AU2017204254A1 - Human monoclonal antibodies against CD20 - Google Patents

Abstract

Isolated human monoclonal antibodies which bind to and inhibit human CD20, and related antibody-based compositions and molecules, are disclosed. The human antibodies can be produced by a transfectoma or in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, non-human transgenic animals and hybridomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.

Description

2017204254 22 Jun2017 HUMAN MONOCLONAL ANTIBODIES AGAINST CD20

Background of the Invention 5 The CD20 molecule (also called human B-lymphocyte-restricted differentiation antigen or Bp35) is a hydrophobic transmembrane protein with a molecular weight of approximately 35 kD located on pre-B and mature B lymphocytes (Valentine et al. (1989),/. Biol. Chem. 264(19):11282-13287; and Einfield et ai. (1988) EMBO J. 7(3):711-717). CD20 is found on the surface of greater than 90% of B cells from 10 peripheral blood or lymphoid organs and is expressed during early pre-B cell development and remains until plasma cell differentiation. CD20 is present on both normal B cells as well as malignant B ceils. In particular, CD20 is expressed on greater than 90% of B cell non-Hodgkin’s lymphomas (NHL) (Anderson et al. (1984) Blood 63(6): 1424-1433), but is not found on hematopoietic stem cells, pro-B cells, normal plasma cells, or other 15 normal tissues (Tedder et ai (1985)7. Immunol. 135(2):973-979).

The 85 amino acid carboxyl-terminal region of the CD20 protein is located within the cytoplasm. The length of this region contrasts with that of other B cell-specific surface structures such as IgM, IgD, and IgG heavy chains or histocompatibility antigens class II a or β chains, which have relatively short intracytoplasmic regions of 3, 3, 28, .15, 20 and 16 amino acids, respectively (Komaromy et at. (1983) NAR 11:6775-6785). Of the last 61 carboxyl-terminal amino acids, 21 are acidic residues, whereas only 2 are basic, indicating that this region has a strong net negative charge. The GenBank Accession No. is NPm690605.

It is thought that CD20 might be involved in regulating an early step(s) in 25 the activation and differentiation process of B cells (Tedder et al. (1986) Eitr.J. Immunol 16:881-887) and could function as a calcium ion channel (Tedder et al. (1990} J. Cell. Biochem. 14D:195).

Despite uncertainly about the actual function of CD20 in promoting proliferation and/or differentiation of B cells, it provides an important target for antibody 30 mediated therapy to control or kill B cells involved in cancers and autoimmune disorders. In particular, the expression of CD20 on tumor cells, e.g., NHL, makes it an important target for antibody mediated therapy to specifically target therapeutic agents -1- 2017204254 22Jun2017 against CD20-positive neoplastic cells. However, while the results obtained to date clearly establish CD20 as a useful target for immunotherapy, they also show that currently available murine and chimeric antibodies do not constitute ideal therapeutic agents. 5 Accordingly, the need exists for improved therapeutic antibodies against CD20 which axe effective in preventing and/or treating a range of diseases involving cells expressing CD20.

Summary of the Invention 10 The present invention provides improved antibody therapeutics for treating and/or preventing diseases associated with cells expressing CD20, including tumor-related diseases, and immune diseases, including autoimmune diseases. The antibodies encompassed by the invention are improved in that they are fully human and, thus, are potentially less immunogenic in patients. 15' As exemplified herein, the human antibodies of the invention mediate . killing of B cells expressing· CD20 by a variety of mechanisms. In one embodiment, , human antibodies of the invention induce complement dependent cytotoxicity (CDC), e.g., at least about 20% CDC mediated lysis, preferably about 30% CDC mediated lysis, and more preferably 40-50% mediated lysis in cells, such as chronic B-lymphocytic 20 leukaemia (B-CLL) cells. In another embodiment, human antibodies of the invention induce apoptosis of cells expressing CD20. In another embodiment, human antibodies of the invention induce homotypic adhesion of cells expressing CD20. Furthermore, the • human antibodies of the invention may induce antibody dependent cellular cytotoxicity (ADCC) of cells expressing CD20 in the presence of human effector cells (e.g., 25 monocytes, mononuclear cells, NK cells and PMNs). Furthermore, human antibodies of the invention may induce phagocytosis of cells expressing CD20 in the presence of macrophages. The human monoclonal antibodies of the invention may work by one or more of these mechanisms. Examples of cells which can be killed by human antibodies of the present invention include, but are not limited to, B cells expressing CD20, such as 30 tumorigenic B cells and B cells involved in immune diseases. In a particular embodiment, the human antibodies are used to mediate killing of B lymphocytes in the treatment of lymphoma, e.g., B cell non-Hodgkin’s lymphoma.

Human antibodies of the invention include IgGl (e.g., IgGl,K), IgG3 (e.g., IgG3,ic ) and IgG4 (e.g., IgG4,K) antibodies. However, other antibody isotypes are 35 also encompassed by the invention, including IgG2, IgM, IgAl, IgA2, secretory IgA, 1

IgD, and IgE, The antibodies can be whole antibodies or antigen-binding fragments thereof including, for example, Fab, F(ab‘)2, Fv, single chain Fv fragments or bispecific antibodies. Furthermore, the antigen-binding fragments include binding-domain -2- 2017204254 22Jun2017 immunoglobulin fusion proteins comprising (i) a binding domain polypeptide (such as a heavy chain variable region or a light chain variable region) that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 5 constant region fused to the CH2 constant region. Such binding-domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939.

Particular human antibodies of the present invention include those referred to as ΠΒ8, 2F2, and 7D8, encoded by human heavy chain and human kappa 10 light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NOs:L 5, or 9 and SEQ ID NOs:3,7, or 11, respectively, and conservative sequence modifications thereof. In another embodiment, the human antibodies are characterized by having human heavy chain and human kappa light chain variable regions comprising the amino acid sequences as set forth in SEQ ID NOs:2,6, 15 or 10 and SEQ ID NOs:4, 8, or 12, respectively, and conservative sequence modifications thereof.

In yet another embodiment, the human antibodies are characterized by having human heavy chain and human kappa light chain variable regions which are at least 90% homologous, preferably at least 95% homologous, and more preferably at 20 least 98%, or at least 99% homologous to the amino acid sequences as set forth in SEQ ED NO;2 and SEQ ID NO:4, respectively; SEQ ID NO:6 and SEQ ED NQ:8, respectively; or SEQ ID NO: 10 and SEQ ID NO: 12, respectively.

Other particular- human antibodies of the invention include those which comprise a CDR domain having a human heavy and light chain CDR1 region, a human 25 heavy and light drain CDR2 region, and a human heavy and light chain CDR3 region, wherein (a) the CDR1, CDR2, and CDR3 human heavy chain regions comprise an amino acid sequence selected from the group consisting of the amino acid sequences CDR1, CDR2, and CDR3 shown in Figures 53, 55, or 57 (SEQ ID NOs:13-15,19-21, 30 and 25-27), and conservative sequence modifications thereof, and (b) the CDR1, CDR2, and CDR3 human light chain regions comprise an amino acid sequence selected from the group consisting of the amino acid sequences CDR1, CDR2, and CDR3 shown in Figures 53, 55, or 57 (SEQ ID NOs: 16-18,22-24, and 28-30), and conservative sequence modifications thereof. 35 Also included within the present invention are antibodies which dissociate from CD20 with a dissociation equilibrium constant (KD) of approximately 1-10 nM or less. Such antibodies also include those which do not cross-react with related cell-surface antigens and thus do not inhibit their function. -3- 2017204254 22 Jun2017

In another embodiment, human anti-CD20 antibodies of the present invention can be characterized by one or more of the following properties: a) specificity for human CD20; b) a binding affinity to CD20 (KD) of about 10 nM or less, preferably, 5 about 5 nM or less and, more preferably, about 1-3 nM or less as determined by the binding experiment disclosed in Example 5 (Figure 9) herein; c) a dissociation rate constant (kd) from CD20 of about 10'4 sec'l or less, preferably, about 10"5 sec'l or less and, more preferably, about 10~6 sec'or less, as determined by the dissociation rate experiment disclosed in Example 5 (Figure 9) herein; 10 d) the ability to mediate a high level of CDC on either CD55/59 negative or CD55/59 positive cells; e) the ability to translocate into lipid rafts upon binding to CD20; f) the ability to inhibit the growth of cells which express CD20; g) the ability to induce apoptosis of cells which express CD20; 15 h) the ability to induce homotypic adhesion of cells which express CD20; i) the ability to induce ADCC of cells which express CD20 in the presence of effector cells; j) the ability to prolong survival of a subject having tumor cells which express CD20; 20 k) the ability to deplete cells which express CD20; and/or

1) the ability to deplete cells which express low levels of CD20 (CD20|OW cells).

