Reshaped human antibody to human medulloblastoma cells

Abstract

The present invention discloses reshaped antibody to human medulloblastoma cells comprising: (A) an L chair comprising: (1) a human L chain C region, and (2) an L chain V region comprising human L chain FRs and L chain CDRs of mouse monoclonal antibody ONS-M21 to human medulloblastoma cells; and, (B) an H chain containing: (1) a human H chain C region, and (2) an H chain V region comprising human H chain FRs and H chain CDRs of mouse monoclonal antibody ONS-M21 to human medulloblastoma cells. Since the majority of this reshaped human antibody is derived from a human antibody and mouse CDRs have a low level of antigenicity, the reshaped human antibody of the present invention has a low level of antigenicity in humans, and is therefore expected to be useful as a therapeutic agent and diagnostic tool for brain tumors such as medulloblastoma which strongly express antigen that is recognized by this antibody.

1 . An L chain variable region (V region) of an antibody to human medulloblastoma cells, comprising three complementarity determining regions (CDRs) having the amino acid sequences defined below: 
 CDR1: Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala    CDR2: Ser Ala Ser Tyr Arg Tyr Ser    CDR3; Gln Gln Tyr Asn Ser Tyr Pro Arg Ala or a portion thereof and four framework regions (FRs).    
     
     
         2 . An L chain of antibody to human medulloblastoma cells comprising the L chain variable region (V region) of claim I and human L chain constant region (C region).  
     
     
         3 . An L chain according to  claim 2  wherein the FRs of said L chain V region are derived from a mouse antibody.  
     
     
         4 . An L chain according to  claim 2  wherein said L chain V region has the amino acid sequence indicated in SEQ ID NO:26.  
     
     
         5 . An L chain according to  claim 2  wherein the FRs of said L chain V region are derived from a human antibody.  
     
     
         6 . An L chain according to  claim 2  or  5  wherein the FRs of said L chain V region are derived from a human antibody REI.  
     
     
         7 . An L chain according to  claim 5  or  6  wherein the amino acid at position 46 in the second FR of said L chain V region is praline.  
     
     
         8 . An L chain according to  claim 5  or 6 wherein the amino acids at positions 42, 43 and 46 in the second FR of said L chain V region are glutamine, serine and proline, respectively.  
     
     
         9 . An L chain according to  claim 2  wherein said L chain V region includes either set of four FRs having the following amino acid sequences: 
 (1) FRI: Asp Ile Gln met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 
 FR2: Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Leu Ile Tyr  
 FR3: Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser reu Gln Pro Clu Asp lie Ala Txr Tyr Tyr Cys  
 FR4: Phe Gly Gln Gly Thr Lys Val Glu Ile Lys  
 
 (2) FR1: Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 
 FR2: Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr  
 FR3: Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys  
 FR4: Phe Gly Gln Cly Thr Lys Val Glu Ile Lys  
 
 
     
     
         10 . An L chain according to  claim 2  wherein said human L chain C region is a KC region.  
     
     
         11 . An H chain V region of an antibody to human medulloblastoma cells containing three CDRs having the amino acid sequences defined below: 
 CDR1; Asp Thr Tyr Ile His    CDR2: Arg Ile Asp Pro Ala Asp Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln Gly CDR3: Ala Tyr Tyr Val Asn Gln Asp Tyr or a portion thereof and four FRs.    
     
     
         12 . An H chain of antibody to human medulloblastoma cells comprising the H chain V region of  claim 11  and a human H C region.  
     
     
         13 . An H chain according to  claim 12  wherein the FRs of said H chain V region are derived from a mouse antibody.  
     
     
         14 . The H chain according to  claim 12  wherein said H chain V region has the amino acid sequence indicated in SEQ ID NO:27.  
     
     
         15 . An H chain according to  claim 12  wherein the FRs of said H chain V region are derived from a human antibody.  
     
     
         16 . An H chain according to  claim 12  or  15  wherein the FRs of said H chain V region are derived from a human antibody of subgroup I.  
     
