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Mol Cancer Ther. 2004;3:1329-1334
© 2004 American Association for Cancer Research

Novel peptide ligands for integrin {alpha}4ß1 overexpressed in cancer cells

Masahito Mikawa, Henry Wang, Linlang Guo, Ruiwu Liu, Jan Marik, Yoshikazu Takada, Kit Lam and Derick Lau

University of California at Davis Cancer Center, Sacramento, California

Requests for reprints: Derick Lau, Division of Hematology/Oncology, University of California at Davis Cancer Center, 4501 X Street, Sacramento, CA 95817. Phone: 916-734-3772; Fax: 916-734-7946. E-mail: derick.lau{at}ucdmc.ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using the "one-bead one-peptide" combinatorial technology, a library of random cyclic octapeptides and nonapeptides, consisting of natural and unnatural amino acids, was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment and proliferation of bronchioloalveolar carcinoma cells (H1650), employing a "cell growth on bead" assay. Consensus peptide sequences of cNleDXXXXc and cXNleDXXXXc (where Nle is norleucine) were identified. With alanine scanning and site-directed deletion, a typical ligand consisted of a motif of -NleDI/V/Nle- with two flanking cysteines. These peptide ligands were specific for promoting cell attachment of the H1650 cells and the cells of lymphoid cancers (Jurkat and Raji) but not other selected human cell lines of lung cancer and fibroblast. In an antibody blocking assay, integrin {alpha}4ß1, which was overexpressed in H1650, Jurkat, and Raji, was identified as a putative receptor for these peptide ligands. Using Chinese hamster ovary cells transfected with either wild-type or mutant integrin {alpha}4, a critical binding site for these peptides was localized to the glycine residue at position 190 of integrin {alpha}4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is one of the most common cancers and is the leading cause of cancer death in the United States (1). The outlook for patients with lung cancer is poor, because they commonly present with advanced disease and their median survival is ~10 months even with chemotherapy (2, 3). Molecularly targeted therapy has become a reality in the treatment of several cancers (4). Rituximab, a monoclonal antibody targeting the cell surface antigen CD20, is being used to treat B-cell lymphoma (5). Trastuzumab, a monoclonal antibody targeting the cell surface receptor of HER-2/neu (erbB-2), has been used for treatment of breast cancer (6). More recently, an antibody against the epidermal growth factor receptor, cetuximab, has been approved for treatment of refractory colon cancer (7). We reason that unique surface receptor(s) also exist for lung cancer cells and that peptide ligands specific for these cell surface receptors could be identified. Our hope is to identify these peptide ligands and to develop them as diagnostic and therapeutic agents for this devastating disease (8).

Recently, using the technology of combinatorial chemistry and a "cell growth on bead" assay, we have identified a novel cyclic peptide, D-cysteine-L-asparagine-L-glycine-L-arginine-L-glycine-L-glutamate-L-glutamine-D-cysteine (cNGRGEQc), specific for promoting attachment and growth of cells of non-small cell lung cancer on polyethylene glycol–linked polystyrene beads (9). Furthermore, we have identified integrin {alpha}3ß1 as a putative cell surface receptor for this peptide ligand. While screening for peptide ligands specific for lung cancer cells, we have also identified a group of novel cyclic peptides specific for integrin {alpha}4 overexpressed in a cell line of bronchioloalveolar carcinoma, a histologic subtype of lung cancer. We are reporting the identification and characterization of these novel peptide ligands for promoting adhesion of cancer cells, which overexpressed integrin {alpha}4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
Human cell lines including H1650 (bronchioloalveolar carcinoma), A549 (lung adenocarcinoma), Calu-1 (squamous lung carcinoma), DMS-114 (small cell lung carcinoma), Jurkat (T-cell leukemia), Raji (Burkitt lymphoma), and Hs68 (newborn foreskin fibroblast) were obtained from American Tissue Culture Collection (Manassas, VA). The Chinese hamster ovary (CHO) lines have been described by Irie et al. and were obtained from Dr. Y. Takada (University of California at Davis, Sacramento, CA). The lung cancer and fibroblast cell lines were maintained in DMEM, the CHO cells in DMEM in sodium pyruvate (110 mg/mL) and G418 (100 µg/mL), and the Jurkat and Raji cells in RPMI 1640. All the culture media were supplemented with fetal bovine serum (10%), penicillin (100 units/mL), and streptomycin (100 µg/mL; Life Technologies, Grand Island, NY).

