AU2017202590A1 - Immunoglobulin variants and uses thereof - Google Patents

Abstract

Abstract. The invention provides humanized and chimeric anti-CD20 antibodies for treatment of B-Cell associated diseases such as CD20 positive malignancies and autoimmune diseases, in particular dosage regimen. The present invention is directed to CD-20 antibodies with Fc region substitutions S298A or E333A or K334A or K326A or K326W or K322A, wherein these substitutions improve ADCC or CDC activity of the antibody. Certain aspects of the invention are directed to the use of the CD-20 antibody Rituximab@ (Rituxan@) in the treatment of diseases such as rheumatoid arthritis. The present antibodies are used in treating CD 20 positive cancers such as non-Hodgkin's lymphoma, treating autoimmune disease and inducing apoptosis in B-Cells. The invention is also directed to combination therapies with further chemotherapeutic agents such as doxorubicin, cyclophosphamide, prednisolone or CHOP, with further anti-rheumatic agents or with further immunosuppressive agents such as methotrexate. The invention also encompasses methods of producing CD-20 antibodies using vectors and host cells comprising nucleotide sequences encoding the CD-20 antibodies.

Description

IMMUNOGLOBULIN VARIANTS AND USES THEREOF FIELD OF THE INVENTION

The invention relates to anti-CD2(J antibodies and their use in the treatment of B-cell related diseases.

BACKGROUND OF THE INVENTION

Lymphocytes are one of several populations of white blood cells: they specifically recognise and respond to foreign antigen. The three major classes of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells) and natural killer (NK.) cells. B lymphocytes are the cells responsible for antibody production and provide humoral immunity, B cells mature within the bone marrow and leave the marrow expressing an antigen-binding antibody on their cell surface. When a naive B cell first encounters the antigen for which its membrane-bound antibody is specific, the cell begins to divide rapidly and its progeny differentiate into memory B cells and effector cells called “plasma cells”. Memory B cells have a longer life span and continue to express membrane-bound antibody with the same specificity as the original parent cell. Hasma cells do not produce membrane-bound antibody but instead produce secreted form of (he antibody. Secreted antibodies arc the major effector molecules of humoral immunity .

The CD20 antigen (also called human B-lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 LD located on pre-B and mature B lymphocytes (Valentine et al. J. Biol. Chem. 264(19):11232-11287 (1989); and Einfeld etal EMBO J, 7(3):711-717 (1988)). The antigen is also expressed on greater rhan 90% of B cell non-Hodgkin's lymphomas (NHL) (Anderson et al. Blood 63(6):1424-1433 (1984)), but is not found on hematopoietic stem cells. pro-B cells, normal plasma cells or other normal tissues (Tedder et al. J. Immunol. 135(2):973-979 (1985)). CD20 is thought to regulate an early stcp(s) in the activation process for cell cycle initiation and differentiation (Tedder etal., supra) and possibly functions as a calcium ion channel (Tedder et al J. Cell. Biochem. 1419:195 (1990)).

Given the expression of C.D20 in B cell lymphomas, this antigen has been a useful therapeutic target to treat such lymphomas. There arc more than 300,000 people in the United States with B-cell NHL and more than 56,000 new cases are diagnosed each year. For example, the riluximab (RITUXAN®) antibody which is a genetically engineered chimeric murine/human monoclonal antibody directed against human CD20 antigen (commercially available from Gcnentech, Inc., South San Francisco, California, U.S.) is used for the treatment of patients with relapsed or refractory low-grade or follicular, CD20 positive, B cell non-Hodgkin's lymphoma. Rituxtmab is the antibody referred to as “C2B8" in US Patent No. 5,736,137 issued April 7, 1998 (Anderson etal.) and in U.S Pat No. 5,776,456. In vitro mechanism of action studies have demonstrated that RITUXAN® binds human complement and lyses lymphoid B cell lines through complement-dependent.cytotoxicity (CPC) (Rcff etal. Blood 83(2):435-445 (1994)). Additionally, it has significant activity1 jn assays for antibody-dependent cellular cytotoxicity (AJDCC). In vivo predinica! studies have shown that RITUXAN® depletes B cells from the peripheral blood, lymph nodes, and bone marrow of cynoinoigus monkeys, presumably through complement and cell-mediated processes (Rcff et al.

Blood 83 (2): 435-445 (1994)). Other anti-CD20 antibodies indicated for the treatment of NHL include the murine antibody Zevalin™ which is linked to the radioisotope, Yttrium-90 (IDEC Pharmaceuticals, San Diego, CA), Bexxar™ which is another fully murine antibody conjugated to 1-131 (Corixa, WA). A major limitation in the use of murine antibodies in human therapy is the human anti-mouse antibody (HAMA) response (see, e.g., Miller, R.A. et al. "Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma" Blood, 62: 988-995,1983; and Schroff, R.W., et al. "Human anti-murine immunoglobulin response in patients receiving monoclonal antibody therapy"

Cancer Res., 45:879-885, 1985). Even chimeric molecules, where the variable (V) domains of rodent antibodies are fused to human constant (C) regions, are still capable of eliciting a significant immune response (HACA, human anti-chimeric antibody) (Neuberger et al. Nature (Lond.), 314:268-270,1985). A powerful approach to overcome these limitations in the clinical use of monoclonal antibodies is "humanization" of the murine antibody or antibody from a non-human species (Jones et al. Nature (Lond), 321:522-525,1986; Riechman et al., Nature (Lond), 332:323-327,1988).

Thus, it is beneficial to produce therapeutic antibodies to the CD20 antigen that create minimal or no antigenicity when administered to patients, especially for chronic treatment. The present invention satisfies this and other needs. The present invention provides anti-CD20 antibodies that overcome the limitations of current therapeutic compositions as well as offer additional advantages that will be apparent from the detailed description below.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

SUMMARY OF THE INVENTION

The present invention provides CD20 binding antibodies or functional fragments thereof, and their use in the treatment of B-cell associated diseases. These antibodies are monoclonal antibodies. In specific embodiments, the antibodies that bind CD20 are humanized or chimeric. The humanized 2H7 variants include those that have amino acid substitutions in the FR and affinity maturation variants with changes in the grafted CDRs. The substituted amino acids in the CDR or FR are not limited to those present in the donor or recipient antibody. In other embodiments, the anti-CD20 antibodies of the invention further comprise changes in amino acid residues in the Fc region that lead to improved effector function including enhanced CDC and/or ADCC function and B-cell killing (also referred to herein as B-cell depletion). Other anti-CD20 antibodies of the invention include those having specific changes that improve stability. In a specific embodiment, the humanized 2H7 variants with increased stability are as described in example 6 below. Fucose deficient variants having improved ADCC function in vivo are also provided. In one embodiment, the chimeric anti-CD20 antibody has murine V regions and human C region. One such specific chimeric anti-CD20 antibody is Rituxan® (Rituximab®; Genentech, Inc.).

In a preferred embodiment of all of the antibody compositions and methods of use of this invention, the humanized CD20 binding antibody is 2H7.vl6 having the light and heavy chain amino acid sequence of SEQ ID NO. 21 and 22, respectively, as shown in FIG. 6 and FIG. 7. When referring to the polypeptide sequences in Figures 6, 7 and 8, it should be understood that the first 19 or so amino acids that form the secretory signal sequence are not present in the mature polypeptide. The V region of all other variants based on version 16 will have the amino acid sequences of v 16 except at the positions of amino acid substitutions which are indicated in the disclosure. Unless otherwise indicated, the 2H7 variants will have the same L chain as that of vl6.

The invention provides a humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody is effective to deplete primate B cells in vivo, the antibody comprising in the H chain Variable region (VH) at least a CDR3 sequence of SEQ ID NO. 12 from an anti-human CD20 antibody and substantially the human consensus framework (FR) residues of human heavy chain subgroup III (VhIII). In one embodiment, the primate B cells are from human and Cynomolgus monkey. In one embodiment, the antibody further comprises the H chain CDR1 sequence of SEQ ID NO. 10 and CDR2 sequence of SEQ ID NO. 11.

In another embodiment, the preceding antibody comprises the L chain CDR1 sequence of SEQ ID NO. 4, CDR2 sequence of SEQ ID NO. 5, CDR3 sequence of SEQ ID NO. 6 with substantially the human consensus framework (FR) residues of human light chain κ subgroup I (VkI). In a preferred embodiment, the FR region in VL has a donor antibody residue at position 46; in a specific embodiment, FR2 in VL has an amino acid substitution of leuL46pro (Leu in the human κΐ consensus sequence changed to pro which is present in the corresponding position in m2H7). The VH region further comprises a donor antibody residue at at least amino acid positions 49, 71 and 73 in the framework. In one embodiment, in the VH, the following FR positions in the human heavy chain subgroup III are substituted: AlaH49Gly in FR2; ArgH71Val and AsnH73Lys in FR3. In other embodiments, the CDR regions in the humanized antibody further comprise amino acid substitutions where the residues are neither from donor nor recipient antibody.

The antibody of the preceding embodiments can comprise the Vh sequence of SEQ ID NO.8 of vl6, as shown in FIG. IB. In a further embodiment of the preceding, the antibody further comprises the VL sequence of SEQ ID NO.2 of vl6, as shown in FIG. 1A.

In other embodiments, the humanized antibody is 2H7.v31 having the light and heavy chain amino acid sequence of SEQ ID NO. 21 and 23, respectively, as shown in FIG. 6 and FIG. 8; 2H7.v31 having the heavy chain amino acid sequence of SEQ ID NO. 23 as shown in FIG. 8; 2H7.v96 with the amino acid substitutions of D56A and N100A in the H chain and S92A in the L chain of vl6.

In separate embodiments, the antibody of any of the preceding embodiments further comprises at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity over the original or parent antibody from which it was derived, v.16 being the parent antibody being compared to in most cases, and Rituxan in other cases. One such antibody with improved activity comprises the triple Alanine substitution of S298A/E333A/K334A in the Fc region. One antibody having S298A/E333A/K334A substitution is 2H7.v31 having the heavy chain amino acid sequence of SEQ ID NO. 23. Antibody 2H7.vl 14 and 2H7.vl 15 show at least 10-fold improved ADCC activity as compared to Rituxan.

In another embodiment, the antibody further comprises at least one amino acid substitution in the Fc region that decreases CDC activity as compared to the parent antibody from which it was derived which is vl6 in most cases. One such antibody with decreased CDC activity as compared to vl6 comprises at least the substitution K322A in the H chain. The comparison of ADCC and CDC activity can be assayed as described in the examples.

In a preferred! embodiment, the antibodies of die invention arc fill] length antibodies wherein the VH region is joined to a human IgG heavy chain constant region. In preferred embodiments, the IgG is human IgGl Or IgG3.

In one embodiment, the CD20 binding antibody is conjugated to a cytotoxic agent. In preferred embodiments the cytotoxic agent is a toxin or a radioactive isotope.

Tn one embodiment, the antibodies of the invention for use in therapeutic or diagnostic purposes are produced in CHO cells.

Also provided is a composition comprising an antibody of any one of the preceding embodiments, and a carrier. Jn one embodiment, the carrier is a pharmaceutically acceptable carrier. These compositions can be provided in an article of manufacture or a kit.

The invention also provided a liquid formulation comprising a humanized 2H7 antibody at 20mg/mL antibody, lOmM histidine sulfate pH5.8, fiOmg/mJ sucrose (6%), 0.2 rng/ml polysorbate 20 (0.02%).

The invention also provides an isolated nucleic add that encodes any of the antibodies disclosed herein, including an expression vector for expressing the antibody.

Another aspect of the invention are host cells comprising the preceding nucleic acids, and host cells that produce the antibody. In a preferred embodiment of the latter, the host ceil is a CHO cell. A method of producing these antibodies is provided, the method comprising culturing the host cell that produces die antibody and recovering the antibody from the cell culture.

Yet another aspect of the invention is an article of manufacture comprising a container and a composition contained therein, wherein the composition comprises an antibody of any of the preceding embodiments. For use in treating NHL, the article of manufacture further comprises a package insert indicating that the composition is used to treat non-Hodgkin1 s lymphoma Λ further aspect of the invention is a method of inducing apoptosis in B cells in vjvo, comprising contacting B cells with the antibody of any of the preceding, thereby killing the B cells.

The invention also provide? methods of treado.g the diseases disclosed herein by administration of a CP20 binding antibody or functional fragment thereof, to a mamma! such as a human patient suffering from the disease. In any of the methods for treating an autoitumune disease or a CD20 positive cancer, in one embodiment, lire antibody is 2H7.vl 6 having the light and heavy chain amino acid sequence of SEQ ID NO. 21 and 22, respectively, as shown in FIG. 6 and FIG. 7. Thus, one embodiment is a method of treating a CD20 positive cancer, comprising administering to a patient suffering from the cancer, a therapeutically effective amount of a humanized CD20 binding antibody of the invention. In preferred embodiments, the CD20 positive cancer is a B cell lymphoma or leukemia including non-Hodgkin’s lymphoma (NHL) or lymphocyte predominant Hodgkin’s disease (LPHD), chronic lymphocytic leukemia (CLL) or SLL. In one embodiment of the method of treating a B cell lymphoma Or leukemia, the antibody is administered at a dosage range of about 275-375mg/m2. In additional embodiments, the treatment method further comprises administering to the patient at least one chemotherapeutic agent, wherein for non-Hodgkin’s lymphoma (NHL), The chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, vincristine and prednisolone.

Also provided is a method of treating an autoimmune disease, comprising administering to a patient suffering from the autoimmune disease, a therapeutically effective amount of the humanized CD20 binding antibody of any one of the preceding claims. The autoimmune 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 (JIT), thrombotic thrombocytopenic purpura (ΤΤΕ), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Revnaud’s syndrome, Sjorgen’s syndrome and glomerulonephritis. Where the autoimmune disease is rheumatoid arthritis, the antibody can be administered in conjunction with a second therapeutic agent which is preferably methotrexate.

In these treatment, methods, the CD20 binding antibodies can be administered alone or in conjunction with a second therapeutic agent such as a second antibody, or a chemotherapeutic agent or an immunosuppressive agent The second antibody can be one that binds CD20 or a different B cell antigen, or a NK or T cell antigen. Tn one embodiment, the second antibody is a radiolabeled anti-CD20 antibody. In Other embodiments, the CD20 binding antibody is conjugated to a cytotoxic agent including a toxin or a radioactive isotope.

It) another aspect, the invention provides a method of treating an autoimmune disease selected from the group consisting of Dermatomyositis, Wegner’s granulomatosis, 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 (GVHJD), IgM mediated, thrombotic thrombocytopenic purpura (TIP), Hashimoto's Thyroiditis, autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs, NS1P, Guillain-Barre Syndrome, large vessel vasculitis, giant cell (Takayasu's) arteritis, medium vessel vasculitis, Kawasaki's Disease, polyarteritis nodosa, comprising administering to a patient suffering from the disease, a therapeutically effective amount of a CD20 binding antibody. In one embodiment of litis method, the CD20 binding antibody is Rituxan®.

The invention also provides an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 24 of the Cynomolgus monkey CJD20 (shown in MG. 19). or a degenerate variant of this sequence.

One embodiment is an isolated nucleic acid comprising a sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO. 25 (shown MG. 20), or SBQ ID NO. 25 (FIG. 20) with conservative amino acid substitutions. Another embodiment is a vector comprising the preceding nucleic acid, including an expression vector for expression ill a host cell. Included as well is a host cell comprising the vector. Also provided is an isolated polypeptide comprising the amino acid sequence [SEQ TD NO. 25; FIG- 20] of the Cynomolgus monkey CD20-

BRIEF DESCRIPTION OF THE FIGURES FIG. 1A is a sequence alignment comparing the amino add sequences of the light chain variable domain (VjQ of each of murine 2H7 (SEQ ID NO. 1), humanized 2H7, variant (SEQ ID NO. 2). and human kappa light chain subgroup Ϊ (SEQ ID NO. 3). The CDRs of Vr. of 2H7 and hu2H7.vl6 are as follows: CDR1 (SEQ ID NO.4), CDK2 (SEQ ID NO.5 ), and CDR3 (SEQ ID NO.6). KIG, IB is a sequence alignment which compares the VH sequences of murine 2H7 (SRQ ID NO. 7), humanized 2H7, v 16 variant (SRQ ID NO. 8), and the human consensus sequence of heavy chain subgroup Π1 (SEQ ID NO. 9). The CDRs of V,, of 2H7 and hu2H7.v!6 are as follow: CDRt (SEQ ID NO. 10). CDR2 (SEQ ID NO-i 1). and CDR3 (SEQ ID NO. 12).

Tn FIG. 1A and FIG. 1R, the CDR t, CDR2 and CDR3 in each chain are enclosed within brackets, flanked by the framework regions. FR1-FR4. as indicated. 2H7 refers to the murine 2H7 antibody. The asterisks in between two rows of sequences indicate the positions that, are different, between the two sequences. Residue numbering is according to Rabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Rethcsda. Md. (1991), with insertions shown, as a, b, c, d, and c. FIG. 2 shows the sequence of phagemid pVX4 (SEQ ID NO. 13) used for construction of 2H7 Fab plasmids (see Example 1) as well as the amino acid sequences of the L chain (SBQ ID NO. 14) and H chain (SEQ ID NO.15) of the Fab for the CDR-grafted atiti-IFN-α humanized antibody. FIG, 3 shows the sequence of the expression plasmid which encodes the chimeric 2H7.v6.S Fab (SEQ ID NO. 16). The amino add sequences of the L chain (SEQ ID NO. 17) and H chain (SEQ ID NO-18) arc shown, FIG. 4 shows the sequence of the plasmid pDRl (SEQ ID NO.19; 5391 bp) for expression of immunoglobulin light chains as described in Example 1. pDRI contains sequences encoding an irrelevant antibody, the light chain of a humanized anii-CD3 antibody (Shalaby et al., I. Exp. Med. 175! 217-225 (1992)), the start, and stop codons for which arc indicated in bold and underlined. FIG. 5 shows die sequence of plasmid pDR2 (SEQ ID NO-20; 6135 bp) for expression of immunoglobulin heavy chains as described in Example 1. pDR2 contains sequences encoding an irrelevant antibody, the heavy chain of a humanized anti-CD3 antibody (Shalaby et al., supra), the start and stop codons for which are indicated in bold and underlined. FIG. 6 shows the amino acid sequence of the 2H7.vl6 complete L chain (SBQ ID NO.21). The first 19 amino acids before DIQ are the secretory signal sequence not present in the mature polypeptide chain. FIG. 7 shows the amino acid sequence of the 2H7.vl6 complete H chain (SEQ TD NO-22). The first 19 amino acids before EVQ before are the secretory signal sequence not present in the mature polypeptide chain. Aligning the Vu sequence in FIG. IB (SEQ ID NO. 8) with the complete H chain sequence, the human γΐ constant region is from amino acid position 114-471 in SEQ ID NO. 22. FIG, 8 shows the amino acid sequence of the 2H7.v31 complete H chain (SEQ TD NO.23). The first 19 amino acids before EVQ before are die secretory signal sequence not present in the mature polypeptide chain. The L chain is the same as for 2H7.v 16 (see FIG. 6). FIG. 9 shows die relative stability of 2H7.vl6 and 2B.7.v73 IgG variants as described in Example 6. Assay results were normalized to die values prior to incubation and reported as percent remaining after incubation. FIG. 10 is a flow chart summarizing the amino acid changes from the murine 2H7 to a subset of humanized versions up to v75. FIG. 11 is a sum maty of mean absolute 13-ccll count rCD3-/CD40+J in all groups (2H7 study and Rituxan study combined), as described in Example 10. FIG. 12 shows the results of a representative ADCC assay on fuco.sc deficient 2H7 variants as described in Example 11. FIG. ] 3 shows the results of the Annexin V staining plotted as a function of antibody concentration. Ramos cells were treated with an irrelevant TgG1 control antibody (Herceptin®; circles), Riruximab (squares), or rhuMAb 2H7.v! 6 (triangles) in the presence of a orosslioking secondary antibody and were analyzed by FACS. Figures 13-15 are described in Example 13. FIG. 14 shows the results of the Annexiil V and propidium iodide double-staining arc plotted as a function of antibody concentration. Ramos cells were treated with an irrelevant IgGl control antibody (Hcnxprin®; circles), Rituxjmab (squares), or rhuMAb 2H7.V1.6 (triangles) in the presence of a orosslinking secondary antibody and were analyzed by FACS. FIG. 15 shows the counts (per 10 s) of live, unstained cells are plotted as a function of antibody concentration. Ramos cells were heated with an irrelevant IgGl control antibody (Herceptin®; circles), Rituximab (squares), or rhuMAb 2H7.v1<5 (triangles) in the presence of a orosslinking secondary antibody and were analyzed by FACS. i’IGs. 16, i 7, 18 show inhibition of Raji cell tumor growth in nude mice, as described in Example 14. Animals were treated weekly (as indicated by vertical arrows; t»=8 mice per group) for 6 weeks with PBS (control) or with Rituxan® or rhuMAb 2H7.v 16 at 5 mg/kg (FIG. 16), 0.5 mg/kg (FIG. 17), or 0.05 mg/kg (FIG. 18). FIG. 19 shows the nucleotide (SEQ ID NO. 24) and amino acid (SEQ ID NO. 25) sequences of Cynomolgus monkey CD20, as described in .Example 15. FIG. 20 shows the ami.no acid sequence for cynomolgus monkey CD20 (SEQ ID NO. 25). Residues that differ from human CD20 are underlined and the human residues (SEQ TD NO- 26) are indicated directly below the monkey residue. The putative extracellular domain of the monkey CD20 is in bold type. FIG. 21 shows the results of Cynomolgus monkey ceils expressing CD20 binding to hu2H7.vl6, ,v31, and Rituxan, as described in Example 15. The antibodies were assayed for the ability to bind and displace FlTC-conjugated murine 2H7 binding to cynomolgus CD20. FIG. 22 shows dose escalation schema for rheumatoid arthritis phase ΙΛ1 clinical trial. MG. 23 shows the vector for expression of 2H7.vl6 in CHO cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The “CD20" antigen is a nan-glycosylated, transmembrane phosphoprotem with a molecular weight, of approximately 35 kD that, is found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation; it is not found on human stem cells, lymphoid progenitor cells or normal plasma cells. CD20 is present on both normal B cells as well as malignant B cells. Other names for CD20 in the literature include "B -1 ym phocy te-rest ri cted differentiation antigen” and "Bp35H, The CJD20 antigen is described in, for example, Clark and Ledbetter, Adv. Can. Res. 52:81-149 (1989) and Valentine et at. J. Biol Chem. 264(19):11282-11287(1989).

