Amy Volk

Anti-TNF-α antibody allows healing of joint damage in polyarthritic transgenic mice

Anti-tumor-necrosis-factor-α (TNF-α) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-α (hTNF-α) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-α antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-α treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-α prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-α and local expression of various proinflammatory mediators were all diminished by anti-TNF-α treatment, confirming a critical role of hTNF-α in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-α treatment was observed in young mice but not in aged mice.

Critical review of preclinical approaches to evaluate the potential of immunosuppressive drugs to influence human neoplasia.

Many immunosuppressive drugs are associated with an increased risk of B-cell lymphoma, squamous cell carcinoma, and Kaposi sarcoma. Thirteen immunosuppressive drugs have been tested in 2-year carcinogenicity studies (abatacept; azathioprine; busulfan; cyclophosphamide; cyclosporine; dexamethasone; everolimus; leflunomide; methotrexate; mycophenolate mofetil; prednisone; sirolimus; and tacrolimus) and in additional models including neonatal and genetically modified mice; chemical, viral, ultraviolet, and ionizing radiation co-carcinogenesis, and in models with transplanted tumor cells. The purpose of this review is to outline the mechanisms by which immunosuppressive drugs can influence neoplasia, to summarize the available preclinical data on the 13 drugs, and to critically review the performance of the models. A combination of primary tumor and metastasis assays conducted with transplanted cells may provide the highest value for hazard identification and can be applied on a case-by-case basis. However, for both small molecules and therapeutic proteins, determining the relative risk to patients from preclinical data remains problematic. Classifying immunosuppressive drugs based on their mechanism of action and hazard identification from preclinical studies and a prospective pharmacovigilance program to monitor carcinogenic risk may be a feasible way to manage patient safety during the clinical development program and postmarketing.

CNTO 530 functions as a potent EPO mimetic via unique sustained effects on bone marrow proerythroblast pools.

Anemia as associated with numerous clinical conditions can be debilitating, but frequently can be treated via administration of epoetin-alfa, darbepoietin-alfa, or methoxy-PEG epoetin-beta. Despite the complexity of EPO-EPO receptor interactions, the development of interesting EPO mimetic peptides (EMPs) also has been possible. CNTO 530 is one such novel MIMETIBODY Fc-domain dimeric EMP fusion protein. In a mouse model, single-dose CNTO 530 (unlike epoetin-alfa or darbepoietin-alfa) bolstered red cell production for up to 1 month. In 5-fluorouracil and carboplatin-paclitaxel models, CNTO 530 also protected against anemia with unique efficiency. These actions were not fully accounted for by half-life estimates, and CNTO 530 signaling events therefore were studied. Within primary bone marrow erythroblasts, kinetics of STAT5, ERK, and AKT activation were similar for CNTO 530 and epoetin-alfa. p70S6K activation by CNTO 530, however, was selectively sustained. In vivo, CNTO 530 uniquely stimulated the enhanced formation of PODXL(high)CD71(high) (pro)erythroblasts at frequencies multifold above epoetin-alfa or darbepoietin-alfa. CNTO 530 moreover supported the sustained expansion of a bone marrow-resident Kit(neg)CD71(high)Ter119(neg) progenitor pool. Based on these distinct erythropoietic and EPOR signaling properties, CNTO 530 holds excellent promise as a new EPO mimetic.

Immunotoxicologic effects of cyclosporine on tumor progression in models of squamous cell carcinoma and B-cell lymphoma in C3H mice

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.

Differential Effects of Long-lived Erythropoietin Receptor Agonists in Rats

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. Here, we report on experiments designed to study how the pharmacokinetic profiles of EPO receptor agonists, ranging from the short-lived epoetin-? to the long-lived EPO-MIMETIBODYTM constructs CNTO 530 and CNTO 531, influence the pharmacodynamic response in rats. Rats received a single dose of an EPO-R agonist and the effects on reticulocytes, red blood cells and hemoglobin were measured over time. The increase in red blood cells and hemoglobin were negatively correlated with clearance. At doses that cause a similar effect on reticulocytes, very long-lived EPO-R agonists caused prolonged production of red blood cells. In conclusion, we have shown that very long-lived EPO-R agonists cause prolonged production of red blood cells and increase in hemoglobin that is independent of their in vitro potency or the peak release of reticulocytes. These data suggest that EPO may be a survival factor for reticulocytes.

