Cell-binding Peptide Research Articles

High-Throughput Discovery of Synthetic Surfaces That Support Proliferation of Pluripotent Cells

Synthetic materials that promote the growth or differentiation of cells have advanced the fields of tissue engineering and regenerative medicine. Most functional biomaterials are based on a handful of peptide sequences derived from protein ligands for cell surface receptors. Because few proteins possess short peptide sequences that alone can engage cell surface receptors, the repertoire of receptors that can be targeted with this approach is limited. Materials that bind diverse classes of receptors, however, may be needed to guide cell growth and differentiation. To provide access to such new materials, we utilized phage display to identify novel peptides that bind to the surface of pluripotent cells. Using human embryonal carcinoma (EC) cells as bait, approximately 3 x 10(4) potential cell-binding phage clones were isolated. The pool was narrowed using an enzyme-linked immunoassay: 370 clones were tested, and seven cell-binding peptides were identified. Of these, six sequences possess EC cell-binding ability. Specifically, when displayed by self-assembled monolayers (SAMs) of alkanethiols on gold, they mediate cell adhesion. The corresponding soluble peptides block this adhesion, indicating that the identified peptide sequences are specific. They also are functional. Synthetic surfaces displaying phage-derived peptides support growth of undifferentiated human embryonic stem (ES) cells. When these cells were cultured on SAMs presenting the sequence TVKHRPDALHPQ or LTTAPKLPKVTR in a chemically defined medium (mTeSR), they expressed markers of pluripotency at levels similar to those of cells cultured on Matrigel. Our results indicate that this screening strategy is a productive avenue for the generation of materials that control the growth and differentiation of cells.

CEL-2000: A therapeutic vaccine for rheumatoid arthritis arrests disease development and alters serum cytokine/chemokine patterns in the bovine collagen type II induced arthritis in the DBA mouse model

The mouse model of collagen induced arthritis (CIA) effectively mimics human disease and thus is useful for testing and development of rheumatoid arthritis (RA) therapies. We developed a Ligand Epitope Antigen Presentation System (LEAPS) peptide hetero-conjugate vaccine containing an epitope of human collagen type II (CEL-2000) that acted as a therapeutic vaccine in the collagen induced arthritis (CIA) mouse model. LEAPS technology converts a small peptide containing a disease specific epitope into an immunogen by attaching it to an immune or T cell binding peptide (I/TCBL). For CEL-2000, a peptide from human collagen type II (254–273) is attached to the I/TCBL peptide from human β2 microglobulin (J). Treatment with CEL-2000 limited disease (CIA) progression, as demonstrated by reduced Arthritic Index (AI) score, and footpad swelling. Efficacy was confirmed by histopathological microscopic examination of tissues at the end of the study. CEL-2000 limited disease progression as well or better than the etanercept (Enbrel) therapeutic control with significantly better histopathological results than the etanercept treated mice. Most interestingly, CEL-2000 therapy modulated serum cytokine levels with an increase in IL-12p70 and IL-10, which are not seen with etanercept therapy, and reduced IL-17 and TNF-α, also seen with etanercept, among other cytokines studied. CEL-2000 was safe and well tolerated for the mice that received 5 injections given every 2 weeks in a 90 day study supporting its potential usage for long term therapy. These studies demonstrate that fewer treatments with CEL-2000 provide therapy at least as effective as etanercept by specifically modulating the disease producing autoimmune response.

Novel β-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy

Novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained decapeptide (CX10C) libraries constructed in fusion to the amino-terminus of P99 β-lactamase molecules were used for identifying β-lactamase-linked cancer cell-specific ligands. The size and quality of both libraries were comparable to the standards of other reported phage display systems. Using the single-round panning method based on phage DNA recovery, we identified several β-lactamase fusion peptides that specifically bind to live human breast cancer MDA-MB-361 cells. The β-lactamase fusion to the peptides helped in conducting the enzyme activity-based clone normalization and cell-binding screening in a very time- and cost-efficient manner. The methods were suitable for 96-well readout as well as microscopic imaging. The success of the biopanning was indicated by the presence of ∼40% cancer cell-specific clones among recovered phages. One of the binding clones appeared multiple times. The cancer cell-binding fusion peptides also shared several significant motifs. This opens a new way of preparing and selecting phage display libraries. The cancer cell-specific β-lactamase-linked affinity reagents selected from these libraries can be used for any application that requires a reporter for tracking the ligand molecules. Furthermore, these affinity reagents have also a potential for their direct use in the targeted enzyme prodrug therapy of cancer.

