Jennifer Cochran is an associate professor of bioengineering, and has a secondary appointment in chemical engineering. Her research group uses interdisciplinary approaches in chemistry, engineering, and biophysics to study complex biological systems and develop new technologies for basic science and biomedical applications. Professor Cochran's research is driven by the philosophy that in order to effectively control physiological processes it is necessary to understand the molecular mechanisms that drive these processes. Her group is interested in elucidating molecular details of receptor-mediated cell signaling events; at the same time developing protein and peptide-based tools that will allow manipulation of cellular processes on a molecular level. For biomedical applications, rational design and combinatorial methods are used to create designer protein therapeutics and diagnostic agents for applications such as regenerative medicine and cancer imaging and therapy.
Co-Director, Stanford-NIST Pre-Doctoral Training Grant (2015 - Present)
Director, Stanford-NIH Biotechnology Predoctoral Training Grant (2014 - Present)
Director of Graduate Studies, Stanford Bioengineering (2014 - Present)
Honors & Awards
Howard Temin Award, NIH / National Cancer Institute (2004)
Translational Partnership Award, Wallace H. Coulter Foundation (2006, 2007)
Kimmel Scholars Award, Sidney Kimmel Foundation (2007)
Mallinckrodt Faculty Scholar Award, Edward Mallinckrodt Jr. Foundation (2007)
McCormick Award, McCormick Foundation (2007)
Hellman Faculty Scholar Award, Hellman Foundation (2008)
Martin D. Abeloff Scholar Award, V Foundation (2008)
Postdoctoral Fellow, MIT, Biological Engineering
Ph. D., MIT, Biological Chemistry (2001)
B.S., University of Delaware, Biochemistry (1995)
Current Research and Scholarly Interests
The Cochran laboratory uses interdisciplinary approaches in chemistry, engineering, and biophysics to study complex biological systems. Our main goals are to develop new technologies for basic science and biomedical applications. Clinical applications of our research involves wound healing, cardiac tissue regeneration, ocular disease, and cancer imaging and therapy. Our research is driven by the philosophy that in order to control physiological processes it is necessary to understand the molecular mechanisms that drive these processes. We are interested in elucidating molecular details of receptor-mediated cell signaling events; at the same time developing protein and peptide-based tools that will allow us to manipulate cellular processes on a molecular level. For biomedical applications, we are combining rational design and combinatorial methods to create designer protein therapeutics and diagnostic agents. Examples of our work are highlighted here: http://med.stanford.edu/ism/2013/august/flashlight.html and http://news.stanford.edu/news/2014/september/metastasis-protein-therapy-092114.html.
Independent Studies (11)
- Bioengineering Problems and Experimental Investigation
BIOE 191 (Aut, Win, Spr, Sum)
- Directed Investigation
BIOE 392 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Sum)
- Directed Study
BIOE 391 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Bioengineering Problems and Experimental Investigation
- Prior Year Courses
High-throughput analysis and protein engineering using microcapillary arrays.
Nature chemical biology
2016; 12 (2): 76-81
We describe a multipurpose technology platform, termed μSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. μSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With μSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the μSCALE platform.
View details for DOI 10.1038/nchembio.1978
View details for PubMedID 26641932
- An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis NATURE CHEMICAL BIOLOGY 2014; 10 (11): 977-983
Beyond antibodies: using biological principles to guide the development of next-generation protein therapeutics.
Current opinion in biotechnology
2013; 24 (6): 1072-1077
Protein-based biologics, which leverage the inherent affinity and specificity of protein-protein interactions, offer an effective strategy for targeting and modulating disease pathways. Despite the broad diversity of the proteome, monoclonal antibodies have been the major focus of such drug discovery efforts. While antibodies have shown great clinical value, the breadth and complexity of human disease highlight the need for alternatives that expand the therapeutic repertoire beyond this single class of proteins. The elucidation of molecular mechanisms underlying human disease has provided new opportunities for protein-based drugs to address challenging clinical problems. Natural ligands and receptors, which inherently modulate complex biological processes, have emerged as promising candidates for protein-based drug discovery efforts. Protein engineering strategies, guided by biological principles, are allowing ligands and receptors to be developed as next-generation therapeutics with improved safety and efficacy.
View details for DOI 10.1016/j.copbio.2013.03.017
View details for PubMedID 23587963
Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma.
Proceedings of the National Academy of Sciences of the United States of America
2013; 110 (36): 14598-14603
Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvβ3, αvβ5, and α5β1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5β1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.
View details for DOI 10.1073/pnas.1311333110
View details for PubMedID 23950221
Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and alpha(v)beta(3) integrin
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (34): 14067-14072
Significant cross-talk exists between receptors that mediate angiogenesis, such as VEGF receptor-2 (VEGFR2) and α(v)β(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)β(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)β(3) integrin. These engineered scVEGF mutants were specific for α(v)β(3) integrin and did not bind to the related integrins α(v)β(5), α(iib)β(3), or α(5)β(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)β(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)β(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.
View details for DOI 10.1073/pnas.1016635108
View details for Web of Science ID 000294163500045
View details for PubMedID 21825147
Engineering hepatocyte growth factor fragments with high stability and activity as Met receptor agonists and antagonists
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (32): 13035-13040
The Met receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) play an important role in mediating both tumor progression and tissue regeneration. The N-terminal and first Kringle domains (NK1) of HGF comprise a naturally occurring splice variant that retains the ability to activate the Met receptor. However, NK1 is a weak agonist and is relatively unstable, limiting its therapeutic potential. Here, we engineered NK1 mutants with improved biochemical and biophysical properties that function as Met receptor agonists or antagonists. We first engineered NK1 for increased stability and recombinant expression yield using directed evolution. The NK1 variants isolated from our library screens acted as weak Met receptor antagonists due to a mutation at the NK1 homodimerization interface. We introduced point mutations that restored this NK1 homodimerization interface to create an agonistic ligand, or that further disrupted this interface to create more effective antagonists. The rationally engineered antagonists exhibited melting temperatures up to approximately 64 °C, a 15 °C improvement over antagonists derived from wild-type NK1, and approximately 40-fold improvement in expression yield. Next, we created disulfide-linked NK1 homodimers through introduction of an N-terminal cysteine residue. These covalent dimers exhibited nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. Moreover, covalent NK1 dimers formed from agonistic or antagonistic monomeric subunits elicited similar activity, further signifying that NK1 dimerization mediates agonistic activity. These engineered NK1 proteins are promising candidates for therapeutic development and will be useful tools for further exploring determinants of Met receptor activation.
View details for DOI 10.1073/pnas.1102561108
View details for Web of Science ID 000293691400022
View details for PubMedID 21788476
Engineered Proteins Pull Double Duty
SCIENCE TRANSLATIONAL MEDICINE
2010; 2 (17)
Myriad bi-specific proteins have been developed that recognize two different clinical targets, with the goal of achieving enhanced therapeutic effects as compared with proteins that interact with only one target. These engineered proteins are starting to enter clinical testing for a variety of biomedical applications, particularly cancer treatment.
View details for DOI 10.1126/scitranslmed.3000276
View details for Web of Science ID 000277303400001
View details for PubMedID 20371477
Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects
2009; 69 (6): 2435-2442
There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with high affinity ( approximately 10 to 30 nmol/L) to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or (64)Cu-1,4,7,10-tetra-azacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) to their N termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. NIR fluorescence and microPET imaging both showed that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 hour postinjection for two high-affinity (IC(50), approximately 20 nmol/L) (64)Cu-DOTA-conjugated knottin peptides was 4.47% +/- 1.21% and 4.56% +/- 0.64% injected dose/gram (%ID/g), compared with a low-affinity knottin peptide (IC(50), approximately 0.4 mumol/L; 1.48 +/- 0.53%ID/g) and c(RGDyK) (IC(50), approximately 1 mumol/L; 2.32 +/- 0.55%ID/g), a low-affinity cyclic pentapeptide under clinical development. Furthermore, (64)Cu-DOTA-conjugated knottin peptides generated lower levels of nonspecific liver uptake ( approximately 2%ID/g) compared with c(RGDyK) ( approximately 4%ID/g) 1 hour postinjection. MicroPET imaging results were confirmed by in vivo biodistribution studies. (64)Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers.
