Optimized serum stability and specificity of an αvβ6 integrin-binding peptide for tumor targeting
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100657
ISSN1083-351X
AutoresIan I. Cardle, Michael C. Jensen, Suzie H. Pun, Drew L. Sellers,
Tópico(s)Immunotherapy and Immune Responses
ResumoThe integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy. The integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy. From 2009 through 2015, pancreatic, liver, lung, and esophageal cancers had the lowest survival rates of any cancer and are projected to contribute to 38% of cancer-related deaths in 2020 (1Siegel R.L. Miller K.D. Jemal A. Cancer statistics, 2020.C. A. Cancer J. Clin. 2020; 70: 7-30Crossref PubMed Scopus (7921) Google Scholar). Patients are often asymptomatic at early stages with these cancers, preventing timely diagnosis and thereby limiting effective treatment options at later stages of disease. Consequentially, there is a significant need for targeted diagnostics and therapeutics that could identify and treat these cancers at early stages to improve patient outcomes. Integrins are a family of heterodimeric transmembrane receptors that interact with proteins in the extracellular matrix and on other cells to mediate cell adhesion and migration. While integrins are involved in a variety of healthy biological functions, including embryogenesis, tissue regeneration, and immune cell trafficking (2Huttenlocher A. Horwitz A.R. Integrins in cell migration.Cold Spring Harb. Perspect. Biol. 2011; 3: a005074Crossref PubMed Scopus (499) Google Scholar), their aberrant expression and activity can drive cancer initiation and metastasis (3Hamidi H. Ivaska J. Every step of the way: Integrins in cancer progression and metastasis.Nat. Rev. Cancer. 2018; 18: 533-548Crossref PubMed Scopus (409) Google Scholar, 4Munshi H.G. Stack M.S. Reciprocal interactions between adhesion receptor signaling and MMP regulation.Cancer Metastasis Rev. 2006; 25: 45-56Crossref PubMed Scopus (101) Google Scholar, 5Khan Z. Marshall J.F. The role of integrins in TGFβ activation in the tumour stroma.Cell Tissue Res. 2016; 365: 657-673Crossref PubMed Scopus (70) Google Scholar). Integrins have thus garnered considerable interest as diagnostic and therapeutic targets for cancer (6Sun C.-C. Qu X.-J. Gao Z.-H. Arginine-glycine-aspartate–binding integrins as therapeutic and diagnostic targets.Am. J. Ther. 2016; 23: e198-e207Crossref PubMed Scopus (19) Google Scholar, 7Raab-Westphal S. Marshall J.F. Goodman S.L. Integrins as therapeutic targets: Successes and cancers.Cancers (Basel). 2017; 9: 110Crossref PubMed Scopus (123) Google Scholar). One such integrin, αvβ6, is an epithelial-restricted integrin involved in wound healing that has low basal expression in healthy tissue (8Breuss J.M. Gallo J. DeLisser H.M. Klimanskaya I.V. Folkesson H.G. Pittet J.F. Nishimura S.L. Aldape K. Landers D.V. Carpenter W. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling.J. Cell Sci. 1995; 108: 2241-2251Crossref PubMed Google Scholar). αvβ6 is broadly upregulated in many solid tumor types, including pancreatic (9Reader C.S. Vallath S. Steele C.W. Haider S. Brentnall A. Desai A. Moore K.M. Jamieson N.B. Chang D. Bailey P. Scarpa A. Lawlor R. Chelala C. Keyse S.M. Biankin A. et al.The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy.J. Pathol. 2019; 249: 332-342Crossref PubMed Scopus (32) Google Scholar), liver (10Patsenker E. Wilkens L. Banz V. Österreicher C.H. Weimann R. Eisele S. Keogh A. Stroka D. Zimmermann A. Stickel F. The αvβ6 integrin is a highly specific immunohistochemical marker for cholangiocarcinoma.J. Hepatol. 2010; 52: 362-369Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Peng Z.