pubmed.ncbi.nlm.nih.gov

Apomab, a fully human agonistic antibody to DR5, exhibits potent antitumor activity against primary and metastatic breast cancer - PubMed

Apomab, a fully human agonistic antibody to DR5, exhibits potent antitumor activity against primary and metastatic breast cancer

Irene Zinonos et al. Mol Cancer Ther. 2009 Oct.

Abstract

Apomab, a fully human agonistic DR5 monoclonal antibody, triggers apoptosis through activation of the extrinsic apoptotic signaling pathway. In this study, we assessed the cytotoxic effect of Apomab in vitro and evaluated its antitumor activity in murine models of breast cancer development and progression. MDA-MB-231-TXSA breast cancer cells were transplanted into the mammary fat pad or directly into the tibial marrow cavity of nude mice. Apomab was administered early, postcancer cell transplantation, or after tumors progressed to an advanced stage. Tumor burden was monitored progressively using bioluminescence imaging, and the development of breast cancer-induced osteolysis was measured using microcomputed tomography. In vitro, Apomab treatment induced apoptosis in a panel of breast cancer cell lines but was without effect on normal human primary osteoblasts, fibroblasts, or mammary epithelial cells. In vivo, Apomab exerted remarkable tumor suppressive activity leading to complete regression of well-advanced mammary tumors. All animals transplanted with breast cancer cells directly into their tibiae developed large osteolytic lesions that eroded the cortical bone. In contrast, treatment with Apomab following an early treatment protocol inhibited both intraosseous and extraosseous tumor growth and prevented breast cancer-induced osteolysis. In the delayed treatment protocol, Apomab treatment resulted in the complete regression of advanced tibial tumors with progressive restoration of both trabecular and cortical bone leading to full resolution of osteolytic lesions. Apomab represents a potent immunotherapeutic agent with strong activity against the development and progression of breast cancer and should be evaluated in patients with primary and metastatic disease.

PubMed Disclaimer

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1

Activity of Apomab against breast cancer cells and normal cells in vitro. A, seven human breast cancer cell lines were seeded in 96-well plates at 1 × 104 cells per well and treated with increasing doses of Apomab plus anti-human IgG Fc, as indicated. Cell viability was assessed by crystal AlamarBlue staining 24 h after treatment. Apomab reduced cell viability in three of the seven cell lines tested, with the MDA-MB231-TXSA being the most sensitive. In contrast, the remaining four cell lines were relatively resistant to Apomab, even after cross-linking with anti-Fc. B, normal human bone cells (osteoblasts) from two independent donors NHB1 and NHB2, normal human foreskin fibroblasts (HFF), normal human gingival fibroblasts (HGF), embryonic fibroblasts (MRC-5), and normal human mammary epithelial cells (MCF-10A) were seeded into 96-well plates and treated with increasing doses of Apomab plus anti-Fc, as indicated. Cell viability was assessed by AlamarBlue assay or crystal violet staining 24 h after treatment. Data points show means of quadruplicate results from a representative experiment, repeated at least twice. Points, mean of quadruplicate wells and are expressed as a percentage of the number of control cells; bars, SD. C, cells were treated with 100 ng/mL of Apomab plus 100 ng/mL of anti-Fc for 24 h. The cells were lysed and subjected to gel electrophoresis and immunoblot analysis.

Figure 2
Figure 2

Proapoptotic activity of Apomab against MDA-MB231-TXSA breast cancer cells in vitro. A, MDA-MB-231-TXSA-TGL cells were treated with increasing doses of Apomab alone or Apomab plus equivalent doses of anti-Fc, as indicated. Cell viability was assessed by AlamarBlue assay 24 h after treatment and expressed as percentage of control. B, MDA-MB231-TXSA-TGL cells were seeded on chamber slides at 5 × 104 cells per chamber and were treated for 24 h with Apomab at 100 ng/mL plus anti-Fc. Cells were fixed with methanol and incubated with 4′,6-diamidino-2-phenylindole before washing in PBS and mounting on PBS/glycerin. 4′,6-Diamidino-2-phenylindole staining was visualized by fluorescence microscopy. C, top, MDA-MB231-TXSA cells were treated with Apomab plus anti-Fc for 1,3,6,9, and 12h.Celllysates were used to determine caspase-3-like activity, using the caspase-3-specific fluorogenic substrate, zDEVD-AFC, as described in the Materials and Methods. Bottom, MDA-MB231-TXSA cells were treated for 12 h with 100 ng/mL of Apomab plus anti-Fc alone or were coincubated with the broad specificity caspase inhibitors z-VAD-fmk (50 μmol/L). To exclude possible toxic effects of the inhibitor, cells were also treated with the inhibitor alone. Cell viability was determined using the AlamarBlue assay and expressed as percentage of control. Columns, means of quadruplicate results from a representative experiment repeated at least twice; bars, SD. D MDA-MB231 -TXSA-TGL cells were seeded at 2 × 106 per T25 flask and were either left untreated or treated with Apomab plus anti-Fc at a concentration of 100 ng/mL each. Cells were then lysed and protein was isolated at 1, 3, 6, 9, and 12 h after treatment. Cell lysates were analyzed by PAGE and transferred to polyvinylidene difluoride membranes for immunodetection. The caspase-8, caspase-9, caspase-10, caspase-3, poly ADP ribose polymerase (PARP), and Bid antibodies detect both full-length and processed forms of the antigen.

