Research Article For reprint orders, please contact: [email protected]
In vitro assessment of the effects of anti-HER2 monoclonal antibodies on proliferation of HER2-overexpressing breast cancer cells Background: HER2 proto-oncogene is critical in the biology of breast cancer and an important therapeutic target of monoclonal antibodies (mAbs). We have recently established a panel of anti-HER2 mAbs recognizing different epitopes within the extracellular domain of HER2. Materials & methods: In the present study the antiproliferative effect of these mAbs was investigated on HER2-overexpressing human breast cancer cell line BT474, using radioactive thymidine incorporation assay. Results: Our results demonstrated that while two of the mAbs (1T0 and 2A8) inhibited cell proliferation dose dependently, similar to trastuzumab, six mAbs (1F2, 1B5, 1H9, 4C7, 1H6 and 2A9) augmented cell proliferation. Treatment of BT474 cells with different combinations of two mAbs induced either synergistic inhibitory or stimulatory effects. Discussion: Our findings indicate that combination of some stimulatory mAbs could completely abolish the inhibitory effect of other mAbs against HER2. Employment of some combinations of mAbs with significant synergistic inhibitory function may improve the therapeutic efficacy of HER2-specific mAbs. KEYWORDS: breast cancer n HER2 n monoclonal antibodies
The proto-oncogene HER2 is located on chromosome 17 and encodes a 185 kDa transmembrane molecule that belongs to the EGF receptor family of receptor tyrosine kinases, and also includes HER1, HER3 and HER4 [1,2]. HER2 is overexpressed in breast, ovarian and prostate cancer [3,4]. Formation of homodimers and heterodimers of HER2 (on HER2-overexpressing tumor cells) transmits biochemical signals essential for stimulation of intrinsic tyrosine kinase activity leading to cell growth and proliferation [5]. The first monoclonal antibody (mAb) capable of blocking EGF receptor activity was reported in 1983 [6]. With the advent of trastuzumab (Herceptin®, Genentech Inc., CA, USA), a humanized anti-HER2 mAb that has been shown to significantly improve survival of patients with HER2-positive disease, several novel HER2-targeting agents have been identified and developed [7,8]. Despite the great promise of targeted therapy for the treatment of patients with breast cancer, de novo and acquired resistance remain major obstacles in the clinic [9]. Trastuzumab alone or in combination with chemotherapy has been shown to be an active therapy in HER2-positive metastatic breast cancer patients [10,11]. Pertuzumab is another humanized mAb that binds to an epitope on domain II of the extracellular region of HER2, different from the binding site of trastuzumab on domain IV [12]. It blocks ligand-activated signaling transduced from
HER2/HER1 and HER2/HER3 heterodimers and, to a lesser extent, HER2 homodimers. Antitumoral activity of pertuzumab has been shown both in vitro and in vivo models [12]. Combination of pertuzumab and trastuzumab has recently demonstrated improved survival in patients with breast cancer [13]. Based on these findings, the US FDA has recently approved pertuzumab in combination with trastuzumab for patients with HER2-positive metastatic breast cancer [14]. The promising results obtained from pertuzumab alone, and in combination with trastuzumab, have paved the way for production of new mAbs with distinct epitope specificities and improved functional activities. Investigation of the functional activities of such mAbs may lead to identification of novel antibodies with a more efficient antitumor activity in patients with HER2-positive malignancies. In the present study, we present data on the antiproliferative activity of a new panel of anti-HER2 extracellular domain (ECD) mAbs.
10.2217/IMT.13.156 © 2014 Future Medicine Ltd
Immunotherapy (2014) 6(1), 43–49
Fathollah Tahmasebi1‡, Tohid Kazemi2,3‡, Mohammad Mehdi Amiri3‡, Jalal Khoshnoodi3, Jafar Mahmoudian1, Ali Ahmad Bayat1, Mahmood Jeddi-Tehrani1, Hodjatallah Rabbani1 & Fazel Shokri*1,3 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 2 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Author for correspondence: Tel.: +98 21 22432020 Fax: +98 21 22432021 [email protected] ‡ Authors contributed equally 1
Materials & methods Production of mouse anti-HER2 mAbs Generation and selection of the mAbs has recently been reported [1]. Briefly, 6–8-week-old female BALB/c mice were immunized with both HER2-expressing NIH-3T3 (prepared in our laboratory) and purified recombinant HER2 ECD (Bender MedSystems, Vienna, Austria).
part of
ISSN 1750-743X
43
100 80
1H9
1B5
1F2
1H6
4C7
2A9
Tahmasebi, Kazemi, Amiri et al.
