Optimized serum stability and specificity of an αvβ6 integrin-binding peptide for tumor targeting

Abstract

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. 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. 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A. that integrin with nanomolar for of pancreatic Cancer Res. 18: PubMed Scopus Google Scholar). of A20FMDV2 with two peptide and thereby tumor but peptide from healthy tissue and J. Marshall J.F. in vivo imaging of integrin αvβ6 with an improved and its in a pancreatic tumor Res. PubMed Scopus Google Scholar, N. The of of an integrin peptide on and tumor PubMed Scopus Google Scholar). these we to an A20FMDV2 peptide with and amino acid modifications that the stability. Here, we the of cyclized A20FMDV2 variants with selective amino acid modifications and their in that these peptide variants have prolonged stability in serum and their binding affinity for αvβ6+ some of these improved αvβ6 specificity the original A20FMDV2 peptide, the for in vivo A20FMDV2 has a with the at the of by a D. R. W. S. A. S. R. J. N. D. D. E. of a on foot-and-mouth disease PubMed Scopus Google Scholar, D. C. Violette S.M. Weinreb P.H. M.J. Marshall J.F. of in αvβ6 integrin Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), and have that the is for αvβ6 amino acids at the and of the peptide are as for binding A. S. of the of foot-and-mouth disease virus for 2006; PubMed Scopus Google Scholar). we that the and C-terminal of the peptide could serum stability without peptide binding to αvβ6. is a for and their P. V. J. therapeutic and 2010; PubMed Scopus Google Scholar), and cysteine perfluoroarylation is a cyclization that to cysteine on Y. H. A to peptide PubMed Scopus Google Scholar). has this for peptide cyclization with a and increased serum stability and affinity of compared with with cyclization C. an peptide through optimization of cyclization 2016; PubMed Scopus Google Scholar, the 2019; PubMed Scopus Google Scholar). synthesized six A20FMDV2 peptide with cysteine substitutions at and C-terminal for cyclization by to and the peptide on the of for with to cell binding by were synthesized with a C-terminal to a that could for selective modification of at the The binding of cyclized A20FMDV2 to αvβ6 with the and cell cell the integrin M.J. Y. Integrins and can mediate and Scopus Google Scholar), but the cells the αvβ6 the we binding of the and to cells with affinity and specificity to the original A20FMDV2 peptide peptide and exhibited binding to cells but poor to cells at that in A20FMDV2 is for αvβ6 the peptide cyclized cysteine substitutions to the binding to is that and are for the of the in A20FMDV2 D. A. S. S. M.J. Marshall J.F. disease virus a highly with its integrin for PubMed Scopus Google Scholar), the cysteine substitutions in the peptide and their cyclization the that the and retained the binding of the original A20FMDV2 peptide, we with these variants for of serum stability. the original A20FMDV2 peptide is by in serum a at K. Desai A. Marshall J.F. of the peptide A20FMDV2 modification of and C-terminal J. 2017; PubMed Scopus Google Scholar), we that variants have prolonged serum stability because of stability from their we the and in serum at for to 6 and the of peptide and any at by the cyclized and that are in serum for C-terminal of the outside the region A and had prolonged peptide compared with versus that the cyclization the C-terminal the for to the and were of amino acids their cyclized a peptide and to the compared with the of the the cyclized peptide in we could any peptide at the and 6 a at the but it and we could the amino acid The is from an have from the that the cyclization of a peptide from to is for and that peptide cyclization and were outside the C-terminal we to these two with amino acid the and we to d-amino acids and for additional amino acid modification other have increased serum stability of with d-amino acids R. K. D. E. A. F. acid stability and of a Sci. S. A. PubMed Scopus Google Scholar), we the of with d-alanine in an to of the C-terminal as the C-terminal is by many E. C. A. S. A. J.M. and on the the Scholar), we it with the as a to peptide modifications in the of and for to the binding of cyclized by the the cyclized region with this modification has affinity for other Z. K. V. P. and in the of and Scholar). to the of for which the as the the cyclized region and the C-terminal contribute to peptide we these amino acids with d-alanine and for additional stability. to these of were synthesized for binding detailed we compared the cell binding of these variants the original A20FMDV2 peptide