CD20 ANTIBODIES AND USES THEREOF

• US 20110195021A1
• Filed: 02/10/2011
• Published: 08/11/2011
• Est. Priority Date: 02/10/2010
• Status: Active Grant

• Current Assignee: ImmunoGen Incorporated
• Original Assignee: ImmunoGen Incorporated
• Inventors: Rui, Lingyun, Park, Peter U., DECKERT, Jutta
• Granted Patent: US 9,173,961 B2
• Time in Patent Office: Days
• US Class Current: 424/1.49
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 39/39541 against normal tissues, cells
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6819 Plant toxins
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61K 51/1027 against receptors, cell-surface antigens or cell-surface determinants
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 31/22 : for herpes viruses
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/14 : of the thyroid hormones, e.g. T3, T4
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 : Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 16/2887 : against CD20
• CPC Class Codes: C07K 16/30 : from tumour cells
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/30 : characterized by aspects of specificity or valency
• CPC Class Codes: C07K 2317/73 : Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/732 : Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/734 : Complement-dependent cytotoxicity [CDC]
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/55 : containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: C12N 2501/48 : Regulators of apoptosis
• CPC Class Codes: G01N 33/57426 : leukemia

Abstract

CD20 is a transmembrane protein of the tetra-spanin family expressed on the surface of B-cells and has been found on B-cells from peripheral blood as well as lymphoid tissues. CD20 expression persists from the early pre-B cell stage until the plasma cell differentiation stage. Conversely, it is not found on hematopoietic stem cells, pro-B cells, differentiated plasma cells or non-lymphoid tissues. In addition to expression in normal B-cells, CD20 is expressed in B-cell derived malignancies such as non-Hodgkin'"'"'s lymphoma (NHL) and B-cell chronic lymphocytic leukemia (CLL). CD20 expressing cells are known to play a role in other diseases and disorders, including inflammation. The present invention includes anti-CD20 antibodies, forms and fragments, having superior physical and functional properties; immunoconjugates, compositions, diagnostic reagents, methods for inhibiting growth, therapeutic methods, improved antibodies and cell lines; and polynucleotides, vectors and genetic constructs encoding same.

Claims

1. An antibody or fragment thereof that specifically bind to a CD20, wherein said antibody or fragment is capable of inducing apoptosis, antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

2. The antibody or fragment of claim 1, wherein said antibody or fragment is murine, non-human, humanized, chimeric, resurfaced or human.

3. The antibody or fragment of claim 1, wherein the antibody or fragment is monoclonal or single-chain.

5. The antibody or fragment thereof of claim 1, obtained from eukaryotic or prokaryotic host cells selected from the group consisting of mammalian, yeast, insect, plant or bacterial cells.

6. The antibody or fragment thereof of claim 5, wherein said mammalian cells are CHO, NS0, SP2/0, PER.C6 or HEK-293 cells.

7. The antibody or fragment thereof of claim 5, wherein said yeast cells are Pichia pastoris or Saccharomyces cerevisiae cells.

8. The antibody or fragment thereof of claim 5, wherein said insect cells are Sf-9 cells.

9. The antibody or fragment thereof of claim 5, wherein said plant cells are Lemna cells.

10. The antibody or fragment thereof of claim 5, wherein said bacterial cells are E. coli cells.

11. The antibody or fragment of claim 1, wherein the antibody or fragment specifically bind to at least one amino acid comprising the amino acid residues located between the third and fourth transmembrane domain of the CD20.

12. A conjugate comprising the antibody or fragment of claim 1 linked to a cytotoxic agent.

13. The conjugate of claim 12, wherein said cytotoxic agent is selected from the group consisting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocarmycin, duocarmycin analog, calicheamicin, dolastatin, dolastatin analog, aristatin, tomaymycin derivative and leptomycin derivative or a prodrug of the conjugate.

14. A pharmaceutical composition comprising the antibody or fragment of claim 1 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition comprising the conjugate of claim 12 and a pharmaceutically acceptable carrier.

