{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T08:11:46Z","timestamp":1761293506408,"version":"build-2065373602"},"reference-count":52,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2017,2,17]],"date-time":"2017-02-17T00:00:00Z","timestamp":1487289600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["PHS NRSA 5T32 CA09302 (to A.P.S.)"],"award-info":[{"award-number":["PHS NRSA 5T32 CA09302 (to A.P.S.)"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100004410","name":"European Molecular Biology Organization","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100004410","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Bioengineering &amp; Transla Med"],"published-print":{"date-parts":[[2017,3]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>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 \u03b1<jats:sub>v<\/jats:sub>\u03b2<jats:sub>3<\/jats:sub> 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\u2010fold higher than the parental antagonist, and 14\u2010fold higher than the natural bivalent VEGF ligand. Increased VEGFR2 binding affinity correlated with the ability to more effectively inhibit VEGF\u2010mediated 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\u2010specific antagonist that we previously designed to simultaneously bind to and inhibit VEGFR2 and \u03b1<jats:sub>v<\/jats:sub>\u03b2<jats:sub>3<\/jats:sub> integrin. The resulting dual\u2010specific protein bound to human and murine endothelial cells with relative affinities of 120\u2009\u00b1\u200910 pM and 360\u2009\u00b1\u200950 pM, respectively, which is at least 30\u2010fold tighter than wild\u2010type VEGF (3.8\u2009\u00b1\u20090.5 nM). Finally, we demonstrated that this engineered high\u2010affinity dual\u2010specific 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.<\/jats:p>","DOI":"10.1002\/btm2.10051","type":"journal-article","created":{"date-parts":[[2016,12,24]],"date-time":"2016-12-24T21:28:45Z","timestamp":1482614925000},"page":"81-91","source":"Crossref","is-referenced-by-count":8,"title":["Engineered ligand\u2010based VEGFR antagonists with increased receptor binding affinity more effectively inhibit angiogenesis"],"prefix":"10.1002","volume":"2","author":[{"given":"Shiven","family":"Kapur","sequence":"first","affiliation":[{"name":"Dept. of Bioengineering Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adam P.","family":"Silverman","sequence":"additional","affiliation":[{"name":"Dept. of Bioengineering Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anne Z.","family":"Ye","sequence":"additional","affiliation":[{"name":"Dept. of Bioengineering Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Niv","family":"Papo","sequence":"additional","affiliation":[{"name":"Dept. of Bioengineering Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Darren","family":"Jindal","sequence":"additional","affiliation":[{"name":"Dept. of Bioengineering Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark S.","family":"Blumenkranz","sequence":"additional","affiliation":[{"name":"Dept. of Ophthalmology Byers Eye Institute, Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer R.","family":"Cochran","sequence":"additional","affiliation":[{"name":"Dept. of Bioengineering Stanford University Stanford CA 94303"},{"name":"Dept. of Chemical Engineering Stanford University Stanford CA 94303"},{"name":"Stanford Cancer Institute Stanford University Stanford CA 94303"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2017,2,17]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1517\/14656566.5.5.1175"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.172398299"},{"key":"e_1_2_6_4_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.172398399"},{"key":"e_1_2_6_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/cdd.2014.81"},{"key":"e_1_2_6_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2013.03.017"},{"key":"e_1_2_6_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature10975"},{"key":"e_1_2_6_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1636"},{"key":"e_1_2_6_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt725"},{"key":"e_1_2_6_10_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.256.5064.1677"},{"key":"e_1_2_6_11_1","doi-asserted-by":"publisher","DOI":"10.1210\/er.2003-0027"},{"key":"e_1_2_6_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrc2442"},{"key":"e_1_2_6_13_1","doi-asserted-by":"publisher","DOI":"10.1186\/2040-2384-2-27"},{"key":"e_1_2_6_14_1","doi-asserted-by":"publisher","DOI":"10.1096\/fj.08-113860"},{"key":"e_1_2_6_15_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10456-009-9141-9"},{"key":"e_1_2_6_16_1","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a009183"},{"key":"e_1_2_6_17_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12013-008-9040-5"},{"key":"e_1_2_6_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/nm0603-669"},{"issue":"112","key":"e_1_2_6_19_1","first-page":"re21","article-title":"VEGF receptor signal transduction","volume":"2001","author":"Matsumoto T","journal-title":"Sci STKE."