Claims
- 1. A non-naturally occurring HER3 variant polypeptide consisting essentially of amino acid 20 to amino acid 329 of HER3 polypeptide sequence of SEQ ID NO: 2; wherein the HER3 variant polypeptide:
(a) specifically binds to the heregulin polypeptide of SEQ ID NO: 4; and (b) exhibits an impaired ability to interact with the HER2 polypeptide of SEQ ID NO: 6.
- 2. A non-naturally occurring HER3 variant polypeptide consisting essentially of amino acid 19 to amino acid 329 of HER3 polypeptide sequence of SEQ ID NO: 2; wherein the HER3 variant polypeptide has the ability to inhibit the interaction between wild type HER3 having the polypeptide sequence of SEQ ID NO: 2 and heregulin having the polypeptide sequence of SEQ ID NO: 4.
- 3. The HER3 variant polypeptide of claim 1 further comprising one or more amino acid substitutions at the following residue position(s) in SEQ ID NO: 2: E43; N44; K51; E64; V66; and V110.
- 4. A HER3 variant polypeptide comprising an amino acid sequence which differs from the native sequence HER3 polypeptide sequence of SEQ ID NO: 2 and has one or more of amino acid substitutions at the following residue position(s) in SEQ ID NO: 2: E43; N44; K51; E64; V66; and V110.
- 5. An isolated nucleic acid comprising a nucleotide sequence encoding the HER3 variant of claim 4.
- 6. A vector comprising the nucleic acid of claim 5.
- 7. A host cell comprising the vector of claim 6.
- 8. The host cell of claim 7 wherein said host cell is E. coli.
- 9. A method of making HER3 variant polypeptide, comprising the steps of: providing a host cell comprising the vector of claim 6; (b) providing culture media; (c) culturing the host cell in the culture media under conditions sufficient to express the HER3 variant polypeptide; (d) recovering the HER3 variant polypeptide from the host cell or culture media; and (e) purifying the HER3 variant polypeptide.
- 10. The HER3 variant polypeptide of claim 4, wherein the HER3 variant polypeptide is conjugated or linked to one or more polyol groups.
- 11. The HER3 variant polypeptide of claim 4, wherein the HER3 variant polypeptide has a substitution at V110.
- 12. The HER3 variant polypeptide of claim 11, wherein the HER3 variant polypeptide has an alanine substitution at V110.
- 13. The HER3 variant polypeptide of claim 4, wherein the HER3 variant polypeptide has a substitution at E64.
- 14. The HER3 variant polypeptide of claim 4, wherein the HER3 variant polypeptide comprises a soluble, extracellular domain HER3 polypeptide.
- 15. The isolated HER3 variant polypeptide of claim 1, wherein the HER3 variant polypeptide does not have any portion of the amino acid sequence consisting of amino acid 330 to amino acid 1342 of HER3 polypeptide sequence of SEQ ID NO: 2.
- 16. A method of identifying a compound which specifically binds the heregulin binding domain in a HER3 variant polypeptide comprising amino acid 20 to amino acid 329 of HER3 polypeptide sequence of SEQ ID NO: 2 comprising contacting the HER3 variant polypeptide with a test compound under conditions favorable to binding and determining whether the test compound specifically binds to the HER3 variant polypeptide such that a compound which binds to the HER3 variant polypeptide can be identified.
- 17. The method of claim 16, further comprising determining whether the test compound inhibits the heregulin induced tyrosine kinase activity associated with a HER3 polypeptide having the sequence shown in SEQ ID NO: 2 comprising the steps of:
contacting mammalian cells that express HER3 receptor with heregulin in the presence and absence of the test compound; and monitoring the mammalian cells for the tyrosine kinase activity associated with the HER3 polypeptide, wherein an inhibition in tyrosine kinase activity in the presence of the test compound as compared to the absence of the test compound identifies the test compound as an inhibitor of heregulin induced tyrosine kinase activity.
- 18. The method of claim 16, further comprising determining whether the test compound enhances the heregulin induced tyrosine kinase activity associated with a HER3 polypeptide having the sequence shown in SEQ ID NO: 2 comprising the steps of:
contacting mammalian cells that express HER3 receptor with heregulin in the presence and absence of the test compound; and monitoring the mammalian cells for the tyrosine kinase activity of the HER3 polypeptide, wherein an increase in tyrosine kinase activity in the presence of the test compound as compared to the absence of the test compound identifies the test compound as an enhancer of heregulin induced tyrosine kinase activity.
- 19. A method of determining whether a test compound modulates the interaction between heregulin polypeptide of SEQ ID NO: 4 and a HER3 variant polypeptide comprising amino acid 20 to amino acid 329 of HER3 polypeptide sequence of SEQ ID NO: 2 comprising the steps of:
(a) contacting the HER3 variant polypeptide with a test compound under conditions favorable to binding; (b) contacting the HER3 variant polypeptide of step (a) with heregulin under conditions favorable to binding; (c) comparing the binding interaction between the HER3 variant polypeptide and heregulin of step (b) with the binding interaction between HER3 variant polypeptide and heregulin in the absence of the test compound such that a compound which modulates the interaction between heregulin and the HER3 variant polypeptide can be identified.
- 20. A method of inhibiting the interaction between a heregulin polypeptide having the sequence shown in SEQ ID NO: 4 and HER3 polypeptide having the sequence shown in SEQ ID NO: 2 comprising exposing the heregulin polypeptide to a non-naturally occurring HER3 variant polypeptide comprising amino acid 20 to amino acid 329 of HER3 polypeptide sequence of SEQ ID NO: 2; wherein the HER3 variant polypeptide specifically binds to the heregulin polypeptide of SEQ ID NO: 4; and exhibits an impaired ability to interact with the HER2 polypeptide of SEQ ID NO: 6.
- 21. The method of claim 20, wherein the HER3 variant polypeptide has one or more of amino acid substitutions at the following residue position(s) in SEQ ID NO: 2: E64 and
- 22. The method of claim 20, wherein the HER3 variant polypeptide has an amino acid substitution at V110.
- 23. The method of claim 20, wherein the amino acid substitution is V91A.
- 24. The method of claim 20, wherein the HER3 variant polypeptide is conjugated or linked to one or more polyol groups.
- 25. The method of claim 24, wherein the polyol group is polyethylene glycol.
RELATED APPLICATIONS
[0001] This application claims priority under Section 119(e) from U.S. Provisional Application Serial No. 60/308,431 filed Jul. 27, 2001, the contents of which are incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60308341 |
Jul 2001 |
US |