Development of a murine model of lymph node metastases suitable for immunotoxicity studies.

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.

Tissue Factor (TF) Expression and Angiogenesis in Tumor Progression and Inhibition of Tumor Growth by Anti-TF Antibodies in Human TissueFactor Knock-In Mice

Background: Tissue factor (TF) serves as the primary initiator of the extrinsic pathway of blood coagulation and mediates signaling via the protease activated receptor-2 (PAR2). TF is over-expressed in several tumor types and may facilitate tumor progression and angiogenesis. To test the hypothesis that inhibition of TF may have an anti-tumor effect, we induced pulmonary adenomas (PA) in human TF knock in (huTF-KI) mice with urethane and studied the relationship between expression of TF and mutations in K-ras with tumor progression and the effect of a monoclonal anti-TF antibody on growth of a transplantable lymphoma. Methods: huTF-KI mice received 10 weekly intraperitoneal (i.p.) injections of urethane and samples of lung were collected at six week intervals between weeks 10 and 28. Expression of TF and von Willibrand factor (vWF) in PA were studied by immunohistochemistry (IHC) and mutations in K-ras were studied by laser capture microdissection and polymerase chain reaction (LCM-PCR). Results: IHC showed that expression of TF and vWF increased as pulmonary epithelial hyperplasias and PA progressed. Dual staining TF and vWF showed that areas of high expression of TF correlated with the tumor vasculature and LCM-PCR showed that mutation of K-ras correlated with expression of TF and the angiogenic switch in PA. Finally, an anti-huTF monoclonal antibody slowed the growth of transplantable urethane-induced lymphomas. Conclusion: Taken together, these data suggest that expression of TF plays an important role in tumor progression and the angiogenic switch. These data also suggest that anti-TF antibodies may be a viable tumor immunotherapy

Development of a squamous cell carcinoma mouse model for immunotoxicity testing

Abstract An important component of safety assessment of new pharmaceuticals is evaluation of their potential to increase the risk of developing cancer in humans. The traditional 2-year rodent bioassay often is not feasible or scientifically applicable for evaluation of biotherapeutics. Additionally, it has poor predictive value for non-genotoxic immunosuppressive compounds. Thus, there is a need for alternative testing strategies. A novel 3-stage tumor model in syngeneic C3H/HeN mice was evaluated here to study the effects of immunosuppressive drugs on tumor promotion and progression in vivo. The model employed a skin squamous cell carcinoma cell line (SCC VII) due to the increased prevalence of squamous cell carcinoma (SCC) in humans associated with immunosuppression after transplants. Local invasion, colonization and tumor progression were evaluated. The validation set of immunosuppressive drugs included: Cyclosporin (CSA), cyclophosphamide (CTX), azathioprine, etanercept, abatacept and prednisone. Local invasion was evaluated by histological assessment as well as fluorescence trafficking from Qdot®-labeled tumor cells from the site of inoculation to the draining lymph node. Colonization was evaluated by lung colony counts following intravenous inoculation. Tumor progression was assessed by morphometric analysis of lesion area, angiogenesis and growth fraction of established metastatic neoplasia. Immunosuppressive drugs in the validation set yielded mixed results, including decreased progression. The methods and results described herein using an in vivo syngeneic mouse tumor model can provide insight about the assessment of immunosuppressive drugs in carcinogenicity risk assessment.

