Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Investigation of anomalous charge variant profile reveals discrete pH-dependent conformations and conformation-dependent charge states within the CDR3 loop of a therapeutic mAb.

During the development of a therapeutic monoclonal antibody (mAb-1), the charge variant profile obtained by pH-gradient cation exchange chromatography (CEX) contained two main peaks, each of which exhibited a unique intrinsic fluorescence profile and demonstrated inter-convertibility upon reinjection of isolated peak fractions. Domain analysis of mAb-1 by CEX and liquid chromatography-mass spectrometry indicated that the antigen-binding fragment chromatographed as two separate peaks that had identical mass. Surface plasmon resonance binding analysis to antigen demonstrated comparable kinetics/affinity between these fractionated peaks and unfractionated starting material. Subsequent molecular modeling studies revealed that the relatively long and flexible complementarity-determining region 3 (CDR3) loop on the heavy chain could adopt two discrete pH-dependent conformations: an "open" conformation at neutral pH where the HC-CDR3 is largely solvent exposed, and a "closed" conformation at lower pH where the solvent exposure of a neighboring tryptophan in the light chain is reduced and two aspartic acid residues near the ends of the HC-CDR3 loop have atypical pKa values. The pH-dependent equilibrium between "open" and "closed" conformations of the HC-CDR3, and its proposed role in the anomalous charge variant profile of mAb-1, were supported by further CEX and hydrophobic interaction chromatography studies. This work is an example of how pH-dependent conformational changes and conformation-dependent changes to net charge can unexpectedly contribute to perceived instability and require thorough analytical, biophysical, and functional characterization during biopharmaceutical drug product development.

VISTA is an acidic pH-selective ligand for PSGL-1.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.

Characterization of Mauritian Cynomolgus Macaque FcγR Alleles Using Long-Read Sequencing.

The FcγRs are immune cell surface proteins that bind IgG and facilitate cytokine production, phagocytosis, and Ab-dependent, cell-mediated cytotoxicity. FcγRs play a critical role in immunity; variation in these genes is implicated in autoimmunity and other diseases. Cynomolgus macaques are an excellent animal model for many human diseases, and Mauritian cynomolgus macaques (MCMs) are particularly useful because of their restricted genetic diversity. Previous studies of MCM immune gene diversity have focused on the MHC and killer cell Ig-like receptor. In this study, we characterize FcγR diversity in 48 MCMs using PacBio long-read sequencing to identify novel alleles of each of the four expressed MCM FcγR genes. We also developed a high-throughput FcγR genotyping assay, which we used to determine allele frequencies and identify FcγR haplotypes in more than 500 additional MCMs. We found three alleles for FcγR1A, seven each for FcγR2A and FcγR2B, and four for FcγR3A; these segregate into eight haplotypes. We also assessed whether different FcγR alleles confer different Ab-binding affinities by surface plasmon resonance and found minimal difference in binding affinities across alleles for a panel of wild type and Fc-engineered human IgG. This work suggests that although MCMs may not fully represent the diversity of FcγR responses in humans, they may offer highly reproducible results for mAb therapy and toxicity studies.

Bioavailability of protein therapeutics in rats following inhalation exposure: Relevance to occupational exposure limit calculations.

Protein therapeutics represent a rapidly growing proportion of new medicines being developed by the pharmaceutical industry. As with any new drug, an Occupational Exposure Limit (OEL) should be developed to ensure worker safety. Part of the OEL determination addresses bioavailability (BA) after inhalation, which is poorly understood for protein therapeutics. To explore this, male Sprague-Dawley rats were exposed intravenously or by nose-only inhalation to one of five test proteins of varying molecular size (10-150 kDa), including a polyethylene glycol-conjugated protein. Blood, lung tissue and bronchoalveolar lavage (BAL) fluid were collected over various time-points depending on the expected test protein clearance (8 minutes-56 days), and analyzed to determine the pharmacokinetic profiles. Since the BAL half-life of the test proteins was observed to be > 4.5 h after an inhalation exposure, accumulation and direct lung effects should be considered in the hazard assessment for protein therapeutics with lung-specific targets. The key finding was the low systemic bioavailability after inhalation exposure for all test proteins (∼≤1%) which did not appear molecular weight-dependent. Given that this study examined the inhalation of typical protein therapeutics in a manner mimicking worker exposure, a default 1% BA assumption is reasonable to utilize when calculating OELs for protein therapeutics.

