The IMiD target CRBN determines HSP90 activity toward transmembrane proteins essential in multiple myeloma.

The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.

Author Correction: Consistent success in life-supporting porcine cardiac xenotransplantation.

In this Letter, Mayuko Kurome and Valeri Zakhartchenko have been added to the author list (affiliated with Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany). The author list and 'Author contributions' section have been corrected online; see accompanying Amendment.

Site-Selective Protein Conjugation by a Multicomponent Ugi Reaction.

The chemical bioconjugation of proteins has seen tremendous applications in the past decades, with the booming of antibody-drug conjugates and their use in oncology. While genetic engineering has permitted to produce bespoke proteins featuring key (un-)natural amino acid residues poised for site-selective modifications, the conjugation of native proteins is riddled with selectivity issues. Chemoselective strategies are plentiful and enable the precise modification of virtually any residue with a reactive side-chain; site-selective methods are less common and usually most effective on small and medium-sized proteins. In this context, we studied the application of the Ugi multicomponent reaction for the site-selective conjugation of amine and carboxylate groups on proteins, and antibodies in particular. Through an in-depth mechanistic methodology work supported by peptide mapping studies, we managed to develop a set of conditions allowing the highly selective modification of antibodies bearing N-terminal glutamate and aspartate residues. We demonstrated that this strategy did not alter their affinity toward their target antigen and produced an antibody-drug conjugate with subnanomolar potency. Excitingly, we showed that the high site selectivity of our strategy was maintained on other protein formats, especially on anticalins, for which directed mutagenesis helped to highlight the key importance of a single lysine residue.

An Approach to Controlling Inflammation and Coagulation in Pig-to-Baboon Cardiac Xenotransplantation.

INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

Consistent success in life-supporting porcine cardiac xenotransplantation.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.

Structural and Functional Analysis of a Highly Active Designed Phosphotriesterase for the Detoxification of Organophosphate Nerve Agents Reveals an Unpredicted Conformation of the Active Site Loop.

Neurotoxic organophosphorus compounds (OPs) pose a severe threat if misused in military conflicts or by terrorists. Administration of a hydrolytic enzyme that can decompose the circulating nerve agent into non-toxic metabolites in vivo offers a potential treatment. A promising candidate is the homo-dimeric phosphotriesterase originating from the bacterium Brevundimonas diminuta (BdPTE), which has been subject to several rational and combinatorial protein design studies. A series of engineered versions with much improved catalytic efficiencies toward medically relevant nerve agents was described, carrying up to 22 mutations per enzyme subunit. To provide a basis for further rational design, we have determined the crystal structure of the highly active variant 10-2-C3(C59V/C227V)─stabilized against oxidation by substitution of two unpaired Cys residues─in complex with a substrate analogue at 1.5 Å resolution. Unexpectedly, the long loop segment (residues 253-276) that covers the active site shows a totally new conformation, with drastic structural deviations up to 19 Å, which was neither predicted in any of the preceding protein design studies nor seen in previous crystallographic analyses of less far evolved enzyme versions. Inspired by this structural insight, additional amino acid exchanges were introduced and their effects on protein stability as well as on the catalytic efficiency toward several neurotoxic OPs were investigated. Somewhat surprisingly, our results suggest that the presently available engineered version of BdPTE, in spite of its design on the basis of partly false structural assumptions, constitutes a fairly optimized enzyme for the detoxification of relevant OP nerve agents.

Engineered Protein Scaffolds as Next-Generation Therapeutics.

The concept of engineering robust protein scaffolds for novel binding functions emerged 20 years ago, one decade after the advent of recombinant antibody technology. Early examples were the Affibody, Monobody (Adnectin), and Anticalin proteins, which were derived from fragments of streptococcal protein A, from the tenth type III domain of human fibronectin, and from natural lipocalin proteins, respectively. Since then, this concept has expanded considerably, including many other protein templates. In fact, engineered protein scaffolds with useful binding specificities, mostly directed against targets of biomedical relevance, constitute an area of active research today, which has yielded versatile reagents as laboratory tools. However, despite strong interest from basic science, only a handful of those protein scaffolds have undergone biopharmaceutical development up to the clinical stage. This includes the abovementioned pioneering examples as well as designed ankyrin repeat proteins (DARPins). Here we review the current state and clinical validation of these next-generation therapeutics.

FerryCalin: an Engineered Lipocalin Protein Directed against the Transferrin Receptor with Potential for Brain Drug Delivery.

