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Her-2 binding antagonists




Title: Her-2 binding antagonists.
Abstract: There is disclosed a a pharmaceutical composition for treating solid tumors that overexpress HER-2, comprising an agent selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1, (b) an isolated and glycosylated polypeptide having from about 300 to 419 amino acids taken from the sequence of SEQ ID NO:2, wherein the C terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present, (c) a monoclonal antibody that binds to the ECD of HER-2, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone, and pharmaceutically acceptable carrier. Also disclosed are prognostic and diagnostic assays. ...


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USPTO Applicaton #: #20100267027
Inventors: Gail M. Clinton, Adam Evans, William D. Henner


The Patent Description & Claims data below is from USPTO Patent Application 20100267027, Her-2 binding antagonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

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This application is a continuation of U.S. patent application Ser. No. 09/506,079, entitled HER-2 BINDING ANTAGONISTS, filed Feb. 16, 2000, which is incorporated herein by reference in its entirety.

FEDERAL FUNDING ACKNOWLEDGMENT

This work was supported at least in part by a grant from the Department of Defense (DOD) Breast Cancer Research Program. The United States Government has certain rights in this invention.

TECHNICAL

FIELD OF THE INVENTION

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The present invention provides a HER-2 binding antagonist. Specifically, intron retention has generated a novel HER-2 antagonist polypeptide that binds to the HER-2 receptor.

BACKGROUND

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The HER-2/neu (erbB-2) oncogene encodes a receptor-like tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994) and in mammalian development (Lee et al., Nature 378:394-398, 1995). The sequence of the HER-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA from placenta (Coussens et al., Science 230:1132-1139, 1985) and from a gastric carcinoma cell line (Yamamoto et al., Nature 319:230-234, 1986). The HER-2 mRNA was shown to be about 4.5 kb (Coussens et al., Science 230:1132-1139, 1985; and Yamamoto et al., Nature 319:230-234, 1986) and encodes a transmembrane glycoprotein of 185 kDa in normal and malignant human tissues (p185HER-2) (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994). The function of the HER-2 gene has been examined mainly by expressing the cDNA corresponding to the 4.5 kb transcript in transfected cells and from the structure and biochemical properties of the 185 kDa protein product. P185HER-2 consists of a large extracellular domain, a transmembrane segment, and an intracellular domain with tyrosine kinase activity (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994). Overexpression of p185HER-2 causes phenotypic transformation of cultured cells (DiFiore et al., Science 237:178-182, 1987; and Hudziak et al., Proc. Natl. Acad. Sci. USA 84:7159-7163, 1987) and has been associated with aggressive clinical progression of breast and ovarian cancer (Slamon et al., Science 235:177-182, 1987; and Slamon et al., Science 244:707-712, 1989). p185HER-2 is highly homologous to the EGFR. However, a ligand that directly binds with high affinity to p185HER-2 has not yet been identified. Moreover, the signaling activity of HER-2 may be mediated through heterodimerization with other ligand-binding members of the EGFR family (Carraway and Cantley, Cell 78:5-8, 1994; Earp et al., Breast Cancer Res. Treat. 35:115-132, 1995; and Qian et al., Oncogene 10:211-219, 1995).

Divergent proteins, containing regions of the extracellular domains of HER family RTKs, are generated through proteolytic processing of full length receptors (Lin and Clinton, Oncogene 6:639-643, 1991; Zabrecky et al., J. Biol. Chem. 266:1716-1720, 1991; Pupa et al., Oncogene 8:2917-2923, 1993; Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996; and Vecchi and Carpenter, J. Cell Biol. 139:995-1003, 1997) and through alternative RNA processing (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990; Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993; and Lee and Maihle, Oncogene 16:3243-3252, 1998). The extracellular domain of p185HER-2 is proteolytically shed from breast carcinoma cells in culture (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990; Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993; and Lee and Maihle, Oncogene 16:3243-3252, 1998), and is found in the serum of some cancer patients (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992) where it is may be a serum marker of metastatic breast cancer (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992) and may allow escape of HER-2-rich tumors from immunological control (Baselga et al., J. Clin. Oncol. 14:737-744, 1966; and Brodowicz et al., Int. J. Cancer 73:875-879, 1997).

