Centyrin ligands for extrahepatic delivery of siRNA.

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

Bioanalytical workflow for novel scaffold protein-drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin-drug conjugate in plasma and tissue samples using liquid chromatography-tandem mass spectrometry.

AIM: Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates. METHODOLOGY: Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed. Tryptic peptides generated from a region of the Centyrin that does not contain a conjugation site, and another that has the conjugation site with the linker-payload attached were used as surrogates of the total and conjugated Centyrin, respectively. CONCLUSION: The methods were successfully applied to analysis of samples from mice to quantify the plasma and tissue concentrations. This same workflow can potentially be applied to other PDCs and site-specific antibody-drug conjugates.

LC/MS/MS Bioanalysis of Protein-Drug Conjugates-The Importance of Incorporating Succinimide Hydrolysis Products.

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity. Existing approaches to quantify the conjugated species of ADCs involve a ligand binding assay (LBA) for conjugated antibody or hybrid LBA/liquid chromatography/tandem mass spectrometry (LC/MS/MS) for quantitation of conjugated drug. In our current work for a protein-drug conjugate (PDC) using the Centyrin scaffold, a similar concept to ADCs but with smaller protein size, an alternative method to quantify the conjugate by using a surrogate peptide approach, was utilized. The His-tagged proteins were isolated from biological samples using immobilized metal affinity chromatography (IMAC), followed by trypsin digestion. The tryptic peptide containing the linker attached to the payload was used as a surrogate of the conjugate and monitored by LC/MS/MS analysis. During method development and its application, we found that hydrolysis of the succinimide ring of the linker was ubiquitous, taking place at many stages during the lifetime of the PDC including in the initial drug product, in vivo in circulation in the animals, and ex vivo during the trypsin digestion step of the sample preparation. We have shown that hydrolysis during trypsin digestion is concentration-independent and consistent during the work flow-therefore, having no impact on assay performance. However, for samples that have undergone extensive hydrolysis prior to trypsin digestion, significant bias could be introduced if only the non-hydrolyzed form is considered in the quantitation. Therefore, it is important to incorporate succinimide hydrolysis products in the quantitation method in order to provide an accurate estimation of the total conjugate level. More importantly, the LC/MS/MS-based method described here provides a useful tool to quantitatively evaluate succinimide hydrolysis of ADCs in vivo, which has been previously reported to have significant impact on their stability, exposure, and efficacy.

Evaluation of a Centyrin-Based Near-Infrared Probe for Fluorescence-Guided Surgery of Epidermal Growth Factor Receptor Positive Tumors.

Tumor-targeted near-infrared fluorescent dyes have the potential to improve cancer surgery by enabling surgeons to locate and resect more malignant lesions where good visualization tools are required to ensure complete removal of malignant tissue. Although the tumor-targeted fluorescent dyes used in humans to date have been either small organic molecules or high molecular weight antibodies, low molecular weight protein scaffolds have attracted significant attention because they penetrate solid tumors almost as efficiently as small molecules, but can be infinitely mutated to bind almost any antigen. Here we describe the use of a 10 kDa protein scaffold, a Centyrin, to target a near-infrared fluorescent dye to tumors that overexpress the epidermal growth factor receptor (EGFR) for fluorescence-guided surgery (FGS). We have developed and optimized the dose and time required for imaging small tumor burdens with minimal background fluorescence in real-time fluorescence-guided surgery of EGFR-expressing tumor xenografts in murine models. We demonstrate that the Centyrin-near-infrared dye conjugate (CNDC) binds selectively to human EGFR+ cancer cells with an EC50 of 2 nM, localizes to EGFR+ tumor xenografts in athymic nude mice and that uptake of the dye in xenografts is significantly reduced when EGFR are blocked by preinjection of excess unlabeled Centyrin. Taken together, these data suggest that CNDCs can be used for intraoperative identification and surgical removal of EGFR-expressing lesions and that Centyrins targeted to other tumor-specific antigens should prove similarly useful in fluorescence guided surgery of cancer. In addition, we demonstrate that the CNDC is detected in the NIR region of the spectrum and can be utilized for fluorescence-guided surgery (FGS). In addition, we propose that with its eventual complete clearance from EGFR-negative tissues and its quantitative retention in the tumor mass for >24 h, a Centyrin-targeted NIR dye should provide excellent tumor contrast when injected at least 6-8 h before initiation of cancer surgery in human patients.