},{"key":"e_1_2_6_20_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.18.11197"},{"key":"e_1_2_6_21_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.273.18.11115"},{"key":"e_1_2_6_22_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.95.8.4625"},{"key":"e_1_2_6_23_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M204107200"},{"key":"e_1_2_6_24_1","doi-asserted-by":"publisher","DOI":"10.4161\/cc.7.3.5234"},{"key":"e_1_2_6_25_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20060807"},{"key":"e_1_2_6_26_1","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.107.160937"},{"key":"e_1_2_6_27_1","doi-asserted-by":"publisher","DOI":"10.1161\/CIRCRESAHA.107.155655"},{"key":"e_1_2_6_28_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1016635108"},{"key":"e_1_2_6_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(04)88027-3"},{"key":"e_1_2_6_30_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt0697-553"},{"key":"e_1_2_6_31_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2011-11-390922"},{"key":"e_1_2_6_32_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.271.10.5638"},{"issue":"4","key":"e_1_2_6_33_1","first-page":"519","article-title":"A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor","volume":"67","author":"Passaniti A","year":"1992","journal-title":"Lab Invest."},{"key":"e_1_2_6_34_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0607542104"},{"key":"e_1_2_6_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2004.03.005"},{"key":"e_1_2_6_36_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.94.3.861"},{"key":"e_1_2_6_37_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2016.05.002"},{"issue":"8","key":"e_1_2_6_38_1","first-page":"1625","article-title":"A model of angiogenesis in the mouse cornea","volume":"37","author":"Kenyon BM","year":"1996","journal-title":"Invest Ophthalmol Vis Sci."},{"key":"e_1_2_6_39_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.368"},{"issue":"1","key":"e_1_2_6_40_1","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1177\/112067211002000106","article-title":"VEGF Trap(R1R2) suppresses experimental corneal angiogenesis","volume":"20","author":"Oliveira HB","year":"2010","journal-title":"Eur J Ophthalmol."},{"issue":"4","key":"e_1_2_6_41_1","first-page":"955","article-title":"Expression of integrins and MMPs during alkaline\u2010burn\u2010induced corneal angiogenesis","volume":"43","author":"Zhang H","year":"2002","journal-title":"Invest Ophthalmol Vis Sci."},{"key":"e_1_2_6_42_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4471-4372-7_14"},{"key":"e_1_2_6_43_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibtech.2010.03.007"},{"key":"e_1_2_6_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molimm.2015.01.003"},{"key":"e_1_2_6_45_1","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1996.0049"},{"key":"e_1_2_6_46_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.94"},{"key":"e_1_2_6_47_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0969-2126(97)00284-0"},{"key":"e_1_2_6_48_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M210931200"},{"key":"e_1_2_6_49_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M308681200"},{"key":"e_1_2_6_50_1","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(79)90157-X"},{"key":"e_1_2_6_51_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.141.7.1659"},{"key":"e_1_2_6_52_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI44762"},{"key":"e_1_2_6_53_1","doi-asserted-by":"publisher","DOI":"10.1161\/01.RES.0000118600.91698.BB"}],"container-title":["Bioengineering &amp; Translational Medicine"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fbtm2.10051","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/aiche.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/btm2.10051","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T21:12:33Z","timestamp":1760044353000},"score":1,"resource":{"primary":{"URL":"https:\/\/aiche.onlinelibrary.wiley.com\/doi\/10.1002\/btm2.10051"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,2,17]]},"references-count":52,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2017,3]]}},"alternative-id":["10.1002\/btm2.10051"],"URL":"https:\/\/doi.org\/10.1002\/btm2.10051","archive":["Portico"],"relation":{},"ISSN":["2380-6761","2380-6761"],"issn-type":[{"type":"print","value":"2380-6761"},{"type":"electronic","value":"2380-6761"}],"subject":[],"published":{"date-parts":[[2017,2,17]]}}}