A truncated extracellular domain of HER-2 is also the product of a 2.3 kb alternative transcript generated by use of a polyadenylation signal within an intron (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993). The alternative transcript was first identified in the gastric carcinoma cell line, MKN7 (Yamamoto et al., Nature 319:230-234, 1986; and Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993) and the truncated receptor was located within the perinuclear cytoplasm rather than secreted from these tumor cells (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993). However, no particular therapeutic, diagnostic or research utility has been ascribed to this truncated extracellular domain polypeptide. A truncated extracellular domain of the EGFR, generated by alternative splicing (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990) is secreted, exhibits ligand-binding, and dimerization properties (Basu et al., Mol. Cell. Biol. 9:671-677, 1989), and may have a dominant negative effect on receptor function (Basu et al., Mol. Cell. Biol. 9:671-677, 1989; and Flickinger et al., Mol. Cell. Biol. 12:883-893, 1992).

Therefore, there is a need in the art to find molecules that bind to cellular HER-2 and particularly molecules that bind to different sites than humanized antibodies to HER-2 (e.g., HERCEPTIN®). Such molecules would be useful therapeutic agents for various cancers that overexpress HER-2.

SUMMARY

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OF EXEMPLARY EMBODIMENTS

The present invention provides an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1. Preferably, the isolated polypeptide is from about 69 to 79 amino acids in length. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD or HER-2).

The present invention further provides an isolated DNA sequence that codes on expression for a polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1. Preferably, the isolated polypeptide is from about 69 to 79 amino acids in length. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD or HER-2). The present invention further provides a transfected cell comprising an expression vector having a DNA sequence that codes on expression for a polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1.

The present invention further provides an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO:2, wherein the C terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present. Preferably, the isolated polypeptide is from about 350 to 419 amino acids in length and four N-linked glycosylation sites are present. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD or HER-2).

The present invention further provides an isolated DNA sequence that codes on expression for a polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO:2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present. Preferably, the isolated polypeptide is from about 350 to 419 amino acids in length and four N-linked glycosylation are present. The present invention further provides a transfected cell comprising an expression vector having a DNA sequence that codes on expression for a polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO:2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present.

The present invention provides a method for treating a solid tumor characterized by overexpression of HER-2, comprising administering an agent that binds to the extracellular domain (ECD) of HER-2, wherein the agent is selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1, (b) an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO. 2, wherein the C-terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present, (c) a monoclonal antibody that binds to the ECD of HER-2, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone. Preferably, the solid tumor that overexpresses HER-2 is selected from the group consisting of breast cancer, small cell lung carcinoma, ovarian cancer and colon cancer. Preferably, the agent is the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1. Most preferably, the agent is a combination of the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1 and the monoclonal antibody that binds to the ECD of HER-2.

The present invention further provides a pharmaceutical composition for treating tumors that overexpress HER-2, comprising an agent selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1, (b) an isolated and glycosylated polypeptide having from about 80 to 419 amino acids taken from the sequence of SEQ ID NO:2, wherein the C terminal 79 amino acids are present, and wherein at least three N-linked glycosylation sites are present, (c) a monoclonal antibody that binds to the ECD of HER-2, and (d) combinations thereof, with the proviso that the agent cannot be the monoclonal antibody alone, and pharmaceutically acceptable carrier. Preferably, the agent is the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1. Most preferably, the agent is a combination of the isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1 and the monoclonal antibody that binds to the ECD of HER-2.

The present invention further provides a method for targeting a therapeutic agent to solid tumor tissue, wherein the solid tumor tissue is characterized by overexpression of HER-2, comprising attaching the therapeutic agent to an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular domain ECD of HER-2 with an affinity binding constant of at least 108 M−1. Preferably, the isolated polypeptide is from about 69 to 79 amino acids in length. Preferably, the isolated polypeptide binds to a site on the ECD of HER-2 that is different from the site of binding of HERCEPTIN® (a marketed humanized monoclonal antibody that is used for the treatment of cancer and that binds to the ECD or HER-2).

The present invention further provides a method for determining the prognosis of tumor treatment in a patient for a tumor that overexpresses HER-2, comprising: (a) obtaining a bodily fluid sample from the patient, wherein the bodily fluid is selected from the group consisting of blood, serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilical cord tissue, amniotic fluid, chorionic villi tissue, and combinations thereof; and (b) measuring the amount of p68HER-2 expressed using an anti-p68HER-2 antibody-based assay, wherein the assay is selected from the group consisting of ELISA, immunoprecipitation, immunohistocytochemistry, and Western analysis. Preferably, the method for determining the prognosis of tumor treatment further comprises measuring the amount of p185HER-2 ECD in the bodily fluid, and determining a ratio between the amount of p68HER-2 and p185HER-2.

