Jeffrey A. Nemeth

CNTO 95, a fully human monoclonal antibody that inhibits αv integrins, has antitumor and antiangiogenic activity in vivo

Integrins of the αv family, such as αvβ3 and αvβ5, are implicated in tumor‐induced angiogenesis; but their role in tumor growth has not been fully explored. CNTO 95 is a fully human antibody that recognizes the αv family of integrins and is likely to be less immunogenic in humans compared to chimeric or humanized antibodies. CNTO 95 bound to purified αvβ3 and αvβ5 with a Kd of approximately 200 pM and to αv integrin–expressing human cells with a Kd of 1–24 nM. In vitro, CNTO 95 inhibited human melanoma cell adhesion, migration and invasion at doses ranging 7–20 nM. In a rat aortic ring sprouting assay, CNTO 95 (approx. 70 nM) completely inhibited sprouting. Using a human melanoma xenograft model in nude mice wherein CNTO 95 recognized αvβ3 and αvβ5 on human tumor cells but not mouse angiogenic integrins, CNTO 95 (10 mg/kg, 3 times/week) inhibited growth of human melanoma tumors in nude mice by approximately 80% (p = 0.0005), suggesting that CNTO 95 inhibited human tumor growth independently of its antiangiogenic activity. In a nude rat human xenograft model where CNTO 95 binds and blocks both tumor and host integrins, this antibody (10 mg/kg once/week) reduced final tumor weight by >99% (p < 0.0001). Based on these preclinical data, a dose‐escalating phase I clinical trial in cancer patients has been initiated. To our knowledge, CNTO 95 is the first fully human MAb to αv integrins that has potent antitumor and antiangiogenic properties in in vivo preclinical models.

Phase I Evaluation of a Fully Human Anti–αv Integrin Monoclonal Antibody (CNTO 95) in Patients with Advanced Solid Tumors

Purpose: A fully human monoclonal antibody to anti–αv integrins (CNTO 95) has been shown to inhibit angiogenesis and tumor growth in preclinical studies. We assessed the safety and pharmacokinetics of CNTO 95 in patients with advanced refractory solid tumors. Experimental Design: In this phase I trial, CNTO 95 (0.1, 0.3, 1.0, 3.0, and 10.0 mg/kg) was infused on days 0, 28, 35, and 42, and clinical assessments, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), and [18F]-2-fluorodeoxyglucose positron emission tomography (FDG-PET) were done. Patients achieving stable disease or better were eligible for extended dosing every 3 weeks for up to 12 months. Results: Among the 24 enrolled patients, CNTO 95 was associated with one episode of grade III and four episodes of grade II infusion-related fever (all responded to acetaminophen). Of the six patients who received extended dosing, one patient (10.0 mg/kg), with cutaneous angiosarcoma, had a 9-month partial response. Pre- and post-treatment lesion biopsies confirmed tumor cell αv integrin expression, as well as CNTO 95 penetration of the tumor and localization to tumor cells in association with reduced bcl-2 expression. A lesion in one patient (10.0 mg/kg) with stable ovarian carcinosarcoma was no longer detectable by FDG-PET by day 49. Exposure to CNTO 95 seemed to increase in a greater-than-dose-proportional manner; dose-dependent mean half-life ranged from 0.26 to 6.7 days. Conclusions: CNTO 95 was generally well tolerated. Six patients received extended therapy, including one patient with a prolonged response. Biopsy data confirmed tumor localization and pharmacodynamic activity.

CNTO 328, a monoclonal antibody to IL-6, inhibits human tumor-induced cachexia in nude mice.

IL‐6 is a multifunctional cytokine implicated in several cancers. IL‐6 is a growth factor for certain tumors and contributes to drug resistance, cachexia and bone resorption. Cachexia is characterized by progressive weight loss and depletion of host reserves of adipose tissue and skeletal muscle. We have developed CNTO 328 (cCLB8), a human–mouse chimeric MAb to IL‐6 (Kd approx. 10−12 M) that inhibits IL‐6 function. A phase I study with CNTO 328 in multiple myeloma patients demonstrated that the antibody was safe and had a circulating half‐life of approximately 17 days. Since IL‐6 is implicated in cachexia, we hypothesized that CNTO 328 could inhibit tumor‐induced cachexia. We used 2 human tumor‐induced cachexia models in nude mice. In the first model, human melanoma cells were inoculated in female nude mice. Control treated animals lost 19% (±7.7%) body weight from day 0 to day 31, whereas CNTO 328 (10 mg/kg)–treated animals lost only 1.5% (±1.3%) body weight from day 0 to day 31 (p = 0.023). In the second cachexia model, human prostate tumor cells were injected into male nude mice. By day 29, control treated animals lost 6% (±3.5%) body weight, whereas CNTO 328 (10 mg/kg)–treated animals gained 7% (±4%) body weight (p = 0.01). Since CNTO 328 blocks human IL‐6 but not mouse IL‐6, the data indicate that tumor cell–secreted IL‐6 directly contributes to body weight loss, highlighting the potential role for CNTO 328 as an anticachectic agent.