Random Peptide Bead Library
An "one-bead one-peptide" combinatorial library, containing TentaGel S-NH2 polystyrene beads (Rapp Polymere GmbH, Tubingen, Germany) grafted with amino-polyethylene glycol and conjugated with random peptides of a general structure of NH2-c(X)nc-COOH-, was synthesized using Fmoc chemistry and a split-synthesis approach as described previously by Lam et al. (10, 11). For this random library, n represents six to seven amino acids in length; c denotes D-cysteine at the NH2 and COOH termini providing intramolecular cyclization by disulfide bonding; and X denotes 1 of 17 natural L-amino acids, excluding L-cysteine, L-arginine, and L-lysine, and 7 unnatural amino acids of L-hydroxyproline (Hyp), L-{alpha}-aminoisobutyric acid (Aib), L-norleucine (Nle), L-phenylglycine, L-norvaline, L-cyclohexylglycine, and L-4-benzoylphenylalanine. (Note: L-cysteine was excluded to avoid intramolecular cyclization, and the positive charged amino acids, L-arginine and L-lysine, were excluded to minimize nonspecific cell attachment.) Therefore, for a random combinatorial library of c(X)6-7c synthesized from 24 amino acids, there were, theoretically, 247 = 4.8 x 109 permutations of peptide sequences.

"Cell Growth on Bead" Assay
A "cell growth on bead" assay was used as described previously for screening the random library for peptides essential for attachment and growth of the H1650 cells on the TentaGel beads (9). Cultured cells were harvested with trypsin/EDTA, washed, and resuspended as single cells in culture medium. Typically, ~150,000 beads with random peptides were mixed with ~1 million suspended cells in 15 mL of medium distributed in six 3-cm culture dishes. The dishes were incubated in a tissue culture incubator with 5% CO2 at 37°C for 48 to 72 hours. Beads covered with a monolayer of cells were removed and attached cells were stripped off with 8 mol/L guanidine hydrochloride. The amino acid sequence of each isolated peptide bead was determined with an automated Procise 494 protein sequencer (Applied Biosystems, Foster City, CA) using a method as described previously by Liu and Lam (12). Consensus peptide sequences identified were synthesized on beads in large quantity and cell attachment to these beads was confirmed with H1650. The structures of these peptides were modified to yield a model ligand peptide, D-cysteine-L-Nle-L-aspartate-L-Nle-L-threonine-L-Hyp-D-arginine-D-cysteine (cNleDNleTHyprc or pM2), with high capacity and specificity for promoting attachment of the H1650 cells and with resistance to proteolysis. The pM2 beads were subsequently used to test for cell type specificity and structure-activity relationship.

Alanine Scanning and Site-Directed Deletion Analysis
To determine the structure-activity requirements of the consensus peptide ligand, the strategies of alanine scanning (13) and site-directed deletion were employed. The model ligand peptide, pM2, was synthesized with Fmoc chemistry (11) on TentaGel beads with each amino acid (except the cysteine) sequentially substituted with L-alanine. In addition, truncated pM2 peptides, with amino acids deleted sequentially from the COOH terminus, were synthesized on beads. Percentage of the peptide beads with cell attachment was evaluated with the H1650 cells using the "cell growth on bead" assay.

Antibody Blocking Assay
To determine whether a particular integrin was a putative receptor for pM2, a panel of antibodies against {alpha} and ß integrins, including {alpha}1 to {alpha}6 and {alpha}v and ß1 to ß5 (Chemicon, Temecula, CA), was tested to block cell attachment to the pM2 beads. Suspended H1650 cells were incubated with each antibody at 37°C with agitation for 30 minutes. Peptide beads were then added to the culture, which was incubated at 37°C for 72 hours in a tissue culture incubator. The number of beads with cell attachment was counted under a microscope and relative percentage of beads with cell attachment was calculated as follows: B / A x 100, where A is percentage of beads with cell attachment in medium without antibody and B is percentage of beads with cell attachment in medium with antibody.

Inhibition Assay with Soluble Peptides
To determine if pM2 was biologically active from the beads, free pM2 was synthesized and tested for competitive inhibition of attachment of H1650 to the model pM2 beads. The peptides of pM2 and its analogue of cNleANleTHyprc (pM2-A), the latter containing A instead of D at position 3 to serve as a negative control, were synthesized on rink amide-MBHA resins (GL Biochem Ltd., Shanghai, China) with Fmoc chemistry as described above. The peptides were subsequently cleaved from the resins and the free peptides were purified as described previously (14). The free pM2 and pM2-A were readily soluble in an aqueous solution. Inhibition of the soluble peptides on attachment of H1650 to the pM2 and pM2-A beads was done as described for the antibody blocking assay.