1H6
2F9G5
2A8
120
1T0
Trastuzumab
Stimulation (%)
Research Article
60 40
Inhibition (%)
20 0 20 40 60
Figure 1. Assessment of tumor proliferation inhibitory or stimulatory activity of anti-HER2 monoclonal antibodies. A total of 10 µg/ml concentration of each monocolonal antibody was added to BT‑474 cells. Cells were then incubated with 3H-thymidine and radioactive thymidine incorporation was measured by a scintillation counter. The percentage of proliferation inhibition or stimulation was calculated as described in the ‘Materials & methods’ section. An isotype-matched irrelevant monocolonal antibody (2F9G5) was used as a negative control. Error bars represent standard deviations calculated from triplicate cultures.
Subsequently, splenocytes from hyperimmunized mice were harvested and fused with mouse myeloma cell line SP2/0 (National Cell Bank of Iran, Tehran, Iran). Fused cells were grown in hypoxanthine–aminopterin–thymidine
Stimulation (%)
60
Trastuzumab
1T0
1B5
40
20
Inhibition (%)
0
20
40
10 µg/ml 2.5 µg/ml 0.6 µg/ml
60
Figure 2. Dose-dependency assessment of inhibitory (1T0) and stimulatory (1B5) function of monocolonal antibodies by radioactive thymindine incorporation assay. A total of 10, 2.5 and 0.6 µg/ml concentrations of each monocolonal antibody were added to BT‑474 cells. After incubation and addition of 3H-thymidine, radioactive thymidine incorporation was measured by a scintillation counter. The percentage of proliferation stimulation and proliferation inhibition was calculated as described in the ‘Materials & methods’ section. Trastuzumab was used as a control inhibitory monocolonal antibody. Error bars represent standard deviations calculated from triplicate cultures.
44
Immunotherapy (2014) 6(1)
(HAT selective medium; Sigma, MO, USA) and cloned by limiting dilution assay. Supernatants of growing cells were screened by an indirect ELISA and all positive hybridomas were selected for further subcloning, by limiting dilution assay.
Purification & characterization of mAbs All positive clones were injected intraperitoneally to BALB/c mice 1 week after injection of 0.5 ml Pristine (Sigma) to obtain ascitic fluid. Ascitic fluids were harvested and mAbs purified using HiTrap SPG column (GE Healthcare, Freiburg, Germany). All mAbs were subsequently assessed for epitope specificity, binding affinity, isotype and surface binding using ELISA, western blotting and flow cytometry techniques, as reported elsewhere [1]. Assessment of tumor proliferation by radioactive thymidine incorporation assay Suppressive or stimulatory potential of the antiHER2 mAbs was tested on HER2-overexpressing cell line BT‑474. BT‑474 cells were seeded in 96-well flat-bottom tissue culture plates (Nunc, Roskilde, Denmark) in RPMI-1640 culture medium (Gibco, NY, USA), supplemented with 15% fetal calf serum (Gibco) 10 µg/ml insulin (Exir Pharmaceutical Co., Iran), penicillin (100 IU/ml) and streptomycin (100 IU/ ml) (Biosera, Ringmer, East Sussex, UK) and were allowed to recover and adhere overnight at 37°C in a humidified 5% CO2 incubator. Antibodies were then added to wells at 10 µg/ ml concentration and the cells were incubated for further 24 h. After incubation, 3H-thymidine (PerkinElmer, MA, USA) was added at 0.5 µCi per well for 8 h. Cells were then harvested and transferred to scintillation fluid for measurement of 3H-thymidine incorporation by a b-counter (Wallac 1410 liquid scintillation counter, Pharmacia, Sweden). All experiments were performed in triplicate. The following formula was used to estimate the proliferation inhibition/stimulation rate: Proliferation inhibition/stimulation (%) = CPM without antibody - CPM with antibody # 100 CPM without antibody
