Jennifer R. Cochran

All Publications

• An engineered interleukin-1 decoy cytokine inhibits receptor signaling and proliferation in lung adenocarcinoma BIOENGINEERING & TRANSLATIONAL MEDICINE McIntosh, B. J., Hartmann, G. G., Yamada-Hunter, S. A., Liu, P., Williams, C. F., Sage, J., Cochran, J. R. 2023

  View details for DOI 10.1002/btm2.10573

  View details for Web of Science ID 001031916000001

• Targeted TLR9 Agonist Elicits Effective Antitumor Immunity against Spontaneously Arising Breast Tumors. Journal of immunology (Baltimore, Md. : 1950) Miller, C. L., Sagiv-Barfi, I., Neuhöfer, P., Czerwinski, D. K., Bertozzi, C. R., Cochran, J. R., Levy, R. 2023

  Abstract

  Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.

  View details for DOI 10.4049/jimmunol.2200950

  View details for PubMedID 37256255

• Mutational screens highlight glycosylation as a modulator of colony-stimulating factor 3 receptor (CSF3R) activity. The Journal of biological chemistry Hollander, M. J., Malaker, S. A., Riley, N. M., Perez, I., Abney, N. M., Gray, M. A., Maxson, J. E., Cochran, J. R., Bertozzi, C. R. 2023: 104755

  Abstract

  The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia (CNL). However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in CNL and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues - the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at wild-type threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain (IL-31Rα). Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.

  View details for DOI 10.1016/j.jbc.2023.104755

  View details for PubMedID 37116708

• Enhanced safety and efficacy of protease-regulated CAR-T cell receptors. Cell Labanieh, L., Majzner, R. G., Klysz, D., Sotillo, E., Fisher, C. J., Vilches-Moure, J. G., Pacheco, K. Z., Malipatlolla, M., Xu, P., Hui, J. H., Murty, T., Theruvath, J., Mehta, N., Yamada-Hunter, S. A., Weber, E. W., Heitzeneder, S., Parker, K. R., Satpathy, A. T., Chang, H. Y., Lin, M. Z., Cochran, J. R., Mackall, C. L. 2022

  Abstract

  Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.

  View details for DOI 10.1016/j.cell.2022.03.041

  View details for PubMedID 35483375

• An engineered ligand trap inhibits leukemia inhibitory factor as pancreatic cancer treatment strategy. Communications biology Hunter, S. A., McIntosh, B. J., Shi, Y., Sperberg, R. A., Funatogawa, C., Labanieh, L., Soon, E., Wastyk, H. C., Mehta, N., Carter, C., Hunter, T., Cochran, J. R. 2021; 4 (1): 452

  Abstract

  Leukemia inhibitory factor (LIF), a cytokine secreted by stromal myofibroblasts and tumor cells, has recently been highlighted to promote tumor progression in pancreatic and other cancers through KRAS-driven cell signaling. We engineered a high affinity soluble humanLIF receptor (LIFR) decoy that sequesters humanLIF and inhibits its signaling as a therapeutic strategy. This engineered 'ligand trap', fused to an antibody Fc-domain, has ~50-fold increased affinity (~20 pM) and improved LIF inhibition compared to wild-type LIFR-Fc, potently blocks LIF-mediated effects in pancreatic cancer cells, and slows the growth of pancreatic cancer xenograft tumors. These results, and the lack of apparent toxicity observed in animal models, further highlights ligand traps as a promising therapeutic strategy for cancer treatment.

  View details for DOI 10.1038/s42003-021-01928-2

  View details for PubMedID 33846527

• Systemic delivery of a targeted synthetic immunostimulant transforms the immune landscape for effective tumor regression. Cell chemical biology Miller, C. L., Sagiv-Barfi, I., Neuhöfer, P., Czerwinski, D. K., Artandi, S. E., Bertozzi, C. R., Levy, R., Cochran, J. R. 2021

  Abstract

  Promoting immune activation within the tumor microenvironment (TME) is a promising therapeutic strategy to reverse tumor immunosuppression and elicit anti-tumor immunity. To enable tumor-localized immunotherapy following intravenous administration, we chemically conjugated a polyspecific integrin-binding peptide (PIP) to an immunostimulant (Toll-like receptor 9 [TLR9] agonist: CpG) to generate a tumor-targeted immunomodulatory agent, referred to as PIP-CpG. We demonstrate that systemic delivery of PIP-CpG induces tumor regression and enhances therapeutic efficacy compared with untargeted CpG in aggressive murine breast and pancreatic cancer models. Furthermore, PIP-CpG transforms the immune-suppressive TME dominated by myeloid-derived suppressor cells into a lymphocyte-rich TME infiltrated with activated CD8+ T cells, CD4+ T cells, and B cells. Finally, we show that T cells are required for therapeutic efficacy and that PIP-CpG treatment generates tumor-specific CD8+ T cells. These data demonstrate that conjugation to a synthetic tumor-targeted peptide can improve the efficacy of systemically administered immunostimulants and lead to durable anti-tumor immune responses.

  View details for DOI 10.1016/j.chembiol.2021.10.012

  View details for PubMedID 34774126

• Engineering a potent receptor superagonist or antagonist from a novel IL-6 family cytokine ligand. Proceedings of the National Academy of Sciences of the United States of America Kim, J. W., Marquez, C. P., Sperberg, R. A., Wu, J., Bae, W. G., Huang, P., Sweet-Cordero, E. A., Cochran, J. R. 2020

  Abstract

  Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.

  View details for DOI 10.1073/pnas.1922729117

  View details for PubMedID 32522868

• Structure and Functional Binding Epitope of V-domain Ig Suppressor of T Cell Activation. Cell reports Mehta, N. n., Maddineni, S. n., Mathews, I. I., Andres Parra Sperberg, R. n., Huang, P. S., Cochran, J. R. 2019; 28 (10): 2509–16.e5

  Abstract

  V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint protein that inhibits the T cell response against cancer. Similar to PD-1 and CTLA-4, a blockade of VISTA promotes tumor clearance by the immune system. Here, we report a 1.85 Å crystal structure of the elusive human VISTA extracellular domain, whose lack of homology necessitated a combinatorial MR-Rosetta approach for structure determination. We highlight features that make the VISTA immunoglobulin variable (IgV)-like fold unique among B7 family members, including two additional disulfide bonds and an extended loop region with an attached helix that we show forms a contiguous binding epitope for a clinically relevant anti-VISTA antibody. We propose an overlap of this antibody-binding region with the binding epitope for V-set and Ig domain containing 3 (VSIG3), a purported functional binding partner of VISTA. The structure and functional epitope presented here will help guide future drug development efforts against this important checkpoint target.

  View details for DOI 10.1016/j.celrep.2019.07.073

  View details for PubMedID 31484064

• Antitumor activity of an engineered decoy receptor targeting CLCF1-CNTFR signaling in lung adenocarcinoma. Nature medicine Kim, J. W., Marquez, C. P., Kostyrko, K. n., Koehne, A. L., Marini, K. n., Simpson, D. R., Lee, A. G., Leung, S. G., Sayles, L. C., Shrager, J. n., Ferrer, I. n., Paz-Ares, L. n., Gephart, M. H., Vicent, S. n., Cochran, J. R., Sweet-Cordero, E. A. 2019

  Abstract

  Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.

  View details for DOI 10.1038/s41591-019-0612-2

  View details for PubMedID 31700175

• Integrin-targeted cancer immunotherapy elicits protective adaptive immune responses. journal of experimental medicine Kwan, B. H., Zhu, E. F., Tzeng, A., Sugito, H. R., Eltahir, A. A., Ma, B., Delaney, M. K., Murphy, P. A., Kauke, M. J., Angelini, A., Momin, N., Mehta, N. K., Maragh, A. M., Hynes, R. O., Dranoff, G., Cochran, J. R., Wittrup, K. D. 2017; 214 (6): 1679-1690

  Abstract

  Certain RGD-binding integrins are required for cell adhesion, migration, and proliferation and are overexpressed in most tumors, making them attractive therapeutic targets. However, multiple integrin antagonist drug candidates have failed to show efficacy in cancer clinical trials. In this work, we instead exploit these integrins as a target for antibody Fc effector functions in the context of cancer immunotherapy. By combining administration of an engineered mouse serum albumin/IL-2 fusion with an Fc fusion to an integrin-binding peptide (2.5F-Fc), significant survival improvements are achieved in three syngeneic mouse tumor models, including complete responses with protective immunity. Functional integrin antagonism does not contribute significantly to efficacy; rather, this therapy recruits both an innate and adaptive immune response, as deficiencies in either arm result in reduced tumor control. Administration of this integrin-targeted immunotherapy together with an anti-PD-1 antibody further improves responses and predominantly results in cures. Overall, this well-tolerated therapy achieves tumor specificity by redirecting inflammation to a functional target fundamental to tumorigenic processes but expressed at significantly lower levels in healthy tissues, and it shows promise for translation.

