An orally administered enzyme therapeutic for homocystinuria that suppresses homocysteine by metabolizing methionine in the gastrointestinal tract.

Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions. We describe the preclinical development of CDX-6512, a methionine gamma lyase (MGL) enzyme that was engineered for stability and activity within the GI tract for oral administration to locally degrade methionine. CDX-6512 is stable to low pH and intestinal proteases, enabling it to survive the harsh GI environment without enteric coating and to degrade methionine freed from dietary protein within the small intestine. Administering CDX-6512 to healthy non-human primates following a high protein meal led to a dose-dependent suppression of plasma methionine. In Tg-I278T Cbs-/- mice, an animal model that recapitulates aspects of HCU disease including highly elevated serum homocysteine levels, oral dosing of CDX-6512 after a high protein meal led to suppression in serum levels of both methionine and homocysteine. When animals received a daily dose of CDX-6512 with a high protein meal for two weeks, the Tg-I278T Cbs-/- mice maintained baseline homocysteine levels, whereas homocysteine levels in untreated animals increased by 39%. These preclinical data demonstrate the potential of CDX-6512 as an oral enzyme therapy for HCU.

Oral enzyme therapy for maple syrup urine disease (MSUD) suppresses plasma leucine levels in intermediate MSUD mice and healthy nonhuman primates.

Maple syrup urine disease (MSUD) is an inborn error of branched-chain amino acid metabolism affecting several thousand individuals worldwide. MSUD patients have elevated levels of plasma leucine and its metabolic product α-ketoisocaproate (KIC), which can lead to severe neurotoxicity, coma, and death. Patients must maintain a strict diet of protein restriction and medical formula, and periods of noncompliance or illness can lead to acute metabolic decompensation or cumulative neurological impairment. Given the lack of therapeutic options for MSUD patients, we sought to develop an oral enzyme therapy that can degrade leucine within the gastrointestinal tract prior to its systemic absorption and thus enable patients to maintain acceptable plasma leucine levels while broadening their access to natural protein. We identified a highly active leucine decarboxylase enzyme from Planctomycetaceae bacterium and used directed evolution to engineer the enzyme for stability to gastric and intestinal conditions. Following high-throughput screening of over 12 000 enzyme variants over 9 iterative rounds of evolution, we identified a lead variant, LDCv10, which retains activity following simulated gastric or intestinal conditions in vitro. In intermediate MSUD mice or healthy nonhuman primates given a whey protein meal, oral treatment with LDCv10 suppressed the spike in plasma leucine and KIC and reduced the leucine area under the curve in a dose-dependent manner. Reduction in plasma leucine correlated with decreased brain leucine levels following oral LDCv10 treatment. Collectively, these data support further development of LDCv10 as a potential new therapy for MSUD patients.

Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and alphavbeta3 integrin.

Significant cross-talk exists between receptors that mediate angiogenesis, such as VEGF receptor-2 (VEGFR2) and α(v)ß(3) integrin. Thus, agents that inhibit both receptors would have important therapeutic potential. Here, we used an antagonistic VEGF ligand as a molecular scaffold to engineer dual-specific proteins that bound to VEGFR2 and α(v)ß(3) integrin with antibody-like affinities and inhibited angiogenic processes in vitro and in vivo. Mutations were introduced into a single-chain VEGF (scVEGF) ligand that retained VEGFR2 binding, but prevented receptor dimerization and activation. Yeast-displayed scVEGF mutant libraries were created and screened by high-throughput flow cytometric sorting to identify several variants that bound with high affinity to both VEGFR2 and α(v)ß(3) integrin. These engineered scVEGF mutants were specific for α(v)ß(3) integrin and did not bind to the related integrins α(v)ß(5), α(iib)ß(3), or α(5)ß(1). In addition, surface plasmon resonance and cell binding assays showed that dual-specific scVEGF proteins can simultaneously engage both receptors. Compared to monospecific scVEGF mutants that bind VEGFR2 or α(v)ß(3) integrin, dual-specific scVEGF proteins more strongly inhibited VEGF-mediated receptor phosphorylation, capillary tube formation, and proliferation of endothelial cells cultured on Matrigel or vitronectin-coated surfaces. Moreover, dual specificity conferred strong inhibition of VEGF-mediated blood vessel formation in Matrigel plugs in vivo, whereas monospecific scVEGF mutants that bind VEGFR2 or α(v)ß(3) integrin were only marginally effective. Instead of relying on antibody associating domains or physical linkage, this work highlights an approach to creating dual-specific proteins where additional functionality is introduced into a protein ligand to complement its existing biological properties.

Identification of bacterial protease domains that cleave human IgM.

