Teresa L. Born

A Poxvirus Protein That Binds to and Inactivates IL-18, and Inhibits NK Cell Response

IL-18 induces IFN-γ and NK cell cytotoxicity, making it a logical target for viral antagonism of host defense. We demonstrate that the ectromelia poxvirus p13 protein, bearing homology to the mammalian IL-18 binding protein, binds IL-18, and inhibits its activity in vitro. Binding of IL-18 to the viral p13 protein was compared with binding to the cellular IL-18R. The dissociation constant of p13 for murine IL-18 is 5 nM, compared with 0.2 nM for the cellular receptor heterodimer. Mice infected with a p13 deletion mutant of ectromelia virus had elevated cytotoxicity for YAC-1 tumor cell targets compared with control animals. Additionally, the p13 deletion mutant virus exhibited decreased levels of infectivity. Our data suggest that inactivation of IL-18, and subsequent impairment of NK cell cytotoxicity, may be one mechanism by which ectromelia evades the host immune response.

Native IL-32 is released from intestinal epithelial cells via a non-classical secretory pathway as a membrane-associated protein

Although IL-32 has been shown to be induced under various pathological conditions, a detailed understanding of native IL-32 intracellular distribution and mechanism of release from cells has not been reported. We examined the expression of IL-32 in the intestinal epithelial cell line HT-29 following TNFα and IFNγ co-stimulation. The subcellular localization of induced IL-32 was associated with the membrane of lipid droplet-like structures and vacuolar structures that co-localized with markers of endosomes and lysosomes. Prolonged co-stimulation resulted in cell death and appearance of IL-32 in the culture medium. IL-32 released from co-stimulated HT-29 cells was found in a detergent-sensitive particulate fraction, and in a step density gradient the IL-32 particulate was buoyant, suggesting association with a membrane-bound vesicle. Upon Triton X-114 partitioning, most of the IL-32 partitioned to the detergent phase, suggesting hydrophobic characteristics. When IL-32-containing vesicles were subjected to protease K treatment, a protease resistant ∼12kDa fragment was generated from ∼24kDa IL-32. We propose that under these conditions, native IL-32 is released via a non-classical secretory route perhaps involving multi-vesicular bodies and exosomes. Demonstration of membrane association for both intracellular and released IL-32 suggests this unique cytokine may have a complex biosynthetic pathway and mechanism of action.

Tumor necrosis factor, tumor necrosis factor inhibition, and cancer risk.

Abstract Objective: Tumor necrosis factor (TNF) is a highly pleiotropic cytokine with multiple activities other than its originally discovered role of tumor necrosis in rodents. TNF is now understood to play a contextual role in driving either tumor elimination or promotion. Using both animal and human data, this review examines the role of TNF in cancer development and the effect of TNF and TNF inhibitors (TNFis) on malignancy risk. Research design: A literature review was performed using relevant search terms for TNF and malignancy. Results: Although administration of TNF can cause tumor regression in specific rodent tumor models, human expression polymorphisms suggest that TNF can be a tumor-promoting cytokine, whereas blocking the TNF pathway in a variety of tumor models inhibits tumor growth. In addition to direct effects of TNF on tumors, TNF can variously affect immunity and the tumor microenvironment. Whereas TNF can promote immune surveillance designed to eliminate tumors, it can also drive chronic inflammation, autoimmunity, angiogenesis, and other processes that promote tumor initiation, growth, and spread. Key players in TNF signaling that shape this response include NF-κB and JNK, and malignant-inflammatory cell interactions, each of which may have different responses to TNF signaling. Focusing on rheumatoid arthritis (RA) patients, where clinical experience is most extensive, a review of the clinical literature shows no increased risk of overall malignancy or solid tumors such as breast and lung cancers with exposure to TNFis. Lymphoma rates are not increased with use of TNFis. Conflicting data exist regarding the risks of melanoma and nonmelanoma skin cancer. Data regarding the risk of recurrent malignancy are limited. Conclusions: Overall, the available data indicate that elevated TNF is a risk factor for cancer, whereas its inhibition in RA patients is not generally associated with an increased cancer risk. In particular, TNF inhibition is not associated with cancers linked to immune suppression. A better understanding of the tumor microenvironment, molecular events underlying specific tumors, and epidemiologic studies of malignancies within specific disease indications should enable more focused pharmacovigilance studies and a better understanding of the potential risks of TNFis.

Demonstration of Functional Similarity of Proposed Biosimilar ABP 501 to Adalimumab.

