Tumor-associated macrophages promote invasion while retaining Fc-dependent anti-tumor function.

Tumor-associated macrophages (TAMs) have been shown to promote tumor progression, and increased TAM infiltration often correlates with poor prognosis. However, questions remain regarding the phenotype of macrophages within the tumor and their role in mAb-dependent cytotoxicity. This study demonstrates that whereas TAMs have protumor properties, they maintain Fc-dependent anti-tumor function. CD11b(+)CD14(+) TAMs isolated from primary human breast tumors expressed activating FcγRs. To model breast cancer TAMs in vitro, conditioned medium from breast cancer cells was used to drive human peripheral monocyte differentiation into macrophages. Tumor-conditioned macrophages were compared with in vitro derived M1 and M2a macrophages and were found to promote tumor cell invasion and express M2a markers, confirming their protumor potential. However, unlike M2a macrophages, tumor-conditioned macrophages expressed FcγRs and phagocytosed tumor cells in the presence of a tumor Ag-targeting mAb, unmasking an underappreciated tumoricidal capacity of TAMs. In vivo macrophage depletion reduced the efficacy of anti-CD142 against MDA-MB-231 xenograft growth and metastasis in SCID/beige mice, implicating a critical role for macrophages in Fc-dependent cell killing. M-CSF was identified in tumor-conditioned media and shown to be capable of differentiating macrophages with both pro- and anti-tumor properties. These results highlight the plasticity of TAMs, which are capable of promoting tumor progression and invasion while still retaining tumoricidal function in the presence of tumor-targeting mAbs.

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.

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.

Anti-integrin monoclonal antibody CNTO 95 enhances the therapeutic efficacy of fractionated radiation therapy in vivo.

Selective targeting of up-regulated integrins on tumor cells is a novel antiangiogenesis strategy for treating solid tumors. CNTO 95 is a fully human anti-alpha(v) integrin monoclonal antibody and has shown antitumor activity when used as a single agent in preclinical studies. We previously showed that radiation combined with an integrin alpha(v)beta(3) antagonist cRGD peptide increased the therapeutic efficacy of radiation in preclinical tumor models. We hypothesized that the combination of radiation and CNTO 95 would synergistically enhance the efficacy of radiation therapy. The in vitro studies showed that CNTO 95 radiosensitized and induced apoptosis in M21 cells in vitronectin-coated dishes. In mice bearing established human cancer xenograft tumors, CNTO 95 alone had only a moderate effect on tumor growth. The combined therapy of CNTO 95 and fractionated radiation significantly inhibited tumor growth and produced the longer tumor growth delay time in multiple tumor models. Maintenance dosing of CNTO 95 following irradiation contributed to efficacy and was important for continued inhibition of tumor regrowth. Immunohistochemistry studies showed that the combined use of CNTO 95 and radiation reduced the alpha(v) integrin and vascular endothelial growth factor receptor expression and the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. CNTO 95 alone and in combination with radiation did not produce any obvious signs of systemic toxicity. These results show that CNTO 95 can potentiate the efficacy of fractionated radiation therapy in a variety of human cancer xenograft tumor types in nude mice. These findings are very promising and may have high translational relevance for the treatment of patients with solid tumors.

Circulating human interleukin-8 as an indicator of cancer progression in a nude rat orthotopic human non-small cell lung carcinoma model.

Clinically relevant animal models of human cancer are necessary for the evaluation of putative therapeutics. We hypothesized that circulating human lung cancer-associated proteins would correlate with physiologic measurements from an orthotopic H460 human non-small cell lung carcinoma model that we developed in immunodeficient rats. Physiologic measurements and serum samples were collected over time. Serum interleukin-8 (IL-8), p53, vascular endothelial growth factor, and matrix metalloproteinase-9 were quantitated for correlation with physiologic measurements. Matrix metalloproteinase-9 and p53 were not significantly detectable. Circulating vascular endothelial growth factor was detected at high levels in some tumor-bearing animals. Human IL-8 was detectable in all tumor-bearing animals and correlated positively with markers of respiratory acidosis (pH, P = 0.012; TCO(2), P = 0.024; pCO(2), P = 0.007; and HCO(3)(-), P = 0.029) and with surface body temperature (P = 0.001) beginning on day 16 after implantation. IL-8 levels negatively correlated with survival (P < 0.001), indicating an association with tumor burden. Circulating human IL-8 might be a useful, clinically relevant circulating tumor protein marker due to its positive correlation with multiple physiologic variables associated with lung cancer progression.

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.

Phase I evaluation of a fully human anti-alphav integrin monoclonal antibody (CNTO 95) in patients with advanced solid tumors.

PURPOSE: A fully human monoclonal antibody to anti-alpha(v) 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 [(18)F]-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 alpha(v) 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.

