Therapeutic Uses of Humanized Antibodies Against ALPHA-4 Integrin

Abstract

The invention provides method of treatment using humanized immunoglobulins that specially bind to alpha-4 integrin. The methods are useful for treatment of asthma, atherosclerosis, AIDS dementia, diabetes, inflammatory bowel disease, rheumatoid arthritis, transplant rejection, graft versus host disease, tumor metastasis, nephritis, atopic dermatitis, psoriasis, myocardial ischemia, and acute leukocyte mediated lung injury.

1 - 26 . (canceled) 
     
     
         27 . A method for producing a humanized immunoglobulin comprising culturing a recombinant cell under conditions suitable for production of the immunoglobulin wherein the recombinant cell comprises, in operable linkage with a promoter, (a) a nucleic acid encoding a humanized light chain or (b) a nucleic acid encoding a humanized heavy chain or both (a) and (b) wherein:
 (a) is a nucleic acid encoding a humanized light chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the mouse 21-6 immunoglobulin light chain variable domain designated SEQ. ID. No. 2, and a variable region framework from a human kappa light chain variable region framework sequence provided that at least one position selected from a first group consisting of L45, L49, L58 and L69 (Kabat numbering convention) is occupied by the same amino acid residue present in the equivalent position of the mouse 21-6 immunoglobulin light chain variable region framework; and   (b) is a nucleic acid encoding a humanized heavy chain comprising three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of the mouse 21-6 immunoglobulin heavy chain variable domain designated SEQ. ID. No. 4, and a variable region framework from a human heavy chain variable region framework sequence provided that at least one position selected from a second group consisting of H27, H28, H29, H30, H44, H71 (Kabat numbering convention) is occupied by the same amino acid residue present in the equivalent position of the mouse 21-6 immunoglobulin heavy chain variable region framework, and   purifying the immunoglobulin from the cultured recombinant cell.   
     
     
         28 . The method of  claim 27  wherein the humanized light chain variable region framework is from an RE1 variable region framework sequence (SEQ. ID No:6) provided that at least one position is selected from the first group, and provided that at least one position selected from a third group consisting of positions L104, L105 and L107 (Kabat numbering convention) is occupied by the same amino acid residue present in the equivalent position of a kappa light chain from any human immunoglobulin other than RE1 (SEQ. ID No:6). 
     
     
         29 . The method of  claim 28 , wherein the humanized heavy chain variable region framework is from a 21/28′CL variable region framework sequence (SEQ. ID No:10). 
     
     
         30 . The method of  claim 29 , wherein the humanized light chain variable region framework comprises at least three amino acids from the mouse 21.6 immunoglobulin at positions in the first group and three amino acids from the kappa light chain from the human immunoglobulin other than REI at positions in the third group, and the humanized heavy chain variable region framework comprises at least five amino acids from the mouse 21.6 immunoglobulin at positions in the second group. 
     
     
         31 . The method of  claim 30 , wherein the humanized light chain variable region framework is identical to the RE1 light chain variable region framework sequence except for the at least three positions from the first group and the three positions from the third group, and the heavy chain variable region framework is identical to the 21/28′CL heavy chain variable region framework sequence (SEQ. ID No:10) except for the at least five positions from the second group. 
     
     
         32 . The method of  claim 31 , wherein the at least three positions from the first group are positions L45, L58 and L69, and at the least five positions from the second group are positions H27, H28, H29, H30 and H71. 
     
     
         33 . The method of  claim 32 , wherein the humanized light chain comprises complementarity determining regions that are identical to the corresponding complementarity determining regions of the mouse 21-6 heavy chain, and the humanized heavy chain comprises complementarity determining regions that are identical to the corresponding complementarity determining regions of the mouse 21-6 heavy chain, except that the CDR3 region of the humanized heavy chain may or may not comprise a phenylalanine residue at position H98. 
     
     
         34 . The method of  claim 33 , wherein the CDR3 of the humanized heavy chain comprises a phenylalanine residue at position H98. 
     
     
         35 . The method of  claim 27 , wherein the amino acid sequence of the mature light chain variable region is
 (a) the sequence designated La in  FIG. 6  (SEQ ID NO: 7) or   (b) Lb in  FIG. 6  (SEQ ID NO: 8).   
     
     
         36 . The method of  claim 27 , wherein the amino acid sequence of the mature heavy chain variable region is
 (a) the sequence designated Ha in  FIG. 7  (SEQ ID NO: 11).   (b) the sequence designated Hb in  FIG. 7  (SEQ ID NO: 12).   (c) is the sequence designated Hc in  FIG. 7  (SEQ. ID NO:13).   
     
     
         37 . The method of  claim 35 , wherein the amino acid sequence of the mature heavy chain variable region is
 (a) the sequence designated Ha in  FIG. 7  (SEQ. ID NO:11), or   (b) the sequence designated Hb in  FIG. 7  (SEQ. ID NO:12),   (c) the sequence designated Hc in  FIG. 7  (SEQ. ID NO:13).   
     
     
         38 . The method of  claim 37  wherein the immunoglobulin has the sequence designated Ha in  FIG. 7  (SEQ. ID NO:11), or the sequence designated Hc in  FIG. 7  (SEQ. ID NO:13), and a constant region domain. 
     
     
         39 . The method of  claim 27  wherein (a) and (b) are comprised on separate expression vectors. 
     
     
         40 . The method of  claim 27 , wherein (a) and (b) are comprised on the same expression vector. 
     
     
         41 . The method of  claim 27 , wherein the recombinant cell is a recombinant eukaryotic cell. 
     
     
         42 . The method of claim  21 , wherein the eukaryotic cell is a yeast cell, CHO cell, COS cell, HeLa cell, myeloma cell, B-cell, or hybridoma. 
     
     
         43 . The method of  claim 27 , wherein the recombinant cell is a recombinant procaryotic cell. 
     
     
         44 . The method of claim  23 , wherein the procaryotic cell is an  Escherichia coli, Bacillus subtilus, Salmonella, Serratia , and  Pseudomonas.