NO20150245L - Immunoglobulin variants and uses thereof - Google Patents

Description

Field of the invention

The invention relates to anti-CD20 antibodies and their use in the treatment of B-cell-related diseases.

Background for the invention

Lymphocytes are one of several populations of white blood cells, they recognize and respond specifically to foreign antigens. The three main classes of lymphocytes are B lymphocytes (B cells), T lymphocytes (T cells) and natural killer (NK) cells. B lymphocytes are the cells responsible for antibody production and provide humoral immunity. B cells mature in the bone marrow and leave the marrow while expressing an antigen-binding antibody on their cell surface. When a naïve B cell first encounters the antigen for which its membrane-bound antibody is specific, the cell begins to divide rapidly and the new cells differentiate into memory B cells and effector cells called plasma cells. Memory B cells have a longer lifespan and continue to express membrane-bound antibody with the same specificity as the original parent cell. Plasma cells do not produce membrane-bound antibody, but instead produce a separable form of the antibody. Secreted antibodies are the most important effector molecules in humoral immunity.

The CD20 antigen (also called human B lymphocyte-restricted differentiation antigen, Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al, J. Biol. Chem. 264(19 ): 11282-11287 (1989), and Einfeld et al, EMBO J. 7(3):711-717 (1988)). The antigen was also expressed on more than 90% of B-cell non-Hodgkin's lymphomas (NHL) (Anderson et al, Blood 63(6): 1424-1433 (1984)), but is not found on hematopoietic stem cells, pro- B cells, normal plasma cells or other normal tissues (Tedder et al, J. Immunol. 135(2):973-979 (1985)). CD20 is thought to regulate an early step in the activation process for cell initiation and differentiation (Tedder et al, supra) and possibly acts as a calcium ion channel (Tedder et al, J. Cell. Biochem. 14D:195 (1990)).

Given the expression of CD20 in B-cell lymphomas, this antigen has been a useful therapeutic target for treating such lymphomas. In the United States, more than 300,000 people have B-cell NHL and more than 56,000 new cases are diagnosed each year. For example, the rituximab (Rituxan) antibody, which is a genetically engineered chimeric murine/human monoclonal antibody directed against human CD20 antigen (commercially available from Genentech, Inc., South San Francisco, California, US), is used in the treatment of patients with relapsed or refractory lower-grade or follicular, CD20-positive, B-cell non-Hodgkin's lymphoma. Rituximab is the antibody referred to as C2B8 in US Patent Document No. 5,736,137, granted April 7, 1998 (Anderson et al.) and in US Patent Document No. 5,776,456. In v/seed mechanism in wake studies have shown that Rituxan binds human complement and lyses lymphoid B-cell lines via complement-dependent cytotoxicity (CDC) (Reff et al, Blood 83(2):435-445 (1994)). In addition, it has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC). Preclinical studies in vivo have shown that Rituxan removes B cells from peripheral blood, lymph nodes and bone marrow in cynomolgus monkeys, probably via complement- and cell-mediated processes (Reff et al, Blood 83(2): 435-445 (1994)). Other anti-CD20 antibodies indicated for the treatment of NHL include the murine antibody zevalin conjugated to the radioisotope yttrium-90 (IDEC Pharmaceuticals, San Diego, (CA), Bexxar which is another fully murine antibody conjugated to 1 -131 (Corixa, WA).

A major limitation in the use of murine antibodies in human therapy is the human anti-mouse antibody (HAMA) response (see e.g. Miller, R.A. et al. Monoclonal antibody therapeutic trials in seven patients with T-cell lymphoma Blood, 62: 988-995, 1983; and Schroff, R.W., et al. Human anti-murine immunoglobulin response in patients receiving monoclonal antibody therapy Cancer Res., 45:879-885, 1985). Even chimeric molecules in which variable (V) domains of rodent antibodies are fused to human constant (C) regions are still able to elicit a significant immune response (HACA, human antichimeric antibody) (Neuberger et al. Nature (Lond) .) 314:268-270, 1985). A promising approach to avoid these limitations in the clinical application of monoclonal antibodies is to humanize the murine antibody or antibody from a non-human species (Jones et al, Nature (Lond), 321:522-525, 1986; Riechman et al , Nature (Lond), 332:323-327, 1988).

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

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 FR and

affinity maturation variants with changes in the transplanted CDRs. The substituted amino acids in the CDR or FR are not limited to those present in the donor or

the recipient antibody. In other embodiments, the anti-CD20 antibodies of the invention comprise additional changes in amino acid residues in the FC region that lead to improved effector function, including improved CDC and/or ADCC function and B cell killing (also referred to herein as B cell depletion). Other anti-CD20 antibodies of the invention include those having specific changes that improve stability. In a specific embodiment, the humanized 2H7 variants with increased stability are as described in Example 6 below. Fucose-deficient variants that have improved ADCC function in vivo are also provided. In one embodiment, the chimeric anti-CD20 antibody has murine V regions and human C region. One such specific chimeric anti-CD20 antibody is Rituxan (Rituximab, Genentech, Inc.).

In a preferred embodiment of all the antibody preparations and methods for use of this invention, the humanized CD20 antibody 2H7.vl6 which has the light chain and heavy chain amino acid sequence in respectively SEQ ID NO:21 and 22, 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 which 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 sequence of vl6, except at the positions for amino acid substitutions indicated herein. Unless otherwise indicated, the 2H7 variants will have the same L chain as that of vl6.

The invention provides a humanized antibody that binds human CD20, or an antigen-binding fragment thereof, wherein the antibody is effective in depleting primate B cells in vivo, wherein the H-chain variable region (VH) of the antibody comprises at least one CDR3 sequence according to SEQ ID NO: 12 from an anti-human CD20 antibody and substantially the human consensus framework (FR) residues in human heavy chain subgroup III (VHIH). 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 according to SEQ ID NO: 10 and the CDR2 sequence in SEQ ID NO: 11.1 another embodiment comprises the preceding antibody L-chain CDR1 sequence in SEQ ID NO: 4, The CDR2 sequence of SEQ ID NO: 5, the CDR3 sequence of SEQ ID NO: 6 with substantially the human consensus framework (FR) residues of human light chain K subgroup I (VkI). In a preferred embodiment, the FR region of the VLen has the donor antibody residue at position 46, in a specific embodiment FR2 of the VLen has an amino acid substitution of leuL46pro (Leu in the human K1 consensus sequence changed to pro which is present at 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, the following FR positions in the human heavy chain subgroup III in VH are substituted: AlaH49Gly in FR2, ArgH71Val and AsnH73Lys in FR3.1 other embodiments comprise (the CDR regions of the humanized antibody further amino acid substitutions where the residues are neither from donor nor recipient antibody.

The antibody in the preceding embodiments may comprise the VH sequence in SEQ ID NO: 8 for v16, as shown in fig. IB. In a further embodiment of the foregoing, the antibody further comprises the VL sequence in SEQ ID NO: 2 in vl6, as shown in fig. IA.

In other embodiments, the humanized antibody is 2H7.v31 which has the light chain and heavy chain amino acid sequence in, respectively. SEQ ID NO: 21 and 23, 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 amino acid substitutions of D65A and N100A in the H chain and S92A in the L chain of vl6.

In separate embodiments, the antibody of any of the preceding embodiments further comprises at least one amino acid substitution in the Fc region that enhances ADCC and/or CDC activity relative to the original or parent antibody from which it is derived, wherein v. 16 is parent -the antibody to which it is compared in most cases, and Rituxan in other cases. One such antibody with improved activity comprises the triple Alanine substitution with S298A/E333A/K334A in the Fc region. One antibody having the S298A/E333A/K334A substitution is 2H7.v31 having the heavy chain amino acid sequence of SEQ ID NO: 23. Antibodies 2H7.v 114 and 2H7.vll5 show at least 10-fold improved ADCC activity compared to Rituxan.

In another embodiment, the antibody further comprises at least one amino acid substitution in the Fc region that decreases CDC activity compared to the parent antibody from which it was derived, which is v16 in most cases. One such antibody with reduced CDC activity compared to vl6 comprises at least substitution K322A in the H chain. The comparison of ADCC and CDC activity can be analyzed as described in the examples.

In a preferred embodiment, the antibodies according to the invention are full-length antibodies in which the VH region is linked to a human IgG heavy chain constant region. In preferred embodiments, the IgG is human IgG1 or IgG3.

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

In one embodiment, the antibodies according to the invention for use for therapeutic or diagnostic purposes are produced in CHO cells.

Also provided is a preparation comprising an antibody according to any of the preceding embodiments and a carrier. In one embodiment, the carrier is a pharmaceutically acceptable carrier. These preparations may be provided in a manufactured article or kit.

The invention also provides a liquid formulation comprising a humanized 2H7 antibody at 20 mg/ml antibody, 10 mM histidine sulfate pH 5.8, 60 mg/ml sucrose (6%), 0.2 mg/ml polysorbate-20 (0, 02%).

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

Another aspect of the invention is host cells that comprise the preceding nucleic acids and host cells that produce the antibody. In a preferred embodiment of the previous one, the host cell is a CHO cell. A method for producing these antibodies is provided, the method comprising culturing the host cell that produces 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 preparation, wherein the preparation comprises an antibody according to any of the preceding embodiments. For use in the treatment of NHL, the manufactured article further includes written instructions indicating that the preparation is used to treat non-Hodgkin's lymphoma.

A further aspect of the invention is a method for inducing apoptosis in B cells in vivo, which comprises contacting B cells with the antibody according to any of the above embodiments, thereby killing the B cells.

The invention also provides methods of treating the diseases described herein by administering a CD20 antibody or functional fragment thereof to a mammal such as a human patient suffering from the disease. In any of the methods for treating an autoimmune disease or a CD20-positive cancer, the antibody is in one embodiment 2H7.vl6 having the light chain and heavy chain amino acid sequences of SEQ ID NO: 21 and 22, respectively, as shown in FIG. 6 and fig. 7. Thus, one embodiment is a method of treating a CD20-positive cancer, which comprises 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 for treating a B-cell lymphoma or leukemia, the antibody is administered in a dosage range of about 275-375 mg/m . In further embodiments, the method of treatment further comprises administering to the patient at least one chemotherapeutic agent, wherein the chemotherapeutic agent for non-Hodgkin's lymphoma is selected from the group consisting of doxorubicin, cyclophosphamide, vincristine and prednisolone.

Also provided is a method of treating an autoimmune disease, comprising administering to a patient suffering from the autoimmune disease a therapeutically effective amount of the humanized CD20 binding antibody according to any one of the preceding claims. The autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA neuropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjøgren's syndrome and glomerulonephritis. Where the autoimmune disease is rheumatoid arthritis, the antibody may be administered together with a second therapeutic agent which is preferably methotrexate.

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

In another aspect, the invention provides a method of treating an autoimmune disease selected from the group consisting of dermatomyositis, Wegener's granulomatosis, ANCA, aplastic anemia, autoimmune hemolytic anemia (AIHA), factor VIII deficiency, hemophilia-A, autoimmune neutropenia, Castleman's syndrome, Goodpasture's syndrome, organ transplant rejection, graft versus host disease (GVHD), IgM-mediated thrombotic thrombocytopenic purpura (TTP), Hashimoto's thyroiditis, autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-graft), vs. NSIP, Guillain-Barre syndrome, large vessel vasculitis, giant cell (Takayasus) arthritis, interstitial vasculitis, Kawasaki disease, polyarthritis nodosa, comprising administering to a patient suffering from said disease a therapeutically effective amount of a CD20-binding antibody. In one embodiment of this method, the CD20 binding antibody is rituxan.

The invention also provides an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO:24 of Cynomolgus monkey CD20 antigen (shown in Fig. 19), or a degenerate variant of this sequence. One embodiment is an isolated nucleic acid comprising a sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO:25 (shown in Fig. 20) or in SEQ ID NO:25 (Fig. 20) with conservative amino acid substitutions. Another embodiment is a vector comprising the above nucleic acid, including an expression vector for expression in a host cell. Also included is a host cell comprising a vector. Also provided is an isolated polypeptide comprising the amino acid sequence [SEQ ID NO: 25, fig. 20] to Cynomolgus monkey CD20.

Brief description of the figures

FIG. IA is a sequence comparison comparing the variable light chain (VL) domain amino acid sequences of each murine 2H7 (SEQ ID NO: 1), humanized 2H7.vl6 variant (SEQ ID NO: 2) and human kappa light chain subgroup I (SEQ ID NO: 3) . The CDRs in VLi 2H7 and hu2H7.vl6 are as follows: CDR1 (SEQ ID NO: 4), CDR2 (SEQ ID NO: 5) and CDR3 (SEQ ID NO: 6). FIG. IB is a sequence comparison comparing the VH sequences of murine 2H7 (SEQ ID NO: 7), humanized 2H7.vl6 variant (SEQ ID NO: 8) and the human heavy chain subgroup III consensus sequence (SEQ ID NO: 9). The CDRs in the VH of 2H7 and hu2H7.vl6 are as follows: CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11) and CDR3 (SEQ ID NO: 12).

In FIG. 1A and FIG. IB CDR1, CDR2 and CDR3 are present in each chain within parentheses, flanked by the framework regions FR1-FR4 as indicated. 2H7 refers to the murine 2H7 antibody. The asterisks between two rows of sequences indicate the positions that differ between the two sequences. Residue numbering is according to Kabat et al., Sequences of Immunological Interest. 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), with deposits shown as a, b, c, d and e. FIG. 2 shows the sequence of the phagemid pVX4 (SEQ ID NO: 13) used for the construction of 2H7-Fab plasmids (see example 1) in addition to the amino acid sequences of the L-chain (SEQ ID NO: 14) and the H-chain (SEQ ID NO: 15) for Fab for the CDR-grafted anti-IFN-α humanized antibody. FIG. 3 shows the sequence of the expression plasmid encoding the chimeric 2h7.v6.8 Fab (SEQ ID NO: 16). The amino acid sequence of the L chain (SEQ ID NO: 17) and the H chain (SEQ ID NO: 18) is shown. FIG. 4 shows the sequence of the plasmid pDRl (SEQ ID NO: 19, 5391 bp) for the expression of immunoglobulin light chains as described in example 1. pDRl contains sequences that code for an irrelevant antibody, the light chain for a humanized anti-CD3 antibody (Shalaby et al. , J. Exp. Med. 175: 217-225 (1992)), start and stop codons for these are indicated in bold and underlined. FIG. 5 shows the sequence of plasmid pDR2 (SEQ ID NO:20, 6135 bp) for the expression of immunoglobulin heavy chains as described in example 1. pDR2 contains sequences that code for an irrelevant antibody, the heavy chain of a humanized anti-CD3 antibody (Shalaby et al. , above), where start and stop codons for these are indicated in bold and underlined. FIG. 6 shows the amino acid sequence of the complete L chain of 2H7.vl6 (SEQ ID NO:21). The first 19 amino acids before DIQ are the secretory signal sequence that is not present in the mature polypeptide chain. FIG. 7 shows the amino acid sequence of the complete H chain of 2H7.vl6 (SEQ ID NO:22). The first 19 amino acids before EVQ are the secretory signal sequence that is not present in the mature polypeptide chain. By compiling the VH sequence in fig. IB (SEQ ID NO:8) with the complete H sequence is present the human yl constant region from amino acid position 114-471 in (SEQ ID NO:22). FIG. 8 shows the amino acid sequence of the complete H chain of 2H7.v31 (SEQ ID NO:23). The first 19 amino acids before EVQ are the secretory signal sequence that is not present in the mature polypeptide chain. The L-chain is the same as for 2H7.vl6 (see Fig. 6). FIG. 9 shows the relative stability of the IgG variants 2H7.vl6 and 2H7.v73 as described in Example 6. Analysis results were normalized to the values before incubation and reported as percent remaining after incubation. FIG. 10 is a sequence map summarizing the amino acid changes from the murine 2H7 to a subset of humanized versions up to v75. FIG. 11 is a summary of mean absolute B-cell count [CD3-/CD40+] in all groups (2H7 study and Rituxan study combined) as described in Example 10. FIG. 12 shows the results from a representative ADCC analysis on fucose-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. Ramos cells were treated with an irrelevant IgG1 control antibody (Herceptin, circles), Rituximab (squares) or rhuMAb 2H7.vl6 (triangles) in the presence of a cross-linking secondary antibody and analyzed by FACS.

Figures 13-15 are described in example 13.

FIG. 14 shows the results of annexin-V and propidium iodide double staining, plotted as a function of antibody concentration. Ramos cells were treated with an irrelevant IgG1 control antibody (Herceptin, circles), Rituximab (squares) or rhuMAb 2H7.vl6 (triangles) in the presence of a cross-linking secondary antibody and were analyzed by

FAX.

FIG. 15 shows the counts (per 10 s) of live unstained cells plotted as a function of antibody concentration. Ramos cells were treated with an irrelevant IgG1 control antibody (Herceptin, circles), Rituximab (squares) or rhuMAb 2H7.vl6 (triangles) in the presence of a cross-linking secondary antibody and were analyzed by

FAX.

FIG. 16, 17, 18 show inhibition of Raji cell tumor growth in nude mice as described in Example 14. They 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.vl6 at 5 mg/kg (Fig. 16), 0.5 mg/kg (Fig. 17) or 0.05 mg/kg (Fig. 18). FIG. 19 shows the nucleotide sequence (SEQ ID NO:24) and the amino acid sequence (SEQ ID NO:25) of cynomolgus monkey CD20 as described in example 15. FIG. 20 shows the amino acid sequence of cynomolgus monkey CD20 (SEQ ID NO:25). Residues different from human CD20 are underlined, and the human residues (SEQ ID NO:26) are indicated immediately below the monkey residue. The putative extracellular domain of monkey CD20 is in bold. FIG. 21 shows the results from cynomolgus monkey cells expressing CD20 and binding to hu2H7.vl6, .v31 and Rituxan, as described in Example 15. The antibodies were analyzed for the ability to bind and displace FITC-conjugated murine 2H7 binding to cynomolgus CD20. FIG. 22 shows a dose escalation scheme for rheumatoid arthritis phase I/II clinical trial.

FIG. 23 shows the vector for expression of 2H7.vl6 in CHO cells.

Detailed description of preferred embodiments

The CD20 antigen is a non-glycosylated transmembrane phosphoprotein with a molecular weight of approximately 35 kD that is found on the surface of more than 90% of B cells from peripheral blood or lymphoid organs. CD20 is expressed during early pre-B cell development and was expressed until plasma differentiation, it is not found on human stem cells, lymphoid progenitor cells or normal plasma cells. CD20 is present both on normal B cells as well as on malignant B cells. Other names for CD20 in the literature include B-lymphocyte (h)differentiation antigen and Bp35. The CD20 antigen is described, for example, in Clark and Ledbetter, Adv. Can. Res. 52-81-149 (1989) and Valentine et al. J. Biol. Chem.264(19):11282-11287(1989).

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

The biological activity of the CD20-binding and humanized CD20-binding antibodies according to the invention will at least include binding of the antibody to human CD20, more preferably binding to human and other primate CD20 (including cynomolgus monkeys, rhesus monkeys, chimpanzees). The antibodies will bind CD20 with a Kd value no greater than 1x10", preferably a Kd value no greater than about 1x10"<9>, and will be capable of killing or depleting B cells in vivo, preferred by at least 20% when compared to the appropriate negative control not treated with such antibody. B-cell depletion may result from one or more of ADCC, CDC, apoptosis, or other mechanisms. In some embodiments of disease treatment described herein, specific effector functions or mechanisms may be preferred over others, and certain variants of humanized 2H7 are preferred to achieve these biological functions, such as ADCC.