Immunoglobulin-degrading proteases are secreted by pathogenic bacteria to weaken the host immune response, contributing to immune evasion mechanisms during an infection. Proteases specific to IgG and IgA immunoglobulin classes have previously been identified and characterized, and only a single report exists on a porcine specific IgM-degrading enzyme. It is unclear whether human pathogens also produce enzymes that can break down human IgM. Here, we have identified four novel IgM-degrading proteases from different genera of human-infecting bacterial pathogens. All four protease domains cleave human IgM at a conserved and unique site in the constant region of IgM. These human IgM proteases may be a useful biochemical tool for the study of early immune responses and have therapeutic potential in IgM-mediated disease.

Optimizing human α-galactosidase for treatment of Fabry disease.

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease. GLAv05 and GLAv09 were identified after screening more than 12,000 GLA variants through 8 rounds of directed evolution. Both GLAv05 and GLAv09 exhibit increased stability at both lysosomal and blood pH, stability to serum, and elevated enzyme activity in treated Fabry fibroblasts (19-fold) and GLA-/- podocytes (10-fold). GLAv05 and GLAv09 show improved pharmacokinetics in mouse and non-human primates. In a Fabry mouse model, the optimized variants showed prolonged half-lives in serum and relevant tissues, and a decrease of accumulated Gb3 in heart and kidney. To explore the possibility of diminishing the immunogenic potential of rhGLA, amino acid residues in sequences predicted to bind MHC II were targeted in late rounds of GLAv09 directed evolution. An MHC II-associated peptide proteomics assay confirmed a reduction in displayed peptides for GLAv09. Collectively, our findings highlight the promise of using directed evolution to generate enzyme variants for more effective treatment of lysosomal storage diseases.

111In-labeled cystine-knot peptides based on the Agouti-related protein for targeting tumor angiogenesis.

Agouti-related protein (AgRP) is a 4-kDa cystine-knot peptide of human origin with four disulfide bonds and four solvent-exposed loops. The cell adhesion receptor integrin α(v)ß(3) is an important tumor angiogenesis factor that determines the invasiveness and metastatic ability of many malignant tumors. AgRP mutants have been engineered to bind to integrin α(v)ß(3) with high affinity and specificity using directed evolution. Here, AgRP mutants 7C and 6E were radiolabeled with (111)In and evaluated for in vivo targeting of tumor integrin α(v)ß(3) receptors. AgRP peptides were conjugated to the metal chelator 1, 4, 7, 10-tetra-azacyclododecane- N, N', N″, N'''-tetraacetic acid (DOTA) and radiolabeled with (111)In. The stability of the radiopeptides (111)In-DOTA-AgRP-7C and (111)In-DOTA-AgRP-6E was tested in phosphate-buffered saline (PBS) and mouse serum, respectively. Cell uptake assays of the radiolabeled peptides were performed in U87MG cell lines. Biodistribution studies were performed to evaluate the in vivo performance of the two resulting probes using mice bearing integrin-expressing U87MG xenograft tumors. Both AgRP peptides were easily labeled with (111)In in high yield and radiochemical purity (>99%). The two probes exhibited high stability in phosphate-buffered saline and mouse serum. Compared with (111)In-DOTA-AgRP-6E, (111)In-DOTA-AgRP-7C showed increased U87MG tumor uptake and longer tumor retention (5.74 ± 1.60 and 1.29 ± 0.02%ID/g at 0.5 and 24 h, resp.), which was consistent with measurements of cell uptake. Moreover, the tumor uptake of (111)In-DOTA-AgRP-7C was specifically inhibited by coinjection with an excess of the integrin-binding peptidomimetic c(RGDyK). Thus, (111)In-DOTA-AgRP-7C is a promising probe for targeting integrin α(v)ß(3) positive tumors in living subjects.

Cystine-knot peptides engineered with specificities for α(IIb)ß(3) or α(IIb)ß(3) and α(v)ß(3) integrins are potent inhibitors of platelet aggregation.

A truncated form of the Agouti-related protein (AgRP), a member of the cystine-knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg-Gly-Asp integrin recognition motif, and randomized the neighboring residues to create a library of approximately 20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high-throughput fluorescence-activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α(IIb)ß(3), a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K(D)) constants for α(IIb)ß(3) integrin ranging from 60 to 90 nM, and did not bind to α(v)ß(3), α(v)ß(5), or α(5)ß(1) integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α(IIb)ß(3) and α(v)ß(3) integrins with K(D) values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α(v)ß(5) or α(5)ß(1) integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA-approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine-knot peptides can be generated with high affinity and specificity to closely-related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors.

APOE immunotherapy reduces cerebral amyloid angiopathy and amyloid plaques while improving cerebrovascular function.

