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.

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.

Characterization of MK-4166, a Clinical Agonistic Antibody That Targets Human GITR and Inhibits the Generation and Suppressive Effects of T Regulatory Cells.

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregsin vitro In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. ©2017 AACR.

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.

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.

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.