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Binding peptides specific for the extracellular domain of ErbB2 and uses therefor

a technology of erbb2 and erbb2, which is applied in the field of peptides and peptidomimetics, can solve the problems of poor prognosis, lack of specificity and associated toxicities to normal tissues, and achieve the effects of improving pharmacokinetic properties and facilitating detection, prevention and treatmen

Inactive Publication Date: 2006-12-07
UNIVERSITY OF VERMONT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about identifying small peptides that specifically bind to the extracellular domain of a protein called ErbB2, which is overexpressed in many forms of cancer. These peptides can be used for detection, prevention, and treatment of disorders associated with overexpression of ErbB2. The peptides have superior pharmacokinetic properties and can be used as therapeutic agents or in gene therapy. The invention provides a composition comprising the peptides and functional equivalents, as well as a pharmaceutically acceptable carrier and other therapeutic agents. The peptides inhibit the phosphorylation of ErbB2, which is important for the development of cancer.

Problems solved by technology

One major drawback of most cancer therapeutics is the lack of specificity and associated toxicity to normal tissues.
It is overexpressed on the surface of breast cancer cells in approximately 30% of newly diagnosed patients and is associated with a poor prognosis.

Method used

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  • Binding peptides specific for the extracellular domain of ErbB2 and uses therefor
  • Binding peptides specific for the extracellular domain of ErbB2 and uses therefor
  • Binding peptides specific for the extracellular domain of ErbB2 and uses therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

Identification of Peptide Phage from Random and Biased Peptide Phage Libraries

[0252] Clones were identified by screening random as well as biased peptide phage library. Peptides identified by screening random libraries include those having the following sequences:

CB022701-20DTDMCWWWSREFGWECAGAG(SEQ ID NO: 37)(E-20)CB051701-19SLALCLSEGVLLGADCRVLF(SREQ ID NO: 38)(C-19)CB051701-25WSSMCGDPTIADWLWCFSDA.(SEQ ID NO: 39)(C-25)

[0253] The following example describes the methodology used to identify four clones from four biased peptide phage libraries. The libraries were enriched for the sequence of the EC-1 phage clone (SEQ ID NO:1). The four libraries had the following design:

X4CLNPEESTWGFCRSAG,(SEQ ID NO: 29)WTGWCX5STWGFCRSAG,(SEQ ID NO: 30)WTGWCLNPEEX5CRSAG,(SEQ ID NO: 31)andWTGWCLNPEESTWGFCX4,(SEQ ID NO: 32)

where X=any amino acid.

[0254] The peptides so identified had the following sequences:

02-124WTGWCLNPEESTWGFCRSAG(SEQ ID NO: 33)02-137WTGWCLSPEESTWGFCRSAG(SEQ ID NO: 34)02-140WT...

example 2

Binding of Peptide Phage to Membrane Lysates from ErbB2 Expressing Cells

[0258] The above identified peptide phage specifically bind to native, intact ErbB2 extracted from the membranes of human breast cancer cells that overexpress ErbB2. Plates were coated with 1 μg of an antibody that binds to the intracellular domain of ErbB2, followed by 100 μl of solubilized membrane preparations from two different breast cancer cell lines that overexpress ErbB2 (BT474 and SKBR3), and probed with CB1 peptide-phage, control library phage, and 9G6 anti-ErbB2. MCF7 cells serve as a (relative) negative target control, as MCF7 breast cancer cells express only a low level of ErbB2. (See FIG. 2A.) The ELISA data indicates that the EBP gives a signal higher than the anti-ErbB2 antibody used as a positive control in the assay.

[0259] Peptide phage derived from the EC-1 biases library similarly bind specifically to membrane lysates from ErbB2 expressing SKBR3 cells but not to lysates from minimally expre...

example 3

Effect of Phagepeptide on ErbB2 Phosphorylation

[0261] ErbB2 overexpressing SKBR3 cells were treated with the EC-1 free peptide to determine its effect on ErbB2 activation. When ErbB2 is activated it becomes phosphorylated on specific tyrosine residues (i.e., pY1248 and pY877). This is turn triggers downstream signal transduction events, culminating in increased cellular proliferation. Viable SKBR3 cells were treated with 25 μM EC-1 peptide for 15 minutes. At 0.5, 2, 4, 8, 22 and 48 hours thereafter, cell lysates were prepared and run on a Western blot using phospho-specific ErbB2 antibodies. (See FIG. 6A.) Densitometric analysis of the Western blots showed that EC-1 peptide inhibits 40% of the phosphorylation of residues pY1248 and pY877 after 0.5 hrs. The results demonstrate that 25 μM EC-1 inhibits the phosphorylation of ErbB2 for at least 8 hours after treatment. (See FIG. 6B.) The peptide had no effect on total ErbB2 expression.

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Abstract

The invention provides methods and compositions for diagnosing and treating subjects using EBPs. Specifically disclosed are peptides and peptidomimetics that bind selectively to the extracellular domain of ErbB2. These compositions are useful in the prevention and treatment of disorders characterized by ErbB2 overexpression (e.g., breast cancer).

Description

RELATED APPLICATIONS [0001] This application is a continuation of application Ser. No. 10 / 272,437, filed on Oct. 15, 2002, now allowed, which claims priority under 35 U.S.C. §119 to U.S. provisional application Ser. No. 60 / 329,183 filed Oct. 12, 2001, the entire contents of each is incorporated by reference herein.GOVERNMENT SUPPORT [0002] This work was funded in part by grant number 1 R01 CA80790-01, from the National Institutes of Health, and grant number DAMD17-94-J-4373 from the Department of Defense. Accordingly, the United States Government may have certain rights to this invention.FIELD OF THE INVENTION [0003] The invention relates to peptides and peptidomimetics that bind to the extracellular domain of ErbB2, and their use in diagnosis, prevention and treatment of disorders associated with overexpression of ErbB2 (e.g., breast cancer). BACKGROUND OF THE INVENTION [0004] One major drawback of most cancer therapeutics is the lack of specificity and associated toxicity to norma...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/12A61K38/10C40B40/10A61K38/00A61K47/48C07K14/475
CPCA61K38/00C07K14/475A61K47/48246A61K47/64
Inventor KRAG, DAVIDPERO, STEPHANIEOLIGINO, LYN
Owner UNIVERSITY OF VERMONT
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