-W. Ikenaga N. Liu S.B. Sverdlov D.Y. Vaid K.A. Dixit R. Weinreb P.H. Violette S. Sheppard D. Schuppan D. Popov Y. Integrin αvβ6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.Hepatology. 2016; 63: 217-232Crossref PubMed Scopus (63) Google Scholar), lung (12Elayadi A.N. Samli K.N. Prudkin L. Liu Y.-H. Bian A. Xie X.-J. Wistuba I.I. Roth J.A. McGuire M.J. Brown K.C. A peptide selected by biopanning identifies the integrin αvβ6 as a prognostic biomarker for nonsmall cell lung cancer.Cancer Res. 2007; 67: 5889-5895Crossref PubMed Scopus (135) Google Scholar, 13Yan P. Zhu H. Yin L. Wang L. Xie P. Ye J. Jiang X. He X. Integrin αvβ6 promotes lung cancer proliferation and metastasis through upregulation of IL-8-mediated MAPK/ERK signaling.Transl. Oncol. 2018; 11: 619-627Crossref PubMed Scopus (16) Google Scholar), esophageal (14Koopman Van Aarsen L.A. Leone D.R. Ho S. Dolinski B.M. McCoon P.E. LePage D.J. Kelly R. Heaney G. Rayhorn P. Reid C. Simon K.J. Horan G.S. Tao N. Gardner H.A. Skelly M.M. et al.Antibody-Mediated blockade of integrin αvβ6 by a transforming growth factor-β–regulated mechanism.Cancer Res. 2008; 68: 561-570Crossref PubMed Scopus (105) Google Scholar), cervical (15Hazelbag S. Kenter G.G. Gorter A. Dreef E.J. Koopman L.A. Violette S.M. Weinreb P.H. Fleuren G.J. Overexpression of the αvβ6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival.J. Pathol. 2007; 212: 316-324Crossref PubMed Scopus (128) Google Scholar), breast (16Moore K.M. Thomas G.J. Duffy S.W. Warwick J. Gabe R. Chou P. Ellis I.O. Green A.R. Haider S. Brouilette K. Saha A. Vallath S. Bowen R. Chelala C. Eccles D. et al.Therapeutic targeting of integrin αvβ6 in breast cancer.JNCI J. Natl. Cancer Inst. 2014; 106dju169Crossref PubMed Scopus (83) Google Scholar), head and neck (17Hsiao J.-R. Chang Y. Chen Y.-L. Hsieh S.-H. Hsu K.-F. Wang C.-F. Tsai S.-T. Jin Y.-T. Cyclic αvβ6-targeting peptide selected from biopanning with clinical potential for head and neck squamous cell carcinoma.Head Neck. 2010; 32: 160-172PubMed Google Scholar), colon (18Yang G.-Y. Guo S. Dong C.-Y. Wang X.-Q. Hu B.-Y. Liu Y.-F. Chen Y.-W. Niu J. Dong J.-H. Integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression.World J. Gastroenterol. 2015; 21: 7457-7467Crossref PubMed Scopus (16) Google Scholar), ovarian (19Ahmed N. Riley C. Rice G.E. Quinn M.A. Baker M.S. αvβ6 integrin-A marker for the malignant potential of epithelial ovarian cancer.J. Histochem. Cytochem. 2002; 50: 1371-1379Crossref PubMed Scopus (83) Google Scholar), stomach (20Zhuang Z. Zhou R. Xu X. Tian T. Liu Y. Liu Y. Lian P. Wang J. Xu K. Clinical significance of integrin αvβ6 expression effects on gastric carcinoma invasiveness and progression via cancer-associated fibroblasts.Med. Oncol. 2013; 30: 580Crossref PubMed Scopus (20) Google Scholar), and oral cancers (21Regezi J.A. Ramos D.M. Pytela R. Dekker N.P. Jordan R.C.K. Tenascin and β6 integrin are overexpressed in floor of mouth in situ carcinomas and invasive squamous cell carcinomas.Oral Oncol. 2002; 38: 332-336Crossref PubMed Scopus (61) Google Scholar), and its overexpression often correlates with a poor prognosis (22Bates R.C. Bellovin D.I. Brown C. Maynard E. Wu B. Kawakatsu H. Sheppard D. Oettgen P. Mercurio A.M. Transcriptional activation of integrin β6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma.J. Clin. Invest. 2005; 115: 339-347Crossref PubMed Scopus (287) Google Scholar, 23Desnoyers A. González C. Pérez-Segura P. Pandiella A. Amir E. Ocaña A. Integrin ανβ6 protein expression and prognosis in solid tumors: A meta-analysis.Mol. Diagn. Ther. 2020; 24: 143-151Crossref PubMed Scopus (4) Google Scholar). The role of αvβ6 in tumorigenesis is correspondingly extensive; αvβ6 binds to fibronectin and tenascin for cell adhesion and migration (14Koopman Van Aarsen L.A. Leone D.R. Ho S. Dolinski B.M. McCoon P.E. LePage D.J. Kelly R. Heaney G. Rayhorn P. Reid C. Simon K.J. Horan G.S. Tao N. Gardner H.A. Skelly M.M. et al.Antibody-Mediated blockade of integrin αvβ6 by a transforming growth factor-β–regulated mechanism.Cancer Res. 2008; 68: 561-570Crossref PubMed Scopus (105) Google Scholar, 24Busk M. Pytela R. Sheppard D. Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.J. Biol. Chem. 1992; 267: 5790-5796Abstract Full Text PDF PubMed Google Scholar, 25Thomas G.J. Poomsawat S. Lewis M.P. Hart I.R. Speight P.M. Marshall J.F. αvβ6 integrin upregulates matrix metalloproteinase 9 and promotes migration of normal oral keratinocytes.J. Invest. Dermatol. 2001; 116: 898-904Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), it activates protransforming growth factor beta to promote the epithelial-to-mesenchymal transition (22Bates R.C. Bellovin D.I. Brown C. Maynard E. Wu B. Kawakatsu H. Sheppard D. Oettgen P. Mercurio A.M. Transcriptional activation of integrin β6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma.J. Clin. Invest. 2005; 115: 339-347Crossref PubMed Scopus (287) Google Scholar, 26Thomas G.J. Hart I.R. Speight P.M. Marshall J.F. Binding of TGF-β1 latency-associated peptide (LAP) to ανβ6 integrin modulates behaviour of squamous carcinoma cells.Br. J. Cancer. 2002; 87: 859-867Crossref PubMed Scopus (45) Google Scholar, 27Annes J.P. Chen Y. Munger J.S. Rifkin D.B. Integrin ανβ6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1.J. Cell Biol. 2004; 165: 723-734Crossref PubMed Scopus (354) Google Scholar, 28Shi M. Zhu J. Wang R. Chen X. Mi L. Walz T. Springer T.A. Latent TGF-β structure and activation.Nature. 2011; 474: 343-349Crossref PubMed Scopus (574) Google Scholar), and it mediates secretion of matrix metalloproteinases that remodel the extracellular matrix for cancer growth and invasion (18Yang G.-Y. Guo S. Dong C.-Y. Wang X.-Q. Hu B.-Y. Liu Y.-F. Chen Y.-W. Niu J. Dong J.-H. Integrin αvβ6 sustains and promotes tumor invasive growth in colon cancer progression.World J. Gastroenterol. 2015; 21: 7457-7467Crossref PubMed Scopus (16) Google Scholar, 29Thomas G.J. Lewis M.P. Hart I.R. Marshall J.F. Speight P.M. αvβ6 integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9.Int. J. Cancer. 2001; 92: 641-650Crossref PubMed Scopus (128) Google Scholar, 30Gu X. Niu J. Dorahy D.J. Scott R. Agrez M.V. Integrin αvβ6-associated ERK2 mediates MMP-9 secretion in colon cancer cells.Br. J. Cancer. 2002; 87: 348-351Crossref PubMed Scopus (40) Google Scholar, 31Morgan M.R. Thomas G.J. Russell A. Hart I.R. Marshall J.F. The integrin cytoplasmic-tail motif EKQKVDLSTDC is sufficient to promote tumor cell invasion mediated by matrix metalloproteinase (MMP)-2 or MMP-9.J. Biol. Chem. 2004; 279: 26533-26539Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Given these qualities, the integrin αvβ6 has become the focus of considerable research efforts in the last two decades as a potential target for cancer imaging and therapy (32Liu H. Wu Y. Wang F. Liu Z. Molecular imaging of integrin αvβ6 expression in living subjects.Am. J. Nucl. Med. Mol. Imaging. 2014; 4: 333-345PubMed Google Scholar, 33Bandyopadhyay A. Raghavan S. Defining the role of integrin αvβ6 in cancer.Curr. Drug Targets. 2009; 10: 645-652Crossref PubMed Scopus (140) Google Scholar). Peptides are attractive targeting ligands for cancer because of their chemical synthesis and small size, enabling inexpensive production, ease of modification, and enhanced solid tumor penetration compared with antibodies (34Ladner R.C. Sato A.K. Gorzelany J. de Souza M. Phage display-derived peptides as therapeutic alternatives to antibodies.Drug Discov. Today. 