera were stored at -20C prior to use for screening by ELISA.(38) After five booster injections, the production of hybridomas was performed according to a standard protocol.(37) Briefly, 3 days after the primary booster injection, splenocytes from immunized mice and SP2/0 myeloma cells were cultured in complete DMEM/HEPES/pyruvate medium (PAA) supplemented with 20% FBS (PAA) and 1% antibiotic-antimycotic (PAA). Cell fusion was performed at 37C with a ratio of myeloma/spleen cells as low as 1:10 in the presence of polyethylene glycol (PEG, MW: 4000; Merck, Darmstadt, Germany). The hybridomas were harvested and plated into five 96-well plates. After incubation with hypoxanthine, aminopterin, and thymidine (HAT, Invitrogen, Carlsbad, CA) medium and feeding over a period of around 2 weeks, the hybridomas were ready for screening. The wells were fed and 2 days later, aliquots of the hybridoma cell culture supernatants were tested in the screening assay using an indirect enzyme linked immunosorbent assay (ELISA). Growing hybridomas (25–50% confluent) from wells with positive culture supernatant in the 96-well plate (master well) were expanded to 24-well plates by resuspending the cells in the master well. Then, after 2 to 3 days, when the cells in the 24-well plate were 25–50% confluent, they were used in cloning by limiting dilution.(37) ELISA assay

Polystyrene 96-well plates were coated with 100 mL recombinant HER2 (500 ng/mL; eBioscience, San Diego, CA) in PBS overnight at 4C. For titration assay, 96-well plates were coated with 100 mL synthetic peptide in PBS. The plates were washed three times with PBS-T20 washing buffer and blocked with 200 mL of 3% skim milk in PBS for 30 min at room temperature (RT). Subsequently, the plates were loaded with 100 mL aliquots of hybridoma media and were incubated for 1 h at RT, followed by three washes. After washing with PBS-T20, 100 mL anti-mouse IgG HRP conjugate (1:2500 dilutions, Promega, Madison, WI) was added to each well and incubated for 1 h at RT. Then, 100 mL per well of tetramethylbanzedine (TMB) substrate (Sigma-Aldrich, St. Louis, MO) was added and color was developed for 10 min. The reaction was stopped by adding an equal volume of 1 N HCl, and the absorbance was measured at 450 nm by ELISA reader (PowerWave XS, Bio-Tek Instruments, Winooski, VT). The sera of non-immunized mice and mouse anti-HER2

NEW ANTI-HER2 MONOCLONAL ANTIBODIES

monoclonal antibody (Invitrogen, Paisley, United Kingdom) were used as negative and positive control in 1:1000 dilutions in PBS, respectively.(39) Charcterization of anti-HER2 peptide MAbs Isotyping. The supernatant culture of isolated positive clones was isotyped using Mouse MAb Isotyping kit (Roche, Nutley, NJ). The assay was performed according to the manufacturer’s instructions.(40) Purification of MAbs and SDS-PAGE analyses. The su-

pernatants of isolated clones with IgG isotype were affinity purified by MAbTrap Kit (GE Healthcare, Piscataway, NJ). Before purification, the supernatants were centrifuged

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(10,000 g for 15 min) and filtered (0.4 mm filter). The IgG fractions from protein-G column were collected into 1 mL tubes containing neutralizing buffer. Purified MAbs were stored in PBS (pH 7.4) containing 0.02% NaN3. Furthermore, the antibody concentration of each tube was determined using Bradford assay. Moreover, for testing the purity of collected fractions, equal amounts of them (50 mg) were run in 12% SDS-PAGE. Resolved proteins were visualized by Coomassie blue staining. Immunoblotting. 40 mL of HER2 recombinant protein (50 mg/mL) were run in 12% SDS-PAGE. In SDS-PAGE analyses mouse anti-HER2 antibody (1:100 dilutions, Invitrogen) was used as positive control. Then, gels were transferred to nitrocellulose membrane (Sigma) using a

FIG. 1. Summary of bioinformatics analysis that were used for HER2 peptide selection from its ECD subdomain III. (A) The predicted peptides P1C and P2C using integrated strategy. (B) Screenshot of SUPERFICIAL showing 3D view of conformational B-cell epitope. (C) Predicted peptides using PEPOP. (D) T-cell epitope prediction. (E) Sequence of discontinuous peptide 626 that was linked to MVF protein by the GPSL linker.

