Bio

Bio


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.

Academic Appointments


Honors & Awards


  • Martin D. Abeloff Scholar Award, V Foundation (2008)
  • Hellman Faculty Scholar Award, Hellman Foundation (2008)
  • McCormick Award, McCormick Foundation (2007)
  • Mallinckrodt Faculty Scholar Award, Edward Mallinckrodt Jr. Foundation (2007)
  • Kimmel Scholars Award, Sidney Kimmel Foundation (2007)
  • Translational Partnership Award, Wallace H. Coulter Foundation (2006, 2007)
  • Howard Temin Award, NIH / National Cancer Institute (2004)

Professional Education


  • Postdoctoral Fellow, MIT, Biological Engineering
  • Ph. D., MIT, Biological Chemistry (2001)
  • B.S., University of Delaware, Biochemistry (1995)

Research & Scholarship

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. One such example is highlighted in a recent press release: http://med.stanford.edu/ism/2013/august/flashlight.html.

Teaching

2013-14 Courses


Postdoctoral Advisees


Publications

Journal Articles


  • Beyond antibodies: using biological principles to guide the development of next-generation protein therapeutics. Current opinion in biotechnology Kariolis, M. S., Kapur, S., Cochran, J. R. 2013; 24 (6): 1072-1077

    Abstract

    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 Science Moore, S. J., Hayden Gephart, M. G., Bergen, J. M., Su, S., Rayburn, H., et al 2013; published ahead of print August 15, 2013
  • 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 Papo, N., Silverman, A. P., Lahti, J. L., Cochran, J. R. 2011; 108 (34): 14067-14072

    Abstract

    Significant cross-talk exists between receptors that mediate angiogenesis, such as VEGF receptor-2 (VEGFR2) and ?(v)?(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 ?(v)?(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 ?(v)?(3) integrin. These engineered scVEGF mutants were specific for ?(v)?(3) integrin and did not bind to the related integrins ?(v)?(5), ?(iib)?(3), or ?(5)?(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 ?(v)?(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 ?(v)?(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 Jones, D. S., Tsai, P., Cochran, J. R. 2011; 108 (32): 13035-13040

    Abstract

    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 epidermal growth factor mutants with faster binding on-rates correlate with enhanced receptor activation FEBS LETTERS Lahti, J. L., Lui, B. H., Beck, S. E., Lee, S. S., Ly, D. P., Longaker, M. T., Yang, G. P., Cochran, J. R. 2011; 585 (8): 1135-1139

    Abstract

    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

  • Engineered Proteins Pull Double Duty SCIENCE TRANSLATIONAL MEDICINE Cochran, J. R. 2010; 2 (17)

    Abstract

    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 CANCER RESEARCH Kimura, R. H., Cheng, Z., Gambhir, S. S., Cochran, J. R. 2009; 69 (6): 2435-2442

    Abstract

    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

  • Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging. PloS one Moore, S. J., Leung, C. L., Norton, H. K., Cochran, J. R. 2013; 8 (4)

    Abstract

    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 ?v?3 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

  • Knottins: Disulfide-bonded Therapeutic and Diagnostic Peptides. Drug Discovery Today: Technologies Moore, S., J., Leung, C., L., Cochran, J., R. 2012; 9: e3-e11
  • Diffusion of Protein through the Human Cornea OPHTHALMIC RESEARCH Charalel, R. A., Engberg, K., Noolandi, J., Cochran, J. R., Frank, C., Ta, C. N. 2012; 48 (1): 50-55

    Abstract

    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

  • Discovery of Improved EGF Agonists Using a Novel In Vitro Screening Platform JOURNAL OF MOLECULAR BIOLOGY Lui, B. H., Cochran, J. R., Swartz, J. R. 2011; 413 (2): 406-415

    Abstract

    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 Hartmann, L., Watanabe, K., Zheng, L. L., Kim, C., Beck, S. E., Huie, P., Noolandi, J., Cochran, J. R., Ta, C. N., Frank, C. W. 2011; 98B (1): 8-17

    Abstract

    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 Web of Science ID 000291598900002

  • Functional Mutation of Multiple Solvent-Exposed Loops in the Ecballium elaterium Trypsin Inhibitor-II Cystine Knot Miniprotein PLOS ONE Kimura, R. H., Jones, D. S., Jiang, L., Miao, Z., Cheng, Z., Cochran, J. R. 2011; 6 (2)

    Abstract

    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 ?(v)?(3) and ?(v)?(5) integrins with affinities in the low nanomolar range, but bound weakly to the related integrins ?(5)?(1) and ?(iib)?(3). In addition, the engineered knottin peptide inhibited tumor cell adhesion to vitronectin, an extracellular matrix protein that binds to ?(v)?(3) and ?(v)?(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