Mcl-1 is regulated by IL-6 and mediates the survival activity of the cytokine in a model of late stage prostate carcinoma.

The proinflammatory cytokine interleukin-6 (IL-6) has been considered a positive growth factor in late stage prostate cancer (PC) cells and a potential target for therapeutic interference. We studied the effects of inhibition of IL-6 in LNCaP-IL6+ cells, a model system for advanced PC, which produce IL-6. By using the chimeric anti-IL-6 antibody, CNTO 328, we showed that the autocrine IL-6 loop is responsible for decreased sensitivity of LNCaP-IL-6+ cells to die by apoptosis. Dysregulation of Bcl-2 family members could be implicated in the acquisition of resistance to apoptosis in malignant cell lines. Myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of this family that is overexpressed in the IL-6 selected cells compared with control. Specific knock-down of Mcl-1 gene expression by siRNA yielded an increase in apoptosis of LNCaP-IL-6+ cells. Interestingly, inactivation of IL-6 autocrine loop was not able to increase apoptosis levels in the absence of Mcl-1, thus suggesting this molecule as a mediator of the survival action of IL-6. Finally, using selective kinase inhibitors we provide evidence for the involvement of p38 and ERK1/2 mitogen-activated protein kinases pathways in the IL-6-mediated regulation of Mcl-1. In conclusion, these data suggest that endogenous IL-6 acts as an antiapoptotic factor in LNCaP-IL-6+ cells and that Mcl-1 is critical for its survival activity. CNTO 328, in our experimental conditions, is able to render LNCaP-IL-6+ cells more sensitive to apoptosis. These data support the concept of anti-IL-6 therapy in human PC.

Inhibition of interleukin-6 with CNTO328, an anti-interleukin-6 monoclonal antibody, inhibits conversion of androgen-dependent prostate cancer to an androgen-independent phenotype in orchiectomized mice.

Initially, prostate cancer is androgen dependent. However, most cases progress to an androgen-independent state through unknown mechanisms. Interleukin-6 (IL-6) has been associated with prostate cancer progression including activation of the androgen receptor (AR). To determine if IL-6 plays a role in the conversion of prostate cancer from androgen dependent to androgen independent, we established androgen-dependent LuCaP 35 human prostate cancer xenografts in nude mice, castrated the mice, and blocked IL-6 activity using a neutralizing antibody (CNT0328) for a period of 18 weeks. IL-6 inhibition increased survival of mice and inhibited tumor growth, as reflected by decreased tumor volume and prostate-specific antigen levels, compared with that in mice receiving isotype control antibody. To test the effect of IL-6 inhibition on the conversion from androgen dependent to androgen independent, tumor cells from the treated mice were assessed for their androgen dependence both in vitro and by implanting them into sham-operated or orchiectomized mice. Tumor cells derived from the isotype-treated animals converted to androgen-independent state, whereas tumor cells from the anti-IL-6 antibody-treated mice were still androgen dependent in vitro and in vivo. Although there was no difference in AR levels between the androgen-independent and androgen-dependent tumors, IL-6 inhibition promoted both apoptosis and inhibited cell proliferation in tumors and blocked the orchiectomy-induced expression of histone acetylases, p300 and CBP, which are AR cofactors. These data show that IL-6 contributes to the development of androgen independence in prostate cancer and suggest that it mediates this effect, in part, through modulation of p300 and CBP.

Alpha-v integrins as therapeutic targets in oncology.

Integrins are heterodimeric cell adhesion receptors that mediate intercellular communication through cell-extracellular matrix interactions and cell-cell interactions. Integrins have been demonstrated to play a direct role in cancer progression, specifically in tumor cell survival, tumor angiogenesis, and metastasis. Therefore, agents targeted against integrin function have potential as effective anticancer therapies. Numerous anti-integrin agents, including monoclonal antibodies and small-molecule inhibitors, are in clinical development for the treatment of solid and hematologic tumors. This review focuses on the role of alpha(v) integrins in cancer progression, the current status of integrin-targeted agents in development, and strategies for the clinical development of anti-integrin therapies.

Absence of adverse effects in cynomolgus macaques treated with CNTO 95, a fully human anti-alphav integrin monoclonal antibody, despite widespread tissue binding.

PURPOSE: CNTO 95 is a fully human anti-alphav 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.

Matrix metalloproteinase activity, bone matrix turnover, and tumor cell proliferation in prostate cancer bone metastasis.