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Table 1. Immunization Schedule for Mouse Monoclonal Antibody Development Day

Manipulation

Adjuvant/site

Description

0

- /eye CFA/IP

25

Control serum collection Primary injection ELISA

36

First booster

IFA/IP

53

ELISA

-/-

60

Second booster

IFA/IP

74

ELISA

-/-

83

Third booster

IFA/IP

110 119

Fourth booster ELISA

IFA/IP -/-

137

Fifth booster

IFA/IP

151

Final booster

IFA/IP

Pre-immune bleed (*0.1 mL/mouse) Immunize with 0.1 mg antigen First test-bleed (*0.1 mL/mouse) Boost with 0.1 mg antigen 2nd test-bleed (*0.1 mL/mouse) Boost with 0.1 mg antigen 3rd test-bleed (*0.1 mL/mouse) Boost with 0.1 mg antigen Death of mouse 3 4th test-bleed (*0.1mL/mouse) Boost with 0.1 mg antigen Boost with 0.1 mg antigen

7

-/-

Antigen preparation, such as peptide synthesize, occurs before day 0.

Trans-Blots Turbo Transfer System (Bio-Rad, Hercules, CA). The membrane was blocked with 3% skim milk in TBS overnight at 4C. The blots were incubated in blocking buffer containing corresponding antibodies against HER2 receptor (1:1000 dilutions) for 1 h at RT. After washing with PBST20, anti-mouse IgG HRP conjugate (diluted 1:10,000– 20,000 in blocking buffer; Promega) was added to the membrane for 1 h at RT. After extensive washes, the antibodyprotein complexes were detected using 3, 3’-diaminobenzidine (DAB, Sigma) and H2O2 (Merck, Kenilworth, NJ). Immunofluorescence assay. SK-BR-3 cells with at least 70% viability were diluted in PBS such that each drop received 20,000–25,000 cells. Then, one drop of the diluted

SK-BR-3 cell suspension was put on glass slides. The slides were fixed for 20 min by cold acetone and were washed twice with PBS (pH 7.4). In the next step, for indirect immunofluorescence assay (IFA), the fixed cells were blocked with 50 mL 3% skim milk-PBS-T20 for 30 min. Overexpressed HER2 receptor on the surface of SK-BR-3 cells was detected using the MAbs generated in the study. Therefore, 50 mL purified MAbs from hybridoma supernatants were added to each respective spot and incubated 30 min at RT. In the control slides, sera from non-immunized mice and mouse anti-HER2 antibody (Invitrogen) were used as negative and positive control, respectively. After washing the slides with PBS-T20 for 3 min, 50 mL of secondary antibody, goat polyclonal secondary antibody to mouse IgG-Fc-FITC (1:100 dilutions; Abcam, Cambridge, MA) were added and incubated 30 min at RT in the dark. Subsequently, each slide was washed as described before, but in the dark. Finally, the slides were mounted by adding 90% glycerol in PBS.(35) Cell viability assay. 180 mL of SK-BR-3 suspension cells (5 · 103 cell/mL) were seeded per well in 96-well plates and cultured overnight at 37C with 5% CO2. Then, 20 mL of twofold serially diluted concentrations of purified MAbs in DMEM from hybridoma supernatants were added to each well and incubated for 72 h. Negative and positive control wells were sera from non-immunized mice and Herceptin (50 mg/mL, Roche), respectively. Next, 20 mL of 5 mg/mL 3[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT, Merck) were loaded to each well and incubated for an additional 4 h. After that, culture medium was discarded and 150 mL dimethyl sulfoxide (DMSO) were added to each well and the absorbance was measured at OD 570 nm by a multiwell scanning spectrophotometer (Stat Fax-2100). The inhibitory growth rate was calculated as follows: 1-(experimental OD value- blank OD value/ negative control OD value- blank OD value) · 100%. The experiments were performed in triplicate for three independent experiments.(28,35) Statistical analysis

Data were expressed as mean – standard error. Comparison between groups was made by the ANOVA single factor. P < 0.05 was considered statistically significant.

FIG. 2. Absorbance of 10 stable clones that were used for cloning by limiting dilution. 5H10 and 5H11 contain antibodies with high specificity for recombinant HER2 protein.

