Supplementary Figure 2 from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

Supplementary Methods, Figure Legends, and References from Context-Dependent Role of Angiopoietin-1 Inhibition in the Suppression of Angiogenesis and Tumor Growth: Implications for AMG 386, an Angiopoietin-1/2–Neutralizing Peptibody

a Coxon, James Bready, Hosung Min, Stephen Kaufman, Juan Leal, Dongyin Yu, Tani Ann Lee, g Sun, Juan Estrada, Brad Bolon, James McCabe, Ling Wang, Karen Rex, Sean Caenepeel, Paul Hughes, Cordover, Haejin Kim, Seog Joon Han, Mark L. Michaels, Eric Hsu, Grant Shimamoto, Russell Cattley, Hurh, Linh Nguyen, Shao Xiong Wang, Anthony Ndifor, Isaac J. Hayward, Beverly L. Falcón, d M. McDonald, Luke Li, Tom Boone, Richard Kendall, Robert Radinsky, and Jonathan D. Oliner

a Coxon, James Bready, Hosung Min, Stephen Kaufman, Juan Leal, Dongyin Yu, Tani Ann Lee, g Sun, Juan Estrada, Brad Bolon, James McCabe, Ling Wang, Karen Rex, Sean Caenepeel, Paul Hughes, Cordover, Haejin Kim, Seog Joon Han, Mark L. Michaels, Eric Hsu, Grant Shimamoto, Russell Cattley, Hurh, Linh Nguyen, Shao Xiong Wang, Anthony Ndifor, Isaac J. Hayward, Beverly L. Falcón, d M. McDonald, Luke Li, Tom Boone, Richard Kendall, Robert Radinsky, and Jonathan D. Oliner

Abstract AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. Mol Cancer Ther; 9(10); 2641–51. ©2010 AACR.

RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting "huRANKL" mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.

Introduction: Ovariectomy (OVX) results in bone loss caused by increased bone resorption. RANKL is an essential mediator of bone resorption. We examined whether the RANKL inhibitor osteoprotegerin (OPG) would preserve bone volume, density, and strength in OVX rats.Materials and Methods: Rats were OVX or sham-operated at 3 mo of age. Sham controls were treated for 6 wk with vehicle (Veh, PBS). OVX rats were treated with Veh or human OPG-Fc (10 mg/kg, 2/wk). Serum RANKL and TRACP5b was measured by ELlSA. BMD of lumbar vertebrae (L-1-L-5) and distal femur was measured by DXA. Right distal femurs were processed for bone histomorphometry. Left femurs and the fifth lumbar vertebra (L-5) were analyzed by mu CT and biomechanical testing, and L-6 was analyzed for ash weight.Results: OVX was associated with significantly greater serum RANKL and osteoclast surface and with reduced areal and volumetric BMD. OPG markedly reduced osteoclast surface and serum TRACP5b while completely preventing OVX-associated bone loss in the lumbar vertebrae, distal femur, and femur neck. Vertebrae from OPG-treated rats had increased dry and ash weight, with no significant differences in tissue mineralization versus OVX controls. mu CT showed that trabecular compartments in OVX-OPG rats had significantly greater bone volume fraction, vBMD, bone area, trabecular thickness, and number, whereas their cortical compartments had significantly greater bone area (p < 0.05 versus OVX-Veh). OPG improved cortical area in L5 and the femur neck to levels that were significantly greater than OVX or sham controls (p < 0.05). Biomechanical testing of L5 and femur necks showed significantly greater maximum load values in the OVXOPG group (p < 0.05 versus OVX-Veh). Bone strength at both sites was linearly correlated with total bone area r(2) = 0.54-0.74, p < 0.0001), which was also significantly increased by OPG (P < 0.05 versus OVX).Conclusions: OPG treatment prevented bone loss, preserved trabecular architecture, and increased cortical area and bone strength in OVX rats.

Receptor activator of NF-κB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. The effects of RANKL are inhibited by a soluble decoy receptor called osteoprotegerin (OPG). Total ablation of RANKL in knockout mice leads to high bone mass, lymph node agenesis, and altered lymphocyte differentiation. In contrast, RANKL inhibition via OPG suppresses bone resorption but not inflammation in animal models of inflammatory bone loss. This suggests that the immune phenotype of RANKL knockout mice is related to total RANKL ablation. We hypothesized that prenatal RANKL inhibition via OPG overexpression would suppress bone resorption without influencing lymph node formation or subsequent immune responses. Transgenic rats were created, wherein soluble OPG was overexpressed by 100-fold vs wild type (WT) controls, by gestational day 11 (i.e., before lymph node formation). The structure of lymph nodes, spleen, and thymus of OPG-transgenic (OPG-Tg) animals were comparable to those of age-matched WT rats at gestational day 19 and in adulthood. The OPG-Tg neonates had elevated bone mass, confirming the prenatal inhibition of RANKL. Adult OPG-Tg rats and OPG-Tg mice exhibited no significant functional alterations relative to WT controls when subjected to immune challenges to test for altered innate and humoral responses (e.g., contact hypersensitivity to oxazolone, IgM response to Pneumovax, IgG response to keyhole limpet hemocyanin, or cytokine response to LPS). In summary, prenatal RANKL inhibition did not impair lymph node development, nor did continuous life-long RANKL inhibition cause obvious changes in innate or humoral immune responses in mice or rats.

