AU2011226858B2 - Immunoglobulin variants and uses thereof - Google Patents

Abstract

The invention provides humanized and chimeric anti-CD20 antibodies for treatment of CD20 positive malignancies and autoimmune diseases.

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant (s): Genentech, Inc. Invention Title: Immunoglobulin variants and uses thereof The following statement is a full description of this invention, including the best method for performing it known to us: - -- - - ~ ~ IPE /S 13 JUL 2004 Attorney Docket No. P1990R3 5 TIMMUNOGLOBLIN VARIANTS AND USES THEREOF FRELD OF THE INVENTION 10 The invention relates to anti-CD20 antibodies and their use in the treatment of B-cell related diseases. BACKGROUND OF TIHE INVENTION Lymphocytes are one of several populations of white blood cells; they specificaUy recognize and 15 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 hunoral Imnunity. B cells mature within the bone marrow and leave the marrow expressing an artigcn-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 20 differentiate intu memory B cells and effcctor 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 cc]). Plasma cells do not produce membrane-bound antibody but instead produce secreted form of the antibody. Secreted antibodies are the major effector molecules of humoral immunity. The CD20 antigen (also called human B-lymphocyte-restricted differentiation antigen, Bp35) is a 25 hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature 13 lymphocytes (Valentine et aL J, Bio. Chen. 264(19):11282-11287 (1989); and Elnfeld et at EMRO J. 7(3):711-717 (1988)). The antigen is also expressed on gmater than 90% of B cell non-Hodgkin's lymphomas (N9L) (Anderson et aL Blood 63(6):1424-1433 (1984)), but is not found on hematopoictic sten cells. pro-B cells, normal plasma cells or other normal tissues (Tedder et aL J. Immuna. 135(2):973-979 30 (1985)). CD20 is thought to regulate an early stcp(s) in the activation process for cell cycle initiation and differentiation (Tedder at al., supra) and possibly functions as a calcium ion channel (Tedder et aL J. Call. Biochem. 14D:195 (1990)). Given the expression of CD20 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 35 and more titan 56,000 new cases are diagnosed each year. For example, the rituximab (RITUXAN@) antibody which is a genetically engineered chicric marine/human monoclonal antibody directed against human CD20 antigen (commercially available from Genentech, 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. Rituximab is the antibody referred to as "C2B8" in US Patent No. 5,736,137 40 issued April 7, 1998 (Anderson eral.) and in US Pat No. 5,776.456. In vitro mechanism of action studies have demonstrated that RITUXAN@ binds human complcment and lyses lymphoid B cell lines through cumpicnient-dependent cytotoxicity (CDC) (Rcff et al. Blood 83(2):435-445 (1994)). Additionally, it has significant activiry'in assays for antibody-dependent cellular cytotoxicity (A)CC). In ViVo preclinical studies have shown that RITUXAN@ depletes B cells from the peripheral blood, lymph nodcs, and bone 45 marrow of cynomoilgus monkeys, presumably through complement and cell-mediated processes (Reff at al. la AMMEH. GE V67*RCVD AT 4126120052:48:59 PM[IEastem Daylight Tie] IVR:USP1TO.EFRIF-l26'IDNIS:2130827* CSID:650 952 9881 * DURATION (mss):24.56 - 2 Blood 83 (2): 435-445 (1994)). Other anti-CD20 antibodies indicated for the treatment of NHL include the murine antibody ZevalinTM which is linked to the radioisotope, Yttrium-90 (IDEC Pharmaceuticals, San Diego, CA), BexxarTM 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 5 (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 10 270,1985). 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 el 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 15 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 20 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. 25 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 30 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 2841745_l (GHMatters) P76023.AU.1 -3 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.). 5 In a preferred embodiment of all of the antibody compositions and methods of use of this invention, the humanized CD20 binding antibody is 2H7.v16 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 10 sequences of v16 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 v16. 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 15 substantially the human consensus framework (FR) residues of human heavy chain subgroup Ill (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 CDR I 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 20 (FR) residues of human light chain K 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 KI 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 25 substituted: AlaH49GIy 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 v16, as shown in FIG. lB. In a further embodiment of the preceding, the antibody further comprises the VL sequence of 30 SEQ ID NO.2 of v16, as shown in FIG. IA. 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 NI OOA in the H chain and S92A in the L chain of v 16. 35 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 2841745_1 (GHMaters) P76023.AU.1 - 3a 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.vI 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 5 that decreases CDC activity as compared to the parent antibody from which it was derived which is v16 in most cases. One such antibody with decreased CDC activity as compared to v16 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. 2841745_1 (GHMatters) P78023AU.1 - -. - - - - _ _ _ __ - - IPEA/US 1 3 JUL 2004 Attorney Docket No. P1990R3 5 In a preferred ermbodiment, the antibodies of the invention arc full length antibodies wherein the Vu region is joined to a human IgG heavy chain constant region. In preferred embodiments, the IgG is human IgGI or IgG3, In one embodiment, the CD20 binding antibody is conjugated to a cytotoxic agent, In preferred embod jments the cytotoxic agent is a toxin or a radioactive isotope. 10 in one embodiment, the antibodies of the invention for use in therapeutic or diagnostic purposes arc produced in CHO cells. Also provided is a composition comprising an antibody of any one of the preceding embodiments, and a carrie. In one embodiment the carrier is a pharmaceutically acceptable carrier. These compositions can be provided in an article of manufacture or a kit. IS The invention also provided a liquid formulation comprising a humanized 2H7 antibody at 20mg/mLL antibody, I 0mM histidine sulfate pH5.8, 60mg/mi sucrose (6%), 0.2 mg/mi polysurbate 20 (0.02%). The invention also provides an isolated nucleic acid that encodes any of the antibodies disclosed herein, including an expression vector for expressing the antibody. 20 Another aspect of the invention are host cells comprising the preceding nuclcic acids, and host cells that produce the antibody. In a preferred embodiment of the latter, the host cell is a CHO cell. A method of producing these antibodies is provided, the method comprising culturing the host cell that povduces the 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 25 composition contained therein, wherein the composition comprises an antibody of any of the prccding embodiments. For use in treating NHL, the article of manufacture further comprises a package insert indicating that the composition is used to treat non-Hodgkin's lymphoma. A further aspect of the invention is a method of inducing apoptosis in R cells In vivo, comprising contacting B cells with the antibody of any of the preceding, thereby killing the 3 cells. 30 The invention also provides methods of treating the diseases disclosed herein by administration of a Cl)20 bJnding antibody or functional fragment thereof. to a mamral such as a human patient suffering from the disease, in any of the methods for treating an autoinmune disease or a CD20 positive cancer, in one embodiment, the antibody is 2H7.v16 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 345 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 40 dosage range of about 275-375mg/m 2 . In additional embodiments, the treatment method further comprises administering to the patient at least one chemotherapeutic agent, wherein for non-Hodgkin's lymphoma (NIL). the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, vincristine and prednisolone. 4 E 5167'RCVD AT 412612005 2:48:59 PM [Eastem Daylight The)' SVR:USPT0-EFXRF.i26' DNIS:2730827' CSD:650 952 9881 DURATION (mm.ss):24.56 ~ IPEAIUS 13 JUL2O4 Aiturney Docket No. P1990R3 5 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 onc of the preceding claims. The autoimmune discuse is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrmmbotic 10 thrombocytopenic purpura (TTP), autoimmune thromhncytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathics, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrmne, SIorgen's syndrome and glomerulonephritis. Where the atloimmune disease is rheumatoid arthritis. the antibody can be administered In conjunction with a second therapeutic agent which is preferably methotrcxate. 15 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 celi antigen. In one embodiment, the sccond antibody is a radiolabeled anti-CD20 antibody. In other embodiments, the CD2O binding antibody is conjugated to a cytotoxic agent including a toxin or a 20 radioactive isotope. In 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, Autoimmunc neutropenia, Castleman's syndrome. Goodpasture's syndrome, solid organ transplant rejection, graft versus host disease (CVHD). IgM 25 mediated. thrombotic thrombocytopenic purpura (TTP), Hashimoto's Thyruiditis, autoimmune hepatitis, lymphold Interstitial pneumonitis (HlV), bronchiolitis obliterans (non-transplant) vs. NSIP, Gmilain-Barre Syndrome, large vessel vasculitis, giant cell (Takayasu's) artertis, medium vessel vasculitis, Kawasaki's Discasc, polyarteritjs nodosa, comprising administering to a patient suffering fTom the disease, a therapeutically effective amount of a CD20 binding antibody. In one embodiment of this method, the CD20 30 binding antibody is Rituxan@. The Invention also provides an isolated nucleic acid comprising the nucleotidc scqucncc of SEQ ID NO.: 24 of the Cynomolgus monkey CD20 (shown In MG. 19). or a degenerate variant of this scqucncc. One embodhnent is an isolated nucleic acid comprising a sequence that encodes a polypeptide with the amino acid sequence of SEQ ID NO. 25 (shown IG. 20), or SEQ ID NO. 25 (FIG. 20) with conservative 35 amino acid substitutions. Another embodiment is a vector comprising the preceding nucleic acid, including an expression vector for expression in a host cell. Included as well is a host cell comprising the vector. Also provided is an isolated polypeptide comprising the amino acid sequence fSEQ 7D NO. 2.5; FTG. 20) of the Cynomolgus monkey CD20. 40 BRIEF DESCRIPTION OF THE FIGURES FiG. IA is a sequence alignment comparing the amino acid sequences of the light chain variable domain (V..) of each of urine 2H7 (SEQ ID NO, 1), huaized 2H7. v16 variant (SEQ ID NO. 2 ), and human kappa light chain subgroup I (SEQ ID NO. 3). The CDRs of Vr. of 2H7 and hu2H7.v16 are as follows: CDRI (SIQ 1D NO.4), CDR2 (SEQ ID NO.5 ), and CDR3 (SEQ ID NO.6). 5 GE 6167* RCVD AT 412612005 2:48:59 PM Eastem Daylight Time t SVR:USPTOEFXRF-1126' DNIS:2730827* CSID:650 952 9881 'DURATION (mmss):24-56 -- PEA/S 3 JUL 2004 Attorney Docket No. P1990R 3 5 FIG. IB is a sequence alignment which compares the VH sequences of murine 2117 (SEQ ID NO. 7), humanized ZH7.v 16 variant (SFQ iD NO. 8), and the htunan consensus sequence of heavy chain subgroup T11 (SEQ TD NO. 9). The CDRs of Y of 2H7 and hu2U7.v16 are as follow: CDR I (SEQ ID NO.10). CDR2 (SEQ ID NO.11), and CDR3 (SEQ ID NO.12). Tn FIG. I A and FIG. I B, the CDR 1, CDR2 and CDR3 in each chain are enclosed within brackets. 10 flanked by the framework regions, FRI -FR4, as indicated. 2H7 refers to the murinc 2H7 antibody. The asterisks in between two rows of sequences indicate the positions that ans different. between the two sequences. Residue numbering is according to Kabat et al., Scquences of Immunological Tnterest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with insertions shown as a, b, c, d. and c. 15 FIG. 2 shows the sequence of phagemid pVX4 (SEQ ID NO.13) used for construction of 2H7 Pab plasmids (see Example 1) as well as the amino acid acquences of the L chain (SEQ ID NO.14) and H chain (SEQ ID NO.15) of the Fab for the CDR-grafted anti-lFN-a humanized antibody. FIG. 3 shows the sequence of the expression plasmid which encodes the chimeric 2H7.v6.8 Pab (SEQ ID NO.16). The amino acid sequences of the L chain (SEQ ID NO.17) and H chain (SEQ TD NO.18) 20 am shown. 1iGo. 4 shows the sequence of the plasmid pDR1 (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 anti-CD3 antibody (Shalaby et al.,.T. Exp. Med. 175: 217-225 (1992)), the stan and stop codons for which are indicated in bold and underUned. 2.9 FIG. 5 shows the sequence of plasnid pDR2(SEQ TD NO.20t 6135 bp) for expression of immunoglobulin heavy chains as described in Example 1. pDR2 contains acqucncca encnding 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 hold and underlined. FIG. 6 shows the amino acid sequence of the 2H7.v16 complete L chain (SEQ ID NO.21). The 30 first 19 amino acids before DIQ are the secretary signal sequence not present in the mature polypcptide chain. FG. 7 shows the amino acid sequence of thc 2H7.vl 6 complete H chain (SEQ TD NO.22). The first 19 amino acids before EVQ before are-the secretary signal sequence not present in the mature polypeptidc chain. Aligning the V 11 sequence in FIG. 1B (SEQ ID NO. 8) with the complete H chain 35 sequence, the human yl constaLnt region is from amino acid position 114-471 in SEQ 1D 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 the secrtory signal sequence not present In the mature polypeptide chain. The L chain is the same as for 2H7.v 16 (sec FIG. 6). 10. 9 shows die relative stability of 2H7.v16 and 2H7.v73 IgG variants as described in Example 6. 40 Assay results wcre normalized to the values prior to incubation and reported as percent remaining after incubation. FIGl. 10 is a flow chart summarizing the amino acid changes from the murinc 2H7 to a subset of humanized versions up to v75. E 7167'RCVD AT 412612005 2:48:59 PM [Eastem Dayght Time]* SVR:USPTO-EFXRF-1126 5 DNIS:2730827*CS!D:650 952 9881 *DURATION (mnss):24-56 Attorney Docket No. PI990R3 ip A US 13 JULZ004 5 FIG. I I is a summary of mean absolute 3-cell count rCD3-/C)40+j in all groups (2H7 study and Rituxan study combined), as described in Example 10, FIG. 12 shows the results of a rep scntative ADCC assay on ficosc deficient 2H7 variants as described in Example 11. FIG. 13 shows the results of the Annexin V staining plotted as a function of antibody concentration. 10 Ramos ecls were Ircated with an irrelevant TgG1 control antibody (Herceptin@; circles), Rituxinimab (squares), or rhuMAb 2H7.v 16 (trianglCs) in the presence of a crosslinrdng secondary antibody and were analyzed by FACS. Figures 13-15 are described in Example 13. FIG. 14 shows the results of the Annexin V and propidium iodide doublc.staining are plotted as a function of antibody concentration. Ramos cells were treated with an irrelevant IgGl control antibody 15 (Herceptin@: circles), Rituximab (squares), or rhuMAb 2H7.v16 (triangles) in the presence of a crosslinking secondary antibody and were analy7ed by FACS. FTG. 15 shows the counts (per 10 m) of live, unstained cells are plotted as a function of antibody concentration. Ramos cells were treated with no irrelevant TgOl control antibody (FHerceptin@; circles), Rituximab (squares), or rhuMAb 2H7.v16 (triangles) in the presence of a crosslinking secondary antibody 20 and were analyzed by FACS. liGs. 16, 17, 18 show inhibition of Raji cell tumor growth in nudc mice, as described in Example 14. Animals were treated weekly (as indicated by vertical arrows; n=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). 25 FIG. 19 shows the nucleotide (SEQ TD NO. 24 ) and amino acid (SEQ TD NO. 25 ) sequences of Cynomoigus monkey CD20, as described in Example 15. FIG. 20 shows the anino acid sequence for cynomolgus monkey CD2O (SEQ 1D NO. 25). Residues that differ from human CD20 are underlined and the human residues (SEQ TD NO. 26) are indicated dircctly below die monkey residue. The putative extracellular domain of the monkey CD20 is in bold type. 30 FIG. 21 shows the results of Cynomolgus monkey cells expressing CD20 binding to hu2H7.vl 6, .v31, and Rituxan, as described in Example 15. The antibodica wer= vawnyed for the ability to bid and displace FITC-conjugated murinc 2H7 binding to cynomolgus CD20. FG. 22 shows dose escalation schema for rheumatoid arthritis phase /11 clinical trial. FiG. 23 shows the vector for expression of 2H7.vl 6 in CHO cells. 35 DETAILED DESCRIPTION OF THE PREFERRED EMBOD1MENTS The "CD20" antigen is a non-glycosylated, transmembrane phosphoprotein with a molecular weight of approximately 35 k that is found on the surface of greater than 90% of B cells fom peripheral blood or lyimphoid organs. CD20 is expressed during early pre-13 cell development and remains until plasma cell 40 diffrcntiation; 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 "BJ-lyMphocyte-restrCted differentiation antigen" and "Bp35". The CD20 antigen is described in, for example, Clark and Jdbetter, Adv. Carm. Res, 52:81-149 (1989) and Valentine et a. J. BioL Chem. 264(19):l1282-11287 (1989). E8167' RCVD AT 4126120052:48:59 PM [Eastem Dayllghtilme] t SVR:USPTO.EFXRF1126'DNIS:2730821 CSID:650 952 9881 'DURATION (mm-ss):24.56 -- P W-- UUW . F at -F ., %W Attorney Docket No. P1 990R3 IKEAN S 13 JUL2004 5 The term "antibody" is used in the broadest scnse and specifically covers monoclonal antibodies (including full length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as thcy exhibit the desired biological activity or function. The biological activity of the CD20 binding and humanized CD20 binding antibodies of the invention will include at least binding of the antibody ro human CD20. more preferably binding to human 10 and other primate CD20 (including cynomolgus monkey, rhesus monkey, chimpanzees). The antibodies would bind CD20 with a Kd value of no higher than I x 10. preferably a Kd value no higher than about I x J0, and he 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 -15 brain, specific cffector fnctions or mechanisms may he 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 20 antibody fragments. "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This fragment consists of a dimcr of one heavy- and onc light-chain variable region domain in tight. non-covalent 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 finding and confer 25 antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three 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 heroin refers to an antibody obtained from a population of substantially homogeneous antibodies, Le., the individual antibodies comprising the population are identical 30 except for possible naturally occurring mutations that may bc present in minor amounts. Monoclonal antibodies are higidy specific. being directed against a single untigcnic site. Furthermore, In contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different dctcrminants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a 35 substantially homogenous 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 el 0L, Narture 256:495 (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 40 in Clackson et at., Nature 352:624-628 (1991) and Marks et aL, J. Mo. Blal. 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 the intact full length molecule from which 8 AM D.EE ENO67RCVDAT 412612005 2:48:59PM[Eastem Daylightime]' SVR:USPT0-EFXRF-il26'DNIS:2130827' CS1D:650 9529881 *DURATION (mmss):24-56 IrE/VWL 1 J JULZUU4 Attorney Docket No. P1990R3 S 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 10 I I0-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 s-sheet configuration, connected by three hypervariablc regions, which form loops connecting, and in some cases forming part of, the P-sheet 15 structure. The hypervariable regions In each chain are held together in close proximity by tie FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodics (see Kabat et al., Sequences of Proteins of immunological 19terest 5th Ed. Public Health Service, National Tnstitutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an atrigen, but exhibit various effector functions, such as participation of the 20 antibody in antibody dependent cellular cytotaxicity (AICC), The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which arc 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), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35B (H 1), 50-65 (H2) and 95-102 (H3) in the VW 25 (Kahat et al,, Seauences of Proteins of Immunological Inter 5th Ed. Public Health Servicc, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariablc loop" (e.g. residues 26-32 (1,]), 50-52 (1.2) and 91-96 (L3) in the VL, and 26-32 (HI), 52A-55 (H2) and 96-101 (W3) In the VH (Chothia and Lesk I. Mol. Blot 196:901-917 (1987)). As referred to herein, the "consensus sequence" or consensus V domain sequence is an artificial 30 sequence derived from a comparison of the amino acid sequenucs 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. X and the human 14 chain subgroup MT1 V domains were prepared. The consensus V sequence does not have any known antibody binding specificity or affinity. 35 "Chimeric" antibodies (immunoglobulins) have a portion of the heavy and/or light chain identical with or homologous to 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 or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as frag ments of such antibodies, so long as they exhibit the desired biological 40 activity (U.S. Patent No. 4,816,567: and Morrison et al, Proc. VaIl. Acad, Sci. USA 81.6851-6855 (1984)). J-Iumanized antibody as used herein is a subset of chimeric antibodies. "Humanized" forms of non-human (e.g., marine) antibodies arc chimcric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are 9 AMID SHEU E 10167'RCVD AT 412612005 2:48:59 PM [Eastem Dayllght Time]* SVR:USPTOEFXRF126*DNIS:27308271 CSID:650 9529881 *DURATION (mm-ss):2446 . - .. __.- - -T - - Attorney Docket No. P1990R3 IPEA/US 13 JUL 2004 5 human imniunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of the rcciplent 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 may comprise residues which are not found in the 10 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 hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions arc those of a human imniunoglobulin sequence although the FR regions may include one or more amino acid 15 substitutions that improve binding affinity. The number of these amino acid substitutions in the 17R arc typically no more than 0 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. Fur further details, see Joncs et al., Nature 321:522-525 (1986); Reichmann et aL, Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Bio. 2;593-596 (1992). 20 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: C1 q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation. 25 "Anlibody-depcndcnt ccll-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK) cells, ncutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen hearing target cell and subsequently kill the target ccll with cytotoxins. The antibodies "arm" the cytotoxic colIs and are absolutely required for such killing. The primary cells for mediating ADCC. NK cells, express 30 PcyRhU only. whereas monocytes express FvyRl, FeyRI and FcvRM. FcR expression on hemttopoictic cells is summarized in Table 3 on page 464 of Ravetch and Kinet. Annu. Rev. Inununol 9:457-92 (1991). To assss ADCC activity of a molecule of interest, an in viiro ADCC assay, such as that described in US Patent No. 5,500.362 or 5,821,337 may be performed. Useful effector cells for such assays include peripheral bkxd monunuclear uclls (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC 35 activity of the molecule of interest may be assessed in vivo, e.g., In a animal model such as that disclosed in Clynes el al. PNAS (USA) 95-652-656 (1998). "Vc receptor" or "eR" describes a receptor that binds to the 1c region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FeR is one which binds an IgGC antibody (a gamma receptor) and includes receptors of the FeyRI, FcYRHU, and FcyRM subclasses, including 40 allclic variants and alternatively spliced forms of these receptors. FoyRI receptors include FcyR TA (an "activating neceptor") and PcyRilB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FeyRIIA contains an imnunorcCCptor tyrosine-based activation motif (lTAM) in its cytoplasm.ic domain, Inhibiting receptor FcyRITIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (see 10 E 11167*RCVD AT 4126J20052:48:59 PM[EastemDaylightimej SVR:USPTO-EFXRF.1126'DNIS:273082P CSID:6509529881 DURATION (nm.ss):24-56 WFJVUS 13 JUL 2004 Attorney Docker No. P1990R3 5 review M. in Dabron, Anna. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravctch and Kinet, Annu Rev. Immwwl 9:457-92 (1991); Capel et aL, Imnunomneihod 4:23-34 (1994). and de Haas et aL, J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those to be identified in the future, arc encompassed by the term "FcR" herein. The term also includes the neonatal mccptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer er aL, J. Immunol. 117:587 (1976) and Kim J0 et al, J. Immwol. 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 ar aL J. BioL Chem. 9(2): 6591-6604 (200 1). "Human effector cells" are leu.kocytes which express one or more FcRs and perform effector 15 functions. Preferably, the cells express at least FcyRTH and pcrtbrm AIDCC effector function, Examples of human leukocytes which mediate ADCC Include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being preferrd- The effector cells may be isolated frm a native source, e.g. from blood. "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell In the presence 20 of complement. Activation of the classical complmcrent pathway Is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subelass) which are bound to their cognate antigen. To assess complement activation. a CDC assay, e.g. as described in Gazzano-Santoro et at., J. ImmiwwL Methods 202:163 (1996), may be performed. Polypeptide variants with altered Fe region amino acid sequences and increased or decreased Clq 25 binding capability are described in US patent No. 6,194,55J B 1 and WO99/51642. -ne contents of those patent publications arc specifically incorporated herein by reference. See, also, Idusogic ey aL J. Immunol. 164: 417FP4184 (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 Fe region, wherein about 80-100% 30 (and preferably about 90-99%) of the antibody in the composition comprises a mature core carbohydrate tructurc which tuoks fucoac, attached to the Fo region of the glycoprotoin. Such compositions were demonstrated herein to exhibit a surprising improvement in binding to FcyRmA(Fl 58), which is not as effective as FeyRIIA (V158) in interacting with human Igo. Thus, the compositions herein are anticipated to be superior to previously described anti-CD20 antibody compositions, especially for therapy of human 35 patients who express FcyRIHA (F158). FeyRJA (F158) is more common thanx FoyRIIIA (V158) In normal, healthy African Americans and Caucasians. Scc Lchrnbcchcr et aL Blood 94:4220 (1999). The present application further demonstrates the synergistic increase in FcyRM binding and/or ADCC function thnt results from combining the glycosylation variations herein with amino acid sequence modification(s) in the Fc region of the glycoproteJn. 