  View details for DOI 10.1084/jem.20160831

  View details for PubMedID 28473400

• High-throughput analysis and protein engineering using microcapillary arrays. Nature chemical biology Chen, B., Lim, S., Kannan, A., Alford, S. C., Sunden, F., Herschlag, D., Dimov, I. K., Baer, T. M., Cochran, J. R. 2016; 12 (2): 76-81

  Abstract

  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 Kariolis, M. S., Miao, Y. R., Ii, D. S., Kapur, S., Mathews, I. I., Giaccia, A. J., Cochran, J. R. 2014; 10 (11): 977-983

  Abstract

  Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

  View details for DOI 10.1038/NCHEMBIO.1636

  View details for Web of Science ID 000343621500022

  View details for PubMedCentralID PMC4372605

• Engineered knottin peptide enables noninvasive optical imaging of intracranial medulloblastoma. Proceedings of the National Academy of Sciences of the United States of America Moore, S. J., Hayden Gephart, M. G., Bergen, J. M., Su, Y. S., Rayburn, H., Scott, M. P., Cochran, J. R. 2013; 110 (36): 14598-14603

  Abstract

  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

  View details for PubMedCentralID PMC3767496

• De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8. Nature communications Roy, A., Shi, L., Chang, A., Dong, X., Fernandez, A., Kraft, J. C., Li, J., Le, V. Q., Winegar, R. V., Cherf, G. M., Slocum, D., Poulson, P. D., Casper, G. E., Vallecillo-Zúniga, M. L., Valdoz, J. C., Miranda, M. C., Bai, H., Kipnis, Y., Olshefsky, A., Priya, T., Carter, L., Ravichandran, R., Chow, C. M., Johnson, M. R., Cheng, S., Smith, M., Overed-Sayer, C., Finch, D. K., Lowe, D., Bera, A. K., Matute-Bello, G., Birkland, T. P., DiMaio, F., Raghu, G., Cochran, J. R., Stewart, L. J., Campbell, M. G., Van Ry, P. M., Springer, T., Baker, D. 2023; 14 (1): 5660

  Abstract

  The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

  View details for DOI 10.1038/s41467-023-41272-z

  View details for PubMedID 37704610

  View details for PubMedCentralID 4717663

• Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro. Nature communications Zeng, L., Liu, Y., Nguyenla, X. H., Abbott, T. R., Han, M., Zhu, Y., Chemparathy, A., Lin, X., Chen, X., Wang, H., Rane, D. A., Spatz, J. M., Jain, S., Rustagi, A., Pinsky, B., Zepeda, A. E., Kadina, A. P., Walker, J. A., Holden, K., Temperton, N., Cochran, J. R., Barron, A. E., Connolly, M. D., Blish, C. A., Lewis, D. B., Stanley, S. A., La Russa, M. F., Qi, L. S. 2022; 13 (1): 2766

  Abstract

  A major challenge in coronavirus vaccination and treatment is to counteract rapid viral evolution and mutations. Here we demonstrate that CRISPR-Cas13d offers a broad-spectrum antiviral (BSA) to inhibit many SARS-CoV-2 variants and diverse human coronavirus strains with >99% reduction of the viral titer. We show that Cas13d-mediated coronavirus inhibition is dependent on the crRNA cellular spatial colocalization with Cas13d and target viral RNA. Cas13d can significantly enhance the therapeutic effects of diverse small molecule drugs against coronaviruses for prophylaxis or treatment purposes, and the best combination reduced viral titer by over four orders of magnitude. Using lipid nanoparticle-mediated RNA delivery, we demonstrate that the Cas13d system can effectively treat infection from multiple variants of coronavirus, including Omicron SARS-CoV-2, in human primary airway epithelium air-liquid interface (ALI) cultures. Our study establishes CRISPR-Cas13 as a BSA which is highly complementary to existing vaccination and antiviral treatment strategies.

  View details for DOI 10.1038/s41467-022-30546-7

  View details for PubMedID 35589813

• Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors. Science advances Grosskopf, A. K., Labanieh, L., Klysz, D. D., Roth, G. A., Xu, P., Adebowale, O., Gale, E. C., Jons, C. K., Klich, J. H., Yan, J., Maikawa, C. L., Correa, S., Ou, B. S., d'Aquino, A. I., Cochran, J. R., Chaudhuri, O., Mackall, C. L., Appel, E. A. 2022; 8 (14): eabn8264

  Abstract

  Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.

  View details for DOI 10.1126/sciadv.abn8264

  View details for PubMedID 35394838

• Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nature medicine Theruvath, J., Menard, M., Smith, B. A., Linde, M. H., Coles, G. L., Dalton, G. N., Wu, W., Kiru, L., Delaidelli, A., Sotillo, E., Silberstein, J. L., Geraghty, A. C., Banuelos, A., Radosevich, M. T., Dhingra, S., Heitzeneder, S., Tousley, A., Lattin, J., Xu, P., Huang, J., Nasholm, N., He, A., Kuo, T. C., Sangalang, E. R., Pons, J., Barkal, A., Brewer, R. E., Marjon, K. D., Vilches-Moure, J. G., Marshall, P. L., Fernandes, R., Monje, M., Cochran, J. R., Sorensen, P. H., Daldrup-Link, H. E., Weissman, I. L., Sage, J., Majeti, R., Bertozzi, C. R., Weiss, W. A., Mackall, C. L., Majzner, R. G. 1800

  Abstract

  The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called 'Don't eat me' signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic 'Eat me' signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

  View details for DOI 10.1038/s41591-021-01625-x

  View details for PubMedID 35027753

• Heterogeneous delivery across the blood-brain barrier limits the efficacy of an EGFR-targeting antibody drug conjugate in glioblastoma. Neuro-oncology Marin, B., Porath, K. A., Jain, S., Kim, M., Conage-Pough, J. E., Oh, J., Miller, C. L., Talele, S., Kitange, G. J., Tian, S., Burgenske, D. M., Mladek, A. C., Gupta, S. K., Decker, P. A., McMinn, M. H., Stopka, S. A., Regan, M. S., He, L., Carlson, B. L., Bakken, K., Burns, T. C., Parney, I. F., Giannini, C., Agar, N. Y., Eckel-Passow, J. E., Cochran, J. R., Elmquist, W. F., Vaubel, R. A., White, F. M., Sarkaria, J. N. 2021

  Abstract

  BACKGROUND: Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important.METHODS: PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH and phosphoproteomics.RESULTS: Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment.CONCLUSIONS: Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

  View details for DOI 10.1093/neuonc/noab133

  View details for PubMedID 34050676

• VISTA immune-checkpoint blunts radiotherapy induced anti-tumor immune response. Nambiar, D. K., Mehta, N., Maddineni, S., Cao, H., Viswanathan, V., Cheunkarndee, T., Cochran, J. R., Quynh Thu Le AMER ASSOC CANCER RESEARCH. 2021

  View details for Web of Science ID 000641160600078

• LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nature chemical biology Ahn, G., Banik, S. M., Miller, C. L., Riley, N. M., Cochran, J. R., Bertozzi, C. R. 2021

  Abstract

  Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.

  View details for DOI 10.1038/s41589-021-00770-1

  View details for PubMedID 33767387

• Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naive human antibody library. Protein science : a publication of the Protein Society Bell, B. N., Powell, A. E., Rodriguez, C., Cochran, J. R., Kim, P. S. 2021

  Abstract

  Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naive antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naive antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naive antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/pro.4044

  View details for PubMedID 33586288

• Use of Outpatient-Derived COVID-19 Convalescent Plasma in COVID-19 Patients Before Seroconversion. Frontiers in immunology Wirz, O. F., Roltgen, K., Stevens, B. A., Pandey, S., Sahoo, M. K., Tolentino, L., Verghese, M., Nguyen, K., Hunter, M., Snow, T. T., Singh, A. R., Blish, C. A., Cochran, J. R., Zehnder, J. L., Nadeau, K. C., Pinsky, B. A., Pham, T. D., Boyd, S. D. 2021; 12: 739037

  Abstract

  Background: Transfusion of COVID-19 convalescent plasma (CCP) containing high titers of anti-SARS-CoV-2 antibodies serves as therapy for COVID-19 patients. Transfusions early during disease course was found to be beneficial. Lessons from the SARS-CoV-2 pandemic could inform early responses to future pandemics and may continue to be relevant in lower resource settings. We sought to identify factors correlating to high antibody titers in convalescent plasma donors and understand the magnitude and pharmacokinetic time course of both transfused antibody titers and the endogenous antibody titers in transfused recipients.Methods: Plasma samples were collected up to 174 days after convalescence from 93 CCP donors with mild disease, and from 16 COVID-19 patients before and after transfusion. Using ELISA, anti-SARS-CoV-2 Spike RBD, S1, and N-protein antibodies, as well as capacity of antibodies to block ACE2 from binding to RBD was measured in an in vitro assay. As an estimate for viral load, viral RNA and N-protein plasma levels were assessed in COVID-19 patients.Results: Anti-SARS-CoV-2 antibody levels and RBD-ACE2 blocking capacity were highest within the first 60 days after symptom resolution and markedly decreased after 120 days. Highest antibody titers were found in CCP donors that experienced fever. Effect of transfused CCP was detectable in COVID-19 patients who received high-titer CCP and had not seroconverted at the time of transfusion. Decrease in viral RNA was seen in two of these patients.Conclusion: Our results suggest that high titer CCP should be collected within 60 days after recovery from donors with past fever. The much lower titers conferred by transfused antibodies compared to endogenous production in the patient underscore the importance of providing CCP prior to endogenous seroconversion.

