Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting

Production of cell-targeting vectors in part involves the addition of new targeting ligands to the vector to mediate binding to the cells of interest. For viral vectors, the ideal approach is to genetically engineer new ligands into the capsid proteins of the virus to generate a single agent to mediate therapy. Although this is ideal, this insertion of an exogenous ligand from one structural context into the differing structural context of a capsid protein can ablate the function of the ligand or disrupt viral assembly and function. To address this context problem for adenoviral vectors, we have engineered a "context-specific" peptide-presenting phage library. We have displayed a 12-amino-acid (12-mer) random peptide library between the H and I sheets of the fiber protein of adenovirus type 5 on the pIII protein of fd bacteriophage. This library was used for peptide selection against C2C12 mouse skeletal muscle cells. Five rounds of selection combined with four rounds of clearing on nontarget cells selected one primary peptide designated 12.51, which bound target C2C12 cells approximately 100-fold better than the positive control RGD peptide. Translation of 12.51 back into the fiber protein produced a ligand-modified adenoviral vector that mediated 14-fold-better transduction of target C2C12 cells. These data suggest context-specific peptide-presenting libraries may allow selection of compatible peptide ligands for functional translation into viral vectors for retargeting.     

Peptide-mediated transcytosis of phage display vectors in MDCK cells

Delivery of therapeutic macromolecules and gene vectors to certain tissues is hampered by endothelial or epithelial barriers. We show here that the transport of phage particles across epithelial cells can be facilitated by peptide ligands selected from a phage library of random peptides. Using MDCK cells, we identified a polycationic peptide sequence, RYRGDLGRR, containing a putative integrin-binding (RGD) motif that enhanced basal-to-apical transcytosis of peptide-bearing phage 1000- to 10,000-fold compared with phage with no peptide insert. Both the synthetic peptide RYRGDLGRR and the integrin-binding peptide GRGDSP inhibited phage transcytosis suggesting the involvement of integrins. Confocal immunofluorescence microscopy showed that following internalization at the basal cell surface, phage particles were delivered to the apical cytoplasm and released at the apical cell surface. These data suggest the feasibility of using short peptides for targeting transcytotic pathways and facilitating delivery of macromolecules across cellular barriers.     

High-throughput Pyrosequencing of a phage display library for the identification of enriched target-specific peptides

Gene therapy clinical trials have highlighted the importance of specific cellular/tissue targeting of gene delivery vectors. Phage display libraries are powerful tools for the selection of novel peptide ligands as targeting moieties because of their high-throughput screening potential. However, a severe rate-limiting step in this procedure in terms of time, numbers, and cost is the sequence identification of selected phages. Here we describe the application of Pyrosequencing technology for sequencing phage isolates after panning a random 7-mer peptide expressing phage library against the A549 bronchial epithelial cell line to search for enrichment of possible targeting peptides. Pyrosequencing allows sequencing of 96 phages at one time in approximately 45 min at only a sixth of the cost of conventional sequencing methods. Using this technology, we have identified four sequences of interest. A phage binding assay revealed that three of the four sequences show a significant increase in binding abilities and specificity for A549 cells when compared to an unrelated cell line.     

Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library

The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.     

Peptide ligands incorporated into the threefold spike capsid domain to re-direct gene transduction of AAV8 and AAV9 in vivo

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes.     

Efficient isolation of peptide ligands for the endothelial cell protein C receptor (EPCR) using candidate receptor phage display biopanning

Phage display biopanning has been used for a number of applications including ligand generation for targeted drug delivery, targeting gene therapy vectors and identification of protein-protein interaction sites. In this study, a random phage display library was used to isolate peptide ligands to the endothelial protein C receptor (EPCR), identifying 74 different peptide sequences and several motifs. Binding to EPCR was characterized by a solid phase binding assay, demonstrating that 95% of isolated peptides were specific for EPCR. Several homologies with potential relevance to EPCR biology were identified, the most notable being leukolysin (MT-MMP6) and cerastocytin.     