and the and selective binding to cells with binding to cells the binding of these and as as of the and and from the affinity of the original A20FMDV2 peptide for the compared with and their we these to of the of the binding from the nanomolar for A20FMDV2 R. S.B. Marshall J.F. S. of the αvβ6 integrin binding and of the foot-and-mouth disease virus derived peptide 2016; PubMed Scopus Google Scholar). from in the binding versus and and for peptide were the of the cyclization and modifications on binding affinity. and exhibited binding affinity for and the with citrulline, d-alanine affinity. for and for d-alanine the cyclized region binding that these amino acids are for αvβ6 The of these cyclization and modifications on peptide specificity for the αvβ6 integrin in a later that the retained their binding affinity for αvβ6+ we with these for and of serum stability. the serum stability of the by peptide for of the a compared with the original and that were 6 were at later and the peptide an of of of the of the to a for as a for and and at between the cysteine that the the of the increased for which that the cyclization than the the cyclized we of for some of the A and as as C-terminal at for the While peptide these the of cyclized in serum improved serum stability. are we to the of in serum of the and by were in and as were for the for While some of the are because of in peptide of serum we were to the to a with the the and were found to in with serum of between and the of peptide in serum for and at and that have basal broadly that and substitutions at the peptide and substitutions with and d-alanine in the cyclized region and have on peptide serum stability. significant of the that by in the of the of the in the of the of the peptide. that of from the is a for these and that the for the the cyclized could by the in the the we were in the with to the stability of A20FMDV2 have to for in other C. D. R. and as in PubMed Scopus Google Scholar). synthesized of citrulline, as substitutions for the and The have of C-terminal as some of these were synthesized we discovered the of d-amino acids at the these of binding potential we that these modifications peptide affinity for αvβ6. and the to retained some selective binding to these cells at low and to the highly of the in integrin these are have of binding for an peptide in the with Y. W. Y. L. H. Y. of Cancer Ther. 2016; PubMed Scopus (32) Google Scholar). the binding we with a is with a of and there are integrins that the αvβ6, and F. J. A. K. H. the role of integrins in (Basel). 2017; 9: PubMed Scopus Google Scholar), it is that for diagnostic and therapeutic are highly specific for their targeted integrin with The cell we the integrin and thus the nonspecific binding of A20FMDV2 to other integrins to αvβ6. we to the nonspecific integrin binding of the original A20FMDV2 peptide and the and to which the and integrins but the integrin H. D. S. S. D. Marshall J.F. K. a to αvβ6 integrin the of a foot-and-mouth disease Biol. PubMed Scopus Google Scholar). to cells as a to peptide and modifications integrin this we binding of A20FMDV2 and and to cells at peptide and by that and nonspecific binding for with an and to the original A20FMDV2 peptide are with by et D. Marshall J.F. M.J. and of in the and of the of integrin αvβ6 PubMed Scopus Google Scholar), which that cyclization of the A20FMDV2 peptide at the of the peptide, of αvβ6 The that found to A20FMDV2 specificity a but we that this cyclization in serum nonspecific binding for on to that C-terminal with has that integrin and nonspecific on binding to the original A20FMDV2 peptide, While some of these to the binding we with these it for their low nonspecific for the peptide has nonspecific binding binding these two with thus that the of the the of the and the of the d-alanine substitutions and the nonspecific integrin binding for the and with some improved αvβ6 integrin specificity compared with the original A20FMDV2 peptide. with their greatly serum the and the potential to αvβ6 targeting for in vivo diagnostic and therapeutic The of for cancer targeting is limited by their from in and of are thus often to to the tumor which can the and of that these The A20FMDV2 peptide, its affinity and specificity for the integrin αvβ6, is such peptide that from poor its the for cancer imaging and therapy. of A20FMDV2 to its is because of the highly and of the its acid sequence a by the and a that a D. R. W. S. A. S. R. J. N. D. D. E. of a on foot-and-mouth disease PubMed Scopus Google Scholar, D. C. Violette S.M. Weinreb P.H. M.J. Marshall J.F. of in αvβ6 integrin Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The