2004; 9: 525-529Crossref PubMed Scopus (227) Google Scholar, 35Vlieghe P. Lisowski V. Martinez J. Khrestchatisky M. Synthetic therapeutic peptides: Science and market.Drug Discov. Today. 2010; 15: 40-56Crossref PubMed Scopus (1003) Google Scholar, 36Firer M.A. Gellerman G. Targeted drug delivery for cancer therapy: The other side of antibodies.J. Hematol. Oncol. 2012; 5: 70Crossref PubMed Scopus (176) Google Scholar, 37Liu H. Zhao Z. Zhang L. Li Y. Jain A. Barve A. Jin W. Liu Y. Fetse J. Cheng K. Discovery of low-molecular weight anti-PD-L1 peptides for cancer immunotherapy.J. Immunother. Cancer. 2019; 7: 270Crossref PubMed Scopus (27) Google Scholar). A20FMDV2 is a 20-amino acid, arginine–glycine–aspartate (RGD)–containing peptide derived from the G–H loop of the capsid protein viral protein 1 from foot-and-mouth disease virus (FMDV) serotype O1 that binds integrin αvβ6 with low nanomolar affinity and high specificity (38Logan D. Abu-Ghazaleh R. Blakemore W. Curry S. Jackson T. King A. Lea S. Lewis R. Newman J. Parry N. Rowlands D. Stuart D. Fry E. Structure of a major immunogenic site on foot-and-mouth disease virus.Nature. 1993; 362: 566-568Crossref PubMed Scopus (300) Google Scholar, 39DiCara D. Rapisarda C. Sutcliffe J.L. Violette S.M. Weinreb P.H. Hart I.R. Howard M.J. Marshall J.F. Structure-Function analysis of Arg-Gly-Asp helix motifs in αvβ6 integrin ligands.J. Biol. Chem. .. 2007; 282: 9657-9665Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 40Hausner S.H. DiCara D. Marik J. Marshall J.F. Sutcliffe J.L. Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: Generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin αvβ6 expression with positron emission tomography.Cancer Res. 2007; 67: 7833-7840Crossref PubMed Scopus (92) Google Scholar). With its favorable binding properties and demonstrated preclinical safety (41Slack R.J. Hafeji M. Rogers R. Ludbrook S.B. Marshall J.F. Flint D.J. Pyne S. Denyer J.C. Pharmacological characterization of the αvβ6 integrin binding and internalization kinetics of the foot-and-mouth disease virus derived peptide A20FMDV2.Pharmacology. 2016; 97: 114-125Crossref PubMed Scopus (20) Google Scholar, 42Keat N. Kenny J. Chen K. Onega M. Garman N. Slack R.J. Parker C.A. Lumbers R.T. Hallett W. Saleem A. Passchier J. Lukey P.T. A microdose PET study of the safety, immunogenicity, biodistribution, and radiation dosimetry of 18F-FB-A20FMDV2 for imaging the integrin αvβ6.J. Nucl. Med. Technol. 2018; 46: 136-143Crossref PubMed Scopus (17) Google Scholar), A20FMDV2 has been used in many cancer research applications, including imaging of αvβ6+ tumors in mice and humans (40Hausner S.H. DiCara D. Marik J. Marshall J.F. Sutcliffe J.L. Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: Generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin αvβ6 expression with positron emission tomography.Cancer Res. 2007; 67: 7833-7840Crossref PubMed Scopus (92) Google Scholar, 43Hausner S.H. Bold R.J. Cheuy L.Y. Chew H.K. Daly M.E. Davis R.A. Foster C.C. Kim E.J. Sutcliffe J.L. Preclinical development and first-in-human imaging of the integrin αvβ6 with [18F]αvβ6-Binding peptide in metastatic carcinoma.Clin. Cancer Res. 2019; 25: 1206-1215Crossref PubMed Scopus (35) Google Scholar), αvβ6-specific drug delivery in vitro and in vivo (44Moore K.M. Desai A. Delgado B.d.L. Trabulo S.M.D. Reader C. Brown N.F. Murray E.R. Brentnall A. Howard P. Masterson L. Zammarchi F. Hartley J.A. van Berkel P.H. Marshall J.F. Integrin αvβ6-specific therapy for pancreatic cancer developed from foot-and-mouth-disease virus.Theranostics. 