View details for DOI 10.1158/0008-5472.CAN-08-2495
View details for Web of Science ID 000264541300037
View details for PubMedID 19276378
In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2016; 138 (24): 7496-7499
Chemoenzymatic modification of proteins is an attractive option to create highly specific conjugates for therapeutics, diagnostics, or materials under gentle biological conditions. However, these methods often suffer from expensive specialized substrates, bulky fusion tags, low yields, and extra purification steps to achieve the desired conjugate. Staphylococcus aureus sortase A and its engineered variants are used to attach oligoglycine derivatives to the C-terminus of proteins expressed with a minimal LPXTG tag. This strategy has been used extensively for bioconjugation in vitro and for protein-protein conjugation in living cells. Here we show that an enzyme variant recently engineered for higher activity on oligoglycine has promiscuous activity that allows proteins to be tagged using a diverse array of small, commercially available amines, including several bioorthogonal functional groups. This technique can also be carried out in living Escherichia coli, enabling simple, inexpensive production of chemically functionalized proteins with no additional purification steps.
View details for DOI 10.1021/jacs.6b03836
View details for Web of Science ID 000378584600013
View details for PubMedID 27280683
- Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free Protein Synthesis MOLECULAR CANCER THERAPEUTICS 2016; 15 (6): 1291-1300
- Degradable Acetalated Dextran Microparticles for Tunable Release of an Engineered Hepatocyte Growth Factor Fragment ACS BIOMATERIALS-SCIENCE & ENGINEERING 2016; 2 (2): 197-204
- Engineering growth factors for regenerative medicine applications ACTA BIOMATERIALIA 2016; 30: 1-12
- Biocompatibility of poly(ethylene glycol) and poly(acrylic acid) interpenetrating network hydrogel by intrastromal implantation in rabbit cornea JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 2015; 103 (10): 3157-3165
Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction.
2015; 45: 56-63
Hepatocyte growth factor (HGF) has been shown to have anti-fibrotic, pro-angiogenic, and cardioprotective effects; however, it is highly unstable and expensive to manufacture, hindering its clinical translation. Recently, a HGF fragment (HGF-f), an alternative c-MET agonist, was engineered to possess increased stability and recombinant expression yields. In this study, we assessed the potential of HGF-f, delivered in an extracellular matrix (ECM)-derived hydrogel, as a potential treatment for myocardial infarction (MI). HGF-f protected cardiomyocytes from serum-starvation and induced down-regulation of fibrotic markers in whole cardiac cell isolate compared to the untreated control. The ECM hydrogel prolonged release of HGF-f compared to collagen gels, and in vivo delivery of HGF-f from ECM hydrogels mitigated negative left ventricular (LV) remodeling, improved fractional area change (FAC), and increased arteriole density in a rat myocardial infarction model. These results indicate that HGF-f may be a viable alternative to using recombinant HGF, and that an ECM hydrogel can be employed to increase growth factor retention and efficacy.
View details for DOI 10.1016/j.biomaterials.2014.12.021
View details for PubMedID 25662495
View details for PubMedCentralID PMC4326250
Interpenetrating polymer network hydrogel scaffolds for artificial cornea periphery.
Journal of materials science. Materials in medicine
2015; 26 (2): 107-?
Three-dimensional scaffolds based on inverted colloidal crystals (ICCs) were fabricated from sequentially polymerized interpenetrating polymer network (IPN) hydrogels of poly(ethyleneglycol) and poly(acrylic acid). This high-strength, high-water-content IPN hydrogel may be suitable for use in an artificial cornea application. Development of a highly porous, biointegrable region at the periphery of the artificial cornea device is critical to long-term retention of the implant. The ICC fabrication technique produced scaffolds with well-controlled, tunable pore and channel dimensions. When surface functionalized with extracellular matrix proteins, corneal fibroblasts were successfully cultured on IPN hydrogel scaffolds, demonstrating the feasibility of these gels as materials for the artificial cornea porous periphery. Porous hydrogels with and without cells were visualized non-invasively in the hydrated state using variable-pressure scanning electron microscopy.
View details for DOI 10.1007/s10856-015-5442-2
View details for PubMedID 25665845
A Chemically Cross-Linked Knottin Dimer Binds Integrins with Picomolar Affinity and Inhibits Tumor Cell Migration and Proliferation
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2015; 137 (1): 6-9
Molecules that target and inhibit αvβ3, αvβ5, and α5β1 integrins have generated great interest because of the role of these receptors in mediating angiogenesis and metastasis. Attempts to increase the binding affinity and hence the efficacy of integrin inhibitors by dimerization have been marginally effective. In the present work, we achieved this goal by using oxime-based chemical conjugation to synthesize dimers of integrin-binding cystine knot (knottin) miniproteins with low-picomolar binding affinity to tumor cells. A non-natural amino acid containing an aminooxy side chain was introduced at different locations within a knottin monomer and reacted with dialdehyde-containing cross-linkers of different lengths to create knottin dimers with varying molecular topologies. Dimers cross-linked through an aminooxy functional group located near the middle of the protein exhibited higher apparent binding affinity to integrin-expressing tumor cells compared with dimers cross-linked through an aminooxy group near the C-terminus. In contrast, the cross-linker length had no effect on the integrin binding affinity. A chemical-based dimerization strategy was critical, as knottin dimers created through genetic fusion to a bivalent antibody domain exhibited only modest improvement (less than 5-fold) in tumor cell binding relative to the knottin monomer. The best oxime-conjugated knottin dimer achieved an unprecedented 150-fold increase in apparent binding affinity over the knottin monomer. Also, this dimer bound 3650-fold stronger and inhibited tumor cell migration and proliferation compared with cilengitide, an integrin-targeting peptidomimetic that performed poorly in recent clinical trials, suggesting promise for further therapeutic development.
View details for DOI 10.1021/ja508416e
View details for Web of Science ID 000348483500002
View details for PubMedID 25486381
View details for PubMedCentralID PMC4304478
Applications of Yeast Surface Display for Protein Engineering.
Methods in molecular biology (Clifton, N.J.)
2015; 1319: 155-175
The method of displaying recombinant proteins on the surface of Saccharomyces cerevisiae via genetic fusion to an abundant cell wall protein, a technology known as yeast surface display, or simply, yeast display, has become a valuable protein engineering tool for a broad spectrum of biotechnology and biomedical applications. This review focuses on the use of yeast display for engineering protein affinity, stability, and enzymatic activity. Strategies and examples for each protein engineering goal are discussed. Additional applications of yeast display are also briefly presented, including protein epitope mapping, identification of protein-protein interactions, and uses of displayed proteins in industry and medicine.
View details for DOI 10.1007/978-1-4939-2748-7_8
View details for PubMedID 26060074
View details for PubMedCentralID PMC4544684
An engineered dimeric fragment of hepatocyte growth factor is a potent c-MET agonist
2014; 588 (24): 4831-4837
Hepatocyte growth factor (HGF), through activation of the c-MET receptor, mediates biological processes critical for tissue regeneration; however, its clinical application is limited by protein instability and poor recombinant expression. We previously engineered an HGF fragment (eNK1) that possesses increased stability and expression yield and developed a c-MET agonist by coupling eNK1 through an introduced cysteine residue. Here, we further characterize this eNK1 dimer and show it elicits significantly greater c-MET activation, cell migration, and proliferation than the eNK1 monomer. The efficacy of the eNK1 dimer was similar to HGF, suggesting its promise as a c-MET agonist.
View details for DOI 10.1016/j.febslet.2014.11.018
View details for Web of Science ID 000346577000040
View details for PubMedID 25451235
- Cystine-knot peptides: emerging tools for cancer imaging and therapy EXPERT REVIEW OF PROTEOMICS 2014; 11 (5): 561-572
- Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma. Proceedings of the National Academy of Sciences of the United States of America 2013; 110 (36): 14598-14603
A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging
2013; 44 (2): 673-681
A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α(v)β(3) integrin receptor with high affinity and specificity. In the current study, an (18)F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-(18/19)F-fluoropropionate ((18/19)F-NFP) to produce the fluorinated peptide, (18/19)F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and (19)F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress α(v)β(3) integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with (18)F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and (19)F-FP-AgRP-7C specifically competed with (125)I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC(50)) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that (18)F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of (18)F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. (18)F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.
