Biophysical characterization of PVR family interactions and therapeutic antibody recognition to TIGIT.

The clinical successes of immune checkpoint blockade have invigorated efforts to activate T cell-mediated responses against cancer. Targeting members of the PVR family, consisting of inhibitory receptors TIGIT, CD96, and CD112R, has been an active area of clinical investigation. In this study, the binding interactions and molecular assemblies of the PVR family receptors and ligands have been assessed in vitro. Furthermore, the anti-TIGIT monoclonal antibody BMS-986207 crystal structure in complex with TIGIT was determined and shows that the antibody binds an epitope that is commonly targeted by the CD155 ligand as well as other clinical anti-TIGIT antibodies. In contrast to previously proposed models, where TIGIT outcompetes costimulatory receptor CD226 for binding to CD155 due to much higher affinity (nanomolar range), our data rather suggest that PVR family members all engage in interactions with relatively weak affinity (micromolar range), including TIGIT and CD155 interactions. Thus, TIGIT and other PVR inhibitory receptors likely elicit immune suppression via increased surface expression rather than inherent differences in affinity. This work provides an improved foundational understanding of the PVR family network and mechanistic insight into therapeutic antibody intervention.

Residue-Level Characterization of Antibody Binding Epitopes Using Carbene Chemical Footprinting.

Characterization of antibody binding epitopes is an important factor in therapeutic drug discovery, as the binding site determines and drives antibody pharmacology and pharmacokinetics. Here, we present a novel application of carbene chemical footprinting with mass spectrometry for identification of antibody binding epitopes at the single-residue level. Two different photoactivated diazirine reagents provide complementary labeling information allowing structural refinement of the antibody binding interface. We applied this technique to map the epitopes of multiple MICA and CTLA-4 antibodies and validated the findings with X-ray crystallography and yeast surface display epitope mapping. The characterized epitopes were used to understand biolayer interferometry-derived competitive binding results at the structural level. We show that carbene footprinting provides fast and high-resolution epitope information critical in the antibody selection process and enables mechanistic understanding of function to accelerate the drug discovery process.

Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and N-Glycosylated Antibodies.

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody-drug conjugates (ADCs) with payloads attached to the C'E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans. Depending on the conjugation site, glycans had both positive and negative effects on plasma stability of ADCs. The findings highlight the importance of consideration for selection of conjugation site to achieve desirable physicochemical properties and plasma stability.

Humanization of a strategic CD3 epitope enables evaluation of clinical T-cell engagers in a fully immunocompetent in vivo model.

T-cell engagers (TCEs) are a growing class of biotherapeutics being investigated in the clinic for treatment of a variety of hematological and solid tumor indications. However, preclinical evaluation of TCEs in vivo has been mostly limited to xenograft tumor models in human T-cell reconstituted immunodeficient mice, which have a number of limitations. To explore the efficacy of human TCEs in fully immunocompetent hosts, we developed a knock-in mouse model (hCD3E-epi) in which a 5-residue N-terminal fragment of murine CD3-epsilon was replaced with an 11-residue stretch from the human sequence that encodes for a common epitope recognized by anti-human CD3E antibodies in the clinic. T cells from hCD3E-epi mice underwent normal thymic development and could be efficiently activated upon crosslinking of the T-cell receptor with anti-human CD3E antibodies in vitro. Furthermore, a TCE targeting human CD3E and murine CD20 induced robust T-cell redirected killing of murine CD20-positive B cells in ex vivo hCD3E-epi splenocyte cultures, and also depleted nearly 100% of peripheral B cells for up to 7 days following in vivo administration. These results highlight the utility of this novel mouse model for exploring the efficacy of human TCEs in vivo, and suggest a useful tool for evaluating TCEs in combination with immuno-oncology/non-immuno-oncology agents against heme and solid tumor targets in hosts with a fully intact immune system.

Improved therapeutic index of an acidic pH-selective antibody.