BackgroundDue to the complex molecular structure and proprietary manufacturing processes of monoclonal antibodies (mAbs), differences in structure and function may be expected during development of biosimilar mAbs. Important regulatory requirements for approval of biosimilar products involve comprehensive assessments of any potential differences between proposed biosimilars and reference mAbs, including differences in all known mechanisms of action, using sensitive and relevant methods. Any identified structural differences should not result in differences in biofunctional or clinical activity.ObjectiveA comprehensive assessment comparing the Amgen biosimilar candidate ABPxa0501 with FDA-licensed adalimumab (adalimumab [US]) and EU-authorized adalimumab (adalimumab [EU]) was conducted to demonstrate similarity in biofunctional activity.MethodsThe functional similarity assessment included testing of binding kinetics to soluble tumor necrosis factor α (TNFα) and relative binding to transmembrane TNFα. The neutralization of TNFα-induced caspase activation, TNFα- and lymphotoxin-α (LTα)-induced chemokine production, and cytotoxicity was also tested. Binding to Fc-gamma receptors FcγRIa, FcγRIIa (131H), FcγRIIIa (158V and 158F), and neonatal Fc receptor (FcRn) was compared with the reference mAbs, as was antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity.ResultsThe data demonstrate that ABPxa0501 is similar to both adalimumab (US) and adalimumab (EU) with respect to evaluated biofunctional activities.ConclusionSimilarity in biofunctional activity is a critical component of the totality of evidence required for demonstration of biosimilarity. The functional similarity demonstrated for ABPxa0501 comprehensively assesses the known mechanisms of action of adalimumab, supporting the conclusion that ABPxa0501, adalimumab (US), and adalimumab (EU) are likely to be clinically similar.

Developing the Totality of Evidence for Biosimilars: Regulatory Considerations and Building Confidence for the Healthcare Community

Biosimilars are highly similar versions of approved branded biologics. Unlike generics, they are not exact replicas of reference products. Minor differences between biosimilars and reference products in some aspects are expected; likewise, biosimilar products will differ from each other. The objective of this review is to discuss the challenges associated with the development and approval of biosimilar products that are unique because of their complex structure and specialized manufacturing processes, which can impact not only efficacy but also immunogenicity and safety. Regulatory guidelines recommend a totality-of-evidence approach focused on stepwise development that involves demonstration of structural similarity and functional equivalence. Structural and functional characteristics of the proposed biosimilar are compared with the reference product; similarity of these functions forms the foundation of the biosimilar development program, including potential animal studies, a human pharmacokinetics/pharmacodynamics equivalence study, and a clinical study to confirm similar efficacy, safety, and immunogenicity. The clinical study should be performed in a sensitive population using appropriate endpoints to allow detection of any clinically meaningful differences between the biosimilar and the reference product if such differences exist. In conclusion, development of biosimilars is focused on the minimization of potential differences between the proposed biosimilar and reference product and the establishment of a robust manufacturing process to consistently produce a high-quality biosimilar product.

489PFUNCTIONAL SIMILARITY ASSESSMENT RESULTS COMPARING BEVACIZUMAB TO BIOSIMILAR CANDIDATE ABP 215

ABSTRACT Aim: ABP 215 is being developed as a biosimilar to bevacizumab, a recombinant monoclonal antibody that binds vascular endothelial growth factor (VEGF) and inhibits binding to receptors. Although bevacizumab and intended biosimilars share the same amino acid sequence, differences will likely exist in product quality attributes due to differences in expression systems, bioprocess and purification. Equivalence of product quality attributes, especially functional equivalence, is of primary importance during stepwise development of a biosimilar in order to provide confidence for similar clinical safety and efficacy in patients. Functional equivalence to bevacizumab is also a critical consideration when considering extrapolation of indications for the candidate biosimilar product. The aim of these studies is to examine functional simiarlity of ABP 215 to bevacizumab. Methods: Comparative assessment of biological activity included testing binding of ABP 215 and bevacizumab to VEGF and VEGF isoforms by surface plasmon resonance and to cell-surface expressed FcRn and to FcgRIIIa by AlphaLISA. The inhibition of proliferation and VEGFR2 autophosphorylation was compared in human umbilical vein endothelial cells. Anti-tumor activity was compared in A431 and Colo205 tumor xenograft models. Results: Equilibrium binding affinity (Kd) to VEGF was similar between ABP 215 (117 pM) and bevacizumab (112 pM). Binding to VEGF165 and VEGF121 was also similar. Binding (3 lots each) to FcRn was similar between ABP 215 (95-102%) and bevacizumab (114-127%) as was binding to FcgRIIIa comparing ABP 215 (100-115%) to bevacizumab (100-105%). Potency in proliferation inhibition was similar across three lots each of bevacizumab (87-98%) compared to ABP 215 (92-101%). Inhibition of autophosphorylation (IC50) was similar for a single lot each of ABP 215 (0.0871 µg/ml) compared to bevacizumab (0.0858 µg/ml). The effects of ABP 215 and bevacizumab were also similar in vivo, in terms of inhibition of both tumor growth and tumor-associated vasculature in A431 and Colo205 models. Conclusions: ABP 215 appears to be highly similar to bevacizumab in multiple sensitive preclinical pharmacologic assessments. Disclosure: T.L. Born: author is a stockholder and emplyoee of of Amgen; Q. H uynh, A. Mathur, J. Velayudhan, J. Canon, K. Reynhardt, T.J. Goletz and R.A. Markus: Amgen employee and stockholder