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, 123 (20), 3128-38

Novel anti-B-cell Maturation Antigen Antibody-Drug Conjugate (GSK2857916) Selectively Induces Killing of Multiple Myeloma

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Novel anti-B-cell Maturation Antigen Antibody-Drug Conjugate (GSK2857916) Selectively Induces Killing of Multiple Myeloma

Yu-Tzu Tai et al. Blood.

Abstract

B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer.

Figures

Figure 1
Figure 1
BCMA is specifically and highly expressed on the cell membrane of patient MM cells. (A) BCMA mRNA was quantitated by real-time qRT-PCR in CD138+ cells from 5 MM patients and 3 normal donors (NPC). 18S is an internal control for normalization. P < .04 (B) BCMA mRNA was measured in paired CD138+ and pDCs from MM patients and normal donors (N1-3) (left and middle panel), as well as matched CD138+ cells, pDCs, and CD138-negative (CD138) cells from 3 MM patients (MM1-3, right panel). (C) MM cell lines were immunostained with a novel humanized anti-BCMA J6M0 mAb (solid line) or isotype control human IgG (dashed line). (D) Dual staining by J6M0-PerCP-Cy5.5 and CD38-PE Cy7 was done in MM1S and CD138+ patient MM cells. Immunostaining with J6M0 mAb (solid line) or isotype control human IgG (iso IgG1, dashed line) was performed in CD138+ cells from additional MM patients (E), as well as paired CD138+ cells and pDCs from a MM patient (F). qRT-PCR, quantitative RT-PCR.
Figure 2
Figure 2
J6M0-mcMMAF selectively inhibits MM cell viability and colony formation via caspase 3/7-dependent apoptosis. (A) J6M0 ADCs (-mcMMAF or -vcMMAE) were added in the culture for 6 days followed by luminescence-based viability assays. Gray bars represent the normalized mean fluorescence intensity of BCMA expression in each cell line. Black triangles and red circles represent the growth inhibitory IC50 for J6M0-mcMMAF and J6M0-vcMMAE, respectively, calculated from triplicate measurements. (B) ANBL6 cells were treated for 3 days with J6M0-mcMMAF/J6M0-vcMMAE, or iso-mcMMAF/iso-vcMMAE (isotype control ADCs), in the presence of absence of BMSCs. (C) CD138+ patient MM cells were incubated for 3 days with indicated ADCs followed by annexin V/PI staining and flow cytometry analysis. CD138+ cells from additional MM patients were treated with J6M0-mcMMAF for 3 days, followed by viability (D) and caspase 3/7 activity (E) assays. (F) J6M0-mcMMAF was added to MM cell culture in methylcellulose for 21 days to determine effects on colony formation. Representative colony formation of INA6 and OPM2 cells are shown on the right.
Figure 3
Figure 3
J6M0-mcMMAF broadly inhibits MM cell growth and survival without targeting BMSCs and effector cells. (A) Various MM cell lines (left panel) and patient MM cells (right panel) were cultured for 3 days, alone or with BMSCs, in the presence of J6M0-mcMMAF followed by luminescent viability assays. (B) Three MM cell lines were treated with serial dilutions of J6M0-mcMMAF or 10 μg/mL iso-mcMMAF (iso 10) overnight followed by caspase 3/7 activity assays. (C) NK cells were treated with J6M0-mcMMAF for 4 days, alone or with BMSCs. MM1Sluc cells, alone or with BMSCs, were included to show differential toxicity. (D) PBMCs, pretreated with or without lenalidomide (Len, 2μM), were treated for 3 days with J6M0-mcMMAF with or without BMSCs and in the presence or absence of OPM2 cells. (E) Monocytes (CD14+) were treated with J6M0-mcMMAF for 4 days, in the presence or absence of BMSCs or H929 MM cells.
Figure 4
Figure 4
J6M0-mcMMAF significantly improved potency and efficacy of ADCC against autologous MM cells. (A) Target cells (MM1Sluc, OPM2, and patient MM cells MM1-2) were labeled with calcein-AM, washed, and incubated with indicated mAbs and NK effector cells in triplicates. Percent lysis was calculated at the end of ADCC assay, based on calcein-AM release. (B) Autologous ADCC activity against CD138+ patient cells was determined using PBMCs or CD138-negative cells from the same MM patient at an E:T ratio of 20:1. All data represent mean percent lysis ± standard deviation.
Figure 5
Figure 5
Lenalidomide further enhances J6M0-mcMMAF–induced MM cell lysis. PBMCs were preincubated with or without Len (2 μM) before adding into calcein-AM–based ADCC assay with J6M0-mcMMAF or J6M0. Target cells included MM1Sluc, XG-1 (A), ANBL6 (B), CD138+ patient MM cells (C), and MM1S cells (D) in the presence or absence of BMSCs. J6M0-mcMMAF–induced ADCC against MM1S cells was also assayed in the supernatant from MM1S cell culture (D). (E) MM1R cells were treated with each drug, alone or together, for 3 days followed by a luminescent viability assay.
Figure 6
Figure 6
J6M0-mcMMAF rapidly eradicates MM tumors established in mice and significantly prolongs survival. (A) SCID mice with palpable H929 tumors (∼150 mm3) were randomized into groups (n = 5 each) and then treated twice weekly for 4 total doses by intraperitoneal injection. (B) SCID mice with palpable OPM2 tumors were randomized into groups (n = 5 each) and then treated twice weekly for a total of 2 or 4 doses by intraperitoneal injection. (C) NK-deficient SCID-beige mice were inoculated IV with MM1Sluc cells. At a mean bioluminescence (BLI) of 3E ± 06 indicating MM1Sluc tumor growth, mice were randomized into groups (n = 8 each) and treated with J6M0-mcMMAF, J6M0, iso-mcMMAF, or vehicle (PBS), twice weekly for a total of 9 doses by intraperitoneal injection. Survival of mice was examined using log-rank (Mantel-Cox) analysis. *P < .0002.
Figure 7
Figure 7
J6M0-mcMMAF promotes phagocytosis of MM cells by macrophages. (A) Long bone tissue sections from vehicle (A-G), iso-mcMMAF (4 mg/kg) (H-N), J6M0 (4 mg/kg) (a-g), J6M0-mcMMAF (0.4 mg/kg) (h-n), and J6M0-mcMMAF (4 mg/kg) (o-u) treated animals stained by H&E (A,H,a,h,o) and by IHC with human IgG (hIgG: B,I,b,i,p), J6M0 (C,J,c,j,q for BCMA), murine IgG (mIgG: D,K,d,k,r), murine anti-human CD138 antibody (E,L,e,l,s), rat IgG, and hematoxylin counterstain (rIgG: F,M,f,m,t), or F4/80 and hematoxylin counterstain (G,N,g,n,u for MΦ). Scale bar = 100 µm. ADCP at 4 hours was determined by flow cytometry using PKH67-labeled MM cells (B, MM1S; C, INA6, RPMI8226, H929) as targets and monocyte-derived macrophages as effector cells at an E:T ratio of 4:1. Experiments were done in triplicates. Percent phagocytosis was measured as the number of dual-positive (PKH67+CD11b+) cells divided by the total number of PKH67+ cells. Data shown are mean ± standard deviation from 2 independent experiments. *P < .02.

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