Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity

Abstract

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cell-mediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. In nonhuman primates, GA101 demonstrates superior B cell-depleting activity in lymphoid tissue, including in lymph nodes and spleen. Taken together, these results provide compelling evidence for the development of GA101 as a promising new therapy for the treatment of B-cell disorders.

Figure 1

GA101 induces superior PS exposure and cell death induction compared with rituximab in the annexin V/PI FACS assay. Z138 NHL cells were seeded and treated with 10 μg/mL GA101 or rituximab for 24 hours. The graphs depict the mean percentage of total annexin V–positive, PI-negative (AnnV⁺) cells and annexin V/PI double-positive cells (AnnV⁺/PI⁺; n = 3). (A) Cell death induction by GA101 compared with B-ly1, camptothecin, and the type I anti-CD20 antibody rituximab. (B) Cell death induction by GA101 can be reduced to the level of rituximab by reintroducing the L11V mutation in the elbow-hinge region of the antibody.

Figure 2

GA101 exhibits characteristics typical of a type II anti-CD20 antibody. (A) Fluorescence intensity at the EC₅₀ value (half-maximal binding) of GA101 compared with rituximab. Titration of a panel of NHL cell lines with Cy-5–labeled GA101 (■) shows that only half the amount of antibody is bound to CD20 on the cells, compared with Cy-5 labeled rituximab (▩) at the EC₅₀ concentration. (B) GA101 does not mobilize CD20 into lipid rafts. On binding of GA101 to CD20 in Ramos cells, CD20 is mainly found in the Triton X-100–soluble fraction (S) and not in the Triton X-100–insoluble pellet (P) representing lipid rafts (top row). In contrast, binding of rituximab resulted in the distribution of CD20 into the Triton X-100–insoluble pellet fraction (S; top row). The distribution of Lyn as a typical lipid raft marker and CD71 as a nonlipid raft marker is not affected (bottom 2 rows). (C) Confocal microscopy: Ramos cells were stained for 30 minutes at 37°C with Cy-3–labeled rituximab or GA101 (red fluorescence, middle panel) and costained with the Alexa 488-labeled lipid raft marker cholera toxin subunit B (CTB-Alexa 488, green fluorescence, left panel), which binds to the membrane ganglioside GMI in lipid rafts. The overlay (right panel) confirms that rituximab binding to CD20 results in accumulation of CD20 clusters in lipid rafts as shown by colocalization with CTB-Alexa 488 (yellow fluorescence), whereas CD20 molecules do not redistribute to lipid rafts on binding of GA101 and do not colocalize with CTB-Alexa 488. Pictures were captured with a Leica TCS SP2 confocal microscope with an HCX PL APO CS 63.0×/1.32 OIL UV objective (numeric aperture 1.32) in glycerol and aquired with Leica Confocal Software Version 2.61. Image manipulation was performed with Metamorph Version 7.0r3 and Jasc Paint Shop Pro. (D) CDC assays (LDH release) with Z138 mantle cell lymphoma cells. In the presence of physiologic concentrations of human unspecific IgG (10 mg/mL RedImmune), the type II anti-CD20 antibody GA101 (black) mediates greatly reduced CDC induction compared with rituximab (gray; n = 3). (E) GA101 induces rapid and pronounced homotypic aggregation of SU-DHL4 cells, whereas rituximab induces only a weak aggregation. Pictures were taken 24 hours after the addition of antibody. Pictures were captured with a Zeiss Axiovert 135 microscope with a Zeiss Fluar 5×/0.25 objective (numeric aperture 0.25) in RPMI 1640, 10% FCS, 2mM L-Glutamin on non fixed Ramos cells and a Cool SNAP K4 camera (Visitron Systems GmbH). Image acquisition and manipulation were performed with Metamorph Version 7.0r3 and Jasc Paint Shop Pro.

Figure 3

The type II anti-CD20 antibody GA101 mediates superior direct cell death induction in normal and malignant B cells. (A) GA101 induces PS exposure and cell death in the annexin V/PI FACS assay at levels superior to those induced by the type II anti-CD20 antibody B1 and rituximab. Z138 NHL cells were seeded and treated with 10 μg/mL GA101, nonglycoengineered GA101 (wt GA101), B1, rituximab, or camptothecin as control for 24 hours. The graph shows the mean percentage of total annexin V–positive cells, that is, annexin V/PI double-positive (AxV⁺/PI⁺) and annexin V–positive, PI-negative cells (AxV⁺/PI⁻; n = 3). (B) NHL cells were seeded and either left untreated (□) or treated with 10 μg/mL GA101 (■) or rituximab (▩), respectively, for 72 hours. The graph shows the percentage of total annexin V–positive cells for a cell line panel of 3 Burkitt lymphoma, 4 DLBCL, and 1 MCL cell lines from 1 representative experiment. (C) GA101 (black curve) induces increased cell death compared with rituximab in purified human nonmalignant B cells isolated from 2 healthy donors (gray curve), as measured by annexin V/PI double-positive staining at 36 hours. All measurements were performed in duplicate; the mean of replicate samples from multiple donors (n = 2) and SD are shown. (D) Murine purified B cells from human CD20 transgenic mice were treated as indicated for 36 hours ex vivo with GA101 or rituximab either with or without prior mitogenic stimulation by IgM or CD40L, and cell death induction was measured by annexin V/PI staining as described in “Annexin V/PI FACS assay.” Two animals were used per stimulation. All measurements were performed in duplicate; the mean of replicate samples from multiple animals (n = 2) and SD are shown.