Probing sequence variation in the receptor-targeting domain of feline leukemia virus envelope proteins with peptide display libraries

Determinants of cellular tropism and receptor targeting lie within a short peptide in the Vr1 region of the envelope (Env) proteins of feline leukemia virus (FeLV) subgroups A and C. Libraries of FeLV Env proteins with random amino acid substitutions in the peptide were screened for their ability to deliver a marker gene to D17 and AH927 cells. Screening on D17 canine cells yielded D17-specific Env proteins that used the FeLV-C receptor. Screening on AH927 cells yielded Env proteins with a broader host range, with maximal titers on AH927 cells and similar or lower titers on other cells. These Env proteins used an unidentified non-FeLV receptor for entry. The A5 isolate obtained from the AH927 screen was readily concentrated to yield titers of 10(5) on human PC-3 prostate tumor cells. The sequence divergence observed among targeting peptides of library-selected Env proteins was greater than that found in parental FeLV isolates. Substitution analyses of a conserved R in the middle of the targeting peptide held constant during screening indicated that maximal titers were obtained only when R was present in both a D17 selected isolate and an AH927 selected isolate. The ability to isolate Env proteins with unique tropisms dependent on the cells on which the library is screened has direct implications for targeting gene delivery vectors.     

Isolation of targeted AAV2 vectors from novel virus display libraries

Random peptide ligands displayed on viral capsids are emerging tools for selection of targeted gene transfer vectors even without prior knowledge of the potential target cell receptor. We have previously introduced adeno-associated viral (AAV)-displayed peptide libraries that ensure encoding of displayed peptides by the packaged AAV genome. A major limitation of these libraries is their contamination with wild-type (wt) AAV. Here we describe a novel and improved library production system that reliably avoids generation of wt AAV by use of a synthetic cap gene. Selection of targeted AAV vectors from wt-containing and the novel wt-free libraries on cell types with different permissivity for wt AAV2 replication suggested the superiority of the wt-free library. However, from both libraries highly specific peptide sequence motifs were selected which improved transduction of cells with moderate or low permissivity for AAV2 replication. Strong reduction of HeLa cell transduction compared to wt AAV2 and only low level transduction of non-target cells by some selected clones showed that not only the efficiency but also the specificity of gene transfer was improved. In conclusion, our study validates and improves the unique potential of virus display libraries for the development of targeted gene transfer vectors.     

Adenoviruses Using the Cancer Marker EphA2 as a Receptor In Vitro and In Vivo by Genetic Ligand Insertion into Different Capsid Scaffolds

Adenoviral gene therapy and oncolysis would critically benefit from targeted cell entry by genetically modified capsids. This requires both the ablation of native adenovirus tropism and the identification of ligands that remain functional in virus context. Here, we establish cell type-specific entry of HAdV-5-based vectors by genetic ligand insertion into a chimeric fiber with shaft and knob domains of the short HAdV-41 fiber (Ad5T/41sSK). This fiber format was reported to ablate transduction in vitro and biodistribution to the liver in vivo. We show that the YSA peptide, binding to the pan-cancer marker EphA2, can be inserted into three positions of the chimeric fiber, resulting in strong transduction of EphA2-positive but not EphA2-negative cells of human melanoma biopsies and of tumor xenografts after intratumoral injection. Transduction was blocked by soluble YSA peptide and restored for EphA2-negative cells after recombinant EphA2 expression. The YSA peptide could also be inserted into three positions of a CAR binding-ablated HAdV-5 fiber enabling specific transduction; however, the Ad5T/41sSK format was superior in vivo. In conclusion, we establish an adenovirus capsid facilitating functional insertion of targeting peptides and a novel adenovirus using the tumor marker EphA2 as receptor with high potential for cancer gene therapy and viral oncolysis.     

A novel peptide, THALWHT, for the targeting of human airway epithelia

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies confirmed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non-airway-derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non-viral and viral gene therapy vectors.     

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.     

Efficient accommodation of recombinant, foot-and-mouth disease virus RGD peptides to cell-surface integrins.

The engineering of either complete virus cell-binding proteins or derived ligand peptides generates promising nonviral vectors for cell targeting and gene therapy. In this work, we have explored the molecular interaction between a recombinant, integrin-binding foot-and-mouth disease virus RGD peptide displayed on the surface of a carrier protein and its receptors on the cell surface. By increasing the number of viral segments, cell binding to recombinant proteins was significantly improved. This fact resulted in a dramatic growth stimulation of virus-sensitive BHK(21) cells but not virus-resistant HeLa cells in protein-coated wells. Surprisingly, growth stimulation was not observed in vitronectin-coated plates, suggesting that integrins other than alpha(v)beta(3) could be involved in binding of the recombinant peptide, maybe as coreceptors. On the other hand, both free and cell-linked integrins did not modify the enzymatic activity of RGD-based enzymatic sensors that contrarily, were activated by the induced fit of anti-RGD antibodies. Those findings are discussed in the context of a proper mimicry of the unusually complex architecture of this cell-binding site as engineered in multifunctional proteins.     