Although therapeutically efficacious, ipilimumab can exhibit dose-limiting toxicity that prevents maximal efficacious clinical outcomes and can lead to discontinuation of treatment. We hypothesized that an acidic pH-selective ipilimumab (pH Ipi), which preferentially and reversibly targets the acidic tumor microenvironment over the neutral periphery, may have a more favorable therapeutic index. While ipilimumab has pH-independent CTLA-4 affinity, pH Ipi variants have been engineered to have up to 50-fold enhanced affinity to CTLA-4 at pH 6.0 compared to pH 7.4. In hCTLA-4 knock-in mice, these variants have maintained anti-tumor activity and reduced peripheral activation, a surrogate marker for toxicity. pH-sensitive therapeutic antibodies may be a differentiating paradigm and a novel modality for enhanced tumor targeting and improved safety profiles.

High-Throughput Surface Plasmon Resonance Biosensors for Identifying Diverse Therapeutic Monoclonal Antibodies.

Identification of antibodies targeting diverse functional epitopes on an antigen is highly crucial for discovering effective therapeutic candidates. Employing a traditional stepwise antibody "screening funnel" as well as prioritizing affinity-based selections over epitope-based selections, result in lead antibody panels lacking epitope diversity. In the present study, we employed an array-based surface plasmon resonance (SPR) platform to perform high-throughput epitope binning analysis on a large number of monoclonal antibodies (mAbs) generated in the early drug discovery process. The mAb panel contained clones from different antibody generation techniques and diverse transgenic mouse strains. The epitope binning results were analyzed in unique ways using various visualizations in the form of dendrograms and network plots, which assisted in determining diversity and redundancy in the mAb sample set. The binning data were further integrated with affinity information to evaluate the performance of seven different transgenic mouse strains. The combination of epitope binning results with binding kinetics and sequence analysis provided an effective and efficient way of selecting high affinity antibodies representing a diverse set of sequence families and epitopes.

Antibody blockade of CD96 by distinct molecular mechanisms.

The molecular interactions of mouse CD96 to CD155 ligand and to two surrogate antibodies have been investigated. Biophysical and structural studies demonstrate that CD96 forms a homodimer but assembles as 1:1 heterodimeric complexes with CD155 or with one of the surrogate antibodies, which compete for the same binding interface. In comparison, the other surrogate antibody binds across the mouse CD96 dimer and recognizes a quaternary epitope spanning both protomers to block exposure of the ligand-binding site. This study reveals different blocking mechanisms and modalities of these two antibodies and may provide insight into the functional effects of antibodies against CD96.

The role of ligand-binding assay and LC-MS in the bioanalysis of complex protein and oligonucleotide therapeutics.

Ligand-binding assay (LBA) and LC-MS have been the preferred bioanalytical techniques for the quantitation and biotransformation assessment of various therapeutic modalities. This review provides an overview of the applications of LBA, LC-MS/MS and LC-HRMS for the bioanalysis of complex protein therapeutics including antibody-drug conjugates, fusion proteins and PEGylated proteins as well as oligonucleotide therapeutics. The strengths and limitations of LBA and LC-MS, along with some guidelines on the choice of appropriate bioanalytical technique(s) for the bioanalysis of these therapeutic modalities are presented. With the discovery of novel and more complex therapeutic modalities, there is an increased need for the biopharmaceutical industry to develop a comprehensive bioanalytical strategy integrating both LBA and LC-MS.

Automated and Faster Affinity Capture Method for Biotransformation Assessment of Site-Specific Antibody Drug Conjugates.