The human anti-CD20 antibodies of the present in vention can be derivatized, finked to or co-expressed to other binding specificities. In a particular 25 embodiment, the invention provides a bispecific or multispecific molecule comprising at least one first binding specificity for CD20 (e.g., a human anti-CD20 antibody or mimetic thereof), and a second binding specificity for a human effector cell, such as a binding specificity for an Fc receptor (e.g., a human Fey receptor, such as FcyRI, or a human Fca receptor) or a T cell receptor, e.g., CD3. 30 Accordingly, the present invention includes bispecific and multispecific molecules that bind to both human CD20 and to an Fc receptor or a T cell receptor, e.g., CD3. Examples of Fc receptors are, e.g., a human IgG receptor, e.g., an Fc-gamma receptor (FcyR), such as FcyRI (CD64), FcyRIl (CD32), and FcyRIII (CD16). Other Fc receptors, such as human IgA receptors (e.g., FcaRI), also can be targeted. The Fc 35 receptor is preferably located on the surface of an effector cell, e.g., a monocyte, macrophage or an activated mononuclear cell. In a preferred embodiment, the bispecific and multispecific molecules bind to an Fc receptor at a site which is distinct from the immunoglobulin Fc (e.g., IgG or IgA) binding site of the receptor. Therefore, the .4- binding of the bispecific and multispecific molecules is not blocked by physiological levels of immunoglobulins. 2017204254 22Jun2017

In yet another aspect, human anti-CD20 antibodies of the invention are derivatized. linked to or co-expressed with another functional molecule, e.g., another 5 peptide or protein (e.g., a Fab* fragment). For example, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., to produce a bispecific or a multispecific antibody), a cytotoxin, cellular ligand or antigen (e.g., to produce an immunoconjugate, such as an immunotoxin). An 10 antibody of the present invention can be linked to other therapeutic moieties, e.g., a radioisotope, a small molecule anti-cancer drug, an anti-inflammatory agent, or an immunosuppressive agent. Accordingly, the present invention encompasses a large variety of antibody conjugates, bispecific and multispecific molecules, and fusion proteins, all of which bind to CD20 expressing cells and which can be used to target 15 other molecules to such cells.

In still another aspect, the invention provides compositions, e.g., pharmaceutical and diagnostic compositions/kits, comprising a pharmaceutically acceptable carrier formulated along with one or a combination of human monoclonal antibodies of the invention. In a particular embodiment, the composition includes a 20 combination of antibodies which bind to distinct epitopes or which possess distinct functional characteristics, such as inducing CDC and inducing apoptosis.

Human antibodies, iimmmoconjugates, bispecific and multispecific molecules and compositions of the present invention can be used in a variety of methods for inhibiting growth of cells expressing CD20 and/or killing cells expressing CD20 by 25 contacting the cells wi th an effective amount of the antibody, immunconjugate, bispecific/multispecific molecule or composition, such that the growth of the cell is inhibited and/or the cell is killed. In one embodiment, the method includes killing of the cell expressing CD20 in the presence of effector cells, for example, by CDC, apoptosis, ADCC, phagocytosis, or by a combination of two or more of these mechanisms. The 30 cells are preferably killed or inhibited without killing or inhibiting the activity of cells which do not express CD20 but which may, for example, express a structurally related cell-surface antigen (i.e., without cross-reactivity to related but functionally distinct cell surface antigens). Cells expressing CD20 which can be inhibited or ldlled using the human antibodies of the invention include, for example, tumorigenic B cells. 35 Accordingly, human antibodies of the present invention can be used to treat and/or prevent a variety of diseases involving cells expressing CD20 by administering the antibodies to patients suffering from such diseases. Exemplary diseases that can be treated (e.g., ameliorated) or prevented include, but are not limited -5- to, tumorigenic diseases and immune diseases, e.g., autoimmune diseases. Examples of tumorigenic diseases which can be treated and/or prevented include B cell lymphoma, e.g., NHL, including precursor B ceil lymphoblastic leulcemia/lymphoma and mature B cell neoplasms, such as B cell chronic lymhocytic leukemia (CLL)/smatl lymphocytic 5 lymphoma (SLL), B cell prolymphocytic leulcemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell 10 myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's 2017204254 22Jun2017 macroglobulinemia, and anaplastic large-cell lymphoma (ALCL). Examples of immune disorders in which CD20 expressing B cells are involved which can be treated and/or prevented include psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, 15 respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud’s syndrome, Sjogren’s syndrome, juvenile onset diabetes, Reiter’s disease, Behcet’s disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immime-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic 20 purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves’ disease, Hashimoto’s thyroiditis,

Wegener’s granulomatosis, Omenn’s syndrome, chronic renal failure, acute infectious mononucleosis, HIV, and herpes vims associated diseases. Further examples are severe 25 acute respiratory distress syndrome and choreoretinitis. Yet further examples are diseases and disorders caused by infection of B-cells with virus, such as Epstein-Barr virus (EBV).

In a particular embodiment of the invention, the subject being administered the antibody is additionally treated with a chemotherapeutic agent, 30 radiation, or an agent that modulates, e.g., enhances or inhibits, the expression or activity of an Fc receptor, e.g., an Fca receptor or an Fey receptor, such as a cytokine. Typical cytokines for administration during treatment include granulocyte colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF). Typical therapeutic agents 35 include, among others, anti-neoplastic agents such as doxorubicin, cisplatin, bleomycin, carraustine, chlorambucil, and cyclophosphamide. -6- 2017204254 22 Jun2017

In yet another aspect, the present invention provides a method for detecting in vitro or in vivo the presence of CD20 in a sample or individual, e.g., for diagnosing a CD20-related disease, preferably at an early stage. This can also be useful for monitoring the disease and effect of treatment and for determining and adjusting the 5 dose of the antibody to be administered. The in vivo method can be performed using imaging technique such as PET (positron emission tomography) or SPECT (single photon emission computed tomography). In one embodiment, this is achieved by contacting a sample to be tested, optionally along with a control sample, with a human monoclonal antibody of the invention under conditions that allow for formation of a 10 complex between the antibody and CD20. Complex formation is then detected (e.g., using an FACS analysis or Western blotting). When using a control sample along with the test sample, complex is detected in both samples and any statistically significant difference in the fonnation of complexes between the samples is indicative of the presence of CD20 in the test sample. 15 In yet another aspect, the invention provides a transgenic non-human animal, such as a transgenic mouse, which express human monoclonal antibodies that bind to CD20. In a particular embodiment, the transgenic non-human animal is a transgenic mouse having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an. antibody of the invention. 20 The transgenic non-human animal can be immunized with a purified or enriched preparation of CD20 antigen and/or cells expressing CD20. Preferably, the transgenic non-human animal, e.g., the transgenic mouse, is capable of producing multiple isotypes of human monoclonal antibodies to CD20 (e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination and isotype switching. Isotype switching may occur 25 by, e.g., classical or non-classical isotype switching.

Accordingly, in yet another aspect, the Invention provides isolated B cells from a transgenic non-human animal as described above, e.g., a transgenic mouse, which expresses human auti-CD20 antibodies. The isolated B cells can then be immortalized by fusion to an immortalized cell to provide a source (e.g., a hybridoma) 30 of human anti-CD20 antibodies. Such hybridomas (i.e., which produce human anti- CD20 antibodies) are also included within the scope of the invention.