  View details for DOI 10.3389/fimmu.2021.739037

  View details for PubMedID 34594341

• Identification of N-Terminally Diversified GLP-1R Agonists Using Saturation Mutagenesis and Chemical Design. ACS chemical biology Longwell, C. K., Hanna, S., Hartrampf, N., Sperberg, R. A., Huang, P., Pentelute, B. L., Cochran, J. R. 2020

  Abstract

  The glucagon-like peptide 1 receptor (GLP-1R) is a class B G-protein coupled receptor (GPCR) and diabetes drug target expressed mainly in pancreatic beta-cells that, when activated by its agonist glucagon-like peptide 1 (GLP-1) after a meal, stimulates insulin secretion and beta-cell survival and proliferation. The N-terminal region of GLP-1 interacts with membrane-proximal residues of GLP-1R, stabilizing its active conformation to trigger intracellular signaling. The best-studied agonist peptides, GLP-1 and exendin-4, share sequence homology at their N-terminal region; however, modifications that can be tolerated here are not fully understood. In this work, a functional screen of GLP-1 variants with randomized N-terminal domains reveals new GLP-1R agonists and uncovers a pattern whereby a negative charge is preferred at the third position in various sequence contexts. We further tested this sequence-structure-activity principle by synthesizing peptide analogues where this position was mutated to both canonical and noncanonical amino acids. We discovered a highly active GLP-1 analogue in which the native glutamate residue three positions from the N-terminus was replaced with the sulfo-containing amino acid cysteic acid (GLP-1-CYA). The receptor binding and downstream signaling properties elicited by GLP-1-CYA were similar to the wild type GLP-1 peptide. Computational modeling identified a likely mode of interaction of the negatively charged side chain in GLP-1-CYA with an arginine on GLP-1R. This work highlights a strategy of combinatorial peptide screening coupled with chemical exploration that could be used to generate novel agonists for other receptors with peptide ligands.

  View details for DOI 10.1021/acschembio.0c00722

  View details for PubMedID 33307682

• PET reporter gene imaging and ganciclovir-mediated ablation of chimeric antigen receptor T-cells in solid tumors. Cancer research Murty, S., Labanieh, L., Murty, T., Gowrishankar, G., Haywood, T., Alam, I. S., Beinat, C., Robinson, E., Aalipour, A., Klysz, D. D., Cochran, J. R., Majzner, R. G., Mackall, C. L., Gambhir, S. S. 2020

  Abstract

  Imaging strategies to monitor chimeric antigen receptor (CAR) T-cell biodistribution and proliferation harbor the potential to facilitate clinical translation for the treatment of both liquid and solid tumors. Additionally, the potential adverse effects of CAR T-cells highlight the need for mechanisms to modulate CAR T-cell activity. The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene has previously been translated as a positron emission tomography (PET) reporter gene for imaging of T-cell trafficking in brain tumor patients. The HSV1-TK enzyme can act as a suicide gene of transduced cells through treatment with the prodrug ganciclovir (GCV). Here we report the molecular engineering, imaging, and GCV-mediated destruction of B7H3 CAR T-cells incorporating a mutated version of the HSV1-tk gene (sr39tk) with improved enzymatic activity for GCV. The sr39tk gene did not affect B7H3 CAR T-cell functionality and in vitro and in vivo studies in osteosarcoma models showed no significant effect on B7H3 CAR T-cell antitumor activity. PET/CT imaging with 9-(4-[18F]-fluoro-3-[hydroxymethyl]butyl)guanine [18F]FHBG of B7H3-sr39tk CAR T-cells in an orthotopic model of osteosarcoma revealed tumor homing and systemic immune expansion. Bioluminescence and PET imaging of B7H3-sr39tk CAR T-cells confirmed complete tumor ablation with intraperitoneal GCV administration. This imaging and suicide ablation system can provide insight into CAR T-cell migration and proliferation during clinical trials while serving as a suicide switch to limit potential toxicities.

  View details for DOI 10.1158/0008-5472.CAN-19-3579

  View details for PubMedID 32958548

• An engineered antibody binds a distinct epitope and is a potent inhibitor of murine and human VISTA. Scientific reports Mehta, N., Maddineni, S., Kelly, R. L., Lee, R. B., Hunter, S. A., Silberstein, J. L., Parra Sperberg, R. A., Miller, C. L., Rabe, A., Labanieh, L., Cochran, J. R. 2020; 10 (1): 15171

  Abstract

  V-domain immunoglobulin (Ig) suppressor of T cell activation (VISTA) is an immune checkpoint that maintains peripheral T cell quiescence and inhibits anti-tumor immune responses. VISTA functions by dampening the interaction between myeloid cells and T cells, orthogonal to PD-1 and other checkpoints of the tumor-T cell signaling axis. Here, we report the use of yeast surface display to engineer an anti-VISTA antibody that binds with high affinity to mouse, human, and cynomolgus monkey VISTA. Our anti-VISTA antibody (SG7) inhibits VISTA function and blocks purported interactions with both PSGL-1 and VSIG3proteins. SG7 binds a unique epitope on the surface of VISTA, which partially overlaps with other clinically relevant antibodies. As a monotherapy, and to a greater extent as a combination with anti-PD1, SG7 slows tumor growth in multiple syngeneic mouse models. SG7 is a promising clinical candidate that can be tested in fully immunocompetent mouse models and its binding epitope can be used for future campaigns to develop species cross-reactive inhibitors of VISTA.

  View details for DOI 10.1038/s41598-020-71519-4

  View details for PubMedID 32938950

• Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nature methods Su, Y., Walker, J. R., Park, Y., Smith, T. P., Liu, L. X., Hall, M. P., Labanieh, L., Hurst, R., Wang, D. C., Encell, L. P., Kim, N., Zhang, F., Kay, M. A., Casey, K. M., Majzner, R. G., Cochran, J. R., Mackall, C. L., Kirkland, T. A., Lin, M. Z. 2020

  Abstract

  Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

  View details for DOI 10.1038/s41592-020-0889-6

  View details for PubMedID 32661427

• Multi-phase catheter-injectable hydrogel enables dual-stage protein-engineered cytokine release to mitigate adverse left ventricular remodeling following myocardial infarction in a small animal model and a large animal model. Cytokine Steele, A. N., Paulsen, M. J., Wang, H. n., Stapleton, L. M., Lucian, H. J., Eskandari, A. n., Hironaka, C. E., Farry, J. M., Baker, S. W., Thakore, A. D., Jaatinen, K. J., Tada, Y. n., Hollander, M. J., Williams, K. M., Seymour, A. J., Totherow, K. P., Yu, A. C., Cochran, J. R., Appel, E. A., Woo, Y. J. 2020; 127: 154974

  Abstract

  Although ischemic heart disease is the leading cause of death worldwide, mainstay treatments ultimately fail because they do not adequately address disease pathophysiology. Restoring the microvascular perfusion deficit remains a significant unmet need and may be addressed via delivery of pro-angiogenic cytokines. The therapeutic effect of cytokines can be enhanced by encapsulation within hydrogels, but current hydrogels do not offer sufficient clinical translatability due to unfavorable viscoelastic mechanical behavior which directly impacts the ability for minimally-invasive catheter delivery. In this report, we examine the therapeutic implications of dual-stage cytokine release from a novel, highly shear-thinning biocompatible catheter-deliverable hydrogel. We chose to encapsulate two protein-engineered cytokines, namely dimeric fragment of hepatocyte growth factor (HGFdf) and engineered stromal cell-derived factor 1α (ESA), which target distinct disease pathways. The controlled release of HGFdf and ESA from separate phases of the hyaluronic acid-based hydrogel allows extended and pronounced beneficial effects due to the precise timing of release. We evaluated the therapeutic efficacy of this treatment strategy in a small animal model of myocardial ischemia and observed a significant benefit in biological and functional parameters. Given the encouraging results from the small animal experiment, we translated this treatment to a large animal preclinical model and observed a reduction in scar size, indicating this strategy could serve as a potential adjunct therapy for the millions of people suffering from ischemic heart disease.

