Humanized antibodies specific for hsp65-derived peptide-6, methods and uses thereof

Abstract

The present invention relates to humanized antibodies that specifically bind a polypeptide comprising peptide-6 as denoted by SEQ ID NO. 15, that is an HSP65 derived peptide. More specifically, the invention relates to humanized anti-peptide-6 antibodies, compositions, methods and uses thereof for the treatment of immune-related disorders, specifically, inflammatory disorders such as arthritis, IBD, psoriasis, diabetes and MS. The invention further provides combined compositions and kit combining the humanized antibodies of the invention and at least one anti-inflammatory agent, as well as uses of the humanized antibodies in diagnostic kits and methods.

1 . A composition comprising a pharmaceutically acceptable carrier, excipient or diluent, and, as an active ingredient, at least one humanized antibody or antigen-binding fragment thereof that specifically binds a polypeptide comprising SEQ ID NO: 15, the humanized antibody comprising:
 a) a heavy chain variable region comprising an amino acid sequence which is at least about 70% identical to SEQ ID NO. 1, said heavy chain variable region comprising Cys at position H22, Ser at H23, Gly at H26, Phe at H27, Ser at H28, Leu at H29, Ser at H30, Thr at H31, Ser at H32, Asn at H33, Met at H34, Gly at H35, Val at H35A, Gly at H35B, Leu at H48, His at H50, Ile at H51, Leu at H52, Trp at H53, Asn at H54, Asp at H55, Ser at H56, Lys at H57, Tyr at H58, Tyr at H59, Asn at H60, Pro at H61, Ala at H62, Leu at H63, Lys at H64, Ser at H65, Cys at H92, Met at H95, Gly at H96, Gly at H97, Tyr at H98, Tyr at H99, Gly at H100, Asn at H100A, Tyr at H100B, Gly at H100C, Tyr at H100D, Tyr at H100E, Ala at H100F, Met at H100G, Asp at H101 and Tyr at H102, and, optionally, at least one of Leu at H49, Tyr at H74, Ile at H11, Ser at H41 and Ser at H108; and   b) a light chain variable region comprising an amino acid sequence which is at least about 70% identical to SEQ ID NO. 2, said light chain variable region comprising Gln at position L1, Cys at L23, Thr at L24, Ala at L25, Ser at L26, Ser at L27, Ser at L27A, Val at L28, Ser at L29, Ser at L30, Ser at L31, Tyr at L32, Leu at L33, His at L34, Trp at L47, Ser at L50, Thr at L51, Ser at L52, Asn at L53, Leu at L54, Ala at L55, Ser at L56, Tyr at L71, Cys at L88, His at L89, Gln at L90, Tyr at L91, His at L92, Arg at L93, Ser at L94, Pro at L95, Pro at L96 and Thr at L97 and, optionally, at least one of Met at L21, Ile at L10 and Ala at L80;   wherein the positions are determined according to the Kabat numbering system, and wherein said active ingredient is presented in an amount effective for the treatment and amelioration of an immune-related disorder.   
     
     
         2 . The composition according to  claim 1 , wherein said heavy chain variable region comprises an amino acid sequence which is at least about 70% identical to SEQ ID NO. 1, said heavy chain variable region comprising a substitution in at least one position selected from the group consisting of H10, H11, H12, H13, H15, H19, H41, H49, H74, H75, H79, H81, H82, H82A, H82C, H84, H85 and H108, and wherein said light chain variable region comprises an amino acid sequence which is at least about 70% identical to SEQ ID NO. 2, said light chain variable region comprising a substitution in at least one position selected from the group consisting of L10, L11, L13, L15, L19, L21, L22, L42, L43, L60, L70, L72, L78, L79, L80, L83 and L100, wherein the positions are determined according to the Kabat numbering system. 
     
     
         3 . The composition according to  claim 2 , wherein said humanized antibody comprising:
 a) a heavy chain variable region as shown in SEQ ID NO: 1, except that H10 is Ala, H11 is Ile or Leu, H12 is Val, H13 is Lys, H15 is Thr, H19 is Thr, H41 is Ser or Ala, H49 is Leu or Ala, H74 is Tyr or Ser, H75 is Lys, H79 is Val, H81 is Thr, H82 is Met, H82A is Thr, H82C is Met, H84 is Pro, H85 is Val and H108 is Ser or Leu; and   b) a light chain variable region as shown in SEQ ID NO: 2, except that L10 is Ile or Thr, L11 is Leu, L13 is Leu, L15 is Pro, L19 is Ala, L21 is Met or Leu, L22 is Ser, L42 is Lys, L43 is Ala, L60 is Ser, L70 is Asp, L72 is Thr, L78 is Leu, L79 is Gln, L80 is Ala or Pro, L83 is Phe and L100 is Gln;   wherein the positions are determined according to the Kabat numbering system.   
     
     
         4 . The composition according to  claim 2 , wherein said humanized antibody comprising:
 a) a heavy chain variable region comprising an amino acid sequence that is at least about 60% identical to any one of SEQ ID NO. 21, 22, 23 and 20, wherein said heavy chain variable region comprises a substitution in at least one position selected from the group consisting of H10, H11, H12, H13, H15, H19, H41, H49, H74, H75, H79, H81, H82, H82A, H82C, H84, H85 and H108; and   b) a light chain variable region comprising an amino acid sequence that is at least about 60% identical to any one of SEQ ID NO. 26, 24 and 25, wherein said light chain variable region comprises a substitution in at least one position selected from the group consisting of L10, L11, L13, L15, L19, L21, L22, L42, L43, L60, L70, L72, L78, L79, L80, L83 and L100;   wherein the positions are determined according to the Kabat numbering system.   
     
