Anti-apoptotic protein antibodies

Abstract

Single-domain antibodies that bind pro-apoptotic proteins Bax and caspase-3 are identified and isolated. These single-domain antibodies may be used to modulate the activity of Bax and caspase-3, thereby modulating the symptoms and steps of oxidative stress and/or cell apoptosis, including Bax dimerization, mitochondrial permeabilization and the release of apoptotic proteins.

We claim: 
     
         1 . An isolated single-domain antibody having an amino acid sequence that comprises at least one of SEQ. ID NO: 7 or SEQ. ID NO: 8. 
     
     
         2 . An isolated single-domain antibody having a binding affinity for caspase-3 and having an amino acid sequence encoded by a nucleic acid sequence that comprises at least one of SEQ ID NO: 16 or SEQ ID NO:17. 
     
     
         3 . A single-domain antibody as claimed in  claim 1  that is fused to a polypeptide. 
     
     
         4 . A single-domain antibody as claimed in  claim 1  that is fused to an antibody domain. 
     
     
         5 . A single-domain antibody as claimed in  claim 4  wherein the antibody domain is an Immunoglobulin (Ig) light chain variable domain (VL), an Ig heavy chain variable domain (VH), a camelid (camels and llamas) heavy chain antibody variable domain (VHH), a nurse shark and wobbegong shark Ig new antigen receptor(IgNAR) variable domain (VH), or a T cell receptor variable domain. 
     
     
         6 . A single-domain antibody as claimed in  claim 3  wherein the polypeptide is a marker protein. 
     
     
         7 . A single-domain antibody as claimed in  claim 3  wherein the polypeptide is a signal protein or sequence determining cellular localization. 
     
     
         8 . A single-domain antibody as claimed in  claim 7  wherein the signal protein is a mitochondrial transport signal protein. 
     
     
         9 . A single-domain antibody as claimed in  claim 3  wherein the polypeptide has the ability to cross cell membranes. 
     
     
         10 . A single-domain antibody as claimed in  claim 9  wherein the polypeptide is a membrane translocating sequence or protein transduction domain. 
     
     
         11 . A single-domain antibody as claimed in  claim 9  wherein the polypeptide is an internalizing protein. 
     
     
         12 . An immunoliposome comprising a single-domain antibody as claimed in  claim 1 . 
     
     
         13 . A multimer comprising at least two single-domain antibodies as claimed in  claim 1 . 
     
     
         14 . A multimer as claimed in  claim 13  comprising five single-domain antibodies. 
     
     
         15 . A method for modulating the symptoms of apoptosis in a cell and/or modulating the effects of oxidative stress in a cell, in vitro or ex vivo, the method comprising the steps of exposing the cell to at least one single domain antibody that comprises at least one of SEQ ID NO: 7 or SEQ ID NO: 8 and allowing binding of the at least one single-domain antibody to caspase-3. 
     
     
         16 . A method as claimed in  claim 15  further comprising the steps of delivering a nucleic acid sequence which encodes the at least one single-domain antibody into the cell and allowing expression of the at least one single-domain antibody in the cell. 
     
     
         17 . A method as claimed in  claim 16  further comprising the steps of fusing the single-domain antibody to a polypeptide which permits membrane translocation or internalization of the single-domain antibody into the cell. 
     
     
         18 . A method for identifying a second single-domain antibody having a binding affinity for an apoptotic protein, said method comprising the steps of:
 a) providing a first single-domain antibody as defined in  claim 1 ;   b) constructing an antibody library based on a structure of the first single domain antibody; and   c) screening the antibody library for single-domain antibodies having a binding affinity for the apoptotic protein.