High molecular weight kininogen binds to laminin - characterization and kinetic analysis

High molecular weight kininogen (HK) is an abundant plasma protein that plays a central role for the function of the kallikrein/kinin/kininogen system. Thus, cleavage of HK by kallikrein liberates bradykinin, which stimulates vascular repair and a two-chain protein, activated HK (HKa), which induces apoptosis in proliferating endothelial cells. The localization of these events remains obscure, although the basement membrane may be of importance. Analyzing the interaction between HK and HKa and selected basement membrane proteins, we observed that they bound to the major noncollageneous proteins laminin, but not to vitronectin or fibronectin coated on microtiter plates. The binding to laminin was Zn2+ independent. However, at low but not at high concentrations of albumin, Zn2+ increased the affinity for the binding by abolishing an inhibitory effect of Ca2+. Recombinant human kininostatin encompassing the amino acid sequence, Arg439-Ser532 but not the endothelial cell binding peptide sequence (His479-His498; HKH20) within kininostatin inhibited the binding of HKa to laminin. This established that the amino acid sequence Arg439-Lys478 in domain 5 of HK is of importance for its binding to laminin. Extensive proteolytic cleavage of HK and HKa with kallikrein abolished the binding to laminin, releasing a 12 kDa anti-kininostatin reacting peptide. On the basis of these results, we propose that the binding of HK to laminin is a primary event, which secures proper localization of the cleavage products for subsequent interaction with the endothelium to promote inflammatory and pro- and anti-angiogenic activities.

Effects of high metastatic potential hepatocellular carcinoma cell-binding peptide on the invasion and metastasis of liver cancer

To evaluate the effects of specific peptide (AWYPLPP peptide) binding to high metastatic potential human hepatocellular carcinoma (HCC) cells on the invasion and metastasis of liver cancer. The effects of AWYPLPP peptide on the invasion, migration, proliferation and adhesion of high metastatic potential human HCC cell line (HCCLM3) were evaluated in vitro by Matrigel invasion assay, migration assay, MTT assay and adhesion assay. The effect of AWYPLPP peptide on lung metastasis of HCC in vivo was evaluated in male nude mice with subcutaneously implanted HCCLM3 cells. Incubation with the AWYPLPP peptide, but not the control peptide, resulted in a concentration-dependent increase of invasion ability in HCCLM3 cells at the concentration of 0.1 to 100 micromol/L. At any concentration used for the invasion assay, the peptide had no effect on cell migration, proliferation and adhesion. After 30 days of transplantation, eight of nine (88.9%) mice in the AWYPLPP peptide group showed obvious lung metastasis. The metastatic rate of lung metastasis was significantly increased in the AWYPLPP peptide group compared with that in the control group. There was no significant difference among the weights of primary tumor in the PBS, control peptide and AWYPLPP peptide groups. AWYPLPP peptide can promote in vitro invasion and in vivo lung metastasis of high metastatic potential human HCC cells. Identification of the receptor for AWYPLPP peptide binding may provide new insights into the molecular mechanism underlying HCC invasion and metastasis as well as new targets for intervention.

Involvement of osteopontin as a core protein in cholesterol gallstone formation

Matrix proteins are considered to be essential for biomineralization and to be important factors in the formation and growth of gallstones. Osteopontin (Opn) is a noncollagenous, acidic bone-matrix glycoprotein, which is sialated and phosphorylated and which has a cell-binding peptide sequence of glycine-arginine-glycine-aspartate-serine (GRGDS). To investigate the role of Opn in cholesterol gallstone formation, we have studied the involvement of Opn in cholesterol gallstone formation in the human gallbladder wall, in the stones, and in the mouse gallbladder using a gallstone experimental model. Immunohistochemical staining was used in the human gallbladder wall and human gallstones and the determination of mRNA expression by reverse transcriptase-PCR was used in the mouse gallbladder of a gallstone experimental model. The epithelium of stone-laden gallbladders demonstrated high Opn reactivity, as did the core of the stones. Microscopically detected early stones without macroscopic evidence of lithiasis showed the same immunoreactivity as larger stones. Stone-laden gallbladders were infiltrated by macrophages showing intense Opn expression. In gallstone-forming mice, the expression of Opn mRNA and its protein were significantly increased in the gallbladder wall in the early phase of a lithogenic diet intake, before the initiation of inflammation. These results suggest that Opn is possibly involved as a core protein in the formation of cholesterol gallstones.

