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ABP 798 is a biosimilar to Rituxan® (rituximab reference product [RP]). Non-clinical assessments relevant to the primary and secondary mechanisms of action (MOA) contribute to the totality of the evidence (TOE) in supporting biosimilarity and are critical in providing scientific evidence for extrapolation of indications. Similarity of ABP 798 with rituximab RP was investigated across a range of biological activities which have potential impact on pharmacokinetics and clinical efficacy with non-clinical assessments relevant to MOA such as CD20 internalization, trogocytosis, binding to primary human natural killer (NK) cells as well as the ability to induce antibody-dependent cellular phagocytosis (ADCP) in peripheral blood mononuclear cells. Additionally, in vitro synergy of ABP 798 or RP with chemotherapeutic agents, in vivo xenograft studies in mice, and toxicological assessments in cynomolgus monkeys (including B cell depletion and toxicokinetics) were also conducted. Results from these non-clinical assessments contribute to the TOE supporting the biosimilarity between ABP 798 and rituximab RP across a range of primary and secondary MOAs and support justification for extrapolation to all indications of use for ABP 798 for which the RP is approved.

ABP 215 (MVASI™) is the first approved biosimilar to Avastin® (bevacizumab). It is approved in the USA and the European Union (EU) for all bevacizumab indications in these jurisdictions except for ovarian cancer in the USA due to orphan drug exclusivity. ABP 215 was shown to be structurally, functionally and clinically (pharmacokinetic, efficacy and safety) similar to the bevacizumab reference product; the pharmacokinetic study was conducted in healthy adult men (n = 202); safety and efficacy were evaluated in patients with advanced nonsquamous non-small-cell lung cancer (n = 642). Together, these results comprise the totality of evidence that provides scientific justification for extrapolation to all approved indications of the reference product and supports the clinical value of ABP 215 as an additional treatment option.

Antibody therapeutics with poor solubility in the subcutaneous matrix may carry unintended risks when administered to patients. The objective of this work was to estimate the risk of antibodies that precipitate in vitro at neutral pH by determining the impact of poor solubility on distribution of the drug from the injection site as well as immunogenicity in vivo. Using fluorescence imaging in a mouse model, we show that one such precipitation-prone antibody is retained at the injection site in the subcutaneous space longer than a control antibody. In addition, we demonstrate that retention at the injection site through aggregation is concentration-dependent and leads to macrophage association and germinal center localization. Although there was delayed disposition of the aggregated antibody to draining lymph nodes, no overall impact on the immune response in lymph nodes, systemic exposure of the antibody, or enhancement of the anti-drug antibody response was evident. Unexpectedly, retention of the precipitated antibody in the subcutaneous space delayed the onset of the immune response and led to an immune suppressive response. Thus, we conclude that precipitation due to poor solubility of high doses of antibody formulations delivered subcutaneously may not be of special concern in terms of exposure or immunogenicity.

Background ABP 980 has been developed as a biosimilar to Herceptin® (trastuzumab). Comprehensive analytical characterization incorporating orthogonal analytical techniques was used to compare ABP 980 to trastuzumab reference products sourced from the United States (US) and the European Union (EU). Methods Physicochemical property comparisons included the following: primary structure related to amino acid sequence and post-translational modifications, including glycans; higher-order structure; product-related substances and impurities, including size and charge variants; subvisible and submicron particles, and protein content. In addition, functional similarity was assessed for Fab-mediated, Fc-mediated, and combined Fab- and Fc-mediated activities. Results ABP 980 has the same amino acid sequence as and similar post-translational modification profiles to trastuzumab (US) and trastuzumab (EU). Importantly, ABP 980 was found to be highly similar to trastuzumab for all functional activities related to the mechanism(s) of action. Higher-order structure, product-related substances and impurities, particles and aggregates were also highly similar between ABP 980 and trastuzumab. Where minor differences were noted, they were evaluated and found unlikely to impact clinical performance. The totality of evidence, including the pharmacokinetic clinical similarity of ABP 980, further supports that ABP 980 is highly similar to trastuzumab. Conclusion Based on the comprehensive analytical similarity assessment, ABP 980 is analytically highly similar to the reference product, trastuzumab.

ABP 215 is a biosimilar product to bevacizumab. Bevacizumab acts by binding to vascular endothelial growth factor A, inhibiting endothelial cell proliferation and new blood vessel formation, thereby leading to tumor vasculature normalization. The ABP 215 analytical similarity assessment was designed to assess the structural and functional similarity of ABP 215 and bevacizumab sourced from both the United States (US) and the European Union (EU). Similarity assessment was also made between the US- and EU-sourced bevacizumab to assess the similarity between the two products. The physicochemical properties and structural similarity of ABP 215 and bevacizumab were characterized using sensitive state-of-the-art analytical techniques capable of detecting small differences in product attributes. ABP 215 has the same amino acid sequence and exhibits similar post-translational modification profiles compared to bevacizumab. The functional similarity assessment employed orthogonal assays designed to interrogate all expected biological activities, including those known to affect the mechanisms of action for ABP 215 and bevacizumab. More than 20 batches of bevacizumab (US) and bevacizumab (EU), and 13 batches of ABP 215 representing unique drug substance lots were assessed for similarity. The large dataset allows meaningful comparisons and garners confidence in the overall conclusion for the analytical similarity assessment of ABP 215 to both US- and EU-sourced bevacizumab. The structural and purity attributes, and biological properties of ABP 215 are demonstrated to be highly similar to those of bevacizumab.

