An adapted consensus protein design strategy for identifying globally optimal biotherapeutics.

Biotherapeutic optimization, whether to improve general properties or to engineer specific attributes, is a time-consuming process with uncertain outcomes. Conversely, Consensus Protein Design has been shown to be a viable approach to enhance protein stability while retaining function. In adapting this method for a more limited number of protein sequences, we studied 21 consensus single-point variants from eight publicly available CD3 binding sequences with high similarity but diverse biophysical and pharmacological properties. All single-point consensus variants retained CD3 binding and performed similarly in cell-based functional assays. Using Ridge regression analysis, we identified the variants and sequence positions with overall beneficial effects on developability attributes of the CD3 binders. A second round of sequence generation that combined these substitutions into a single molecule yielded a unique CD3 binder with globally optimized developability attributes. In this first application to therapeutic antibodies, adapted Consensus Protein Design was found to be highly beneficial within lead optimization, conserving resources and minimizing iterations. Future implementations of this general strategy may help accelerate drug discovery and improve success rates in bringing novel biotherapeutics to market.

Optimization of a transient antibody expression platform towards high titer and efficiency.

Transient gene expression (TGE) using mammalian cells is an extensively used technology for the production of antibodies and recombinant proteins and has been widely adopted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major workhorses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used, and post-transfection culture conditions, we arrived at an uniquely optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We further investigated the amount of coding DNA used in TGE and the influence of kinetics and size of the transfection complex on the in vitro efficiency of the transfection. We present here the first report of an optimized TGE platform using Filler DNA in an early drug discovery setting for the screening and production of therapeutic mAbs.

The oxidase activity of vascular adhesion protein-1 (VAP-1) is essential for function.

Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

Bioluminescent method for assaying multiple semicarbazide-sensitive amine oxidase (SSAO) family members in both 96- and 384-well formats.

Vascular adhesion protein-1 (VAP-1), also known as semicarbazide-sensitive amine oxidase (SSAO) or copper-containing amine oxidase (AOC3, EC 1.4.3.6), catalyzes oxidative deamination of primary amines. One endogenous substrate has recently been described (Siglec 10), and although its mechanism of action in vivo is not completely understood, it is suggested to play a role in immune cell trafficking, making it a target of interest for autoimmune and inflammatory diseases. Much of the enzymology performed around this target has been conducted with absorbance, fluorescent, or radiometric formats that can have some limitations for high-throughput screening and subsequent compound profiling. The authors present the use of a bioluminescent assay, originally developed for monoamine oxidase enzymes, in a high-throughput format. It can be used for related SSAOs such as AOC1 given their substrate similarity with VAP-1. The authors also demonstrate that it is compatible with different sources of VAP-1, both purified recombinant and VAP-1 overexpressed on live cells.

Agonist versus antagonist induce distinct thermodynamic modes of co-factor binding to the glucocorticoid receptor.

The glucocorticoid receptor (GR) is involved in the transcriptional regulation of genes associated with inflammation, glucose homeostasis, and bone turnover through the association with ligands, such as corticosteroids. GR-mediated gene transcription is regulated or fine-tuned via the recruitment of co-factors including coactivators and corepressors. Current therapeutic approaches to targeting GR aim to retain the beneficial anti-inflammatory activity of the corticosteroids while eliminating negative side effects. Towards achieving this goal the experiments discussed here reveal a mechanism of co-factor binding in the presence of either bound agonist or antagonist. The GR ligand binding domain (GR-LBD(F602S)), in the presence of agonist or antagonist, utilizes different modes of binding for coactivator versus corepressor. Coactivator binding to the co-effector binding pocket of GR-LBD(F602S) is driven both by favorable enthalpic and entropic interactions whereas corepressor binding to the same pocket is entropically driven. These data support the hypothesis that ligand-induced conformational changes dictate co-factor binding and subsequent trans-activation or trans-repression.

Development of a high-throughput assay to measure histidine decarboxylase activity.

Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.

Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase.

Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1-313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14-313) by thrombin digestion during purification. The Pim-1 (14-313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 A resolution and belong to space group P6(5), with unit-cell parameters a = b = 95.9, c = 80.0 A, beta = 120 degrees and one molecule per asymmetric unit.

Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase.

Pim-1 kinase is a member of a distinct class of serine/threonine kinases consisting of Pim-1, Pim-2, and Pim-3. Pim kinases are highly homologous to one another and share a unique consensus hinge region sequence, ER-PXPX, with its two proline residues separated by a non-conserved residue, but they (Pim kinases) have <30% sequence identity with other kinases. Pim-1 has been implicated in both cytokine-induced signal transduction and the development of lymphoid malignancies. We have determined the crystal structures of apo Pim-1 kinase and its AMP-PNP (5'-adenylyl-beta,gamma-imidodiphosphate) complex to 2.1-angstroms resolutions. The structures reveal the following. 1) The kinase adopts a constitutively active conformation, and extensive hydrophobic and hydrogen bond interactions between the activation loop and the catalytic loop might be the structural basis for maintaining such a conformation. 2) The hinge region has a novel architecture and hydrogen-bonding pattern, which not only expand the ATP pocket but also serve to establish unambiguously the alignment of the Pim-1 hinge region with that of other kinases. 3) The binding mode of AMP-PNP to Pim-1 kinase is unique and does not involve a critical hinge region hydrogen bond interaction. Analysis of the reported Pim-1 kinase-domain structures leads to a hypothesis as to how Pim kinase activity might be regulated in vivo.