Discovery of RXFP2 genetic association in resistant hypertensive men and RXFP2 antagonists for the treatment of resistant hypertension.

Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.

An Engineered Mouse Model That Generates a Diverse Repertoire of Endogenous, High-Affinity Common Light Chain Antibodies.

Bispecific antibodies have gained increasing popularity as therapeutics as they enable novel activities that cannot be achieved with monospecific antibodies. Some of the most popular bispecific formats are molecules in which two Fab arms with different antigen specificities are combined into one IgG-like molecule. One way to produce these bispecific molecules requires the discovery of antibodies against the two antigens of interest that share a common light chain. Here, we present the generation and characterization of a common light chain mouse model, in which the endogenous IGKJ cluster is replaced with a prearranged, modified murine IGKV10-96/IGKJ1 segment. We demonstrate that genetic modification does not impact B-cell development. Upon immunization with ovalbumin, the animals generate an antibody repertoire with VH gene segment usage of a similar diversity to wildtype mice, while the light chain diversity is restricted to antibodies derived from the prearranged IGKV10-96/IGKJ1 germline. We further show that the clonotype diversity of the common light chain immune repertoire matches the diversity of immune repertoire isolated from wildtype mice. Finally, the common light chain anti-ovalbumin antibodies have only slightly lower affinities than antibodies isolated from wildtype mice, demonstrating the suitability of these animals for antibody discovery for bispecific antibody generation.

VHH CDR-H3 conformation is determined by VH germline usage.

VHHs or nanobodies are single antigen binding domains originating from camelid heavy-chain antibodies. They are used as diagnostic and research tools and in a variety of therapeutic molecules. Analyzing variable domain structures from llama and alpaca we found that VHHs can be classified into two large structural clusters based on their CDR-H3 conformation. Extended CDR-H3 loops protrude into the solvent, whereas kinked CDR-H3 loops fold back onto framework regions. Both major families have distinct properties in terms of their CDR-H3 secondary structure, how their CDR-H3 interacts with the framework region and how they bind to antigens. We show that the CDR-H3 conformation of VHHs correlates with the germline from which the antibodies are derived: IGHV3-3 derived antibodies almost exclusively adopt a kinked CDR-H3 conformation while the CDR-H3 adopts an extended structure in most IGHV3S53 derived antibodies. We do not observe any bias stemming from V(D)J recombination in llama immune repertoires, suggesting that the correlation is the result of selection processes during B-cell development. Our findings demonstrate a previously undescribed impact of germline usage on antigen interaction and contribute to a better understanding on how properties of the antibody framework shape the immune repertoire.

Revealing the Hidden Sensitivity of Intrinsically Disordered Proteins to their Chemical Environment.

Intrinsically disordered protein-regions (IDRs) make up roughly 30% of the human proteome and are central to a wide range of biological processes. Given a lack of persistent tertiary structure, all residues in IDRs are, to some extent, solvent exposed. This extensive surface area, coupled with the absence of strong intramolecular contacts, makes IDRs inherently sensitive to their chemical environment. We report a combined experimental, computational, and analytical framework for high-throughput characterization of IDR sensitivity. Our framework reveals that IDRs can expand or compact in response to changes in their solution environment. Importantly, the direction and magnitude of conformational change depend on both protein sequence and cosolute identity. For example, some solutes such as short polyethylene glycol chains exert an expanding effect on some IDRs and a compacting effect on others. Despite this complex behavior, we can rationally interpret IDR responsiveness to solution composition changes using relatively simple polymer models. Our results imply that solution-responsive IDRs are ubiquitous and can provide an additional layer of regulation to biological systems.

Structure of human DPEP3 in complex with the SC-003 antibody Fab fragment reveals basis for lack of dipeptidase activity.

Dipeptidase 3 (DPEP3) is one of three glycosylphosphatidylinositol-anchored metallopeptidases potentially involved in the hydrolytic metabolism of dipeptides. While its exact biological function is not clear, DPEP3 expression is normally limited to testis, but can be elevated in ovarian cancer. Antibody drug conjugates targeting DPEP3 have shown efficacy in preclinical models with a pyrrolobenzodiazepine conjugate, SC-003, dosed in a phase I clinical trial (NCT02539719). Here we reveal the novel atomic structure of DPEP3 alone and in complex with the SC-003 Fab fragment at 1.8 and 2.8 Å, respectively. The structure of DPEP3/SC-003 Fab complex reveals an eighteen-residue epitope across the DPEP3 dimerization interface distinct from the enzymatic active site. DPEP1 and DPEP3 extracellular domains share a conserved, dimeric TIM (ß/α)8-barrel fold, consistent with 49% sequence identity. However, DPEP3 diverges from DPEP1 and DPEP2 in key positions of its active site: a histidine to tyrosine variation at position 269 reduces affinity for the ß zinc and may cause substrate steric hindrance, whereas an aspartate to asparagine change at position 359 abolishes activation of the nucleophilic water/hydroxide, resulting in no in vitro activity against a variety of dipeptides and biological substrates (imipenem, leukotriene D4 and cystinyl-bis-glycine). Hence DPEP3, unlike DPEP1 and DPEP2, may require an activating co-factor in vivo or may remain an inactive, degenerate enzyme. This report sheds light on the structural discriminants between active and inactive membrane dipeptidases and provides a benchmark to characterize current and future DPEP3-targeted therapeutic approaches.

