Lipocalin muteins with binding-affinity for glypican-3 (gpc-3) and use of lipocalin muteins for target-specific delivery to cells expressing gpc-3

Abstract

The present invention relates to novel, specific-binding therapeutic and/or diagnostic lipocalin muteins directed against Glypican-3 (GPC-3). The invention also relates to nucleic acid molecules encoding such muteins and to methods for generation of such muteins and nucleic acid molecules. In addition, the invention also is directed to pharmaceutical compositions comprising such muteins and practical uses of these lipocalin muteins. In addition, the present invention provides methods of using muteins of human lipocalin 2 (Lcn2 or NGAL) for target-specific delivery of therapeutic moieties or detectable labels to cells expressing GPC-3, and related therapeutic and diagnostic utilization.

1 - 87 . (canceled) 
     
     
         88 . A method of treating GPC-3-expressing cancers, comprising contacting cancer cells that express GPC-3 with at least one hNGAL lipocalin mutein, wherein said hNGAL mutein is capable of being internalized from said cells' surface, and wherein said hNGAL mutein is capable of binding GPC-3 with an affinity by K D  of about 10 nM or lower. 
     
     
         89 . The method of  claim 88 , wherein said hNGAL mutein has an affinity by K D  of about 1 nM or lower. 
     
     
         90 . The method of  claim 89 , wherein said hNGAL mutein has an affinity by K D  of about 0.3 nM or lower. 
     
     
         91 . The method of  claim 90 , wherein said hNGAL mutein has an affinity by K D  of about 0.1 nM or lower. 
     
     
         92 . The method of  claim 90 , wherein said hNGAL mutein is capable of competing for binding to GPC-3 in a competition assay with an IC 50  value of about 1 nM or lower. 
     
     
         93 . The method of  claim 88 , wherein said hNGAL mutein has mutated amino acids at one or more positions corresponding to position 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 106, 125, 127, 132, and/or 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 1). 
     
     
         94 . The method of  claim 88 , wherein said hNGAL mutein has at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 2-6 or a fragment or variant thereof. 
     
     
         95 . The method of  claim 88 , wherein said hNGAL mutein has at least 75% identity to the sequence of mature hNGAL (SEQ ID NO: 1). 
     
     
         96 . The method of  claim 88 , wherein the cancer comprises hepatocellular cancer (HCC), melanoma, thyroid cancer, lung squamous cell carcinoma, Wilms' tumor, neuroblastoma, hepatoblastoma, or testicular germ-cell tumors. 
     
     
         97 . The method of  claim 93 , for contributing to killing a cancer cell. 
     
     
         98 . The method of  claim 94 , wherein the cancers comprise hepatocellular cancer (HCC), melanoma, thyroid cancer, lung squamous cell carcinoma, Wilms' tumor, neuroblastoma, hepatoblastoma, or testicular germ-cell tumors. 
     
     
         99 . An hNGAL mutein or fragment or variant thereof that is capable of being internalized from a GPC-3-expressing cell's surface. 
     
     
         100 . A method for target-specific delivery of a therapeutic moiety or detectable label to cells that express GPC-3, comprising contacting said cells with at least one hNGAL lipocalin mutein, wherein said hNGAL mutein is capable of being internalized from said cells' surface, wherein said mutein is capable of binding GPC-3 with an affinity by K D  of about 10 nM or lower, and wherein said hNGAL mutein is conjugated or coupled to said therapeutic moiety or detectable label. 
     
     
         101 . The method of  claim 100 , wherein the therapeutic moiety is immunomodulatory. 
     
     
         102 . The method of  claim 100 , wherein the therapeutic moiety acts as a therapeutic agent. 
     
     
         103 . The method of  claim 102 , wherein the therapeutic agent comprises a toxin and/or radioisotope. 
     
     
         104 . The method of  claim 103 , wherein the toxin is an immunotoxin. 
     
     
         105 . The method of  claim 100 , for contributing to killing a cancer cell. 
     
     
         106 . The method of  claim 105 , wherein the cancer comprises hepatocellular cancer (HCC), melanoma, thyroid cancer, lung squamous cell carcinoma, Wilms' tumor, neuroblastoma, hepatoblastoma, or testicular germ-cell tumors. 
     
     
         107 . The method of  claim 100 , wherein the detectable label is a diagnostic for detecting cancers associated with GPC-3 overexpression.