View details for DOI 10.1007/s00726-012-1391-y
View details for Web of Science ID 000313794600036
View details for PubMedID 22945905
Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging.
2013; 8 (4)
Cystine-knot miniproteins, also known as knottins, have shown great potential as molecular scaffolds for the development of targeted therapeutics and diagnostic agents. For this purpose, previous protein engineering efforts have focused on knottins based on the Ecballium elaterium trypsin inhibitor (EETI) from squash seeds, the Agouti-related protein (AgRP) neuropeptide from mammals, or the Kalata B1 uterotonic peptide from plants. Here, we demonstrate that Agatoxin (AgTx), an ion channel inhibitor found in spider venom, can be used as a molecular scaffold to engineer knottins that bind with high-affinity to a tumor-associated integrin receptor.We used a rational loop-grafting approach to engineer AgTx variants that bound to αvβ3 integrin with affinities in the low nM range. We showed that a disulfide-constrained loop from AgRP, a structurally-related knottin, can be substituted into AgTx to confer its high affinity binding properties. In parallel, we identified amino acid mutations required for efficient in vitro folding of engineered integrin-binding AgTx variants. Molecular imaging was used to evaluate in vivo tumor targeting and biodistribution of an engineered AgTx knottin compared to integrin-binding knottins based on AgRP and EETI. Knottin peptides were chemically synthesized and conjugated to a near-infrared fluorescent dye. Integrin-binding AgTx, AgRP, and EETI knottins all generated high tumor imaging contrast in U87MG glioblastoma xenograft models. Interestingly, EETI-based knottins generated significantly lower non-specific kidney imaging signals compared to AgTx and AgRP-based knottins.In this study, we demonstrate that AgTx, a knottin from spider venom, can be engineered to bind with high affinity to a tumor-associated receptor target. This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging.
View details for DOI 10.1371/journal.pone.0060498
View details for PubMedID 23573262
- Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging. PloS one 2013; 8 (4)
- Engineering Multivalent and Multispecific Protein Therapeutics Engineering in Translational Medicine edited by Cai, W. Springer.. 2013: 1
- Surface Modification of High-Strength Interpenetrating Network Hydrogels for Biomedical Device Applications Handbook of Biofunctional Surfaces edited by Knoll, W. Pan Stanford Publishing.. 2013: 407–446
Diffusion of Protein through the Human Cornea
2012; 48 (1): 50-55
To determine the rate of diffusion of myoglobin and bovine serum albumin (BSA) through the human cornea. These small proteins have hydrodynamic diameters of approximately 4.4 and 7.2 nm, and molecular weights of 16.7 and 66 kDa, for myoglobin and BSA, respectively.Diffusion coefficients were measured using a diffusion chamber where the protein of interest and balanced salt solution were in different chambers separated by an ex vivo human cornea. Protein concentrations in the balanced salt solution chamber were measured over time. Diffusion coefficients were calculated using equations derived from Fick's law and conservation of mass in a closed system.Our experiments demonstrate that the diffusion coefficient of myoglobin is 5.5 ± 0.9 × 10(-8) cm(2)/s (n = 8; SD = 1.3 × 10(-8) cm(2)/s; 95% CI: 4.6 × 10(-8) to 6.4 × 10(-8) cm(2)/s) and the diffusion coefficient of BSA is 3.1 ± 1.0 × 10(-8) cm(2)/s (n = 8; SD = 1.4 × 10(-8) cm(2)/s; 95% CI: 2.1 × 10(-8) to 4.1 × 10(-8) cm(2)/s).Our study suggests that molecules as large as 7.2 nm may be able to passively diffuse through the human cornea. With applications in pharmacotherapy and the development of an artificial cornea, further experiments are warranted to fully understand the limits of human corneal diffusion and its clinical relevance.
View details for DOI 10.1159/000329794
View details for Web of Science ID 000305551100009
View details for PubMedID 22398578
- Knottins: Disulfide-bonded Therapeutic and Diagnostic Peptides. Drug Discovery Today: Technologies 2012; 9: e3-e11
In-111-Labeled Cystine-Knot Peptides Based on the Agouti-Related Protein for Targeting Tumor Angiogenesis
JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY
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)β(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)β(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)β(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)β(3) positive tumors in living subjects.
View details for DOI 10.1155/2012/368075
View details for Web of Science ID 000303728900001
View details for PubMedID 22570527
ENGINEERING KNOTTINS AS NOVEL BINDING AGENTS
METHODS IN ENZYMOLOGY: PROTEIN ENGINEERING FOR THERAPEUTICS, VOL 203, PT B
2012; 503: 223-251
Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface.
View details for DOI 10.1016/B978-0-12-396962-0.00009-4
View details for Web of Science ID 000300198000009
View details for PubMedID 22230571
Discovery of Improved EGF Agonists Using a Novel In Vitro Screening Platform
JOURNAL OF MOLECULAR BIOLOGY
2011; 413 (2): 406-415
Directed evolution is a powerful strategy for protein engineering; however, evolution of pharmaceutical proteins has been limited by the reliance of current screens on binding interactions. Here, we present a method that identifies protein mutants with improved overall cellular efficacy, an objective not feasible with previous approaches. Mutated protein libraries were produced in soluble, active form by means of cell-free protein synthesis. The efficacy of each individual protein was determined at a uniform dosage with a high-throughput protein product assay followed by a cell-based functional assay without requiring protein purification. We validated our platform by first screening mock libraries of epidermal growth factor (EGF) for stimulation of cell proliferation. We then demonstrated its effectiveness by identifying EGF mutants with significantly enhanced mitogenic activity at low concentrations compared to that of wild-type EGF. This is the first report of EGF mutants with improved biological efficacy despite much previous effort. Our platform can be extended to engineer a broad range of proteins, offering a general method to evolve proteins for improved biological efficacy.
View details for DOI 10.1016/j.jmb.2011.08.028
View details for Web of Science ID 000296404100010
View details for PubMedID 21888916
- Toward the development of an artificial cornea: Improved stability of interpenetrating polymer networks JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART B-APPLIED BIOMATERIALS 2011; 98B (1): 8-17
Toward the development of an artificial cornea: improved stability of interpenetrating polymer networks.
Journal of biomedical materials research. Part B, Applied biomaterials
2011; 98 (1): 8-17
A novel interpenetrating network (IPN) based on poly(ethylene glycol) (PEG) and poly(acrylic acid) was developed and its use as an artificial cornea was evaluated in vivo. The in vivo results of a first set of corneal inlays based on PEG-diacrylate precursor showed inflammation of the treated eyes and haze in the corneas. The insufficient biocompatibility could be correlated to poor long-term stability of the implant caused by hydrolytic degradation over time. Adapting the hydrogel chemistry by replacing hydrolysable acrylate functionalities with stable acrylamide functionalities was shown to increase the long-term stability of the resulting IPNs under hydrolytic conditions. This new set of hydrogel implants now shows increased biocompatibility in vivo. Rabbits with corneal inlay implants are healthy and have clear cornea and non-inflamed eyes for up to 6 months after implantation.
View details for DOI 10.1002/jbm.b.31806
View details for PubMedID 21504051
Engineered epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation
2011; 585 (8): 1135-1139
Receptor tyrosine kinases (RTKs) regulate critical cell signaling pathways, yet the properties of their cognate ligands that influence receptor activation are not fully understood. There is great interest in parsing these complex ligand-receptor relationships using engineered proteins with altered binding properties. Here we focus on the interaction between two engineered epidermal growth factor (EGF) mutants and the EGF receptor (EGFR), a model member of the RTK superfamily. We found that EGF mutants with faster kinetic on-rates stimulate increased EGFR activation compared to wild-type EGF. These findings support previous predictions that faster association rates correlate with enhanced receptor activity.