  View details for DOI 10.1016/j.cyto.2019.154974

  View details for PubMedID 31978642

• CD52 Is Elevated on B cells of SLE Patients and Regulates B Cell Function. Frontiers in immunology Bhamidipati, K. n., Silberstein, J. L., Chaichian, Y. n., Baker, M. C., Lanz, T. V., Zia, A. n., Rasheed, Y. S., Cochran, J. R., Robinson, W. H. 2020; 11: 626820

  Abstract

  Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B cell dysregulation and breaks in tolerance that lead to the production of pathogenic autoantibodies. We performed single-cell RNA sequencing of B cells from healthy donors and individuals with SLE which revealed upregulated CD52 expression in SLE patients. We further demonstrate that SLE patients exhibit significantly increased levels of B cell surface CD52 expression and plasma soluble CD52, and levels of soluble CD52 positively correlate with measures of lupus disease activity. Using CD52-deficient JeKo-1 cells, we show that cells lacking surface CD52 expression are hyperresponsive to B cell receptor (BCR) signaling, suggesting an inhibitory role for the surface-bound protein. In healthy donor B cells, antigen-specific BCR-activation initiated CD52 cleavage in a phospholipase C dependent manner, significantly reducing cell surface levels. Experiments with recombinant CD52-Fc showed that soluble CD52 inhibits BCR signaling in a manner partially-dependent on Siglec-10. Moreover, incubation of unstimulated B cells with CD52-Fc resulted in the reduction of surface immunoglobulin and CXCR5. Prolonged incubation of B cells with CD52 resulted in the expansion of IgD+IgMlo anergic B cells. In summary, our findings suggest that CD52 functions as a homeostatic protein on B cells, by inhibiting responses to BCR signaling. Further, our data demonstrate that CD52 is cleaved from the B cell surface upon antigen engagement, and can suppress B cell function in an autocrine and paracrine manner. We propose that increased expression of CD52 by B cells in SLE represents a homeostatic mechanism to suppress B cell hyperactivity.

  View details for DOI 10.3389/fimmu.2020.626820

  View details for PubMedID 33658999

  View details for PubMedCentralID PMC7917337

• Defining the features and duration of antibody responses to SARS-CoV-2 infection associated with disease severity and outcome. Science immunology Röltgen, K. n., Powell, A. E., Wirz, O. F., Stevens, B. A., Hogan, C. A., Najeeb, J. n., Hunter, M. n., Wang, H. n., Sahoo, M. K., Huang, C. n., Yamamoto, F. n., Manohar, M. n., Manalac, J. n., Otrelo-Cardoso, A. R., Pham, T. D., Rustagi, A. n., Rogers, A. J., Shah, N. H., Blish, C. A., Cochran, J. R., Jardetzky, T. S., Zehnder, J. L., Wang, T. T., Narasimhan, B. n., Gombar, S. n., Tibshirani, R. n., Nadeau, K. C., Kim, P. S., Pinsky, B. A., Boyd, S. D. 2020; 5 (54)

  Abstract

  SARS-CoV-2-specific antibodies, particularly those preventing viral spike receptor binding domain (RBD) interaction with host angiotensin-converting enzyme 2 (ACE2) receptor, can neutralize the virus. It is, however, unknown which features of the serological response may affect clinical outcomes of COVID-19 patients. We analyzed 983 longitudinal plasma samples from 79 hospitalized COVID-19 patients and 175 SARS-CoV-2-infected outpatients and asymptomatic individuals. Within this cohort, 25 patients died of their illness. Higher ratios of IgG antibodies targeting S1 or RBD domains of spike compared to nucleocapsid antigen were seen in outpatients who had mild illness versus severely ill patients. Plasma antibody increases correlated with decreases in viral RNAemia, but antibody responses in acute illness were insufficient to predict inpatient outcomes. Pseudovirus neutralization assays and a scalable ELISA measuring antibodies blocking RBD-ACE2 interaction were well correlated with patient IgG titers to RBD. Outpatient and asymptomatic individuals' SARS-CoV-2 antibodies, including IgG, progressively decreased during observation up to five months post-infection.

  View details for DOI 10.1126/sciimmunol.abe0240

  View details for PubMedID 33288645

• Structural Basis of the Differential Binding of Engineered Knottins to Integrins alphaVbeta3 and alpha5beta1. Structure (London, England : 1993) Van Agthoven, J. F., Shams, H., Cochran, F. V., Alonso, J. L., Kintzing, J. R., Garakani, K., Adair, B. D., Xiong, J., Mofrad, M. R., Cochran, J. R., Arnaout, M. A. 2019

  Abstract

  Targeting both integrins alphaVbeta3 and alpha5beta1 simultaneously appears to be more effective in cancer therapy than targeting each one alone. The structural requirements for bispecific binding of ligand to integrins have not been fully elucidated. RGD-containing knottin 2.5F binds selectively to alphaVbeta3 and alpha5beta1, whereas knottin 2.5D is alphaVbeta3 specific. To elucidate the structural basis of this selectivity, we determined the structures of 2.5F and 2.5D as apo proteins and in complex with alphaVbeta3, and compared their interactions with integrins using molecular dynamics simulations. These studies show that 2.5D engages alphaVbeta3 by an induced fit, but conformational selection of a flexible RGD loop accounts for high-affinity selective binding of 2.5F to both integrins. The contrasting binding of the highly flexible low-affinity linear RGD peptides to multiple integrins suggests that a "Goldilocks zone" of conformational flexibility of the RGD loop in 2.5F underlies its selective binding promiscuity to integrins.

  View details for DOI 10.1016/j.str.2019.06.011

  View details for PubMedID 31353240

• An engineered dimeric fragment of hepatocyte growth factor improves corneal epithelial wound healing in vitro Carter, K., Ye, A., Fernandes-Cunha, G., Cochran, J. R., Myung, D. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019

  View details for Web of Science ID 000488628107322

• Next-generation protein therapeutics: Challenges and opportunities Cochran, J. AMER CHEMICAL SOC. 2019

  View details for Web of Science ID 000478860500908

• Engineering ligand-based receptor agonists or antagonists as next-generation protein therapeutics Cochran, J. AMER CHEMICAL SOC. 2018

  View details for Web of Science ID 000447600001370

• Targeting the CLCF1-CNTFR signaling axis using directed evolution for lung cancer therapy Marquez, C., Kim, J., Giaccia, A., Cochran, J., Sweet-Cordero, A. AMER ASSOC CANCER RESEARCH. 2018

  View details for DOI 10.1158/1538-7445.AM2018-3918

  View details for Web of Science ID 000468819502175

• Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein JOURNAL OF BIOLOGICAL CHEMISTRY Mitchell, A. C., Kannan, D., Hunter, S. A., Sperberg, R., Chang, C. H., Cochran, J. R. 2018; 293 (14): 4969–80

  Abstract

  Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.

  View details for PubMedID 29386351

  View details for PubMedCentralID PMC5892588

• Photoactive Split Green Fluorescent Protein: Engineering a New Optogenetic and Imaging System Romei, M. G., Longwell, C. K., Cochran, J. R., Boxer, S. G. CELL PRESS. 2018: 177A–178A

  View details for Web of Science ID 000430439600139

• Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein ACS CHEMICAL BIOLOGY Mitchell, A. C., Alford, S. C., Hunter, S. A., Kannan, D., Sperberg, R., Chang, C. H., Cochran, J. R. 2018; 13 (1): 66–72

  Abstract

  Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.

  View details for PubMedID 29125730

• High-throughput screening technologies for enzyme engineering CURRENT OPINION IN BIOTECHNOLOGY Longwell, C. K., Labanieh, L., Cochran, J. R. 2017; 48: 196–202

  Abstract

  Emerging technologies are enabling ultra-high-throughput screening of combinatorial enzyme libraries to identify variants with improved properties such as increased activity, altered substrate specificity, and increased stability. Each of these enzyme engineering platforms relies on compartmentalization of reaction components, similar to microtiter plate-based assays which have been commonly used for testing the activity of enzyme variants. The technologies can be broadly divided into three categories according to their spatial segregation strategy: (1) cells as reaction compartments, (2) in vitro compartmentalization via synthetic droplets, and (3) microchambers. Here, we discuss these emerging platforms, which in some cases enable the screening of greater than 10 million enzyme variants, and highlight benefits and limitations of each technology.