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and greatly influences the development of amyloid-ß (Aß) pathology. Our current study investigated the potential therapeutic effects of the anti-human APOE antibody HAE-4, which selectively recognizes human APOE that is co-deposited with Aß in cerebral amyloid angiopathy (CAA) and parenchymal amyloid pathology. In addition, we tested whether HAE-4 provoked brain hemorrhages, a component of amyloid-related imaging abnormalities (ARIA). ARIA is an adverse effect secondary to treatment with anti-Aß antibodies that can occur in blood vessels with CAA. We used 5XFAD mice expressing human APOE4 +/+ (5XE4) that have prominent CAA and parenchymal plaque pathology to assess the efficacy of HAE-4 compared to an Aß antibody that removes parenchymal Aß but increases ARIA in humans. In chronically treated 5XE4 mice, HAE-4 reduced Aß deposition including CAA compared to a control antibody, whereas the anti-Aß antibody had no effect on CAA. Furthermore, the anti-Aß antibody exacerbated microhemorrhage severity, which highly correlated with reactive astrocytes surrounding CAA. In contrast, HAE-4 did not stimulate microhemorrhages and instead rescued CAA-induced cerebrovascular dysfunction in leptomeningeal arteries in vivo. HAE-4 not only reduced amyloid but also dampened reactive microglial, astrocytic, and proinflammatory-associated genes in the cortex. These results suggest that targeting APOE in the core of both CAA and plaques could ameliorate amyloid pathology while protecting cerebrovascular integrity and function.

Interrogating and predicting tolerated sequence diversity in protein folds: application to E. elaterium trypsin inhibitor-II cystine-knot miniprotein.

Cystine-knot miniproteins (knottins) are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI) as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering.

Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys.

Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.

Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Developing therapeutic proteins by engineering ligand-receptor interactions.

Ligand-receptor interactions govern myriad cell signaling pathways that regulate homeostasis and ensure that cells respond properly to stimuli. Growth factors, cytokines and other regulatory elements use these interactions to mediate cell responses, including proliferation, migration, angiogenesis, immune responses and cell death. Proteins that inhibit these processes have potential as therapeutics for cancer and autoimmune disorders, whereas proteins that stimulate these processes offer promise in regenerative medicine. Although much of the focus in this area over the past decade has been on monoclonal antibodies, recently there has been increased interest in the use of non-antibody proteins as therapeutic agents. Here, we review recent advances and accomplishments in the use of rational and combinatorial protein engineering approaches to developing ligands and receptors as agonists and antagonists against clinically important targets.

Quenched autoligation probes.

Methods are described for preparation and use of quenched autoligation (QUAL) probes. These modified oligonucleotide fluorescent probes can be used to detect DNA and RNA in solution, on solid surfaces, and in fixed and living bacterial and human cells. They are quenched probes, and thus provide a "lighting up" signal in a single step, without removing unbound or unreacted probes from the analyte. QUAL probe signals can be detected by fluorescence spectrometer, fluorescence microscope, or flow cytometry. These probes can distinguish between very small variations, including single nucleotide differences, in nucleic acid targets. The described method includes a description of how to prepare the needed dabsyl quencher linker, how to prepare the QUAL probes by DNA synthesizer, and how to employ them in detecting nucleic acids in solution and in detecting RNAs in bacterial and human cells.

New, stronger nucleophiles for nucleic acid-templated chemistry: Synthesis and application in fluorescence detection of cellular RNA.

Nucleic acid-templated chemistry is a promising strategy for imaging genetic sequences in living cells. Here we describe the synthesis of two new nucleophiles for use in templated nucleophilic displacements with DNA probes. The nucleophilic groups are phosphorodithioate and phosphorotrithioate; we report on synthetic methods for introducing these groups at the 3'-terminus of oligonucleotides. Both new nucleophiles are found to be more highly reactive than earlier phosphoromonothioates. This increased nucleophilicity is shown to result in more rapid templated reactions with electrophilic DNA probes. The new probes were demonstrated in detection of specific genetic sequences in solution, with clear signal over background being generated in as little as 20 min. The probes were also tested for imaging ribosomal RNA sequences in live Escherichia coli; useful signal was generated in 20 min to 1h, approximately one quarter to one-half the time of earlier monothioate probes, and the signal-to-noise ratio was increased as well.

Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation.

The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid ß (Aß) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aß pathology in mice producing both human Aß and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aß deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aß pathology and may represent a selective approach to treat AD.

Quenched autoligation probes allow discrimination of live bacterial species by single nucleotide differences in rRNA.

