Article Figures & Data
Figures
- Fig. 1.
SDS-PAGE of the purified F(ab′)2 preparations of the anti-Her-2 monoclonal antibodies. The mixture of F(ab′)2 preparations was analyzed by SDS-PAGE under nonreducing conditions using the PhastGel System. Gels were stained with Coomassie Blue. Lanes 1 and 6, standard high molecular mass protein markers with molecular weights of 212, 170, 116, 76, and 53 kDa; Lane 2, Herceptin, IgG; Lane 3, F(ab′)2 fragments of Herceptin; Lane 4, a mixture of HER-50, HER-66, and HER-70 IgGs; Lane 5, mixtures of F(ab′)2 fragments of HER-50, HER-66, and HER-70. F(ab′)2 fragments have a molecular mass of ∼100 kDa, whereas the whole IgG is ∼150 kDa. Scanning the gel by using the Pharmacia LKB UltroScan XL indicated that the F(ab′)2 fragments of the mixture F(ab′)2 and Herceptin F(ab′)2 preparations tested were >95% pure.
- Fig. 2.
F(ab′)2 fragments of anti-Her-2 monoclonal antibodies do not induce antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) against BT474 cells in vitro. A (ADCC), flow cytometric analysis was performed on the 5-(and-6)-carboxyfluorescein diacetate, succinimidylester-stained effector cells (mouse spleen cells) using propidium iodide to stain BT474 cells treated with a mixture of F(ab′)2 fragments (1 μg/105 cells) from HER-50, HER-66, and HER-70 (•); the same molar amount of a mixture IgG1s (○); or F(ab′)2 (▾) and IgG (▿) of the isotype-matched control at the same molar concentrations. Negative control BT474 cells were left untreated (▪), and positive control cells were treated with 10 mg/ml NaN3 (□). The E:T ratios were 10:1, 50:1, and 100:1. B (CDC), target BT474 cells were treated with F(ab′)2 fragments (1 μg/105 cells) or the same molar amount of IgGs and mixed with 1:10-, 1:50-, and 1:100-fold dilutions of mouse serum as a source of complement for the CDC assays. Mixture of F(ab′)2 fragments from HER-50, HER-66, and HER-70, •; mixture of HER-50, HER-66, and HER-70 IgG1s, ○; or F(ab′)2 (▾) and IgG (▿) of the isotype-matched control at the same molar concentrations. Negative control BT474 cells were left untreated (▪), and positive control cells were treated with 10 mg/ml NaN3 (□). Both panels depict one representative experiment of three experiments performed. The difference between the percentage of lysis by the mixtures of F(ab′)2 versus IgGs is statistically significant (P = 0.082). Relative to the isotype-matched control, the effect of the IgG mixture treatment is statistically significant (P = 2.12 × 10−3). The F(ab′)2 mixture is not statistically significant from its isotype-matched control (P = 0.67).
- Fig. 3.
F(ab′)2 fragments of HER-50, HER-66, and HER-70 inhibit the secretion of vascular endothelial growth factor (VEGF) by BT474 cells in vitro. Cell supernatants were harvested from 5 × 105 BT474 cells treated with various concentrations of a mixture of F(ab′)2 fragments (•) or IgGs at the same molar concentrations (○) and the F(ab′)2 (▾) and IgG (▿) of the isotype-matched control at the same molar concentrations. VEGF levels were measured using a competitive ELISA kit. This assay can detect 0.195–200 ng/ml VEGF. Untreated BT474 cells secrete approximately 80 ng VEGF/ml/5 × 105 cells over 72 h (▪), and cells treated with 10 mg/ml NaN3 inhibited VEGF secretion (□). The difference between the effect of mixtures of the F(ab′)2 versus IgGs is not statistically significant (P = 0.87). Relative to their isotype-matched controls, the effect of IgG1 mixture and F(ab′)2 mixture is statistically significant (P < 6.95 × 10−4. This is a representative experiment of three such experiments performed.
- Fig. 4.