  View details for PubMedID 28624724

• Heterochiral Knottin Protein: Folding and Solution Structure BIOCHEMISTRY Mong, S. K., Cochran, F. V., Yu, H., Graziano, Z., Lin, Y., Cochran, J. R., Pentelute, B. L. 2017; 56 (43): 5720–25

  Abstract

  Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective β-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate β-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.

  View details for PubMedID 28952732

• A novel protein-engineered hepatocyte growth factor analog released via a shear-thinning injectable hydrogel enhances post-infarction ventricular function. Biotechnology and bioengineering Steele, A. N., Cai, L., Truong, V. N., Edwards, B. B., Goldstone, A. B., Eskandari, A., Mitchell, A. C., Marquardt, L. M., Foster, A. A., Cochran, J. R., Heilshorn, S. C., Woo, Y. J. 2017

  Abstract

  In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 μg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 μg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;9999: 1-11. © 2017 Wiley Periodicals, Inc.

  View details for DOI 10.1002/bit.26345

  View details for PubMedID 28574594

• Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions BIOTECHNOLOGY JOURNAL Lim, S., Glasgow, J. E., Interrante, M. F., Storm, E. M., Cochran, J. R. 2017; 12 (5)

  Abstract

  Yeast surface display, a well-established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N- or C-terminus of the yeast Aga2p mating protein. Historically, yeast-displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N-terminus and one to the C-terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody-staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein-protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co-expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.

  View details for DOI 10.1002/biot.201600696

  View details for Web of Science ID 000400613400008

  View details for PubMedID 28299901

• Measurements of translation initiation from all 64 codons in E. coli NUCLEIC ACIDS RESEARCH Hecht, A., Glasgow, J., Jaschke, P. R., Bawazer, L. A., Munson, M. S., Cochran, J. R., Endy, D., Salit, M. 2017; 45 (7): 3615-3626

  Abstract

  Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

  View details for DOI 10.1093/nar/gkx070

  View details for Web of Science ID 000399448400011

  View details for PubMedID 28334756

• Targeting ligand-receptor interactions for development of cancer therapeutics. Current opinion in chemical biology Kim, J. W., Cochran, J. R. 2017; 38: 62-69

  Abstract

  The biological importance and druggable properties of receptors and their cognate ligands have designated them as especially useful clinical targets. This significance continues to expand as new molecular insights underlying disease pathophysiology are uncovered. While both ligands and receptors have been exploited as drug targets, their differing biochemical properties require nuanced considerations for drug development, including where in the body they are located and how they are regulated on a cellular and molecular level. In this review we will discuss ligands and receptors as therapeutics targets, including their biodistribution and biological function. We provide examples of monoclonal antibodies (mAbs) used to modulate the activity of these targets, and discuss approaches for using engineered versions of ligands and receptors themselves for therapeutic intervention in cancer.

  View details for DOI 10.1016/j.cbpa.2017.03.010

  View details for PubMedID 28371692

• Engineered ligand-based VEGFR antagonists with increased receptor binding affinity more effectively inhibit angiogenesis. Bioengineering & translational medicine Kapur, S., Silverman, A. P., Ye, A. Z., Papo, N., Jindal, D., Blumenkranz, M. S., Cochran, J. R. 2017; 2 (1): 81-91

  Abstract

  Pathologic angiogenesis is mediated by the coordinated action of the vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling axis, along with crosstalk contributed by other receptors, notably αvβ3 integrin. We build on earlier work demonstrating that point mutations can be introduced into the homodimeric VEGF ligand to convert it into an antagonist through disruption of binding to one copy of VEGFR2. This inhibitor has limited potency, however, due to loss of avidity effects from bivalent VEGFR2 binding. Here, we used yeast surface display to engineer a variant with VEGFR2 binding affinity approximately 40-fold higher than the parental antagonist, and 14-fold higher than the natural bivalent VEGF ligand. Increased VEGFR2 binding affinity correlated with the ability to more effectively inhibit VEGF-mediated signaling, both in vitro and in vivo, as measured using VEGFR2 phosphorylation and Matrigel implantation assays. High affinity mutations found in this variant were then incorporated into a dual-specific antagonist that we previously designed to simultaneously bind to and inhibit VEGFR2 and αvβ3 integrin. The resulting dual-specific protein bound to human and murine endothelial cells with relative affinities of 120 ± 10 pM and 360 ± 50 pM, respectively, which is at least 30-fold tighter than wild-type VEGF (3.8 ± 0.5 nM). Finally, we demonstrated that this engineered high-affinity dual-specific protein could inhibit angiogenesis in a murine corneal neovascularization model. Taken together, these data indicate that protein engineering strategies can be combined to generate unique antiangiogenic candidates for further clinical development.

  View details for DOI 10.1002/btm2.10051

  View details for PubMedID 28516164

• Engineering High Affinity Protein-Protein Interactions Using a High-Throughput Microcapillary Array Platform. ACS chemical biology Lim, S., Chen, B., Kariolis, M. S., Dimov, I. K., Baer, T. M., Cochran, J. R. 2017; 12 (2): 336-341

  Abstract

  Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein-protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.

  View details for DOI 10.1021/acschembio.6b00794

  View details for PubMedID 27997117

• Where is the Future of Drug Discovery for Cancer? CELL Superti-Furga, G., Cochran, J., Crews, C. M., Frye, S., Neubauer, G., Prinjha, R., Shokat, K. 2017; 168 (4): 564-565

  Abstract

  With both small molecules and biologics succeeding in trials and in the clinic, the scope of drug discovery in cancer is changing. We asked a group of researchers to share their visions for how to identify new targets and how to approach taming them.

  View details for Web of Science ID 000396277600006

  View details for PubMedID 28187277

• Inhibition of the GAS6/AXL pathway augments the efficacy of chemotherapies JOURNAL OF CLINICAL INVESTIGATION Kariolis, M. S., Miao, Y. R., Diep, A., Nash, S. E., Olcina, M. M., Jiang, D., Jones, D. S., Kapur, S., Mathews, I. I., Koong, A. C., Rankin, E. B., Cochran, J. R., Giaccia, A. J. 2017; 127 (1): 183-198

  Abstract

  The AXL receptor and its activating ligand, growth arrest-specific 6 (GAS6), are important drivers of metastasis and therapeutic resistance in human cancers. Given the critical roles that GAS6 and AXL play in refractory disease, this signaling axis represents an attractive target for therapeutic intervention. However, the strong picomolar binding affinity between GAS6 and AXL and the promiscuity of small molecule inhibitors represent important challenges faced by current anti-AXL therapeutics. Here, we have addressed these obstacles by engineering a second-generation, high-affinity AXL decoy receptor with an apparent affinity of 93 femtomolar to GAS6. Our decoy receptor, MYD1-72, profoundly inhibited disease progression in aggressive preclinical models of human cancers and induced cell killing in leukemia cells. When directly compared with the most advanced anti-AXL small molecules in the clinic, MYD1-72 achieved superior antitumor efficacy while displaying no toxicity. Moreover, we uncovered a relationship between AXL and the cellular response to DNA damage whereby abrogation of AXL signaling leads to accumulation of the DNA-damage markers γH2AX, 53BP1, and RAD51. MYD1-72 exploited this relationship, leading to improvements upon the therapeutic index of current standard-of-care chemotherapies in preclinical models of advanced pancreatic and ovarian cancer.

  View details for DOI 10.1172/JCI85610

  View details for Web of Science ID 000392271300021

  View details for PubMedID 27893463

  View details for PubMedCentralID PMC5199716

• CAR T-cell immunotherapy of MET-expressing malignant mesothelioma ONCOIMMUNOLOGY Thayaparan, T., Petrovic, R. M., Achkova, D. Y., Zabinski, T., Davies, D. M., Klampatsa, A., Parente-Pereira, A. C., Whilding, L. M., van der Stegen, S. C., Woodman, N., Sheaff, M., Cochran, J. R., Spicer, J. F., Maher, J. 2017; 6 (12)

  View details for DOI 10.1080/2162402X.2017.1363137

  View details for Web of Science ID 000419128300001

• Engineered Proteins for Visualizing and Treating Cancer Cochran, J. R., Natl Acad Engn NATL ACADEMIES PRESS. 2017: 101–6

  View details for Web of Science ID 000431842000016

• Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment TRENDS IN PHARMACOLOGICAL SCIENCES Kintzing, J. R., Interrante, M. V., Cochrane, J. R. 2016; 37 (12): 993-1008

  Abstract

  Protein-based therapeutics have been revolutionizing the oncology space since they first appeared in the clinic two decades ago. Unlike traditional small-molecule chemotherapeutics, protein biologics promote active targeting of cancer cells by binding to cell-surface receptors and other markers specifically associated with or overexpressed on tumors versus healthy tissue. While the first approved cancer biologics were monoclonal antibodies, the burgeoning field of protein engineering is spawning research on an expanded range of protein formats and modifications that allow tuning of properties such as target-binding affinity, serum half-life, stability, and immunogenicity. In this review we highlight some of these strategies and provide examples of modified and engineered proteins under development as preclinical and clinical-stage drug candidates for the treatment of cancer.

