PSMA-reactive NB7 single domain antibody fragment: A potential scaffold for developing prostate cancer theranostics.

INTRODUCTION: Single domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their 211At-labeled analogues. METHODS: [*I]SGMIB, iso-[*I]SGMIB, [211At]SAGMB, and iso-[211At]SAGMB conjugates of NB7 sdAb were synthesized and their binding affinity, cell uptake and internalization were assessed in PSMA+ PC3 PIP and PSMA- PC3 flu cells. Biodistribution studies were performed in mice bearing PSMA+ PC3 PIP xenografts. First, a single-label experiment evaluated the tissue distribution of a NB7 bearing a His6-tag (NB7H6) and labeled with iso-[125I]SGMIB. Three paired-label experiments then were performed to compare: a) NB7 labeled using [*I]SGMIB and iso-[*I]SGMIB, b) 131I- vs 211At-labeled NB7 conjugates and c) [125I]SGMIB-NB7H6 to the small molecule PSMA inhibitor [131I]YF2. RESULTS: All NB7 radioconjugates bound specifically to PSMA with dissociation constants, Kd, in the low nM range (1.4-6.4 nM). An initial biodistribution study demonstrated good tumor uptake for iso-[125I]SGMIB-NB7H6 (7.2 ± 1.5 % ID/g at 1 h) and no deleterious effect of the His6-tag on renal activity levels, which declined to 3.1 ± 1.1 % ID/g by 4 h. Paired-label biodistribution found no distinction between the two SGMIB isomer NB7 conjugates with the [131I]SGMIB-NB7-to-iso-[125I]SGMIB-NB7 tumor uptake ratios not significantly different from unity: 1.06 ± 0.08 at 1 h, 1.04 ± 0.12 at 4 h, and 1.07 ± 0.09 at 24 h. Both isomer conjugates cleared rapidly from normal tissues and exhibited very low uptake in thyroid, lacrimal and salivary glands. Paired-label biodistribution of [131I]SGMIB-NB7H6 and [211At]SAGMB-NB7H6 demonstrated similar tumor uptake and kidney clearance for the two radioconjugates. However, levels of 211At in thyroid, stomach, salivary and lacrimal glands were significantly higher (P < 0.05) that those for 131I suggesting greater dehalogenation for [211At]SAGMB-NB7H6. Finally, co-administration of [125I]SGMIB-NB7H6 and [131I]YF2 demonstrated good tumor uptake for both with considerably more rapid renal clearance for the NB7 radioconjugate. CONCLUSION: NB7 radioconjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. We conclude that NB7 is a potentially useful scaffold for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches.

Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers.

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.

Developing a dual VEGF/PDL1 inhibitor based on high-affinity scFv heterodimers as an anti-cancer therapeutic strategy.

Cancer progression is enhanced by the interaction of programmed death-ligand 1 (PDL1), which is associated with inhibition of the immune response against tumors, and vascular endothelial growth factor (VEGF), which inhibits immune cell activity while inducing angiogenesis and proliferation of cancer cells. Dual inhibition of PDL1 and VEGF may therefore confer a synergistic anti-cancer therapeutic effect. We present a novel strategy for developing a therapeutic that simultaneously binds and inhibits both PDL1 and VEGF. We generated a bi-specific protein, designated DuRan-Bis, comprising a single chain variable fragment (scFv)-based inhibitor of PDL1 fused to an scFv-based inhibitor of VEGF, with the latter being attached to an Fc fragment. We found that DuRan-Bis binds to both PDL1 and VEGF with high affinity. Compared to treatments with mono-specific proteins, alone or in combination, the DuRan-Bis chimera showed superior inhibition of the proliferation of glioblastoma cells. In comparison to treatment with immune cells alone, a combination of immune cells with DuRan-Bis decreased the viability of head and neck cancer cells. To the best of our knowledge, this study is the first to use a single polypeptide chain scFv-scFv-Fc scaffold for engineering a high-affinity bi-specific inhibitor of PDL1 and VEGF.

In vitro inhibition of cancer angiogenesis and migration by a nanobody that targets the orphan receptor Tie1.

The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.