The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), multispeeific antibodies (e.g.. bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity or function.

The biological activity of the C.D20 binding and humanized CD20 binding antibodies of the invention will include at least binding of the antibody to human CD20. more preferably binding to human and other primate. CD20 (including cynomolgus monkey, rhesus monkey, chimpanzees). The antibodies would bind CD20 with a Kj value of no higher than 1 x 10'?. preferably a Ku value no higher than about I x 10'9, and be able to kill or deplete B cells in Vivo, preferably by at least 20% when compared to the appropriate negative control which is not treated with such an antibody. B cell depletion can be a result of one or more of ADCC, CDC, apoptosis, or other mechanism. In some embodiments of disease treatment herein, specific effector functions or mechanisms may be desired over others and certain variants of the humanized 2H7 are preferred to achieve those biological functions, such as ADCC. "Antibody fragments" comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2. and Fv fragments: diabodies; linear antibodies: single-chain antibody molecules: and multispecific antibodies formed from antibody fragments. "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, iton-covalcnt association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to die antibody. However, even a single variable domain (or half of an Fv comprising only tliree CDRs specific for an antigen) .has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, /.<?., the individual antibodies comprising the population arc identical except for possible naturally occurring mutations (hat may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic sire. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different, antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of die antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et «/,, Nature 250:495 (1975), or may be made by recombinant DNA methods (see, e.g., U-S- Patent No. 4,816,567). The "monoclonal antibodies" may also be isolated from, phage antibody libraries using the techniques described in Claekson era/., Nature 352:624-628 (1991) and Marks etal., J. Mol. Biol. 222:581-597 (1991), for example, 'Functional fragments” of the CD20 binding antibodies of the invention are those fragments that retain binding to CD20 with substantially the same affinity as (lie intact Ml length molecule from which they are derived and show biological activity including depicting B cells as measured by in vitro or in vivo assays such as those described herein.

The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs. largely adopting a β-sheef. configuration, connected by three hypcrvariable regions, which form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by tJie FRs and. with the hypervariablc regions from die other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et ah, Sequences of Proteins of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health. Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypcrvariable region generally comprises amino acid residues from a “complementarity determining region” or “CDR” (e.g. around about residues 24-34 (LI), 30-56 (L2) and 89-97 (L3) in the Vu and around about 3I-35B (HI), 50-65 (H2) and 95-102 (H3)in the VH (Kabat, et a!„ Sequences of Proteins of immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a “hypcrvariable loop” (e.g. residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the VL, and 26-32 (HI), 52Λ-55 (H2) and 96-101 (M3) in die VH (Chothia and Lesk J. Mo,l. Blot. 196:901-917 ()987)).

As referred to herein, the “consensus sequence” or consensus V domain sequence is an artificial sequence derived from a comparison of the amino acid sequences of known human immunoglobulin variable region sequences. Based on these comparisons, recombinant nucleic acid sequences encoding the V domain amino acids that are a consensus of the sequences derived from, the human, κ and the human H chain subgroup ΠΤ V domains were prepared. The consensus V sequence does not have any known antibody binding specificity or affinity. "Chimeric" antibodies (immunoglobulins) have a portion of the heavy and/or light chain identical, with or homologous ro corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with o.r homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as web as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567: arid Morrison et <d„ Froc. Nall, Acad. Sci. USA 81:6851-6855 (1984)). Humanized antibody as used herein is a subset of chimeric antibodies. "Humanized" forms of non-human (e.g., murine) antibodies arc chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of rh.e recipient are replaced by hypcrvariablc region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies Olay comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypcrvariable loops correspond to those of a non-human immunoglobulin and all or substantially al I of the FR regions arc those of a human immunoglobulin sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 rn the H chain, and in the L chain, no more than 3. The humanized antibody optionally also will comprise al least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. Nature 321:522-575 (1986); Reichmann et al, Nature 332:323-329 (1988): and Presta, Curr. Op. Struct. Biol 2:593-596 (1992),

Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding: antibody-dependent cell-mediated cytotoxicity (AJDCC); phagocytosis; down regulation of cell Surface receptors (e.g. B cell receptor); and B cell activation. “Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Jig bound onto Fc receptors (FcRs) present on certain cytotoxic cells («.#. Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigenbearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic ceils and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express PcyRTTJ only, whereas monocytes express FcyRI, FcyRH and FcyRUT. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annie Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay. Such as that described in UK Patent No. 5,500.362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e,g., in a animal model such as that disclosed in Clynes et al. FNAS (USA) 95:652-656 (1998). "Fc receptor" or "FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRlI, and FcyRIlT subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRH receptors include FcyRTlA (an "activating receptor") and PcyRlIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoTcecptor tyrosine-based activation motif (ΓΓΛΜ) in its cytoplasmic domain. Inhibiting receptor FcyRTTB contains an imraunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (see review M. in Dacron, Amu. Rev. Immunol. 15:203-234 (1997)). FcRs are .reviewed in Ravctch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Cape) ewl, hnmummethodn 4:25-34 (1994): and de Haas etal., J. !^ab. Clin. Mad. )2(5:330-4) (1995). Other FcRs, including those to be identified in the future, arc encompassed by the term "FcR” herein, The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 117:587 (1976) and Kim clal. J. Immunol. 24:249 (1994)). WOOO/42072 (Presta.) describes antibody variants with improved or diminished binding to FcRs. The content of that patent publication is specifically incorporated herein by reference. See, also, Shields at al. J. Biol. Chem. 9(2): 6591-6604 (2001). “Human effector cells"’ are leukocytes which express one or more FcRs and perform effector functions, Preferably, the cells express at least FcyRTJI and perform ADCC effector function. Examples of human leukocytes which mediate ADCC include peripheral blood mononuclear cells (FBMC), natural killer (NK) cells, monocytes, cytotoxic Tcells and neutrophils; with PRMCs and NK cells being preferred. The effector cells may be isolated from a native source, e.g. from blood. “Complement dependent cytotoxicity” or “CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, e.g. as described in Gazzaru)-Santoro et at.. J. Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region atnjno acid sequences and increased or decreased Clq binding capability are described in US patent No. 6.194,55.1 B1 and WC99/5 J 642, The contents of those patent publications arc specifically incorporated herein by reference. See, also, Idusogic etal. J. Immunol 164:4178-4184(2000).

The N-glycosylation site in IgG Is at Asn297 in the. CH2 domain. The present invention also provides compositions of a CD20-binding, humanized antibody having a Fc region, wherein about 80-100% (and preferably about 90-99%) of the antibody in the composition comprises a mature core carbohydrate structure which lacks Fucohc, attached to the Fc region of the glycoprotein. Such compositions wore demonstrated herein to exhibit a surprising improvement in binding to FcyRI3IA(F158), which is not as effective as FcyRTIJA (V158) in interacting with human IgG. Thus, the compositions herein are anticipated to be superior to previously described anti-CD20 antibody compositions, especially for therapy of human patients who express FcyRIIIA. (F1.58). FcyRTUA (F158) is more common than FcyRJHA (V158) in normal, healthy African Americans and Caucasians. See Lchmbcehcr et aL Blood 94:4220 (1999). The present application further demonstrates the synergistic increase in FcvRTTI binding and/or ADCC function thftt results front combining the glycosylation variations herein with amino acid sequence modification(s) in the l-’c region of the glycoprotein.

An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment arc materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least ] 5 residues of Ν'-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAOE under reducing or nonreducing conditions using Coomassie blue or, preferably, stiver stain, Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished front the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the antibody where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, inchide a promoter, optionally an. operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylalion signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, .DNA for a presequcncc or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence: or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and. in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to bo contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers arc used in accordance with conventional practice. “Vector' includes shuttle and expression vectors- Typically, tile plasmid construct will also include an origin of replication («.#„ the ColEl origin of replication.) and a selectable marker (e.g., ampicillin or tetracycline resistance), for .replication and selection, respectively, of the plasmids in bacteria. An ’expression vector” refers to a vector that contains the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment of the invention, in bacterial or eukaryotic cells. Suitable vectors are disclosed below.

The cell that produces a humanized CD20 binding antibody of the invention will include the bacterial and eukaryotic host cells into which nucleic acid encoding the antibodies have been introduced. Suitable host cells arc disclosed below.

The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody. The label may itself be detectable by itself (e.g.r radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.

An "autoimmune disease” herein is a non-maJignant disease or disorder arising from and directed against an individual’s own (seiO antigens and/or tissues.

As used herein, “B ceil depletion” refers to a reduction in B cell levels in an animal or human after drug or antibody treatment, as compared to the B cell level before treatment R cell levels are measurable using well known assays such as those described in the Experimental Examples. B ceil depiction can be complete or partial. In one embodiment, the depletion of CD20 expressing B cells is at leasr 25%, Not to be limited by any one mechanism, possible mechanisms of B-cell depletion include ADCC, CDC, apoptosis, modulation of calcium flux or a combination of two or more of the preceding.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The terra is intended to include radioactive isotopes (e.g., )131. I1», Y* and Rc]M). chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. A "chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkalyzing or alkylating agents such as thiotepa and cyciosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimincs and methylamelarnincs including altrctamine, triethylenemclaminc, trictylenephosphoramide, triethylcnethiophosphaoramide and fritnethyloiomelamine; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamidc, estramustine, ifosfemidc, mechlorethamine, mecblorethaminc oxide hydrochloride, melphalan, novcmbichin, phencstcrinc, prednimustine, trofosfamide, uracil mustard; nifrosureas such as carmuslinc, chlorozotocin, tbtcmusfine, lomustine, nimustine, ranimustine: antibiotics such as aclaciaomysins, actinomycin, authramycin, azaserixte, bleomycins, cactinomycin, calicheamicin, carabicin. canninomycin, carzinophilin, chromomycins, daclirtumyein, daunorubicin, dctorubicin, 6-diazo-5-oxo-l^nor)eucine, doxorubicin (Adriamycin), epirubiein, esorubicin, idarubicin, marecllomycin, mitomycins, mycophenoHc acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamyein, rodombtein, streptonigrin, strcptozocin. tubercidin, ubenimex, zinostatin, zorubicin: anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabinc, azacitidinc, 6-azauridinc, carmofur, eytarabinc, didcoxyuridinc, doxifluridine, enocitabine, floxuridinc, 5-FU; androgens such as ealusterone, dromostattolone propionate, epiliostanol, mepitiostanc, testolaetonc; anti-adrenals such as aminoglutethimide, raitotane, trilostane; folic acid replenish® such as frolinic acid; aceglatone; aldophosphamidc glycoside; aminolevulinic acid; amsacrine; bestrabueil; bisamrene; edatraxate; defofamine; demecolcine; riiaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate.; hydroxyurea; lentmao; lonidamjne; mitoguazone; mitoxantrone; mopidamol: nitracrine; pentostatin; phenamet; pirambicin; podophyllinie acid; 2-cthylhydrazidc; procarbazine; PSK®; razoxane; sizofiran; spirogermamum; tenuazorac acid; triaziquone; 2, 2',2’i-trichlorotriethyiamine; urethan; vindesine; dacarbazine; manoomustine; rndtobronitol; mitolactoU pipobroman; gacytosine; arabinostde ("Ara-C"): thiotepa; taxoids, e.g. paclitaxel (TAXOL'\ Bristol-Myers Squibb Oncology, Princeton, Ni) and doxetaxel (TAXOTERE®, Rhflne-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-lhioguanine; mcreaptopurinc; methotrexate: platinum analogs Such as cisplatin and carboplatin; platinum; etoposide (V?-1<5); sfosfamlde; mitomycin C; mitoxantrone; vincristine; vinblastine; vinorclbims; navelbifie; novautronc; feniposidc; dauiiomycin; aminopteriti; xcloda; ibandronate; CPT-1 1; lopoisomerasc inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamidns; capecilabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above, Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-iinidazoles, 4-hydroxytamoxifcn, trioxilenc, keoxifenc. LY117018, onapristonc, and toremifene (Fareston); anti-androgens such as flutamide, nilutamide, bicalutarnidc, leuprolide, and goserelin; other chemotherapeutic agents such as prednisolone, Pharmaceutically acceptable salts, acids or derivatives of any of the above are included. "Treating" or “treatment” or “alleviation" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) tbe targeted pathologic condition or disorder. A subject is successfully “treated" for a CD20 positive cancer or an autoimmune disease if, after receiving a therapeutic amount of a CD20 binding antibody of the invention according to the methods of the present invention, the subject shows observable and/or measurable reduction In or absence of one or more signs and symptoms of the particular disease. For example, for cancer, reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (('.«?,, slow to some extent and preferably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; increase in length of remission, and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. Reduction of the signs or symptoms of a disease may also be felt by the patient. Treatment can achieve a complete response, defined as disappearance of all signs of cancer, or a partial response, wherein the size of the tumor is decreased, preferably by more than 50 percent, more preferably by 75%. A patient is also considered treated if the patient experiences stable disease. In a preferred embodiment, the cancer patients are still progression-free in the cancer after one year, preferably after 15 months, These parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician of appropriate skill in the art. A ‘'therapeutically effective amount" refers to an amount of an antibody or a drug effective to “treat” a disease or disorder in a subject. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce tbe tumor size; inhibit (f.e., slow to some extentand preferably stop) cancer cel] infiltration into peripheral organs; inhibit (/. e., slow to some extent, and preferably slop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent One or more of the symptoms associated with the cancer. See preceding definition of “treating”. "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Compositions and Methods of the Invention

The invention provides .humanized antibodies that bind human CD2D, and preferably other primate. CD20 as well, comprising a H chain having at least one, preferably two or all of the H chain CDRs of a nonhuman species anti-human CD20 antibody (donor antibody), and substantially all of the framework residues of a human consensus antibody as the recipient antibody. The donor antibody can be from various nonhuman species including mouse, rat. guinea pig, goat, rabbit, horse, primate but most frequently will be a murine antibody. “Substantially all" in this context is meant that the recipient FR regions in the humanized antibody may include one or more amino acid substitutions not originally present in die human consensus FR sequence. These FR changes may comprise residues not found jo tile recipient or (be donor antibody.

Tn one embodiment, the donor antibody is the murine 2H7 antibody, the V region including the CDR and FR sequences of each of the H and L chains of which are shown in FTG. 1A and IB, In a specific embodiment, the residues for the human Fab framework correspond to the consensus sequence of human Vκ subgroup I and of V„ subgroup 111, these consensus sequences are shown in Figure 1A and Figure IB, respectively. The humanized 2H7 antibody of the invention will have at least one of the CDRs in the H chain of the murine donor antibody. In one embodiment, the humanized 2H7 antibody that binds human CD20 comprises the CDRs of both the H and L chains of the donor antibody.

In a full length antibody, the humanized C.D20 binding antibody of the invention will comprise a humanized V domain joined to a C domain of a human immunoglobulin. Tn a preferred embodiment, the H chain C region is from human IgG, preferably TgGl or IgG3. The L chain C domain is preferably from human κ chain.

Unless indicated otherwise, a humanized 2H7 antibody version heroin will have the V and C domain sequences of 2H7.V16 L chain (FIG. 6, SEQ ID NO, 21) and H chain (FIG 7., SBQ ID NO. 22) except at the positions of amino acid substitutions or changes indicated in the experimental examples below.

The humanized CD20 binding antibodies will bind at least human CD20 and preferably bind other primate CD20 such as that of monkeys including cynomolgus and rhesus monkeys, and chimpanzees. The sequence of the cynomolgus monkey CD20 is disclosed in Example 15 and Figure 19

The biological activity of the CD20 binding antibodies and humanized CD20 binding antibodies of the invention will include at least binding of the antibody to human CD20, more preferably binding to human and primate CD20 (including cynomolgus monkey, rhesus monkey, chimpanzees), with a value of no higher than 1 -χ 10'8, preferably a Kj value no higher <hau about 1 a 109, even untie piefeiahly a Ka value no higher than about 1 x 10‘m, and be able to kill or deplete 11 cells in vitro or in vivo, preferably by at least 20% when compared to the baseline level or appropriate negative control which is not treated with such an antibody.

The desired level of B cell depletion will depend on the disease. For the treatment of a CD20 positive cancer, it may be desirable to maximize the depletion of (he B cells which arc the target of the anti-CD20 antibodies of the invention. Thus, for the treatment of a CD20 positive B cell neoplasm, it is desirable that the B cell depletion be sufficient to at least prevent progression of the disease which can be assessed by the physician of skill in the art, c.g., by monitoring tumor growth (size), proliferation of the cancerous cell type, metastasis, other signs and symptoms of the particular cancer. Preferably, the B cell depletion is sufficient to prevent, progression of disease for at least. 2 months, more preferably 3 months, even more preferably 4 months, more preferably 5 months, even more preferably 6 or more months. In even more preferred embodiments, the B cell depletion is sufficient to increase the time in remission by at least 6 months, more preferably 9 months, more preferably one year, more preferably 2 years, more preferably 3 years, even more preferably 5 or more years. In a most preferred embodiment, the B cell depletion is sufficient u> cure the disease. In preferred embodiments, the B cell depletion in a cancer patient is at least about 75% and more preferably, 80%, 85%, 90%, 95% , 99% and even 100% of the baseline level before treatment.

For treatment of an autoimmune disease, it may be desirable to modulate the extent of B cell depletion depending on the disease and/or Cite severity of (he condition in the individual patient, by adjusting the dosage of CD20 binding antibody. Thus, B cell depietion can but does not have to be complete. Or, total B cell depiction may be desired in initial treatment but in subsequent treatments, the dosage may be adjusted to achieve only partial depletion. In one embodiment, the B cell depletion is at least 20%, i.e., 80% or less of CD20 positive B cells remain as compared to the baseline level before treatment. In other embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70% or greater. Preferably, the B cell depletion is suffieientto halt progression of the disease, more preferably to alleviate the signs and symptoms of the particular disease under treatment, even more preferably to cure tile disease.

The invention also provides bispecific CD20 binding antibodies wherein one arm of tbe antibody has a humanized H and L chain of the humanized CD20 binding antibody of the invention, and the Otiler arm has V region binding specificity for a second antigen. In specific embodiments, the second antigen is selected from the group consisting of CD3, CD64. CD32A, CD 16, NKG2D or other NK activating ligands.

In comparison with Rituxan (rituximab), vl6 exhibits about 2 to 5 Ibid increased ATXZC potency , ~3-4 fold decreased CDC than Rituxan.

Antibody production

Monoclonal antibodies

Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DMA methods (U.S, Patent No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or arc capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or snore substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if tbe parental myeloma cells lack the enzyme hypoxanthine guanine phospboribosyl transferase (HGPRT or HPRT), tbe selective culture medium for the hybridomas typically will include hypoxanthine, aminoptciin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are (hose that fuse efficiently, support stable high-level production of antibody by (he selected antibody-producing cells, and arc sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution

Center, San Diego, California USA, and 5P-2 and derivatives e g,, X63-Ag8-653 cells available from the American lype Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromycloma cell lines also have been described for the production of human monoclonal antibodies (Kozbot,J. irhmunoL, 133:3001 (1984): and Brodeur at at.. Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc„ New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunopreciphation or by an in vitro binding assay, such as radioimmunoassay (R.IA) or enzyme-linked immunosorbent assay (ETJSA).

The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson ct al, Anal Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Coding, Monoclonal Antibodies: Principles and Practice, pp.59 -103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MBM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g, by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or scrum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g„ using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures by using oligonucleotide probes that arc capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli- cells, simian CDS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in foe recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skcrra ct al, Cutr. Opinion in Immunol, 5:256-262 (1993) and Pliickthun, Immunol. Revs., 130:151-188 (1992) .

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCaffcrty et al, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991) and Marks etal, J. Mol Biol, 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Βία/Tschnology, 10:779-783 (1992)). as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse ei al. Nuc. Acids. Pcs., 21:2265-2266 (1993) ). Thus, these techniques ate viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and Q.) sequences for the homologous murine sequences (U.S, Patent No. 4,816,567; and Morrison, etai, Proc, Nat! A cud. Sci. USA, 81:6851 (1984)). or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The nonimmunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

Humanized antibodies

Methods for humanizing non-human antibodies have been described in the art, Preferably, a humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These ηοη-human aniino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al, Nature, 321:522-525 (1986); Reichmann etai, Nature, 332:323-327 (1988): Verhocycn et at., Science, 239:1534-1536 (1988)), by substituting hypervariablc region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariablc region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important, to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human therapeutic use. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims etai, J, Immunol., 151:2296 (1993); Chotfria etai, J. Mol. Biol., 196:901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et at, Proc. Natl. Acad. Sci. USA, 89:4285 (1992): Presta et at., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled ill the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of ihc likely role of the residues in the functioning of the candidate immunoglobulin sequence, Le., the analysis of residues that influence the ability of the candidate immu noglobulin to bind its antigen. To this way, I;R residues can be selected and combined front the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target ar)tigcn(s), is achieved. In general, the bypcrvariable region residues are directly and most substantially involved in influencing antigen binding.

The humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agentfs) in order to generate an immu noconjugate. Alternatively, (he humanized antibody may be an full length antibody, such as an full length JgGI antibody.

Human antibodies and phage display methodology

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals {e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chai n joining region (.TH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. Sec, e.g., Jakobovits e.t ai, Proc. Natl. Acad. Sr.L USA. 90:2551 (1993); Jakobovits et at, Nature, 362:255-25¾ (1993): Bruggemann e.t ai, Year in· fmmurto.., 7:33 (1993); U.S. Patent Nos. 5,545,806,5,569,825,5,591,669 (all ol'GenPhann): 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCaffert.y etal., Nature 348:552-553 [Ί990]) can he used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coal protein gene of a filamentous bacteriophage, such as Μ13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Tiuis, the phage mimics some of the properties of the B-ccll. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Gackson etal.. Nature, 352/624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V gcncs.dcrivcd from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks el ai, J. Mol, Biol, 222:581-597 ¢1.991), or Griffith a at, EMBOJ. .12:725-734 (1993). See, also, U.S.

Patent Nos. 5,565,332 and 5,573,9()5.

As discussed above, human antibodies may also be generated by in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275). A Mibody fragments in certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows lbr rapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g.. Morimoto et at., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et at., Science, 229:81 (1985», However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. colt, thus allowing the facile production of large amounts of these fragments. Antibody fragments can he isolated from the antibody phage libraries discussed above. Alternatively, Fab'-5H fragments can be directly recovered from fi, coli and chemically coupled to formF(ab')2 fragments (Carter et at., Bio/Technology 10:193-167 (1992)). According to another approach. F(ab')x fragments can be isolated directly from recombinant host cell culture. Fab and F(ab‘» fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869.046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5.587,458. Fv and sFv are the only species with intact combining sites that arc devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may he constructed to yield fusion of an effector protein at either the amino or the carbosy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a ‘'linear antibody”, e.g., as described in U.S. Patent 5,641,870 for example. Such linear antibody fragments may be monospecific or bispceific.

Bispecific antibodies

Bispceific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the CD20 protein. Other such antibodies may combine a CD20 binding site with a binding site for another protein, Alternatively, an ami-CD20 arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3), or Fe receptors for IgO (FeyR), such as FcyRT (CD64), FcyRJJ (CD32) and FcyPJII (CD 16), or NKG2D or other NK cell activating ligand, so as to focus and localize cellular defense mechanisms to the CD20-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CD20. These antibodies possess a CD20-binding arm and an arm which binds the cytotoxic agent (e.g. saporin. anli-inlerfcron-α. vinca alkaloid, riein A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')t bispecific antibodies), WO 96/16673 describes a bispecific anti-ErbB2/ant.i-FcyRJII antibody and U.S. Patent No. 5,837.234 discloses a bispecific anti-ErbB2/anti-FcyRT antibody. A bispecific anti-lirbB2/Fca antibody is shown in WO98/02463, U.S, Patent No. 5,821,337 teaches a bispecific anti-KrbB2/anti-CD3 antibody.

Methods for making bispecific antibodies are known in the art. Traditional production of ful) length hi sped lie antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different, specificities (Millstein el at., Nature. 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, tlicsc hybridomas (quadromaa) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures ara disclosed in WO 93/08829, and in Traunccker e.t al, EM BO./., 10:3655-3659(1991).

According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion Is with an Ig heavy chain constant domain, comprising at least part of the .lunge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain bonding, present in at least one of the fusions. DMAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co transfected into a suitable host cell. This provides for greater flexibility in adjusting the mutual proportions of the tliree polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is. however, possible to insert the coding sequences for two or all three polypeptide Chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when Lite ratios have no significant affect on the yield of the desired chain combination.

In a preferred embodiment of this approach, the bispecific antibodies arc composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It. was found lhat this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94/04690, For further details of generating bispecific antibodies see. for example. Surcsh et aL. Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which arc recovered from recombinant cell culture- The preferred interface comprises at least a part of the Ch3 domain. In this method, one or more small amino acid side chains from cite interface of the first antibody molecule arc replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-produefs such as homodimers.

Bispecific antibodies include cross-linked or "beteroconjugate" antibodies. For example, one of the antibodies in the beteroconjugate can be coupled to avidin, the otiter to biolin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and HP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents ate well known in the art, and are disclosed in U.S. Patent No. 4,676,980, along with a number of cross-linking techniques,

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan ei al. Science, 229: 81 (1985) describe a procedure wherein intact antibodies are pratcolyrjeajly cleaved to gcncrare F(ab')2 fragments. These fragments arc reduced in the presence of the dithiol completing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intcrmolecular disulfide formation. The Fab' fragments generated are diets converted to thiomlrobcnzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the F'ab'-thiol by reduction with mercaptocthylamijje and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab'-SW fragments from E. colt, which can be chemically coupled to form bispecific antibodies, Shalaby etal,./. Exp. Med., 175: 217-225 (1992) describe the production of a folly humanized bispecific antibody Flab’)? molecule. Bach Fab' fragment was separately secreted from E. coti and subjected to directed chemical coupling in vitro to form the bispecific antibody. Πιc bispecific antibody thus formed was able to bind to cells overexpressing the EfbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny at al., J. Immunol, 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.

The antibody homodimers were reduced at the hinge region m form monomers and then re-oxidizcd to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.

The "diabody" technology described by Bollinger el at., Proc. Natl, Acad. Set USA, 90:6444-6448 (1993) has provided mi alternative mechanism for making bispecific antibody fragments The frngrnp.nrs comprise, a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the Vh and Vt. domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy lor making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber etal, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies arc contemplated. For example, trispecific antibodies can be prepared. Tutt etalL J. Immunol- 147: 60 (1991).

Multivalent Antibodies A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind- The antibodies of the present invention can be multi valent antibodies (which are other than of the lgM class) with three or more antigen binding sites (e.g. tctravalcnt antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region, in rbis scenario, the antibfidy will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding .sites, The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(Xl)„-VD2-(X2)„-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide ehain(s) may comprise: VH-CHl-flexiblc Hnker-VH-CHl-Fc region chain; or VH-CHI-VfJ-CHl-Fc region chain. The multivalent antibody herein preferably further comprises at least, two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

Other amino (tcid· sequence modifications

Amino acid sequence modification(s) of the CD20 binding antibodies described herein are contemplated, For example, it may be desirable to improve the binding affinity and/or other biological properties of She antibody. Amino acid sequence variants of the anti-CD20 antibody are prepared by introducing appropriate nucleotide changes into the anti-CD20 antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the anli-CD20 antibody. Any combination of deletion, insertion, and substitution is made to arrive at die final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the anti-CD20 antibody, such as changing the number or position of glycosylation sites. A useful method for identification of certain residue.·: or regions of the anti CD2.0 antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells in Science, 244:1081-1085 (1989). Here, a residue or group of target residues are identified (e.%., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with CD20 antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or oilier variants at, or for, the sites of substitution, Thus, while the site tor introducing an amino acid sequence variation is predetermined, the nature of the .mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at the target codon or region and the expressed anli-CD20 antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as welt as intrascqucnce insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-CD20 antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertionai variants of the anti-CD20 antibody molecule include the fusion to the N- or C-tcrminus of the anti-CD20 antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the seruoi half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the a.nti-CD20 antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include tile hypervariable regions, but FR alterations arc also contemplated. Conservative substitutions are shown in rhe Tabic below under the heading of "preferred substitutions”. If sucli substitutions result in a change in biological activity, then mote substantial changes, denominated "exemplary substitutions" in the Table, or as further described below in reference to amino acid classes, may be introduced and the products screened. TABLE of Amino Acid Substitutions

Substantial modifications in the biological properties of the antibody arc accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone, in the area of Uic substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobieily of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues arc divided into groups based on common side-chain properties: (1) hydrophobic: norlcucine, met, ala. val, leu. ile; (2) neutral, hydrophilic: cys, ser, thr; (3) acidic: asp, glu: (4) basic: asn, gin. his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp. lyr, phc.

Non-conservativc substitutions will entail exchanging a member of one of these classes for another class.

Any Cysteine residue not involved in maintaining the proper conformation Ol'the anti-CD20 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an fv fragment). A particularly preferred type of substitutional variant involves substituting one or more hypcrvariable region residues of a parent antibody {e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated, A convenient way for generating such substitutional varianrs involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g. <5-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated arc displayed in a monovalent fashion from filamentous phage particles as fusions to the gene Til product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g. binding affinity) as herein disclosed. In order to identity candidate hypcrvariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and human CD20. Such contact, residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the pane) of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that arc not present in the antibody.

Glycosylation of antibodies is typically either N-linked or Q-Hnked- N-linked refers to the attachment of the carbohydrate moiety to the side chain, of an asparagine residue. The tripeptide sequences asparag ϊne-X-serlne and asparagine-X-tbreonine, where X is any amino acid except, proline, are the \ recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of cither of these tripeptide sequences in a polypeptide creates a potential glycosylation site, O-linked glycusylation refers to the attachment of one of the sugars N-accylgalactosamine. galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-bydroxypralinc or 5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptidc sequences (for N-linked glycosylation sites), The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules encoding amino acid sequence variants of the anti-CD20 antibody are prepared by a variety of methods known in die art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-CD20 antibody.

It may be desirable to modify tile antibody of the invention with respect ro effector function, e.g. so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodirncric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et at.,, J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol 148:2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctiona] cross-linkers as described in Woll'f et aL Cancer Research 53:2560-2565 (1993). Alternatively, at) antibody can be engineered which has dual Fc regions and may thereby have enhanced complement mediated lysis and ADCC Capabilities. See Stevenson et. at. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of tile antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.5. Patent 5.739,277, for example. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an TgG molecule (e.g., TgGi, IgC^· IfiGj, or IgG.,) that is responsible for increasing the in vivo serum half-life of the TgG molecule.

Other antibody modifications

Other modifications of the antibody are contemplated herein, For example, the antibody may be linked to one of a variety of nonpvotemaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylen.es, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in mierocapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelafin-mierocapsules and poly-(raelhylmclhaeylatc) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particle* and nanocapsules), or in tnacroemulsions. Such Techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

Screening for antibodies with the desired properties

Antibodies with certain biological characteristics may be selected as described in the Experimental Examples.

The growth inhibitory effects of an anti-CD20 antibody of the invention may be assessed by methods known in the art, e.g., using cells which express CD20 either endogenously or following transfection with the CD2Q gene. For example, tumor cell lines and CD20-franslectcd ceils may treated with an anli-CD20 monoclonal antibody of the invention at various concentrations lor a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing ^H-tbymidinc uptake by the ceils treated in the presence or absence an anti-CD20 antibody of the invention. After antibody treatment, the cells arc harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line.

To select for antibodies which induce cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. CD20-exprassing tumor cells arc incubated with medium alone or medium containing of the appropriate monoclonal antibody at. e.g, about J Opg/ml . The cel Is are incubated lor a 3 day time period. Followi ng each treatment, cells are washed and aliquoted into 35 min strainer-capped 12 x 75 tubes (1ml per rube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive FT (10ug/ntl). Samples may be analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuesi software (Bceton Dickinson). Those antibodies which induce statistically significant levels of cell death as determined by Pi uptake may be .selected as cell death-inducing antibodies.

To screen for antibodies which bind to an epitope on CD20 bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (l 988), can be performed. This assay can be used to determine if a test antibody binds the same site or epitope as an anti-CD20 antibody of the invention. Alternatively, or additionally, epitope mapping can be performed by methods known in tlie art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initailly tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of CD20 can be used in Competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

Vectors, Host Cells and Recombinant Methods

The invention also provides an isolated nucleic add encoding a humanized CD20 binding antibody, vectors and host cells comprising the nucleic acid, and recombinant, techniques for the production of the antibody. ! For recombinant production of the antibody, die nucleic acid encoding it is isolated and inserted into a replicable vector lor further cloning (amplification of the DNA) or for expression. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g.. by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer clement, a promoter, and a transcription termination sequence. (1) Signal sequence component

The CD20 binding antibody of this invention may be produced rccombmantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at. the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (r.<?., cleaved by a signal peptidase) by die host cell. For prokaryotic host cells that do not recognize and process the native CD20 binding antibody signal sequence, die signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase. Ipp, or heat-stable enterotoxin 11 leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertase leader, « factor leader (including Saccliaromyces and Kluyveromyces «-factor leaders), or acid phosphatase leader, the C- albicans glucoamyla.se leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNA encoding the CD20 binding antibody, (it) Origin of replication

Both expression and cloning vectors contain a nucleic acid sequence tliat enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from (lie plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV4Q, polyoma, adenovirus, VSV or BPV) arc useful for cloning vectors in mammalian ceils. Generally , the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter). (Hi) Selection gene component

Expression and cloning vectors may contain a selection gene, also termed a selectable marker.

Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampieillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemose for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the CD20 binding, antibody nucleic acid, such as DHFR, thymidine kinase, mctallothioncin-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a cujture medium that, contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host ceil when wild-type DHFR is employed is the Chinese, hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096).

Alternatively, host cells (particularly wild-type hosts that, contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding CD20 binding antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3’-phosphotransfcrasc (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965.399. A suitable selection gene for use in yeast is tire trp 1 gene present in the yeast plasmid YRp7 (Srinchcomb et al, Nature, 282:39 (1979)). 'Die irpl gene provides a selection marker for a mutant Strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PBP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp\ lesion in the yeast, host cell genome then provides an effective environment for detecting transformation by growth in the absence of lryplophan. Similarly, Ae«2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDl can be used for transformation of Kiuyveromyces yeasts. Alternatively, an expression system for large-scale production of .recombinant calf chymusin was reported for K. lactis. Van den Berg, BiofTechmlogy, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kiuyveromyces have also been disclosed. Fleer et al, Bw/Tecknalogy, 9:968-975 (1991). (iv) Promoter component

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is opcrably linked to the nucleic acid encoding the CD20 binding antibody. Promoters suitable for use with prokaryotic hosts include the pkoA promoter , β-lactamasc and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S-D-) sequence operably linked to tile DNA encoding the CD20 binding antibody.

Promoter sequences arc known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated- Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may he any nucleotide . At the 3’ end of most eukaryotic genes is an AATA A A sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences arc suitably Inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyccraldehyde-3-phosphate dehydrogenase, hexokinasc, pyruvate decarboxylase, phospbofructokinase, glucoseO-phosphate isomerase. 3-phosphoglycerate mutase, pyruvate kinase, triosephosphale isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, arc the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradafive enzymes associated with nitrogen metabolism, metal lothioncin, glyceraldehyde-A-pbosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,637, Yeast enhancers also are advantageously used with yeast promoters. CD2U binding antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as Adenovirus 2), bovine papilloma vims, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindlH E restriction fragment- A system for expressing DNA in mammalian hosts using the bovine papilloma vims as a vector is disclosed in U.S. Patent No. 4,419,446. Λ modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et aL. Nature 297:598-601 (1982) on expression of human β-intertcron cDN A in mouse cel Is under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. (v) Enhancer element component

Transcription of a DNA encoding the CD20 binding antibody of this invention by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein. and insulin). Typically, however, one will use an enhancer from a eukaryotic cell vims. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5’ or 3' to the CD20 binding antibody-encoding sequence, but is preferably located at a site S’ from the promoter. (vi) Transcription termination component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as poiyadenylatcd fragments in the untranslated portion of the mRNA encoding CD20 binding antibody. One useful transcription termination component is the bovine growth hormone polyadenylalion region. See W094/11026 and the expression vector disclosed therein. (vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria·, such as Gram-negative or Gram-positive organisms, for example, Enlerobacteriaceae such a&amp; Escherichia, 4.1»,, E. coll. Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimitrium, Serrat.ia., e.g,. Serratio, marcescam, and Shigella, as well as Bacilli such as B. subtilis and B. Ucheniformis (e.g., ti. Ucheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Slrcpn.vnyc.es. One preferred E. colt cloning host is E. colt 294 (ATCC 31,446), although other strains such as E. coli B, &amp; coli X1776 (ATCC 31,537), and E. coli W3! 10 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (c.g,, a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation.

Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, c.g., U.S. 5.648,237 (Carter et. al.), U.S. 5,789.199 (.Toly et ah), and U.S. 5.840.523 (Simmons et. al.) which describes translation initiation region (T1R) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E. coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g,, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for CD20 binding antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomycespombe; Kluyve.romyces hosts such as, e.g.. K. laclis, K. fragtlis (ATCC 12,424), K. Mgaricus (ATCC 16,045), K. wicke.ramii (ATCC 24,178), K. wultii (ATCC 56,500), K. drosophilamm (ATCC 36.906), K. thermotolerans, and K. marxianus: yarrowia (EF 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniornyc.es occidentalis-, and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocktdiumu and Aspergillus hosts such as A. nidukms and A. niger.

Suitable host cells for the expression of glycosylated CD20 binding antibody are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodopterafrugipenla (caterpillar), Andes aegypti (mosquito), Aeries albopictus (mosquito), Drosophila mekmagasier (fruitfly).. and Bombyx man. have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californico. NPV and the Bm-5 strain of Bombyx more NPV, and such viruses may be used a? the virus herein according ro the present invention, particularly lbr transfection of Spodopterafrugiperdci cells.

Plant cell cultures of cotton, com, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture {tissue culture) has beeonie a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CVI line transformed by SV40 (C.OS-7, ATCC CR.L 1651)1 human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham el al., J, Gen Virol. 36:59 (1977)): baby hamster kidney cells (BHK, ATCC CCT. 10); Chinese hamster ovary cclls/-DHFR (C.HO, Urlaub et of.,

Pmc. Natl, Acad. ScL USA 77:4216 (1980)); mouse senoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (llELA, ATCC CCL 2): canine kidney cells (MDCK, ATCC CCT- 34); buffalo rat. liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, MB 8065); mouse mammary tumor (MMT 060562, ATCC CCLS1); TR1 cells (Mather el al, Amah N.Y. Acad $ci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for CD20 binding antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. (viii) Culturing the host celts

The host cells used to produce the CD20 binding antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco’s Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham el al, Meth, Enz. 58:44 (1979), Barnes et al, Anal BiochemA<n:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762: 4.560,655: o.r 5,122,469; WO 90/03430; WO 87/00195; or U.S, Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be Supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEFES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected .for expression, and will be apparent, to the ordinarily skilled artisan. (be) Purification of antibody

When using recombinant techniques, the antibody can be produced intiacellularly, in the periplasmio space, or directly secreted into the medium. If the antibody is produced intraeelhilarly, as a first step, the particulate debris, cither host Cells or lysed fragments, arc removed, tor example, by centrifugation or ultrafiltration. Carter e.t al, Bio/Te.chnology 10:163-167 (1992) describe a procedure for Isolating antibodies which are secreted to the periplasmic space of E. coll Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenyl methylsujfcmylfluoridc (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems arc generally first concentrated using a commercially available protein concentration filter, for example, an'Ainicon or Milliporc Pellicon ultrafiltralion unit. Λ protease inhibitor such as PIVTSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells ca.ii be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present, in the antibody.

Protein A can be used to purify antibodies that arc based on human yl, y2, or γ4 heavy chains (landmark et ol, J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isolypes and for human γ3 (Ouss ft al, EMBOJ. 5:15671575 (1986)). llie matrix to which the affinity ligand is attached is most often agarose, but ofher matrices are available. Mechanically stable matrices such as controlled pore glass or po!y(.sfyretiedivi®yi)beozeiie allow for faster flow rates and shorter processing times than can be achieved with agarose. Where, (lie antibody comprises a Ch3 domain, the Bakerbond ABX™resin (J. T- Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-cxchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation arc also available depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (<?.#., from about 0-D.25M salt).

Antibody conjugates

The antibody may be conjugated to a cytotoxic agent such as a toxin or a radioactive isotope. In certain embodiments, tlie toxin is calicheamicin. a maytansinoid, a dolastatin, auristatin 12 and analogs or derivatives thereof, are preferable.