Sites in the AAV5 capsid tolerant to deletions and tandem duplications

Gene therapy vectors based on adeno-associated virus (AAV) have shown much promise in clinical trials for the treatment of a variety of diseases. However, the ability to manipulate and engineer the viral surface for enhanced efficiency is necessary to overcome such barriers as pre-existing immunity and transduction of non-target cells that currently limit AAV applications. Although single amino acid changes and peptide insertions at select sites have been explored previously, the tolerance of AAV to small deletions and tandem duplications of sequence has not been globally addressed. Here, we have generated a large, diverse library of >10⁵ members containing deletions and tandem duplications throughout the viral capsid of AAV5. Four unique mutants were identified that maintain the ability to form viral particles, with one showing improved transduction on both 293T and BEAS-2B cells. This approach may find potential use for the generation of novel variants with improved and altered properties or in the identification of sites that are tolerant to insertions of targeting ligands.     

Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX

The utility of the present generation of adenovirus (Ad) vectors for gene therapy applications could be improved by restricting native viral tropism to selected cell types. In order to achieve modification of Ad tropism, we proposed to exploit a minor component of viral capsid, protein IX (pIX), for genetic incorporation of targeting ligands. Based on the proposed structure of pIX, we hypothesized that its C terminus could be used as a site for incorporation of heterologous peptide sequences. We engineered recombinant Ad vectors containing modified pIX carrying a carboxy-terminal Flag epitope along with a heparan sulfate binding motif consisting of either eight consecutive lysines or a polylysine sequence. Using an anti-Flag antibody, we have shown that modified pIXs are incorporated into virions and display Flag-containing C-terminal sequences on the capsid surface. In addition, both lysine octapeptide and polylysine ligands were accessible for binding to heparin-coated beads. In contrast to virus bearing lysine octapeptide, Ad vector displaying a polylysine was capable of recognizing cellular heparan sulfate receptors. We have demonstrated that incorporation of a polylysine motif into the pIX ectodomain results in a significant augmentation of Ad fiber knob-independent infection of CAR-deficient cell types. Our data suggest that the pIX ectodomain can serve as an alternative to the fiber knob, penton base, and hexon proteins for incorporation of targeting ligands for the purpose of Ad tropism modification.     

双靶向MMP-14和金属离子的结合肽筛选与分子模拟

以基质金属蛋白酶-14(MMP-14)催化结构域为靶标,通过噬菌体随机十二肽库 筛选和分子模拟、细胞免疫荧光、金属离子亲和层析以及体外细胞作用测定等技 术,进行了双靶向MMP-14和金属离子小分子结合多肽的筛选与研究.经4轮筛选, 噬菌体得到有效富集并获得13条不同的多肽序列.序列分析显示,可能的一致序列 有：AHQLH、HHXH、EI/LPLL/I.分子模拟与对接进一步确认一致序列AHQLH、HHTH 、LPLL与MMP-14催化结构域的氨基酸120~125区域良好分子对接并具有一定的专 一性,多条MMP-14结合肽不仅靶向MMP-14,同时结合金属离子.细胞生物学研究确 认,所测定的结合肽噬菌体对MMP-14诱导表达的MG63细胞具有良好的结合作用,揭 示结合肽对MMP-14的靶向结合特性,并且合成的AHQLH、LPLL一致序列多肽对MG63 细胞活力具有一定的抑制能力.这些新的和具有一定MMP-14专一性的一致序列可望 用于靶向MMP-14抗肿瘤药物的研发和利用.     

Heparan sulfate proteoglycan binding properties of adeno-associated virus retargeting mutants and consequences for their in vivo tropism

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues.     

Identification using phage display of peptides promoting targeting and internalization into HPV-transformed cell lines

'High-risk' human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumours therapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, we selected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalently on pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16 transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel of normal or transformed cell lines. Two of the three selected peptides targeted cells only when presented on phage particles in a constrained conformation. However, all three peptides retained their targeting capacity when presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form. These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervical cancer.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

The protein invasin expressed on the cell surface of the pathogenic bacteria Yersinia pseudotuberculosis mediates the entry of this bacterium into cultured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protein constructs consist of part of the outer membrane domain of the invasin protein, six proline spacers, and a decamer of random peptides flanked by cysteine residues (CX(10)C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designated as ESPEL (E. coli Surface Peptide Expression Library). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bacterial clones were identified whose binding to human cells was mediated by peptides expressed on the bacterial cell surface. Flow cytometric analysis showed that both the identified bacterial clones and these corresponding chemically synthesized peptides bound to human cells specifically. The techniques described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors.     

Combinatorial discovery of tumor targeting peptides using phage display

Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.