As exemplified herein, human antibodies of the invention can be obtained directly from hybridomas which express the antibody, or can be cloned and recombinantly expressed in a host cell (e.g., a CHO cell, a NS/0 cell or a lymphocytic 35 cell). Further examples of host cells are microorganisms, such as E. coli, and fungi, such as yeast. Alternatively, they can be produced recombinantly in a transgenic nonhuman animal or plant. Accordingly, in another aspect, the present invention provides methods for producing human monoclonal antibodies which bind to human CD20. In -7- one embodiment, the method includes immunizing a transgenic non-human animal, e.g., a transgenic mouse, as previously described (e.g., having a genome comprising a human heavy chain transgene and a human light chain transgene encoding all or a portion of an anti~CD20 antibody), with a purified or enriched preparation of human 5 CD20 antigen and/or cells expressing human CD20. B cells (e.g., splenic B cells) of the animal are then obtained and fused with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies against CD20, 2017204254 22 Jun2017

In yet another aspect, the invention provides nucleic acid molecules encoding human anti-CD2Q antibodies (e.g., variable regions thereof), as well as 10 recombinant expression vectors which include the nucleic acids of the invention, and host cells transfected with such vectors. Methods of producing the antibodies by culturing these host cells are also encompassed by the invention. Particular nucleic acids provided by the invention comprise the nucleotide sequences shown in SEQ ED NOs:l, 5, or 9 and SEQ ID NOs:3, 7, or 11, encoding the heavy and light chains, 15 respectively, of human anti-CD20 antibodies 2F2, 7D8, and 1 IBS.

Other features and advantages of the instant invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings 20 Figure 1 shows the ρΟΟΝγΙ f/variabie-heavy vector used for recombinant production of the human monoclonal antibodies 2F2 and 1 IBS.

Figure 2 shows the pCONx/variable-light vector used for recombinant production of 2F2 and Π B8.

Figure 3 shows the double-gene cloning vector (ρΟΟΝγ1ί/'/ι2Ρ2) used 25 for recombinant production of 2F2 and 11B8.

Figure 4 is a graph comparing the binding of human monoclonal antibodies 2F2, 7D8, and 11B8 to Raji, Daudi, and CD20 transfected NS/'O cells and parental NS/Q cells using flow cytometry.

Figures 5A and 5B show the binding of 2F2 to PBMCs from three 30 human donors using flow cytometry.

Figure 6 is a graph comparing the binding affinity of l25I-labeled 2F2 and 125I-labeled 11B8 to Ramos-EHRB cells.

Figures 7A and 7B show the binding of 12sI-labeled 2F2 and 125I-labeled 11B8 compared to 125I~labeled rituximab (chimeric anti-CD20 antibody, IDEC) and 35 125I-labeled B1 (the term B1 corresponds to the unlabeled form of Bexxar™, which is a !J1I-labeled murine anti-human CD20 antibody, Coulter) to Ramos-EHRB cells (A) and Daudi cells (B). -8-

Figure 8 is a graph comparing the dissociation rates of 125I-labeled 11B8T, 125i-labeled 2F2,I25I-labeled rituximab (RIT), and l25I-labeled Bl. 2017204254 22 Jun2017

Figure 9 shows the dissociation rates of the F(ab’)2 fragments of 2F2, 1 IB ST. and rituximab in Ranios-EHRB cells. 5 Figures 10A and 1 OB show the CDC by 2F2T, 11B8T, 7D8, rituximab, and an isotype control antibody (HuMab-KLH) of Daudi cells (A) and SU-DHL-4 cells (B) at different time points (functional off-rate) using flow cytometry.

Figures 1JA-E show the Idnetics of CDC induced by 2F2 and rituximab in different cell lines using flow cytometry. 10 Figures 12A-D show CDC induced by 2F2 and rituximab in different cell lines as a function of the concentration of complement (normal human serum (NHS)) at two different antibody concentrations using flow cytometry.

Figures 13A-D show concentration-dependent induction of CDC by 2F2 and rituximab in different cell lines using flow cytometry.

15 Figures 14A and 14B show concentration-dependent induction of CDC by 2F2, 2F2T, 1ΓΒ8Τ, Bl, and rituximab in Daudi cells (A) andRaji cells (B).

Figures ISA and J5B are graphs comparing CDC of Daudi cells (cells expressing low levels of CD55/59) by human monoclonal antibodies 2F2, 7D8, and 11B8 and rituximab; (A) shows percent lysis of unwashed cells and (B) shows percent 20 lysis of cells which were washed before the addition of serum.

Figures 16A and 16B are graphs comparing CDC of Raji cells (cells expressing high levels of CD55/59) by human monoclonal antibodies 2F2, 7D8, and 1 IBS and rituximab; (A) shows percent lysis of cells not blocked with anti-CD55 and anti-CD59 antibodies, and (B) shows percent lysis of cells blocked with anti-CD55 and 25 anti-CD59 antibodies.

Figures 17A-C show the role of CD55 and CD59 in CDC induced by . 2F2 and rituximab in Raji cells. (A) shows the percentage of lysed cells upon addition of anti-CD55 antibody, (B) shows the percentage of lysed cells upon addition of anti-CD59 antibody, and (C) shows the percentage of lysed cells upon addition of both anti-30 CD55 antibody and anti-CD59 antibody.

Figures 18A-D show the binding of complement factor Clq by 2F2 and rituximab in different cell lines as determined by flow cytometry.

Figures 19A-D show the deposition of complement factor fragment C4c by 2F2 and rituximab in different cell lines as determined by flow cytometry.

35 Figure 20 shows lysis of ARH-77 cells by 2F2, rituximab, and 11B8T in the presence of PMNs, MNCs, plasma or whole blood.

Figure 21 shows lysis of B-CLL cells by 2F2, rituximab, and 1 IB ST in the presence of PMNs, MNCs, plasma or whole blood. -91 2017204254 22 Jun2017

Figure 22 shows lysis of HCL (hairy cell leukemia) cells by 2F2, rituximab, and 11B8T in the presence of PMNs, MNCs, plasma or whole blood.

Figure 23 shows lysis of B-ALL cells by 2F2, and rituximab, in the presence of PMNs, MNCs, plasma or whole blood, 5 Figure 24 shows lysis of follicular lymphoma (FL) cells by 2F2, rituximab, and 11B8T in the presence of PMNs, MNCs. plasma or whole blood.

Figure 25 shows lysis of mantle cell lymphoma cells by 2F2, rituximab, and 11B8T in the presence of PMNs, MNCs, plasma or whole blood.

Figure 26 shows concentration dependent lysis of ARH-77 cells by 2F2 10 and rituximab in the presence of whole blood.

Figure 27 shows MNC-mediated lysis of ARH-77 cells by 2F2T, 11B8T, and rituximab.

Figure 28 shows MNC-mediated lysis ofRaji cells by 2F2T, 11B8T, and rituximab. 15 Figures 29A, B, and C are graphs showing clustering of CD20 in the lipid rafts upon incubation with 2F2, 7D8, or 11B8 using FRET analysis and Triton-X insolubility assay.

Figure 30 shows clustering of CD20 in the lipid rafts upon incubation with 2F2, rituximab, or 11B8T using FRET analysis. 20 Figure 31 shows the proportion of GD20 remaining in the insoluble raft fraction after treatment with Triton X-100 (TX) and incubation with 2F2, rituximab, or 11BST.

Figure 32 shows the distribution of CD20 between the raft and non-raft membrane fractions upon stimulating Daudi cells with 2F2, rituximab, or 11B8T. 25 Figures 33A-G show apoptosis of Daudi cells by 2F2, 7D8, and 11B8 using flow cytometry. ; Figure 34 shows induction of apoptosis of Raji cells by 2F2, 11B8T, rituximab, orBl using flow cytometry.

