Ganglioside-associated recombinant antibodies and the use thereof in the diagnosis and treatment of tumors

Abstract

The present invention is related with the obtaining of modified antibodies by means of the DNA recombinant technology from the murine monoclonal antibody P3 (MAb P3) produced by the hybridoma cell line deposited under Budapest Treaty with accession number ECACC 94113026 and from its anti-idiotype murine monoclonal antibody 1E10(MAbai 1E10) produced by the hybridoma cell line with deposit number ECACC 97112901, with the objective of achieving monoclonal antibodies which preserve the biological function of specific binding to the antigen of the original antibodies, but being at the same time less immunogenic. The chimeric antibodies of the invention contain the variable domains of the murine immunoglobulin and the constant regions of the human immunoglobulin; and those humanized, besides containing the constant regions of the human immunoglobulins, they are modified in the region of the murine frameworks (FRs) and in particular in those zones that could be in an antigenic site for the T cells, so several positions of the FRS are human as well. These antibodies can be used in the diagnosis and therapy of different types of tumors. The present invention is also related with use of the antibodies for therapeutical and diagnostic purposes.

1 . A chimeric monoclonal antibody derived from the murine monoclonal antibody Mab P3 that recognizes gangliosides containing N-glycolylated sialic acid, and which is produced by the hybridoma cell line with deposit number ECACC 94113026, wherein the hypervariable domains of its heavy and light chains comprise the following sequences: 
 Heavy Chain                                              CDR1:   RYSVH   (SEQ ID NO: 1)                       CDR2:   MIWGGGSTDYNSALKS   (SEQ ID NO: 2)                   CDR3:   SGVREGRAQAWFAY   (SEQ ID NO: 3)                                 Light Chain                                              CDR1:   KASQDVSTAVA   (SEQ ID NO: 4)                       CDR2:   SASYRYT   (SEQ ID NO: 5)                   CDR3:   QQHYSTPWT   (SEQ ID NO: 6)                                 
     
     
         2 . A chimeric monoclonal antibody according to  claim 1  wherein the framework regions (frs) of its heavy and light chains comprise the following sequences: 
 Heavy Chain  
                           FR1:             QVQLKESGPGLVAPSQSLSITCTVSGFSLS   (SEQ ID NO: 7)           FR2:     WVRQPPGKGLEWLG   (SEQ ID NO: 8)           FR3:     RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR   (SEQ ID NO: 9)           FR4:     WGQGTLV   (SEQ ID NO: 10)                                 
 Light Chain  
                           FR1:             DIVMTQSHKFMSTSVGDRVSITC   (SEQ ID NO: 11)           FR2:     WYQQKPGQSPKLLIY   (SEQ ID NO: 12)           FR3:     GVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC   (SEQ ID NO: 13)           FR4:     FGGGTKL   (SEQ ID NO: 14)                                 
 
     
     
         3 . A monoclonal antibody according to  claim 1 , wherein for its humanization and preserving the binding properties to the antigen, it includes at least one of the following substitutions: 
 Light Chain:    Position 8: His by Pro    Position 9: Lys by Ser    Position 10: Phe by Ser    Position 11: Met by Leu    Positionn 13: Thr by Ala    
     
     
         4 . A monoclonal antibody according to  claim 1 , wherein the constant region of the heavy chain comprises the amino acid sequence of gamma-1 chain and the constant region of the light chain comprises the amino acid sequence of a kappa chain, both derived from human immunoglobulins.  
     
     
         5 . A cell line that produces any of the monoclonal antibodies of  claim 1 .  
     
     
         6 . A pharmaceutical composition for the treatment of malignant tumors of breast and melanomas, their metastases and relapses which comprises any of the monoclonal antibodies of  claim 1 .  
     
     
         7 . A pharmaceutical composition for “in vivo” localization e identification of malignant tumors of breast and melanomas, their metastases and relapses which comprises any of the monoclonal antibodies of  claim 1 .  
     
     
         8 . Use of the monoclonal antibody of any one of the  claim 1  for the manufacture of a medicament useful for the treatment of malignant tumors of breast and melanomas, their metastases and relapses.  
     
     
         9 . A chimeric monoclonal antibody derived from the murine anti-idiotypic monoclonal antibody 1E10 that recognizes murine MAb P3 and produced by the hybridoma cell line with deposit number ECACC 97112901, wherein the hypervariable domains of its heavy and light chains comprise the following sequences: 
 Heavy Chain                                              CDR1:   SYDIN   (SEQ ID NO: 15)                       CDR2:   WIFPGDGSTKYNEKFKG   (SEQ ID NO: 16)                   CDR3:   EDYYDNSYYFDY   (SEQ ID NO: 17)                                 Light Chain                                              CDR1:   RASQDISNYLN   (SEQ ID NO: 18)                       CDR2:   YTSRLHSG   (SEQ ID NO: 19)                   CDR3:   QQGNTLPWT   (SEQ ID NO: 20)                                 
     
     
         10 . A monoclonal antibody according to  claim 9  wherein the framework regions (FRs) of its heavy and light chains comprise the following sequences: 
 Heavy Chain  
                           FR1:             QVQLQQSGAELVKPGASVKLSCKASGYTFT   (SEQ ID NO: 21)           FR2:     WVRQRPEQGLEWIG   (SEQ ID NO: 22)           FR3:     KATLTTDKSSSTAYMQLSRLTSEDSAVYFCAR   (SEQ ID NO: 23)           FR4:     WGQGTTLTV   (SEQ ID NO: 24)                                 
 Light Chain  
                           FR1:             DIQMTQTTSSLSASLGDRVTISC   (SEQ ID NO: 25)           FR2:     WYQQKPDGTVKLLIY   (SEQ ID NO: 26)           FR3:     VPSRFSGSGSGTDYSLTISNLEQEDIATYFC   (SEQ ID NO: 27)           FR4:     FGGGTKLESK   (SEQ ID NO: 23)                                 
 
     
     
         11 . A monoclonal antibody according to  claim 9 , wherein for its humanization and preserving the binding properties to the antigen, it includes at least one of the following substitutions: 
 Light Chain:    Position 7: Thr by Ser    Position 8: Thr by Pro    Position 15: Leu by Val    Heavy Chain    Position 5: Gln by Val    Position 40: Arg by Ala    Position 42: Glu by Gly    Position 87 (83 according to Kabat's numbering): Thr by Arg    
     
     
         12 . A monoclonal antibody according to  claim 9  wherein the constant region of the heavy chain comprises the amino acid sequence of gamma-1 chain and the constant region of the light chain comprises the amino acid sequence of a kappa chain, both derived from of human immunoglobulins.  
     
     
         13 . A cell line that produces any of the monoclonal antibodies of  claim 9 .  
     
     
         14 . A pharmaceutical composition for the treatment of malignant tumors of breast and melanomas, their metastases and relapses which comprises any of the monoclonal antibodies of  claim 9 .  
     
     
         15 . A pharmaceutical composition for “in vivo” localization e identification of malignant tumors of breast and melanomas, their metastases and relapses, comprising any of the monoclonal antibodies of claims which comprises any of the monoclonal antibodies of  claim 9 .  
     
     
         16 . Use of the monoclonal antibody of any one of the  claim 9  for the manufacture of a medicament useful for the treatment of malignant tumors of breast and melanomas, their metastases and relapses.