Estudo clínico da aplicação de matriz inorgânica de osso associado a peptídeo sintético de adesão celular (MIO/P-15), PepGen P-15®, em lesões periodontais avançadas de cães

Os mecanismos biológicos desenvolvidos para aumentar a qualidade da regeneração óssea e da reparação tecidual de sítios periodontais específicos continuam a ser um desafio e têm sido complementado pela capacidade de adesão celular do colágeno do tipo I, promovida por um peptídeo sintético de adesão celular (P-15), associado a uma matriz inorgânica de osso (MIO) para formar MIO/P-15. O objetivo deste estudo foi avaliar a perda do nível clínico de inserção e a resposta da bolsa periodontal em dentes após 3 e 6 meses da aplicação de enxerto com MIO/P-15. Vinte e um cães do Hospital Veterinário da Universidade de São Paulo foram anestesiados para realização de tratamento periodontal e 132 faces dentais com perda de nível clínico de inserção foram tratadas, sendo que 36,4% (48 faces) receberam o peptídeo de adesão celular e 63,6% (84 faces) compuseram o grupo controle que recebeu tratamento convencional (retalho muco-gengival e aplainamento radicular). O procedimento foi documentado através de radiografia intra-oral e todas as sondagens de bolsas periodontais foram fotografadas. Depois de 3 e de 6 meses, os animais foram re-anestesiados a fim de se obter novas avaliações, radiografias, fotografias e sondagens periodontais. As 48 faces com perda de nível clínico de inserção que receberam material de enxertia apresentaram taxa de 40% de recuperação do nível clínico de inserção após 6 meses. O grupo controle de faces dentais não apresentou alteração do nível clínico de inserção. A face palatina foi a que apresentou melhor taxa de regeneração (40%) e os dentes caninos e molares mostraram as melhores respostas (57,14% e 65%, respectivamente). Não houve sinais de infecção pós-cirúrgica relacionadas à falta de higienização oral dos animais. Pode-se concluir que o MIO/P-15 auxilia na regeneração e re-aderência das estruturas periodontais, incluindo osso alveolar. Sua aplicação mostrou-se fácil e prática e a incidência de complicações pós-cirúrgicas foi baixa. Ainda assim, mais estudos e pesquisas são necessários para que se avalie a quantidade e a qualidade do osso e do ligamento periodontal formados.

Targeted Induction of Lung Endothelial Cell Apoptosis Causes Emphysema-like Changes in the Mouse

Pulmonary gas exchange relies on a rich capillary network, which, together with alveolar epithelial type I and II cells, form alveolar septa, the functional units in the lung. Alveolar capillary endothelial cells are critical in maintaining alveolar structure, because disruption of endothelial cell integrity underlies several lung diseases. Here we show that targeted ablation of lung capillary endothelial cells recapitulates the cellular events involved in cigarette smoke-induced emphysema, one of the most prevalent nonneoplastic lung diseases. Based on phage library screening on an immortalized lung endothelial cell line, we identified a lung endothelial cell-binding peptide, which preferentially homes to lung blood vessels. This peptide fused to a proapoptotic motif specifically induced programmed cell death of lung endothelial cells in vitro as well as targeted apoptosis of the lung microcirculation in vivo. As early as 4 days following peptide administration, mice developed air space enlargement associated with enhanced oxidative stress, influx of macrophages, and up-regulation of ceramide. Given that these are all critical elements of the corresponding human emphysema caused by cigarette smoke, these data provide evidence for a central role for the alveolar endothelial cells in the maintenance of lung structure and of endothelial cell apoptosis in the pathogenesis of emphysema-like changes. Thus, our data enable the generation of a convenient mouse model of human emphysema. Finally, combinatorial screenings on immortalized cells followed by in vivo targeting establishes an experimental framework for discovery and validation of additional ligand-directed pharmacodelivery systems.