Preferred drugsfloxins include DNA damaging agents, inhibitors of microtubule polymerization or depolymerization and antimetabolites. Preferred classes of cytotoxic agents include, for example, file enzyme inhibitors such as dihydrofolats reductase inhibitors, and thymidylate synthase inhibitors, DNA intcrcalators, DNA cleavers, lopoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the podophyllotoxins and differentiation inducers. Particularly useful members of those classes include, for example, methotrexate, methopterin, dicbloromethotrexate, 5-fluorouracil, 6-niercapt.opurine, cytosine arabinoside, melpMaft, leurosine, leurosidejne, actinomycin, daunorubicin, doxorubicin, N-(5,5-diaectoxypentyDdoxorubicin, morpholino-doxorubicin, l-(2-choroehtliyl)-l,2-dimethanesulfonyl hydrazide, Nfi-acetyl spermidine, aminopterin methopterin, esperamicin, mitomycin C, mitomycin A, actinomycin, bleomycin, carminomyein, aminopterin, tallysoittycin, podophylj.otoxin and podophyllotoxin derivatives such as eloposide or ctoposide phosphate, vinblastine, vincristine, vindcsinc. taxol, taxotere, retinoic acid, butyric acid. NB'aeety! spermidine, camptotheein, calicheamicin. bryostatins, cephalostatins, ansamitocin, aetosin, maytaiistnoids such as DM-!, maytansine, maytansinol, N-desmeihyJ.-4,5-desepoxymaytansino.(, C-19-dechioromaytansinol, C-20-hydroxymaylansinol, C-20-demetboxymaytansinol, C-9-SH maytansinol, C-14-alkoxymethylmaytansinol, C-14-hydroxy or acetyloxymetblmaytansmol, C-15-hydroxyi'aectyloxymaytansinol, C-15-melliOxy may tan sin ol. C-18-N-demethylmaylansinol and 4,5-dcoxy maytansinol, auristatins such as auristatin E, Μ, PHF. and PE: dolostatins such as doiostatin A, doiostatin B, doiostatin C, doiostatin D, doiostatin E (20-epi and 11-epi), doiostatin G, doiostatin H, doiostatin 1. doiostatin I, doiostatin 2, doiostatin 3, doiostatin 4, doiostatin 5, doiostatin 6, doiostatin 7. doiostatin 8, doiostatin 9, doiostatin 10, deo-dolostatio 10, doiostatin 11, doiostatin 12, doiostatin 13, doiostatin 14, doiostatin 15, doiostatin 16. doiostatin 17, and doiostatin 18; cephalostatins such as cephalostatin 1, cephalostatin 2, ccphalo.statin 3, cephalostatin 4, cephalostatin 5. cephalostatin 6, ccphalostatin 7,25'-epi-cepba)ostati.il 7, 20-epi-cephaiostalin 7, cephalostatin 8, cephalostatin 9, cephalostatin 10, cephalostatin 11.,cephalostatin 12,cephalostatin 13,cephalostatin 14. cephalostatin l5.cephalostatin 16,cephalostatin 17, cephalostatin 18, and cephalostatin 19..

Maytaminoids are mitototic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maylemts serrate (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maylansinol esters (U.S. Patent Net. 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in U-S- Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260.608; 4.265,814; 4,294,757; 4,307,016; 4,308,268: 4,308,269; 4,309,428; 4,313,946; 4,315,929;4,317,821; 4.322,348; 4,331,598; 4,361,650; 4,364,866:4,424,219; 4,450,254; 4,362,663; and 4,371,533, the disclosures of which are hereby expressly incorporated by reference.

Maytansine and maytansinoids have been conjugated to antibodies specifically binding to tumor cell antigens. Irnmunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in 11-5. Patent Nos. 5,208,070, 5,416,064 and European Patent HP 0 425 235 III, the disclosures of which are hereby expressly incorporated by reference. Liu et a!., Proc. Natl, Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against human colorectal cancer. The conjugate was found to be highly cytotoxic towards cultured colon cancer cells, and showed antitumor activity in an in vivo tumor growth assay. Chari etal. Cancer Research 52:127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated via a disulfide linker to the murine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody ΤΑ. 1 that binds the HER-2/raeit oncogene.

There are many linking groups known in the ait for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent No. 5,208,020 or EP Patent 0 425 235 B1, and Chari etal Cancer Research 52: 127-131 (1992). The linking groups include disufide groups, thinether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and fhipether groups being preferred.

Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such asN-suCCinimidyi-3-(2-pyridyldithio) propionate (SPDF), succinimidyl-4-(N-maleiimdomethyl) cyclohexane- 1-carboxylatc, iminolhiolar.e (ΓΙ'), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (Such as glutareldehyde), bis-arido compounds (Such as bis (p-azidobenzoy!) hexanediamine), bis-diazonium derivatives (such as bis-(p-diaMmiurnbcnzoyl)-clhylertedianiine). diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2.4-dinitrobenzene), Particularly preferred coupling agents include N-SUccinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson ct aL. Rinc/wm.,!. 173:723-737 11978J) and N-SuCCinimidyl-4-(2-pyridy)thio)pentanoate (SPP) to provide for a disulfide linkage.

The linker may be attached to the maytansinoid molecule at various positions, depending on the type of the link. For example, an ester linkage may be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction may occur at the C-3 position having a hydroxyl group, the C-14 position modified with hyrdoxymethyl, die C-15 position modified with a hydroxyl group, and the C-20 position having a hydroxyl group, la a preferred embodiment, the linkage is formed at the C-3 position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immuaocoiljugate of interest, comprises an CD20 binding antibody conjugated to one or mere ealichcamiein molecules. The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. patents 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770.701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin which may be used include, but are not limited to, γι’, a5‘, 0¾1. N-acetyl-γι', PSAG and θ’ι (Hinman etal. Cancer Research 53:3336-3342 (1993), Lode etaL Cancer Research 58; 2925-2928 (1998) and die aforementioned U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be conjugated is QFA which is ail sintifolsitft Both caliohRamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internalization greatly enhances their cytotoxic effects.

Radioactive isotopes

For selective destruction of the tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugatcd anti-CD20 antibodies. Examples include Arn, I131, l125, Y°°, Rel8s, Re18*, Sml5'\ Bi212, P32, Pb212 and radioactive isotopes of Lu. When the conjugate is used lbr diagnosis, it may comprise a radioactive atom for scintigraphic studies, for example tc"m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, niuOgen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynrbesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as fc·59™ or 1IB, .Re186, RcIKK and In'11 can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The TODOGEN method (Fraker et al (3978) Biochem. Biophys, Res. Commun. 80; 49-57 can be used to incorporate iodine-123- “Monoclonal Antibodies in Immunoscintigraphy" (Chatai.CRC Press 1989) describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunetional protein coupling agents sucli as N-succinimidy)-3-(2-pyriciyldithio) propionate (SPDP), suecinimidyl-4-(N-malcimidomcthyl) cyclohexane-)-carboxylate, iminothiolane (IT), bifunctional derivatives of iniidoesters (such as dimethyl ndipimidate HCL), active esters (such as disuccinimidyl subsrate), aldehydes (such as glularcldehydc), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediaminc), bis-diazonium derivatives (such as bis-(p-diazoniiiinben2oyl)-cthylenedianiine), ditsocyanales (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 155-difluoro-2,4-ditiitrobcnacnc). For example, a ricin imimmotoxin can be prepared as described in Vitetta e.t al. Science 238: 1098 (1987). Carbon-14-labeled l-isolhiocyanatobcnzyl-3-mcthyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. The linker may be a “clcavablc linker” facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari el aL Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020) may beused-

Thcrapcutic Uses of the CD20 binding Antibodies

The CD20 binding antibodies of the invention arc useful to treat a number of malignant and non-malignaut diseases including autoimmune diseases and related conditions, and CD20 positive cancers including B cell lymphomas and leukemias. Stem cells (B-cell progenitors) in bone marrow lack the CD20 antigen, allowing healthy B-ce!J.s to regenerate after treatment and return to normal levels within several months.

Autoimmune diseases or autoimmune related conditions include arthritis (rheumatoid arthritis-juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis including atopic dermatitis; chronic autoimmune urticaria, po 1 ymyosilis/dermatomyosilis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (1BD) (Crohn’s disease, ulcerative colitis), respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (STJR), lupus (including nephritis, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lyinphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including ANCA), aplastic anemia, Coombs positive anemia, Diamond Rlackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (ΛΙΗΑ), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CN$ inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anri-pbosphulipid antibody syndrome, allergic neuritis, Bechet disease. Castlcman’s syndrome, Goodpasture’s Syndrome, Lambert-F.aton Myasthenic Syndrome, Reynaud’s syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including pretreatment for high panel reactive antibody titers, TgA deposit in tissues, etc), graft versus host disease (GVHD), pemphigoid bullous, pemphigus (aJJ including vulgaris, foliaceus), autoimmune polyendocrinopathics, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies orTgM mediated neuropathy, idiopathic thrombocytopenic purpura (TTP), thrombotic ihrobocytopcnic purpura (TTP), autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashiffloto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism. Addison’s disease. Grave’s disease, autoimmune polyglandular syndromes (or polyglandular endoerinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (JDDM) and Sheehan's syndrome; autoimmune hepatitis, Lymphoid interstitial pneumonitis (HJ.V), bronchiolitis obliterans (ton-transplant) vs NSIP, Guillain-Barre’ Syndrome. Large Vessel Vasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu’s) Arteritis), Medium Vessel Vasculitis (including Kawasaki’s Disease and Polyarteritis Nodosa), ankylosing spondylitis, Berger’s Disease (IgA nepliropathy), Rapidly Progressive Glomerulonephritis, Primary biliary cirrhosis, Ceiiac sprue (gluten enteropathy), Cryoglobulinemia, ALS, coronary artery disease. CD20 positive Cancers arc those comprising abnormal proliferation of cells that express CD20 on the cell surface. The CD20 positive B cell neoplasms include CD20-pos.itive Hodgkin's disease including lymphocyte predominant. Hodgkin's disease (LPHD); non-Hodgkin’s Jymphoma (NHL); follicular center coll (FCC) lymphomas; acute lymphocytic leukemia (ALL); chronic lymphocytic leukemia (CLL); Hairy cell leukemia, The non-Hodgkins lymphoma include low grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic lymphoma (SLL), intermediate grade/follicular NHL, intermediate grade diffuse NHL. high grade iromunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky disease NHL plasmacytnid lymphocytic lymphoma, mantle cell lymphoma, AIDS- related lymphoma and Waldenstrom's macroglobulincmia. Treatment of relapses of these cancers arc also contemplated. LPHD is a type of Hodgkin's disease that tends to relapse frequently despite radiation or chemotherapy treatment and is characterized by CD20-positive malignant cells. CLL is one of four major types of leukemia. A cancer of mature B-ceils called lymphocytes, CLL is .manifested by progressive accumulation of cells in blood, bone marrow and lymphatic tissues.

In specific embodiments, the humanized CD20 binding antibodies and functional fragments thereof arc used to treat non-Hodgkin’s lymphoma (NHL), lymphocyte predominant Hodgkin's disease (LPHD), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia, rheumatoid arthritis and juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE) including lupus nepliritis, Wegener’s disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ΓΓΡ), thrombotic throbocytopcnic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Raynaud's syndrome, Sjorgcrt’s syndrome and glomerulonephritis. 'Hie humanized CD20 binding antibodies or functional fragments thereof are useful as a single-agent treatment in, c.g.r for relapsed or refractory low-grade or follicular, CD20-posilivc. B-ccll NHL, or can be administered to patients in conjunction with other drugs in a multi drug regimen.

Indolent lymphoma is a slow-growing, incurable disease in which the average patient survives between six and 10 years following numerous periods of remission and relapse. In one embodiment, the humanized CD20 binding antibodies or functional fragments thereof are used to treat indolent NHL.

The parameters for assessing efficacy or success of treatment of the neoplasm will be known to the physician of skill in the appropriate disease. Generally, the physician of skill will look for reduction in the signs and symptoms of the specific disease. Parameters can include median lime to disease progression, time in remission, stable disease.

The following references describe lymphomas and CLL, their diagnoses, treatment and standard medical procedures for measuring treatment efficacy. Candles GP, Lister, TA, Sklar JL: The Lymphomas. W.R.Saunders Company. Philadelphia, 1998; van Besien K and Cabanillas, P: Clinical Manifestations, Staging and Treatment of Non-Hodgkin’s Lymphoma, Chap. 70, pp 1293-1338. in: Hematology, Basic Principles and Practice, 3rd ed. Hoffman et a), (editors). Churchill Livingstone, Philadelphia, 2000; and Rai, K and Patel, DrChronic Lymphocytic Leukemia, Chap. 72, pp 1350-1362, in: Hematology, Busk Principles and Practice, 3rd ed. Hoffman et at (editors). Churchill Livingstone, Philadelphia, 2000.

The parameters for assessing efficacy or success of treatment of an autoimmune or autoimmune related disease will be known to the physician of skill in the appropriate disease. Generally, the physician of skill will look for reduction in the signs and symptoms of the specific disease. The following arc by way of examples.

Tn one embodiment, the antibodies of the invention are useful to treat rheumatoid arthritis. R A is characterized by inflammation of multiple joints, cartilage loss and bone erosion that leads to joint destruction and ultimately reduced joint function. Additionally, since RA is a systemic disease, it can have effects in other tissues such as the lungs, eyes and bone marrow. Hewer than 50 percent of patients who have had RA for more than 10 years can continue to work or function normally on a day-to-day basis.

The antibodies can be used as first-line therapy in patients with early RA (i.c., methotrexate (MTX) naive) and as monotherapy, or in combination with, e.g,, MTX or cyclophosphamide. Or, the antibodies can be used in treatment as second-line therapy for patients who were DMARD and/or MTX refractory, and as monotherapy or in combination with, e.g., MTX. The humanized CD20 binding antibodies arc useful to prevent and control joint damage, delay structural damage, decrease pain associated with inflammation in RA, and generally reduce the signs and symptoms in moderate to severe RA. The RA patient can be treated with the humanized CD20 antibody prior to, after or together with treatment with other drugs used in treating RA (see combination therapy below), in one embodiment, patients who had previously failed disease-modifying antirhcumatic drugs and/or had an inadequate response to methotrexate alone axe treated with a humanized CD20 binding antibody of the invention. In one embodiment of this treatment, the patients are in a 17-day treatment regimen receiving humanized CD20 binding antibody alone (lg iv infusions on days 1 and 15); CD20 binding antibody plus cyclophosphamide (750mg iv infusion days 3 and 17); or CD20 binding antibody plus methotrexate.

One method of evaluating treatment efficacy in RA is based on American College of Rheumatology (ACR) criteria, which measures the percentage of improvement in tender and swollen joints, among other things. The RA patient can be scored at lor example, ACR 20 (20 percent improvement) compared with no antibody treatment (e.g„ baseline before treatment) or treatment with pJace.bo, Other ways of evaluating the efficacy of antibody treatment include X-ray scoring such as the Sharp X-ray score used to score structural damage such as bone erosion and joint space narrowing. Patients can also be evaluated for the prevention of or improvement in disability based on Health Assessment Questionnaire fHAQJ score, AIMS score, SF-36 at time periods during or after treatment The ACR 20 criteria may include 20% improvement in both tender (painful) joint count and swollen joint count plus a 20% improvement in at least. 3 of 5 additional measures: Ϊ. patient's pain assessment, by visual analog scale (VAS), 2. patient's global assessment of disease activity (VAS), 3. physician’s global assessment of disease activity (VAS), 4. patient’s self-assessed disability measured by the Health Assessment Questionnaire, and 5- acute phase reactants, CRP or RSR,

The ACR 50 and 70 are defined analogously. Preferably, the patient is administered an amount of a C.D20 binding antibody of the invention effective to achieve at least a score of ACR 20, preferably at least ACR 30, more preferably at leasr ACR50, even more preferably at least ACR7Q, most preferably at least ACR 75 and higher.

Psoriatic arthritis has unique and distinct radiographic features. For psoriatic arthritis, joint erosion and joint space narrowing can be evaluated by the Sharp score as well. The humanized CD20 binding antibodies of the invention can be used to prevent the joint damage as well as reduce disease signs and symptoms of the disorder.

Yet another aspect of the invention is a method of treating Lupus or SLE by administering to the patient suffering from SLE, a therapeutically effective amount of a humanized CD20 binding antibody of the invention. SLEDAJ scores provide a numerical quantitation of disease activity, The SLEDAI is a weighted index of 24 clinical and laboratory parameters known to correlate with disease activity, with a numerical range of 0-103. see Bryan Gescuk &amp; John Davis, “Novel therapeutic agent for systemic lupus erythematosus" in Current Opinion in Rheumatology 2002, 14:515-521. Antibodies to double-stranded DNA are believed to cause renal flares and other manifestations of lupus. Patients undergoing antibody treatment can be monitored fur time to renal flare, which is defined as a significant, reproducible increase in serum creatinine, urine protein or blood in the urine. Alternatively or in addition, patients can be monitored for levels of antinuclear antibodies and antibodies to double-stranded DNA. Treatments for ST.F. include high-dnsc corticosteroids and/or cyclophosphamide (HDCC).

Spondyloarthropathies are a group of disorders of the joints, including ankylosing spondylitis, psoriatic arthritis and Crohn's disease. Treatment success can be determined by validated patient and physician global assessment measuring tools.

Various medications arc used to treat psoriasis; treatment differs directly in relation to disease severity. Patients with a more mild form of psoriasis typically utilize topical treatments, such as topical steroids, anthralin, calcipolriene, clobetasol, and tazarotene, to manage the disease while patients with moderate and severe psoriasis arc more likely to employ systemic (methotrexate, retinoids, cyclosporine, PUVA and UVB) therapies. Tars are also used. These therapies have a combination of safety concerns, time consuming regimens, or inconvenient processes of treatment. Furthermore, some require expensive equipment and dedicated space in the office setting. Systemic medications can. produce serious side effects, including hypertension, hyperlipidemia, bone marrow suppression, liver disease, kidney disease and gastrointestinal upset. Also, the use of phototherapy can increase tile incidence of skin cancers. In addition to the inconvenience and discomfort associated with the use of topical therapies, phototherapy and systemic treatments require cycling patients on and off therapy and monitoring lifetime exposure due to their side effects.

Treatment efficacy for psoriasis is assessed by monitoring changes in clinical signs and symptoms of the disease including Physician’s Global Assessment (PGA) changes and Psoriasis Area and Severity Index (PAST) scores, Psoriasis Symptom Assessment (PSA), compared with the baseline condition. The patient can be measured periodically throughout treatment on the Visual analog scale used to indicate the degree of ilcliing experienced at specific time points.

Patients may experience an infusion reaction or infusion-related symptoms with their first infusion of a therapeutic antibody. These symptoms vary in severity and generally are reversible with medical intervention. These symptoms include but are not limited to, flu-like fever, ehiils/rigors, nausea, urticaria, headache, bronchospasm, angioedema. It would be desirable for the disease treatment methods of the present invention to minimize infusion reactions. Thus, another aspect of the invention is a method of treating the diseases disclosed by administering a humanized CD20 binding antibody wherein the antibody has reduced or no complement dependent cytotoxicity and results in reduced infusion related symptoms as compared to treatment with Rituxan®. In one embodiment, the humanized CD20 binding antibody is 2H7.vl 16.

Dosage

Depending on the indication to be treated and factors relevant to the dosing that a physician of skill in the field would be familiar with, the antibodies of the invention will be administered at a dosage that is efficacious for the treatnrinnt nf that indication while minimizing toxicity and side effects. For the treatment of a CD20 positive cancer or an autoimmune disease, the therapeutically effective dosage will be in the range of about 250nig/m2 to about 40t) mg/m2 or 500 mg/m2, preferably about 250-375mg/mz. In one embodiment, the dosage range is 275-375 mg/m2. In one embodiment of the treatment of a CD20 positive ΰ cell neoplasm, the antibody is administered at a range of 300-375 mg/m2. For the treatment of patients suffering from B-celi lymphoma such as non-Hodgkins lymphoma, in a specific embodiment, the anti-CD20 antibodies and humanized anti-CD20 antibodies of the invention will be administered to a human patient at a dosage of lOrog/kg or 375mg/m2. For treating NHL, one dosing regimen would be to administer one dose of the antibody composition a dosage of lOmg/kg in the first week of treatment, followed by a 2 week interval, then a second dose of the same amount of antibody is administered, Generally, NHL patients receive such treatment once during a year but. upon recurrence of the lymphoma, such treatment can be repeated. In another dosing regimen, patients treated with low-grade NHL receive four weeks of a version of humanized 2H7. preferably vl6 (375 mg/m2 weekly) followed at week five by three, additional courses of the antibody plus standard CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or CVP (cyclophosphamide, vincristine, prednisone) chemotherapy, which was given every three weeks for three cycles.

For treating rheumatoid arthritis, in one embodiment, the dosage range for the humanized antibody is I25mg/m2 (equivalent to about 200mg/dosc) to 600rog/m2, given in two doses, e.g., the first dose of 200mg is administered on day one followed by a second dose of 200mg on day 15. In different embodiments, the dosage is 250mg/dose, 27.5mg, 3G0mg. 325mg, 350mg, 375mg, 400mg, 425 mg. 450mg, 475mg, 500mg, 525mg, 550itig, 575mg, SQQmg.

In treating disease, the CD20 binding antibodies of the invention can be administered to the patient chronically or intermittently, as determined by the physician of skill in the disease. Λ patient administered a drug by intravenous infusion or subcutaneously may experience adverse events such as fever, chills, burning sensation, asthenia and headache. To alleviate or minimize such adverse events, the patient may receive an initial conditioning dose(s) of the antibody followed by a therapeutic dose, ‘life conditioning dosc(s) will be lower than the therapeutic dose to condition the patient to tolerate .higher dosages.

Route of admin istration

The CD20 binding antibodies are administered to a human patient in accord with known methods, such as by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by subcutaneous, intramuscular, intraperitonea!, intracerobrospinal, intra-articutar, intrasynovial, intrathecal, or inhalation routes, generally by intravenous or subcutaneous administration.

In on embodiment, the humanized 2H7 antibody is administered by intravenous infusion with 0.9% sodium chloride solution as an infusion vehicle.

