Humanized pcrv antibody having anti-pseudomonal activity

Abstract

Provided are a humanized monoclonal antibody against PcrV or a part thereof, and a pharmaceutical composition containing the same as an active ingredient, as an effective means for therapy of infection, particularly infection with Pseudomonas aeruginosa. Concretely, the humanized monoclonal antibody of the present invention has an excellent inhibitory activity on the cytotoxicity with respect to a target cell of Pseudomonas aeruginosa. Also, the humanized monoclonal antibody of the present invention has a high affinity for PcrV.

1 . A humanized monoclonal antibody against PcrV or a part thereof, having at least one feature selected from (A) to (D):
 (1) inhibiting 50% or more of cytotoxicity to leukocyte cell of  Pseudomonas aeruginosa  at a concentration of 1 nM to 200 nM in vitro;   (2) inhibiting 50% or more of cytotoxicity to myeloma cell of  Pseudomonas aeruginosa  at a concentration of 1 nM to 50 nM in vitro;   (3) having a dissociation constant (Kd) with PcrV of 2×10 −9  (M) or less; and   (4) having its epitope at positions of 136 to 233 in amino acid sequence of SEQ ID NO: 1.   
     
     
         2 . A humanized monoclonal antibody or a part thereof, having in the complementarity determining region (CDR) the amino acid sequence of the CDR of the monoclonal antibody produced by the deposited hybridoma accession number FERM ABP-11805. 
     
     
         3 . A humanized monoclonal antibody against PcrV or a part thereof, having
 1) in a complementarity determining region, a heavy chain variable region including the following amino acid sequences: SFTSYWMH (SEQ ID NO: 15), INPSNGRTNYNEKFNT (SEQ ID NO: 16), YGNYVVYYTMDY (SEQ ID NO: 17) and 2) in a complementarity determining region, a light chain variable region including the following amino acid sequences: SASTSVSYME (SEQ ID NO: 18), TTSKLAS (SEQ ID NO: 19), HQWRNYPFT (SEQ ID NO: 20).   
     
     
         4 . A humanized monoclonal antibody against PcrV or a part thereof, having
 1) in a complementarity determining region, a heavy chain variable region including the following amino acid sequences: SFTSYWMH (SEQ ID NO: 15), INPSNGRTNYNEKFNT (SEQ ID NO: 16), YGNYVVYYTMDY (SEQ ID NO: 17) or a heavy chain variable region including a substitution, addition or deletion of one or several amino acid in at least one of SEQ ID NOS: 15, 16 or 17, and   2) in a complementarity determining region, a light chain variable region including the following amino acid sequences: SASTSVSYME (SEQ ID NO: 18), TTSKLAS (SEQ ID NO: 19), HQWRNYPFT (SEQ ID NO: 20) or a light chain variable region including a substitution, addition or deletion of one or several amino acids in at least one of SEQ ID NOS: 18, 19 or 20, and at least one feature selected from (1) to (4):
 (1) inhibiting 50% or more of cytotoxicity to leukocyte cell of  Pseudomonas aeruginosa  at a concentration of 1 nM to 200 nM in vitro; 
 (2) inhibiting 50% or more of cytotoxicity to myeloma cell of  Pseudomonas aeruginosa  at a concentration of 1 nM to 50 nM in vitro; 
 (3) having a dissociation constant (Kd) with PcrV of 2×10 −9  (M) or less; and 
 (4) having its epitope at positions of 136 to 233 in amino acid sequence of SEQ ID NO: 1. 
   
     
     
         5 . A humanized monoclonal antibody against PcrV or a part thereof, having 1) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27, and 2) a light chain variable region having the amino acid sequence of SEQ ID NO: 28. 
     
     
         6 . A pharmaceutical composition comprising the antibody or a part thereof according to  claim 1 , as an active ingredient and a pharmaceutically acceptable carrier. 
     
     
         7 . A polynucleotide encoding a heavy chain variable region or a light chain variable region of an antibody according to  claim 3 . 
     
     
         8 . An expression vector comprising of the polynucleotide according to  claim 7 . 
     
     
         9 . A pharmaceutical composition comprising the antibody according to  claim 2  as an active ingredient and a pharmaceutically acceptable carrier. 
     
     
         10 . A pharmaceutical composition comprising the antibody according to  claim 3  as an active ingredient and a pharmaceutically acceptable carrier. 
     
     
         11 . A pharmaceutical composition comprising the antibody according to  claim 4  as an active ingredient and a pharmaceutically acceptable carrier. 
     
     
         12 . A pharmaceutical composition comprising the antibody according to  claim 5  as an active ingredient and a pharmaceutically acceptable carrier. 
     
     
         13 . A polynucleotide encoding a heavy claim variable region or a light chain variable region according to  claim 4 . 
     
     
         14 . A polynucleotide encoding a heavy claim variable region or a light chain variable region according to  claim 5 . 
     
     
         15 . An expression vector comprising the polynucleotide according to  claim 13 . 
     
     
         16 . An expression vector comprising the polynucleotide according to  claim 14 . 
     
     
         17 . A part of a monoclonal antibody that is a Fab, F(ab′) 2  or single chain Fv portion of a humanized monoclonal antibody according to  claim 1 . 
     
     
         18 . A disulfide stabilized antibody comprising the Vh and Vl of the monoclonal antibody of  claim 1  in which an amino acid within the each of the Vh and Vl has been substituted by a cysteine amino acid, and the so modified Vh and Vl are joined by a disulfide bond between said cysteine residues. 
     
     
         19 . A peptide comprising one or more of the complementarity determining region peptides of the monoclonal antibody of  claim 1 . 
     
     
         20 . The peptide of  claim 19 , in which the complementarity determining region peptides have an amino acid sequence selected from the group consisting of SEQ ID NOS: 15-19 and 20. 
     
     
         21 . A diabody comprising an scFv of the monoclonal antibody of  claim 1  and a second scFv having specificity for an epitope different from an epitope presented by positions of 136 to 233 in the amino acid sequence of SEQ ID NO: 1.