Bioorthogonal PEGylation Prolongs the Elimination Half-Life of N-TIMP2 While Retaining MMP Inhibition.

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of the matrix metalloproteinase (MMP) family of proteins, whose members are key regulators of the proteolysis of extracellular matrix components and hence of multiple biological processes. In particular, imbalanced activity of matrix metalloproteinase-14 (MMP-14) may lead to the development of cancer and cardiovascular and other diseases. This study aimed to engineer TIMP2, one of the four homologous TIMPs, as a potential therapeutic by virtue of its ability to bind to the active-site Zn2+ of MMP-14. However, the susceptibility to degradation of TIMP2 and its small size, which results in a short circulation half-life, limit its use as a therapeutic. PEGylation was thus used to improve the pharmacokinetic profile of TIMP2. PEGylation of the MMP-targeting N-terminal domain of TIMP2 (N-TIMP2), via either cysteine or lysine residues, resulted in a significant decrease in N-TIMP2 affinity toward MMP-14 or multisite conjugation and conjugate heterogeneity, respectively. Our strategy designed to address this problem was based on incorporating a noncanonical amino acid (NCAA) into N-TIMP2 to enable site-specific mono-PEGylation. The first step was to incorporate the NCAA propargyl lysine (PrK) at position S31 in N-TIMP2, which does not interfere with the N-TIMP2-MMP-14 binding interface. Thereafter, site-specific PEGylation was achieved via a click chemistry reaction between N-TIMP2-S31PrK and PEG-azide-20K. Inhibition studies showed that PEGylated N-TIMP2-S31PrK did indeed retain its inhibitory activity toward MMP-14. The modified protein also showed improved serum stability vs non-PEGylated N-TIMP2. In vivo pharmacokinetic studies in mice revealed a significant 8-fold increase in the elimination half-life of PEGylated N-TIMP2 vs the non-PEGylated protein. This study shows that site-specific bioorthogonal mono-PEGylation extends the half-life of N-TIMP2 without impairing its biological activity, thereby highlighting the advantage of this strategy for generating potent PEGylated proteins.

Modifying pH-sensitive PCSK9/LDLR interactions as a strategy to enhance hepatic cell uptake of low-density lipoprotein cholesterol (LDL-C).

LDL-receptor (LDLR)-mediated uptake of LDL-C into hepatocytes is impaired by lysosomal degradation of LDLR, which is promoted by proprotein convertase subtilisin/kexin type 9 (PCSK9). Cell surface binding of PCSK9 to LDLR produces a complex that translocates to an endosome, where the acidic pH strengthens the binding affinity of PCSK9 to LDLR, preventing LDLR recycling to the cell membrane. We present a new approach to inhibit PCSK9-mediated LDLR degradation, namely, targeting the PCSK9/LDLR interface with a PCSK9-antagonist, designated Flag-PCSK9PH, which prevents access of WT PCSK9 to LDLR. In HepG2 cells, Flag-PCSK9PH, a truncated version (residues 53-451) of human WT PCSK9, strongly bound LDLR at the neutral pH of the cell surface but dissociated from it in the endosome (acidic pH), allowing LDLR to exit the lysosomes intact and recycle to the cell membrane. Flag-PCSK9PH thus significantly enhanced cell-surface LDLR levels and the ability of LDLR to take up extracellular LDL-C.

A KLK4 proteinase substrate capture approach to antagonize PAR1.

Proteinase-activated receptor-1 (PAR1), triggered by thrombin and other serine proteinases such as tissue kallikrein-4 (KLK4), is a key driver of inflammation, tumor invasiveness and tumor metastasis. The PAR1 transmembrane G-protein-coupled receptor therefore represents an attractive target for therapeutic inhibitors. We thus used a computational design to develop a new PAR1 antagonist, namely, a catalytically inactive human KLK4 that acts as a proteinase substrate-capture reagent, preventing receptor cleavage (and hence activation) by binding to and occluding the extracellular R41-S42 canonical PAR1 proteolytic activation site. On the basis of in silico site-saturation mutagenesis, we then generated KLK4S207A,L185D, a first-of-a-kind 'decoy' PAR1 inhibitor, by mutating the S207A and L185D residues in wild-type KLK4, which strongly binds to PAR1. KLK4S207A,L185D markedly inhibited PAR1 cleavage, and PAR1-mediated MAPK/ERK activation as well as the migration and invasiveness of melanoma cells. This 'substrate-capturing' KLK4 variant, engineered to bind to PAR1, illustrates proof of principle for the utility of a KLK4 'proteinase substrate capture' approach to regulate proteinase-mediated PAR1 signaling.