  View details for DOI 10.1016/j.tips.2016.10.005

  View details for Web of Science ID 000389393400003

  View details for PubMedID 27836202

• Eradication of large established tumors in mice by combination immunotherapy that engages innate and adaptive immune responses. Nature medicine Moynihan, K. D., Opel, C. F., Szeto, G. L., Tzeng, A., Zhu, E. F., Engreitz, J. M., Williams, R. T., Rakhra, K., Zhang, M. H., Rothschilds, A. M., Kumari, S., Kelly, R. L., Kwan, B. H., Abraham, W., Hu, K., Mehta, N. K., Kauke, M. J., Suh, H., Cochran, J. R., Lauffenburger, D. A., Wittrup, K. D., Irvine, D. J. 2016

  Abstract

  Checkpoint blockade with antibodies specific for cytotoxic T lymphocyte-associated protein (CTLA)-4 or programmed cell death 1 (PDCD1; also known as PD-1) elicits durable tumor regression in metastatic cancer, but these dramatic responses are confined to a minority of patients. This suboptimal outcome is probably due in part to the complex network of immunosuppressive pathways present in advanced tumors, which are unlikely to be overcome by intervention at a single signaling checkpoint. Here we describe a combination immunotherapy that recruits a variety of innate and adaptive immune cells to eliminate large tumor burdens in syngeneic tumor models and a genetically engineered mouse model of melanoma; to our knowledge tumors of this size have not previously been curable by treatments relying on endogenous immunity. Maximal antitumor efficacy required four components: a tumor-antigen-targeting antibody, a recombinant interleukin-2 with an extended half-life, anti-PD-1 and a powerful T cell vaccine. Depletion experiments revealed that CD8(+) T cells, cross-presenting dendritic cells and several other innate immune cell subsets were required for tumor regression. Effective treatment induced infiltration of immune cells and production of inflammatory cytokines in the tumor, enhanced antibody-mediated tumor antigen uptake and promoted antigen spreading. These results demonstrate the capacity of an elicited endogenous immune response to destroy large, established tumors and elucidate essential characteristics of combination immunotherapies that are capable of curing a majority of tumors in experimental settings typically viewed as intractable.

  View details for DOI 10.1038/nm.4200

  View details for PubMedID 27775706

  View details for PubMedCentralID PMC5209798

• Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles CURRENT OPINION IN CHEMICAL BIOLOGY Kintzing, J. R., Cochran, J. R. 2016; 34: 143-150

  Abstract

  Inhibitor cystine-knots, also known as knottins, are a structural family of ultra-stable peptides with diverse functions. Knottins and related backbone-cyclized peptides called cyclotides contain three disulfide bonds connected in a particular arrangement that endows these peptides with high thermal, proteolytic, and chemical stability. Knottins have gained interest as candidates for non-invasive molecular imaging and for drug development as they can possess the pharmacological properties of small molecules and the target affinity and selectively of protein biologics. Naturally occurring knottins are clinically approved for treating chronic pain and GI disorders. Combinatorial methods are being used to engineer knottins that can bind to other clinically relevant targets in cancer, and inflammatory and cardiac disease. This review details recent examples of engineered knottin peptides; their use as molecular imaging agents, therapeutics, and drug delivery vehicles; modifications that can be introduced to improve peptide folding and bioactivity; and future perspectives and challenges in the field.

  View details for DOI 10.1016/j.cbpa.2016.08.022

  View details for Web of Science ID 000389103600019

  View details for PubMedID 27642714

• Integrin-Targeting Knottin Peptide-Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation. Angewandte Chemie (International ed. in English) Cox, N., Kintzing, J. R., Smith, M., Grant, G. A., Cochran, J. R. 2016; 55 (34): 9894-9897

  Abstract

  Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins. This KDC binds to tumor cells with low-nanomolar affinity, is internalized by an integrin-mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine-resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug-conjugate toolkit to include non-antibody protein scaffolds.

  View details for DOI 10.1002/anie.201603488

  View details for PubMedID 27304709

• In Vivo Site-Specific Protein Tagging with Diverse Amines Using an Engineered Sortase Variant JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Glasgow, J. E., Salit, M. L., Cochran, J. R. 2016; 138 (24): 7496-7499

  Abstract

  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 Currier, N. V., Ackerman, S. E., Kintzing, J. R., Chen, R., Interrante, M. F., Steiner, A., Sato, A. K., Cochran, J. R. 2016; 15 (6): 1291-1300

  Abstract

  Antibody-drug conjugates (ADC) have generated significant interest as targeted therapeutics for cancer treatment, demonstrating improved clinical efficacy and safety compared with systemic chemotherapy. To extend this concept to other tumor-targeting proteins, we conjugated the tubulin inhibitor monomethyl-auristatin-F (MMAF) to 2.5F-Fc, a fusion protein composed of a human Fc domain and a cystine knot (knottin) miniprotein engineered to bind with high affinity to tumor-associated integrin receptors. The broad expression of integrins (including αvβ3, αvβ5, and α5β1) on tumor cells and their vasculature makes 2.5F-Fc an attractive tumor-targeting protein for drug delivery. We show that 2.5F-Fc can be expressed by cell-free protein synthesis, during which a non-natural amino acid was introduced into the Fc domain and subsequently used for site-specific conjugation of MMAF through a noncleavable linker. The resulting knottin-Fc-drug conjugate (KFDC), termed 2.5F-Fc-MMAF, had approximately 2 drugs attached per KFDC. 2.5F-Fc-MMAF inhibited proliferation in human glioblastoma (U87MG), ovarian (A2780), and breast (MB-468) cancer cells to a greater extent than 2.5F-Fc or MMAF alone or added in combination. As a single agent, 2.5F-Fc-MMAF was effective at inducing regression and prolonged survival in U87MG tumor xenograft models when administered at 10 mg/kg two times per week. In comparison, tumors treated with 2.5F-Fc or MMAF were nonresponsive, and treatment with a nontargeted control, CTRL-Fc-MMAF, showed a modest but not significant therapeutic effect. These studies provide proof-of-concept for further development of KFDCs as alternatives to ADCs for tumor targeting and drug delivery applications. Mol Cancer Ther; 15(6); 1291-300. ©2016 AACR.

  View details for DOI 10.1158/1535-7163.MCT-15-0881

  View details for Web of Science ID 000377427600014

• Degradable acetalated dextran microparticles for tunable release of an engineered hepatocyte growth factor fragment. ACS biomaterials science & engineering Suarez, S. L., Muñoz, A., Mitchell, A., Braden, R. L., Luo, C., Cochran, J. R., Almutairi, A., Christman, K. L. 2016; 2 (2): 197-204

  Abstract

  Injectable biomaterials are promising as new therapies to treat myocardial infarction (MI). One useful property of biomaterials is the ability to protect and sustain release of therapeutic payloads. In order to create a platform for optimizing the release rate of cardioprotective molecules we utilized the tunable degradation of acetalated dextran (AcDex). We created microparticles with three distinct degradation profiles and showed that the consequent protein release profiles could be modulated within the infarcted heart. This enabled us to determine how delivery rate impacted the efficacy of a model therapeutic, an engineered hepatocyte growth factor fragment (HGF-f). Our results showed that the cardioprotective efficacy of HGF-f was optimal when delivered over three days post-intramyocardial injection, yielding the largest arterioles, fewest apoptotic cardiomyocytes bordering the infarct and the smallest infarcts compared to empty particle treatment four weeks after injection. This work demonstrates the potential of using AcDex particles as a delivery platform to optimize the time frame for delivering therapeutic proteins to the heart.

  View details for DOI 10.1021/acsbiomaterials.5b00335

  View details for PubMedID 29333489

  View details for PubMedCentralID PMC5761072

• Degradable Acetalated Dextran Microparticles for Tunable Release of an Engineered Hepatocyte Growth Factor Fragment ACS BIOMATERIALS-SCIENCE & ENGINEERING Suarez, S. L., Munoz, A., Mitchell, A. C., Braden, R. L., Luo, C., Cochran, J. R., Almutairi, A., Christman, K. L. 2016; 2 (2): 197-204

  Abstract

  Injectable biomaterials are promising as new therapies to treat myocardial infarction (MI). One useful property of biomaterials is the ability to protect and sustain release of therapeutic payloads. In order to create a platform for optimizing the release rate of cardioprotective molecules we utilized the tunable degradation of acetalated dextran (AcDex). We created microparticles with three distinct degradation profiles and showed that the consequent protein release profiles could be modulated within the infarcted heart. This enabled us to determine how delivery rate impacted the efficacy of a model therapeutic, an engineered hepatocyte growth factor fragment (HGF-f). Our results showed that the cardioprotective efficacy of HGF-f was optimal when delivered over three days post-intramyocardial injection, yielding the largest arterioles, fewest apoptotic cardiomyocytes bordering the infarct and the smallest infarcts compared to empty particle treatment four weeks after injection. This work demonstrates the potential of using AcDex particles as a delivery platform to optimize the time frame for delivering therapeutic proteins to the heart.