Flow Cytometry for Detection of {alpha}4 and ß1 Integrins
Flow cytometry was used to compare the levels of expression of {alpha}4 and ß1 integrins among the selected cell lines as described previously (15). Cells were incubated with a mouse anti-human {alpha}4 (Serotec, Raleigh, NC) or ß1 antibody (Chemicon), 2 µg/mL, for 30 minutes on ice. After washing, the cells were incubated with a FITC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes. Cells were analyzed for level of fluorescence using a Coulter EPICS XL flow cytometer (Beckman Coulter, Miami, FL).

Adhesion to pM2 Beads of CHO Cells with Wild-type or Mutant Integrin {alpha}4
To determine the potential binding site of {alpha}4 integrin for pM2 in initiating cell attachment, the model of CHO cells with transfected wild-type (CHO-{alpha}4) or mutant human integrin {alpha}4 was employed. This model was established by Irie et al. by transfecting CHO, a cell line devoid of endogenous integrin {alpha}4 expression, with cDNA of human wild-type integrin {alpha}4 or integrin {alpha}4 substituted with alanine sequentially (alanine scanning mutagenesis) from residues 108 to 268. These residues are believed to contain the putative binding sites for vascular cell adhesion molecule-1 and fibronectin (16, 17). The CHO cell lines harboring the mutant integrin {alpha}4 at residues 176 to 203 were tested in this study. The levels of expression of integrin {alpha}4 in the CHO cells and CHO-{alpha}4 cells were verified with flow cytometry as described above. Cell adhesion to the pM2 beads of these CHO cells was studied using the "cell growth on bead" assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Peptide Ligands for H1650
Using the "cell growth on bead" assay to screen a "one-bead one-peptide" random library of cyclic octapeptides and nonapeptides for attachment of H1650 cells, 72 beads with cell attachment were isolated from screening ~4.7 million of peptide beads. The peptide sequences of these beads were determined and 12 beads with the most common consensus sequences of amino acids were identified: cNleDIDLLc, cNleDITPLc, cGNleDINleHypAibc, cFNleDIDAHc, cNleDINleEFGc, cFNleDIYGSc, cFNleDINeGTc, cNleDNleYEAibc, cNleDNleTHypGc, cNleDNleHPHypMc, cNleDVVDTSc, and cNleDVDNleHGc. These consensus peptide beads were synthesized in large quantities and their capacity of promoting cell attachment and growth was confirmed with the H1650 cells. These sequences contained a motif of -NleDX-, where D is aspartic acid and X is isoleucine, Nle, or valine. Attachment of one to three cells to the peptide beads was observable within 6 hours (Fig. 1). By transferring beads with attached cells to a fresh culture plate, cell proliferation on the beads was observed in 24 hours and confluency of cells on beads was commonly observed in 96 to 120 hours (Fig. 2).



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  		Figure 1. Attachment of H1650 cells to TentaGel beads conjugated with the pM2 peptide, cNleDNleTHyprc, after 6 hours of incubation. Arrow, attachment of single cell to a bead.

 


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  		Figure 2. Peptide beads isolated from Fig. 1 and incubated for 96 hours. Note that the beads are covered with cells, indicating cell proliferation on beads.

 
To obtain a peptide homologous with the peptide ligands identified in above but with enhanced capacity of attracting cell attachment and with resistance to proteolytic digestion, the peptide sequence of D-cysteine-L-Nle-L-aspartate-L-Nle-L-threonine-L-Hyp-glycine-D-cysteine (cNleDNleTHypGc or pM1) was modified with the glycine replaced with D-arginine to yield the peptide of cNleDNleTHyp{gamma}c or pM2. The modified peptide, pM2, was resistant to proteolytic digestion in human plasma. In addition, pM2 showed higher capacity of promoting cell attachment than pM1. Cell type specificity of attachment to pM2 beads was tested with a panel of human cell lines. Cell attachment was consistently observed with H1650, Jurkat (T-cell leukemia), and Raji (Burkitt lymphoma) but not with A549 (lung adenocarcinoma), Calu-1 (squamous lung carcinoma), DMS-114 (small cell lung carcinoma), and Hs68 (fibroblast; Table 1). It was hypothesized that these findings were due to overexpressions of {alpha}4 and ß1 integrins in H1650 cells as compared with that of other lung cancers. To prove this hypothesis, flow cytometry was used to compare the levels of expression of {alpha}4 and ß1 integrins among the selected cell lines. Cell attachment to the pM2 beads correlated with the level of expression of integrin {alpha}4 (Table 1). Integrin {alpha}4 was expressed at a relatively high level with the H1650, Jurkat, and Raji cells but at a low level with A549, Calu-1, and DMS-114 cells. Relative high levels of integrin ß1 were detected in all these cell lines (data not shown).