16. A diagnostic reagent comprising the conjugate of claim 12, wherein said antibody or fragment is labeled.

17. The diagnostic reagent of claim 16, wherein said label is selected from the group consisting of a radiolabel, a fluorophore, a chromophore, an imaging agent and a metal ion.

18. The antibody or fragment of claim 1, wherein the antibody or fragment specifically bind to at least one amino acid comprising the amino acids of SEQ ID NO:

45, or a sequence corresponding to any one of GI 21330989, GenBank Protein ID 23110989.

19. The antibody or fragment of claim 1, wherein said antibody or fragment is capable of inducing death of a cell expressing a CD20.

20. The antibody or fragment of claim 1, wherein said antibody or said fragment is a Type III antibody, having the capability of substantially inducing apoptosis in the absence of cross-linking agents, and having the capability of causing redistribution of CD20 into a lipid raft and substantially inducing complement dependent cytotoxicity (CDC).

21. The antibody or fragment of claim 1, wherein the antibody or fragment thereof specifically bind to said CD20 in a Western blot.

22. The antibody or fragment of claim 1, wherein the antibody or fragment thereof specifically bind to said CD20 in an ELISA.

23. The antibody or fragment thereof of claim 1, wherein the antibody or fragment thereof specifically bind to said CD20 in a flow cytometry assay.

24. The antibody or fragment of claim 1, wherein said antibody or fragment comprises a Fab, a Fab′

, a F(ab′

)₂, a Fd, a single chain Fv or scFv, a disulfide linked Fv, a V-NAR domain, a IgNar, an intrabody, an IgGΔ

CH₂, a minibiody, a F(ab′

)₃, a tetrabody, a triabody, a diabody, a single-domain antibody, DVD-Ig, Fcab, mAb², a (scFv)₂, or a scFv-Fc.

25. The antibody or fragment of claim 1, comprising an immunoglobulin heavy chain constant domain.

26. The antibody or fragment of claim 25, wherein said immunoglobulin heavy chain constant domain is selected from the group consisting of an IgG1 constant domain, an IgG2 constant domain, an IgG3 constant domain and an IgG4 constant domain.

27. The antibody of claim 19, wherein said cell is derived from a B cell.

28. The antibody of claim 19, wherein said cell is a cancer cell.

29. The antibody of claim 28, wherein said cancer cell is selected from the group consisting of B cell lymphomas, NHL, precursor B cell lymphoblastic leukemia/lymphoma and mature B cell neoplasms, B cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), low-grade, intermediate-grade and high-grade (FL), cutaneous follicle center lymphoma, marginal zone B cell lymphoma, MALT type marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, splenic type marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt'"'"'s lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom'"'"'s macroglobulinemia, and anaplastic large-cell lymphoma (ALCL).

30. The antibody or fragment of claim 19, wherein said cell influences an unwanted immune response.

31. The antibody or fragment of claim 19, wherein said cell influences inflammation.

32. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 48% of Ramos lymphoma cells, wherein said killing comprises apoptosis in the absence of a cross-linking agent.

33. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 48% of Ramos lymphoma cells, wherein said killing comprises apoptosis in the absence of a cross-linking agent;

and wherein said antibody is capable of killing Ramos lymphoma cells with an EC₅₀ of about 0.018 m/mL or lower, wherein said killing comprises CDC.

34. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 48% of Ramos lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent;

and wherein said antibody or fragment is capable of killing Ramos lymphoma cells with an EC₅₀ of about 0.018 μ

g/mL or lower, wherein said killing comprises CDC; and

wherein said antibody or fragment is capable of translocating CD20 into a lipid raft compartment of a membrane of said cells.

35. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 53% of Raji lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

36. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 34% of WSU-DLCL-2 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

37. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 34% of WSU-DLCL-2 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent;

and wherein said antibody or said fragment is capable of killing WSU-DLCL-2 lymphoma cells with an EC₅₀ of about 0.58 μ

g/mL or lower, wherein said killing comprises CDC.

38. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 12% of Jeko-1 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

39. The antibody of claim 1, wherein said antibody or fragment is capable of killing at least about 40% of Granta-519 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

40. The antibody of claim 1, wherein said antibody or fragment binds to an epitope on CD20, wherein said epitope does not comprise or require the amino acid residue proline at position 170 and/or 172, and/or wherein said epitope comprises or requires the amino acid residues asparagine at position 163 and/or 166.