View details for DOI 10.1016/j.febslet.2011.03.044
View details for Web of Science ID 000289505400004
View details for PubMedID 21439278
Preliminary evaluation of Lu-177-labeled knottin peptides for integrin receptor-targeted radionuclide therapy
EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING
2011; 38 (4): 613-622
Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, (177)Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts.Knottins 2.5D and 2.5F were synthesized using solid phase peptide synthesis, folded in vitro, and site-specifically coupled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at their N terminus for (177)Lu radiolabeling. The stability of the radiopeptides (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F was tested in both phosphate-buffered saline (PBS) and mouse serum. Cell uptake assays of the radiolabeled peptides were performed in U87MG integrin-expressing human glioma cells. The biodistribution studies of both (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F were examined in U87MG tumor-bearing athymic nu/nu mice. Radiation absorbed doses for the major tissues of a human adult male were calculated based on the mouse biodistribution results.DOTA-2.5D and DOTA-2.5F were labeled with (177)Lu at over 55% efficiency. High radiochemical purity for both radiocomplexes (> 95%) could be achieved after high performance liquid chromatography (HPLC) purification. Both radiopeptides were stable in PBS and mouse serum. Compared to (177)Lu-DOTA-2.5D (0.39 and 0.26 %ID/g at 2 and 24 h, respectively), (177)Lu-DOTA-2.5F showed much higher tumor uptake (2.16 and 0.78 %ID/g at 2 and 24 h, respectively). It also displayed higher tumor to blood ratios than that of (177)Lu-DOTA-2.5D (31.8 vs 18.7 at 24 h and 52.6 vs 20.6 at 72 h). Calculation of radiodosimetry for (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F suggested that tumor and kidney were tissues with the highest radiation absorbed doses. Moreover, (177)Lu-DOTA-2.5F had a higher tumor to kidney radiation absorbed dose ratio than that of (177)Lu-DOTA-2.5D.Cystine knot peptides can be successfully radiolabeled with (177)Lu for potential therapeutic applications. Knottin 2.5F labeled with (177)Lu exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.
View details for DOI 10.1007/s00259-010-1684-x
View details for Web of Science ID 000288255500002
View details for PubMedID 21153409
Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein
2011; 6 (2)
The Ecballium elaterium trypsin inhibitor (EETI-II), a 28-amino acid member of the knottin family of peptides, contains three interwoven disulfide bonds that form multiple solvent-exposed loops. Previously, the trypsin binding loop of EETI-II has been engineered to confer binding to several alternative molecular targets. Here, EETI-II was further explored as a molecular scaffold for polypeptide engineering by evaluating the ability to mutate two of its structurally adjacent loops.Yeast surface display was used to engineer an EETI-II mutant containing two separate integrin binding epitopes. The resulting knottin peptide was comprised of 38 amino acids, and contained 11- and 10-residue loops compared to wild-type EETI-II, which naturally contains 6- and 5-residue loops, respectively. This knottin peptide bound to α(v)β(3) and α(v)β(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins α(5)β(1) and α(iib)β(3). In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to α(v)β(3) and α(v)β(5) integrins. A (64)Cu radiolabeled version of this knottin peptide demonstrated moderate serum stability and excellent tumor-to-muscle and tumor-to-blood ratios by positron emission tomography imaging in human tumor xenograft models. Tumor uptake was ∼3-5% injected dose per gram (%ID/g) at one hour post injection, with rapid clearance of probe through the kidneys.We demonstrated that multiple loops of EETI-II can be mutated to bind with high affinity to tumor-associated integrin receptors. The resulting knottin peptide contained 21 (>50%) non-native amino acids within two mutated loops, indicating that extended loop lengths and sequence diversity were well tolerated within the EETI-II scaffold. A radiolabeled version of this knottin peptide showed promise for non-invasive imaging of integrin expression in living subjects. However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop.
View details for DOI 10.1371/journal.pone.0016112
View details for Web of Science ID 000287482800005
View details for PubMedID 21364742
PET Imaging of Integrin Positive Tumors Using F-18 Labeled Knottin Peptides
2011; 1: 403-412
Purpose: Cystine knot (knottin) peptides, engineered to bind with high affinity to integrin receptors, have shown promise as molecular imaging agents in living subjects. The aim of the current study was to evaluate tumor uptake and in vivo biodistribution of (18)F-labeled knottins in a U87MG glioblastoma model.Procedures: Engineered knottin mutants 2.5D and 2.5F were synthesized using solid phase peptide synthesis and were folded in vitro, followed by radiolabeling with 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP). The resulting probes, (18)F-FP-2.5D and (18)F-FP-2.5F, were evaluated in nude mice bearing U87MG tumor xenografts using microPET and biodistribution studies.Results: MicroPET imaging studies with (18)F-FP-2.5D and (18)F-FP-2.5F demonstrated high tumor uptake in U87MG xenograft mouse models. The probes exhibited rapid clearance from the blood and kidneys, thus leading to excellent tumor-to-normal tissue contrast. Specificity studies confirmed that (18)F-FP-2.5D and (18)F-FP-2.5F had reduced tumor uptake when co-injected with a large excess of the peptidomimetic c(RGDyK) as a blocking agent.Conclusions: (18)F-FP-2.5D and (18)F-FP-2.5F showed reduced gallbladder uptake compared with previously published (18)F-FB-2.5D. (18)F-FP-2.5D and (18)F-FP-2.5F enabled integrin-specific PET imaging of U87MG tumors with good imaging contrasts. (18)F-FP-2.5D demonstrated more desirable pharmacokinetics compared to (18)F-FP-2.5F, and thus has greater potential for clinical translation.
View details for DOI 10.7150/thno/v01p0403
View details for Web of Science ID 000299121000034
View details for PubMedID 22211146
- Rational and Combinatorial Methods for Creating Designer Protein Interfaces Comprehensive Biomaterials edited by Ducheyne, H., Hutmacher, G. Elsevier.. 2011: 1
- Cystine-knot peptides engineered with specificities for alpha(IIb)beta(3) or alpha(IIb)beta(3) and alpha(v)beta(3) integrins are potent inhibitors of platelet aggregation. J Mol Recognit. 2011; 24 (1): 127-35
- Targeting of Cancer Cells Using Quantum Dot-Polypeptide Hybrid Assemblies That Function as Molecular Imaging Agents and Carrier Systems ADVANCED FUNCTIONAL MATERIALS 2010; 20 (23): 4091-4097
PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model with a Novel Cu-64-DOTA-Knottin Peptide
2010; 70 (22): 9022-9030
Due to the high mortality of lung cancer, there is a critical need to develop diagnostic procedures enabling early detection of the disease while at a curable stage. Targeted molecular imaging builds on the positive attributes of positron emission tomography/computed tomography (PET/CT) to allow for a noninvasive detection and characterization of smaller lung nodules, thus increasing the chances of positive treatment outcome. In this study, we investigate the ability to characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors are first identified with small animal CT followed by characterization with the use of small animal PET with a novel 64Cu-1,4,7,10-tetra-azacylododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-knottin peptide that targets integrins upregulated during angiogenesis on the tumor associated neovasculature. The imaging results obtained with the knottin peptide are compared with standard 18F-fluorodeoxyglucose (FDG) PET small animal imaging. Lung nodules as small as 3 mm in diameter were successfully identified in the transgenic mice by small animal CT, and both 64Cu-DOTA-knottin 2.5F and FDG were able to differentiate lung nodules from the surrounding tissues. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumors combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared with FDG (6.01±0.61 versus 4.36±0.68; P<0.05). Ex vivo biodistribution showed 64Cu-DOTA-knottin 2.5F to have a fast renal clearance combined with low nonspecific accumulation in the thorax. Collectively, these results show 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer.
