Antibody preparation

Abstract

An IgG antibody is provided having a binding affinity for the CD3 antigen complex in which in the heavy chain has a variable region framework together with at least one CDR selected from the amino acid sequences of SEQ ID No 2, 4 and 6 and respective conservatively modified variants thereof and the light chain has a variable region framework together with at least one CDR selected from the amino acid sequences of SEQ ID No 8, 10 and 12 and respective conservatively modified variants thereof characterised in that the heavy chain variable region framework corresponds in sequence to the human type sequence and the light chain variable region framework includes one or more of the specific amino acids characteristic of the rodent type sequence. The novel antibody is capable of being expressed by mammalian cell expression systems at enhanced yields.

1 . An IgG antibody having a binding affinity for the CD3 antigen complex in which in the heavy chain has a variable region framework together with at least one CDR selected from the amino acid sequences of SEQ ID No 2, 4 and 6 and respective conservatively modified variants thereof and the light chain has a variable region framework together with at least one CDR selected from the amino acid sequences of SEQ ID No 8, 10 and 12 respective conservatively modified variants thereof 
 characterised in that the heavy chain variable region framework corresponds in sequence to the human type sequence and the light chain variable region framework includes one or more of the specific amino acids characteristic of the rodent type sequence.    
     
     
         2 . An IgG antibody as claimed in  claim 1  characterised in that the light chain variable region includes sufficient amino acids specific to the rodent type sequence such that the light and heavy chains asssociate more strongly than when the light chain variable region is of the corresponding fully human type.  
     
     
         3 . An IgG antibody as claimed in  claim 1  or  claim 2  characterised in that the light chain variable region includes sufficient amino acids specific to the rodent type sequence such that when DNA encoding for the antibody is expressed in PEE12 cells, an antibody yield of in excess of 50 μg/ml is obtained.  
     
     
         4 . An antibody as claimed in any one of  claims 1  to  3  characterised in that the light chain variable region corresponds entirely to the rat sequence.  
     
     
         5 . An antibody as claimed in any one of the preceding claims characterised in that the light chain variable region corresponds to that of SEQ ID No 14 or that sequence altered such that one or more, but not all, of the amino acids Gln-1, Ala-2, Val-3, Val-4, Ala-7, Asn-8, Thr-12, Leu-14, Ser-16, Lys-19, Leu-20, Leu-39, Tyr-40, Glu-41, Ser-44, Met-48, Tyr-50, Phe-75, His-79, Asn-80, Val-81, Ala-82, Ile-83, Ile-88 and Phe-90 are substituted by corresponding human amino acids selected from Asp-1, Phe-2, Met-3, Leu-4, Pro-7, His-8, Glu-12, Pro-14, Lys-15, Ile-19, Ile-20, Gln-39, Arg-40, Pro-41, Ala-44, Val-48, Phe-50, Ser-75, Ser-79, Gly-80, Leu-81, Gln-82, Thr-83, Asp-88 and Tyr-90.  
     
     
         6 . An antibody as claimed in any one of the preceding claims characterised in that the heavy chain and/or light chain each have all three of their respective CDRs of SEQ ID No 2, 4 and 6 and SEQ. ID. No 8, 10 and 12.  
     
     
         7 . An antibody as claimed in any one of the preceding claims characterised in that the antibody is aglycosylated.  
     
     
         8 . An antibody as claimed in any one of the preceding claims characterised in that the CDRs are arranged in the heavy chain in the sequence in the order: human framework region 1/SEQ ID No 2/human framework region 2/SEQ ID No 4/human framework region 3/SEQ ID No 6/human framework region 4 in a leader to constant domain (n-terminal to C-terminal) direction and in the light chain in the sequence: rodent framework region 1/SEQ ID No 8/rodent framework region 2/SEQ ID No 10/rodent framework region 3/SEQ ID No 12/rodent framework region 4 in a leader to constant domain direction.  
     
     
         9 . An antibody as claimed in any one of the preceding claims characterised in that the human framework regions comprise amino acid sequences SEQ ID No 21, 22, 23 and/or 24  
     
     
         10 . An antibody as claimed in any one of the preceding claims characterised in that the one or more preferred CDRs of the heavy chain of the rat anti-CD3 antibody are present in a human variable domain framework which has the amino acid sequence reading in the leader to constant region direction comprising 
 SEQ ID No 21/CDR/SEQ ID No 22/CDR/SEQ ID No 23/CDR/SEQ ID No 24.    
     
     
         11 . An antibody as claimed in any one of the preceding claims characterised in that the antibody constant region is of human type.  
     
     
         12 . An antibody as claimed in  claim 11  characterised in that the antibody constant region corresponds to that of human origin.  
     