Quenched autoligation (QUAL) probes are a class of self-reacting nucleic acid probes that give strong fluorescence signal in the presence of fully complementary RNAs and selectivity against single nucleotide differences in solution. Here, we describe experiments designed to test whether QUAL probes can discriminate between bacterial species by the detection of small differences in their 16S rRNA sequences. Probes were introduced into live cells using small amounts of detergent, thus eliminating the need for fixation, and fluorescence signal was monitored both by microscopy and by flow cytometry without any washing steps. The effects of probe length, modified backbone, probe concentration and growth state of the bacteria were investigated. The data demonstrate specific fluorescence discrimination between three closely related bacteria, Escherichia coli, Salmonella enterica and Pseudomonas putida, based on single nucleotide differences in their 16S rRNA. Discrimination was possible with cells in mid-log phase or in lag phase. These results suggest that QUAL probes may be useful for rapid identification of microorganisms in laboratory and clinical settings.

Engineered ligand-based VEGFR antagonists with increased receptor binding affinity more effectively inhibit angiogenesis.

Pathologic angiogenesis is mediated by the coordinated action of the vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling axis, along with crosstalk contributed by other receptors, notably αvß3 integrin. We build on earlier work demonstrating that point mutations can be introduced into the homodimeric VEGF ligand to convert it into an antagonist through disruption of binding to one copy of VEGFR2. This inhibitor has limited potency, however, due to loss of avidity effects from bivalent VEGFR2 binding. Here, we used yeast surface display to engineer a variant with VEGFR2 binding affinity approximately 40-fold higher than the parental antagonist, and 14-fold higher than the natural bivalent VEGF ligand. Increased VEGFR2 binding affinity correlated with the ability to more effectively inhibit VEGF-mediated signaling, both in vitro and in vivo, as measured using VEGFR2 phosphorylation and Matrigel implantation assays. High affinity mutations found in this variant were then incorporated into a dual-specific antagonist that we previously designed to simultaneously bind to and inhibit VEGFR2 and αvß3 integrin. The resulting dual-specific protein bound to human and murine endothelial cells with relative affinities of 120 ± 10 pM and 360 ± 50 pM, respectively, which is at least 30-fold tighter than wild-type VEGF (3.8 ± 0.5 nM). Finally, we demonstrated that this engineered high-affinity dual-specific protein could inhibit angiogenesis in a murine corneal neovascularization model. Taken together, these data indicate that protein engineering strategies can be combined to generate unique antiangiogenic candidates for further clinical development.

Quenched probes for highly specific detection of cellular RNAs.

Nucleic acid-based RNA detection is a promising field in molecular biotechnology that is leading to the rapid and accurate identification of microorganisms, diagnosis of infections and imaging of gene expression. The specificity of short synthetic DNA probes raises the hope of distinguishing small differences in sequence, ultimately achieving single nucleotide resolution. Recent work using quenched fluorescently labeled oligonucleotide probes as sensors for RNA in bacterial and human cells has overcome several difficult hurdles on the way to these goals, including delivery of probes to live cells, accessing RNA sites containing a high degree of secondary structure, and eliminating many sources of background. Two new classes of quenched oligonucleotide probes, molecular beacons and quenched auto-ligation probes, have shown the most promise for in situ RNA detection. High-specificity detection, at the single-nucleotide resolution level, is now possible in solution with these classes of probes. However, for applications in intact cells, signal and background issues still need to be addressed before the full potential of these methods is achieved.

Evaluation of a (64)Cu-labeled cystine-knot peptide based on agouti-related protein for PET of tumors expressing alphavbeta3 integrin.

UNLABELLED: Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to alpha(v)beta(3) integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects. METHODS: Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetra-azacyclododecane-N,N',N''N'''-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with (64)Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of (64)Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine. RESULTS: AgRP-7C and DOTA-AgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 +/- 4 and 14 +/- 2 nM, respectively). DOTA-AgRP-7C was labeled with (64)Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, (64)Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 +/- 0.93 percentage injected dose per gram [%ID/g] and 2.37 +/- 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of (64)Cu-DOTA-AgRP-7C was confirmed in vitro and in vivo by showing that a large molar excess of the unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications. CONCLUSION: A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the alpha(v)beta(3) integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.

Engineered cystine-knot peptides that bind alpha(v)beta(3) integrin with antibody-like affinities.

The alpha(v)beta(3) integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4-kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to alpha(v)beta(3) integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a six amino acid loop of AgRP with a nine amino acid loop containing the Arg-Gly-Asp integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting to identify clones with high affinity to detergent-solubilized alpha(v)beta(3) integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing alpha(v)beta(3) integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown to bind specifically to alpha(v)beta(3) integrins and had only minimal or no binding to alpha(v)beta(5), alpha(5)beta(1), and alpha(iib)beta(3) integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally occurring ligand for alpha(v)beta(3) and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second-generation libraries by individually randomizing these loops in one of the high-affinity integrin-binding variants. Screening of these loop-randomized libraries against alpha(v)beta(3) integrins resulted in peptides that retained high affinities for alpha(v)beta(3) and had increased specificities for alpha(v)beta(3) over alpha(iib)beta(3) integrins. Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target.