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  		Table 1. Cell type–specific attachment to pM2 beads and relative level of integrin {alpha}4 expression of human cell lines

 
Structure-Activity Relationship Studies
By alanine scanning, the amino acids of L-Nle at position 2 and L-aspartate (D) at position 3 of pM2 were absolutely required for promoting cell attachment of H1650 to beads (Table 2). Substitution of amino acids at positions 4 to 6 with L-alanine essentially did not affect cell attachment. By site-directed deletion, the extent of attachment of the H1650 cells to the native and modified pM2 beads is shown in Table 3. The extent of cell attachment decreased progressively as amino acids were deleted sequentially from the COOH terminus. Furthermore, deletion of D-cysteine from the NH2 terminus almost abolished cell attachment to most beads. The peptide of cNleDNlec maintained ~50% of biological activity. However, peptide of cNleDc lost almost all of its activity. Thus, the structure-activity requirements for pM2 are summarized as follows: (a) The motif for cell attachment is NleDNle. (b) The cyclic structure provided by the two D-cysteines is essential for biological activity. (c) The minimal length of the peptide for promoting cell attachment is three amino acids with two flanking cysteines.


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  		Table 2. Cell attachment of H1650 to TentaGel beads conjugated with the peptide pM2 and its analogues modified with alanine scanning (mean ± SD, n = 3)

 

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  		Table 3. Cell attachment of H1650 to TentaGel beads conjugated with the peptide pM2 and its analogues modified by sequential deletion of amino acids from the COOH terminus (mean ± SD, n = 3)

 
Identification of Putative Receptor for Peptide Ligand
We hypothesized that integrin was the receptor for pM2 for promoting cell attachment. A panel of antibodies to {alpha} and ß integrin subunits, including {alpha}1 to {alpha}6, {alpha}v, and ß1 to ß5, were tested to block attachment of H1650 cells to the pM2 beads. At a concentration of 1 µg/mL, antibody to {alpha}4 totally blocked cell attachment, and antibody to ß1 only partially blocked cell attachment to the peptide beads. The other antibodies did not affect cell attachment at all. The combination of antibodies to {alpha}4 and ß1 subunits was additive in blocking cell attachment. The antibody to {alpha}4 blocked cell attachment in a dose-dependent manner as shown in Fig. 3. The concentration of {alpha}4 antibody that inhibited attachment of cells to 50% of the peptide beads (IC50) was 2.2 ng/mL. The IC50 for the antibody to ß1 could not be determined because this antibody blocked cell attachment only partially even at a concentration up to 1 µg/mL. These data indicated that integrin {alpha}4ß1 was likely a receptor for the pM2 peptide ligand.



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  		Figure 3. Dose-response curve of inhibition of cell attachment to pM2 beads by an antibody to integrin {alpha}4 (mean ± SD, n = 4).

 
Biological Activity of Soluble pM2 Peptide
The biological activity of soluble pM2 and pM2-A peptides was tested by competitive inhibition of attachment of H1650 cells to the pM2 beads. The pM2-A peptide at concentration as high as 1 mmol/L did not inhibit cell attachment at all. The soluble pM2 inhibited cell attachment to the pM2 beads in a concentration-dependent manner as shown in Fig. 4. The concentration of soluble pM2 that inhibited attachment of H1650 cells to 50% of the pM2 beads (IC50) was 10 µmol/L.



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  		Figure 4. Dose-response curve of inhibition of cell attachment to pM2 beads by the soluble pM2 and pM2-A peptides (mean ± SD, n = 4).