During our initial attempts to produce transgenic rats, we found that an anaesthetic combination typically used for embryo transfer (intramuscular injection of ketamine [90 mg/kg] with xylazine [10 mg/kg]) yielded extensive variation in both the depth and length of anaesthesia. In the present prospective study, we compared the reproductive outcomes afforded by using either isoflurane (5% for induction, 2% for maintenance, carried in 2 l/min of oxygen) with morphine (5 mg/kg s.c., given immediately after isoflurane induction) or ketamine/xylazine in adult (250–300 g), pseudopregnant Sprague-Dawley rats. Each animal was anaesthetized with either isoflurane/morphine or ketamine/xylazine, after which 30 microinjected eggs were transferred into the left uterine horn. The mean pregnancy rate for isoflurane/morphine (15%) was 50% greater than that achieved with ketamine/xylazine (10%). The mean number of live pups (just over five per litter) was comparable for both regimens. All rats given isoflurane/morphine quickly achieved a surgical depth of anaesthesia and experienced a rapid postoperative recovery (3–5 min). In contrast, 25% of rats injected with ketamine/xylazine did not reach a depth of anaesthesia within 10 min that was sufficient for laparotomy, and all that were anaesthetized successfully required an extended postoperative recovery period (60–90 min). These data show that isoflurane/morphine is well tolerated by microinjected embryos and suggest that its use during embryo transfer may provide a means for both reducing the number of pseudopregnant females used and increasing the speed with which rat transgenic projects are completed.

The purpose of this study was to examine the response of rats of different genetic backgrounds to various superovulatory hormonal treatments. Immature Sprague Dawley (SD), FBNF1, and F344 female rats (30 to 35 days of age) were used for this study as representatives of outbred, hybrid, and inbred strains respectively. Animals from each strain were allocated into four groups of hormone treatments as follows: 1) 30 IU pregnant mare serum gonadotrophin (PMSG) intraperitoneally (i.p.) followed 52 h later with 25 IU human chorionic gonadotrophin (HCG) i.p.; 2) 15 IU PMSG i.p. followed 52 h later with 7.5 IU HCG i.p.; 3) 1.0 IU follicle stimulating hormone (FSH) daily via Alzet mini-pumps for 60 h; and 4) 1.0 IU FSH daily via Alzet mini-pumps for 54 h followed by 10 mg luteinizing hormone (LH). The efficacies of the hormone treatments were evaluated using the following criteria: % mated, % ovulated, total oocytes per female, and % fertilized. The % mated of SD rats treated with PMSG(30)+HCG(25) was significantly higher (P < 0.05) than that of animals treated with PMSG(15)+HCG(7.5); in addition, the total oocytes per female was significantly higher (P < 0.05) for SD animals receiving PMSG(30)+HCG(25) than all other treatments. The % ovulated of SD rats was significantly lower (P < 0.05) in response to FSH alone as compared to all other treatments. The % ovulated for FBNF1 rats was significantly greater (P < 0.05) in response to both PMSG+HCG treatments as compared to FSH and FSH+LH. The % ovulated and % fertilized were significantly lower (P < 0.05) in F344 rats treated with FSH alone as compared to all other treatments. F344 rats produced significantly (P < 0.05) more oocytes per female in response to both PMSG+HCG treatments as compared to FSH and FHS+LH. The % ovulated of SD and F344 rats were significantly higher (P < 0.05) than that of FBNF1 rats in response to FSH and FSH+LH. SD rats produced a significantly greater (P < 0.05) number of oocytes per female than did FBNF1 rats in response to PMSG(30)+HCG(25) and a significantly greater (P < 0.05) number of oocytes per female as compared to those of FBNF1 and F344 rats in response to FSH and FSH+LH. The % fertilized of SD and FBNF1 rats were significantly higher (P < 0.05) than that of F344 rats in response to PMSG(15)+HCG(7.5). Our study demonstrates that treatment with PMSG+HCG is an effective method of eliciting superovulatory responses according to most criteria examined. We have also shown that outbred (SD) rats generally produced more oocytes per female in response to hormonal stimulation than did inbred and hybrid rats. Our results indicate that different strains of rats have various degrees of hormone sensitivity and response to different superovulation protocols.