Monoclonal antibodies to progastrin and their uses

Abstract

The present disclosure is directed to progastrin monoclonal antibodies, fragments thereof, compositions comprising progastrin monoclonal antibodies, and methods of making and using progastrin monoclonal antibodies and compositions thereof. The present disclosure is directed to methods of treating colorectal cancer with progastrin monoclonal antibodies and compositions comprising progastrin monoclonal antibodies or fragments thereof. The present disclosure is further directed to methods comprising detection of progastrin, including methods of diagnosing colorectal cancer and methods of monitoring efficacy of anti-cancer therapy in subjects suffering from colorectal cancer.

What is claimed is: 
     
         1 . A monoclonal antibody that specifically binds human progastrin (hPG). 
     
     
         2 . The monoclonal antibody of  claim 1  which specifically binds an N-terminal region of hPG. 
     
     
         3 . The monoclonal antibody of  claim 2  which is obtainable using an immunogen comprising a peptide antigen having an amino acid sequence corresponding to SEQ ID NO:25. 
     
     
         4 . The monoclonal antibody of  claim 2  that competes for binding hPG with a reference anti-hPG monoclonal antibody obtainable from a hybridoma selected from the group consisting of 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, and 1C11F5E8. 
     
     
         5 . The monoclonal antibody of  claim 2  which comprises VL and VH CDRs corresponding in sequence to the VL and VH CDRs of the monoclonal antibodies obtainable from a hybridoma selected from the group consisting of: 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, and 1C11F5E8. 
     
     
         6 . The monoclonal antibody of  claim 2  which comprises VL and VH CDR shaving sequences selected from one of the following groups of VL and VH CDR sequences:
 (i) VH CDRI.3, VH CDR 2.3, VH CDR 3.3, VL CDR 1.3, VL CDR 2.3, and VL CDR 3.3; 
 (ii) VH CDR 1.4, VH CDR 2.4, VH CDR 3.4, VL CDR 1.4, VL CDR 2.4, and VL CDR 3.4; 
 (iii) VHCDR 1.16, VHCDR2.16, VHCDR3.16, VLCDR 1.16, VLCDR2.16, and, VLCDR3.16; and
 (iv) VH CDR 1.19, VH CDR 2.19, VH CDR 3.19, VL CDR 1.19, VL CDR2.19, and VL CDR 3.19. 
 (v) 
 
 
     
     
         7 . The monoclonal antibody of any one of  claims 1 - 6 , which is humanized. 
     
     
         8 . The monoclonal antibody of  claim 7  which comprises VH and VL chain shaving sequences selected from one of the following groups of VH and VL sequences:
 (i) hVH 3 (SEQ ID NO:21) and hVL 3 (SEQ ID NO:22); 
 (ii) hVH 4 (SEQ ID NO:23) and hVL 4 (SEQ ID NO:24); 
 (iii) hVH 16a (SEQ ID NO:84) and hVL 16a (SEQ ID NO:85); 
 (iv) hVH 16b (SEQ ID NO:86) and hVL 16b (SEQ ID NO:87); 
 (v) hVH 16c (SEQ ID NO:88) and hVL 16c (SEQ ID NO:89); 
 (vi) hVH 19a (SEQ ID NO:90) and hVL 19a (SEQ ID NO:91); 
 (vii) hVH 19b (SEQ ID NO:92) and hVL 19b (SEQ ID NO:93); and 
 (viii) hVH 19c (SEQ ID NO:94) and hVL 19c (SEQ ID NO:95). 
 
     
     
         9 . The monoclonal antibody of  claim 1  which specifically binds a C-terminal region of hPG. 
     
     
         10 . The monoclonal antibody of  claim 9  which is obtainable using an immunogen comprising a peptide antigen having an amino acid sequence corresponding to SEQ ID NO:27. 
     
     
         11 . The monoclonal antibody of  claim 9  which competes for binding hPG with a reference anti-hPG monoclonal antibody obtainable from a hybridoma selected from the group consisting of 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7, 1F11F5E10, 1F11F5G9, and 1A11F2C9. 
     
     
         12 . The monoclonal antibody of  claim 9  which comprises VL and VH CDRs corresponding in sequence to the VL and VH CDRs of the monoclonal antibodies obtainable from a hybridoma selected from the group consisting of: 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7, 1F11F5E10, 1F11F5G9, and 1A11F2C9. 
     
     
         13 . The monoclonal antibody of  claim 9  which comprises VH and VL CDR shaving sequences selected from one of the following groups of VH and VL CDR sequences:
 (i) VH CDR 1.8, VH CDR 2.8, VH CDR 3.8, VL CDR 1.8, VL CDR 2.8, and VL CDR 3.8; and 
 VH CDR 1.13, VH CDR 2.13, VH CDR 3.13, VL CDR 1.13, VL CDR 2.13, and VL CDR 3.13. 
 