BT474 cells are killed in vitro by both the mixture of IgGs and their F(ab′)2 fragments. BT474 cells were treated with varying concentrations of the mixture of F(ab′)2 fragments (•), IgGs (○) at the same molar concentrations, and F(ab′)2 (▾) and IgG (▿) of the isotype-matched control at the same molar concentrations. Untreated BT474 cells were used as a negative control, and positive control cells were treated with 10 mg/ml NaN3 (□) for 72 h and then pulsed with [3H]thymidine for 6 h. Growth inhibition, expressed as the IC50, was calculated by comparing [3H]thymidine incorporation in treated versus untreated cells. The difference between the IC50 values of the F(ab′)2 fragment mixture and the monoclonal antibody mixture is not statistically significant (P = 0.3). Relative to the isotype-matched control, the IC50 values of F(ab′)2 mixture and IgG mixture are statistically significant (P < 0.02). This is one representative experiment of three such experiments performed.
- Fig. 5.
Biodistribution of 125I-HER-50 IgG1 or 125I-HER-50 F(ab′)2 in tumor-bearing mice. After i.p. injection of a dose of 33.3 μg/animal (4.8 × 108 cpm) of radiolabeled HER-50 IgG (A) or its F(ab′)2 fragment (B), levels of radioactivity were measured at 24 (hatched box), 48 (white box), and 72 h (gray box) using a gamma counter. The results are expressed relative to the injected dose and organ weight or ml of serum (absolute percentage of initial injected dose/g tissue or ml of serum). This is one representative experiment of three such experiments performed.
- Fig. 6.
The mixture of F(ab′)2 fragments is less effective than the mixture of anti-Her-2 monoclonal antibodies in vivo. Because of the differences in half-lives between the IgGs and the F(ab′)2 values in vivo, 5-fold greater amounts of the F(ab′)2 monoclonal antibodies were administered. Groups of five preirradiated severe combined immunodeficient mice with 200-mm3 s.c. BT474 breast tumors received i.p. injection with one dose of 1500 μg (75 μg/g mouse) of a mixture of F(ab′)2 fragments (•), 300 μg (15 μg/g mouse) of a mixture of the three IgGs (○), 75 μg/g mouse of F(ab′)2 fragments of the isotype-matched control (▾), 75 μg/g mouse of F(ab′)2 fragments of Herceptin (▿), and 15 μg/g mouse Herceptin IgG (□). The mean tumor volume (mm3) ± SD for the five mice in each group was plotted. The difference between the effects of the mixture of our murine IgGs and Herceptin IgG versus the control are significant (Ps < 1.3 × 10−5). The difference between the effects of the mixture of F(ab′)2 and Herceptin F(ab′)2 versus the control is not significant (Ps < 0.1). The differences between the effects of the mixture of F(ab′)2 and IgG and between Herceptin F(ab′)2 and Herceptin IgG are significant (Ps < 7.6 × 10−4). Animals were euthanized when tumor burdens exceeded 1500 mm3 or 10% of total body weight.
Tables
- Table 1
Characterization of the anti-Her-2 F(ab′)2 fragments and IgGs
Antibody Direct immunofluorescence Pharmacokinetics parameters in SCID micec Relative binding affinity (×10−9m)a % Positive cells (100 μg/105 cells) MFIb t1/2 (h) AUC (μg-h/ml) MRT (h) FCR (day−1) F(ab′)2 HER-50 0.23 ± 0.12 97.3 ± 1.6 181.3 ± 24.2 25.6 ± 20.9 1456.8 ± 9.6 91.8 ± 0.1 1.54 ± 0.035 HER-66 5.88 ± 9.88 97.1 ± 1.5 155.1 ± 25.8 N/D N/D N/D N/D HER-70 2333.3 ± 1247.2 90.53 ± 1.95 52.1 ± 19.4 N/D N/D N/D N/D Herceptin 0.5 ± 0.2 97.48 ± 0.54 157.7 ± 12.4 N/D N/D N/D N/D IgG1 HER-50 0.1 ± 0d 93.37 ± 4.2 160.9 ± 51.9 149.8 ± 100.9 30847.3 ± 950.9 222.9 ± 14.9 0.075 ± 0.004 HER-66 6.07 ± 8.43 96.83 ± 1.44 209.2 ± 61.9 N/D N/D N/D N/D HER-70 1433.3 ± 1144.1 82.15 ± 2.25 69.0 ± 52.9 N/D N/D N/D N/D Herceptin 0.1 ± 0 97.6 ± 0.85 212.9 ± 64.0 N/D N/D N/D N/D -
a Cells (105 cells/ml) were treated with different concentrations of FITC-anti-Her-2 F(ab′)2 and IgGs, followed by FACScan analysis. The percentage of positive cells was plotted versus the F(ab′)2or IgG concentrations to determine the concentration necessary to reach 50% saturation of cells (i.e., the relative binding affinity).
