Immunoglobulin variants and uses thereof

1. A humanized antibody that binds human CD20, the humanized antibody comprising the heavy and light chain amino acid sequences of SEQ ID NO. 39 and 40, respectively, wherein the binding affinity to human CD20 of the humanized antibody is higher than the binding affinity to human CD20 of an antibody comprising the heavy chain amino acid sequence of SEQ ID NO. 39 with sequence modifications in the variable region wherein position 49 is Ala, position 71 is Arg, and position 73 is Asn, and the light chain amino acid sequence of SEQ ID NO. 40 with a sequence modification in the variable region wherein position 46 is Leu, wherein the amino acid positions are numbered according to the numbering system of Kabat.

2. An antigen-binding fragment of a humanized antibody that binds human CD20, wherein the antigen-binding fragment comprises the CDR1 sequence of SEQ ID NO. 10, the CDR2 sequence of SEQ ID NO. 11, and the CDR3 sequence of SEQ ID NO. 12 in the heavy chain, and the CDR1 sequence of SEQ ID NO. 4, the CDR2 sequence of SEQ ID NO. 5, and the CDR3 sequence of SEQ ID NO. 6 in the light chain; wherein the humanized antibody comprises the heavy and light chain amino acid sequences of SEQ ID NO. 39 and 40, respectively, and wherein the binding affinity to human CD20 of the humanized antibody is higher than the binding affinity to human CD20 of an antibody comprising the heavy chain amino acid sequence of SEQ ID NO. 39 with sequence modifications in the variable region wherein position 49 is Ala, position 71 is Arg, and position 73 is Asn, and the light chain amino acid sequence of SEQ ID NO. 40 with a sequence modification in the variable region wherein position 46 is Leu, wherein the amino acid positions are numbered according to the numbering system of Kabat.

3. The humanized antibody of claim 1 or the antigen-binding fragment of claim 2 conjugated to a cytotoxic agent.

4. The humanized antibody or the antigen-binding fragment of claim 3 wherein the cytotoxic agent is a radioactive isotope or a toxin.

5. A composition comprising the humanized antibody of claim 1 or the antigen-binding fragment of claim 2 , and a pharmaceutically acceptable carrier.

6. An article of manufacture comprising a container and a composition contained therein, wherein the composition comprises the humanized antibody of claim 1 or the antigen-binding fragment of claim 2 .

7. The article of manufacture of claim 6 , further comprising a package insert indicating that the composition can be used to treat non-Hodgkin's lymphoma.

8. A method of producing a humanized antibody comprising the heavy and light chain sequences of SEQ ID NO. 39 and 40, respectively, comprising culturing a cell that produces the humanized antibody and recovering the humanized antibody from the cell culture; and wherein the binding affinity to human CD20 of the humanized antibody is higher than the binding affinity to human CD20 of an antibody comprising the heavy chain amino acid sequence of SEQ ID NO. 39 with sequence modifications in the variable region wherein position 49 is Ala, position 71 is Arg, and position 73 is Asn, and the light chain amino acid sequence of SEQ ID NO. 40 with a sequence modification in the variable region wherein position 46 is Leu, wherein the amino acid positions are numbered according to the numbering system of Kabat.

9. A liquid formulation comprising a humanized antibody or an antigen-binding fragment thereof at 20 mg/mL, 10 mM histidine sulfate at pH5.8, 60 mg/ml sucrose, and 0.2 mg/ml polysorbate 20, wherein the humanized antibody comprises the heavy and light chain sequences of SEQ ID NO. 39 and 40, respectively, wherein the antigen-binding fragment comprises the CDR1 sequence of SEQ ID NO. 10, the CDR2 sequence of SEQ ID NO. 11, and the CDR3 sequence of SEQ ID NO. 12 in the heavy chain, and the CDR1 sequence of SEQ ID NO. 4, the CDR2 sequence of SEQ ID NO. 5, and the CDR3 sequence of SEQ ID NO. 6 in the light chain; and wherein the binding affinity to human CD20 of the humanized antibody is higher than the binding affinity to human CD20 of an antibody comprising the heavy chain amino acid sequence of SEQ ID NO. 39 with sequence modifications in the variable region wherein position 49 is Ala, position 71 is Arg, and position 73 is Asn, and the light chain amino acid sequence of SEQ ID NO. 40 with a sequence modification in the variable region wherein position 46 is Leu, wherein the amino acid positions are numbered according to the numbering system of Kabat.

10. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequences of SEQ ID NO. 42 and 43, respectively, wherein the antigen-binding fragment comprises the three heavy chain CDRs of SEQ ID NO. 42 and the three light chain CDRs of SEQ ID NO. 43.

11. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequences of SEQ ID NO. 46 and 47, respectively, wherein the antigen-binding fragment comprises the three heavy chain CDRs of SEQ ID NO. 46 and the three light chain CDRs of SEQ ID NO. 47.

12. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequences of SEQ ID NO. 48 and 47, respectively, wherein the antigen-binding fragment comprises the three heavy chain CDRs of SEQ ID NO. 48 and the three light chain CDRs of SEQ ID NO. 47.

13. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequences of SEQ ID NO. 49 and 47, respectively, wherein the antigen-binding fragment comprises the three heavy chain CDRs of SEQ ID NO. 49 and the three light chain CDRs of SEQ ID NO. 47.

14. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequences of SEQ ID NO. 50 and 47, respectively, wherein the antigen-binding fragment comprises the three heavy chain CDRs of SEQ ID NO. 50 and the three light chain CDRs of SEQ ID NO. 47.

15. A composition comprising the humanized antibody or the antigen-binding fragment of claim 11 , and a pharmaceutically acceptable carrier.

16. A composition comprising the humanized antibody or the antigen-binding fragment of claim 10 or 14 , and a pharmaceutically acceptable carrier.

17. A humanized antibody that binds human CD20 or an antigen-binding fragment thereof, produced by a method comprising expressing a nucleic acid encoding a humanized antibody comprising the heavy and light chain amino acid sequences of SEQ ID NO. 39 and 40, respectively, in a host cell, and recovering the humanized antibody or an antigen-binding fragment thereof expressed in the host cell, wherein the antigen-binding fragment comprises the CDR1 sequence of SEQ ID NO. 10, the CDR2 sequence of SEQ ID NO. 11, and the CDR3 sequence of SEQ ID NO. 12 in the heavy chain, and the CDR1 sequence of SEQ ID NO. 4, the CDR2 sequence of SEQ ID NO. 5, and the CDR3 sequence of SEQ ID NO. 6 in the light chain; and wherein the humanized antibody encoded by the nucleic acid has a binding affinity to human CD20 that is higher than the binding affinity to human CD20 of an antibody comprising the heavy chain amino acid sequence of SEQ ID NO. 39 with sequence modifications in the variable region wherein position 49 is Ala, position 71 is Arg, and position 73 is Asn, and the light chain amino acid sequence of SEQ ID NO. 40 with a sequence modification in the variable region wherein position 46 is Leu, wherein the amino acid positions are numbered according to the numbering system of Kabat.

18. The humanized antibody of claim 1 or the antigen-binding fragment of claim 2 , wherein the binding affinity is determined by Scatchard analysis.

19. The humanized antibody of claim 1 or the antigen-binding fragment of claim 2 , wherein the binding affinity is determined by a cell-based enzyme-linked immunosorbent assay (ELISA).