amino acids in the and that the the affinity and specificity of A20FMDV2 for αvβ6 D. A. S. S. M.J. Marshall J.F. disease virus a highly with its integrin for PubMed Scopus Google Scholar), and thus these amino acids the of these that are for the this we synthesized a of than A20FMDV2 variants that were cyclized by cysteine perfluoroarylation with and with d-amino acids and to improve the A20FMDV2 serum stability without sacrificing its affinity and specificity for αvβ6. From this we variants and that in serum for and binding affinity and specificity to αvβ6+ with the αvβ6 specificity than the original A20FMDV2 peptide. these in vivo of the cyclization and modifications herein could greatly improve the of the A20FMDV2 peptide for cancer diagnosis and Despite to the A20FMDV2 peptide, there are opportunities for peptide optimization and serum stability we of in as as C-terminal at to for the cyclized et K. Desai A. Marshall J.F. of the peptide A20FMDV2 modification of and C-terminal J. 2017; PubMed Scopus Google that this and C-terminal can with d-amino acid to improve A20FMDV2 serum stability without binding affinity. that the can with acid and that the can with acid without reducing αvβ6 these modifications with the cyclization and modifications could to the of A20FMDV2 peptide that for in the for this as of this amino acid for a of in of A20FMDV2 variants that we explore in this is their potential for has that of with derived from can in that are by of serum that S. N. of acid with acid in PubMed Scopus Google Scholar). to with with d-amino acids found to this immune that the as to While the A20FMDV2 peptide to in a N. J. K. N. W. A. J. A of the and of for imaging the integrin 2018; PubMed Scopus Google Scholar), its from for with and the of d-amino acids and the A20FMDV2 sequence in this the potential of this peptide, its for including and were from and were from and and were from The from and with and were from serum and were from and were from serum from from serum for stability by and to for were to the and serum and at were by the of and of synthesized are in with a peptide by peptide on a at The in for to of in in at for by with to amino acids in and at for with the for the amino acid in the which to to the for were for and because of the that of these amino and of the from the in for at with in the for were in and at for at A of retained and by to and of peptide. some of the a of the peptide because of with the on were in The were in and in to the peptide were at in in and to by on an with a a of and a to of in with were for of these The of were by and were of the by and were and at cyclization of with as C. an peptide through optimization of cyclization 2016; PubMed Scopus Google Scholar), but with some peptide in with of and in and to the an at with the cyclization in and peptide with a from the in and cyclization by by and cyclized were and at The and cell in binding were from The cell a from and of and by of cells with two the integrin and a and the other the integrin and a cells were by and cell and cells were in with and cells were in with and and were with to binding to extracellular integrin peptide were in at and the a were at and for binding of to cells were with in for at for cell peptide were in with and were at with serum to the in a and with peptide for at were with and with in for at were as and in for on an were by and of cell were as of were to A20FMDV2 binding to binding and in 6 were and from serum as C. an peptide through optimization of cyclization 2016; PubMed Scopus Google Scholar, the 2019; PubMed Scopus Google but with some to serum during peptide were to in and in serum for at in an of the and in an of serum proteins were by at for and the with any to the for and as The with the and on a The peptide in and for A of peptide in and by at the were and in of on were a the 2019; PubMed Scopus Google Scholar). from serum stability were as and at a of were in for and for at the of of were on a with a were a to of in with and to the peptide for of peptide. were to that of a that the peptide with the were in 6 The that the of this are in this and the for this and are from the on C. J. has in and a C. J. is a and in as a of the and is a of the of C. J. some of which are to and The other that have of interest with the of this are to of for and and of of for of the peptide to the of in of of for the cell D. L. S. and D. L. S. and C. and C. and D. L. S. and S. H. P. D. L. S. H. and C. and D. L. S. H. and C. J. and by the of D. L. D. L. and S. H. and by a The is the of the and the of the of and

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