  • 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. Silverman AP, Kariolis, MS, Cochran, JR 2011; 24 (1): 127-35
  • PET Imaging of Integrin Positive Tumors Using F-18 Labeled Knottin Peptides THERANOSTICS Liu, S., Liu, H., Ren, G., Kimura, R. H., Cochran, J. R., Cheng, Z. 2011; 1: 403-412

    Abstract

    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

  • PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model with a Novel Cu-64-DOTA-Knottin Peptide CANCER RESEARCH Nielsen, C. H., Kimura, R. H., Withofs, N., Tran, P. T., Miao, Z., Cochran, J. R., Cheng, Z., Felsher, D., Kjaer, A., Willmann, J. K., Gambhir, S. S. 2010; 70 (22): 9022-9030

    Abstract

    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 Willmann, J. K., Kimura, R. H., Deshpande, N., Lutz, A. M., Cochran, J. R., Gambhir, S. S. 2010; 51 (3): 433-440

    Abstract

    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

  • 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 Jiang, L., Kimura, R. H., Miao, Z., Silverman, A. P., Ren, G., Liu, H., Li, P., Gambhir, S. S., Cochran, J. R., Cheng, Z. 2010; 51 (2): 251-258

    Abstract

    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

  • Phage Display and Molecular Imaging: Expanding Fields of Vision in Living Subjects. Biotechnology and Genetic Engineering Reviews. Cochran, F., V., Cochran, J., R. 2010; 27: 57-94
  • A Dual-Labeled Knottin Peptide for PET and Near-Infrared Fluorescence Imaging of Integrin Expression in Living Subjects. Bioconjugate chemistry Kimura, R. H., Miao, Z., Cheng, Z., Gambhir, S. S., Cochran, J. R. 2010

    Abstract

    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 PubMedID 20131753

  • An Engineered Knottin Peptide Labeled with F-18 for PET Imaging of Integrin Expression BIOCONJUGATE CHEMISTRY Miao, Z., Ren, G., Liu, H., Kimura, R. H., Jiang, L., Cochran, J. R., Gambhir, S. S., Cheng, Z. 2009; 20 (12): 2342-2347

    Abstract

    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 Kimura, R. H., Levin, A. M., Cochran, F. V., Cochran, J. R. 2009; 77 (2): 359-369

    Abstract

    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 Lahti, J. L., Silverman, A. P., Cochran, J. R. 2009; 5 (9)

    Abstract

    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 Myung, D., Farooqui, N., Zheng, L. L., Koh, W., Gupta, S., Bakri, A., Noolandi, J., Cochran, J. R., Frank, C. W., Ta, C. N. 2009; 90 (1): 70-81

    Abstract

    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

  • Antibodies specifically targeting a locally misfolded region of tumor associated EGFR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Garrett, T. P., Burgess, A. W., Gan, H. K., Luwor, R. B., Cartwright, G., Walker, F., Orchard, S. G., Clayton, A. H., Nice, E. C., Rothacker, J., Catimel, B., Cavenee, W. K., Old, L. J., Stockert, E., Ritter, G., Adams, T. E., Hoyne, P. A., Wittrup, D., Chao, G., Cochran, J. R., Luo, C., Lou, M., Huyton, T., Xu, Y., Fairlie, W. D., Yao, S., Scott, A. M., Johns, T. G. 2009; 106 (13): 5082-5087

    Abstract

    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 Silverman, A. P., Levin, A. M., Lahti, J. L., Cochran, J. R. 2009; 385 (4): 1064-1075

    Abstract

    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

  • Developing therapeutic proteins by engineering ligand-receptor interactions TRENDS IN BIOTECHNOLOGY Jones, D. S., Silverman, A. P., Cochran, J. R. 2008; 26 (9): 498-505

    Abstract

    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 BIOTECHNOLOGY PROGRESS Myung, D., Duhamel, P., Cochran, J. R., Noolandi, J., Ta, C. N., Frank, C. W. 2008; 24 (3): 735-741

    Abstract

    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

  • Design and fabrication of an artificial cornea based on a photolithographically patterned hydrogel construct BIOMEDICAL MICRODEVICES Myung, D., Koh, W., Bakri, A., Zhang, F., Marshall, A., Ko, J., Noolandi, J., Carrasco, M., Cochran, J. R., Frank, C. W., Ta, C. N. 2007; 9 (6): 911-922

    Abstract

    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 BIOCHEMISTRY Hanick, N. A., Rickert, M., Varani, L., Bankovich, A. J., Cochran, J. R., Kim, D. M., Surh, C. D., Garcia, K. C. 2007; 46 (33): 9453-9461