Conformational flexibility of an anti-IL-13 DARPin†.

Designed ankyrin repeat proteins (DARPin®) are artificial non-immunoglobulin binding proteins with potential applications as therapeutic molecules. DARPin 6G9 binds interleukin-13 with high affinity and blocks the signaling pathway and as such is promising for the treatment of asthma and other atopic diseases. The crystal structures of DARPin 6G9 in the unbound form and in complex with IL-13 were determined at high resolution. The DARPin competes for the same epitope as the IL-13 receptor chain 13Rα1 but does not interfere with the binding of the other receptor chain, IL-4Rα. Analysis of multiple copies of the DARPin molecule in the crystal indicates the conformational instability in the N-terminal cap that was predicted from molecular dynamics simulations. Comparison of the DARPin structures in the free state and in complex with IL-13 reveals a concerted movement of the ankyrin repeats upon binding resulted in the opening of the binding site. The induced-fit mode of binding employed by DARPin 6G9 is very unusual for DARPins since they were designed as particularly stable and rigid molecules. This finding shows that DARPins can operate by various binding mechanisms and suggests that some flexibility in the scaffold may be an advantage.

Engineering a targeted delivery platform using Centyrins.

Targeted delivery of therapeutic payloads to specific tissues and cell types is an important component of modern pharmaceutical development. Antibodies or other scaffold proteins can provide the cellular address for delivering a covalently linked therapeutic via specific binding to cell-surface receptors. Optimization of the conjugation site on the targeting protein, linker chemistry and intracellular trafficking pathways can all influence the efficiency of delivery and potency of the drug candidate. In this study, we describe a comprehensive engineering experiment for an EGFR binding Centyrin, a highly stable fibronectin type III (FN3) domain, wherein all possible single-cysteine replacements were evaluated for expression, purification, conjugation efficiency, retention of target binding, biophysical properties and delivery of a cytotoxic small molecule payload. Overall, 26 of the 94 positions were identified as ideal for cysteine modification, conjugation and drug delivery. Conjugation-tolerant positions were mapped onto a crystal structure of the Centyrin, providing a structural context for interpretation of the mutagenesis experiment and providing a foundation for a Centyrin-targeted delivery platform.

Fusion to a highly stable consensus albumin binding domain allows for tunable pharmacokinetics.

A number of classes of proteins have been engineered for high stability using consensus sequence design methods. Here we describe the engineering of a novel albumin binding domain (ABD) three-helix bundle protein. The resulting engineered ABD molecule, called ABDCon, is expressed at high levels in the soluble fraction of Escherichia coli and is highly stable, with a melting temperature of 81.5°C. ABDCon binds human, monkey and mouse serum albumins with affinity as high as 61 pM. The solution structure of ABDCon is consistent with the three-helix bundle design and epitope mapping studies enabled a precise definition of the albumin binding interface. Fusion of a 10 kDa scaffold protein to ABDCon results in a long terminal half-life of 60 h in mice and 182 h in cynomolgus monkeys. To explore the link between albumin affinity and in vivo exposure, mutations were designed at the albumin binding interface of ABDCon yielding variants that span an 11 000-fold range in affinity. The PK properties of five such variants were determined in mice in order to demonstrate the tunable nature of serum half-life, exposure and clearance with variations in albumin binding affinity.

Induced conformational change in human IL-4 upon binding of a signal-neutralizing DARPin.

The crystal structure of DARPin 44C12V5 that neutralizes IL-4 signaling has been determined alone and bound to human IL-4. A significant conformational change occurs in the IL-4 upon DARPin binding. The DARPin binds to the face of IL-4 formed by the A and C α-helices. The structure of the DARPin remains virtually unchanged. The conformational changes in IL-4 include a reorientation of the C-helix Trp91 side chain and repositioning of CD-loop residue Leu96. Both side chains move by >9 Å, becoming buried in the central hydrophobic region of the IL-4:DARPin interface. This hydrophobic region is surrounded by a ring of hydrophilic interactions comprised of hydrogen bonds and salt bridges and represents a classical "hotspot." The structures also reveal how the DARPin neutralizes IL-4 signaling. Comparing the IL-4:DARPin complex structure with the structures of IL-4 bound to its receptors (Hage et al., Cell 1999; 97, 271-281; La Porte et al., Cell 2008, 132, 259-272), it is found that the DARPin binds to the same IL-4 face that interacts with the junction of the D1 and D2 domains of the IL-4Rα receptors. Signaling is blocked since IL-4 cannot bind to this receptor, which it must do first before initiating a productive receptor complex with either the IL-13α1 or the γc receptor.