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Table 2. Antibody Concentration in Each Eluted Purified Supernatant Culture Sup culture

Elution1

Elution2

Elution3

Elution4

Elution5

Elution6

1A11 5H5 5H11

33.15152 35.12121 46.18182

44.21212 22.09091 48.45455

34.9697 32.24242 34.66667

13.60606 26.33333 31.93939

13 21.48485 24.66667

24.36364 27.09091 22.09091

Results

Generation of anti-HER2 MAbs

Prediction and HER2 epitope selection

The synthetic chimeric peptide (KLLSLIKGVIVHRLEGVE-GPSL-DPASNTAPSAFDPE) was used as antigen to immunize BALB/c mice for antibody production. During immunization, the specific antibody titer was controlled by testing serum samples using ELISA with HER2 ECD recombinant protein to assay their immunogenicity and generation of antibodies against the chimeric peptide. In this research, the immunization schedule was done according to Table 1. When the antibody titer reached >1:1600, splenocyte and myeloma fusion was carried out. Subsequently, cloning by limiting dilution and screening was done. A total of 53 hybridoma supernatant cultures was positive in the first ELISA screen (data not shown). For expansion and cloning to establish the cell line and to make sure the monoclonality of the hybridomas, 20 of the best candidates were chosen. In the end, 10 stable clones (clones 1A11, 2H4, 3H2, 4H11, 5H5, 5H6, 5H8, 5H9, 5H10, and 5H11) were obtained for further characterization. The findings of present work using ELISA showed that the supernatants derived from the two hybridomas (5H10, 5H11) had antibodies with high specificity for recombinant HER2 protein (Fig. 2).

In our previous studies, the epitope selection from subdomain III of HER2 ECD was performed.(31,32) By using an integrated strategy, the best peptides were the linear B-cell epitopes P5: 379-394 and P15:491-506 and conformational B-cell epitopes P1C: 378-393 and P2C: 500-510 (Fig. 1A). The peptides P1C and P2C overlap with S75-S78 and S99S100 predicted by PEPOP, respectively. Furthermore, peptides P4: PESFDGD-X-TAPLQ, P5: PESFDGDP X TAPLQ, P6: ESFDGDP X NTAPLQP, P7: PESFDGDP-X-NTAPLQ, P8: ESFDG-XX-TAPLQPEQL, and P9: ESFDGDP-XNTAPLQP were predicted by SUPERFICIAL software. In SUPERFICIAL software, predicted conformational peptides were linear B-cell epitopes that were linked by the linkers (Fig. 1B). In addition, PEPOP selected 12 ‘‘discontinuous– continuous peptides’’ from 3D structure of subdomain III of HER2 ECD (PDB: 1S78) (Fig. 1C). Moreover, T-cell epitope prediction results indicated only four peptides that contain epitopes for B-cells and both of MHC class molecules: 626: DPASNTAPSAFDPE, 1402: FDPEDPASNTAPQPEQQ, 123: HTANRPEDEVGEGLACHQ, and 729: QGHTHSWHTANRPEDE. These four peptides containing both B- and T-cell epitopes were compared based on their average antigenicity. Based on the findings, the peptide 626 was chosen for incorporating to MVF protein sequence 288302 using the ‘‘GPSL’’ linker (Fig. 1D, E) to make a synthetic chimeric peptide antigen.

FIG. 3. Running IgG supernatant culture on SDS-PAGE compared to positive control (IgG mouse anti HER2). Left to right: positive control, molecular weight marker, elution2 of 1A11, elution1 of 5H5, elution2 of 5H11.

HER2 peptide MAb characterization

Isotyping showed that 2H4, 3H2, 4H11, 5H6, 5H8, 5H9, 5H10 produced IgM antibody, while clones 1A11, 5H5, and 5H11 produced IgG antibody. Antibodies derived from 1A11 hybridoma supernatants was IgG3 isotype and the supernatants derived from 5H5 and 5H11 were IgG1 isotype. For further studies, protein-G affinity chromatography was used to purify the IgG antibodies from hybridoma supernatant cultures. Every supernatant culture had 6 · 1 mL eluted solution. The purity of these eluted solutions was determined by

FIG. 4. Immunoblotting of 5H5, 5H11, and 1A11 containing IgG isotype MAb with anti-mouse IgG HRP conjugate.

