Article Figures & Data
Figures
- Figure 1.
Antibody-binding assay comparing binding of GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) to CD20-expressing NHL cells Z138 (A) and SU-DHL4 (B). Cells were incubated for 30 minutes at 4°C with increasing concentrations of CD20 antibodies followed by staining using a FITC-labeled secondary antibody and flow cytometry analysis. Dead cells were excluded by PI staining. Calculated EC50 values using SU-DHL4: GA101, 3.7 nmol/L; rituximab, 7.4 nmol/L; ofatumumab, 4.1 nmol/L. Statistical analysis corresponding to comparison of the maximal binding on Z138 cells: GA101 versus rituximab, P = 0.0028; GA101 versus ofatumumab, P = 0.0013; rituximab versus ofatumumab, P = 0.0006; SUDHL4 cells: GA101 versus rituximab, P = 0.0009; GA101 versus ofatumumab, P = 0.0037; rituximab versus ofatumumab, P = 0.9233. C, redistribution of CD20 on Z138 cells by GA101, rituximab, and ofatumumab: top, overlay of GA101–Alexa Fluor 568- and ofatumumab-Alexa Fluor 488–bound CD20 complexes; bottom, overlay of GA101-Alexa Fluor 568–bound CD20 complexes and rituximab–Alexa Fluor 488–bound CD20 complexes. The average fluorescence intensity and SDs of one of three independent experiments were calculated from the triplicates of each experiment. MFI, mean fluorescence intensity.
- Figure 2.
Binding of the complement component C1q to CD20 antibody-coated dishes (A) and CDC induced by GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) in two NHL cell lines, SU-DHL4 (B) and Z138 (C). Rituximab and ofatumumab bound significantly higher amounts of C1q and induced higher CDC after 2 hours of incubation with rabbit complement and different CD20 antibody concentrations. The average CDC and SDs were calculated from the triplicates of each experiment. The data from one of three independent experiments are shown. Calculated EC50 values for CDC with SU-DHL4: GA101, 6.3 nmol/L; rituximab, 0.42 nmol/L; ofatumumab, 0.48 nmol/L. Calculated EC50 values for CDC with Z138: GA101, >200 nmol/L; rituximab, 1.2 nmol/L; ofatumumab, 0.7 nmol/L.
- Figure 3.
A, GA101-, rituximab-, and ofatumumab-mediated direct cell death assessed in four CD20-expressing cell lines, Raji, SU-DHL4, Wil2S, and Z138. Cells were incubated for 24 hours with CD20 antibodies (10 μg/mL) and subsequently stained with Annexin V–FITC and PI to detect apoptotic cells by flow cytometry. In three of four cell lines tested (Raji, Wil2S, and Z138), GA101 induced significantly stronger Annexin V+/PI+ cells compared with rituximab and ofatumumab. The data from one of three independent experiments carried out for each cell line are shown. The average and SDs were calculated from the triplicates in each experiment. Statistical analysis, Student t test, ***, P < 0.0001; *, P ≤ 0.05. B, time-lapse imaging of direct cell-death induction in Z138 lymphoma cells treated with rituximab, ofatumumab, or GA101. Images were taken at time = 0 (i and iv), time = 2 hours (ii and v), and time = 5.5 hours (iii and vi). Fluorescent (i)–(iii) represent Annexin V (Ann V) FLUOS (detects phosphatidylserine exposure, green) and PI (detects loss of membrane integrity, red). Corresponding transmission images are shown in (iv)–(vi). Control- (buffer only), rituximab-, and ofatumumab-treated cells display limited direct cell-death induction. In contrast, incubation with GA101 leads to profound cell death within 5 to 6 hours. See also Supplementary Video S1.
- Figure 4.