Figure 35A shows induction of apoptosis of Daudi cells by 2F2T, 30 11B8T, rituximab, or B1 using flow cytometry.

Figure 35B shows early stage and late stage apoptosis of Daudi cells by human monoclonal antibodies 2F2T, 11B8T, rituximab, and B1 using flow cytometry.

Figures 36A-E show homotypic adhesion of Ramos-EHRB cells by 2F2, 7D8, and 11B8 using light microscopy. 35 Figure 3.7 show homotypic adhesion of Daudi cells by 2F2, rituximab, and B1 using light microscopy.

Figure 38 is a graph showing the percent survival of SOD mice injected with Daudi cells and treated with 2F2 or 7D8. -10- 2017204254 22 Jun2017

Figure 39 shows the percent survival of SCID mice injected with Tanoue cells and treated with 2F2, rituximab, or Bl.

Figure 40 shows the percent survival of SCID mice injected with Daudi cells and treated with different concentrations of 2F2 or rituximab. 5 Figure 41 shows the percent survival of SCED mice injected with Daudi

cells and treated with 11B8T or B L

Figure 42 sho ws bioluminescence imaging of tumor cells in SCID mice on day 39 (31 days after treatment with 10 /ig of Bl, rituximab, 11B8T, 2F2T, or huIgGl). The bioluminescence is represented in red color (the dark areas in the mice) 10 (light intensity >50 photons per 5 min) as overlay on the black and white body image of the mice.

Figure 43 shows the tumor mass in each mouse quantified on day 25, 32, 39, and 4 6 following administration, on day 8, of 10 jig of Bl, rituximab, 11B8T, 2F2T, or huIgGl by integrating the light signals over the body surface. 15 Figures 44A-C show flow cytometric analysis of CD20+ cells in peripheral blood of cynomolgus monkeys following intravenous administration of 2F2 or rituximab at different dosages, 4 x 1.25 mk/kg (A), 4 x 6.25 mg/kg (B), or 4 x 12.50 mg/kg (C).

Figures 45A-C show flow cytometric analysis of CD2T cells in 20 peripheral blood of cynomolgus monkeys following intravenous administration of 2F2 or rituximab at different dosages, 4 x 1.25 mk/kg (A), 4 x 6.25 mg/kg (B), or 4 x 12.50 mg/kg (C).

Figures 46A-C show flow cytometric analysis of CD20+ cells in lymph node of cynomolgus monkeys following intravenous administration of2F2 or 25 ' rituximab at different dosages, 4 x 1.25 mk/kg (A), 4 x 6.25 mg/kg (B), or 4 x 12.50 mg/kg (C).

Figures 47A-C show flow cytometric analysis of CD20lowCD23+CD40hl=h expressing cells in peripheral blood of cynomolgus monkeys following intravenous administration of 2F2 or rituximab at different dosages, 4 x 1.25 30 mlc/kg (A), 4 x 6.25 mg/kg (B), or 4 x 12.50 mg/kg (C).

Figures 48A-E show binding of rituximab (A), 2F2 (B), 11B8 (C), Bl (D), or an isotype control antibody (E) to CHO cells expressing wild type (WT) CD20, mutant CD20 (AxP), or both WT CD20 and mutant CD20 (AxP) as determined by flow cytometry. 35 Figures 49A-F show percentage binding of 2F2,11B8T, Bl. or rituximab to mutant P172S vs. WT CD20 (A), percentage binding of 2F2T, 11B8T,

Bl, CAT (CAT 13.6E12, a mouse monoclonal IgG2A anti-CD20 antibody, Diatec.Com), a control isotype antibody (KXH) or rituximab to mutant CD20 (AxP) vs. -11 - 2017204254 22 Jun2017 WT CD20 (B), percentage binding of 2F2,11B8T, B1 or rituximab to mutant N166D vs. WT CD20 (C), percentage binding of2F2T, CAT or rituximab to mutant N166D vs. WT CD20 ¢0) , percentage binding of 2F2T, 2F2, HB8T, B1 or rituximab to mutant N163D vs. WT CD20 (E), and percentage binding of 2F2T, CAT or rituximab 5 to mutant N163D vs. WT CD20 (F).

Figure 50 shows binding of 2F2T, 7D8, and isotype control antibody, as determined by ELIS A, to three anti-idiotypic antibodies, anti-2F2 sab 1.1, anti-2F2 sab 1.2, and anti-2F2 sab 1.3. raised against 2F2.

Figure 51 shows binding of 1 IB ST, as determined by ELISA, to anti-10 idiotypic antibodies, anti-11B8T sab 2.2, anti-11B8T sab 2.3, anti-11B8T sab 2.4, anti-11B8T sab 2.5, and anti-11B8T sab 2.6, raised against 11B8T, but no binding to the anti-idiotypic anti-2F2 antibodies.

Figures 52A-C show dose-dependent binding of 2F2T, as detennined by ELISA, to three anti-idiotypic antibodies, anti-2F2 sab 1.1 (A), anti-2F2 sab 1.2 (B), 15 and anti-2F2 sab 1.3 (C), raised against 2F2.

Figure 53 shows the amino acid sequence (SEQ ID NO :2) of die heavy chain V region and the amino acid sequence (SEQ ID NO:4) of the light (kappa) chain V region of human monoclonal antibody 2F2 with CDR regions designated.

Figure 54 shows the nucleotide sequence (SEQ ID NO:l) of the heavy 20 chain V region and the nucleotide sequence (SEQ ID NO:3) of the light (kappa) chain V region of human monoclonal antibody 2F2.

Figure 55 shows the amino acid sequence (SEQ ID NO:6) of the heavy chain. V region and the amino acid sequence (SEQ ID NO :8) of the light (kappa) chain V region of human monoclonal antibody 7D8 with CDR regions designated. 25 Figure 56 shows the nucleotide sequence (SEQ ID NO:5) of the heavy chain V region and the nucleotide sequence (SEQ ID NO:7) of the light (kappa) chain V region of human monoclonal antibody 7D8.

Figure 57 shows the amino acid sequence (SEQ ID NO: 10) of the heavy chain V region and the amino acid sequence (SEQ ID NO: 12) of the light (kappa) chain 30 V region of human monoclonal antibody 11B8 with CDR regions designated.

Figure 58 shows the nucleotide sequence (SEQ ID NO :9) of the heavy · chain V region and the nucleotide sequence (SEQ ID NO:ll) of the light (kappa) chain V region of human monoclonal antibody 11B8. 3 5 Detailed Description of the Invention

The present invention provides improved antibody-based therapies for treating and diagnosing a variety of disorders involving cells expressing CD20,

Therapies of the invention employ isolated human monoclonal antibodies which -12- specifically bind to an epitope present on CD20. Isolated human monoclonal antibodies encompassed by the present invention include IgA, IgGl-4, IgB, IgM, and IgD antibodies.

In one embodiment the antibody is an IgGl antibody, more particularly an IgGLtc or IgGl ,X isotype. In another embodiment the antibody is an IgG3 antibody, more particularly an IgG3,K or IgG3,X isotype. In yet another embodiment the antibody is an IgG4 antibody, more particularly an IgG4,K or IgG4,X isotype. In still another embodiment the antibody is an IgAl or IgA2 antibody.

In still another embodiment the antibody is an IgM antibody.