40 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 intcrferc with diagnostic or therapeutic uss for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the antibody will hc purified (I) to greater than 95% by weight of antibody as determined by the Lowry method, and most II AMENDED SHEET E12167'RCVD AT 4126120052:48:59PM[EasternDayight Time' SVR:USPTO-EFXRF-1126*DNIS:2730827'CSID:6509529881 DURATION (m.ss):24.56 [rTJip t) J LJL LUU4 Attorney Docket No. P1990R3 5 prcrerably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PACIE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver 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 10 by at least one purification step. An "isolated" nucleic acid molecule Is a nuclcic 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 tho 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 from the nucleic acid molecule 15 as it exists In natural cells. However, an isolated nuclcic 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 NA sequences necessary for the expression of an operably linked coding scquence in a particular host organism. The control sequences that are suitable for 20 prokaryotes, fur example, include a promoter, optionally an. operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation 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 presequcnce 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 25 pnnnotCr 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 faciMltate 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 be contiguous. Linking Is accomplished by ligation at convenient restriction sites, If such sites do not exist, the 30 synthetic oligonucleotide adapters or linkcrs arc used in accordance with conventional practice. "Vectr" includes shutrle and expresion vectors. Typically, die plasmid construct will also include an origin of replication (e.g., the CoIBi orgIn 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 31 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 cukaryotic host cells into which nucleic acid encoding the antibodies have been introduced. Suitable host cells are disclosed below. 40 The word 'label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody. Thc label may itself bc detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alreration of a substrate compound or composition which is detectable. 12 AM-D SEET E 13167 'RCVD AT 412612005 2:48:59 PM [Eastem Daylight Time] *SVR:USPTO.EFXRF-iI26'DNIS:2730827 CSID:650 952 9881 *DURATION m.ss):24.56 KAENS. 13 JUL2004 Attorney Docket No. P1 990R3 5 An 'autuimniune disease" herein is a non-malignant disease or disorder arising from and directed against an individual's own (sell) antigens and/or tissues. As used herein, "B cell depletion" refers to a reduction in B cell levels in an~ animal or human after drug or antibody treatment 1 as compared to the 13 cell level before treatment. H cell levels are measurable using well known assays suchi as those described in the Experimental Examples. B ceU depletion can bc 10 complete or partial. in one emnbodimnt, the depletion of CD2O expressing B cells Is at least 2575, Not to be limited by any one mechanism, possible mnechdnisms 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. Tho tem Is intended to Include radioactive isotopes (e.g., 3. 15 1"'O and Re'"'), 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, lixampics of chemotherapeutic agents include alkalyzing or alkylating agents sucb as thiotepa and cyclosphosphrimide (CYTOXANTM); alkyl sulfonatces such as busulfan, inxprosulfun and piposulfan; ciziridines such as 20) hcnrzodopa, carboquono. incturedopa, and uredopa; ethylenimincs and niethylanielamincs including altrctamine. trlethylenemlaminc, trictylenephosphoramlde. triethylcnethiophosphaoraniide and trrmethylolomelarninc; nitrogen mustardIs such as chlorambucil, cblornaphazine, cholophospharnidc, ostramustine, ifuslinnidc, inechlorethamine, nechlorethaminc oxide hydrochloride, nteiphalan, novembichin, phcncstcrinc, prednimustine, trofosfamide, uracil mustard; nitrosureas such as camiuslinc, ehlorwtocin, 25 fotemustlne, lomustIne, nimuslinc, ra~nimustinc; antibiotics such a6 aciacinoniysins, actinomnycin, authramycln, azaserino, blomycin$, cactinomycin, calicheamnicin, carabicin. cmnmnomycir, carzinophitin, chromomycins, ductinomycin, daunorubicin, dctorubiciD, 6-dliazo-5-4,xo-b.-norleuclme doxorubicin (Adriatnyclfl). epirublolin. esorubicin, idaz-ubicin, wareoellomycin, witomycin-, niycaphenollc acid, nogalaaiycin, olivoinycins, pePlormYcin, potliromycin, puronlycin, quelamycin, rodorubicin, styeponlgrift, 30 strcptozucin, tubcrcidin, ubenimcx, zinostatin, worubicin; anti-metabolites such as methotrexate and 5 fluorouracil (59-FL]); folic acid analogues such as denopterin, nietbotrexate, pteropterlin, trimnetexalo-; purinc: analogs such as fludarabine, 6-viercaptopurino, thianlprine, thioguanine; pyriniidinc analog% such a% ancitabinc, azacitidine, 6-azauridine, carniofur, cytarabine, didcoxywoidinc, doxifluridine, enocitahine, tloxuridinc, 5-FU; androgens such as calusteroie. drocuostanolone propionate, epitiostaaol, nitpitiostanc, 35 :tulactonc; anti-adronalb Such AS luninoglutetliiilido, mtotane0, IWosI=e folk acid replenisber Such as frolinic acid; aceglatone; aldophosphamide glycosidei aminatcvulinlc acid; amsacrine; bcstrabuoit; bisantee edatraxate; defofamine; demnecolcine; dia7.iquone; elfornithine; elliptinium acetate, etogtucid, Salliumn aitrale; hydroxyurean; lentinae; lonldarnijne; xnitoguazon; nitoxantrone- mopidamol: nitracrine. pentostatin; phenamnet- pirarubicin; podophyltinie acid; 2-cthylhydrazidc; prnearbazinc; PSK®D; nizoxane; 40 sizliraf spirgetniaihtfl teflttzoik acid tjajquone; 2, 2',2"-tricblorotrletbyiamlne-: uretha;vnele dacarbAzinc; niaoomustine; mniobrooitol; ,nitolactol; pipobrornan; gacytoslne; arabInoside ('Ama-C"): thiolepa; taxoids, e.g. pactitaxel (TAX01.:'r, Bristol-Myers Squibb Oncology. ]Princeton, NJ) and doxetaxel (lAXOTLIRBO, Rh~ne-Foulenc Rorer, Antony, rance); chioramnbucil: gemcitabine; 6-thioguanine; mercaptcopurinc; metbotmeAute; platinum analogs such as cisplatin and carhoplatin; platinum; etoposide (WP 13 AMM S"IT GE 14167' RC VD AT 412005 2:48:59 PM [Eastern Daylight Time] SYR:USPTO*EFXRFII126, ONIS:27308271 C$10:650 952 9881 'DURATION (mm.ss):2446 I V sI ww %#. w F T - -T IPEA/US 1 3 JUL 2004 Attorney Docket No. P1990R3 5 16); ifosfinilde; mitomycin C mitoxantrone; vincristinu; vinblastine; vinorcibine; navelbine; novantronc; tcniposidc; daunomycin; aminopterin; xcloda; ibandronate: CPT-l J; topoisomerasc inhibitor RFS 2000; difluoromethylornithine (DMPO); retinoic acid; esperamicins; capecitabine; nnd pharmaceutically accept-ible salts, acids or derivatives of any of the above, Also included in this definition arc anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogcns including for example 10 tamoxifen. raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydrmxytamoxifcn, trioxifenc, keoxifenc, LY I 17018, onapristonc, and toremifene (Fareston); anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelio; other chemothcrpeutic 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 thcrapeudc treatment a nd prophylactic or 15 proventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. A subject is successMly "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 20 of cancer cells or absence of the cancer cells: reduction in the tumor size; inhibition (L e., 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, 25 defined as disappearance of all signs of cancer, or a partial response, wherein the size of the rumor is decreased. preferably by more than 50 percent. inure preferably by 75%. A patient is also considered treated ifthe 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 assess Ilg successful treatment and Improvement in the disease are readily measurable by routine procedures familiar 31) to a physician of appropriate skill in the art. A "therpeutically effective amount" rcfrs to an amount of an antibody or a drug effectivc to "great" a disease or disorder in a subject. In the case of cancer, the therapetiically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and 35 preferably stop) 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 40 cyclic in nature. Compositions and Methods of the Invention The Invention provides bumanized antibodies that bind human CD20, and preferably other primate CD20 as well, comprising a 11 chain having at least one, prcferably two or all of the H chala CDRs of a non 14 AMENDW SfEET E 15161 *RCVD AT 41262005 2:48:59 PM Eastem Daylightilmej' SVR:USPTO-EFXRF-1126*DNIS:2730827 CSID:650 952 9881 *DURATION m-ss):24-6 Attorney Docket No. P1990R3 PEAAS 13 JUL 2004 5 human 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 non human species including mouse, rat, guinea pig, goat, rabbit, horse, primate but most frequently will bc a murinc 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 the human consensus JO FR sequence. These FR changes may comprise residues not found in the recipient or the donor antibody. In one embodiment, the donor antibody is the murinc 2H7 antibody, the V region including the CDR and ER sequences of each of the H and L chains of which are shown in FIG. IA and lB, In a specific embodiment, the residues for the human Fab framework correspond to the consensus sequence of human Vic subgroup I and of V 1 1 subgroup Ill , these consensus sequences are shown in Figure IA and Figure 1B. 15 respectively. The humanized 2H7 antibody of the invention will have at least one of the CDRs In the H chain of the marine 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 CD20 binding antibody of the invention will comprise a humanized V domain joined to a C domain of a human immunoglobulin. In a preferred embodiment, the H 20 chain C region is from human IgG, preferably TgGI or IgG3. The L chain C domain Is preferably from human ir chain. Unless indicated otherwise, a humanized 2H7 antibody version herein will have the V and C domain sequences of 2H7.v16 L chain (FIG. 6, SEQ TD NO, 21) and H chain (FIG 7., SBQ .U) NO. 22) except at the positions of amino acid substitutions or changes indicated in the experimental examples below. 25 The humanized CD20 binding antibodies will hind at least human CD20 and preferably hind other primate CD20 such as that of monkeys including cynomolgus and rhesus m onkeys, 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 3() human and primate CD20 (iocluding cynomolgus monkey, rhesus monkey, chimpanzees), with a K1 value of no higher than I x 10 prefrably a Kd valum no higher than about I A 1o', cveni unne pIerriubly a Kd vulue no higher than about 1 x 10-'l, and be able to kill or deplete B3 cells in vitro or in vivo, prvfcrably by at last 20% when compared to the baseline level or appropriate negative control which is not treated with such an antibody. 35 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 the 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 suffcient 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 40 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 ycars, more preferably 3 15 1RM SHEET Ei16167' RCVD AT42612005 2:48:59 PM[EastemnDaylightime]* SVR:,USPTO.EFXRF-il26'DNIS:21308271'CSID:6509529881 *DURATI0N (mm-ss):24.56 Attorney Docket No. P1990R3 1 JUL2004 5 years, even more preferably 5 or more years. In a most preferred embodiment. the B cell depletion is sufficient to 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 autoJmmune disease, it may be desirable to modulate the extent of B cell 10 depletion depending on the disease and/or the scvcrity of the condition in the individual patient, by adjusting the dosage of CD20 binding antibody. Thus, B cell depletion can bur does not have to be complete. Or, total B cell depiction may be dcsired in hitial 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 lcss of CD20 positive B cells remain as compared to the baseline level before treatment. In other 15 embodiments, B cell depletion is 25%, 30%, 40%, 50%, 60%, 70% or greater. Preferably, the i cell depletion Is sufficient to halt progression of the disease, more preferably to alleviate the signs and symptoms of the particular disease uider treatment, even more preferably to cure the disease. The invention also provides bispecific CD20 binding antibodies wherein one arm of the antibody has a humanized H and L chain of the humanized CD20 binding antibody of the invention, and the other arm 20 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, CD16, NKG2D or other NK activating ligands. In comparison with Rituxan (rituximab), v6 exhibits about 2 to 5 fold increased ADCC potency, -3-4 fold decreased CDC than Rituxan. 25 Antibody production Monoclonal antibodies Monoclonal antibodies may be made using the hybridorna method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is 30 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 A nrlodies. Principles and Practice, pp.59-103 (Academic Press, 1986)). 35 The hybridoma cells thus prepared are seeded and grown in a suitable culture medium whicb medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental nyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HPRT or BPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT 40 medium), which substances prevent the growth of HGPRT-dcficicnt cells. Preferred fusion partner myeloma cells am those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and arc sensitive to a selective medium that sclcts against the unfused parental cells. Preferred myeloma cell lines are urine myeloma lines, such as those derived from MOPC-21 and MPC- I mouse tumors available from the Salk Institute Ccil Distribution 16 E 17167'RCVD AT 426l2OO5 2:48:59 PM Eastem Daylight Time] ISVRUP TOEFXRF-1I261 DNIS:2730827' CSID:650 952 9881 'DURATION (mmss):24-56 11 1 WW of 9 4 btJ -TI 16 Attorney Dockct No. P1990R3 13 JUL 2004 5 Center, San Diego, California USA, and SP-2 and derivatives e.g., X63.-Ag8-653 cells available from the American Type CuTruxe Collection, Rockville, Maryland USA. Human mycloma and mouse-human hcteromycinma cell lines also have been described for the production of human monoclonal antibodies (Kuz.or,J. mnmunol, 133:3001 (1984). and Brodeur et at, Monoclonal Antibody Production Techniques and Applications, pp. 51--63 (Marcel Dekker, Inc., New York, 1987)). 10 Culture medium in which hybridoma cells are growing Is assayed for production of monclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is dctcrmined by immunnprecipitation or by an in vitro binding assay, such as radioinmunoassay (RIA) or enzyme-linked immunosorbent assay (ET.JSA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard 15 analysis described in Munson er at., Anal. Blochem., 107:220 (1980). Once hybridoma cells that produce antibodies of the desired specificity, affiolty, and/or activity are identified, the clones may he subcloned by limiting dilution procedures and grown by standard methods (Coding, Monoclonal Andbodies.- Principles and Practice, pp.59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1 640 medium. In addition, the 20 bybridoma 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 ar suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, far example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, 25 hydroxylapatite chromatography, gel clectrophorcsis, dialysis, etc. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g.. by using oligonucleotide probes that are capable of binding specifically to gencs 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 exprssion vectors, which are then transfected into host 30 cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Rcvicw articles on recombinant expression in bacteria of DNA encoding the antibody include Skcrra er at, Curr. Opinion in ImmunoL, 5:256-262 (1993) and Plackthun, Immunol. Revs., 130:151-188 ((992). 35 In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty er al, Nature, 348:552-554 (1990). Clackson at at, Nature, 352:624-628 (1991) and Marks er at., J. Mot Blot, 222:581-597 (1991) describe the isolation of marine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks 40 cr at, Bio/iechnology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et a.. Nuc. Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolution of monoclonal antibodies. 17 AMENDED SHEET 18167' RCVD AT 42612005 2:48:59 PM Eastem Daylght Time] SVR:USPTO-EFXRF-1126'DNIS:2730827'CSID:650 952 9881 'DURATION (mm.ss):2446 rI IIUQ UJ /14 U "tLJ Attorney Docket No. P 1990R3 IP A US 1 3 JUL2004 5 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 C1.) sequences for the homologous urine sequences (U.S. Patent No. 4,816,567; and Morrison, et at, Proc. Nat/ Acad. 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 non 10 immunoglobulin polypeptide sequences can substitute for the constant domains or 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 antigon-combining site having specificity for a differnt antigen. 15 Humanized antibodies Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or mom amino acid residues introduced into it from a source which is non human. Thesc non-human amino acid residues are often referred to as "import" residues, which are typically takcn from ao "Import' variable domain. Humanization can be esseotially performed following the method 20 of Winter and co-workers (Jones et aL, JVature, 321:522-525 (1986); Reichmann et at, Nature, 332:323-327 (1988): Vcrhocycn et aL, Science, 239:1534-1536 (1988)). by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "liumnanized" 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, 25 humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly somc 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 aotigenicity 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 30 sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The buman 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 er at, J, Immunot., 151:2296 (1993); Chothia etaL, .J. Mot RIot., 196:901 (1987)). Another mietbod uses a particular framework region derived from the consensus sequence of all human antibodies of a particular 35 subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Cartcr et at, Proc. Natl. Acad. SciL USA, 89:4285 (1992): Presta el aL., 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, 40 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 in the art. Computer programs are available which illustrate and display probable three-dinensional confonnational structures of selected candidate inmunoglobulin sequences. Inspection of these displays permits analysis of 18 AMENDED SWEET E19167* RCVD AT 412612005 2:48:59 PM Eastem Daylight Time]SVR:USPTEFXRF-1l26 DNIS:2730827' CSID:650 952 9881 * DURATION (mm.ss):24-56 Attorney Docket No. P1990R3 MENUS 13 JUL2004 5 the likely role of the residues In the functioning of the candidate immunoglobulin sequence, ie., the analysis of residues that influence the ability of the candidate Immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined fmm the recipient and import sequCnces so that the desired antibody characteristic, such as increased alTinity for the target antigcn(s), is achieved. In general, the bypcrvariable region residues are directly and most substantially involved in influencing antigen binding. 10 '1ic humanized antibody may be an antibody fragment, such as a Fab.. which is optionally conjugatcd with one or more cytotoxic agent(s) in odcr to generate an immunoconjugate. AlternativeJy, the humanized antibody may be an full length antibody, such as an full length IgG I antibody. Hwnan antibudies and phage display methodology IS As an alternative to humanization. human antibodies can be generated. For example, It Is now possible to produce transgcnic animals (e.g., mice) that are capable, upon immunization, of producitg 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-chain joining region (JH) gene In chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. 20 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. See, e.g., Jakobovits el at, Proc. Natd. Acad. Sri. USA. 90:2551 (1993); Jakobovits et at., Nature, 362:255-258 (1993); Bmuggemann er aL, Year in Immune., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,82.5, 5,591,669 (ali of GenPharm); 5,545,807; and WO 97/17852. 25 Alternatively, phagc display technology (McCafferty et al., Nature 348:552-553 [19901) can he used to produce human antibxlics and antibody fragments In vitro, from inununoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this tcchniquc, antihody V domain genes are cloned in-frame Into either a mvjor or minor coat protein gene of a filamentous bacteriophage, such as M 13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the 30 filamentous particle contains a single-stranded DNA copy of the phage genome, scications based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. 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 35 display. Clackson et at, Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes.dcrivcd from the spleens of immunized mice. A repertoire of V genes from uniwimunized 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 et al., J. Mol. Biol, 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See, also, U.S. 40 Patent Nos. 5,565,332 and 5,573,905. 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), 19 AMENDED S E 20167*RCVD AT 42612005 2:48:59 PM Eastem Daight Time] SVR:USPTO-EFXRF-il26 DNIS:27308271 CSID:650 952 9881 'DURATION (mm-ss):2446 Attorney Docket No. I1 990R3 IPENUS 13 JUL 2004 5 Antibody fragments In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance. and may lead to improvcd access to solid tumors. . Various techniques have been developed for the production of antibody fragments. Traditionally, 10 these fragments were derived via proteolytic digestion of intact antibodies (see, e.g.. Morimoto et at , Journal of Biuchemical and Biophysical Methods 24:107-117 (1992); and Brennan er 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 S. coli, thus allowing the facile production of large amounts of these fragments. Antfibody fragments can be isolated from the antibody 15 phage libraries discussed above. Alternatively, Fab'-SH fragments can bc directly recovered from E, coli ond chemically coupled to form F(ab) 2 fragments (Cartcr et at, Bio/Technology 10:)63-167 (1992)). According to another approach, F(ah') fragments can be isolated directly from recombinant host cell culture. Fab and F(ab) 2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope rciduos are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody 20 fraginents will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a singlc 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 are 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 carboxy terminus of an sFv. See 25 Antibody Enginccring, cd. 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 monospecifie or bispccific. Bispecific antibodies 30 BisPecific 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. Alternativcly, an ant i-CD20 arm may be combined with an arm which binds to a triggering molecule on a Icukocyte such as a '1-cell receptor molecule (e.g. CD3), or Fc receptors for IgG (FcyR), such as FcyRT (CD64). FcyRfl (CD32) 35 and FcyRlIlI (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, anti-interferon-t, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody 40 fragments (e.g. F(ab')2 bispecific antibodies). WO 96/16673 describes a bispecific anti-ErhB2/anti-Fc7rRII antibody and U.S. Patent No. 5,837.234 discloses a bispecific anti-ErbB2/anti-PcyRT antibody. A bispecific anti-lBrbB2/Fcot antibody is shnwn in W098/02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-PrhB2/anti-CD3 antibody. 20 AMENDED SHEET 'E 21167 RCVDAT412612005 2:48:59 PM [Eastem DaylightTime]ISVR:USPTO-EFXRF26 DNIS:2730827* CSID:650 952 9881 'DURATION (mm.ss):24-56 1PEI5 1 3 JUL 200 Attorney Docket No. P1990R3 5 Methods for making bispecific antibodies are known in the art. Traditional production of full length hispecilic antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificitics (Millstein er al., Natrre. 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chaJns, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the 10 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 are disclosed in WO 93/08829, and in Trauncuker eral., EMBO J., 10:3655-3659 (1991). According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigcn combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the 15 fusion is with an ig heavy chain constant domain. comprising at least part of the thing, CH2, and Ch3 regions. It is preferred to have the first heavy-chain constant region (C41) containing the site necessary for light chain bonding, present in at least one of the fusions, DNAs 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 ccll. This provides for greater flexibility in adjusting the mutual 20 proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yicld of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all threc polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratio$ results in high yields or when the mtios have no significant affect on the yield of the desired chain combination. 25 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 an, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. kt was found that this asymmetric structure facilitates the separation of the desired bispocific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of 30 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 er 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 percenrage of heterodimers which arc recovered 35 from recombinant cell culture. The preferred interface comprises at least a part of the C 11 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule arc replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory cavitiess" 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 40 mcchpnism for increasing the yield of the heterodimer over other unwanted cnd-products such as hoinodimers. Bispccific antibxlics include cross-linkcd or "hetcroconjugate" antibodies. For example, one of the antibodies in the hetCroconjugate can he coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and 21 AME ED SHEET E22167RCVDAT4/26120052:48:59PM [Eastem Daylight Time' SVR:USPTO.EFXRF-1126* DNIS:27308271CSID:650 952 9881 *DURATION (mm-1ss):24-56 , W. - -T'W . - I Attorney Docket No. Pi990R3 IPENUS 13 JUL2004 5 Ihr treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089). HEcterOconjugate antibodies may he made using any convenient cross~linking methods. Suitable cross-linking agents are 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 exampic, bispecific antibodies can be prepared using chemical linkage. Brennan ei al.. 10 Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium amscnite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobcnzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptocthylamine and is mixed with an 13 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'-Sf fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby er a., . Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab) 2 molecule. Each Fab fragment was separately 20 -screted from E. coli and subjected to directed chemical coupling in vitro to form the bispecifie antibody. Thc bispecific antibody thus formed was able to bind to colls ovcrcxpressing the ErbD2 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 25 recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et at, J. ImmunoL, 148(5):1547-1553 (1992). The Icucine zipper peptides from the Fos and Jun proteins were linked to the Pab' portions of two different antibodies by gene fusion. The antibody hoinodimers were reduced at the hingc region to form monomers and then re-oxidized to form the Antibody heterodimers. This method can also be utilized for the production of antibody homodimers. 3 The "dlabody" technology described by Hollinger et at, Proc. Nat!. Acad Sci USA, 90:6444-6448 (1993) ha prmvided Fin alternative mechanism for making hispeiflic antihody fmngmerntn The frgmenas e.nmprise a