The present invention further provides an assay for cancer treatment, prognosis or diagnosis in a patient comprising: (a) obtaining a bodily fluid sample from the patient, wherein the bodily fluid is selected from the group consisting of blood, serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilical cord tissue, amniotic fluid, chorionic villi tissue, and combinations thereof; (b) determining whether a particular ECDIIIa variant sequence is present in the bodily fluid sample with a sequence identity assay; and (c) correlating the presence of the ECDIIIa variant sequence to cancer treatment and diagnosis using an historical database. Preferably, the sequence identity assay is selected from the group consisting of DNA sequencing, PCR assays, ELISA immunologic assays, immunoassays, hybridization assays, and combinations thereof.

The present invention further provides an assay for cancer treatment, prognosis or diagnosis in a patient comprising: (a) obtaining a bodily fluid sample from the patient, wherein the bodily fluid is selected from the group consisting of blood, serum, urine, lymph, saliva, tumor tissue, placental tissue, umbilical cord tissue, amniotic fluid, chorionic villi tissue, and combinations thereof; (b) determining whether an amount of an p68HER-2 ECDIIIa variant is present in the bodily fluid sample using an anti-p68HER-2 antibody-based assay, wherein the assay is selected from the group consisting of ELISA, immunoprecipitation, immunohistocytochemistry, and Western analysis; and (c) correlating the presence or amount of the p68HER-2 ECDIIIa variant to cancer treatment and diagnosis using an historical database.

The present invention further provides for the above-mentioned cancer treatment, prognostic or diagnostic assays, further comprising measuring the amount of p185HER-2 ECD in the bodily fluid sample.

The present invention further provides for the above-mentioned cancer treatment, prognostic or diagnostic assays further comprising measuring the amount of p185HER-2 ECD in the bodily fluid sample, and determining a ratio between the amount of p185HER-2 ECD and a particular p68HER-2 ECDIIIa variant.

The present invention further provides for antibodies specific for ECDIIIa variants of the sequence in SEQ ID NO:1 or SEQ ID NO:2, below.

BRIEF DESCRIPTION OF THE DRAWINGS

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FIG. 1 shows a nucleotide sequence and amino acid of the insert in the extracellular domain of HER-2. The HER-2 ECD coding sequence from exon 1-9 (primers A and B) was amplified by PCR from a cDNA library from SKOV-3 cells. A product of ˜1420 by was found to be HER-2-specific by Southern blot analysis. This product was subcloned and the nucleotide sequence was determined. In panel A, a nucleotide sequence (287 bp; SEQ ID NO:16) is shown for the 275 by insert (within the open-ended boxes) plus the immediately adjacent 5′ and 3′ sequences (framed by the open-ended boxes). The 275 by insert sequence, using the numbering of Coussens et al. (Science 230:1132-1139, 1985), is located between nucleotide residues 1171 and 1172 and following amino acid residue 340 in p185HER-2. SEQ ID NO:17 (276 bp) shows the 275 by insert sequence plus the immediately 5′ nucleotide (“G”). The consensus 5′ and 3′ splice sites at the arrows are shown in larger print. The inserted sequence is in-frame with 5′ HER-2 exon sequence and is deduced to encode a 79 amino acid extension (SEQ ID NO:11) following Arg 340 (R340). The novel 79 acid sequence (SEQ ID NO:11) encoded by the insert is proline-rich (19%) and has a consensus asparagine linked glycosylation site, which is underlined. A stop codon was found at nucleotides 236-238 within the inserted sequence. In panel B, the predicted product of the alternative transcript is a truncated secreted protein which contains subdomains I and II identical to p185 and is missing the transmembrane domain and cytoplasmic domain. If fully glycosylated, the expected size is 65-70 kDa. This polypeptide product is referred to as p68HER-2. Thus, the product will be a truncated secreted protein which is missing the transmembrane domain and cytoplasmic domain found in p185HER-2.