The transferrin receptor (TfR) mediates transcytosis across the blood-brain barrier (BBB), which offers a promising approach for the non-invasive delivery of therapeutics into the brain parenchyma. Employing the recombinant homodimeric murine TfR ectodomain, prepared in a biochemically functional state, we have selected a cognate Anticalin via phage display and bacterial cell surface display from a random library based on the human lipocalin 2 (Lcn2). After affinity maturation, several engineered lipocalin variants were identified that bind murine TfR in a non-competitive manner with the natural ligand (transferrin ⋅ Fe3+ ), among those an Anticalin - dubbed FerryCalin - exhibiting a dissociation constant (KD ) of 3.8 nM. Epitope analysis using the SPOT technique revealed a sequential epitope in a surface region of TfR remote from the transferrin-binding site. Due to the fast kon rate and short complex half-life, as evidenced by real-time surface plasmon resonance (SPR) measurements, FerryCalin, or one of its related mutants, shows characteristics as a potential vehicle for the brain delivery of biopharmaceuticals.

cART Restores Transient Responsiveness to IFN Type 1 in HIV-Infected Humanized Mice.

The lack of a human immunodeficiency virus (HIV) cure has heightened interest in immunotherapy. As such, type I interferons (IFNs), in particular, IFN alpha (IFN-α), have gained renewed attention. However, HIV pathogenesis is driven by sustained IFN-mediated immune activation, and the use of IFNs is rather controversial. The following questions therein remain: (i) which IFN-α subtype to use, (ii) at which regimen, and (iii) at what time point in HIV infection it might be beneficial. Here, we used IFN-α14 modified by PASylation for its long half-life in vivo to eventually treat HIV infection. We defined the IFN dosing regimen based on the maximum increase in interferon-stimulated gene (ISG) expression 6 h after its administration and a return to baseline of ubiquitin-specific protease 18 (USP18) prior to the next dose. Notably, USP18 is the major negative regulator of type I IFN signaling. HIV infection resulted in increased ISG expression levels in humanized mice. Intriguingly, high baseline ISG levels correlated with lower HIV load. No effect was observed on HIV replication when PASylated IFN-α14 was administered in the chronic phase. However, combined antiretroviral therapy (cART) restored responsiveness to IFN, and PASylated IFN-α14 administered during analytical cART interruption resulted in a transiently lower HIV burden than in the mock-treated mice. In conclusion, cART-mediated HIV suppression restored transient IFN responsiveness and provided a potential window for immunoenhancing therapies in the context of analytical cART interruption. IMPORTANCE cART is highly efficient in suppressing HIV replication in HIV-infected patients and has resulted in a dramatic reduction in morbidity and mortality in HIV-infected people, yet it does not cure HIV infection. In addition, cART has several disadvantages. Thus, the HIV research community is exploring novel ways to control HIV infection for longer periods without cART. Here, we explored novel, long-acting IFN-α14 for its efficacy to control HIV replication in HIV-infected humanized mice. We found that IFN-α14 had no effect on chronic HIV infection. However, when mice were treated first with cART, we observed a transiently restored responsiveness to INF and a transiently lower HIV burden after stopping cART. These data emphasize (i) the value of cART-mediated HIV suppression and immune reconstitution in creating a window of opportunity for exploring novel immunotherapies, (ii) the potential of IFNs for constraining HIV, and (iii) the value of humanized mice for exploring novel immunotherapies.

Complement inhibition at the level of C3 or C5: mechanistic reasons for ongoing terminal pathway activity.

Blocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a "C3 bypass" activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.

Molecular Design of 68Ga- and 89Zr-Labeled Anticalin Radioligands for PET-Imaging of PSMA-Positive Tumors.

Anticalin proteins directed against the prostate-specific membrane antigen (PSMA), optionally having tailored plasma half-life using PASylation technology, show promise as radioligands for PET-imaging of xenograft tumors in mice. To investigate their suitability, the short-circulating unmodified Anticalin was labeled with 68Ga (τ1/2 = 68 min), using the NODAGA chelator, whereas the half-life extended PASylated Anticalin was labeled with 89Zr (τ1/2 = 78 h), using either the linear chelator deferoxamine (Dfo) or a cyclic derivative, fusarinine C (FsC). Different PSMA targeting Anticalin versions (optionally carrying the PASylation sequence) were produced carrying a single exposed N- or C-terminal Cys residue and site-specifically conjugated with the different radiochelators via maleimide chemistry. These protein conjugates were labeled with radioisotopes having distinct physical half-lives and, subsequently, applied for PET-imaging of subcutaneous LNCaP xenograft tumors in CB17 SCID mice. Uptake of the protein tracers into tumor versus healthy tissues was assessed by segmentation of PET data as well as biodistribution analyses. PET-imaging with both the 68Ga-labeled plain Anticalin and the 89Zr-labeled PASylated Anticalin allowed clear delineation of the xenograft tumor. The radioligand A3A5.1-PAS(200)-FsC·89Zr, having an extended plasma half-life, led to a higher tumor uptake 24 h p.i. compared to the 68Ga·NODAGA-Anticalin imaged 60 min p.i. (2.5% ID/g vs 1.2% ID/g). Pronounced demetallation was observed for the 89Zr·Dfo-labeled PASylated Anticalin, which was ∼50% lower in the case of the cyclic radiochelator FsC (p < 0.0001). Adjusting the plasma half-life of Anticalin radioligands using PASylation technology is a viable approach to increase radioisotope accumulation within the tumor. Furthermore, 89Zr-ImmunoPET-imaging using the FsC radiochelator is superior to that using Dfo. Our strategy for the half-life adjustment of a tumor-targeting Anticalin to match the physical half-life of the applied radioisotope illustrates the potential of small binding proteins as an alternative to antibodies for PET-imaging.

