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Activation of PKG1α is a compelling strategy for the treatment of cardiovascular diseases. As the main effector of cyclic guanosine monophosphate (cGMP), activation of PKG1α induces smooth muscle relaxation in blood vessels, lowers pulmonary blood pressure, prevents platelet aggregation, and protects against cardiac stress. The development of activators has been mostly limited to cGMP mimetics and synthetic peptides. Described herein is the optimization of a piperidine series of small molecules to yield activators that demonstrate

Antibody-drug conjugates (ADCs) represent a rapidly evolving area of drug development and hold significant promise. To date, nine ADCs have been approved by the US Food and Drug Administration (FDA). These conjugates combine the target specificity of monoclonal antibodies with the anticancer activity of small-molecule therapeutics (also referred to as payload). Due to the complex structure, three analytes, namely ADC conjugate, total antibody, and unconjugated payload, are typically quantified during drug development; however, the benefits of measuring all three analytes at later stages of clinical development are not clear. The cytotoxic payloads, upon release from the ADC, are considered to behave like small molecules. Given the relatively high potency and low systemic exposure of cytotoxic payloads, drug-drug interaction (DDI) considerations for ADCs might be different from traditional small molecule therapeutics. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) convened an ADC working group to create an IQ ADC database that includes 26 ADCs with six unique payloads. The analysis of the ADC data in the IQ database, as well as nine approved ADCs, supports the strategy of pharmacokinetic characterization of all three analytes in early-phase development and progressively minimizing the number of analytes to be measured in the late-phase studies. The systemic concentrations of unconjugated payload are usually too low to serve as a DDI perpetrator; however, the potential for unconjugated payloads as a victim still exists. A data-driven and risk-based decision tree was developed to guide the assessment of a circulating payload as a victim of DDI.

[Image: see text] The robustness of good laboratory practice and clinical data is reliant upon a clear understanding of the bioanalytical assays. One of the most important components of ligand-binding based assays is critical reagents used to directly or indirectly measure biologic markers or signals. High quality, reproducible, sustainable critical reagents through the development lifecycle could avoid unnecessary rework, multiple validations, cross-validations, and ensure consistency of the data. Numerous analytical methods (UPLC-size exclusion chromatography, cation exchange chromatography, biacore/octet, and high-resolution mass spectrometry) have been evaluated by using current critical reagents. A comprehensive analytical toolbox of biochemical and biophysical methods has been employed to evaluate the quality of critical reagents and explore potential issues if there are any. Moving forward, this “tiered approach” of critical reagents characterization will be used not only to establish critical quality attributes for new reagents but also to evaluate stability in support of reagents recertification.

A liquid chromatography–tandem mass spectrometry assay was developed and qualified for the multiplexed quantitation of a small molecule stimulator of soluble guanylate cyclase (sGC) and its target engagement biomarker, 3′,5′-cyclic guanosine monophosphate (cGMP), in ocular tissues and plasma from a single surrogate matrix calibration curve. A surrogate matrix approach was used in this assay due to the limited quantities of blank ocular matrices in a discovery research setting. After optimization, the assay showed high accuracy, precision, and recovery as well as parallelism between the surrogate matrix and the biological matrices (rabbit plasma, vitreous, and retina–choroid). This assay provided pharmacokinetic and target engagement data after intravitreal administration of the sGC stimulator. The nitric oxide-sGC-cGMP pathway is a potential target to address glaucoma. Increasing sGC-mediated production of cGMP could improve aqueous humor outflow and ocular blood flow. The sGC stimulator showed dose-dependent exposure in rabbit vitreous, retina–choroid, and plasma. The cGMP exhibited a delayed yet sustained increase in vitreous humor but not retina–choroid. Multiplexed measurement of both pharmacokinetic and target engagement analytes reduced animal usage and provided improved context for interpreting PK and PD relationships.