2020; 10: 2930-2942Crossref PubMed Scopus (13) Google Scholar), and engineering chimeric antigen receptors for αvβ6-directed adoptive T-cell immunotherapy (45Whilding L.M. Parente-Pereira A.C. Zabinski T. Davies D.M. Petrovic R.M.G. Kao Y.V. Saxena S.A. Romain A. Costa-Guerra J.A. Violette S. Itamochi H. Ghaem-Maghami S. Vallath S. Marshall J.F. Maher J. Targeting of aberrant αvβ6 integrin expression in solid tumors using chimeric antigen receptor-engineered T cells.Mol. Ther. 2017; 25: 259-273Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Recent studies also show the utility of the peptide for imaging idiopathic pulmonary fibrosis and those associated with connective tissue disease, radiation therapy, and severe acute respiratory syndrome coronavirus 2 infection (46Lukey P.T. Coello C. Gunn R. Parker C. Wilson F.J. Saleem A. Garman N. Costa M. Kendrick S. Onega M. Kang'ombe A.R. Listanco A. Davies J. Ramada-Magalhaes J. Moz S. et al.Clinical quantification of the integrin αvβ6 by [18F]FB-A20FMDV2 positron emission tomography in healthy and fibrotic human lung (PETAL Study).Eur. J. Nucl. Med. Mol. Imaging. 2020; 47: 967-979Crossref PubMed Scopus (20) Google Scholar, 47Saleem A. Helo Y. Win Z. Dale R. Cook J. Searle G.E. Wells P. Integrin αvβ6 positron emission tomography imaging in lung cancer patients treated with pulmonary radiation therapy.Int. J. Radiat. Oncol. 2020; 107: 370-376Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 48Foster C.C. Davis R.A. Hausner S.H. Sutcliffe J.L. αvβ6-Targeted molecular PET/CT imaging of the lungs after SARS-CoV-2 infection.J. Nucl. Med. 2020; 61: 1717-1719Crossref PubMed Scopus (10) Google Scholar). However, the clinical translation of A20FMDV2 has been limited, in part, by poor metabolic stability of the peptide that impairs its pharmacokinetics (40Hausner S.H. DiCara D. Marik J. Marshall J.F. Sutcliffe J.L. Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: Generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin αvβ6 expression with positron emission tomography.Cancer Res. 2007; 67: 7833-7840Crossref PubMed Scopus (92) Google Scholar, 49Kimura R.H. Teed R. Hackel B.J. Pysz M.A. Chuang C.Z. Sathirachinda A. Willmann J.K. Gambhir S.S. Pharmacokinetically stabilized cystine Knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer.Clin. Cancer Res. 2012; 18: 839-849Crossref PubMed Scopus (80) Google Scholar). Modification of A20FMDV2 with two short PEG chains (∼1 kDa each) reduces peptide degradation and thereby increases tumor retention but also slows peptide clearance from healthy tissue and increases renal retention (50Hausner S.H. Abbey C.K. Bold R.J. Gagnon M.K. Marik J. Marshall J.F. Stanecki C.E. Sutcliffe J.L. Targeted in vivo imaging of integrin αvβ6 with an improved radiotracer and its relevance in a pancreatic tumor model.Cancer Res. 2009; 69: 5843-5850Crossref PubMed Scopus (75) Google Scholar, 51Hausner S.H. Bauer N. Hu L.Y. Knight L.M. Sutcliffe J.L. The effect of Bi-terminal PEGylation of an integrin αvβ₆-targeted 18F peptide on pharmacokinetics and tumor uptake.J. Nucl. Med. 2015; 56: 784-790Crossref PubMed Scopus (31) Google Scholar). For these reasons, we sought to engineer an A20FMDV2 peptide with chemistries and amino acid modifications that increase the peptide's inherent metabolic stability. Here, we report the design of cyclized A20FMDV2 variants with selective amino acid modifications and their characterization in vitro. We demonstrate that these peptide variants have prolonged stability in serum and retain their binding affinity for αvβ6+ cells. Importantly, some of these optimized peptides demonstrate improved αvβ6 specificity over the original A20FMDV2 peptide, further increasing the benefit for future in vivo application. A20FMDV2 has a hairpin loop structure with the RGD motif at the tip of hairpin turn followed by a 310 helix (Fig. 1A) (38Logan D. Abu-Ghazaleh R. Blakemore W. Curry S. Jackson T. King A. Lea S. Lewis R. Newman J. Parry N. Rowlands D. Stuart D. Fry E. Structure of a major immunogenic site on foot-and-mouth disease virus.Nature. 