IL-2/CD25: A Long-Acting Fusion Protein That Promotes Immune Tolerance by Selectively Targeting the IL-2 Receptor on Regulatory T Cells.

Low-dose IL-2 represents an immunotherapy to selectively expand regulatory T cells (Tregs) to promote tolerance in patients with autoimmunity. In this article, we show that a fusion protein (FP) of mouse IL-2 and mouse IL-2Rα (CD25), joined by a noncleavable linker, has greater in vivo efficacy than rIL-2 at Treg expansion and control of autoimmunity. Biochemical and functional studies support a model in which IL-2 interacts with CD25 in the context of this FP in trans to form inactive head-to-tail dimers that slowly dissociate into an active monomer. In vitro, IL-2/CD25 has low sp. act. However, in vivo IL-2/CD25 is long lived to persistently and selectively stimulate Tregs. In female NOD mice, IL-2/CD25 administration increased Tregs within the pancreas and reduced the instance of spontaneous diabetes. Thus, IL-2/CD25 represents a distinct class of IL-2 FPs with the potential for clinical development for use in autoimmunity or other disorders of an overactive immune response.

Solution structures of the SH3 domains from Shank scaffold proteins and their interactions with Cav1.3 calcium channels.

Shank proteins are abundant scaffold proteins in the postsynaptic density (PSD) region of brain synapses. Mutations in Shank proteins are associated with autism, schizophrenia, and Alzheimer's disease. To gain insights into Shank protein interactions at the PSD, we determined the solution structures of the src homology 3 (SH3) domains of all three mammalian Shank proteins. Our findings indicate that they have identical and typical SH3 folding motifs, but unusual target-binding pockets. An investigation into the interaction between the Shank SH3 domains and the proline-rich region of the Cav1.3 calcium channel revealed an atypical interaction in which the highly acidic specificity binding pocket of the SH3 domains binds to a Cav1.3 region containing a cluster of three Arg residues. Our study provides insights into Shank SH3-mediated interactions.

Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcγR Engagement.

While engagement of the inhibitory Fcγ-receptor (FcγR) IIB is an absolute requirement for in vivo antitumor activity of agonistic mouse anti-CD40 monoclonal antibodies (mAbs), a similar requirement for human mAbs has been disputed. By using a mouse model humanized for its FcγRs and CD40, we revealed that FcγRIIB engagement is essential for the activity of human CD40 mAbs, while engagement of the activating FcγRIIA inhibits this activity. By engineering Fc variants with selective enhanced binding to FcγRIIB, but not to FcγRIIA, significantly improved antitumor immunity was observed. These findings highlight the necessity of optimizing the Fc domain for this class of therapeutic antibodies by using appropriate preclinical models that accurately reflect the unique affinities and cellular expression of human FcγR.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.

Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid.

Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions.

The structure of the death receptor 4-TNF-related apoptosis-inducing ligand (DR4-TRAIL) complex.

The structure of death receptor 4 (DR4) in complex with TNF-related apoptosis-inducing ligand (TRAIL) has been determined at 3 Šresolution and compared with those of previously determined DR5-TRAIL complexes. Consistent with the high sequence similarity between DR4 and DR5, the overall arrangement of the DR4-TRAIL complex does not differ substantially from that of the DR5-TRAIL complex. However, subtle differences are apparent. In addition, solution interaction studies were carried out that show differences in the thermodynamics of binding DR4 or DR5 with TRAIL.

Comparison of Ensemble and Single Molecule Methods for Particle Characterization and Binding Analysis of a PEGylated Single-Domain Antibody.