In one embodiment, the human antibodies are produced in a non-human transgenic animal, e.g, a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies to CD20 by undergoing V-D-J recombination and iso type switching. Accordingly, aspects of the invention include not only antibodies, antibody fragments, and pharmaceutical compositions thereof, but also non-human transgenic animals, B cells, host cell transfectomas, and hybridomas which produce monoclonal antibodies. Such transgenic animal can also be a transgenic rabbit for producing polyclonal antibodies such as disclosed in US 2003/0017534. Accordingly, the invention also encompasses human polyclonal antibodies which specifically bind to CD20. In one embodiment the invention relates to polyclonal antibodies which bind to an epitope on CD20 (i) which does not comprise or require the amino acid residue proline at position 172; (ii) which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172; (iii) which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166; (iv) which does not comprise or require the amino acid residue proline at position 172, but which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166; or (v) which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172, but which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166. hi another embodiment the invention relates to human polyclonal antibodies which have one or more of the following characteristics: (i) bind to mutant P172S CD20 (proline at position 172 mutated to serine) with at least the same affinity as to human CD20; (ii) bind to mutant AxP (alanine at position 170 mutated to serine, and proline at position 172 mutated to serine) with at least the same affinity as to human CD20; (iii) show a reduced binding of 50% or more to mutant N166D (asparagine at position 166 mutated to aspartic acid) to human CD20 at an antibody concentration of 10 ju-g/ml; and/or (iv) show a reduced binding of 50% or more to mutant N163D (asparagine at position 163 mutated to aspartic acid) compared to human CD20 at an antibody concentration of 10 pg/ml -13- 2017204254 22 Jun2017 in yet another embodiment the invention the invention relates to human polyclonal antibodies which bind to an epitope in the small first extracellular loop of human CD2Q. In still another embodiment the invention also encompasses human polyclonal, antibodies which bind to a discontinuous epitope on CD20. In a further 5 embodiment the invention relates to human polyclonal antibodies which bind a discontinuous epitope on CD20, which has part of the first small extracellular loop and part of the second extracellular loop, hi still a further embodiment the invention relates to human polyclonal antibodies which bind to a discontinuous epitope on CD20, which has residues AGIYAP of the small first extracellular loop and residues 10 MES LNFERAHTPYI of the second extracellular loop.

Methods of using the antibodies of the invention to detect a cell expressing CD20 are encompassed by the invention. Methods of using the antibodies of the invention to block or inhibit CD20 induced activities, e.g., proliferative and/or differentiation activities, are also provided and are useful in the treatment of disorders 15 associated with CD20, such as tumorigenic diseases (e.g., B cell lymphoma) and autoimmune diseases (e.g., RA, Chrohn’s disease and Wegener’s granulomatosis). hi order that the present invention may he more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. 20 The terms "CD20” and “CD20 antigen” are used interchangeably herein, and include any variants, isoforms and species homologs of human CD20 which are naturally expressed by cells or are expressed on cells transfected with the CD20 gene. Binding of an antibody of the invention to the CD20 antigen mediate the killing of cells expressing CD20 (e.g., a tumor cell) by inactivating CD20. The killing of the cells 25 expressing CD20 may occur by one or more of the following mechanisms: complement dependent cytotoxity (CDC) of cells expressing CD20; apoptosis of cells expressing CD20; effector cell phagocytosis of cells expressing CD20; or effector cell antibody dependent cellular cytotoxicity (ADCC) of cells 30 expressing CD20.

Synonyms of CD20, as recognized in the art, include B-lymphocyte antigen CD20, B-lymphocyte surface antigen Bl, Leu-16, Bp35, BM5, and LF5.

As used herein, the term “inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the cell growth when contacted with an 35 anti-CD20 antibody as compared to the growth of the same cells not in contact with an anti-CD20 antibody, e.g., the inhibition of growth of a cell culture by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Such a decrease in cell -14- 2017204254 22Jun2017 growth can occur by a variety of mechanisms, e.g., effector cell phagocytosis, ADCC, CDC, and/or apoptosis.

The term “raft” refers to the sphingolipid- and cholesterol-rich membrane microdomains located in the outer leaflet area of the plasma membrane of a cell. The 5 ability of certain proteins to associate within such domains can effect the protein’s function. For example, the translocation of CD20 molecules into lipid rafts, after being bound by human antibodies of the present invention, creates a high density of CD20 antigen-antibody complexes in the plasma membranes. Such a high density of CD20 antigen-antibody complexes can enable effi cient activation of the complement system 10 during CDC.

The terni “antibody” as referred to herein includes whole antibodies and any antigen binding fragment (ie., "antigen-binding portion”) or single chain thereof.

An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion 15 thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as Vh) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated hei-ein as Vl) and a light chain constant region. The Vh and Vl regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more 20 conserved, termed framework regions (FR). Each Vh and Vl is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order; FR1, CDR1, FR2, CDR2, FRS, CDR3, FR4. The variable regions of the heavy and light chains, contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to 25 host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CD20). It has been shown that the 30 antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigenbinding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the Vl, Vh, Cl and CHi domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; 35 (iii) a Fd fragment consisting of the Vh and Chi domains; (iv) a Fv fragment consisting of the Vl and Vh domains of a single arm of an antibody, (v) a dAb fragment (Ward et at, (1989) Nature 341:544-546), which consists of a Vh domain; (vi) an isolated complementarity determining region (CDR), and (vii) a combination of two or more -15- 2017204254 22 Jun2017 isolated CDRs which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, Vl and Vh, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the Vl and Vh regions pair to form 5 monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. A further example is binding-domain immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is 10 fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CHS constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The binding-domain immunoglobulin fusion proteins are further disclosed in 15 US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term “epitope” means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings 20 of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

The term "discontinuous epitope," as used herein, means a 25 conformational epitope on a protein antigen which is formed from at least two separate regions in the primary sequence of the protein.

The term "bispecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding 30 specificities. For example, the molecule may bind to, or interact with, (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term "multispecific molecule" or "heterospecific molecule" is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. For example, the molecule may bind to, or interact with, (a) a cell surface 35 antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the invention includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific molecules which are directed to cell -16- 2017204254 22 Jun2017 surface antigens, such as CD20, and to other targets, such as Fc receptors on effector cells.

The term "bispecific antibodies" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single 5 polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger, P,, et al. (1993) Proc. Natl. Acad. Set. USA 90:6444-6448; Poljak, R.J., et . al. (1994) Structure 2:1121-1123). 10 Tire term “human antibody derivatives” refers to any modified form of the antibody, e.g., a conjugate of the antibody and another agent or antibody.

As used herein, a human antibody is “derived from” a particular germline sequence if the antibody is obtained from a system using human immunoglobulin sequences, e.g., by immunizing a transgenic mouse carrying human immunoglobulin 15. genes or by screening a human immunoglobulin gene library, and wherein the selected human antibody is at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by the germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence will display no more than 10 amino 20 acid differences, more preferably, no more than 5, or even more preferably, no more than 4, 3,2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

As used herein, the term "heteroantibodies" refers to two or more antibodies, derivatives therefrom, or antigen binding regions linked together, at least two 25 of which have different specificities. These different specificities include a binding specificity for an Fc receptor on an effector cell, and a binding specificity for an antigen or epitope on a target cell, e.g., a tumor cell.

The term "human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline 30 immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another 35 mammalian species, such as a mouse, have been grafted onto human framework sequences. -17- 2017204254 22Jun2017

The terms "monoclonal antibody'1 or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal 5 antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, e.g,, a transgenic mouse, having a genome comprising a human heavy chain transgene 10 and a light chain transgene fused to an immortalized cell.

The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or tranachiOmosomal for human immunoglobulin genes or a hybridoma prepared 15 therefrom (described further in Section I, below), (b) antibodies isolated from a host cell transformed to express the antibody, e.g,. from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human 20 antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the Vh and Vl regions of the recombinant antibodies are 25 sequences that, while derived from and related to human germline Vh and VL sequences, may not naturally exist within the human antibody germline repertoire in viva.

The term “transfectoma”, as used herein, includes recombinant eukaryotic host cell expressing the antibody, such as CHG cells, NS/0 cells, HEK293 cells, plant cells, or fungi, including yeast cells . 30 As used herein, a "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal. 35 As used herein, a "heterohybrid antibody refers to an antibody having a light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. -18- 2017204254 22 Jun2017

Examples of heterohybrid antibodies include chimeric and humanized antibodies, discussed supra.