Structural basis of Alzheimer ß-amyloid peptide recognition by engineered lipocalin proteins with aggregation-blocking activity.

We describe the structural analysis of two Anticalin® proteins that tightly bind Aß40, a peptide involved in the pathophysiology of Alzheimer's disease. These anticalins, US7 and H1GA, were engineered on the basis of the human lipocalin 2, thus yielding compact single-domain binding proteins as an alternative to antibodies. Albeit selected under different conditions and mutually deviating in 13 amino acid positions within the binding pocket (of 17 mutated residues in total), both crystallised anticalins recognize the same epitope in the middle of the ß-amyloid peptide. In the two complexes with the Aß40 peptide, its central part comprising residues LysP16 to LysP28 shows well defined electron density whereas the flanking regions appear structurally disordered. The compact zigzag-bend conformation which is seen in both structures may indicate a role during conversion of the soluble monomeric form into pathogenic Aß state(s) and, thus, explain the aggregation-inhibiting effect of the anticalins. In contrast to solanezumab, which targets the same Aß region in a different conformation, the anticalin H1GA does not show cross-reactivity with sequence-related human plasma proteins. Consequently, anticalins offer promising reagents to prevent oligomerization of Aß peptides to neurotoxic species in vivo and their small size may enable new routes for brain delivery.

Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Post-VX exposure treatment of rats with engineered phosphotriesterases.

The biologically stable and highly toxic organophosphorus nerve agent (OP) VX poses a major health threat. Standard medical therapy, consisting of reactivators and competitive muscarinic receptor antagonists, is insufficient. Recently, two engineered mutants of the Brevundimonas diminuta phosphotriesterase (PTE) with enhanced catalytic efficiency (kcat/KM = 21 to 38 × 106 M-1 min-1) towards VX and a preferential hydrolysis of the more toxic P(-) enantiomer were described: PTE-C23(R152E)-PAS(100)-10-2-C3(I106A/C59V/C227V/E71K)-PAS(200) (PTE-2), a single-chain bispecific enzyme with a PAS linker and tag having enlarged substrate spectrum, and 10-2-C3(C59V/C227V)-PAS(200) (PTE-3), a stabilized homodimeric enzyme with a double PASylation tag (PAS-tag) to reduce plasma clearance. To assess in vivo efficacy, these engineered enzymes were tested in an anesthetized rat model post-VX exposure (~ 2LD50) in comparison with the recombinant wild-type PTE (PTE-1), dosed at 1.0 mg kg-1 i.v.: PTE-2 dosed at 1.3 mg kg-1 i.v. (PTE-2.1) and 2.6 mg kg-1 i.v. (PTE-2.2) and PTE-3 at 1.4 mg kg-1 i.v. Injection of the mutants PTE-2.2 and PTE-3, 5 min after s.c. VX exposure, ensured survival and prevented severe signs of a cholinergic crisis. Inhibition of erythrocyte acetylcholinesterase (AChE) could not be prevented. However, medulla oblongata and diaphragm AChE activity was partially preserved. All animals treated with the wild-type enzyme, PTE-1, showed severe cholinergic signs and died during the observation period of 180 min. PTE-2.1 resulted in the survival of all animals, yet accompanied by severe signs of OP poisoning. This study demonstrates for the first time efficient detoxification in vivo achieved with low doses of heterodimeric PTE-2 as well as PTE-3 and indicates the suitability of these engineered enzymes for the development of highly effective catalytic scavengers directed against VX.

Direct Affinity Purification of Long-Acting PASylated Proteins with Therapeutic Potential Using L-Prolinamide for Mild Elution.