Domain antibodies (dAbs) are single immunoglobulin domains that form the smallest functional unit of an antibody. This study investigates the behavior of these small proteins when covalently attached to the polyethylene glycol (PEG) moiety that is necessary for extending the half-life of a dAb. The effect of the 40 kDa PEG on hydrodynamic properties, particle behavior, and receptor binding of the dAb has been compared by both ensemble solution and surface methods [light scattering, isothermal titration calorimetry (ITC), surface Plasmon resonance (SPR)] and single-molecule atomic force microscopy (AFM) methods (topography, recognition imaging, and force microscopy). The large PEG dominates the properties of the dAb-PEG conjugate such as a hydrodynamic radius that corresponds to a globular protein over four times its size and a much reduced association rate. We have used AFM single-molecule studies to determine the mechanism of PEG-dependent reductions in the effectiveness of the dAb observed by SPR kinetic studies. Recognition imaging showed that all of the PEGylated dAb molecules are active, suggesting that some may transiently become inactive if PEG sterically blocks binding. This helps explain the disconnect between the SPR, determined kinetically, and the force microscopy and ITC results that demonstrated that PEG does not change the binding energy.

Role of Self-Association and Supersaturation in Oral Absorption of a Poorly Soluble Weakly Basic Drug.

PURPOSE: Precipitation of weakly basic drugs in intestinal fluids can affect oral drug absorption. In this study, the implications of self-association of brivanib alaninate in acidic aqueous solution, leading to supersaturation at basic pH condition, on its solubility and oral absorption were investigated. METHODS: Self-association of brivanib alaninate was investigated by proton NMR spectroscopy, surface tension measurement, dynamic light scattering, isothermal titration calorimetry, and molecular modeling. Drug solubility was determined in various pH media, and its tendency to supersaturate upon pH shift was investigated in buffered and biorelevant aqueous solutions. Pharmacokinetic modeling of human oral drug absorption was utilized for parameter sensitivity analyses of input variables. RESULTS: Brivanib alaninate exhibited continuous, and pH- and concentration-dependent self-association. This phenomenon resulted in positive deviation of drug solubility at acidic pH and the formation of a stable supersaturated drug solution in pH-shift assays. Consistent with the supersaturation phenomenon observed in vitro, oral absorption simulations necessitated invoking long precipitation time in the intestine to successfully predict in vivo data. CONCLUSIONS: Self-association of a weakly basic drug in acidic aqueous solution can increase its oral absorption by supersaturation and precipitation resistance at the intestinal pH. This consideration is important to the selection of parameters for oral absorption simulation.

Investigation of a Degradant in a Biologics Formulation Buffer Containing L-Histidine.

PURPOSE: An unknown UV 280 nm absorbing peak was observed by SEC for protein stability samples formulated in L-histidine during a stress stability study. Understanding the source would enhance the confidence in the SEC results. We identified the unknown peak, studied the cause, and evaluated ways to eliminate it. METHODS: The unknown peak was fractionated by preparative size exclusion chromatography separations, and subsequently analyzed by Hydrophilic Interaction Chromatography (HILIC) coupled with Time-of-Flight (TOF) high resolution mass spectrometry. The possible degradation was also studied with the presence of different excipients, including metal cations, chelating agents, and amino acids. RESULTS: The unknown peak was identified to be trans-urocanic acid, a degradant of histidine, based on evidences from HILIC retention time, UV profile, accurate mass measurement, trans-cis isomerization, and pI measurement. The degradation from histidine to urocanic acids was not affected by the presence of Fe(2+), but slightly activated by Mn(2+). The chelating agents, EDTA and DTPA, counteracted the Mn(2+) effects. This degradation was evidenced to be caused by contamination. Adding alanine or cysteine as an excipient was found to reduce this degradation by 97 and 98%, respectively. CONCLUSIONS: L-histidine formulation buffer can be contaminated to induce histidine degradation to trans-urocanic acid, which shows a large UV 280 nm absorbing peak at the total permeation volume under SEC conditions. Amino acids alanine and cysteine effectively inhibit this histidine degradation.

Detection of drug specific circulating immune complexes from in vivo cynomolgus monkey serum samples.