Combination Therapy

In treating the B cell neoplasms described above, the patient can be treated with the CD20 binding antibodies of the present invention in conjunction with one or more therapeutic agents such as a chemotherapeutic agent in u multidrtig regimen. The CD20 binding antibody can be administered concurrently, sequentially, or alternating with the chemotherapeutic agent, or after non-responsiveness with other therapy. Standard chemotherapy for lymphoma treatment may include cyclophosphamide, cytarabine, mclphalan and initoxaot.rone plus melphalan. CHOP is one of the roost common chemotherapy regimens for treating Non-Hodgkin's lymphoma. Tire following arc the drugs used in the CHOP regimen: cyclophosphamide (brand names cyLoxan, neosar); adriamycin (doxorubicin / hydroxydoxorubicif)): vincristine (Oncovin); and prednisolone (sometimes called Deltasone or Orasone). In particular embodiments, the CD20 binding antibody is administered to a patient in need thereof in combination with one or more of the following chemotherapeutic agents of doxorubicin, cyclophosphamide, vincristine and prednisolone. Tn a specific embodiment, a patient suffering from a lymphoma (such as a non-Hodgkin’s lymphoma) is treated with an anti-CD20 antibody of the present invention in conjunction with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. In another embodiment, the cancer patient can be treated with a humanized CD20 binding antibody of the invention in combination with CVP (cyclophosphamide, vincristine, and prednisone) chemotherapy. In a specific embodiment, the patient suffering from CD20-po$itive NHL is treated with humanized 2H7.vl6 in conjunction with CVP. Tn a specific embodiment of the treatment of CLL, the CP20 binding antibody is administered in conjunction with chemotherapy with one or both of fludarabinc and cytoxan.

In treating the autoimmune diseases or autoimmune related conditions described above, the patient can be treated with the CJD20 binding antibodies of the present invention in conjunction with a second therapeutic agent, such as an immunosuppressive agent, such as in a multi drug regimen. The CD2Q binding antibody can be administered concurrently, sequentially or alternating with the immunosuppressive agent or upon non-responsiveness with other therapy. The immunosuppressive agent can be administered at the same or lesser dosages than as set forth in the art. The preferred adjunct immunosuppressive agent will depend on many factors, including the type of disorder being treated as well as the patient’s history. ‘'Immunosuppressive agent" as used herein for adjunct therapy refers to substances that act to suppress or mask the immune system of a patient Such agents would include substances that suppress cytokine production, down regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include steroids such as glucocorticosteroids, c.g., prednisone, methylprerinisolone, and dexametbasone; 2-am.ino-6-aryl-5-substirut.ed pyrimidines (see TJ.S. Pat. No. 4,665,077), azathioprine (or cyclophosphamide, if there is an adverse reaction to azathioprine); bromocryptine; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat, No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; cytokine or cytokine receptor antagonists including anti-intcrfcron-γ, -β, or -a antibodies; anti-tumor necrosis factor-α antibodies; anti-luinor necrosis factor-β antibodies; anti-intcrlcukin-2 antibodies and anti-IL-2 receptor antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7/26/90); streptokinase; TGF-β; streptodomasc; RNA or DNA from the host; FK506; RS-61443: deoxysperguaiin; rapamycin; T-cell receptor (U.S. Pat. No. 5,114,72)); T-ceJI receptor fragments (Offner et «/.-, Science. 251:430-432 (1991); WO 90/11294: and WO 91/01133); and T cell receptor antibodies (HP 340,109) such as TI0B9.

For the treatment of rheumatoid arthritis, the patient can be treated with a CD20 antibody of the invention in conjunction with any one or more of the following drugs: DMARDS (disease-modifying anti-rheumatic dregs (e.g., methotrexate), NSAl orNSAID (non-steroidal anti-inflammatory drugs), HUM1RA™ (adalimunxab; Abbott laboratories), ARAVA® (leflunomide), REMTCADE® (infliximab; Centocor Inc., of Malvern, pa), ENBRF.L (etancrccpt; Immunex, WA), COX-2 inhibitors. DMARDs commonly used in RA are hydroxycloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab, azathioprine, D-penicillaminc, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunnadsorption. Adaiimumab is a human monoclonal antibody that, binds to TNFa. Infliximab is a chimeric monoclonal antibody that binds to TNFa, Btanercept is an •’immunoadbesin" fusion protein consisting of the extracellular ligand binding portion of the human 75 kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion of a human TgGl. For conventional treatment of RA, see, e.g., "Guidelines for the management of rheumatoid arthritis” Arthritis &amp; Rheumatism 46(2): 328-346 (February. 20()2). Ill a specific embodiment, the RA patient is treated with a CD20 antibody of the invention in conjunction with methotrexate (MTX). An exemplary dosage of MTX is about 7.5-25 mg/kg/wk. MTX can he administered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's disease, the patient can he treated with a CD20 binding antibody of the invention in conjunction with, for example. Remicade© (infliximab; from Centocor Inc., of Malvern, Pa.), liNBREL (etanercept; Imirmncx, WA),

Treatments for SLE include high-dose corticosteroids and/or cyclophosphamide (HDCC).

For the treatment of psoriasis, patients Can be administered a CD20 binding antibody in conjunction with topical treatments, such as topical steroids, anthralin, calcipotricne, clobetasol, and tazarotene, or with methotrexate, retinoids, cyclosporine, PtJVA and UVR therapies. In one embodiment, the psoriasis patient is treated with the CD20 binding antibody sequentially or concurrently with cyclosporine.

Pharmaceutical Formulations

Therapeutic formulations of the CD20-binding antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 1 6th edition, Osol, A, Ed. (J 980)), in the form of lyophilized formulations or aqueous solutions. Acceptable earners, excipients, or stabilizers are nontoxie to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadccyldimcthylbenzyl ammonium chloride; hexamethoxiiurn chloride; bcnzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paruben; catechol; resorcinol; cyclohcxanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as scrum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protcin complexes); and/or non-ionic surfactants such as TWEEN™, PLURONTCS™ or polyethylene glycol (ΡΕΩ).

Exemplary anti-CD20 antibody formulations are described in W098/56418, expressly incorporated herein by reference. Another formulation is a liquid multidose formulation comprising the anli-CD20 antibody at 40 ing/tnL, 25 ruM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0-02% polysorbatc 20 at pH 5.0 that has a minimum shelf lift; of two years storage at. 2-8°C. Another anti-CD20 formulation of interest comprises tOmg/mJL antibody in 9,0 mg/mT. sodium chloride, 7.35 mg/ttiL sodium, citrate di hydrate, 0.7mg/mL polysorbatc 80, and Sterile Water for Injection, pH 6.5. Yet another aqueous pharmaceutical formulation comprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.$. preferably at pH5-5, polysorbate as a surfactant in a an amount of about 0.01 -0.1 % v/v, trehalose at an amount of about 2-10% w/v, and benzyl alcohol as a preservative (U.S. 6,171,586). Lyophilized formulations adapted for subcutaneous administration are described in WD97/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein.

One formulation for the humanized 2H7 variants is antibody at 12-14 mg/mf, in IDniM histidine, 6% sucrose, 0.02% polysorbats 20, pH 5.8.

In a specific embodiment, 2H7 variants and in particular 2H7.vl6 is formulated at 20ntg/mL antibody in lOmM histidine sulfate, bOntg/rnl sucrose., 0.2 mg/ml polysorbate 20, and Sterile Water for Injection, al pH5.8.

The formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, if may be desirable to further provide a cytotoxic agent, chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. one which acts or T cells, such as cyclosporin or an antibody that b.i nds T cells, c.g. one which binds LFA-1). The effective amount, of such oilier agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein or about from 1 to 99% of the heretofore employed dosages.

The active ingredients may also be entrapped in microcapsu]es prepared, for example, by coacervation techniques or by interfacia! polymerization, for example, hydroxymethylcellulose or gclatin-microcapsuies and poly-(mcthylmetbaeylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, mierocmul.sions, nano-particles and nanocapsules) or in macroemul.sions. Such techniques arc disclosed in Remington'.v Pharmaceutical Science.·: Kith edition, Osol, A. lid. <J 980).

Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include .semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamie acid and. ethyl-L-glutamate, ποη-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolidc acetate), and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

Articles of Manufacture and Kits

Another embodiment of the invention is an article of manufacture containing materials useful for the treatment of autoimmune diseases and related conditions and CD20 positive cancers such as non-Hodgkin's lymphoma. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic, The container holds a composition which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least otie active agent in the composition is a CD20 binding antibody of the invention. The label or package insert indicates that the composition is used for treating the particular condition. The label or package insert will further comprise instructions for ad ministering the antibody composition to the patient. Package insert refers to instructions customarily included in commercial packages of therapeutic products, that contain information about tile indications, usage, dosage, administration, contraindications and/or warnings concerning the use of Such therapeutic products. In one embodiment, the package insert indicates that the composition is used for treating non-Hodgkins' lymphoma.

Additionally, the article of manufacture may further comprise a second container comprising a pharmaceuticaily-acceptable buffer, such as bacteriostatic water for injection (BWFT), phosphate-buffered saline. Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, c.g.. for B-cell killing assays, as a positive control for apoptosis assays, for purification or irnmunoprecipitation of CD20 from cells. For isolation and purification of CD20, the kit can contain an anti-CD20 antibody coupled to beads (e,g„ sepharose beads). Kits can be provided which contain the antibodies for detection and quantitation of CD20 in vitro, e.g. in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one a«ti-CD20 antibody of the invention. Additional containers may be included that contain, e.g„ diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

Cynomolgus monkey CD20

The invention also provides an isolated nucleic acid comprising the nucleotide sequence of SBQ ID NO,: 24 of the Cynomolgus monkey CD20 as shown in FIG. 19- In one embodiment, the nucleic acid is a cDNA. In One embodiment, the nucleic arid encoding the monkey CD20 is in an expression vector for expression in a host cell. The nucleotide sequence of SHQ ID NO.: 24 in the expression vector is operably linked to an expression control sequence such as a promoter or promoter and enhancer, The expression control sequence can be can be the native sequence normally associated with the Cynomolgus CD20 gene, or heterologous to the gene. Also provided is an isolated polypeptide comprising the amino acid sequence ISBQ ID NO. 25; FIG. 19 &amp; 201 of the Cynomolgus monkey CD20, as well as host cells containing the Cynomolgus CD20 nucleic acid. Ift. one aspect the host cells are eukaryotic cells, e,g., CHO cells. Fusion proteins comprising the Cynomolgus CD20 amino acid sequence or fragments of the sequence are also contemplated.

Experimental Examples

Example 1

Humanization of 2H7 anti-CD20 murine monoclonal antibody

Humanization of the murine anti-human CD20 antibody, 2B7 (also referred to herein as m2H7, in. for murine), was carried out in a series of site-directed mutagenesis steps. The murine 2H7 antibody variable region sequences and the chimeric 2H7 with the mouse V and human C have been described, see, c.g., TI.S. patents 5,846,818 and 6.204,023. The CDR residues of 2H7 were identified by comparing the amino acid sequence of the murine 2H7 variable domains (disclosed in IJ.S, 5,846,818) with the sequences of known antibodies (Kabatetal., Sequences of proteins of immunological interest, Ed. 5, Public Health Service, Natlonal Institutes of Health, Betbesda, MD (1991)). The CDR regions for the light and heavy chains were defined based on sequence hypervariability (Rabat et al., supra) and are shown in Fig. J A and Fig. IB, respectively. Using synthetic oligonucleotides (Tabic 1), site-directed mutagenesis (Kunkel, Prac. Natl. Arad. Set. 82:488-492 (5985)) was used to introduce all six. of tfie murine 2H7 CDR regions into a complete human Fab framework corresponding to a consensus sequence VKI,VH1U (VL kappa subgroup I, Vh subgroup 111) contained on plasmid pVX4 (Fig. 2).

The phagemiri pVX4 (Fig. 2) was used for mutagenesis as well as for expression of F(ab)s in Hi. coli. Based on the phagemid pb0720, a derivative of pB0475 (Cunningham et ah. Science 243: 1330-1336 (1989)), pVX4 contains a DNA fragment, encoding a humanized consensus κ-subgroup T light chain (VLKt-Cl) and a humanized consensus subgroup ΠΙ heavy chain (VhIH-Cji 1) anti-JFN-ct (interferon a) antibody. pVX4 also has an alkaline phosphatase promotor and Shinc-Dalgamo sequence both derived from another previously described pUGI 19-based plasmid. pAK2 (Carter et ah, Free. Natl. Acad. Set. USA 89: 4285 (1992)). A unique Spel restriction site was introduced between the DNA encoding for the F(ab) light and heavy chains. The first 23 amino acids in both antj-IFN-oc heavy and light chains are the 5ίΠ secretion signal sequence (Chang et al., Gene 55: 189-196 (1987)).

To construct the CDR-swap version of 2H7 (2H7.v2), site-directed mutagenesis was performed on a dcoxyuridine-containipg template of pVX4; all six CDRs of anti-TFN-cc were changed to the murine 2B7 CDRs. The resulting molecule is referred to as humanized 2H7 version 2 (2H7.v2), or the "CDR-swap version” of 2H7: it has the m2B7 CDR residues with the consensus human FR residues shown in Figures 1Λ and IB. Humanized 2H7.v2 was used for further humanization.

Table 1 shows the oligonucleotide sequence used to create each of the murine 2H7 (m2H7) CDRs in the H and I» chain. For example, the CDR-H1 oligonucleotide was used to recreate the m2H7 H chain CDR1. CDR-U1, CPRH2 and CDR-H3 refers to the H chain CDR1, CDR2 and CDR3. respectively; similarly, CDR-L1, CDR-L2 and CDR-L3 refers to each of the L chain CDRs. The substitutions in CDR-H2 were done in two steps with two oligonucleotides, CDR-H2A and CDR-H2B.

Table 1. Oligonucleotide sequences used for construction of the CDR-swap of murine 2H7 CDRs into a human framework in pVX4. Residues changed by each

oligonucleotide are underlined.

For comparison with humanized constructs, a plasmid expressing a chimeric 2H7 Fab (containing murine Vj_ and Vh domains, and human Cl and CH| domains) was constructed by sife-dircctcd mutagenesis (KunkeJ. supra) using synthetic oligonucleotides to introduce the murine framework residues into 2H7,v2. The sequence of the resulting plasmid construct, for expression of the chimeric Fab known as 2H7.v6.8, is shown in Fig. 3. Each encoded chain of the Fab has a 23 amino acid 5711 secrerion signal sequence as described for pVX4 (Fig.2) above.

Based on a sequence comparison of the murine 2H7 framework residues with the human ν*Ι,νΗΠΙ consensus framework (Figures IA and IB) and previously humanized antibodies (Carter et al., Proc. Neal. Acad. Sci. USA 89:4285-4289 (1992)), several framework mutations were introduced into the 21)7. v2 Fab construct by site-directed mutagenesis. These mutations result in a change of certain human consensus framework residues to those found in the murine 2H7 framework, at sites that might affect CDR conformations or antigen contacts. Version 3 contained Vh(R71 V, N73K), version 4 contained Vh(R71 V), version 5 contained Vjj(R7lV, N73K) and Vl(L46P), and version 6 contained Vh(R71V, N73K.) and Vi.(L46P>T47W),

Humanized and chimeric Fab versions of rr»2H7 antibody were expressed in E. coli and purified as follows. Plasmids were transformed into E. coli strain XT.-J Blue (Stratagenc, San Diego, CA) for preparation of double-and single-stranded DNA. For each variant, both light and heavy chains were completely sequenced using the dideoxynucleotide method (Sequenase, U.S. Biochemical Corp.). Plasmids were transformed into E. coli strain 16C9, a derivative of MM294, plated onto LB plates containing 5 ftg/ml carbenicillin, and a single colony selected for protein expression. The single colony was grown in 5 ml LB-100 pg/inl carbenicillin for 5-8 h at 37° €. The 5 ml culture was added to 500 ml AP5-100 pg/ml carbenicillin and allowed to grow for 16 h in a 4 L baffled shake flask at 37UC. APS media consists of: 1.5g glucose. 1 i .0 Bycase SF, 0.6g yeast extract (certified). 0. J.9g anhydrous MgSOa, 1 -07g NFL Cl, 3.73g KCl, 1,2g NaCl, 120 mJ 1 M triethanolamine, pH 7.4, to 1 L water and then sterile filtered through 0.1 pm Scalkccn filter.

Cells were harvested by centrifugation in a 1 L centrifuge bottle (Nalgene) at 3000xg and the supernatant removed. After freezing for 1 h. tile pellet was resuspended in 25 ml cold 10 niM MES-10 mM EDTA. pH 5.0 (buffer A). 250 μΐ of 0.1M PMSF (Sigma) was added to inhibit proteolysis and 3.5 ml of stock 10 mg/ml hen egg white lysozyme (Sigma) was added to aid lysis of the bacterial ceil wail. After gentle shaking on ice for 1 h, the sample was centrifuged at 40,000xg for 15 min. The supernatant was brought to 50 ml with buffer A and loaded onto a 2 ml DEAE column equilibrated with buffer A. The flowthrough was then applied to a protein G-Sepharosc CL-4B (Pharmacia) column (0.5 ml bed volume) equilibrated with buffer A. The column was washed with 10 ml buffer A and eluted with 3 ini 0.3 M glycine, pH 3.0, into 1,25 mJJ. M Tris, pH 8.0. The F(ab) was then buffer exchanged into PBS using a Ccntrieon-30 (Amicon) and concentrated to a final volume of 0.5 mJ. SDS-PAGE gels of ail F(ab)s were run to ascertain purity and the molecular weight of each variant was verified by elcctrospray mass spectrometry.

In cell-based ELISA binding assays (described below), the binding of Fabs, including chimeric 2H7 Fab, to CD20 was difficult to detect. Therefore, the 2H7 Fab versions were reformatted as full-length IgOI antibodies For assays and further mutagenesis.

Plasmids for expression of full-length IgG’s were constructed by Subcloning the Vl and Vh domains of chimeric 2H7 (v6.8) Fab as well as humanized Fab versions 2 to 6 into previously described pRK vectors for mammalian cell expression (Gorman et ah, DNA Fret. Eng. Tech. 2:3-10 (1990)). Briefly, each Fab construct was digested with EcoRV and Blpl to excise a Vh fragment, which was cloned into the EcoRVIBlpl sites of plasmid pDRl (Fig. 4) for expression of the complete light chain (Vg-Q, domains). Additionally, each Fab construct was digested with Pvull and Apal to excise a VH fragment, which was cloned into die PvuUJApal sites of plasmid pDR2 (Fig. 5) for expression of the complete heavy chain (VI i-CHi-hinge-CH2-CH3 domains). For each IgG variant, transient transfections were performed by cotransfecting a light-chain expressing plasmid and a heavy-chain expressing plasmid into an adenovirus-transformed human embryonic kidney ceil line, 293 (Graham et al., J, Gen. Virol., 36:59-74, (1977)).

Briefly, 293 cells were split on the day prior to transfection, and plated in scrum-containing medium. On the following day, double-stranded DNA prepared as a calcium phosphate precipitate was added, followed by pAdVAntage™ DNA (Prorocga, Madison, WI), and cells were incubated overnight at 37°C. Cells were cultured in serum-free medium and harvested after 4 days. Antibodies were purified from culture supernatants using protein A-Sepharose C.L-4U, then buffer exchanged into 10 «)M sodium succinate, 14!) mM NaCI, pH 6.0. and concentrated using a Centricon-lO (Amicon). Protein concentrations were determined by quantitative amino acid analysis.

To measure relative binding affinities to the CD20 antigen, a cell-based ELISA assay was developed. Human B-lymplioblastoid WJL2-S cells (ATCC CRL 8885, American Type Culture Collection, Rockville, MD) were grown in RPMf f 640 supplemented with 2 mM L-glutaminc, 20 mM HEPRS, pH 7-2 and 10% heat-inactivated fetal bovine serum in a humidified 5% C02 incubator. The colls were washed with PBS containing 1 % FBS (assay buffer) and seeded at 250-300,000 cell/well jn 96-well round bottom plates (Nunc, Roskilde, Denmark). Two-fold serially diluted standard (15-6-1000 ng/ml of 2H7 v6,8 chimeric IgG) and threefold serially diluted samples (2.7-2000 ng/ml) in assay buffer were added to the plates. The plates were buried in ice and incubated for 45 min. To remove the unbound antibody, 0.1 mL assay buffer were added to the wells. Plates were centrifuged and supernatants were removed. Cells were washed two more limes with 0.2 mL assay buffer. Antibody bound to the plates was detected by adding peroxidase conjugated goat anti-human Fc antibody (Jackson ImmunoResearch, West Grove, PA) to the plates. After a 45 min incubation, cell? were washed as described before. TMB substrate (S^'.SjS'-tetrame.lhyl benzidine;

Kirkegaa.rd &amp; Perry Laboratories. Gaithersburg, MD) was added to the plates. The reaction was stopped by adding 1 M phosphoric acid. Titration curves were fit with a four-parameter nonlinear regression curvefitting program (KaleidaGraph, Synergy software, Reading, PA). The absorbance al the midpoint of the titration curve (mid-OD) and its corresponding concentration of the standard were determined. Then die concentration of each variant at this mid-OD was determined, and the concentration of the standard was divided by that of each variant, Hence the values are a ratio of the binding of each variant relative to the standard. Standard deviations in relative affinity {equivalent concentration) were generally +/-10% between experiments.