    Abstract

    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

  • Directed evolution of the epidermal growth factor receptor extracellular domain for expression in yeast PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS Kim, Y. S., Bhandari, R., Cochran, J. R., Kuriyan, J., Wittrup, K. D. 2006; 62 (4): 1026-1035

    Abstract

    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

  • Improved mutants from directed evolution are biased to orthologous substitutions Protein Engineering, Design and Selection Cochran JR, Kim YS, Lippow SM, Rao B, Wittrup KD 2006; 19: 245-53
  • Fine epitope mapping anti-epidermal growth factor receptor antibodies through random mutagenesia and yeast surface display JOURNAL OF MOLECULAR BIOLOGY Chao, G., Cochran, J. R., Wittrup, K. D. 2004; 342 (2): 539-550

    Abstract

    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 Johns, T. G., Adamas, T. E., Cochran, J. R., Hall, N. E., Hoyne, P. A., Olsen, M. J., Kim, Y. S., Rothacker, J., Nice, E. C., Walker, F., Ritter, G., Jungbluth, A. J., Old, L. J., Ward, C. W., Burgess, A. W., Wittrup, K. D., Scott, A. M. 2004; 279 (29): 30375-30384

    Abstract

    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 Cochran, J. R., Kim, Y. S., Olsen, M. J., Bhandari, R., Wittrup, K. D. 2004; 287 (1-2): 147-158

    Abstract

    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 NATURE BIOTECHNOLOGY Feldhaus, M. J., Siegel, R. W., Opresko, L. K., Coleman, J. R., Feldhaus, J. M., Yeung, Y. A., Cochran, J. R., Heinzelman, P., Colby, D., Swers, J., Graff, C., Wiley, H. S., Wittrup, K. D. 2003; 21 (2): 163-170

    Abstract

    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 Ge, Q., Stone, J. D., Thompson, M. T., Cochran, J. R., Rushe, M., Eisen, H. N., Chen, J. Z., Stern, L. J. 2002; 99 (21): 13729-13734

    Abstract

    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 BIOPHYSICAL JOURNAL Stone, J. D., Cochran, J. R., Stern, L. J. 2001; 81 (5): 2547-2557

    Abstract

    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 Cameron, T. O., Stone, J. D., Cochran, J. R., Stern, L. J. 2001; 22 (9): 479-480

    View details for Web of Science ID 000170753800003

    View details for PubMedID 11556322

  • Receptor proximity, not intermolecular orientation, is critical for triggering T-cell activation JOURNAL OF BIOLOGICAL CHEMISTRY Cochran, J. R., Cameron, T. O., Stone, J. D., Lubetsky, J. B., Stern, L. J. 2001; 276 (30): 28068-28074

    Abstract

    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 Cochran, J. R., Aivazian, D., Cameron, T. O., Stern, L. J. 2001; 26 (5): 304-310

    Abstract

    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 Cameron, T. O., Cochran, J. R., Yassine-Diab, B., Sekaly, R. P., Stern, L. J. 2001; 166 (2): 741-745

    Abstract

    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 Cochran, J. R., Stern, L. J. 2000; 7 (9): 683-696

    Abstract

    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 IMMUNITY Cochran, J. R., Cameron, T. O., Stern, L. J. 2000; 12 (3): 241-250

    Abstract

    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

Books and Book Chapters


  • Surface Modification of High-Strength Interpenetrating Network Hydrogels for Biomedical Device Applications Handbook of Biofunctional Surfaces Myung, D., Kourtis, L., Noolandi, J., Cochran, J., Ta, C., N., Frank, C., W. edited by Knoll, W. Pan Stanford Publishing.. 2013: 407-446
  • Engineering Multivalent and Multispecific Protein Therapeutics Engineering in Translational Medicine Liu, C., J., Cochran, J., R. edited by Cai, W. Springer.. 2013: 1
  • Rational and Combinatorial Methods for Creating Designer Protein Interfaces Comprehensive Biomaterials Lui, B., H., Cochran, J, R. edited by Ducheyne, H., Hutmacher, G. Elsevier.. 2011: 1
  • Yeast Surface Display Therapeutic Antibodies: from Theory to Practice Lahti, J., L., Cochran, J., R. edited by An, Z., Strohl, W. John Wiley & Sons, Inc.. 2009: 1
  • Cell Surface Display Systems for Protein Engineering Protein Engineering and Design Moore, S., J., Olsen, M., J., Cochran, J., R., Cochran, F., V. edited by Park, Sheldon, J., Cochran, Jennifer, R. Taylor and Francis, Boca Raton.. 2009: 1
  • Protein Engineering and Design edited by J., R., Park, Jennifer Taylor and Francis, Boca Raton.. 2009

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