Traditionally the biotransformation of antibody drug conjugates (ADCs) has been evaluated by affinity capture on streptavidin magnetic beads coated with a biotinylated capture reagent. To reduce the complexity of the analyte, the affinity captured ADCs are digested with enzymes ("on-bead" or after elution), and/or interchain disulfides are reduced to generate LC and HC fragments prior to mass spectrometry analysis. The "on-bead" enzymatic digestion with IdeS and PNGase F is not efficient and requires longer incubation times to achieve complete Fc and N-glycan removal. This results in a prolonged sample preparation time (7-18 h) and is not suitable for labile ADCs due to the possibility of assay-induced artifacts. To address these challenges, we developed an affinity capture method, where the ADCs are first captured onto streptavidin cartridges coated with a biotinylated generic capture reagent, followed by a 15 min "on-cartridge" digestion with IdeS or PNGase F. The ADCs are then eluted and directly analyzed by LC-HRMS. This method was successfully applied for the biotransformation assessment of site-specific ADCs with payload conjugated on the Fab or Fc. The reduced complexity of the analyte (Fc and N-glycan removal) combined with HRMS enabled sensitive and accurate identification of minor mass change catabolites and changes in the DAR distribution. This automated cartridge-based affinity capture method is fast with a total sample preparation time of less than 4 h (hands-on time of less than 1 h) and can be utilized for any human mAb/ADC independent of isotype (IgG1, IgG2, and IgG4).

Structures of mouse and human GITR-GITRL complexes reveal unique TNF superfamily interactions.

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor-ligand interface and receptor-receptor interface; the unique C-terminal receptor-receptor enables higher order structures on the membrane. Human GITR-GITRL has potential to form a hexameric network of membrane complexes, while murine GITR-GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor-receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries.

Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.

Universal Affinity Capture Liquid Chromatography-Mass Spectrometry Assay for Evaluation of Biotransformation of Site-Specific Antibody Drug Conjugates in Preclinical Studies.

Antibody drug conjugates (ADCs) can undergo in vivo biotransformation (e.g., payload metabolism, deconjugation) leading to reduced or complete loss of activity. The location/site of conjugation of payload-linker can have an effect on ADC stability and hence needs to be carefully optimized. Affinity capture LC-MS of intact ADCs or ADC subfragments has been extensively used to evaluate ADC biotransformation. However, the current methods have certain limitations such as the requirement of specific capture reagents, limited mass resolution of low mass change metabolites, low sensitivity, and use of capillary or nanoflow LC-MS. To address these challenges, we developed a generic affinity capture LC-MS assay that can be utilized to evaluate the biotransformation of any site-specific ADC independent of antibody type and site of conjugation (Fab and Fc) in preclinical studies. The method involves a combination of some or all of these steps: (1) "mono capture" or "dual capture" of ADCs from serum with streptavidin magnetic beads coated with a generic biotinylated antihuman capture reagent, (2) "on-bead" digestion with IdeS and/or PNGase F, and (3) reduction of interchain disulfide bonds to generate ∼25 kDa ADC subfragments, which are finally analyzed by LC-HRMS on a TOF mass spectrometer. The advantages of this method are that it can be performed using commercially available generic reagents and requires sample preparation time of less than 7 h. Furthermore, by reducing the size of intact ADC (∼150 kDa) to subfragments (∼25 kDa), the identification of conjugated payload and its metabolites can be achieved with excellent sensitivity and resolution (hydrolysis and other small mass change metabolites). This method was successfully applied to evaluate the in vitro and in vivo biotransformation of ADCs conjugated at different sites (LC, HC-Fab, and HC-Fc) with various classes of payload-linkers.

Design and characterization of mouse IgG1 and IgG2a bispecific antibodies for use in syngeneic models.

The development of antibody therapeutics relies on animal models that accurately recapitulate disease biology. Syngeneic mouse models are increasingly used with new molecules to capture the biology of complex cancers and disease states, and to provide insight into the role of the immune system. The establishment of syngeneic mouse models requires the ability to generate surrogate mouse counterparts to antibodies designed for humans. In the field of bispecific antibodies, there remains a dearth of technologies available to generate native IgG-like mouse bispecific antibodies. Thus, we engineered a simple co-expression system for one-step purification of intact mouse IgG1 and IgG2a bispecific antibodies from any antibody pair. We demonstrated proof of concept with CD3/CD20 bispecific antibodies, which highlighted both the quality and efficacy of materials generated by this technology.