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FIG. 5. Immunofluorescence results of purified MAbs. (A) Reactivity of purified MAbs (5H5 and 5H11) with SKBR3 cells (· 100 zoom). (B) Negative control (· 100 zoom).

SDS-PAGE analyses. After determination of antibody concentrations in eluted solutions, each of them that had the maximum Ab concentration was chosen for SDS-PAGE experiments (Table 2). As shown in Table 2, E1 of 5H5 and E2 of 1A11 and 5H11 had the highest Ab concentration for running on SDS-PAGE gels (Fig. 3). In SDS-PAGE analyses, mouse anti-HER2 antibody (1:100 dilutions, Invitrogen) was used as positive control and 5H5, 1A11, and 5H11 indicated similar bands to it. In SDS-PAGE gel experiments, all purified MAbs consisted predominantly of two bands compromising heavy (50 kDa) and light (22 kDa) chains with no major contaminating proteins. In Western blot analysis, 40 mL of HER2 recombinant protein (50 mg/mL) were probed with hybridoma media from selected clones (1:100 dilutions). We found that 5H5 and 5H11 purified IgG MAbs derived from hybridoma supernatants recognize HER2 protein in approximately 180 kDa. However, 1A11 cannot recognize HER2 protein in Western blot analysis (Fig. 4), possibly because of differences in epitope recognition by this MAb rather than other MAbs. Next, we examined the specificity of anti-HER2 antibodies using immunofluorescent microscopy. As shown in Figure 5A, HER2 overexpressed SK-BR-3 cells were immunostained with purified IgG MAbs derived from 5H5, 1A11, and 5H11 hybridoma supernatants. The immunore-

activity was observed on the surface of SK-BR-3 cells, while the negative control slide displayed no immunofluorescent signal (Fig. 5B). IFA results showed that the purified IgG MAbs derived from hybridoma supernatants could recognize the HER2 receptor expressed in the SKBR-3 cell line. Collectively, the Western blot and IFA results demonstrated the specificity of the generated antiHER2 antibodies. Reactivity of purified anti-HER2 MAbs against chimeric peptide and titration

After determining the concentration of purified MAbs derived from 1A11, 5H5, and 5H11 supernatants using Bradford assay, their two-fold serial dilutions were prepared. Then, the absorbance of them against chimeric peptide was measured using ELISA as described in Materials and Methods. 1A11 (IgG3 isotype) MAb (20 mg/mL) had 0.099 absorbance. Also, 5H5 and 5H11 (IgG1 isotype) MAbs (36 and 22 mg/mL) had 1.6731 and 0.906 absorbance, respectively. These results compared to the positive control indicated that 5H5 and 5H11 MAbs had the highest reactivity to chimeric peptide KLLSLIKGVIVHRLEGVE-GPSL-DPASNTAPSAFDPE and 1A11 MAb indicated less reactivity to the peptide (Fig. 6).

FIG. 6. Titration of 5H5, 1A11, and 5H11 (0.3–36 mg/mL) MAbs against chimeric peptide KLLSLIKGVIVHRLEGVEGPSL-DPASNTAPSAFDPE. Each point is presented by mean – SEs of two independent experiments.

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FIG. 7. Growth inhibitory effects of purified MAbs with different doses (2.5–20 mg/mL) on SK-BR-3 cells by MTT assay. Data are representative of three independent experiments. SEs are shown by error bars. Significant differences are represented by asterisk ( p < 0.05) and double asterisk ( p < 0.01). Effects of anti-HER2 MAbs on breast cancer (SK-BR-3) cell proliferation

To assay the effect of purified anti-HER2 MAbs on cell proliferation, SK-BR-3 cancer cell line was treated with different doses (2.5–20 mg/mL) of purified anti-HER2 MAbs derived from 1A11, 5H5, and 5H11 hybridoma supernatants. As shown in Figure 7, treatment of purified MAbs resulted in a dose-dependent inhibition of SK-BR-3 cell proliferation using MTT test. The growth inhibitory rates for 5H11 MAb were 22, 35, 39, and 47% at the corresponding concentrations of 2.5, 5, 10, and 20 mg/mL, respectively. Furthermore, at 20 mg/mL concentration, the growth inhibitory rate of 5H11 MAb (47%) was the highest compared to 1A11 (-10%) and 5H5 (40%) MAbs (Fig. 7). Discussion