FIG. 2 shows the detection of alternative HER-2 transcripts containing the ECDIIIa sequence by Northern blot analysis. PolyA+ mRNA (2.5 μg) from different human fetal tissues (Clontech) or isolated from HEK-293 cells was resolved in a formalin agarose gel and transferred to a BRIGHTSTAR® membrane (Ambion) in 10×SSC. The membrane was hybridized with a 32P-labeled antisense RNA probe complimentary to the ECDIII sequence, stripped and reprobed with a 32P labeled cDNA probe specific for the 5′ HER-2 exon sequence. The membranes were washed under high stringency conditions and analyzed by phosphorimaging (Molecular Dynamics).

FIG. 3 shows a sequence-specific reactivity of anti-ECDIIIa with a protein of ˜68 kDa in a human embryonic kidney cell line (HEK293). Cell extract protein (20 μg) and 20 μl of media conditioned by HEK-293 cells were Western blotted and probed with anti-ECDIIIa diluted 1:10,000 (lanes 1 and 2) or with anti-ECDIIa diluted 1:10,000 containing 50 μg/ml purified His-tagged ECDIIIa peptide (lanes 3, 4).

FIG. 4 shows the expression of p185HER-2, relative to p68ECDIIIa expression, is markedly elevated in carcinoma cell lines in which the HER-2 gene is amplified. Cell extracts (15 μg of protein) from human embryonic kidney cell line (HEK293), nontumorigenic ovarian surface epithelial cell line (IOSEVAN), ovarian carcinoma cell line with HER-2 gene amplification (SKOV-3), nontumorigenic breast epithelial cell line (HBL100), and breast carcinoma cell lines with HER-2 gene amplification (BT474 and SKBR-3), were resolved by SDS-PAGE in 7.5% acrylamide gels and analyzed as a Western blot. The Western blot was probed with both antibodies specific for p68HER-2 (anti-ECDIIIa) and for p185HER-2 (anti-neu(C)).

FIG. 5 shows that p68ECDIIIa binds to p185HER-2. In panel A: Two mg of SKBR-3 cells extracted in nondenaturing buffer were immunoprecipitated with 5 μl anti-neu(N) specific for the N-terminal sequence of p68HER-2 and p185HER-2, or with 5 μl anti-neu(C) specific for the C-terminus of p185HER-2 and then probed as a Western blot with both anti-ECDIIIa specific for p68HER-2 and with anti-neu(C) specific for p185HER-2. In panel B: 100 μg of 17-3-1 cell extract were incubated in duplicate with 50 μl packed volume of NiNTA agarose (Qiagen) coupled to 20 μg of His-tagged ECDIIIa or to 20 μg His-tagged CREB fragment in 200 μl of wash buffer (20 mM Tris pH 8.0, 300 mM NaCl) at room temperature for 1 hr with shaking. The resin was then washed 4 times with 500 μl of wash buffer and proteins were eluted by incubation with 50 μl SDS-sample buffer at 100° C. for 2 min. Eluted proteins were analyzed by Western blot analysis using antibodies against the C-terminus of p185HER-2, anti-neu(C). In panel C: Monolayers of ˜105 3T3 cells or HER-2 transfected 17-3-1 cells in 12 well plates were washed twice with PBS and then incubated with 0.5 ml of serum-free media with 1% BSA and 39, 75, 150, and 300 nM of purified recombinant His-tagged ECDIIIa for 2 hrs at 4° C. Cells were washed 1 time in PBS containing 1% BSA and twice in PBS and then were extracted in denaturing buffer. Equal aliquots (20 μg protein) were analyzed by western blotting with antibodies specific for ECDIIIa (anti-ECDIIIa) or, in the upper panel, with antibodies specific for p185HER-2 (anti-neu(C)).




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stats Patent Info
Application #
US 20100267027 A1
Publish Date
10/21/2010
Document #
File Date
12/31/1969
USPTO Class
Other USPTO Classes
International Class
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Drawings
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Solid Tumors

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Chemistry: Molecular Biology And Microbiology   Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip   Involving Nucleic Acid  

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20101021|20100267027|her-2 binding antagonists|There is disclosed a a pharmaceutical composition for treating solid tumors that overexpress HER-2, comprising an agent selected from the group consisting of (a) an isolated polypeptide having from about 50 to 79 amino acids taken from the sequence of SEQ ID NO:1, wherein the polypeptide binds to the extracellular |Oregon-Health-&-Science-University
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