"Antibody fragments" include a portion of a full-length antibody, generally the antigen-binding region or the variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments, dialegmes, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain and a light chain variable region domain with tight, non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops each from the H and L chain) emerge which contribute the amino acid residues for antigen binding and which transfer antigen binding specificity to the antibody. Nevertheless, 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 this occurs with a lower affinity than for the entire binding site.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in smaller amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The term "monoclonal" denotes that the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by a particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may is produced v ed by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or be prepared by recombinant DNA methods (see, for example, U.S. patent document no. 4 816 567). The "monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352-624-628 (1991) and Marks et al., J. Mol. Biol. 222-581-597 (1991), for example.

"Functional fragments" of the CD20-binding antibodies of the invention are those fragments which maintain binding to CD20 with substantially the same activity as the intact full-length molecule from which they are derived and exhibit biological

activity including depletion of 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 are highly divergent in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. Nevertheless, the variability is not evenly distributed over the 110 amino acids of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FR) of 15-30 amino acids, interrupted by shorter regions of extreme variability called "hypervariable regions", each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely occupying a P-face configuration, linked by three hypervariable regions that form loops that bind together and, in some cases, form part of the P-face structure . The hypervariable regions of each chain are held together by FRenes and contribute together with the hypervariable regions from the other chain to the formation of the antigen-binding site of antibodies (see Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region", when used herein, refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (at approximately residues 24 - 34 (LI), 50 - 56 (L2) and 89 - 97 (L3) of VL, and at approximately 31 - 35B ( Hl), 50 - 65 (H2) and 95 - 102 (H3) in VH (Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991) ) and/or the residues from a hypervariable loop (for example residues 26 - 32 (LI), 50 - 52 (L2) and 91 - 96 (L3) in VLog 26 - 32 (Hl), 52A - 55 (H2) and 96 -101 (H3) in VH (Chothia and Lesk J. Mol. Biol, 196 : 901 - 917

(1987)).

As referred to herein, the "consensus sequence" or consensus V domain sequence is an artificial sequence derived from a comparison of the amino acid sequences of known human immunoglobulin variant homology sequences. Based on these comparisons, recombinant nucleic acid sequences encoding V domain amino acids that are a consensus of the sequences derived from the human k and the human H chain subgroup III-V domain were prepared. The consensus V sequence has no known antibody binding specificity or affinity.

"Chimeric" antibodies (immunoglobulins) have part of the heavy chain and/or light chain identical to or homologous to corresponding sequences in antibodies that are

derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species, or belonging to another antibody class or subclass, in addition to fragments of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al., Proe. Nat. Acad. Sei. USA 81:6851-6855 (1984)). Humanized antibodies used here are a subset of chimeric antibodies.

"Humanized" forms of non-human (eg, murine) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues in the recipient are replaced with hypervariable region residues from a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate that has the desired specificity, affinity and capacity. In some cases, Fv framework region (FR) residues in the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in the recipient antibody or in the donor antibody. These modifications are performed to further refine antibody performance such as binding affinity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, where all or substantially all of the hypervariable loops correspond to those in a non-human immunoglobulin and all or substantially all of the FR regions are those from a human immunoglobulin sequence, even whether the FR regions may include one or more amino acid substitutions that improve bmcmig affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody will possibly also comprise at least one part of an immunoglobulin constant region (Fc), typically that from a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986), Reichmann et al., Nature 332:323-329 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

Antibody "effector functions" refer to the biological activities that can be linked to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody and vary by antibody subtype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (eg B-cell receptor) and B-cell activation.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted IgG binds to Fc receptors (FcRs) present on certain cytotoxic cells (eg, natural killer (NK) cells, neutrophils and macrophages) and which these cytotoxic effector cells able to bind specifically to

an antigen-bearing target cell and then kill the target cell with cytotoxins. The "arm" of the antibodies and the cytotoxic cells are absolutely necessary for such killing. The primary cells to mediate ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Fox. Immunol 9:457 - 92

(1991). To examine ADCC activity for a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. patent no. 5 500 362 or 5 821 337 be carried out. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be examined in vivo, for example in an animal model, such as that disclosed in Clynes et al. PNAS (USA) 95:652 - 656 (1998). "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) on its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See review in Daéron, Annu. Rev. Immunol 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Fox. Immunol 9:457-92 (1991), Capel et al. Immunomethods 4:25 - 34

(1994), and de Haas et al., J. Lab. Clin. With. 126:330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to fetuses (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)) .

WO 00/42072 (Presta) describes antibody variants with improved or reduced binding to FcRer. The contents of this patent publication are specifically incorporated herein by reference. See also Shields et al. J. Biol. Chem. 9(2): 6591 - 6604 (2001). "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least Fcylll and perform ADCC function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils with PBMC and NK cells being preferred. The effector cells can be isolated from a native source, for example from blood. "Complement dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) bound to their associated antigen. To examine complement activation, a CDC assay, for example as described in Gazzano-Santoro et al., J. Immunol Methods 202:163 (1996), can be performed.

Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding ability are described in U.S. Pat. patent document no. 6 194 551B1 and WO 99/51642. The contents of these patent publications are specifically incorporated herein by reference. See also Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

The N-glycosylation site in IgG is on Asn297 in the CH2 domain. The present invention also provides preparations of a CD20-binding, humanized antibody having an Fc region in which approximately 80-100% (and preferably approximately 90-99%) of the antibody in the preparation comprises a mature chemical carbohydrate structure that lacks fucose, bound to the Fc- region of the glycoprotein. Such preparations were shown here to exhibit a surprising improvement in binding to Fc^RIIIA(F158) which is not as efficient as Fc^RIIIA(V158) in reacting with human IgG. Thus, the preparations here are expected to be superior to previously described anti-CD20 antibody preparations, especially for therapy of human patients expressing Fc^RIIIA(F158). Fc(RIIIA (F158) is more common than Fc(RIIIA (V158) in normal, healthy African Americans and in the white population. See Lehrnbecher et al. Blood 94:4220 (1999). The present application further demonstrates the synergistic increase in Fc^RIII -binding and/or ADCC function resulting from combining the glycosylation variations herein with amino acid sequence modifications in the Fc region of the glycoprotein.

An "isolated" antibody is one that has been identified and separated and/or recovered from a component found in its natural environment. Contaminating components in its natural environment are materials that would interfere with diagnostic or therapeutic applications of the antibody, and may include enzymes, hormones, and other proteinaceous or ildce proteinaceous dissolved components. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight relative to antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to to obtain at least 15 residues of N-terminal or internal amino acid sequence using a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using coomassie blue or, preferably, silver staining. Isolated antibody includes the antibody in situ in recombinant cells since at least one component of the antibody's natural environment will not be present. Nevertheless, usually isolated antibody will be prepared by means of at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminating nucleic acid molecule with which it is usually associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or setting in which it is found in nature. Isolated nucleic acid molecules are therefore different from the nucleic acid molecule that exists in natural cells. Nevertheless, an isolated nucleic acid molecule includes a nucleic acid molecule present in cells that normally express the antibody where, for example, the nucleic acid molecule is present at a chromosomal location different from that in natural cells.

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

A nucleic acid is "operably linked" when it is placed in a functionally dependent relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. In general, "operably linked" means that the DNA sequences that are linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Binding is performed by ligation at appropriate restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice. "Vector" includes shuttle and expression vectors. Typically, the plasmid construction will also include an initiation site for replication (for example the ColEl initiation site for replication) and a selectable marker (for example ampicillin or tetracycline resistance), for respectively replication and selection of the plasmids in bacteria. An "expression vector" refers to a vector containing the necessary control sequences or regulatory elements for expression of the antibodies including antibody fragment according to the invention in bacterial cells or eukaryotic cells. Appropriate vectors are indicated below.

The cell that produces a humanized CD20-binding antibody according to the invention will include the bacterial and eukaryotic host cells into which nucleic acids encoding the antibodies have been introduced. Suitable host cells are indicated below.

The word "label", as used herein, refers to a detectable compound or preparation conjugated directly or indirectly to the antibody. The label may itself be detectable in itself (eg radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical change of a substrate compound or preparation that is detectable.

An "autoimmune disease" here is a non-malignant disease or disorder arising from and directed against an individual's own (self) 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, compared to the B cell level before treatment. B-cell levels are measurable using well-known assays such as those described in the Experimental Examples. B-cell depletion can be complete or partial. In one embodiment, the depletion of CD20-expressing B cells is at least 25%. Without being limited by a single mechanism, possible mechanisms of B cell depletion include ADCC, CDC, apoptosis, calcium current modeling, or a combination of two or more of the foregoing.

The term "cytotoxic agent", as used herein, refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (for example I131,1<12>5,Y9<0>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. Examples of chemotherapeutic agents include alkalizing or alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN), alkylsulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benzodopa, carboquone, meturedopa and uredopa, ethylenemines and methylmelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine, nitrogen mustards such as chlorambucil, chlornafazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquine, fenesterin, prednimustine, trophosphamide, uracil mustard, nitrosoureas such as carmustine, chlorzotosin, fotemustine, lomustine, nimustine, ranimustine, antibiotics such as aclasinomycins , actinomycin, autramycin, azaserin, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carcinophilin, chrommycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (adriamycin), epirubicin, esorubicin, idarubicin, m arcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, anti-metabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogues such such as denopterin, methotrexate, pteropterin, trimetrexate, purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine, pyrrinidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, (udeoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU, androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostan, testolactone, antiadrenals such as aminoglutethimide, mitotane, trilostane, folic acid substitutes such as folic acid, aceglatone, aldophosphamide glycoside, ammolevulinic acid, amsacrine, bestrabusil, bisantren, edatraksate, defofamine, demecolcin, diaziquone, elfornitine , elliptinium acetate, etogluside, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, fenamet, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK, razoxane, sizofiran, spirogermanium, tenuazonic acid, triazicon, 2,2<\>2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), thiotepa, taxoids such as paclitaxel (TAXOL, Bristol-Myers Squibb Oncology, Princeton, NJ) and doxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogues such as cisplatin and carboplatin, platinum, etopside (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinblastine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS 2000, (kfluromethylormtin (DMFO), retinoic acid, esperamycins, capecitabine and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone production on tumors such as antiestrogens including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston ), antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, other chemotherapeutic agents such as prednisolone. Pharmaceutically acceptable salts, acids or derivatives of any of the above are included. "Treat" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventive measures, where the aim is to prevent or slow down (reduce) the current, pathological condition or disorders. A subject is successfully "treated" for a CD20-positive cancer or an autoimmune disease if, after receiving a therapeutic amount of a CD20-binding antibody of the invention according to the method of the present invention, the subject shows observable and/or measurable reduction in or absence of one or more signs and symptoms of the particular disease. For example, for cancer, reduction in the number of cancer cells or absence of the cancer cells, reduction in tumor size, inhibition (ie to some extent slowing down the extent and preferably stopping) of tumor metastases, inhibition, to some extent, of tumor growth, increase in the length of remission and /or relief to some degree, one or more of the symptoms associated with the specific form, reduced morbidity and mortality and improvement in quality of life. Reduction of the signs or symptoms of a disease may also be felt by the patient. Treatment can achieve a complete response, defined as the disappearance of all signs of cancer, or a partial response, in which the size of the tumor is reduced, preferably by more than 50%, more preferably by 75%. A patient is also considered to be treated if the patient experiences a stable disease. In a preferred embodiment, the cancer patients are still progression-free in relation to the cancer after one year, preferably after 15 months. These parameters for assessing successful treatment and improvement in the disease are easily measurable using routine procedures known to a physician suitably skilled in the art.

A "therapeutically effective amount" refers to an amount of an antibody or drug that is effective in "treating" a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells, reduce tumor size, inhibit (ie slow to some extent and preferably stop) cancer cell infiltration in peripheral organs, inhibit (ie slow to some extent and preferably stop) tumor metastasis, inhibit , to some extent, tumor growth and/or alleviate to some extent one or more of the symptoms associated with the cancer. See previous definition of "treatment". "Chronic" administration refers to the administration of the agent(s) in a continuous manner as opposed to an acute manner, to maintain the original therapeutic effect (activity) for an extended period of time. "Interrupted" administration is treatment that is not done consecutively without interruption, but rather is cyclical in nature.

Preparations and methods according to the invention

The invention provides humanized antibodies that bind human CD20 and preferably other primate CD20 in addition, comprising an H chain having at least one, preferably two or all of the H chain CDRs of an antihuman CD20 antibody from a non-human species ( 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 often it will be a murine antibody. "Substantially entire" in this context means that the recipient FR regions of the humanized antibody may include one or more amino acid substitutions not originally present in the human consensus FR sequence. These FR changes may include residues that are not found in the recipient or in the donor antibody.

In one embodiment, the donor antibody is the murine 2H7 antibody, the V region including the CDR and FR sequences of each of the H and L chains shown in Figures IA and IB. In a specific embodiment, the residues of the human Fab framework correspond to the consensus sequence of human VK subgroup I and VH subgroup III, where these consensus sequences are shown in Figure IA and Figure IB, respectively. The humanized 2H7 antibody according to the invention will have at least one of the CDRs in the H chain of the murine donor antibody. In one embodiment, the humanized 2H7 antibody that binds human CD20 comprises the CDRs in both the H and L chains of the donor antibody.

In a full-length antibody, the humanized CD20-binding antibody according to the invention will comprise a humanized V-domain linked to a C-domain from a human immunoglobulin. In a preferred embodiment, the H chain C region is from human IgG, preferably IgG1 or IgG3. The L chain C domain is preferably from human K chain.

Unless otherwise indicated, a humanized 2H7 antibody version herein will have the V and C domain sequences of 2H7.vl6 L chain (Figure 6, SEQ ID NO: 21) and -H chain (Figure 7, SEQ ID NO : 22) except at the positions of amino acid substitutions or changes indicated in the experimental examples below.

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

The biological activity of the CD20-binding antibodies and humanized CD20-binding antibodies according to the invention will at least include binding of the antibody to human CD20, more preferably binding to human CD20 and primate CD20 (including cynomolgus monkeys, rhesus monkeys, chimpanzees) with a Kj value which is no higher than 1 x 10" , preferably a Kd value no higher than about 1 x IO"<9>, even more preferably a Kd value no higher than about 1 x IO"<10>, and be capable of killing or depleting B cells in vitro or in vivo, preferably by at least 20% compared to baseline levels or appropriate negative control not treated with such antibody.

The desired level of B-cell depletion will depend on the disease. For the treatment of a CD20-positive form of cancer, it will be desirable to maximize the depletion of the B cells which are the target of the anti-CD20 antibodies according to the invention. For the treatment of a CD20-positive B-cell neoplasm, it is therefore desirable that the B-cell depletion is sufficient to at least prevent progression of the disease which can be assessed by the experienced doctor in the field, for example by monitoring tumor growth (size), proliferation of the cancerous cell type, metastasis, other signs and symptoms of the particular type of 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 1 year, more preferably 2 years, more preferably 3 years, even more preferably 5 or more years. In a most preferred embodiment, the B-cell depletion is sufficient 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 pre-treatment baseline level.

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

The invention also provides bispecific CD20-binding antibodies where one arm of the antibody has a humanized H and L chain in the humanized CD20-binding antibody according to the invention, and the other arm has V region binding specificity for another antigen. In specific embodiments, the second antigen is selected from the group consisting of CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.

Compared to Rituxan (rituximab), vl6 exhibits approximately 2 to 5 times increased ADCC potency, approximately 3 to 4 times decreased CDC than Rituxan.

Antibody production

Monoclonal antibodies

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

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

The hybridoma cells that are produced in this way are seeded and grown in a suitable culture medium, where this medium preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells (also referred to as fusion partner). For example, if the parent myeloma cells lack the enzyme hypoxanthine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for the hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium) where these substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parent cells. Preferred myeloma cell lines are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA and SP-2 and derivatives, such as X63-Ag8 -653 cells available from the American Type Culture Collection, Rockville, Maryland USA. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol, 133:3001 (1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are grown is examined for the production of monoclonal antibodies directly against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by in v/fra binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The Bmcmi<g>affinity of the monoclonal antibody can be determined, for example, by the Scatchard assay described in Munson et al., Anal. Biochem., 107:220 (1980).

Once hybridoma cells producing antibodies with the desired specificity, affinity and/or activity are identified, the clones can be subcloned using limiting dilution procedures and cultured using standard procedures (Goding, Monoclonal Antibodies, Principles and Practice, pp.59 - 103 (Academic Press, 1986)). Suitable culture medium for this purpose includes, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells can be grown in vivo as ascites tumors in an animal, for example by i.p. injection of the cells into mice.

The monoclonal antibodies that are secreted by the subclone are suitably separated from the culture medium, the ascites fluid or the serum by means of conventional antibody purification procedures such as, for example, affinity chromatography (for example by using protein A or protein G Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis , dialysis etc.

DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes capable of binding specifically to genes encoding heavy chains and light chains of murine antibodies). The hybridoma cells serve as a preferred source for such DNA. Once isolated, the DNA can be placed into expression vectors that are then transfected into host cells, such as in E. cø// cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to achieve the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al, Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), Clackson et al, Nature, 352:624- 628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describes respectively the isolation of murine and human antibodies by using subject libraries. Later publications describe the production of low-affinity (nM range) human antibodies by chain transfer (Marks et al, Bio/Technology, 10:779-783 (1992)), in addition to combinatorial infection and in vivo recombination as a strategy for constructing very large subject libraries (Waterhouse et al, Nuc. Acids. Res., 21:2265-2266

(1993)). Thus, these techniques are good alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

DNA encoding antibodies can be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CL) sequences with the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison et al, Proe .Nati.Acad.Sei.USA, 81:6851

(1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for an iMce immunoglobulin polypeptide (heterologous polypeptide). The Ildce immunoglobulin polypeptide sequences may be substituted with constant domains of an antibody, or they may be substituted with variable domains of an antigen-binding site of an antibody to form a chimeric bivalent antibody comprising an antigen-binding site having specificity for an antigen, and another antigen-binding site that has specificity for a different antigen.

Humanized antibodies

Procedures for humanizing non-human antibodies have been described in the field. Preferably, a humanized antibody has one or more amino acid residues introduced originating from a non-human source. These non-human amino acid residues are often referred to as "imported" residues that are typically taken from an "imported" variable domain. Humanization can essentially be performed by following the procedure of Winter et al (Jones et al, Nature, 321:522-525

(1986); (Reichmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536 (1988)), by substituting hypervariable region sequences with the corresponding sequences for a human antibody. Such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less than an intact human variable domain has been substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted with residues from analogous sites in rodent antibodies.

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

It is also important that the antibodies are humanized while retaining a high bmchn<g>affinity for the antigen and other advantageous biological properties. To achieve this goal, according to a preferred method, humanized antibodies are produced by means of a process of analysis of the parent sequences and various conceptual humanized products by using three-dimensional models of the parent sequences and the humanized sequences. Three-dimensional immunoglobulin models are widely available and are known to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional core formation structures of selected kanchda immunoglobulin sequences. Inspection of these possible analyzes of the probable role of the residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, the FR residue can be selected and combined from the recipient sequences and the imported sequences, so that the desired antibody characteristics, such as increased affinity for the target antigen(s) are achieved. In general, the hypervariable region residues are directly and very significantly involved in influencing antigen binding.

The humanized antibody may be an antibody fragment such as a Fab which is optionally conjugated with one or more cytotoxic agents to generate an immunoconjugate. Alternatively, the humanized antibody may be a full-length antibody, such as a full-length IgG1 antibody.