Figure 3
Figure 3

Antitumor activity of Apomab as a single agent against orthotopic mammary tumors. A, early treatment protocol: 8-wk-old female nude mice were injected with 1 × 106 MDA-MB-231-TXSA-TGL cells into the mammary fat pad, as described in the Materials and Methods. Seven days after cancer cell transplantation, mice were randomized into two groups of six mice per group, which received either vehicle or Apomab at 10 mg/kg i.p. once weekly until termination of the experiment. Mice were imaged weekly using the Xenogen IVIS 100 bioluminescence imaging system. Representative whole body images of vehicle and Apomab-treated animals during the course of the experiments are shown. All vehicle-treated animals were humanely killed on day 19 due to high tumor load, whereas all Apomab-treated animals exhibited complete responses and remained free of tumor with weekly treatment until day 90 when the experiment was terminated. In a separate cohort of animals (n = 4), where Apomab treatment was discontinued on day 21, animals also remained free of mammary tumors, with no evidence of recurrence. B, delayed treatment protocol. Mammary tumors were allowed to progress to an advanced stage of ~ 1,000 mm3 and mice were randomized into two groups of six mice per group. Apomab was administered on day 19 at either 3.0 or 10 mg/kg i.p once weekly until termination of the experiment. Representative whole body images and quantification of bioluminescence signal demonstrating complete regression of tumors in mice treated with Apomab at both doses are shown. C, tumor burden as measured by BLI is well correlated with tumor volume of palpable tumors. BLI measurements are expressed as the sum of integrated photon counts per second and data are expressed as mean ± SEM (solid line). Tumor dimensions were measured by digital caliper, and tumor volumes were calculated as [Length × (Width)2/2] measured by calipers and expressed as mm3 (dotted line) D, histologic examination of representative sections from the mammary tumors 48 h after treatment indicated that Apomab induced apoptosis in a substantial proportion of the tumor cells, with intense TUNEL-positive staining of tumor cells when compared with vehicle-treated animals.

Figure 4
Figure 4

Apomab inhibits tumor growth in bone and protects against breast cancer–induced osteolysis. Early treatment protocol: MDA-MB231-TXSA-TGL breast cancer cells were injected directly into the tibial marrow cavity of 4-wk-old female athymic mice (1 × 105/10 μL injection), as described in the Materials and Methods. A, tibial tumors were allowed to establish for 14 d and mice were randomized into two groups (n = 10 per group), which received either vehicle or Apomab at 10 mg/kg once weekly. Representative whole body BLIs are shown, with the graph representing the average tumor signal over time, measured as mean photon counts per second. B, three-dimensional micro-CT images from representative animals taken at day 27 showed extensive osteolysis in the tibiae of the vehicle-treated animals, whereas Apomab treatment offered complete protection from cancer-induced osteolysis. The graph shows the average total bone volume in each group for both the tumor-bearing and nontumor-bearing tibiae; *, P < 0.001 with respect to the nontumor-bearing control. Bone volume was measured from 750 micro-CT sections of the proximal tibia, starting from the growth plate and using the program CTan. Data shown in each case are the average bone volume from all animals in that group: points, means; bars, SEM.

Figure 5
Figure 5

Apomab treatment reduces tumor burden in bone and induces massive apoptosis of tumor cells in the bone marrow. Delayed treatment protocol: A, cells were injected intratibially and tumors were allowed to progress to an advanced stage before Apomab therapy so that by day 26, BLI and live micro-CT analysis showed high tumor load and extensive osteolysis. Animals (n = 6 per group) were treated with Apomab on day 26 at 10 mg/kg i.p. once weekly, and tumor regression was monitored by BLI at the indicated times. Shown are representative bioluminescence whole body images, demonstrating a striking reduction in tumor burden in the tibiae only 2 d after Apomab treatment, with all animals remaining tumor free thereafter with weekly treatment. B, histologic examination of representative sections from decalcified tibial sections 48 h after treatment indicated that Apomab induced apoptosis in a substantial proportion of the intraosseus and extraosseous tumor mass, with intense TUNEL-positive staining of tumor cells when compared with vehicle-treated animals.

Figure 6
Figure 6

Apomab treatment reduces tumor burden and promotes healing of osteolytic lesions. Animals were treated as described in Fig. 6. BLI and live micro-CT imaging were done on all animals before treatment on day 26 and the effect of Apomab therapy at 2 wk (A), 5 wk (B), and 9 wk (C) after Apomab treatment was assessed in the same animals. BLI showed complete tumor regression, whereas comparison of longitudinal and cross-sectional two-dimensional and three-dimensional live micro-CT images before and after treatment showed marked resolution of osteolytic lesions. Shown also are the corresponding histologic sections from decalcified tibiae of Apomab-treated animals stained with H&E or TRAP for the detection of new bone formation and the presence of osteoclasts on the bone surface.

Similar articles

Cited by

References

    1. Mundy GR. Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer. 2002;2:584–93. - PubMed
    1. Ashkenazi A, Pai RC, Fong S, et al. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest. 1999;104:155–62. - PMC - PubMed
    1. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor–related apoptosis-inducing ligand in vivo. Nat Med. 1999;5:157–63. - PubMed
    1. Bouralexis S, Findlay DM, Evdokiou A. Death to the bad guys: targeting cancer via Apo2L/TRAIL. Apoptosis. 2005;10:35–51. - PubMed
    1. Emery JG, McDonnell P, Burke MB, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem. 1998;273:14363–7. - PubMed

Publication types

MeSH terms

Substances