An "isolated antibody," as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities 5 (e.g., an isolated antibody that specifically binds to CD20 is substantially free of antibodies that specifically bind antigens other than CD20). An isolated antibody that specifically binds to an epitope, isoform or variant of human CD20 may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., CD20 species homologs). Moreover, an isolated antibody may be substantially free of other cellular 10 material and/or chemicals. In one embodiment of the invention, a combination of “isolated” monoclonal antibodies having different specificities are combined in a well defined composition.

As used herein, "specific binding" refers to antibody binding to a predetermined antigen. Typically, the antibody hinds with an affinity corresponding to a 15 ICd of about 1 x 10'7 M or less, and binds to the predetermined antigen with an affinity corresponding to a Kd that is at least two orders of magnitude lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases "an antibody recognizing an antigen” and " an antibody specific for an antigen" are used interchangeably herein with the term 20 "an antibody which binds specifically to an antigen".

As used herein, the term “ka” (sec'1), as used herein, is intended to refer to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the k0ff value.

The term "ka" (M"1 x sec'1), as used herein, is intended to refer to the 25 association rate constant of a particular antibody-antigen interaction.

The term "KD" (M), as used herein, is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.

The term “Ka” (M'1), as used herein, is intended to refer to the association equilibrium constant of a particular antibody-antigen interaction and is 3 0 obtained by dividing the ka by the kd-

As used herein, "isotype" refers to the antibody class (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

As used herein, "isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig 35 classes.

As used herein, "nonswitched isotype" refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the CH gene encoding the nonswitched isotype is typically the first CH gene immediately -19- 2017204254 22Jun2017 downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non-classical isotype switching may occur by, for example, homologous 5 recombination between human σμ and human Σμ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, may occur and effectuate isotype switching.

As used herein, the term "switch sequence" refers to those DNA sequences responsible for switch recombination. A "switch donor" sequence, typically a 10 μ switch region, will be 5' (i.e., upstream) of the construct region to be deleted during the switch recombination. The "switch acceptor" region will he between the construct region to be deleted and the replacement constant region (e.g., γ, ε, etc.). As there is no specific site where recombination always occurs, the final gene sequence will typically not be predictable from the construct. 15- As used herein, "glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin (antibody) protein. A glycosylation pattern of a heterologous antibody can be characterized as being,substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the non-human transgenic animal, 20 when one of ordinary skill in the art would recognize tire glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the non-human transgenic animal than to the species from which the CH genes of the transgene were derived.

The term "naturally-occurring" as used herein as applied to an object 25 refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

The term "rearranged" as used herein refers to a configuration of a heavy 30 chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete Vh or VL domain, respectively. A rearranged immunoglobulin (antibody) gene locus can be identified by comparison to germline DNA; a rearranged locus will have at least one recombined heptamer/nonamer homology element. 35 The term "unrearranged" or "germline configuration" as used herein in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment. -20- 2017204254 22Jun2017

The term "nucleic acid molecule", as used herein, is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.

The term "isolated nucleic acid molecule,” as used herein in reference to 5 nucleic acids encoding whole antibodies or antibody portions (e.g., VH, Vl, CDR3) that bind to CD20, is intended to refer to a nucleic acid molecule in which the nucleotide sequences encoding the intact antibody or antibody portion axe free of other nucleotide sequences encoding whole antibodies or antibody portions that bind antigens other than CD20, which other sequences may naturally flank the nucleic acid in human genomic 10 DNA. In one embodiment, the human anti-CD20 antibody includes the nucleotide or amino acid sequence of 2F2, 7D8, or 11B8, as well as heavy chain (Vh) and light chain (Vl) variable regions having the sequences shown in SEQ ID NOs: 1,5, or 9, and SEQ ID NOs; 3, 7, or Π, respectively.

As disclosed and claimed herein, the sequences set forth in SEQ ID NOs: 15 1-30 include “conservative sequence modifications,” i.e., nucleotide and amino acid sequence modifications which do not significantly affect or alter the binding characteristics of the antibody encoded by the nucleotide sequence or containing the amino acid sequence. Such conservative sequence modifications include nucleotide and amino acid substitutions, additions and deletions. Modifications can be introduced into 20 SEQ ID NOs:l-30 by standard techniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains 25 (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g,, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, iso leucine) and aromatic side chains (e.g-., tyrosine, phenylalanine, 30 tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a human anti-CD20 antibody is preferably replaced with another amino acid residue from the same side chain family.

The present invention also encompasses “derivatives” of fire amino acid sequences as set forth in SEQ ID NOs: 1-30 and conservative sequence modifications 35 thereof, wherein one or more of the amino acid residues have been derivatised, e.g., by acylation or glycosylation, without significantly affecting or altering the binding characteristics of the antibody containing the amino acid sequences. -21- 2017204254 22 Jun2017

Furthermore, the present invention comprises antibodies in which alterations have been made in the Fc region in order to change the functional or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of Clq binding and CDC or of FcqR binding and ADCC. Substitutions can, 5 for example, be made in one or more of the amino acid residues at positions 234,235, 236,237,297, 318,320 and 322 of the heavy chain constant region, thereby causing an alteration in an effector function while retaining the ability to bind to the antigen as compared with the unmodified antibody, cf. US 5,624,821 and US 5,648,260.

The in vivo half-life of tire antibodies can also be improved by modifying 10 the salvage receptor epitope of the Ig constant domain or an Ig-like constant domain such that the molecule does not comprise·an intact CH2 domain or an intact Ig Fc region, cf. US 6,121,022 and US 6,194,551. The in vivo half-life can furthermore be increased by making mutations in the Fc region, e.g., by substituting threonine for leucine at position 252, by substituting threonine for serine at position 254, or by 15 substituting threonine for phenylalanine at position 256, cf. US 6,277,375.

Furthermore, the glycosylation pattern of the antibodies can be modified in order to change the effector function of the antibodies. For example, the antibodies can be expressed in a transfectoma which does not add the fucose unit normally attached to Asn at position 297 of the Fc region in order to enhance the affinity of the Fc region 20 for FcyRIII which, in turn, will result in an increased ADCC of the antibodies in the presence of NK cells, cf. Shield et al. (2002) JBC, 277:26733. Furthermore, modification of galactosylation can be made in order to modify CDC.

Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a anti-CD20 antibody coding sequence, such as by 25 saturation mutagenesis, and the resulting modified anti-CD20 antibodies can be screened for binding activity.

Accordingly, antibodies encoded by the (heavy and light chain variable region) nucleotide sequences disclosed herein and/or containing the (heavy and light chain variable region) amino acid sequences disclosed herein (i.e,, SEQ ID NOs: 1-30) 30 include substantially similar antibodies encoded by or containing similar sequences which have been conservatively modified. Further discussion as to how such substantially similar antibodies can be generated based on the partial (i.e., heavy and light chain variable regions) sequences disclosed herein as SEQ ID Nos: 1-30 is provided below. 35 For nucleic acids, the term "substantial homology" indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about -22- 2017204254 22 Jun2017 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

For nucleotide and amino acid sequences, the term "homology" indicates 5 the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate insertions or deletions. Alternatively, substantial homology exists when Hie DNA segments will hybridize under selective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of hie number 10 of identical positions shared by the sequences (z.a, % homology = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the 15 non-limiting examples below.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two 20 nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller {Comput. Appl. Biosci,, 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch 25 (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16,14,12,10, 8,6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention can 30 further be used as a "query sequence" to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J, Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to the nucleic 35 acid molecules of the invention. BLAST protein searches can he performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, -23- 2017204254 22 Jun2017 (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can he used. See http://www.ncbi.nhn.nili.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or in a 5 partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See, F. Ausubel, et at, ed. Current 10 Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures thereof, may be mutated in accordance with standard techniques to 15 provide gene sequences. For coding sequences, these mutations, may affect amino acid sequence as desired, ha particular, DNA sequences substantially homologous to or derived from native V, D, J,.constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or mo dified from another sequence). 20 A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription of regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding 25 regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting ano ther nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into 30 which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g,, bacterial vectors having a bacterial origin of replication, and epxsomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be 35 integrated into tire genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). -24- 2017204254 22 Jun2017

Bi general, expression vectors of utility in recombinant DNA techniques are often in the form, of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral 5 vectors (e.g., replication defective retroviruses, adenoviruses and adeno-asso dated viruses), which serve equivalent functions.