1993; 362: 566-568Crossref PubMed Scopus (300) Google Scholar, 39DiCara D. Rapisarda C. Sutcliffe J.L. Violette S.M. Weinreb P.H. Hart I.R. Howard M.J. Marshall J.F. Structure-Function analysis of Arg-Gly-Asp helix motifs in αvβ6 integrin ligands.J. Biol. Chem. .. 2007; 282: 9657-9665Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and previous reports have demonstrated that the extended RGDLXXL motif is most critical for αvβ6 binding, whereas amino acids at the N terminus and C terminus of the peptide are not as critical for binding (52Burman A. Clark S. Abrescia N.G.A. Fry E.E. Stuart D.I. Jackson T. Specificity of the VP1 GH loop of foot-and-mouth disease virus for αv integrins.J. Virol. 2006; 80: 9798-9810Crossref PubMed Scopus (89) Google Scholar). Accordingly, we postulated that chemistries involving the N- and C-terminal amino-acid positions of the peptide could increase serum stability without negatively affecting peptide binding to αvβ6. Cyclization is a well-established technique for stabilizing peptides and improving their pharmacokinetic profiles (35Vlieghe P. Lisowski V. Martinez J. Khrestchatisky M. Synthetic therapeutic peptides: Science and market.Drug Discov. Today. 2010; 15: 40-56Crossref PubMed Scopus (1003) Google Scholar), and cysteine perfluoroarylation is a facile cyclization approach that uses perfluoroaromatic molecular linkers to staple together cysteine thiol moieties on unprotected peptides (53Spokoyny A.M. Zou Y. Ling J.J. Yu H. Lin Y.-S. Pentelute B.L. A perfluoroaryl-cysteine SNAr chemistry approach to unprotected peptide stapling.J. Am. Chem. Soc. 2013; 135: 5946-5949Crossref PubMed Scopus (269) Google Scholar). Our laboratory has previously used this technique for peptide cyclization with a decafluorobiphenyl (DFBP) linker (Fig. 1B) and demonstrated increased serum stability and affinity of DFBP-cyclized peptides compared with counterparts with disulfide, amide, or triazole cyclization (54Ngambenjawong C. Pineda J.M.B. Pun S.H. Engineering an affinity-enhanced peptide through optimization of cyclization chemistry.Bioconjug. Chem. 2016; 27: 2854-2862Crossref PubMed Scopus (13) Google Scholar, 55Sellers D.L. Tan J.-K.Y. Pineda J.M.B. Peeler D.J. Porubsky V.L. Olden B.R. Salipante S.J. Pun S.H. Targeting ligands deliver model drug cargo into the central nervous system along autonomic neurons.ACS Nano. 2019; 13: 10961-10971Crossref PubMed Scopus (6) Google Scholar). We therefore synthesized six unique A20FMDV2 peptide sequences with cysteine substitutions primarily at N- and C-terminal amino-acid positions for cyclization by DFBP to stabilize and close the hairpin peptide structure (Fig. 1C). Biotin was conjugated on the N terminus of all peptides for labeling with streptavidin-AF647 to assess cell binding by flow cytometry. Peptides were also synthesized with a C-terminal lysine to mimic a prospective methyltrityl-lysine that could be added for selective modification or synthesis of branched peptides at the lysine side chain. The binding of cyclized A20FMDV2 sequences to αvβ6 was evaluated with the matched erythroleukemia K562 and K562 αvβ6:mCherry cell lines. Both cell lines endogenously express the α5β1 integrin (56Hewish M.J. Takada Y. Coulson B.S. Integrins α2β1 and α4β1 can mediate SA11 rotavirus attachment and entry into cells.J. Virol. 