Human antibodies and phage display methodology

As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (eg mice) which, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody nm<g>chain joining<g>sre<g>ion (JH) gene in chimeric and germline mutant mice leads to complete inhibition of endogenous antibody production. Transfer of the human gonad immunoglobulin assembly into such a germline mutant mouse will lead to the production of human antibodies as a result of antigen challenge. See e.g. Jakobovits et al., Proe. Nati. Acad. Pollock. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggeman et al., Year in Immuno, 7:33

(1993); U.S. Patent Document No. 5,545,806, 5,591,669 (all from GenPharm); 5,545,807 and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature 348:552-553

[1990]) be used to produce human antibodies and antibody fragments in vitro from the variable (V) domain gene repertoire of immunoglobulins from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into either a large or small coat protein gene from a filamentous bacteriophage, such as Ml3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also lead to selection of the gene encoding the antibody exhibiting those properties. Thus, the phage also mimics some of the properties of the B cell. Phage display can be carried out in a number of different formats which are reviewed in e.g. Johnson, Kevin S. and Chiswell, David J., Current in Structural Biology 3:564-571 (1993). Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a heterogeneous collection of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies against a heterogeneous collection of antigens (including (self-antigens)) can be isolated by 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. patent documents no. 5 565 332 and 5 573 905.

As discussed above, human antibodies can also be generated using in vitro seed-activated B cells (see U.S. Patent Nos. 5,567,610 and 5,229,275).

Antibody fragments

In certain cases, it is advantageous to use antibody fragments instead of whole antibodies. The smaller size of the fragments allows for rapid removal and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24:107-117

(1992); and Brennan et al, Science, 229:81 (1985)). Nevertheless, these fragments can now be produced directly using recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli thereby allowing the convenient production of large quantities of the fragments. Antibody fragments can be isolated from the antibody tag libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al, Bio/Technology 10:163-167 (1992). In accordance with a In another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased half-life in vivo that include receptor binding epitopesides are described in U.S. Patent No. 5,869,046. Other techniques for the preparation antibody fragments will be available to those skilled in the art. In other embodiments, the antibody selected is a single-chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894 and U.S. Patent No. 5,587 458. Fv and sFv are the only entities with intact combination sites that are free of constant regions, and thus are suitable for reduced non-specific binding during in vivo use. sFv fusion proteins can be engineered to provide fusion of an effector protein either at amino acids minal or carboxy terminus of an sFv. See Antibody Engineering, publisher Borrebaeck, above. Antibody fragments can also be a "linear antibody, for example as described in U.S. Patent No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

Bispecific antibodies

Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes on

the CD20 protein. Other such antibodies may combine a CD20 binding site with a binding site for another protein. Alternatively, an anti-CD20 arm can be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T-cell receptor molecule (eg, CD3) or Fc receptors for IgG (FcγR), such as FcγRI ( CD64), FcγRII (CD32) and FcγRIII (CD 16) or NKG2D or other NK cell activating ligand, to focus and localize cellular defense mechanisms against the CD20-expressing cell. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD20. These antibodies contain a CD20-binding arm and an arm that binds the cytotoxic agent (eg, saporin, anti-interferon-a, vinca alkaloid, ricin-A chain, methotrexate, or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2-bispecific antibodies).

WO 96/16673 discloses a bispecific anti-ErbB2/anti-FcγRIII antibody and U.S. Pat. U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcYRI antibody. A bispecific anti-ErbB2/Fca antibody is disclosed in WO 98/02463. U.S. patent document no. 5,821,337 describes a bispecific anti-ErbB2/anti-CD3 antibody.

Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromes) produce a potential mixture of 10 different antibody molecules of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually carried out by means of affinity chromatography steps, is quite difficult and the product yield is low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

According to another approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferred is the fusion with an Ig heavy chain constant domain comprising at least one part of the CH2 and CH3 hinge regions. It is preferred that the first heavy chain constant region (CH1) contains the site necessary for light chain binding present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into an appropriate host cell. This provides greater flexibility in the adjustment of the mutual proportions for the three polypeptide fragments in embodiments when different ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody. It is nevertheless possible to insert the coding sequence for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal proportions leads to high yields or when the conditions have no significant effect on the yield of the desired chain combination.

In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain - light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure promotes the separation of the desired bispecific compound from undesired immunoglobulin chain combinations since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy means of separation. This approach is disclosed in WO 94/04690. For further details regarding the generation of bispecific antibodies, see, for example, Suresh et al. Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. patent document no.

5,731,168 the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. The preferred interface comprises at least part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (eg tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the larger side chains are formed at the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (eg, alanine or threonine). This provides a mechanism to increase the yield of the heterodimer relative to other undesired end products such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be linked to avidin and the other to biotin. Such antibodies have been proposed, for example, to attack immune system cells to unwanted cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. patent document no. 4 676 980 together with a number of patent applications.

Techniques for producing bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared by using chemical coupling. Brennan et al. Science, 229: 81 (1985) describes a procedure in which intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize nearby dithiols and prevent intermolecular disulfide formation. The Fab' fragments produced are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab' thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies that are produced can be used as agents for the selective immobilization of enzymes.

Recent advances have promoted the direct recovery of the Fab'-SH fragment from E. Coli which can be chemically linked to form bispecific antibodies. Shalaby et al. J. Exp. Med., 175: 217-225 (1992) describes the preparation of a fully humanized bispecific antibody F(ab') 2 molecule. Each Fab' fragment was separately isolated from E. Coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus generated was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, in addition to triggering the lyrical activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been prepared using leucine zippers. Kostelny et al. J. Immunol, 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were bound to the Fab' part of two different antibodies by means of gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used in the preparation of antibody homodimers. The "Dialegeme" technology described by Hollinger et al. Pro. Nati. Acad. Pollock. USA 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. The fragments comprise a VH linked to a VL by means of a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment to thereby form two antigen binding sites. Another strategy to prepare bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valences are included. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147:60 (1991).

Multivalent antibodies

A multivalent antibody can be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies according to the present invention can be multivalent antibodies (which are not of the IgM class) with three or more antigen-binding sites (for example tetravalent antibodies) which can be easily produced by means of recombinant expression of nucleic acids which code for the polypeptide chains of the antibody. The multivalent antibody may 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-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains) where the polypeptide chain/chains comprise two or more variable domains. For example, the polypeptide chain(s) may comprise VD1- (Xl)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, XI and X2 represent an amino acid or polypeptide, and n is 0 or 1. For example, the polypeptide chain(s) may comprise: VH-CH1 flexible linker-VH-CH1-Fc region chain, or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody here preferably comprises a further at least two (and preferably four) light chain variable domain polypeptides. For example, the multivalent antibody herein may comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides here comprise a light chain variable domain and optionally a further CL domain.

Other amino acid sequence modifications

Amino acid sequence modifications of the CD20 binding antibodies described herein are encompassed. 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 introducing appropriate nucleotide changes into the anti-CD20 antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues in the amino acid sequence of the anti-CD20 antibody. Any combination of deletion, insertion and substitution is performed to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes can also alter post-translational processes of the anti-CD20 antibody, such as changing the number or position of glycosylation ethers.

A useful method for identifying certain residues or regions of the anti-CD20 antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells in Science, 244:1081-0185

(1989). Here, a residue or group of target residues is identified (for example charged residues such as arg, asp, his, lys and glu) and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to influence the interaction of the amino acids with CD20 -antigen. Those amino acid locations that show functional sensitivity to the substitutions are then improved by introducing additional or other variants at, or instead of, the sites of substitution. While the site of introduction of an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the induction of a mutation at a given site, ala-scanning or random mutagenesis is performed on 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 polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal inserts include an anti-CD20 antibody with an N-terminal methionyl residue or the antibody fused to a cytotoxic polypeptide. Other insert variants of the anti-CD20 antibody molecule include the fusion of the N- or C-terminus of the anti-CD20 antibody to an enzyme (eg for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the anti-CD20 antibody molecule replaced by another residue. The sites of greatest interest for substitution mutagenesis include the hypervariable regions, but FR changes are also encompassed. Conservative substitutions are shown in the table below under the heading "preferred substitutions". If such substitutions lead to a change in biological activity, then more extensive changes, called "exemplary substitutions" in the table, or as further described below with reference to amino acid classes, can be introduced and the products screened.

Extensive modifications in the biological properties of the antibody are effected by selecting substitutions that differ substantially in their effectiveness in maintaining (a) the structure of the polypeptide backbone in the region of the substitution, for example as a planar conformation or a helical conformation (b) the charge or the hydrophobicity of the molecule at the target site or (c) the extent of the side chain. Naturally occurring residues are divided into groups based on shared side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile,

(2) neutral hydrophilic: cys, ser, thr,

(3) acids: asp, glu,

(4) basic: asn, gin, his, light, arg,

(5) residues affecting chain orientation: gly, pro and

(6) aromatic, trp, tyr, phe.

Non-conservative substitutions would involve swapping a member of one of these classes for another class.

Any cysteine residue not involved in maintaining the appropriate conformation of the anti-CD20 antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent improper Loys binding. Conversely, modifications may be introduced into the antibody to improve its stability (especially where the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of a substitution variant involves substituting one or more hypervariable region residues in a parent antibody (eg, a humanized or human antibody). In general, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, multiple hypervariable region sites (eg 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions with the product of gene III in M13 packaged within each particle. The phage display variants are then screened for their biological activity (eg binding affinity) as disclosed herein. In order to identify candidate hypervariable region sites for modification, genome-wide scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be advantageous to analyze a loystal structure of the antigen-antibody complex to identify points of contact between the antibody and human CD20. Such contact residues and neighboring residues are candidates for substitution according to the techniques blk used here. Immediately such variants have been generated from the panel of variants subjected to screening as described here, 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 changes the original glycosylation pattern of the antibody. By change is meant the removal of one or more carbohydrate units that are found on the antibody and/or the introduction of one or more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the linkage of the carbohydrate unit on the side chain to an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagm-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate unit to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugar units N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, usually serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Introduction of glycosylation sites on the antibody blk conveniently carried out by changing the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The change can also be effected by the addition of, or the substitution with, one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

Nucleic acid molecules that code for amino acid sequence variants of the anti-CD20 antibody blk are produced using a number of different 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 production by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of a previously produced variant or a non-variant -version of the anti-CD20 antibody.

It may be desirable to modify the antibody according to the invention with regard to effector function, for example to improve antigen-dependent, cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) for the antibody. This can be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residues can be introduced into the Fc region to thereby allow cross-chain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capacity and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with improved antitumor activity can also be prepared by using heterobifunctional cross-linkers as described in Wolff et al. Cancer Research 53: 2560-2565 (1993). Alternatively, an antibody having two Fc regions could be developed and thereby have improved complement-mediated lysis and ADCC capacities. See Stevenson et al. Anti-Cancer Drug Design 3: 219-230 (1989).

To increase the half-life of the antibody in serum, one can incorporate a rescue receptor-binding epitope into the antibody (especially an antibody fragment) as described in U.S. Pat. patent document no. 5 739 277 for example. As used herein, the term "rescue receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (eg, IgGi, IgG2, IgG3, or IgG4) that is responsible for increasing the half-life of the IgG molecule in vivo in serum.

Other antibody modifications

Other modifications of the antibody are covered here. For example, the antibody can be linked to one of a number of different non-protein polymers, for example polyethylene glycol, polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. The antibody can also be entrapped in microcapsules, prepared for example by means of preservation techniques or by means of interphase polymerization (for example hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacylate) microcapsules) in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in (Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., ed. (1980).

Screening for antibodies with the desired properties

Antibodies with certain biological characteristics can be selected as described in the examples.

The growth inhibitory effects of an anti-CD20 antibody according to the invention can be investigated using methods known in the field, for example by using cells that express CD20 either endogenously or after transfection with the CD20 gene. For example, tumor cell lines and CD20-transfected cells can be treated with a monoclonal anti-CD20 antibody according to the invention at various concentrations for a few days (eg 2-7 days) and stained with crystal violet or MTT or analyzed using another colorimetric analysis. Another method for measuring proliferation would be to compare H-thymidine uptake for the cells that have been treated in the presence or absence of an anti-CD20 antibody according to the invention. After antibody treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA is quantified in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line.

To select for antibodies that induce cell death, loss of membrane integrity, as indicated by, for example, propidium iodide (PI), trypan blue or 7AAD uptake, can be examined relative to control. A P1 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 the appropriate monoclonal antibody at, for example, about 10 µg/ml. The cells are incubated for a 3-day time period. After each treatment, cells are washed and distributed into 35 mm filter-stoppered 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) to remove cell clumps. The tubes then receive PI (10Hg/ml). Samples can be analyzed using a FACSCAN flow cytometer and FACSCONVERT-CellQuest software (Becton Dickinson). Those antibodies that induce statistically significant levels of cell death as determined by P1 uptake may be selected as cell death-inducing antibodies.

To screen for antibodies that bind to an epitope on CD20 bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. Harlow and David Lane (1988) be carried out. This assay can be used to determine whether a test antibody binds to the same site or epitope as an anti-CD20 antibody of the invention. Alternatively, or in addition, epitope mapping can be performed using methods known in the art. For example, the antibody sequence can be mutagenized, such as in Alanm scanning, to identify contact residues. The mutant antibody is initially tested for binding with polyclonal antibody to ensure proper folding. In another method, peptides corresponding to different regions of CD20 can be used in competitive analyzes with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

Vectors, host cells and recombinant methods

The invention also provides an isolated nucleic acid encoding a humanized CD20-binding antibody, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody.

For recombinant production of the antibody, the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for expression. DNA encoding the monoclonal antibody can be readily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

(i) Signal sequence component

The CD20-binding antibody according to the invention can be produced recombinantly, not only directly, but also as a fusion polypeptide with a heterologous polypeptide which is preferably a signal sequence or other polypeptide which has a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence chosen is preferably one that is recognized and processed (ie cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native CD20-binding antibody signal sequence, the signal sequence is substituted with a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin-II leaders. For secretion in yeast, the native signal sequence can be substituted with, for example, the yeast invertase leader, the α-factor leader (including the Saccharomyces and Kluyveromyces α-factor leaders) or the acid phosphatase leader, the C. a/6/caHs-glucoamylase leader or the signal described in WO 90/13646.1 mammalian cell expression mammalian signal sequences in addition to viral secretory leaders such as the herpes simplex gD signal are available.

DNA for such a precursor region is ligated in reading frame with DNA that codes for the CD20-binding antibody.

(ii) Origin of replication

Both expression vectors and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In cloning vectors, this sequence is generally one that enables the vector to replicate independently of the host's chromosomal DNA and includes the origin of replication or autonomously replicating sequences. Such sequences are well known for a number of bacteria, yeast and viruses. The start site for replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the start site in the 2^ plasmid is suitable for yeast, and various start sites in viruses (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of the replication component is not required for mammalian expression vectors (the origin in SV40 can typically be used only because it contains the early promoter).

(iii) Selection gene component

Expression vectors and cloning vectors can contain a selection gene which is also called a selectable marker. Typical selection genes code for proteins that (a) transfer resistance to antibiotics or other toxins, for example ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies or (c) supply necessary nutrients that are not available from the complex medium, e.g. the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme uses a drug to stop the growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survives the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

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

For example, cells transformed with the DHFR selection gene are first identified by growing all the transformants in a culture medium containing methotrexate (Mtx), which is a competitive antagonist of DHFR. An appropriate host cell when wild-type DHFR is used is the Chinese hamster ovary (CHO) cell line lacking DHFR activity (eg, ATCC CRL-9096).

Alternatively, host cells (especially wild-type hosts containing endogenous DHFR) can be transformed or co-transformed with DNA sequences encoding CD20-binding antibody, wild-type DHFR protein and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH) be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycoside antibiotic, e.g. kanamycin, neomycin or G418. See U.S. patent no. 4 965 199. A useful selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1. Jones, Genetics 85:12 (1977). The presence of the trplesion in the yeast host cell genome thus provides an efficient environment for detecting transformation by growth in the absence of tryptophan. Similarly, Lett2-deficient yeast strains (ATCC 20 622 or 38 626) are covered by known plasmids carrying the Let/2 gene.

In addition, vectors derived from the circular plasmid pKD1 of 1.6 µm can be used for transformation of Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multicopy expression vectors for the secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed. Fleer et al., Bio/Technology, 9:968-975 (1991).

(iv) Promoter component

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

Promoter sequences are known for eukaryotes. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence that can be found 70 to 80 bases upstream from the start of transcription from many genes is a CNCAAT region where N can be any nucleotide. At the 3' end of most eukaryotic genes there is an AATAAA sequence which can be the signal for the addition of the poly-A tail to the 3' end of the coding sequence. All of these sequences can conveniently be inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters that are inducible promoters that have the additional advantage that transcription is controlled by growth conditions are the promoter regions of alcohol dehydrogenase-2, isocytochrome-C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes that are responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73 657. Yeast enhancers are also advantageously used together with yeast promoters.

For example, CD20-binding antibody transcription from vectors in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus-2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B- virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, for example the actin promoter or an immunoglobulin promoter, from heat shock promoters, provided that such promoters are compatible with the host cell systems.

The early and late promoter from the S V40 virus is conveniently provided as an SV40 restriction fragment which also contains the virus origin of replication from S V40. The rapid early promoter from human cytomegalovirus is conveniently provided as an Hm(Un-E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papillomavirus 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 et al., Nature 297:598-601 (1982) on expression of human β-interferon cDNA in mouse cells under the control of a thymiclin kinase promoter from herpes simplex virus Alternatively, the long terminal repeat from Rous Sarcomavirus can be used as the promoter.

(v) Enhancer element component

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

(vi) Transcription termination component

Expression vectors that are used in eukaryotic host cells (yeast cells, fungal cells, insect cells, plant cells, animal cells, human cells or cells with nuclei from other multicellular organisms) will also contain sequences that are necessary for the termination of transcription and to stabilize mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding CD20-binding antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region, see WO 94/11026 and the expression vector disclosed therein.

(vii) Selection and transformation of host cells

Suitable host cells for cloning or expressing DNA in the vectors herein are the prokaryotic cells, yeast cells or the higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example Enterobacteriaceae such as Escherichia, for example ii. Coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans and Shigella in addition to Bacilli such as B. subtilis and B. licheniformis (for example B. licheniformis 41P notified in DD 266 710 published April 12, 1989), Pseudomonas such as P. aeruginosa and Streptomyces. A preferred £.Cø// cloning host is E. Coli 294 (ATCC 31 446), although other strains such as E. Coli B, E. Coli X1776 (ATCC 31 537) and E. Coli W3110 (ATCC 27 325) is appropriate. These examples are illustrative rather than limiting.

Full-length antibody, antibody fragments, and antibody-fusion proteins can be produced in laboratories, especially when glycosylation and Fc effector function are not required, such as when the therapeutic antibody is conjugated to a cytotoxic agent (eg, a toxin) and the immunoconjugate itself shows effectiveness in tumor cell destruction. Full-length antibodies have a longer half-life in circulation. Production in E. Coli is faster and more cost-effective. For expression of the antibody fragment and polypeptide in bacteria, see, for example, U.S. Pat. 5,648,237 (Carter et al.), U.S. Pat. 5,789,199 (Joly et al.) and U.S. Pat. 5,840,523 (Simmons et al.) which describes translation initiation region (TIR) and signal sequences to optimize expression and secretion, these patents being incorporated herein by reference. After expression, the antibody is isolated from Æ.cø/z cell mass in a soluble fraction and can be purified via, for example, a protein A or G column depending on the isotype. Final purification can be carried out in the same way as the process for purifying antibody expressed in, for example, CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for CD20-binding antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. Nevertheless, a number of other families, species and strains are readily available and useful herein, such as Schizosaccharomyces pombe, Kluyveromyces hosts such as, for example, K. lactis, K. fragilis (ATCC 12 424), K. bulgaricus (ATCC 16 045), K. wickeramii (ATCC 24 178), K. waltii (ATCC 56 500), K. drosophilarum (ATCC 36 906), K. thermotolerans and K. marxianus, yarrowia (EP 402 226), Pichiapastoris (EP 183 070), Candida , Trichoderma reesia (EP 244 234), Neurospora crassa, Schwanniomyces such as Schwanniomyces occidentalis and filamentous fungi such as for example Neurospora, Penicillium, Tolypocladium and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated CD20-binding antibodies are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Countless baculovirus strains and variants and corresponding insect facultative host cells from hosts such as Spodoptera frugiperda (butterfly caterpillar), Åedes aegypti (mosquito), Åedes albopictus (mosquito), Drosophila melanogaster (banana fly) and Bombyx mori have been identified. A number of virus strains for transfection are widely available, for example the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus here according to the present invention, especially for transfection of Spodoptera frigiperda- cells.