  View details for DOI 10.1021/acsbiomaterials.5b00335

  View details for Web of Science ID 000369876700007

  View details for PubMedCentralID PMC5761072

• Engineering growth factors for regenerative medicine applications ACTA BIOMATERIALIA Mitchell, A. C., Briquez, P. S., Hubbell, J. A., Cochran, J. R. 2016; 30: 1-12

  Abstract

  Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.

  View details for DOI 10.1016/j.actbio.2015.11.007

  View details for Web of Science ID 000368563600001

  View details for PubMedCentralID PMC6067679

• Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations PEPTIDE, PROTEIN AND ENZYME DESIGN Hunter, S. A., Cochran, J. R. 2016; 580: 21-44

  Abstract

  Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented.

  View details for DOI 10.1016/bs.mie.2016.05.002

  View details for Web of Science ID 000383905300003

  View details for PubMedID 27586327

• Engineering growth factors for regenerative medicine applications. Acta biomaterialia Mitchell, A. C., Briquez, P. S., Hubbell, J. A., Cochran, J. R. 2016; 30: 1-12

  Abstract

  Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.Growth factors are promising therapeutic proteins that have the ability to modulate morphogenetic behaviors, including cell survival, proliferation, migration and differentiation. However, the translation of growth factors into clinical therapies has been hindered by properties such as poor protein stability, low recombinant expression yield, and non-physiological delivery, which lead to suboptimal efficacy and adverse side effects. To address these needs, researchers are employing clever molecular and material engineering and design strategies to both improve the intrinsic properties of growth factors and effectively control their delivery into tissue. This review highlights examples of interdisciplinary tools and technologies used to augment the therapeutic potential of growth factors for clinical applications in regenerative medicine.

  View details for DOI 10.1016/j.actbio.2015.11.007

  View details for PubMedID 26555377

  View details for PubMedCentralID PMC6067679

• 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 Zheng, L. L., Vanchinathan, V., Dalal, R., Noolandi, J., Waters, D. J., Hartmann, L., Cochran, J. R., Frank, C. W., Yu, C. Q., Ta, C. N. 2015; 103 (10): 3157-3165

  Abstract

  We evaluated the biocompatibility of a poly(ethylene glycol) and poly(acrylic acid) (PEG/PAA) interpenetrating network hydrogel designed for artificial cornea in a rabbit model. PEG/PAA hydrogel measuring 6 mm in diameter was implanted in the corneal stroma of twelve rabbits. Stromal flaps were created with a microkeratome. Randomly, six rabbits were assigned to bear the implant for 2 months, two rabbits for 6 months, two rabbits for 9 months, one rabbit for 12 months, and one rabbit for 16 months. Rabbits were evaluated monthly. After the assigned period, eyes were enucleated, and corneas were processed for histology and immunohistochemistry. There were clear corneas in three of six rabbits that had implantation of hydrogel for 2 months. In the six rabbits with implant for 6 months or longer, the corneas remained clear in four. There was a high rate of epithelial defect and corneal thinning in these six rabbits. One planned 9-month rabbit developed extrusion of implant at 4 months. The cornea remained clear in the 16-month rabbit but histology revealed epithelial in-growth. Intrastromal implantation of PEG/PAA resulted in a high rate of long-term complications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3157-3165, 2015.

  View details for DOI 10.1002/jbm.a.35453

  View details for Web of Science ID 000360501300005

  View details for PubMedCentralID PMC4552592

• 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 Zheng, L. L., Vanchinathan, V., Dalal, R., Noolandi, J., Waters, D. J., Hartmann, L., Cochran, J. R., Frank, C. W., Yu, C. Q., Ta, C. N. 2015; 103 (10): 3157-3165

  Abstract

  We evaluated the biocompatibility of a poly(ethylene glycol) and poly(acrylic acid) (PEG/PAA) interpenetrating network hydrogel designed for artificial cornea in a rabbit model. PEG/PAA hydrogel measuring 6 mm in diameter was implanted in the corneal stroma of twelve rabbits. Stromal flaps were created with a microkeratome. Randomly, six rabbits were assigned to bear the implant for 2 months, two rabbits for 6 months, two rabbits for 9 months, one rabbit for 12 months, and one rabbit for 16 months. Rabbits were evaluated monthly. After the assigned period, eyes were enucleated, and corneas were processed for histology and immunohistochemistry. There were clear corneas in three of six rabbits that had implantation of hydrogel for 2 months. In the six rabbits with implant for 6 months or longer, the corneas remained clear in four. There was a high rate of epithelial defect and corneal thinning in these six rabbits. One planned 9-month rabbit developed extrusion of implant at 4 months. The cornea remained clear in the 16-month rabbit but histology revealed epithelial in-growth. Intrastromal implantation of PEG/PAA resulted in a high rate of long-term complications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 3157-3165, 2015.

  View details for DOI 10.1002/jbm.a.35453

  View details for PubMedID 25778285

• Delivery of an engineered HGF fragment in an extracellular matrix-derived hydrogel prevents negative LV remodeling post-myocardial infarction. Biomaterials Sonnenberg, S. B., Rane, A. A., Liu, C. J., Rao, N., Agmon, G., Suarez, S., Wang, R., Munoz, A., Bajaj, V., Zhang, S., Braden, R., Schup-Magoffin, P. J., Kwan, O. L., DeMaria, A. N., Cochran, J. R., Christman, K. L. 2015; 45: 56-63

  Abstract

  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 Parke-Houben, R., Fox, C. H., Zheng, L. L., Waters, D. J., Cochran, J. R., Ta, C. N., Frank, C. W. 2015; 26 (2): 107-?

  Abstract

  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 Kim, J. W., Cochran, F. V., Cochran, J. R. 2015; 137 (1): 6-9

  Abstract

  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.) Cherf, G. M., Cochran, J. R. 2015; 1319: 155-175

  Abstract

  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 FEBS LETTERS Liu, C. J., Jones, D. S., Tsai, P., Venkataramana, A., Cochran, J. R. 2014; 588 (24): 4831-4837

  Abstract

  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

• An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis. Nature chemical biology Kariolis, M. S., Miao, Y. R., Jones, D. S., Kapur, S., Mathews, I. I., Giaccia, A. J., Cochran, J. R. 2014; 10 (11): 977-983

  Abstract

  Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.

  View details for DOI 10.1038/nchembio.1636

  View details for PubMedID 25242553

• Cystine-knot peptides: emerging tools for cancer imaging and therapy EXPERT REVIEW OF PROTEOMICS Ackerman, S. E., Currier, N. V., Bergen, J. M., Cochran, J. R. 2014; 11 (5): 561-572

  Abstract

  Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature's repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

  View details for DOI 10.1586/14789450.2014.932251

  View details for Web of Science ID 000342232200004

• Cystine-knot peptides: emerging tools for cancer imaging and therapy. Expert review of proteomics Ackerman, S. E., Currier, N. V., Bergen, J. M., Cochran, J. R. 2014; 11 (5): 561-72

  Abstract

  Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature's repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

  View details for DOI 10.1586/14789450.2014.932251

  View details for PubMedID 25163524

• A Bioengineered Peptide that Localizes to and Illuminates Medulloblastoma: A New Tool with Potential for Fluorescence-Guided Surgical Resection. Cureus Ackerman, S. E., Wilson, C. M., Kahn, S. A., Kintzing, J. R., Jindal, D. A., Cheshier, S. H., Grant, G. A., Cochran, J. R. 2014; 6 (9)

  Abstract

  Tumors of the central nervous system are challenging to treat due to the limited effectiveness and associated toxicities of chemotherapy and radiation therapy. For tumors that can be removed surgically, extent of malignant tissue resection has been shown to correlate with disease progression, recurrence, and survival. Thus, improved technologies for real-time brain tumor imaging are critically needed as tools for guided surgical resection. We previously engineered a novel peptide that binds with high affinity and unique specificity to αVβ3, αVβ5, and α5β1 integrins, which are present on tumor cells, and the vasculature of many cancers, including brain tumors. In the current study, we conjugated this engineered peptide to a near infrared fluorescent dye (Alexa Fluor 680), and used the resulting molecular probe for non-invasive whole body imaging of patient-derived medulloblastoma xenograft tumors implanted in the cerebellum of mice. The engineered peptide exhibited robust targeting and illumination of intracranial medulloblastoma following both intravenous and intraperitoneal injection routes. In contrast, a variant of the engineered peptide containing a scrambled integrin-binding sequence did not localize to brain tumors, demonstrating that tumor-targeting is driven by specific integrin interactions. Ex vivo imaging was used to confirm the presence of tumor and molecular probe localization to the cerebellar region. These results warrant further clinical development of the engineered peptide as a tool for image-guided resection of central nervous system tumors.