V

11 connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the Vni and Vt. domains of one fragment are forced to pair with the complementary VL and V domains of another fragment, thereby forming two antigen-binding sites, Another strategy for 35 making bispecific antibody fragments by the use of single-chain Fv (sFv) diners has also been reported. See Grubcr er at, J. ImmunoL, 152:5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepamd, Tutt et at J. ImrnunoL 147: 60 (1991). 40 Multivalent Antibodies A multivalent antilxxly may be internali7ed (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 multivalent antibodies (which are other than of the 1gM class) with three or more antigen binding sites (e.g. tctravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding 22 AMEND SHEET 23167*RCVDATC4612005 2:48:59 PM[Eastem DaylightiTimel SVR:USPT0-EFXRF-il26* DNIS:2130827*CSID:6509529881 *DURATIO0*m0s:24-56 Rf\/UI ' JUL LUU1 Attorney Docket Nu. P1990R3 5 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 this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-rerminal to the Pc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalcnt antibody 10 comprises at least one polypeptide chain (and preflrably two polypeptide chains), whemin the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptidc chain(s) may comprise VDI (Xl).-VD2-(X2).-Fc. whercin VDI is a first variable domain, VD2 is a second variable domain, -c is one polypeptide chain of an Fc region, X I and X2 represent an amino acid or polypeptidc, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH I-Fc region chain; or Vii 15 CH I-VH-CH11-lc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The inultivalcnt antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. Tihe light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain. 20 Other amino acid sequence modifications Amino acid sequence modification(s) of the CD20 binding antibodies described herein arc contemplated. For example, it may be desirable to Improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the anti-CD20 antibody are prepared by 25 introducing appropriate nuclcotide 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 anti-CD20 antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of 30 the unti-CD20 antibody, such as changing the number or position of glycosylation sites. A useful mathad for identification of certain rcniduen or regionn of the anti CD20 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.R., charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino 35 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 other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutati.on per se need not be predctermined. For example, to analyze the performance of a inutation at a given site, ala scanning or 40 random mutagenesis is conducted at the target codon or region and the expressed anti-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 polypcptidcs containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an anti-CD20 antibody 23 AMENDED SEET E 24167'RCVD AT 412612005 2:48:59 PM [Easte Daylight Time] SVR:USPTO.EFXRF1126' DNIS:21308271 CSID:650 952 9881 'DURATION 0m.ss):24-56 A attorney Docket No. P1990R3 5 with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insertional variants of the anti-CD20 nntibody moleculk include the fusion to the N- or C-terminus of the antd-CD20 antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. Another type of varianit is an amino acid substitution variant. These variants have at least one 10 amino acid residue in the anti-CD20 antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis inchade the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in the Table below under the heading of "preferred substitutions". If such substitutions result in a change In biological activity, then more substantial changes, denominated "exemplary substitutions" in the Table, or as futther described below in reference to amino acid I5 classes, may be introduced and the products screened. TABLE efAmino Acid Substuiaons Original Residue Exemplary Preferred Substituo Snbstitutions Ala (A) val, leu; ile val Arg (R) lys; gin; asn lys Asn (N) gin; his; asp, lys; arg 8in Asp (D) glu; asn Slu Cys (C) scr ala ser Gin (Q) asn; glu asn Clu (2) asp; gin asp Gly (G) ala ala His (H) asn; gIn; lys; arg arg Tlc (T) len: val: met; ala; phe: norleucine leti T.eu (L) norleucine; ile; val; met; ala; phe ile T.ys (TC) arg; gin; asn arg met (M) leu; phe; ile leu Phe (1 ieu; val; ile: ala; tyr tyr Pro (P) ala ala Scr (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norieucine Ieu 24 AMENDED SEET E 251671 RCVD AT 4126f2005 2:48:59 PM [Eastem Daylight Time] SVR:USPTEFXRF-1126 DNS:273Q821 CSID:650 952 9881 DURATION M-ss):24-56 PEA/US 13 JUL 2004 Attorney Docket No. P1990R3 5 Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring 10 residues are divided into groups based on common side-chain properties: (1) hydrophobic: noricucine, met, ala. val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asan, g1n, his, lys, arg; 15 (5) residues that influence chain orientation: gly, pro; and (6) amomatic: trp, tyr, phc. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Any cystcinc residue not Involved in maintaining the proper conformation of the anti-CD20 20 antibody also may be substituted, generally with scrine, 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 iFv fragment). A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues ofa parent antibody (e.g. a humanized or human antibody). Generally, the 25 resulting variant(s) sclccted 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 variants Involves affinity maturation using phage display. Briefly, scveral hypervariable region sites (e-g. 0 7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated arm displayed in a monovalent fashion from filamcntous phage particles as fusions to the gene Ill 30 product of M1 3 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 identify candidate hypervariable region sites for modification, alanine scaniolg mutagenesis can be performed to identify hypervariablo 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 35 the antibody and human CD20. Such contact residues and neighboring residues arc candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel 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 40 antibody. By altering is meant deleting one or more carbohydrate moieties found Jn 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 0-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the 25 AMENDED SMET E 26167'RCVD AT 4126120052:48:59PM[EastemDaylight Tmej SVR:USPTOEFXRF.126'DNIS:2730827'CSID:6509529881 'DURATION (nss):2446 0 V11V "%J,, I &jI. VT L- %0 Attorney Docket No. P I 990R3 5 recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of cither of these trpeptide sequcuecs in a polypeptide creates a potential glycosylation site. O-linked glycosylation 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-hydroxyprolinc or 5 hydroxylysine may also be used. 10 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.-dcscribed tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more scrine or threonine residues to the sequence of the original antibody (for 0-linked giycosylation sites). Nucleic acid molecules encoding amino acid sequence variants of the anti-CD20 antibody are 15 prepared by a variety of methods known in the 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 oligonuclcotide-mediated (or sitc-directed) mutagenesis, PCR mnutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the anti-CD20 antibody. It may be desirable to modify the antibody of the invention with respect to effector function, e.g. so 20 as to enhance antigen-dependent cell-mnediated cyotoxicity (ADCC) and/or complement dependent cytoroxicily (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 homodimcric antibody thus generated may have improved Internulization capability and/or increased 25 complement-mediated cel killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. ImmenoL 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et at Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement mediated lysis and 30 ADCC capabilitics. See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989). To increase the serum half life of the antibody, one may incorporate a salvage receptor binding opitope into the antibody (especially an antibody fragment.) as described in U.S. Patent 5,739,277, for example. As used herein, the term "salvage receptor binding epitupc" refers to an epitope of the Pc region of an TgG molecule (e.g., 1g., IgG2. Ig-, or igG 4 ) that Is responsible for increasing the In vivo serum half-life 35 of the TgG molecule. Other anfibudy modifications Other modifications of the antibody are contemplated herein, For example, the antibody may be linked to one of a variety of nonproteinaceous polymers. e.g., polyethylene glycol, polypropylene glycol, 40 polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (lr example, hydroxymethylcellulose or gelatin-microcapsules and poly (ncthylmcthacylatc) microcapsules, respectively), in colloidal drug delivery systems (for example, 26 AMENDED MEET 3E 27167RCVD AT 412612005 2:48:59 PM [Eastem Daylight Time]' SVR:USPTO-EFXRF.1126*DMS:2730827' CSID:6509529881 "DURATION (mMss):24.56 IPE IS 13 JUL 2004 Attorney Docket No. P1990R3 5 liposones, albumin ncrosphCres, microemulsions, nano-particles and nanovapsules), or in macroenulsions. Such techniques are disclosed in Rembtgton's Pharmaceutical Sciences, l6th edition, Oslo, A., Ed., (1980). Screenktgfor atibodles with th desired properties Antibodies with certain biological characteristics may be selected as described in the Experimental 10 Examples. The growth inhibitory cffccts 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 CD20 gene. For example, tumor cell lines and CD20-transfectcd cells may tated with an anti-CD20 monoclonal antibody of the invention at various concentrations for a few days (e.g., 2-7) days 15 and stained with crystal violet or MTT or analyzed by some other cokorimetric assay. Another method of measuring proliferation would be by comparing 'H-thymidinc uptake by the cells trcatcd in the presence or absence an anti-CD20 antibody of the invention. After antibody treatment, the cells arm 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 20 growth of that cell line. To select for antibodies which Induce cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (P1). 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-expressing tumor cells are incubated with medium alone or medium containing of the appropriate monoclonal antibody at. e.g, about Z5 I0Ig/ml. The cells are Incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 min strainer-capped 12 x 75 tubes (1 ml per tube, 3 tubes per treatmuem group) for removal of cell clumps. Tubes then receive PT (10tg/ml). Samples nay be analyzed using a FACSCANTM flow cytometer and FACSCONVERT"m CellQuest software (Bcton Dickinson). Those antibodies which induce statistically significant levels of cell dcath as determined by PT uptake may be selected as cell death-inducing 310 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 Lzborary Manual, Cold Spring Harbor Laboratory, Ed Hartow and David Lane (1988), 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 35 additionally, epitope mapping can be performed by methods known in the 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 cpitope. 40 Vctors, Host Cells and Recombinant Methods The invention also provides an isolated nucleic acid encoding a humanized CD20 binding antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody. 27 AMENDED SEET E 28167'RCVD AT 412612005 2:48:59 PM [Eastem Daylight Time]' SVR:USPTOMXRF-i26' DNIS:273D821 CSID:650 952 9881 *DURATION (mm-ss):24-56 f Uii U VU / /9 "v TL-% Attorney Docket No. P1990R3 A S 13 JUL2004 5 For recombinant production of the antibody, the nucleic acid encoding It is isolated and inserted into a replicable vector for 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 gcnes encoding the heavy and light chains of the antibody). Many vectors are available. Thc vector components generally include, but are not limited 10 to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. (1) Signal sequence component The CD20 binding antibody of this invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterolugous polypeptide, which is preferably a signal sequence or other 1S 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 (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native CD20 finding antibody signal sequence. the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline pbusphatase, penicillinase, lipp, or heat-stable 20 cnterotoxin 11 leaders. For yeast secretion the native signal sequence may be substituted by, e.g., the yeast invertasc leader, tz factor leader (including Sarcharomyces and Kluyveromyces ot-factor leaders), or acid phosphatase leader, the C. albicans glucoamylasc 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. 25 The DNA for such precursor region is ligated in reading frame to DNA encoding the CD20 binding antibody. (ii) Origin of replicaion Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloolng vectors this sequence is one that enables 30 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 the plasmid pBR322 Is suitable for most Gram-negative bacteria, the 24t plasmid origin is suitable for yeast, and various viral origins (SV40, polyona, adenovirus, VSV or BPV) ar useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not 35 needed for mammalian expression vector, (the SV40 origin may typically be used only because it contains the early promoter). (iii) Selection gene component F.xpression and cloning vectors may contain a selection gene, also termed a selectable mnarker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., A() ampicillin, neomycin, methobtrxatc, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacill. 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 28 AM t 29167'RCVDAT04262005 2:48:59 PM Eastem Daylight Time]' SVR:USPTO.EFXRF-126'DNIS:2730827' CSID:650 952 9881 'DURATION (mm-ss):24-56 PEA/US 13 JUL2004 Attorney Docket No. P1990R3 5 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 kinasc, mctallothioncin-1 and -11, preferably primate mctallothionein genes, adenosine deaninase, 10 ornithine decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell when wild-type DHPR Is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCC CRL-9096). 15 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 Y-phosphutransferasc (APH) can be selected by cell growth in medium containing a selection agent for the scicetable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199. 20 A suitable selection gene for use in yeast Is the trpl genc present in the yeast plasmid YRp7 (Stinchcomb et al, Nature, 282:39 (1979)). T17he Irpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in bypophan, for example, ATCC No. 44076 or PBP4- 1. Jones, Genetics, 85:12 (1977). Thc prcscnce of the rp 1 lesion in the yeast host cell genome then provides an cffcctive cnvironment for detecting transformation by growth in the absence of tryplophan. Similarly, Leu2-deficient 25 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 p~m circular plasmid pKD) can be used for transformation of Ktuyveronmyces yeasts. Alternatively, an expression system for large-scale production of ecombinant calf chymosin was reported for K. lactis. Van den Bcrg, Blo/TechnolORy, 8:135 (1990). Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of 30 Kluyveromyces have also been disclosed. Fleer et aL. Bio/rechnology, 9:968-975 (1991). (iv) Promoter component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to thc nucleic acid encoding thc CD20 binding antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter , -lactamasc and lactose promoter systems, alkaline 35 phosphatasc 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 the DNA encoding the CD20 binding antibody. Promoter sequences arc known for cukaryotes. Virtually all cukaryotic gcnes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another 40 sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA 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. 29 AME] NDES EET E 387'RCVD AT 41262O05 2:48:59 PM [Eastem Day~ghtTile'SVR:USPTO.EFXRF-il26'DNIS:21308271'0SID:650 952 9881 'DURATION (Tm-ss):24.56 I 111W %#W9.f "I ' "" Attorney Docket No. P1990R3 lfENUS 13 JUL2004 5 Examples of suitable promoter sequences for use with yeast hosts include tbc promoters for 3 phosphoglycerate kinuse or other glycolytic enzymes, such as enolase, glyccraldehyde-3-phosphate dchydrognCaase. hexokinase, pyruvate decarboxylase, phosphofructokinase, glucosc-6-phosphate isomcrase, 3-phosphoglyceratc mutase, pyruvate kinase, triosephosphatc isomerase, phosphoglucose isomerase, and glucokinase. 10 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, degradative enzymes associated with nitrogen metabolism, metallothioncin, glyceraldehyde.3-phosphate debydrogenasc, and enzyines responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 15 73,657, Yeast enhancers also are advantageously used with yeast promoters. CD20 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 virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian 20 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 a. 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 HindI E restriction fragment. A system for expressing DNA 25 in mammalian hosts using the bovine papilloma virus as a vector is disclosed In U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes C aL, Nature 297:598..601 (1982) on expression of human s-intcrfcron cDNA in mouse cells 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. 30 (v) Enhancer element component Transcription of a DNA encoding the CD20 binding antibody of this invention by higher cukaryoten is often increased by Inserting an enhancer sequence into the vector. Many enhancer sequences arc now known from mammalian genes (globin, elastase, albumin, a-fctoprotcin. and insulin). Typically, however. one will use an enhancer rnom a eukaryotle cell virus. Examples include the SV40 enhancer on the 35 latc 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. Sc also Yaniv, Nature 297:17-18 (1982) on enhancing clcments 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 5' from the promoter. 40 (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 cDNAX. These regions contain 30 3A1DED 9WET 31161'RCVD AT 412612005 2:48:59 PM[EastemnDaylight Time] SVR:USPT0-EFXRF-il26'ODNIS:21308271'CSID:650 952 9881 'DURATI0N (mm-ss):24.56 Attorney Dcket No. P 1990R3 E 3-JUL2004 . nucleoride segments transcribed as polyadenylated fragments in the uitranslated portion of the mRNA encoding CD20 binding antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See W094/11026 and the expression vector disclosed therein. (vii) Selection and transformation of host cells Suitable host cells for cloning or exprCssIng the DNA in the vectors herein are the prokaryote, 10 yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceac such as Escherichia. e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteux, Salmonella, e.g., Salmonella typhimuriun, Serratia, e.g.. Serratla marcescans, and Shigella, as well as Bacilli such as B. subritis and B. licheniformls (e.g., B. licheniformis 41 P disclosed In DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, 15 and Streptomyces. One prcfermd E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and F. call W3 110 (ATCC 27,325) are suitable. '11ese examples are illustrative rather than limiting. Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, In particular when glycosyladton and Fe effector function are not needed, such as when the therapeutic 20 antibody is conjugated to a cytotoxic agent (e.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, sec, c.g., U.S. 5.648,237 (Carter ct. al.), U.S. 5,789,199 (Toly et al.), and U.S. 5.840,523 (Sinunons et al.) which describes translation initiation region (TIR) and signal sequences for 25 optimizing expression and secretion, these patents Incorporated herein by reference. After expression, the antibody is Isolated from the E. coli ccli 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, cukaryotic nilcrbes such as filamentous fungi or yeast are suitable 30 cloning or expression hosts for CD20 binding andbody-encoding vectors. Saccharomyces cerevislae, 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 Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g.. K. tactis, K figill.s (ATCC 12,424), K. bulgarivus (ATCC 16,045), K. wickeramit (ATCC 24,178), K. watii (ATCC 56,500), K. drosophilarum 35 (ATCC 36.906), K . the rmnrolerans, and K. marxianus; yarmwia (BP 402,226); PIchia paxtoris (EP I83,070); Candika; Trichoderma reesia (EP 244,234): Neuwspora crassa; Schwanniomyces such as Scrwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospom, Penleillium, Tuoypocladium, and Aspergillos hosts such as A. nidulms and A. niger. Suitable host cells for the expression of glycosylated CD20 binding antibody are derived from 40 multicellular organisms. Examples of invertebrate cells include plat and insect cells. Numerous baculoviral strains and variants and corresponding permissive sect host cells from hosts such as Spodopterafirugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogas/er (fmitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L- variant of Autographa californica NPV and the Bm-5- strain of Bombyx mort 31 AMENDE SHEET E 32671'RCVD AT41262005 2:48:59 PM Eastem DayflghtTime] ISVR:USPTO-EFXRF-il26 DNIS:27308271 CSID:650 952 9881 'DURATION (mmss):24.56 iPEAUS 13 JUL 2004 Attorney Docket No. PI990R3 5 NPV. and such viruses may bc used as the virus herein according to the present invention, particularly for transftection of Spodopterafragiperda cells. Plant cell cultures of cotton, corn, 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 10 (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651): human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et at., J. Gen ViroL 36:59 (1977)): baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hrunster ovary eclls/-DHFR (CHO, Urlaub etat. Proc. Natl A cad. ScL USA 77:4216 (1980)) ; mouse sCrtoi cells (TM4, Mather. BioL Reprod. 23:243-2-51 I5 (1980)); monkey kidney cells (CV I ATCC CCL 70); African green monkey kidney cells (VERO-76. ATCC CRL-1587); human cervical carcinoma cells (lIILA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MlvT 060562, ATCC CCL 1); TRI cells (Mather e at., Annal N.Y. Aca. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human bepatoma 2f) lino (Hep G2). Host cells are transformed with the abovc-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 lwsf cells 25 The host cells used to produce the CD20 binding antibody of this invention may he cultured In a variety of media. Conunercially available media such as Ham's F 10 (Sigma), Minimal Essential Medium ((MuM). (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 et aL., Meth. EnZ. 58:44 (1979), Barnes er al., Anal Blochem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 30 4.560,655: or 5,122,469; WO 90/03430; WO 87100195; 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), buffer (such as HEPES), nuclcotides (such as adenosine and thymidinc), antibiotics (such as GENTAMYCINrm drug), trace elements (defined as inorganic compounds 35 usually present at final concentrations in the micoamolar 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 wt. Thc 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. (ir) Purification of antibody 40 When using recombinant techniques. the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly. as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter er at., RBo/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the 32 AMENDED SHEET E33167'RCVDAT426120052:48:59 PM [Eastem Daylight Time]' SVR:USPTO.EFXRFi26'DNIS:2730821'CSID:6509529881 'DURATION (m.ss):24.56 Attorney Docket No. P1990R3 IPEA S 13 JUL 20Ot 5 presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyifluoride (PMSF) over abou( 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium. supemafants from such expression systems arc generally first concentrated using a commercially available protein concentration filter, for example, an' Amicon or Milliporc Pellicon ultrafiltratiun unit A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit pr~teolysis and antibiotics may he 10 included to prevent the growth of adventitious contaminants. The antibody composition prepared from the cells ca be purified using, for example. hydroxylapatite chromatography, gcl clectrmphoresis, 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 Fe domain that is present in the antibody. 15 Pmtein A can be used to purify antibodies that are based on human 7l, y2, or -y4 heavy chains (Lindmark et al., .1. Irnmanol Meth. 62:1-13 (1983)). Protein G is recommended for all mouse Isotypes and for human y3 (Cuss er al, FMRO J. 5:15671575 (1986)). 'The matrix to which the affinity ligand is attached is most often agamsc, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)beozene allow for faster flow rates and shorter processing times than can be achieved 20 with agarose. Where the antibody comprises a C 1 1 3 domain, the Bakerbond ABXThre-sin (J. T. Baker, Phillipsburg. NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reversc Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSErm chromatography on an anion or cation exchange resin (such as a polyaspartc acid columno), chromatofocusing, SDS-PACE, and ammonium sulfate precipitation arc also 25 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 clution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about U-0.25M salt). 30 Antibody coniugates The antibody may be conjugated to a cytotoxic agent such as a toxin or a radioactive Isotope. In certain cmbodiments, the toxin Is calicheamilcin. a maytansinold, a dolastatin, auristatin E and analogs or derivatives thereof, are preferable. 35 Preferred drugst(oxins include DNA damaging agents, Inhibitors of microtubule polymerization or dcpolymeri7ation and antimetabolites. Preferred classes of cytotoxic agents include, for example, the enzyme Inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA Imercalators, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mltomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, diynenes, the 40 poduphyllotoxins and differentiation inducers. Particularly useful members of those classes include, for example, methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cyrosine arabinoside, melphalan, leurosine, leuros ideine, actinonycin, daunorubicin, doxorubicin, N-(5,5 diacctoxypcntyl)doxorubicin, morpholino-doxorubicin, 1-(2-choroehthyl).-1,2-dinethanesulfonyI hydrazide, N.-aceryl sparmidine, aminopterin methopterin, esperamicir, mitomnycin C, mitomycin A, actinomycin, 33 AMENDED ET E34167RCVD AT 41262Q05 2:48:59 PM [Eastem Daylight le]SVR:USPTO.EFXRF.i26'DNIS:27308271 CSID:650 952 9881 'DURATION (mm-ss):2446 Attorney Docket No. P1990R3 PEA/US 13 JUL 2004, 5 hicomycin. carminomycin, aminopterin, tallysonycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or ctoposide phosphate, vinblustine, vincristlie, vindesinc, taxol, taxotere, retinoic acid, butyric acid, Neacetyl spermidine, eamptothecin, calicheamicin. bryostatins, cephalostatins, ansamitacin, actosin, maytansinoids such as DM- 1, maytansine, maytansinol, N-desmetliyl-4,5-desepoxymaytansino.t, C 19-dechloromaytansinol, C-20-hydroxymaytansinol, C-20-demethoxymaytansinol, C-9-SHT- maytansinol, C 0 14-alkoxymethylmaytansinol, C- I4-hydroxy or acetyloxymethimaytansinol, C-15 hydroxy/acetyloxymaytansinol, C- 15-methoxymaytaminol. C-1 8-N-demetbylinaytansinol and 4,5 dcoxymaytansinol, auristatins such as auristatin E, M, PHE and PE: dolostatins such as dolostarin A, dolostatin B, dolostatin C, dolostatin D, dolostatin E (20-epi and I 1-epi), dolostatin G, dolostatin H, dolostatin 1, dolostatin 1, dolostatin 2, dolostatin 3, dolostatin 4, dolostatin 5, dolostatin 6, dolostatin 7. 15 dolostatin 8, dolostatin 9, dolostatin 10, deo-dolostato 10, dolostatin 1I, dolostatin 12, dolostatin 13, dolostatin 14, dolostatin 15, dolostatin 16. dolostatin 17, and dolostatin 18; cephalostatins such as cephalostatin 1, cephalostatin 2, ccphalostatin 3, cephalostatin 4, cephalostatin 5, cephalostatin 6, ccphalostatin 7, 25'-epi-cepbalostatin 7, 20-epi-cephalostatin 7, cephalstatin 8, cephalostatin 9, cephalostatin 10, cephalostadn I icephalostatin 12,cephalostatin 13,cephalostatin 14, cephalostatin 20 15.cephalostatin 16,cephalostatin. 17, cephalostatin 18, and cephalostatin 19.. Maytansinoids are mitototic Inhibitors which act by inhibiting tubulin polymcrization. Maytansine was first isolated from the east African shrub Maylenu serrata (U.S. Patent No. 3,896,111). Subscqucntly, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinl esters (U.S. Patent No. 4,151,042). Synthetic maytansinal and derivatives and analogues 25 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 30 cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for cxample, in I I.S. Patent Nns. 5,20R,020, 5,416,064 and Puropean Patent F.P 0 425 2.5 111, the disclosures of which are hereby cxpressly incorporated by reference. Liu et at, Proc. Nat. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprising a maytansinoid designated DM1 linked to the monoclonal antibody C242 directed against buman colorectal cancer. The conjugate was found to be highly cytotoxic 35 towards cultured colon cancer cells, and showed antituwor activity in an in vivo turmor growth assay. Chari et aL Canc Research 52:12 7 -131 (1992) describe oiunioconjugates In which a maytansinoid was conjugated via a disulfide linker to the urine antibody A7 binding to an antigen on human colon cancer cell lines, or to another murine monoclonal antibody TA. 1 that binds the HER-2/neu ancogenc. There are many linking groups known In the art for making antibody-maytansinoid conjugates, 40 including, for example, those disclosed in U.S. Patent No. 5,208,020 or EP Patent 0 425 235 B1, and Chari et al. CancerResearch 52: 127-131 (1992). The linking groups include disufide group., thinether groups, acid labile groups, photolabile groups, peptidase labile groups, or esterase labile groups, as disclosed in the above-identified patents, disulfide and thioether groups being preferred. 34 EM DED 10 35167 RCVD AT 412612005 2:48:59 PM [EastemnDaylight Time]'SVR:USPTO-EFXRF-1I26'DNIS:2130827' CSID:650 952 9881 'DURAilN (mm.ss):24-56.