Calvarial bone regeneration by a combination of natural anorganic bovine‐derived hydroxyapatite matrix coupled with a synthetic cell‐binding peptide (PepGen™): an experimental study in rats

The aim of this study was to evaluate histologically the effect of natural anorganic bovine-derived hydroxyapatite matrix (ABM) coupled with a synthetic cell-binding peptide on the healing of critical size calvarial defects in rats. Sixteen 4-month-old rats were used in the study. A 5 mm trephine defect was created in each parietal bone of every animal. One defect was left untreated (control) while the contralateral defect was treated with a natural ABM coupled with a synthetic cell-binding peptide (test). At 60 and 120 days post-operatively, groups of eight animals were sacrificed and 7-10-microm-thick decalcified sections were produced from both test and control sides. Three sections, 100 mum apart, representing the central area of each defect were selected for the histometric analysis. Histological analysis showed limited bone formation in both control and test defects at both observation periods. The control defects healed with fibrous connective tissue occupying the midportion of the defect and minimal new bone formation at the periphery. In the test defects, the major part of the defect was occupied by graft particles embedded in connective tissue. After 60 days of healing the residual defects accounted up to 94.6% of the original defect dimensions in the control specimens and 90.6% in the test specimens. The differences between test and control defects were not statistically significant (P=0.06). After 120 days of healing, the residual defects accounted up 89.9% of the original defect dimensions in the control specimens and 85% in the test specimens. The difference was not statistically significant (P=0.33). The ABM coupled with a synthetic cell-binding peptide failed to substantially promote new bone formation in rat calvarial defects.

P.174 Conservative-surgical treatment of parodontitis

Augmentation of the craniofacial region is necessary for many aesthetic and reconstructive procedures. Tissue engineering offers a new option to supplement existing treatment regimens. In this procedure, materials composed of hydroxyapatite (HA), of synthetic or natural origin, are used as scaffolds. The aim of this study was to evaluate the effects of three HA materials on cultured human osteoblasts in vitro.Explant cultures of cells from human alveolar bone were established. Human osteoblasts were cultured on the surface of HA calcified from red algae (C GRAFT™/Algipore®), deproteinized bovine HA (Bio-Oss®) and bovine HA carrying the cell binding peptide P-15 (Pep Gen P-15™). Cultured cells were evaluated with respect to cell attachment, proliferation and differentiation. Cells were cultured for 6 and 21 days under osteogenic differentiation conditions, and tissue-culture polystyrene dishes were used as control. The ability of cells to proliferate and form extracellular matrix on these scaffolds was assessed by a DNA quantification assay, protein synthesis analysis and by scanning electron microscopical examination. Osteogenic differentiation was screened by the expression of alkaline phosphatase. The osteoblastic phenotype of the cells was monitored using mRNA levels of the bone-related proteins including osteocalcin, osteopontin and collagen Type I.We found that cells cultured on C GRAFT™/Algipore® and Pep Gen P-15™ showed a continuous increase in DNA content and protein synthesis. Cells cultured on Bio-Oss®showed a decrease in DNA content from Day 6 (P < 0.05) to Day 21 (P < 0.0001) and protein synthesis on Day 21 (P < 0.005). Alkaline phosphatase activity increased in cells grown on C GRAFT™/Algipore® and Pep Gen P-15™ in contrast to cells grown on Bio-Oss®, in which the lowest levels of activity could be observed on Day 21 (P < 0.05). Reverse transcriptase polymerase chain reaction analysis confirmed the osteoblastic phenotype of the cells grown on all three materials throughout the whole culture period.The results of our in vitro study show that the differences in metabolic activity of cells grown on HA materials are directly related to the substrate on which they are grown. They confirm the excellent properties of HA carrying the cell binding peptide P-15 and HA calcified from red algae as used in maxillofacial surgery procedures.