Therapies based on monoclonal antibodies (mAbs) have delivered an impressive success in the clinics due to their exquisite specificity, potential for agonistic or antagonistic responses, tunable effector function, and optimal pharmacokinetic properties. Building on these inherent antibody properties, the design and development of antibody-drug conjugates (ADCs) with improved or gained therapeutic activity and safety has been successfully demonstrated in oncological applications. There is enormous potential for this new type of hybrid biologics but there are also significant engineering, manufacturing and bioanalytical challenges. In this manuscript, we highlight the range and diversity of assays that are critical to characterize the individual components of ADCs-linker, carrier, and payload. We discuss a series of in vitro and in vivo preclinical experimental approaches we implemented to characterize two anti-inflammatory steroid bearing ADCs, and an ADC bearing a modified glucagon-like peptide 1 receptor/glucagon receptor co-agonist peptide.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

IgG4 antibodies are evolving as an important class of cancer immunotherapies. However, human IgG4 can undergo Fab arm (half molecule) exchange with other IgG4 molecules in vivo. The hinge modification by a point mutation (S228P) prevents half molecule exchange of IgG4. However, the experimental confirmation is still expected by regulatory agencies. Here, we report for the first time the extensive analysis of half molecule exchange for a hinge-modified therapeutic IgG4 molecule, pembrolizumab (Keytruda) targeting programmed death 1 (PD1) receptor that was approved for advanced melanoma. Studies were performed in buffer or human serum using multiple exchange partners including natalizumab (Tysabri) and human IgG4 pool. Formation of bispecific antibodies was monitored by fluorescence resonance energy transfer, exchange with Fc fragments, mixed mode chromatography, immunoassays, and liquid chromatography-mass spectrometry. The half molecule exchange was also examined in vivo in SCID (severe combined immunodeficiency) mice. Both in vitro and in vivo results indicate that the hinge modification in pembrolizumab prevented half molecule exchange, whereas the unmodified counterpart anti-PD1 wt showed active exchange activity with other IgG4 antibodies or self-exchange activity with its own molecules. Our work, as an example expected for meeting regulatory requirements, contributes to establish without ambiguity that hinge-modified IgG4 antibodies are suitable for biotherapeutic applications.

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Aim: Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC–MS. Methods & results: We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC–MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold. Conclusion: Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.

As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. Full characterization of these ADCs enabled procession to in vitro proof of concept, wherein ADCs 1-22 and 1-37 were demonstrated to afford potent, targeted delivery of glucocorticoids to a representative cell line, as measured by changes in glucocorticoid receptor-mediated gene mRNA levels. These activities were found to be antibody-, linker-, and payload-dependent. Preliminary mechanistic studies support the notion that lysosomal trafficking and enzymatic linker cleavage are required for activity and that the utility for the pyrophosphate linker may be general for internalizing ADCs as well as other targeted delivery platforms.

Meeting abstracts LAG-3 has been shown to act as an inhibitory molecule involved in the regulation of T cell activation, proliferation and homeostasis. Exhausted T cell populations that evolve in the tumor microenvironment or during chronic viral infections show coordinated expression of LAG-3 and

Quantitation of therapeutic monoclonal antibodies (mAb) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for pharmacokinetic (PK) studies is becoming an essential complement to traditional antibody-based ligand binding assays (LBA). Here we show an automated method to perform LC-MS/MS-based quantitation, with IgG1 conserved peptides, a heavy isotope labeled mAb internal standard, and anti-human Fc enrichment. All reagents in the method are commercially available with no requirement to develop novel assay-specific reagents. The method met traditional quantitative LC-MS/MS assay analytical characteristics in terms of precision, accuracy, and specificity. The method was applied to the pharmacokinetic study of a mAb dosed in cynomolgus monkey, and the results were compared with the immunoassay data. This methodology has the potential to benefit and accelerate the early biopharmaceutical development process, particularly by enabling PK analysis across species and candidate molecules with minimal method development.

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2-8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.

Background Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. Objective Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. Methods The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. Results Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. Conclusions and clinical relevance These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination.