- -W -- w, t rE Attorney Docket No. P1990R3 I-"A/US 1 3 JUL 200 5 Conjugates of the antibody and maytansinoid may be made using a variety of bifunctional protein coupling agents such as N-succinniidyl-3-(2-pyridyldithio) propionate (SPDP), succininidyl-4-(N malcinidomethyl) cyclohexane- I -carboxylatc, iminothiolane (f), bifunctional derivatives of imidnesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberatc), akichydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium 10 derivatives (such as bis-(p.dia7Aniumbcnzoyl)-ethylenediatine). dilsocyanates (such as toluene 2,6 diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2.4-dinitrobenzeine). Particularly preferred coupling agents include N-succinimidyl-3.(2-pyridyldithio) propionate (SPDP) (Carlsson el at, Rinchem. J. 173:723-737 11978]) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage. 15 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, the C-15 position modified with a hydroxyl group, and the C 20 position having a hydroxyl group. In a preferred embodiment, the linkage is formed at the C-3 position 20 of maytansinol or a maytansinol analogue. Calichcamicin Another immunoconjugate of Jnterest comprises an CD20 binding antibody conjugated to (me or more calichcamicin molecules. The calicheamicin family of antibiotics are capable of producing double 25 stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamiclo family, see US. 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, Y', X2. 1 , , N-acetyl-y,', PSAG and W', (Hinman et aL Cancer Research 53: 3336-3342 (1993), Lode et al CancerResearch 58: 2925-2928 (1998) and the aforementioned 30 U.S. patents to American Cyanamid). Another anti-tumor drug that the antibody can be coqjugated is QFA whlch is an ontifointp. Rnth enlichenmrin nd QFA hive intmcclhlnr sites of action atnd tin not rearlily cross the plasma membrane. Therefore, cellular uptake of these agents through antibody mediated internall7ation greatly enhances their cytotoxic effects. 35 Radioactive i.wtopes For selective destruction of thc tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugated anti-CD20 antibodies. Examples include AtmJ, 1, , Yo, Re", Rem, Sm'I, B1 2 2 Pn, Pb 2 n and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may comprise a radioactive atom for scintigraphic studies, for 40 example te""' or Im, or a spin label for nuclear magnedc resonance (NMR) imaging (also known as magnetic resonance imaging, roi), such as iodine-123 again, iodine-131, indium-I 11. fluorine-19. carbon 13, nitrogen-I5, oxygen-17, gadolinium, manganese or iron. The radio- or other labels may be incorporated in the conjugate In known ways. For example. the peptide way be biosynresized or may be synthesized by chemical amino acid synthesis using suitable amino 35 AME FXRF-i24 :36161' RCVD AT 412612005 2:48:59 PM [asternDaylght ime'8YR:USPTOEfYXRF*1J26' DNS:2730827 CSID:650 952 9881 DURATION (nss):24406 Attorney Docket No. P1990R3 IP US 1 3 JUL 2004 5 acid preursors involving, for example, fluorine-19 In placc of hydrogen. Labels such as to""' or 112, .Rc'", Rc'"" and In" can be attached via a cysteine tsidue in the peptide. Yttrium-90 can be attached via a lysine residue. The JODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123. "Monoclonal Antibodies In limunoscintigraphy" (Chatal.CRC Press 1989) describes other methods in detail. 10 Conjugates of the antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succininidyl-3-(2-pyridyldithio) prmpionate (SPDP), succinimidyl-4-(N maleimidomethyl) cyclohexane-J-carboxylate, iminothiolane (IT), bifunctional derivatives of Imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldchydc), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediaminc), bis-diazoniun 15 derivatives (such as bis-(p-diazoniunbenzoyl)-cthylenediamine), dilsocyanates (such as tolyEne 2,6 (iisocyanate), and bs-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobcnzenc). For example, a ricin Immunotoxin can be prepared as described in Vitettaer at Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanaobenzyl-3-mcthyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of rmdionucleotide to the antibody. See W094/11026. Thc linker may he a 20 "cleavablc linkcr" tcilitating 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 et aL Cancer Research 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used. Therapeutic Uses of the CD20 binding Antibodies 25 The CD20 binding antibodies of the invention arc useful to treat a number of malignant and non malignant diseases including autoimmune diseases and rclatcd conditions, and CD20 positive cances including B cell lymphomas and leukemias. Stem cells (B-cell progenitors) in bone marrow lack the CD24) antigen. allowing healthy B-cells to regenerate after treatment and return to normal levels within several months. 30 Autoimmune diseases or autoimmune related conditions include arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis including topic dcrmatitis; chronic autoiammunc urticaria, polynyositis/dermatomyositis, toxic epidernal necrolysis, systemic sclcroderma and sclerosis, rsponses associated with inflammatory bowel disease (IB)) (Crohn's disease, ulcerative colitis), respiratory distress syndrome, adult respiratory distress syndrome (ARDS), 35 meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glom.erulonephritis. allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflarmatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLF), lupus (including nephritis, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis, allergic cncephalonyelitis, Immune responses associated with acute and delayed hypersensitivity mediated by 40 cytokincs and T-lymphocytes, tuberculosis. sarcoldosis. granulonatosis Including Wegener's granulomatosis, agranulocytosis, vasculitis (including ANCA), aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AlHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIll deficiency, hemophilia A, autoimmune ncutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory 36 AMENDED SHEET E 3161*RCVD AT 4/26/2052:48:59PM[EastemDaylight Tme)*SVR:USPT0OFXRF-il26*DNIS:2130821* CSID:6509529881 *DURATION (mss):24-56 Attorney Docket No. PI990R3 PENUS 1 3 JUL 2004 5 disorderss, multiple organ injury syndrome, myusthenia gravis, antigen-antibody complex mediated diseases, anti-gloncrular basement membrane disease, anri-phospholipid antibody syndrome, allergic neuritis. Boehct disease, Castlcman's syndrome, Goodpasture's Syndrome, Lambert-Enton Myasthcnic Syndrome, Reynaud's syndrome. Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including pretreatment ror high panel reactive antibody titcrs, TgA deposit in tissues, etc), graft versus host disease 10 (GVHD), pemphigoid bullous, pcmphigus (a)) including vulgaris, foliaccus), autoimmune polyendocrinopathics, Reiter's disease, stiff-man syndrome, giant cell arthritis, immune complex nephritis, IgA nephropathy, IgM polyncuropathies or TgM mediated neuropathy, idiopathic thrombocytopcnic purpura (ITP), thrombotic lhrobocytopcnic purpura (TTP), autoimmune thrombocytopenia, autoimmunc disease of the testis and ovary including autoimune orcbitis and oophoritis, primary hypothyroidism; autoimmune 15 endocrine discascs including autoimmune thyroiditis, chronic thyrniditis (Hashimoto's Thyroiditis), subacute thyroiditis, idinpathic hypothyroidism. Addison's discasc, Grave's disease, autoimmune polyglandular syndromes (or polyglandular enducrinopathy syndromes), Type I diabetes also referred to as insulin dependent diabetes nellitus (JDDM) and Sheehan's syndrome; autoimmune hepatitis, Lymphoid interstitial pneumonitJs (HIV), bronchiolitis obliterans (non-transplant) vs NSTP, Guillain-Barre' Syndrome. Large 20 Vcssel Vasculltis (including Polymnyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis), Medium Vessel Vasculids (including Kawasaki's Disease and Polyarteritis Nodosa), ankylosing spondylitis, lierger's Disease (lgA nephropathy), Rapidly Progressive Gloincmruloncphritis, Primary biliary cirrhosis, Celiac sprue (gluten enteropathy), Cryoglobulinemia, ALS, coronary artery disease. CD20 positive cancCrs are thosc comprising abnormal proliferation of cells that express CD20 on 25 the cll surface. The CD20 positive B cell neoplasms include CD20-positive Hodgkin's disease including lymphocyte predominant Hodgkin's disease (TYHD); non-Hodgkin's lymphoma (NltL); follicular center coil (FCC) lymphomas; acute lymphocytic leukemia (AT.L); chronic lymphocytic Jeukenia (CLL); Hairy celi leukemia, The non-Hodgkins lyrnphoma include low grade/follicular non-Hodgkin's lymphoma (NUL), small lymphocytic lymphoma (SIL), intermediate grade/follicular NHL, intermediate grade diffuse NAIL. 30 high grade jlmmunoblastic NT-L, high grade lymphoblastic NT-L, high grade small non-cleaved cell NHL, bulky di.n&a NH1. plasnw.ynid lymphocytic lymphoma, mantle cell lymphoma. A ir)S- rnlnted lymphoma and Waldensmmos macroglobulincmia. Treatment of rclapscs of thcsc cancers arc also contcmplated. LPD 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 35 lcukem ia. A cancer of mature B-cells called lymphocytes, CLL Is manifested by progressive accumulation of cls in blood, bone marrow and lymplialic tissues. In specific cmbodimentS, the humanized CD20 binding antibodies and functional fragments thereof are 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 40 rhuniatoid arthritis, systemic lupus crythcmatosus (SLE) including lupus nephritis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (lTP), thrombotic throbocytopcnic purpura ('Tlr?). autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA ncphmpathy, IgM polyneuropathles, wyasthenia gravis, vasculitis, diabetes mellitus, Rcynaud's syndrome, Sjorgcn's syndromac and glumerulonephrits. 37 AMEND ET E 38167'RCVD AT 42612005 2:48:59 PM [Eastem Daylight TimeI'SVR:USPTOEFXRF-i26' DNIS:2730827' CSID:650 952 9881 'DURATION (mm-ss):24-56 Attorney Docket No. P I 99R3 3 JUL 2004 5 The humanized CD20 binding antibudics or functional fragments thereof are useful as a single agent treatment in, c.g., for relapsed or refractory low-grade or follicular, CD20-positivc, 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 10 humanized CD20 binding antibodies or functional fragments thereof are used to treat indolent NiL. 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 time to disease progression, time in remission, stable disease. 15 The following references describe lympbomas and CLL, their diagnoses, treatment and standard medical procedures for measuring treatment efficacy. Canellos UP, Lister, TA, Sklar JL: The Lymphomas. W.B.Saunders Company, Philadelphia, 1998; van Besien K and Cabanillas, 1': Clinical Manifestations, Staging and Treatment of Non-Hodgkin's Lymphoma, Chap. 70, pp 1293-1338, in: HematoloRy, Basic Principles and Practice, 3rd ed. Hoffman at al. (editors). Churchill Livingstone, Philadelphia, 2000; and Rai, 20 K and Patel, D:Chronic Lymphocytic Leukemia, Chap. 72, pp 1350.. 1362, in: Hematology, Basic Principles and Practice, 3rd ed. Hoffman et al. (editors). Churchill Livingstone, Philadelphia, 2000. Thc parameters for assessing efficKay or success of treatment of an autoimmune or auttoimmunc related disease will be known to the physician of skil in the appropriate disease. Generally, the physician of skill will look for reduction in the signs and symptoms of the specific disease. The following are by way of 25 examples. Tn one embodiment, the antibodies of the invention are useful to treat rheumatoid arthritis. RA 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. I'ewer than 50 percent of patients who 30 have had RA for more than 10 years can continue to work or function normally on a day-to-day basis. The antibodies can he 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 humanji.ed CD20 binding antibodies are useful to 35 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 discasc-modifying antirheumatic drugs and/or had an inadequate response to rnethotrexate alone are treated 40 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 (ig iv infusions on days I and 15); CD20 binding antibody plus cyclophosphamide (750mg iv infusion days 3 and 17); or CD20 binding antibody plus methotrexatc. 38 AMED 2 E 39161RCVD AT 41202052:48:59 PM[Eastem Daylightime)*ISVR:USPT0-EFXRF.Il26'DNIS:21308271 CSID:650 952 9881 1*DURATION mss):24.56 Attorney Docker No. P1990R3 IP U 13 JUL 2004 5 One method of evaluating treatment efficacy in RA is based on American College of Rheunatology (ACR) criteria, which measures the percentage of improvement in tender and swollen joints, among other things.'Ihe RA patient can be scored at for example, ACR 20 (20 percent improvement) compared with no antibody treatment (e.g,, baseline before treatment) or treatment with placebo. Other ways of evaluating the efficacy of antibody treatment include X-ray scoring such as the Sharp X-ray score used to scorc structural 10 damage such as bone crosion and joint space narrowing. Patients can also be evaluated for the prevention of or Improvement in disability based on Health Assessment Questionnaire [HAQ] 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: 1. patient's pain assessment by visual analog scale (VAS), 15 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 F.SR. 20 The ACR 50 and 70 are defined analogously. Preferably, the patient Is administered an amount of a CD20 binding antibody of the invention effective to achieve at least a score of ACR 20, preferably at least ACR 30, more preferably at least ACR50, even more preferably at least ACR70, most preferably at least ACR 75 and higher. 25 Psoriatic arthrids 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. Yct another aspect of the invention is a method of treating Lupus or SLE by administering to de 30 patient suffering from ST, a therapeutically effective amount of a humanized C020 binding antibody of the invention. SLEDAI scores provide a numerical quaotitatlon of disease activity. The SLEDAT is a weighted index of 24 clinical and laboratory parameters known to correlate with disease activity, with a numerical range of 0-103, sea Bryan Gescuk & John Davis, "Novel therapeutic agent for systemic Jupus erythematosus" in Current Opinion in Rheumatology 2002, 14:515-521. Antibodies to doubje-stranded DNA 35 am believed to cause renal flares and other manifestations of lupus. Patients undergoing antibody treatment can be monitored for time to renal flare, which is defined as a significant, reproducible increase in scrum creatinine, urine protein or blood in the urine. Altcmativcly or in addition, patients can be monitored for lcvcls of antinuclear antibodies and antibodies to double-stranded DNA. Treatments for SLE include high dose corticosteroids and/or cyclophosphamide (HDCC). 40 Spondyloarthropathics are a group of disorders of the joints, including ankylosing spondylitis, psoriatic arthritis and Crohn's discasc. Treatment success can be determined by validated patient and physician global assessment measurinig tools. Various medications tire used to treat psoriasis; treatment differs directly in relation to disease svcerity. Parents with a more mild form of psorlasis typically utilize topical treatments, such as topical 45 steroids, anthralin, calcipotriene, clobetasol, and tazarotene, to manage the disease while patients with moderate and severe psoriasis are more likely to employ systemic (methotrexate. retinoids, cyclosporine, 39 AMENDED iE 40167 'RCVD AT 412612005 2:48:59 PM[LEastem Daylight Time]' SVR:USPTO-EFXRF1i26' DNIS:273827'CSID:650 952 9881 'DURATION (mm-ss):24.56 Attorney Docket No. P I990R3 3 JUL 2004 5 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, byperlipidcmia, bone marrow supprmssion, liver disease, kidney disease and gastrointestinal upset. Also, the use of phototherapy can increase the incidence of skin cancers. In addition 10 to the inconvenience and discomfort associated with the use of topical therapies, phorotherapy 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 Aea and Severity 15 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 itching experienced at specific time points. Patients may experience an infusion reaction'or infusion-rlated symptoms with their first infusion of a therapeutic antibody. These symptoms vary in severity and gencrally are reversible with medical 2) intervention. These symptoms Ioclude but are not limited to, flu-like fever, chillsrigors, nausea, urticaria, headache, bronchospasm, angioedcma. 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 25 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 30 In the field would be familiar with, the antibodies of the invention will be administered at a dosage that is efficacious for the treatment of thnt indication which ninim-izing toxicity and side effects. For the treatment of a CD20 positive cancer or an autoirmune disease, the therapeuticaJly effective dosage will be in the range of about 250mg/m 2 to about 400 mg/mz or 500 mg/m 2 preferably about 250-375mg/m 2 . In one embodiment, the dosage range is 275-375 mg/mi. In one embodiment of the treatment of a CD20 positive 13 35 cell neoplasm, the antibody is administered at a range of 300-375 mg/rm 2 . For the treatment of patients suffering from B-cell lymphoma such as non-Hodgkins lymphoma, in a specific embodinent, the anti-CD 2 0 antibodies and hunanized anti-CD20 antibodies of the invention will be administered to a human patient at a dosage of 10Ig/kg or 375mg/rm. Por treating NHL, one dosing regimen would be to administer one dose of the antibody compositions a dosage of 10mg/kg in the first week of treatment, followed by a 2 week interval, 40 then a second dose of tihe 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 v1 6 (375 mg/m2 weekly) followed at week five by three additional courses of the antibody plus standard CHOP (cyclophosphamide, doxorubicin, vincristinc and prednisone) or CVP 40 AMENDED %WE E41167'RCVDAT4126120052:48:59PM[EastemDaylightrunel t SVR:USPTO-EFXRF-Il26aDNIS:2730827*CSID:6509529881 aDURATION (mmss):2446 Attorney Docket No. P1990R3 IPEAU 13 JUL 2004 5 (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 I 25mg/ni2 (equivalent to about 200mg/dose) to 600mg/m 2 , 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 10 cmbodimcnts, the dosage Is 250mg/dose, 2 7 5mg. 300mg. 325mg, 350mg, 375mg, 400mg, 425mg. 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg. 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. A patient administered a drug by intravenous infusion or subcutaneously may experience adverse 15 events such as fcvcr, chills, burning sensation, asthenia and headache. To alleviate or mininze such adverse events, the patient may receive an initial conditioning dose(s) of the antibody followed by a therapeutic dose. The conditioning dose(s) will he lower than the therapeutic dose to condition the patient to tolerate higher dosages. 20 Roue of adndnistration The CD20 binding antibodies are administered to a human patient in accord with known methods, such as by intmvcnous administrtion, e.g., as a bolus or by continuous infusion over a period of time, by subcutaneous, intramuscular, intraperitoneal, intracerohrospinal, intra-articular, intrasynovial, intrthecal, or inhalation routes, generally by intravenous or subcutaneous administration. 25 In on embodiment, the humanizted 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 30 antibodies of the present invention in conjunction with one or inore therapeutic agents such as a cbemothernpontio agent in u multidrug regimen. The CD20 binding antibody can be administerd concurrently, sequentially, or alternating with the cbemother-apeutic agent, or after non-responsivcncss with other therapy. Standard chemotherapy fur lymploma treatment may include cyclophosphamide, cytarabine, mclphalan and Inoxantrone phls melphalan. CHOP is one of the most common chemotherapy regimens 35 for treating Non-HOdgkin'S lympboma. The following are the drugs used In the CHOP regimen: cyclophosphamide (brand names cytoxan, neosar); adriamycin (doxorubicin / hydroxydoxorubcei); vincristine (Oncovin)t and prednisolone (sometimes called Deltasone or Orasone). In particular embodiments, the CD20 binding antibody is administered to a patient in oeed thereof in combination with one or more of the following chemotherapeutic agents of doxorubicin. cyclophosphamide, vincristinc and 40 prednisolone, In a specific embodiieflt, 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 cyclophosphamided, 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) cbemotherapy. in a specific embodiment, the patient 41 AMEND SEW E 42167'RCVD AT 412612005 2:48:59 PM [Eastem Daylight Time]* SVR:USPTO-EFXRF*1i26* DNIS:27308271 CSID:650 952 9881 'DURATION (mm.ss):24-6 Attorney Docket No. Pl 990R3 PEA/S 13 JUL2004 5 suffering from CD20-positive NHL is treated with humanized 217.y16 In conjunction with CVP. Tn a specific embodiment of the treaunent of CLL, the CD20 binding antibody is administered in conjunction with chemotherapy with one or both of fludarabine and cytoxan. in treating the autoimmune diseases or autnimmune related conditions described above, the patient can be treated with the CD20 binding antibodies of the present invention in conjunction with a second 10 therapeutic agent, such as an imuirnosuppressive agent, such as in a muhi drug regimen. The CT)20 binding antibody can bc administered concurrently, sequentially or alternating with the immunosuppressive agent or upon non-responsiveness with other therapy. The immunosuppressive agent can be administcrd at the same or lesser dosages than as set forth in the art. The prefered adjunct Inununosuppressive agent will depend on many factors, including the type of disorder being t=ated as well as the patients history. 15 "lmmunosuppressive 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, e.g., prednisone, methylprednisolone, and dexanetbasone; 2-amino-6-aryl-5-substituted pyrimiidines (see U.S. Pat. No. 4,665,077), a7.athioprine 20 (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; cytokinc or cytokine receptor antagonists including anti intcrferon-y, -P, or -a antibodies; anti-tumor necrosis factor-a antibodies; anti-tumor necrosis factor-P antibodies; anti-interlcukin-2 antibodies and anti-1L-2 receptor antibodies; anti-L3T4 antibodies; 25 heterologous anti-lymphocyte globulin; pan-T antibodies, prmferably anti-CD3 or unti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 90/08187 published 7126/90); streptokinasc; TGF-; streptodornasc; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycint T-cell receptor (U.S. Pat. No. 5,114,721); T-cell receptor fragments (Offner er aL, Science 251430..432 (1991); WO 9011J294; and WO 91/01133); and r cell receptor antibodies (PP 340,109) such as 30 TIOB9. 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-mldifying anti rheumatic drugs (e.g., methotrexate), NSAI or NSAID (non-steroidal anti.inflammatory drugs), HUM1 RATM (adalinunab; Abbott Laboratorics), ARAVA@ (leflunomide), REMTCADE@ (inflixiniabt 35 Centocnr Tnc., of Malvern, Pa), ENBRPT. (etancrccpt; Immunex, WA), COX-2 inhibitors. DMARDs commonly used in RA am hydroxycloroquine, sulfasalazinc, methotrexate, leflunornide, etanereept, infliximab, azathloprlne, D-penicillaminc, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, Staphylococcal protein A immunnadsorption. Adaliumab is a human monoclonal antibody that binds to TNFa. Infliximab is a chimeric monoclonal antibody that binds to TNFa. Etanemept is an 40 *inntnoadhesin" fusion protein consisting of the extracellular ligand binding portion of the human 75 kD (p75) tumor necrosis factor receptor (TNFR) linked to the Fe portion of a human TgG1. For conventional treatment of RA, see. e.g., "Guidelines for the management of rheumatoid arthritis" Arthritis & Rheumatism 46(2): 328-346 (February, 2002). In a specific embodiment, the RA patient is treated with a CD20 antibody 42 WENDE9U n E43167 'RCVD AT 412612005 2:48:59 PM[Eastem DayighlTime] SVRUSPTOOFRF-il26'DNIS:21308271'CSID:650 952 9881 ' DURATlON (mmss):24.56 'W a F ww 'W r I &I Up - s., vf Attorney Docket No. P1 990R3 IPEA/US 1 3 JUL 2004 5 of the Invention in conjunction with methotrexate (MTX). An exemplay dosage of MTX is about 7.5 25 mg/kg/wk. MTX can be adminislercd orally and subcutancously. For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's discase, 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.), UNBREL (etanereept; munex, WA). 10 Treatments for SLE include high-dose corticostcroids and/or cyclophosphamide (HDCC). For the treatment of psoriasis, patients can be administered a CD20 binding antibody in conjunction with topical treatmncnts, such as topical steroids, anthralin, calcipotricne, clobetasol, and tazarotene, or with methotruxate, retinoids, cyclosporine, PUVA and UVB therapies. In one embodiment, the psoriasis patient is treated with the CD20 binding antibody sequentially or concurrently with cyclosporine. 15 Pharmacetical 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 carricra, excipients or stabilizers (Remington's Pharmaceusicad Sciences 16th 20 edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methioninec; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzcthonium chloridc; phcnol, butyl or ben7yl alcohol; a Ikyl parabens such as 25 methyl or propyl paraben; catechol; resorcinol; cyclohcxanal; 3-pentanol; and n-cresol); low molecular weight (lcss than about 10 residues) polypcptidcs; proteins, such as scrum albumin, gelatin, or immunoglohulins; hydrophilic polymers such as olyvinylpyrrolidonc; amino acids such as glycine, glutamiine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as 3(1 sucrosc, mannitol, trchalOse or sorbitol; salt-forming counter-ions such as sodium; mctal complcxcs (e.g. Zn protein complcxcs); and/or non-ionic surfactants such as 'TWPNTM. PLURONTCSTM or polycthylcnc glycol (PEG). Exemplary anti-CD20 antibody formulations are described in WO98/56418, expressly Incorporated herein by reference. Another formulation is a liquid multidose formulation comprising the antl-CD20 35 antibody at 40 mg/niL, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbatc 20 at pH 5.0 that has a minimum shelf life of two years storage at 2-8"C. Another anti-CD20 formulation of Interest comprises 10mg/mL antibody in 9.0 mg/mi. sodium chloride, 7.35 mg/iL sodium citrate dihydrate, 0,7mg/mT. polysorbate 80, and sterile Water for Tnjection, pH 6.5. Yet another aqueous pbarmaceutical formulation comprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.5. preferably at pH5.5, 40 polysorbate as a surfactant in a un 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 W097/04801. Such lyophilized forimulations 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. 43 E 44167'RCVD AT 4126205 2:48:59 PM [Eastem Dayight Time)* SVR:USPTO-EFXRF*1i26' DNIS:2730827'CSID:650 952 9881 'DURATION (m.ss):2446 Attorney Docket No. PI1990R3 IPEA/US 13 JUL 20( 5 One formulation for the humanized 2H7 variants is antibody at 12-14 mg/mU in 10 mM histidine, 6% sucrose, 0.02% polysorbate 20. pH 5.8. Tn a specific embodiment, 2H7 variants and in particular 2H7.v16 is formulated at 20mg/mL antibody in 10mM hlstidinc sulfate. 60mg/ml sucrose., 0.2 mg/mi polysorbate 20, and Sterile Water for tInjction, at pH5.8. 10 . The formulation herein may also contain more than one active compound as necessary for the particular indication being trcated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a cytotoxic agent, chemotherapeutic agent, cytokinc or immunosuppressive agent (e.g. one which acts on T cells, such as cyclosporin or an antibody that binds T cells, e.g. one which binds LFA- I). The effective amount of such other agents IS 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 I to 99% of the heretofore employed dosages, The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin 20 microcapsules and poly-(methylmethacylate) mlcrocapsutles, respectively, in colloidal drug delivery systems (for example, liposomes, albumnb milcrospheres, miemcmulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques arc disclosed in Reingtonr's Pharmaceutical Sciences 16th edition, Osol, A. .Id. (1980). Sustained-relcasc preparations may be prepared. SuiTable examples of sustained-rcleasc 25 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 nicrocapsules. Examples of sustained rclease matrices include polycsters, hydrogcls (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and. ethyl-L glutamate, non-degradable ethylene-vinyl acetate. degradable lactic acid-glycolic acid copolymers such as 30 the LUPRON DEPOT"N injectablee microspheres composed of lactic acid-glycolic acid copolymer and lcuprolide acetatc), and poly-D-(-)-3-hydrxybutyric acid. The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. 35 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 40 containers may be formed front a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the condition and may havc a stcrilc 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 one active agent in the composition is a CD20 binding antibody of the invention. The label 44 -EE SHEET E45167' RCVD AT 412612005 2:48:59 PM EastemDayllght Time]' SVR:USPTO-EFXRFi26' DNIS:2730827 CSID:650 952 9881 'DURATION (mm.ss):24-56 Attorney Dockct No. Pl 990R3 IPEA/US 1 3 JUL2004 5 or package insert indicates that the composition is used for treating the particular condition. Thc label or package insert will furtber comprise instructions for administering the antibody composition to the patient. Package insert refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concCring the use of such therapeutic products. Tn one embodiment, the package insert indicates 10 that the composition is used for treating non-Hodgkins' lyniphoma. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bactcriostatic water for injection (BWF), 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, dilucnts, filters, needles, and syringes. 15 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 lnmunoprecipitation 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 vi/ro, e.g. in an EL1SA or a Western blot. As with the article of manufacture, the kit comprises a 20 container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-CD20 antibody of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. Thc labcl or package insert may provide a description of the composition as well as instructJons for the intended in vitro or diagnostic use. 25 Cynomolgus monkey CD20 Thc invention also provides an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO.: 24 of the Cynomolgus monkey CD20 as shown in FIG. 19. Tn one embodiment, the nucleic acid is a cDNA. In one embodiment, the nucleic acid encoding the monkey CD20 is in an expression vector for expression in a bst ecl. The nucleotide sequence of SRQ ID NO.: 24 in the expression vector in operably 30 linked to an expression control sequence such as a promoter or promoter and enhancer. Thc expression control sequence can be can be the native scqucnce normally associated with the Cynomolgus CD20 gene, or hctemlogous to the gene. Also provided is an isolated polypeptide comprising the amino acid sequence ISEBQ ID NO. 25; FIG. 19 & 201 of the Cynomolgus monkey CD20, as well as host cells containing the Cynomolgus CD20 nucleic acid. In. one aspect tie host cells are eukaryotic cells, e.g., CHO cells. Fusion 35 proteins comprising the Cynomolgus CD20 amino acid sequence or fragments of the sequence are also contemplated. Experimental Examples Example 1 40 Humanization of 2H7 anti-CD20 morine monoclonal antibody Humanization of die murine anti-human CD20 antibody, 2H7 (also referred to heroin as m2H7, m for urinee, was carried out in a series of siie-directed mutagenesis steps. The urine 2H7 antibody variable region sequences and the chimeric 2-7 with the mousc V and human C have been described, see, 45 e.g., U.S patents 5,846,818 and 6.204.023. The CDR residues of 2H7 were Identified by conparing the 45 .M . S SH EET E 4616P RCVD AT 4/26120O52:48:59 PM[Eastem Daylight Time]' SVR:USPTO.EFXRF-1l26' DNIS:21308271'C81D:650 952 9881 ' DURATION (mm-ss):24.56 Attorney Docket No. P1990R3 JPEA/US 1 3 JUL 2004 5 amino acid sequence of the urine 2H7 variable domains (disclosed in U.S. 5,846,818) with the sequences of known antibodies (Kabat et al., Sequences of proteins of immunological interest, Ed. 5. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The CDR regions for the light and heavy chains were defined based on sequence hypervariability (Kabat et al., supra) and are shown in Fig. IA and Flig. I B, rCspcCtively. Using synthetic oligonucleoides (Table 1), site-directed iutagencsis (Kunkel, Proc. 10 Natt A rad. Sci. 82:488-492 (1985)) was used to introduce all six of the murinc 2H7 CDR. regions into a complete human Fab framework corresponding to a consensus sequence VIj,Va1ll (VL kappa subgroup 1, V11 subgroup ILI) contained on plasmid pVX4 (Fig. 2). The phagemid pVX4 (Ig. 2) was used for mutagenesis as well as for expression of F(ab)s in U. coil. Based on the phagem-dd pb0720, a derivative of pBD475 (Cunningham et aL. Science 243: 1330-1336 15 (1989)), pVX4 contains a DNA fragment encoding a humanized consensus -subgroup T light chabi (VLICd C.) and a humanized consensus subgroup III heavy chain (Vali-C, I1) anti-TFN-a (interferon a) antibody. pVX4 also has an alkaline phosphatase promotor and Shinc-Dalgamo sequence both derived from another previously described pUC1 19-based plasmuid, pAK2 (Carter et al., Proc. Nat]. Acad. Scl. USA 89:4285 (1992)). A unique Spel restriction site was introduced between the DNA encoding for the F(ab) light and 20 heavy chains. *The first 23 amino acids in both anti-.FN-ta heavy and light chains arc the 5tJY 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-containing template of pVX4; all six CDRs of anti-TFN-oc were changed to the urine 2H7 CT)Rs. The resulting molcculc is referred to as humanized 2H7 version 2 (2H7.v2), or the "CDR-swap 25 version" of 2H7; it has the m2H7 CDR resid ues with the consensus human FR residues shown in Figures IA and I B. Humanized 2H7.v2 was used for further humanization. Table I shows the oligonucleotidc sequence used to create each of the murine 2H7 (m2H7) CDRs in the H and T. chain. For example, the CDR-H I oligonuelcotide was used to recreate the ni2H7 H chain CDR1, CDR-111, CDR-H2 and CDR-H3 refers to the H chain CDR 1, CDR2 and CDR3. respectively; 30 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 oligonuclootidos, 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 oligonucleotido arm underlined. Substitution O]igonucleotide sequence CDR-Mil C TAC ACC TiC ACO _AQ TAT AAC AT CAC TOG OTC CG (SEQ ID NO. 27) CDR-H2A G A'T AAT CCT GAC AAC QQQ QA_ ACG AGC TAT AAC CAG AAG TTC AAG GOC CG (SEQ TD NO. 28) CDR-H21B G3AA TGM OTT GCA GCG ATC IT CCT U(Q AAC GGC GAC AC (SEQ ID NO. 29) CDR-H3 AT TAT TOT OCT CGA GTG QTQ TAC T A C A CA TQ TAC = GAC QIL TCG CGT CAA GGA (SEQ ID NO. 30) CDR-L1 C TGC ACA GCC AGC I TCr GTC AGC TAT ATG CAT TO (SEQ ID NO. 31) CDR-L2 AA CA CfG AT TAC QCT CCA TQ AAC CTC QCG TCT GGA GTC C (SEQIDN. 32) CDR-L3 TAT TAC TGT CAA CAG TMQ Ag =Q AT CC =Q ACA TTT GGA CAG (SEQ 1V NO. 33) 46 E 47167'RCVD AT 4312005 2:48:59 PM [astem Daylight Timel SVR:USPTOEXRF.1126'DNIS:27308271 CSID:650 952 9881 'DURATION nm-ss):2446 Attorncy Docket No. P1 990R3 1PEA/US 13 JUL 2004 For comparison with humanized constructs, a plasmid expressing a chimeric 2H7 Fab (containing murine VL and VH domains, and human CL and CHi domains) was constructed by site-directed mutagenesis (Kunkel. supra) using synthetic oligonucleotides to introduce the murine framcwork residues into 2Ji7.v2. The sequence of the resulting plasmid construct for expression of the chimeric Fab known as 2H7.v6.8. is 10 shown in Pig. 3. Each encoded chain of the Fab has a 23 amino acid SrU secretion signal sequence as described for pVX4 (Fig.2) above. Based cm a sequence comparison of the murine 21H7 framework residues with the human VVJ,VnIll consensus framework (Figures IA and I B) and previously humanized antibodies (Carter et al., Proc. Nari. Acad. Sci. USA 89:4285-4289 (1992)), several framework mutations were introduced into the 21)7.v2 Fab 15 construct by site-directed mutagcncsis. These mutations result in a change of certain human consensus framework residues to those found in the murine 2117 framework, at sites that might affect CDR confirmations or antigen contacts. Version 3 contained VH(R7]V, N73K), version 4 contained VH(R71V), version 5 contained V(R71V, N73K) and VL(L46P), and version 6 contained VH(R71V. N73K) and V:.(IA6P, I A7W). 20 Humanized and chimeric Fab versions of m2ff7 antibody were expressed in F. coli and purified as follows. Plasmids were transformed into . coli strain XT.-J Blue (Stratagenc, San Diego, CA) for preparation of double-and singlc-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 g/ml 25 carbenicillin. and a single colony selected for protein expression. The single colony was grown in 5 ml LB 100 jpg/ml carbenicillin for 5-8 h at 37* C. The 5 mi culture was added to 500 ml APS-1 00 pg/ml carbenicillin and allowed to grow for 16 h in a 4 L baffled shake flask at 37uC. AP5 media con)sisis of: 1.5g glucose. 11.0 Hycase SP, 0.6g yeast extract (certified), 0. 19g anhydrous MgSO4, 1.07g NTiCl, 3.73g KCI, 1.2g NaCl, 120 ml) M triethanolaminc, pH 7.4, to 1 L water and then sterie filtered through 0.1 pm 3) Scalkccn filter. Cells were harvested by centrifugation in a I L centrifuge bottle (Nalgene) at 3000xg and the supernatant removed. After freezing for I h, the pellet was resuspended in 25 ml cold 10 mM MBS-10 mM LiYrA. pH 5.0 (huffir A). 250 pW 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 cell wall. After 35 gentle shaking on ice for I h, the sample was centrifuged at 40,OODxg for 15 min. The supernatant was brought to 50 ml with buffer A and loaded onto a 2 ml DLEAE column equilibrated with buffer A. The flow through was then applied to a protein G-Sepharose CL-413 (Pbarmacia) column (0.5 ml bed volume) oquilibrated with buffer A. Thc column was washed with 10 ml buffer A and eluted with 3 ml 0.3 M glycine, pH 3.0, into 1.25 mil 1 M Tris, pH 8.0. The F(ab) was then buffer exchanged into PBS using a 40 Centricon-30 (Ampicon) and concentrated to a final volume of 0.5 ml. SDS-PAGE gels of all F(ab)s were run to ascertain purity and the molecular weight of each variant was verified by electruspray mass spectrometry. 47 AMENDED SHEFT E 48167'RCVD AT 412012005 2:48:59 PM Eastem Daylight Time] IVR:USPTO0EFXRF1i26' DNIS:2730827' CSID:650 952 9881 'DURATION (mm-ss):24.56 Attorney Docket No. P1990R3 S 1 3 JUL 2004 5 In cell-based ELISA binding assays describedd 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 IgG1 antibodies for assays and further mutagenesis. Plasmids for expression of full-length TgG's were constructed by subuloning the VL and VH domains of chimcric 2H7 (v6.8) Fab as well as buianized Fab versions 2 tu 6 into previously described 10 pRK vectors for mammalian ccl1 expression (Corman et al., DNA Prvt Eng. Tech. 2:3-10 (1990)). Briefly, cach Fab construct waN digested with EcoRV and Blpl to excise a V, fragment, which was cloned into the rRV/BlpI sites of plusmid pDR1 (Fig. 4) for expression of the complete light chain (Vl-CL domains). Additionally, each Fab construct was digested with Pvull and Apal to excise a Vm fragment, which was cloned into the PuWl/Apal sites of plasmid pDR2 (Fig. 5) for expression of the complete heavy chain (VIi IS CH -hinge-CH 2 -CH3 domabis). Fur cach IgG variant, transient transfectioos were performed by cotransfecting a light-chain expressing plasmid and a heavy-chain expressing plasmid into an adenovirus transformed human embryonic kidney cell line, 293 (Graham et al., J. Gen. ViroL, 36:59-74, (1977)). Briefly, 293 cells were split on the day prior to transfcetion, and plated in scrum-contnining medium. On the following day, double-stranded DNA prcparcd as a calcium phosphate precipitate was added, followed by 20 pAdVAntageTm DNA (Promega, Madison, WI), and cells werc incubated overnight at 37C. Cells were cultured in serum-free medium and harvested after 4 days. Antibodies were purified from culture supernatants using protein A-Sepharose CL-41U. then buffer exchanged into 10 mM sodium succinate, 140 mM NaCI, pH 6.0, and concentrated using a Centricon-10 (Amicon). Protein concentrations were determined by quantitative ami0o acid analysis. 25 To measure relative binding affinities to the CD20 antigen, a cell-based ELISA assay was developed. Human B-lymplioblastoid WIL2-S cells (ATCC CRL 8885, American Type Culture Collection, Rockville, MD) were grown in RPMT 1640 supplemented with 2 mM L-glutaminc, 20 mM HEPES, pH 7.2 and 10% heat-inactivated fetal bovine serum in a humidified 5% CO 2 incubator. The colls were washed with PBS containing 1% FBS (assay buffer) and seeded at 250-300,000 cell/wel I in 96-well round bottom plates 30 (Nunc, Roskilde, Denmark). Two-fold serially diluted standard (15.6-1000 ng/mi of 2H7 v6.8 chinmeric lgG) and threefold serially diluted samples (2.7-2000 ng/ml) in assay buffer were added to tbe plates. The plates were buried in ice and incubated for 45 min. To remove the unbound antibody. 0.1 nIL assay buffer were added to the wells. Plates were centrifuged and supernatants were removed, Cells were washed two more times with 0.2 mL assay buffer. Antibody bound to the plates was dctceted by adding peroxidase conjugated 35 goat anti-human Fc antibody (Jackson immunoResearch, West Grove, PA) to the plates. After a 45 min incubation, cells were washed as described before. TMB substrate (3,3',5,'-tetrmnethyl benzidine: Kirkegaard & Perry Laboratories, Gailthersburg, MD) was added to the plates. The reaction was stopped by adding I M phosphoric acid. Titration curves we= fit with a four-parameter nonlinear regression curve fitting program (KaleidaGraph, Synergy software, Reading, PA). The absorbance at the midpoint of the 40 titration curve (mid-OD) and its corresponding concentration of the standard were dctcrmined. Then the concentration of each variant at this mid-OD was determined, and the concentration of the standard was divided by that of cach variant, Hence the values are a ratio of the binding of each variant relative to the 48 AMENDED 9 E 49167'RCVD AT 4126l2O52:48:59 PM [Eastem Daylight ilme) SVR:USPT0-EFXRF-il26*DNIS:2736827*CSID:650 952 9881 * DURATI0N (mss):24-56 Attorney Docket No. P1 990R3 EANS 13 JUL 200. 5 standard. Standard deviations in relative affinity (equivalent concentration) were general +/- 10% between cxpcrimcnts. As shown in Table 2, bindIng of the CDR-swap variant (v.2) was extrcrncly reduced compared to chimcric 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 21-7, additional 10 mutations and combinatioms of mutations were constructed by site-direct Mutagcnesis to produce variants 7 to 17 as indicated in Table 3. In particular. these included Vo mutations A49G, F67A, 169L, N73K, and L78A: and VL mutations M4L, M331, and F7 IY. Versions 36 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 15 rmework residues to murine framework residues (Table 3), was therefore chosen as the humanized form for additional characterization. Tablc 2. Relative binding affinity of humanized 2H7 TgC 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 20 concentration of the variant required for equivalent binding; hence a ratio <1 indicates weaker affinity for (be variant. Standard deviation in relative affinity determination avcragcd +/. 10%. Framework substitutions in the variabic domains are relative to the CDR-swap version according to the numbering system of Kabat (Kabat ct al., supm). 2H7 Heavy chain (V1) Light Chain (V 1 ) Relative version substitutions substitutions binding 6.8 (Chimera) (Chimera) -I 2 (CDR swap) (CDR swap) 0.01 3 R71V, N73K (CDR swap) 0.21 4 R71V (CDR swap) 0.21 5 R7IV, N73K L46P 0.50 6 R7]V,N73K L46P, L47W 0.58 7 R71V L46P 0,33 8 R71V, L78A TA6P 0.19 9 R71V,F67A LA6P 0.07 J0 R71V.F67A.169L L46P 0.12 il R71V, P67A, L78A L46P 0.19 12 R71V L46P, M4L 0.32 13 R71V L46P, M331 0.31 14 R71V L46P, F71Y 0.25 15 R71V L46P, M4L, M331 0.26 16 R71VN73K,A49G L46P 0.65 17 R71V, N73K, A490 L46P, L47W 0.67 25 49 E 50107RCVD AT 41262005 2:48:59 PM [Eastern Daylight Time] ISVR:USPTO-EFXRF-i6*DNIS:2730827 CSID:650 952 9881 'DURATION (mm-ss):24-56 Attorney Docket No. P1 990R3 IPEA/US 1 3 JUL 2004 5 Table 3 Oligonucleotide sequences used for construction of mutations VH(A490, R71 V, N73K) and VT,(TA6P) in humanized 2H7 version 16 (2H7.v 16). Underlined codons encode the Indicated amino acid substitutions. For V 1 1 (R71V, N73K) and V, (.A6P), the oligos are shown as the sensc strand since these wcrc used for mutagenesis on the Fab template, whilc for VH (A49G), the oligo is shown as the anti-scnse strand, since this was used with the pRK (IgG heavy chain) template. The protein sequence of version 16 Js 10 shown in Fig. 6 and Fig. 7. Substitution Oligonucleotidc sequence

