Sh2 domain variants

Abstract

The present invention relates to variant SH2 domains for binding a phosphotyrosine (pTyr)-containing peptide. The variant SH2 domains of the present invention include a parent SH2 domain having at least one amino acid substitution in a pre-defined region of 15 amino acid positions of the parent SR2 domain, wherein said at least one amino acid substitution increases the affinity of the variant SH2 domain for the pTyr-containing peptide relative to the parent SH2 domain. The present application relates also to methods of using the variant SH2 domains in the treatment of protein kinase-associated disorders, or the diagnosis or prognosis of protein kinase-associated disorders, for isolating and measuring the concentration of pTyr-containing molecules, and as reagents in research.

1 .- 19 . (canceled) 
     
     
         20 . A method for preventing or inhibiting the effects of a tyrosine kinase in a cell, the method comprising delivering or introducing a variant SH2 domain into the cell, the variant SH2 domain varying from a parent SH2 domain, so that relative to the parent SH2 domain, the variant SH2 domain comprises a modified phosphotyrosine (pTyr) binding region substituted at at least one amino acid position in a pre-defined region of 15 amino acid positions of the parent SH2 domain, the substituted amino acids increasing the binding affinity of the modified pTyr binding region for a pTyr-containing peptide relative to an unmodified pTyr binding region of the parent SH2 domain. 
     
     
         21 . The method of  claim 20 , wherein the variant SH2 domain is provided within a carrier that allows transportation across the cell. 
     
     
         22 . The method of  claim 20 , wherein the polypeptide is provided as a fused product comprising the polypeptide and a cell membrane penetrating molecule. 
     
     
         23 . (canceled) 
     
     
         24 . A method of assessing the presence of pTyr-containing peptides in a sample, the method comprising (a) contacting the sample to a variant SH2 domain, such that a pTyr-containing peptide/variant SH2 domain complex is formed if the pTyr-containing peptides are present in the sample, the variant SH2 domain varying from a parent SH2 domain, so that relative to the parent SH2 domain, the variant SH2 domain comprises a modified phosphotyrosine (pTyr) binding region substituted at at least one amino acid position in a pre-defined region of 15 amino acid positions of the parent SH2 domain, the substituted amino acids increasing the binding affinity of the modified pTyr binding region for a pTyr-containing peptide relative to an unmodified pTyr binding region of the parent SH2 domain; and (b) detecting the formation of the complex, thereby detecting the presence of the pTyr-containing peptides in the sample. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . A method for isolating pTyr-containing peptides from a sample, the method comprising: (a) contacting the sample to a variant SH2 domain, such that a pTyr-containing peptide/variant SH2 domain complex is formed if the pTyr-containing peptides are present in the sample, the variant SH2 domain varying from a parent SH2 domain, so that relative to the parent SH2 domain, the variant SH2 domain comprises a modified phosphotyrosine (pTyr) binding region substituted at at least one amino acid position in a pre-defined region of 15 amino acid positions of the parent SH2 domain, the substituted amino acids increasing the binding affinity of the modified pTyr binding region for a pTyr-containing peptide relative to an unmodified pTyr binding region of the parent SH2 domain; and (b) releasing the pTyr-containing peptides from the complex, thereby isolating the pTyr-containing peptides. 
     
     
         28 . The method of  claim 27 , wherein the method further comprises determining the concentration of the pTyr-containing peptides in the sample by measuring the amount of pTyr-containing peptides released. 
     
     
         29 . A method of determining the concentration of pTyr-containing peptides in a sample comprising: (a) immobilizing a variant SH2 domain on a resin, the variant SH2 domain varying from a parent SH2 domain, so that relative to the parent SH2 domain, the variant SH2 domain comprises a modified phosphotyrosine (pTyr) binding region substituted at at least one amino acid position in a pre-defined region of 15 amino acid positions of the parent SH2 domain, the substituted amino acids increasing the binding affinity of the modified pTyr binding region for a pTyr-containing peptide relative to an unmodified pTyr binding region of the parent SH2 domain, (b) passing the sample through the resin with the bound variant SH2 domain, (c) releasing any pTyr-containing peptide bound to the resin by adding a solvent that removes the ability for the variant SH2 domain to bind to the pTyr-containing peptide thereby creating elution fractions, and (d) determining the concentration of the pTyr-containing peptides present in the elution fractions. 
     
