Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies.

Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

Site-Specific Antibody Conjugation Using Modified Bisected N-Glycans: Method Development and Potential toward Tunable Effector Function.

Antibody-drug conjugates (ADCs) have garnered worldwide attention for disease treatment, as they possess high target specificity, a long half-life, and outstanding potency to kill or modulate the functions of targets. FDA approval of multiple ADCs for cancer therapy has generated a strong desire for novel conjugation strategies with high biocompatibility and controllable bioproperties. Herein, we present a bisecting glycan-bridged conjugation strategy that enables site-specific conjugation without the need for the oligosaccharide synthesis and genetic engineering of antibodies. Application of this method is demonstrated by conjugation of anti-HER2 human and mouse IgGs with a cytotoxic drug, monomethyl auristatin E. The glycan bridge showed outstanding stability, and the resulting ADCs eliminated HER2-expressing cancer cells effectively. Moreover, our strategy preserves the feasibility of glycan structure remodeling to fine-tune the immunogenicity and pharmacokinetic properties of ADCs through glycoengineering.

Structural insights into selective small molecule activation of PKG1α.

cGMP-dependent protein kinase I-α (PKG1α) is a target for pulmonary arterial hypertension due to its role in the regulation of smooth muscle function. While most work has focused on regulation of cGMP turnover, we recently described several small molecule tool compounds which were capable of activating PKG1α via a cGMP independent pathway. Selected molecules were crystallized in the presence of PKG1α and were found to bind to an allosteric site proximal to the low-affinity nucleotide binding domain. These molecules act to displace the switch helix and cause activation of PKG1α representing a new mechanism for the activation and control of this critical therapeutic path. The described structures are vital to understanding the function and control of this key regulatory pathway.

Long-chain polyunsaturated lipids associated with responsiveness to anti-PD-1 therapy are colocalized with immune infiltrates in the tumor microenvironment.

The programmed cell death protein-1 (PD-1) is highly expressed on the surface of antigen-specific exhausted T cells and, upon interaction with its ligand PD-L1, can result in inhibition of the immune response. Anti-PD-1 treatment has been shown to extend survival and result in durable responses in several cancers, yet only a subset of patients benefit from this therapy. Despite the implication of metabolic alteration following cancer immunotherapy, mechanistic associations between antitumor responses and metabolic changes remain unclear. Here, we used desorption electrospray ionization mass spectrometry imaging to examine the lipid profiles of tumor tissue from three syngeneic murine models with varying treatment sensitivity at the baseline and at three time points post-anti-PD-1 therapy. These imaging experiments revealed specific alterations in the lipid profiles associated with the degree of response to treatment and allowed us to identify a significant increase of long-chain polyunsaturated lipids within responsive tumors following anti-PD-1 therapy. Immunofluorescence imaging of tumor tissues also demonstrated that the altered lipid profile associated with treatment response is localized to dense regions of tumor immune infiltrates. Overall, these results indicate that effective anti-PD-1 therapy modulates lipid metabolism in tumor immune infiltrates, and we thereby propose that further investigation of the related immune-metabolic pathways may be useful for better understanding success and failure of anti-PD-1 therapy.

Optimization and Mechanistic Investigations of Novel Allosteric Activators of PKG1α.

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 in vitro phosphorylation of vasodilator-stimulated phosphoprotein as well as antiproliferative effects in human pulmonary arterial smooth muscle cells. Hydrogen/deuterium exchange mass spectrometry experiments with the small molecule activators revealed a mechanism of action consistent with cGMP-induced activation, and an X-ray co-crystal structure with a construct encompassing the regulatory domains illustrated a binding mode in an allosteric pocket proximal to the low-affinity cyclic nucleotide-binding domain.

Risk-Based Pharmacokinetic and Drug-Drug Interaction Characterization of Antibody-Drug Conjugates in Oncology Clinical Development: An International Consortium for Innovation and Quality in Pharmaceutical Development Perspective.

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.

A Surrogate Matrix-Based Approach Toward Multiplexed Quantitation of an sGC Stimulator and cGMP in Ocular Tissue and Plasma.

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.

A Tiered Approach for Characterization to Ensure Quality, Reproducibility, and Long-Term Stability of Critical Reagents in Regulated Bioanalysis to Support PK/ADA/NAb Assays for Biologics and Vaccines Programs.

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.

Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells.

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.

Epitopes and Mechanism of Action of the Clostridium difficile Toxin A-Neutralizing Antibody Actoxumab.

The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial pathogen Clostridium difficile and are responsible for the pathology associated with C. difficile infection (CDI). The antitoxin antibodies actoxumab and bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab was recently approved by the FDA for reducing the recurrence of CDI. We have previously shown that a single molecule of bezlotoxumab binds to two distinct epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, preventing toxin binding to host cells. In this study, we characterize the binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. Using a combination of approaches including a number of biophysical techniques, we show that there are two distinct actoxumab binding sites within the CROP domain of TcdA centered on identical amino acid sequences at residues 2162-2189 and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at higher antibody:toxin concentration ratios. Actoxumab prevented the association of TcdA with target cells demonstrating that actoxumab neutralizes toxin activity by inhibiting the first step of the intoxication cascade. This mechanism of neutralization is similar to that observed with bezlotoxumab and TcdB. Comparisons of the putative TcdA epitope sequences across several C. difficile ribotypes and homologous repeat sequences within TcdA suggest a structural basis for observed differences in actoxumab binding and/or neutralization potency. These data provide a mechanistic basis for the protective effects of the antibody in vitro and in vivo, including in various preclinical models of CDI.

Automated DBS microsampling, microscale automation and microflow LC-MS for therapeutic protein PK.

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.

Discovery of Pyrophosphate Diesters as Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody-Drug Conjugates.

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.

Comprehensive Analysis of the Therapeutic IgG4 Antibody Pembrolizumab: Hinge Modification Blocks Half Molecule Exchange In Vitro and In Vivo.

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.

A functional approach reveals a genetic and physical interaction between ribonucleotide reductase and CHK1 in mammalian cells.

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.

Generic automated method for liquid chromatography-multiple reaction monitoring mass spectrometry based monoclonal antibody quantitation for preclinical pharmacokinetic studies.

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.

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

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 ß-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.

The identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors.

The ribonucleotide reductase (RNR) enzyme is a heteromer of RRM1 and RRM2 subunits. The active enzyme catalyzes de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. Complexity in the generation of physiologically relevant, active RRM1/RRM2 heterodimers was perceived as limiting to the identification of selective RRM1 inhibitors by high-throughput screening of compound libraries and led us to seek alternative methods to identify lead series. In short, we found that gemcitabine, as its diphosphate metabolite, represents one of the few described active site inhibitors of RRM1. We herein describe the identification of novel 5'-amino gemcitabine analogs as potent RRM1 inhibitors through in-cell phenotypic screening.

Developability studies before initiation of process development: improving manufacturability of monoclonal antibodies.

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.

Biomarkers of Therapeutic Response in the IL-23 Pathway in Inflammatory Bowel Disease.

OBJECTIVES: 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). METHODS: 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. RESULTS: 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 3ß (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 REG3ß/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. CONCLUSIONS: 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.

Biomarkers of disease and treatment in murine and cynomolgus models of chronic asthma.

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.