Inhibition of Interleukin-6 Signaling with CNTO 328 Enhances the Activity of Bortezomib in Preclinical Models of Multiple Myeloma

Purpose: Inhibition of the proteasome leads to the activation of survival pathways in addition to those that promote cell death. We hypothesized that down-regulation of interleukin-6 (IL-6) signaling using the monoclonal antibody CNTO 328 would enhance the antitumor activity of the proteasome inhibitor bortezomib in multiple myeloma by attenuating inducible chemoresistance. Experimental Design: The cytotoxicity of bortezomib, CNTO 328, and the combination, along with the associated molecular changes, was assessed in IL-6–dependent and IL-6–independent multiple myeloma cell lines, both in suspension and in the presence of bone marrow stromal cells and in patient-derived myeloma samples. Results: Treatment of IL-6–dependent and IL-6–independent multiple myeloma cell lines with CNTO 328 enhanced the cytotoxicity of bortezomib in a sequence-dependent fashion. This effect was additive to synergistic and was preserved in the presence of bone marrow stromal cells and in CD138+ myeloma samples derived from patients with relative clinical resistance to bortezomib. CNTO 328 potentiated bortezomib-mediated activation of caspase-8 and caspase-9 and the common downstream effector caspase-3; attenuated bortezomib-mediated induction of antiapoptotic heat shock protein-70, which correlated with down-regulation of phosphorylated signal transducer and activator of transcription-1; and inhibited bortezomib-mediated accumulation of myeloid cell leukemia-1, an effect that was associated with down-regulation of phosphorylated signal transducer and activator of transcription-3. Conclusions: Taken together, our results provide a strong preclinical rationale for the clinical development of the bortezomib/CNTO 328 combination for patients with myeloma.

Prostatic ductal system in rats: Regional variation in stromal organization

The rat prostate is composed of a complex system of branching ducts which terminate proximally at the urethra. It has been recognized that epithelial cells lining the ducts respond differently to androgen in various regions of the ducts, with responses ranging from proliferation to apoptosis, but the cellular mechanisms underlying these effects are unclear. Interaction between prostatic stroma and epithelium is essential to normal prostate growth and development, and the prostatic stroma is thought to be the first site of androgen action. Therefore we have examined the organization and distribution of stromal cell types along the rat prostatic ductal system. Using immunohistochemical techniques, we observed abundant fibrous tissue surrounding the distal region of the ducts, with a sparse, discontinuous smooth muscle layer. The intermediate region was surrounded by a continuous layer of smooth muscle one to two cells thick, which increased to greater than four layers thick at the proximal region. Fibrous tissue was located in interductal spaces and occasionally interspersed within the muscle layers in both regions. These observations indicate that regional variations in the distribution of stromal cell types exist and suggest that their corresponding secretory products could be responsible for the various effects of androgen on the epithelium in the rat prostatic ductal system.

Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death

Interleukin (IL)‐6‐mediated signalling attenuates the anti‐myeloma activity of glucocorticoids (GCs). We therefore sought to evaluate whether CNTO 328, an anti‐IL‐6 monoclonal antibody in clinical development, could enhance the apoptotic activity of dexamethasone (dex) in pre‐clinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL‐6‐dependent and ‐independent human myeloma cell lines (HMCLs), including a bortezomib‐resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Experiments with pharmacologic inhibitors revealed a role for the p44/42 mitogen‐activated protein kinase pathway in IL‐6‐mediated GC resistance. Although CNTO 328 alone induced minimal cell death, it potentiated dex‐mediated apoptosis, as evidenced by increased activation of caspases‐8, ‐9 and ‐3, Annexin‐V staining and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex‐mediated apoptosis was preserved in the presence of human bone marrow stromal cells. Importantly, the increased activity of the combination was also seen in plasma cells from patients with GC‐resistant myeloma. Taken together, our data provide a strong rationale for the clinical development of the CNTO 328/dex regimen for patients with myeloma.

Prostatic ductal system in rats : Tissue-specific expression and regional variation in stromal distribution of transforming growth factor-β1

Regional variations in stromal‐epithelial interactions, mediated through soluble growth factors, may be responsible for differences in epithelial growth and death observed between regions of the rat prostatic ductal system. Since transforming growth factor‐β1 (TGF‐β1) can induce prostatic epithelial cell death in vitro and in vivo, we examined the localization and production of TGF‐β1 with respect to the functional regions of the rat prostatic ductal system.