BACKGROUND: Administration of a biotherapeutic can result in the formation of anti-drug antibodies (ADAs). The resulting ADA can potentially form immune complexes (ICs) with the drug leading to altered pharmacokinetic (PK) profiles and/or adverse events. Furthermore the presence of such complexes may interfere with accurate PK assessment, and/or detection of ADA in immunogenicity assays. Here, we present two assays to detect the presence of drug-ADA immune complexes in cynomolgus monkeys. RESULTS: Serum samples were analyzed for IC formation in vivo. 8/8 tested animals were positive for drug specific IC. Depending on the time point tested 4/8 or 7/8 animals tested positive for ADA during drug dosing. All 8 animals were confirmed positive for ADA during the washout phase, indicating drug interference in the bridging assay. Relative amount of IC over time was determined and its correlation with PK and ADA was then assessed. Multivariate data analysis demonstrates good correlation between signals obtained from the anti-drug and FcγRIIIa based capture assays, although due to its biological characteristic FcγRIIIa based assay captured only a subset of drug specific IC. In one animal IC remained in circulation even when the drug levels decreased below detection limit. CONCLUSION: Results from this study indicate the presence of IC during administration of an immunogenic biotherapeutic. Potential application of these assays includes detection of ADA in an IC during high drug levels. The results on the kinetics of IC formation during ADA response can complement the understanding of PK and ADA profiles. Moreover, the presence of IC indicates possible ADA interference in standard PK assays and potential underestimation of total drug exposure in toxicology studies. In addition this study also highlights the need to understand downstream in vivo consequences of drug-ADA IC as no animals under investigation developed adverse events.

Engineering of a novel anti-CD40L domain antibody for treatment of autoimmune diseases.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

Application of a kosmotrope-based solubility assay to multiple protein therapeutic classes indicates broad use as a high-throughput screen for protein therapeutic aggregation propensity.

Aggregation propensity is a critical attribute of protein therapeutics that can influence production, manufacturing, delivery, and potential activity and safety (immunogenicity). It is therefore imperative to select molecules with low aggregation propensity in the early stages of drug discovery to mitigate the risk of delays or failure in clinical development. Although many biophysical methods have been developed to characterize protein aggregation, most established methods are low-throughput, requiring large quantities of protein, lengthy assay times, and/or significant upstream sample preparation, which can limit application in early candidate screening. To avoid these limitations, we developed a reliable method to characterize aggregation propensity, by measuring the relative solubility of protein therapeutic candidates in the presence of the kosmotropic salt ammonium sulfate. Manual bench-scale and automated plate-based methods were applied to different protein therapeutic formats including Adnectins, domain antibodies, PEGylated Adnectins, Fc fusion proteins, and monoclonal antibodies. The kosmotrope solubility data agreed well with the aggregation propensity observed by established methods, while being amenable to high-throughput screening because of speed, simplicity, versatility and low protein material requirements. The results suggest that kosmotrope-based solubility assessment has broad applicability to selecting protein therapeutic candidates with low aggregation propensity and high "developability" to progress into development.

MIRG Survey 2011: snapshot of rapidly evolving label-free technologies used for characterizing molecular interactions.

The field of label-free biophysical technologies used to quantitatively characterize macromolecular interactions with each other and with small molecules has grown enormously in the last 10 years. The most widely used analytical technologies for characterizing biomolecular interactions are surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), biolayer interferometry (BLI), and analytical ultracentrifugation (AUC). Measuring interaction parameters accurately and quantitatively is challenging, as it requires specialized expertise, training, and instrumentation. The Molecular Interaction Research Group (MIRG) conducted an online survey designed to capture the current profile of label-free technologies, including ITC, SPR, and other biosensors used in academia and the pharmaceutical industry sector. The main goal of the survey was to take a snapshot of laboratory, instrumentation, applications for measuring various biophysical parameters, confidence in data interpretation, data validation and acceptability, and limitations of using various technologies. Through this survey, we anticipate that the participating laboratories will be able to gauge their own capabilities and gain insights into the relative success of the different technologies that they use for characterizing molecular interactions.

ABRF-MIRG benchmark study: molecular interactions in a three-component system.

Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.