A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv).

Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.

Interleukin 1/Toll-like receptor-induced autophosphorylation activates interleukin 1 receptor-associated kinase 4 and controls cytokine induction in a cell type-specific manner.

IRAK4 is a central kinase in innate immunity, but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown, and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues Thr-342, Thr-345, and Ser-346 is required for full activity, and the death domain regulates the activation of IRAK4. Using antibodies against activated IRAK4, we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and Toll-like receptor agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, although complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-κB, or MAPK activation, there is complete ablation of these processes in IRAK4-deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848-induced IL-6 production with minimal effect on NF-κB or MAPK activation. Taken together, these studies define the mechanism of IRAK4 activation and highlight the differential role of IRAK4 kinase activity in different human cell types as well as the distinct roles IRAK4 scaffolding and kinase functions play.

Fc-GDF15 glyco-engineering and receptor binding affinity optimization for body weight regulation.

GDF15 is a distant TGF-ß family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.

Discovery and optimization of a novel anti-GUCY2c x CD3 bispecific antibody for the treatment of solid tumors.

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

H6PDH interacts directly with 11beta-HSD1: implications for determining the directionality of glucocorticoid catalysis.

Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11beta-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system. Interestingly, both full-length H6PDH and the truncated NTD are secreted into the culture medium in the absence of 11beta-HSD1. Purified full-length H6PDH is a bi-functional enzyme with glucose-6-phosphate dehydrogenase (G6PDH) activity as well as 6-phosphogluconolactonase (6PGL) activity. Using co-immunoprecipitation experiments with purified H6PDH and 11beta-HSD1, and with cell lysates expressing H6PDH and 11beta-HSD1, we observe direct physical interaction between the two enzymes. We also show the modulation of 11beta-HSD1 directionality by H6PDH using overexpression and siRNA knockdown systems. The NTD retains the ability to interact with 11beta-HSD1 physically as well as modulate 11beta-HSD1 directionality indicating that the NTD of H6PDH is sufficient for the regulation of the 11beta-HSD1 activity.

Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

Methods for the differential integrative omic analysis of plasma from a transgenic disease animal model.

Multitiered quantitative analysis of biological systems is rapidly becoming the desired approach to study hierarchical functional interactions between proteins and metabolites. We describe here a novel systematic approach to analyze organisms with complex metabolic regulatory networks. By using precise analytical methods to measure biochemical constituents and their relative abundance in whole plasma of transgenic ApoE*3-Leiden mice and an isogenic wild-type control group, simultaneous snapshots of metabolic and protein states were obtained. Novel data processing and multivariate analysis tools such as Impurity Resolution Software (IMPRESS) and Windows-based linear fit program (WINLIN) were used to compare protein and metabolic profiles in parallel. Canonical correlations of the resulting data show quantitative relationships between heterogeneous components in the TG animals. These results, obtained solely from whole plasma analysis allowed us, in a rapid manner, to corroborate previous findings as well as find new events pertaining to dominant and peripheral events in lipoprotein metabolism of a genetically modified mammalian organism in relation to ApoE3, a key mediator of lipoprotein metabolism.

Integrative biological analysis of the APOE*3-leiden transgenic mouse.

Integrative (or systems biology) is a new approach to analyzing biological entities as integrated systems of genetic, genomic, protein, metabolite, cellular, and pathway events that are in flux and interdependent. Here, we demonstrate the application of intregrative biological analysis to a mammalian disease model, the apolipoprotein E3-Leiden (APO*E3) transgenic mouse. Mice selected for the study were fed a normal chow diet and sacrificed at 9 weeks of age-conditions under which they develop only mild type I and II atherosclerotic lesions. Hepatic mRNA expression analysis showed a 25% decrease in APO A1 and a 43% increase in liver fatty acid binding protein expression between transgenic and wild type control mice, while there was no change in PPAR-alpha expression. On-line high performance liquid chromatography-mass spectrometry quantitative profiling of tryptic digests of soluble liver proteins and liver lipids, coupled with principle component analysis, enabled rapid identification of early protein and metabolite markers of disease pathology. These included a 44% increase in L-FABP in transgenic animals compared to controls, as well as an increase in triglycerides and select bioactive lysophosphatidylcholine species. A correlation analysis of identified genes, proteins, and lipids was used to construct an interaction network. Taken together, these results indicate that integrative biology is a powerful tool for rapid identification of early markers and key components of pathophysiologic processes, and constitute the first application of this approach to a mammalian system.

Novel synthesis and structural characterization of a high-affinity paramagnetic kinase probe for the identification of non-ATP site binders by nuclear magnetic resonance.

To aid in the pursuit of selective kinase inhibitors, we have developed a unique ATP site binder tool for the detection of binders outside the ATP site by nuclear magnetic resonance (NMR). We report here the novel synthesis that led to this paramagnetic spin-labeled pyrazolopyrimidine probe (1), which exhibits nanomolar inhibitory activity against multiple kinases. We demonstrate the application of this probe by performing NMR binding experiments with Lck and Src kinases and utilize it to detect the binding of two compounds proximal to the ATP site. The complex structure of the probe with Lck is also presented, revealing how the probe fits in the ATP site and the specific interactions it has with the protein. We believe that this spin-labeled probe is a valuable tool that holds broad applicability in a screen for non-ATP site binders.

Investigation of leucine-rich repeat kinase 2 : enzymological properties and novel assays.

Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity,may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used,for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2.Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (Lantha-ScreenTM) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRETassays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation,G2019S, results in a robust enhancement of kinase activity with LRRKtideas the substrate. This mutation also affects binding of ATP to LRRK2,with wild-type binding being tighter (Km,app of 57 lm) than with theG2019S mutant (Km,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.

Mass spectrometry-compatible silver staining.

INTRODUCTIONThe ammoniacal silver staining method is one of the most sensitive methods used to detect proteins on an SDS-PAGE gel. However, this and other standard silver staining methods are not compatible with mass spectrometry (MS), which is fast becoming the best way to identify proteins isolated on 2D gels. Because the proteins in gels to be analyzed by mass spectroscopy cannot be modified, many of the common sensitizing agents (e.g., glutaraldehyde and strong oxidizing agents) cannot be used. This method is compatible with MALDI and ESI-MS, and it shows an increased ability to deal with semipreparative protein loads without negative staining as compared with other silver staining methods. However, this process is less sensitive than standard silver staining methods.

Staining membrane-bound proteins with coomassie blue r250.

INTRODUCTIONCoomassie Blue R250 permanently stains membrane-bound proteins and is compatible with PVDF and nitrocellulose membranes, but it is incompatible with nylon membranes. This technique is relatively insensitive, with a detection limit of ~1.5 µg of protein. One drawback of Coomassie Blue staining is that it produces a high background that can make interpretation of results difficult.

Staining membrane-bound proteins with ponceau s.

INTRODUCTIONBecause Ponceau S is relatively insensitive (~1 µg of protein), only the most abundant proteins will be visible. However, it is a reversible stain that can be removed completely with H(2)O prior to processing the blots. After staining, a soft lead pencil can be used to record the presence of visible proteins and molecular-weight markers, which will help when aligning the proteins detected on the membrane by western analysis with those in a total protein-stained gel or membrane. Ponceau S is compatible with both nitrocellulose and PVDF membranes.

Staining membrane-bound proteins with colloidal gold.

INTRODUCTIONColloidal Gold is the most sensitive staining technique for proteins bound on membranes, detecting as little as 1-3 ng of protein. Protein spots are permanently stained a dark red after incubation with the Colloidal Gold solution. Colloidal Gold staining can detect proteins on both nitrocellulose and PVDF membranes, but it is not recommended for nylon membranes.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: SDS-PAGE of Proteins.

INTRODUCTIONFollowing first-dimension IEF and equilibration of the IPG gel strips, the proteins are separated on the basis of their molecular weight in the second dimension on an SDS-PAGE gel. Systems for this separation are available from a variety of suppliers and are commonly found in many protein chemistry laboratories. This protocol describes a method for placement of the IPG strip and gives some recommended electrophoresis conditions for these second-dimension gels.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins.

INTRODUCTIONThis protocol describes a method for rehydration of IPG gel strips in preparation for their use for isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. Following rehydration, IEF can be performed using either a flatbed unit or a self-contained instrument.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Isoelectric Focusing of Proteins in a Multipurpose Flatbed Electrophoresis Unit.

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. This method serves as the first dimension of the 2D separation. The method described in this protocol utilizes a flatbed unit; however, self-contained instruments for IEF are also available.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IEF of Proteins in an IEF-Dedicated Electrophoresis Unit.

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels, providing the first dimension of the 2D separation. In this protocol, the IPG gels are focused using self-contained instruments for IEF. These high-voltage systems allow fewer manipulations of the IPG gels, resulting in less error, strip mix-up, contamination, air contact, or urea crystallization. Because rehydration and IEF can be performed consecutively within a single unit, these two steps can be performed unattended overnight. Finally, faster separations and sharper focusing are possible due to the higher voltage available in these instruments.

Phosphoprotein Staining with the GelCode Phosphoprotein Staining Kit.

INTRODUCTIONThe phosphorylation state of a protein has an important role in the regulation of a wide variety of cellular processes. As a result, there has been a great deal of interest in detecting phosphorylated proteins. The method presented here uses the GelCode phosphoprotein staining kit (Pierce Chemical Company). This method depends on the hydrolysis of the phosphoprotein phosphoester linkage using sodium hydroxide in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-molybdate complex is stained with Methyl Green. After destaining, the phosphoproteins are colored green to green-blue. The detection limit is in the nanogram range, but depends on the degree of phosphorylation of the protein. This method will detect the phosphoproteins phosvitin and ß-casein in the 40-80 ng/band and 80-160 ng/band range, respectively. The method presented here is for staining minigels. Volumes will need to be increased for larger gels.

Preparation of Vertical SDS Slab Gels: Casting a Single Homogeneous Gel.

INTRODUCTIONFollowing the separation of proteins by IEF, the second dimension is carried out by SDS-PAGE. This protocol details the method for casting single homogeneous SDS-PAGE gels. Homogeneous gels (with the same %T and %C throughout) offer the best resolution for a particular molecular-weight range and are commonly used because they are the easiest to pour reproducibly. The second-dimension gels can be conveniently prepared in three different formats (i.e., sizes): minigels, for use with 7-cm IEF first-dimension gels; standard gels, for use with 11-, 13-, and 18-cm IEF first-dimension gels; and large-format gels, for use with 18- and 24-cm IEF first-dimension gels. All of the gels use a common set of reagents, listed below, but differ slightly in the equipment required.