Screening and identification of a novel hepatocellular carcinoma cell binding peptide by using a phage display library

The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develop a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide (WH-16) was synthesized. The competitive ELISA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.

Effects of a Putty‐Form Hydroxyapatite Matrix Combined With the Synthetic Cell‐Binding Peptide P‐15 on Alveolar Ridge Preservation

Various grafting materials have been used for preservation of the dimensions of the residual alveolar ridge following tooth extraction. The purpose of this study was to evaluate clinical, histomorphometric, and radiographic healing 4 months after tooth extraction with or without placement of a putty-form anorganic bovine-derived hydroxyapatite matrix combined with a synthetic cell-binding peptide P-15 (Putty P15) to determine the effect on alveolar ridge preservation following exodontia. Twenty-four consecutive subjects in need of extraction of maxillary premolars were recruited. Recruited subjects were randomly assigned to the test (Putty P15 and bioabsorbable collagen wound dressing material) or control (bioabsorbable collagen wound dressing material only) group. Data were recorded at 1, 2, 4, 8, and 16 weeks after ridge preservation procedures. At 16 weeks, a reentry surgery was performed, clinical measurements were repeated, and bone core biopsies were obtained for histomorphometric analysis prior to dental implant placement. The control group had a mean reduction in ridge height of -0.56 +/- 1.04 mm, whereas alveolar ridge height appeared to remain unchanged in the test group (0.15 +/- 1.76). The test group showed a mean reduction in ridge width of -1.31 +/- 0.96 mm, whereas the mean value for the control group was -1.43 +/- 1.05 mm. No statistical significance was observed between the groups. Mean bone density was significantly superior in the test group (2.08 +/- 0.65 versus 3.33 +/- 0.65). Histomorphometric analyses revealed similar percentages of bone vitality (test: 29.92% +/- 8.46%; control: 36.54% +/- 7.73%). Comparable percentages of bone marrow and fibrous tissue also were observed (test: 65.25% +/- 6.41%; control: 62.67% +/- 7.41%). Only 6.25% of the Putty P15 particles remained at 4 months in the analyzed biopsies. A favorable response was observed when Putty P15 was applied to extraction sockets, suggesting that it may be useful for alveolar ridge preservation prior to dental implant placement.

Characterizing the Mycobacterium tuberculosis Rv2707 protein and determining its sequences which specifically bind to two human cell lines

The Rv2707 gene encoding a putative alanine- and leucine-rich protein was found to be present in all Mycobacterium tuberculosis complex strains (by PCR) and its transcription was shown by RT-PCR in all but M. bovis and M. microti. Antibodies raised against Rv2707 peptides specifically recognized the native protein by Western blot and were able to locate this protein on the M. tuberculosis membrane by immunoelectron microscopy. A549 and U937 cells lines were used in binding assays involving synthetic peptides covering the whole Rv2707 protein. High A549 cell-binding peptide 16083 (281 QEEWPAPATHAHRLGNWLKAY 300) was identified. Peptides 16072 (61 LFGPDTLPAIEKSALSTAHSY 80) and 16084 (301 RIGVGTTTYSSTAQHSAVAA 320) presented high specific binding to both A549 and U937 cells. Cross-linking assays revealed that peptide 16084 specifically bound to a 40-kDa and a 50-kDa U937 cell membrane protein. High activity binding peptides (HABPs) 16083 and 16084 were able to inhibit M. tuberculosis invasion of A549 cells. Our results suggest that these sequences could be part of the binding sites used by the bacillus for interacting with target cells, and thus represent good candidates to be tested in a future subunit-based, multiepitope, antituberculosis vaccine.