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

Nevertheless, interest has been greatest in vertebrate cells and the propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proe. Nati. Acad. Sei. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), dog kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL3A, AATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al ., Annals N.Y. Acad, Sci. 383:44-68 (1982)), MRC 5 cells, FS4 cells and a human hepatoma line (Hep G2).

Host cells are transformed with the above-described expression or cloning vectors for CD20-binding antibody production and cultured in appropriate nutrient media, modified appropriately to induce promoters, select transformants or amplify the genes encoding the desired sequences.

(viii) Cultivation of the host cells

The host cells which are used to produce the CD20-binding antibody according to this invention can be cultured in a number of different media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition can any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657 866, 4,927,762, 4,560,655, or 5,122,469, WO 90/03430, WO 87/00195, or U.S. Patent Re. 30,985 are used as culture media for the host cells.Either of these media may be supplemented as necessary with hormones and/or or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN drug ), trace substances (defined as inorganic compounds that are usually present at final concentrations in the micromolar range) o g glucose or a similar energy source. Any other necessary additives may also be included in appropriate concentrations that will be known to those skilled in the art. The culture conditions, such as temperature, pH and the like, are those previously used for the host cell selected for expression and will be obvious to those skilled in the art.

(ix) Purification of antibody

When recombinant techniques are used, the antibody can be produced intracellularly in the periplasmic space or is directly secreted into the medium. If the antibody is produced intracellularly, as a first step the particulate debris, either host cells or lysed fragments, is removed, for example by centrifugation or ultrafiltration. Carter et al. Bio/Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. Coli. Briefly, cell mixture is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) over approximately 30 minutes. Cell waste can be removed by centrifugation. Where the antibody is secreted into the medium, generally supernatants from such an expression system are first concentrated using a commonly available protein concentration filter, for example an Amicon or Millipore-Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the previous steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of harmful contaminants.

The antibody preparation produced from the cells can be purified by, for example, using hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, where affinity chromatography is the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein-A can be used to purify antibodies based on human γ, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein-G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow faster solidification rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX resin (J.T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin-SEPHAROSE, chromatography on an anion- or cation-exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

After any preceding purification step, the mixture comprising the antibody of interest and impurities may be subjected to low pH hydrophobic chromatography using an elution buffer at a pH between about 2.5-4.5, preferably carried out at low salt concentrations (eg from approximately 0-0.25 M salt).

Antibody conjugates

The antibody can be conjugated to a cytotoxic agent such as a toxin or a radioactive isotope. In certain embodiments, toxins calicheamicin, a maytansinoid, a dolastatin, auristatin-E, and analogs or derivatives thereof are preferred.

Preferred drugs/toxins include DNA-damaging agents, inhibitors of myotubule polymerization or depolymerization, and antimetabolites. Preferred classes of cytotoxic agents include, for example, the enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors, DNA intercalating agents, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vincal drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drugs, the diynenes, the podophyllotoxins, and differentiation inducers . Particularly useful members of these classes include, for example, methotrexate, metopterin, dichlormethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosidein, actinomycin, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, morpholino- doxorubicin, l-(2-chloroethyl), 1,2-dimethansulfonylhydrazide, N - acetylspermidine, aminopterin metopterin, esperamycin, mitomycin-C, mitomycin-A, actinomycin, bleomycin, carminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etopside or etopside phosphate , vinblastine, vincristine, vindesine, taxol, taxotes, retinoic acid, butyric acid, N -acetylspermidine, camptothecin, cahcheamicin, bryostatins, cephalostatins, ansamitocin, actosin, maytansinoids such as DM-1, maytansine, maytansinol, N-desmethyl-4,5- desepoxymaytansinol, C-19-dechloro-maytansinol, C-20-hydroxymaytansinol, C-20-demethoxymaytansinol, C-9-SH-maytansinol, C-14-alkoxymethylmaytansinol, C-14-hydroxy- or acetyloxy methylmaytansinol, C-15-hy(oxy/acetyloxymaytansinol, C-15-methoxymaytansinol, C-18-N-demethylmaytansinol and 4,5-deoxymaytansinol, auristatins such as auristatin E, M, PHE and PE, dolostatins such as dolostatin -A, dolostatin-B, dolostatin-C, dolostatin-D, dolostatin-E (20-epi and 11-epi), dolostatin-G, dolostatin-H, dolostatin-I, dolostatin-1, dolostatin-2, dolostatin- 3, dolostatin-4, dolostatin-5, dolostatin-6, dolostatin-7, dolostatin-8, dolostatin-9, dolostatin-10, deo-dolostatin-10, dolostatin-11, dolostatin-12, dolostatin-13, dolostatin- 14, dolostatin-15, dolostatin-16, dolostatin-17 and dolostatin-18, cephalostatins such as cephalostatin-1, cephalostatin-2, cephalostatin-3, cephalostatin-4, cephalostatin-5, cephalostatin-6, cephalostatin-7,25 '-epi-cephalostatin-7, 20-epi-cephalostatin-7, cephalostatin-8, cephalostatin-9, cephalostatin-10, cephalostatin-11, cephalostatin-12, cephalostatin-13, cephalostatin-14, cephalostatin-15, cephalostatin- 16, cephalostatin-17, cephalostatin-18 and cephalostatin -19.

Maytansinoids are mitotic inhibitors that work by inhibiting tubulin polymerisation. Maytansin was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Eventually, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinole esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and derivatives and analogs thereof are disclosed, for example, in U.S. Pat. patent document no. 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, 4 313 915, 4 9 , 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 contents of which are hereby incorporated by reference.

Maytansine and maytansinoids have been conjugated to antibodies that specifically bind to tumor cell antigens. Immunoconjugates containing maytansinoids and their therapeutic use are disclosed, for example, in U.S. Pat. patent no. 5 208 020, 5 416 064 and in European patent EP 0 425 235 B1, the contents of which are incorporated by reference here. Liu et al. Pro. Nati. Acad. Pollock. USA 93:8618-8623 (1996) describes immunoconjugates comprising a maytansinoid designated DM1 bound to the monoclonal antibody C242 directed against human colon cancer. The conjugate was found to be highly cytotoxic against cultured colon cancer cells and showed antitumor activity in an in v/vo tumor growth assay. Chari et al. Cancer Research 52:127-131 (1992) describes immunoconjugates in which a maytansinoid was conjugated via a (hsulfi(linker) to the murine antibody A7 which binds to an antigen on human ovarian cancer cell lines or to another murine monoclonal antibody TA. 1 which binds HER- 2/wett oncogene.

There are many linkers known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Pat. patent document no. 5 208 020 or EP patent 0 425 235 Bl and Chari et al. Cancer Research 52: 127-131 (1992). The linking groups include disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidaselabile groups or esteraselabile groups, as disclosed in the above-given patents, disulfide and thioether groups being preferred.

Conjugates of the antibody and the maytansinoid can be prepared by using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolan (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as (hsuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis -diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-(linitrobenzene). In particular preferred coupling agents include N-succinimidyl-3-(2-pyridylthio)propionate (SPDP) (Carlsson et al. Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate ( SPP) to provide a disulfide bond.

The linker can be connected to the maytansinoid molecule at different positions, depending on the type of connection. For example, an ester linkage can be formed by reaction with a hydroxyl group using conventional coupling techniques. The reaction can occur at the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, 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 of maytansinol or a maytansinol analog.

Calicheamicin

Another immunoconjugate of interest comprises a CD-20 binding antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at subpicomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Patent No. 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 belonging to the American Cyanamid Company). Structural analogs of calicheamicin that may be used include, but are not limited to, y/, o^<1>, a3<!>, N-acetyl-y/, PSAG, and 2<1>! (Hinman et al. Cancer Research 53:3336-3342 (1993), Lode et al. Cancer Research 58:2925-2928 (1998) and the aforementioned U.S. patents to American Cyanamid). Another antitumor drug with which the antibody can be conjugated is QFA which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. The cellular uptake of these agents via antibody-mediated internalization therefore strongly increases their cytotoxic effects.

Radioactive isotopes

For selective destruction of the turome, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radio-conjugated anti-CD-20 antibodies. Examples include At211,1<13>1,1125, Y9<0>, Re<186>, Re<188>, Sm , , P , Pb and radioactive isotopes of Lu. When the conjugate is used for diagnosis, it may include a radioactive atom for scintigraphic examinations, for example tc<99m>or I<123>, or a spin label for nuclear magnetic resonance (NMR) imaging, (also known as magnetic resonance imaging, rnri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radiolabels or other labels may be incorporated into the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using appropriate amino acid precursors involving, for example, fluorine-19 instead of hydrogen. Tags, such as tc or I , Re , Re and Inlukan are attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used to incorporate iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

Conjugates of the antibody and cytotoxic agents can be prepared by using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyl-thio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-l- carboxylate, iminothiol (IT), bifunctional derivatives of imido esters (such as dimethyl dipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-(hazoniumbenzoyl)-ethylene(hamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-( linitrobenzene).For example, a ricin immunotoxin can be prepared as described in Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for conjugation of radionucleotide to the antibody See WO 94/11026 Link ren may be a "cleavable linker" that promotes release of the cytotoxic drug into the cell. For example, an acid-labile linker, peptidase sensitive linker, photolabile linker, dimethyl linker or (hsmphidine-containing linker (Chari et al. Cancer Research 52: 127-131 (1992), U.S. Patent No. 5,208,020) may be used.

Therapeutic applications of the CD-20 binding antibodies

The CD-20 binding antibodies of the invention are useful in treating a number of malignant and non-malignant diseases including autoimmune diseases and related conditions and CD-20 positive cancers including B-cell lymphomas and leukemias. Stem cells (B cell precursors) in the bone marrow lack the CD-20 antigen, which allows healthy B cells to regenerate after treatment and return to normal levels within several months.

Autoimmune diseases or autoimmune-related conditions include arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis including atopic dermatitis, chronic autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), respiratory distress syndrome, adult respiratory stress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), lupus (including nephritis, non-renal, discoid, alopecia), pediatric diabetes, multiple sclerosis, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokine are and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including ANCA), aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia, including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia-A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ damage syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease , anti-phospholipid antibody syndrome, allergic neuritis, Bechet's disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton Myasthenic syndrome, Reynaud's syndrome, Sjøgren's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including pretreatment for high paneheactive antibody titers, IgA deposition in tissues, etc. .), graft versus host disease (GVHD), bullous pemphigoid, pemphigus ( all including vulgaris, foliaceus), autoimmune polyendocrinopathies, Reiter's disease, Stiffman's syndrome, giant cell arteritis, immune complex nephritis, IgA neuropathy, IgM polyneuropathies or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune disease of the testes and ovaries including autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune, polyglandular syndromes (or polyglandular endocrinopathic syndromes), type-I diabetes, also referred to as insulin-dependent diabetes mellitus (IDDM) and Sheehan's syndrome, autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-graft) versus NSIP, Guillain-Barre syndrome, large vessel vasculitis ( including polymyalgia rheumatica etc g large cell (Takayasus) arteritis), medium vessel vasculitis (including Kawasaki disease and polyarthritis nodosa), ankylosing spondylitis, Berger's disease (IgA neuropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobulinemia, ALS, coronary artery disease. CD-20-positive cancers are those that involve abnormal proliferation of cells that express CD-20 on the cell surface. The CD-20-positive B-cell neoplasms include CD-20-positive Hodgkin's disease including lymphocyte predominant Hodgkin's disease (LPHD), non-Hodgkin's lymphoma (NHL), follicular center cell (FCC) lymphomas, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia. Non-Hodgkins lymphoma includes low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), intermediate/follicular NHL, intermediate-grade diffuse NHL, low-grade immunoblastic NHL, low-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, "bulky disease"- NHL, plasmacytoi(lymphocytic lymphoma, mantle cell lymphoma, AIDS-related lymphoma and Waldenström's macroglobulinemia. Treatment of recurrence of these cancers is also covered. LPHD is a type of Hodgkin's disease that tends to recur frequently despite radiation or chemotherapy treatment and is characterized by CD-20-positive, malignant cells. CLL is one of four main types of leukemia. A cancer of mature B cells called lymphocytes, CLL is manifested by the progressive accumulation of cells in the blood, bone marrow and lymphatic tissue.

In specific embodiments, the humanized CD-20 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 pediatric rheumatoid arthritis, systemically lupus erythematosus (SLE) including lupus nephritis, Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA neuropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, diabetes mellitus, Reynaud's syndrome, Sjøgren's syndrome and glomerulonephritis.

The humanized CD-20-binding antibodies or functional fragments thereof are useful as a single-agent treatment in, for example, relapsed or refractory lower-grade or follicular CD-20-positive, B-cell NHL, or can be administered to patients together with other drugs in a multidrug regimen.

Indolent lymphoma is a slowly developing, incurable disease in which the average patient survives between 6 and 10 years after many periods of remission and relapse. In one embodiment, the humanized CD-20 binding antibodies, or functional fragments thereof, are used to target indolent NHL.

The parameters for examining the effectiveness or success of treatment of the neoplasm will be known to a physician skilled in the relevant disease. In general, the experienced physician will look for a reduction in signs and symptoms for the specific disease. Parameters may include average time to disease progression, remission time, stable disease.

The following references describe lymphomas and CLL, their diagnoses, treatment, and standard medical procedures for measuring treatment effectiveness. Canellos GP, Lister TA, Sklar JL, The Lymphomas. W.B. Saunders Company, Philadelphia, 1998, van Besien K and Cabanillas, F: Clinical Manifestations, Staging and Treatment of Non-Hodgkin's Lymphoma, Chap. 70, pp. 1293-1338, in: Hematology, Basic Principles and Practice, 3rd ed. Hoffman et al. (publishers). Churchill Livingstone, Philadelphia, 2000 and Rai, K and Patel, D: Chronic Lymphocytic Leukemia, Chapter 72, pp. 1350-1362, in: Hematology, Basic Principles and Practice, 3rd ed. Hoffman et al. (publishers). Churchill Livingstone, Phildelphia, 2000.

The parameters for assessing the effectiveness or success of treatment of an autoimmune or autoimmune-related disease will be known to an experienced physician for the appropriate disease. In general, the experienced physician will look for a reduction in signs and symptoms for the specific disease. The following is given for the example.

In one embodiment, the antibodies according to the invention are useful for treating rheumatoid arthritis. RA is characterized by inflammation in many joints, cartilage loss and bone erosion, which leads to joint destruction and ultimately reduced joint function. In addition, since RA is a systemic disease, it can have effects on other tissues such as the lungs, eyes and bone marrow. Fewer than 50 percent of patients who have had RA for more than 10 years can continue to work or function normally on a day-to-day basis. The antibodies can be used as a first-line therapy in patients with early RA (ie methotrexate)

(MTX) naive) and as monotherapy or in combination with, for example, 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, for example, MTX. The humanized CD-20 binding antibodies are useful in preventing and controlling joint damage, delaying structural damage, reducing pain associated with inflammation in RA, and generally reducing the signs and symptoms of moderate to severe RA. The RA patient can be treated with the humanized CD-20 antibody before, after or together with treatment with other drugs used to treat RA (see combination therapy below). In one embodiment, patients who were previously unsuccessfully treated with disease-modifying antirheumatic drugs and/or had an insufficient response to methotrexate alone, are treated with a humanized CD-20-binding antibody according to the invention. In one embodiment of this treatment, the patients are given a 17-day treatment regimen of humanized CD-20 binding

antibody alone (lg iv infusions on days 1 and 15), CD-20-binding antibody plus cyclophosphamide (750 mg iv infusion on days 3 and 17) or CD-20-binding antibody plus methotrexate.

One method for evaluating treatment effectiveness in RA is based on criteria from the American College of Rheumatology (ACR), which measure the percentage of improvement in sore and swollen joints, among other things. The RA patient may be assigned a value of, for example, ACR 20 (20 percent improvement) compared to no antibody treatment (eg, pre-treatment baseline) or placebo treatment. Other ways to evaluate the effectiveness of antibody therapy include X-ray scoring such as "Sharp-X-ray" imaging that was used to score structural damage such as bone erosion and joint space narrowing. Patients may also be evaluated for the reduction or improvement in disability based on Health Assessment Questionnaire [HAQ] scores, AIMS scores, SF-36 at time periods during and after treatment. ACR-20 criteria may include 20% improvement in both tender (painful) joint count and swollen joint count plus 20% improvement in at least 3 of 5 additional measures:

1. the patient's pain assessment using a visual analogue scale (VAS),

2. the patient's global assessment of disease activity (VAS),

3. the doctor's global assessment of disease activity (VAS),

4. the patient's self-assessed disability measured using the Health Assessment Questionnaire,

and

5. acute phase reactants, CRP or ESR.

ACR-50 and -70 are defined analogously. Preferably, the patient is administered an amount of a CD-20-binding antibody according to the invention which is effective to achieve at least an ACR 20 titer, preferably at least ACR 30, more preferably at least ACR50, even more preferably at least ACR70, most preferably at least ACR75 and higher. Psoriatic arthritis has unique and distinct radiographic features. For psoriatic arthritis, joint erosion and narrowing of the joint opening can be evaluated with the Sharp vercht device in addition. The humanized CD-20-binding antibodies according to the invention can be used to prevent joint damage in addition to reducing disease signs and symptoms of the disorder.

Yet another aspect of the invention is a method of treating lupus or SLE by administering to the patient suffering from SLE a therapeutically effective amount of a humanized CD-20 binding antibody of the invention. SLEDAI value assignment provides a numerical quantification of disease activity. The SLEDAI is a weighted index of 24 clinical and laboratory parameters known to correlate with disease activity, with a numerical range of 0-103. See Bryan Gescuk & John Davis, "Novel therapeutic agent for systemic lupus erythematosus" in Current Opinion in Rheumatology 2002, 14:515-521. Antibodies against double-stranded DNA are thought to cause more recent envenoming and other manifestations of lupus. Patients undergoing antibody therapy may be monitored for time to renal clearance, which is defined as a significant, reproducible increase in serum creatinine, urinary protein, or blood in the urine. Alternatively or additionally, patients may be monitored for levels of antinuclear antibodies and antibodies to double-stranded DNA. Treatments for SLE include high-dose corticosteroids and/or cyclophosphamide (HDCC).

Spondyloarthropathies are a group of joint disorders, including ankylosing spondylitis, psoriatic arthritis and Crohn's disease. Treatment success can be determined using global validated patient survey measurement tools and physician survey measurement tools.

Different medications are used to treat psoriasis, and treatment is different in direct relation to the severity of the disease. Patients with milder forms of psoriasis typically use topical treatments such as topical steroids, anthralin, calcipotriene, clobetasol and tazarotene, to manage the disease, while patients with moderate and severe psoriasis more often use systemic (methotrexate, retinoids, cyclosporine, PUVA and UVB) therapies . Tars are also used. These therapies have a combination of safety measures, time-consuming regimens, or inappropriate processes for treatment. Furthermore, some require expensive equipment and prepared space in the office setting. Systemic drugs can cause serious side effects including hypertension, hyperlipidaemia, bone marrow suppression, liver disease, kidney disease and intestinal disturbance. The use of phototherapy can also increase the incidence of skin cancers. In addition to the impracticality and discomfort associated with the use of topical therapies, phototherapy and systemic treatments require patients to be taken on and off therapy and lifetime exposure monitoring due to their side effects.