BACKGROUND: The metastasis of prostate cancer to bone is associated with a substantial increase in bone matrix turnover. Matrix metalloproteinases (MMPs) play roles in both normal bone remodeling and invasion and metastasis of prostate cancer. This study was designed to determine the role of MMP activity in prostate cancer that has metastasized to bone. METHODS: Single human fetal bone fragments were implanted subcutaneously in immunodeficient mice. Four weeks later, PC3 human prostate cancer cells were injected directly into some of the implants, and daily treatment was begun with batimastat (a broad-spectrum MMP inhibitor). There were six mice (i.e., six implants) in each of four experimental arms: bone alone with and without batimastat and bone injected with PC3 cells with and without batimastat. Bone implants were harvested after 14 days of treatment and analyzed for MMP expression, bone histomorphometry, osteoclast counts, blood vessel density, and tumor cell proliferation and apoptosis. Complementary data were obtained from bone biopsy samples from patients and a bone organ coculture system. All statistical tests were two-sided. RESULTS: MMPs were detected in tumor and stromal cells of clinical specimens and experimental bone implants. In vivo, MMP inhibition reduced the number of osteoclasts per millimeter in PC3-injected implants-from 8.2 (95% confidence interval [CI] = 7.9 to 8.5) to 3.0 (95% CI = 2.3 to 3.7) (P =.006). In addition, it prevented degradation of marrow trabeculae within the bone implants (cross-sectional area of implant occupied by mineralized trabeculae: untreated implant = 29.1% [95% CI = 27.1% to 31.1%], PC3-injected implant = 14.0% [95% CI = 10.9% to 17.1%] [P =.005 versus untreated], and batimastat-treated PC3-injected implant = 27.2% [95% CI = 22.4% to 32.0%] [P =.03 versus PC3 injected alone]). MMP inhibition reduced proliferating tumor cells from 20.8% (95% CI = 19.9% to 21.7%) to 7.4% (95% CI = 5.2% to 9.6%) (P =.006), without affecting angiogenesis or apoptosis. In vitro, MMP inhibition had no toxic effect on PC3 cells but prevented calcium release from bone fragments cocultured with PC3 cells. CONCLUSIONS: MMP activity appears to play an important role in bone matrix turnover when prostate cancer cells are present in bone. Bone matrix turnover and metastatic tumor growth appear to be involved in a mutually supportive cycle that is disrupted by MMP inhibition.

Inhibition of alpha(v)beta3 integrin reduces angiogenesis, bone turnover, and tumor cell proliferation in experimental prostate cancer bone metastases.

The growth of metastatic prostate cancer cells in the bone involves an intimate interaction between the tumor cells and various elements of the bone microenvironment, resulting in increased rate of bone turnover and rapid tumor growth. The alpha(v)beta3 integrin has been shown to play an important role in tumor growth and angiogenesis, and is known to be critical to osteoclast formation and activity. This study was designed to examine the role of alpha(v)beta3 expressed by cells native to the bone in the growth and pathogenesis of prostate cancer bone metastases. Human prostate cancer cells which do not express alpha(v)beta3 or alpha(IIb)beta3 integrins were injected directly into human bone fragments previously implanted subcutaneously in SCID mice (SCID-human-bone model). At the same time treatment with anti-beta3 antibody fragment (m7E3 F(ab')2) i.p. at 300 microg/dose 3 x per week was initiated and continued for 2 weeks. In this system, m7E3 F(ab')2 only recognizes human bone-derived alpha(v)beta3. Antibody inhibition of alpha(v)beta3 integrin in vivo resulted in a specific reduction in the proportion of antigenically-human blood vessels within tumor-bearing bone implants (from 73.5% +/- 3.93 in controls to 17.74% +/- 5.64 in treated animals). Proliferation of the alpha(v)beta3-negative tumor cells was also reduced, although the overall vessel density was maintained by compensating mouse vasculature. Blockage of human bone-derived alpha(v)beta3 also significantly reduced the recruitment of osteoclasts in response to tumor cells, as well as degradation of calcified bone tissue. Together these observations confirm the importance of alpha(v)beta3 in bone metabolism and angiogenesis, and point to the role of these processes in controlling growth of metastatic prostate cancer cells in the bone.

Cyclophosphamide creates a receptive microenvironment for prostate cancer skeletal metastasis.

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

JAK1 activates STAT3 activity in non-small-cell lung cancer cells and IL-6 neutralizing antibodies can suppress JAK1-STAT3 signaling.