Internalization assessment of surface HER2 by mAbs BT‑474 cells were incubated in six-well plates at 80% confluency at 37°C in presence or future science group
Inhibitory effect of combinations of anti-HER2 monoclonal antibodies
future science group
100
*
*
80
*
60 40 20
1T
0
+
2A 8
ab +
8 2A
+ 0 1T
tra
tra st
st
uz
um
uz um
ab
2A 8
0 1T
ab
0 tu zu m
Assessment of the effect of each mAb on tumor cell proliferation In the first step, we assessed the pro- or antiproliferative effect of these mAbs on HER2 overexpressing cell line BT‑474. The results are summarized in Figur e 1 and indicate that these mAbs had different effects on proliferation of BT‑474. Accordingly, we categorized these mAbs in to two groups; inhibitory and stimulatory mAbs. The results showed that two mAbs (1T0 and 2A8) inhibited proliferation of BT‑474 and six mAbs (1F2, 4C7, 1B5, 1H9, 2A9 and 1H6) enhanced cell proliferation from 20 to 150%. To assess whether the inhibitory or stimulatory function of mAbs is dose-dependent, the tumor cells were incubated with 10, 2.5 and 0.6 µg/ml of 1T0 and 1B5 mAbs. The results demonstrated a dose-dependent pattern of inhibition (1T0) and stimulation (1B5) of
120
as
Results Production, purification & characterization of mAbs Production and characterization of the mAbs have been reported [1]. Briefly, eight mAbs were generated by subcloning of selected hybridoma cells. All mAbs displayed IgG isotype (six IgG1 and two IgG2a) with binding affinities ranging from 1 × 107 to 1 × 109 M-1. None of the mAbs significantly cross-inhibited each other or trastuzumab, indicating recognition of distinct epitopes on HER2 ECD [1].
Assessment of the effect of different combinations of mAbs on tumor cell proliferation Since each mAb recognizes a distinct nonoverlapping epitope [1], we assessed whether combination of two mAbs could induce either a synergistic or antagonistic inhibitory or stimulatory effect. Combination of the inhibitory mAbs resulted in either additive or synergistic inhibition of tumor cells as compared with each mAb alone (Figure 3). In the next series of experiments, we assessed the effect of different combinations of the inhibitory and stimulatory mAbs on tumor cell proliferation. As shown in Figur e 4 , combination of stimulatory mAbs with inhibitory mAbs lead to either inhibitory or stimulatory outcome, depending on the selected mAbs. For instance, combination of 2A8 (inhibitory) and 1F2 (stimulatory) reverted the growth-stimulatory effect of 1F2 mAb. The inhibitory effect of 2A8 plus 1F2 was higher than 2A8 alone. Similar results were obtained for some other combinations such as 2A8 plus 1H6 and 2A8 plus 4C7. Other combinations, including trastuzumab plus 4C7 and 1T0 plus 4C7, showed that the stimulatory effect of some mAbs could be reverted when combined with
Tr
Statistics Statistical analysis was performed using the SPSS statistical package (SPSS, IL, USA). The results were evaluated by Mann–Whitney U test where appropriate. Probability values of less than 0.05 were considered significant. The results, presented in charts, represent mean ± standard deviation.
mAbs (Figure 2). Trastuzumab was employed as an inhibitory control mAb.
Inhibition (%)
absence of 10 µg/ml of anti-HER2 mAbs for 24 h. Cells were then detached from wells by incubation on ice for 15 min with 0.05% trypsin, 5.3 mM EDTA in phosphate-buffered saline and washed by an equal volume of culture medium containing fetal bovine serum. For flow cytometry, cells were incubated 1 h on ice with fluorescein isothiocyanate-labeled noncompetitive mAb (1T0) [1]. The cells were finally scanned by a flow cytometer (Partec, Nuremberg, Germany). The Flomax f low cytometry analysis software (Partec) was used to analyze the data.