41. The antibody of claim 1, wherein said antibody or fragment binds to an epitope of CD20, wherein said epitope does not comprise or require the amino acid residue proline at position 170 and/or 172, and/or wherein said epitope comprises or requires the amino acid residues asparagine at position 163 and/or 166;

and wherein said antibody or fragment is capable of killing at least about 48% of Ramos lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

42. The antibody of claim 1, wherein said antibody or fragment binds to an epitope of CD20, wherein said epitope does not comprise or require the amino acid residue proline at position 170 and/or 172, and/or wherein said epitope comprises or requires a CD20 epitope having the amino acid residues asparagine at position 163 and/or 166;

and wherein said antibody or fragment is capable of killing at least about 53% of Raji lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

43. The antibody of claim 1, wherein said antibody or fragment binds to an epitope on CD20, wherein said epitope does not comprise or require the amino acid residue proline at position 170 and/or 172, and/or wherein said epitope comprises or requires the amino acid residues asparagine at position 163 and/or 166;

and wherein said antibody or fragment is capable of killing at least about 34% of WSU-DLCL-2 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

44. The antibody of claim 1, wherein said antibody or fragment binds to an epitope on CD20, wherein said epitope does not comprise or require the amino acid residue proline at position 170 and/or 172, and/or wherein said epitope comprises or requires the amino acid residues asparagine at position 163 and/or 166;

and wherein said antibody or fragment is capable of killing at least about 12% of Jeko-1 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

45. The antibody of claim 1, wherein said antibody or fragment binds to an epitope on CD20, wherein said epitope does not comprise or require the amino acid residue proline at position 170 and/or 172;

and/or wherein said epitope comprises or requires the amino acid residues asparagine at position 163 and/or 166; and

wherein said antibody or fragment is capable of killing at least about 40% of Granta-519 lymphoma cells, wherein said killing comprises apoptosis in the absence of cross-linking agent.

73. The antibody or fragment thereof of claim 1, wherein said antibody or fragment is obtained from a transgenic expression host, wherein said host consists of a transgenic goat, chicken, cow, or egg.

74. A method for inhibiting the growth of a cancer cell comprising contacting said cell with the antibody or fragment of claim 1.

78. A method for treating a patient having a cancer comprising administering to said patient an effective amount of the conjugate of claim 19.

80. A method for diagnosing a subject suspected of having a cancer, said method comprising:

administering to said subject the diagnostic reagent of claim 16; and

detecting the distribution of said reagent within said subject.

81. The method of diagnosis of claim 80, wherein said cancer is a cancer selected from the group consisting of B cell lymphomas, NHL, precursor B cell lymphoblastic leukemia/lymphoma and mature B cell neoplasms, B cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), low-grade, intermediate-grade and high-grade (FL), cutaneous follicle center lymphoma, marginal zone B cell lymphoma, MALT type marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, splenic type marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt'"'"'s lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom'"'"'s macroglobulinemia, and anaplastic large-cell lymphoma (ALCL).

84. A polynucleotide encoding the antibody or fragment of any one of claims 1, 4, 18 and 48.

85. A polynucleotide encoding a light or heavy chain of the antibody or fragment of any one of claims 1, 4, 18 and 48.

86. A vector comprising the polynucleotide of claim 85.

87. The vector of claim 86, wherein said vector is an expression vector capable of expressing said antibody or antibody fragment.

88. A host cell comprising the expression vector of claim 87.

90. A method of treating a patient having a cancer comprising administering to said patient an effective amount of the antibody or fragment of claim 1, wherein said antibody or fragment is linked to a radiolabeled compound.

91. The conjugate of claim 12, wherein said linking of antibody or fragment to said cytotoxic agent is through a linker selected from the group consisting of a disulfide group, a thioether group, an acid labile group, a photolabile group, a peptidase labile group and an esterase labile group.

92. The conjugate of claim 91, wherein the linker is selected from the group consisting of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP), 2-iminothiolane and acetylsuccinic anhydride.