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma.

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

The pro-apoptotic domain of BIM protein forms toxic amyloid fibrils.

BIM is a key apoptotic protein, participating in diverse cellular processes. Interestingly, recent studies have hypothesized that BIM is associated with the extensive neuronal cell death encountered in protein misfolding diseases, such as Alzheimer's disease. Here, we report that the core pro-apoptotic domain of BIM, the BIM-BH3 motif, forms ubiquitous amyloid fibrils. The BIM-BH3 fibrils exhibit cytotoxicity, disrupt mitochondrial functions, and modulate the structures and dynamics of mitochondrial membrane mimics. Interestingly, a slightly longer peptide in which BIM-BH3 was flanked by four additional residues, widely employed as a model of the pro-apoptotic core domain of BIM, did not form fibrils, nor exhibited cell disruptive properties. The experimental data suggest a new mechanistic role for the BIM-BH3 domain, and demonstrate, for the first time, that an apoptotic peptide forms toxic amyloid fibrils.

Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies.

Receptor tyrosine kinases (RTKs) are major players in signal transduction, regulating cellular activities in both normal regeneration and malignancy. Thus, many RTKs, c-Kit among them, play key roles in the function of both normal and neoplastic cells, and as such constitute attractive targets for therapeutic intervention. We thus sought to manipulate the self-association of stem cell factor (SCF), the cognate ligand of c-Kit, and hence its suboptimal affinity and activation potency for c-Kit. To this end, we used directed evolution to engineer SCF variants having different c-Kit activation potencies. Our yeast-displayed SCF mutant (SCFM) library screens identified altered dimerization potential and increased affinity for c-Kit by specific SCF-variants. We demonstrated the delicate balance between SCF homo-dimerization, c-Kit binding, and agonistic potencies by structural studies, in vitro binding assays and a functional angiogenesis assay. Importantly, our findings showed that a monomeric SCF variant exhibited superior agonistic potency vs. the wild-type SCF protein and vs. other high-affinity dimeric SCF variants. Our data showed that action of the monomeric ligands in binding to the RTK monomers and inducing receptor dimerization and hence activation was superior to that of the wild-type dimeric ligand, which has a higher affinity to RTK dimers but a lower activation potential. The findings of this study on the binding and c-Kit activation of engineered SCF variants thus provides insights into the structure-function dynamics of ligands and RTKs.

Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer.

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA+ cells and that accumulate specifically in PSMA+ tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA+ cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

PRSS3/Mesotrypsin and kallikrein-related peptidase 5 are associated with poor prognosis and contribute to tumor cell invasion and growth in lung adenocarcinoma.

Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.

An Engineered Variant of the B1 Domain of Protein G Suppresses the Aggregation and Toxicity of Intra- and Extracellular Aß42.

Intra- and extraneuronal deposition of amyloid ß (Aß) peptides have been linked to Alzheimer's disease (AD). While both intra- and extraneuronal Aß deposits affect neuronal cell viability, the molecular mechanism by which these Aß structures, especially when intraneuronal, do so is still not entirely understood. This makes the development of inhibitors challenging. To prevent the formation of toxic Aß structural assemblies so as to prevent neuronal cell death associated with AD, we used a combination of computational and combinatorial-directed evolution approaches to develop a variant of the HTB1 protein (HTB1M2). HTB1M2 inhibits in vitro self-assembly of Aß42 peptide and shifts the Aß42 aggregation pathway to the formation of oligomers that are nontoxic to neuroblastoma SH-SY5Y cells overexpressing or treated with Aß42 peptide. This makes HTB1M2 a potential therapeutic lead in the development of AD-targeted drugs and a tool for elucidating conformational changes in the Aß42 peptide.