V

1 (R71V, N73K) CT TTC ACT ATA AGT QE GYAC AAG TCC AAA AAC ACA TT (SEQ ID NO. 34) Vj(A490) GCCAGGATAGATGOCGCCAACCCCACCAGGCC (SQ iD NO. 35) VL (L46P) AAGCTCCG AAACCACTGATTTACGCr (SEQ ID NO. 36) Extample 2 Antigen-binding determinants (paratope) of 2H7 Alanine substitutions (Cunningham & Wells, Science 244: 1081-1.085 (1989) were made in J5 2H7.v16 or 2117.v17 in order to test the contributions of individual side chains of the antibody in binding to CD20. JgG variants were expressed in 293 cells from pDR1 and pDR2 vectors, purified, and assayed for relative binding affinity as described above. Scvcral alanine substitutions resulted in significant decreases in relative binding to CD20 on WIL-2S cells (Table 4). 20 Table 4. Effects of alanine substitutions in the CDR regions of homanizcd 2H7.vl 6 measured using cell based HLTSA (W).L2-S cells). The relative binding is expressed as the concentration of the 2H7.v16 parent over the concentration of the variant required for equivalent binding;. huece 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 25 2117.v16 according (o the numbering system of Kabat (Kabat ct al., supm). NBD means no detectable binding. The two numbers for version 45 are from separate experiments. S2H7 CDR I1avv chain ILiht chain kelative binding version location kubstitutiuns substitutions 16 - - - -1 140 Hi G26A - 0.63 141 H1 Y27A - .47 34 H1 T28A 0.86 35 11 F29A - 0.07 36 -H 30A - 0.81 37 1 S31A - 0.97 142 111 Y32A - 0.63 143 Hi N33A - NDB 144 HI M34A - 1.2 145 HI H35A - <0.25 146 H2 A500 - 0.31 147 H2 151A - 0.65 50 AMEND SHEET 51167'RCVDAT412612005 2:48:59 PM [Eastem DaylightTimej SVR:USPTO.EFXRF-l26' DNIS:2730821 CSID:650 952 9881 DURATION (mm-ss):24.56 II'WMIU 13 JUL LUU Attorney Docket No. P 1990R3 38 12 Y52A -0.01 148 H2 PS2aA 0 .66 39 1-12 G53A 0 .89 67 H2 N54A -1.4 40 H2 G55A Q .79 41 H2 D56A - 2.0 99 H-2 '157A - 0.61 90 H-2 S58A - 0.92 1 F2 Y59A . 0.74 ___H2 N60A - 0.80 3 H52 61 IA - 0.83 4 H2 K62A 0 .44 5 112 F63A D .51 3l H2 V7A 0 .96 149 H2 K64A 0 .82 151. H2___ 065A ____ 1.2 153 H3 V95A -0.89 42 __. __ V96A -0.98 43 H3 Y97A 0 .63 44 H-3 Y98A 0 .40 45 H3 S99A -. 84; 0.92 46 H3 lOQ0A - .81 47 IH3 S I DaA -. 85 48 __ 1H13 Yl00bA -. 78 49 J3 WIDOcA .02 59 H3 Y1O0dA _______0.98 60 p,3 1:1400A ND)B 61 H3 D101A -0.31 1.51 H3 V102A - .I 117 1l R____ 24A .OM 118 Li A250 0.86 119 LI - S26A 0.98 120 LI - S27A .. 98 121 LI - S28A 1.0 122 LI - V29A 0.41 0 L1 - -530A 0.96 I 1l -_____ Y32A 1.0 123 1l - M33A M.0 124 1l - H34A 0.21 125 2 - A00 0.92 126 ____ P51 A 0.88 52 [2 -S52A 0.80 53 L2 Z N53A 0.76 54 L2 L54A 0.60 127 L2 -A5G 11i 128 L2 S.56A 11. 51 -MU SH E 52167' RCVD AT 412612005 2:48:59 PM [Eastern Daylight Time]'I SVR:USPTO.EFXRF*1126' DNIS:2730827P CSID:650 952 9881 1 DURATION (mm.ss):24.56 Attorney Dticket No. P1I990R3 lP ALS 13 JUL 2004 129 T-3 !2189A .- 46 130 L3 Q ___ 90A <0.22 55 1.2 -W91A 0.88 56 W. S 92A 1.1 57 L3 93A 0.36 58 W. N 94A 0.61 131 -- P9A NTB 132 L3 - 96A .18 1-33 3 r.0A.22 Example 3 Additional mutations witim 2H7 CDR reions Substitutions of additional residues and combi nations of substitutions at CDR positions thirt were identified as important by Ala-scanning wcre also tested. Several combination variants, particularly v-96 10 nppearcd to bind mome tightly than v. 16. Table 5. Effects of combinations of mutations and non-alanine substitutions in ti1c CDR regions of humanized 2M.06 weasurcd using cell-based ELISA (WIL2-S cells), The rr-ativc binding to CD20 is expressed as the concentration of the 217VM6 parnt over dhe concentration of the variant required for 15 equivalent binding- hcnce a ratio <1 indicates wcakcr affinity for the variant; a ratio >1 indicates higher affinity for the variant. Standard deviation in relative afiity determination averaged +/- M0A. Frwmcwork substitutions in the variable domains are relative to 2B.7.vl6 according to the numbering system of Kabat (IKahat et at., supraz). 2H-7 iHravy chain jight chain Ielative binding vmrsion I substitutions Is wstitrons 16 - 1 96 D50A, K1 004 S92A 3.5 97 _S99TNI 00CYY100bT _______ 0.99 98 S990, N I OS, YiO I __________ 1.6 9q 1100, Y1O0bl ______ _ 0.80 101 N54S D56A -1.7 102 N.54K, D56A 0_________.48 103 D56ANl00A _ _______2.1 104 S99Tt NI 1000 _________.81 105 S99Ci,NIODS _ _______1.1 106 111100 ______ 1 167 S I WaCI.Y IOOhS _______ 136 D56A, N IOOA SS6A, S92A 2.6 137 D56A, NlOOA A55G, 592A 2.1 156 fl6 iOAS26A, S56A, S92A 2.1 107 D56A, Ni OQA. Y I O0bI S92A not cxprcsscd 18S2 ;Y27W ____________________ 52 ~WD MS E531671 RCVD AT MOM100 2:48:59 PM PEasr Daylght Time' SYR:USPTOEFXRF4116 I DNMS:27308271 CSID:650 952 9881 1DURATION (mnss):24.56 IPE/US 3JU20 Atrnrncy Docket No. P I99OR3 3JL20 IHI Y27F ________ I1R4 1-29Y __ _ _ _ _ _ IS M 29W ____ 196S Y321; __ _ __ 187 Y32W____ ___ 18K8 N.33Q 18H9 N33D _________ 190 N33Y 191 N33S _________ 208 H35S _____ 209 50OS ________ 210 A5OR _____J_ 211 A50V__ _ _ _ _ _ __ _ _ _ _ 212 &50_________ ___.__ 168 Y.52W__ _ _ _ _ __ _ _ _ 19 Y52F 0________ .7.5 170 N54D_________ 0.25 171 N548 ________ 1.2 172 1)56K 1________ 173 D56R_____ ___ 174 D56H 1.5 175 1)56E3________ 1.2 213 D56S ________ 14 D56G 215 D56N 216 D56Y 176 Y59W 177 Y59F____ ___ ISO K62R____ ___ 181 X62V)________ ______ 178 F03W_____ ___ 179 F63Y________ ____ ___ 157 Y97W 0________ .04 158 Y97F _________1.2 159 Y98W 0________ .64 160 V98F 0________ .88 106 NI0O0 161 W I Q(CY 0.__ _ __ _ .5 162 W100civ 0________ .27 161 FlO0cY 0________ .59 164 F I00cW 0.71 161 DIMiN 0__ _ _ _ _ .64 166 S990, N 100G, S1O0aD,Y100b deleted 0_________ .99 217 VIU2Y ____ ____1.0 207 - H34Y___ ____ .53 -MN 9W E 2123 RCVD AT 4126)2005 4:38:33 PM [Eastern Daylght ThIiSV:UPTO.EFRF4125 IDNIS:2730827' CS!D:550 952 9881 1DURATION (mmss):0748 IffJ-NUS 13 JUL ZUU4 Attorney Docker No, F I990R3 192 - Q89E 193 -________________ Q89N 194 - Q90E 195 - Q90N 196 -_______________W91Y 197 -____________ __ W91 F ____ 205 - _____________ _ S92N 206 -S92(3_______ 198 -________________F93Y_______ 199 - _____9______ 3W _ _____ 204 __________________ 3S, N94Y 200) P96L _______ 201 -P96Y___ ____ 202 - _ __5 20.3 -P96R Exampvle4 Mufttons sit sitepg of framework humanization sobsftit~ns Substitutions of additional residues at frameawork positinns that were changed diuri ng humanization 10 were also tested In the 2H7,vl6 background. In particular, alternative framework substitutions that werei neither fund in thc mrwinc 2H7 parent nor tbc human consensus framcwork wcrc made tit VJ(P46) and

V

11 (049. V71, and K73). These s~ubstitutionls generally led to little chringe io relative, bindig (Table 6), IndicAtig that thero is urn flexibility in framework residues at these positons. J5 Table 6. Rclative binding ini a Lcu]-based (WIL2-S) assay of framework substitutions. i1gG variants are shown with mutarions with respect to the 2H7.v16 background. The tulative binding is cxprcsscd as the concentrationt of die 2H-7.v6.8 chimera over the concentration of the variant required for e~quivalrnt. binding; licrnce a ratio <1 Indicates weaker affinity for the variant; a ratio >1 indicates~ higher affinity far the variant 20 Standard deviation in relative affinity determination averaged +/- 10%. Framework substitutions in the variable domains are relative to 21{7.Yl 6 according to the numbering system of Kabat (Kabat et al.. supra). (*) Variants that were assayed with 2147.016 a% the standard comparator- rcliativc values are nornmalized to that (if thc chimera. 2H7 lea vy ch1i n ight chain Rcuicbning v~sihi suniitutions ubstjltions (?, ialie (chimecra)- 16 U_____________ _________ .64 78 K73R 0.72 79 K73H 0.49 80 K73Q D.58 al V711 ___________0.42 82 V71T _______ ___0.58 3 V71 A_______ __ 84 049S _________ _0.32 AMEM&i 9W WGE 3)232 RCVD AT 412 W2005 4:38:33 PM lpastem Dayflght Time] I SVR:USPTQ.EFXRF1251 DNIS27308271 CSID:650 952 98811 'DURATION (mss):0748 1PEWIS 13 JUL 2004 A ttorney Dockct Nn.- P1I990R3 .95 049L______ _____ __ 86 ________________4F 0.22 87 -P46V 0.51 88 P______________40T ________ 108 .049A, V71T, 1(73R S92A, M32L, P461 ().026* 109 Cu49A, A490, V71T. K73R S92A, M32L. P46f 0.026* 110 X73R 56A. NIOOA S92A, M321., Not cxprQ-ssed H]l G49A, V7 ITK73R _____________ 112 049A, A5OG, V7 IT, K73R 0._______ 12* (*) Variants that were. assaycd with 2T17.v 16 as the- standard comparator; rweiutivc values arc narmalized to that of th imera, Example 15 Humanized 21R7 variants with enhanced effecter functionIs Bccausc 2117 can mediate lysis of B-cells through hodi complenient-depestdent cytotoxdcity (~CDC) 10 amwd antibody-dependent cellular cytotoxicity (ADCC). we sought to produce variants of humanized 2H7.v 16 with improved CDC and ADCC activity. Mutations of certain residues wit~n the Fek- regions of other mnibodies have been described (Idusoaie el al.,. Jmmwwlo. 166:2571-2575 (2001)) for improving CDC through enhanced binding to (he complement component Clq. Mtitntlons have also been described (Shields in at.b 1. Hkio. Chemn. 276:6591-6A (2001); Pmesa ot al., Bioclzem Soc. Trams. 30:.487-490 (2002)) for 15 j improving ADCC through enhanced IgG binding to activating Fcyrcceptors% and reduced IgG binding to inhlibitory Fcy receptors, lfo particular, three mutations have been identified for improving CDC and AI)CC activity: S298A/E333A/K334A (albso referred to herein as a triple Ala Mutant or variant; numbering in the Fe rcglon is according to the EU numnbcring system; Kabat et al.. su~pra) as described (Idujsoge et al.. supra (200 1 ) Shiclds ct al., supra). 20 Tn order to enhance CODC and ADCC activity of 2H7, a triple Alam mutant of the 2H7 Fe was constructed. A humanized variant of rhe ansi-HER2 antibody 4d5 has been produced with mutations S298A/E3133A1K334A and Is known as 4D1'cl 10 (Le., anti-p'"HER2 Yg(;l (S298A/KR333A/K3.14A); Shields et t.s upra). A pIitsnid, p4D5Fcl10 encoding antibody 4D5Fc1 10 (Shieldr et a!., supra) was digested with Apal and !iisll, and the P7c-fragment (containing mutations S298 A/F333A/K334A) was 25 ligatedl into the ApuIllfindIJ sites of the 2137 heavy-chain vector pDR2-v16., to produce pDR2-v31. The amino acid ..lqucntcc oftb vI)Ytsiohi 31. complete H chain is sbown in Fig. 8. The T. chain is the same as that of v 6. Although the constant doins of the Fc regions of IgCU antibodies are relatively conserved within a given species, aliclic variations exist (roviewed by Lefranc and Lefranc. In 'The /urna-rl IgG subdasses. 30 mnlecular analysis~ of strlscture, funcrion, aid regule4 iort, pp. 43-78, F. Shakib (ed.), Pergasnzhon ProSs. Ox ford (1990)). Table 7. Effects of substitaitiotis in the Pc region on CD20 binding. Rclativec binding to CD20 was measured in a cell-based (WIL2-S) assay of framework substitutions- Fc mutations (*) ire indicated by EU 35 numbering (Knibat, supra) and are relative to the 2H7.v 16 parent The combination of three Ala changes in 55 AMEXDB MEET AGE 4123' RCVD AT 41263205 4:38:33 PM lEast Dayllgt Time)' SVR:USPTOEFXRF*125' DNIS:27308271 CSIDISD0 952 9881 1 DURATION (mmss):0748 -- ac c rn---. -- -~ - -- ItNUS 1 3 JUL2004 Attorney Dnckct No. P1 990R3 5 the Vc region of v.31 is described as "Fc I ICD." TgG variants are shown with mutations with respect to the 2H-7.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 <I indicatess weaker affinity for- the variant, Standard deviation in relative affinity dctcrmination averaged +/- 10%. 2H7 Pc Relative version Substitutions* binding ~6 .0.65 31 5298A, E333A, K334A 0.62 10 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 other processes that may affect product quality, in a suitable ij formularion buffer. In 2R7.v16, several residues were identified as possible sources of instability: VT. (M32) and Vi (M34, N100), Therefore, mutations were ltroduced tit these sites for comparison with v1 6. Table 8. Relative binding of 2H7 variants designed for enbanced stability and/or effector function, to CD20 In a cell-hased (WTL2-S) assay. JgG variarts are shown with mutations with respect to the 2H7.v16 20 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 <I 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 Rystem of Kabat and Fc mutations (*) are indicated by EU numbering (Kabal et al., supra), ('*) Variants that were measured 25 with 2H7.v 16 us the blandard compactor; relative values are nurmalizcd to that of the chimera. Additional Fe mutations wcrc combined with stability or affinity-enalncing mutations to alter or onhanco effector functions based on previously rcparted mutations (Idusogic ct al. (2000); Idusogle et al. (2001); Shields et aL (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 30 enhance CDC activity; and E356D/M358L to test the effects of ailotypic changes I the Fc region. None of these mutations caused significant differences in CD20 binding affinity. 2.H'7 Heavy chain eight chain c changes * Rative version (VI) changes (V.) changes |binding 6.8 (chimera) (chimera)