The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the 10 particular subject cell blit to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within tire scope of the term "host cell" as used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, NS/0 cells, and lymphocytic cells. 15 As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and nonmammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

The terms “transgenic, non-human animal” refers to a non-human animal 20 having a genome comprising one or more human heavy and/or light chain transgenes or transchromosomes (either integrated or non-integrated into the animal’s natural genomic DNA) and which is capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and either a human heavy chain transgene or human heavy chain transchromosome, such that the mouse produces 25 human anti-CD20 antibodies when immunized with CD20 antigen and/or cells expressing CD20. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAb mice, such as HCo7 or HCol2 mice, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (e.g., KM) mice as described 30 in WO 02/43478. Such transgenic and transchromosomal mice are capable of producing multiple isotypes of human monoclonal antibodies to CD20 (e.g., IgG, IgA and/or IgE) by undergoing V-D-J recombination and isotype switching.

Various aspects of the invention are described in further detail in the following subsections. 35 -25- 2017204254 22Jun2017 I. Production of Human Antibodies to CD20

Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 5 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody cm be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes.

The preferred animal system for preparing hybridomas that secrete 10 human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure, immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

In a preferred embodiment, human monoclonal antibodies directed 15 against-CD20 can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as “transgenic mice.”

The HuMAb mouse contains a human immunoglobulin gene miniloei that 20 encodes unrearranged human heavy (μ and y) and κ light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al. (1994) Nature 368 (6474): 856-859). Accordingly, the. mice exhibit reduced expression of mouse IgM or κ and in response to immunization, the introduced human heavy and light chain transgenes, undergo class switching and 25 somatic mutation to generate high affinity human IgG tc monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern.

Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci 764: 536-546). The preparation of HuMAb mice is described in detail in Taylor, L. 30 et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al (1993) International Immunology 5: 647-656; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg et al, (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13:65-93; Harding, 35 F. and Lonberg, N. (1995) Ann. N. Y. Acad. Sci 764:536-546; Fishwild, D. et al. (1996)

Nature Biotechnology 14:845-851. See further, US Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to Lonberg and Kay, as well as US 5,545,807 to Surard et al:, WO -26- 2017204254 22Jun2017 9S/24884, WO 94/25585, WO 93/1227, WO 92/22645, WO 92/03918 and WO 01/09187.

The KM mouse contains a human heavy chain transchromosome and a human kappa light chain transgene. The endogenous mouse heavy and light chain genes 5 also have been disrupted in the KM mice such that immunization of the mice leads to production of human immunoglobulins rather than mouse immunoglobulins.

Construction of KM mice and their use to raise human immunoglobulins is described in detail in WO 02/43478. 10 Immunizations

To generate folly human monoclonal antibodies to CD20, transgenic or transchromosoraal mice containing human immunoglobulin genes (e.g., HCol2, HCo7 or KM mice) can be immunized with an enriched preparation of CD20 antigen and/or cells expressing CD20, as described, for example, by Lonberg et al. (1994), supra; 15 Fishwild et al (1996), supra, and WO 98/24884. Alternatively, mice can be immunized with DNA encoding human CD20. Preferably, the mice will be 6-16 weeks of age upon foe first infusion. For example, an enriched preparation (5-50 pg) of the CD20 antigen can be used to immunize the HnMAb mice intrap eritoneally. In the event that immunizations using a purified or enriched preparation of the CD20 antigen do not 20 result in antibodies, mice can also be immunized with cells expressing CD20, e.g., a cell line, to promote immune responses.

Cumulative experience with various antigens has shown that the HuMAb •transgenic mice respond best when initially immunized intraperitoneally (EP) or subcutaneously (SC) with CD20 expressing cells in complete Freund's adjuvant, 25 followed by every other week IP immunizations (up to a total of 10) with CD20 expressing cells in PBS. The immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds. The plasma can be screened by FACS analysis (as described below), and mice with sufficient titers of anti-CD20 human immunoglobulin can be used for fusions. Mice can be 30 boosted intravenously with CD20 expressing cells 3 days before sacrifice and removal of the spleen.

Generation of Hyhridomas Producing Human Monoclonal Antibodies to CD20

To generate hyhridomas producing human monoclonal antibodies to 35 human CD20, splenocytes and lymph node cells from immunized mice can be isolated and fused to an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hyhridomas can then be screened for the production of antigen-specific antibodies. For example, single cell suspensions of splenic lymphocytes from -27- 2017204254 22 Jun2017 immunized mice can be fused to SP2/Q-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG (w/v). Cells can be plated at approximately 1 x 10s per well in flat bottom microtiter plate, followed by a two week incubation in selective medium containing besides usual reagents 10% fetal Clone Serum, 5-10% origen 5 hybridoma cloning factor (IGEN) and IX HAT (Sigma). After approximately two weeks, cells can be cultured in medium in which the HAT is replaced with HT.

Individual wells can then be screened by ELISA for human kappa-light chain containing antibodies and by FACS analysis using CD20 expressing cells for CD20 specificity.

Once extensive hybridoma growth occurs, medium can be observed usually after 10-14 10 days. The antibody secreting hybridomas can be replated, screened again, and if still positive for human IgG, anti-CD20 monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate antibody in tissue culture medium for characterization. 15 Generation of Transfectomas Producing Human Monoclonal Antibodies to CD20

Human antibodies of the invention also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S. (1985) Science 229:1202). 20 For example, in one embodiment, tire gene(s) of interest, e.g., human antibody genes, can be ligated into an expression vector such as a eukaryotic expression plasmid such as used by the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art. The purified plasmid with the cloned antibody genes can be introduced in eukaryotic host 25 cells such as CHO cells, NS/0 cells or HEK293 cells or alternatively other eukaryotic cells like a plant derived cells, fungi or yeast cells. The method used to introduce these genes could be methods described in the art such as electroporation, lipofectine, lipofectamine or other. After introducing these antibody genes in the host cells, cells expressing the antibody can be identified and selected. These cells represent the 30 transfectomas which can then be amplified for their expression level and upscaled to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and/or cells.

Further Recombinant Means for Producing Human Monoclonal Antibodies to CD20 35 Alternatively, the cloned antibody genes can be expressed in other expression systems, including prokaryotic cells, such as microorganisms, such as E. coli, for the production of single chain Fv antibodies, algi, as well as insect cells.

Furthermore, the antibodies can be produced in transgenic non-human animals, such as -28- 2017204254 22 Jun2017 in milk from sheep and rabbits or in eggs from, hens, or in transgenic plants. See e.g. Verma, R., et al (1998). Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems. J.ImmunolMeth. 216:165-181; Pollock, et al (1999). Transgenic milk as a method for the production of recombinant antibodies. 5 JJmm.unol.Metk. 231;147-157; and Fischer, R., et al. (1999). Molecular farming of recombinant antibodies in plants. Biol.Chem. 380:825-839.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through amino 10 acid residues that are located in the six heavy and light chain complementarity