Use of Anorganic Bovine‐Derived Hydroxyapatite Matrix/Cell‐Binding Peptide (P‐15) in the Treatment of Class II Furcation Defects: A Clinical and Radiographic Study in Humans

This study compared clinical and radiographic findings for the treatment of Class II furcation defects in human mandibular molars using anorganic bovine-derived hydroxyapatite matrix (ABM)/cell-binding peptide (P-15) or open flap debridement (OFD). Twelve subjects showing two comparable Class II furcation defects in their mandibular molars were enrolled. The defects in each subject were assigned randomly to the test (ABM/P-15) or the control (OFD) group. Clinical measurements and standardized radiographs were taken at baseline and 6 to 7 months after surgery. There were no statistically significant differences between the test and control groups for any clinical or radiographic parameter (P >0.05). On comparing the baseline and final measurements, the gain in horizontal clinical attachment level and reduction in gingival recession were significant only in the test group (P < or =0.02), whereas the gain in the vertical clinical attachment level was significant in both groups (P < or =0.04). In the test group, four of 12 sites showed complete closure, and five showed partial closure; in the control group, three defects showed complete closure, and four showed partial closure (P = 0.42). Subtraction radiography revealed similar gains in bone height and increases in mean bone density with both treatments (P >0.05). ABM/P-15 yielded favorable results in the treatment of Class II furcation defects over a 6-month evaluation period; however, there was no difference compared to OFD. Further studies using a larger sample size are needed to confirm the present findings.

Silk Fibroin Microfluidic Devices.

Silk Fibroin Microfluidic Devices.

Cytophobic Surface Modification of Microfluidic Arrays for In Situ Parallel Peptide Synthesis and Cell Adhesion Assays

A combination of PEG-based surface passivation techniques and spatially addressable SPPS (solid-phase peptide synthesis) was used to demonstrate a highly specific cell-peptide adhesion assay on a microfluidic platform. The surface of a silicon-glass microchip was modified to form a mixed self-assembled monolayer that presented PEG moieties interspersed with reactive amino terminals. The PEG provided biomolecular inertness and the reactive amino groups were used for consequent peptide synthesis. The cytophobicity of the surface was characterized by on-chip fluorescent binding assays and was found to be resistant to nonspecific attachment of cells and proteins. An integrated system for parallel peptide synthesis on this reactive amino surface was developed using photogenerated acid chemistry and digital microlithography. A constant synthesis efficiency of >98% was observed for up to 7mer peptides. To demonstrate specific cell adhesion on these synthetic peptide arrays, variations of a 7mer cell binding peptide that binds to murine B lymphoma cells were synthesized. Sequence-specific binding was observed on incubation with fluorescently labeled, intact murine B lymphoma cells, and key residues for binding were identified by deletional analysis.

Molecular Changes in the Vasculature of Injured Tissues

We have explored molecular specialization of the vasculature of regenerating wound tissue in the skin and tendons to identify a different repertoire of markers from that obtained by studying tumor vasculature. We screened a phage-displayed peptide library for peptides that home to wounds in mice and identified two peptides that selectively target phage to skin and tendon wounds: CARSKNKDC (CAR) and CRKDKC (CRK). CAR is homologous to heparin-binding sites in various proteins and binds to cell surface heparan sulfate and heparin. CRK is similar to a segment in thrombospondin type 1 repeat. Intravenously injected CAR and CRK phage, as well as fluorescein-labeled CAR and CRK peptides, selectively accumulated at wound sites, where they partially co-localized with blood vessels. The CAR peptide showed a preference for early stages of wound healing, whereas the CRK favored wounds at later stages of healing. The CAR peptide was internalized into the target cells and delivered the fluorescent label into the cell nuclei. These results identify new molecular markers in wound tissues and show that the expression of these markers in wound vasculature changes as healing progresses. The peptides recognizing these markers may be useful in delivering treatments into regenerating tissues.