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b MFI, mean fluorescence intensity; SCID, severe combined immunodeficient; t1/2 (h), half-life; AUC (μg-h/ml), area under the curve; MRT (h), mean residence time; FCR (day−1), fractional catabolic rate; N/D, not done.
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c The results are expressed as percentage of initial radioactivity relative to the amount of whole body radioactivity after i.p. injection of 100 μg of 125I-radiolabeled protein (33.3 μg/animal, with a radioactive load of 0.3–1 × 107 cpm). The pharmacokinetic parameters were determined using a noncompartmental model with the PKCALC program.
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d SDs are based on at least three experiments.
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- Table 2
A mixture of three anti-Her-2 F(ab′)2 fragments induces similar growth inhibition and apoptosis as the mixture of three anti-Her-2 IgGs in vitro
Antibody IC50 × 10−8 (m)a % Annexin-V+ cellsb % PI+ cellsb,c % Total dead cells F(ab′)2 Mixture 0.43 ± 0.32d 28.0 ± 0.03 31.6 ± 14.5 59.6 ± 7.3 Herceptin 1.3 ± 0.0 15.9 ± 2.9 12.1 ± 3.1 28 ± 3.0 RFT5 (control) 582.5 ± 428.3 2.3 ± 1.6 1.8 ± 1.7 4.1 ± 1.7 IgG1 Mixture 0.1 ± 0.04 28.4 ± 0.7 38.2 ± 5.9 66.6 ± 3.3 Herceptin 0.3 ± 0.1 36.4 ± 2.2 10.0 ± 8.5 46.4 ± 4.3 RFT5 (control) 233.8 ± 167.8 0.7 ± 0.2 0.7 ± 0.3 1.4 ± 0.3 Other None NA 0.6 ± 0.2 0.64 ± 0.12 1.24 ± 0.2 NaN3e 0.004 ± 0.003 43.8 ± 9.0 49.2 ± 11.9 93 ± 5.3 -
a Cells (2.5 × 105 cells/ml) were incubated for 72 h at 37°C with different concentrations of anti-Her-2 monoclonal antibodies or F(ab′)2s and then pulsed with [3H]thymidine for 6 h. IC50 values represent the concentration of monoclonal antibodies required to kill 50% of cells. The difference between the IC50in each treatment group versus untreated control is statistically significant, with Ps < 0.01.
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b Cells (2.5 × 105 cells/ml) were incubated for 4 h at 37°C with 300 μg/ml anti-Her-2 monoclonal antibodies or F(ab′)2s and then stained with annexin-V FITC plus propidium iodide (50 μg/ml) and analyzed by FACScan. The percentage of total dead cells is the sum of the percentage of annexin-V-FITC-positive plus propidium iodide-positive cells. The difference between the percentage of annexin-V-positive cells in each treatment group versus untreated control is statistically significant, with Pvalues < 0.04. The difference between the percentage of propidium iodide-positive cells in each treatment group versus untreated control is statistically significant, with Pvalues < 0.01.
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c PI, propidium iodide; NA, not applicable (because the untreated control values were taken as 100%).
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d SD are based on at least three experiments.
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e Positive control.
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Volume 10, Issue 10