Treatment efficacy for psoriasis is examined by monitoring changes in clinical signs and symptoms of the disease including Physician's Global Assessment (PGA) changes and Psoriasis Area and Severity Index (PASI) ratings, Psoriasis Symptom Assessment (PSA), compared to baseline. The patient may be measured periodically throughout the treatment on the visual analogue scale which is used to indicate the degree of itching experienced at specific times.

Patients may experience an infusion reaction or infusion-related symptoms with their first infusion of a therapeutic antibody. These symptoms vary in severity and are generally reversible with medical interventions. These symptoms include, but are not limited to, flu-like fever, chills/twitches, nausea, urticaria, headache, bronchospasm, angioedema. It would be desirable for the disease treatment method according to the present invention to minimize infusion reactions. Thus, another aspect of the invention is a method of treating the diseases indicated by administering a humanized a CD-20 binding antibody wherein the antibody has reduced or no complement-dependent cytotoxicity and leads to reduced infusion-related symptoms compared to treatment with Rituxan. In one embodiment, the humanized CD-20 binding antibody is 2H7.vl 16.

Dosage

Depending on the indication to be treated and factors relevant to the dosage that a physician experienced in the field will be familiar with, the antibodies according to the invention will be administered at a dosage that is effective for the treatment of this indication, while toxicity and side effects will be minimized. For the treatment of a CD-20 positive cancer or an autoimmune disease, the therapeutically effective dosage will be in the range of about 250 mg/m to about 400 mg/m or 500 mg/m, preferably about 250-375 mg/m. In one embodiment, the dosage range is 275-

375 mg/m . In one embodiment of the treatment of a CD-20 positive B cell neoplasm, the antibody is administered in a range of 300-375 mg/m . For the treatment of patients suffering from B-cell lymphoma, such as non-Hodgkin's lymphoma, the anti-CD-20 antibodies and the humanized anti-CD-20 antibodies of the invention in a specific embodiment will be administered to a human patient at a dosage of 10 mg/kg or 375 mg/m . For the treatment of NHL, a dosage regimen would be to administer a dose of the antibody preparation as a dosage of 10 mg/kg in the first week of treatment, followed by a 2 week interval, and then a second dose of the same amount of the antibody is administered. In general, NHL patients receive such treatment once in a year, but in the event of recurrence of the lymphoma, such treatment may be repeated. In another dosing regimen, patients treated with lower-grade NHL receive four weeks of a version of humanized 2H7, preferably vl6 (375 mg/m weekly) followed in week five by three additional runs of the antibody pulse standard CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) or CVP (cyclophosphamide, vincristine, prednisone) chemotherapy, which was given every three weeks for three cycles.

In one embodiment, to treat rheumatoid arthritis, the dosage range of the humanized antibody is 125 mg/m (equivalent to about 200 mg/dose) to 600 mg/m , given in two doses, for example the first dose of 200 mg is administered on day one, followed by a second dose of 200 mg on day 15.1 different embodiments, the dosage is 250 mg/dose, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg,

475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg.

In the treatment of the disease, the CD-20-binding antibodies according to the invention can be administered to the patient chronically or intermittently, as determined by the doctor who has experience with the disease.

A patient who is administered a drug by intravenous infusion or subcutaneously may experience adverse events such as fever, chills, burning sensation, asthenia and headache. To alleviate or minimize such adverse events, the patient may receive an initial conditioning dose(s) of the antibody followed by a therapeutic dose. The conditioning dose(s) will be lower than the therapeutic dose to condition the patient to tolerate higher doses.

Administration route

The CD-20 binding antibodies are administered to a human patient in accordance with known methods, such as by intravenous administration, for example as a bolus or by continuous infusion over a period of time, either subcutaneously, intramuscularly, intraperitoneally, intracerebrospinally, intra-articularly , intrasynovially, intrathecally or by inhalation, generally by intravenous or subcutaneous administration.

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

Combination therapy

When treating the B-cell neoplasms described above, the patient can be treated with the CD-20-binding antibodies according to the present invention together with one or more therapeutic agents such as a chemotherapeutic agent in a multi-drug regimen. The CD-20 binding antibody may be administered concurrently, sequentially or alternately with the chemotherapeutic agent, or after non-responsiveness to other therapy. Standard chemotherapy for lymphoma treatment may include cyclophosphamide, cytarabine, melphalan, and mitoxantrone plus melphalan. CHOP is one of the most commonly used chemotherapy regimens to treat non-Hodgkin's lymphoma. The following are the drugs used in the CHOP regimen: cyclophosphamide (trade name cytoxan, neosar), adriamycin (doxombicin/hydroxydoxorubicin), vincristine (oncovin), and prednisolone (sometimes called deltasone or orasone). In particular embodiments, the CD-20-binding antibody is administered to a patient in need thereof in combination with one or more of the following chemotherapeutic agents of doxorubicin, cyclophosphamide, vincristine and prednisolone. In a specific embodiment, a patient suffering from a lymphoma (such as a non-Hodgkin's lymphoma) is treated with an anti-CD20 antibody of the present invention in conjunction with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) therapy. In another embodiment, the cancer patient can be treated with a humanized CD-20 binding antibody according to the invention together with CVP (cyclophosphamide, vincristine and prednisone) chemotherapy. In a specific embodiment, the patient suffering from CD-20 positive NHL is treated with humanized 2H7.vl6 together with CVP. In a specific embodiment of the treatment of CLL, the CD-20 binding antibody is administered along with chemotherapy with one or both of fludarabine and cytoxam.

In treating the autoimmune diseases or autoimmune-related conditions described above, the patient may be treated with the CD-20 binding antibodies of the present invention together with a second therapeutic agent, such as an immunosuppressive agent, such as in a multidrug regimen. The CD-20 binding antibody may be administered simultaneously, sequentially or alternately with the immunosuppressive agent or in case of non-responsiveness with other therapy. The immunosuppressive agent can be administered at the same or at lower dosages than those determined in the field. The preferred adjunctive immunosuppressive agent will depend on many factors, including the type of disorder being treated as well as the patient's history.

"Immunosuppressant" as used herein for adjunctive 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, downregulate or suppress self-antigen expression, or mask the MHC antigen. Examples of such agents include steroids such as glucocorticosteroids, for example prednisone, methylprednisolone and dexamethasone, 2-amino-6-aryl-5-substituted pyrimidines (see U.S. Patent No. 4,665,077), azathioprine (or cyclophosphamide, if there is a adverse reaction to azathioprine), bromocriptine, glutaraldehyde (which masks the MHC antigen, as described in U.S. Patent No. 4,120,649), anti-idiotypic antibodies to MHC antigens and MHC fragments, cyclosporine-A, cytokine or cytokine receptor antagonists including anti-interferon-(- 3 or - V antibodies, antitumor necrosis factor - V antibodies, antitumor necrosis factor - 3 antibodies, anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies, anti-L3T4 antibodies, heterologous anti- lymphocyte globulin, pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies, soluble peptide containing an LFA-3 binding domain (WO 90/08187 published 7/26/90), streptokinase, TGF - 3 , streptodornas e, host RNA or DNA, FK506, RS-61443, deoxy-spergualin, rapamycin, T-cell receptor (U.S. U.S. Patent No. 5,114,721) T-cell receptor fragments (Offner et al., Science 251:430-432 (1991), WO 90/11294 and WO 91/01133) and T-cell receptor antibodies (EP 340109) such as T10B9.

For the treatment of rheumatoid arthritis, the patient can be treated with a CD-20 antibody according to the invention together with any one or more of the following drugs: DMARDS (disease-modifying anti-rheumatic drugs (for example methotrexate), NSAIDs or NSAIDs (non-steroidal anti-inflammatory drugs drugs), HUMIRA (adalimumab, Abbott Laboratories), ARA VA (leflunomide), REMICADE (irifliximab, Centocor Inc., Malvern, Pa), ENBREL (etanercept, Immunex, WA), COX-2 inhibitors. DMARDs commonly used in RA are hydroxychloroquine, sulfasalazine, methotrexate, leflunomide, etanercept, infliximab, azathioprine, D-penicillamine, Gold (oral), Gold (intramuscular), minocycline, cyclosporine, staphylocoldc<p>rotem-A immunoadsorption Adalimumab is a human monoclonal antibody that binds to TNF V. Infliximab is a chimeric monoclonal antibody that binds to TNF V. Etanercept is an "irmunadhesin" fusion protein consisting of the extracellular ligand-binding portion of the human 75 kD (p75 ) the tumor necrosis factor receptor (TNFR) bound to the Fc portion of a human IgG1. For conventional treatment of RA, see, for example, "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 CD-20 antibody according to the invention together with methotrexate (MTX). An example of a dose of MTX is approximately 7.5-25 mg/kg per week. MTX can be administered orally and subcutaneously.

For the treatment of ankylosing spondylitis, psoriatic arthritis and Crohn's disease, the patient can be treated with a CD-20 binding antibody according to the invention together with, for example, Remicade (infliximab, fira Centocor Inc, Malvern, Pa.), ENBREL (etanercept, Immunex, WA).

Treatments for SLE include high-dose corticosteroids and/or cyclophosphamide

(HDCC).

For the treatment of psoriasis, patients may be administered a CD-20-binding antibody together with topical treatments, such as topical steroids, anthralin, calcipotriene, clobetasol and tazarotene or with methotrexate, retioids, cyclosporine, PUVA and UVB therapies. In one embodiment, the psoriasis patient is treated with the CD-20 binding antibody sequentially or concurrently with cyclosporine.

Pharmaceutical formulations

Therapeutic formulations of the CD-20 binding antibodies used in accordance with the present invention are prepared for storage by mixing an antibody having the desired degree of purity with any pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol , A. ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyl(limethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol), low molecular weight polypeptides (less than about 10 residues), proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as olivinyl pyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming e counterions such as sodium, metal complexes (for example Zn-protein complexes) and/or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG).

Examples of anti-CD20 antibody formulations are described in WO 98/56418, which is incorporated herein by reference. Another formulation is a liquid multidose formulation comprising the anti-CD20 antibody at 40 mg/ml, 25 mM acetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate-20 at pH 5.0 which has a minimum shelf life of two years when stored at 2-8 °C. Another anti-CD-20 formulation of interest comprises 10 mg/ml antibody in 9.0 mg/ml sodium chloride, 7.35 mg/ml sodium citrate dihydrate, 0.7 mg/ml polysorbate-80 and sterile water for injection, pH 6.5. Yet another pharmaceutical formulation comprises 10-30 mM sodium acetate from about pH 4.8 to about pH 5.5, preferably at pH 5.5, polysorbate as a surfactant in an amount of about 0.01-0.1% v /v, trehalose at an amount of about 2-10% w/v, and benzyl alcohol as a preservative (U.S. 6,171,586). Lyophilized formulations adapted for subcutaneous administration are described in WO 97/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation may be administered subcutaneously to the mammal to be treated herein.

A formulation for the humanized 2H7 variants is antibody at 12-14 mg/ml in 10 mM histidine, 6% sucrose, 0.02% polysorbate-20, pH 5.8.

In a specific embodiment, 2H7 variants, and particularly 2H7.vl6, are formulated at 20 mg/ml antibody in 10 mM Mstidine sulfate, 60 mg/ml sucrose, 0.2 mg/ml polysorbate-20, and sterile water for injection at pH 5 ,8.

The formulation herein may also contain more than one active compound necessary for the particular indication being treated, 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, cytokine or immunosuppressive agent (for example one that acts on T cells, such as cyclosporine or an antibody that binds T cells, for example one that binds LFA- 1). The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with acl administration as described here or approximately from 1 to 99% of the dosages used up to this point.

The active ingredients can also be entrapped in microcapsules prepared for example by means of coacervation techniques or by means of interphase polymerization, for example hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacylate) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980).

Sustained release preparations can be prepared. Suitable examples of sustained release compositions include semi-permeable matrices of solid, hydrophobic polymers containing the antagonist, where the matrices are in the form of molded articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Patent 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 LUPRON DEPOT (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for intravenous administration must be sterile. This can easily be achieved by filtration through sterile filtration membranes.

Crafted items and sets

Another embodiment of the invention is an article of manufacture containing materials useful in the treatment of autoimmune diseases and related conditions and CD-20 positive cancers such as non-Hodgkin's lymphoma. The article of manufacture comprises a container and a mark or package insert on or associated with the container. Suitable containers include, for example, bottles, tubes, syringes, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a preparation effective to treat the condition and may have a sterile port of entry (for example, the container may be an intravenous solution pack or a tube having a seal that can be pierced by a hypodermic injection needle). At least one active agent in the preparation is a CD-20-binding antibody according to the invention. The label or package insert indicates that the preparation is used to treat the particular condition. The label or package insert will further include instructions for administering the antibody preparation to the patient. Package inserts refer to instructions that are usually included in commercial packages of therapeutic products and that contain information about the indications, use, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In one embodiment, the package insert indicates that the preparation is used to treat non-Hodgkin's lymphoma.

In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), 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, suppressants, filters, needles, and syringes.

Kits are also provided that are useful for various purposes, such as for B-cell killing assays, as a positive control for apoptosis assays, for purification or immunoprecipitation of CD-20 from cells. For isolation and purification of CD-20, the kit may contain an anti-CD20 antibody coupled to beads (eg Sepharose beads). Kits can be provided containing the antibodies for detection and quantification of CD-20 in vitro, for example in an ELISA or in a Western blot. As with the article of manufacture, the kit includes a container and a label or package insert on or associated with the container. The container contains a preparation comprising at least one anti-CD20 antibody according to the invention. Additional containers may be included containing, for example, blocking agents and buffers, control antibodies. The label or package insert may provide a description of the preparation in addition to instructions relating to the intended in v/fra use or diagnostic use.

Cynomolgus monkey-CD-20

The invention also provides an isolated nucleic acid comprising the nucleotide sequence of SEQ ID NO: 24 of cynomolgus monkey CD-20 as shown in Figure 19.1, in one embodiment the nucleic acid is a cDNA. In one embodiment, the nucleic acid encoding monkey CD-20 is in an expression vector for expression in a host cell. The nucleotide sequence of SEQ ID NO: 24 in the expression vector is operably linked to an expression control sequence such as a promoter or promoter and enhacer. The expression control sequence can be the native sequence normally associated with the cynomolgus CD-20 gene or heterologous to the gene. Also provided is an isolated polypeptide comprising the amino acid sequence [SEQ ID NO: 25, Figures 19 and 20] of cynomolgus monkey CD-20, in addition to host cells containing the cynomolgus CD-20 nucleic acid. In one aspect, the host cells are eukaryotic cells, such as CHO cells. Fusion proteins comprising the cynomolgus CD-20 amino acid sequence or fragments thereof are also encompassed.

EXAMPLES

Example 1

Humanization of Murine 2H7 Anti-CD-20 Monoclonal Antibody Humanization of the murine anti-human CD-20 antibody 2H7 (also referred to herein as m2H7, m for murine) was performed in a series of site-directed mutagenesis steps. The variable region sequences of the murine 2H7 antibody and the chimeric 2H7 with mouse-V and human-C have been described, see, for example, U.S. Pat. U.S. Patent Nos. 5,846,818 and 6,204,023. The CDR residues of 2H7 were identified by aligning the amino acid sequence of the murine 2H7 variable domains (disclosed in U.S. Patent No. 5,846,818) with the sequences of known antibodies (Kabat et al., Sequences of proteins of immunological interest, ed. 5. Public Health Sevice, 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 Figure IA and Figure IB, respectively. Using synthetic oligonucleotides (Table 1), site-directed mutagenesis (Kunkel, Proe. Nati. Acad. Sei. 82:488-492 (1985)) was used to introduce all six murine 2H7 CDR regions into a fully human Fab framework corresponding to a consensus sequence VJ, VHIH (VL-kappa subgroup-I, VH-subgroup-II) contained in plasmid pVX4 (Figure 2).

The phagemid pVX4 (figure 2) was used for mutagenesis as well as for expression of F(ab) in E. coli. Based on phagemid pb0720, which is a derivative of pB0475 (Cunningham et al., Science 243:1330-1336 (1989)), pVX4 contains a DNA fragment encoding a humanized consensus K-subgroup I light chain-( VLKI-CL) and a humanized consensus subgroup-III heavy chain (VHIII-CH1) anti-IFN-a (interferon a) antibody. pVX4 also has an alkaline phosphatase promoter and a Shine-Dalgamo sequence both derived from another previously described pUC1 19-based plasmid, pAK2 (Carter et al., Proe. Nati. Acad. Sei. USA 89:4285 (1992) ). A unique Spel restriction site was introduced between the DNA encoding the F(ab) light and heavy chains. The first 23 amino acids of both the anti-IFN-α heavy and light chains are the StII secretion signal sequence (Chang et al., Gene 55:189-196 (1987)).

To construct the CDR-swapping version of 2H7 (2H7.v2), site-directed mutagenesis was performed on a deoxyuridine-containing template of pVX4, all six CDRs of the anti-IFN-α were changed to the murine 2H7 CDRs. The resulting molecule is referred to as humanized 2H7 version-2 (2H7.v2) or the "CDR swapped version" of 2H7, it has the m2H7 CDR residues with the human consensus FR residues shown in Figures IA and IB. Humanized 2H7.v2 was used for further humanization.

Table 1 shows the oligonucleotide sequence used to prepare each of the murine 2H7 (m2H7) CDRs in the H chain and the L chain. For example, the CDR-H1 oligonucleotide was used to recreate the m2H7-H chain CDR1. CDR-H1, CDR-H2 and CDR-H3 refer to H-chain CDR1, -CDR2 and -CDR3, respectively, and correspondingly CDR-L1, CDR-L2 and CDR-L3 refer to each of the L-chain CDRs . The substitutions in CDR-H2 were performed in two steps with two oligonucleotides, CDR-H2A and CDR-H2B.

For comparison with humanized constructs, a plasmid expressing a chimeric 2H7-Fab (containing murine VL and VH domains and human CL and CHr domains) was constructed by site-directed mutagenesis (Kunkel, supra) using synthetic oligonucleotides to introducing the murine framework residues into 2H7.v2. The sequence of the resulting plasmid construct for expression of the chimeric Fab known as 2H7.v6.8 is shown in Fig. 3. Each encoded chain in the Fab has a Stll secretion signal sequence of 23 amino acids as described for pVX4 (Fig. 2) above. .

Based on a sequence comparison of the murine 2H7 framework residues with the human VKI,VHIII consensus framework (Figures IA and IB) and previously humanized antibodies (Carter et al., Proe. Nati. Acad. Sei. USA 89:4285-4289 (1992 )), several framework mutations were introduced into the 2H7.v2-Fab construct by site-directed mutagenesis. These mutations lead to a change of certain human consensus framework residues to those found in the murine 2H7 framework at sites that may affect CDR conformations or antigen contacts. Version-3 contained VH(R71V, N73K), version-4 contained VH(R71V), version-5 contained VH(R71V,N73K) and VL(L46P) and version-6 contained VH(R71V, N73K) and VL(L46P , L47W).

Humanized and chimeric Fab versions of m2H7 antibody were expressed in E. coli and purified as follows. The plasmid was transformed into E. coli strain XL-1-Blue (Stratagene, San Diego, CA) for production of double-stranded and single-stranded DNA. For each variant, both light and heavy chains were completely sequenced using the (hdeoxynucleotide method (Sequenase, U.S. Biochemical Corp.). Plasmids were transformed into E. cø// strain 16C9, a derivative of MM294, plated on LB plates containing 5 µg/ml carbenicillin and a single colony was selected for protein expression.The single colony was grown in 5 ml LB 100 µg per ml carbenicillin for 5 hours at 37°C.