Murine gammaherpesvirus-68 (MHV-68) is not horizontally transmitted amongst laboratory mice by cage contact

Abstract Murine gammaherpesvirus-68 (MHV-68), a natural pathogen of mice, is being evaluated as a model of Epstein Barr Virus (EBV) infection for use in investigation of the effects of immunomodulatory therapy on herpesvirus pathogenesis in humans. Immunosuppressive agents are used for treatment of a variety of autoimmune diseases as well as for prevention of tissue rejection after organ transplantation and can result in recrudescence of latent herpesvirus infections. Prior to examination of MHV-68 as a suitable model for EBV, better characterization of the MHV-68 model was desirable. Characterization of the MHV-68 model involved development of assays for detecting virus and for demonstration of safety when present in murine colonies. Limited information is available in the literature regarding MHV-68 transmission, although recent reports indicate the virus is not horizontally spread in research facilities. To further determine transmission potential, immunocompetent and immunodeficient mice were infected with MHV-68 and co-habitated with naïve animals. Molecular pathology assays were developed to characterize the MHV-68 model and to determine viral transmission. Horizontal transmission of virus was not observed from infected animals to naïve cagemates after fluorescence microscopy assays and quantitative PCR (qPCR). Serologic analysis complemented these studies and was used as a method of monitoring infection amongst murine colonies. Overall, these findings demonstrate that MHV-68 infection can be controlled and monitored in murine research facilities, and the potential for unintentional infection is low.

Abstract B58: Early safety assessment of single and combination therapy using TDAR

The discovery of targeted therapies against tumors offers promise of therapeutic benefit to patients however, it poses challenges in assessing immunologic and toxicologic risk during drug development. We determined that early safety assessment of combination therapy, prior to later stage toxicology studies, can be used to support Go/No-Go NME decision and thereby reduce time, efficiency, and resources used to choose and develop lead candidates. The rodent T-cell-dependent antibody response (TDAR) assay is used to assess the effect of candidate therapeutic agents on the immune system by measuring primary and secondary IgM and IgG antibody responses to exogenous antigen challenge. TDAR responses require intact function of multiple immune cells including antigen presenting cells and T and B lymphocytes, as well as a cytokine-dependent isotype class switch from IgM to IgG, resulting in production of an antigen-specific antibody response. Alterations in the amount of antibody produced therefore can reflect effects on any or all cell populations involved in TDAR. TDAR is commonly used in preclinical drug development especially where increased cause for concern exists (ICH guideline S8). Development of combination therapy that engages multiple targets impacting the immune system poses unique opportunity for increased efficacy but also unique risk for increased immunotoxicity including immune stimulation. For this work, a mouse TDAR evaluating primary and secondary KLH antibody responses in a KLH-Specific IgM and IgG sandwich enzyme-linked immunosorbent assay (ELISA) was first validated and then used to assess immunotoxicologic potential of multiple single immunosuppressive agents (cyclophosphamide (CTX), abatacept, azathioprine (AZT), etanercept, cyclosporine (CsA), and prednisone) and biological therapies (A, B, C, D, E, and F) and combination biologic therapies ( A+B, C+D, E+F). Doses of 100 mg/kg CsA, 250 mg/kg abatacept, 125 mg/kg etanercept, 100 mg/kg AZT, 20 mg/kg prednisone administered subcutaneously (s.c.) on Days 1 and 3 had mild immunosuppressive effects. 200 mg/kg of CTX administered s.c. had an expected robust immunosuppressive effect that was statistically significant than control. Combination of biologic therapies did not result in enhanced TDAR immunotoxicity compared to single biologic therapy alone for the molecules evaluated. Tier 1 immunotoxicology assessments similar to those in standard toxicity studies were added to the TDAR assessment. Together, these data support the use of the TDAR assay for early safety assessment of potential combination therapies against tumors. Citation Format: Amy L. Volk, Mindi Walker, Kerry Brosnan, Dorie Capaldi, Patricia Rafferty, Daniel Weinstock, Eva Emmell. Early safety assessment of single and combination therapy using TDAR. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B58.