ADCC induced by standard doses of GA101 (black squares), GA101 WT (open squares), rituximab (open diamonds), and ofatumumab (open triangles). Cells were incubated for 4 hours in the presence of the CD20 antibodies and human PBMCs as effectors (E:T, 25:1) and percentage of ADCC was calculated by measuring lactate dehydrogenase release in cell supernatants. PBMCs expressing the V158/V158 FcγRIIIa receptor were incubated with Z138 (A) and SU-DHL4 (B) cell lines. PBMCs expressing the F158/F158 FcγRIIIa receptor were incubated with Z138 (C) and SU-DHL4 (D) cell lines. High doses of GA101 (black squares), GA101 WT (open squares), rituximab (open diamonds), and ofatumumab (open triangles) were added to Z138 cell lines incubated in the presence of PBMCs expressing the V158/V158 FcγRIIIa receptor (E). GA101 induces higher levels of ADCC compared with rituximab and ofatumumab even at high antibody concentrations. The average and SDs were calculated from the triplicates of each experiment. The data from one of three independent experiments are shown. Calculated EC50 values: (A): GA101, 16 pmol/L; rituximab, 269 pmol/L; ofatumumab, approximately 262 pmol/L; GA101 WT, 302 pmol/L; (B): GA101, <2 pmol/L; rituximab, 38.7 pmol/L; ofatumumab, 47.3 pmol/L; GA101 WT, 57.3 pmol/L; (C): GA101, 12 pmol/L; rituximab, approximately 78 pmol/L; ofatumumab, approximately 69.3 pmol/L; GA101 WT, 182.7 pmol/L; (D): GA101, 2 pmol/L; rituximab, approximately 35 pmol/L; ofatumumab, approximately 23 pmol/L; GA101 WT, 79.3 pmol/L; (E): GA101, approximately 30 pmol/L; rituximab, approximately 39 pmol/L; ofatumumab, approximately 36 pmol/L; GA101 WT, 140 pmol/L. F, ADCP of Raji cells by human MDMs polarized to M1 or M2c subtypes. Raji cells were incubated with M1 or M2c macrophages for 1 hour (E:T, 3:1) in the presence of increasing concentrations of the CD20 antibodies. Analysis of phagocytosed target cells, assessed by flow cytometry, showed that all three antibodies induced comparable levels of ADCP. M2c displayed superior phagocytic activity compared with M1 macrophages. Calculated EC50 values: M1: GA101, 28 pmol/L; rituximab, 32.7 pmol/L; ofatumumab, 38 pmol/L; M2c: GA101, 8 pmol/L; rituximab, 11.3 pmol/L; ofatumumab, 8 pmol/L. G, ADCP of Raji cells by human M2c macrophages (E:T, 3:1) in presence of 10 mg/mL competing human IgG (Redimune) and 1 μg/mL CD20 antibodies for 4 hours. Analysis of phagocytosed target cells, assessed by flow cytometry, showed that all three antibodies induced comparable levels of ADCP in presence of competing human IgGs.
- Figure 5.
Whole-blood B-cell depletion mediated by GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) in heparin-treated whole-blood samples: F/F donor (A), F/V donor (B), V/V donor (C). The average B-cell depletion and SDs were calculated from the triplicates of each experiment. The data from one of three independent experiments for each genotype are shown. Average values of triplicates corresponding to EC50 values, percentage maximal killing and statistical analysis conducted for each donor and genotypes (3 donors/genotype, total of 9 experiments) are included in the Supplementary Table S1.
- Figure 6.
A, antitumor activity of GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) in a subcutaneous SU-DHL4 model (six once-weekly 30 mg/kg, i.p. doses commencing on day 25 after tumor inoculation; median and interquartile range; n = 10 animals/group). B, antitumor activity of GA101, rituximab, and ofatumumab (five once-weekly 30 mg/kg, i.p. doses) given as second-line treatment following first-line rituximab therapy (two once-weekly doses of 10 mg/kg, i.p. starting on day 25 after tumor inoculation; median and interquartile range; n = 7–9 animals/group). C, antitumor activity of GA101, rituximab, and ofatumumab (30 mg/kg, i.p. once-weekly) in subcutaneous RL model (median and interquartile range; n = 10 animals/group). In the rituximab and ofatumumab groups, mice received an injection on days 14, 21, and 28. In the GA101 group, mice received an injection on days 14, 21, 28, and 35.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 100K
- Supplementary Figure Legend - PDF file - 43K
- Supplementary Video Legend - PDF file - 39K
- Supplementary Figure 1 - PDF file - 62K, Internalization of GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) upon binding to (A) SU-DHL4 cells and (B, C) whole blood derived from two CLL patients. Samples were incubated for 0.5, 2, 4, or 7 h in (A) and for 0.5, 1, 2, 3 and 5 h in (B, C) with Alexa Fluor (trademark) 488-labeled GA101, rituximab, or ofatumumab at 37{degree sign}C, washed and incubated in the presence or absence of anti-Alexa Fluor 488 for 30 min at 4{degree sign}C. The remaining fluorescence indicates the amount of labeled antibody that is not accessible to the quenching anti-Alexa Fluor 488 antibody and thus corresponds to internalized antibody. The average fluorescence intensity and standard deviations were calculated from duplicates of the experiment with SU-DHL4 cells. Due to low number of primary CLL samples the average fluorescence intensity in B and C correspond to single values.
- Supplementary Figure 2 - PDF file - 307K, Whole-blood B-cell depletion mediated by GA101 (black squares), rituximab (open diamonds), and ofatumumab (open triangles) in lepirudin-treated (A, B) and heat-inactivated (HI) (C) whole-blood samples. GA101 mediated superior B-cell depletion in human whole-blood samples in all experimental conditions (A-C). Heat inactivation of human serum revealed that, in contrast to GA101, ofatumumab and rituximab rely more strongly on complement for efficient B-cell depletion (C). The average B-cell depletion and standard deviations were calculated from the triplicates of each experiment.
- Supplementary Video 1 - WMV file - 14044K, Time-lapse imaging of direct cell death induction in Z138 lymphoma cells treated with either rituximab, ofatumumab, or GA101. Images were taken over 5.5 h of treatment. Annexin V FLUOS (detects phosphatidylserine exposure, green) and PI (detects loss of membrane integrity, red).
Volume 12, Issue 10