This work describes the production and characterization of novel murine MAbs specific to human HER2 protein. Due to the low affinity of peptide immunogens to the native proteins (weak immunogen), generation of highly specific antibodies is challenging.(41) We have overcome this challenge by developing a bioinformatics method to identify discontinuous peptides containing both of B- and T-cell epitopes.(31,32) Participation of B- and T-cell epitopes is necessary for generating effective immune response to foreign proteins.(42) Furthermore, we incorporated the selected peptide to the MVF protein as a T-cell epitope in a single chimeric peptide. Similar to our work, the research groups of Dakappagari, Garrett, and Wang used peptide constructs that were incorporated to MVF.(19,43,44) In the present work, the synthetic peptide KLLSLIKGVIVHRLEGVE-GPSL-DPASNTAPSAFDPE was used as antigen for generation of three IgG isotype monoclonal antibodies (1A11, 5H5, and 5H11) and seven IgM isotype MAb using a standard hybridoma technology.(37) Moreover, in bioinformatics methods that were fully described in our previous studies,(31,32) we selected the best peptides from HER2 ECD subdomain III that were different from epitopes of two broadly used anti-HER2 monoclonal antibodies, herceptin and pertuzumab.(35) It is because of that that anti-HER2 antibodies targeting different epitopes demonstrated distinct biological effects on cancer cell proliferation.(28,30) Furthermore, clinical applications reported

that drug resistance to herceptin treatment started to appear in patients with significant therapeutic effect.(21) These reports persuaded investigators to conduct more research and to develop novel MAbs for HER2. Thus, three clones of murine MAbs 1A11, 5H10, and 5H11 (IgG isotype), which were against HER2 ECD subdomain III, were developed. In the next step, to determine whether purified MAbs can recognize HER2 protein, we tested their specificity in Western blotting and immunofluorescence assays using overexpressing HER2 cancer cell line SKBR3. We found that two purified MAbs (5H5 and 5H11) specifically react with recombinant HER2 protein by immunofluorescence and Western blot analyses. Both of these MAbs were able to bind to native and denatured HER2 protein, but 1A11 purified MAb could not bind to HER2 protein in Western blot analysis and immunofluorescence assays. It is because of that that the 1A11 purified MAb is not an antibody against HER2 ECD or if it is, 1A11 MAb is not a high affinity anti-HER2 MAb. Also, in titration assays, 1A11 MAb demonstrated less reactivity than 5H5 and 5H11 to the synthetic peptide. Therefore, the titration results were supported by immunofluorescence and Western blot analyses. Finally, we examined the growth inhibitory rates of purified MAbs binding to subdomain III using MTT assay on HER2 overexpressing cancer cell line SKBR3. The results showed that the cell proliferation was significantly inhibited by 5H5 (40%) and 5H11 (47%) in a dose-dependent manner. Similarly, Zhang and colleagues reported the production of anti-HER2 MAb muA21 and found that it could prevent the proliferation of SKBR3 by about 36% in 5.4 mg/mL.(28) In spite of our study, the muA21 MAb was able to bind to HER2 ECD subdomain I. Taken together, we produced and characterized three novel IgG isotype MAbs against HER2 ECD subdomain III. Furthermore, these MAbs were already shown to be helpful for detection by immunofluorescence microscopy, therefore they could be valuable tools in cell biology studies and diagnostic tests for breast cancer. In addition, their ability for growth inhibition on human breast cancer SKBR3 cells would be useful for cancer treatment and can be humanized to be used as passive immunotherapy agents. Moreover, the synthetic and chimeric peptide used in the current work was able to induce the immune system to generate antibodies, especially

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IgG isotype. Hence, the synthetic peptide may be of particular importance for making cancer peptide vaccines in active immunotherapy studies. Aknowledgment

We thank Azadeh Miriyan and Ali Sharifi for their technical assistance. This work was supported by Isfahan University of Medical Sciences, Iran. Author Disclosure Statement

The authors have no financial interests to disclose. References

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Address correspondence to: Dr. Mehrnaz Keyhanfar Department of Biotechnology Faculty of Advanced Sciences and Technologies University of Isfahan Isfahan, 81746-73441 Iran E-mail: [email protected] Received: November 25, 2014 Accepted: March 17, 2015