Research Article
Figure 3. Comparison of the synergistic inhibitory effects of trastuzumab, 1T0 and 2A8 monocolonal antibodies alone and in combination with each other. A total of 10 µg/ml concentration of each monocolonal antibody alone or a combination of 5 µg/ml of each monocolonal antibody was added to BT‑474 cells. After incubation and addition of 3H-thymidine, radioactive thymidine incorporation was measured by a scintillation counter and the percentage of proliferation inhibition was calculated as explained in the ‘Materials & methods’ section. Error bars represent standard deviations calculated from triplicate cultures. *Data showing significant differences (p
In vitro assessment of the effects of anti-HER2 monoclonal antibodies on proliferation of HER2-overexpressing breast cancer cells Background: HER2 proto-oncogene is critical in the biology of breast cancer and an important therapeutic target of monoclonal antibodies (mAbs). We have recently established a panel of anti-HER2 mAbs recognizing different epitopes within the extracellular domain of HER2. Materials & methods: In the present study the antiproliferative effect of these mAbs was investigated on HER2-overexpressing human breast cancer cell line BT474, using radioactive thymidine incorporation assay. Results: Our results demonstrated that while two of the mAbs (1T0 and 2A8) inhibited cell proliferation dose dependently, similar to trastuzumab, six mAbs (1F2, 1B5, 1H9, 4C7, 1H6 and 2A9) augmented cell proliferation. Treatment of BT474 cells with different combinations of two mAbs induced either synergistic inhibitory or stimulatory effects. Discussion: Our findings indicate that combination of some stimulatory mAbs could completely abolish the inhibitory effect of other mAbs against HER2. Employment of some combinations of mAbs with significant synergistic inhibitory function may improve the therapeutic efficacy of HER2-specific mAbs. KEYWORDS: breast cancer n HER2 n monoclonal antibodies
The proto-oncogene HER2 is located on chromosome 17 and encodes a 185 kDa transmembrane molecule that belongs to the EGF receptor family of receptor tyrosine kinases, and also includes HER1, HER3 and HER4 [1,2]. HER2 is overexpressed in breast, ovarian and prostate cancer [3,4]. Formation of homodimers and heterodimers of HER2 (on HER2-overexpressing tumor cells) transmits biochemical signals essential for stimulation of intrinsic tyrosine kinase activity leading to cell growth and proliferation [5]. The first monoclonal antibody (mAb) capable of blocking EGF receptor activity was reported in 1983 [6]. With the advent of trastuzumab (Herceptin®, Genentech Inc., CA, USA), a humanized anti-HER2 mAb that has been shown to significantly improve survival of patients with HER2-positive disease, several novel HER2-targeting agents have been identified and developed [7,8]. Despite the great promise of targeted therapy for the treatment of patients with breast cancer, de novo and acquired resistance remain major obstacles in the clinic [9]. Trastuzumab alone or in combination with chemotherapy has been shown to be an active therapy in HER2-positive metastatic breast cancer patients [10,11]. Pertuzumab is another humanized mAb that binds to an epitope on domain II of the extracellular region of HER2, different from the binding site of trastuzumab on domain IV [12]. It blocks ligand-activated signaling transduced from
HER2/HER1 and HER2/HER3 heterodimers and, to a lesser extent, HER2 homodimers. Antitumoral activity of pertuzumab has been shown both in vitro and in vivo models [12]. Combination of pertuzumab and trastuzumab has recently demonstrated improved survival in patients with breast cancer [13]. Based on these findings, the US FDA has recently approved pertuzumab in combination with trastuzumab for patients with HER2-positive metastatic breast cancer [14]. The promising results obtained from pertuzumab alone, and in combination with trastuzumab, have paved the way for production of new mAbs with distinct epitope specificities and improved functional activities. Investigation of the functional activities of such mAbs may lead to identification of novel antibodies with a more efficient antitumor activity in patients with HER2-positive malignancies. In the present study, we present data on the antiproliferative activity of a new panel of anti-HER2 extracellular domain (ECD) mAbs.
10.2217/IMT.13.156 © 2014 Future Medicine Ltd
Immunotherapy (2014) 6(1), 43–49
Fathollah Tahmasebi1‡, Tohid Kazemi2,3‡, Mohammad Mehdi Amiri3‡, Jalal Khoshnoodi3, Jafar Mahmoudian1, Ali Ahmad Bayat1, Mahmood Jeddi-Tehrani1, Hodjatallah Rabbani1 & Fazel Shokri*1,3 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 2 Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran *Author for correspondence: Tel.: +98 21 22432020 Fax: +98 21 22432021 [email protected] ‡ Authors contributed equally 1
Materials & methods Production of mouse anti-HER2 mAbs Generation and selection of the mAbs has recently been reported [1]. Briefly, 6–8-week-old female BALB/c mice were immunized with both HER2-expressing NIH-3T3 (prepared in our laboratory) and purified recombinant HER2 ECD (Bender MedSystems, Vienna, Austria).
part of
ISSN 1750-743X
43
100 80
1H9
1B5
1F2
1H6
4C7
2A9
Tahmasebi, Kazemi, Amiri et al.