  View details for DOI 10.7759/cureus.207

  View details for PubMedID 28729960

  View details for PubMedCentralID PMC5515084

• A Bioengineered Peptide that Localizes to and Illuminates Medulloblastoma: A New Tool with Potential for Fluorescence-Guided Surgical Resection Cureus Ackerman, S. E., Wilson, C. M., Kahn, S. A., Kintzing, J. R., Jindal, D. A., Cheshier, S. H., Grant, G. A., Cochran, J. R. 2014

  View details for DOI 10.7759/cureus.207

• 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 Sciences of the United States of America Moore, S. J., Hayden Gephart, M. G., Bergen, J. M., Su, Y. S., Rayburn, H., Scott, M. P., Cochran, J. R. 2013; 110 (36): 14598-14603

  Abstract

  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

  View details for PubMedCentralID PMC3767496

• A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging AMINO ACIDS Jiang, H., Moore, S. J., Liu, S., Liu, H., Miao, Z., Cochran, F. V., Liu, Y., Tian, M., Cochran, J. R., Zhang, H., Cheng, Z. 2013; 44 (2): 673-681

  Abstract

  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. 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 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

  View details for PubMedCentralID PMC3616073

• 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): e60498

  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 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

  View details for PubMedCentralID PMC3616073

• Engineering Multivalent and Multispecific Protein Therapeutics Engineering in Translational Medicine Liu, C., J., Cochran, J., R. edited by Cai, W. Springer.. 2013: 1
• 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
• 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

  View details for PubMedCentralID PMC3569487

• Knottins: Disulfide-bonded Therapeutic and Diagnostic Peptides. Drug Discovery Today: Technologies Moore, S., J., Leung, C., L., Cochran, J., R. 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 Jiang, L., Miao, Z., Kimura, R. H., Silverman, A. P., Ren, G., Liu, H., Lu, H., Cochran, J. R., Cheng, Z. 2012

  Abstract

  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

  View details for PubMedCentralID PMC3336217

• ENGINEERING KNOTTINS AS NOVEL BINDING AGENTS METHODS IN ENZYMOLOGY: PROTEIN ENGINEERING FOR THERAPEUTICS, VOL 203, PT B Moore, S. J., Cochran, J. R. 2012; 503: 223-251

  Abstract

  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 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

• 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 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

  View details for PubMedCentralID PMC3161552

• 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

  View details for PubMedCentralID PMC3156163

• 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; 98 (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 PubMedID 21504051

• 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

• 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

  View details for PubMedCentralID PMC3118396

• Preliminary evaluation of Lu-177-labeled knottin peptides for integrin receptor-targeted radionuclide therapy EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Jiang, L., Miao, Z., Kimura, R. H., Liu, H., Cochran, J. R., Culter, C. S., Bao, A., Li, P., Cheng, Z. 2011; 38 (4): 613-622

  Abstract

  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 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 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

  View details for PubMedCentralID PMC3041754

• 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

  View details for PubMedCentralID PMC3248644

• 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
• 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
• Targeting of Cancer Cells Using Quantum Dot-Polypeptide Hybrid Assemblies That Function as Molecular Imaging Agents and Carrier Systems ADVANCED FUNCTIONAL MATERIALS Atmaja, B., Lui, B. H., Hu, Y., Beck, S. E., Frank, C. W., Cochran, J. R. 2010; 20 (23): 4091-4097

  Abstract

  We report a highly tunable quantum dot (QD)-polypeptide hybrid assembly system with potential uses for both molecular imaging and delivery of biomolecular cargo to cancer cells. In this work, we demonstrate the tunability of the assembly system, its application for imaging cancer cells, and its ability to carry a biomolecule. The assemblies are formed through the self-assembly of carboxyl-functionalized QDs and poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) diblock copolypeptide molecules, and they are modified with peptide ligands containing a cyclic arginine-glycine-aspartate [c(RGD)] motif that has affinity for αvβ3 and αvβ5 integrins overexpressed on the tumor vasculature. To illustrate the tunability of the QD-polypeptide assembly system, we show that binding to U87MG glioblastoma cells can be modulated and optimized by changing either the conditions under which the assemblies are formed or the relative lengths of the PEGLL and PLL blocks in the PEGLL-PLL molecules. The optimized c(RGD)-modified assemblies bind integrin receptors on U87MG cells and are endocytosed, as demonstrated by flow cytometry and live-cell imaging. Binding specificity is confirmed by competition with an excess of free c(RGD) peptide. Finally, we show that the QD-polypeptide assemblies can be loaded with fluorescently labeled ovalbumin, as a proof-of-concept for their potential use in biomolecule delivery.

  View details for DOI 10.1002/adfm.201000732

  View details for Web of Science ID 000285392900007

  View details for PubMedCentralID PMC4349486

• Targeting of Cancer Cells Using Quantum Dot-Polypeptide Hybrid Assemblies that Function as Molecular Imaging Agents and Carrier Systems. Advanced functional materials Atmaja, B., Lui, B. H., Hu, Y., Beck, S. E., Frank, C. W., Cochran, J. R. 2010; 20 (23): 4091-4097

  Abstract

  We report a highly tunable quantum dot (QD)-polypeptide hybrid assembly system with potential uses for both molecular imaging and delivery of biomolecular cargo to cancer cells. In this work, we demonstrate the tunability of the assembly system, its application for imaging cancer cells, and its ability to carry a biomolecule. The assemblies are formed through the self-assembly of carboxyl-functionalized QDs and poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) diblock copolypeptide molecules, and they are modified with peptide ligands containing a cyclic arginine-glycine-aspartate [c(RGD)] motif that has affinity for αvβ3 and αvβ5 integrins overexpressed on the tumor vasculature. To illustrate the tunability of the QD-polypeptide assembly system, we show that binding to U87MG glioblastoma cells can be modulated and optimized by changing either the conditions under which the assemblies are formed or the relative lengths of the PEGLL and PLL blocks in the PEGLL-PLL molecules. The optimized c(RGD)-modified assemblies bind integrin receptors on U87MG cells and are endocytosed, as demonstrated by flow cytometry and live-cell imaging. Binding specificity is confirmed by competition with an excess of free c(RGD) peptide. Finally, we show that the QD-polypeptide assemblies can be loaded with fluorescently labeled ovalbumin, as a proof-of-concept for their potential use in biomolecule delivery.

  View details for DOI 10.1002/adfm.201000732

  View details for PubMedID 25750609

  View details for PubMedCentralID PMC4349486

• 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

  View details for PubMedCentralID PMC3057960

• 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

• 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; 21 (3): 436-444

  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 DOI 10.1021/bc9003102

  View details for Web of Science ID 000275711600004

  View details for PubMedCentralID PMC3004996

• 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

• 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 DOI 10.1021/bc9003102

  View details for PubMedID 20131753

  View details for PubMedCentralID PMC3004996

• 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

• Phage display and molecular imaging: expanding fields of vision in living subjects BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS, VOL 27 Cochran, F. V., Cochran, J. R. 2010; 27: 57-93

  Abstract

  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

• 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
• 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

  View details for PubMedCentralID PMC2804269

• 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

  View details for PubMedCentralID PMC2725296

• 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

• 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; 90A (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 Web of Science ID 000266648700007

  View details for PubMedCentralID PMC2856598

• 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 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

  View details for PubMedCentralID PMC2833353

• 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

  View details for PubMedCentralID PMC2925133

• 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 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
• 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
• 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 234th National Meeting of the American-Chemical-Society Myung, D., Duhamel, P., Cochran, J. R., Noolandi, J., Ta, C. N., Frank, C. W. WILEY-BLACKWELL. 2008: 735–41

  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 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 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

• BIOT 66-A novel, biomimetic hydrogel construct to repair the cornea: Molecular design and biological response Myung, D., Cochran, J. R., Noolandi, J., Ta, C. N., Frank, C. W. 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 Cochran, J. R., Kim, Y., Lippow, S. M., Rao, B., Wittrup, K. D. 2006; 19 (6): 245-253

  Abstract

  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 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

• 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