The function of CD4+ T cells is dependent on Ca2+ influx through Ca2+ release–activated Ca2+ (CRAC) channels formed by ORAI proteins. To investigate the role of ORAI1 in proinflammatory Th1 and Th17 cells and autoimmune diseases, we genetically and pharmacologically modulated ORAI1 function. Immunization of mice lacking Orai1 in T cells with MOG peptide resulted in attenuated severity of experimental autoimmune encephalomyelitis (EAE). The numbers of T cells and innate immune cells in the CNS of ORAI1-deficient animals were strongly reduced along with almost completely abolished production of IL-17A, IFN-γ, and GM-CSF despite only partially reduced Ca2+ influx. In Th1 and Th17 cells differentiated in vitro, ORAI1 was required for cytokine production but not the expression of Th1- and Th17-specific transcription factors T-bet and RORγt. The differentiation and function of induced regulatory T cells, by contrast, was independent of ORAI1. Importantly, induced genetic deletion of Orai1 in adoptively transferred, MOG-specific T cells was able to halt EAE progression after disease onset. Likewise, treatment of wild-type mice with a selective CRAC channel inhibitor after EAE onset ameliorated disease. Genetic deletion of Orai1 and pharmacological ORAI1 inhibition reduced the leukocyte numbers in the CNS and attenuated Th1/Th17 cell-mediated cytokine production. In human CD4+ T cells, CRAC channel inhibition reduced the expression of IL-17A, IFN-γ, and other cytokines in a dose-dependent manner. Taken together, these findings support the conclusion that Th1 and Th17 cell function is particularly dependent on CRAC channels, which could be exploited as a therapeutic approach to T cell–mediated autoimmune diseases.

The Ash1 protein is a daughter cell-specific repressor of HO gene transcription in Saccharomyces cerevisiae. Both ASH1 mRNA and protein are localized to the incipient daughter cell at the end of mitosis; Ash1 then inhibits HO transcription in the daughter cell after cytokinesis. Mother cells, in contrast, contain little or no Ash1 and thus are able to transcribe HO. We show that deletion of PHO85, which encodes a cyclin-dependent protein kinase, causes reduced transcription of HO and that this reduction is dependent on ASH1. In pho85 mutants, Ash1 protein is no longer asymmetrically localized and is present, instead, in both mother and daughter cells. Initially, it appears to be localized properly but then persists as daughter cells mature into mother cells. In contrast, ASH1 mRNA is localized appropriately to daughter cells in pho85 mutants. We observe that Ash1 protein is phosphorylated by Pho85 in vitro and that Ash1 stability increases in a pho85 mutant. These data suggest that phosphorylation of Ash1 by Pho85 governs stability of Ash1 protein.

Pho85 is a cyclin-dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression. Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known. When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28-dependent cyclins, Cln1 and Cln2. To identify potential targets of Pcl2-Pho85, we performed a two-hybrid screen using the Pcl2 cyclin as bait and recovered the transcription factor Swi5 as a Pcl2-interacting protein. We performed both biochemical and genetic tests to discover the biological significance of the interaction between Pcl2 and Swi5 seen in the two-hybrid assay. We found that Swi5 interacts in vitro with Pho85 cyclins and is phosphorylated in vitro by the Pho80-Pho85 kinase. We discovered that a subset of genes that are controlled by Swi5 and a homologous transcription factor, Ace2, was misregulated in a pho85 deletion strain; expression of the ASH1 and CTS1 genes was reduced in an ace2 deletion strain, whereas expression of both genes was increased in an ace2Delta pho85Delta double mutant. We also found that overexpression of SWI5 caused cell lethality in a pho85 deletion strain. Our results are consistent with misregulation of Swi5 activity in vivo in the absence of Pho85 and implicate Swi5 as a potential substrate of Pho85 cyclin-dependent kinase complexes.

There is a need for methods to improve the diagnosis, patient staging and evaluation of therapeutic response in patients with autoimmune conditions to improve patient care. Inflammatory bowel disease (IBD) and rheumatoid arthritis (RA) are two inflammatory diseases characterized by involvement of innate and adaptive immune components that change the metabolic state of their respective target tissues, thus providing an opportunity for molecular imaging probes to detect such changes. Optimally, such probes and the imaging methods employed would be non-invasive, robust and reproducible, give a quantitative result, report on the status of the affected tissue(s) and respond to the effects of a therapeutic molecule. Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are nuclear imaging approaches that have the potential to satisfy such requirements. In this review, the work to date and the potential of PET and SPECT imaging probes in these two inflammatory conditions, IBD and RA, are discussed.

Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is necessary to first obtain a complete picture of the spatiotemporal expression of the Wnt signaling factors with respect to the different tissue layers of the gut. Here, we offer an in depth in situ gene expression study of Wnt ligands, frizzled receptors, and frizzled related modulators over several days of chicken gut development. These data show some expected locations of Wnt signaling as well as a surprising lack of expression of factors in the hindgut. This paper describes the first comprehensive characterization of the dynamic expression of Wnt signaling molecules during gut development. These data form the basis for future studies to determine the role of Wnt signaling in the developing gastrointestinal tract.