Blockade of interleukin-6 signalling with siltuximab enhances melphalan cytotoxicity in preclinical models of multiple myeloma

Signalling through the interleukin (IL)‐6 pathway induces proliferation and drug resistance of multiple myeloma cells. We therefore sought to determine whether the IL‐6‐neutralizing monoclonal antibody siltuximab, formerly CNTO 328, could enhance the activity of melphalan, and to examine some of the mechanisms underlying this interaction. Siltuximab increased the cytotoxicity of melphalan in KAS‐6/1, INA‐6, ANBL‐6, and RPMI 8226 human myeloma cell lines (HMCLs) in an additive‐to‐synergistic manner, and sensitized resistant RPMI 8226.LR5 cells to melphalan. These anti‐proliferative effects were accompanied by enhanced activation of drug‐specific apoptosis in HMCLs grown in suspension, and in HMCLs co‐cultured with a human‐derived stromal cell line. Siltuximab with melphalan enhanced activation of caspase‐8, caspase‐9, and the downstream effector caspase‐3 compared with either of the single agents. This increased induction of cell death occurred in association with enhanced Bak activation. Neutralization of IL‐6 also suppressed signalling through the phosphoinositide 3‐kinase/Akt pathway, as evidenced by decreased phosphorylation of Akt, p70 S6 kinase and 4E‐BP1. Importantly, the siltuximab/melphalan regimen demonstrated enhanced anti‐proliferative effects against primary plasma cells derived from patients with myeloma, monoclonal gammopathy of undetermined significance, and amyloidosis. These studies provide a rationale for translation of siltuximab into the clinic in combination with melphalan‐based therapies.

Effects of Siltuximab on the IL-6 Induced Signaling Pathway in Ovarian Cancer

Purpose: To explore potential therapeutic strategies for interrupting the interleukin-6 (IL-6) signaling pathway, we measured IL-6 expression in ovarian cancer tissues, and evaluated the effects of a monoclonal anti-IL-6 antibody; siltuximab (CNTO 328), on levels of IL-6–induced Stat3 phosphorylation, Stat3 nuclear translocation, and Stat3 downstream antiapoptotic genes. We then looked for enhancing paclitaxel sensitivity in multidrug-resistant ovarian cancer cell lines. Experimental Design: Expressions of IL-6 in ovarian cancer patient specimens were assessed by immunohistochemistry. Effects of siltuximab on IL-6–induced activation of Stat3 in an ovarian cancer cell line were determined by Western blot and real-time analysis of Stat3 nucleocytoplasmic translocation. Influence of combination of siltuximab and paclitaxel on tumor growth was evaluated in a xenograft mouse mode in vivo. Results: Metastatic and drug-resistant recurrent tumors have significantly higher IL-6 expression when compared with the matched primary tumors. Siltuximab specifically suppressed IL-6–induced Stat3 phosphorylation and Stat3 nuclear translocation. Treatment with siltuximab significantly decreased the levels of Stat3 downstream proteins such as MCL-1, Bcl-XL, and survivin. Treatment with siltuximab reduced expression of multiple IL-6–induced genes in these cell lines. Furthermore, siltuximab increased the cytotoxic effects of paclitaxel in a paclitaxel resistant ovarian cancer cell line in vitro, but combination therapy with siltuximab did not have a significant effect on paclitaxel resistant tumor growth in vivo. Conclusions: These results show that siltuximab effectively block the IL-6 signaling pathways and IL-6–induced gene expression. Blockage of IL-6 signaling may provide benefits for the treatment of ovarian cancer. Clin Cancer Res; 16(23); 5759–69. ©2010 AACR.

Absence of Adverse Effects in Cynomolgus Macaques Treated with CNTO 95, a Fully Human Anti-αv Integrin Monoclonal Antibody, Despite Widespread Tissue Binding

Purpose: CNTO 95 is a fully human anti-αv integrin monoclonal antibody that inhibits macaque and rodent angiogenesis and inhibits human tumor growth in rodents. The purpose of these studies was to evaluate the preclinical safety of long-term administration of CNTO 95 in cynomolgus macaques. Experimental Design: The in vitro binding profiles of CNTO 95 to human and macaque tissues and the in vivo binding to macaque tissues was evaluated by immunohistochemistry. The preclinical safety of CNTO 95 (10 and 50 mg/kg, i.v.) was evaluated in macaques treated once per week for up to 6 months. Safety was evaluated by clinical observations, ophthalmic and physical examinations (including heart rate, blood pressure, and electrocardiogram), clinical pathology (including coagulation parameters), and comprehensive anatomic pathology. The effect of CNTO 95 (50 mg/kg, i.v.) on incisional wound healing was evaluated in macaques. Results: The tissue binding studies showed that CNTO 95 bound with mild to moderate intensity to macaque and human endothelial cells, epithelial cells, and vascular smooth muscle cells in most normal tissues examined. CNTO 95 showed strong to intense staining to the positive control tissue, human placenta. Despite the widespread binding to normal tissues, treatment of cynomolgus macaques with CNTO 95 produced no signs of toxicity and no histopathologic changes in any of the tissues examined (including ovaries and bone growth plates). CNTO 95 did not impair wound healing. Conclusion: These studies show that CNTO 95 is safe and, unlike some other angiogenesis inhibitors, does not seem to inhibit normal physiologic angiogenesis.