Both insufficient plasma half-life (circulation for only few hours or less) and laborious downstream purification can be bottleneck for biological drug development. We report a novel strategy for the efficient and gentle affinity purification of pharmacologically relevant proteins modified by PASylation for prolonged action in vivo. We previously described antibodies specific for Pro/Ala-rich sequences (PAS) covering a range of binding characteristics. Our present approach relies on a chromatography matrix functionalized with a low-affinity PAS-specific antibody Fab fragment for specific adsorption of the PASylated protein from a macromolecular mixture. With the complete absence of hydrophobic/aromatic or ionic groups in the PAS sequence epitope, binding is mediated by Van der Waals contacts and distinct hydrogen bonds only. Surprisingly, selective competitive elution is achieved by application of the highly soluble and biologically inactive imino acid derivative L-prolinamide. Based on the specific but strongly dynamic biomolecular interaction, our procedure allows the direct one-step purification of PASylated proteins from a cell extract or culture supernatant while avoiding harsh elution conditions as they are often needed for conventional affinity chromatography.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

The extracellular region of bovine milk butyrophilin exhibits closer structural similarity to human myelin oligodendrocyte glycoprotein than to immunological BTN family receptors.

Bovine butyrophilin (BTN1A1) is an abundant type I transmembrane glycoprotein exposed on the surface of milk fat globules. We have solved the crystal structure of its extracellular region via multiple wavelength anomalous dispersion after incorporation of selenomethionine into the bacterially produced protein. The butyrophilin ectodomain exhibits two subdomains with immunoglobulin fold, each comprising a ß-sandwich with a central disulfide bridge as well as one N-linked glycosylation. The fifth Cys residue at position 193 is unpaired and prone to forming disulfide crosslinks. The apparent lack of a ligand-binding site or receptor activity suggests a function predominantly as hydrophilic coat protein to prevent coagulation of the milk fat droplets. While there is less structural resemblance to members of the human butyrophilin family such as BTN3A, which play a role as immune receptors, the N-terminal bovine butyrophilin subdomain shows surprising similarity to the human myelin oligodendrocyte glycoprotein, a protein exposed on the surface of myelin sheaths. Thus, our study lends structural support to earlier hypotheses of a correlation between the consumption of cow milk and prevalence of neurological autoimmune diseases and may offer guidance for the breeding of cattle strains that express modified butyrophilin showing less immunological cross-reactivity.

Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro.

Highly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M-1 min-1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC-MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(-) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(-) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

Translating the Concept of Bispecific Antibodies to Engineering Heterodimeric Phosphotriesterases with Broad Organophosphate Substrate Recognition.

We have adopted the concept of bispecific antibodies, which can simultaneously block or cross-link two different biomolecular targets, to create bispecific enzymes by exploiting the homodimeric quaternary structure of bacterial phosphotriesterases (PTEs). The PTEs from Brevundimonas diminuta and Agrobacterium radiobacter, whose engineered variants can efficiently hydrolyze organophosphorus (OP) nerve agents and pesticides, respectively, have attracted considerable interest for the treatment of the corresponding intoxications. OP nerve agents and pesticides still pose a severe toxicological threat in military conflicts, including acts of terrorism, as well as in agriculture, leading to >100000 deaths per year. In principle, engineered conventional homodimeric PTEs may provoke hydrolytic inactivation of individual OPs in vivo, and their application as catalytic bioscavengers via administration into the bloodstream has been proposed. However, their narrow substrate specificity would necessitate therapeutic application of a set or mixture of different enzymes, which complicates biopharmaceutical development. We succeeded in combining subunits from both enzymes and to stabilize their heterodimerization by rationally designing electrostatic steering mutations, thus breaking the natural C2 symmetry. The resulting bispecific enzyme from two PTEs with different bacterial origin exhibits an ultrabroad OP substrate profile and allows the efficient detoxification of both nerve agents and pesticides. Our approach of combining two active sites with distinct substrate specificities within one artificial dimeric biocatalyst-retaining the size and general properties of the original enzyme without utilizing protein mixtures or much larger fusion proteins-not only should facilitate biological drug development but also may be applicable to oligomeric enzymes with other catalytic activities.

Scavenging Bacterial Siderophores with Engineered Lipocalin Proteins as an Alternative Antimicrobial Strategy.

Iron acquisition mediated by siderophores, high-affinity chelators for which bacteria have evolved specific synthesis and uptake mechanisms, plays a crucial role in microbiology and in host-pathogen interactions. In the ongoing fight against bacterial infections, this area has attracted biomedical interest. Beyond several approaches to interfere with siderophore-mediated iron uptake from medicinal and immunochemistry, the development of high-affinity protein scavengers that tightly complex the siderophores produced by pathogenic bacteria has appeared as a novel strategy. Such binding proteins have been engineered based on siderocalin-also known as lipocalin 2-an endogenous human scavenger of enterobactin and bacillibactin that controls the systemic spreading of commensal bacteria such as Escherichia coli. By using combinatorial protein design, siderocalin was reshaped to bind several siderophores from Pseudomonas aeruginosa and, in particular, petrobactin from Bacillus anthracis, none of which is recognized by the natural protein. Such engineered versions of siderocalin effectively suppress the growth of corresponding pathogenic bacteria by depriving them of their iron supply and offer the potential to complement antibiotic therapy in situations of acute or persistent infection.