--

1 16 -_- 0.65 62 - M321 - 0.46 63 M341 - - 0.49 64 N100A 65 _ 10A L47W - 0.74 66 S99A L47W - .62 67 N54A 56 AGE 5123'RCVD AT 412612005 4:38:33 PM Eastem Daylightiiej'SVR:USPTO.EFXRF.i25 DNIS:2730821 CSID:650 952 9881 1DURATION (mm.ss):D748 lPEA/lS 1 3 JUL 2004 Attorney Docket No. PI 990113 M M321 -0.48 69 -M32.L -____ 0.52 70 Ni OOA -____ S'298A, UE333A, K334A 0.80 71 NMOOD -S298A 1333A, K334A 0.44 72 NIOOA 32I 0.59 73 INIOOA M2L -0.53 74 N 1100A M321 S298A, H333A, K334A 0.61 75 N100A M32L S298A, E33 3A, K334A 0.60 1II - -_____ F3 5 6D, M.45 T. ().60-83 114 D56A, N I 00A 32L. S92A 5299A, E-333A, K334 A I_____ 1 15 D56A, N I OOA 321, S92A S298A, E333A, K334A, P-3561). M358E- ______ 116 TD56A. Ni OOA M321. S92A S298A. K334A, K322A I .2* 34 D6,N I OOA 32T.., 892A .3156!), M3581., D)265Aj * 135 D50A, N lOA M321., S92A P5613, M3587-, D26SA, K320W ______*t 138 DS56A. N 1. OA M32L. S92A S298A, Li333A, K334A, K326A 12 119 156A. NI OOA M32L. S92A S298A, B333A, K334A, K326A, E356N, M358L ii I54 - D265A 155 1______- S298A, K322A, K334A 7e (**) Variants that were measured with 2H7.vl (5 as comparator, relative binding values are nrimali7ed to that of the chimera. To testbte effects of stability mutations on the rate of protein degradation, 21{7,v 16 aind 2H7.v73 were rormulatud at 12- 14 ing/mIL in 10 mM histidine. 6% sucrose. 0.02% polysorbate 20, pH .5.83and Incubated at 4 0 C for 16 dys. Tbc incubated samples wcru tbcn assayed for chwigcs in charge variants by inn cxchange chromatography, aggregation and fragmentation by A=z exclusion chromatography, and 10 relative hind ing by testing in a cell-based (W1L2-S) amsmay. Thle results (Fig. 9) show that 2J47 v.73 has greater stability compared to 2H7 v. 16 with respect to losses In the ftactioti of main peak by ion exchange chrmatography under accelerated stability conditions. No signifkcnt differences were seen with respect to aggregation, fragmentation, or binding affinity. 15 Excample 7 Scatchard nnalysis of antibody binding to CD20 on WIL2-S cells Equilibrium dissociation conistants (Ko) were determined for 2117 TgO variant-'binding to WIL2-S ecIK uting radiulabeled 2H7 IgO. igo variants were produced In CHO celik. Rituxn® (source for at) experiments itt Gencnteh, S. Sea Francisco, CA) anid murine 2H17 (J)BD PhajfMingen, -San Diego, CA) were 20 used for comptirisoil with humanmized varlaots. The murine 2H17 antibody is also available from other sourcess, c.g., eBioscience, and Calbiochern (both of San Diego, CA), Accun, te Cheiedcal & Scientific Corp,, (Wcetthury, NY), Ancll (Bayport, MN), and VinCi-ilocbein (Vinci, Italy), Au diltons were peformed In binding assay buffer (DMEM media containing 1% bovinc scrumn albumin, 25mTM HEFES pH 7.2, and 0,0 1% :mdiumn uz7idc). Aliquots (0.025 niL) of 115I-2H47.vl6 (lodiated with laCcoperoxidase) at a 2.5 concentration of 0.8 nM were di.4nnsed into wclls of a V-bttorn 96-wcIl microassay plate, and scrial dilutions (0.05 niL) of cold antibody wcrc added and mixed. WEL2-S celtt (6D000l( cell% in 0.025 niL) were thon acded. The plate war, sealed and incubated at room temperature for 24h, then centrifuged for 15 min at 3,500 RPM. The supernatant was then aspirated and the cell pellet was washed and centrifuged. The 57 AMEMW E AGE 6123'RCVD AT 412512005 4:38:33 PM lPaster Daylight Time) SVR:USPTO.EFXRF1125 OMS8273D8271 CSID:65D 952 9881 1'DURATION (mniss):0743 .. .. ... .... , . .. ..... . - . - - -- -- R n r W W I j U .LJL.VU Attorney Docket No. P I 990R 3 5 supernatant was again aspirated, and the pellets were dissolved in IN NaOH and transferred to tubes for gamma counting, The data were used for Scatchard analysis (Munson and Rodbard, ArtaL. Biwchm, 107:220-239 (1980)) using the program Ligand (McPhersort, Comput. Programs Binmed. 17: 107-114 (1983)). The results, shown it Table 9, Indicate that humanircd 2H7 variants had similar CD20 binding affinity as compared to inurine 2H7, and similar binding affinity to Rituxan@. It is expected that 2H7.v31 ID will have very similar K 4 to v.16 on the basis of the binding shown in Table 7 above. Table 9. Equilibrium binding affinity of 2H7 variants from Scatchard analysis Antibody variant K 1 (nM) n Rituxan 0.9920U.49 3 2H7 (murine) 1.23±0,29 3 2H7.v16 0.8420.37 4 2H7.v73 1.22±0.39 4 2H7.v75 1.09 0,17 4 Example 8 15 Complement Dependent Cytotoxicity (CDC) Assays 2117 lgG variunts wcrc assayed for their ability to mediate complcment-dcpendent lysis of WIL2-S cells, a CD20 expressing lymphoblastoid B-cell line, essentially as described (Tdusngie et al., J. Immunol, 164:4178-4184 (2000); Idusugic et al., J. ImmunoL 166:2571-2575 (2001)). Antibodies were serially diluted 1:3 from a 0.1 mg/mL stock solution, A 0.D5 mL aliquot of each dilution was added to a 96-well tissue 20 culture plate that contained 0.05 mL of a solution of normal human complement (Quidel, Sun Diego, CA) To this mixture, 50,000 WIL2-S cells were added in a 0.05 mL volume. After incubation for 2h at 37"C, 0.03 mL of a solution of Alamar blue (Accurmed International, Westlake, OH) was added, and incubation was continued for an additional 18h at 37'C. Covers were then removed fron the plates, and they were shaken for 15 min at room temperature on an orbital shaker. Relative fluorescent units (RPU) were mad 25 using a 530 nnm excitation filter and a 590 nm emission filter. An ECso was calculated by fitting RFU as a function of concentration for each antibody using KajeldaGraph software. The results (Table 10) show surprising improvement in CDC by humanized 2H7 antibodies, with rClativC potency similar to Rituxan@ for v.73, 3-fold morc patent than RirnxanO for v.75, and 3-fod weaker than Rituxan@ for v.16. .30 Table 10. CDC activity of 217 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. Antibody variant In EC(variant)/EC Rituxan) Rituxan@ 4 -1 2H7.v16 4 3.72 4.08 2H7.v31* 4 2.21 2117.v73 4 1.05 2H7.v75 4 0.33 58 AMENDED 1E N 41!E712390 RVD AT 41260205 4:38:33 PM[E astem Daylght Time) SVR:USPT0-EFXRF-il2P IDNIS:2730827* C$1D:650 952 9881 ' DURATI0N(111-ss4):01.48 iPEANS 1 3 JUL2004 Attorney Docket No. P1990R3 2H7.v96* 4 .956 2H7.vil4* 4 0.378 2H7.v115* 4 0.475 2H7.v I 16* i 100 2H7.v 135* 12 0.42 5 Example Antibody Dependent Cellular Cytotoxicity (ADCC) Assays 2H7 Ig variants were assayed for their ability to ruediate Natural-Killer cell (NK ccli) lysis of J0 WIL2-S cells, aCD20 expressing lymphoblastoid B-cell line, essentafly as described (Shicids ct al., J. BioL C/wm. 276:6591-6604 (2001)) using a lactate dehydrogenase (LDH) readout. NK cells were prepared from 100 mL of heparinized blood, diluted with 100 mL of PBS (phosphate buffered saline), obtained from normal human donors who had been isotyped for FYRJ.I, also known as CD16 (Koene et aL, Blood 90:1 109-11 14(1997)). In this experiment, the NK cells were from human donors hererozygous for CD16 15 (PISSN158). The diluted blood was layered over 15 mL of lymphocyte separation medium (1CN Biochumical. Aurora, Ohio) and centrifuged for 20 min at 2000 RPM. White cells at the interface between laycr were dispensed to 4 clean 50-mL tubes, which were filled with RPMI medium containing 15% fital calf serum. Tubes were centrifuged for 5 min at 1400 RPM and the supernatant discarded. Pellets were resuspended in MACS buffer (0.5% BSA, 2mM RDTA), and NK cells were purified using beads (NK Cell 20 Isolation Kit, 130-046-502) according to the manufacturer's protocol (MillenyJ Biotech,). NK cells were diluted in MACS buffer to 2x10 6 cells/mL. Scrial dilutions of antibody (0.05 mL) in assay medium (F12/DMIJM 50:50 wIthout glycine, J mM HF.PUS buffer til 7,2, pennlcillin/Streptomycin (100 units/mL; CribcO), 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 25 assay buffer to a concentration of 4 x 10 5 nL, WJL2-S cells (0.05 m.. per well) were mixed with diluted antibndy in the 96-wull plate and incubated for 30 min at room temperature to allow binding of antibody to C)20 (opsonization). 'thie ADCC reaction was initiater1 by adlins 0 I mT. nf NW rnlls tn cch we11. In Cnntrol wclls, 7% Triton X-100 was added. The plate was then incubated for 4h at 37"C. Levcls of LDH released were 30 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 10s. The plate was then covered with aluminum foil and incubated in the dark at mo'rn temperature for IS min. Optical density at 490 wn was then read and use to calculate % lysis by dividing by the total LDY1 measured in control wells. Lysix was plotted as a function of antibody concentration, and a 4 35 parameter curve fit (KaleldaGraph) was used to determine EC 5 0 concentrations. The results showed that humanized 2H7 antibodles were active in ADCC, with relative potency 20 fold higher than RituxanO for v.31 and v.75, 5-fold more potent than Rituxan® for v.16, and almost 4-fold higher than Rituxan@ for v.73. 59 AMENDED SEfET GE 8123'RCVD AT 4126005 4:38:33 PM [aste DaylightTime]'CVR:USPTQ-EFXRF-1125' DNIS:2730821 CSID:650 952 9881 'DURATION (mss):0748 lPEA/US 13 JUL2004 Auorney Docket No. P1990R3 5 Table 11. ADCC activity of 2H7 untibodies on WIL2-S cells compared to 2H7.vl 6, based on n experiments. (Vulues >l indicate lower potency than 2H7.v16, and values <l indicate greater potency.) Antibody variant n EC_(variant)/BC__ (2H7.v6) Rituxan@ 4 5.3 2H7.v16 5 1 2H7.v3l 1 0.24 2H7.v73 5 J.4 2H7.v75 4 0.25 Additional ADCC assays were carried out to compare combination-variants of 2H7 with Rituxan@. 10 Thc results of these assays Indicated that 2H7.vl 14 and 2H7.vl 15 have >10-fold improved ADCC potency as ctmpared to Rituxan@ (Table 12). Table 12. ADCC activity of 2H7 antibodies on WIL2-S cells uomparcd to Rituxan@), based on n experiments (Values >1 indicate lower potency than Rituxan®, and values <1 indicate greater potency). 15 Antihody variant FC50(variant)C50(Rituxan) RituxanoD -2 -1 2H7 v.16 2 0.52 2H7 v.96 .2 0.58 217.v 14 -2 0.093 2H7.v115 2 0.083 2H7.vl 16 20.30 Examiple 10 In vivo effects of 2H7 variants In a pilot study in cynomolgus monkeys 2H7 variants, produced by transient transfection of C110 cells, were tested in norma! male 20 cynomolgus (Macacafascicularis) monkeys in order to evaluate their in vivo acdvides. Other anti-CD20 antibodies, such as C2B8 (Rituxan@P) have demonstrated an ability to deplete B-cells in normal primates (Rrfl et al.. Blood 83: 435-445 (1994)). In onc study, humanized 2147 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 25 uIg/kg hu2H7.v16, (3) 10 me/kg hu2H7.v16, (4) 0.05 mg/kg .hu2H7.v31, and (5) 10 mg/kg hu2H7.v31. Antibodles were administered intravenously at a concentration of 0, 0.2, or 20 mg/mL, for a total of two doses, one on day I of the rtudy, and another on day 8. te first day of dosing Is designated day 1 and the previous day is designated day -1; the first day of recovery (for 2 animals in each group) is designated as day i1. Blood samples wcre collected on days -19, -12, 1 (prior to dosing), and at 6h, 24h, and 72h 30 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). Peripher B-cell concentrations were determined by a FACS method that counted CD3-/CD40+ cells. The percent o(CD3-CD40+ B cells of total lymphocytes in monkey samples were obtained by the following gating strategy. The lymphocyte population was marked on the forward scater/ side scatter 60 AMENDED SHEET GE 923'RCVD AT 412612005 4:38:33 PM [astem Daylight Time] SVR:USPT0-EFXR1125I'DN18:2730821'CSID:65 952 9881 'DURATION (mm-ss):0748 IPEAUS 13 JUL 2004 Attorney Docket No. P1990R3 5 sattergrnm to define Region I (R I). Using events in R I, fluorecence intensity dot plots were displayed for CD40 and CD3 markers. Fluorescently labeled isotype controls were used to determine respective cutoff points for CD40 and CD3 positivity. 1lie results indicated that both 2H7.v16 and 2H7.v3 I were capable of producing full peripheral Bl ecll depletion at the 10 mg/kg dose and partial peripheral B-cell depletion at the 0,05 mg/kg dose (Fig. 11). JO The time course and extent of B-cell depletion measured during the first 72h of dosing were similar for the two antibodies. Subsequent analysis of the recovery animals Indicated that animals treated with 2H7.v31 showed a prolonged depletion of B-cells as compared to thosc dosed with 2H7.v16. In particular, recovery animals treated with 10 mg/kg 2H7.v] 6, B-cells showed substantial 13-cell recovery at some time between sampling on Day 10 and on Day 36. However, for recovery animals (Tmatcd with 10 mg/kg 2H7.v3 1, B-cells 15 did not show recovery until some time between Day 36 and Day 67 (Fig. I1). This suggests a greater duration of full depletion by about one month for 2H7.v31 compared to 2H7.v 16. No toxicity was observed in the monkey study at low or high dose and the gross pathology was normal. In other studies, v16 was well tolerated op to the highest dose evaluatcd of (I00mg/kgx2 = 1200 mg/mn 2 A2) following i.v. administration of 2 doses given 2 weeks apart in these monkeys. 20 Data in Cynomolgus monkeys with 2H7.v 16 versus R ituxan@ 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 infusIon reactions than one with greater CDC activity. R5arnaie 11 Fucose deficient 2H7 variant antibodies with enhanced effector funclon Normal CHO and HEK293 cells add fucose to IgO ollgosaccharlde to a high degree (97-989). IgG from sora are also highly fucosylated. DP12, a dihydrofolate reductase minus (DHFR) CHO cell line that is fucosylation competent, and 30 Lec 3. a cell line that is deficient in protein fucosylation were used to produce antibodies for this study. The CHO cell line Pm-Lec 3.6a (Lec 13), was obtained from Professor Pamela Stanley of Albert Einstein College of Medicine of Yeshiva University. Parental lines am Pro- (proline Auxotroph) and Oat- (glycine, adenosine, thynidine auxotroph). The CHO-DP 12 cell linc is a derivative of the CHO-KI cell line (ATCC #CCL-6)), which is dihydrofolate reductase deficient, and has a reduced requirement for insulin. Ce lines 35 were transfcted with cDNA using the Superfect method (Qiaget, Valencia, CA). Selection of the Lec13 cells expressing transfected antibodies was performed using puromycin dihydrochlorlde (Calbiocheam, San Diego, CA) at 10 sg/mi In growth medium containing: MEM Alpha Medium with L-glutamine, ribunucleosides and deoxyribonuclensides (GIBCO-BRL, Gaithersburg. MD), supplemented with 10% inactivated 11BS (GIBCO), 10 rnM HEPFS, and IX penicillin/streptomycin (G1BCO). 'Tlie CHO cells were 40 similarly selcctcd in growth medium containing Ham's Fl 2 without GI-T: Low Glucnse DMEM without Glycine with NaHCO3 supplemented with 5% FBS (GLBCO), 10 mM HEIPS, 2 mM L-giutamine, IX GH'T(glycle, hypoxanthine,thymnidine), and IX penicillin/stroptomycin. Colonics formed within two to three weeks and were pooled for expansion and protein expression. The call pnnls were seeded initially at 3 x 106 cells/10 cm plate for small batch protein expression. The cells 61 MEND9E1 AGEiDl23*RCVDAT4/2612OO54:38:33PM[iEas'item~aylightTime]'SVR:,USPTO.EFXRF-il25'DNIS:213082P'CSID:6509529881 'DURATION (mm-Ss):0748 Attorney Docket No. Fl 990R3 5 wore con verted to serum-free media once they grew to 90-95% confluency and after 3-5 days ccil supernatants were collected and tested it an Fe IgG- and intact TgG-ELISA to estimate protein expression levels. Lecd3 and CHO cells were seeded at approximately 8 x 10 6 cells/15 cm plate one day prior to converting to PS24 production medium. supplemented with 10 mg/L recombinant human insulin and Img/L trace clements. 10 Lec13 cells and DP 12 celis 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 protese inhibitors PMSF and aprotinin (Sigma, St. Louis, MO) were added and the supernatants were concentrated 5-fold on stirred cells using MWCO30 filters (Amicon, Beverly, MA) prior to immediate purification using protein G chromatography (Amersham Pharmacia Biotech. Piscataway, NJ)). All proteins 15 were buffer exchanged into phosphate-buffered saline (PBS) using Centripricp-30 concentrators (Amicon) and analyzed by SDS-polyacrylamidde gel clctrophorcsis. Protein concentrations were determined using A280 and verified using amino acid composition analysis. The CHO cells were transfccted with vectors expressing humanized 2H7vl 6, 2H7v.31 and selected as described. The 2H7v.16 antibody retains the wild type Fc region while v.31 (sec Example 5, Table 7 20 above) has on Fe region wherein 3 amino acid changes were mude (S298A, E333A, K334A) which results in higher affinity for the FcyRma receptor (Shields et al. J. Biol. Chem. 276 (9):6591-6604 (2001)). Following tnsfection and selection, individual colonies of culls were isolated and evaluated for protein expression lcvcl 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. Ccls were grown, 25 transferred to serum five medium for a period of 7 days, then the medium was collected, loaded onto a protein A column and the antibody was cluted using standard techniques. The final concentration of the antibody was determined using an Elisa that measures intact antibody. All proteins were buffer exchanged Into phnsphate-buffered salinc (PBS) using Centripriep-30 concentrators. (Amicon) and analyzed by SDS polyacrylamide gel electrophoresis. 30 Matrix-Assisted Laser Desorption/Joniatlen Tirne-of-flight (MALDI-TOF) Mass Spectral Analysis rf'Asparagine-Linked Oligosaccharides: N-finked olinossecharides were released from recombinant glycoproleius using the procedure of Papac e( al., Glycobiology 8, 445-454 (1998). Briefly, the wells of a 96 well PVDF-lined nicrotitre plate (MJilipore, Bedford, MA) were conditioned with 100 pl methanol that was drawn tUvrugh the PDVJP membranes by applying vacuum to the Millipore Multiscreen vacuum manifold. 35 The conditioned PVDF membrunes were washed with 3 X 250 sl water. Between all wash steps the wells were drained completely by applying gentle vacuum to the manifold. Tbe membranes were washed with reduction and carboxymethylation buffer (RCM) consisting of 6 M guanidine hydrochloride, 360 mM Tris, 2 mM F.DTA, pH 3.6. Glycoprotein samples (50 p&g) wese applied to individual wells, again drawn through the PVDF membranes by gentle vacuum and the wells were washed with 2 X 50 i of RCM buffer. The 40 immobilized samples were reduced by adding 50 d of a 0.1 M dithiothreltol (DTI') solution to each well and incubating the mierotitre plate at 37*C for I hr. DTT was removed by vacuum and the wells wore washed 4 x 250 i water. Cysteine residues were carboxylmethylated by the addition of 50I pL of a 0.1 M indoacetic acid (IAA) solution which was freshly prepared in I M NaOH and diluted to 0.1 M with RCM buffer. Carboxymethylation was accomplished by incubation for 30 min in the dark at ambient temperature. 62 AMENDED SKET GE 11123'RCVD AT 402612005 4:38:33 PM [Eastem DayIght TimelI S UPTOEFXRFIlF5 DNIS:2730827 CSID:650 952 9881 *DURATIQN (mmss):0748 IM r.IPEA/US 13 JUL2004 Attorney Docket No. P1990R3 5 Vacuum was applied to the plate to remove the IAA solution and the wells were washed with 4 x 250 pl purified water. The PVDF membranes were blocked by the addition of 100 pl of 1% PVP360 (polyvinylpyrrolidJne 360,000 MW) (Sigma) solution and incubation for 1 hr at ambient temperature. The PVP-30 solution was removed by gentle vacuum and the wells were washed 4 x 250 I water. hie PNGasc F (New England Biolabs, Beverly, MA) digest solution, 25 I of a 25 Unit/mI solution in 10 mM Tris 10 acetate, pH 8.4, was added to each well and the digest procccded for 3 hr at 37 0 C. After digestion, the samples were transfcrrcd to 500 il Eppendori 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 br at ambient temperature to convert the oligosaccharides from glycosylanines to the hydroxyl form. Prior to MALDE-TOF mass spectral analysis, the recased oligosaccbarldes were desulted using a 0.7-ml bed of cation exchange main (AG50W-XS rsin 15 in the hydrogen form) (Blo-Rad, Hercules, CA) scurried packed into compact reaction tubes (US liochemical, Cleveland, OH). For MALDT-TOF mass spectral analysis of the samples in the positive mode, the desated oligosaccharides (0.5 gi aliquots) were applied to the stainless target with 0.5 I of the 2.5 dihydroxybcnrxoic acid matrix (sDIB) that was prepared by dissolving 2 mg 2,5 dibydruxybenzoic acid with 0.1 ig of 5 20 methoxyslicylic acid In 1 ml of ethanol/I0 mM sodium chloride 1:1 (v/v). The sample/matrix mixture was dried by vacuum, Vor analysis in the negative mode, the desalted N-linked oligosaccharides (0.5 id aliquots) were applied to the stainless target along with 0.5 p& 2',4','-trihydroxyacetophcnonc matrix (THAP) prepared in 1:3 (v/v) acetonitrile/13.3 mM ammonium citrate buffer. Thie sample/matrix mixture was vacuum dried and then allowed to absorb atmospheric moisture prior to analysis. Released oligosaccharides 25 wero analyzed by MALDI-TOP on a PerSeptive Biosystems Voyager-DR mass spectrometer. The mass speermmeter was operated at 20 kV either in the positive or negative mode with the linear configuration and utililing 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 uslng a 19 point Savitsky-Golay algorithm before the masses 30 were assigned. Integration of the mass spectral data was achieved using Caesar 7.0 data analysis software package (SciBridgc Software). Natural killer (NK) cell andbody dependent cytoxicity assays. ADCC assays were performed as described in Example 9. NK 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. 