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with KD in the sub-nanomolar range. However, it also exhibits significant (KD of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies.

A two-in-one antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions.

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.

Selection and screening using antibody phage display libraries.

Phage display is a powerful tool to isolate specific binders from a large and diverse combinatorial library. Here we provide a step-by-step protocol in how to set up a successful phage panning experiment in order to isolate useful antibodies. The protocol includes testing antigens for their suitability in the phage panning procedure and optimizing the panning conditions and alternative screening methods to minimize nonspecific binding. We describe example phage panning experiments starting from the library transformation to the phage clone screening.

Stability of phosphatidylethanol species in spiked and authentic whole blood and matching dried blood spots.

BACKGROUND: Phosphatidylethanol (PEth) is currently under investigation as a highly sensitive and specific marker of alcohol misuse. As its stability in blood samples has not systematically been investigated, a study was performed to determine the stability of major PEth species in spiked and authentic whole blood and also in matching dried blood spots (DBS) at different conditions. METHODS: To PEth-free blood from teetotalers, low and high concentrations of two major PEth (18:1/18:1 and 16:0/18:1) species were added chosen on the basis of concentrations determined from authentic samples which were collected from the subjects undergoing alcohol detoxification treatment. Effects of sampling (EDTA or heparinized tubes), temperature, and time (≤30 days) were investigated. Processed samples (two at each condition, respectively) were subjected to LC gradient separation using multiple reaction monitoring. Stability was assessed using the critical difference or a periodic analysis result that was within 15 % of the initial concentration. Reaction kinetics of degradation was investigated with rate constants being checked for an Arrhenius relationship. RESULTS: PEth was stable in dried blood spot (DBS) stored either at room temperature or frozen, whereas it was not stable in whole blood except in samples stored at -80 °C. Activation energies increased in the following order: spiked heparinized blood < spiked EDTA blood < authentic EDTA blood. CONCLUSIONS: PEth is a labile analyte which is predominantly degraded by hydrolysis. Only at -80 °C, stability in whole blood can be ascertained, and analysis should be performed within 30 days. EDTA should be preferred over heparin as an additive. DBS is able to stabilize PEth thus partly resolving pre-analytical difficulties of PEth measurement.

Structure and conservation of the periplasmic targeting factor Tic22 protein from plants and cyanobacteria.

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis.

LC-MS/MS analysis of phosphatidylethanol in dried blood spots versus conventional blood specimens.

Phosphatidylethanol (PEth), which is formed extrahepatically by the action of phospholipase D on phosphatidylcholine in the presence of ethanol, has been suggested as a promising marker of alcohol misuse. Analysis of dried blood spots (DBS) is particularly advantageous for the determination of delicate analytes such as PEth. Therefore, measurement of PEth species (18:1/18:1, 16:0/18:1) in DBS versus whole blood was performed to ascertain whether respective results are directly comparable. Samples were obtained from subjects (n = 40) undergoing alcohol detoxification treatment. Analysis involved liquid-liquid extraction from both, DBS and whole blood (100 µL, respectively), with phosphatidylpropanol as the internal standard. Extracts were subjected to LC gradient separation using multiple reaction monitoring of deprotonated molecules. Results from measurements of corresponding DBS and whole blood specimens were compared by estimating the respective mean values and by a Bland and Altman analysis. Concentrations of PEth 18:1/18:1 ranged from 46.1 to 3,360 ng/mL in whole blood (mean, 461.7 ng/mL) and from 35.8 to 3,360 ng/mL in DBS (mean, 457.6 ng/mL); for PEth 16:0/18:1, concentrations were from 900 to 213,000 ng/mL (mean, 23,375 ng/mL) and 922-213,000 ng/mL (mean, 23,470 ng/mL) in blood and DBS, respectively. Estimated mean differences were -4.3 ng/mL for PEth 18:1/18:1 and 95.8 ng/mL for PEth 16:0/18:1. The Bland-Altman plot of both PEth species showed that the variation around the mean difference was similar all through the range of measured values and that all differences except one were within the limits of agreement. It could be shown that the determination of PEth species in DBS is as reliable as in whole blood samples. This assay may facilitate monitoring of alcohol misuse.

