Cell/tissue-specific cell-penetrating antibodies

Abstract

Presented herein are cyclic peptides that specifically bind to a cell surface molecule, thereby allowing cell/tissue-specific targeting. The cyclic peptides can be attached to an agent, for example, a polypeptide such as an antibody, e.g., a cell-penetrating antibody. Cyclic peptide-containing cell/tissue-specific cell-penetrating antibodies described herein are capable of targeted delivery in a cell type-specific or tissue-specific (i.e., cell/tissue-specific) manner. The cell/tissue-specific cell-penetrating antibodies described herein can be used as an effective anticancer agent for cancer that overexpresses a cell membrane protein that specifically binds to the fused cyclic peptides.

What is claimed: 
     
         1 . A cell/tissue-specific cell-penetrating antibody comprising a light chain comprising a light chain variable region (“VL”), a heavy chain comprising a heavy chain variable region (“VH”), and a cyclic peptide that specifically binds to a cell surface molecule. 
     
     
         2 . The antibody of  claim 1 , wherein the cell surface molecule is selected from the group consisting of cell membrane protein epithelial cell adhesion molecule (EpCAM), integrin αvβ5, integrin αvβ3, integrin αvβ6, and epidermal growth factor receptor (EGFR). 
     
     
         3 . The antibody of  claim 1 , wherein the amino acid sequence of the cyclic peptide comprises: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 110) 
                 
                     
                   X1-X2-Leu-X4-Cys5-X6-Gly-X8-X9-Cys10-Trp-X12 
                 
             
                
                
               
            
           
         
         wherein X1 is any one of Glu, His, Asp, Lys, Asn, and Arg; X2 is any one of His, Asn, and Gly; X4 is any one of His, Leu, Gln, Arg, and Trp; X6 is any one of Leu and Ile; X8 is any one of Ser, Asn, and Arg; X9 is any one of Leu and Ile; and X12 is any one of Pro and Ser. 
       
     
     
         4 . The antibody of  claim 3 , wherein the amino acid sequence of the cyclic peptide is selected from the group consisting of SEQ ID NOs: 14 to 20 and 29 to 37. 
     
     
         5 . The antibody of  claim 4 , wherein the cyclic peptide is fused to the light chain and comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 28 and 38 to 48. 
     
     
         6 . The antibody of  claim 1 , wherein the amino acid sequence of the cyclic peptide comprises: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 100) 
                 
                     
                   Asp-Gly-X3-X4-X5-Cys6-Arg-Gly-Asp-Cys10-X11- 
                 
                     
                     
                 
                     
                   X12-X13-X14-X15 
                 
             
                
                
                
                
               
            
           
         
         wherein X3 is any one of Glu, Val, Gly, Gln, and Asp; X4 is any one of Arg and Lys; X5 is any one of Asn, Ser, Ala, Gln, Leu, and Thr; X11 is any one of Ile and Phe; X12 is any one of Asp and Glu; X13 is any one of Gly, Asp, Pro, Val and Ala; X14 is any one of Gln, Ser, Pro, and Ala; and X15 is any one of Pro, Gln, Asp, Leu, Val, and Ser. 
       
     
     
         7 . The antibody of  claim 6 , wherein the amino acid sequence of the cyclic peptide is selected from the group consisting of SEQ ID NOs: 50 to 57. 
     
     
         8 . The antibody of  claim 7 , wherein the cyclic peptide fused to the light chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 59 to 66. 
     
     
         9 . The antibody of  claim 1 , wherein the amino acid sequence of the cyclic peptide is selected from the group consisting of SEQ ID NOs: 73 to 76 and 78 to 85. 
     
     
         10 . The antibody of  claim 9 , wherein the cyclic peptide fused to the light chain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 86 to 89 and 91 to 98. 
     
     
         11 . The antibody of  claim 1 , wherein the antibody is of full-length immunoglobulin format. 
     
     
         12 . The antibody of  claim 11  comprising an endosomal escape motif in the light chain variable region (VL) and/or the heavy chain variable region (VH) of the antibody, wherein the endosomal escape motif induces penetration of the antibody into the cytosol of the cell. 
     