View details for DOI 10.1158/0008-5472.CAN-10-1338
View details for Web of Science ID 000284213300008
View details for PubMedID 21062977
Targeted Contrast-Enhanced Ultrasound Imaging of Tumor Angiogenesis with Contrast Microbubbles Conjugated to Integrin-Binding Knottin Peptides
JOURNAL OF NUCLEAR MEDICINE
2010; 51 (3): 433-440
Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. The purpose of this study was to develop and test a new class of targeting ligands for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis with small, conformationally constrained peptides that can be coupled to the surface of ultrasound contrast agents.Directed evolution was used to engineer a small, disulfide-constrained cystine knot (knottin) peptide that bound to alpha(v)beta(3) integrins with a low nanomolar affinity (Knottin(Integrin)). A targeted contrast-enhanced ultrasound imaging contrast agent was created by attaching Knottin(Integrin) to the shell of perfluorocarbon-filled microbubbles (MB-Knottin(Integrin)). A knottin peptide with a scrambled sequence was used to create control microbubbles (MB-Knottin(Scrambled)). The binding of MB-Knottin(Integrin) and MB-Knottin(Scrambled) to alpha(v)beta(3) integrin-positive cells and control cells was assessed in cell culture binding experiments and compared with that of microbubbles coupled to an anti-alpha(v)beta(3) integrin monoclonal antibody (MB(alphavbeta3)) and microbubbles coupled to the peptidomimetic agent c(RGDfK) (MB(cRGD)). The in vivo imaging signals of contrast-enhanced ultrasound with the different types of microbubbles were quantified in 42 mice bearing human ovarian adenocarcinoma xenograft tumors by use of a high-resolution 40-MHz ultrasound system.MB-Knottin(Integrin) attached significantly more to alpha(v)beta(3) integrin-positive cells (1.76 +/- 0.49 [mean +/- SD] microbubbles per cell) than to control cells (0.07 +/- 0.006). Control MB-Knottin(Scrambled) adhered less to alpha(v)beta(3) integrin-positive cells (0.15 +/- 0.12) than MB-Knottin(Integrin). After blocking of integrins, the attachment of MB-Knottin(Integrin) to alpha(v)beta(3) integrin-positive cells decreased significantly. The in vivo ultrasound imaging signal was significantly higher after the administration of MB-Knottin(Integrin) than after the administration of MB(alphavbeta3) or control MB-Knottin(Scrambled). After in vivo blocking of integrin receptors, the imaging signal after the administration of MB-Knottin(Integrin) decreased significantly (by 64%). The imaging signals after the administration of MB-Knottin(Integrin) were not significantly different in the groups of tumor-bearing mice imaged with MB-Knottin(Integrin) and with MB(cRGD). Ex vivo immunofluorescence confirmed integrin expression on endothelial cells of human ovarian adenocarcinoma xenograft tumors.Integrin-binding knottin peptides can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Our results demonstrate that microbubbles conjugated to small peptide-targeting ligands provide imaging signals higher than those provided by a large antibody molecule.
View details for DOI 10.2967/jnumed.109.068007
View details for Web of Science ID 000275133100026
View details for PubMedID 20150258
- A Dual-Labeled Knottin Peptide for PET and Near-Infrared Fluorescence Imaging of Integrin Expression in Living Subjects BIOCONJUGATE CHEMISTRY 2010; 21 (3): 436-444
Evaluation of a Cu-64-Labeled Cystine-Knot Peptide Based on Agouti-Related Protein for PET of Tumors Expressing alpha(v)beta(3) Integrin
JOURNAL OF NUCLEAR MEDICINE
2010; 51 (2): 251-258
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.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.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.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.
View details for DOI 10.2967/jnumed.109.069831
View details for Web of Science ID 000274152800028
View details for PubMedID 20124048
A Dual-Labeled Knottin Peptide for PET and Near-Infrared Fluorescence Imaging of Integrin Expression in Living Subjects.
Previously, we used directed evolution to engineer mutants of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin that bind to alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity, and showed that Cy5.5- or (64)Cu-DOTA-labeled knottin peptides could be used to image integrin expression in mouse tumor models using near-infrared fluorescence (NIRF) imaging or positron emission tomography (PET). Here, we report the development of a dual-labeled knottin peptide conjugated to both NIRF and PET imaging agents for multimodality imaging in living subjects. We created an orthogonally protected peptide-based linker for stoichiometric coupling of (64)Cu-DOTA and Cy5.5 onto the knottin N-terminus and confirmed that conjugation did not affect binding to alpha(v)beta(3) and alpha(v)beta(5) integrins. NIRF and PET imaging studies in tumor xenograft models showed that Cy5.5 conjugation significantly increased kidney uptake and retention compared to the knottin peptide labeled with (64)Cu-DOTA alone. In the tumor, the dual-labeled (64)Cu-DOTA/Cy5.5 knottin peptide showed decreased wash-out leading to significantly better retention (p < 0.05) compared to the (64)Cu-DOTA-labeled knottin peptide. Tumor uptake was significantly reduced (p < 0.05) when the dual-labeled knottin peptide was coinjected with an excess of unlabeled competitor and when tested in a tumor model with lower levels of integrin expression. Finally, plots of tumor-to-background tissue ratios for Cy5.5 versus (64)Cu uptake were well-correlated over several time points post injection, demonstrating pharmacokinetic cross validation of imaging labels. This dual-modality NIRF/PET imaging agent is promising for further development in clinical applications where high sensitivity and high resolution are desired, such as detection of tumors located deep within the body and image-guided surgical resection.
View details for DOI 10.1021/bc9003102
View details for PubMedID 20131753
- Phage Display and Molecular Imaging: Expanding Fields of Vision in Living Subjects. Biotechnology and Genetic Engineering Reviews. 2010; 27: 57-94
Phage display and molecular imaging: expanding fields of vision in living subjects
BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, VOL 27
2010; 27: 57-93
In vivo molecular imaging enables non-invasive visualization of biological processes within living subjects, and holds great promise for diagnosis and monitoring of disease. The ability to create new agents that bind to molecular targets and deliver imaging probes to desired locations in the body is critically important to further advance this field. To address this need, phage display, an established technology for the discovery and development of novel binding agents, is increasingly becoming a key component of many molecular imaging research programs. This review discusses the expanding role played by phage display in the field of molecular imaging with a focus on in vivo applications. Furthermore, new methodological advances in phage display that can be directly applied to the discovery and development of molecular imaging agents are described. Various phage library selection strategies are summarized and compared, including selections against purified target, intact cells, and ex vivo tissue, plus in vivo homing strategies. An outline of the process for converting polypeptides obtained from phage display library selections into successful in vivo imaging agents is provided, including strategies to optimize in vivo performance. Additionally, the use of phage particles as imaging agents is also described. In the latter part of the review, a survey of phage-derived in vivo imaging agents is presented, and important recent examples are highlighted. Other imaging applications are also discussed, such as the development of peptide tags for site-specific protein labeling and the use of phage as delivery agents for reporter genes. The review concludes with a discussion of how phage display technology will continue to impact both basic science and clinical applications in the field of molecular imaging.
View details for Web of Science ID 000286179900003
View details for PubMedID 21415893
An Engineered Knottin Peptide Labeled with F-18 for PET Imaging of Integrin Expression
2009; 20 (12): 2342-2347
Knottins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded beta-sheet fold. Previously, directed evolution of the Ecballium elaterium trypsin inhibitor (EETI-II) knottin led to the identification of a mutant that bound to tumor-specific alpha(v)beta(3) and alpha(v)beta(5) integrin receptors with low nanomolar affinity. The objective of this study was to prepare and evaluate a radiofluorinated version of this knottin (termed 2.5D) for microPET imaging of integrin positive tumors in living subjects. Knottin peptide 2.5D was prepared by solid-phase synthesis and folded in vitro, and its free N-terminal amine was reacted with N-succinimidyl-4-18/19F-fluorobenzoate (18/19F-SFB) to produce the fluorinated peptide 18/19F-FB-2.5D. The binding affinities of unlabeled knottin peptide 2.5D and 19F-FB-2.5D to U87MG glioblastoma cells were measured by competition binding assay using 125I-labeled echistatin. It was found that unlabeled 2.5D and 19F-FB-2.5D competed with 125I-echistatin for binding to cell surface integrins with IC(50) values of 20.3 +/- 7.3 and 13.2 +/- 5.4 nM, respectively. Radiosynthesis of 18F-FB-2.5D resulted in a product with high specific activity (ca. 100 GBq/micromol). Next, biodistribution and positron emission tomography (PET) imaging studies were performed to evaluate the in vivo behavior of 18F-FB-2.5D. Approximately 3.7 MBq 18F-FB-2.5D was injected into U87MG tumor-bearing mice via the tail vein. Biodistribution studies demonstrated that 18F-FB-2.5D had moderate tumor uptake at 0.5 h post injection, and coinjection of a large excess of the unlabeled peptidomimetic c(RGDyK) as a blocking agent significantly reduced tumor uptake (1.90 +/- 1.15 vs 0.57 +/- 0.14%ID/g, 70% inhibition, P < 0.05). In vivo microPET imaging showed that 18F-FB-2.5D rapidly accumulated in the tumor and quickly cleared from the blood through the kidneys, allowing excellent tumor-to-normal tissue contrast to be obtained. Collectively, 18F-FB-2.5D allows integrin-specific PET imaging of U87MG tumors with good contrast and further demonstrates that knottins are excellent peptide scaffolds for development of PET probes with potential for clinical translation.