High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2) antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF) to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33.

GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.

Conserved properties of polypeptide transport-associated (POTRA) domains derived from cyanobacterial Omp85.

Proteins of the Omp85 family are conserved in all kingdoms of life. They mediate protein transport across or protein insertion into membranes and reside in the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Omp85 proteins contain a C-terminal transmembrane beta-barrel and a soluble N terminus with a varying number of polypeptide-transport-associated or POTRA domains. Here we investigate Omp85 from the cyanobacterium Anabaena sp. PCC 7120. The crystallographic three-dimensional structure of the N-terminal region shows three POTRA domains, here named P1 to P3 from the N terminus. Molecular dynamics simulations revealed a hinge between P1 and P2 but in contrast show that P2 and P3 are fixed in orientation. The P2-P3 arrangement is identical as seen for the POTRA domains from proteobacterial FhaC, suggesting this orientation is a conserved feature. Furthermore, we define interfaces for protein-protein interaction in P1 and P2. P3 possesses an extended loop unique to cyanobacteria and plantae, which influences pore properties as shown by deletion. It now becomes clear how variations in structure of individual POTRA domains, as well as the different number of POTRA domains with both rigid and flexible connections make the N termini of Omp85 proteins versatile adaptors for a plentitude of functions.

pH sensitivity of the GTPase Toc33 as a regulatory circuit for protein translocation into chloroplasts.

The properties of membrane-embedded GTPases are investigated to understand translocation of preprotein across the outer envelope of chloroplasts. The homo- and heterodimerization events of the GTPases had been established previously. We show that the hydrolytic activity of the GTPase Toc33 is pH insensitive in the homodimeric conformation but has a bell-shaped pH optimum in the monomeric conformation. Further, Toc33 GTPase homodimerization and protein translocation into chloroplasts are pH sensitive as well. pH sensitivity might serve to regulate translocation; alternatively, the documented pH sensitivity might reflect a mechanistic requirement for GTPase silencing during translocation as the GTPase switches between homo- and heterodimeric conformations.

On the significance of Toc-GTPase homodimers.

Precursor protein translocation across the outer chloroplast membrane depends on the action of the Toc complex, containing GTPases as recognizing receptor components. The G domains of the GTPases are known to dimerize. In the dimeric conformation an arginine contacts the phosphate moieties of bound nucleotide in trans. Kinetic studies suggested that the arginine in itself does not act as an arginine finger of a reciprocal GTPase-activating protein (GAP). Here we investigate the specific function of the residue in two GTPase homologues. Arginine to alanine replacement variants have significantly reduced affinities for dimerization compared with wild-type GTPases. The amino acid exchange does not impact on the overall fold and nucleotide binding, as seen in the monomeric x-ray crystallographic structure of the Arabidopsis Toc33 arginine-alanine replacement variant at 2.0A. We probed the catalytic center with the transition state analogue GDP/AlF(x) using NMR and analytical ultracentrifugation. AlF(x) binding depends on the arginine, suggesting the residue can play a role in catalysis despite the non-GAP nature of the homodimer. Two non-exclusive functional models are discussed: 1) the coGAP hypothesis, in which an additional factor activates the GTPase in homodimeric form; and 2) the switch hypothesis, in which a protein, presumably the large Toc159 GTPase, exchanges with one of the homodimeric subunits, leading to activation.

The GTPase cycle of the chloroplast import receptors Toc33/Toc34: implications from monomeric and dimeric structures.

Transport of precursor proteins across chloroplast membranes involves the GTPases Toc33/34 and Toc159 at the outer chloroplast envelope. The small GTPase Toc33/34 can homodimerize, but the regulation of this interaction has remained elusive. We show that dimerization is independent of nucleotide loading state, based on crystal structures of dimeric Pisum sativum Toc34 and monomeric Arabidopsis thaliana Toc33. An arginine residue is--in the dimer--positioned to resemble a GAP arginine finger. However, GTPase activation by dimerization is sparse and active site features do not explain catalysis, suggesting that the homodimer requires an additional factor as coGAP. Access to the catalytic center and an unusual switch I movement in the dimeric structure support this finding. Potential binding sites for interactions within the Toc translocon or with precursor proteins can be derived from the structures.