As shown in Table 2, binding of the CDR-swap variant (v.2) was extremely reduced compared to chimeric 2H7 (v.6.8). However, versions 3 to 6 showed improved binding. To determine the minimum number of mutations that might be required to restore binding affinity to that of chimeric 2H7, additional mutations and combinations of mutations were constructed by site-direct mutagenesis to produce variants 7 to 17 as indicated in Table 2. In particular, these included VH mutations A49G, F67A, I69L, N73K, and L78A; and VL mutations M4L, M33I, and F71Y. Versions 16 and 17 showed the best relative binding affinities, within 2-fold of that of the chimeric version, with no significant difference (s.d. = +/-10%) between the two. To minimize the number of mutations, version 16, having only 4 mutations of human framework residues to murine framework residues (Table 2), was therefore chosen as the humanized form for additional characterization.

Table 2. Relative binding affinity of humanized 2H7 IgG variants to CD20 compared to chimeric 2H7 using cell-based ELISA. The relative binding is expressed as the concentration of the chimeric 2H7 over the concentration of the variant required for equivalent binding; hence a ratio <1 indicates weaker affinity for the variant. Standard deviation in relative affinity determination averaged +/-10%. Framework substitutions in the variable domains are relative to the CDR-swap version according to the numbering system of Kabat (Kabat et al., supra).

Table 3 Oligonucleotide sequences used for construction of mutations VH(A49G, R71V, fJ73K) and VI,(L46F) in humanized 2H7 version 16 (2H7.vlG). Underlined codons encode the indicated amino acid substitutions. For V,, (R71V, N73K) and VL (L46P), the oiigos are shown as the sense strand since these were used for mutagenesis on the Fab template, while for VH (A49G), die oligo is shown as the anti-sense strand, since this was used with the pR.K (lg<3 heavy chain) template. The protein sequence of version 16 is shown in Fig. 6 and Fig. 7.

Example 2

Antigen-binding determinants (paratope) of 2H7

Alanine substitutions (Cunningham <&amp; Wells, Science 244: 1081-1083 (1989) were made in 2H7.v16 or 2H7.v 17 in order to test the contributions of individual side chains of the antibody in binding to CD20, IgC variants were expressed in 293 cells from pDRl and pD.R2 vectors, purified, and assayed for relative binding affinity as described above. Several alanine substitutions resulted in significant decreases in relative binding to CD20 on W1L-2S cells (Table 4).

Table 4, F.ffects of. alanine substitutions in the CDR regions of humanized 2H7.v16 measured using cell-based KUSA (W1L2-S cells). The relative binding is expressed as the concentration of the 2H7.vl6 parent over the concentration of the variant required for equivalent binding; hence a ratio < I indicates weaker affinity for the variant: a ratio >1 indicates higher affinity for the variant. Standard deviation in relative affinity determination averaged */- 10%, Framework substitutions in the variable domains are relative to 2M7.v ] 6 according to die numbering System of Kabat (Kabat ct ah, supra). NBD means no detectable binding. The two numbers for version 45 arc from separate experiments.

Example 3

Additional mutations within 2H? CDR regions Substitutions of additional residues and combinations of substitutions at CDR positions that were identified as important by Ala-scanning were also tested. Several combination variants, particularly v.96 appeared to bind more tightly than v.16.

Table S. Effects of combinations of mutations and non-alanine substitutions in the CDR regions of humanized 2H7.vl6 measured using cell-based ELISA (WIL2-S cells). The relative binding to CD20 is expressed as the concentration of the 2H7.vl6 parent over the concentration of the variant required for equivalent binding; hence a ratio <1 indicates weaker affinity for the variant; a ratio >1 indicates higher affinity for the variant. Standard deviation in relative affinity determination averaged +/- 10%, Framework substitutions in the variable domains are relative to 2H7.V16 according to the numbering system of Kabat (Kabat et a!„ supra).

Example 4

Mutations at sites of framework humanization substitutions

Substitutions of additional residues at framework positions that were changed during humanization were also tested in the 2H7,vl6 background. In particular, alternative framework substitutions that were neither found in the murine 2H7 parent nor the human consensus framework were made at Vj_(P46) and V,i(G49. V71, and K73).

These substitutions generally led to little change in relative binding (Table 6), indicating that there is some flexibility in framework residues at these positions.

Table ft. Relative binding in a cell-based (W1L2-S) assay of framework substitutions. IgG valiants are shown with mutations with respect to the 2Η7.νΙό baekground. The relative binding is expressed as the concentration of the 2H7.V(>.8 chimera over the concentration of the variant required for equivalent binding; hence a ratio <1 indicates weaker affinity forthe variant; a ratio >1 indicates higher affinity tor the variant. Standard deviation in relative affinity determination averaged +/- 10%. Framework substitutions in the variable domains are relative to 2H7.v16 according to the numbering system of Kabul (Kabal et al., supra). <*) Variants that were assayed with 2H7.v16 as the standard comparator: relative values are normalized to that of the chimera.

(*) Variants that were assayed with 2H7.v16 as the standard comparator; relative values arc normalized to that of the chimera,

Examnle 5

Humanized 2H7 variants with enhanced effector ftmetions

Because 2H7 can mediate lysis of B-cells through both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), we sought to produce variants of humanized 2H7.vl6 with improved CDC and ADCC activity. Mutations of certain residues within the Fc regions of other antibodies have been described (Idusogieet ah, A JmmunoL 166:2571-2575 (2001)) for Improving CDC through enhanced binding to the complement component Clq. Mutations have also been described (Shields ct al,, J, Biot, Chttm. 276:6591-6604 (2001); Presta et a!., Biockem. Soc, Trans. 30:487-490 (2002)) for improving ADCC through enhanced JgQ binding to activating Fey receptors and reduced TgG binding to inhibitory Fey receptors, In particular, three mutations have been identified for improving CDC and ADCC activity: S298A/E333A/K334A (also referred to herein as a triple Ala mutant or variant; numbering in the Fc region is according to the EU numbering system; Kabal et al.. supra) as described (Idusogie et al.. supra (2001); Shields ct al.. supra).

Tn order to enhance CDC and ADCC activity of 2H7, a triple Ala mutant of the 2H7 Fc was constructed. A humanized variant of the ant.i-HER2 antibody 4d5 has been produced with mutations 5298A/E333A/K334A and is known as 4D5hJcl 10 (*>., anrj-pwHER2 IgOI (S298A/F.333A/K334A); Shields et al.. supra). A plasmid, p4D5Fcll<) encoding antibody 4D5Fc110 (Shields et al., supra) was digested with Apal and Uindlll, and the Fc-fragmenr (containing mutations S298A/E333A/K334A) was ligated into the ApaVliindUl sites of the 2H7 heavy-chain vector pDR2-v16, to produce pDR2-v31. The amino acid sequence of the version 31 complete H chain is shown in Fig. 8. The T, chain is the same as that of vl 6.

Although the constant domains of die Fc region of IgGl antibodies are relatively conserved within a given species, allelic variations exist (reviewed by Lefranc and Lefranc, in The human IgG subclasses: molecular analysis of structure, function, and regulation, pp. 43-78, F. Shakib (ed.), Fergiunmon Press, Oxford (1990)).

Table 7. Effects of substitutions in the Fc region on CD20 binding. Relative binding to CD20 was measured in a cell-based (W1L2-S) assay of framework substitutions. Fc mutations (*) are indicated by EU numbering (Kabat, supra) and ore relative to the 2H7.V 16 parent. The combination of three Ala changes in the i;c region of v.31 is described as "Fol 10." TgO variants are shown with mutations with respect to the 2H7.v I ft background, The relative binding is expressed as the concentration of the 2H7.v6.8 chimera over the concentration of the variant required for equivalent binding; hcncc a ratio <1 indicates weaker affinity for the variant Standard deviation in relative affinity determination averaged +/- 10%.

Example 6

Humanized 2H7 variants with enhanced stability

For development as therapeutic proteins, it is desirable to choose variants that remain stable with respect to oxidation, deamidation, or otheT processes that may affect product quality, in a suitable formulation buffer. Tn 2H7.v1ft, several residues were identified as possible sources of instability: VT, (M32) and VH (M34, N100), Therefore, mutations were introduced at these sites for comparison with v16.

Table 8, Relative binding of 2H7 variants designed for enhanced stability and/or effector function, to CD20 in a coll-based (WTL2-S) assay. IgG variants are shown with mutations with respect to the 2H7.v 16 background. The relative binding is expressed as the concentration of the 2H7.v6.8 chimera over the concentration of the variant required for equivalent binding; hence a ratio <! indicates weaker affinity for the variant Standard deviation in relative affinity determination averaged +/- 10%. Framework substitutions in the variable domains arc relative to 2H7.v 16 according to the numbering system of Rabat, and Fc mutations <*) are indicated by EU numbering (Kabal el al., supra). (**) Variants that wore measured with 2H7.vl6as the standard comparator, relative values are normalized to that of the chimera.

Additional Fe mutations were combined with stability or affinity-enhancing mutations to alter or enhance effector functions based on previously reported mutations (Idusogic ct al. (2000);

Idusogie et al. (2001); Shields et a).. (2001.)), These changes include S298, E333A, K334A as described in Example 5; K322A to reduced CDC activity; D265A to reduce ADCC activity; K326A or K326W to enhance CDC activity; and E356D/M358L to lest the effects of allotypic changes in die Fc region. None of these mutations caused significant differences in CD20 binding affinity,

(**) Variants that were measured with 2H7.v16 as comparator; relative binding values are normalized to that of the chimera.

To test (be effects of stability mutations on the rate of protein degradation, 2H.7,vl6 and 2H7,v73 were formulated at 12-34 mg/mL in 10 mM histidine. 6% sucrose, 0.02% polysorbate 20. pH 5.8 and incubated at 40°C for J 6 days. The incubated samples were then assayed for changes in charge variants by ion exchange chromatography, aggregation and fragmentation by size exclusion chromatography, and relative binding by tasting in a cell-based (WTL2-S) assay.

The results (Fig. 9) show that. 2H7 v.73 has greater stability compared to 2H7 v. 16 with respect to losses in the fraction of main peak by ion exchange chromatography under accelerated stability conditions. No significant differences were seen with respect to aggregation, fragmentation, or binding affinity.

Example 7

Scatchard analysis of antibody binding to CD20 on WIL2-S cells

Equilibrium dissociation constants (1¾) were determined for 2H7 TgO variants binding to WTL2-S cells using radiolabeled 2H7 IgG. IgG variants were produced in CHQ cells. Rituxan® (source for al,l experiments is Gcncnlech, S. San.Francisco, CA) and murine 2H7 (BD PharMingen, San Diego, CA) were used for comparison will» humanized variants, The murine 2H7 antibody is also available from other .sources, c.g., cBioscicnce, and Calbiochem (both of San Diego, CA), Accurate Chemical &amp; Scientific Coup., (Wcsthury, NY), Anccll (Bayport, MN), and Vinci-Biochem (Vinci, Italy), All dilutions were performed in binding assay buffer (DMEM media containing 1 % bovine scrum albumin, 25 mM HEPES pH 7.2, and 0,01 % sodium azide). Aliquots (D.025 mL) of lMl-2H?.vl6 (iodinated with lactoperoxidase) at a concentration of 0.R nM were dispensed into wells of a V-bottom 96-wcll raicroassay plate, and serial dilutions (0.05 mL) of cold antibody were added and mixed. WIL2-S cells (0(),000 cells in 0.025 mL) were then added. The plate was sealed and incubated at room temperature for 24b, then centrifuged for IS min at. 3,500 RPM. The supernatant was then aspirated and the cell pellet was washed and centrifuged. The supernatant was again aspirated, and the pellets were dissolved id IN NaOH and transferred to tubes for gamma counting. The data were used tor Seatchard analysis (Munson and Rodbard, Anal, liiudwm. 107:220-239 (1980)) using the program Ligand (McPherson, Comput. Programs Ewmed. 17: Ι07Ί 14 (1983)). The results, shown in Table 9, indicate that humanized 2H7 variants had similar CD20 binding affinity as compared to murine 2H7, and similar binding affinity to Rituxan®, It is expected dial 2H7.v31 will have very similar IC, to v,16 on the basis of the binding shown in Table 7 above.

Table 9, Equilibrium binding affiniry of 2H7 variants from Seatchard analysis

Example 8

Complement Dependent Cytotoxicity (CDC) Assays 2147 lgG variants were assayed for their ability to mediate complement-dependent, lysis of WJL2-S cells, a CD20 expressing lymphoblastoid B-cell line, essentially as described (Idusogie et al., J. Immunol. 164:4178-4184 (2000); Idusogie ct ah, J. Immunol. 166:2571 -2575 (2001)). Antibodies were serially diluted ! :3 from a 0.1 mg/mL stock solution. A tt.05 mL aliquot of each dilution was added (o a 96-well tissue culture plate that contained 0.05 mL of a solution of normal human complement (Quidel, San Diego, CA)

To this mixture, 50,000 WIL2-S cells were added in a 0,05 UlL volume. After Incubation for 2h at. 37°C, 0.05 mL of a solution of Alamar blue (Accumed International, Westlake, OH) was added, and incubation was continued for an additional I8h at 37rtC. Covers were then removed from the plates, and they were shaken for 15 min at room temperature on an orbital shaker. Relative fluorescent units (RFU) were lead using a 530 tun excitation filter and a 590 nm emission filter. An EQ® was calculated by fitting RFU as a function of concentration for each antibody using KaleidaGrapli software.

The results (Table 10) show surprising improvement in CDC by humanized 2H7 antibodies, with relative potency similar to Rituxan® for v.73,3-fold more potent than Rituxan® for v.75, and 3-fold weaker than Rituxan® for v.16.

Table 10, CDC activity of 2H7 antibodies compared to Rituxan. Numbers >1 indicate less potent CDC activity than Rituxan® and numbers <1 indicate more potent activity than Rituxan®. Antibodies were produced from stable CHO lines, except that those indicated by (*) were produced transiently .

Example 9

Antibody Dependent Cellular Cytotoxicity (ADCC) Assays 2H7 IgG variants were assayed for their ability to mediate NaUiral-Kiiler cell (NK cell) lysis of WIL2-S cells, a C.D20 expressing lymphohfastoid R-eelS line, essentially as described (Shields ct ah, J. Biol.

Ckem, 276:(1591-6(504 (2001)) using a lactate dehydrogenase (LDH) readout. NK cells were prepared from 100 rtiL of heparinized blood, diluted with 100 mL of PBS (phosphate buffered saline), obtained from normal human donors who Had been isotyped for FcyRJJI, also known as CD 16 (Koenc et al., Blood 90:1109-1114 (1997)). In ibis experiment, the NK cells were from human donors heterozygous forCDJ.6 (P158/VI58). Tire diluted blood was layered over 15 mL of lymphocyte separation medium (ICN Biochemical, Aurora. Ohio) and centrifuged for 20 min at 2000 RPM, White cells at the interface between layers were dispensed to 4 clean 50-nxL lubes, which were filled with RPMI medium containing 15% fetal calf scrum. Tubes were centrifuged for 5 min at 1400 RPIVT and the supernatant discarded. Pellets were resuspended in MACS buffer (0.5% BSA, 2mM EDTA), and NK cells were purified using beads (NK Cell Isolation Kit, 130-046-502) according to tbs manufacturer’s protocol (MUtenyj Biotech,), NK cells were diluted in MACS buffer to ZxlO6 celts/mL.

Serial dilutions of antibody (0.05 mL) in assay medium (F12/DMBM 50:50 without glycine, 1 tnM HEPliS buffer pH 7,2, Peonicjllin/Streptonrycin (tOO units/mL; Gibco), glutamine, and 1% heat-inactivated fetal bovine serum) were added to a 96-well round-bottom tissue culture plate. WTL2-S cells were diluted in assay buffer to a concentration of 4 x lOVmL. WIL2-S ceils (0-05 n.xL per well) were mixed with diluted antibody in the 96-well plate and incubated for 30 min at room temperature to allow binding of antibody to CD20 (opsonization).

The ADCC reaction was initiated by adding 0 i inT. (if NK colls to each well. In control wells, ?.%

Triton X-100 was added. The [date was then incubated for 4h at 37°C. Levels of LDH released were measured using a cytotoxicity (LDH) detection kit (Kit#1644793, Roche Diagnostics. Indianapolis, Indiana.) following the manufacturers instructions. 0.1 mL of LDH developer was added to each well, followed by mixing for 1 Os. The plate was then covered with aluminum foil and incubated in the dark at room ' temperature for 15 mid. Optical density at 490 lira was then read and use to calculate % lysis by dividing by the total LDH measured in control wells. Lysis was plotted as a function of antibody concentration, and a 4-parameter curve fit (KideidaOraph) was used to determine ECjo concentrations.

The results showed that humanized 2H7 antibodies were active in ADCC, with relative potency 20-fold higher than Riluxan® lor v,31 and v.75, 5-foid more potent than Kiruxnn® for v.16, and almost 4-fold higher than Ritux&amp;n® for v.73,

Table 11, ADCC activity of 2H7 antibodies on W1L2-S cells compared to 2H7.vJ 6, based on n experiments. (Values >1 indicate Iowet potency than 2H7.v 16, and values <1 indicate greater potency.)

Additional ADCC assays were carried out to compare combination-variants of 2H7 with Rituxan©. The results of these assays indicated that 2H7.vl 14 and 2H7.vl 15 have>lO-fold improved ADCC potency as compared to Rituxan® (Table 12).

Table 12. ADCC activity of 207 antibodies oil W1L2-S cells compared to Rituxan®, based on. n experiments (Values >i indicate lower potency than Rituxan®, and values <1 indicate greater potency).

Example 10

Jj> vivo effects of 2H7 variants in a pilot study in cynomolgus monkeys 2H7 variants, produced by transient transfec tion of Cl 1.0 ceils, were tested in normal mala cynomolgus (Macacafasciculark) monkeys in order to evaluate their in vivo activities. Other anti-CD20 antibodies, such as C2B8 (Rituxan®) have demonstrated an ability to deplete B-cells in normal primates (Reff et ah. Blood 83; 435-445 (1994)).

In one study, humanized 2H7 variants were compared. Tn a parallel study, Rituxan® was also tested in cynomolgus monkeys. Four monkeys were used in each of five dose groups: (1) vehicle, (2) 0.05 nig/kg Iiu2H7.vl6, (3) 10 mg/kg hu2H7.vl6, (4) 0.05 tng/kg hu2H7.v31, and (5) 10 mg/kg hu2H7.v31. Antibodies were administered intravenously at a concentration of 0, 0.2, or 20 mg/mL, for a total of two doses, ooe on day I of the study, and another on day 8, The first day of dosing is designated day 1 and the previous day is designated day -1; the first day of rectivery (for 2 animals in each group) is designated as day 11. Blood samples were collected on days -19,-12, t (prior to dosing), and at. 6h, 24h, and 72h following the first dose. Additional samples were taken on day 8 (prior to dosing), day 10 (prior to sacrifice of 2 animals/group), and on days 36 and 67 (for recovery animals).

Peripheral B-cell concentrations were determined by a FACS method that counted CD37CD4Q+ cells. The percent of CD3-CJD40+1} cells of total lymphocytes in monkey samples were obtained by the following gating strategy. The lymphocyte population was marked on the forward scatter/ side scatter scattcrgram to define Region I (R i). Using events in RI, fluorescence intensity dot plots were displayed for C.D40 and CD3 markers, Fluoresecntly labeled isotype controls were used to determine respective cutoff points for CD40 and CD3 positivity. 'llte results indicated that both 2H7.V16 and 2H7.v31 were capable of producing full peripheral B-ccll depletion at the IOmg/kg dose and partial peripheral B-cell depletion at the 0.05 mg/kg dose (Fig. Π). TIic time course and extent of B-cell depletion measured during the first 72h of dosing were similar for the two antibodies. Subsequent analysis «if the recovery animals indicated that animals treated with 2H7.v31 showed a prolonged depletion of B-cells as compared to those dosed with 2H7.vl6, In particular, recovery animals treated with 10 mg/kg 2H7.vl6, B-cells showed substantial B-cell recovery at some time between sampling on Day It) and on Day 36. However, for recovery animals treated with 10 mg/kg 2H7.v31, B-cells did not show recovery until some time between Day 36 and Day 67 (Fig. 11), This suggests a greater duration of full depletion by about one month for 2Η7·ν31 compared to 2H7,vl6.

No toxicity was observed in The monkey study at low or high dose and the gross pathology was normal. In other studies, vi6 was well tolerated op to the highest dose evaluated of (100mg/kgx2 = 1200 mg/itf x2) following i.v. administration of 2 doses given 2 weeks apart in these monkeys.

Data in Cynomolgus monkeys with 2H7.v 16 versus Rituxan® suggests that a 5-fold reduction in CDC activity does not adversely affect potency. An antibody with potent ADCC activity but reduced CDC activity may have more favorable safety profile with regard to first inftision reactions than one with greater CDC activity.