93. The conjugate of claim 91, wherein the linker is selected from the group consisting of N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), κ

-maleimidoundecanoic acid N-succinimidyl ester (KMUA), β

-maleimidopropanoic acid N-succinimidyl ester (BMPS), γ

-maleimidobutyric acid N-succinimidyl ester (GMBS), ε

-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-(α

-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(β

-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), N-(p-maleimidophenyl)isocyanate (PMPI), N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA), and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

4. An antibody or fragment thereof produced by the hybridoma (ATCC Accession No. PTA-10486) or hybridoma (ATCC Accession No. PTA-10487).

46. An antibody or fragment thereof comprising at least one complementarity-determining region having an amino acid sequence selected from the group consisting of SEQ ID NOS:

25-30 and 17-22.

47. The antibody or fragment of claim 46, wherein said antibody or said fragment binds a CD20.

48. An antibody or fragment thereof comprising at least one heavy chain and at least one light chain, wherein said heavy chain comprises three sequential complementarity-determining regions having the amino acid sequences of SEQ ID NOS:

20-22 or 28-30, and wherein said light chain comprises three sequential complementarity-determining regions having the amino acid sequences of SEQ ID NOS;

17-19 or 25-27.

49. The antibody or fragment of claim 48, wherein said heavy chain has at least 90% sequence identity to the amino acid sequence represented by SEQ ID NO:

47, 34, 35 or 36.

50. The antibody or fragment of claim 48, wherein said heavy chain has at least 95% sequence identity to the amino acid sequence represented by SEQ ID NO:

47, 34, 35 or 36.

51. The antibody or fragment of claim 48, wherein said heavy chain has the amino acid sequence of SEQ ID NO:

47, 34, 35 or 36.

52. The antibody or fragment of claim 48, wherein said light chain has at least 90% sequence identity to an amino acid sequence represented by SEQ ID NO:

46, 32 or 33.

53. The antibody or fragment of claim 48, wherein said light chain has at least 95% sequence identity to said amino acid sequence represented by SEQ ID NO:

46, 32 or 33.

54. The antibody or fragment of claim 48, wherein said light chain has an amino acid sequence of SEQ ID NO:

46, 32 or 33.

55. The antibody or fragment of claim 48, wherein the amino acid sequence of said heavy chain comprises from 1-5 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

47, 34, 35 or 36.

56. The antibody or fragment of claim 48, wherein the amino acid sequence of said heavy chain comprises from 1-3 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

47, 34, 35 or 36.

57. The antibody or fragment of claim 48, wherein the amino acid sequence of said heavy chain comprises from 3 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

47, 34, 35 or 36.

58. The antibody or fragment of claim 48, wherein the amino acid sequence of said heavy chain comprises from 2 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

47, 34, 35 or 36.

59. The antibody or fragment of claim 48, wherein the amino acid sequence of said heavy chain comprises from 1 conservative substitution as compared to the amino acid sequence represented by SEQ ID NOS:

47, 34, 35 or 36.

60. The antibody or fragment of claim 48, wherein the amino acid sequence of said light chain comprises from 1-5 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

46, 32 or 33.

61. The antibody or fragment of claim 48, wherein the amino acid sequence of said light chain comprises from 1-3 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

46, 32 or 33.

62. The antibody or fragment of claim 48, wherein the amino acid sequence of said light chain comprises from 3 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

46, 32 or 33.

63. The antibody or fragment of claim 48, wherein the amino acid sequence of said light chain comprises from 2 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

46, 32 or 33.

64. The antibody or fragment of claim 48, wherein the amino acid sequence of said light chain comprises from 1 conservative substitutions as compared to the amino acid sequence represented by SEQ ID NOS:

46, 32 or 33.

65. An improved antibody or fragment that specifically bind to a CD20, prepared by:

(a) providing a DNA encoding an antibody or fragment thereof comprising at least one sequence selected from the group consisting of SEQ ID NOS;

32-36, 46, 47, 17-22, 25-30 and 1-7;

(b) introducing at least one nucleotide mutation, deletion or insertion into said DNA such that the amino acid sequence of said antibody or antibody fragment encoded by said DNA is changed;

(c) expressing said antibody or antibody fragment;

(d) screening said expressed antibody or antibody fragment for said improvement, whereby said improved antibody or antibody fragment is prepared.

