CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

Many immunosuppressive drugs are associated with an increased risk of neoplasia, principally non-melanoma skin cancers and B-cell lymphomas. However, only 6 of the 13 immunosuppressive drugs tested in 2 year bioassays increased the incidence of neoplasia. For example, the 2-year bioassays conducted with cyclosporine (CSA), an International Agency for Research on Cancer (IARC) Group 1 human carcinogen, were negative. The purpose of these investigations was to use transplanted tumor models in immunocompetent, syngeneic mice to gain insight into the failure of the 2-year bioassay to show an increased incidence of neoplasia with CSA. C3H HeN mice were used in a battery of assays with a transplanted squamous cell carcinoma (SCC VII cells) or a B-cell, lymphoma (38C13 cells) cells to study effects of CSA on local growth and metastases, experimental metastases, and progression of established metastases. Mice received CSA twice weekly by subcutaneous (SC) injection at doses of 0.5, 5, or 50 mg/kg; controls received the CSA vehicle. CSA had a modest inhibitory effect on SC tumors initiated by 38C13 cells and on intramuscular tumors initiated by SCC VII cells. CSA also decreased the number of lung colonies and decreased the size, growth fraction and vascularity of established lung metastases initiated by SCC VII cells. In contrast, CSA increased progressive growth of metastases to the sentinel lymph node from an intramuscular SCC VII tumor, but had no effect cellular traffic to the node. In conclusion, CSA at doses up to 50 mg/kg did not facilitate tumor progression and it partially inhibited tumor growth, suggesting that suppression of tumor progression may partially explain the failure of CSA to act as a carcinogen in 2 year bioassays.

Non‐selective immunosuppressive drugs are associated with an increased risk of neoplasia. Although selective immunosuppressants, e.g., monoclonal antibodies, will likely pose less of a hazard, concerns remain about an increased risk of neoplasia, particularly squamous cell carcinoma. The 2 year bioassay has been the industry standard for evaluating carcinogenicity. However, it has poor predictive value for immunosuppressive drugs, accurately identifying a hazard with only 5 of the 13 drugs tested. Thus, there is need for an alternative testing strategy. We have developed a novel 3‐stage model to study tumor promotion and progression in vivo. We chose a murine squamous skin cell carcinoma (SCC VII) cell line to study local invasion and dissemination, colonization, and tumor progression in C3H‐HeN, SCID and SCID/bg mice in the presence and absence of cyclosporin A. Local invasion and dissemination were evaluated by following emigration of quantum dot labeled tumor cells to the draining lymph node. Lung colony counts were made after iv injection of tumor cells and angiogenesis and growth fraction were evaluated morphometrically. Immunosuppression, was associated with increased local growth and lung colonization but with decreased angiogenesis and growth rates. These results suggest that a series of models described here may offer a viable alternative to the 2 year bioassay.

INTRODUCTION:Immunosuppressive drugs are associated with an increased risk of infections and in some cases neoplasia, particularly non-melanoma skin cancers. This paper describes the development of a model to test the effects of immunosuppressive drugs on local invasion and metastases of a squamous cell carcinoma in syngeneic, immunocompetent mice. METHODS:SCC VII cells were labeled with 655 quantum dots (QDs), injected intramuscularly into C3H HEN mice and traffic and progressive growth in the draining popliteal lymph node were evaluated. RESULTS:SCC VII cells express RAE-1, an NKG2D ligand, and were sensitive to natural killer (NK) cells in vitro. QDs were stable in SCC VII cells and showed no evidence of toxicity to the cells. In vivo, confocal microscopy showed that QD-labeled SCC VII cells could migrate to the draining node and microfluorimetry showed progressive traffic of QDs to the node. There was no evidence of systemic toxicity of QDs. Primary immunosuppression in SCID and SCID-beige mice and treatment of normal mice with immunosuppressive agents (anti-asialoGM1 and cyclophosphamide) can enhance traffic of QDs and/or metastases to the draining lymph node. In contrast, cyclosporine had no effect on traffic or metastases. CONCLUSION:This model of local invasion and metastases may be useful in immunotoxicology for identifying and characterizing the hazard posed by selective immunosuppressive drugs.