The 5 ml culture was added to 500 ml AP5 lOO^ig per ml of carbenicillin and allowed to grow for 16

hours in a shaken 4-liter bottle at 37 °C. AP5 media consists of: 1.5 g glucose, 11.0 Hycase SF, 0.6 g yeast extract (certified), 0.19 g anhydrous MgSO 4 , 1.07 g NH 4 Cl , 3.73 g KC 1 , 1.2 g NaCl, 120 ml 1 M triethanolamine, pH 7.4, to 11 water and then sterile filtered through a 0.1 µm Sealkeen filter.

Cells were harvested by centrifugation in a 1 liter centrifuge bottle (Nalgene) at 3000 xg and the supernatant was removed. After freezing for 1 hour, the pellet was resuspended in 25 ml of cold 10 mM MES-10 mM EDTA, pH 5.0 (buffer-A). 250 µl of 0.1 M PMSF (Sigma) was added to inhibit proteolysis and 3.5 ml of stock solution 10 mg/ml hen egg white lysozyme (Sigma) was added to aid in lysis of the bacterial cell wall. After gentle shaking on ice for 1 hour, the sample was centrifuged at 40,000 xg for 15 min. The supernatant was brought to 50 ml with buffer-A and applied to a 2 ml DEAE column equilibrated with buffer-A. The flow-through was then applied to a protein G-sepharose-CL-4B (Pharmacia) column (0.5 ml base volume) equilibrated with buffer-A. The column was washed with 10 ml of buffer A and eluted with 3 ml of 0.3 M glycine, pH 3.0 into 1.25 ml of 1 M tris, pH 8.0. F(ab) was then buffer exchanged into PBS using a Centricon-30 (Amicon) and concentrated to a final volume of 0.5 ml. SDS-PAGE gels of all F(ab) were run to ensure purity and the molecular weight of each variant was verified by electrospray mass spectrometry.

In cell-based ELISA binding assays (described below), the binding of Fabs, including chimeric 2H7 Fab, to CD20 was difficult to detect. Therefore, the 2H7 Fab versions were reformatted as full-length IgG1 antibodies for assays and further mutagenesis.

Plasmids for expression of full-length IgGs were constructed by subcloning the VL and VH domains of chimeric 2H7 (v6.8) Fab as well as humanized Fab versions 2 to 6 into previously described pRK vectors for mammalian cell expression ( Gorman et al., DNA Prot. Eng. Tech. 2:3-10 (1990)). Briefly, each Fab construct was digested with EcoRV and BlpI to excise a VL fragment that was cloned into the EcoRV/5/pI sites of plasmid pDRl

(Fig. 4) for expression of the complete light chain (VL-CL domains). In addition, each Fab construct was digested with Pvull and ApaI to cut out a VH fragment that was cloned into the PvullApaI set, in plasmid pDR2 (Fig. 5) to express the complete heavy chain (VH-CH1- hinge CH2-CH3 domains). For each IgG variant, transient transfections were performed by cotransfecting a light chain-expressing plasmid and a heavy chain-expressing plasmid into an adenovirus-transformed human embryonic kidney cell line, (Graham et al., J. Gen. Virol., 36:59-74, (1977) ). Briefly, 293 cells were split the day before transfection and plated in serum-containing medium. On the following day, double-stranded DNA prepared as a calcium phosphate precipitate was added, followed by pAdVAntage DNA (Promega, Madison, WI) and cells were incubated overnight at 37°C. Cells were grown in serum-free medium and harvested after 4 days. Antibodies were purified from culture supernatants using protein A-Sepharose-CL-4B and then the buffer was changed to 10 mM sodium succinate, 140 mM NaCl, pH 6.0 and concentrated using a Centricon-10 (Amicon). Protein concentrations were determined by quantitative amino acid analysis.

To measure relative bmclm<g>affinities to the CD20 antigen, a cell-based ELISA assay was developed. Human B-lymphoblastoid WIL2-S cells (ATCC CRL 8885, American Type Culture Collection, Rockville, MD) were grown in RPMI 1640 supplemented with 2 mM L-glutamine, 20 mM HEPES, pH 7.2, and 10% heat-inactivated fetal bovine serum in a humidified 5% CO2 incubator. The cells were washed with PBS containing 1% FBS (analysis buffer) and seeded at 250-300,000 cells per well in 96-well round-bottom plates (Nunc, Roskilde, Denmark). Two-fold serially diluted standard (15.6-1000 ng/ml with 2H7 v6.8 chimeric IgG) and three-fold serially diluted samples (2.7-2000 ng/ml) in assay buffer were added to the plates. The plates were buried in ice and incubated for 45 min. To remove the unbound antibody, 0.1 ml of assay buffer was added to the wells. Plates were centrifuged and supernatants were removed. Cells were washed two or more times with 0.2 ml assay buffer. Antibody bound to the plates was detected by adding peroxidase-conjugated goat anti-human FC antibody (Jackson ImmunoResearch, West Grove, PA) to the plates. After incubation for 45 minutes, the cells were washed as described previously. TMB substrate (3,3\5,5'-tetramethylbenzidine, Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the plates. The reaction was stopped by adding 1 M phosphoric acid. Titration curves were fitted with a four-parameter nonlinear regression curve fitting program (KaleidaGraph, Synergy software, Reading, PA). The absorbance at the midpoint of the titration curve (mid-OD) and its corresponding concentration of the standard were determined. Then the concentration of each variant of this mid-OD was determined and the concentration of the standard was divided by that of each variant. Thus, the values are a ratio of the binding of each variant relative to the standard. Standard deviation of relative affinity (equivalent concentration) was generally +/- 10% between experiments.

As shown in Table 2, binding of the CDR swap variant (v.2) was extremely reduced compared to chimera 2H7 (v.6.8). Nevertheless, versions 3 to 6 showed improved binding. To determine the minimum number of mutations necessary to restore bmclm<g>affinity to that of chimera 2H7, additional mutations and combinations of mutations were constructed by site-directed mutagenesis to produce variants 7 to 17 as shown in Table 2. In particular, these included VH mutations A49G, F67A, I69L, N73K and L78A, and VL mutations M4L, M33I and F71Y. Versions 16 and 17 showed the best relative bmcmi<g>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, which has only 4 mutations of human framework residues to murine framework residues (Table 2), was therefore chosen as the humanized form for further characterization.

Table 2. Relative bmclm<g>affinity of humanized 2H7 IgG variants to CD20

compared to chimera 2H7 using cell-based ELISA. The relative binding is expressed as the concentration of the chimeric 2H7 above the concentration of the variant required for equivalent binding, thus a ratio < 1 indicates weaker affinity for the variant. Standard deviation in relative affinity determination averaged +/- 10%.

Framework substitutions in the variable domains are relative to the CDR swap version according to the numbering system of Kabat (Kabat et al., supra). Table 3. Oligonucleotide sequences used for the construction of mutations VH(A49G, R71V, N73K) and VL(L46P) in humanized 2H7 version-16 (2H7.vl6). Underlined codons code for the indicated amino acid substitutions. For VH (R71V, N73K) and VL(L46P), the oligos are shown as the sense strand since these were used for mutagenesis on the Fab template, while for VH (A49G) the oligo is shown as the antisense strand, since this was used with pRK (IgG heavy chain) template. The protein sequence of version-16 is shown in Figure 6 and Figure 7.

Example 2

Antigen-binding determinants (paratope) for 2H7 Alanine substitutions (Cunningham & Wells, Science 244: 1081-1085 (1989)) were performed in 2H7.vl6 or 2H7.vl7 to test the contribution of individual side chains of the antibody in binding to CD20. IgG variants were expressed in 293 cells from pDR1 and pDR2 vectors, purified and analyzed for relative bmdin<g>affinity as described above Several alanine substitutions led to a significant decrease in relative binding to CD20 on WIL-2S cells (Table 4).

Table 4. Effects of alanine substitutions in CDR regions in humanized 2H7.vl6 measured using cell-based ELISA (WIL2-S cells). The relative binding is expressed as the concentration of the native 2H7.vl6 above the concentration of the variant required for equivalent binding, thus a ratio < 1 indicates weaker affinity for the variant; a ratio > 1 indicates higher affinity for the variant. Standard deviation in relative affinity determination averaged +/-10%. Framework substitutions in the variable domain are relative to 2H7.vl6 according to the numbering system of Kabat (Kabat et al., supra). NBD means no detectable binding. The two numbers for version-45 are from separate experiments.

Example 3

Additional mutations in 2H7 CDR regions

Substitutions of additional residues and combinations of substitutions at CDR positions identified as important by Ala scanning were also tested. Several combination variants, especially v.96 appeared to bind more tightly than v.16.

Table 5. Effects of combinations of mutations and iMce-alanine substitutions in the CDR regions of humanized 2H7.vl6 measured using self-based ELISA (WIL2-S cells). The relative binding to CD20 is expressed as the concentration of the original 2H7.vl6 above the concentration of the variant required for equivalent binding, thus a ratio < 1 indicates weaker affinity for the variant, a ratio > 1 indicates higher affinity for the variant. Standard deviation in relative affinity determination averaged +/- 10%. Framework substitutions in the variable domain are relative to 2H7.vl6 according to the numbering system of Kabat (Kabat et al., supra).

Example 4

Mutations at sites with framework humanization substitutions Substitutions of additional residues at framework positions that were altered during humanization were also tested in the 2H7.vl6 background. In particular, alternative framework substitutions that were not found in either the original murine 2H7 or the human consensus framework were made at VL(P46) and VH(G49, V71 and K73).

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

Table 6. Relative binding in a cell-based (WIL2-S) analysis of framework substitutions. IgG variants are shown with mutations with respect to the 2H7.vl6 background. The relative binding is expressed as the concentration of the chimeric 2H7.v6.8 above the concentration of the variant required for equivalent binding, thus a ratio <1 indicates weaker affinity for the variant; a ratio >1 indicates higher affinity for the variant. Standard deviation in relative affinity determination averaged +/- 10%. Framework substitutions in the variable domains are relative to 2H7.vl6 according to the numbering system of Kabat (Kabat et al., supra).

(<*>) Variants that were analyzed with 2H7.vl6 as the standard comparison, relative values are normalized to that of the chimera.

Example 5

Humanized 2H7 variants with improved effector functions Because 2H7 can mediate lysis of B cells via both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC), we sought to produce variants of humanized 2H7.vl6 with improved CDC and ADCC activity. Mutations of certain residues within the Fc regions of other antibodies have been described (Idusogie et al., J. Immunol. 166:2571 (2001)) to enhance CDC via enhanced binding to the complement component Clq. Mutations have also been described (Shields et al. al., J. Biol. Chem. 276:6591 (2001); Presta et al., Biochem. Soc. Trans. 30:487-490 (2002)) to enhance ADCC via enhanced IgG binding to activating Fcγ receptors and reduced IgG binding to inhibitory Fcγ receptors. In particular, three mutations have been identified to enhance CDC and ADCC activity: S298A/E333A/K3334A (also referred to herein as a triple Ala mutant or variant, numbering in Fc- the region is according to the EU numbering system, Kabat et al., supra) as described (Idusogie et al., supra (2001); Shields et al., supra).

To improve CDC and ADCC activity for 2H7, a triple Ala mutant of 2H7-Fc was prepared. A humanized variant of the anti-HER2 antibody 4d5 has been prepared with mutations S298A/E333A/K334A and is known as 4D5Fcl 10 (ie, anti-pl85HER2-IgGl (S298A/E333A/K334A), Shields et al., supra). A plasmid p4D5FcllO encoding antibody 4D5Fc 110 (Shields et al., supra) was digested with Med and HindlU, and the Fc fragment (containing mutations S298A/E333A/K334A) was ligated into ApaI/HindlH sites in the 2H7 heavy chain vector pDR2 -vl6 to make pDR2-v31. The amino acid sequence of the complete H chain of version 31 is shown in Figure 8. The L chain is the same as that of vl6.

Although the constant domain in the Fc region of IgG 1 antibodies is relatively conserved within a given species, allelic variation occurs (reviewed by Lefranc and Lefranc, in The human IgG subclasses: molecular analysis of structure, function, and regulation, pp. 43-78, F. Shakib (ed.), Pergammon Press, Oxford (1990)).

Table 7. Effects of substitutions in the Fc region on CD20 binding. Relative binding to CD20 was measured in a cell-based (WIL2-S) framework substitution assay. Fc mutations (<*>) are indicated by EU numbering (Kabat, supra) and are relative to the original 2H7.vl6. The combination of three Ala changes in the Fc region of v.31 is described as "Fel 10". IgG variants are shown with mutations with respect to the 2H7.vl6 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, thus a ratio <1 indicates weaker affinity for the variant. Standard deviation in relative affinity determination averaged +/- 10%.

Example 6

Humanized 2H7 variants with improved stability

For development as therapeutic proteins, it is desirable to select variants that remain stable with respect to oxidation, deamidation or other processes that may affect product quality, in an appropriate formulation buffer. 12H7.vl6, several residues have been identified as possible sources of instability: VL (M32) and VH (M34, N100). Therefore, mutations were introduced at these sites for comparison with vl6.

Table 8. Relative binding of 2H7 variants designed for improved stability and/or effector function to CD20 in a cell-based (WIL2-S) assay. IgG variants are shown with mutations with respect to the 2H7.vl6 background. The relative binding is expressed as the concentration of the chimeric 2H7.v6.8 above the concentration of the variant required for equivalent binding, thus indicating a ratio <1 weaker affinity for the variant. Standard deviation in relative affinity determination averaged +/- 10%. Framework substitutions in the variable domains are relative to 2H7.vl6 according to the numbering system of Kabat and Fc mutations (<*>) are indicated by EU numbering (Kabat et al., supra). (<**>) Variants measured with 2H7.vl6 as standard comparison, relative values are normalized to those of the chimera.

Additional Fc mutations were combined with stability- or affinity-enhancing mutations to alter or enhance effector functions based on previously reported mutations (Idusogie et al. (2000); Idusogie et al. (2001); Shields et al. (2001)). These changes included S298, E333A, K334A as described in Example 5, K322A to reduce CDC activity, D265A to reduce ADCC activity, K326A or K326W to enhance CDC activity and E356D/M358L to test the effects of allotypic changes in the Fc region. None of these mutations caused significant differences in CD20-bmclm<g>affinity.

To test the effects of stability mutations on the rate of protein degradation, 2H7.vl6 and 2H7.v73 were formulated at 12-14 mg/ml in 10 mM histidine, 6% sucrose, 0.02% polysorbate-20, pH 5.8 and incubated at 40 °C for 16 days. The incubated samples were then analyzed for changes in charge variants by ion exchange chromatography, aggregation and fragmentation by size exclusion chromatography and relative binding by testing in a cell-based (WIL2-S) assay.

The results (Fig. 9) show that 2H7v.73 has greater stability compared to 2H7v. 16 with respect to loss in the fraction in the main peak by ion exchange chromatography under accelerated stability conditions. No significant differences were seen with respect to aggregation, fragmentation or binding affinity.

Example 7

Scatchard analysis of antibody binding to CD20 on WIL2-S cells Equilibrium dissociation constants (Kd) were determined for 2H7-IgG variants that bind to WIL2-S cells using radiolabeled 2H7-IgG. IgG variants were produced in CHO cells. Rituxan (source for all experiments is Genentech, S. San Francisco, CA) and murine 2H7 (BD PharMingen, San Diego, CA) were used for comparison with humanized variants. The murine 2H7 antibody was also available from other sources, e.g. eBioscience and Calbiochem (both in San Diego, CA), Accurate Chemical & Scientific Corp., (Westbury, NY), Ancell (Bayport, MN), and Vinci-Biochem (Vinci, Italy). All dilutions were performed in binding assay buffer (DMEM medium containing 1% bovine serum albumin, 25 mM HEPES pH 7.2 and 0.01% sodium azide). Volumes (0.025 ml) of I-2H7.vl6 (iodinated with lactoperoxidase) at a concentration of 0.8 nM were added to wells of a V-bonded 96-well microassay plate and serial dilutions (0.05 ml) of cold antibody were added and mixed. WIL2-S cells (60,000 cells in 0.025 ml) were then added. The plate was sealed and incubated at room temperature for 24 hours, then centrifuged for 15 minutes at 3,500 RPM. The supernatant was then aspirated and the cell pellet was washed and centrifuged. The supernatant was again aspirated and the pellets were dissolved in IN NaOH and transferred to tubes for gamma counting. The data were used for Scatchard analysis (Munson and Rodbard, Anal. Biochem. 107:220-239 (1980) using the program Ligand (McPherson, Comput. Programs Biomed. 17:107-114 (1983)). These results which are shown in Table 9 indicates that humanized 2H7 variants had similar CD20-bin(lmg affinity as compared to murine 2H7, and similar binclmg affinity to Rituxan. It is expected that 2H7.v31 will have very similar Kdmed v. 16 on the basis of the binding that is shown in Table 7 above.

Example 8

Complement-Dependent Cytotoxicity (CDC) Assays 2H7-IgG variants were assayed for their ability to mediate complement-dependent lysis of WIL2-S cells, which are CD20-expressing lymphoblastoid B cell lines, essentially as described (Idusogie et al., J. Immunol. 164:4178-4184 (2000); Idusogie et al., J. Immunol. 166:2571-2575 (2001)). Antibodies were serially diluted 1:3 from a stock solution of 0.1 mg/ml. A volume of 0.05 ml of each dilution was added to a 96-well tissue culture plate containing 0.05 ml of a normal human complement solution (Quidel, San Diego, CA). To this mixture, 50,000 WIL2-S cells were added in a volume of 0.05 ml. After incubation for 2 h at 37 °C, 0.05 ml of a solution of Alamar boat (Accumed International, Westlake, OH) was added and incubation was continued

for a further 18 hours at 37 °C. The covers were then removed from the plates and they were shaken for 15 minutes at room temperature on an orbital shaker. Relative fluorescence units (RFU) were read using an excitation filter of 530 nm and an emission filter of 590 nm. An EC50 value was calculated by fitting the RFU as a function of concentration for each antibody using KaleidaGraph software.

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

Table 10. CDC activity for 2H7 antibodies compared to Rituxan. Values

> 1 indicates less strong CDC activity than Rituxan and values < 1 indicate stronger activity than Rituxan. Antibodies were prepared from stable CHO lines, except that those indicated by (<*>) were transiently prepared.

Example 9

Antibody-dependent cellular cytotoxicity (ADCC) assays

2H7 IgG variants were analyzed for their ability to mediate natural killer (NK) cell lysis of WIL2-S cells, which is a CD20-expressing lymphoblastoid B cell line, essentially as described by (Shields et al., J. Biol. Chem. 276:6591-6604

(2001)) by using a lactate dehydrogenase (LDH) solution. NK cells were prepared from 100 ml of heparinized blood, diluted with 100 ml of PBS (phosphate-buffered saline), obtained from normal human donors that had been isotyped for FcγRIII, also known as CD16 (Koene et al., Blood 90:1109-1114 ( 1997)). In this experiment, the NK cells were from human donors who were heterozygous for CD16 (F158/V158). The diluted blood was overlaid with 15 ml of lymphocyte separation medium (ICN Biochemical, Aurora, Ohio) and centrifuged for 20 minutes at 2000 RPM. White cells in the transition between layers were transferred to 4 clean 50 ml tubes, which were filled with RPMI medium containing 15% fetal calf serum. Tubes were centrifuged for 5 min at 1400 RPM and the supernatant was discarded. Pellets were resuspended in MACS buffer (0.5% BSA, 2 mM EDTA) and NK cells were purified using beads (NK cell isolation kit, 130-046-502) according to the manufacturer's protocol (Miltenyi Biotech). NK cells were diluted in MACS buffer to 2x10<6> cells/ml.