Figure 4

Superior ADCC and B cell–depleting activities of GA101 compared with rituximab. GA101 exhibits a more potent ADCC-inducing ability than rituximab, both in the presence and absence of nonspecific human IgG. Representative ADCC assay with Raji cells as target cells and NK cells from human PBMCs (F/V 158) as effector cells (calcein release, E/T ratio = 20:1) in the absence (A) and presence (B) of physiologic concentrations of nonspecific human IgG (20 mg/mL RedImune; n = 3). In the absence of nonspecific IgG, GA101 (black) was approximately 35-fold more potent in terms of EC₅₀ values than rituximab (gray) at inducing ADCC. In the presence of nonspecific IgG, GA101 (black) still exhibited significant ADCC-inducing activity, whereas that of rituximab (gray) was completely abolished. (C). Enhanced B cell–depletion activity of GA101, as demonstrated in a whole blood B cell–depletion assay with whole blood from a healthy donor (CD16 genotype F158/F158). Representative results for the depletion of CD19-positive B cells are depicted here. GA101 (black) was approximately 25-fold more potent in terms of EC₅₀ values and 1.9-fold more effective (in terms of absolute B-cell depletion) compared with rituximab (gray). Evaluation of relative B-cell depletion was performed using the B-/T-cell ratio set to 0% for untreated control samples (n = 4). (D) Representative whole blood B cell–depletion assay with whole blood from a B-CLL patient. GA101 (black) was more effective at depleting B cells compared with rituximab (gray) and the CD52 antibody alemtuzumab (dotted line). Because alemtuzumab also depletes T cells, the evaluation of relative B-cell depletion was performed based on the absolute B-cell counts. The B-cell numbers were then set to 100% for maximal depletion, and results were compared between the antibodies (n = 4).

Figure 5

Superior antitumor efficacy of GA101 compared with rituximab in human lymphoma xenograft models. (A) Established subcutaneous SU-DHL4 (DLBCL) tumors (250 mm³; n = 10 per group) were treated with 1 mg/kg (dotted lines), 10 mg/kg (short dashed lines), and 30 mg/kg (solid lines) GA101 (every 7 days, 3 times, intravenously; black) compared with identical doses of rituximab (dark gray) and vehicle control (light gray). GA101 treatment resulted in a dose-dependent inhibition of tumor growth that was superior to that of rituximab. A total of 10 of 10 mice showed complete tumor remission and 9 of 10 mice showed long-term survival (> 90 days; cure) after treatment with 30 mg/kg GA101; and 1 of 10 mice showed complete tumor remission after treatment with 10 mg/kg GA101. In the rituximab-treated groups, no complete tumor remission was observed. Data are mean ± SD. (B) Established subcutaneous SU-DHL4 xenografts (n = 10 per group) were treated with rituximab (30 mg/kg every 7 days, intravenously) as single-agent first-line therapy (days 22-35). Xenografts progressing under first-line rituximab treatment (30 mg/kg every 7 days) were subsequently randomized and reassigned to the following treatment groups with weekly dosing (from days 35-60): vehicle (gray curve), rituximab (30 mg/kg every 7 days; dark gray curve), or GA101 (30 mg/kg every 7 days; black curve). SU-DHL4 tumor progression (advanced xenografts; 750 mm³) was effectively controlled through the use of GA101 as a second-line therapy, whereas rituximab treated-tumors remained refractory. (C) Treatment of the aggressive orthotopic disseminated Z138 (MCL) model was initiated 29 days after intravenous injection of tumor cells (n = 10 per group). Treatment with 10 mg/kg GA101 (every 7 days, 6 times, intravenously; black line) resulted in increased overall and median survival, compared with 10 mg/kg rituximab treatment (dark gray line; P < .008) and vehicle control (light gray line). ↑ indicates the treatment time points.

Figure 6

Superior B-cell depletion in cynomolgus monkeys with GA101 treatment compared with rituximab. The efficacy of GA101 at depleting B cells in cynomolgus monkeys was compared with that of rituximab in groups of 3 animals. GA101 (2 × 10 mg/kg, ■; and 30 mg/kg, ) was compared with rituximab (2 × 10 mg/kg, ) and vehicle (□) after 2 intravenous doses administered on days 0 and 7 to male and female cynomolgus monkeys (n = 3 per group; 1 female, 2 males). Peripheral blood and lymph node B-cell numbers were evaluated at baseline (day −7) and on the indicated days by flow cytometric analysis. B-cell numbers were evaluated in the spleens of the treated animals on day 35. (A) Mean B-cell numbers expressed as B-/T-cell ratios in peripheral blood of cynomolgus monkeys treated with GA101 and rituximab. (B) Mean B-cell numbers expressed as B-/T-cell ratios in the lymph nodes of cynomolgus monkeys treated with GA101 and rituximab. (C) Mean B-cell numbers expressed as B-/T-cell ratios in the spleens of cynomolgus monkeys treated with GA101 and rituximab on day 35. GA101 treatment resulted in statistically superior depletion of total B cells from lymph nodes, compared with rituximab, from days 9 to 35, with a decrease in B-cell numbers of more than 95%. Data are mean ± SD.