scFv Antibodies Which Pass Epithelial and/or Endothelial Layers

Abstract

scFv antibodies which specifically bind selected antigens and are obtainable by a method comprising (i) selecting from a pool of soluble and stable antibody frameworks a soluble and stable framework matching best the framework of a non-human antibody against the antigen with a certain binding specificity, (ii) either providing said soluble and stable framework with CDRs that bind specifically to said antigen, or mutating the framework of said non-human antibody towards the sequence of said soluble and stable framework, to generate scFv antibodies, (iii) testing the generated antibody for solubility and stability, and testing the generated antibody for antigen binding, and (iv) selecting an scFV that is soluble, stable and binds to the antigen specifically. Also provided are pharmaceutical compositions comprising said scFv antibody, methods of treatment and diagnosis for diseases related to over expression of antigens that are specifically bound by said antibody.

1 . An antigen-binding polypeptide represented by the formula:
 Y-L-Z; or Z-L-Y;   with Y being [F1-CDR1-F2-CDR2-F3-CDR3F4] and Z being [F5-CDR1-F6-CDR2-F7-CDR3F8];   wherein framework regions (F1-F4) of Y are at least 85% identical to the framework regions of the human light chain framework set forth in SEQ ID NO: 1;   framework regions (F5-F6) of Z are at least 85% identical to the framework regions of the human heavy chain framework set forth in SEQ ID NO: 10;   CDRs (CDRs1-3) of Y are derived from one or more non-human donor CDRs capable of binding a target antigen;   CDRs (CDRs4-6) of Z are derived from one or more non-human donor CDRs capable of binding the target antigen; and   L is a flexible polypeptide linker.   
     
     
         2 . An antibody comprising:
 (a) a light chain variable domain (VL) having three non-human VL CDR regions and VL framework regions of at least about 85% similarity to VL framework regions of a light chain variable domain selected from the group consisting of: SEQ ID NOs: 1, 2, 3, 4, 5, 6, and 7; and   (b) a heavy chain variable domain (VH) having three non-human VH CDRs and VH framework regions of at least about 85% similarity to VH framework regions of a heavy chain variable domain selected from the group consisting of: SEQ ID NOs: 8, 9, 10 and 11.   
     
     
         3 . The antibody of  claim 2 , comprising a light chain variable domain (VL) framework of at least about 95% similarity to the framework sequence SEQ ID NO: 1 and a heavy chain variable domain (VH) framework of at least about 95% similarity to the framework sequence of SEQ ID NO: 10. 
     
     
         4 . An isolated polynucleotide molecule encoding the antigen-binding polypeptide of  claim 1  or the antibody of  claim 2 . 
     
     
         5 . A cloning or expression vector comprising the isolated polynucleotide molecule according to  claim 4 . 
     
     
         6 . A suitable host cell transformed with an expression vector according to  claim 5 . 
     
     
         7 . The host cell of  claim 6 , being a prokaryotic or eukaryotic cell, in particular an  E. coli , yeast, plant, insect or a mammalian cell. 
     
     
         8 . A pharmaceutical composition comprising the antigen-binding polypeptide of  claim 1  or the antibody of  claim 2 . 
     
     
         9 . The pharmaceutical composition of  claim 8 , being formulated for topical application to mucous membranes, skin or eye. 
     
     
         10 . The pharmaceutical composition of  claim 8 , being formulated for intramuscular, subcutaneous, intravenous, intraaterial, intrathecal or intraperitoneal injection. 
     
     
         11 . The pharmaceutical composition of claim comprising the antigen-binding polypeptide of  claim 1  formulated to achieve an intraocular concentration of at least 100 ng/ml or more. 
     
     
         12 . The pharmaceutical composition according to  claim 11 , formulated for topical administration to yield an intraocular concentration of 100 ng/ml or more based on a cellular or animal model system. 
     
     
         13 . The pharmaceutical composition according to  claim 11 , formulated to remain soluble at high concentrations. 
     
     
         14 . The pharmaceutical composition of  claim 10  formulated for topical application to eye and capable of passing through the cornea and into an intraocular space in the absence of penetration enhancer. 
     
     
         15 . A method for treating an eye disease or disorder comprising administering a polypeptide of  claim 1  to a patient in need thereof.