 
Identification of Binding Site of Integrin {alpha}4 for pM2
The profile of cell attachment to the pM2 beads for CHO, CHO-{alpha}4, and the mutant CHO-{alpha}4 created by alanine scanning mutagenesis is shown in Fig. 5. Essentially no attachment to pM2 beads was observed with the parental CHO cells. On the other hand, cell attachment to all the pM2 beads was observed with the CHO-{alpha}4. For the CHO-{alpha}4 mutants, cell attachment was observed on 80% to 100% of the pM2 beads. The only exception is the G190A mutant, in which the glycine was replaced with alanine at residue 190 of integrin {alpha}4. These findings indicated that this glycine residue was critical for binding of pM2 to integrin {alpha}4 for promoting cell attachment.



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  		Figure 5. Profile of cell attachment to pM2 beads of parental CHO, CHO-{alpha}4, and CHO-{alpha}4 mutants created by alanine scanning mutagenesis at residues 176 to 203 of integrin {alpha}4 (mean ± SD, n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Peptide ligands for various cell surface receptors have been identified using phage libraries. The best known peptide identified by this technique is the tripeptide of arginine-glycine-aspartate, which is a ligand for {alpha}vß3 and {alpha}5ß1 integrins (18, 19). The sequence of arginine-glycine-aspartate has been identified as an essential site of several natural extracellular matrix, including vitronectin, laminin, fibrinogen, and fibronectin, for binding to their receptors (20–23). Whereas the technique of phage display provides the advantage of identification of ligands for cellular receptors in vivo, the "one-bead one-peptide" method, on the other hand, provides the ease of in vitro study and allows the use of random peptides containing unnatural amino acids (8). In this study, we have identified peptide ligands containing D- and synthetic unnatural amino acids. These peptides are resistant to proteolytic enzymes, which very likely would enhance biological stability in vivo. The inclusion of these unnatural amino acids also exponentially increases the number of permutations of random peptides. In our study, the library of random cyclic octapeptides and nonapeptides contained 246 + 247 = (1.91 x 108 + 4.59 x 109) ~= 4.8 x 109 possible permutations of peptide sequences using 24 different amino acids. Of 4.7 million peptide beads screened in this study, which represented only a small fraction of possible permutations of the library, we isolated 72 beads with cell attachment. On sequencing, 12 peptides with consensus motifs of -NleDI-, -NleDNle-, and -NleDV- were identified. Using the techniques of alanine walk and sequential deletion, the motif of -NleDI/V/Nle- was confirmed. The glycine residue of position 190 of integrin {alpha}4 has been identified as a potential binding site for the pM2 model peptide. It is likely that additional motifs could have been identified if more random peptide beads had been screened.

Cell type–specific peptide ligands for lung cancer cells have been identified using the "one-bead one-peptide" combinatorial library and the "cell growth on bead" assay established in our laboratory. Previously, we reported that a cyclic peptide, cNGRGEQc, was identified for promoting adhesion of non-small cell lung cancer cells overexpressing integrin {alpha}3ß1 (9). In this study, we have additionally identified a series of cyclic octapeptides and nonapeptides with a general structure of cNleDXXXXc or cXNleDXXXXc in a bronchioloalveolar carcinoma cell line, H1650, overexpressing {alpha}4ß1. Because there is a lack of availability of appropriate bronchioloalveolar carcinoma cell lines, we were unable to verify whether these peptides were specific for bronchioloalveolar carcinoma or {alpha}4ß1 expression was unique in this cell type of lung cancer. In an immunohistochemical study by Koukoulis et al. (24) of seven cases of bronchioloalveolar carcinoma lung cancer, no expression of {alpha}4 was observed, but strong expression of integrin ß1 was detected. Additional studies with a larger sample size are necessary to confirm these observations.

Although it is uncertain if the pM2 peptide is specific for bronchioloalveolar carcinoma cells in general, it is obvious that this peptide is specific for cancer cells overexpressing integrin {alpha}4. This notion is supported by the fact that the pM2 peptide specifically promoted attachment of integrin {alpha}4 expressing cells of H1650 (bronchioloalveolar carcinoma), Jurkat (T-cell leukemia), and Raji (Burkitt lymphoma). Recently, a series of peptides containing the motifs of LDI/V/F has also been identified for {alpha}4ß1 integrin of Jurkat cells on screening "one-bead one-peptide" libraries (25). The pM2 analogues identified in the current study seem to have pharmacokinetic advantages because they contain D- and unnatural amino acids and are more resistant to proteolytic digestion in human plasma. Thus, these peptides may be more ideal for development into diagnostic and therapeutic agents.