     
         30 . The method of  claim 28 , wherein the concentration of pTyr-containing peptides is determined through high performance liquid chromatography (HPLC). 
     
     
         31 . The method of  claim 24 , wherein the variant SH2 domain is bound to an affinity column or onto a lateral flow strip. 
     
     
         32 . (canceled) 
     
     
         33 . A method of manufacturing a variant SH2 domain having enhanced binding affinity for a pTyr-containing peptide relative to a parent SH2 domain, the method comprising substituting at least one amino acid residue in 15 pre-defined amino acid positions of the parent SH2 domain that correspond to the positions of Arg18 (position 1), Lys19 (position 2), Ala21 (position 3), Arg38 (position 4), Ser40 (position 5), Glu41 (position 6), Thr42 (position 7), Thr43 (position 8), Ala46 (position 9), Ser48 (position 10), Leu49 (position 11), Ser50 (position 12), Lys63 (position 13), His64 (position 14), and Lys66 (position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned with SEQ ID NO:1, the substituted amino acids increasing the binding affinity of the modified pTyr binding region for the pTyr-containing peptide relative to an unmodified pTyr binding region of the parent SH2 domain. 
     
     
         34 . (canceled) 
     
     
         35 . The method of  claim 29 , wherein the concentration of pTyr-containing peptides is determined through high performance liquid chromatography (HPLC). 
     
     
         36 . The method of  claim 27 , wherein the variant SH2 domain is bound to an affinity column or onto a lateral flow strip. 
     
     
         37 . The method of  claim 28 , wherein the variant SH2 domain is bound to an affinity column or onto a lateral flow strip. 
     
     
         38 . The method of  claim 29 , wherein the variant SH2 domain is bound to an affinity column or onto a lateral flow strip. 
     
     
         39 . The method of  claim 20 , wherein the pre-defined region of 15 amino acid positions of the parent SH2 domain correspond to the positions of Arg18 (position 1), Lys19 (position 2), Ala21 (position 3), Arg38 (position 4), Ser40 (position 5), Glu41 (position 6), Thr42 (position 7), Thr43 (position 8), Ala46 (position 9), Ser48 (position 10), Leu49 (position 11), Ser50 (position 12), Lys63 (position 13), His64 (position 14), and Lys66 (position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned with SEQ ID NO: 1. 
     
     
         40 . The method of  claim 24 , wherein the pre-defined region of 15 amino acid positions of the parent SH2 domain correspond to the positions of Arg18 (position 1), Lys19 (position 2), Ala21 (position 3), Arg38 (position 4), Ser40 (position 5), Glu41 (position 6), Thr42 (position 7), Thr43 (position 8), Ala46 (position 9), Ser48 (position 10), Leu49 (position 11), Ser50 (position 12), Lys63 (position 13), His64 (position 14), and Lys66 (position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned with SEQ ID NO:1. 
     
     
         41 . The method of  claim 27 , wherein the pre-defined region of 15 amino acid positions of the parent SH2 domain correspond to the positions of Arg18 (position 1), Lys19 (position 2), Ala21 (position 3), Arg38 (position 4), Ser40 (position 5), Glu41 (position 6), Thr42 (position 7), Thr43 (position 8), Ala46 (position 9), Ser48 (position 10), Leu49 (position 11), Ser50 (position 12), Lys63 (position 13), His64 (position 14), and Lys66 (position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned with SEQ ID NO:1. 
     
     
         42 . The method of  claim 29 , wherein the pre-defined region of 15 amino acid positions of the parent SH2 domain correspond to the positions of Arg18 (position 1), Lys19 (position 2), Ala21 (position 3), Arg38 (position 4), Ser40 (position 5), Glu41 (position 6), Thr42 (position 7), Thr43 (position 8), Ala46 (position 9), Ser48 (position 10), Leu49 (position 11), Ser50 (position 12), Lys63 (position 13), His64 (position 14), and Lys66 (position 15) of SEQ ID NO:1 when the parent SH2 domain is aligned with SEQ ID NO:1.