Residue-level determinants of angiopoietin-2 interactions with its receptor Tie2.

We combined computational and experimental methods to interrogate the binding determinants of angiopoietin-2 (Ang2) to its receptor tyrosine kinase (RTK) Tie2-a central signaling system in angiogenesis, inflammation, and tumorigenesis. We used physics-based electrostatic and surface-area calculations to identify the subset of interfacial Ang2 and Tie2 residues that can affect binding directly. Using random and site-directed mutagenesis and yeast surface display (YSD), we validated these predictions and identified additional Ang2 positions that affected receptor binding. We then used burial-based calculations to classify the larger set of Ang2 residues that are buried in the Ang2 core, whose mutations can perturb the Ang2 structure and thereby affect interactions with Tie2 indirectly. Our analysis showed that the Ang2-Tie2 interface is dominated by nonpolar contributions, with only three Ang2 and two Tie2 residues that contribute electrostatically to intermolecular interactions. Individual interfacial residues contributed only moderately to binding, suggesting that engineering of this interface will require multiple mutations to reach major effects. Conversely, substitutions in substantially buried Ang2 residues were more prevalent in our experimental screen, reduced binding substantially, and are therefore more likely to have a deleterious effect that might contribute to oncogenesis. Computational analysis of additional RTK-ligand complexes, c-Kit-SCF and M-CSF-c-FMS, and comparison to previous YSD results, further show the utility of our combined methodology.

Depsipeptides Featuring a Neutral P1 Are Potent Inhibitors of Kallikrein-Related Peptidase 6 with On-Target Cellular Activity.

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease that belongs to the family of tissue kallikreins (KLKs). Many KLKs are investigated as potential biomarkers for cancer as well as therapeutic drug targets for a number of pathologies. KLK6, in particular, has been implicated in neurodegenerative diseases and cancer, but target validation has been hampered by a lack of selective inhibitors. This work introduces a class of depsipeptidic KLK6 inhibitors, discovered via high-throughput screening, which were found to function as substrate mimics that transiently acylate the catalytic serine of KLK6. Detailed structure-activity relationship studies, aided by in silico modeling, uncovered strict structural requirements for potency, stability, and acyl-enzyme complex half-life. An optimized scaffold, DKFZ-251, demonstrated good selectivity for KLK6 compared to other KLKs, and on-target activity in a cellular assay. Moreover, DKFZ-633, an inhibitor-derived activity-based probe, could be used to pull down active endogenous KLK6.

Combinatorial engineering of N-TIMP2 variants that selectively inhibit MMP9 and MMP14 function in the cell.

Developing selective inhibitors for proteolytic enzymes that share high sequence homology and structural similarity is important for achieving high target affinity and functional specificity. Here, we used a combination of yeast surface display and dual-color selective library screening to obtain selective inhibitors for each of the matrix metalloproteinases (MMPs) MMP14 and MMP9 by modifying the non-specific N-terminal domain of the tissue inhibitor of metalloproteinase-2 (N-TIMP2). We generated inhibitor variants with 30- to 1175-fold improved specificity to each of the proteases, respectively, relative to wild type N-TIMP2. These biochemical results accurately predicted the selectivity and specificity obtained in cell-based assays. In U87MG cells, the activation of MMP2 by MMP14 was inhibited by MMP14-selective blockers but not MMP9-specific inhibitors. Target specificity was also demonstrated in MCF-7 cells stably expressing either MMP14 or MMP9, with only the MMP14-specific inhibitors preventing the mobility of MMP14-expressing cells. Similarly, the mobility of MMP9-expressing cells was inhibited by the MMP9-specific inhibitors, yet was not altered by the MMP14-specific inhibitors. The strategy developed in this study for improving the specificity of an otherwise broad-spectrum inhibitor will likely enhance our understanding of the basis for target specificity of inhibitors to proteolytic enzymes, in general, and to MMPs, in particular. We, moreover, envision that this study could serve as a platform for the development of next-generation, target-specific therapeutic agents. Finally, our methodology can be extended to other classes of proteolytic enzymes and other important target proteins.