Members of the signal transducer and activator of transcription (STAT) family of transcription factors are potential targets for the treatment and prevention of cancers including non-small-cell lung cancer. STAT proteins can be phosphorylated and activated by diverse upstream kinases including cytokine receptors and tyrosine kinases. We examined STAT protein activation in lung cancer cell lines including those with activating mutations in the EGFR and examined upstream kinases responsible for STAT3 phosphorylation and activation using small molecules, antibodies, and RNA interference. We found more pronounced STAT3 activation in cells with activating EGFR mutations, yet inhibition of EGFR activity had no effect on STAT3 activation. Inhibition of JAK1 with small molecules or RNA interference resulted in loss of STAT3 tyrosine phosphorylation and inhibition of cell growth. An interleukin-6 neutralizing antibody, siltuximab (CNTO 328) could inhibit STAT3 tyrosine phosphorylation in a cell-dependent manner. Siltuximab could completely inhibit STAT3 tyrosine phosphorylation in H1650 cells, and this resulted in inhibition of lung cancer cell growth in vivo. Combined EGFR inhibition with erlotinib and siltuximab resulted in dual inhibition of both tyrosine and serine STAT3 phosphorylation, more pronounced inhibition of STAT3 transcriptional activity, and translated into combined effects on lung cancer growth in a mouse model. Our results suggest that JAK1 is responsible for STAT3 activation in lung cancer cells and that indirect attacks on JAK1-STAT3 using an IL-6 neutralizing antibody with or without EGFR inhibition can inhibit lung cancer growth in lung cancer subsets.

Down-regulation of suppressor of cytokine signaling-3 causes prostate cancer cell death through activation of the extrinsic and intrinsic apoptosis pathways.

Suppressor of cytokine signaling-3 (SOCS-3) acts as a negative feedback regulator of the Janus-activated kinase/signal transducers and activators of transcription factors signaling pathway and plays an important role in the development and progression of various cancers. To better understand the role of SOCS-3 in prostate cancer, SOCS-3 expression was down-regulated in DU-145, LNCaP-IL-6+, and PC3 cells by consecutive SOCS-3 small interfering RNA transfections. SOCS-3 mRNA and protein expression as measured by quantitative reverse transcription-PCR and Western blot, respectively, were decreased by approximately 70% to 80% compared with controls. We observed a significant decrease in cell proliferation and viability in all SOCS-3-positive cell lines but not in the parental LNCaP cell line, which is SOCS-3 negative. In this study, we show that down-regulation of SOCS-3 leads to an increased cell death in prostate cancer cell lines. We found a considerable increase in the activation of the proapoptotic caspase-3/caspase-7, caspase-8, and caspase-9. A significant up-regulation of cleaved poly(ADP-ribose) polymerase and inhibition of Bcl-2 expression was observed in all SOCS-3-positive cell lines. Overexpression of Bcl-2 could rescue cells with decreased SOCS-3 levels from going into apoptosis. Tissue microarray data prove that SOCS-3 is highly expressed in castration-refractory tumor samples. In conclusion, we show that SOCS-3 is an important protein in the survival machinery in prostate cancer and is overexpressed in castration-resistant tumors. SOCS-3 knockdown results in an increase of cell death via activation of the extrinsic and intrinsic apoptosis pathways.

Regulation of growth of prostate cancer cells selected in the presence of interleukin-6 by the anti-interleukin-6 antibody CNTO 328.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional regulator of cellular events in prostate cancer. LNCaP-IL-6+ cells selected in the presence of IL-6 were taken for assessment of effects of the chimeric monoclonal anti-IL-6 antibody CNTO 328. METHODS: Cell viability was assessed after treatment with CNTO 328 by the ATP assay. Expression of Bcl-2 and Bax and activation of signaling pathways were evaluated by Western analysis. Nude mice were inoculated with LNCaP-IL-6+ cells and treated with CNTO 328. The tumors were analyzed by immunohistochemistry for expression of Ki-67, tissue transglutaminase, and vascular endothelial growth factor. RESULTS: CNTO 328 caused a statistically significant inhibition of cell viability. The protein levels of Bcl-2 and the phosphorylation of ERK1/2 mitogen-activated protein kinases were decreased by the anti-IL-6 antibody. Treatment with CNTO 328 yielded an increase in the phosphorylation of signal transducers and activators of transcription factor 3. The mean tumor volume in animals inoculated with LNCaP-IL-6+ cells and treated with CNTO 328 was insignificantly lower than that in animals treated with the control antibody. There was a statistically significant decrease in the percentage of Ki-67-positive cells in CNTO 328-treated tumors. CONCLUSION: CNTO 328 has a potential in prostate cancer therapy and could be further tested in various combination experimental treatments.

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 (K(d) 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.

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

Integrins of the alphav family, such as alphavbeta3 and alphavbeta5, 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 alphav family of integrins and is likely to be less immunogenic in humans compared to chimeric or humanized antibodies. CNTO 95 bound to purified alphavbeta3 and alphavbeta5 with a Kd of approximately 200 pM and to alphav 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 alphavbeta3 and alphavbeta5 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 alphav integrins that has potent antitumor and antiangiogenic properties in in vivo preclinical models.