     
         5 . The composition according to  claim 4 , wherein said heavy chain variable region is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 20, and wherein said light chain variable region is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 24 and SEQ ID NO: 25. 
     
     
         6 . The composition according to  claim 5 , wherein the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 24, and SEQ ID NO: 25. 
     
     
         7 . The composition according to  claim 6 , wherein the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is SEQ ID NO: 26. 
     
     
         8 . The composition according to  claim 6 , wherein the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is SEQ ID NO: 25. 
     
     
         9 . The composition according to  claim 6 , wherein the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is SEQ ID NO: 24. 
     
     
         10 . The composition according to  claim 6 , wherein the heavy chain variable region is SEQ ID NO: 22 and the light chain variable region is selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26. 
     
     
         11 . The composition according to  claim 10 , wherein the heavy chain variable region is SEQ ID NO: 22 and the light chain variable region is SEQ ID NO: 25. 
     
     
         12 . The composition according to  claim 1 , wherein said active ingredient is presented in an amount effective for the treatment and amelioration of Behçet's syndrome. 
     
     
         13 . The composition according to  claim 1 , wherein said active ingredient is presented in an amount effective for the treatment and amelioration of uveitis. 
     
     
         14 . A method for the treatment or amelioration of an immune-related disorder, said method comprising the step of administering to a subject in need thereof a therapeutically effective amount of at least one humanized antibody or antigen-binding fragment thereof that specifically binds a polypeptide comprising SEQ ID NO: 15, or of a composition comprising the same, wherein said humanized antibody comprises:
 a) a heavy chain variable region comprising an amino acid sequence which is at least about 70% identical to SEQ ID NO. 1, said heavy chain variable region comprising Cys at position H22, Ser at H23, Gly at H26, Phe at H27, Ser at H28, Leu at H29, Ser at H30, Thr at H31, Ser at H32, Asn at H33, Met at H34, Gly at H35, Val at H35A, Gly at H35B, Leu at H48, His at H50, Ile at H51, Leu at H52, Trp at H53, Asn at H54, Asp at H55, Ser at H56, Lys at H57, Tyr at H58, Tyr at H59, Asn at H60, Pro at H61, Ala at H62, Leu at H63, Lys at H64, Ser at H65, Cys at H92, Met at H95, Gly at H96, Gly at H97, Tyr at H98, Tyr at H99, Gly at H100, Asn at H100A, Tyr at H100B, Gly at H100C, Tyr at H100D, Tyr at H100E, Ala at H100F, Met at H100G, Asp at H101 and Tyr at H102, and, optionally, at least one of Leu at H49, Tyr at H74, Ile at H11, Ser at H41 and Ser at H108; and   b) a light chain variable region comprising an amino acid sequence which is at least about 70% identical to SEQ ID NO. 2, said light chain variable region comprising Gln at position L1, Cys at L23, Thr at L24, Ala at L25, Ser at L26, Ser at L27, Ser at L27A, Val at L28, Ser at L29, Ser at L30, Ser at L31, Tyr at L32, Leu at L33, His at L34, Trp at L47, Ser at L50, Thr at L51, Ser at L52, Asn at L53, Leu at L54, Ala at L55, Ser at L56, Tyr at L71, Cys at L88, His at L89, Gln at L90, Tyr at L91, His at L92, Arg at L93, Ser at L94, Pro at L95, Pro at L96 and Thr at L97 and, optionally, at least one of Met at L21, Ile at L10 and Ala at L80;   wherein the positions are determined according to the Kabat numbering system.   
     
     
         15 . The method according to  claim 14 , wherein said heavy chain variable region comprises an amino acid sequence which is at least about 70% identical to SEQ ID NO. 1, said heavy chain variable region comprising a substitution in at least one position selected from the group consisting of H10, H11, H12, H13, H15, H19, H41, H49, H74, H75, H79, H81, H82, H82A, H82C, H84, H85 and H108, and wherein said light chain variable region comprises an amino acid sequence which is at least about 70% identical to SEQ ID NO. 2, said light chain variable region comprising a substitution in at least one position selected from the group consisting of L10, L11, L13, L15, L19, L21, L22, L42, L43, L60, L70, L72, L78, L79, L80, L83 and L100, wherein the positions are determined according to the Kabat numbering system. 
     
     
         16 . The method according to  claim 15 , wherein said humanized antibody comprising:
 a) a heavy chain variable region comprising an amino acid sequence that is at least about 60% identical to any one of SEQ ID NO. 21, 22, 23 and 20, wherein said heavy chain variable region comprises a substitution in at least one position selected from the group consisting of H10, H11, H12, H13, H15, H19, H41, H49, H74, H75, H79, H81, H82, H82A, H82C, H84, H85 and H108; and   b) a light chain variable region comprising an amino acid sequence that is at least about 60% identical to any one of SEQ ID NO. 26, 24 and 25, wherein said light chain variable region comprises a substitution in at least one position selected from the group consisting of L10, L11, L13, L15, L19, L21, L22, L42, L43, L60, L70, L72, L78, L79, L80, L83 and L100;   wherein the positions are determined according to the Kabat numbering system.   
     
     
         17 . The method according to  claim 16 , wherein said heavy chain variable region is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 20, and wherein said light chain variable region is selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 24 and SEQ ID NO: 25. 
     
     
         18 . The method according to  claim 17 , wherein the heavy chain variable region is SEQ ID NO: 21 and the light chain variable region is SEQ ID NO: 26. 
     
     
         19 . The method according to  claim 14 , for the treatment and amelioration of Behçet's syndrome. 
     
     
         20 . The method according to  claim 14 , for the treatment and amelioration of uveitis.