Example 11

Kucose deficient 2H7 variant antibodies with enhanced, effector function

Normal CHO and HEK293 cells add fucose to igG oligosacdiaride to a high degree (97-98%). IgG from sera are also highly tucosylafcd. DPI2, a dihydrofolate reductase minus (DHFR') CHO cell line that is fueosylation competent, and Led 3, &amp; cell line that is deficient in protein fueosylation were used to produce antibodies for this Study. The CHO cell line Pro-Lec13.6a (Lccl3), was obtained from Professor Pamela Stanley of Albert Einstein College of Medicine of Yeshiva University, Parental lines are Pro- (praline auxotroph) and Gat- (glycine, adenosine, thymidine auxotroph). The CHQ-DP12 cell line is a derivative of the CHO-K.1 ceil line (ATCC #CCL-63), which is dihydrofolate reductase deficient, and has a reduced requirement for insulin. Cell lines were transfected with oDNA using llte Superfect method (Qiagen, Valencia, CA). Selection of the Led 3 ceils expressing transfected antibodies was performed using puromycin dihydrochiorlde (Calbtocheiu, San Diego, CA) at 10 itg/nal in growth medium containing: MEM Alpha Medium with L-gluUtmine, ribonucleosides and deoxyribonucleosides (G1BCO-BRL, Gaithersburg, MD), supplemented with 10% inactivated PBS (G1BCO), 10 mM HEPES, and 1X pcnicillin/streptomycin (G1BCO). The CHO cells were similarly selected in growth medium containing Ham's Ft 2 without OflT: 1 w Glucose DMEM without Glycine with NaHC03 supplemented with 5% FBS (GJLBCO), 10 mM HEPliS, 7 mM L-gJutamine, 1X GH'Ifgiycine, hypoxant.hine,thymidine), and IX pcnicillin/streptomycin.

Colonics formed within two to three weeks and were pooled for expansion and protein expression. The cell pools were seeded initially at 3 x 106 celis/10 cm plate for small batch protein expression. The cells were converted to serum-free media once they grew to 90-95% oonfluency and after 3-5 days ceil supernatants were collected and tested ill an Fc IgG- and intact. TgG-ELlSA to estimate protein expression levels. Led 3 and CHO ceils were seeded at approximately 8 x 10° ee!!s/15 cm plate one day prior to converting to PS24 production medium, supplemented with 10 mg/L recombinant human insulin and 1 mg/L trace elements.

Lecl3 cells and DP 12 cells remained in serum-free production medium for 3-5 days. Supernatants were collected and clarified by centrifugation in 150 ml conical tubes to remove cells and debris. The protease inhibitors PMSF and aprotinjo (Sigma, St. Louis, MO) were added and the supernatants were concentrated 5-fo)d on stirred cells using MWCO30 filters (Amicon, Beverly, MA) prior to immediate purification using protein G chromatography (Amersham Pharmacia Biotech. Piseataway, NJ)). All proteins were buffer exchanged into phosphate-buffered saline (PBS) using Centripricp-30 concentrators (Amicon) and analysed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined using A280 and verified using amino acid composition analysis.

The CHO cells were transfected with vectors expressing humanized 2H7v16, 2H7v.31 and selected as described. The 2H7v,16 antibody retains the wild type Fc region while v.3l (see Example 5, Table 7 above) has an Fc region wherein 3 amino acid changes were made (S29SA, E333A, K334A) which results in higher affinity for the FcyRTTTa Teceptor (Shields et al. J. Biol- Chem. 276 (9):6591-6604 (2001)), Following transfection and selection, individual colonics of cells were isolated and evaluated for protein expression level and the highest producers were subjected to methotrexate selection to select for cells that had amplified the plasmid copy number and which therefore produced higher levels of antibody. Cells were grown, transferred to scrum free medium for a period of 7 days, then the medium was collected, loaded onto a protein A column and the antibody was eluted using standard techniques. The final concentration of the antibody was determined using an Elisa that measures intact antibody. All proteins were buffer exchanged into phosphate-buffered saline (PBS) using Ccntripriep-30 concentrators. (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis.

Matrix-Assisted Laser Desorptiott/JonizAtipn Time-of-flighi (MALD1-TOF) Mass Spectral Analysis of Asparagine-Unhid Oligosaccharides: N-liitked oligosaccharides were released from recombinant glycoproteins using the procedure of Papac e( aL Gfycobtok?gy 8,445-454 (1998). Briefly, the wells of a 96 well PVDF-lincd microtitre plate (MJlJi.po.re, Bedford, MA) were conditioned with 100 μΐ methanol tliat was drawn through the PDVF membranes by applying vacuum to the Millipore Multiscreen vacuum manifold. The conditioned PVDF membranes were washed with 3 X 250 μΐ water. Between all wash steps the wells were drained completely by applying gentle vacuum to the manifold. The membranes were washed with reduction and earboxymethylatiot) buffer (,RCM) consisting of 6 M guanidine hydrochloride, 360 mM Tris, 2 itiM EDTA, pH 8.6. Glycoprotein samples (50 pg) were applied to individual wells, again, drawn, through the PVDF membranes by gentle vacuum and the wells were washed with 2 X 50 ui of BCM buffer. The immobilized samples were reduced by adding 50 μΐ of a 0.1 M dilhiothreitol (DTT) solution to each well and incubating the microtitre plate at 37°C For 1 lir. DTT was removed by vacuum and the wells were washed 4 x 250 μΐ water. Cysteine residues were cafrwxylmethylated by the addition of 50 μΐ of a 0.1 M iodoucclio add (IAA) solution which was freshly prepared in 1 M NaOH and diluted to 0-1 M with ROM buffer. Carboxymethylation was accomplished by incubation for 30 min in the dark al ambient temperature.

Vacuum was applied to the plate to remove the IAA solution aod the wells were washed with 4 x 250 μΐ purified water. The PVDF membranes were blocked by the addition of 100 ul of 1 % PVP3M) (pnlyvinylpyiTolidine 360,000 MW) (Sigma) solution and incubation for 1 hr at ambient temperature. The RVR-SOO solution was removed by gentle vacuum and the wells were washed 4 x 250 μ! water. The PNGasc F (New Ungland Biolabs, Beverly, MA) digest solution» 25 μΐ of a 25 Unit/ml solution in 10 mM Tris acetate, pH 8.4, was added to each well and the digest proceeded for 3 hr at 37°C. After digestion, the samples were transferred to 500 μΙ Eppendorf tubes and 2.5 pIL of a 1,5 M acetic acid solution was added to each sample. The acidified samples were incubated for 3 hr at ambient temperature to convert the oligosaccharides from glycosylatnihes to the hydroxy! form. Prior to MAl.Dl-TOF mass spectral analysis, the released oligosaccharides were desalted using a 0-7-tnl bed of cation exchange resin (AG50W-X8 resin in the hydrogen form) (Bio-Rad, Hercules, CA) slurried packed into compact reaction tubes (US Biochemical, Cleveland, OH).

For MALDT-TOF mass spectral analysts of the samples in the positive mode, the desalted oligosaccharides (0.5 μΐ aliquots) were applied to the stainless target with 0.5 μΐ of the 2.5 dihydroxybenzoic acid matrix (sDHB) that was prepared by dissolving 2 mg 2,5 dihydroxybenzoic acid with 0.1 mg of 5-tnctlinxyslicylic acid it) 1 ml of elhanol/t 0 mM sodium chloride 1:1 (v/v). The sample/matrjx. mixture was dried by vacuum, For analysis in the negative mode, the desalted N-linkcd oligosaccharides (0,5 fU aliquots) were applied to the stainless target along with 0.5 μΙ 2’,4’,6’-trihydroxyacclophcnonc matrix (THAP) prepared in 1:3 (v/v) acetonitrile/13.3 mM ammonium citrate buffer. The santpic/matrix mixture was vacuum dried and then allowed to absorb atmospheric moisture prior to analysis. Released oligosaccharides were analyzed by MALDI-TOPon a FcrSepiive BioSystcms Voyager-DF. mass spectrometer. The mass spectrometer was operated at 20 kV either in the positive or negative mode with the linear configuration and utilizing delayed extraction. Data were acquired using a laser power of 1300 and in the data summation mode (240 scans) to improve the signal to noise. The instrument was calibrated with a mixture of standard oligosaccharides and the data was smoothed using a 1.9 point Savitsky-Golay algorithm before the masses were assigned. Integration of the mass spectral data was achieved using Caesar 7.0 data analysis software package (Sci Bridge Software).

Natural killer (NK) cell antibody dependent cytoxkity assays. ADCC assay's were, performed as described in Example 9, JNK. to target cell (WIL2-S) ratio was 4 to 1, assays were run for 4 hours, and toxicity was measured as before using lactose dehydrogenase assay. Target cells were opsottked with (be concentrations of antibody indicated for 30 min prior to addition of NK. cells. The Riluxan© antibody used was from GenentecJ) (S. San Francisco, CA). Figure l2shows the results of a representative ADCC assay.

The results show that underfucosylated. anfbodies mediate NK cell target cell killing more efficiently than do antibodies with a full complement of fucose. The underfucosylated antibody, 2H7v.3l, is most efficient at mediating target coll killing. This antibody is effective at lower concentrations and is capable of mediating killing of a greater percentage of target ceils at higher concentrations than are the other antibodies. The activity of the antibodies is as follows: Lee 13-derived2H7 v31>Lec 13 derived 2H7v1fi> Dpi 2 derived 2H7v31> Dpi 2 derived 2H7v16 > or = to Rituxan. The protein and carbohydrate alterations arc additive. Comparison of the carbohydrate found on native IgC from the Lee 13-produced and CHO- produced IgG showed no appreciable differences in the extent of galactosyladon and hence the results can be attributed solely to the presence/absence of fucose.

Example 12

Fucose-deficient 2H7 variant antibodies with enhanced ADCC in vivo

This example describes ADCC activity in vivo of the fticose-defioicnt humanized 2117 variants including v. 16 and v.31 produced in Lcci3 compared to normal fucusylated counterparts produced in DP12, in mice expressing human CDIfi [FcRyili] and human CD20.

Generation oflmCD20Tg+ huCD16TR+ rnCDiSA mice

Human CD20 transgenic mice were generated from human CD20JBAC DNA (Invitrogen, Carlsbad, CA). Mice were screened based on the FACS analysis Of human CD2Q expression. HuCD20 Tg* mice were then crossed with huCD16Tg+mCD).6'/" mice to generate huCD20Tg1 huCD 16Tg+mCD 1 6'1' mice.

In vivo treatment

Ten to 100 pg of each of the 2H7 variants or Rituxan® is administrated to huCD20TgTujCD 16Tg+mCD16'1 mice via intraperiioneal injections. Equal amount of isotypc-mutched antibodies will be applied similarly to the negative control group of animals.

Mouse lymphocytes, preparation

Mouse lymphocytes fiOftt whole blood, spleen, lymph nodes and bone marrow are prepared according to standard protocol described in “Current Protocols in Immunology, edited by John Coligan. Ada Kruisbeek, David Margulies, lithan Sbevach and Warren Strobcr, 1994". FACS analysis

Half million cells are washed and resuspended in 100 μΐ. of FACS buffer, which is phosphate buffered saline with 1% BSA, containing 5 μΐ of staining or control antibody. All the staining antibodies, including isotype controls, are. obtained from PharMIngen, Sait Diego, CA, Human CD20 expression is assessed by staining with Rituxan® along with FITC-conjugafed anti-human IgGl secondary antibody. FACS analysis is conducted using FACScan and Cell Quest (Recton Dickinson imnuinocylomelry Systems, Son Jose, CA), All the lymphocytes are defined in the forward and side light scatterings, while all the B lymphocytes are defined with the expression of B22Q on the cell surface. B cell depletion and recovery are assessed by analyzing peripheral B cell counts and analysis of hCD20+ B cells by FACS in the spleen, lymph node and bone marrow on a daily basis for the first week after injection and thereafter on a weekly basis. Serum levels of the injected 2H7 variant antibody are monitored.

The results of this in vivo assay confirms the In vitro findings on the Increased ADCC activity and greater B cell depletion of fucose-deftcient 2H7 variants over wild-type (with resepet to fucosylation) glycosylation counterparts.

Example 13 Apoptosis Activity

Anti-CTCO antibodies including Rituxan® have been shown to induce apoptosis in vitro when crosslinkcd by a secondary antibody or by chemical means (Shan ctal., Blood 9:1644-1652 (1998): Byrd el ill,. Blood 99:1038-43 (2002): Pederson et al., Blood 99:1314-19 (2002)). When chemically crosslinkcd, murine 2117 dimers induced apoptosis of Daudi cells (Ghetie et al., Proc Nail Acad Sci USA 94:7509-14 (1997)). Crossfinking with a secondary antibody also induced apoptosis with the murine 2H7 antibody (Shan ct al., 1998). These activities are believed to be physiologically relevant because a variety of mechanisms could lead to crosslinking of anti-CP20 antibodies bound to cell-surface CD20 in vivo.

RhuMAb 2H7,vl6 [humanized 2H7 v]6; RhuMAb stands for recombinant human monoclonal antibody I and Rituxan® were compared in apoptosis assays in vitro using a secondary crosslinking antibody. Ramos cells (CRL-1596, ATCC. Manassas, VA), a CD20-expressing. human B lymphocyte cell line, were used to measure the ability of the anti-CD20 monoclonal antibodies rhuMAb 2)i7.vl6 and Rituximab versus a negative-control antibody, Trastuzumab (fferceptin®, Genentech, South San Francisco, CA), to Induce apoptosis as measured through Annexin V staining and propidium iodide dye exclusion (Vybraot® Apoptosis Assay Kit, Molecular Probes, Seattle, WA). The Ramos cells were cultured in RPM1-1640 medium (Gibco, Rockville, MD) containing i 0% fetal bovine scrum (Biosourcc Internationa], Camarillo, CA) and 2 mM L-glutasnke (Gibco). Prior to being assayed, the cells were washed twice in fresh media and then adjusted to a cell concentration of 2 X HI6 per rnL. Cells (150 pJL) were added to 96-well assay plates (Recton Dickinson, Palo Alto, CA) which contained 150 pL of a predetermined amount of control IgGl, rhuMAb 2H7.V 16', or Rituxbnab, along with F(ab)‘2 goat anti-human Fc (Pierce Biotechnology. Rockford, IL). The final IgG concentrations were 100, 10, 1.0, 0.1,0.01 and 0.001 nM, and the F(ab)’2 goat anti-luunan Fc antibody concentration was set at. twice the respective sample antibody concentration. Each dilution was set up in triplicate. After a 24-hotir incubation at. 37° C, the cells were washed twice with PBS and then stained with Annexin V and propidium iodide according to the manufacturer’s recommendations. The staining patterns of the Ramos cells were analyzed by flow cytometry using a l-ACscan Flow Cytometer (Beeton Dickinson, San Jose, CA), and data were collected for 10 s-periods. Tne data were reduced using the CellquestPro software (Bccton Dickinson). Ramos ceils that were positive for (1) Annexin V staining, (2) Annexin V and propiduim iodide double-staining, and (3) the number of unstained live cells, were counted and plotted using KaleidaGraph software (Synergy Software, Reading, PA).

Both rhuMAb 2H7,vl6 and Rituximab induced apoptosis of Ramos cells when crosslinked with anti-human Fc and as compared to an irrelevant IgGl control antibody (Figures 13-15). The apoptotic activity of (rhuMAb 2H7) was slightly lower than that of Rituximab. At 10 nM concentrations of crosslinked rhuMAb 2H7, Rituximab, and control IgGl antibody, fractions of Annexin V stained cells were 18.5, 16.5, 2.5%, respectively, fractions of doubly labeled cells were 29, 38, and 16%,and numbers oflive cells counted per 10 s were 5200, 3100, and 8600.

These in vitro data demonstrate that apoptosis is one potential mechanism tor in vivo B cell depiction, in vivo croaslinking of rhuMAb 2H7 or Rituximab bound to cell-surface CD20 may occur through FtryR on the surfaces of immune effector cells.

Example 14

In Vivo Suppression, of Tumor Growth

The ability of rhtiMAb 2H7,vi 6 to inhibit the growth of the Raji human B-cellg. a lyjnphoma eel] line (ATCC CCL 86), was evaluated in Balb/c nude (athyittic) mice. The Raji cells express CD2Q and have been reported to grow in nude mice, producing metastatic disease; tumor growth is inhibited by Rituxan® (Clynes et al.. Nature Medicine 6,443-446 (2000)), Fifty-six 8-10 week old, Balb/c nude mice were divided into 7 groups (Λ-G) with each group consisting of 8 mice. On day 0, each mouse received a subcutaneous injection of 5 X 10° Raji B-lymphuma cells in the flank. Beginning at day 0, each mouse received either 100 til, of the negative-control solution (PBS; phosphate-buffered saline), Rituxan® or 2H7.vl6. Dosage was dependent on weight and drug delivery was intravenously via the tail vein. Group A mice received PBS. Groups B-D received Rituxan® at 5.0, mg/kg, 0.5 mg/kg, and 0,05 mg/kg respectively. Groups E-G mice received 2H7 v.16 at.5.0 mg/kg, 0-5 mg/kg, and 0.05 mg/kg respectively. The injections were repeated every week for 6 weeks. At weekly intervals during treatment, each mouse was inspected for the presence of palpable tumors at the site of injection, and the volume of the tumors if present were measured and recorded. Λ final inspection was made at week 8 (after a two-week interval of no treatments).

The results of this study showed that both rhuMAb 2H7.v16 and Rituxan® and were effective at. inhibiting subcutaneous Raji-cell tumor growth in nude mice (FIGs. 16-18). Turnor growth was observed in the PBS control group beginning at 4 weeks. However, no tumor growth was observed in groups treated with Rituxan® or 2H7.vI6at5 mg/kg or 0.5 mg/kg for the 8-week duration of the study. In the low-dose 0.05 mg/kg treatment groups, tumors were observed in one animal in the 2H7 group and in one animal in the Rituxan® group (FIG. 18).

Example 15

Cloning of Cynomolgus monkey CD20 a»d antibody binding

The CD20 DNA sequence for cynomolgus monkey (Macaca fescicularis) was determined upon the isolation of cDNA encoding 0020 from a cynomolgus spleen cDNA library. Λ SUPERSCRIPT™ Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat# 18248-013, Invitrogen, Carlsbad, CA) was used with slight modifications to construct the library. The cDNA library was ligated into a pRR5E vector using restriction sites Xho 1-and Not 1. mRNA was isofated from spleen tissue ((California Regional Research Primate Center, Davis, CA). Primers to amplify cDNA encoding CD20 were designed based on non-coding sequences of human CD20. N-termioai region primer 5'-AGTTTTGAGAGCAAAATG-3' (SEQ ID NO. 37) and C-tcmiinal region primer 5’-AAGCTATGAACACTAATG-3' (SEQ TD NO. 38) were used to clone by polymerase chain reaction (PCR) the cDNA encoding cynomolgus monkey CD20. The PCR reaction was carried out using Platinum Taq DNA Polymerase Higlt Fidelity according to the manufacturers recommendation (Gibco, Rockville, MD). The PCR product was subcloned into pCR ®2.1-TOPO ® Vector (Invitrogen) and transformed into XL-1 blue E. coli (Straiagene. La Jolla. CA). Plasmid DNA containing ligated PCR products was isolated from individual clones and sequenced.

The amino acid sequence -for cynomolgus monkey CD20 is shown in Figure 19. Figure 20 shows a comparison of cynomolgus and human CD20. The cynomolgus monkey CD20 is 97.3% similar to human CD20 with 8 differences. The extracellular domain contains one change at VI57A. while the remaining 7 residues can be found in the cytoplasmic or transmembrane regions.

Antibodies directed against human CD20 were assayed Ι'ρτ the ability to bind and displace FITC-conjugatcd murine 2H7 binding to cynuntolgus monkey cells expressing CD20. Twenty milliliters of blood were drawn from 2 cyoomolgus monkeys (California Regional Research Primate Center, Davis, CA) into sodium heparin and shipped directly to Cienentech Tnc., On the same day, the blood samples were pooled and diluted 1:1 by the addition of 40 ml of phosphate buffered saline (PBS). 20 ml of diluted blood was layered on 4 x 20 ml of Ficoll-Paque ™Plus (Amcrsham Bioscicnces, Uppsala, Sweden) in 50 ml conical tubes (Cat#352098, Falcon, Franklin Lakes, NJ) and eemrifuged at 1300 rpm lor 30 minutes R.T. in a Sorv.il 7 centrifuge. (Dupont, Newtown, CT). The PBMC layer was isolated and washed in PBS. Red blood cells ware lysed in a 0,2% NaCl solution, restored to isolonicity with an equivalent volume of a 1 .<5% NaCJ solution, and centrifuged for 10 minutes at 1000 RPM, The PBMC pellet was resuspended in RPM11640 (Gibco, Rockville, MD) containing 5% fetal bovine serum (FBS) and dispensed into a 10 cm tissue culture dish lor 1 hour at 37° C. The ηοη-adherent B and T cell populations were removed by aspiration, centrifuged and counted. A total of 2.4 x I07 cells were recovered. The resuspended PBMC were distributed into twenty 12 x 75 mm culture tubes (Cat#352053, Falcon), with each tube containing lx 10° cells in a voJtmte of 0.25 ml. Tubes were divided into four sets of five tubes. To each set was added either media (RPM11640,5% FBS), titrated amounts of control human IgGi antibody, Riuixan®1,2H7.vl6, or2H7.v3I, The filial concentration of each antibody was 30, 10, 3.3 and 1.1 nM. In addition, each tube also received 20 til of Fluorescein Tsothiocyanate (FITC)-conjugated anti-human CD20 (Cat#555622. BD Biosciences, San Diego, CA). The cells were gently mixed, incubated for 1 hour on ice and then washed twice in cold PBS. The cell surface Staining was analyzed on a Epic XL-MCL (Coulter, Miami, FI,), the geometric means derived, plulted (KaleidaGraph™, Synergy Software,, Reading, PA) versus antibody concentrations.

