Humanized antibodies

An isolated humanized monoclonal antibody or antigen-binding fragment thereof that binds to a target antigen prepared by a method comprising,

• (1) obtaining the sequence of a non-human donor antibody that binds to said target antigen, and determining the donor CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 sequences of said non-human donor antibody;
• (2) obtaining the sequences of a human germline VL and a human germline VH, and determining the germline framework and germline CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 sequences of said human VL and VH;
• (3) aligning each of the non-human donor CDR-L1, CDR-L2, and CDR-L3 sequences with the corresponding germline CDR sequence from said human VL, and each of the non-human CDR-H1 and CDR-H2 sequences with corresponding germline CDR sequence from said human VH;
• (4) identifying positions in CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 where human germline residue is the same as, or different from, the corresponding non-human donor residue;
• and
• (5) generating the isolated humanized monoclonal antibody or antigen-binding fragment thereof by changing a non-human donor residue of the donor CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 to the corresponding human germline residue if the human germline residue at the position is different from the corresponding non-human residue, wherein:
• (a) said isolated humanized monoclonal antibody or antigen-binding fragment thereof comprises (i) a VH domain comprising a human germline VH framework sequence from step (2) and a VL domain comprising a human germline VL framework sequence from step (2);
• and (ii) a CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2 from step (5);
• and (iii) a CDR-H3;
• (b) for each position within said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2, the residue is either human germline residue from said human germline VL or VH, or corresponding residue from said non-human donor antibody;
• (c) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 each comprises at least one more human germline residue as compared to the corresponding non-human donor CDR,
• (d) said CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 each comprises at least one more non-human donor residue as compared to the corresponding human germline VH or VL CDR;
• and
• (e) for each position within CDR-H3, the residue is any one of the 20 natural amino acid residues.