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Fc-engineered CD54 antibody MSH-TP15 for myeloma therapy
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Figure 5. In vivo anti-tumor efficacy of the MSH-TP15 monoclonalantibody variants tested in two myeloma xenograft models. (A) 48 hours (h) after intraperitoneal (i.p.) injection of 2x107 INA-6.Tu1_red cells in ten SCID/beige mice per group, animals were treated twice weekly either with vehicle (phosphate buffered saline con- trol [PBS ctrl]; dotted black line), MSH-TP15 (black line), MSH-TP15 Fc-engineered (Fc-eng.) (dotted grey line) or MSH-TP15 Fc knockout (Fc k.o.) (grey line). All mice received one dose of 10 mg/kg and six doses of 5 mg/kg by i.p. injection, time points are marked with ↓. (B) Left graph shows the human interleukin 6 receptor (huIL-6R) concentration in final sera of all mice measured by enzyme-linked immunosorbent assay. Correlation between huIL-6R concentration and tumor weight of the explanted tumors of PBS and MSH-TP15 Fc k.o. treated mice is shown in the right graph. (C) Survival and (D) tumor volume of five mice per group injected sub- cutaneously with 5x106 INA-6.Tu1_red cells 10 days prior start of twice weekly i.p. treatment with five doses of 10 mg/kg (left graphs), 1 mg/kg (middle) or 0.1 mg/kg (right) MSH-TP15 (black line) and MSH-TP15 Fc-eng. (dotted grey line). Animals in the MSH-TP15 Fc k.o. group (grey line) received 10 mg/kg and PBS treated animals (dotted black line) served as control. ↓ time point of treatment. Tumor volume was calculated by regular caliper measurement of subcutaneous tumors.
from healthy donors, which highly expressed FcγRII/CD32 and FcγRI/CD64 as well as lower levels of FcγRIIIa/CD16a (Online Supplementary Figure S2). In order to determine ADCP, CD14-stained macrophages were incubated with CFSE-labeled Raji cells at an E:T ratio of 1:3 in the presence of the indicated mAb. CD14/CFSE double positive and CD19 negative cells were defined as phagocytosed tumor cells by flow cytometry. Significant ADCP activity was observed for MSH-TP15 Fc-eng. and the positive control rituximab, while the MSH-TP15 Fc k.o. completely lacked activity and ADCP by MSH-TP15 was moderate (Figure 4D).
The significant recruitment of NK cells and macrophages for ADCC and ADCP of myeloma cells by MSH-TP15 Fc-eng. most likely reflects the improved affin- ity of this variant to FcγRIIIa and FcγRIIa (Figure 1D and E).
MSH-TP15 and MSH-TP15 Fc-engineered have significant in vivo anti-myeloma activity
The MSH-TP15 mAb variants were tested in INA- 6.Tu1_red myeloma xenograft models in immunodefi- cient SCID/beige mice. In the first experiment, 48 h after i.p. injection of tumor cells treatment with MSH-TP15
mAb or vehicle control (phosphate buffered saline [PBS]) was started. Within 65 days, control animals and MSH- TP15 Fc k.o. treated mice needed to be sacrificed due to tumor size, resulting in a median survival of 39 and 32 days, respectively (Figure 5A). Human IL-6 receptor (huIL- 6R) secreted by the INA-6.Tu1_red cells was detectable in final sera of these mice and its concentration correlated with explanted tumor weight (Figure 5B). In contrast, ani- mals treated with MSH-TP15 or MSH-TP15 Fc-eng. did not develop any tumors until the end of the experiment on day 90 (P<0.001 vs. control mice) and no huIL-6R was measured (Figure 5B). Thus, treatment with both, MSH- TP15 and MSH-TP15 Fc-eng., completely prevented the development of myeloma in this model.
Next, in order to compare the efficacy of MSH-TP15 and MSH-TP15 Fc-eng. in more detail, doses of 10, 1 or 0.1 mg/kg were administered to s.c. tumor bearing mice. MSH-TP15 Fc k.o. was given at 10 mg/kg and PBS-treated animals served as control (Figure 5C and D). Compared to the control mice that needed to be sacrificed after 22 days, MSH-TP15 Fc k.o. treated mice reached a median survival of 27 days (Figure 5C). Direct, Fab-mediated anti-myelo- ma effects may account for the slightly delayed tumor
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