Cysteine engineered fibronectin type iii domain binding molecules

Abstract

Cysteine engineered monospecific and bispecific EGFR and/or c-Met FN3 domain containing molecules comprising one or more free cysteine amino acids are prepared by mutagenizing a nucleic acid sequence of a parent molecule and replacing one or more amino acid residues by cysteine to encode the cysteine engineered FN3 domain containing monospecific or bispecific molecules; expressing the cysteine engineered FN3 domain containing molecules; and recovering the cysteine engineered FN3 domain containing molecule. Isolated cysteine engineered monospecific or bispecific FN3 domain containing molecules may be covalently attached to a detection label or a drug moiety and used therapeutically.

1 .- 39 . (canceled) 
     
     
         40 . A protein comprising an amino acid sequence that is at least 87% identical to SEQ ID NO: 27, wherein the protein has at least one cysteine substitution at a position that corresponds to a position selected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the amino acid sequence of SEQ ID NO: 27. 
     
     
         41 . The protein of  claim 40 , wherein the protein is chemically-conjugated to a thiol-reactive reagent. 
     
     
         42 . The protein of  claim 41 , wherein the thiol-reactive reagent is a maleimide moiety. 
     
     
         43 . The protein of  claim 42 , wherein the maleimide moiety is selected from the group consisting of NEM, PEG24-maleimide, fluorescein maleimide, MMAE, and MMAF. 
     
     
         44 . The protein of  claim 40 , further comprising a half-life extending moiety attached to the protein at one of the at least one cysteine substitutions. 
     
     
         45 . The protein of  claim 44 , wherein the half-life extending moiety is CD8 binding molecule, albumin, an albumin variant, an albumin binding molecule, a polyethylene glycol (PEG), CD8, CD8 variant, or at least a portion of an Fc region of an immunoglobulin. 
     
     
         46 . The protein of  claim 45 , wherein the albumin binding molecule is an albumin binding FN3 domain. 
     
     
         47 . The protein of  claim 40 , wherein the protein further comprises a protein that is at least 83% identical to SEQ ID NO: 41. 
     
     
         48 . An isolated polynucleotide encoding the protein of  claim 40 . 
     
     
         49 . A vector comprising the polynucleotide of  claim 48 . 
     
     
         50 . An isolated host cell comprising the vector of  claim 49 . 
     
     
         51 . A method of producing the protein according to  claim 40 , wherein a cell comprising a polynucleotide encoding the protein is cultured under conditions to produce the protein. 
     
     
         52 . The method of  claim 51 , further comprising introducing the polynucleotide encoding the protein into the cell prior to the culturing step. 
     
     
         53 . The method of  claim 51 , further comprising purifying the protein. 
     
     
         54 . A method of treating cancer in a subject, the method comprising administering the protein of  claim 47  to the subject. 
     
     
         55 . The method of  claim 54 , wherein the cancer is a EGFR and/or c-Met-expressing tumor. 
     
     
         56 . The method of  claim 54 , wherein the cancer is epithelial cell cancer, breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, colorectal cancer, anal cancer, prostate cancer, kidney cancer, bladder cancer, head and neck cancer, ovarian cancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer, vaginal cancer, cervical cancer, cancer of the spleen, testicular cancer, gastric cancer, cancer of the thymus, colon cancer, thyroid cancer, liver cancer, or sporadic or hereditary papillary renal carcinoma (PRCC).