Serial dilutions of antibody (0.05 ml) in assay medium (F12/DMEM 50:50 without glycine, 1 mM HEPES buffer pH 7.2, penicillin/streptomycin (100 units/ml; Gibco), glutamine, and 1% heat-inactivated fetal bovine serum ) was added to a 96-well round tissue culture plate. WIL2-S cells were diluted in assay buffer to a concentration of 4 x 10<5>/ml. WIL2-S cells (0.05 ml per well) were mixed with diluted antibody in the 96-well plate and incubated for 30 min at room temperature to allow binding of antibody to CD20 (opsonization).

The ADCC reaction was started by adding 0.1 ml of NK cells to each well. In control wells, 2% Triton X-100 was added. The plate was then incubated for 4 hours at 37°C. Levels of LDH released were measured using a cytotoxicity (LDH) detection kit (Kit #1644793, Roche Diagnostics, Indianapolis, Indiana) following the manufacturer's instructions. 0.1 ml of LDH developer was added to each well followed by mixing for 10 sec. The plate was then covered with aluminum foil and incubated in the dark at room temperature for 15 min. Optical density at 490 nm was then read and used to calculate % lysis by dividing by the total LDH measured in control wells. Lysis was plotted as a function of antibody concentration and a 4-parameter curve fit (KaleidaGraph) was used to determine EC50 concentrations.

The results showed that humanized 2H7 antibodies were active in ADCC with relative potency 20-fold higher than Rituxan for v.31 and v.75, 5-fold stronger than Rituxan for v.16 and almost 4-fold stronger than Rituxan for v.73.

Additional ADCC assays were performed to compare combination variants of 2H7 with Rituxan. The results of these assays indicated that 2H/.vl 14 and 2H7.vl 15 have more than 10-fold improved ADCC potency compared to Rituxan (Table 12).

Example 10

In vivo effects of 2H7 variants in a pilot study in cynomolgus monkeys 2H7 variants produced by transient transfection of CHO cells were tested in normal cynomolgus monkeys {Macaca fascicularis) to evaluate their in vivo activities. Other anti-CD20 antibodies, such as C2B8 (Rituxan) have shown an ability to deplete B cells in normal primates (Reff et al., Blood 83:435-445 (1994)).

In one study, humanized 2H7 variants were compared. In a parallel investigation, Rituxan was also tested in cynomolgus monkeys. Four monkeys were used in each of five dosage groups: (1) vehicle, (2) 0.05 mg/kg hu2H7.vl6, (3) 10 mg/kg hu2H7.vl6, (4) 0.05 mg/kg hu2H7. v31 and (5) 10 mg/kg hu2H7.v31. Antibodies were administered intravenously at a concentration of 0, 0.2, or 20 mg/ml in a total of two doses, one on day 1 of the study and another on day 8. The first day of dosing is designated day 1 and the previous day is thus day -1, the first day of recovery (for 2 animals in each group) is designated day 11. Blood samples were collected on days -19, -12, 1 (before dosing) and at 6 hours, 24 hours and 72 hours after the first dose. Additional samples were taken on day 8 (before dosing), day 10 (before killing 2 animals per group) and on days 36 and 67 (for recovering animals).

Peripheral B-cell concentrations were determined using a FACS method that counted CD3-/CD40+ cells. The percentage of CD3-CD40+ B cells of total lymphocytes in monkey samples was obtained using the following entry strategy. The lymphocyte population was marked on the forward scatter/side scatter scatter diagram to define Region 1 (R1). Using events in RI, fluorescence intensity dotplots were displayed for CD40 and CD3 markers. Fluorescently labeled isotype controls were used to determine respective cut-off points for CD40 and CD3 positivity.

The results indicated that both 2H7.vl6 and 2H7.v31 were able to produce full peripheral B cell depletion at the 10 mg/kg dose and partial peripheral B cell depletion at the 0.05 mg/kg dose (Fig. 11). The time course and extent of B-cell depletion measured during the first 72 hours of dosing were similar for the two antibodies. Subsequent analyzes of the recovering animals indicated that animals treated with 2H7.v31 showed a prolonged depletion of B cells compared to those dosed with 2H7.vl6. In particular, recovering animals treated with 10 mg/kg 2H7.vl6 showed substantial B-cell recovery at a time point between sampling on day 10 and on day 36. For recovering animals treated with 10 mg/kg 2H7.v31, however, B -cells recovery until a time between day 36 and day 67 (Figure 11). This suggests a longer duration of full depletion of approximately one month for 2H7.v31 compared to 2H7.vl6.

No toxicity was observed in the monkey study at low or high doses and the overall pathology was normal. In other investigations, vi6 was well tolerated up to the highest dose assessed (100 mg/kg x 2 = 1200 mg/m x 2) after i.v. administration of two doses given 2 weeks apart to these monkeys.

Data from cynomolgus monkeys with 2H7.vl6 relative to Rituxan suggest that a 5-fold reduction in CDC activity does not adversely affect potency. An antibody with strong

ADCC activity but reduced CDC activity may have a more favorable safety profile with respect to first infusion reactions than one with higher CDC activity.

Example 11

Fucose-deficient 2H7 variant antibodies with improved effector function Normal CHO and HEK293 cells attach fucose to IgG oligosaccharide to a high degree (97-98%). IgG from serum is also highly fucosylated.

DP12, which is a dihydrofolate reductase minus (DHFR ) CHO cell line that is fucosylation competent, and Lee 13, which is a cell line that lacks protein fucosylation were used to produce antibodies for this investigation. The CHO cell line Pro-Lecl3.6a (Lecl3) was obtained from Professor Pamela Stanley of the Albert Einstein College of Medicine of Yeshiva University. Maternal lines are Pro- (proline auxotroph) and Gat-(glycine, adenosine, thymidine auxotroph). The CHO-DP12 cell line is a derivative of the CHO-K1 cell line (ATCC#CCL-61), which lacks dihydrofolate reductase and has a reduced need for insulin. Cell lines were transfected with cDNA using the Superfect method (Qiagen, Valencia, CA). Selection of the Lecl3 cells expressing transfected antibodies was performed using puromycm dihydrochloride (Calbiochem, San Diego, CA) at 10 ng/ml in growth medium containing: MEM-alpha medium with L-glutamine, ribonucleosides and deoxyribonucleosides (GIBCO-BRL) , Gaithersburg, MD), supplemented with 10% inactivated FBS (GIBCO), 10 mM HEPES, and IX penicillin/streptomycin (GIBCO). The CHO cells were similarly selected in growth medium containing Hams F12 without GHT: Low glucose DMEM without glycine with NaHCO 3 supplemented with 5% FBS (GIBCO), 10 mM HEPES, 2 mM L-glutamine, IX GHT (glycine, hypoxanthine, thymidine) and IX penicillin/streptomycin.

Colonies formed within two to three weeks and were pooled for expansion and protein expression. The pooled cells were initially seeded at 3 x 106 cells per 10 cm plate for small scale protein expression. The cells were transferred to serum-free medium once they had grown to 90-95% confluence and after 3-5 days cell supernatants were collected and tested in an Fc-IgG and intact IgG-ELISA to calculate protein expression levels. Lee 13 and CHO cells were seeded at approximately 8x10<6> cells per 15 cm plate one day before conversion to PS24 production medium supplemented with 10 mg/l recombinant human insulin and 1 mg/l trace elements.

Lecl3 cells and DP12 cells remained in serum-free production medium for 3-5 days. Supernatants were collected and clarified by centrifugation in 150 ml conical tubes to remove cells and debris. The protease inhibitors PMSF and 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 rapid purification using protein G chromatography ( Amersham Pharmacia Biotech, Piscataway, NJ)). All proteins were buffer exchanged to phosphate-buffered saline (PBS) using Centripriep-30 concentrators (Amicon) and analyzed by SDS-polyacrylamide gel electrophoresis. Protein concentrations were determined using A280 and confirmed using amino acid composition analysis.

The CHO cells were transfected with vectors expressing humanized 2H7vl6, 2H7v.31 and selected as described. The 2H7vl6 antibody maintains the wild-type Fc region while v.31 (see Example 5, Table 7 above) has an Fc region in which 3 amino acid changes were made (S298A, E333A, K334A) leading to higher affinity for the FcγRIIIa receptor ( Shields et al. J. Biol. Chem. 276(9):6591-6604 (2001)). After transfection and selection, individual colonies of cells were isolated and assessed for protein expression level and those that produced the most were subjected to methotrexate selection to select for cells that had increased plasmid copy number and therefore produced higher levels of antibody. Cells were cultured, transferred to serum-free medium for a period of 7 days and then the medium was collected, applied to a protein A column and the antibody eluted using standard techniques. The final concentration of the antibody was determined by using an Elisa that measures intact antibody. All proteins were buffer exchanged to phosphate buffered saline (PBS) using Centripriep-30 concentrators (Amicon) and analyzed on SDS-polyacrylamide gel electrophoresis.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectral Analysis of Asparagine-Linked Oligosaccharides: N-linked oligosaccharides were released from recombinant glycoproteins using the procedure of Papac et al, Glycobiology 8, 445-454 (1998) . Briefly, the wells of a 96-well PVDF-coated microtiter plate (Millipore, Bedford, MA) were conditioned with 100 µl methanol drawn through the PDVF membranes by applying vacuum on the Millipore-Multiscreen vacuum manifold. The conditioned PVDF membranes were washed with 3 x 250 µl of water. Between all washing steps, the wells were drained completely by gently applying vacuum to the manifold. The membranes were washed with reduction and carboxymethylation buffer (RCM) consisting of 6 M guanidine hydrochloride, 360 mM Tris, 2 mM EDTA, pH 8.6. Glycoprotein samples (50 ng) were added to individual wells, again pulled through the PVDF membranes using gentle vacuum and the wells were washed with 2 x 50 RCM buffer. The immobilized samples were reduced by adding 50 µl of a 0.1 M dithiothreitol (DTT) solution to each well and incubating the microtiter plate at 37 °C for 1 hour. DTT was removed by vacuum and the wells were washed 4 x 250 µl water. Cysteine residues were carboxymethylated by the addition of 50 with a 0.1 M iodoacetic acid (IAA) solution freshly prepared in 1 M NaOH and diluted to 0.1 M with RCM buffer. Carboxymethylation was carried out by incubation for 30 min in the dark at ambient temperature. Vacuum was applied to the plate to remove the IAA solution and the wells were washed with 4 x 250 purified water. The PVDF membranes were blocked by adding 100 µl of 1% PVP360 (polyvmylpyrrolidine 360000 MW) (Sigma) solution and incubated for 1 hour at ambient temperature. The PVP-360 solution was removed by light vacuum and the wells were washed 4 x 250 µl water. The PNGase-F (New England Biolabs, Beverly, MA) digestion solution, 25 µl with a 25 unit/ml solution in 10 mM Tris-acetate, pH 8.4 was added to each well and the digestion continued for 3 h at 37 °C. After digestion, the samples were transferred to 500 µl Eppendorf tubes and 2.5 µl of a 1.5 M acetic acid solution was added to each sample. The acidified samples were incubated for 3 hours at ambient temperature to convert the oligosaccharides from glycosylamines to the hydroxyl form Prior to MALDI-TOF mass spectral analysis, the released oligosaccharides were desalted using a volume of 0.6 mL of cation exchange resin (AG50W-X8 resin in the hydrogen form) (Bio-Rad, Hercules, CA) that was lightly packed in compact reaction tubes (US Biochemical, Cleveland, OH).

For MALDI-TOF mass spectral analysis, the samples were in the positive mode, the desalted oligosaccharides (0.5 µl volumes) were applied to the unstained target with 0.5 µl of the 2,5-(lmy(oxybenzoic acid) matrix (sDHB) as was prepared by dissolving 2 mg of 2,5-dihydroxybenzoic acid with 0.1 mg of 5-methoxysilicylic acid in 1 ml of ethanol/10 mM sodium chloride 1:1 (v/v). The sample/matrix mixture was vacuum dried. For analysis in the negative mode, the desalted N-linked oligosaccharides (0.5 µl volumes) were applied to the unstained target together with 0.5 µl 2\ 4', 6'-trm<y>(loxyacetophenone) matrix (THAP) prepared in 1 :3 (v/v) acetonitrile/13.3 mM ammonium citrate buffer. The sample/matrix mixture was vacuum dried and then allowed to absorb atmospheric moisture before analysis. Released oligosaccharides were analyzed by MALDI-TOF on a PerSeptive-BioSystems-Voyager- DE mass spectrometer The mass spectrometer was operated at 20 kV in either the positive or negative mode with the linear configuration and v ed to use delayed extraction. Data were collected using a laser power of 1300 and in data summarization mode (240 scans) to improve signal to noise. The instrument was calibrated with a mixture of standard oligosaccharides and the data were fitted using a 19-point Savitsky-Golay algorithm before masses were assigned. Integration of the mass spectral data was achieved using the Caesar 7.0 data analysis software package (SciBridge Software).

Natural killer (NK) cell antibody-dependent cytotoxicity assays

ADCC assays were performed as described in Example 9. The ratio of NK cells to target cell (WIL2-S) was 4 to 1, assays were run for 4 hours and toxicity was measured as before using lactose dehydrogenase assay. Target cells were opsonized with the concentrations of antibody indicated for 30 min before addition of NK cells. The Rituxan antibody used was from Genentech (S. San Francisco, CA). Figure 12 shows the results for a representative ADCC assay.

The results show that underfucosylated antibodies mediate NK cell target cell killing more efficiently than antibodies with a full complement of fucose. The underfucosylated antibody, 2H7v.31, is most effective in mediating target cell killing. This antibody is effective at lower concentrations and able to mediate killing of a greater percentage of target cells at higher concentrations than other antibodies are. The activity of the antibodies is as follows: Lee 13-derived 2H7v31>Lec 13-derived 2H7vl6>Dpl2-derived 2H7v31>Dpl2-derived 2H7vl6> or = with Rituxan. The protein and carbohydrate changes are additive. Comparison of the carbohydrate found on native IgG from the Lee 13-produced and CHO-produced IgG showed no significant differences in the extent of galactosylation and thus the results can be attributed only to the presence/absence of fucose.

Example 12

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

This example describes in vivo ADCC activity of the fucose-deficient humanized 2H7 variants including v. 16 and v. 31 produced in Lee 13 compared to normal fucosylated counterparts produced in DP12, in mice expressing human CD16[FcRyIII] and human CD20.

Generation of huCD20Tg<+>huCD16Tg<+>mCDlGf' mice

Human CD20 transgenic mice were generated from human CD20-BAC DNA (Invitrogen, Carlsbad, CA). Mice were screened based on the FACS analysis of human CD20 expression. HuCD20 Tg<+->mice were then crossed with huCD16Tg+mCD16 "" mice to obtain huCD20Tg<+>huCD16Tg<+>mCD^ mice.

In vivo treatment

Ten to 100 µg of each of the 2H7 variants or Rituxan were administered to huCD20Tg<+>huCD16Tg<+>mCD16"</>" mice via intraperitoneal injections. Equal amounts of isotype-matched antibodies will be added correspondingly to the negative control group of animals.

Preparation of mouse lymphocytes

Mouse lymphocytes from whole blood, spleen, lymph nodes and bone marrow were prepared according to the standard protocol described in "Current Protocols in Immunology, published by John Coligan, Ada Kruisbeek, David Margulies, Ethan Shevach and Warren Strober, 1994".

FACS analysis

Half a million cells were washed and resuspended in 100 µl FACS buffer which is phosphate buffered saline with 1% BSA containing 5 µl staining or control antibody. All staining antibodies including isotype controls were obtained from PharMingen, San Diego, CA. Human CD20 expression was examined by staining with Rituxan together with FITC-conjugated anti-human IgG1 secondary antibody. FACS analysis was performed using FACScan and Cell Quest (Becton Dickinson Immunocytometry Systems, San Jose, CA). All lymphocytes are defined in the forward light scatters and side light scatters, while all B lymphocytes are defined by the expression of B220 on the cell surface.

B cell depletion and recovery were examined by analyzing peripheral B cell counts and analysis of hCD20+ B cells by FACS in the spleen, lymph node and bone marrow on a daily basis for the first week after injection and then on a weekly basis. Serum levels of the injected 2H7 variant antibody were monitored.

The results of this in vitro analysis confirm the in vitro findings of increased ADCC activity and higher B cell depletion of fucose-deficient 2H7 variants relative to wild-type (with respect to fucosylation) glycosylation counterparts.

Example 13

Apoptosis activity

Anti-CD20 antibodies including Rituxan have been shown to induce apoptosis in vitro when cross-linked to a secondary antibody or by chemical means (Shan et al., Blood 9:1644-1652 (1998); Byrd et al., Blood 99 :1038-43 (2002); Pederson et al., Blood 99:1314-19 (2002)). When chemically cross-linked murine 2H7 dimers induced apoptosis in Daudi cells (Ghetie et al., Proe Nati Acad Sei USA 94:7509-14 (1997)). Cross-linking with a secondary antibody also induced apoptosis with the murine 2H7 antibody (Shan et al., 1998). These activities are believed to be physiologically relevant because a variety of mechanisms can lead to loys binding of anti-CD20 antibodies bound to cell surface CD20 in vivo.

RhuMAb 2H7.vl6 [humanized 2H7 v 16; RhuMAb stands for recombinant human monoclonal antibody] and Retuxan were compared in an in vitro apoptosis assay using a secondary cross-linking antibody. Ramos cells (CRL-1596, ATCC, Manassas, VA), which is a CD20-expressing human B-lymphocyte cell line, were used to measure the potency of the anti-CD20 monoclonal antibodies rhuMAb 2H7.vl6 and Rituximab versus a negative control antibody , Trastuzumab (Herceptin, Genentech, South San Francisco, CA), to induce apoptosis as measured by Annexin-V staining and propidium iodine dye exclusion (Vybrant Apoptosis Assay Kit, Molecular Probes, Seattle, WA). The Ramos cells were grown in RPMI-1640 medium (Gibco, Rockville, MD) containing 10% fetal bovine serum (Biosource International, Camarillo, CA) and 2 mM L-glutamine (Gibco). Before being analyzed, the cells were washed twice in fresh medium and then adjusted to a cell concentration of 2 x 106 per ml. Cells (150 µl) were added to 96-well assay plates (Becton Dickinson, Palo Alto, CA) containing 150 µl of a predetermined amount of control IgGl, rhuMAb 2H7.vl6, or Rituximab together with F(ab),2 -goat-anti-human-Fc (Pierce Biotechnology, Rockford, IL). The final IgG concentrations were 100, 10, 1.0, 0.1, 0.001 and 0.001 nM and the F(ab),2-goat anti-human Fc antibody concentration was set at twice the respective sample antibody concentration. Each dilution was set up in triplicate. After a 24 h incubation at 37 °C, the cells were washed twice with PBS and then stained with Annexin-V and propidium iodide according to the manufacturer's recommendations. The staining patterns of the Ramos cells were analyzed by flow cytometry using a FACscan flow cytometer (Becton Dickinson, San Jose, CA) and data were collected in 10 second periods. Data were reduced using Cellquest-pro software (Becton Dickinson). Ramos cells positive for (1) Annexin-V staining, (2) Annexin-V and propi(humiodide double staining, and (3) the number of unstained live cells were counted and plotted using KaleidaGraph software (Synergy Software, Reading, PA).