     
         13 . The antibody of  claim 12 , wherein the VL comprises a complementarity determining region (CDR) 1, CDR2, and CDR3, and the VH comprises a CDR1, CDR2, and CDR3, and wherein acid sequence of the endosomal escape motif comprises:
 1) Trp-Tyr-Trp-X (SEQ ID NO: 69) CDR3 of VL and/or VH, wherein X is selected from the group consisting of methionine (Met), isoleucine (Ile), and leucine (Leu); and   2) aspartic acid (Asp) or glutamic acid (Glu) as the first amino acid VL and/or VH.   
     
     
         14 . The antibody of  claim 13 , wherein affinity of the antibody for heparan sulfate proteoglycan (HSPG) is reduced or abolished. 
     
     
         15 . The antibody of  claim 13 , wherein the light chain variable region (VL) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 8. 
     
     
         16 . The antibody of  claim 13 , wherein the heavy chain variable region (VH) of the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 and 12. 
     
     
         17 . The antibody of  claim 13 , wherein the light chain variable region (VL) of the antibody comprises an amino acid sequence represented by SEQ ID NO: 8, and the heavy chain variable region (VH) of the antibody comprises an amino acid sequence represented by SEQ ID NO: 12. 
     
     
         18 . A cyclic peptide comprising: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 110) 
                 
                     
                   X1-X2-Leu3-X4-Cys5-X6-Gly-X8-X9-Cys10-Trp-X12 
                 
             
                
                
               
            
           
         
         wherein X1 is any one of Glu, His, Asp, Lys, Asn, and Arg; X2 is any one of His, Asn, and Gly; X4 is any one of His, Leu, Gln, Arg, and Trp; X6 is any one of Leu and Ile; X8 is any one of Ser, Asn, and Arg; X9 is any one of Leu and Ile; and X12 is any one of Pro and Ser, wherein the cyclic peptide specifically binds to EpCAM. 
       
     
     
         19 . The cyclic peptide of  claim 18 , wherein the amino acid sequence of the cyclic peptide is selected from the group consisting of SEQ ID NOs: 14 to 20 and 29 to 37. 
     
     
         20 . A cyclic peptide comprising: 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 100) 
                 
                     
                   Asp-Gly-X3-X4-X5-Cys6-Arg-Gly-Asp-Cys10-X11- 
                 
                     
                     
                 
                     
                   X12-X13-X14-X15 
                 
             
                
                
                
                
               
            
           
         
         wherein X3 is any one of Glu, Val, Gly, Gln, and Asp; X4 is any one of Arg and Lys; X5 is any one of Asn, Ser, Ala, Gln, Leu, and Thr; X11 is any one of Ile and Phe; X12 is any one of Asp and Glu; X13 is any one of Gly, Asp, Pro, Val and Ala; X14 is any one of Gln, Ser, Pro, and Ala; and X15 is any one of Pro, Gln, Asp, Leu, Val, and Ser, wherein the cyclic peptide specifically binds αvβ5 or αvβ3 integrin. 
       
     
     
         21 . The cyclic peptide of  claim 20 , wherein the amino acid sequence of the cyclic peptide is selected from the group consisting of SEQ ID NOs: 50 to 57. 
     
     
         22 . A cyclic peptide of the amino acid sequence selected from the group consisting of SEQ ID NOs: 73 to 76 and 78 to 85. 
     
     
         23 . A polypeptide comprising the cyclic peptide of  claim 18 . 
     
     
         24 . A polypeptide comprising the cyclic peptide of  claim 20 . 
     
     
         25 . A polypeptide comprising the cyclic peptide of  claim 22 . 
     
     
         26 . A polynucleotide encoding the cell/tissue-specific cell-penetrating antibody of  claim 1 . 
     
     
         27 . A method of making a cell/tissue-specific cell-penetrating antibody comprising:
 introducing a vector comprising the polynucleotide of  claim 26  into a host cell;   expressing the vector in the host cell; and   recovering the expressed antibody.   
     
     
         28 . A pharmaceutical composition comprising the cell/tissue-specific cell-penetrating antibody of  claim 1  as an active ingredient and a pharmaceutically acceptable carrier. 
     
     
         29 . A method of preventing or treating a disease or condition in a subject comprising administering a therapeutically effective amount of the cell/tissue-specific cell-penetrating antibody of  claim 1  to the subject. 
     
     
         30 . The method of  claim 29 , wherein a disease or condition is a cancer.