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NK group 2 member A (NKG2A), an immune checkpoint inhibitor, is an emerging therapeutic target in immuno-oncology. NKG2A forms a heterodimer with CD94 on the cell surface of NK and a subset of T cells and recognizes the nonclassical human leukocyte antigen (HLA-E) in humans. Therapeutic blocking antibodies that block the ligation between HLA-E and NKG2A/CD94 have been shown to enhance antitumor immunity in mice and humans. In this study, we illustrate the practical utilities of mass spectrometry (MS)-based protein footprinting in areas from reagent characterization to antibody epitope mapping. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the higher-order structure characterization of NKG2A in complex with CD94 provides novel insights into the conformational dynamics of NKG2A/CD94 heterodimer. To fully understand antibody/target interactions, we employed complementary protein footprinting methods, including HDX-MS and fast photochemical oxidation of proteins (FPOP)-MS, to determine the binding epitopes of therapeutic monoclonal antibodies targeting NKG2A. Such a combination approach provides molecular insights into the binding mechanisms of antibodies to NKG2A with high specificity, demonstrating the blockade of NKG2A/HLA-E interaction.

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

The emergence and spread of multi-drug-resistant strains of Staphylococcus aureus in hospitals and in the community emphasize the urgency for the development of novel therapeutic interventions. Our approach was to evaluate the potential of harnessing the human immune system to guide the development of novel therapeutics. We explored the role of preexisting antibodies against S. aureus α-hemolysin in the serum of human individuals by isolating and characterizing one antibody with a remarkably high affinity to α-hemolysin. The antibody provided protection in S. aureus pneumonia, skin, and bacteremia mouse models of infection and also showed therapeutic efficacy when dosed up to 18 h post-infection in the pneumonia model. Additionally, in pneumonia and bacteremia animal models, the therapeutic efficacy of the α-hemolysin antibody appeared additive to the antibiotic linezolid. To better understand the mechanism of action of this isolated antibody, we solved the crystal structure of the α-hemolysin:antibody complex. To our knowledge, this is the first report of the crystal structure of the α-hemolysin monomer. The structure of the complex shows that the antibody binds α-hemolysin between the cap and the rim domains. In combination with biochemical data, the structure suggests that the antibody neutralizes the activity of the toxin by preventing binding to the plasma membrane of susceptible host cells. The data presented here suggest that protective antibodies directed against S. aureus molecules exist in some individuals and that such antibodies have a therapeutic potential either alone or in combination with antibiotics.

NORE1A is a Ras-binding protein that belongs to a group of tumor suppressors known as the Ras association domain family. Their growth- and tumor-suppressive function is assumed to be dependent on association with the microtubule cytoskeleton. However, a detailed understanding of this interplay is still missing. Here, we show that NORE1A directly interacts with tubulin and is capable of nucleating microtubules. Strikingly, the ability to stimulate nucleation is regulated in a dual specific way either via phosphorylation of NORE1A within the Ras-binding domain by Aurora A kinase or via binding to activated Ras. We also demonstrate that NORE1A mediates a negative effect of activated Ras on microtubule nucleation. On the basis of our results, we propose a novel regulatory network composed of the tumor suppressor NORE1A, the mitotic kinase Aurora A, the small GTPase Ras, and the microtubule cytoskeleton.

The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next-generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower-affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

Therapeutic antibodies are often engineered or selected to have high on-target binding affinities that can be challenging to determine precisely by most biophysical methods. Here, we explore the dynamic range of the kinetic exclusion assay (KinExA) by exploiting the interactions of an anti-DKK antibody with a panel of DKK antigens as a model system. By tailoring the KinExA to each studied antigen, we obtained apparent equilibrium dissociation constants (K(D) values) spanning six orders of magnitude, from approximately 100 fM to 100 nM. Using a previously calibrated antibody concentration and working in a suitable concentration range, we show that a single experiment can yield accurate and precise values for both the apparent K(D) and the apparent active concentration of the antigen, thereby increasing the information content of an assay and decreasing sample consumption. Orthogonal measurements obtained on Biacore and Octet label-free biosensor platforms further validated our KinExA-derived affinity and active concentration determinations. We obtained excellent agreement in the apparent affinities obtained across platforms and within the KinExA method irrespective of the assay orientation employed or the purity of the recombinant or native antigens.

A class of putative Ras effectors called Ras association domain family (RASSF) represents non-enzymatic adaptors that were shown to be important in tumour suppression. RASSF5, a member of this family, exists in two splice variants known as NORE1A and RAPL. Both of them are involved in distinct cellular pathways triggered by Ras and Rap, respectively. Here we describe the crystal structure of Ras in complex with the Ras binding domain (RBD) of NORE1A/RAPL. All Ras effectors share a common topology in their RBD creating an interface with the switch I region of Ras, whereas NORE1A/RAPL RBD reveals additional structural elements forming a unique Ras switch II binding site. Consequently, the contact area of NORE1A is extended as compared with other Ras effectors. We demonstrate that the enlarged interface provides a rationale for an exceptionally long lifetime of the complex. This is a specific attribute characterizing the effector function of NORE1A/RAPL as adaptors, in contrast to classical enzymatic effectors such as Raf, RalGDS or PI3K, which are known to form highly dynamic short-lived complexes with Ras.