Both rhuMAb 2H7.vl6 and Rituximab induced apoptosis in Ramos cells when cross-linked with anti-human Fc and as compared to an irrelevant IgG1 control antibody (Figures 13-15). The apoptotic activity of (rhuMAb 2H7) was somewhat lower than that of Rituximab. At 10 nM concentrations of cross-linked rhuMAb 2H7, Rituximab and control IgGl antibody, fractions of Annexin-V stained cells were 18.5, 16.5, 2.5%, respectively, fractions of double-labelled cells were 29, 38 and 16% and the number of living cells counted per 10 sec was 5200, 3100 and 8600.

These in v/fra data demonstrate that apoptosis is one potential mechanism for in vivo B-cell depletion. In v/vo-loyssbmding of rhuMAB 2H7 or Rituximab bound to cell surface CD20 can take place via FcyR on the surfaces of immune effector cells.

Example 14

In v/v suppression of tumor growth

The ability of rhuMAb 2H7.vl6 to inhibit the growth of the human Raji-B cells, which is a lymphoma cell line (ATCC CCL 86), was evaluated in Balb/c nude (athymic) mice. The Raji cells express CD20 and have been reported to grow in nude mice where they lead to metastatic disease, tumor growth being inhibited by Rituxan (Clynes et al., Nature Medicine 6, 443-446 (2000)). Fifty-six 8-10 week old Balb/c nude mice were divided into 7 groups (A-G) where each group consists of 8 mice. On day 0, each mouse received a subcutaneous injection of 5 x 10<6>Raji B lymphoma cells in the flank. Starting on day 0, each mouse received either 100 of the negative control solution (PBS, phosphate buffered saline), Rituxan, or 2H7.vl6. Dosage was dependent on weight and drug delivery was intravenous via the needle vein. Group-A mice received PBS. Groups B-D received Rituxan at 5.0 mg/kg, 0.5 mg/kg and 0.05 mg/kg. Groups E-G mice received 2H7v. 16 at respectively 5.0 mg/kg, 0.5 mg/kg and 0.05 mg/kg. The injections were repeated every week for 6 weeks. At weekly intervals during treatment, each mouse was inspected for the presence of obvious tumors at the site of injection, and the volume of the tumors if present was measured and recorded. A final inspection was done at week 8 (after a two-week interval with no treatments).

The results of this study showed that both rhuMAb 2H7.vl6 and Rituxan were effective in inhibiting subcutaneous Raji cell tumor growth in nude mice (FIGS 16-18). Tumor growth was observed in the PBS control group from the fourth week. Nevertheless, no tumor growth was observed in groups treated with Rituxan or 2H7.vl6 at 5 mg/kg or 0.5 mg/kg during the 8 week duration of the study. In the low dose treatment groups with 0.05 mg/kg, tumor was observed in one animal in the 2H7 group and in one animal in the Rituxan group (FIG. 18)

Example 15

Cloning of cynomolgus monkey CD20 and antibody binding The cynomolgus monkey (Macaca fascicularis) CD20 DNA sequence was determined by the isolation of cDNA encoding CD20 from a cynomolgus spleen cDNA library. A SUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat. No. 18248-013, Invitrogen, Carlsbad, CA) was used with slight modifications to construct the library. The cDNA library was ligated into a pRK5E vector using the restriction sites Xho I and Not I. mRNA was isolated from spleen tissue (California Regional Research Primate Center, Davis, CA). Primers to amplify cDNA encoding CD20 were designed based on non-coding sequences from human CD20. N-terminal region primer 5'-AGTTTTGAGAGCAAAATG-3' (SEQ ID NO. 37) and C-terminal region primer 5'-AAGCTATGAACACTAATG-3' (SEQ ID NO. 38) were used by polymerase chain reaction (PCR) to clone cDNA which encodes cynomolgus monkey CD20. The PCR reaction was performed using Platinum-Taq-DNA polymerase-High Fidelity according to the manufacturer's recommendation (Gibco, Rockville, MD). The PCR product was subcloned into pCR 2.1-TOPO vector (Invitrogen) and transformed into XL-1-blue E. coli (Stratagene, La Jolla, CA). Plasmid DNA containing ligated PCR products was isolated from individual clones and sequenced.

The amino acid sequence for cynomolgus monkey CD20 is shown in Figure 19. Figure 20 shows a comparison of cynomolgus CD20 and human CD20. Cynomolgus monkey CD20 is 97.3% similar to human CD20 with 8 differences. The extracellular domain contains one change at V157A while the remaining 7 residues 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 cynomolgus monkey cells expressing CD20. Twenty mL of blood was drawn from 2 cynomolgus monkeys (California Regional Research Primate Center, Davis, CA) into sodium heparin and shipped directly to Genentech Inc. On the same day, blood samples were pooled and diluted 1:1 by adding 40 mL of phosphate-buffered saline

(PBS). 20 ml of diluted blood was plated onto 4 x 20 ml Ficoll-Paque™Plus (Amersham, Biosciences, Uppsala, Sweden) in 50 ml conical tubes (Cat. No. 352098, Falcon, Franklin Lakes, NJ) and centrifuged at 1300 rpm for 30 minutes R.T. in a Sorval 7 centrifuge. (Dupont, Newtown, CT). The PBMC layer was isolated and washed in PBS. Red blood cells were lysed in a 0.2% NaCl solution, brought back to isotonicity with an equivalent volume of a 1.6% NaCl solution, and centrifuged for 10 min at 1000 rpm. The PBMC pellet was resuspended in RPMI 1640 (Gibco, Rockville, MD) containing 5% fetal bovine serum (FBS) and added to a 10 cm tissue culture dish for 1 h at 37°C. The non-adherent 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 into twenty 12 x 75 mm culture tubes (Cat no. 352053, Falcon), where each tube contained 1 x 10<6> cells in a volume of 0.25 ml. Tubes were divided into four sets of five tubes. To each set was added either medium (RPMI1640, 5% FBS), titrated amounts of human control IgGi antibody, Rituxan, 2H7.vl6 or 2H7.v31. The final concentration of each antibody was 30, 10, 3.3 and 1.1 nM. In addition, each tube also received 20 Fluorescein Isothiocyanate (FITC)-conjugated anti-human CD20 (Cat. No. 555622, BD Biosciences, San Diego, CA). The cells were gently mixed, incubated for 1 h on ice and then washed twice in cold PBS. The cell surface staining was analyzed on an Epic-XL-MCL (Coulter, Miami, FL), the geometric means were derived, plotted (KaleidaGraph Synergy Software, Reading, PA) against antibody concentrations.

Data in Figure 21 show that 2H7 v.16 and 2H7 v.31 completely displaced FITC-murine 2H7 binding to cynomolgus monkey cells. Furthermore, Rituxan also displaced FITC murine 2H7 binding thus showing that both 2H7 and Rituxan bind to an overlapping epitope on CD20. In addition, the data show that the IC50 value for 2H7 v. 16, 2H7 v. 31 and Rituxan are similar and falls within the 4-6 nM range.

Example 16

Phase-I/I I study of rhuMAb 2H7 (2H7.vl6) in moderate

to severe rheumatoid arthritis

Protocol Synopsis

A randomized, placebo-controlled, multicenter, blinded phase I/II study of the safety of escalating doses of PRO70769 (rhuMAb 2H7) in subjects with moderate to severe rheumatoid arthritis receiving stable doses of concomitant methotrexate.

Goal

The primary objective of this study is to evaluate the safety and tolerability of escalating intravenous (IV) doses of PRO70769 (rhuMAb 2H7) in subjects with moderate to severe rheumatoid arthritis (RA).

Design of survey

This is a randomized, placebo-controlled, multicenter, blinded phase-I/II, investigator- and subject-blinded study of the safety of escalating doses of PRO70769 in combination with MTX in subjects with moderate to severe RA. The investigation consists of a dose escalation phase and a second phase with enrollment of a larger number of individuals. The sponsor will remain uninvolved in processing.

Individuals with moderate to severe RA who have not responded to one to five disease-modifying antirheumatic drugs or biological agents who, to date, have unsatisfactory clinical responses to treatment with MTX will be enrolled.

Subjects will be required to receive MTX in the range of 10-25 mg weekly for at least 12 weeks prior to study entry and to be on a stable dose for at least 4 weeks prior to receiving their first dose of study drug (PRO70769 or placebo). Individuals may also receive stable doses of oral corticosteroids (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 15 according to the following dose escalation schedule (see

Figure 22).

Dose escalation will take place according to specific criteria and after review of safety data by an internal safety data review committee and assessment of acute toxicity 72 hours after the second infusion in the last individual treated in each ward. After the dose escalation phase, 40 additional subjects (32 active and 8 placebo) will be randomized to each of the following dose levels: 2x50 mg, 2x200 mg, 2x500 mg and 2x1000 mg if the dose levels have been shown to be tolerable during the dose escalation phase. Approximately 205 individuals will be enrolled in the survey.

B-cell counts will be obtained and recorded. B-cell counts will be evaluated using flow cytometry in a 48-week follow-up period after the 6-month efficacy evaluation. B-cell depletion will not be considered a dose-limiting toxicity (DLC) but rather the expected pharmacodynamic outcome of PRO70769 treatment.

In any further investigation, blood for serum and RNA analyses, in addition to urine samples, will be obtained from individuals at various times. These samples may be used to identify biomarkers that may be predictive of response to PRO70769 treatment in individuals with moderate to severe RA.

Results

The primary outcomes of this study are the safety and tolerability of PRO70769 in subjects with moderate to severe RA.

Investigation processing

Wards with subjects will receive two IV infusions of PRO70769 or placebo equivalent at the indicated dose on days 1 and 15 according to the following escalation schedule:

-10 mg PRO70769 or placebo equivalent: 4 subjects active drug, 1 control

- 50 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2 controls

- 200 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2 controls

- 500 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2 controls

- 1000 mg PRO70769 or placebo equivalent: 8 subjects active drug, 2 controls

Efficiency

The efficacy of PRO70769 will be measured using ACR responses. The percentage of individuals achieving an ACR20, ACR50 and ACR70 response will be summarized by treatment group and 95% confidence intervals will be generated for each group. The components of these responses and their changes from baseline will be summarized at treatment and visit.

Conclusion

The above data demonstrate the success of producing humanized CD20-binding antibodies, particularly humanized 2H7 antibody variants, which maintained and even improved their biological properties. The humanized 2H7 antibodies of the invention bound to CD20 with affinities similar to the murine donor and chimeric 2H7 antibodies and were effective in killing B cells in a primate, leading to B cell depletion. Certain variants showed improved ADCC relative to a chimeric anti-CD20 antibody currently used to treat NHL, promoting the application of lower doses of the therapeutic antibody to patients. In addition, whereas it may be necessary for a chimeric antibody having murine FR residues to be administered at a dose effective to achieve complete B-cell depletion to avoid an antibody response thereto, the present humanized antibodies may be administered at dosages that achieve partial or complete B-cell depletion, and for different durations, as desired for the particular disease and patient. In addition, these antibodies showed stability in solution. These properties of the humanized 2H7 antibodies make them ideal for use as immunotherapeutic agents in the treatment of CD20-positive cancers and autoimmune diseases, and these antibodies are not expected to be immunogenic or at least to be less immunogenic than fully murine or chimeric anti- CD20 antibodies in human patients.

References

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

The practice of the present invention will use, unless otherwise indicated, conventional techniques for molecular biology and the like, which are within known techniques in the field. Such techniques are fully explained in the literature. See e.g. Molecular Cloning: A Laboratory Manual, (J. Sambrook et al., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989); Current Protocols in Molecular Biology (F. Ausubel et al., ed. 1987 updated); Essential Molecular Biology (T. Brown ed., IRL Press 1991); Gene Expression Technology (Goeddel ed., Academic Press 1991); Methods for Cloning and Analysis of Eukaryotic Genes (A. Bothwell et al. ed., Bartlett Publ. 1990); Gene Transfer and Expression (M. Kriegler, Stockton Press 1990); Recombinant DNA Methodology II (R. Wu et al., eds., Academic Press 1995); PCR: A Practical Approach (M. McPherson et al., IRL Press at Oxford University Press 1991); Oligonucleotide Synthesis (M. Gait ed., 1984); Cell Culture for Biochemists (R. Adams ed., Elsevier Science Publishers 1990); Gene Transfer Vectors for Mammalian Cells (J. Miller & M. Calos eds., 1987); Mammalian Cell Biotechnology (M. Butler ed., 1991); Animal Cell Culture (J. Pollard et al., eds., Humana Press 1990); Culture of Animal Cells, 2nd edition (R. Freshney et al., eds., Alan R. Liss 1987); Flow Cytometry and Sorting (M. Melamed et al., eds., Wiley-Liss 1990); the Methods in Enzymology series (Academic Press, Inc.); Wirth M. and Hausser H. (1993); Immunochemistry in Practice, 3rd ed. A Johnstone & R. Thorpe, Blackwell Science, Cambridge, MA, 1996; Techniques in Immunocytochemistry, (G. Bullock & P. Petrusz eds., Academic Press 1982, 1983, 1985, 1989); Handbook of Experimental Immunology, (D. Weir & C. Blackwell, eds.); Current Protocols in Immunology (J. Coligan et al. ed. 1991); Immunoassay (E.P. Diamandis & T.K. Christopoulos, Eds., Academic Press, Inc., 1996); Goding (1986) Monoclonal Antibodies: Principles and Practice (2nd edn) Academic Press, New York; Ed Harlow and David Lane, Antibodies A laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988; Antibody Engineering, 2nd ed. (C. Borrebaeck, ed., Oxford University Press, 1995); and the Annual Review of Immunology series; the Advances in Immunology series.

Claims (22)

1. Humanized antibody that binds human CD20 or an antigen-binding fragment thereof, characterized in that the antibody is effective in depleting primate B cells in vivo, where possibly the primate B cells are from humans, where the antibody comprises a) a heavy chain variable region (VH ) comprising CDR1 sequence according to SEQ ID NO. 10; The CDR2 sequence according to SEQ ID NO. llog CDR3 sequence according to SEQ ID NO. 12, and substantially the human consensus framework (FR) residues from human heavy chain subgroup-III (VHIH), wherein the heavy chain variable region further comprises the amino acid substitutions R71V; N73K and A49G, and b) a light chain variable region (VL ) comprising CDR1 sequence according to SEQ ID NO. 4, CDR2 sequence according to SEQ ID NO. 5, CDR3 sequence according to SEQ ID NO. 6 and essentially the human consensus framework (FR) residues of human light chain K-subgroup I (Vk I), where the light chain variable region further comprises the amino acid substitution L46P 2. Antibody or an antigen-binding fragment thereof according to claim 1, where it comprises the VH sequence according to SEQ ID NO. 8. 3. Antibody or an antigen-binding fragment thereof according to claim 1, where it comprises the VL sequence according to SEQ ID NO. 2. 4. Antibody or an antigen-binding fragment thereof according to any one of the preceding claims, where the VH region is linked to a human IgG chain constant region, optionally where the human IgG is IgG1 or IgG3. 5. Antibody or an antigen-binding fragment thereof according to claim 3 where it has the amino acid substitutions D56A and N100A in the H chain and S92A in the L chain. 6. Antibody or an antigen-binding fragment thereof according to any of the preceding claims, wherein it further comprises at least one amino acid substitution in the Fc region that improves ADCC and/or CDC activity, optionally where the amino acid substitutions are S298A/E333A/ K334A; or further comprises at least one amino acid substitution in the Fc region which reduces CDC activity, optionally it comprises at least the substitution K322A. 7. Antibody or an antigen-binding fragment thereof according to any one of the preceding claims, where it is conjugated to a cytotoxic agent, optionally where the cytotoxic agent is a radioactive isotope or a toxin. 8. Isolated nucleic acid or expression vector, characterized in that it codes for the antibody or the antigen-binding fragment thereof according to any of the preceding claims. 9. Host cell, characterized in that it comprises a nucleic acid according to claim 8 or expression vectors that code for the antibody or the antigen-binding fragment thereof, optionally where the host cell produces the antibody or the antigen-binding fragment therein, and where it is optionally a CHO cell. 10. Method for producing antibody or an antigen-binding fragment thereof according to any one of the preceding claims, characterized in that it comprises cultivating the cell that produces the antibody or the antigen-binding fragment thereof according to claim 9 and recovering the antibody or the antigen-binding fragment thereof. 11. Antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for use in a method for treating an autoimmune disease, wherein the method comprises administering to the patient suffering from the autoimmune disease a therapeutically effective amount of antibody, optionally where said autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arteritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, psoriasis , IgA neuropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, ANCA vasculitis, diabetes mellitus, Reynaud's syndrome, Sjøgren's syndrome and glomerulonephritis. 12. Antibody or an antigen-binding fragment thereof for use according to claim 11, wherein the autoimmune disease is multiple sclerosis. 13. Antibody or an antigen-binding fragment thereof for use according to any one of claims 1 to 7 for use in a method for depleting B cells in vivo, wherein the method comprises contacting B cells with the antibody according to any one of the preceding requirements, thereby depleting the B cells. 14. Antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for use in a method for treating a CD20-positive form of cancer, wherein the method comprises administering to the patient suffering from the form of cancer a therapeutically effective amount of antibody, optionally where the CD20-positive form of cancer is a B-cell lymphoma or a leukaemia, where the CD20-positive form of cancer is non-Hodgkin's lymphoma (NHL) or lymphocyte-predominant Hodgkin's disease (LPHD), and where the form of cancer is chronic lymphocytic leukemia or SLL. 15. Antibody or an antigen-binding fragment thereof according to claim 14, where the CD20-positive form of cancer is a B-cell lymphoma or a leukemia and where the antibody is administered in a dosage range from approximately 275 to 375 mg/m <2>. 16. Antibody or an antigen-binding fragment thereof according to claim 14, where the method further comprises administration to the patient of at least one chemotherapeutic agent, possibly where the form of cancer is non-Hodgkin's lymphoma (NHL) and the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide , vincristine and prednisolone. 17. Use of antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for the preparation of a medicament for use in a method for treating an autoimmune disease, wherein the method comprises administering to the patient suffering from the autoimmune disease a therapeutic effective amount of antibody, optionally wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, juvenile rheumatoid arteritis, systemic lupus erythematosus (SLE), Wegener's disease, inflammatory bowel disease, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura ( TTP), autoimmune thrombocytopenia, psoriasis, IgA neuropathy, IgM polyneuropathies, myasthenia gravis, vasculitis, ANCA vasculitis, diabetes mellitus, Reynaud's syndrome, Sjøgren's syndrome and glomerulonephritis. 18. Use according to claim 17, wherein the autoimmune disease is multiple sclerosis. 19. Use of antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for the production of a medicament for use in a method for depleting B cells in vivo, wherein the method comprises contacting B cells with the antibody according to any one of the preceding claims, thereby depleting the B cells. 20. Use of antibody or an antigen-binding fragment thereof according to any one of claims 1 to 7 for the production of a medicament for use in a method for treating a CD20-positive form of cancer, wherein the method comprises administration to the patient suffering from the form of cancer a therapeutically effective amount of antibody, optionally where the CD20-positive cancer is a B-cell lymphoma or a leukemia, optionally where the CD20-positive cancer is non-Hodgkin's lymphoma (NHL) or lymphocyte-predominant Hodgkin's disease (LPHD), and optionally where the cancer is chronic lymphocytic leukemia or SLL. 21. Use of antibody or an antigen-binding fragment thereof according to claim 20, where the CD20-positive form of cancer is a B-cell lymphoma or a leukemia and where the antibody is administered in a dosage range from approximately 275 to 375 mg/m . 22. Use of antibody or an antigen-binding fragment thereof according to claim 21, where the method further comprises administration to the patient of at least one chemotherapeutic agent, possibly where the form of cancer is non-Hodgkin's lymphoma (NHL) and the chemotherapeutic agent is selected from the group consisting of doxorubicin , cyclophosphamide, vincristine and prednisolone.