35 Target cells were opsonized with the concentratloos of anybody indicated for 30 di prior to addition of NK cells. The Rituxan® antibody used was from Genentech (S. San Francisco, CA). Figure 12shows the results of a representative ADCC assay. The results show that underfucosylated anthodies mediate NK cell target cell killing more efficiently than do antibodies with a lill complement of fucose. The underfucosylated antibody, 2H7v.31, is 40 most efficient at mediating target cell killing. This antibody is effective at lower concentrations and is capable of mediating killing of a greater percentage of target celLs at higher concentrations than are the other antihodles. The activity of the antibodies is as follows: Lce 13-derived 2H7 v31>Lec 13 derived 2T17v16> Dp12 derived 2H7v31> Dpl2 derived 2H7v16 > or = to Rituxan. The protein and carbohydrate alterations are additive, Comparison of the carbohydrate found on native IgO from the Lee 13-produced and CHO 63 AMED SEET AGE 1223'RCVD AT 431205 4:38:33 PM [Eastem Daylight Time]' YR:USPTO-EFXRF-I25' DNIS:2730827' CSID:50 952 9881 'DURATION (mm-ss):0748 eKib~W : ut;W "r -- 'i - - I I &-rV V V I J JULMU 4 Attorney Docket No. P1990R3 5 produced IgG showed no appreciable differences In ite extent of galactosylation and hence the results Can be attributed solely to the presencefabriucce of fucose. Examule 12 Facose-deficlent 2H17 wuan~t antibodies with cahanced ADCC In vivo IC0 1iis example describes ADCC activity in vivo of The fcose-deicicnt humanized 2W1 variants including v. 16 and v.31 produced in Lccd 3 compared to Dnral fucu~ylutcd counterparts produced in DPI 2, in mice expressing human CDM( [FoR411] and humun C1320. Generattion' of hmCD2OTg' huCD]6Tg' m(7064 mnice Human CD20) transgenic mice were generated from human CD20 JIAC! DNA (Invitrogen, Carlsbad, 15 CA). Mice wcrc surccricd based on the FACS analysis of human CD20 expression. HuCD2() T,t mice were then crossed with huCDlI 6Tg'~mCD J.6" mice to generate huCD2OTShuCDl 6-gemCD 16* mice. I Wt'o treatment Ten t 100 Fig of each of the 2H7 variants or Rituxan@ Is adxndnistratcd to huCtV2(YrghbuCD I fiTgmCD1 6"" mice via Intraperitoneal injc-tions. Equal amount of isotypc-znatched 20 antibodies will be applied slinarly to the negative control group of animals. Momst? kyvnphcyks preparalion Mouse lymphocytes from whole blood, splccn, lymph ndes and bone marrow ame prcparcd according to standard protocol described in " Current Protocols in Immunology, edited by John Culigan, Ada Truisbeek, TDavid Margulies. LEthma Shevach anjd Wamn Sb-obcr, 1994". r) FA CS anallysis Half million cells wre washed and resuspended in 100 0. of FACS buffer, which is phosphate buffered saline with I1% BSA, containing 5 ji of staining or control antibody. All the stalling antibodies, Including ISCIype controls, are, obtained froin PharMingesn, San Diego, CA. Humn CD20 cxprcssion is assossod by staining with Rituxan@ along with FT'TC-conjugated anti-humian ig01 secondary antibody. 30 ( FACS a nalysis I s co ducted usi ng FACScan a nd CallI Quest (JReCton DIckinson linniu nocytoznetry Sybtcms, Sakn loae, CA). All the lymnphooYteD arm defined in the forward an~d aide light ocatterlogo, while all the BD lymphocytes ame defined with the expression of lB220 on the cell surface. B cell depletion and recovery are assessed by analyzing peripheral H cell counts and analysis of hCD20+ R cells by FAC-S in the spleen, lymph nodc and bm~e marrow on a daily basis for the first week ,15 after illjection and thereafter on a weekly basis. Serum levels of the injected 2157 variatkt antibody are monitored. noe results of this In vivo assay confirms the In v~tr findings on the increased ADCC activity and grcaer B cell depletion of fucose-daficient 211.7 vajviants over wild-type (with resepol to fucosylation) Slycmylatiin counterparts. 40 64 AGE 131232 RCVDAT4I2l2D54:38:33 PM Pastem Dayight Time] t MVR.USM DEXFI DNIS:27308271 C810:650 952 988 11 DURATION jrnss);D7.48 IU~VLJ I JUL LUU4 Attorney Docket No, P1990R3 5 E'ape13 Apoptosis Activity Anti-CD20 antibodies including Rituxan@ have been shown to induce upoptosis in vitro when crosslinked by a xccondury antibody or by chemical means (Shan at al., Blood 9:1644-1652 (1998): Byrd ct 10 al.. Blood 99:1038-43 (2002); Pederson at al., Blood 99:1314-19 (2002)). When chemically crosslinked, murine 2117 dimers Induced apoptosis of Daudi cells (Ghctie et al., Proc Notn Acad Sci USA 94:7509-14 (1997)). Crosslinking with a secondary antibody also induced apoptosis with the urine 2H7 antibody (Shan ct al., 1998). Thesc activities are believed to be physiologically relevant because a variety of mechanisms could lead to crossliiking of anti-CD20 antibodies bound to cell-surfacc CD20 in viva. 15 RhuMAb 2H7.v16 humanized 2H7 v16; RhuMAb stands for recombinant human monoclonal antibody and Rituxan® were compared in apoptosis assays In vir using a secondary crosslinking antibody. Ramos cells (CRL-1596, ATCC, Manassas. VA), a CD20-expressing. human 13 lymphocyte cc11 line, wert used to measure the ability of tie anti-CD20 monoclonal antibodies rhuMAb 2)17.v16 and Rituxiinab versus a negative-control antibody, Trastuzumab (Herceptin@, G3enentech, South San Francisco, CA.), to Induce 20 apoptoslis as measured through Annexin V staining and propidium iodide dye exclusion (Vybract@ Apoptosis Assay Kit, Molecular Probes, Scattle, WA). The Ramos cells were cultured in RPM]- 640 medium (flibco, Rockville, MD) containing 10% fetal bovine scrum (Biosource Intemational, Camarillo, CA) and 2 meM L-glutamine (GIbco), Prior to being assayed, the cells were washed twice in fresh media and then adjusted to a cell concentration of 2 X 106 per mL. Cells (150 L) were added to 96-well assay plates 25 (Becton Dickinson, Palo Alto, CA) which contained 150 pL of a predetermined amount of control IG I, rhuMAb 2H7.v 16, or RJtuXImab, 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 human Fc antibody concentration was set at twice the respective sample antibody concentration. Bach dilution was sct up in triplicate. After a 24-hour incubation at 370 C, the cells were washed twice with PBS 30 and then stained with Annexin V and pmpidium iodide according to the manufacturer's recommendations. le staining patterns of the Rarnos cells were analyzed by flow cytoinetry using a FACscan Flow Cytometer (lcton Dickinson, San Jose, CA), and data were collected for 10 s-periods. T-he data were reduced using the Cellquest Pro software (Becton Dickinson). Ramos cells that were positive for (1> Annexin V staining, (2) Annexin V and propiduim iodide doublc-staining. and (3) the number of unstained live cells, were 35 counted and plotted using KaleidaGraph software (Synergy Software, Reading, PA). Both rhuMAb 2H7.Y16 and Rituxmab induced apoptosis of Ramos cells when crosslinked with anti-human Fc and as compared to an irrelevant IgGi control antibody (Figures 13-15). The apoptotic activity of (rhuMAb 2W47) was slightly lower than that of Rituximab. At 10 nM concentrations of crosslinked rhuMAb 2147, Rituximab, and control IgGI antibody, fractions of Annexin V stained cells were 40 18.5, 16.5, 2.5%, respectively, fractions of doubly labeled cells were 29, 3?, and 16%, and numbers of live cells counted per 10 s were 5200, 3100, and 8600. These in vitro data demonstrate that apoptosis is one potential mechanism for In vivo B cell depletion. In viva crosslinking of rhuMAb 2H7 or Rituximab bound to cell-surface CD20 may occur through Fc'yR on the surfaces of immune effector cells. 65 AME E EET E 14123'RCVD AT4126120054:38:33 PM rasem Daylight Time] BVR:UBPTO.EFXRF-1125' DNIS:273082P1 CSID:650 952 9881 'DURATION (mmss):0748 PR-26-2005 12:066 -KUrl: WENN trtU-l LM OD IPENUS 13 JUL 2004 Attorney Dncker No. P1 990R3 ExamPle 14 In Vivo Suppressionx of Tumor Growth The ability of rhuMAb 2H7.v 16 to inhibit the growth of the Raji human R-cells, a lymphoma cell line (ATCC CCL 86), was evaluated in Balb/c nude (athymic) mice. The Raji cells express CD20 and have. becn reported to grow in nude mice, producing metastatic disease; tumor growth is inhibited by Rhuxan@ 10 (Clynes et al.. Nature Medicine 6, 443-446 (2000)), Fifty-six S-10 week old, Balb/c nude muic were divided into 7 groups (A-G) with each group consisting of 8 micec. On day 0, each mouse reccivcd a subcutaneous Injection of 5 x 104I Raj 13-lymphoma cells in the flank. Beginning at day 0, each mouse received either 100 ul, of the negative-control solution (PBS; phosphatl-buffered saline), Rituxao@ or 2H7.v16. Dosagc was dependent on weight and drug delivery was intravenously via the tail vein, Group A mice reccivcd PBS. 15 Groups B-D received Rituxan@ at 5.0, mg/kg, 0.-5 mg/kg, and 0,05 nig/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. Thc injections were repented every week for 6 weeks. At weekly intervals during treatment, each mouse was inspected for the presence of palpable tumoms at the site of injection, and the volume of the tumors if present wer measured and recorded. A final inspection was made at week 8 (after a two-week interval of no treatments). 20 The results of this study showed that both rhuMAb 2H7.vI 6 and Rituxau@ and were effective at inhibiting subcutaneous Raji-cell tumor growth in nude mice (FIGs. 16-18). Tumor growth was observed in the PBS control gmup beginning at 4 weeks. However, no tumor growth was observed in groups treated with R ituxanO or 2H7.v16 at 5 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 21 RituxanOD group (FIG. 18). ExamPle ,15 Cloning of Cynomnolgus monkey CD20 and antibody binding The CD20 DNA sequence for cynomolgus monkey (Macaca fascicularis) was determined upon the 30 Isolation of cDNA encoding CD20 from a cynomiolgus spleen cDNA library. A SUPERSCRIPTrM 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 pRKSE vector using restriction sites Xhu I-and Not 1. mRNA was isolated from spleen tissue ((California Regional Research Primate Center, Davis, CA). Primers to amplify cDNA encoding CD20 wcre designed based on non-coding 35 sequences of human CD20, N-termloal region primer 5'-AGTT1TGAGAGCAAAATG-3' (SEQ ID NO. 37) and C-rcrninal region primer 5'-AAGCTrATCAACACTAATG-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 Polymneraso High Fidelity according to the manufacturers mcconmendation (Gibco. Rockville. MD), 'Ibe PCR product was subcloned into pCR 02. 1 -TOPO 0 Vector 40 (Invitrogen) and transformed into XL- I bluc E, coli (Siratagene. La Jolla. CA). Plasmid DNA containing ligated PC products was isolated from individual clones and sequenced. The amino acid sequcnec for cynomolgus monkey CD20 Is shown In Figure 19. Figure 20 shows a comparison of cynomnIgus and human CD20. The cynomolgus monkey CD20 is 97.3% similar to human 66 AMENDED SHET GE 15123*RCVD AT 412612005 4:38:33 FM EasternDaylghtTImej SVR:USPT-EFXRF-125' DNIS:2130821'CSID:650 952 9881 'DURATON (m-ss):0748 PR-26-2005 12: 07 FROM: GiENE I tui LtIHL tou I = 1 J L 0 I PEMS 3 JUL 2004 Attorney Docket No. P1990R3 5 CD20 with 8 difFerences. The extracellular domain contains one change at VI 57A, while the remaining 7 rusiduas can be found in the cytoplasmic or transmembrane regions. Antibodies directed against human CD20 were assayed for the ability to bind and displace FITC conjugated murine 2H7 binding to cynumolgus monkey cells expressing CD20. Twenty milliliters of blood were drawn from 2 cynomolgus monkeys (Califbrnia Regional Research Primate Center, Davis, CA) into 10 sodium heparin and shipped directly to Crenentech Inc., On the same day, the blond 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 ImPlus (Amersham Bioscicnces, Uppsala, Sweden) In 50 nil conical tubes (Cat#35209 8 . Falcon, Franklin Lakes, NJ) and centrifuged at 1300 rpm for 30 minutes R.T. in a Sorva 7 centrifuge. (Dupont, Newtowo, CT). The PBMC layer was isolated and washed in PBS. Red blood cells 15 were lysed in a 0.2% NaCl solution, restored to isotunicity with an equivalent volume of a 1.6% NaC solution, and centrifuged for 10 minutes at 1000 RPM. The PBMC pellet was resuspended in RPMI 1640 (Glbco, Rockville, MD) containing 5% fetal bovine serum (FBS) and dispensed into a 10 cm tissue culture dish for 1 hour at 370 C. The non-adlherent B and T cell populations were removed by aspiration, centrifuged and counted. A total of 2.4 x 10' cells were recovered. The resuspended PBMC were distributed 20 into twenty 12 x 75 mm culture tubes (Cat#352053, Falcon), with each tube containing 1 x I0 cells in a volume of 0.25 ml. Tubes were divided into fbur sets of five tubes. To each set was added either media (RPM11640, 5% FBS), thrated amounts of control human IgG2 antibody, Rituxan", 2H7.v 16, or2H7.v31. The final concentration of each antibody was 30, 10, 3.3 and 1.1 nM. Tn addition, each tube also received 20 ul of Fluoresceln lsothiocyanate (FTTC)-conjugated anti-human CD20 (Cat#555622. BD Biosciences, San 25 Diego, CA). The cells were gently mixed, incubated for 1 hour on ice and then washed twice in cold PBS. Tc cell surface staining was analyzed on a Epic XIrMCL (Coulter, Miami, FL), the geometric means derived, plotted (KaleidaGrAph, Synergy Software,. Reading, PA) versus antibody concentrations. Data in Figure 21 showed that 2H7 v.16 and 2H7 v.31 competitively displaced FIIC-murine 2117 binding to cynomolgus monkey cells. Furthermore, RituxanO also displaced FITC-murine 2H7 balding thus 30 demonstrating that both 21H7 and RiLuxan* bind to en overlapping epirope on CD20. Tn addition, the data show that the TC, value for 2H7 v.1 6 , 2117 v.31 and Rituxan are similar and fall in the 4-6 nM range. Example 16 Phase I/1M study of rhuMAb 2117 (2H7.v6) in moderate to seven rheumatoid arthritis 35 Protocol Synopsis A randomized, placebo-controlled, multicenter, blinded phase 1/11 study of the safety of escalating doses of PRO70769 (rhuMAb 2H17) in subjects with moderate to severe rheumatoid arthritis receiving stable doses of concomitant methotrexate. 40 Objectives lie primary objective of this study is to evaluate the safety and tolerability of escalating Intravenous (IV) do4cs of PRO70769 (rhuMAb 2H7) in subjects with moderate to never rheumatoid arthritis (RA). 67 AMENDED SHEET AGE 16123'RCVD AT 42612OO54:38:33 PM [Eastem Daylght Time]' SVR:USPTOEXRF-1I25' DNIS:2730827' CSID:650 952 9881 ' DURATION (mms):0748 PR-26-2005 12:07 FRUM:(:itNltkN LtkHL OQ 01r W IrtvuC 1-3 JUL 2004 A attorney Docket No. Pl990R3 5 Study Design This is a randomized, placbocontrolled, multicenter, blinded Phase 1/11, Investigator- and subject-blinded study of the safety of escalating doses of PRO70769 in combination with MTX In subjects with moderate to sever RA. TIhe study consists of a dose escalation phase and a second phase with enrollment of a larger number of subjects. The Sponsor will remain unblended to treatment assignment. lo Subjects with moderate to severe RA who have failed one to five diseasc-mudifying antirheumatic drugs or biologics 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 mig weekly for at least 12 week prior to study entry and to be on a stable dose for at least 4 weeks before receiving their initial dose of study I 5 drug (PR070769 or placebo). Subjects may also receive stable doses of oral corticosterolds (up to 10 mg daily or prednisone equivalent) and stable doses of nonsteroidal anti-inflammatory drugs (NSAIDs). Subjects will receive two IV infusions of PRO70769 or placebo equivalent at the indicated dose on Days 1 and IS according to the following dose escalatJon plan (see Figure 22). Dosc cscalation will occur according to specific criteria and after review of safety data by an 20 internal safety data review committee and assessment of acute tnxicity 72 hours following the second infusion in the last subject trated 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, 2x5OO mg, and 2x1000 mg, IW the dose levels have been demonstrated to be tolerable during the dose escalation phasc. Approximately 205 subjects will be enrolled in the study. 25 B-ccll counts will be obtained and recorded. B-cell counts will be evaluated using flow cytormctry in a 48-weck follow-up period beyond the 6-moth efficacy evaluation. B-cell depletion will not be considered a dosc-limiting toxicity (DLC), but rather the expected pbarmacodyoatuic outcome of PRO70769 treatment. In an optional substudy, blood for serum and RNA analyses, as well as urine samples will be 0 obtained from subjects at various timcpoints. These samples may be used to identify biomarkers that may be predictive of response to PRO70769 treatment in subjects with moderate to severe RA. Outcome Measures The primary outcome measure for this study is the safety and tolerability of PR070769 in subjects with 35 moderate to severe RA. Study Treatment Cohorts of subjects will receive two IV Inflslons of PRO70769 or placebo equivalent at the indicated dose on Days I and 15 according to the following escalation plaM 40 . 10 rg PRO70769 or placebo equivalent: 4 subjects active drug. I control - 50 mg PRZ070769 or placebo equivalent 8 subjects active drug, 2 control 200 mg PR070769 or placebo equivalent: 8 subjects active drug, 2 control - 500 mg PR070769 or placebo equivalent: 8 subjects active drug, 2 control 1000 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2 control 45 68 AMENDED SHEET AGE 17123' RCVD AT 412612005 4:38:33PM [EasternDaylight Time]' SVR:USPT-EFXRF-I25' DNIS:2730827 CSID:650 952 9881 'DURATION (mm-ss):0748 IPEA/US 1 3 JUL 2004 Attorney Docket No. P1990R3 5 Emeacy The efficacy of PRO70769 will be measured by ACR responses. The percentage of subjects who achieve an ACR20, ACRI0, and ACR70 response will be summarized by treatment group and 95% confidence intervals will be generated for cach gmup. The components of these response and their change rrorm baseline will be summarized by treatment and visit. I0 ConcusPion 'Lh 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 2H7 antibodies of the invention hound to CD20 at affinities similar to the murine donor and 15 chimoric 2H7 antibodies and were effective at B cell killing in a primate, leading to B cell depletion. Certain variants showed enhanced ADCC over a chimeric anti-CD20 antibody currently used to treat NRL, favoring the use of lower doses of the therapeutic antibody in patients. Additional, whereas it way be necessary for a chinoric antibody that has marine FR residues to be administered at a dose effective to achicyc complete B ccl depletion to obviate an antibody response against it, the present humanized antibodies can be 20 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 humunotherapeutic agent in the treatment of CD20 positive cancers and autoimmune diseases; these antibodies am not expected L be immunogenic or will at least be less immunogcnic than fully marine or chimeric noti-CD20 antibodies 25 in human patients. References References cited within this application, including patents. published applications and other publications, are hereby incorporated by reference. Ie practice of the present invention will employ, unless otherwise indicated, conventional 30 techniques of molecular biology and tbe like, which are within the skill of the art. Such techniques noe explained fully in the literature. See e.g., Molecular Cloolna: A Laboratory Manual, (J, Sambrook et aL, Cold Spring Harbor Laboratory, Cold Spring Harbor. N.Y., 1989): Current Protocols in Molecular Biology (F. Ausubel et aL, eds., 1987 updated); Essential Molecular Biology (T. Brown cd., IRL Press 1991); Gene Expression Technology (loeddel ed., Academic Press 1991); Methods for Cnning and Analysis of 35 karytic Genes (A. Bothwell el aL eds., Bartlett Pub]. 1990); Gene Transfer and Expression (M. Krieglcr, Stockton Press J 990); Recombinant DNA Methodolpoy IT (R. Wu et aL cds., Academic Press 1995); PCR: &Praclical Approach (M. McPherson el at., IRL Press at Oxford University Press 1991); Q1iggnuclentide SVpycsj (M. Gait ed., 1984); Cell Culture for Blocheotis (R. Adams ed., Flsevier Science Publishers 1990); .Gcne Transfer Vcutors for Mammalian Cells (J. Miller & M. Calos eds., 1987): Mammallan Cell 40 Biotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard et al. eds., Humana Press 1990); Culture of Animal Cells. 2"d Ed. (R. Freshncy el al. eds., Alan R. Liss 1987); low Cytonietry and Sorting (M, Mclamed er al. cds., Wilcy-Liss 1990); the series Methods in Enzymnology (Academic Press, Inc.):Wirth M. and Hauser H. (1993); Immunochemistry in Practice, 3rd edition. A. Johnstone & R. Thorpe, Blackwell Science. Cambridge, MA, 1996; Techniques in lmmunovtochmistry, (G. Bullock & P. Pctrusz eds., 69 AMENDW ET 3E1B23'RCVDAT4/2620054:38:33PM[EastemDaylIghtTime]'SVR:USPTO-EFXRF.125'DNIS:2730827'CSID:6509529881 *DURATION (mm.ss):048 IPENS 13 J U L2004. Attorney Docket No. P) 990R3 5 Academic Pims 1982, 1983, 1985, 1989); Hlandbook of Experintal Inmunology, (D. Weir & C. Blackwell, eds.); Current Protocols in Immunojorzy (I. Cioligan at al. eds. 1991): Inunoassay (E. P. Diarnianis & 'I'J( Cbj-Istopoulos. eds.. Academic Press, Inc.. 1996); Goding (1986) Monoclimp- AntibadicN: Prineivlcs tind Preclice (2d cd) Acadcrmic Prcss, New York; Ed Harlow and David Lane, Antisies A jpbontiturv Manukti Cold Spring Harbor Laboratary, Cold Spring Harbnr, New York, 1988; Antibody 10 jZn.pi ccrnn, 2 "d edition (C. Borrebacck, ed., Oxford Univerqity Press, 1995); and ibe, series Annual Review or Immunology; thc scrics Advances in Tnimunology. E 19123' RCVD AT 412612005 4:38:33PM [Eastern Daylight Tie BVR:USPTO*EFXRF*11251 DNIS:2730821 CSID:650 952 9881 'DURATION (mm-ss):07.48

