Soluble Form of Carbonic Anhydrase IX (s-CA IX), Assays to Detect s-CA IX, CA IX's Coexpression with Her-2/neu/c-erbB-2, and CA IX-Specific Monoclonal Antibodies to Non-Immunodominant Epitopes
Disclosed herein is the discovery of a soluble MN/CA IX (s-CA IX) in body fluids, such as, urine and serum. Said s-CA IX comprises the extracellular domain of CA IX or portions thereof. The Predominant s-CA IX species is the extracellular domain comprising a proteoglycan-like (PG) domain and carbonic anhydrase (CA) domain, and having a molecular weight of about 50/54 kilodaltons (kd) upon Western blot. A smaller s-CA IX form of about 20 to about 30 kd comprising the CA domain or parts thereof, not linked to the PG domain, has also been found in body fluids. Diagnostic/prognostic methods for precancer and cancer that detect or detect and quantitate said s-CA IX in body fluids, are described. Also disclosed herein is the coexpression of CA IX and HER-2/neu/c-erbB-2 that provides parallel, alternative and potentially synergistic diagnostic/prognostic and therapeutic strategies for precancer and cancer. Further disclosed are new MN/CA IX-specific antibodies generated from MN/CA IX-deficient mice, preferably monoclonal antibodies and immunoreactive fragments and engineered variants thereof. 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 generated from MN/CA IX-deficient mice wherein said antibody binds specifically to MN/CA IX protein or MN/CA IX polypeptide.
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 proteoglycan-like (PG) domain, the transmembrane (TM) domain, and the intracellular (IC) domain.
4 . 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 domain and the proteoglycan-like domain.
5 . The monoclonal antibody of claim 1 which specifically binds the carbonic anhydrase domain of MN/CA IX protein.
6 . The monoclonal antibody of claim 5 , wherein said carbonic anhydrase domain has an amino acid sequence substantially 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 substantially homologous to SEQ ID NO: 67 or SEQ ID NO: 69.
8 . The monoclonal antibody of claim 1 which specifically binds the proteoglycan-like (PG) domain of MN/CA IX protein.
9 . The monoclonal antibody of claim 8 , wherein said proteoglycan-like domain has an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 98, and amino acid sequences substantially homologous to SEQ ID NO: 8 and SEQ ID NO: 98.
10 . The monoclonal antibody of claim 1 which specifically binds the transmembrane domain of MN/CA IX protein.
11 . The monoclonal antibody of claim 10 , wherein said transmembrane domain has an amino acid sequence substantially homologous to SEQ ID NO: 6 or has the amino acid sequence of SEQ ID NO: 6.
12 . The monoclonal antibody of claim 1 which specifically binds the intracellular domain of MN/CA IX protein.
13 . The monoclonal antibody of claim 12 , wherein said intracellular domain has amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is substantially homologous to SEQ ID NO: 7.
14 . The monoclonal antibody of claim 12 , that binds the epitope represented by SEQ ID NO: 68.
15 . The monoclonal antibody of claim 5 , which is produced by the hybridoma VU-V/10, deposited at BCCM™/LMBP in Ghent, Belgium under Accession No. ______.
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 V H and V L 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. ______.
20 . The monoclonal antibody of claim 1 that is not directed to immunodominant epitopes of MN/CA IX.