Humanized, anti-n2 antibodies

Abstract

The present invention encompasses humanized antibodies that specifically bind N2 peptide, methods for the preparation thereof and methods for the use thereof.

1 - 42 . (canceled) 
     
     
         43 . A humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein:
 i. the VH region comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 comprises SEQ ID NO: 3, VH CDR2 comprises SEQ ID NO: 4 and VH CDR3 comprises SEQ ID NO: 5;   ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:
 a. the VH FWR1 comprises SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23; 
 b. The VH FWR2 comprises SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:24 or SEQ ID NO: 50; 
 c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and 
 d. VH FWR4 comprises SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; 
   iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3 wherein the VL CDR1 comprises SEQ ID NO: 6, VH CDR2 comprises SEQ ID NO: 7 and VH CDR3 comprises SEQ ID NO: 8;   iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:
 a. the VL FWR1 comprises SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35; 
 b. VL FWR2 comprises SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36; 
 c. VL FWR3 comprises SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and 
 d. VL FWR4 comprises SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38. 
   
     
     
         44 . The antibody or antigen-binding fragment of  claim 43 , wherein:
 i. the VH region comprises three complementarity determining regions (CDRs) VH CDR1, VH CDR2 and VH CDR3 wherein the VH CDR1 consists of SEQ ID NO:3, VH CDR2 consists of SEQ ID NO: 4 and VH CDR3 consists of SEQ ID NO: 5;   ii. the VH region comprises four framework regions (FWR) VH FWR1, VH FWR2, VH FWR3 and VH FWR4 wherein:
 a. the VH FWR1 consists of SEQ ID NO: 15, SEQ ID NO: 19 or SEQ ID NO: 23; 
 b. The VH FWR2 consists of SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO:24 or SEQ ID NO: 50; 
 c. VH FWR3 comprises SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 51 or SEQ ID NO: 52; and 
 d. VH FWR4 consists of SEQ ID NO: 18, SEQ ID NO: 22, or SEQ ID NO: 26; 
   iii. the VL region comprises three complementarity determining regions (CDRs) VL CDR1, VL CDR2 and VL CDR3 wherein the VL CDR1 consists of SEQ ID NO: 6, VH CDR2 comprises SEQ ID NO: 7 and VH CDR3 consists of SEQ ID NO: 8;   iv. the VL region comprises four framework regions (FWR) VL FWR1, VL FWR2, VL FWR3 and VL FWR4 wherein:
 a. the VL FWR1 consists of SEQ ID NO: 27, SEQ ID NO: 31, or SEQ ID NO: 35; 
 b. VL FWR2 consists of SEQ ID NO: 28, SEQ ID NO: 32, or SEQ ID NO: 36; 
 c. VL FWR3 consists of SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37; and 
 d. VL FWR4 consists of SEQ ID NO: 30, SEQ ID NO: 34, or SEQ ID NO: 38. 
   
     
     
         45 . The antibody or antigen-binding fragment of  claim 43 , wherein the VH region comprises a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49. 
     
     
         46 . The antibody or antigen-binding fragment of  claim 43 , wherein the VL region comprises a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. 
     
     
         47 . The antibody or antigen-binding fragment of  claim 43 , wherein the VH region consists of a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49. 
     
     
         48 . The antibody or antigen-binding fragment of  claim 43 , wherein the VL region consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO:13 and SEQ ID NO: 14. 
     
     
         49 . The antibody or antigen-binding fragment of  claim 43 , wherein the VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 12. 
     
     
         50 . The antibody or antigen-binding fragment of  claim 43 , wherein the VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 13. 
     
     
         51 . The antibody or antigen-binding fragment of claim  1 , wherein the VH region consists of the amino acid sequence of SEQ ID NO: 49 and the VL region consists of the amino acid sequence of SEQ ID NO: 14. 
     
     
         52 . The antibody or antigen-binding fragment of  claim 43 , wherein the VH region consists of the amino acid sequence of SEQ ID NO: 54 and VL region consists of a sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. 
     
     
         53 . A humanized, anti-N2 antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) region comprising a sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49. 
     
     
         54 . The antibody or antigen-binding fragment of  claim 43 , wherein the isotype of the constant region is IgG1, IgG2, IgG3, or IgG4. 
     
     
         55 . The antibody or antigen-binding fragment of  claim 54 , wherein the isotype of the IgG constant region is IgG1. 
     
     
         56 . The antibody or antigen-binding fragment of  claim 54 , wherein the isotype of the IgG constant region is IgG4. 
     
     
         57 . The antibody or antigen-binding fragment of  claim 43  having a heavy chain immunoglobulin constant domain selected from the group consisting of a human IgG1 constant domain and a human IgG4 constant domain. 
     
     
         58 . The antibody or antigen-binding fragment of  claim 43  having a human Ig kappa constant domain. 
     
     
         59 . The antibody or antigen-binding fragment of  claim 43 , wherein the antibody is aglycosylated. 
     
     
         60 . The antibody or antigen-binding fragment of  claim 43  having a human IgG1 constant domain that is aglycosylated by replacing the amino acid corresponding to asparagine (Asn) 297 of the constant region heavy chain with an alternative amino acid residue. 
     
     
         61 . The antibody or antigen-binding fragment of  claim 60 , wherein the Asn 297 is replaced with glutamine, alanine, histidine or glycine. 
     
     
         62 . The antibody or antigen-binding fragment of  claim 61 , wherein the Asn 297 is replaced with glutamine. 
     
     
         63 . The antibody or antigen-binding fragment of  claim 43 , wherein the heavy chain immunoglobulin constant domain is a human IgG4 constant domain wherein serine 228 is replaced with proline. 
     
     
         64 . The antibody or antigen-binding fragment of  claim 43 , which is a scFv, diabody, Fab, minibody or scFv-Fc. 
     
     
         65 . A nucleotide sequence encoding the antibody or antigen-binding fragment of  claim 43 . 
     
     
         66 . A nucleotide sequence encoding a VH region, wherein the VH region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 49. 
     
     
         67 . (canceled) 
     
     
         68 . A nucleotide sequence encoding a VL region, wherein the VL region comprises an amino acid selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14. 
     
     
         69 - 71 . (canceled) 
     
     
         72 . A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antibody or antigen-binding fragment of  claim 43 . 
     
     
         73 . A method of treating an inflammatory disease or disorder comprising administering to a subject an effective amount of the antibody or antigen-binding fragment of  claim 43 . 
     
     
         74 . The method of  claim 73 , wherein the inflammatory disease or disorder is ischemia-reperfusion injury. 
     
     
         75 - 76 . (canceled) 
     
     
         77 . The method of  claim 73 , wherein the ischemia-reperfusion results after myocardial infarction, stroke or a surgical procedure.