Humanized antibodies

1. A method of generating a library comprising a plurality of polypeptides, for selection of an isolated humanized monoclonal antibody that binds to a target antigen, comprising:

(a) 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 antibody;

(b) 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;

(c) aligning each of the non-human donor CDR-L1, CDR-L2, and CDR-L3 sequences with the corresponding germline CDR sequences from said human VL, and each of the non-human CDR-H1 and CDR-H2 sequences with corresponding germline CDR sequences from said human VH;

(d) identifying positions in CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2 where a human germline residue is the same as, or different from, the corresponding non-human donor residue; and

(e) generating a library of polypeptides, each polypeptide comprising an antibody variable domain, wherein said antibody variable domain comprises

(1) a VH domain comprising the human germline VH from step (b);

(2) a VL domain comprising the human germline VL from step (b);

(3) a CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2, wherein for each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2:

(i) if the human germline residue at said position is the same as the corresponding non-human donor residue, all polypeptides in the library comprise the human germline residue at said position;

(ii) if the human germline residue at the position is different from the corresponding non-human donor residue, a portion of the polypeptides in the library comprise the human germline residue at said position, the remainder of the polypeptides comprise the corresponding non-human donor residue at said position;

(iii) 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; and

(iv) 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

(4) a CDR-H3, wherein for each individual position within the CDR-H3, the residue is any one of the 20 natural amino acid residues and

wherein the polypeptide binds said target antigen with a binding affinity (Kd) value that is equal or less than the binding affinity (Kd) value of said non-human donor antibody.

2. The method of claim 1 , wherein for each individual position within CDR-L1, CDR-L2, CDR-L3, CDR-H1, and CDR-H2, if the human germline residue and non-human donor residue are different according to step (d), the percentage of polypeptides comprising the human germline residue at said position is from 15% to 85%, and the remainder of the polypeptides comprise the corresponding non-human donor residue at said position.

3. The method of claim 1 , wherein for each individual position within CDR-H3, each of the 20 natural amino acid residues is represented by at least 0.1% of the polypeptides in the library.

4. The method of claim 1 , wherein said human germline VH framework sequence comprises a VH3, VH1, or VH5 framework sequence.

5. The method of claim 1 , wherein said human germline VH framework sequence comprises a VH germline consensus framework sequence.

6. The method of claim 1 , wherein said human germline VH framework sequence comprises the VH framework sequence of any one of the consensus sequences listed in Table 2 and Table 5.

7. The method of claim 1 , wherein said human germline VL framework sequence comprises a V K or Vλ framework sequence.

8. The method of claim 1 , wherein said human germline VL framework sequence comprises a VL germline consensus framework sequence.

9. The method of claim 1 , wherein said human germline VL framework sequence comprises the VL framework sequence of any one of the human germline sequences listed in Table 3, Table 4 and Table 6.

10. The method of generating a library of claim 1 , wherein said human germline VH framework sequence is derived from the VH framework sequence of human germline DP54.

11. The method of generating a library of claim 1 , wherein said human germline VH framework sequence is derived from the VH framework sequence of SEQ ID NO:74.

12. The method of generating a library of claim 1 , wherein said human germline VL framework sequence is derived from a V K framework sequence.

13. The method of generating a library of claim 1 , wherein said human germline VL framework sequence is derived from the VL framework sequence of human germline DPK9.

14. The method of generating a library of claim 1 , wherein said human germline VL framework sequence is derived from the VL framework sequence of SEQ ID NO:144.

15. The method of generating a library of claim 1 , wherein the polypeptide maintains highly specific binding to said target antigen as compared to binding of said non-human donor antibody to said target antigen.