View details for DOI 10.1021/bc900361g
View details for Web of Science ID 000272690100018
View details for PubMedID 19908826
Engineered cystine knot peptides that bind alpha v beta 3, alpha v beta 5, and alpha 5 beta 1 integrins with low-nanomolar affinity
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
2009; 77 (2): 359-369
There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI-II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin-binding agents. We generated yeast-displayed libraries of EETI-II by substituting its 6-amino acid trypsin binding loop with 11-amino acid loops containing the Arg-Gly-Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high-throughput manner by fluorescence-activated cell sorting to identify mutants that bound to alpha(v)beta(3) integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half-maximal inhibitory concentration values of 10-30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both alpha(v)beta(3) and alpha(v)beta(5) integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to alpha(v)beta(3), alpha(v)beta(5), and alpha(5)beta(1) integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI-II as a scaffold for protein engineering, and highlight the development of unique integrin-binding peptides with potential for translational applications in cancer.
View details for DOI 10.1002/prot.22441
View details for Web of Science ID 000269872900009
View details for PubMedID 19452550
Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein
PLOS COMPUTATIONAL BIOLOGY
2009; 5 (9)
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.
View details for DOI 10.1371/journal.pcbi.1000499
View details for Web of Science ID 000270800100031
View details for PubMedID 19730675
Bioactive interpenetrating polymer network hydrogels that support corneal epithelial wound healing.
Journal of biomedical materials research. Part A
2009; 90 (1): 70-81
The development and characterization of collagen-coupled poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) interpenetrating polymer network hydrogels is described. Quantitative amino acid analysis and FITC-labeling of collagen were used to determine the amount and distribution of collagen on the surface of the hydrogels. The bioactivity of the coupled collagen was detected by a conformation-specific antibody and was found to vary with the concentration of collagen reacted to the photochemically functionalized hydrogel surfaces. A wound healing assay based on an organ culture model demonstrated that this bioactive surface supports epithelial wound closure over the hydrogel but at a decreased rate relative to sham wounds. Implantation of the hydrogel into the corneas of live rabbits demonstrated that epithelial cell migration is supported by the material, although the rate of migration and morphology of the epithelium were not normal. The results from the study will be used as a guide toward the optimization of bioactive hydrogels with promise in corneal implant applications such as a corneal onlay and an artificial cornea.
View details for DOI 10.1002/jbm.a.32056
View details for PubMedID 18481785
View details for PubMedCentralID PMC2856598
- Bioactive interpenetrating polymer network hydrogels that support corneal epithelial wound healing JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A 2009; 90A (1): 70-81
Antibodies specifically targeting a locally misfolded region of tumor associated EGFR
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (13): 5082-5087
Epidermal Growth Factor Receptor (EGFR) is involved in stimulating the growth of many human tumors, but the success of therapeutic agents has been limited in part by interference from the EGFR on normal tissues. Previously, we reported an antibody (mab806) against a truncated form of EGFR found commonly in gliomas. Remarkably, it also recognizes full-length EGFR on tumor cells but not on normal cells. However, the mechanism for this activity was unclear. Crystallographic structures for Fab:EGFR(287-302) complexes of mAb806 (and a second, related antibody, mAb175) show that this peptide epitope adopts conformations similar to those found in the wtEGFR. However, in both conformations observed for wtEGFR, tethered and untethered, antibody binding would be prohibited by significant steric clashes with the CR1 domain. Thus, these antibodies must recognize a cryptic epitope in EGFR. Structurally, it appeared that breaking the disulfide bond preceding the epitope might allow the CR1 domain to open up sufficiently for antibody binding. The EGFR(C271A/C283A) mutant not only binds mAb806, but binds with 1:1 stoichiometry, which is significantly greater than wtEGFR binding. Although mAb806 and mAb175 decrease tumor growth in xenografts displaying mutant, overexpressed, or autocrine stimulated EGFR, neither antibody inhibits the in vitro growth of cells expressing wtEGFR. In contrast, mAb806 completely inhibits the ligand-associated stimulation of cells expressing EGFR(C271A/C283A). Clearly, the binding of mAb806 and mAb175 to the wtEGFR requires the epitope to be exposed either during receptor activation, mutation, or overexpression. This mechanism suggests the possibility of generating antibodies to target other wild-type receptors on tumor cells.
View details for DOI 10.1073/pnas.0811559106
View details for Web of Science ID 000264790600025
View details for PubMedID 19289842
Engineered Cystine-Knot Peptides that Bind alpha(v)beta(3) Integrin with Antibody-Like Affinities
JOURNAL OF MOLECULAR BIOLOGY
2009; 385 (4): 1064-1075
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.
View details for DOI 10.1016/j.jmb.2008.11.004
View details for Web of Science ID 000263073400005
View details for PubMedID 19038268
- Protein Engineering and Design edited by J., R., Park, Jennifer Taylor and Francis, Boca Raton.. 2009
- Cell Surface Display Systems for Protein Engineering Protein Engineering and Design edited by Park, Sheldon, J., Cochran, Jennifer, R. Taylor and Francis, Boca Raton.. 2009: 1
- Yeast Surface Display Therapeutic Antibodies: from Theory to Practice edited by An, Z., Strohl, W. John Wiley & Sons, Inc.. 2009: 1
Developing therapeutic proteins by engineering ligand-receptor interactions
TRENDS IN BIOTECHNOLOGY
2008; 26 (9): 498-505
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.
View details for DOI 10.1016/j.tibtech.2008.05.009
View details for Web of Science ID 000259324200006
View details for PubMedID 18675482
Development of hydrogel-based keratoprostheses: A materials perspective
234th National Meeting of the American-Chemical-Society
WILEY-BLACKWELL. 2008: 735–41
Research and development of artificial corneas (keratoprostheses) in recent years have evolved from the use of rigid hydrophobic materials such as plastics and rubbers to hydrophilic, water-swollen hydrogels engineered to support not only peripheral tissue integration but also glucose diffusion and surface epithelialization. The advent of the AlphaCor core-and-skirt hydrogel keratoprosthesis has paved the way for a host of new approaches based on hydrogels and other soft materials that encompass a variety of materials preparation strategies, from synthetic homopolymers and copolymers to collagen-based bio-copolymers and, finally, interpenetrating polymer networks. Each approach represents a unique strategy toward the same goal: to develop a new hydrogel that mimics the important properties of natural donor corneas. We provide a critical review of these approaches from a materials perspective and discuss recent experimental results. While formidable technical hurdles still need to be overcome, the rapid progress that has been made by investigators with these approaches is indicative that a synthetic donor cornea capable of surface epithelialization is now closer to becoming a clinical reality.