     
     
         14 . A monoclonal antibody of any one of  claims 9 - 13 , which is humanized. 
     
     
         15 . The monoclonal antibody of  claim 14  which comprises VH and VL chains having sequences selected from one of the following groups of VH and VL sequences:
 (i) hVH 8a (SEQ ID NO:75) and hVL 8a (SEQ ID NO:76); 
 (ii) hVH 8b (SEQ ID NO:77) and hVL 8b (SEQ ID NO:78); 
 (iii) hVH 8c (SEQ ID NO:79) and hVL 8c (SEQ ID NO:76); 
 (iv) hVH 13a (SEQ ID NO:80) and hVL 13a (SEQ ID NO:81); and 
 (v) hVH 13b (SEQ ID NO:82) and hVL 13b (SEQ ID NO:83). 
 
     
     
         16 . The monoclonal antibody of  claim 1  which has an affinity for hPG in the range of about 1 pM to about 7 nM. 
     
     
         17 . The monoclonal antibody of  claim 1  that binds to an epitope comprising an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs:28, 29, 30, 31, and 32. 
     
     
         18 . The monoclonal antibody of  claim 1  that binds to an epitope comprising an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs:33, 34, 35, and 36. 
     
     
         19 . The monoclonal antibody of  claim 1  which competes for binding hPG with a reference antibody selected from the group consisting of: anti-hPG MAb1, anti-hPG MAb2, anti-hPG MAb3, anti-hPG MAb4, anti-hPG MAb15, anti-hPG MAb16, anti-hPG MAb17, anti-hPG MAb18, anti-hPG MAb19, and anti-hPG MAb20. 
     
     
         20 . The monoclonal antibody of  claim 1  which competes for binding hPG with a reference antibody selected from the group consisting of: anti-hPG MAb5, anti-hPG MAb6, anti-hPG MAb7, anti-hPG MAb8, anti-hPG MAb9, anti-hPG MAb10, anti-hPG MAb11, anti-hPG MAb12, anti-hPG MAb13, anti-hPG MAb14, anti-hPG MAb21, anti-hPG MAb22, and anti-hPG MAb23. 
     
     
         21 . The monoclonal antibody of  claim 1  which neutralizes hPG activity in an in vitro assay carried out with LS174T cells. 
     
     
         22 . A composition comprising a monoclonal antibody according to  claim 1  and an excipient, carrier, and/or diluent. 
     
     
         23 . The composition of  claim 22  which is formulated for pharmaceutical use. 
     
     
         24 . A polynucleotide encoding a variable light chain for an anti-hPG monoclonal antibody according to  claim 1 . 
     
     
         25 . A polynucleotide encoding a variable heavy chain for an anti-hPG monoclonal antibody according to  claim 1 . 
     
     
         26 . An expression vector comprising a polynucleotide encoding a variable light chain for an anti-hPG monoclonal antibody according to  claim 1 . 
     
     
         27 . An expression vector comprising a polynucleotide encoding a variable heavy chain for an anti-hPG monoclonal antibody according to  claim 1 . 
     
     
         28 . A host cell transformed with pairs of polynucleotides suitable for expressing an anti-hPG monoclonal antibody according to  claim 1 . 
     
     
         29 . A hybridoma capable of secreting an anti-hPG monoclonal antibody according to  claim 1 . 
     
     
         30 . The hybridoma of  claim 29  selected from the group consisting of: 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and 1A11F2C9. 
     
     
         31 . The hybridoma of  claim 29 , which is selected from the group consisting of: 43B9G11, WE5H2G7, 1B4A11D11, 1B6A11F2, 1B11E4B11, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2H9F4B7, 1E9A4A4, 1C8D10F5, 1A7C3F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and 1A11F2C9. 
     
     
         32 . The hybridoma of  claim 29 , which is selected from the group consisting of: 1B4A11D11, 1B6A11F2, 1B11E4B11, 2B4C8C8, 2B11E6G4, and 1E9A4A4. 
     
     
         33 . A method of obtaining an anti-hPG monoclonal antibody comprising:
 (a) culturing a hybridoma of  claim 21  under suitable conditions; and   (b) recovering the anti-hPG monoclonal antibody from the culture medium or the hybridoma cells.   
     
     
         34 . A method of treating colorectal cancer comprising administering to a subject an amount of a humanized neutralizing anti-hPG monoclonal antibody effective to treat the colorectal cancer. 
     