IL-33 is an IL-1-related cytokine which has been implicated in T(h)2-associated biology and allergic diseases in humans and mice. IL-33 stimulates T(h)2 cells, mast cells, eosinophils, basophils, iNKT cells and circulating CD34(+) stem cells to proliferate and produce pro-allergic cytokines such as IL-5 and IL-13. IL-33 mediates its cytokine effects through a receptor consisting of ST2 and IL-1RAcP. Whereas IL-1RAcP is ubiquitously expressed, ST2 expression is cell-type restricted and determines responsiveness to IL-33. Studies employing ST2-deficient mice have reported variable results on the role of this receptor, and consequently IL-33, with regards to allergic lung inflammation. In this study, we demonstrate that IL-33 is important for allergic lung inflammation. Intra-nasal administration of IL-33 triggered an immediate allergic response in the airways, and more importantly, we show that endogenous IL-33 contributes to airway inflammation and peripheral antigen-specific responses in ovalbumin-induced acute allergic lung inflammation using IL-33-deficient mice. Our results suggest that IL-33 is sufficient and required for severe allergic inflammation in the lung and support the concept of IL-33 as a therapeutic target in allergic lung inflammation.

Idiopathic interstitial pneumonias are a group of idiopathic interstitial lung diseases of which idiopathic pulmonary fibrosis (IPF) is the lesion of usual interstitial pneumonia. Although the pathogenic mechanisms remain incompletely understood, disease-specific changes in blood, a readily accessible biospecimen, have not been fully characterized. To identify biomarkers from blood and sera, the immune status of IPF patients and control subjects without structural lung disease was quantified by measuring cell surface markers, mRNA levels, and serum proteins. Statistically significant differences in cellular and molecular markers were observed between the 2 groups. The cytokine receptor IL-17RB was significantly higher in CD14+ peripheral blood mononuclear cells (PBMCs) from IPF patients, whereas expression of the chemokine receptor CXCR4 was lower. Gene expression analyses identified 18 differentially expressed genes out of 195 selected. Of these, EMR1, CCR3, UPAR, FCGR2A, OPN, CEACAM3, CD16a, CD18, CD11b, LTF, and LCN2 were up-regulated, whereas IL-17RB, IL-10, PDGFA, CD301/Clec10a, CD25/IL-2RA, IL-23p19, and IL-15 were down-regulated in IPF. Differentially regulated genes were in the functional areas of inflammation and cell signaling. Serum levels of UPAR and OPN were higher in IPF. These observations reveal significant differences in cell and molecular markers involved in monocyte/macrophage activation and migration, and suggest a role for IL-17RB in IPF.

Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.

Interleukin-23 (IL-23) has emerged as a new therapeutic target for the treatment of inflammatory bowel disease (IBD). As biomarkers of disease state and treatment efficacy are becoming increasingly important in drug development, we sought to identify efficacy biomarkers for anti-IL-23 therapy in Crohn's disease (CD).Candidate IL-23 biomarkers, downstream of IL-23 signaling, were identified using shotgun proteomic analysis of feces and colon lavages obtained from a short-term mouse IBD model (anti-CD40 Rag2(-/-)) treated preventively with monoclonal antibodies (mAbs) to the IL-23 receptor (IL-23R). The biomarkers were then measured in an IBD T-cell transfer model treated therapeutically with a mAb to IL-23 (p19), confirming their association with IBD. To assess the clinical relevance of these markers, we assessed their concentrations in clinical serum, colon tissue, and feces from CD patients.We identified 57 proteins up or downregulated in diseased animals that returned to control values when the mice were treated with mAbs to IL-23R. Among those, S100A8, S100A9, regenerating protein 3β (REG), REG3γ, lipocalin 2 (LCN2), deleted in malignant tumor 1 (DMBT1), and macrophage migration inhibitory factor (MIF) mRNA levels correlated with disease score and dose titration of mAbs to IL-23R or IL-23(p19). All biomarkers, except DMBT1, were also downregulated after therapeutic administration of mAbs to IL-23(p19) in a T-cell transfer IBD mouse model. In sera from CD patients, we confirmed a significant upregulation of S100A8/A9 (43%), MIF (138%), pancreatitis-associated protein (PAP, human homolog of REG3β/γ; 49%), LCN2 (520%), and CCL20 (1280%), compared with control samples, as well as a significant upregulation of S100A8/A9 (887%), PAP (401%), and LCN2 (783%) in human feces from CD patients compared with normal controls.These studies identify multiple protein biomarkers downstream of IL-23 that could be valuable tools to assess the efficacy of this new therapeutic agent.Clinical and Translational Gastroenterology (2012) 3, e10; doi:10.1038/ctg.2012.2; published online 16 February 2012.