Selection of high-affinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions.

Alternative scaffold molecules represent a class of proteins important to the study of protein design and mechanisms of protein-protein interactions, as well as for the development of therapeutic proteins. Here, we describe the generation of a library built upon the framework of a consensus FN3 domain sequence resulting in binding proteins we call Centyrins. This new library employs diversified positions within the C-strand, CD-loop, F-strand and FG-loop of the FN3 domain. CIS display was used to select high-affinity Centyrin variants against three targets; c-MET, murine IL-17A and rat TNFα and scanning mutagenesis studies were used to define the positions of the library most important for target binding. Contributions from both the strand and loop positions were noted, although the pattern was different for each molecule. In addition, an affinity maturation scheme is described that resulted in a significant improvement in the affinity of one selected Centyrin variant. Together, this work provides important data contributing to our understanding of potential FN3 binding interfaces and a new tool for generating high-affinity scaffold molecules.

N-terminal ß-strand swapping in a consensus-derived alternative scaffold driven by stabilizing hydrophobic interactions.

The crystal structure of an N-terminal ß-strand-swapped consensus-derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N-terminal ß-strand swapping, leading to the formation of a stable compact dimer.

C-terminal ß-strand swapping in a consensus-derived fibronectin Type III scaffold.

The crystal structures of six different fibronectin Type III consensus-derived Tencon domains, whose solution properties exhibit no, to various degrees of, aggregation according to SEC, have been determined. The structures of the five variants showing aggregation reveal 3D domain swapped dimers. In all five cases, the swapping involves the C-terminal ß-strand resulting in the formation of Tencon dimers in which the target-binding surface is blocked. All of the variants differ in sequence in the FG loop, which is the hinge loop in the ß-strand-swapped dimers. The six tencon variants have between 0 and 5 residues inserted between positions 77 and 78 in the FG loop. Analysis of the structures suggests that a non-glycine residue at position 77 and insertions of <4 residues may destabilize the ß-turn in the FG loop promoting ß-strand swapping. Swapped dimers with an odd number of inserted residues may be less stable, particularly if they contain proline residues, because they cannot form perfect ß-bridges in the FG regions that link the swapped dimers. The Tencon ß-swapped variants with the longest FG sequences are observed to form higher order hexameric or helical oligomeric structures in the crystal correlating well with the aggregation properties of these domains observed in solution. Understanding the structural basis for domain-swapped dimerization and oligomerization will support engineering efforts of the Tencon domain to produce variants with desired biophysical properties.

Engineering peptide therapeutics using MIMETIBODY™ technology.

The MIMETIBODY™ platform was developed to expand the opportunities for application of biotherapeutics. While the utility of antibodies as antagonists has been well demonstrated, their application as agonists has been more challenging. For steric reasons, antibodies may be less well suited to perform as agonists or as inhibitors of GPCRs. In contrast, many bioactive peptides function as agonists or by interaction with GPCRs but their development as therapeutics has been challenging due to their small size and metabolic lability. The MIMETIBODY™ platform has been used to develop a variety of stable, long-lived molecules with intrinsic activities similar to that of their parent peptides. This chapter describes methods for construction of expression plasmids, expression and purification strategies, and methods for characterizing the activity of these novel proteins.

Inhalation delivery of protein therapeutics.

Inhaled therapeutics are used routinely to treat a variety of pulmonary diseases including asthma, COPD and cystic fibrosis. In addition, biological therapies represent the fastest growing segment of approved pharmaceuticals. However, despite the increased availability of biological therapies, nearly all inhaled therapeutics are small molecule drugs with only a single inhaled protein therapeutic approved. There remains a significant unmet need for therapeutics in pulmonary diseases, and biological therapies with potential to alter disease progression represent a significant opportunity to treat these challenging diseases. This review provides a background into efforts to develop inhaled biological therapies and highlights some of the associated challenges. In addition, we speculate on the ideal properties of a biologic therapy for inhaled delivery.

GLP-1 receptor activation inhibits VLDL production and reverses hepatic steatosis by decreasing hepatic lipogenesis in high-fat-fed APOE*3-Leiden mice.