     
         17 . An H chain according to  claim 12  or  15  wherein the FRs of said H chain V region are derived from a human antibody Eu.  
     
     
         18 . The H chain according to  claim 15  wherein said H chain V region contains four FRs having the following amino acid sequences; 
 FR1: Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys Val Ser Ws Lys Ala Ser Gly Phe Asn Ile Lys  
 FR2: Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly  
 FR3: Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser beu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys Ala Ser  
 FR4: Trp Gly Gln Gly Thr Thr Val Thr Val Sor Ser  
 
     
     
         19 . The H chain according to  claim 15  wherein said human C region is a γ-1C region or γ-4C region.  
     
     
         20 . An antibody to human medulloblastoma cells composed of the L chain as set forth in  claim 2  and the H chain as set forth in  claim 12 .  
     
     
         21 . An antibody according to  claim 20  wherein the FRs of said V region are derived from a mouse antibody.  
     
     
         22 . The antibody as set forth in  claim 20  wherein the FRs of said V region are derived from a human antibody.  
     
     
         23 . A DNA coding for an L chain of an antibody to human medulloblastoma cells comprising an L chain V region containing three CDRs having the amino acids as set forth in  claim 1  or a portion thereof and four FRs, and a human L chain C region.  
     
     
         24 . A DNA according to  claim 23  wherein said L chain V region has a nucleotide sequence indicated in SEQ ID NO:58, 61, 63, 66, 70 or 73.  
     
     
         25 . A DNA coding for an H chain of an antibody to human medulloblastoma cells comprising an H chain V region containing three CDRs having the amino acids as set forth in  claim 11  or a portion thereof and four FRs, and a human H chain C region.  
     
     
         26 . A DNA according to  claim 25  wherein said H chain V region has the nucleotide sequence indicated in SEQ ID NO:80.  
     
     
         27 . A recombinant vector comprising a DNA according to  claim 23  or  24  or a portion thereof.  
     
     
         28 . A recombinant vector comprising a DNA according to  claim 25  or  26  or a portion thereof.  
     
     
         29 . A transformant co-transformed with a recombinant vector according to  claim 27  and the recombinant vector as set forth in  claim 28 .  
     
     
         30 . A process for producing antibody to human medulloblastoma cells using gene recombination technology comprising a culturing a transformant according to  claim 29  and then isolating a target antibody produced.  
     
     
         31 . A single-chain Fv composed by linking an H chain V region according to  claim 11  with an L chain V region as set forth in claim I by means of a peptide linker.  
     
     
         32 . A single-chain Fv according to  claim 31  wherein said linker peptide has the following amino acid sequence: 
       Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 
     
     
         33 . A single-chain Fv according to  claim 31  or  32  comprising an H chain V region having an amino acid sequence of amino acid numbers from 1 to 116 in the amino acid sequence set forth in SEQ ID NO:80, and an L chain V region having an amino acid sequence of amino acid numbers from 1 to 106 in the amino acid sequence set forth in SEQ ID NO:40, 43, 46, 47, 50, 51, 54, 55, 58, 61, 62. 63, 66, 69, 70 or 73.  
     
     
         34 . A single-chain Pv according to  claim 31  or  32  comprising an H chain V region having an amino acid sequence of amino acid numbers from 1 to 116 i:: the amino acid sequence set forth in SEQ ID NO. 80, and an L chain V region having an amino acid sequence of amino acid numbers from 1 to 106 in the amino acid sequence set forth in SEQ ID NO:73.  
     
     
         35 . A single-chain Fv according to  claim 31  having an amino acid sequence as set forth in SEQ ID NO; 39.  
     
     
         36 . A DNA coding for a single-chain Fv according to any one of claims  31  to 35.  
     
     
         37 . A recombinant vector comprising a DNA according to  claim 36 .  
     
     
         38 . A host transfected with a recombinant vector according to  claim 37 .  
     
     
         39 . A process for producing a single-chain Fv comprising culturing a transformant according To  claim 38  and recovering single-chain Fv region from said culture.