In this and previous studies, we have identified cyclic peptides specific for integrins {alpha}3ß1 and {alpha}4ß1. These peptides are cell type specific depending on the expression of a particular type of integrin. In the clinical setting, bronchioloalveolar carcinoma, adenocarcinoma, and squamous carcinoma have been classified under the general category of non-small cell lung cancer and they, traditionally, have been treated in a similar manner. However, it is becoming clear that not all non-small cell lung cancers are alike and that molecular signature targets may provide a more accurate guidance for classification and specific therapy of lung cancers. Cell type–specific expression of integrins such as {alpha}3 or {alpha}4 seems to be viable diagnostic and therapeutic targets. In a recent clinical trial, a response rate of only 10% was observed for non-small cell lung cancer treated with gefitinib (Iressa), an inhibitor of the tyrosine kinase domain of epidermal growth factor receptor (26). It has been found recently that the response to gefitinib was observed mostly in lung cancer harboring mutations of in-frame deletion or substitutions clustered around the ATP binding pocket of the tyrosine kinase binding domain of epidermal growth factor receptor (27). Therefore, gene profiling has been advocated as a more accurate means than the classic histologic observation in classifying and guiding treatment for lung cancer (28, 29).

Preclinical studies have shown the promise and feasibility of cancer therapy with antibodies and radiolabeled arginine-glycine-aspartate peptide targeting integrins (30, 31). In addition, liposome-encapsulated doxorubicin loaded with the Fab' fragment of an antibody to ß1 integrin has been used to target human lung cancer xenografts (32, 33). To study if the peptides with a general structure of cNleDXXXXc indeed are of diagnostic and therapeutic value, we have shown that soluble pM2, but not the control peptide pM2-A, is capable of inhibiting attachment of H1650 to the pM2 beads. Studies are ongoing to further define the biological activity of soluble pM2 in vitro and in vivo.