Data in Figure 21 showed that 2H7 v.16 and 2H7 v.31 competitively displaced FlTC-murine 2117 binding to cynomolgua monkey cells. Furthermore. Rilnxan® also displaced FlTC-murine 2H7 binding thus demonstrating that both 2H7 and Riluxan® bind to an overlapping epitope on CD20. Tn addition, the data Show that the TCa) value for 2H7 v.16, 2H7 v.3l and Rltuxan are similar and fall in the 4-6 nM range.

Example 16

Phase MI study of rhuMAb 2H7 (2H7.v16) in moderate tn severe rheumatoid arthritis Protocol Synopsis A randomized, placebo-controllcd, multiccnter, blinded phase Ml study of the safety of escalating doses of PRO70769 (rhuMAb 2H7) in subjects with moderate to severe, rheumatoid arthritis receiving stable doses of concomitant methotrexate.

Objectives

The primary objective of this study is to evaluate the safety and tolerability of escalating intravenous (IV) doses Ol’ PRO70769 (rhuMAb 2ΪΤ7) in subjects with moderate to sever rheumatoid arthritis (RA).

Study Design

This is a randomized, placebo-controlled, multicenter, blinded Phase I/1I, investigator- and subject-blinded study of the safety of escalating doses of PRO70769 in combination with MTX in subjects with moderate to sever RA, Tile study consists of adose escalation phase and a second phase with enrollment of a larger number of subjects. The Sponsor will remain unblended to treatment assignment.

Subjects with moderate to severe RA who have failed one to five disease-modifying antirheumatic drugs or biologies who currently have unsatisfactory clinical responses to treatment with MTX will be enrolled.

Subjects will be required to receive MTX in the range of 10-25 mg weekly for at least 12 weeks prior to study entry and to be on a stable dose for at least 4 weeks before receiving their initial dose of study drug (PRO70769 or placebo). Subjects may also receive stable doses of oral corticosteroids (tip to 10 mg daily or prednisone equivalent) and stable doses of nonsteroidal anti-inflammatory drugs (NSAlDs).

Subjects will receive two IV infusions of PRO70769 or placebo equivalent at the indicated dose on Days 1 and 15 according to the following dose escalation plan (see Figure 22).

Dose escalation will occur according to specific criteria and after review of safety data by an internal safety data review committee and assessment of acute toxicity 72 hours following the second infusion in the last subject treated in each cohort. After the dose escalation phase, 40 additional subjects (32 active and 8 placebo) will be randomized to each of the following dose levels: 2x50 mg, 2x200 mg, 2x500 mg, and 2x1000 mg, if the dose levels have been demonstrated to be tolerable during the dose escalation phase. Approximately 205 subjects will be enrolled in the study, B-ccll counts will be obtained and recorded. B-ccl) counts will be evaluated using flow cytometry in a 48-week follow-up period beyond the 6-mouth efficacy evaluation- B-cell depletion will not be considered a dose-limiting toxicity (DLC), but rather the expected pharmacodynamic outcome of PRO70769 treatment.

In an optional substudy, blood for scrum and RNA analyses, as well as urine samples will be obtained from subjects at various timepoints. These samples may be used to identify biomarkers that may be predictive of response to PRO70769 treatment, in subjects with moderate t.o severe RA.

Outcome Measures

The primary outcome measure for this study is the safety and tolerability of PRO70769 in subjects with moderate to severe RA.

Study Treatment

Cohorts of subjects will receive two IV infusions of PR.070769 or placebo equivalent at the indicated dose on Days 1 and 15 according to the following escalation plan: 10 mg PRO70769 or placebo equivalent; 4 subjects active drug. 1 control 50 mg PR07Q769 or placebo equivalent: 8 subjects active drug, 2 control 200 mg PRO70769 or placebo equivalent; 8 subjects active drug, 2 control 500 mg PRO70769 or placebo equivalent; 8 subjects active drug, 2 control 5000 mg PRO?0769 or placebo equivalent.· 8 subjects active drug, 2 control

Efficacy

The efficacy of PR070769 wii] bo measured by ACR responses. The percentage of subjects who achieve an ACR20, ACR50, and ACR70 response will be summarized by treatment group and 95% confidence intervals will be generated for each group. The components of these response and their change from baseline will be summarized by treatment and visit.

Conclusion

The data above demonstrated the success in producing humanized CD20 binding antibodies, in particular humanized 2H7 antibody variants·, that maintained and even enhanced their biological properties. The humanized 2M7 antibodies of the invention bound to CJD20 at affinities similar ro the murine donor and chimeric 2H7 antibodies and were effective at B cel! killing in a primate, leading to B cell depletion. Certain variants showed enhanced ADCC over a chimeric ant.i-CD20 antibody currently used to treat. NHL, favoring the use of lower doses of the therapeutic antibody in patients. Additional, whereas it may be necessary for a chimeric antibody that has murine HR residues to be administered at. a dose effective to achieve complete B cell depletion to obviate- an antibody response against it, the present, humanized antibodies can be administered at dosages that achieve partial or complete B cell depletion, and for different durations of time, as desired for the particular disease and patient. Tn addition, these antibodies demonstrated stability in solution. These properties of the humanized 2H7 antibodies make them ideal for use as immunolherapeutic agent in the treatment of CD20 positive cancers and autoimmune diseases; these antibodies are not expected to be immunogenic or will at least be less immunogenic than fully murine or chimeric aoti-CD20 antibodies in human patients.

References

References cited within this application, including patents, published applications and other publications, are hereby incorporated by reference.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology and the like, which are within the skill of die art. Such techniques are explained fully in the literature. See e.g., Molecular Cloning: A Laboratory Manual, (J. Sambrook et al,

Cold Spring Harbor Laboratory, Cold Spring Harbor. N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubel e.t al, eds., 1987 updated); Essential Molecular Biology (T. Brown cd., IPX Press 1991); Gene Expression Technology (Gocddel ed., Academic Press 1991): Methods for Cloning and Analysis of .Eukaryotic Genes (A. Both well et aL eds., Bartlett Publ. 1990); Gene Transfer and Expression (M. Kriegier, Stockton Press 1990); Recombinant DNA Methodology IT (R. Wu et al eds.. Academic Press 1995); PCR: A Practical Approach (M, McPherson el al., IRL Press at Oxford University Press 1991); Qligpiiuclentide Synthesis (M. Gait cd., 1984); Cell Culture for Biochemists t'R. Adams ed-, F.Isevier Science Publishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller &amp; M. Caios eds., 1987}; Mammalian Cell Biotechnology (M. Butler ed., 1991); Animal Cell Culture (X Pollard e.t al eds., Humana Press 1990);

Culture of Animal Cells. 2nl1 Ed. (R. Frcshncy et al. eds., Alan R. Liss 1987); Mow Cytometry and Sorting (M, Melamed era!, eds., Wilcy-Liss 1990): the series Methods in Enzviaoiogy {Academic Press, inc.):Wklh M. and Hauser H. (1993); fmmunochcmi.strv in Practice. 3rd edition, A. Johnstone &amp; R, Thorpe. Blackwell Science, Cambridge, MA, 1996; Techniques in Immunocvtochcmistrv. (G. Bullock &amp; P. Petrus/, eds.,

Academic Press 1982, 1983, 1985, 1989): Handbook of Experimental Immunology, (D. Weir &amp; C. Blackwell, eds.); Current Protocols in Immunology (J. Coligan et al. eds. 1991): Immunoassay (E.P. Diamandis &amp; T.K. Christopoulos, eds.. Academic Press, Inc.. 1996); Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd ed) Academic Press, New York; Ed Harlow and David Lane, Antipodies A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988; Antibody Engineering. 2nd edition (C. Borrebacck, cd., Oxford University Press, 1995); and the series Annual Review of Immunology; the series Advances in Immunology.

The entire disclosure in the complete specification of our Australian Patent Application No. 2003301079, Australian Patent Application No. 2011226858 and Australian Patent Application No. 2015201983 are by this cross-reference incorporated into the present application.

Claims (110)

1. The claims defining the invention are as follows:

   1. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, the antibody comprising the Vh sequence of SEQ ID NO. 8 and the VL sequence of SEQ ID NO. 2.
2. 2. An antibody or an antigen-binding fragment according to claim 1, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 39 and 40, respectively.
3. 3. An antibody or an antigen-binding fragment according to claim 1, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 39 and 40, respectively, and further comprising at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity.
4. 4. An antibody or an antigen-binding fragment according to claim 3, wherein the amino acid substitutions are S298A/E333A/K334A.
5. 5. An antibody or an antigen-binding fragment according to claim 3 or 4, wherein the amino acid substitution in the Fc region improves CDC activity.
6. 6. An antibody or an antigen-binding fragment according to claim 5, wherein the amino acid substitution is K326A or K326W.
7. 7. An antibody or an antigen-binding fragment according to any one of claims 3 to 6, wherein the Fc further comprises the amino acid substitution K326A.
8. 8. An antibody or an antigen-binding fragment according to claim 3, further comprising at least one amino acid substitution in the Fc region that decreases CDC activity.
9. 9. An antibody or an antigen-binding fragment according to claim 8, comprising at least the substitution K322A.
10. 10. An antibody or an antigen-binding fragment according to any one of the preceding claims conjugated to a cytotoxic agent.
11. 11. An antibody or an antigen-binding fragment according to claim 10, wherein the cytotoxic agent is a radioactive isotope or a toxin.
12. 12. A composition comprising an antibody or an antigen-binding fragment according to any one of the preceding claims, and a pharmaceutically acceptable carrier.
13. 13. An article of manufacture comprising a container and a composition contained therein, wherein the composition comprises an antibody or an antigen-binding fragment according to any one of claims 1 to 11.
14. 14. An article of manufacture according to claim 13, further comprising a package insert indicating that the composition can be used to treat non-Hodgkin’s lymphoma or an autoimmune disease.
15. 15. A method of inducing apoptosis in B cells in vivo, comprising contacting B cells with an antibody or an antigen-binding fragment according to any one of claims 1 to 11, thereby killing the B cells.
16. 16. A method of treating a CD20 positive cancer, comprising administering to a patient suffering from the cancer, a therapeutically effective amount of a humanized CD20 binding antibody or an antigen-binding fragment according to any one of claims 1 to 11.
17. 17. A method according to claim 16, wherein the CD20 positive cancer is a B cell lymphoma or leukemia.
18. 18. A method according to claim 17, wherein CD20 positive cancer is non-Hodgkin’s lymphoma (NHL) or lymphocyte predominant Hodgkin’s disease (LPHD).
19. 19. A method according to claim 16, wherein the cancer is chronic lymphocytic leukemia or small lymphocytic lymphoma.
20. 20. A method according to any one of claims 16-19, wherein the antibody comprises the light and heavy chain amino acid sequence of SEQ ID NO. 40 and 39, respectively.
21. 21. A method according to any one of claims 16 to 20, wherein the antibody or the antigen-binding fragment is administered at a dosage range of about 275-375mg/m .
22. 22. A method according to any one of claims 16 to 20, wherein the antibody or the antigen-binding fragment is administered at a dosage range of about 250mg/m2 to about 500 mg/m2.
23. 23. A method according to any one of claims 16 to 20, wherein the patient is administered at least two doses of the antibody or the antigen-binding fragment at 375 mg/m per dose.
24. 24. A method according to claim 23, wherein the two doses are administered two weeks apart.
25. 25. A method according to any one of claims 16 to 24, further comprising administering to the patient at least one chemotherapeutic agent.
26. 26. A method according to claim 25, the cancer is non-Hodgkin’s lymphoma (NHL) and the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, vincristine, prednisolone, and CHOP.
27. 27. A method of treating an autoimmune disease, comprising administering to a patient suffering from the autoimmune disease, a therapeutically effective amount of a humanized CD20 binding antibody or an antigen-binding fragment according to any one of claims 1 to 11.
28. 28. A method according to claim 27, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, 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, ANCA vasculitis, solid organ transplant rejection, graft versus host disease, diabetes mellitus, Reynaud’s syndrome, Sjorgen’s syndrome and glomerulonephritis.
29. 29. A method according to claim 28, wherein the autoimmune disease is rheumatoid arthritis.
30. 30. A method according to claim 29, wherein the patient is suffering from moderate to severe rheumatoid arthritis and has failed treatment with at least one disease-modifying anti-rheumatic drug.
31. 31. A method according to claim 29 or 30, further comprising administering to the patient a second therapeutic agent.
32. 32. A method according to claim 31, wherein the second therapeutic agent is an immunosuppressive agent.
33. 33. A method according to claim 32, wherein the immunosuppressive agent is methotrexate.
34. 34. A method according to claim 29, wherein the humanized CD20 binding antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 39 and 40, respectively.
35. 35. A method according to claim 34, wherein the antibody or the antigen-binding fragment is administered at a dosage selected from 2x1 Omg, 2x50mg, 2x 200mg and 2x500mg.
36. 36. A method according to any one of claims 27-35, wherein the antibody or the antigenbinding fragment is administered by intravenous infusion or subcutaneous administration.
37. 37. A method according to claim 28, wherein the autoimmune disease is multiple sclerosis.
38. 38. An isolated nucleic acid that encodes an antibody or an antigen-binding fragment according to any one of claims 1 to 9.
39. 39. An expression vector encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 9.
40. 40. A host cell comprising a nucleic acid according to claim 39.
41. 41. A host cell according to claim 40, that produces the antibody or the antigen-binding fragment encoded by the nucleic acid.
42. 42. A host cell according to claim 41, which is a CHO cell.
43. 43. A method of producing a humanized antibody or an antigen-binding fragment, comprising culturing a cell that produces the antibody or the antigen-binding fragment according to any one of claims 1-9.
44. 44. A method according to claim 43, further comprising recovering the antibody or the antigen-binding fragment produced by the host cell.
45. 45. An antibody or an antigen-binding fragment produced by a method comprising expressing a nucleic acid encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 9 in a host cell and recovering the antibody or antigen-binding fragment produced by the host cell.
46. 46. An isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO. 24 of the Cynomolgus monkey CD20, or a degenerate variant of this sequence.
47. 47. An isolated nucleic acid comprising a sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO. 25, or SEQ ID NO. 25 with conservative amino acid substitutions.
48. 48. A vector comprising a nucleic acid according to claim 47.
49. 49. A vector according to claim 48, which is an expression vector wherein the nucleic acid is operably linked to an expression control sequence.
50. 50. A host cell comprising a nucleic acid according to claim 46 or 47.
51. 51. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO. 25 of the Cynomolgus monkey CD20.
52. 52. A liquid formulation comprising a humanized 2H7 antibody at 20mg/mL, lOmM histidine sulfate at pH5.8,60mg/ml sucrose, and 0.2 mg/ml polysorbate 20 which antibody comprises the heavy and light chain sequence of SEQ ID NO. 39 and 40, respectively.
53. 53. A method of treating rheumatoid arthritis (RA) in a human subject comprising administering a CD20 antibody to the subject at a dosage selected from the group consisting of 2x50mg, 2x200mg, and 2x500mg.
54. 54. A method according to claim 53, wherein the dosage is 2x50mg.
55. 55. A method according to claim 53, wherein the dosage is 2x200mg.
56. 56. A method according to claim 53, wherein the dosage is 2x500mg.
57. 57. A method according to claim 53, wherein the CD20 antibody is a humanized antibody.
58. 58. A method according to claim 53, wherein the CD20 antibody is a humanized 2H7 antibody.
59. 59. A method according to claim 53, wherein the CD20 antibody is rituximab.
60. 60. A method according to claim 53, wherein the RA is moderate to severe RA.
61. 61. Use of a therapeutically effective amount of a humanized CD20 binding antibody or an antigen-binding fragment according to any one of claims 1 to 11 in the manufacture of a medicament for treating a CD20 positive cancer in a patient suffering from the cancer.
62. 62. The use according to claim 61, wherein the CD20 positive cancer is a B cell lymphoma or leukemia.
63. 63. The use according to claim 62, wherein CD20 positive cancer is non-Hodgkin’s lymphoma (NHL) or lymphocyte predominant Hodgkin’s disease (LPHD).
64. 64. The use according to claim 61, wherein the cancer is chronic lymphocytic leukemia or small lymphocytic lymphoma.
65. 65. The use according to claim 63 or 64, wherein the antibody or the antigen-binding fragment comprises the light and heavy chain amino acid sequence of SEQ ID NO. 40 and 39, respectively.
66. 66. The use according to any one of claims 61 to 65, wherein the antibody or the antigenbinding fragment is administered at a dosage range of about 275-375mg/m2.
67. 67. The use according to any one of claims 61 to 65, wherein the antibody or the antigenbinding fragment is administered at a dosage range of about 250mg/m2 to about 500 mg/m2.
68. 68. The use according to any one of claims 61 to 65, wherein the patient is administered at least two doses of the antibody or the antigen-binding fragment at 375 mg/m2 per dose.
69. 69. The use according to claim 68, wherein the two doses are administered two weeks apart.
70. 70. The use according to any one of claims 61 to 69, further comprising administering to the patient at least one chemotherapeutic agent.
71. 71. The use according to claim 70, the cancer is non-Hodgkin’s lymphoma (NHL) and the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, vincristine, prednisolone, and CHOP.
72. 72. Use of a therapeutically effective amount of a humanized CD20 binding antibody or an antigen-binding fragment according to any one of claims 1 to 11 in the manufacture of a medicament for treating an autoimmune disease in a patient suffering from the autoimmune disease.
73. 73. The use according to claim 72, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), lupus nephritis, ulcerative colitis, 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, ANCA vasculitis, solid organ transplant rejection, graft versus host disease, diabetes mellitus, Reynaud’s syndrome, Sjorgen’s syndrome and glomerulonephritis.
74. 74. The use according to claim 73, wherein the autoimmune disease is rheumatoid arthritis.
75. 75. The use according to claim 74, wherein the patient is suffering from moderate to severe rheumatoid arthritis and has failed treatment with at least one disease-modifying antirheumatic drug.
76. 76. The use according to claim 74 or 75, further comprising administering to the patient a second therapeutic agent.
77. 77. The use according to claim 76, wherein the second therapeutic agent is an immunosuppressive agent.
78. 78. The use according to claim 77, wherein the immunosuppressive agent is methotrexate.
79. 79. The use according to claim 74, wherein the humanized CD20 binding antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 39 and 40, respectively.
80. 80. The use according to claim 79, wherein the antibody or the antigen-binding fragment is administered at a dosage selected from 2x1 Omg, 2x50mg, 2x 200mg and 2x5Q0mg.
81. 81. The use according to any one of claims 72-80, wherein the antibody or the antigenbinding fragment is administered by intravenous infusion or subcutaneous administration.
82. 82. The use according to claim 73, wherein the autoimmune disease is multiple sclerosis.
83. 83. Use of a CD20 antibody in the manufacture of a medicament for treating rheumatoid arthritis (RA) in a human subject, wherein the CD20 antibody is administered to the subject at a dosage selected from the group consisting of 2x50mg, 2x200mg, and 2x500mg.
84. 84. The use according to claim 83, wherein the dosage is 2x50mg.
85. 85. The use according to claim 83, wherein the dosage is 2x200mg.
86. 86. The use according to claim 83, wherein the dosage is 2x500mg.
87. 87. The use according to claim 83, wherein the CD20 antibody is a humanized antibody.
88. 88. The use according to claim 83, wherein the CD20 antibody is a humanized 2H7 antibody.
89. 89. The use according to claim 83, wherein the CD20 antibody is rituximab.
90. 90. The use according to claim 83, wherein the RA is moderate to severe RA.
91. 91. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 41 and 40, respectively.
92. 92. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 42 and 43, respectively.
93. 93. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the Vh sequence of SEQ ID NO.51 and the Vl sequence of SEQ ID NO.52.
94. 94. The humanized antibody of claim 93, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 44 and 45, respectively.
95. 95. A humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody comprises the Vh sequence of SEQ ID NO.51 and the Vl sequence of SEQ ID N0.53.
96. 96. A humanized antibody according to claim 95, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 46 and 47, respectively.
97. 97. A humanized antibody according to claim 95, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 48 and 47, respectively.
98. 98. A humanized antibody according to claim 95, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 49 and 47, respectively.
99. 99. A humanized antibody according to claim 95, wherein the antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 50 and 47, respectively.
100. 100. A humanized antibody according to claim 93 or 95, wherein the VH region is joined to a human IgG chain constant region.
101. 101. An antibody according to claim 100, wherein the human IgG is IgGl or IgG3.
102. 102. An antibody according to claim 101, wherein the human IgG is IgGl.
103. 103. An antibody according to claim 102, wherein the IgGl Fc region comprises the amino acid substitutions S298A/E333A/K334A.
104. 104. A composition comprising a humanized antibody or an antigen-binding fragment according to any one of claims 91-103, and a pharmaceutically acceptable carrier.
105. 105. An isolated nucleic acid that encodes an antibody or an antigen-binding fragment according to any one of claims 91-103.
106. 106. An expression vector encoding an antibody or an antigen-binding fragment according to any one of claims 91-103.
107. 107. A host cell comprising a nucleic acid of claim 105.
108. 108. The host cell of claim 107 which is a CHO cell.
109. 109. A method of producing an antibody or an antigen-binding fragment comprising culturing a host cell that produces the antibody or the antigen-binding fragment according to any one of claims 91-103.
110. 110. The method of claim 109, further comprising recovering the antibody or the antigenbinding fragment produced by the host cell. Ill An antibody or an antigen-binding fragment produced by a method comprising expressing a nucleic acid encoding an antibody or an antigen-binding fragment according to any one of claims 91-103 in a host cell and recovering an antibody expressed by the host cell.