1H6
2F9G5
2A8
120
1T0
Trastuzumab
Stimulation (%)
Research Article
60 40
Inhibition (%)
20 0 20 40 60
Figure 1. Assessment of tumor proliferation inhibitory or stimulatory activity of anti-HER2 monoclonal antibodies. A total of 10 µg/ml concentration of each monocolonal antibody was added to BT‑474 cells. Cells were then incubated with 3H-thymidine and radioactive thymidine incorporation was measured by a scintillation counter. The percentage of proliferation inhibition or stimulation was calculated as described in the ‘Materials & methods’ section. An isotype-matched irrelevant monocolonal antibody (2F9G5) was used as a negative control. Error bars represent standard deviations calculated from triplicate cultures.
Subsequently, splenocytes from hyperimmunized mice were harvested and fused with mouse myeloma cell line SP2/0 (National Cell Bank of Iran, Tehran, Iran). Fused cells were grown in hypoxanthine–aminopterin–thymidine
Stimulation (%)
60
Trastuzumab
1T0
1B5
40
20
Inhibition (%)
0
20
40
10 µg/ml 2.5 µg/ml 0.6 µg/ml
60
Figure 2. Dose-dependency assessment of inhibitory (1T0) and stimulatory (1B5) function of monocolonal antibodies by radioactive thymindine incorporation assay. A total of 10, 2.5 and 0.6 µg/ml concentrations of each monocolonal antibody were added to BT‑474 cells. After incubation and addition of 3H-thymidine, radioactive thymidine incorporation was measured by a scintillation counter. The percentage of proliferation stimulation and proliferation inhibition was calculated as described in the ‘Materials & methods’ section. Trastuzumab was used as a control inhibitory monocolonal antibody. Error bars represent standard deviations calculated from triplicate cultures.
44
Immunotherapy (2014) 6(1)
(HAT selective medium; Sigma, MO, USA) and cloned by limiting dilution assay. Supernatants of growing cells were screened by an indirect ELISA and all positive hybridomas were selected for further subcloning, by limiting dilution assay.
Purification & characterization of mAbs All positive clones were injected intraperitoneally to BALB/c mice 1 week after injection of 0.5 ml Pristine (Sigma) to obtain ascitic fluid. Ascitic fluids were harvested and mAbs purified using HiTrap SPG column (GE Healthcare, Freiburg, Germany). All mAbs were subsequently assessed for epitope specificity, binding affinity, isotype and surface binding using ELISA, western blotting and flow cytometry techniques, as reported elsewhere [1]. Assessment of tumor proliferation by radioactive thymidine incorporation assay Suppressive or stimulatory potential of the antiHER2 mAbs was tested on HER2-overexpressing cell line BT‑474. BT‑474 cells were seeded in 96-well flat-bottom tissue culture plates (Nunc, Roskilde, Denmark) in RPMI-1640 culture medium (Gibco, NY, USA), supplemented with 15% fetal calf serum (Gibco) 10 µg/ml insulin (Exir Pharmaceutical Co., Iran), penicillin (100 IU/ml) and streptomycin (100 IU/ ml) (Biosera, Ringmer, East Sussex, UK) and were allowed to recover and adhere overnight at 37°C in a humidified 5% CO2 incubator. Antibodies were then added to wells at 10 µg/ ml concentration and the cells were incubated for further 24 h. After incubation, 3H-thymidine (PerkinElmer, MA, USA) was added at 0.5 µCi per well for 8 h. Cells were then harvested and transferred to scintillation fluid for measurement of 3H-thymidine incorporation by a b-counter (Wallac 1410 liquid scintillation counter, Pharmacia, Sweden). All experiments were performed in triplicate. The following formula was used to estimate the proliferation inhibition/stimulation rate: Proliferation inhibition/stimulation (%) = CPM without antibody - CPM with antibody # 100 CPM without antibody
Internalization assessment of surface HER2 by mAbs BT‑474 cells were incubated in six-well plates at 80% confluency at 37°C in presence or future science group
Inhibitory effect of combinations of anti-HER2 monoclonal antibodies
future science group
100
*
*
80
*
60 40 20
1T
0
+
2A 8
ab +
8 2A
+ 0 1T
tra
tra st
st
uz
um
uz um
ab
2A 8
0 1T
ab
0 tu zu m