     
         13 . An antibody as claimed in any one of the preceding claims characterised in that the antibody constant region comprises the human type lambda constant region.  
     
     
         14 . An antibody as claimed in any one of the preceding claims characterised in that the rodent light chain variable region is attached to human type lambda constant region.  
     
     
         15 . An antibody as claimed in any one of the preceding claims characterised in that it comprises a rat light chain variable domain framework region of SEQ ID No 14.  
     
     
         16 . An antibody as claimed in any one of the preceding claims characterised in that it comprises a rat human chimeric light chain and lambda constant region amino acid sequence SEQ ID No 18.  
     
     
         17 . An aglycosylated antibody according to any of  claims 1  to  16 , in which the heavy chain constant region is of an IgG1 isotype.  
     
     
         18 . An aglycosylated antibody according any one of the preceding claims in which asparagine residue at position 297 of the constant region heavy chain is replaced by an alternative amino acid residue.  
     
     
         19 . An aglycosylated antibody according to  claim 18 , in which the asparagine residue is replaced by an alanine residue.  
     
     
         20 . An aglycosylated antibody according to any of the preceding claims, in which only one of the arms thereof has an affinity for the CD3 antigen.  
     
     
         21 . An aglycosylated antibody according to  claim 20  which is monovalent.  
     
     
         22 . An aglycosylated antibody according to  claim 21 , in which one half of the antibody consists of a complete heavy chain and light chain and the other half consists of a similar but truncated heavy chain lacking the binding site for the light chain.  
     
     
         23 . An aglycosylated antibody according to any one of the preceding claims characterised in that it is in the form of a pharmaceutical composition comprising a physiologically acceptable diluent or carrier.  
     
     
         24 . An aglycosylated antibody according to any of  claims 1  to  23  for use in therapy.  
     
     
         25 . Use of an aglycosylated antibody according to any of  claims 1  to  24  for the manufacture of a medicament for use in immunosuppression or treating cancer.  
     
     
         26 . Recombinant nucleic acid encoding for an antibody as claimed in any one of the preceding claims.  
     
     
         27 . Recombinant nucleic acid as claimed in  claim 26  characterised in that it comprises a nucleotide sequence of SEQ ID No 13.  
     
     
         28 . Recombinant nucleic acid as claimed in  claim 26  or  27  characterised in that it comprises a nucleotide sequence of SEQ ID No 17.  
     
     
         29 . Recombinant nucleic acid as claimed in any one of  claims 26  to  28  characterised in that it encodes for a peptide of amino acid sequence SEQ ID No 20.  
     
     
         30 . Recombinant nucleic acid as claimed in any one of  claims 26  to  29  characterised in that it encodes for an amino acid sequence reading in the leader to constant region direction 
 SEQ ID No. 25/CDR/SEQ ID No. 26/CDR/SEQ ID No. 27/CDR/SEQ ID No. 28.  
 
     
     
         31 . Recombinant nucleic acid as claimed in any one of  claims 22  to  26  characterised in that it encodes for an amino acid sequence SEQ ID No 14  
     
     
         32 . Recombinant nucleic acid as claimed in any one of  claims 22  to  27  characterised in that it is DNA.  
     
     
         33 . Recombinant nucleic acid characterised in that it encodes for a protein comprising an amino acid sequence SEQ ID No 18  
     
     
         34 . Recombinant DNA of SEQ ID No 17.  
     
     
         35 . A protein expression system characterised in that it comprises a recombinant nucleic acid as claimed in any one of  claims 26  to  34 .  
     
     
         36 . A system as claimed in  claim 35  characterised in that it comprises a vector incorporating nucleic acid as claimed in any one of  claims 26  to  34 .  
     
     
         37 . A system as claimed in  claim 35  or  36  characterised in that it comprises separate constructs of recombinant nucleic acid encoding for heavy and light chains respectively.  
     
     
         38 . A system as claimed in  claim 37  wherein the constructs encode for chains with constant regions.  
     
     
         39 . A prokaryotic or eucaryotic cell expressing an antibody as claimed in any one of  claims 1  to  24 .  
     
     
         40 . A cell as claimed in  claim 39  characterised in that it comprises nucleic acid as claimed in any one of  claims 26  to  34 .  
     
     
         41 . A cell as claimed in  claim 39  or  40  characterised in that it is an immortalised human cell.  
     
     
         42 . A process for producing an antibody as claimed in any one of  claims 1  to  24  characterised in that it comprises culturing a cell as claimed in any one of  claims 39  to  41 .  
     
     
         43 . A method of treating a patient having cancer or requiring immunosuppression which comprises administering to said patient a therapeutically effective amount of a ligand or an antibody or fragment thereof according to any of  claims 1  to  24 .