     
         35 . The method of  claim 34  in which the neutralizing anti-hPG monoclonal antibody competes for binding with an anti-hPG monoclonal antibody obtainable from a hybridoma selected from the group of consisting of: 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 2H9F4B7, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and 1A11F2C9. 
     
     
         36 . The method of  claim 34  in which the neutralizing anti-hPG monoclonal antibody has CDRs corresponding in sequence to the CDRs of an anti-hPG monoclonal antibody selected from the group consisting of: anti-hPG MAb1, anti-hPG MAb2, anti-hPG MAb3, anti-hPG MAb4, anti-hPG MAb5, anti-hPG MAb6, anti-hPG MAb7, anti- hPG MAb8, anti-hPG MAb9, anti-hPG MAb10, anti-hPG MAb11, anti-hPG MAb12, anti-hPG MAb13, anti-hPG MAb15, anti-hPG MAb16, anti-hPG MAb17, anti-hPG MAb18, anti-hPG MAb19, anti-hPG MAb20, anti-hPG MAb21, anti-hPG MAb22, or anti-hPG MAb23. 
     
     
         37 . A method of inhibiting proliferation of colorectal tumor cells, comprising exposing the colorectal tumor cells to a neutralizing anti-hPG monoclonal antibody. 
     
     
         38 . The method of  claim 37  in which the neutralizing anti-hPG monoclonal antibody competes for binding with an anti-hPG monoclonal antibody obtainable from a hybridoma selected from the group of consisting of: 43B9G11, WE5H2G7, 6B5B11C10, 20D2C3G2, 1B4A11D11, 1B6A11F2, 1B11E4B11, 1C10D3B9, 1D8F5B3, 1E1C7B4, 2B4C8C8, 2B11E6G4, 2C6C3C7, 1E9A4A4, 1E9D9B6, 1C8D10F5, 1A7C3F11, 1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9, and 1A11F2C9. 
     
     
         39 . The method of  claim 37  in which the neutralizing anti-hPG monoclonal antibody has CDRs corresponding in sequence to the CDRs of an anti-hPG monoclonal antibody selected from the group consisting of: anti-hPG MAb1, anti-hPG MAb2, anti-hPG MAb3, anti-hPG MAb4, anti-hPG MAb5, anti-hPG MAb6, anti-hPG MAb7, anti- hPG MAb8, anti-hPG MAb9, anti-hPG MAb10, anti-hPG MAb11, anti-hPG MAb12, anti-hPG MAb13, anti-hPG MAb15, anti-hPG MAb16, anti-hPG MAb17, anti-hPG MAb18, anti-hPG MAb19, anti-hPG MAb20, anti-hPG MAb21, anti-hPG MAb22, or anti-hPG MAb23. 
     
     
         40 . The method of  claim 37  which is carried out in vitro. 
     
     
         41 . The method of  claim 37  which is carried out in vivo. 
     
     
         42 . A kit useful for detecting hPG, comprising an N-terminal anti-hPG monoclonal antibody according to  claim 2  and an antibody that specifically binds a C-terminal region of hPG. 
     
     
         43 . The kit of  claim 42 , wherein the antibody that specifically binds a C-terminal region of hPG is a polyclonal antibody. 
     
     
         44 . A kit useful for detecting hPG, comprising a C-terminal anti-hPG monoclonal antibody according to  claim 9  and an antibody that specifically binds an N-terminal region of hPG. 
     
     
         45 . The kit of  claim 44 , wherein the antibody that specifically binds an N-terminal region of hPG is a polyclonal antibody. 
     
     
         46 . A method of diagnosing colorectal cancer in a subject, comprising quantifying the amount of progastrin in a serum sample from the subject using at least one monoclonal antibody according to  claim 1 , wherein a quantity of progastrin in a range of about 20 to 400 pM is indicative of colorectal cancer. 
     
     
         47 . A method of selecting a patient suitable for anti-hPG therapy, comprising quantifying the amount of progastrin in a serum sample from a patient with colorectal cancer using at least one monoclonal antibody according to  claim 1 , wherein a quantity of progastrin in a range of about 20 to 400 pM is indicative of suitability for anti-hPG therapy. 
     
     
         48 . A method of monitoring the effectiveness of a colorectal cancer treatment, comprising quantifying the amount of progastrin in a serum sample from a subject who is being treated for colorectal cancer, as a function of time, using at least one monoclonal antibody according to  claim 1 , where a time-dependent decrease in the subject's serum progastrin level indicates the treatment is effective. 
     
     
         49 . A method of inhibiting a progastrin-dependent signal transduction cascade in a cell, comprising exposing cells that secretes hPG to an amount of a neutralizing anti-hPG monoclonal antibody effective to inhibit progastrin-dependent signal transduction.