Effect of Chirality on Common in Vitro Experiments: An Enantiomeric Pair Analysis.

This analysis elucidates the impact of small molecule architecture on common in vitro ADME assays. In vitro parameters considered in this analysis included Caco-2 permeability/efflux, CYP3A4 inhibition, hERG inhibition, and rat microsomal extraction ratio (ER). The statistical significance and practical meaningfulness of chirality were determined by comparison of the distribution of enantiomers with the experimental variation distribution observed from duplicate measurements. Statistical tools were applied to characterize the role of molecular architecture on the outcome of a given in vitro assay. We found that CYP3A4 inhibition, hERG inhibition, Caco-2 permeability, and efflux are unlikely to be modulated by chirality. However, rat microsomal ER provides a statistically significant, and quantitatively meaningful, chance of being influenced by chirality.

Benefit of Retraining pKa Models Studied Using Internally Measured Data.

The ionization state of drugs influences many pharmaceutical properties such as their solubility, permeability, and biological activity. It is therefore important to understand the structure property relationship for the acid-base dissociation constant pKa during the lead optimization process to make better-informed design decisions. Computational approaches, such as implemented in MoKa, can help with this; however, they often predict with too large error especially for proprietary compounds. In this contribution, we look at how retraining helps to greatly improve prediction error. Using a longitudinal study with data measured over 15 years in a drug discovery environment, we assess the impact of model training on prediction accuracy and look at model degradation over time. Using the MoKa software, we will demonstrate that regular retraining is required to address changes in chemical space leading to model degradation over six to nine months.

Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides.

Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes.

Accelerating the drug optimization process: identification, structure elucidation, and quantification of in vivo metabolites using stable isotopes with LC/MSn and the chemiluminescent nitrogen detector.

Most preclinical leads exhibit poor ADME/PK properties and require optimizing to increase the likelihood of becoming successful pharmaceuticals. As a means of accelerating the evaluation of these leads in vivo, we assessed the use of LC/MS with the chemiluminescent-nitrogen detector (CLND) and a stable isotope to identify and quantify in vivo metabolites and to measure excretion. A 14C-labeled preclinical lead that also contained two chlorine atoms was administered orally to rats, and samples of bile, urine, and plasma were collected and analyzed by LC with radiodetection and by LC/MS-CLND with the chlorine atoms used as tracers. Both methods identified seven metabolites in bile and two metabolites in urine. The amount and abundance of each metabolite was measured, and the results were equivalent for the two methods. Material balance was measured by liquid scintillation counting of the starting samples, by LC/radiodetection, and by LC/MS-CLND. All three methods yielded the same results and showed that the primary route of clearance was metabolism followed by immediate excretion. This study demonstrates that LC/MS-CLND with a stable isotope is a method that can efficiently track and accurately quantify metabolites, making it possible to rapidly study ADME/PK in vivo without radiolabeling.

An automated LC method for the small-scale purification of organic molecules derived from combinatorial libraries.

This paper presented the development of an automated HPLC small-scale purification method for single bead compounds derived from combinatorial libraries. The method was found to produce higher and more consistent recoveries of purified compounds as compared to conventional manual HPLC purification. Using the manual method, the average percentage recovery of one synthetic compound was determined to be 24% and the coefficients of variation (C.V.%) of recovery were found to be greater than 38%. Using the automated system, the average percentages recovery of a standard compound at 600 and 1000 micromol l(-1) were determined to be 72.63+/-10.17% and 81.34+/-4.39%, respectively. This represented an approximate 3-folds increase in percentage recovery compared to that of the manual small-scale purification process. It was also found that the C.V.% of recovery were less than 15% at both concentration levels. The development of this automated method was found to be straightforward. The importance and implications of this study were discussed.