OBJECTIVE: In addition to improve glucose intolerance, recent studies suggest that glucagon-like peptide-1 (GLP-1) receptor agonism also decreases triglyceride (TG) levels. The aim of this study was to evaluate the effect of GLP-1 receptor agonism on very-low-density lipoprotein (VLDL)-TG production and liver TG metabolism. EXPERIMENTAL APPROACH: The GLP-1 peptide analogues CNTO3649 and exendin-4 were continuously administered subcutaneously to high fat diet-fed APOE*3-Leiden transgenic mice. After 4 weeks, hepatic VLDL production, lipid content, and expression profiles of selected genes involved in lipid metabolism were determined. RESULTS: CNTO3649 and exendin-4 reduced fasting plasma glucose (up to -30% and -28% respectively) and insulin (-43% and -65% respectively). In addition, these agents reduced VLDL-TG production (-36% and -54% respectively) and VLDL-apoB production (-36% and -43% respectively), indicating reduced production of VLDL particles rather than reduced lipidation of apoB. Moreover, they markedly decreased hepatic content of TG (-39% and -55% respectively), cholesterol (-30% and -55% respectively), and phospholipids (-23% and -36% respectively), accompanied by down-regulation of expression of genes involved in hepatic lipogenesis (Srebp-1c, Fasn, Dgat1) and apoB synthesis (Apob). CONCLUSION: GLP-1 receptor agonism reduces VLDL production and hepatic steatosis in addition to an improvement of glycemic control. These data suggest that GLP-receptor agonists could reduce hepatic steatosis and ameliorate dyslipidemia in patients with type 2 diabetes mellitus.

Mechanisms of self-association of a human monoclonal antibody CNTO607.

Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irreversibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, (99)FHW(100a), as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular interaction. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 prevents the antibody from precipitating at low concentration yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but significant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific interaction of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation.

Design of novel FN3 domains with high stability by a consensus sequence approach.

The use of consensus design to produce stable proteins has been applied to numerous structures and classes of proteins. Here, we describe the engineering of novel FN3 domains from two different proteins, namely human fibronectin and human tenascin-C, as potential alternative scaffold biotherapeutics. The resulting FN3 domains were found to be robustly expressed in Escherichia coli, soluble and highly stable, with melting temperatures of 89 and 78°C, respectively. X-ray crystallography was used to confirm that the consensus approach led to a structure consistent with the FN3 design despite having only low-sequence identity to natural FN3 domains. The ability of the Tenascin consensus domain to withstand mutations in the loop regions connecting the ß-strands was investigated using alanine scanning mutagenesis demonstrating the potential for randomization in these regions. Finally, rational design was used to produce point mutations that significantly increase the stability of one of the consensus domains. Together our data suggest that consensus FN3 domains have potential utility as alternative scaffold therapeutics.

Structure-based engineering of a monoclonal antibody for improved solubility.

Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.

Cross-interaction chromatography: a rapid method to identify highly soluble monoclonal antibody candidates.

PURPOSE: To develop a high-throughput cross-interaction chromatography screening method to rapidly identify antibody candidates with poor solubility using microgram quantities of purified material. METHODS: A specific recombinant antibody or bulk polyclonal IgG purified from human serum was chemically coupled to an NHS-activated chromatography resin. The retention times of numerous monoclonal antibodies were determined on this resin using an HPLC and compared to the solubility of each antibody estimated by ultrafiltration. RESULTS: Retention times of the antibodies tested were found to be inversely related to solubility, with antibodies prone to precipitate at low concentrations in PBS being retained longer on the columns with broader peaks. The technique was successfully used to screen microgram quantities of a panel of therapeutic antibodies to identify candidates with low solubility in PBS. CONCLUSIONS: The cross-interaction chromatography methods described can be used to screen large panels of recombinant antibodies in order to discover those with low solubility. Addition of this tool to the array of tools available for characterization of affinity and activity of antibody therapeutic candidates will improve selection of candidates with biophysical properties favorable to development of high concentration antibody formulations.

Epitope mapping of anti-interleukin-13 neutralizing antibody CNTO607.

CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODY: factors that influence productivity and product quality.

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide-1-antibody fusion protein (also known as a Glucagon like peptide-1 MIMETIBODY), we have noted that the N-terminal GLP-1 portion of the MIMETIBODY was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O-linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N-terminal clipping had increased level of O-linked glycosylation in the linker region, giving credence to the hypothesis that O-linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N-terminal clipping had longer in vivo serum half-life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules.