Title of Invention

ANTI-CD20 ANTIBODIES AND FUSION PROTEINS THEREOF AND METHODS OF USE

Abstract A humanized anti-CD20 hCD20) monoclonal antibody or antigen-binding fragment thereof comprising the complementarity determining regions (CDRs) of at least one murine anti-CD20 MAb variable region and the framework regions (FRs) of at least one human MAb variable region, wherein said humanized anti-CD20 MAb or fragment thereof retains substantially the B-cell and B-cell lymphoma and leukemia cell targeting of said mataine anti-CD20 MAb.
Full Text

Anti-CD20 Antibodies and Fusion Proteins Thereof and
Methods of Use
BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to humanized chimeric and human anti-CD20 antibodies, particularly monoclonal antibodies (mAbs) therapeutic and diagnostic conjugates of humanized, chimeric and human anti-CD20 antibodies and methods of treating B ceil lymphomas and leukemias and various autoimmmne diseases using htananized, chimeric and human anti-CD20 antibodies. The present invention relates to antibody fusion proteins or fiagmrats thereof comprising at least two anti-CD20 mAbs or firagments tiiereof or at least one anti-CD20 MAb or fragment thereof and at least one second MAb or fiagment diereof^ other than the antiCD20 MAb or firagment thereof- The humanized, chimeric and human anti-CD20 mAbs, fragments thereof, antibody fusion proteins thereof or fragments thereof may be administered alone, as a therapeutic conjugate or in combination with a therapeutic immunoconjugate, with other naked antibodies, or with therapeutic agents or as a diagnostic conjugate. The present invention relates to DNA sequences encoding humanized, chimeric and human anti-CD20 antibodies, and antibody fusion proteins, vectors and host cells containing the DNA sequences, and metliods of making the humanized, chimeric and human anti-CD20 antibodies. 2. Background
The immune system of vertebrates consists of a number of organs and cell types which have evolved to accurately recognize foreign antigens, specifically bind to, and eliminate/destroy such foreign antigens. Lymphocytes, amongst others, are critical to the immune system. Lymphocytes are di\dded into two major sub-populations, T cells and B cells. Although inter-dependent,^ T cells are largely responsible for cell-mediated immunit}^ and B cells are largely responsible for antibody production (humoral iramunity).

In hujnans, each B cell can produce an enormous number of antibod}' molecules. Such antibody production tj'pically ceases (or substantially decreases) wiaen a foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B cell v,ill continue unabated and may^ result in a cancer known as a B cell lymphoma, B-cell lymphomas, such as the B-cell subt5npe of non-Hodgldn's lymphoma, are significant contributors to cancer mortality. The response of B-cell malignancies to various fomis of treataKnt is mixed. For example, in cases in whkii adequate clinical staging of Don-Hod^dn's lynqAoma is possible, field r^iiation therapy can provide satisfectory treatm^it Still, about one-half of the patients die from the disease. Dcvesaetal., J. Natl Cancer Inst. 79:701(1987).
The majority of chronic lymjiiocytic leyriremias are of B-cell lineage. Freedman, Hematol Oncol Oin. North Am, 4:405 (1990). This type of B-ceU mahgnancy is the most common leukemia in the Western world. Goodman er al.. Leukemia and LymphorTia 22:1 (1996). The natural history of chronic lymphocytic leukemia fells into several phases. In the early phase, chronic lymphocytic leukemia is an indolent disease, characterized by the accumulation of small mature functionally-incompetent malignant B-cells having a lengthened life span. Eventually, the doubling time of the malignant B-cells decreases and patients become increasingly symptomatic. While treatment can provide symptomatic relief, the overall survival of the patients is only minimally affected. The late stages of chronic lymphocytic leukemia are characterized by significant anemia and/or thrombocytopenia. At this point, the median survival is less than two years. Foon et al, Aivials Int. Medicine 113:525 (1990). Due to the very low rate of cellular proliferation, chronic lymphocytic leukemia is resistant to cytotoxic drug treatment Traditional methods of treating B-cell malignancies, including chemotherapy and radiotherapy, have limited utility due to toxic side effects. The use of monoclonal antibodies to direct radionuclides, toxins, or other 1her^)eutic agents offers the possibility that such agents can be delivered selectively to tumor sites, thus limiting toxicity to normal tissues. Also, the presence of B-ceil antigens on these B-cell maUgnancies makes them optimal targets for therapy with unconjugated B-cell antibodies, such as against CD19, CD20, CD2L CD23, and CD22 markers on B-ceUs. HLA-DR and other antigens may serve as targets for normal and malignant B-celis, although they are also expressed on other cell types. Further, certain MUCl, MLTC2,

IvfLTCS, and NiUC4 antigens, preferably MUCL are also expressed in different hematopoietic malignancies, including B-ceil mmors expressing CD20 and other B-cell maiicers. Still other antigen targets, such as those associated with the vascular endothelium of tumors, including tenascin, vascular endothelium growth factor-(VEGF), and placental growth factor (PIGF), as well as other categories of antigens associated with B-cell malignancies, such as oiKX)gene products, are also suitable targets for said complementary antibodies fer nse in the presort invention.
B cells comprise cell surfece proteins which can be utilized as maikers for diJGFerendation and identification. One such lumian B-cell maikar is the himian B lymphocyte-restricted differentiation antigen Bp35, referred to as CD20. CD20 is expressed during early pre-B cell development and remains until plasma cell differentiation CD20 is expressed on both normal B cells and rrialignant B cells whose abnormal growth can lead to B-cell lymjiiamas. Antibodies against the CD20 antigen have been investigated for the therapy of B-cell lymphomas. For example, a chimeric anti-CD20 antibody, designated as 'TDEC-C2B8," has activity against B-cell lymphomas when provided as unconjugated antibodies at repeated injections of doses exceeding 500 mg per injection Maloney et al., Blood 84:2457 (1994); Longo, Curr. OpiTL Oncol. 5:353 (1996). About 50 percent of non-Hodgkin's patients, having the low-grade indolent form, treated with this regimen showed responses. Therapeutic responses have also been obtained using ^^^I-labeled Bl anti-CD20 murine monoclonal antibody when provided as repeated doses exceeding 600 mg per injection. Kaminski et al., K Engl J. Med. 329:459 (1993); Press et al, N. Engl. J. Med 52P:1219 (1993); Press et al.. Lancet 346:336 (1995). However, these antibodies, wliether provided as unconjugated forms or radiolabeled forms, have not shown high rates of objective and durable responses in patients with the more prevalent and lethal form of B-cell lymphoma, the intermediate or aggressive type. Therefore, a need exists to develop an immimotherapy for BVell malignancies that achieves a therapeutic response of significant duration.
Additional studies targeting CD20 surface antigen have been demonstrated using an anti-CD20 murine monoclonal antibody, IF 5, which was administered by continuous intravenous infusion to B ceil lymphoma patients. Extremely high levels (>2 grams) of 1F5 were reportedly required to deplete circulating tumor cells, and the results were described as being "transient." Press et al., "Monoclonal Antibody 1F5

(Anti-CD20) Serotherapy of Human B-Cell Lymphomas." Blood 69/2:584-591 (1987). However, a potential problem with tins approach is that non-human monoclonal antibodies (e.g., murine monoclonal antibodies) typically lack human effector functionality, i.e., they are unable to mediate complement-dependent lysis or lyse himian target cells through antibody-d^pfeiKient cellular toxicity or Fc-receptor mediated phagoc5lx)sis. Furthermore, non-human monoclonal antibodies can be recognized by the human host as a fcffdgn prtrtrai aini, tha^fore, repealed ingectioos of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reacti The use of diimeric antibodies is mc^e preferred because they do rM>t elicit as strong a HAMA response as murine antibodies. Chimeric antibodies are antibodies which comprise portions from two or more different species. For example, Liu, A- Y. et al^ "Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20 with Potent Fc-Dependent Biologic Activity" /, IrTtmun. 139/10:3521-3526 (1987), describe a mouse/human chimeric antibody directed against the CD20 antigen. See also, PCT Pubhcation No. WO 88/04936. However, no information is provided as to the ability, efficacy or practicality of using such chimeric antibodies for the treatment of B cell disorders in the reference. It is noted that in vitro functional assays (e.g., complement-dependent lysis (CDC); antibody dependent cellular cytotoxicity (ADCC), etc.) cannot inherentiy predict the in vivo capability of a chimeric antibody to destroy or deplete target cells expressing the specific antigen. See, for example, Robinson, R. D. er a/., "Chimeric mouse-human anti-caxcinoma antibodies that mediate different anti-tumor cell biological activities," Hum. Antibod H)^bridomas 2:84-93 (1991) (chimeric mouse-human antibody having undetectable ADCC activity). Therefore, the potential therapeutic efficacy of a chimeric antibody can only truly be assessed by in vivo experimentation, preferably in the species of interest for the specific therapy.
One approach that has improved the ability of murine monoclonal antibodies to be effective in the treatment of B-cell disorders has been to conjugate a radioactive label or chemotherapeutic agent to the antibody, such that the label or agent is localized at the tumor site. For example, the above-referenced 1F5 antibody and other B-cell antibodies have been labeled with ^^*I and were reportedly evaluated for

biodistribunon in tvv'o patients. See Eary, J. F. et al., "Imaging and Treatment of B-Cell Lymphoma" 1 Nnc. Med 31/8:1257-1268 (1990): see also, Press, 0. W. ei al,, "Treatment of Refiractorv' Non-Hodgkin's Lymphoma ^^ath Radiolabeled MB-1 (Anti-CD37) .Antibody" J. Clin One. 7/8:1027-1038 (1989) Cmdication that one patient treated with "* I-labeled lF-5 achieved a partial response); Goldenberg. D. M. et ai., "Targeting, Dosimetry and Radioimmimothenpy of B-Ceil Lymphomas ^ith ' I-Labeled LL2 Monoclonal Antibody" J. Qm. OBCOL 9/4:548-564 (1991) (three of . ei^ patients receiving multiple ii^ecticHis rqxHted to have developed a HAMA response to this CD22 murine antibody); AppeSoanm, F. R. "Radiolabeled Monoclonal Antibodies in the Treatment ofNonr-Hodgkin's Lymphoma" HemJOncol Climes ofN. A. 5/5:1013-1025 (1991) (review article); Press, O. W, ei al. "Radiolabeled-Antibody Therapy of B-Cell Lymphoma wifli Autologous B Autoimmune diseases are a class of diseases associated with B-cell disorders. Examples include immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, myastheiiia gravis, lupus nephritis, lupus erytiiematosus, and rheumatoid arthritis. The most common treatments are corticosteroids and cytotoxic drugs, which can be very toxic. These drugs also suppress the entire immune system, can result in serious infection, and have adverse affects on the bone marrow, Uver and kidneys. Other therapeutics that have been used to treat Class HI autoimmune diseases to date have been directed against T-cells and macrophages. There is a need for more effective methods of treating autoimmune diseases, particularly Class EQ autoimmxme diseases.

To address the many issues related to B-cell disorders and their treatment the present invention provides humanized, chimeric and human anti-CD20 monoclonal antibodies with "die same complementarity determining regions (CDRs) that bind to the CD20 antigen of the present invention used alone, conjugated to a therapexitic agent or in combination with other treatment modalities, for the treatment of B cell -lymphomas and leukemias and autoimmune disorders in humans and otiier m^mmj^k wrtbout the adverse responses associated with vemg marine antibodies.
SUMMARY OF THE INVENTION
Accordii^y, the present invention jwovides humanized, chimeric and human anti-CD20 antibodies that bind to a human B cell matter, refexred to as CD20, which is usefiil for the treatment and diagnosis of B-cell disorders, such as B-ceU malignancies and autoimmune diseases.
The present invention forther provides methods of treatment of mammalian subjects, such as humans or domestic animals, with one or more humanized, chimeric and human CD20 antibodies, alone, as an antibody fiision protein, as a therapeutic conjugate alone or as part of an antibody fusion protein, in combination, or as a multimodal therapy, with other antibodies, other therapeutic agents or immunomodulators or as an immimoconjugate linked to at least one therapeutic agent therapeutic radionuchde or immimomodulator. These humanized, chimeric and human CD20 antibodies can also be used as a diagnostic imaging agent alone, in combination with other diagnostic imaging agents, and/or in conjtmction with therapeutic ^plications.
The present invention additionally is directed to anti-CD20 mAbs or fragments thereof that contain specific murine CDRs or a combioation of murine CDRs from more than one murine or chimeric anti-CD20 MAb that have specificity for CD20. These niAbs can be humanized, chimeric or human anti-CD20 mAbs.
The present invention is also dhected to antibody fusion proteins comprising at least two anti-CD20inAbs or fragments thereof or a first MAb comprising an anti-CD20mAbs or fragments thereof and a second MAb.
The present invention is further directed to a therapeutic or diagnostic conjugates of the anti-CD20 mAbs or fragments thereof or antibody fusion proteins of the anti-CD20 mAbs or other mAbs or fragments thereof boimd to at least one

therapeutic agent or at least one diagnostic agent .Aatibody fusion proteins with multiple therapeutic agents of the same or different t>'pe are encompassed by the present invention.
The present invention is additionally directed to a method of using the anti-CD20 mAbs or fragments thereof or antibody fusion proteins thereof or fragments thereof for therapy, either alone, in combination with each other, as the antibody component of a therapeutic inununocoiijifegaie with cme or more tha:apeatic agraSs OT each administered in combin^on with one OTDBOTC therapeutic agents or with an immunoconjugate with one or mwe theraperaic agarts.
The presort invention furtho' is directed to a method of using the an±i-CD20 mAbs or fragments thereof or antibody fusion jHOteins thereof or fragments tibexeof as a diagnostic bound to one or more diagnostic agents.
The presait invention additionally is directed to a method of pretargetxDg a cell in a patients sufifering from a B-cell lymphoma or leukemia or an autoimmune disease using an antibody fiision protein or fragment thereof of the present invention. •
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 discloses the V gene sequences cloned by RT-PCR from a hybridoma cell line producing a mtirine anti-CD20, and the deduced amino acid sequences of the variable light (Figure 1 A) and heavy chain (Figure IB) of the A20 antibody, designated as A20Vk and A20VH, respectively. Underlined arrows indicate the sequences of the PCR primers used for cloning. The putative CDR region sequences, as defined by the Kabat numbering scheme, are shown in bold and underlined, Amino acid sequences are given as single-letter codes below the corresponding nucleotide sequence. The Kabat numbering scheme was used for amino acid residues. Amino acid residues numbered by a letter represent the insertion residue according to Kabat, and have the same number as that of the previous residue. For example, residues 82, 82A, 82B and 82C in Figure IB are indicated as 82 A, B, and C, respectively..
Figure 2 discloses the Vk, the variable Ught chain, and the VH, the variable heavy chain, sequences of cA20, a chimeric anti-CD20 antibody. The CDR region sequences are shown in bold and underhned. The amino acid residues and the nucleotides are numbered sequentially and same numbering system is used for

humanized V sequences. The light chain variable region is shown in Fig. 2A and the heavy chain variable region is shoun in Fig. 2B. The numbering system is the same as for Figure 1. The restriction sites used for constructing cA20 are underlined.
Figure 3.shows a comparison of the binding affinities of the chimeric A20 (cA20), and murine A20, (A20), in a cell surface competitive binding assay against ^■^-labled A20. Increasing concentrations of cA20 blocked the binding of radiolabeled A20 to Raji cells (as dqricted by closed circles) in a comparable manziea: as that of murine A20 (dq)icted by closed dianKmd^).
Figiure 4 conq^ies the amiiK) acid seqaences of tte variable heavy chain (VH) and variable light chain (Vt) of human antibodies, and chimeric and humanzied anti-CD20 antibodies. Figure 4A compares the amiDO acid sequences of the variable heavy chain (VH) of the human antibodies, EU and NEWM (TFR4 only), the chiBKaic antibody, (cA20VH) and two humanized antibodies, (hA20VHl and hA20VH2) and Figure 4B compares the amino acid sequences of the variable light chain (Vk) of the human antibody, (REFVk), a chimeric antibody, (cA20Vk), and a humanized antibody, (hA20Vlc). Dots indicate that the residues in A20 are identical to the corresponding residue in the human antibody. The CDRs are identified as a boxed region. The Kabat numbering scheme was used to number the amino acid residues.
Figure 5 discloses the nucleotide sequences of hA20 light chain V genes, QxMOVk) (Figure 5 A), and heavy chain V genes, hA20VHl (Figure 5B) and hA20VH2 (Figure 5C), as well as tiie adjacent flanking sequences of the VKpBR2 (Figure 5A) and VHpBS2 (Figures 5B and 5C) staging vectors, respectively. The non-translated nucleotide sequences are shown hi lowercase. The restriction sites used for subcloning are underlined and indicated. The secretion signal peptide sequence is indicated by a double underline. Numbering of Vk and VH amino acid residues is same as that in Figure 2.
Figure 6 shows the results of a cell surface competitive binding assay to compare the binding activity of two humanized A20 antibodies, (hA20-l and hA20-2), wi& tiaat of A20, cA20 and a chimeric anti-CD20 MAb, c2B8. Figure 6A shows hA20-l (closed triangles) and hA20-2 (closed circles) and the mm±ie anti-CD20 antibody, A20 (closed squares) competed equally well for the binding of ^^I-A20 to Raji cells. Figure 6B shows hA20-l (closed circles), cA20 (closed squares) and c2B8
1 "AC
(closed diamonds) competed equally well for the binding of I-c2B8 to Raji cells.

Figure 7 discloses the constant region of a human IgGl (CH-hinge) (Figure 7A) and the constant region of a human kappa chain (Ck) (Figure 7B).
Figure 8 is a competitive ceil surface binding assay. Ag-binding specificit}' and affinity studies of humanized anti-CD20 Abs (cA20, hA20, and clFS, purified by affinity chromatography on a Protein A column) were evaluated b^^ a cell surface competitive binding assay with murine 2B8 and rituximab (IDEC Pharmaceuticals Q)rp., San Diego, CA). Figure 8 (A) is a conqiadson of the binding activities of cA20 (square), hA20-l (triangle) aiKi hA20-l (drcle) with that of m2B8 (diam Figure 9 is a study comparing tlie binding activities of hA20 with other anti-CD20 Abs, including rituximab and mxarine Bl, by a cdl surface competitive binding assay. A constant amount (100,000 cpm, -10 iCi/ig) of ^^^-labeled rituximab was incubated with Raji cells in the presence of varying concentrations (0.2-700 nM) of competing Abs, hA20 (triangle), mBl (Downward triangle) or rituximab (square) at 4*^0 for 1-2 h.
Figure 10 depicts the cytotoxic effect of crosslinked hA20 and other CD20 Abs on cultured lymphoma cells. Total cell and viable cell cell populations were measured by (A) trypan blue staining and cell counting or (B) Mil assay.
Figure 11 is a graph of in vivo therapy studies with various anti-CD20 and other Abs. Raji cells administered i.v. to SCID mice, to create a Raji lymphoma model of disseminated disease.
Figure 12 is a graph depicting in vivo therapy with hA20 and hLLZ Raji cells administered i.v. to SCID mice, to create a Raji lymphoma model of disseminated disease.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
As discussed above, anti-CD20 antibodies that are unconjugated or labeled with a therapeutic radionuclide, have failed to provide high rates of objective and lasting responses in pati^its with intermediate or aggressive forms of B-cell lymphoma. The present invention provides a humanized, a chimeric and a human anti-CD20 antibody and antibody fusion proteins thereof useful for treatment of

mammalian subjects, humans and domestic animals, alone, as a conjugate or administered in combination Vviih other therapeutic agents, including other naked antibodies and antibody therapeutic conjugates.
The anti-CD20 mAbs of the present invention contain specific murine CDRs or a combination of murine CDRs from more than one murine or chimeric anli-CD20 MAb that have specificity for the CD20 antigen. The anti-ODIO mAbs of the present invaition are humanized, chimeric or human mAbs and they contain the amino acids of the CDRs of a murine aiiti-CD20 MAb and re^adn substantially tte B-cell and B-cdl lymphnnnfl and Icuki^nia cell targeting of the nmrii^ anti-CD20 MAb. The CDRs of ^ light chain variable region of the anti-CD20 MAb cOTuprises CDRl comprising amrnn adds RASSSVSYIH, RASSSLSFMH or RASSSVSYMH; CDR2 comprising annno acids ATSNLAS; and CDR3 comprismg ammo adds QQ'^TSNPPT, HQWSSNPLT OT QQSFSNPPT; and the CDRs of the heavy chain variable region of the anti-CD20 MAb comprises CDRl comprising amino adds SYNMH; CDR2 comprising amino adds AIYPGNGDTSYNQKFKG and CDRS comprising ammo acids STYYGGDWYFDV, STYYGGDWYFNV, SHYGSNYVDYFDV or WYYSNSYWYTDV.
In one embodiment, the humanized and diimeric MAb or fragment thereof does not contain the CDR3 of the heavy chain variable region comprising STYYGGDWYFNV. More preferably, CDRl of "die hght chain variable region does not comprise RASSSLSFMH wien the CDRS of the light chain variable region comprises HQWSSNPLT and the CDRS of the heavy chain variable region comprises SHYGSNYVDYFDV- In another embodmaenL the CDRS of the light chain variable region does not comprise HQWSSNPLT when CDRl of the light chain variable region comprises RASSSLSFMH and when CDRS of the heavy chain variable region comprises SHYGSNYVDYTDV. hi a fiirther embodiment, the CDRS of the heavy chain variable region does not comprise SHYGSNYVDYTDV wiien the CDRl of the hght chain variable region comprises RASSSLSFMH and the CDRS of the light chain variable region comprises HQWSSNPLT. In another embodiment, the CDRl of the hght chain variable region does not comprise RASSSVSYMH when the CDRS of the hght chain variable region comprises QQSFSNPPT and the CDRS of the hea\n/ chain variable region comprises WY'YSNSYWYFDV.
Further, in another embodiment, the anti-CD20 monoclonal antibody (MAb) or fragment thereof does not contain CDRS of the Ught chain variable region of amino

acids QQSFSNPPT wiien CDRl of the light chain variable region comprises RASSSVSYMH and the CDR3 of the hea\y chain variable region comprises WA^SNS^'WYFDV. Additionally, the anti-CD20 M^b does not contain CDR3 of the heavy chain variable region with amino acids V\^YYSNSYWYFDV when lie CDRl of the light chain variable region comprises R.ASSSVSYMH and die CDR3 of the hght chain variable region comprises QQSFSNPPT.
In a preferred embodimCTt the hmnssmsd an±i-CD20 (hCD20) moooclcnai antibody or antigeih-binding fiagment tiiereaf comprising tiie complemCTdcarity detennining r^ons (CDRs) of at least cHie murine anti-CD20 MAb variaWe r^ion aad te firamewoik regions (FRs) of at least cme hiiman MAb variable region, wherein said humanized anti-CD20 MAb or fragment thereof retains substantially the B-ceil and B-cell lymphoma and leukemia cell targeting of said murine anti-CD20 MAb. 11^ humanized antibody's variable region may ccanprise a light chain variable region, a heavy chain variable region or a both light and heavy chain variable regions. The humanized antibody or firagment thereof may fiirther comprise light and teavy chain constant regions of at least one human antibody.
The humanized anti-CD20 MAb or fragment thereof of the present invention comprises the CDRs of a murine anti-CD20 MAb and the firameworic (FR) regions of the light and heavy chain variable regions of a human antibody, "while retaining substantially the B-cell, and B-cell lymphoma and leukemia cell targeting of the parent murine anti-CD20 IVlAb, and wherein the CDRs of the light doain variable region of the murine anti-CD20 MAb comprises CDRl comprising amino acids RASSSVSYIH, CDR2 comprising amino acids ATSNLAS and CDRS comprising QQWTSNPPT and the CDRs of the heavy chain variable region of murine anti-CD20 MAb comprises CDRl comprising amino acids SYNMH, CDR2 comprising amino acids AIYPGNGDTSYNQKFKG and CDR3 comprising amino acids STYYGGDWYFDV. But the humanized anti-CD20 MAb or fi*agment thereof may further contain in the FRs of the light and heavy chain variable regions of the antibody at least one amino acid from the corresponding FRs of the murine MAb. The humanized MAbs may further contain the hght and heavy chain constant regions of a human antibody. Specifically, the humanized anti-CD20 MAb or fi-asment tiiereof contains at least one amino acid residue 1, 5, 27, 30, 38, 48, 67, 68, 70,^95, 115 and 116 of the murine heav^ chain variable region of Fig. 4A, designated as hA20VHl or hA20VH2 and of at least one amino acid

residue 4. 2L 35, 38, 45, 46, 59, 99, 104 and 106 of the murine light chain variable region Fig. 4B, designated L\20VL One or more of the murine amino acid sequences can be maintained in the human FR regions of the lidit and hea\jy variable chains if necessary to maintain proper binding or to enhance binding to the CD20 antigen- More preferably the humanized anti-CD20 MAb. .or figment thereof of the present invention comprises ti^ hA20Vk of Figure 4B and tiie hA2VHl of Figtire 4A. Most preferably, tbe humanized anti-CD20 MAb or fragment dieiecrf of tbe fHieseait inventicMi ccHiqaises the hA20Vk of Figure 4B and the hA2VH2 of Figuffe 4A. This latter sequence ccKitaiDs more human amino acid seqiiKices in tbe FRs of ti^ VH2 chain than the VHL sad thus is more humanized-
The preferred chimeric anti-CD20 (cQD20) MAb or fragment thCTeof of tte
present invention comprises the CDRs of a murine anli-CD20 MAb and the FR legicms
of tbe light and heavy chain variable regions of tbe murine anti-CD 20 MAb, Le.^ tte Fvs
of the parental murine MAb^ and tbe light and heavy chain constant regions of a human
antibody, wberein tbe chimeric anti-CD20 MAb or fragment tbereof retains substantially
the B-cell, and B-cell lymphoma and leukemia cell targeting of the murine anti-CD20
MAb, wherein the CDRs of the light chain variable region of the chimeric anti-CD20
MAb comprise CDRl comprising ammo acids RASSSVSYIH, RASSSLSFMH or
RASSSVSYMH; CDR2 comprising amino acids ATSNLAS; and CDR3 comprising
amino acids QQWTSNPPT, HQWSSNPLT or QQSFSNPPT; and the CDRs of the
heavy chain variable region of the chimeric anti-CD20 MAb conqDiise CDRl
comprising amino acids SYNMH; CDR2 comprising amino acids
AIYPGNGDTSYNQKFKG and CDR3 comprising STYYGGDWYFDV,
STY^^GGDWYFNV, SITx^GSNYVDYFDV or WYYSNSYWYFDV with the
following provisos,
(a) wherein the CDR3 of the heavy chain variable region does not comprise STYYGGDWYFNV, wben tiie CDRl of the li^t chain variable region comprises amino acids RASSSVSYIH, CDR2 of the light chain variable region comprises amino acids ATSNLAS, CDR3 of the light chain variable region comprises amino acids QQWTSNPPT, CDRl of the heavy chain variable region comprises amino acids SYNMH, and CDR2 of the hght chain variable region comprises amino acids AIYPGNGDTSYNQKFKG;

(b) wherein the CDR3 of the heavy chain variable region does not comprise SHYGSN"Y"VT)\TDV, v.hen the CDRl of the hght chain variable region comprises amino acids RASSSLSFMH, CDR2 of die lighi chain variable region comprises amino acids ATSNLAS, CDR3 of the Ught chain variable region comprises amino acids HQWSSNPLT, CDRl of the heavy chain vari^ijfe- region comprises amino acids SYNMH, and CDR2 of the light chain variable region comprises amino acids AIYPGNGDTSYNQKFKG; and
(c) 'w^ierein the CDR3 of ^ bsavy chain variable region does not coaqxise WYYSNSYWYFDV, %vhen the CDRl of the I^ diain variable region comfnises amino acids RASSSVSYMH, CDR2 of the Hght chain variable region comprises amino adds ATSNLAS, CDRS of the light chain vari^i^le r^cai ccwnpiises amino acids QQSFSNPPT, CDRl of the heavy chain variable region comprises amino adds SYNMH, and CDR2 of the light chain variable region comprises amino acids ATYPGNGDTSYNQKFKG.
More preferably the chimeric anti-CD20 MAb or fragment thereof comprisiog the complementarity-determining regions (CDRs) of a murine anti-CD20 MAb and the frameworic (FR) regions of the light and heavy chain variable regions of the murine anti-CD20 MAb and the Ught and heavy chain constant regions of a human antibody, wherein the chimeric anti-CD20 MAb or fragment thereof retains substantially the B-cell, and B-cell lymphoma and leukemia cell targeting of the murine anti-CD20 MAb, v/herein the CDRs of the h^t chain variable region of the chimeric anti-CD20 MAb comprises die CDRs shown in Figs. 4B and 4A, respectively, designated cAlOVk and cA20VH. Most preferably, the chimeric anti-CD20 MAb or fragment thereof comprises the light and heavy chain variable regions of murine anti-CD20 MAb shown in Figs. 4B and 4A, respectively, designated cA20Vk and cA20 VR
The present invention also encompasses a human anti-CD20 MAb or fragment thereof comprising the light and heavy chain variable and constant regions of a human antibody, wherein said human CD20 MAb retains substantially the B-cell, and B-cell lymphoma and leukemia ceU targeting and cell binding characteristics of a murine anti-CD20 MAb, wherein tte CDRs of the Ught chain variable region of the human anti-CD20 MAb comprises the same CDRs as set forth above for the chimeric and humanized anti-CD20 mAbs and as shown in Figs. 4A and 4B.

The present invention is also intended lo encompass antibody fusion proteins or fragments thereof comprising at least nvo anti-CD20 nL^bs or fragments thereof as described above. The antibody fusion protein or fragment thereof of the present invention is also intoided to encompass an antibod\^ fbsion protein or fragment thereof comprising ai le?st one frrst anti-CD20 MAb or fiagmeot thereof as described above and at least one second MAb or fragment tiiereof otha: iban the antiCD20 MAb or fragment described above. More p^ferably this secoiKi MAb is a MAb reactive Avith CD4, CDS, CD8, CD14, CD15, CD19, CD2U CD2Z CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80. CD126, B7, MUCL MUC2, MUC3, MUC4, la, HM1J24, HLA-DR, tenascin, VEGF, PIGF, an oncogene, oncogo^ product, or a combination thereof, and even an anti-CD20 MAb that is differoat th?JTi the anti-CD20 MAb described herein. The antibody fnsion proteins of the present invention may be composed of one CD20 MAb and one or more of the second mAbs to provide specificity to different antigens, and are described in more detail below.
The humanized, chimeric and human anti-CD20 antibody may possess enhanced affinity binding with the epitope, as well as antitumor and anti-B-cell acti\dty, as a result of CDR mutation and manipulation of the CDR and other sequences in the variable region to obtain a superior therapeutic agent for the treatment of B-K:ell disorders, including B-cell lymphomas and leukemias and autoimmune diseases. Modification to the binding specificity, affinity or avidity of an antibody is known and described in "WO 98/44001, as affinit}'" maturation, and this appHcation summarizes methods of modification and is incorporated in its entirety'- by reference.
It may also be desirable to modify' the antibodies of the present invention to improve effector fimction, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antagonist One or more amino acid substitutions or the introduction of cysteine in the Fc region may be made, thereby improving internalization capability and/or increased complement-mediated cell killing and ADCC. See Caron et ai, J. Ex. Med 176:1191-1195 (1991) and Shopes, B.I Immunol 148:2918-2022 (1992), incorporated herein by reference in their entirety. An antibody fusion protein may be prepared that has dual Fc regions with both enhanced con^)lement lysis and ADCC capabihties.

The pitsent invention is also directed to DNA sequences comprising a nucleic acid encoding a MAb or fragment thereof selected from the group consisting
(a) an airti-CD20 IvlAb or fragment thereof as described herein,
(b) an antibody fusion protein or fragment thereof corr^^rising at least two of the anti-CD20 mAbs or fragments tiiereof,
(c) an antibodj' fusion protein or fragmoit thereof comprising at least one j5rst MAb or fragment thereof comprismg the aEfii-CD20 MAb or fragment tfaraeof as described ha:ein and at least one second MAb CH: fragment thereof, otter than tibe antiCD20 MAb or fiagniCTttheieof, and
(d) an antibody fusicai pHTOtein or fragmesot thereof ccanjnising at least one fiist MAb or fr^ment thereof comprising the anli-CD20 MAb or fragment thereof Tmd at least one second MAb or fragmrat 1hereo£ \^iismi ih.e second MAb is a MAb reactive with CD4, CDS, CD8, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUCU>1UC2, MUC3, MUC4, la, HMl-24, HLA-DR, tenascin, VEGF, PIGF, an oncogene, oncogene product, or a combination thereof.
Also encompassed by the present invention are expression vectors comprising the DNA sequences. These vectors contain the light and heavy chain constant regions and the hinge region of the human immunoglobulin., in the case of vectors for use in preparing the humanized chimeric and human anti-CD20 mAbs or antibody fiision proteins thereof or fragments thereof These vectors additionally contain, where required, promoters that express the mAbs in tiae sdected host cell, immunoglobulin enhances and signal or leader sequences. Vectors that are particularly useful in the present invention are pdHL2 or GS, particularly when used to express a chimeric, humanized or human antibodies, such as gigs, where the vector codes for the heavy and U^t chain constant regions and hinge region of IgGl. More preferably, the light and hea\7' chain constant regions and hii^e region are from a human EU myeloma immunoglobulin, where optionally at least one of the amino acid in the allotype positions is changed to that found in a different IgGl allotype, and wherein optionally amino acid 253 of the heavy chain of EU based on the EU number system may be replaced with alanine. See Edeknan et al., Proc. Natl. Acad Sci USA 63: 78-85 (1969), incorporated herein in its entirety by reference.

Host cells containing the DNA sequences encoding the anti-CD20 mAbs or fragments thereof or antibody fusion proteins or ftagments thereof of the present invention or host cells containing the vectoi^ that contain these DNA sequences are encompassed by tl^ present Luventioa Particularly useiul host cells are mammahan cells, more specifically lymphocytic cells, such as myeloma ceUs, discussed in more detail below.
Also encompassed by the present inveaadoa is the HM^KXI of expressing the anti-CD2Q MAb or firagmeait tiiereof or antibody foskm protein or fragment tbereof rrtnyri^nng- (a) tiansfecting a mammalian cell with a DNA sequence of encoding the anti-CnSO mAbs or fragments tiiereof or antibody foskm proteins or fragments thereol and (b) cuituring the cell transfected wifli tbe DNA seqoaice that secretes the anti-CD20 or fragment thereof or antibody fiision proteru or fragment thereof. Known tecbniqiKs may be used that include a selection marker on tbe vectOT so tiiat host cells that express the mAbs and the marker can be easily selected-
The present invention particularly encompasses B-lymphoma cell and leukemia cell targeting diagnostic or therapeutic conjugates comprising an antibody component comprisiog an anti-CD20 lAAb or fragment thereof or an antibody fiision protein or fragment thereof of the present Lavention that binds to the B-lymphoma or leukemia ceU, that is bound to at least one diagnostic or at least one therapeutic agent.
The diagnostic conjugate comprises the antibody component comprising an anti-CD20 MAb or fragment thereof or an antibody fiision protein or fragment thereof Avherein tiie diagnostic agent comprises at least one photoactive diagnostic agent, and more preferably wherein tbe label is a radioactive label with an energy between 60 and 4,000 keV or a non-radioactive label. The radioactive label is preferably a gamma-, beta-, and positron-emitting isotope and is selected from the group consisting of "^L
131i^ 123j^ 124j^ E6Y^ 186^^^ ISSj^^^ 62^.^ 64^^ lU^ 67Q^ SSQ^ 99m^^^ 94^^^ ISy^ U^^
^^, ^^O, ^^r and combinations thereof
The diagnostic conjugate of the present invention also utilizes a diagnostic agent such as a contrast agent ft)r example, such as manganese, iron or gadolinium.
The therapeutic conjugate of the present invention comprises an antibody component comprising an antibody fiision protein or fragment thereof, wlierein each of said mAbs or fragments thereof are bound to at least one therapeutic agent The therapeutic conjugate of preferably is selected from the group consisting of a radioactive

"label, an immunomodulator, a hormone, a photoactive therapeutic agent a c\-ioio>uc agent, wiiich may be a drug or a toxin, and a combination thereof. The drugs usefijl in the present invention are those drugs thai possess the pharmaceutical property selected from the group cor^sisting of antimitotic, antikinase, alkylating, antunetabohte, antibiotic, alkaloid antiangiogenic, apoptotic agents and comtmations thereof. More specifically, these drugs are selected from the group consisting of nitrogen mustards, ethylenimine derivatives- alkyl sulfonates, nitrosoureas, triazeaes, folic acid analogs, COX-2 inhibitors, pyrinridine analogs, purine analogs, antibiotics, oizyines, epipodophyllotoxins, platinum coordinfflon cxxnfkses, vinca alkaloids, substitoted ureas, methyl hydrazine derivatives, adrenocortical suppressants, antagonists, endostatin, taxols, camptothecins, antiiracycliifces, taxai^s, and tteir analogs, and a combination tiiereof. The toxins encompassed by the present invention are selected from the group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g,, onconase^ DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin, and Psendomonas endotoxiiL
Usefiil therapeutic conjugates of the present invention are immunomodulators selected from the group consisting of a cytokine, a stem cell growth factor, a Ijmaphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erj^thropoietin, thrombopoietin and a combination thereof. Specifically useful are lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (EL), colony stimulating factor, such as granoilocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating fector (GM-CSF)), interferon, such as interferons-a, -p or -y? ^^^ st&xn. cell growth factor, such as designated "SI factor". More specifically, unmunomodulator, such as IL-1, IL-2; EL-3, IL-6, IL-10, IL-12, IL-18, IL-21 interferon-y, TNF- Particularly usefiil therapeutic conjugates comprise one or more radioactive labels that have an enerey between 60 and 700 keV. Such radioactive labels are selected from the group consisting of ^Ac, ^^Ga, ^Y, , '^^In, ^^*L ^'^'L ^^^Re, ^^Re, ^^^Lu, ^-P, , ^Cu, ^"Cu, "^-Bi, -^^i, ^^^At and combinations thereof Other useful therapeutic conjxigates are photoactive therapeutic agent, such as a chromogen or dye.

Other useful therapeutic conjugates comprise oligonucleotides, especiaih-antisense oligonucleotides that preferably are directed against oncogenes and oncogene products of B-celi malignancies, such as bcl-2.
The present invention particularly encompasses methods of treating a B-cell lymphoma or leukemia cell disease or an autoimmone disease in a subject-sucfa as a mammal^ including humans, domestic or companion pets, such as dogs and cats. mmpri«dng administoing to the subject a ti^npeoticafly efective amount of an anti-0320 MAb or a fragment thereof of the jweseftf inversion, formulated in a piiamiaceiiticaily acceptable vehicle. This thei^* utili25es a "naked antibody" that dbes not have a therapeutic agent bound to it Tte administration of tte "naked anti-CD20 antibody" can be supplemented by administerii^ to the subject concuirentiy or sequCTtially a thCTapeutically effective amount of anotiier "naked antibody^ that binds to or is reactive with anotha: antigen on flie surfece of the target cell or that has other fimctions, such as effector functions in the Fc portion of the MAb, that is therapeutic and which is discussed herein. Preferred additional mAbs are at least one humanized, chimeric, hxunan or murine (in the case of non-human animals) MAb selected from the group consisting of a MAb reactive with CD4, CDS, CDS, CD14, CD15, CD19, CD20, CD2h CD22, CD23, CD25, CD33, CDS?; CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUCl, la, HM1.24, and HLA-DR, tenascin, VEGF, PIGF, an oncogene, oncogene product, or a combination thereof, fonnulated in a phannaceutically acceptable vehicle.
Both the naked anti-CD20 ther^y alone or in combination with other naked mAbs as discussed above can be fiirther supplemented with the administration, either concurrentiy or sequentially, of a therapeutically effective amount of at least one therapeutic agent, formulated in a pharmaceutically acceptable vehicle. As discussed herein the therapeutic agent may comprises a cytotoxic agent, a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic agent or a combination thareof, formulated in a pharmaceutically acceptable vehicle.
In another therapeutic melhod, both the naked anti-CD20 therapy alone or in combination witii other naked mAbs, as discussed above, can be further supplemented with the administration, either concurrentiy or sequentially, of a therapeutically effective amovmt of at least one therapeutic conjugate, described herein and fonnulated in a phannaceutically acceptable vehicle. The antibody component of the therap^itic

cxinjugate comprises al leasi one humanized chimeric, human or murine (for non-human subjects) ]vlA.b selected from the group consisting of a MAb reactive v^ith CD4, CDS. CDS. CD14. CD15, CD19, CD20. CD21, CD22. CD23. CD25. CD33. CD37. CD38, CD40, CD40L CDA6., CD52, CD54, CD74, CD80, CD126, B7, MUCL MUC2, NfTjC3, MUC4, la, HML24, and HLA-DR, tenascin, VEGF, PIGF, an oncogene, oncogene product, or a combinaticHi thereof, formulated in a pbannaceutically accqytabie veiiicle. As discussed herein tbe tberape^itic agait may comprise a cytotoxic agent a radioactive label, an iHBaauiKHnodulator, a horoKme, a photoactive ttoapcutic agent or a combinatkHitheieol jSsniulated in a phannaceutically acceptable vehicle.
As described herein die presoit invention p^ticQiarfy encompasses a method of treating a B-cell lymphoma or leukemia or an amoimmune disease in a subject comprising administering to a subject a tteT^)euticaily effective amount of an antibody fusion protein or fragment thereof comprising at least two anti-CD20 mAbs or fragments thereof of die present invention or comprising at least one anti-CD20 MAb or frc^ment thereof of the present invention and at least one additional MAb, preferably selected from the group consisting of mAbs reactive with CD4, CDS, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD3S, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUCl, MUC2, MUC3, MUC4, la, HM1.24, and HLA-DR, tenascin, VEGF, PIGF, an oncogene, oncogene product, or a combination thereof, formulated in a phannaceutically acceptable vehicle.
This therapeutic method can further be supplemMited with the administration to the subject concurrently or sequentially of a therapeutically effective amount of at least one therapeutic agent formulated in a phannaceutically acceptable vehicle, wherein the therapeutic agent is preferably a cytotoxic agent, a radioactive label an immunomodulator, a hormone, a photoactive therapeutic agent or a combination thereof-formulated in a phannaceutically acceptable vehicle.
Further, the antibody fiision proteins can be administered to a subject concurrently or sequentially a therapeutically effective amount of a therapeutic conjugate comprising at least one MAb bound to at least one therapeutic agent wherein said MAb component of flie conjugate preferably comprises at least one humanized chimeric, human or murine (for non-human subjects) MAb selected from the group consisting of a MAb reactive with CD4, CD5, CDS, CD14, CD15, CD19, CD20, CD2L CD22,

CD23, CD25, CD33, CD37, CD38, CD¥}, CD40L, CD46, CDS2. CD54, CD74,
CD80, CD126, B7, MUCl, NiUC2, KiUC3, N'fUC4, la, HM124, aiid HLA-DR,
tenascin, VEGF, PIGF, an oncogene, oncogene product or a combination thereof,
formulated in a pharmaceutically accq>table vehicle. The antibody fusion protein itself
can be conjugated to a therapeutic agent and uius provides a vehicle to attach more
than one therapeutic agent to an antibody component and these therapeutic agents can
be a combinatioD of different recited agents or combin^kHis of the same agents, such
as two different therapeutic radioactive labels. Also eaKX«npassed by the jHesejit
inventicm is a tncAod of diagnosing a B-ceil lyn^Aoma or leukemia in a sutgect mmprigrng administering to the subject, such as a rrfflanmal, including humans and domestic and con:^)amon pets, such as dogs, cats, rc4>bits- guinea pigs, a diagnosdc conjugate comprising an anti-CD20 MAb or fragment thoeof or an antibody fusion protein or fragment thereof of the present invention that binds to the lymphoma or leukemia cell, wherein the anti-CD20 MAb or fragment thereof or antibody fusion protein or fragment thereof is bound to at least one diagnostic agent fomaulated in.a pharmaceutically acceptable vehicle. The usefiil diagnostic agents are described herein. 2. Definitions
In the description that follows, a number of terms are used and the following defrnitions axe provided to faciUtate understanding of the present invention.
An antibody, as described herein, refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an inamimoglobulin molecule, like an antibody fragment
An antibody fragment is a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-CD20 monoclonal antibody fragment binds with an epitope of CD20. The term "antibody fragment also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody firagments include isolated firagments consisting of the variable regions, such as the "Fv" firagments consisting of the variable regions of the hea\7 and Ught chains, recombinant single chain polypeptide molecules in which tight and

he^^7 variable reeions axe connected by a peptide linker ("scFv proteins'), and minimal recomition units consisting of the amino acid residues that mimic the hypervariable region.
A naked antibody is generally an entire antibody T;\1iich is not conjugated to a therapeutic agent This is so because the ,Fc pcroon of the antibody molecule pro\ides effector functions, such as complement fixation and ADCC (antibody depCTident ceU cytotoxicity), \^ch set n^chanisms iiso action that may result in cell lysis. Hdweva:, it is possible that the Fc portion is tK>t required for therap^itic function , with other mechanisms, sudi as ^)optosis, coming into play. Nafced antibodies include both polyclonal and monocl(mal antibodies, as weU as certain recombinant antibodies, such as chimeric, humanized or human antibodies.
A chimCTic antibody is a recombinant protein Hat contains the variable domains including the complementarity determining r^ons (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule is derived from those of a human antibody. For veterinary apphcations, the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.
A h;jmani2ed antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, is transferred from the heavy and Hght variable chains of the rodent antibody into human heavy and light variable domains. The constant domains of the antibody molecule is derived from those of a human antibody.
A human antibody is an antibody obtained from transgenic mice that have been "engineered"" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and Hght chain locus are introduced iato strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et aL, Nature Genet. 7:13 (1994), Lonberg et al, Nature 368:856 (1994), and Taylor et al, Ini. Immun 6:579 (1994). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phase displav technolo2v. all of

which are knovvn in the ait. See for example, McCafferty et al, Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in virro, from immunoglobulin variable domain gene repertoires from unimmunized donors. In this technique, antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, 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 jtege geaiome, sdections based on the fnnctional properties of the antibody also result m selection of the gene OKXxiing tbe andbody exhibiting those propaties. In this way, the phage mimics some of tbe properties of the B cell. Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and QiiswdL Current Opinion in Structural Biology 3:5564-571(1993).
Human antibodies may also be generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and 5^9^75, which are incorporated in tiieir entiret>^ by reference.
A therapeutic a^ent is a molecule or atom v^ch is administered separately, concuirentiy or sequentially with an antibody moiety or conjugated to an antibody moiety, i.e., antibody or antibody fragment or a subfragment and is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, to?dns, nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes and radioisotopes.
A diag;nostic agent is a molecule or atom which is administered conjugated to an antibody moiety, i.e., antibody or antibody fragment or subfiragment, and is useful in diagnosing a disease by locating the cells containing the antigen. Useftil diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnetic ions) for magnetic resonance imaging (MRI). U.S. Patent No. 6,331,175 describes MRI technique and the preparation of antibodies conjugated to a MRI enhancing agent and is incorporated in its entirety by reference-Preferably, the diagnostic agents are selected from the group consisting of radioisotopes, enhancing agents for use in magnetic resonance imaging, and fluorescent compounds. In order to load an antibody component with radioactive metals or paramagnetic ions, it may be necessary to react it with a reagent having a

long rail to which are attached a multipiicin- of chelating groups for binding the ions. Such a tail can be a polymer such as a poiylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to n-iiich can be bound chelating groups such as, e.g., ethylenediaminetetraacedc acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crovm ethers, bis-thiosemicarbazones. polyoximes, and like groups known to be useful for this purpose. Chelates are coupled to the peptide antigens using standard chanistries. The cteiate is normally linked to the antibody by a group which enables fonnadon of a hcsyd to the molecule with miTimifll loss of immunoreMTtivity and minimal ^gregation and/or intanal cross-linking, otiier, more unusnaL methods and reagoits for coiqngating chelates to antibodies are disclosed in U.S. Patent 4,824,659 to HawthOTne, aithled "Antibod>^ Conjugates", issued April 25, 1989, the disclosure of which is incorpcffated herein in its entirety by reference. Particularly useful metal-chelaie combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs^ used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as ^"^I, '^^I, *^I, ^^\ ^Cu, ^Cu, ^«F, *^ V 'Ga, ^^Ga, ^^c, ^^Tc, ^^C, ^, ^^O, ^^r , for radio-imaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI, when used along with the antibodies of the invention. MacrocycUc chelates such as NOT A, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuchdes of gaUium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-tj^pe chelates such as macrocyclic polyethers, which are of interest for, stably binding nuclides, such as
TIT
Ra for RATT are encompassed by the invention.
An inununoconjugate is a conjugate of an antibody component with a ther^eutic or diagnostic agent The diagnostic agent can comprise a radioactive or non-radioactive label, a contrast agent (such as for magnetic resonance imaging, computed tomography or ultrasoimd), and the radioactive label can be a gamma-, beta-, alpha-. Auger electron-, or positron-emitting isotope.
An expression vector is a DNA molectdes comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatorv^ elements, including constitutive or inducible promoters, tissue-specific

re^jlator}^ elements and enhancers. Such a gene is said lo be "operably linked to"' the regulatory elements.
A recombinant host may be any prokarv'otic or e-ukarv^otic cell that contains either a cloning vector or expression vector. This term also includes those prokar}'otic or eukaryotic cells, as well as an transgenic animal, 1hat ieve been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell or cells of the host cells. Suitable mammalian host cefls include myeloma cells. such as SP2/0 cells, and NSO cells, as Nvell as Cirinese Haaaster Ovary (CHO) cells, hybridoma cell lines and othea: mammalian host ceU nsefei for expressing antibodies. Also particulariy useful to express mAbs and otter fhsicm proteins, is a hmnan cell hue, PER.C6 disclosed in WO 0063403 A2, which prodiKes 2 to 200-fold more recombinant protein as compared to conventional mammalian cell lines, such as CHO, COS, Vero, Hela, BHK and SP2- cell lines. Special transgenic animals with a modified immun.e system are particulariy useful for making fully human antibodies.
As used herein, the term antibody fiision protein is a recombinantiy produced antigen-binding molecule in which two or more of the same or different single-chain antibody or antibody fragment segments with the same or different specificities are linked. Valency of the fusion protein indicates how many binding arms or sites the fusion protein has to a single antigen or epitope; i.e., monovalent, bivalent, trivalent or muthvalent The multivalency of the antibody fiision protein means that it can take advantage of multiple interactions in binding to an antigen, thus increasing the avidity of binding to the antigen. Specificity indicates how many antigens or epitopes an antibody fusion protein is able to bind; i.e., monospecific, bispecific, tiispecific, multispecific. Using these definitions, a natural antibody, e.g., an IgG, is bivalent because it has two binding arms but is monospecific because it binds to one epitope. Monospecific, multivalent fusion proteins have more than one binding site for an epitope but only binds with one epitope, for example a diabody with two binding site reactive with the same antigen. The fusion protein may comprise a single antibody component, a multivalent or multispecific combination of different antibody components or multiple copies of the same antibody component. The fiision protein may additionally comprise an antibody or an antibody fragment and a therapeutic agent Examples of therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-hnmunomodulator fusion protein") and toxins

("anubody-toxin fusion protein"). One preferred toxin comprises a ribonuclease (TUsase'i, preferably a recombinant KNase.
A multispecific antibody is an antibody that can bind simultaneously to ai least two targets that are of different structure, e.g., two diferenl antigens, two different epitopes on the same antigerL or a hapten and-'or an antigea or epitope. One specificity would be for a B-cell, T-cell, myeloid-, plasma-, and mast-ceU antigen or epitope. Another specificity could be to a difiereair ao^^a CHI the same c^U type, sudi as CD20, CD19, CD2L CD23, CD46, CD80, HIA-DIL CD74, MUCl, and CD22 on B-cells. Muiti^)ecific, muitival^ri antibodies are constructs thai have mwe than one binding site, and the binding sites are of diffeTCTi specificity. For example, a diabody, where one binding site reacts with one antigeaa and the oflier with the other anfigen.
A bispecific antibody is an antibody that can bind simultaneously to two targets which are of different structure. Bispecific antibodies (bsAb) and bispecific antibody firagments (bsFab) have at least one arm that specifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope and at least one other arm that specifically binds to a targetable conjugate that bears a therapeutic or diagnostic agent A variety of bispecific fusion proteins can be produced using molecular engineering. In one form, the bispecific fusion protein is monovalent, consisting o^ for example, a scFv with a single binding; site for one antigen and a Fab fiagment with a single binding site for a second antigen. In another form, the bispecific fusion protein is divalent, consisting of, for example, an IgG with a binding site for one antigen and two scFv with two binding sites for a second antigen.
Caninized or felinized antibodies are recombinant proteins in which rodeni (or another species) complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of rodent (or another species) immunoglobulin into a dog or cat respectively, immunoglobulin variable domain. Domestic animals incliide large animals such as horses, catUe, sheep, goats, llamas, alpacas, and pigs, as well as companion animals. In a preferred embodiment, the domestic animal is a horse.
Companion animals include animals kept as pets. These are primarily dogs and cats, although small rodents, such as guinea pigs, hamsters, rats, and ferrets, are

also included- as are subhuman primates such as monkeys. In a preferred emb^Ddiment the companion animal is a dog or a cat
3. Preparation of Monoclonal Antibodies including Chimeric, Humanized
and Hr.maa Antibodies
Monoclonal antibodies (MAbs) are a homogeneous population of antibodies to a particular antigen and the antibody comprises only ooe type of antigen binding site and lands to only one epitope on an antigenic detenninant Rodent monoclonal antibodies to specific antigens may be obtained by na^hods known to liiose skilled in the art See, for example, KOUCT and Milstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter "Coligan^. Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones wiiich produce antibodies to the antigen, culturing tiie clones that produce antibodies to the antigen, and isolating the antibodies jfrom the hybridoma cultures.
MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al,, "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).
After the initial raising of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art For example, humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immimoglobulin into a human variable domain, and therL substituting human residues in the framework regions of the murine counterparts. The

use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated v^iih the immunogenicitx^ of murine constant regions.
General techniques for cloning murine immunoglobulin variable domains are described^ for example, by the pubUcation of Orlandi et al., Proc. NaflAcad Scl USA 86: 3833 (1989), which is incorporated by reference in its entirety: Techniques for constructing chimeric antibodies are well known to those of skill in the art As an example, Lexmg et al., Hybridoma 73:469 (1994), describe how they produced an LL2 chimera by combining DNA sequences encoding tbe V- and VH domains of LL2 nK>QOclonal antibody, an anti-CD22 antibody, with regsective human K and IgGi constant region domains. This pubUcation also provides the nucleotide sequences of the LL2 light and heavy chain variable regions, V^ and Vj^ re^sectively. Techniques for producing humanized MAbs are described, for example, by Jones et al., Nature 321: 522 (1986), Riechmann et al, Nature 332: 323 (1988), Verhoeyen et al„ Science 239: 1534 (1988), Carter et al,, Proc, Natl Acad ScL USA 89: 4285 (1992), Sandhu, Crit Rev, Biotech 12: 437 (1992), and Singer et aL J- Immm, 150: 2844 (1993), each of which is hereby incorporated by reference.
A chimeric antibody is a recombinant protein thai contains the variable domains including the CDRs derived from one species of animal, such as a rodent antibody, while the remainder of the antibody molecule; i.e., the constant domains, is derived from a human antibody. Accordingly, a chimeric monoclonal antibody can also be humanized by replacing the sequences of the murine FR in the variable domains of the chimeric MAb with one or more different human FR- Specifically, mouse CDRs are transferred from heavy and light variable chains of the mouse immunoglobulin into the corresponding variable domains of a human antibody. As simply transfa~ring mouse CDRs into human FRs often results in a reduction or even loss of antibody affinity, additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomphshed by the replacement of one or more some human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding af&nity to its epitope. See, for example. Tempest et al, Biotechiology 9:266 (1991) and Veriioeyen et al.. Science 239: 1534 (1988). Fxirther, the affinity' of humanized, chimeric and human MAbs to a specific q)itope can be increased by mutagenesis of the CDRs, so that a lower dose of antibody may be as

etiecd ve as a higher dose of a lower afiinirv M\b prior to mutagenesis. See for example, WO0029584A1
Another method for producing flie antibodies of the present invention is by production in the milk of transgenic Uvestock. See. e.g., Colman, A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Patent 5,827,690, both of w%ich are incorporated in their entirety by reference. Two DNA constructs are prq)ared which contain, respectively, DNA segm^its encoding paired ronnuDOglobdin heavy and h^t chains. The DNA segments are cloned into expression vectors whicii contain a jKomoter sequence that is prefeentially expressed in mammar>' ^nfedial cells. Examples include, but are not limited to, promoters from rabbit, cow aid sheep casein genes, the cow a-lactoglobulin gene, the sheep p-lactogiobulin gene and the mouse whey acid protein gene. Preferably, the inserted fragment is flanked on its 3' side by cognate genomic sequences from a mammaiy-specific gene. This provides a polyadenylation site and transcript-stabilizing sequences. The expression cassettes are co-injected into the pronuclei of fertilized, mammahan eggs, which are then implanted into the uterus of a recipient female and allowed to gestate. After birdi, the progeny are screened for the presence of both transgenes by Southern analysis. In order for the antibody to be present, both heavy and Ught chain genes must be expressed concuorently in the same cell. Milk from transgenic females is analyzed for the presence and functionahty of the antibody or antibody fragment using standard immunological methods known in the art The antibody can be purified from the milk using standard methods known in the art
A fully hunaan antibody of the present invention, i.e., hxmaan anti-CD20 MAbs or other human antibodies, such as anti-CD22, anti-CD 19, anti-CD23, or anti-CD21 MAbs for combination therapy with humanized, ctiimeric or human anti-CD20 antibodies, can be obtained from a transgenic non-human animal. See, e.g., Mendez et aL Nature Genetics, 15: 146-156 (1997); U.S. Patent No. 5,633,425, which are incorporated in their entirety by reference. For example, a human antibody can be recovered from a transgenic mouse possessing himian immunoglobulin loci. The mouse humoral immune system is humanized by inactivating the endogenous immimoglobulin genes and introducing human immunoglobulin loci. The human immunoglobulin loci are exceedingly complex and comprise a large number of discrete segments \^iiich together occupy almost 0.2% of the human genome. To

ensure thai transgenic mice are capable of producing adequate repertoires of antibodies, large pordons of human heavy- and light-chain lc>ci must be introduced into the moxose genome. This is accomplished in a stepwise process beginning wath the formation of yeast artificial chromosomes (YACs) containmg either human heavy-or light-chain immunoglobulin loci in germline configuration. Syice each insert is approximately 1 Mb in size, YAC construction requires hcnnologous recombination of overlapping firagments of the inamunoglobuiin loci. The two YACs, one containing the heavy-chain loci and one containing the light-chain lod, are introduced separately into mice via fusion of YAC-containing yeast spteioblas^ w^ mouse eanbryonic stem cells. Embryonic stem cell clones are then microrigected into mouse blastocysts. Resulting chimeric males are screened for their ability to transmit the YAC through their germline and are bred with mice deficient in murine antibody production. Breeding the two transgenic strains, one containing the human heavy-chain loci and the other contaioing the human hght-chain loci, creates progeny ^\hich produce human antibodies in response to immunization.
Further recent methods for producing bispecific mAbs include engineered recombinant mAbs Vtdiich have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald et al, Protein Eng. 10(10):1221-1225,1997. Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g., Coloma etoL^Natwe Biotech, 15:159-163, 1997. A variety of bispecific fusion proteins can be produced using molecular engineering. In one form, the bispecific fiision protein is monovalent consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen. In another form, the bispecific fusion protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.
Bispecific fiision proteins Linking tvv^o or more different single-chain antibodies or antibody fragments are produced in shnilar manner. Recombinant methods can be used to produce a variety effusion proteins. For example a fusion protein comprising a Fab fragment derived from a himianized monoclonal anti-CD20 antibody and a scFv derived from a murine anti-diDTPA can be produced. A flexible

linker, such as GGGS connects the scFv to the constant region of the heaw chain of the anti-CD20 antibody. .Alteroatively. the scFv can be connected to the constant region of the light chain of another humanized antibody. Appropriate linker sequences necessary for the in-frame connection of the heavy chain Fd to the scFv are introduced into the VL and VK domains through PGR reactions. The DNA fragment encoding the scFv is then ligated into a staging vector containing a DNA sequence encoding the CHI domain. The resulting scFv-CHl constraci is excised and ligated into a vector containing a DNA sequence encoding the VH region of an and-CD20 aitibody. The resulting vector can be used to transfect an appxypnate host cell such as a mammalian cell for the expression of the biqjecific fiision protein.
4- Production of Antibody Fragments
Antibody fragments which recognize specific epitopes can be generated by known techniques. The antibody fragments are antigen binding portions of an antibody, such as F(ab*)2, Fab', Fab, Fv, sFv and the like. Other antibody fragments include, but • are not limited to: the F(ab)'2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab' fragments, which can be generated by reducing disulfide bridges of the F(ab)'2 fragments. Alternatively, Fab' expression libraries can be constructed (Huse er a/., \9S9, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity. The present invention encompasses antibodies and antibody fragments.
A single chain Fv molecule (scFv) comprises a VL domain and a VH domain. The VL and VH domains associate to form a target binding site. These two domains are further covalently hnked by a peptide linker (L). A scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-tenninal part of the scFv molecule. Methods for making scFv molecules and designing suitable peptide linkers are desciibed in US Patent No. 4,704,692, US Patent No. 4,946,778, R. Raag and M. Whitlow, 'Single Cliain Fvs/' FASEB Vol 9:73-80 (1995) and R.E. Bird and B.W. Walker, "'Single Chain Antibody Variable Regions:' TIBTECH, Vol 9: 132-137 (1991). These references are incorporated herein by reference.
An antibody Sagment can be prepared by proteolytic hydrolysis of the full length antibody or by expression in E. coli or another host of the DNA coding for the

fragment- .\n antibody fragment can be obtained by pepsin or papain digestion of ftdl length antibodies by conventional methods. For example, an antibody fragment can be produced by en2:>Tnatic cleavage of antibodies v^ith pepsin to pro\ide a 5 S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the snlfriydryl groups resulting from. cleavage of disulfide linkages, to produce 3.5S Fab' monovaleal fragments. Alternatively, an eozymatic cleavage using papain produces two monovalait Fab fragments emd an Fc fragment directly. These m^bods are described, for example, by Goldenberg, U^. Patent Nos. 4,036,945 and 433L647 and referaices contained therein, which patents are incoiporated herein in their entireties by ref^ence. Also, .s^Kisor^eeraI.,ArchBio€he77j.Biopf^s. SP: 230 (1960); Porter, Bzoc/aeni. J. 73: 119 (1959), Edelman et a/., in METHODS IN ENZYMOLOGY VOL. L page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
Another form of an antibod}'" fragment is a peptide coding for a single complementarity-determining region (CDR). A CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable-region. Accordingly, a CDR is sometimes referred to as hypervariable region. A variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et ah, Methods: A Companion to Methods in Enzymology 2: 106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al, "Genetic Manipulation and Expression of Antibodies," in MONOCLONAL ANTIBODIES: PRINCIPLES AKD APPLICATIONS, Birch et ai, (eds.), pages 137-185 (WUey-Liss, Inc. 1995).
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heav^' chain &agments, further cleavage of firagments, or other enzymatic, chemical or genetic techniques may also be lised, so long as the fragments bind to the antigen that is recognized by the intact antibody.

5, Multispecific and multivalent antibodies
The anti-CD20 antibodies, as well as other antibodies v.ath different specificities for use in combination therapy, described herein, can also be made as multispecific antibodies (comprising at least one binding site to a CD20 epitope or antigen and at least one binding site to another epitope on CD20 or another antigen) and multivalent antibodies (comprising multiple binding sites to the same epitope or antigen). Multivalent target binding proteins are (kscribed in US Serial No. 09/^11,610 (Leung et al.), whidi is incorporated heran by refojence in its entiret}'.
Tte present invention provides a bispecific antibody or antibody fi:agment having at least a binding region that specijfically binds a targeted cell marker and at least one other binding region that specifically binds a targetable conjugate. The targetable conjugate comprises a carrier portion which conqjtises or bears at least one epitope recognized by at least one binding region of the bi^)ecrfic antibody or antibody firagmenL
A variety of recombinant methods can be used to produce big)ecific antibodies and antibody fiagments as described above.
An anti-CD20 multivalent antibody is also contemplated in the present invention. This multivalent target binding protein is constructed by association of a first and a second polypeptide. The first polypeptide comprises a first single chain Fv molecule covalentiy linked to a first immimoglobulin-hke domain which preferably is an immvmoglobulin light chain variable region domain. The second polypeptide comprises a second single chain Fv molecule covalentiy linked to a second immunoglobulin-like domain which preferably is an immunoglobulin heavy chain variable region domain. Each of the first and second single chain Fv molecules forms a target binding site, and the first and second inamunoglobulin-like domains associate
to form a third tareet binding site.
«—■ t—•
A single chain Fv molecule with the VL-L-VH configuration, wherein L is a linker, may associate with another single chain Fv molecule with the VH-L-VL configuration to form a bivalent dimer. In this case, the VL domain of the first scFv and the VH domain of the second scFv molecule associate to fonn one target binding site, while the VH domain of the first scFv and the VL domain of the second scFv associate to fomi the other tareet binding site.

.Another embodiment of the present invention is a CD20 bispecific, trivaient targeting protein comprising two heterologous polypeptide chains associated non-covalently to form three blading sites, two of which have affinity for one target and a third which has affinity for a hapten thai can be made and attached to a carrier for a diagnostic and/or therapeutic agent. Preferably, the binding protein has two CD20 binding sites and one CD22 blading site. The bispecific, trivaient targeting agents have two diffCTent scFvs, one scFv contains two VR domaii^ from one antibody connected by a diort linker to the VL domain of ano*ber antfl)ody and the second scFv OMitains two VL domains from tbe first antibody connected by a short linker to the VH domain of the other antibody. The me&ods for geaiCTating multivalent, multispecific ageaits from VH and VL domains provide that individual 6- Diabodies, Triabodies and Tetrabodies
The anti-CD20 antibodies of the present invention can also be used to prepare functional bispecific single-chain antibodies (bscAb), also caUed diabodies, and can be produced in mammalian cells using recombinant methods. See, e.g.. Mack et al., Froc. Natl. Acad Set, 92: 7021-7025, 1995, incorporated. For example, bscAb are produced by joining two single-chain Fv fragments via a glycine-serine linker using recombinant methods. The V light-chain (VL) and V heavy-chain (VH) domains of two antibodies of interest are isolated using standard PCR methods. The VL and V'H cDNA's obtained from each hybridoma are then joined to form a single-chain fragment in a two-step fiision PCR. The first PCR step introduces the (Gly4-Seri)3 linker, and the second step joins the VL and VH amplicons. Each single chain

molecule is then cloned into a bacterial expression vector. Following amplification, one of the single-chain molecules is excised and sub-cloned into the other vector, containing the second single-chain molecule of interest The resulting bscAb fragment is subcloned into an eukar}^otic o^ression vector. Functional protein expression can be obtained by transfecting the vector into Chinese hamster ovary cells. Bispecific fusion proteins are prepared in a similar manner. Bispecific single-chain antibodies and bispecific fiision proteins are included wrflrin 4e scope of the jnesent invention.
For exanq>le, a humanized, chimaic or human anti-(3>20 monoclonal antibody can be used to produce antigen specific diabodies, triabodies. and t^rabodies. The monospecific diabodies, triabodies, and tetiabodies bind selectively to targeted antigens-and as the number of binding sites on the molecule increases, the affinity for the target cell increases and a longer residence tinK is observed at the desired location. For diabodies, the two chains comprising the VH polypeptide of the humanized CD20 MAb connected to the VK polypeptide of the humanized CD20 MAb by a five amino acid residue linker are utilized. Each chain forms one half of the humanized CD20 diabody. In the case of triabodies, the tiiree chains comprising VH polypeptide of the hmnanized CD20 MAb connected to die VK polypeptide of the humanized CD20 MAb by no linker are utilized. Each chain forms one third of the hCD20 triabody.
The ultimate use of the bispecific diabodies described herein is for pre-targeting CD20 positive tumors for subsequent specific delivery of diagnostic or therapeutic agents. These diabodies bind selectively to targeted antigens allo\ving for increased affinitv^ and a longer residence time at the desired location. Moreover, non-antigen bound diabodies are cleared fi-om the body quickly and exposure of normal tissues is mimmized. Bispecific antibody point mutations for enhancing the rate of clearance can be found in US Provisional Application No. 60/361,037 to Qu et al, (Atty Docket No. 18733/1037), which is incorporated herein by reference m its entirety. Bispecific diabodies for affinity enhancement are disclosed in US Application Nos- 10/270,071 (Rossi et aL\ 10/270,073 (Rossi etal.) and 10/328,190 (Rossi et aL), which are incorporated herein by reference in their entirety. The diagnostic and therapeutic agents can include isotopes, drugs, toxins, cytokines, honnones, growth factors, conjugates, radionuclides, and metals. For example,

gadolinium metal is used for magnetic resonance imaging (MRI). Examples of radionuclides are ^^Ac. '^F. ^^Ga, ^^Ga, ^^ ^ Y, "V ^^'L '"L '^L ^^TC, ^^"-TC, =^^e, '^^Re, '"Lu, "Cu, ^Cu, ^'CiL "'^Bi. ^'^Bi, ^^P, '^C. ^^. ^^O. ^^r, and ='V\L Other radionuclides are also available as diagnostic and therapeutic agents, especially those in the energy range of 60 to 4,000 keV.
More recently, a tetravalent tandem diabody (teimed tandab) with dual spedficity has also been reported (Codilovius et al:. Cancer Research (2000) 60; 4336-4341). The bi^pecific tanr^ah is a dimer of two id«itical polypeptides, each containing four variable domains of two diSerent airtibodies (VHI^ VLI:, VH2: ^U) linked in an orientation to facilitate the formation of two potential binding sites for each of the two different specificities upon self-association.
7. Conjugated multivaient and multispecific anti-CD20 antibodies
In another embodiment of the instant invention is a conjugated multivalent anti-CD20 antibody. Additional amdno acid residues may be added to either the N- or C- . teraiiuus of the first or the second polypeptide. The additional amino acid residues may comprise a peptide tag, a signal peptide, a cytokine, an enzyme (for example, a pro-drug activating enzyme), ahonnone, a peptide toxin, such as pseudomonas extoxin, a peptide drug, a cytotoxic protein or other functional proteins. As used herein, a fimctional protein is a protein which has a biological fimction.
In one embodiment, drugs, toxins, radioactive compounds, enzymes, hormones, cytotoxic proteins, chelates, cytokines and other functional agents may be conjugated to the multivalent target binding protein, preferably through covalent attachments to the side chains of the amino acid residues of the multivalent target binding protein, for example amine, carboxyl, phenyl, thiol or hydroxyl groups-Various conventional linkers may be used for this purpose, for example, diisocyanates, diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleimide-hydroxysuccinimide esters, giutaraldehyde and the like. Conjugation of agents to the multivalent protein preferably does not significantly affect the protein's binding specificity or affinity to its target. As used herein, a functional agent is an agent which has a biological function. A preferred functional agent is a cytotoxic aaent.

In still other embodiments, bispecific antibody-directed deliven- of therapeutics or prodrug polymers to in vh-c targets can be combined with bispecific antibody deliver>^ of radionuclides, such that combination chemotherapy and radioimmunotherapy is achieved. Each therapy can be conjugated to the targetable conjugate and administered simultaneously, or the nuclide can be given as part of a first targetable conjugate and the drug given in a later stq) as part of a second targeteble conjugate.
In anotiier embodiment, cytotoxic agents may be coigi^ated to a polymeric caniex, and the polymeric carrier may subsequently be coigiigated to the multivalent target binding protein. For this method, see Ryser et aL, Proc, Neitl. Acad Set USA, 75 J867-3870,1978, US Patent No. 4,699,784 and US PateniNo. 4,046,722, which are incorporated herein by reference. Conjugation preferably does not significantly affect the binding specificity or affinity of the multivalent binding protein.
8. Humanized, Chimeric and Human Antibodies Use for Treatment and Diagnosis
Humanized, chimeric and human monoclonal antibodies, i.e., anti-CD20 MAbs and other MAbs described herein, in accordance with this invention are suitable for use in therapeutic methods and diagnostic methods. Accordingly, the present invention contemplates the administration of the humanized, chimeric and human antibodies of the present invention alone as a naked antibody or administered as a multimodal therapy, temporally according to a dosing regimen^ but not conji^ated to, a therap^rtic agent The efficacy of the naked anti-CD20 MAbs can be enhanced by si^plementing naked antibodies with one or more other naked antibodies, i.e., MAbs to specific antigens, such as CD4, CD5, CDS, CD14, CD15, CD19, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUCl, la, HM1.24, or HLA-DR, tenascuL VEGF, PIGF. an oncogene, an oncogene product, or a combination thereofwith one or more immunoconjugates of anti-CD20, or antibodies to theses recited antigens, conjugated with therapeutic agents, including drugs, toxins, immunomodulators, hormones, therapeutic radionucUdes, etc., witii one or more therapeutic agents, including drugs, toxins, immunomodulators, hormones, therapeutic radionuclides, etc., administered concurrentiy or sequentially or according to a prescribed dosing regimen, with the MAbs. Preferred B-cell antigens include those

equivalent to human CD19, CD20. CD2L CD22, CD23, CD46, CD52, CD74, CD80, and CDS antigens. Preferred T-cell antigens include those equivalent to human CD4, CDS and CD25 (the IL-2 receptor) antigens. An equivalent to HLA-DR antigen can be used in treatment of both B-cell and T-cell disorders. Particularly preferred B-cell antigens are those equivalent to human CD19, CD22, CD2L CD23, CD74, CD80, and HLA-DR antigens. Particulariy preferred T-cell antigens are those equivalent to human CD4. CDS aiKi CD25 antigens. CD46 is an antig^i CHI the surfe::e of cancer cells that block cotnplement-depeaident lysis (CDC).
Further, tte present invention contemplates the administraaion of an immuDOconjugate for diagnostic and therapeutic uses in B cell lymphomas and other disease or disorders. An immunocoryugate, as described herein, is a molecule comprising an antibody component and a therapeutic or diagnostic agent including a peptide which may bear the diagnostic or therapeutic agent An immunoconjugate retains the immunoreactivity of the antibody con:qx>nent, te.. the antibody moiety has about the same or slightiy reduced ability to bind the cognate antigen after conjugation as before conjugation.
A wide variety of diagnostic and therapeutic agents can be advantageously conjugated to the antibodies of the invention. The therapeutic agents recited here are those agents that also are useful for administration separately with the naked antibody as described above. Therapeutic agents include, for example, chemotherapeutic drugs such as vinca alkaloids, anthracyclines, epidophyllotoxinj taxanes, antimetabolites, alkylating agents, antikina.se agents, antibiotics, Cox-2 inhibitors, antimitotics^ antiangiogenic and apoptotoic agents, particularly doxorubicin, methotrexate, taxoL CPT-11, camptothecans, and others from these and other classes of anticancer agents , and the like. Other useful cancer chemotherapeutic drugs for the preparation of immunoconjugates and antibody fusion proteins include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REIVUNGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMANS THE PHARMACOLOGICAL BASIS OF THER^APELTICS, 7th Ed. (MacMiUan Publishmg Co. 1985), as well as revised

editions of these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.
Additionally, a chelator such as DTPA^ DOT A, TET.A^ or NOTA or a suitable peptide, to which a detectable label, such as a fluorescent molecule, or cytotoxic agent, such as a heavy metal or radionuclide, can be conjugated. For example, a therapeuticaUy useful immunoconjugate can be obtained by coigugating a photoactive agent or dye to an antibody composite. Flumesc^it ccHiq>ositions, such as ftucHOchrome, and other chromogens, or dyes, such as porphjnins s^isitive to visible tight have been used to detect and to treat lesions by directing tbe suitable light to die lesion. In ther^y, this has been temied photoradiatioiL phototherapy, or pbotodynamic therapy (Jori et al. (eds.)= PHOTODYKAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodies have been coupled with photoactivated dyes for achieving phototherapy. Mew et oL, J. bnimmoL 730:1473 (1983); idem.. Cancer Res. ^5:4380 (1985); Oseroffer al., Proc. NatL Acad Sci. USA 53:8744 (1986); idem,, Photochem. Photobiol 45:83 (1987); Hasan et al. Prog. Clin. Biol Res. 288:471 (1989); Tatsuta et al, Lasers Surg. Med 9:422 (1989); Pelegrin et al, Cancer 67:2529 (1991). However, these earlier studies did not include use of endoscopic therapy applications, especially with the use of antibody ftagments or subfragments. Thus, the present invention contemplates the therapeutic use of immunoconjugates comprising photoactive agents or dyes.
Also contemplated by the present invention are the use of radioactive and nonradioactive agents as diagnostic agents. A suitable non-radioactive diagnostic agent is a contrast agent suitable for magnetic resonance imaging, computed tomography or ultrasound. Magnetic imaging agents include, for example, non-radioactive metals, such as manganese, iron and gadolinium- complexed with metal-chelate combinations that include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, when used along with the antibodies of the invention. See U.S. Serial No. 09/921J290 filed on October 10, 2001, wtiich is incorporated in its entirety by reference.
Furthermore, a radiolabeled antibody or immunoconjugate may comprise a y-emitting radioisotope or a positron-emitter useful for diagnostic imaging. Suitable radioisotopes, particxilarly in the energy range of 60 to 4,000keV, include '"'l, '^^L
,24^^ S6Y 62^^ 64^^^ UIj^ 67Q^ 68^^ 9^^^^ 94^^^ ,8p_ H^^ 13^^ ,5Q^ 753^^ ^^ ^^

like. See for example, U.S. Paieni Applicaiioa entitled "Labeling Targeting Agents with Gallium-68"- Inventors G.L.Grifiiths 2nd WJ. McBride, (TLIS. Provisional Application No. 60/342J(>4), which discloses positron emitters, such as ^^F, ^^Ga, ^"^c. and the lite, for imaging purposes and which is incorporated in its entirety by reference. Particularly useful therapeutic radionuclides include, but are not limited to, ^^P, ^^P, ^'Sc, "Cu, ^^Cu, ^'Ga, ^Y, "'Ag, '"in, '^% '\ '^Pr, ^^'Sm, '^'Tb. '^Dy, '«Ho. '"Lu, ^"^e, ^**Re, ^«^, ^'^Pb, '^BL "^L ^l4L ^^^ and ^Ac. Particulariy useful diagnostic/detection radionuclides include- but are not limited to, '*F, =Fe, ^Cu, ^Cu, ^Cu, ^'Ga, ^^Ga, ^, ^Zr, ^^c, ^c, ^^c, "V '^L ^^^L
A toxin, such as Pseudomorias exotoxin, may also be c(Miq)k3ced to or form the tha^tpeutic agent portion of an antibody fusion protein of an anti-CD20 antibody of the present invention. Other toxins suitably employed in "fl^ preparation of such conjugates or other fusion proteins, include ricin, abrin, ribonuclease (RNase), DNase L Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin, Pseudomoitos exotoxin, and Psendomonas endotoxin. See, for example, Pastan et al, Cell 47:641 (1986), and Goldenberg, CA - A Cojicer Journal for Cliniciaits 44'A3 (1994). Additional toxins suitable for use in the present invention are known to those of skill in the art and are disclosed in U.S. Patent 6,077,499, v^ch is incorporated in its entirety by reference.
An immunomodulator, such as a cytokine may also be conjugated to, or form the therapeutic agent portion of an antibody fusion protein or be administered with the humanized anti-CD20 antibodies of the present invention. Suitable cytokines for the present invention include, but are not limited to, interferons and iaterleukins, as described below.
An ohgonucleotide, such the antisense molecules inhibiting bcl-2 expression that are described in U.S. 5,734,033 (Reed) which is incorporated by reference in its entirety, may also be conjugated to, or form the therapeutic agent portion of an antibody fusion protein or be administered with the humanized anti-CD20 antibodies of the present invention.

9. preparation ot immunoconjugates
>uiy of the antibodies or antibody fusion proteins of the present invention can be conjugated with one or more therapeutic or diagnostic agents. Generally, one diera^utic or diagnostic agent is attached to each antibody or antibody fragment but more than one therapeutic agent or diagnostic agent can be attached to the same antibody or antibody fragment The antibody ftision proteins of the present invention OHiifHrise two or more antibodies or fragments tiiereof aiwi eadi of the antibodies that conqx)ses this fiision protein can contain a ther^)eutic ageaat or diagnostic agent Additionally, one or more of the antibodies of the antibody fiision protein can have more than one ther^jeutic of diagnostic agent attached. Further, the therapeutic agents do not need to be tiie same but can be difFCTcot therapeutic agents. For example, one can attach a drug and a radioisotope to the same ftision protein. Particularly, an IgG can be radiolabeled with *^^I and attached to a drug. The ^^^I can be incorporated into the tyrosine of the IgG and the drug attached to the epsilon amino groicp of the IgG lysines. Both therapeutic and diagnostic agents also can be attached to reduced SH groups and to the carbohydrate side chains.
Radionuclides suitable for treating a disease tissue substantially decay by beta-particle emission and include, but are not limited to: ^"P, ^^P, ^^Sc, '^e, ^Cu, ^^Cu, ■^Se, '^As, «^Sr, 'Y, ^'MO, ^°^Rh, '"^d, '^'Ag, ^^^I, '^^I, ^^^Pr, ^^^Pr, '^^Pm, '"Sm, '^'Tb, '"Ho, '^^r, '^'Lu, '^^Re, '««Re, '^^e, '^Ir, '^^Au, '^Au, "^'Pb, ^^Pb and -'^Bi. Maximum decay energies of useftil beta-particle-emitting nuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuchdes that substantially decay with Auger-emitting panicles. For example, ^«Co, ^'Ga, ^"^r, ^^c, ^"^-j^ i09p^ m-^^ u9^^_ .25^^ lei^^^
^^ Os and '"Ir. Decay energies of useful Auger-particle-emitting nuclides axe preferably Radionuchdes useful as diagnostic agents utiUzing gamma-ray detection ioclude, but are not limited to: '^Cr, ^^Co, ^^Co. ^^Fe, ^^Cu, ^^Ga. '^Se. ^^Ru, ^Tc.

■^^'^In, '^^"^n, ~L 125L ^^^L ^^TD, '^'"Hg, and-^'ll. Decay energies of useful gamma-ray emitting radionuclides are preferably 20-2000 keV^ more preferably 60-600 keV, and most preferably lQO-300 keV.
Radionuclides useful for positron emission tomography include, but are not limited to: *^, '^Mn, '^Mn, '¥e, 'Co, ^Cu, ^Cu, "^Ga, ^r^ ^^r. '^r, '^b, ^Sr, ^\ ^Zr, 94mTc, 'V ^'^, and ^'^L Total decay energies of useful positron-f^mittTTig radionuclides are preferably Bi^)ecific antibodies of the pffesent invention are usefbl in pietargeting mediods and provide a [Hef€a.red way to dehver two therapeutic agoits or two diagnostic ag^its to a sufagecL U.S. Serial Nos. 09/382,186 and 09/337,756 cfiscioses a m^bod of fKttergeting using a bispectfic antibody, in which the biq)ecific arrtibody is labeled with ^^ and delivered to a subject, followed by a divalent pqitide labeled with ^^™Tc, and are incoiporated herein by reference in their entirety. Pretargeting methods are also described in US Serial Nos. 09/823,746 (Hansen et al.) and 10/150,654 (Goldenberg et alX and US Provisional AppUcation filed January 31, 2003, entitled "Methods and Compositions for Administration of Therapeutic and Diagnostic Agaits, Atty Docket No. 018733/1103 (McBride et aL), which are all also incorporated herein by reference in their entiret5^ The deUvery results in excellent tumor/normal tissue ratios for ^"^I and ^^*^c, thus showiug the utility of two diagnostic radioisotopes. Any combination of known therapeutic agents or diagnostic agents can be used to label the antibodies and antibody fusion proteins. The binding specificity of the antibody component of the MAb conjugate, the efficacy of the therapeutic agent or diagnostic agent and the effector activity of the Fc portion of lie antibody can be deteimined by standard testing of the conjugates.
The invention is directed to a method for pretargeting a cell in a patients suffering from a B-cell lymphoma or leukemia or an autoimmune disease comprising:
(i) administering an antibody fiision protein or fragment thereof that is multispecific having at least one arm that specifically binds the cell and at least one other arm that specifically binds a targetable conjugate; (ii) optionally, administering to the patient a clearing composition, and allowing the composition to cl-ear non-antigen bound antibody fusion protein or Segment thereof fi'om circulation; and (iii) administering to the patient a targetable conjugate comprising a carrier portion which

comprises or bears at least one epitope recognizable by at least one other arm of the antibody fusion protein or fragment thereof, and is conjugated at least one first therapeutic or diagnostic agent Tne antibody fusion protein of me present invention should be multi^>ecific antibody. In a preferred embodiment the antibody is a bispecific antibody, and can be a diabody. The first therapeutic agent is selected fi-om the group consisting of a radioactive label, an immunomodulalor, a hormone, a photoactive liierap&CJiic agrai, a cytotoxic agrait, an oligonucleoti(te aiMi a comlHnation thoreof and -wbea^in the first diagnostic agait is at least one of a ralio^:tive labd, afrfK)toactive (fiagoostic Bgsnt or a ncm-radioactive label.
The antibodyfusionproteinorfi:aginaitthereof alsomay becoxgug^edtoa second tfaer^jesitic, such as at least one radioactive label, an imnaunomoduiator^ a hormone, a photoactive therapeutic agent, a cytotoxic agent an oUgonucleoticfe and a combination tbeieof or may be conjugated the second diagnostic agent, such as at least one of a radioactive label, a photoactive diagnostic agent or a non-radioactive label. In one embodim^it the first and second therapexitic agent or diagnostic agent are the same.
A therapeutic or diagnostic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation. As an alternative, such peptides can be attached to the antibody component using a heterobifiinctional cross-Iinker, such as 7V-succinyl 3-{2-pyridyldithio)propionate (SPDP). Yu et al, Int. J. Cancel' 56: 244 (1994). General techniques for such conjugation are well-known in tiie art See, for example, Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et oL, "Modification of Antibodies by Chemical Methods," in MONOCLONAL .ANITBODIES: PRINCIPLES AND APPLICATIONS, Birch et al (eds.). pages 187-230 (Wiley-Liss, Inc. 1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in MONOCLONAL .ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICS APPLICATION, Ritter et al (eds.), pages 60-84 (Cambridge University Press 1995). Alternatively, the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiet}^ in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same peptide that is bound to a thiol group, or the carbohydrate moiety can be used to bind a difierent peptide.

Methods for conjugating peptides lo antibod}' components via an antibody carbohydrate moiet}^ are Vv■ell-kno\^'n to those of skill in the art See, for example, Shin ei al, M. J. Cancer 41: 832 (1988); Shih et al, Int. J. Cmicer 46: 1101 (1990); and Shih e^a/^, U.S. PateniNo. 5,057313, all of which are Lncorporaied in theii entirety by reference. The general method involves reacting an anubod}' component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine fimctioa and that is loaded with a piiirality of peptide. This reaction results in an initial Sciiff base (imine) linkage, which can be stabilized by rediKtion to a secondary amine to form the final conjure.
The Fc region is absent if die antibody used as tbe antibody component of the immunocorgugate is an antibody fragment However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment See, for example, Leung et al,, J. Immunol 154: 5919 (1995); Hansen et al,, U.S. Patent No. 5,443,953 (1995), Leung et al, U.S. patent No. 6,254,868, all of which are incorporated in their entirety by reference. The engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent
10. Pharmaceutically Acceptable Excipients
The humanized, chimeric and human anti-CD20 mAbs to be delivered to a subject can consist of the MAb alone, immunoconjugate, fusion protein, or can comprise one or more pharmaceutically suitable excipients, one or more additional ingredients, or some combination, of these.
The immunoconjugate or naked antibody of the present invention can be formulated according to known methods to prepare pharmaceutically usefal compositions, whereby the immunoconjugate or naked antibody are combined in a mixture with a pharmaceutically suitable excipient Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well-known to those in the art See, for example, Ansel et al, PHAJRMACEUTICAL-DOSAGE F0KK4S AM) DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON^ S PHARMACEUTICAL SCIENCES, 1 Sth Edition (Mack Publishing Company 1990), and revised editions thereof
The immunoconjugate or naked antibody of the present invention can be formulated for intravenous administration via^ for example, bolus injection or

'Continuous infusion. Preferably, the antibody of the present invention is infxised over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours. For example, the first 25-50 mg could be infused within 30 minutes, preferably even 15 miru and the remainder infused over the next 2-3 hrs. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multi-dose"" containers, with an added preservative. The compositions can take such forms as si^jeosions, soiiitions or emulsions in oily or aqi^ous vehicles, aod can contain fonnulatory agents such as suspending, stabilizing and/or disposing agents. Alternatively, the active ingredient can be in powder fons for coiistitntion with a suhabie vehicle, e.g., sterile pyrogen-free water, before use.
Additional pharmaceutical methods may be employed to coaorrol the duration of action of "flie ther^eutic or diagnostic conjugate or naked antibody. Control release preparations can be prepared through the use of polymers to complex or adsorb the inmiunoconjugate or naked antibody. For example, biocompatible polymers include matrices of poly(ethylene-co-vinyl acetate) and matrices of a polyanhydride copolymer of a stearic acid dimer and sebacic acid. Sherwood er al. Bio/TecJmoIog}' 10: 1446 (1992). The rate of release of an immunoconjugate or antibody from such a matrix depends upon the molecular weight of the immunoconjugate or antibod3% the amount of immunoconjugate, antibody within the matrix, and the size of dispersed particles. Saltzman et aL.Biophys. J. 55: 163 (1989); Sherwood et al.^ supra. Other solid dosage forms are described in Ansel et al, PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'S PIL'\RMACEUnCAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions thereof.
Tlie inununoconjugate, antibody fusion proteins, or naked antibody may also be administered to a mammal subcutaneously or even by other parenteral routes. Moreover, the administration may be by continuous iniusion or by single or multiple boluses. Preferably, the antibody of the present invention us infused over a period of less than about 4 hours, and more preferably, over a period of less than about 3 hours. This is preferably performed by infusing slowly at first. For example, a dose of 25 to 50 mg is infused within 15-30 minutes and the remainder of the dose is infused over a period of up to 2-3 hrs. In general, the dosage of an administered imraunoconjugate.

fusion protein or naked antibody for humans v»ili v^ry depending upon such factors as the patient's age, vveight heighu sex, general roedicaj condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of tmmunoconjugate, antibody fusion protein or naked antibody that is in the range of from about Img/kg to 20 mg'kg as a single intravenous infusion-, aitliough a lower or highpT dosage also may be administered as circumstances dictate. ITjerefore, 1-20 mgfkg for a 70 kg patient, for example, is a dose of 70-1,400 mg.. or 41-824 mg/m" for a 1.7-m patieaiL This dosage may be repeated as needed, for example, once per week fcK 4-10 wedcs, prefCTably once per week for 8 wedcs, and more prefCTably, once per week for 4 wedcs. It may also be given less frequently, such as every other week for several monEtbs. More specifically, an antibody of the present inventkHL such as naked anti-CD20, may be administered as one dosage every 2 or 3 weeks, repeated for a total of at least 3 dosages. Also preferred, the antibodies of the present invention may be administered once per week for 4-8 weeks. In other words, if the dosage is lowered to approximately 200-300 mg/m^ (which is 340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a 70 kg patient), it may be administered once weekly for 4 to 8 weeks. Alternatively, the dosage schedule may be decreased, namely every 2 or 3 weeks for 2-3 months; for example, if the dosage is 300-500 mg/m" (i.e.. 510-850 mg for a 1.7-m patient, or 7.3-12 mg/kg for a 70 kg patient). The dosing schedule can optionally be repeated at other intervals and dosage may be given through various parenteral routes, with appropriate adjustment of the dose and schedule.
For purposes of therapy, the immunoconjugate, ftision protein, or naked antibody is administered to a mammal in a therapeutically effective amoimL A suitable subject for the present invention are usually a human, although a non-himian animal subject is also contemplated. An antibody preparation is said to be administered in a "therapeutically effective amount" if the amount admioistered is physiologically significant An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient manmiai. In particular, an antibody preparation of the present mvention is physiologically significant if its presence invokes an antitumor response or mitigates the signs and symptoms of an autoimmune disease state. A physiologically significant effect could also be the evocation of a humoral and^or cellular immune response in the recipient mammal.

11. Methods of Treatment
The present invention contemplates the use of naked anti-CD20 antibodies of the present invention as the primary^ composition for treatment of B ceil disorders and odier diseases. In particular, the compositions described herein are particularly useful for treatment of various autoimmune as well as indolent forms of B-cell lymphomas, aggressive forms of B-cell lymphomas, chronic lymphatic leukemias. acute hmiphatic laricemias, and WaldenstrOTi's macroglobulinemia. For example, ti^ humanized m^r-CDlO antibody components and immunoconjugates can be used to treat both indcje^it and a^ressive fcwrms of non-Hodgkin's lymphcmia.
The compositions for treatment contain at least one hmnamzei chimeric or hnman monoclonal anti-CD20 antibody alone or in corobroation witii o&er antibodies, such as other humanized, chimeric, or human antibodies, therapeutic agents or immimomodulators. In particular, combiEiation therapy with a fiiUy hmnan antibody is also contemplated and is produced by the methods as set forth above. Naked or conjugated antibodies to the same or different epitope or antigen may be also be combined with one or more of the antibodies of the present invention. For example, a humanized, chimeric or human naked anti-CD20 antibody may be combined wdth another naked humanized, naked chimeric or naked human anti-CD20, a hmnanized, chimeric or human naked anti-CD20 antibody may be combined with an anti-CD20 immunoconjugate, a naked anti-CD20 antibody may be combined with an anti-CD22 radioconjugate or an anti-CD22 naked antibody may be combined with a humanized, chimeric or human anti-CD20 antibody conjugated to an isotope, or one or more chemotherapeutic agents, cytokines, toxins or a combination thereof. A fusion protein of a hinnanized, chimeric or human CD20 antibody and a toxin or immunomodulator, or a fusion protein of at least two different B-cell antibodies (e.g., a CD20 and a CD22 MAb) may also be used in this invention. Many different antibody combinations, targeting at least two different antigens associated with B-cell disorders, as listed already above, may be constructed, either as naked antibodies or as partly naked and partly conjugated with a therapeutic agent or inmiunomodulator, or merely in combiriation with another therapeutic agents, such as a cytotoxic drug or with radiation.
As used herein, the term "immunomodulator" includes c\1:okines, stem cell growth factors, lymphotoxins, such as tumor necrosis factor (TNF), and

hematopoietic factors, such as interieuldns (e.g., interleukin-1 (TL-1), IL.-2, ILo, IL-6, IL-10, IL-12; IL-21 and IL-IS), colony stimulating factors (e.g., granulocyte-colony stimulaiing factor (G-CSF) and granulocyte macrophage-colony stimulaiing factor (GM-CSF))^ interferons {e,g., interferons-a, -P and -y), the stem cell growth factor designated "SI factor/ er>thropoietin and thrombopoietiiL Examples of suitable immunomodulalor moieties include IL-2, IL-6, IL-10, EL-IZ IL-18, IL-21, interferon-y, T^JF-o- and tfae like. Ahematively, subjects can receive naked anti-CD20 aaiflxxlies and a s^)arately administered cytokine^ which can be administered before, conc^HTenrly or aftex administration of the naked anti-CD20 aaidbodies. As discussed supr(L, the anti-CD20 antibody maiy also be conjugated to the immunonK>dulator. The immcEoomodulator may also be conjugated to a hybrid antibody consisting of one or more antibodies binding to different antigens.
Multimodal therapies of the present invention further include immunotherapy witii naked anti-CD20 antibodies supplemented with administration of anti-CD22, anti-CD19, anti-CD21, anti-CD74, anti-CD80, anti-CD23, anti-CD46 or HLA-DR (including the invariant chain) antibodies in the form of naked antibodies, fusion proteios, or as immimoconjugates. The naked anti-CD20 antibodies or fragments thereof may also be supplemented with naked antibodies against a MUCl antigen that is expressed on certain B-cells. These antibodies include polyclonal, monoclonal, chimeric, hmnan or humanized antibodies that recognize at least one epitope on these antigenic detemiinants. Anti-CD19 and anti-CD22 antibodies are known to those of skill in the art See, for example, Ghetie et al., Cancer Res. ¥S:2610 (1988); Hekman et al., Cancer Immunol. Imrminother. 32:364 (1991); Longo, Curr, Opin. Oncol 5:353 (1996) and U.S. Patent Nos. 5,798,554 and 6,187,287, incorporated in their entirety by reference.
In another form of multimodal therapy, subjects receive naked anti-CD20 antibodies, and/or immunoconjugates, in conjunction with standard cancer chemotherapy. For example, "CVB" (L5 g/m~ cyclophosphamide, 200-400 mg/m" etoposide, and 150-200 mg/ra" carmustioe) is a regimen used to treat non-Hodgkin's lymphoma. Patti et al, Eur. J. Haematol 51: 18 (1993). Other suitable combmation chemotherapeutic regimens are well-known to those of skill LQ the art. See, for example, Freedman et al, "Non-Hodgkin's L>Tnphomas," in CANCER MEDICINE, VOLUME 2, 3rd Edition, Holland et al (eds.), pages 2028-2068 (Lea & Febiger

1993). As an ilJusiration, first generarion chemotherapeutic regimens for treatment of interaiediate-grade non-Hodgkin's lymphoma (NHL) include C-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). A useful second generation chemotherapeudc regimen is m-BACOD (methotrexate, bieomycin. doxorabicim cyclophosphamide, vincristine, dexametbasone and l^icovorin), while a smt^ie tbird generation regimai is MACOP-B (metbotrexsae, doxorubicin, cyck^Aosphamide^ vincristiDe, prednisone, bleomycin aiMi leucovoidii)- Additional useftd drugs include phenyl butyrate and brostadn-l. In a prefaced multimodal li^aapy, bodi chemotherapeutic dmgs and cytokines are co-administered \^ath an antibody, immunoconjugale or fusion protein according to tbe pres^ti: invoition- The cytokines, chemotherapeutic drugs and antibody or immunoccHijugate can be administered in any order, or together.
In a preferred embodiment, NHL or the autoimmune disease is treated with 4 weekly Infiisions of the humanized anti-CD20 antibody at a does of 200-400 mg/m" weekly for 4 consecutive weeks (iv over 2-6 hours), repeated as needed over the next monihs/yrs. Preferably, the humanized anti-CD-20 antibody is administered at a dose of 200-300 mg/m^ once every other week or every third week, for 4 to 8 injections. Also preferred, NHL is treated with 4 weekly infusions as above, or injections less frequently as above, but combined with epratuzumAb (anti-CD22 humanized antibody) on the same days, at a dose of 360 mg/m , given as iv infusion over 1 hour, either before, dindng or after the anti-CD20 monoclonal antibody infusion. Or, the antibodies used in combination therapy may also be infused in alternative sequences, such that they are alternated on different weeks, resulting in each being given every other week for a total injection sequence for each of 4 to 8 or more doses. These dosage schedules can then be repeated at different intervals, such as every 3-6 months, depending on the patient's clinical status and response to each therapy regimen. Still preferred, NHL is treated with 4 weekly infusions, or less fre4uent infiisions, of the anti-CD20 antibody as above, combined with one or more injections of CD22 MAb radiolabeled with a therapeutic isotope such as 3^ttrium-90 (at a total dose of Y-90 between 5 and 35 mCL/meter-square as one or more injections over a period of weeks or months). US Serial No. 09/590,284 (Goldenberg et al.) discloses

immunotherapy of autoimmune disorders using an anti-CD22 antibo In addition, a therapeutic composition of the present invention can contain a mixture or hybrid molecules of monoclonal naked anti-CD20 antibodies directed to different non-blocidng CD20 epitopes. Accordingly, die present invantion conteanplates therapeutic compositions comprising a mixture of monoclonal anti-aD20 antibodies that bind at least two CD20 epitopes. Additionally^ the th^ape^c composition desaibed herein may contain a mixture of airti-CD20 antibodies with varying CDR sequaices.
AlthnngH naked anti-CD20 antibodies are die jaimary therapeutic compositions for treatment of B cell lymphoma and autoimmune diseases, the efBcacy of such antibody therapy can be enhanced by supplementing tte naked antibodies, with supplemental agents, such as immunomodulators, like interferons, including IFNP, EFNb and IFNy, kiterleukms mcludhig E^-L IL-2, IL-6, IL-12, IL-15, IL-IS, IL-21, and cytokines including G-CSF and GM-CSF. Accordingly, the CD20 antibodies can be combined not only with antibodies and cytokines, either as mixtures (given separately or ia some predetermined dosing regiment) or as conjugates or fusion proteins to the anti-CD20 antibody, but also can be given as a combiuation with drugs. For example, the anti-CD20 antibody may be combined with CHOP as a 4-drug chemotherapy regimen. Additionally, a naked anti-CD20 antibody may be combined with a naked anti-CD22 antibodies and CHOP or fludarabine as a drug combination for NHL therapy. Immunotherapy of B-cell malignancies using an anti-CD22 antibody is described in US Patent No. 6,183,744 (Goldenberg et al,) and US Serial No. 09/307,816 (Goldenberg et al.\ which are incorporated herein by reference in their entirety. The supplemental therapeutic compositions can be administered before, concurrently or after administration of the anti-CD20 antibodies.
As discussed supra, the antibodies of the present invention can be used for treating B cell lymphoma and leukemia, and other B cell diseases or disorders. For example, anti-CD20 antibodies can be used to treat B-cell related autoimmune diseases, including Class EQ autoimmune diseases such as immxme-mediated thromboc}n:openias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocylopenic purpura, dermatomyositis, Sjogren's syndrome, multiple sclerosis, Sydenham's chorea, myasthenia gravis, sj'stemic lupus erythematosus.

lupus nephritis, rheumatic fever, iheumatoid arthritis, polyglandular syndromes, bullous pemphigoid, diabeies melhrus, Henoch-Schonlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid ardiritis. sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, poh*arteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis ubito^ns,, primary bUiary cinhosis, Hashimoto's thyroiditis, thvTotoxicosis, scteodoona, dironic active hqjatitis, poiymyositis/dennatCHnyosrds, potydiondritis, pannidgus vulgaris, Wegener's granulomatosis, membranous nepioropathy, amyo«n^)hic latoal sdaosis, tabes dorsaiis, giant cell artedtis/polyiiiyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis-
Anti-CD20 antibodies may also induce apoptosis in cells expressing the CD20 antigen. Evidenceof this induction is supported in the literature. For example, it was demonstrated that apoptosis could be induced using lymphoid cells that have Fc-receptors reactive with the IgGl-Fc of CD20MAbs that crosslinked. See Shan etaL^ Cojicer Immunol Immwiotlier. 48(12):673-683 (2000). Further, it was reported that aggregates of a chimeric CD20 MAb, i.e.. homopohoners, uiduced apoptosis. See Ghetie et al.. Blood 97(5): 1392-1398 (2000) and Ghetie et al, Proc. Natl Acad Sci USA 94(14): 7509-7514 (1997).
Antibodies specific to the CD20 surface antigen of B cells can be injected into a mammalian subject which then biad to the CD20 cell surface antigen of both normal and malignant B cells. A mammalian subject includes humans and domestic animals, including pets, such as dogs and cats. The anti-CD20 mAbs of the present invention, i.e., humanized chimeric, human, caninized and felinized, and even murine anti-CD20 mAbs, can be used to treat the non-human mammalian subjects when there is a species crossreactivity for the CD20 antigen. See Examples 10 and 11, below. The murine mAbs, which are immunogenic in humans, are usually less immunogenic in non-himian mammalian subjects. The anti-CD20 antibody bound to the CD20 surface antigen leads to the destruction and depletion of neoplastic B cells. Because both normal and malignant B cells express the CD20 antigen, the anti-CD20 antibody will result in B cell death- However, only normal B cells will repopulate and the malignant B cells will be eradicated or significantly reduced. Additionally, chemical agents or radioactive labels having the potential to destroy the tumor can be

conjugated to the ax}ti-CD20 antibody such that the agent is specifically targeted lo the neoplastic B cells.
12. Expression Vectors
The DNA sequence encoding a humanized, chimeric or human anti-CD20 MAb can be recombinantly engineered into a variety of known host vectors thpit provide ftr r^Hcatioa of the nucleic acid. These vectors can be desigi^i using known methods- to contain tiie elements necessary for directing transcription, transition, or botiL of the nucleic acid in a cell to which it is delivered. Known m^hodology can be used to generate expression constructs the have a {Mrotein-coding sequence opeiably linked with appropriate transcriptJonal/tcapslatioDal control signals. These methods include in vitro recombinant DNA techniques and sjoithedc techniques. For example, see Sambrook et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (New York); Ausubel et al, 1997, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John W^l^y & Sons (New York). Also provided for in this invention is the delivery of a polynucleotide not associated with a vector.
Vectors suitable for use in the instant invention can be viral or non-viral. Particular examples of \dral vectors include adenovirus, AAV, herpes simplex virus, lenti\drus, and retrovirus vectors. An example of a non-viral vector is a plasmid. In a preferred embodiment the vector is a plasmid.
An expression vector, as described herein- is a polynucleotide comprising a gene that is expressed in a host cell. Typically, gene ex^pression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. Such a gene is said to be "operably linked to" the regulatory elements.
Preferably, the expression vector of the instant invention comprises the DNA sequaice encoding a humanized, chimeric or human anti-CD20 MAb, winch includes both the heavy and the light chain variable and constant regions. However, two expression vectors may be usei with one comprising the heavy chain variable and constant regions and the other comprising the Ught chain variable and constant regions. Still preferred, the expression vector further comprises a promoter. Because any strong promoter can be used, a DNA sequence encoding a secretion signal peptide, a genomic

sequence encoding a human IgGl heavy chain constant region, an Ig enhancer element and at least one DNA. sequence encoding a selection marker.
Also contemplated herein is a method for expressing a humanized anti-CD20 MAb- comprising (i) linearizing at least one expression vector comprising a DNA sequencff'encoding a humanized, chimeric, or human anti-CD20 Iv^IAb, (If) transfeciing mammalian cells with at least one of said linearized vector^ (iii) selecting transfected cdls viindi oqpress a marker ger^- and (iv) identifying the cells ^cr^rog die htananized anti-CD20 MAb fiom the transfected cells.
13. Mrthods of Making Anti-CD20 Antibodies
In gODeraL die VK (variable light chain) and VH (variable heaw diam) sequences for an anti-CD20 MAb can be obtained by a variety of molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening. Specifically, the V genes of an anti-CD20 MAb can be cloned by PCR amplification from a cell that expresses a murine or chimeric anti-CD20 MAb, sequenced. To confirm their authenticity, the cloned VL and VH genes can be expressed in cell culture as a chimeric Ab as described by Oriandi et al., (Froc. Natl Acad ScL, USA, 86: 3833 (1989)) which is incorporated by reference. Based on the V gene sequences, a humanized anti-CD20 MAb can then be designed and constructed as described by Leung et al. {MoL Immui'iol^ 32: 1413 (1995)), which is incorporated by reference. cDNA can be prepared from any known hybridoma line or transfected cell hue producing a murine or chimeric anti-CD20 MAb by general molecular cloning techniques (Sambrook et al.. Molecular Cloning, A laborator>^ manual, 2"*^ Ed (1989)). The VK sequence for the MAb may be amplified usiag the primers VKIBACK and VKIFOR (Oriandi ei al., 1989) or the extended primer set described by Leimg et al. {BioTechnigues, 15: 286 (1993)), which is incorporated by reference, while VH sequences can be amplified using the primer pair VHIBACKA^IFOR (Oriandi et al., 1989 above), or the primers annealing to the constant region of murine IgG described by Leung et al (Hybridoma, 13:469 (1994)), which is incorporated by reference. The PCR reaction mixtures containing 10 }il of the first strand cDNA product, 10 til of 1 OX PCR buffer [500 mM KCl 100 mM Tris-HCl (pH 8.3), 15 mM MgClo, and 0.01% (w/v) gelatin] (Peridn Ehner Cetus, Nor^^^alk, CT), 250 (Jvl of each dNTP, 200 nfvl of the primers, and 5 xmits of Taq DNA polymerase (Perkin Elmer Cetus) can be subjected to 30 cycles of PCR- Each PCR cycle preferably

consists of deuaturarion at 94°C for 1 min. annealing ai 50 C for L5 min. and polymerization at 72 C for 1.5 min Amplified VK and VH fragments can be purified on 2% agarose (BioRad, Richmond, CA). Similarly, die humanized V genes can be constracted by a combination of long oligonucleotide template syntheses and PCR amplification as described by Leung et al. {Mol IimnimoL, 32: 1413^ (1995)). See Example 3 for a method for the synthesis of an oligo A and an oligo B on an automated RNA/DNA synthesizes- (Applied Biosystems, foster City, CA) f PCR jxoducts ftr VK can be subcloned into a staging vector, sodi as a pBR327-based staging vector. VKpBR, that contains an Ig promoter, a signal pef^e sequence and ccmvenieni restriction sites to facilitate in-firame ligation of the VK PCR jxoducts. PCR fffoducts for VH can be subcloned into a similar staging vector, such as the pBlxiescxipt-based VHpBS. Individual clones containing the reqjective PCR products may be sequenced by, for example, the method of Sanger et ai (Proa Natl Acad. Sci., USA, 74: 5463 (1977)), which is incorporated by reference.
The DNA sequences described herein are to be taken as including all alleles, mutants and variants thereof, whether occurring naturally or induced.
The expression cassettes containing the VK and VH, together with the promoter and signal peptide sequences can be excised from VKpBR and VHpBS, respectively, by double restriction digestion as HmdTTT-RamHT fragments. The VK and VH expression cassettes can then be hgated into appropriate expression vectors, such as pKh and pGl g, respectively (Leung et al., Hybridoma, 13:469 (1994)). The expression vectors can be co-transfected into an appropriate cell, e.g., myeloma Sp2/0-Agl4 (ATCC, VA). colonies selected for hygromycin resistance, and supernatant fluids monitored for production of a chimeric or humanized anti-CD20 MAb by, for example, an ELISA. assay, as described below. AJtemately, the VK and VH expression cassettes can be assembled in the modified staging vectors, VKpBR2 and VTIpBSl, excised as Xbal/BamHI and XhoI/BamHI fragments, respectively, and subcloned into a single expression vector, such as pdHL2. as described by Gilies et al, (J. Immunol Methods 125:191 (1989) and also shown m Losman et al.. Cancer, 80:2660 (1997)) for the expression in Sp2/0-Agl4 cells. Another vector that is useful in the present invention is the GS vector, as described in Barnes et al, Cyiotechnolog)^ 32:109-123 (2000), wiiich is oreferablv expressed in the NSO cell line and CHO cells. Other appropriate mammaUan

expression s>'Stems are described in Werner et al,^ .Ar2neim.-For5cb.-.Drjg Res. 48(11), Nr. 8,870-880(1998),
Co-transfection and assay for antibodv secretine clones bv ELISA, can be carried out as follows- About 10 jig of VTCpKh (light chain expression vector) and 20 fxg of VHpGIg (heavy chain expression vector) can be used for the transfection of 5 X 10^ SP2y'0 myeloma ceils by electroporation (BioRad, Richmond, CA) according to Co ef a/.. 1 hnmtmol, 148: 1149 (1992) ^^^ch is incoiporated by refereaace- Followiag traosfectkKL cells may be grown in 96-well microtiter plates in conqdeie HSFM medium (Life TedHic>k>gies, Inc., Grand Island, NY) al 37^C, 5%C02. The selection process can be initiated after two days by the addition of hygromydn sdecticHi medium (Calbioctem, San Diego, CA) at a final concCTttration of 500 units/ml of hygromycin. Colonies typically emerge 2-3 wedcs post-electroporation. The cultures can then be expanded for further analysis.
Transfectoma clones that are positive for the secretion of chimeric or humanized heavy chain can be identified by ELISA assay. Briefly, supernatant samples (—100 [d) firom transfectoma cultures are added in triphcate to ELISA microtiter plates precoated with goat anti-human (GAH)-IgG, F(ab*)2 fragment-specific antibody (Jackson ImmunoResearch, West Grove, PA). Plates are incubated for 1 h at room temperature. Unbound proteins are removed by washing three times with wash buffer (PBS containing 0.05% polysorbate 20). Horseradish peroxidase (HRP) conjugated GAH-IgG, Fc fragment-specific antibodies (Jackson ImmunoResearch) are added to the wells, (100 pi of antibody stock diluted x 10"^, supplemented with the unconjugated antibody to a final concentration of 1.0 [ig/ml). Following an iacubation of 1 k the plates are washed, typically three times. A reaction solution, [100 jil, containing 167 pg of orthophenylene-diamine (OPD) (Sigma, St. Loiiis, MO), 0.025% hydrogen peroxide in PBS], is added to the wells. Color is allowed to develop in the dark for 30 minutes. The reaction is stopped by die addition of 50 pi of 4 N HCl solution into each well before measuring absorbance at 490 nm in an automated ELISA reader (Bio-Tek iostraments, Winooski, VT). Bound chimeric antibodies are than determined relative to an irrelevant chimeric antibody standard (obtainable from Scotgen, Ltd, Ediaburg, Scotland).
Antibodies can be isolaied from cell culture media as foliow^s. Transfectoma cultures are adapted to serum-free medium. For production of chimeric antibody, cells are grown as a 500 ml culture m roller bottles using HSFM Cultures are centrifuged

and the supematani filiered through a 0.2 u membrane. The filtered medium is passed through a protein A column (1x3 cm) at a flow rate of 1 roi/mia. The resin is then washed with about 10 column voltimes of PBS and protein A-bound antibody is eluted from tbe column with 0.1 M glycine bufier (pH 3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubes containing 10 ^1 of 3 M Tiis (pH 8.6), and protein concentrations determined from the absorbance at 280/260 nm Peak fractions are pooled, dialyzed against PBS, and the antibody conceatrated, for exanq4e, with the CeaHriccHi 30 (AmiccHi, Berveriy, MA). Tlie antibody conceaatradcai is determined by FT-ISA, as before, aid its coocesitratioa adjusted to about 1 mg/ml tisiiig PBS. Sodium azide, 0.01% (w/v), is conveniently added to the sample as paeservative.
The following are the nucleotide sequences of the primers used to prepare the anti-CD20 antibodies: hA20VKA
5^-CATCTCTGAG CGCATCTGTT GGAGATAGGG TCACTATGAC TTGTAGGGCC AGCTCAAGTG TAAGTTACAT CCACTGGTTC CAGCAGAAAC CAGGGAAAGC ACCTAAACCC TGGATTTATG-3'
hA20VKB
5'-GGTGTCCCTG TCCGATTCTC TGGCAGCGGA TCTGGGACAG
ATTACACm CACCATCAGC TCTCTTCAAC CAGAAGACAT
TGCAACATAT TATTGTCAGC AGTGGACTAG TAACCCACCC
ACGTTCGGTG-3'
hA20VIC4-Backward
5'-CAGCTGACCC AGTCTCCATC ATCTCTGAGC GCATCTGTTG-3'
hA20VKA-Forward
5'-AGGTTCGAAG TGGCAT.A^^AT CCAGGGTTTA GGTGCT-3'
M20]^KB Bachvard
5'-CACTTCGAAC CTGGCTTCTG GTGTCCCTGT CCGATTCTC-3'



}L4201-1FI2B
5'-ATCAG.\AGTT C.\AGGGTAGA GCCA.CAAT.AA CTGCCGACGA ATCCACCA.^T ACAGCCTACA TGGAGCTGAG CAGCCTGAGG TCTGAGGACA CGGCATTTTA TTTTTGTGCA AGATCGACTT ACTACG hA20VH2A Forward
5'-TTCCGGGATA AATAGCTCCC ATCCATTCCA GACCCTG-3^
hA20VH2B Backward
5'-ATCCCGGAAA TGGTGATACT TCCTACAATC AGA-AGTTCAA
GGGTAGAGCCA-3'
The ijQV^ition is further described by reference to the following examples, which are provided for illustration only. The invention is not limited to the examples but rather includes all variations that are evident from the teachings provided herein.
EXAMPLES Example 1. Construction of a humanized anri-CD20 antibody
The VH and VK genes of A20, an anti-CD20 antibody, was obtained by RT-PCR using the primer pairs VHIBACK/ VHIFOR and VKIBACK/ VKIFOR, respectively Orlandi etal, (Proc. Natl Acad. Set, USA, 86: 3833 (1989)). Multiple independent clones were sequenced to eliminate possible errors resulting from the PCR reaction. The cloned murine VH and VK sequences as the final PCR product were designated A20\'Tc (Figure 1 A) and A20VH (Figure IB), respectively. A chimeric A20 (cA20) antibody was constructed and expressed in Sp2/0 cell. The Vk and VH of sequences of cA20 are shown in Figure 2. The cA20 antibody bound to Raji cell and competed with radiolabeled A20 purified from the hybridoma cell culture supernatant (Figure 3). This result confirmed the authenticity of the cloned V genes.
A single light chain and tv^'o hea\y chain variable region sequences encoding the humanized anti-hCD20 (hA20) antibody were designed and constructed. Human REI framework sequences were used for VK (Figure 1 A), and a combination of EU and NEWM fi:amework sequences were used for VH (Figure IB). There are a number

of amino acid changes in each chain outside of the CDR regions when compared to the starting human antibody frameworks. Fne hea\'\* chain of hA20, IL^OVHL contains nine changes, \vhile hA20VH2 contains three changes from the human EU frameworks (Figure 4A). hAlOVnl is preferred because it contains more amino acids from the human antibody framework region than hAIOVnl. The light cfctain of hA20, hA20Vic contains seven amino acid changes from the REI framework (Figure 4B).
ExsBiple 2- Mrfhod of hA20 antibody constmction
Eadi variable diain was constructed in two parts, a 5'- and 3'-halE designated
as "A" and '^B'" respectively. Each half was produced by PCR amplification of a
single strand synttetic oligonucleotide template with two short flanking primers,
using Taq polymerase. The amplified fragments were first cloned into the pCR4 TA
cloning vector fixsm Invitrogen (Carlsbad, CA) and subjected to DNA sequencing.
The templates and primer pairs are listed as follows:
Template Primers
VKA VkA-Backwa^dA^cA-For^^^ard
VKB VkB-BackwardAndB-Forward
VHIA VHA-BackwardAH^lA-Forward
VHIB VHlB-BackwardA^HB-Forward
VH2A VHA-BackwaxdAai2A-Forward
VH2B VH2B-BackwardArHB-Forward
Light cly2in
For constructing the ftill-length DNA of the humanized VK sequence, oligo hA20\TCA (120 mer) and hA20VKB (130 mer) were syTithesized on an automated RNA/DNA synthesizer (Applied Biosystems). hA20VKA and B represent the nt 26-145 and 166-195, respectively, of the hA20 VK. (See Fig. 5A) OHgo hA20VKA and B were cleaved from the support and deprotected by treatment with concentrated ammonium hydroxide. After samples were vacuum-tried and resuspended in 100 ul of water, incomplete oligomers (less than i 00-mer) w^ere removed by centrifugation through a ChormaSpin-100 column (Clontech, Palo Alto, CA). All flanking primers were prepared similarly, except ChromaSpui-30 colimins were used to remove s^mthesis by-products. 1 ul of ChromaSpin column purified hA20VKA was PCR amplified in a reaction volume of 100 ul containing 10 jil of 1 OX PCR buffer [500 mNI

KCL 100 mM Tris-HCl ipH 8.3), 15 niKl MgCh. and 0.01'^Q (W/V) gelatm] (Parkin Elmer Cetus. Nor^^-alk CTi 250 uM of each dNTP, 200 nM of \lo\-Back\\CTd and VlcA-For^^ani and 5 units of Taq DNA pohTnerase (Peikin Elmer Cetus). This reaction mixture was subjected to 30 cycles of PCR reaction consisting of denaturation at 94°C tor 1 min. annealing at 50 C for 1.5 min, and polymerization at 72 C for 15 rm-n hA20VKB was PCR-amplified by the primer pair VkB-Backward and \TcB-Forward iHKia: shmka- ccaidition. The amplified VKA and VKA fragm^its WQ^ purified on 2% agarose (Bk^ad, Richmond, CA). Unique restriction sites were designed at the eaids of each fiagnieni to Militate joiidngthrou^ DNA ligation. The amplified VKA ftagment contamed a Pvull restriction site, CAGCTG, at its 5'-«Ki and a BstBI restriction site, TTCGAA, at the 3'-eni The amplified VKB fragment contained a BstBI restriction site at its 5'-end and a Bglll restriction site, AGATCT, at liie 3'-end. Assembly of the full-length VK chain was accomplished by restriction enzyme digestion of each fiaigment with the appropriate 5'- and 3'-enz>Tnes and ligation into the VKpBR2 vector previously digested with PvuII and BcU (Bell digested end is compatible with that of BgUI). The resulting ligated product contains the A Segment ligated to the PvuII site, the B fragment ligated to the BcU site^ and the A and B fragments joined together at the BstBI site (Figure 5A). VKpBR2 is a modified staging vector of VKpBR (Leung et al,, Hybridoma, 13:469 (1994)), into vAich aXbal restriction site w^as introduced at 14 bases upstream of the translation initiation codon. Upon c-onfixmation of a conrect open reading frame by DNA sequencing, the intact chain was removed fix)m VKpBR2 as a Xbal to BamHI fragment and ligated into the pdHL2 expression vector. The vector containing only VK sequence was designated as hA20VKpdHL2- pdHL2 contains the expression cassettes for both human IgGl C1, C2, C3, and hinge regions (Fig. lA) and the human kappa chain Ck (Fig. 7B) under the control of IgH enhancer and MTi promoter, as well as a mouse dhjr gene, controlled by a weak SV40 promotor, as a marker for selection of transfectants and co-amplification of the trans-genes (Gillies et al., J. Immunol Methods 125:191 (1989): Losraan et al.. Cancer 80:2660 (1997)). By replacing the VK and VH segments of pdHL2, difierent chimeric or humanized Abs can be expressed.

Hecny chain
For the construction of L^OV^HL oligo \Til A (121 mer) and VHIB (151
mer). representing the nt 23-143 and 179-329, respectively, (See Fig. 5B) were
synthesized as described above. Similarly, for hAlOVYH..^ oligo VH2A and \TI2B
were prepared. These ohgos w^ere PCR-amplified by their respective primer pairs as
listed in Example 2. The same construction method as done for VK w-as carried out for
both types of VH= with the following modifications: the 5'-end restrictioB site of the
A fi^gmerfc was Psfl (CTGCAG) and the 3'-end restriction site of B ftagments w^as
B^H (GGTCACC)- These fragm^its were joined together upon ligation into the
VHpBS2 vector at a common Neil site (CCCGG), resulting in ftdl-length VH
sequeiK^s (Figure 5B and 5C) and confirmed by DNA sequencing. VHpBS2 is a
modified staging vector of VHpBS (Leung et ai., Hybridoma, 13:469 (1994))^ into
which a Xhol restriction site was introduced at 16 bases upstream of the transialion
initiation codon. The assembled VH genes were subcloned as XhoI-BamHI restriction
fiagments into the expression vector containing the VK sequence. hA20VKpdHL2.
Since the heavy chain region of pdHL2 lacks a BamHI restriction site, this ligation
required use of the HNB hnker to provide a bridge between the BamHI site of the
variable chain and the Hindlll site present in the pdHL2 vector. The resulting
expression vectors were designated as hA20-lpdHL2 and hA20-2pdHL2.
HNB linker 5'-AGCTTGCGGCCGC-3'
3'-ACGCCGGCGCTAG-5'
Example 3- Transfection and Expression of hA20 Antibodies
Approximately 30 ^g of the expression vectors for hA20 were linearized by digestion with Sail and transfected iato Sp2/0-Agl4 cells by electroporation (450V and 25 uF). The transfected cells were plated into 96-well plates for 2 days and then selected for drug-resistance by adding MTX into the medium at a final concentration of 0.025 uM. MTX-resistant colonies emerged in the wells 2-3 weeks. Supematants from colonies surviving selection were screened for human Ab secretion by ELISA assay. Briefly, 100 jil supematants were added into the wells of a microtiter plate precoated with GAH-IgG. Ffab")! fragment-specific Ab and incubated for 1 h at room temperature. Unbound proteins were removed by washing three times with wash buffer (PBS containing 0.05% polysorbate 20). HRP-conjugated GAH-IgG,, Fc fragment-

specific Ab was added to the wells. Follcv,ing an incubation of 1 L The piaie was washed. The bound HRP-conjugaied Ab was revealed by reading A490nia after the addition of a substrate solution containing 4 mM OPD and 0.04% HoOo. Positive cell clones w^ere expanded and hA20-l and hA20-2 were purified from ceU culture supernatant by affinity chromatography on a Protein A column-
Example 4, J^Bding Activity of Aiiti-CD20 Afltibodies
A cocapetitioG cdl-binding assay was carried out to assess the innnunoreactivity of hA20 r^ative to ib& parent cA20 and the anti-CD20 Ab c2B8. A constant amount of ^'^-labeled murine A20 or c2B8 (100,000 cpm. -10 \iCij\xg) was iinaibated wifli R^ cell in the presCTice of varying concentrations (0J2-700 nM) of hA20-l, -Z murine A20, cA20, or c2B8 at 4*^0 for 1-2 h- Unbound Abs were removed by washing the cells in PBS. The radioactivity associated with cells was determined after washing. As shown in Figure 6, bodi humanized A20 mAbs, hA20-l and hA20-2, exhibited comparable binding activities as A20, tiie murine anti-CD20 MAb, cA20, and c2B8- a chimeric anti-CD20 MAb, when competing with binding of *^I-A20 or ^^-c2B8 to Raji ceUs.
By direct binding of radiolabeled Mabs to Raji cells and Scatchard plot anlaysis, the dissociation constants were measured to be 2.9 and 4.2 nm for cA20 and hA20, respectively, in comparison to 3.9 nM for C2B8- In vitro crosslinking experiments, using a goat anti- human IgG, Fc fragment specific antibody to complex with the antibodies showed similar killing of Raji NIIL cells between cA20 and hA20, aswellasC2B8.
Example 5- Treatment of a Patient with Relapsed Intermediate-Grade Non-Hodgldn^s Lymphoma
A patient with intermediate grade non-Hodgkin's lymphoma has failed prior
aggressive chemotherapy, consisting of CHOP x 6, which led to a complete remission for four months, another course of CHOP x 6, resulting in progression, D-MOPP x 2, resulting in stable disease for three months^ and CVB with peripheral stem cell transplantation, which led to a partial remission for five months. The patient presents wth recurrent lymphoma in a neck lymph node, measurable by computerized tomography and palpation-

iJ_f-
Tlie patient is infused within 3 hrs with 450 mg of hnmanized CD20 monoclonal antibody A20 on days 0, 14. 28. and 42 with no serions adverse effects noted either dming or immediately after the infusions. Eight wrecks later, palpation of Ac neck node enlargement shows a measurable decrease of about 50%. Follow-up measuremciiits made at twenty' weeks post therapy show no evidence of the disease ic the neck- and nowhere else, as confirmed by computed tomography studies of the body. Since new disease is not detected elsewhere, Ae patieaat is consi Example 6- Treatmoit of a patient with chronic idiopathic Hirombocytopenia parpara
A 45-year-old female with chronic idiopathic thrombocytopenia purpura has been treated widi prednisone, gamma globulins, and high dose dexamethasone, but the disease progresses. She undergoes splenectomy, which fails to stabilize the disease. Her platelet coimt falls to less than 30,000/microhter, and hemorrhagic events increase in frequency. The patient is then treated with the humanized CD20 A20 MAb, 500 mg intravenously on the first week, followed by a dose of 250 mg given once every other week for a total of 4 injections. Ten weeks after the last dose of A20 a mariied increase in platelet number is observed, to 150,000/microliter, and the hemorrhagic events disappear. Five months after the last antibody infiision the disease is in remission.
Example 7. Treatment of a patient ^ith progressive rheumatoid arthritis
A 70 year old female, with severe progressive Aeumatoid arthritis of the finger joints, WTists, and elbows, has failed therapy with methotrexate, and obtains only minor relief when placed on Enbrel therapy. The patient is then treated with A20 humanized CD20 MAb, 300 mg intravenously each week, for a period of four weeks. After 3 months, a 40% improvement in measures of disease activity is observed, which is maintamed for 5 months. The patient is again treated with A20, ai the same dose and frequency. The patient continues to improve, and 6 months after the second A20 MAb therapy, a 60% improvement is observed. No human anti-A20 antibodies are observed at any time during, or after the A20 Therapy. Although

normal B-cells are depleted from the blood, no infectious complications, or other drug-related severe toxicit\' is observ'ed.
Example 8, Treatment of a patient with myasthenia gravis
A 65 year old ffiSe has failed all conventional therapy for myasthenia gratis, pnd is admitted to a neurological intensive therapy unit The patient was stabilized by plasma erxdiai^e, and given intravenous immunoglobulin to rediice tiie titer of antiac^kiicdii^ receptor antibody. The patient remained bedridden, aini V/BS Ihen treated with A20 hmnsnzed CD20 MAb, 400 mg intravenously once ev^y other week- for aperiod of trai wedcs. One week after the last dose of A20, no blocwi B-cdis were detectable, and a significant drop in the titer of die anti-acetyldioline antibody was observed. Four months after the last A20 MAb dose the parioit was mobile, and was released from the hospital.
Example 9. Treatment of a Dog with Aggressive Non-Hodgkin's B-cell Lymphoma in Lymph Nodes and Bone Marrow
A 65-pound 7-year old male Golden Retriever is diagnosed witli diffuse large cell aggressive lymphoma. The dog is placed on combination chemotherapy v\ith vincristine, cyclophosphamide, prednisolone, and doxorubicin, with good response. However, the dog subsequently is characterized as having progressive lymphadenopathy, and seven months after this is found to have extensive Ijonphoma infiltration of bone marrow, extensive lymphoadenopathy of neck, chest, abdomen, pelvis, and hepatosplenomegaly.
The dog is given therapy with 1F5 chimeric monoclonal antibody. The dog is infused intravenously with 120 mg of 1F5 antibody, and the treatment is repeated weekly for 4 weeks following this initial treatment. Four months after the final dose of 1F5, a computerized tomography scan of the patient shows no evidence of lymphoma, and all signs and symptoms of the disease were not evident.
Example 10. Treatment of a Dog with Relapsed Intermediate-Grade Non Hodgldn's Lymphoma
A 78-pound, 9-year old, German Shepherd dog with intermediate grade non-Hodgkin's lymphoma receives chemotherapy, which initially leads to a complete

remission for five months, followed by another course of chemotherapy which results in stable disease for six months. The dog then presents with recurrent lymphoma in the chest and in a neck l\Tnph node, both measurable by computerized tomograp.hy and paipatioiL respectively.
The patient is infused with a ^'-labeled immunoconjugate of L243 (HLA-DR) monoclonal antibody weekly for two weeks, at a radiation dose of 8 mCi in 50 mg of antibody protein, in combination witii ti>e A20 humanized CD20 antibody at a dose of 100 mg pes: each weeMy infusion. Three wedcs lata:, palpation of the neck node CTilarffinkSitf shows a measurable decrease, while a repeat conaputeaized tomograjAiy scan of the chest shows a marked reduction in tumor. Follow-up measurements made at ten weeks post tiier^y show evidence of the disease iu the neck or the chest being reduced by a about 60 percent Since new disease is not detected elsewhere, the patient is considered to be in partial remissioiL Follow-up studies every 10-12 weeks confirms a partial remission for at least 7 months post therapy.
Example 11, Treatment of a Cat with Relapsed Lymphoma
A 10-pound, 12-year-old, domestic short hair presents Avith enlargement of a siagle submandibular lymph node. After excision, there is recurrence of the lesion at. 6 months. The lesion is again excised, but then reappears 6 months later. The cat is then given weekly treatments for 4 weeks with an ^ I-labeled immunoconjugate of anti-CD20 Bl monoclonal antibody, at a radiation dose of 15 mCi in 45 mg antibody protein. The treatment is repeated 3 months later. When examined 3 months after the last treatment a marked decrease can be palpated. No recurrence of the disease is observed for over one year.
Example 12. Evalulation of chimeric and humanized anti-CD20 Mabs in human NHL cells in culture or xenografted in SCID mice
The properties of a chimeric (cA20) and humanized (hA20) CD20 antibody was assessed in NHL cell lines. The results demonstrate that cA20 and hA20 behave similarly to Rituximab, staining more than 99% of Raji, Ramos, RL, Daudi and Su-DHL-6 cells and reacting with approximately 5% of lyniphoc}^s (expected % B-cells). In all B-cell lines, specific growth iohibition was seen with the Mabs, but the

level o f inhibition varied between the cell lines, wiA Su-DHL being the most sensitive. In the absence of cross-linking, murine anti-CD20, cAlO, hA20 and riiuximab ail fielded between 77 and 90° o inhibition- ^^^th cross-linking, iniiibition of proliieralaion ranged from 94-98%. Rituximab, cA20, and hA20 were also similar in their ability to induce apoptosis in Raji cells in the presence of a cross-linMng second monoclonal antibod>'.
Also, SCID mice ware injected intravenously witii 2.5X10 Raji cells on day 0. Injections of mnrine, chimeric and humanized anti-CD20 antibodies, and tte cA20 F{ab^ fragmeat were initiated on day-1 with 100 Sg/injection of intact antibody, or 67 Sg/iiqection F(ab")2 fragm^rt, five times per week for two weets, the twice weekly for three weeks. In one study, control mice died of disseminated disease with a median survivial time of 15 days post tumor innoculalion, but median survival was extended to 38 days for cA20, 22.5 days for hA20, and 21 days for murine anti-CD20 treated mice (aU statistically significant by log-rank analysis (p 11).
Example 13, Competitive cell surface binding assay.
Ag-bindiog specificity and affinity studies of humanized anti-CD20 Abs (cA20, hA20, and clF5), purified by affinity chromatography on a Protein A column) were evaluated by a cell surface competitive binding assay with murine 2B8 and ritaximab (IDEC Pharmaceuticals Corp., San Diego, CA) (Figxnre 8). Briefly, a constant amount (100,000 cpm, -10 iCL'ig) of ^^Mabeled (A) m2B8 or (B) rituximab was incubated with Raji cells in the presence of varying concentrations (0.2-700 nM) of competing Abs (cA20, hA20, m2B8, clFS. or rituximab) at 4^C for 1-2 h. Unbound Abs were removed by washing the cells with PBS. Radioactivit}' associated with the cells was determined after washiag. Figure 8 (A) is a comparison of the binding activities of cA20 (square), hA20-l (triangle) and hA20-l (circle) v,dth that of m2B8 (diamond); Figure 8 (B) Compares the binding activities of cA20 (square), clF5 (triangle) and rituximab (diamond).

In another study, the binding activities of hA20 with other anti-CD20 A.bs. rituximab and murine B1 were compared by a cell surface competitive binding assa}" (Figure 9). Briefly, a constant amount (100,000 cpm, --10 iCiAg) of ^^I-Iabeled rituximab was incubated with R^i cells in the presence of varying concentranoBS (02-700 nNf) of competing Abs, hA20 (triangle), mBl (Downward triangle) or rituximab (square) at 4^C for 1-2 L Uabound Abs were removed by washing tbe ceUs with PBS. Racfioactivity associated wife the cells was detenmned after washing- The IC50 values for these three Abs w^ie calculated to be 6.8, 34, and 5, respectively.
Example 14. Cytotoxic effect of crossKnked hA20 and other CD20 Abs on cultured lymphoma cdis.
Raji cells were treated with various CD20 Abs in the presence of a crosslinker (an anti-human IgG, Fc ftagment specific antibody) to complex the CD20 antibodies (Figure 10). A final concentration of 5 ig/ml of hA203 cA20j rituximab, or a positive control Ab, hLLl, was incubated with Raji cells, with 20 ig/ml of tiie crossliaker (red), without the crosslinker (orange), or with an anti-mouse IgG, Fc fragment specific antibody (blue) for 48 h. Total cell and viable cell populations were measured by (A) trypan blue staining and cell counting or (B) MTT assay (B). The data show a similar effect of liA20 and rituximab on Raji NHL cell survival, and that the cytotoxic effect is dependent on the specific crosslinidng of the antibodies.
Example 15- In vivo therapy with hA20 and hLL2.
Raji cells wer administered i.v. to 60 SCID mice, at 2.5 x 10^ cells/100 jj.l/mouse (Figure 12). MAbs were admiiustered i.p. on days 1 to 1 L followed by MAb injections twice per week, for approximately 3 weeks. The body weight of the animals was measured weekly until the study was terminated. The animals were examined daily for paralysis of the hind legs. When paralysis occured, tbe animals were sacrificed and necropsied for visual inspection of disseminated tumor nodules (specifically in lungs, kidneys, and ovaries). Control mice treated with a control humanized IgGl Ab, hMN-14 (an anti-CEA antibody), died of disseminated disease manifested with C>>fS paralysis. The median survival time was 13 days post tumor i.v. inoculation. Median survival in the group treated v»ath h.A20 was extended to about 25 days. This value represents median survival increase of approximately 2 fold

for hA20. Although the group neated with hLL2 alone showed the same median survival time compared to the control mice, treatmeni with combination of hA20 and hLL2 increased the median survival time of the mice to approximaiely 30 days.










We Claim:

6- The humanized antibody or fragment thereof of claim 2, wherein said light chain
variable region comprises CDRl comprising an amino acid sequence selected from the group consisting of RASSSVSYHL RASSSLSFMH and RASSSVSYMH; CDR2 comprising an amino acid sequence of ATSNLAS; and CDR3 comprising an amino acid sequence selected from the group consisting of QQWTSNPPT, HQWSSNPLT and QQSFSNPPT.
7. The humanized antibody or fragment thereof of claim 3, wherein said heavy-
chain variable region comprises CDRl comprising an amino acid sequence of SYNMH; CDR2 comprising an amino acid sequence of .ATYPGNGDTSYNQKFKG and CDR3 comprising an amino acid sequence selected from the group consisting of


9. The humanized antibody or fragment thereof of claim 8, further comprising the
FRs of the light and heavy chain constant regions of a human antibody.
10. The humanized antibody or fragment thereof of claim 8, wherein said CDR3 of
the heavy chain variable region does not comprise STYYGGDWYFNV.
11. The humanized antibody or fragment thereof of claim 8, wherein said CDRl of
the light chain variable region does not comprise RASSSLSFMH when said CDR3 of
the light chain variable region comprises HQWSSNPLT and said CDR3 of the heavy
chain variable region comprises SHYGSNYVDYFDV.
12 The humanized antibody or fragment thereof of claim 8, wherein said CDR3 of the light chain variable region does not comprise HQWSSNPLT when said CDRl of the light chain variable region comprises RASSSLSFMH and said CDR3 of the heavy chain variable region comprises SHYGSNYVDYFDV.
13, The humanized antibody or frgment thereof of claim 8, wherein said CDR3 of the heavy chain variable region does cot comprise SHYGSNYVDYFDV when said

CDRI or the light chain vanable region comprises R.A.SSSLSFMK and said CDR3 of the light chain variable legion comprises HQWSSNPLT.
14. The humanized antibody or fragment thereof of claim 8, wherein said GDRl of tile light chain variable region does not comprise RASSSVSYNffl when said CDP3 of the light chain variable region comprises QQSFSNPPT and said CDR3 of the heavy-chain variable region comprises VVYYSNSYWYFDV.

variable region of the murine aQti-CD20 MAb comprises CDRl comprising an amino acid sequence of RASSSVSYIR, CDR2 comprising an amino acid sequence of ATSNLAS and CDR3 comprising an amino acid sequence of QQWTSNPPT and the CDRs of the heavy chain variable region of murine anti-CD20 MAb comprises CDRl comprising an amino acid sequence of SYNMH, CDR2 comprising an amino acid sequence of AIYPGNGDTSYNQEFKG and CDR3 comprising an amino acid sequence of STYYGGD WYFDV.

IS. The humanised autibody or fragment thereof of claim 1, wherein said FRs of the light and heavy chain variable regions of said humanized antibody comprise al least one amino acid substituted from said corresponding FRs of said murine MAb.

25- A chimeric anti-CD20 (cCD20) monoclonal antibody, (MAb) or fragment
thereof comprising the compiementarit)'-deteimining regions (CDRs) of at least one murine anti-CD20 N'lAb variable region and the framework regions (FRs) of at least one murine anti-CD 20 IvIAb variable region, wherein said chimeric anti-CD20 MAb or frragment thereof retains substantially the B-cell, and B-cell lymphoma and leukemia ceil


(b) wherein said CDR3 of the heavy chain variable region does not comprise
SHYGSNYVDYFDV, when said CDRl of the light chain variable region comprises
amino acids RASSSLSFMH, CDR2 of the light chain variable region comprises amino
acids ATSNLAS, CDR3 of die light chain variable region comprises amino acids
HQWSSNPLT- CDRl of the heavy chain variable region comprises amino acids
SYNMH- and CDR2 of the light chain variable region comprises amino acids
AIYPGNGDTSYNQKFKG; and
(c) wherein said CDR3 of the heavy chain variable region does not comprise
VVYYSNSYWYFDV, when said CDRl of the light chain variable region comprises
amino acids RASSSVSYMH, CDR2 of the light chain variable region comprises amiao
acids ATSNLAS, CDR3 of the light chain variable region comprises amino acids
QQSFSNPPT, CDRl of the heavy chain variable region comprises amino acids
SYNfvlH- and CDR2 of the light chain variable region comprises amino acids
.AIYPGNGDTS^T^QKFKG-

26. The chimeric antibody or fragment thereof of claim 25, further comprising light and heavy chain constant regions of at least one human annbody

30. A human anti-CD20 (huCD20) monoclonal antibody (MAb) or fragment thereof -comprising the light and heavy chain variable regions of a human antibody, wherein said huCD20 MAb retaios substantially the B-cell, and B-cell lymphoma and leukemia cell targeting and cell binding characteristics of a murine anti-CD20 MAb. wherein the CDRs of the light chain variable region of the human anti-CD20 MAb comprises CDRl comprising an amino acid sequence selected from the group consisting of


32. An antibody fusion protein or fragment thereof comprising at least two mAbs or
fragmaits tbereof, wherein said mAbs are selected from said anti-CD20 mAbs of any
one of claims 1-31.
33. An antibody fusion protein or fragment thereof comprising at least one first anti-
CD20 MAb or fragment thereof of anv one of claims 1-31 and at least one second MAb
or fragment thereof, other than the antiCD20 MAb or fragment thereof of any one of
claims 1-31.
34. An antibody fusion protein or fragment thereof of claim 33, whereia said second
MAb is selected from the group consisting of mAbs reactive with CD4, CDS, CDS,
CD14. CD15. CD19, CD2L CD22. CD23, CD25, CD33, CD37, CD38. CD40.
CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUCl, la, HML24, HLA-
DR, tenascin, VEGF, PIGF, an oncogene, an oncogene product, and a combination
Thereof.
35. A DNA sequence comprising a nucleic acid encoding a MAb or fragment thereof
selected from the group consisting
(a) an anti-CD20 MAb or fragment thereof of any one of claims 1-31; (h) an antibody fusion protein or fragment thereof comprising at least two of said aati-CD20 mAbs or fragments thereof.


3 6. An expression vector comprising the DN A sequence of claim 35.
37. An expression vector of claim 36, wherein the vector is pdHL2 or GS.
38. An expression vector of claim 37, wherein the pdHL2 or GS vector, when used
to express a chimeric, humanized or human IgG, codes for the heavy and light chain
constant regions and hinge region of IgGl.
39. An expression vector of claim 38, wherein said heavy chain constant regions and
hinge region shown in Fig. 7A and said light chain constant region is shown in Fig. 7B,.
wherein optionally at least one of the amino acids in the allotype positions is changed to
that found in different IgGl allotype, and wherein optionally amino acid 253 of the
heavy- chain of EU may be replaced with alanine.
40. A host cell comprising the DNA sequence of claim 35.
41. A host cell of claim 40. wherein said cell is a mammalian cell.
42. A host cell of claim 4L wherein said mammalian cell is a lymphocytic cell.


53. A method for the expression of an anti-CD20 MAb or fragment thereof or
antibody fusion protein or fragment thereof comprising:
(a) transfecting a mammalian cell with a DNA sequence of claim 38; and
(b) culturing said cell secreting said anti-CD20 MAb or fragment thereof or
antibody fusion protein or fragment thereof
54. A method for the exnression of an anti-CD20 MAb or fragment thereof or
antibody fusion protein or fragment thereof comprising:


61. A diagnostic conjugate of claim 60, wherein said contrast agent is a metal
comprising manganese, iron or gadolinium.
62, A therapeutic conjugate of claim 55, wherein said antibody component is an
antibody fusion protein or fragment thereof wherein each of said mAbs or fragments
thereof are bound to at least one tharapeutic agent

63. A therapeutic conjugate of claim 55 or 62, said tiierspeuiic agent is selected from the group consisting of a radioactive label, an immunomoduiator, a hormone, a fdiotaaciive therapeutic agent a cytotoxic agent an ohgonucleotide and a combinalion thereol

67. A therapeutic conjugate of claim 64, wherein said drug is selected from the group consisting of nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants, antagonists, endostatin, taxols, camptothecins, doxorubicins and their analogs, and a combination thereof.

69. A therapeutic conjugate of claim 63, wherein said immunomodulator is selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), a stem cell growth factor, erythropoietin, thrombopoietin and a combination thereof.

70. A therapeutic conjugaie of claim 69, wherein said iympboroxixL is rirmnr necrosis factor (TNF), said hematopoietic factor is an interieukin (IL), said colony-stimulating factor is granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-coiony stimulating factor (GM-CSF)), said interferon is interferons-cL, -p or -y, and said stem ceD growth factor is designated "S1 factor".



81. A method of claim 80, wherein said therapeutic agent comprises a cytotoxic agent, a radioactive labeL an immunomodulator, a hormone, a photoactive therapeutic agent, an oligonucleotide or a combination thereof, formulated in a phannaceutically acceptable vehicle.
S2- A method of claim 81, wherein said oligonucleotide is an antisense oligonucleotide.
83- A method of claim of 75. further comprising admioistering to said subject concurrently or sequentially a therapeutically effective amount of a therapeutic conjugate comprising at least one MAb bound to at least one therapeutic agent wherein said MAb comprises at least one humanized, chimeric, human or murine MAb selected from the group consistmg of aMAb reactive with CD4, CDS, CDS, CD14, CD15, CD19, CD20, CD2L CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52,


87. A method of claim 86, wherein said therapeutic agent comprises a cytotoxic agent, a radioactive label, an immunomodnlator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic agent or a combination thereof, formulated in a pharmaceutically acceptable vehicle.
88- A method of claim 86, wherein said oligonucleotide is an antisense oligonucleotide.
89. A method of treating a B-cell lymphoma or leukemia or an autoimmune disease in a subject comprising administering to said subject a therapeutically effective amount of an antibody fusion protein or fragment thereof comprising at least two mAbs or


93. A method of claini of 77, further comprising administering to said subject concurrently or sequentially a therapeutically effective amount of a therapeutic conjugate comprising at least one MAb bound to at least one therapeutic agent, wherein said MAb comprises at least one humanized, chimeric, human or murine MAb selected from the group consisting of aMAb reactive with CD4, CD5, CDS, CD14, CD15, CD19, CD20, CD2L CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, B7, MUCL la, HMl-24, HLA-DR, tenascin, VEGF, PIGF, an oncogene, an oncogene product and a combination thereof formulated in a pharmaceutically acceptable vehicle,
94. A method of claim 93, wherein said therapeutic agent comprises a cytotoxic agent, a radioactive label, an , immunomodulator, a hormone, an enzyme, an ohgonncleotide a photoactive therapeutic agent or a combination thereof, formulated in a pharmaceutically acceptable vehicle.

95, A method of claim 94, wherein said oligonucleotide is an antisense oligonucleotide.

100. A method of claim 98, wherein said drag Is selected from the group consisting of niirogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purine analogs, antibiotics, enzymes, epipodophyllotoxins, platinum coordination complexes, vinca alkaloids, substituted ureas, methyl hydrazine derivatives, adrenocortical suppressants-antagonists, endostatin, taxols, camptothecins, anthracyclines, and their analogs, and a combination thereof.

101. A. method of claim 98, wherein said toxin selected from ihe group consisting of ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), DNase L Staphylococcal emerotoxin-.A- pokeweed antiviral protein, gelonin. diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.


110. A method of diagnosing a B-cell ly'mphoma or leukemia or an autoimmune disease in a subject comprising administering to said subject a diagnostic conjugate comprising an anti-CD20 MAb or fragment thereof or an antibody fusion protein or Sragment thereof of any one of claims 1-34 that binds to said cell, wherein said anti-



121. The method of any of claims 118-120, wherein said first therapeutic agent is selected from the group consisting of a radioactive label, an inimunomodulator, a homione, a photoactive ther^eutic agent, a cytotoxic agent, an oligonucleotide and a combination thereof and wherein said first diagnostic agent is at least one of a radioactive label, a photoactive diagnostic agent or a non-radioactive label.
122- The method of claim 118-121, wherein said antibody fusion protein or fragmcnt thereof is conjugated to a second therapeutic or diagnostic agent
123 The method of claim 122, wherein said second therapeutic agent is selected from the group consisting of a radioactive label, an immunomodulator, a hormone, a photoactive therapeutic agent, a cytotoxic agent an oligonucleotide and a combination thereof and wherein said second diagnostic agent is at least one of a radioactive label, a photoactive diagnostic agent or a non-radioactive label.


Documents:

3585-CHENP-2007 AMENDED PAGES OF SPECIFICATION 29-06-2011.pdf

3585-CHENP-2007 AMENDED CLAIMS 29-06-2011.pdf

3585-chenp-2007 correspondence others 27-12-2010.pdf

3585-chenp-2007 form-3 29-06-2011.pdf

3585-CHENP-2007 OTHER PATENT DOCUMENT 29-06-2011.pdf

3585-CHENP-2007 POWER OF ATTORNEY 29-06-2011.pdf

3585-CHENP-2007 EXAMINATION REPORT REPLY RECEIVED 29-06-2011.pdf

3585-CHENP-2007 FORM-13 15-02-2008.pdf

3585-chenp-2007-abstract.pdf

3585-chenp-2007-assignement.pdf

3585-chenp-2007-claims.pdf

3585-chenp-2007-correspondnece-others.pdf

3585-chenp-2007-description(complete).pdf

3585-chenp-2007-drawings.pdf

3585-chenp-2007-form 1.pdf

3585-chenp-2007-form 26.pdf

3585-chenp-2007-form 3.pdf

3585-chenp-2007-form 5.pdf


Patent Number 248379
Indian Patent Application Number 3585/CHENP/2007
PG Journal Number 28/2011
Publication Date 15-Jul-2011
Grant Date 08-Jul-2011
Date of Filing 16-Aug-2007
Name of Patentee IMMUNOMEDICS, INC
Applicant Address 300 AMERICAN ROAD, MORRIS PLAINS, NJ 07950
Inventors:
# Inventor's Name Inventor's Address
1 QU, ZHENGXING 15 SYCAMORE WAY, WARREN, NJ 07059, USA
2 GOLDENBERG, DAVID, M 1 CHAROLAIS FARM ROAD, MENDHAM, NJ 07945, USA
3 HANSEN, HANS, J 6014 ANGLER DRIVE, PICAYUNE, MS 39466,
PCT International Classification Number C07K 16/28
PCT International Application Number PCT/GB03/00665
PCT International Filing date 2003-02-14
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/416,232 2002-10-07 U.S.A.
2 60/356,132 2002-02-14 U.S.A.