Tumor histopathology is an important method to understand the mechanism of action of anti‐neoplastic therapies and immunotherapeutics in animal models. An experimental metastasis model was established with the MDA‐MB‐231 human breast adenocarcinoma cell line in SCID beige mice. Compared to subcutaneous and orthotopic models, this model allows evaluation of multiple lesions at earlier time points. Mice were injected i.v. with MDA‐MB‐231 cells and sacrificed either 14 or 21 days post tumor cell injection. Lungs were processed for histology and stained with H&E. Multiple focal lesions were observed and these lesions had a random distribution in the lung and were variable in size. The lesions increased in size and number over time. These observations were confirmed by a rank order analysis on disease severity, and morphometry on size of the lesions. Morphometry also revealed a significant increase in proliferating cells within lung lesions at Day 21 compared to Day 14. Numerous infiltrating macrophages could also be identified within the lesions. No evidence of angiogenesis within the lesions was observed in this time course. It is hypothesized that the lesions studied have not yet crossed the angiogenic switch and therefore no new blood vessel formation was observed. Lesions can be further characterized by additional histologic methods.

Abstract CCL2, (CC-chemokine ligand 2 or monocyte chemoattractant protein-1 (MCP-1)), is overexpressed in many human tumors and is believed to exert pro-tumor effects by recruiting monocytes to the tumor, where these cells become tumor associated macrophages (TAMs). TAMs secrete growth factors that stimulate angiogenesis and tumor growth, as well as proteases to promote tumor invasion and metastasis. CCL2 expression levels in primary breast tumors have been correlated with macrophage infiltration and blood vessel density, which in turn is correlated with disease stage and prognosis. These correlations indicate that CCL2 is a key player in tumor macrophage infiltration and/or tumor growth/invasion, and suggest that neutralizing CCL2 could be an effective form of therapy for breast cancer patients.The objective of these studies was to investigate whether CCL2 blockade could inhibit tumor growth in mice bearing human breast tumors. The human breast tumor cell lines MDA-MB-231 (ER-, PR-, Her2-) and MDA-MB-361 (ER+, PR+, Her2+) were implanted orthotopically in immunocompromised mice, and in both models the primary tumors metastasized to lungs and brain. Neutralizing antibodies to human CCL2 (CNTO 888) and to the mouse orthologs, MCP-1 and MCP-5, were administered therapeutically, either as a cocktail (termed CCL2 blockade) or individually to study the relative roles of host vs tumor derived CCL2 in promoting tumor growth.In both tumor models, CCL2 blockade significantly inhibited the growth of established primary tumors in the mammary fat pad. In addition, CCL2 blockade inhibited metastasis to distant sites. As measured by Taqman, visual inspection and immunohistochemistry, mice with MDA-MB-361 tumors treated with CCL2 blockade showed significantly reduced metastasis to lungs and brain, while mice bearing MDA-MB-231 tumors showed significantly reduced metastasis to lungs.To define the relative roles of human tumor-derived CCL2 vs mouse host-derived MCP-1/MCP-5, in vivo monotherapy tumor studies were conducted using the individual neutralizing antibodies. These studies included the mammary fat pad model and a tail vein metastasis model. In both cases, only the treatment with the anti-mouse MCP-1 antibody significantly inhibited primary tumor growth and distant metastasis, indistinguishable from the effect of CCL2 blockade treatment. In the tail vein metastasis model, the antibody treatment resulted in significantly fewer detectable lesions with these lesions showing a significant reduction in both tumor size and growth fraction, suggesting antibody treatment inhibits tumor seeding and growth. Mechanistic studies are in progress to further understand the basis of the anti-tumor effect mediated by the antibody treatment. These results demonstrate that host-derived MCP-1, produced from the tumor microenvironment, plays the critical role in tumor growth and metastasis in these models of human breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6095.

BACKGROUND:The immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases.METHODS:TLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C).RESULTS:There was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFalpha were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy.CONCLUSION:These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.