View details for DOI 10.1021/bp070476n
View details for Web of Science ID 000256593300033
View details for PubMedID 18422366
View details for PubMedCentralID PMC2743969
Design and fabrication of an artificial cornea based on a photolithographically patterned hydrogel construct
2007; 9 (6): 911-922
We describe the design and fabrication of an artificial cornea based on a photolithographically patterned hydrogel construct, and demonstrate the adhesion of corneal epithelial and fibroblast cells to its central and peripheral components, respectively. The design consists of a central "core" optical component and a peripheral tissue-integrable "skirt." The core is composed of a poly(ethylene glycol)/poly(acrylic acid) (PEG/PAA) double-network with high strength, high water content, and collagen type I tethered to its surface. Interpenetrating the periphery of the core is a microperforated, but resilient poly(hydroxyethyl acrylate) (PHEA) hydrogel skirt that is also surface-modified with collagen type I. The well-defined microperforations in the peripheral component were created by photolithography using a mask with radially arranged chrome discs. Surface modification of both the core and skirt elements was accomplished through the use of a photoreactive, heterobifunctional crosslinker. Primary corneal epithelial cells were cultured onto modified and unmodified PEG/PAA hydrogels to evaluate whether the central optic material could support epithelialization. Primary corneal fibroblasts were seeded onto the PHEA hydrogels to evaluate whether the peripheral skirt material could support the adhesion of corneal stromal cells. Cell growth in both cases was shown to be contingent on the covalent tethering of collagen. Successful demonstration of cell growth on the two engineered components was followed by fabrication of core-skirt constructs in which the central optic and peripheral skirt were synthesized in sequence and joined by an interpenetrating diffusion zone.
View details for DOI 10.1007/s10544-006-9040-4
View details for Web of Science ID 000250462200017
View details for PubMedID 17237989
Elucidation of the interleukin-15 binding site on its alpha receptor by NMR
2007; 46 (33): 9453-9461
The cytokine interleukin-15 (IL-15) signals through the formation of a quaternary receptor complex composed of an IL-15-specific alpha receptor, together with beta and gammac receptors that are shared with interleukin-2 (IL-2). The initiating step in the formation of this signaling complex is the interaction between IL-15 and IL-15Ralpha, which is a single sushi domain bearing strong structural homology to one of the two sushi domains of IL-2Ralpha. The crystal structure of the IL2-Ralpha/IL-2 complex has been determined, however little is known about the analogous IL-15Ralpha/IL-15 binding interaction. Here we show that recombinant IL-15 can be overexpressed as a stable complex in the presence of its high affinity receptor, IL-15Ralpha. We find that this complex is 10-fold more active than IL-15 alone in stimulating proliferation and survival of memory phenotype CD8 T cells. To probe the ligand/receptor interface, we used solution NMR to map chemical shifts on 15N-labeled IL-15Ralpha in complex with unlabeled IL-15. Our results predict that the binding surface on IL-15Ralpha involves strands C and D, similar to IL-2Ralpha. The interface, as predicted here, leaves open the possibility of trans-presentation of IL-15 by IL-15Ralpha on an opposing cell.
View details for DOI 10.1021/bi700652f
View details for Web of Science ID 000248692400010
View details for PubMedID 17655329
BIOT 66-A novel, biomimetic hydrogel construct to repair the cornea: Molecular design and biological response
AMER CHEMICAL SOC. 2007
View details for Web of Science ID 000207593903359
Improved mutants from directed evolution are biased to orthologous substitutions
PROTEIN ENGINEERING DESIGN & SELECTION
2006; 19 (6): 245-253
We have engineered human epidermal growth factor (EGF) by directed evolution through yeast surface display for significantly enhanced affinity for the EGF receptor (EGFR). Statistical analysis of improved EGF mutants isolated from randomly mutated yeast-displayed libraries indicates that mutations are biased towards substitutions at positions exhibiting significant phylogenetic variation. In particular, mutations in high-affinity EGF mutants are statistically biased towards residues found in orthologous EGF species. This same trend was also observed with other proteins engineered through directed evolution in our laboratory (EGFR, interleukin-2) and in a meta-analysis of reported results for engineered subtilisin. By contrast, reported loss-of-function mutations in EGF were biased towards highly conserved positions. Based on these findings, orthologous mutations were introduced into a yeast-displayed EGF library by a process we term shotgun ortholog scanning mutagenesis (SOSM). EGF mutants with a high frequency of the introduced ortholog mutations were isolated through screening the library for enhanced binding affinity to soluble EGFR ectodomain. These mutants possess a 30-fold increase in binding affinity over wild-type EGF to EGFR-transfected fibroblasts and are among the highest affinity EGF proteins to be engineered to date. Collectively, our findings highlight a general approach for harnessing information present in phylogenetic variability to create useful genetic diversity for directed evolution. Our SOSM method exploits the benefits of library diversity obtained through complementary methods of error-prone PCR and DNA shuffling, while circumventing the need for acquisition of multiple genes for family or synthetic shuffling.
View details for DOI 10.1093/protein/gzl006
View details for Web of Science ID 000238650200001
View details for PubMedID 16740523
Directed evolution of the epidermal growth factor receptor extracellular domain for expression in yeast
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
2006; 62 (4): 1026-1035
The extracellular domain of epidermal growth factor receptor (EGFR-ECD) has been engineered through directed evolution and yeast surface display using conformationally-specific monoclonal antibodies (mAbs) as screening probes for proper folding and functional expression in Saccharomyces cerevisiae. An EGFR mutant with four amino acid changes exhibited binding to the conformationally-specific mAbs and human epidermal growth factor, and showed increased soluble secretion efficiency compared with wild-type EGFR. Full-length EGFR containing the mutant EGFR-ECD was functional, as assayed by EGF-dependent autophosphorylation and intracellular MAPK signaling in mammalian cells, and was expressed and localized at the plasma membrane in yeast. This approach should enable engineering of other complex mammalian receptor glycoproteins in yeast for genetic, structural, and biophysical studies.
View details for DOI 10.1002/prot.20618
View details for Web of Science ID 000235872700018
View details for PubMedID 16355407
Fine epitope mapping anti-epidermal growth factor receptor antibodies through random mutagenesia and yeast surface display
JOURNAL OF MOLECULAR BIOLOGY
2004; 342 (2): 539-550
Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.
View details for DOI 10.1016/j.jmb.2004.07.053
View details for Web of Science ID 000223684900014
View details for PubMedID 15327953
Identification of the epitope for the epidermal growth factor receptor-specific monoclonal antibody 806 reveals that it preferentially recognizes an untethered form of the receptor
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (29): 30375-30384
The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, an observation often correlated with poor clinical outcome. Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain. Monoclonal antibody (mAb) 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild type (wt) EGFR when overexpressed but does not bind the wt EGFR expressed in normal tissues. Despite only binding to a low proportion of the wt EGFR expressed in A431 tumor cells (approximately 10%), mAb 806 displays robust antitumor activity against A431 xenografts grown in nude mice. To elucidate the mechanism leading to its unique specificity and mode of antitumor activity, we have determined the EGFR binding epitope of mAb 806. Analysis of mAb 806 binding to EGFR fragments expressed either on the surface of yeast or in an immunoblot format identified a disulfide-bonded loop (amino acids 287-302) that contains the mAb 806 epitope. Indeed, mAb 806 binds with apparent high affinity (approximately 30 nm) to a synthetic EGFR peptide corresponding to these amino acids. Analysis of EGFR structures indicates that the epitope is fully exposed only in the transitional form of the receptor that occurs because EGFR changes from the inactive tethered conformation to a ligand-bound active form. It would seem that mAb 806 binds this small proportion of transient receptors, preventing their activation, which in turn generates a strong antitumor effect. Finally, our observations suggest that the generation of antibodies to transitional forms of growth factor receptors may represent a novel way of reducing normal tissue targeting yet retaining antitumor activity.