66. The antibody or fragment of claim 65, wherein said improvement is an increased affinity for CD20.

67. The antibody or fragment of claim 65, wherein said at least one nucleotide mutation, deletion or insertion is made by a method selected from the group consisting of oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, and the use of mutator-strains of E. coli.

69. The antibody or fragment of claim 65, wherein said improvement is increased effector function.

70. The antibody or fragment of claim 65, wherein said improvement is increased CDC activity.

71. The antibody or fragment of claim 65, wherein said improvement is increased ADCC activity.

72. The antibody or fragment of claim 65, wherein said improvement is increased ADCC activity, increased CDC activity and increased effector function.

68. An antibody or fragment thereof comprising a polypeptide comprising at least one member selected from the group consisting of SEQ ID NOs:

1-7 and 17-36.

75. A method for treating a patient having a cancer comprising administering to said patient an effective amount of the antibody or fragment or conjugate thereof that specifically bind to a CD20, wherein said antibody or fragment is capable of inducing apoptosis, antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

76. The method of claim 75 further comprising administering to said patient a therapeutic agent.

77. The method of claim 76 wherein said therapeutic agent is a cytotoxic agent.

79. The method of treatment of claim 75, wherein said cancer is a cancer selected from the group consisting of B cell lymphomas, NHL, precursor B cell lymphoblastic leukemia/lymphoma and mature B cell neoplasms, B cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), low-grade, intermediate-grade and high-grade (FL), cutaneous follicle center lymphoma, marginal zone B cell lymphoma, MALT type marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, splenic type marginal zone B cell lymphoma, hairy cell leukemia, diffuse large B cell lymphoma, Burkitt'"'"'s lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom'"'"'s macroglobulinemia, and anaplastic large-cell lymphoma (ALCL).

82. A method for treating a patient having an autoimmune or inflammatory disease comprising administering to said patient an effective amount of the antibody or fragment or conjugate thereof that specifically bind to a CD20, wherein said antibody or fragment is capable of inducing apoptosis, antibody dependent cell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).

83. The method of treatment of claim 82, wherein said autoimmune or inflammatory disease is a autoimmune or inflammatory disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener'"'"'s disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud'"'"'s syndrome, Crohn'"'"'s diasease, ulcerative colitis, gastritis, Hashimoto'"'"'s thyroiditis, ankylosing spondylitis, hepatitis C-associated cryoglobulinemic vasculitis, chronic focal encephalitis, bullous pemphigoid, hemophilia A, membranoproliferative glomerulnephritis, adult and juvenile dermatomyositis, adult polymyositis, chronic urticaria, primary biliary cirrhosis, neuromyelitis optica, Graves'"'"' dysthyroid disease, bullous pemphigoid, membranoproliferative glonerulonephritis, Churg-Strauss syndrome, asthma, psoriatic arthritis, dermatitis, respiratory distress syndrome, meningitis, encephalitits, uveitis, eczema, atherosclerosis, leukocyte adhesion deficiency, juvenile onset diabetes, Reiter'"'"'s disease, Behcet'"'"'s disease, hemolytic anemia, atopic dermatitis, Wegener'"'"'s granulomatosis, Omenn'"'"'s syndrome, chronic renal failure, acute infectious mononucleosis, HIV and herpes-associated disease, systemic sclerosis, Sjorgen'"'"'s syndrome and glomerulonephritis, dermatomyositis, ANCA, aplastic anemia, autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, Castleman'"'"'s syndrome, Goodpasture'"'"'s syndrome, solid organ transplant rejection, graft versus host disease (GVHD), autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant), Guillain-Barre Syndrome, large vessel vasculitis, giant cell (Takayasu'"'"'s) arteritis, medium vessel vasculitis, Kawasaki'"'"'s Disease, and polyarteritis nodosa.

89. A means for specifically binding to a CD20, wherein said means is capable of inducing apoptosis, ADCC and CDC.