Mn/ca ix-specific monoclonal antibodies generated from mn/ca ix-deficient mice and methods of use

Abstract

Disclosed herein among other MN/CA IX-related inventions are new MN/CA IX-specific antibodies generated from MN/CA IX-deficient mice, preferably monoclonal antibodies and immunoreactive fragments and engineered variants thereof. Subsets of the new antibodies are to either the proteoglycan-like (PG) domain or to the carbonic anhydrase (CA) domain of MN/CA IX, and methods are provided by which antibodies can be prepared to the other MN/CA IX domains. Such new MN/CA IX-specific antibodies, fragments and variants are useful diagnostically/prognostically and therapeutically for cancer and precancer. Particularly preferred are the new monoclonal antibodies, fragments and variants that are specific for the non-immunodominant epitopes of MN/CA IX, which antibodies are, among other uses, useful to detect soluble MN/CA IX (s-CA IX) in body fluids, alone but preferably in combination with antibodies specific to the immunodominant epitopes of MN/CA IX, for example, in a sandwich assay.

1 . A monoclonal antibody or antigen-binding fragment thereof that specifically binds to a non-immuno dominant epitope of MN//CA IX protein or MN/CA IX polypeptide, wherein said antibody is generated from MN/CA IX-deficient mice and wherein said antibody does not compete for binding to MN/CA IX with monoclonal antibodies MN7, M75 or MN12, with the proviso that the antibody does not bind to the proteoglycan-like (PG) domain of MN/CA IX, and wherein said epitope is selected from epitopes
 (a) within the carbonic anhydrase (CA) domain having the amino acid sequence of SEQ ID NO: 9,   (b) within the CA domain having the amino acid sequence of SEQ ID NO: 101,   (c) within the CA domain having the amino acid sequence of SEQ ID NO: 67,   (d) within the CA domain having the amino acid sequence SEQ ID NO: 69,   (e) within the transmembrane domain (TM) having the amino acid sequence of SEQ ID NO: 6, and   (f) within the intracellular domain (IC) having the amino acid sequence of SEQ ID NO: 7.   
     
     
         2 . The monoclonal antibody of  claim 1 , wherein said MN/CA IX-deficient mice have a null mutation in Car9. 
     
     
         3 . The monoclonal antibody of  claim 1  which specifically binds an MN/CA IX domain, wherein said domain is selected from the group consisting of the carbonic anhydrase (CA) domain, the transmembrane (TM) domain, and the intracellular (IC) domain. 
     
     
         4 . (canceled) 
     
     
         5 . The monoclonal antibody of  claim 1  which specifically binds the carbonic anhydrase domain of MN/CA IX protein, and which is produced by the hybridoma VU-V/10, deposited at BCCM/LMBP in Ghent, Belgium under Accession No. LMBP 6009 CB. 
     
     
         6 . The monoclonal antibody of  claim 5 , wherein said carbonic anhydrase domain has an amino acid sequence homologous to SEQ ID NO: 9 or SEQ ID NO: 101. 
     
     
         7 . The monoclonal antibody of  claim 5 , which has an epitope within the carbonic anhydrase domain that has the amino acid sequences of SEQ ID NO: 7 or SEQ ID NO: 69 or an amino acid sequence homologous to SEQ ID NO: 67 or SEQ ID NO: 69. 
     
     
         8 .- 9 . (canceled) 
     
     
         10 . The monoclonal antibody of  claim 1  which specifically binds the transmembrane domain of MN/CA IX protein. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . The monoclonal antibody of  claim 12 , that binds the epitope represented by SEQ ID NO: 68. 
     
     
         15 . (canceled) 
     
     
         16 . The monoclonal antibody of  claim 1  which is selected from the group consisting of antigen-binding fragments comprising MN/CA IX protein binding regions. 
     
     
         17 . The monoclonal antibody of  claim 16  wherein the said fragments are single chain molecules, comprising peptide-linked VH and VL domains. 
     
     
         18 . The monoclonal antibody of  claim 17 , wherein said fragments have been dimerized. 
     
     
         19 . The hybridoma VU-V/10 which produces the monoclonal antibody V/10 and which has been deposited at BCCM/LMBP in Ghent, Belgium under Accession No. LMBP 6009 CB. 
     
     
         20 . (canceled) 
     
     
         21 . The antibody of  claim 1 , which specifically binds to soluble MN/CA IX polypeptide. 
     
     
         22 . The monoclonal antibody of claim  9 , wherein said intracellular domain. has amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is homologous to SEQ ID NO:7, and wherein said antibody specifically binds the epitope represented by SEQ ID NO:68.