View details for DOI 10.1074/jbc.M401218200
View details for Web of Science ID 000222531900063
View details for PubMedID 15075331
Domain-level antibody epitope mapping through yeast surface display of epidermal growth factor receptor fragments
JOURNAL OF IMMUNOLOGICAL METHODS
2004; 287 (1-2): 147-158
Individual domains from extracellular proteins are potential reagents for biochemical characterization of ligand/receptor interactions and antibody binding sites. Here, we describe an approach for the identification and characterization of stable protein domains with cell surface display in Saccharomyces cerevesiae, using the epidermal growth factor receptor (EGFR) as a model system. Fragments of the EGFR were successfully expressed on the yeast cell surface. The yeast-displayed EGFR fragments were properly folded, as assayed with conformationally specific EGFR antibodies. Heat denaturation of yeast-displayed EGFR proteins distinguished between linear and conformational antibody epitopes. In addition, EGFR-specific antibodies were categorized based on their ability to compete ligand binding, which has been shown to have therapeutic implications. Overlapping EGFR antibody epitopes were determined based on a fluorescent competitive binding assay. Yeast surface display is a useful method for identifying stable folded protein domains from multidomain extracellular receptors, as well as characterizing antibody binding epitopes, without the need for soluble protein expression and purification.
View details for DOI 10.1016/j.jim.2004.01.024
View details for Web of Science ID 000221148800013
View details for PubMedID 15099763
Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library
2003; 21 (2): 163-170
A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.
View details for DOI 10.1038/nbt785
View details for Web of Science ID 000180802000029
View details for PubMedID 12536217
Soluble peptide-MHC monomers cause activation of CD8+T cells through transfer of the peptide to T cell MHC molecules
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2002; 99 (21): 13729-13734
T cell receptor (TCR)-mediated activation of CD4(+) T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide-MHC complexes. In contrast, for CD8(+) T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric L(d) and K(b) peptide-MHC complexes and free peptide as stimulators of CD8(+) T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naive and effector CD8(+) T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (K(b)) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naive T cells.
View details for DOI 10.1073/pnas.212515299
View details for Web of Science ID 000178635700071
View details for PubMedID 12374859
T-cell activation by soluble MHC oligomers can be described by a two-parameter binding model
2001; 81 (5): 2547-2557
T-cell activation is essential for initiation and control of immune system function. T cells are activated by interaction of cell-surface antigen receptors with major histocompatibility complex (MHC) proteins on the surface of other cells. Studies using soluble oligomers of MHC-peptide complexes and other types of receptor cross-linking agents have supported an activation mechanism that involves T cell receptor clustering. Receptor clustering induced by incubation of T cells with MHC-peptide oligomers leads to the induction of T-cell activation processes, including downregulation of engaged receptors and upregulation of the cell-surface proteins CD69 and CD25. Dose-response curves for these T-cell activation markers are bell-shaped, with different maxima and midpoints, depending on the valency of the soluble oligomer used. In this study, we have analyzed the activation behavior using a mathematical model that describes the binding of multivalent ligands to cell-surface receptors. We show that a simple equilibrium binding model accurately describes the activation data for CD4(+) T cells treated with MHC-peptide oligomers of varying valency. The model can be used to predict activation and binding behavior for T cells and MHC oligomers with different properties.
View details for Web of Science ID 000171755200010
View details for PubMedID 11606269
- TCR: losing its inhibitions? TRENDS IN IMMUNOLOGY 2001; 22 (9): 479-480
Receptor proximity, not intermolecular orientation, is critical for triggering T-cell activation
JOURNAL OF BIOLOGICAL CHEMISTRY
2001; 276 (30): 28068-28074
Engagement of antigen receptors on the surface of T-cells with peptides bound to major histocompatibility complex (MHC) proteins triggers T-cell activation in a mechanism involving receptor oligomerization. Receptor dimerization by soluble MHC oligomers is sufficient to induce several characteristic activation processes in T-cells including internalization of engaged receptors and up-regulation of cell surface proteins. In this work, the influence of intermolecular orientation within the activating receptor dimer was studied. Dimers of class II MHC proteins coupled in a variety of orientations and topologies each were able to activate CD4+ T-cells, indicating that triggering was not dependent on a particular receptor orientation. In contrast to the minimal influence of receptor orientation, T-cell triggering was affected by the inter-molecular distance between MHC molecules, and MHC dimers coupled through shorter cross-linkers were consistently more potent than those coupled through longer cross-linkers. These results are consistent with a mechanism in which intermolecular receptor proximity, but not intermolecular orientation, is the key determinant for antigen-induced CD4+ T-cell activation.
View details for Web of Science ID 000170093400044
View details for PubMedID 11384988
Receptor clustering and transmembrane signaling in T cells
TRENDS IN BIOCHEMICAL SCIENCES
2001; 26 (5): 304-310
T cells are activated via engagement of their cell-surface receptors with molecules of the major histocompatibility complex (MHC) displayed on another cell surface. This process, which is a key step in the recognition of foreign antigens by the immune system, involves oligomerization of receptor components. Recent characterization of the T-cell response to soluble arrays of MHC-peptide complexes has provided insights into the triggering mechanism for T-cell activation.
View details for Web of Science ID 000168720000013
View details for PubMedID 11343923
Cutting edge: Detection of antigen-specific CD4(+) T cells by HLA-DR1 oligomers is dependent on the T cell activation state
JOURNAL OF IMMUNOLOGY
2001; 166 (2): 741-745
Class I MHC tetramers have proven to be invaluable tools for following and deciphering the CD8(+) T cell response, but the development of similar reagents for detection of CD4(+) T cells based on class II MHC proteins has been more difficult. We evaluated fluorescent streptavidin-based oligomers of HLA-DR1 for use as reagents to analyze Ag-specific human CD4(+) T cells. Staining was blocked at low temperatures and by drugs that disrupt microfilament formation and endocytosis. Cell-associated MHC oligomers were resistant to a surface stripping protocol and were observed by microscopy in intracellular compartments. This behavior indicates that detection of CD4(+) T cells using class II MHC oligomers can depend on an active cellular process in which T cells cluster and/or endocytose their Ag receptors. T cells of identical specificity but in different activation states varied greatly in their ability to be detected by class II MHC oligomers.
View details for Web of Science ID 000166259600005
View details for PubMedID 11145645
A diverse set of oligomeric class II MHC-peptide complexes for probing T-cell receptor interactions
CHEMISTRY & BIOLOGY
2000; 7 (9): 683-696
T-cells are activated by engagement of their clonotypic cell surface receptors with peptide complexes of major histocompatibility complex (MHC) proteins, in a poorly understood process that involves receptor clustering on the membrane surface. Few tools are available to study the molecular mechanisms responsible for initiation of activation processes in T-cells.A topologically diverse set of oligomers of the human MHC protein HLA-DR1, varying in size from dimers to tetramers, was produced by varying the location of an introduced cysteine residue and the number and spacing of sulfhydryl-reactive groups carried on novel and commercially available cross-linking reagents. Fluorescent probes incorporated into the cross-linking reagents facilitated measurement of oligomer binding to the T-cell surface. Oligomeric MHC-peptide complexes, including a variety of MHC dimers, trimers and tetramers, bound to T-cells and initiated T-cell activation processes in an antigen-specific manner.T-cell receptor dimerization on the cell surface is sufficient to initiate intracellular signaling processes, as a variety of MHC-peptide dimers differing in intramolecular spacing and orientation were each able to trigger early T-cell activation events. The relative binding affinities within a homologous series of MHC-peptide oligomers suggest that T-cell receptors may rearrange in the plane of the membrane concurrent with oligomer binding.
View details for Web of Science ID 000089866300004
View details for PubMedID 10980449
The relationship of MHC-peptide binding and T cell activation probed using chemically defined MHC class II oligomers
2000; 12 (3): 241-250
A series of novel chemically defined soluble oligomers of the human MHC class II protein HLA-DR1 was constructed to probe the molecular requirements for initiation of T cell activation. MHC dimers, trimers, and tetramers stimulated T cells, as measured by upregulation of the activation markers CD69 and CD25, and by internalization of activated T cell receptor subunits. Monomeric MHC-peptide complexes engaged T cell receptors but did not induce activation. For a given amount of receptor engagement, the extent of activation was equivalent for each of the oligomers and correlated with the number of T cell receptor cross-links induced. These results suggest that formation or rearrangement of a T cell receptor dimer is necessary and sufficient for initiation of T cell signaling.
View details for Web of Science ID 000086292100002
View details for PubMedID 10755611