Assessment of the effect of each mAb on tumor cell proliferation In the first step, we assessed the pro- or antiproliferative effect of these mAbs on HER2 overexpressing cell line BT‑474. The results are summarized in Figur e 1 and indicate that these mAbs had different effects on proliferation of BT‑474. Accordingly, we categorized these mAbs in to two groups; inhibitory and stimulatory mAbs. The results showed that two mAbs (1T0 and 2A8) inhibited proliferation of BT‑474 and six mAbs (1F2, 4C7, 1B5, 1H9, 2A9 and 1H6) enhanced cell proliferation from 20 to 150%. To assess whether the inhibitory or stimulatory function of mAbs is dose-dependent, the tumor cells were incubated with 10, 2.5 and 0.6 µg/ml of 1T0 and 1B5 mAbs. The results demonstrated a dose-dependent pattern of inhibition (1T0) and stimulation (1B5) of
120
as
Results Production, purification & characterization of mAbs Production and characterization of the mAbs have been reported [1]. Briefly, eight mAbs were generated by subcloning of selected hybridoma cells. All mAbs displayed IgG isotype (six IgG1 and two IgG2a) with binding affinities ranging from 1 × 107 to 1 × 109 M-1. None of the mAbs significantly cross-inhibited each other or trastuzumab, indicating recognition of distinct epitopes on HER2 ECD [1].
Assessment of the effect of different combinations of mAbs on tumor cell proliferation Since each mAb recognizes a distinct nonoverlapping epitope [1], we assessed whether combination of two mAbs could induce either a synergistic or antagonistic inhibitory or stimulatory effect. Combination of the inhibitory mAbs resulted in either additive or synergistic inhibition of tumor cells as compared with each mAb alone (Figure 3). In the next series of experiments, we assessed the effect of different combinations of the inhibitory and stimulatory mAbs on tumor cell proliferation. As shown in Figur e 4 , combination of stimulatory mAbs with inhibitory mAbs lead to either inhibitory or stimulatory outcome, depending on the selected mAbs. For instance, combination of 2A8 (inhibitory) and 1F2 (stimulatory) reverted the growth-stimulatory effect of 1F2 mAb. The inhibitory effect of 2A8 plus 1F2 was higher than 2A8 alone. Similar results were obtained for some other combinations such as 2A8 plus 1H6 and 2A8 plus 4C7. Other combinations, including trastuzumab plus 4C7 and 1T0 plus 4C7, showed that the stimulatory effect of some mAbs could be reverted when combined with
Tr
Statistics Statistical analysis was performed using the SPSS statistical package (SPSS, IL, USA). The results were evaluated by Mann–Whitney U test where appropriate. Probability values of less than 0.05 were considered significant. The results, presented in charts, represent mean ± standard deviation.
mAbs (Figure 2). Trastuzumab was employed as an inhibitory control mAb.
Inhibition (%)
absence of 10 µg/ml of anti-HER2 mAbs for 24 h. Cells were then detached from wells by incubation on ice for 15 min with 0.05% trypsin, 5.3 mM EDTA in phosphate-buffered saline and washed by an equal volume of culture medium containing fetal bovine serum. For flow cytometry, cells were incubated 1 h on ice with fluorescein isothiocyanate-labeled noncompetitive mAb (1T0) [1]. The cells were finally scanned by a flow cytometer (Partec, Nuremberg, Germany). The Flomax f low cytometry analysis software (Partec) was used to analyze the data.
Research Article
Figure 3. Comparison of the synergistic inhibitory effects of trastuzumab, 1T0 and 2A8 monocolonal antibodies alone and in combination with each other. A total of 10 µg/ml concentration of each monocolonal antibody alone or a combination of 5 µg/ml of each monocolonal antibody was added to BT‑474 cells. After incubation and addition of 3H-thymidine, radioactive thymidine incorporation was measured by a scintillation counter and the percentage of proliferation inhibition was calculated as explained in the ‘Materials & methods’ section. Error bars represent standard deviations calculated from triplicate cultures. *Data showing significant differences (p