   Footnotes
 
Grant support: Pamela Pond Scholarship and Joan's Legacy Foundation grant (http://www.joanslegacy.org).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/16/03; revised 8/11/04; accepted 8/27/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5–26.[Abstract/Free Full Text]
  2. Kelly K, Crowley J, Bunn P Jr, et al. Randomized phase III trial of paclitaxel plus carboplatin versus vinorelbine plus cisplatin in the treatment of patients with advanced non-small-cell lung cancer: a Southwest Oncology Group trial. J Clin Oncol 2001;19:3210–8.[Abstract/Free Full Text]
  3. . Schiller JH, Harrington D, Belani CP, et al. Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer. N Engl J Med 2002;346:92–8.[Abstract/Free Full Text]
  4. Goldman JM, Melo JV. Targeting the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1084–6.[Free Full Text]
  5. Davis TA, Grillo-Lopez AJ, White CA, et al. Rituximab anti-CD20 monoclonal antibody therapy in non-Hodgkin's lymphoma: safety and efficacy of re-treatment. J Clin Oncol 2000;18:3135–43.[Abstract/Free Full Text]
  6. Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001;344:783–92.[Abstract/Free Full Text]
  7. Ellis L, Hoff PM. Targeting the epidermal growth factor receptor: an important incremental step in the battle against colorectal cancer. J Clin Oncol 2004;22:1177–9.[Free Full Text]
  8. Aina OH, Sroka TC, Chen ML, Lam KS. Therapeutic cancer targeting peptides. Biopolymers 2002;66:184–99.[CrossRef][Medline]
  9. Lau DH, Guo L, Liu R, Song A, Shao C, Lam KS. Identifying peptide ligands for cell surface receptors using cell-growth-on-bead assay and one-bead one-compound combinatorial library. Biotechnol Lett 2002;24:497–500.[CrossRef]
  10. Lam KS, Salmon SE, Hers EM, Hruby VJ, Kazmierski WM, Knapp RJ. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 1991;354:82–4.[CrossRef][Medline]
  11. Lam KS, Lebl M, Krchnak V. The "one-bead-one-compound" combinatorial library method. Chem Rev 1997;97:411–48.[CrossRef][Medline]
  12. Liu R, Lam KS. Automatic Edman microsequencing of peptides containing multiple unnatural amino acids. Anal Biochem 2001;295:9–16.[CrossRef][Medline]
  13. Cunningham BC, Wells JA. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 1989;244:1081–5.[Abstract/Free Full Text]
  14. Lam KS, Lehman AL, Song A, et al. Synthesis and screening of "one-bead one-compound" combinatorial peptide libraries. Methods Enzymol 2003;369:298–322.[Medline]
  15. Cardarelli PM, Yamagata S, Taguchi I, Gorcsan F, Chiang SL, Lobl T. The collagen receptor {alpha}2ß1, from MG-63 and HT1080 cells, interacts with a cyclic RGD peptide. J Biol Chem 1992;267:23159–64.[Abstract/Free Full Text]
  16. Irie A, Kamata T, Puzon-McLaughlin W, Takada Y. Critical amino acid residues for ligand binding are clustered in a predicted ß-turn of the third N-terminal repeat in the integrin {alpha}4 and {alpha}5 subunits. EMBO J 1995;14:5550–6.[Medline]
  17. Irie A, Kamata T, Takada Y. Multiple loop structures critical for ligand binding of the integrin {alpha}4 subunit in the upper face of the ß-propeller mode 1. Proc Natl Acad Sci 1997;94:7198–203.[Abstract/Free Full Text]
  18. Koivunen E, Wang B, Ruoslahti E. Phage libraries displaying cyclic peptides with different ring sizes: ligand specificities of the RGD-directed integrins. Biotechnology 1995;13:265–70.[CrossRef][Medline]
  19. Koivunen E, Gay DA, Ruoslahti E. Selection of peptides binding to the {alpha}5ß1 integrin from phage display library. J Biol Chem 1993;268:20205–10.[Abstract/Free Full Text]
  20. Ruoslahti E, Hayman EG, Pierschbacher MD. Extracellular matrices and cell adhesion. Arteriosclerosis 1985;5:581–94.[Free Full Text]
  21. Ruoslahti E. RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 1996;12:697–715.[CrossRef][Medline]
  22. Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 1984;309:30–3.[CrossRef][Medline]
  23. Hayman EG, Pierschbacher MD, Ruoslahti E. Detachment of cells from culture substrate by soluble fibronectin peptides. J Cell Biol 1985;100:1948–54.[Abstract/Free Full Text]
  24. Koukoulis GK, Warren WH, Virtanen I, Gould VE. Immunolocalization of integrins in the normal lung and in pulmonary carcinomas. Hum Pathol 1997;28:1018–25.[CrossRef][Medline]
  25. Park S, Renil M, Vikstrom B, et al. The use of one-bead one-compound combinatorial library method to identify peptide ligands for {alpha}4ß1 integrin receptor in non-Hodgkin's lymphoma. Lett Peptide Sci 2002;8:171–8.
  26. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 2003;290:2149–58.[Abstract/Free Full Text]
  27. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.[Abstract/Free Full Text]
  28. Garber ME, Troyanskaya OG, Schluens K, Petersen S, Thaesler Z, Pacyna-Gengelbach M. Diversity of gene expression in adenocarcinoma of the lung. Proc Natl Acad Sci 2001;98:13784–9.[Abstract/Free Full Text]
  29. Gordon GJ, Jensen RV, Hsiao LL, et al. Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma. Cancer Res 2002;62:4963–7.[Abstract/Free Full Text]
  30. Janssen ML, Oyen WJ, Dijkgraaf I, et al. Tumor targeting with radiolabeled {alpha}(v)ß(3) integrin binding peptides in a nude mouse model. Cancer Res 2002;62:6146–51.[Abstract/Free Full Text]
  31. van Hagen PM, Breeman WA, Bernard HF, et al. Evaluation of a radiolabeled cyclic DTPA-RGD analogue for tumor imaging and radionuclide therapy. Int J Cancer 2000;90:186–98.[CrossRef][Medline]
  32. Abra RM, Bankert RB, Chen F, et al. The next generation of liposome delivery systems: recent experience with tumor-targeted, sterically-stabilized immunoliposomes and active-loading gradients. J Liposome Res 2002;12:1–3.
  33. Sugano M, Egilmez NK, Yokota SJ, et al. Antibody targeting of doxorubicin-loaded liposomes suppresses the growth and metastatic spread of established human lung tumor xenografts in severe combined immunodeficient mice. Cancer Res 2000;60:6942–9.[Abstract/Free Full Text]



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O. H. Aina, J. Marik, R. Liu, D. H. Lau, and K. S. Lam
Identification of novel targeting peptides for human ovarian cancer cells using "one-bead one-compound" combinatorial libraries
Mol. Cancer Ther., May 1, 2005; 4(5): 806 - 813.
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