Identification and Evaluation of a New Tumor Cell-Binding Peptide, FROP-1

Peptides are useful tools for the targeted delivery of radionuclides or chemotherapeutic drugs to their site of action within an organism. Given that the peptide receptor is overexpressed at the tumor, therapeutically active doses can be delivered to the tumor with reduced side effects. Because currently known peptides are restricted to a small number of tumors, new molecules and their corresponding receptors have to be identified to enlarge the spectrum of malignancies that can be diagnosed or treated using tumor-targeting peptides. A 12-amino-acid peptide phage display system was applied to identify a new peptide binding to follicular thyroid carcinoma cells. The properties of the radiolabeled peptide were assessed in binding, competition, and internalization experiments in a variety of tumor cell lines including FRO82-2 and MCF-7 cells, and the pharmacokinetic behavior of the radiolabeled peptide was evaluated in tumor-bearing mice. Peptide stability was studied in human serum. After 5 selection rounds, the new peptide, FROP-1 (EDYELMDLLAYL), was identified. It showed binding to follicular thyroid carcinoma as well as anaplastic thyroid carcinoma, mammary carcinoma, cervix carcinoma, prostate carcinoma, and cell lines derived from head and neck tumors, and low affinity could be observed to control cells such as human umbilical vein endothelial cells or immortalized keratinocytes. In MCF7 cells, 78% and 86% of the bound activity was internalized after 10 and 60 min of incubation, respectively. Stability experiments in human serum showed the appearance of a degradation product after 15 min. Tumor uptake of the radioactive labeled peptide increased for 45 min in nude mouse models, reaching an accumulation level of approximately 3.6 percentage injected dose (%ID)/g for FRO82-2 tumors or approximately 3.8 %ID/g for MCF-7 tumors. The target of FROP-1 is most likely a molecule found generally in tumors, making this peptide highly attractive for diagnostic or therapeutic applications. However, modifications are needed to increase stability and affinity.

A mineralization‐associated membrane protein plays a role in the biological functions of the peptide‐coated bovine hydroxyapatite

Anorganic bovine mineral coated with a cell-binding peptide (P-15) is superior to anorganic bovine mineral alone in the treatment of periodontal osseous defects. However, the molecular interactions between P-15 and periodontal ligament fibroblasts remain unclear. We first compared the in vitro osteogenic activities between periodontal ligament fibroblasts cultured with anorganic bovine mineral alone and with the P-15/anorganic bovine mineral combination. We then harvested the periodontal ligament cell lysate, incubated it with various graft materials, and then washed it to remove unbound proteins. The bound proteins were eluted from graft materials and analyzed using electrophoresis, followed by mass spectrometry and then western blotting. Finally, a neutralizing antibody against one bound protein was added to the cell cultures to repeat the osteogenic assays to clarify its role in the in vitro effects of the P-15/anorganic bovine mineral combination. Cells treated with P-15/anorganic bovine mineral were more viable and showed greater osteogenic activities than cells treated with anorganic bovine mineral alone and the no-graft control. Annexin II, a mineralization-associated protein, bound to P-15/anorganic bovine mineral significantly more than to anorganic bovine mineral alone. The addition of neutralizing antibody for annexin II decreased the osteogenic activities of the P-15/anorganic bovine mineral combination. Annexin II of periodontal ligament fibroblasts interacted with the peptide of P-15, and was partially responsible for better in vitro osteogenesis in the P-15 graft.

The Trp Cage Motif as a Scaffold for the Display of a Randomized Peptide Library on Bacteriophage T7

Phage libraries displaying linear or disulfide-constrained peptides often yield weak binders, upon screening against a target, and must be optimized to improve affinity. The disadvantages of libraries based on larger complex proteins, such as single chain antibodies, have stimulated interest in the development of smaller nonimmunoglobulin protein scaffolds. A promising candidate is the Trp cage motif, a 20-residue C-terminal sequence of exendin-4. Amino acid substitution within the Trp cage resulted in a 20-mer peptide recognized as an ultrafast cooperative folding miniprotein, with ideal characteristics for the discovery of small structured nonimmunoglobulin motifs having a stable tertiary structure. Although we were unable to display the Trp cage on M13 phage, successful display was achieved using the lytic T7 phage. Interestingly, mutations were observed at a frequency dependent on display valency. A Trp cage library designed with randomized amino acids at seven solvent-exposed positions was developed from 1.6 x 10(9) primary clones in T7Select10-3b. DNA sequencing of 109 library clones revealed 38% mutants and 16% truncations by TAG codons at randomized positions. Amino acid frequencies were largely within expected bounds and DIVAA analysis revealed that the library had an average diversity of 0.67. Utility of the library was demonstrated by identification of HPQ containing Trp cage miniproteins, which bound streptavidin, and AAADPYAQWLQSMGPHSGRPPPR, which bound to human bronchial epithelial cells. A high complexity library based on the Trp cage miniprotein has demonstrated potential for identifying novel cell and protein binding peptides that could be used for the delivery of therapeutic molecules or as target-specific therapeutic agents.