2000; 74: 228236Crossref Scopus (136) Google Scholar), but only the K562 αvβ6:mCherry cells express the αvβ6 integrin. Of the DFBP-cyclized sequences, we observed binding of the C1C18 DFBP (N1C; A18C), C1C20 DFBP (N1C; T20C), and C2C18 DFBP (A2C; A18C) peptides to K562 αvβ6:mCherry cells with high affinity and specificity comparable to the original A20FMDV2 peptide (Fig. 1D). Interestingly, peptide sequences C1C19 DFBP (N1C; R19C) and C2C19 DFBP (A2C; R19C) exhibited not only high binding to K562 αvβ6:mCherry cells but also poor specificity. Both peptides significantly bound to parental K562 cells at high concentrations, suggesting that Arg19 in A20FMDV2 is important for αvβ6 specificity. Similarly, the peptide cyclized via cysteine substitutions proximal to the RGDLXXL motif, C6C17 DFBP (L6C; V17C), displayed minimal binding to K562 αvβ6:mCherry cells. It is known that Val12 and Val17 are important for the structure of the post-RGD helix in A20FMDV2 (57DiCara D. Burman A. Clark S. Berryman S. Howard M.J. Hart I.R. Marshall J.F. Jackson T. Foot-and-Mouth disease virus forms a highly stable, EDTA-resistant complex with its principal receptor, integrin αvβ6: Implications for Infectiousness.J. Virol. 2008; 82: 1537-1546Crossref PubMed Scopus (62) Google Scholar), so the cysteine substitutions in the C6C17 DFBP peptide and their cyclization likely impaired the 310 helix structure. Given that the DFBP-cyclized C1C18, C1C20, and C2C18 peptides retained the favorable binding properties of the original A20FMDV2 peptide, we moved forward with these variants for characterization of serum stability. As the original A20FMDV2 peptide is degraded by over 50% in normal mouse serum within a 4 h incubation at 37 °C (58Hung K. Harris P.W.R. Desai A. Marshall J.F. Brimble M.A. Structure-activity relationship study of the tumour-targeting peptide A20FMDV2 via modification of Lys16, Leu13, and N- and/or C-terminal functionality.Eur. J. Med. Chem. 2017; 136: 154-164Crossref PubMed Scopus (7) Google Scholar), we hypothesized that our DFBP-cyclized variants would have prolonged serum stability because of added structural stability from their cyclization. To investigate this, we incubated the DFBP-cyclized C1C18, C1C20, and C2C18 peptides in normal mouse serum at 37 °C for up to 6 h and measured the presence of intact peptide and any degradation products at different time points by MALDI-TOF MS (Fig. 2, A–C). As shown, the partially cyclized C1C18 DFBP and C2C18 DFBP peptides formed degradation products that are 385 Da smaller after incubation in serum for 2 h, corresponding C-terminal cleavage of the arginine–threonine–lysine (RTK) group outside the DFBP-cyclized region (Fig. 2, A and C). C1C18 DFBP had prolonged intact peptide presence compared with C2C18 DFBP (6 h versus 2 h), suggesting that the C1C18 cyclization scheme better protects the exocyclic C-terminal RTK group. Importantly, the 385 Da smaller degradation products for both peptides persisted up to the last time point sampled, and no further degradation peaks were observed, demonstrating good protection of amino acids within their cyclized regions. We also assayed a disulfide-cyclized C1C18 peptide (C1C18 S–S) and observed faster degradation to the 385 Da smaller product compared with C1C18 DFBP (Fig. S1), emphasizing the importance of the DFBP molecular linker. To our surprise, the fully cyclized C1C20 DFBP peptide degraded within 2 h in serum; we could not detect any peptide or degradation products at the 4 and 6 h time points (Fig. 2B). We detected a low-intensity 135 smaller Da degradation product at the 2 h time point, but it was near background, and we could not predict the amino acid sequence. The molecular weight difference is near what would be expected from an internal arginine deletion (138 Da smaller), so Arg7 or Arg19 may have been metabolically cleaved from the sequence. These data suggest that the complete cyclization of a peptide from N to C terminus is not always beneficial for stability, and that peptide structure sh
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