An Aß42 variant that inhibits intra- and extracellular amyloid aggregation and enhances cell viability.

Aggregation and accumulation of the 42-residue amyloid ß peptide (Aß42) in the extracellular matrix and within neuronal cells is considered a major cause of neuronal cell cytotoxicity and death in Alzheimer's disease (AD) patients. Therefore, molecules that bind to Aß42 and prevent its aggregation are therapeutically promising as AD treatment. Here, we show that a non-self-aggregating Aß42 variant carrying two surface mutations, F19S and L34P (Aß42DM), inhibits wild-type Aß42 aggregation and significantly reduces Aß42-mediated cell cytotoxicity. In addition, Aß42DM inhibits the uptake and internalization of extracellularly added pre-formed Aß42 aggregates into cells. This was the case in both neuronal and non-neuronal cells co-expressing Aß42 and Aß42DM or following pre-treatment of cells with extracellular soluble forms of the two peptides, even at high Aß42 to Aß42DM molar ratios. In cells, Aß42DM associates with Aß42, while in vitro, the two soluble recombinant peptides exhibit nano-molar binding affinity. Importantly, Aß42DM potently suppresses Aß42 amyloid aggregation in vitro, as demonstrated by thioflavin T fluorescence and transmission electron microscopy for detecting amyloid fibrils. Overall, we present a new approach for inhibiting Aß42 fibril formation both within and outside cells. Accordingly, Aß42DM should be evaluated in vivo for potential use as a therapeutic lead for treating AD.

Targeting the Tie2-αvß3 integrin axis with bi-specific reagents for the inhibition of angiogenesis.

BACKGROUND: Increased activity of the receptor tyrosine kinase Tie2 has been implicated in the promotion of pathological angiogenesis. This activity is mainly mediated through angiopoietin (Ang)1- and Ang2-dependent activation of integrins by Tie2, rendering the Ang/Tie2/integrin axis an attractive putative target for cancer therapeutics. RESULTS: To target this axis, we developed single domain, non-immunoglobulin high-affinity bi-specific protein inhibitors against both Tie2 and αvß3 integrin. We have previously engineered the Ang2-binding domain of Tie2 (Ang2-BD) as a Tie2 inhibitor. Here, we engineered an exposed loop in Ang2-BD to generate variants that include an integrin-binding Arg-Gly-Asp (RGD) motif and used flow cytometry screening of a yeast-displayed Ang2-BD RGD loop library to identify the integrin antagonists. The bi-specific antagonists targeting both Tie2 and αvß3 integrin inhibited adhesion and proliferation of endothelial cells cultured together with the αvß3 integrin ligand vitronectin, as well as endothelial cell invasion and tube formation. The bi-specific reagents inhibited downstream signaling by Tie2 intracellularly in response to its agonist Ang1 more effectively than the wild-type Ang2 BD that binds Tie2 alone. CONCLUSIONS: Collectively, this study-the first to describe inhibitors targeting all the known functions resulting from Tie2/integrin αvß3 cross-talk-has created new tools for studying Tie2- and integrin αvß3-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand-Tie2 and ligand-integrin αvß3 receptor interactions. Given the roles of these pathways in cancer angiogenesis and metastasis, this proof of principle study paves the route to create novel Tie2/integrin αvß3-targeting proteins for clinical use as imaging and therapeutic agents.

A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and αvß3 integrin for osteoporosis therapy.

There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and αvß3 integrin, which act in concert to facilitate bone resorption by osteoclasts. Using functional fluorescence-activated cell sorting (FACS)-based screening assays of random mutagenesis macrophage colony-stimulating factor (M-CSF) libraries against c-FMS and αvß3 integrin, we engineered dual-specific M-CSF mutants with high affinity to both receptors. These bispecific mutants act as functional antagonists of c-FMS and αvß3 integrin activation and hence of osteoclast differentiation in vitro and osteoclast activity in vivo. This study thus introduces a versatile platform for the creation of new-generation therapeutics with high efficacy and specificity for osteoporosis and other bone diseases. It also provides new tools for studying molecular mechanisms and the cell signaling pathways that mediate osteoclast differentiation and function.