Claims (30)

1. An antigen binding fragment of a humanized antibody that binds human CD20, wherein the humanized antibody comprises the VH sequence of the amino acid sequence of SEQ ID NO. 8 and the VL sequence of the amino acid sequence of SEQ ID NO. 2.

2. An antigen binding fragment according to claim 1, wherein the humanized antibody comprises the heavy and light chain amino acid sequence of SEQ ID NO. 39 and SEQ ID NO. 40.

3. An antigen binding fragment according to any one of the preceding claims conjugated to a cytotoxic agent.

4. An antigen binding fragment according to claim 3, wherein the cytotoxic agent is a radioactive isotope or a toxin.

5. A composition comprising an antigen binding fragment according to any one of claims I to 4 and a pharmaceutically acceptable carrier.

6. An article of manufacture comprising a container and a composition contained therein, wherein the composition comprises an antigen binding fragment according to any one of claims 1 to 4.

7. An article of manufacture according to claim 6, further comprising a package insert indicating that the composition can be used to treat a CD20 positive cancer and/or an autoimmune disease.

8. An article of manufacture according to claim 7, wherein the autoimmune disease is multiple sclerosis.

9. An isolated nucleic acid that encodes an antigen binding fragment according to claims I to 4.

10. An expression vector comprising a nucleic acid according to claim 9.

11. A host cell comprising a nucleic acid according to claim 9 or an expression vector according to claim 10.

12. A host cell according to claim 11, which is a CHO cell.

13. A method of producing an antigen binding fragment of a humanized antibody, comprising the step of culturing a host cell that produces the antigen binding fragment according to claim 1 or 2 and recovering the antigen binding fragment produced by the cell. 6055556 1 (GHMatters) P76023.AU.1 KAROLA 72

14. An antigen binding fragment produced by a method comprising expressing a nucleic acid encoding an antigen binding fragment of an antibody comprising the VH and VL sequences of SEQ ID NO. 8 and SEQ ID NO. 2, respectively in a host cell, and recovering the antigen binding fragment produced by the host cell.

15. A method of depleting B cells in a mammal, comprising administering to the mammal a therapeutically effective amount of an antigen binding fragment according to any one of claims I to 4.

16. A method of depleting B cells in a mammal, comprising administering to the mammal a therapeutically effective amount of a humanized antibody that binds human CD20, wherein the humanized antibody comprises the VH sequence of the amino acid sequence of SEQ ID NO. 8 and the VL sequence of the amino acid sequence of SEQ ID NO. 2.

17. Use of an antigen binding fragment of any one of claims 1 to 4 in the manufacture of a medicament for depleting B cells in a mammal.

18. Use of a humanized antibody that binds human CD20 in the manufacture of a medicament for depleting B cells in a mammal, wherein the humanized antibody comprises the VH sequence of the amino acid sequence of SEQ ID NO. 8 and the VL sequence of the amino acid sequence of SEQ ID NO. 2.

19. A method or use according to any one of claims 15 to 18, wherein the mammal is a human.

20. A method or use according to any one of claims 15 to 18, wherein the peripheral B cells are depleted in the mammal.

21. A method or use according to any one of claims 15 to 18, wherein the peripheral B cells are partially depleted in the mammal.

22. A method of treating an autoimmune disease, comprising administering to a patient suffering from the autoimmune disease, a therapeutically effective amount of an antigen binding fragment according to any one of claims I to 4.

23. Use of an antigen binding fragment according to any one of claims 1 to 4 in the manufacture of a medicament for treating an autoimmune disease, comprising administering to a patient suffering from the autoimmune disease, a therapeutically effective amount of the antigen binding fragment.

24. A method or use according to claim 22 or 23, wherein the antigen binding fragment is administered by intravenous administration or by subcutaneous administration.

25. A method or use according to any one of claims 22 to 24, 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 6055556 1 (GHMatters) P76023.AU.1 KAROLA 73 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.

26. A method or use according to claim 25, wherein the autoimmune disease is multiple sclerosis.

27. A method or use according to claim 25, wherein the autoimmune disease is rheumatoid arthritis.

28. A method of treating a CD20 positive cancer, comprising the step of administering to a patient suffering from the cancer, a therapeutically effective amount of an antigen binding fragment according to any one of claims I to 4.

29. Use of an antigen binding fragment according to any one of claims I to 4 in the manufacture of a medicament for treating a CD20 positive cancer, comprising the step of administering to a patient suffering from the cancer, a therapeutically effective amount of the antigen binding fragment.

30. A liquid formulation comprising an antigen binding fragment according to any one of claims 1 to 4 at 20mg/mL, 10mM histidine sulfate at pH5.8, 60mg/ml sucrose and 0.2 mg/ml polysorbate 20. 6055556 1 (GHMatters) P76023.AU.1 KAROLA