determining regions (CDRs). For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific 15 naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033). Such framework 20 sequences can be obtained from public DNA databases that include germline antibody gene sequences. These geimline sequences will differ from mature antibody gene sequences because they will not include completely assembled variable genes, which are formed by V(D)J joining during B cell maturation. Germline gene sequences will also differ from the sequences of a high affinity secondary repertoire antibody at individual 25 evenly across the variable region. For example, somatic mutations are relatively infrequent in the amino tenninal portion of framework region 1 and in the carboxy-temiinal portion of framework region 4. Furthermore, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate 30 an intact recombinant antibody having binding properties similar to those of the original antibody (see WO 99/45962). Partial heavy and light chain sequence spanning the CDR regions is typically sufficient for this purpose. The partial sequence is used to detennine which gennline variable and joining gene segments contributed to the recombined antibody variable genes. The geimline sequence is then used to fill in missing portions 35 of the variable regions. Heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add missing sequences, cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, tire entire variable -29- 2017204254 22Jun2017 region can be synthesized as a set of short, overlapping, oligonucleotides and combined by PCR amplification to create an entirely synthetic variable region clone. This process has certain advantages such as elimination or inclusion or particular restriction sites, or optimization of particular codons. 5 The nucleotide sequences of heavy and light chain transcripts from hybridomas are used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with identical amino acid coding capacities as the natural sequences. The synthetic heavy and kappa chain sequences can differ from the natural sequences in three ways: strings of repeated nucleotide bases are interrupted to facilitate 10 oligonucleotide synthesis and PCR amplification; optimal translation initiation sites are incorporated according to Kozak’s rules (Kozak, 1991, J. Biol. Chem. 266:19867-19870); and Hindlll sites are engineered upstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimized coding and corresponding non-coding, strand sequences are broken down into 30 -15 50 nucleotides approximately at the midpoint of the con-esponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled into overlapping double stranded sets that span segments of 150 - 400 nucleotides. The pools are then used as templates to produce PCR amplification products of 150 -400 nucleotides. Typically, a single variable region oligonucleotide set will be broken 20 down into two pools which are separately amplified to generate two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant region (including the Bbsl site of the kappa light chain, or the Agel site if the gamma heavy chain) in the PCR amplification to 25 generate fragments that can easily be cloned into the expression vector constructs.

The reconstructed heavy and light chain variable regions are then combined with cloned promoter, leader sequence, translation initiation, constant region, 3 ’ untranslated, polyadenylation, and transcription termination, sequences to form expression vector constructs. The heavy and light chain expression constructs can be 30 combined into a single vector, co-transfected, serially transfected, or separately transfected into host cells which are then fused to form a host cell expressing both chains.

Plasmids for use in construction of expression vectors for human IgGic are described below. The plasmids were constructed so that PCR amplified V heavy and 35 V kappa light chain cDNA sequences could be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human, or chimeric IgGl,K or IgG4,K antibodies. Similar plasmids can be constructed for -30- expression of other heavy chain isotypes, or for expression of antibodies comprising lambda light chains. 2017204254 22Jun2017

Thus, in another aspect of the invention, the structural features of the human anti-CD20 antibodies of the invention, e.g., 11B8, 2F2, or 7D8, are used to 5 create structurally related human anti-CD20 antibodies that retain at least one functional property of the antibodies of the invention, such as binding to CD20. More specifically, one or more CDR regions of 2F2,7D8, or 11B8 can be combined recombinantly with known human framework regions and CDRs to create additional, recombinantly-engineered, human anti-CD20 antibodies of the invention. 10 Accordingly, in another embodiment, the invention provides a method for preparing an anti-CD20 antibody comprising: preparing an antibody comprising (1) human heavy chain Framework regions and human heavy chain CDRs, wherein at least one of the human heavy chain CDRs comprises an amino acid sequence selected from the amino acid sequences of 15 CDRs shown in Figures 53, 55, or 57 (or corresponding amino acid residues in SEQ ID NOs:13-15, 19-21, or 25-27); and (2) human light chain framework regions and human light chain CDRs, wherein at least one of the human light chain CDRs comprises an amino acid sequence selected from the amino acid sequences of CDRs shown in Figures 53, 55, or 57 (or corresponding amino acid residues in SEQ ED NOs: 16-18, 22-24, or 20 28-30); wherein the antibody retains the ability to bind to CD20.

The ability of the antibody to bind CD20 can be determined using standard binding assays, such as those set forth in the Examples (e.g., a FACS analysis).

Since it is well known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity/affinity of an 25 antibody for an antigen, the recombinant antibodies of the invention prepared as set forth above preferably comprise the heavy and light chain CDR3s of 2F2,7D8, or 1 IBS. The antibodies further can comprise the CDR2s of 2F2, 7D8, or 11B8. The antibodies further can comprise the CDRls of 2F2, 7D8, or 11B8. Accordingly, the invention further provides anti-CD20 antibodies comprising: (1) human heavy chain framework 30 regions, a human heavy chain CDR1 region, a human heavy chain CDR2 region, and a human heavy chain CDR3 region, wherein the human heavy chain CDR3 region is the CDRS of 2F2, 7D8, or 11B8 as shown in Figures 53, 55, or 57 (or corresponding amino acid residues as shown in SEQ ID NOs: 15, 21, or 27); and (2) human light chain framework regions, a human light chain CDR1 region, a human light chain CDR2 35 region, and a human light chain CDR3 region, wherein the human light chain CDR3 region is the CDR3 of 2F2,7DS, or 1 IBS as shown in Figures 53, 55, or 57 (or corresponding amino acid residues as shown in SEQ ID NOs: 18,24, or 30), wherein the antibody binds CD20. The antibody may further comprise the heavy chain CDR2 and/or -31- 2017204254 22Jun2017 the light chain CDR2 of 2F2,7D8, or 1 IBS. The antibody may further comprise the heavy chain CDR1 and/or the light chain CDR1 of 2F2, 7D8, or 11B8,

Preferably, the CDR1, 2, and/or 3 of the engineered antibodies described above comprise the exact amino acid sequence(s) as those of 2F2, 7D8, or 11B8 5 disclosed herein. However, the ordinarily skilled artisan will appreciate that some deviation from the exact CDR sequences of 2F2, 7D8, or 11B8 may be possible while still retaining the ability of the antibody to bind CD20 effectively (e.g., conservative substitutions). Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 90%, 95%, 98% or 99.5% 10 identical to one or more CDRs of 2F2,7D8, or 1 IBS.

In addition to simply binding CD20, engineered antibodies such as those described above may be selected for their retention of other functional properties of antibodies of the invention, such as: (I) low dissociation rate from CD20; 15 (2) high affinity binding to CD2G; (3) binding to a unique epitope on CD20, and/or binding in a specific orientation to CD20, and/or binding to a specific form of CD20; (4) mediation of a high level of CDC on either CD55/59 negative or CD55/59 positive cells; 20 (5) translocation into lipid rafts upon binding to CD20; (6) inhibition of the growth of cells which express CD20; (7) inducement of apoptosis of cells which express CD20; (8) inducement of homotypic adhesion of cells which express CD20; (9) prolonged survival of a subject having tumor cells which express 25 CD20; (10) mediation of ADCC of CD20 targets when mixed with appropriate effector cells; (II) ability to deplete cells which express CD20; and/or (12) ability to deplete cells which express low levels of CD20 (CD20low 30 cells).

Characterization of Binding of Human Monoclonal Antibodies to CD20

To purify human anti-CD20 antibodies, selected hybridomas can be grown in two-liter spinner-flasks for monoclonal antibody purification. Supernatants 35 can be filtered and concentrated before affinity chromatography with protein A- sepharose (for IgGl isotype antibodies) (Pharmacia, Piscataway, NJ) or anti-human IgG coated sepharose or protein G-sepharose in case of IgG3 isotype antibodies. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to - 32 - ensure purity. The buffer solution can be exchanged into PBS, and the concentration can be determined by OD280 using 1.43 extinction coefficient. The monoclonal antibodies can be afiquoted and stored at -80 °C. 2017204254 22 Jun2017

To determine if the selected human anti-CD20 monoclonal antibodies 5 bind to unique epitopes, site-directed or multi-site directed mutagenesis can be used.

To determine the isotype of purified antibodies, isotype ELISAs can be performed. Wells of microtiter plates can be coated with 10 μg/ml of anti-human Ig overnight at 4 °C. After blocking with 5% BSA, the plates are reacted with 10 pg/ml of monoclonal antibodies or purified isotype controls, at ambient temperature for two 10 hours. The wells can then be reacted with either human IgGl, IgG2, IgG3 or IgG4 or human IgM-specific alkaline phosphatase-conjugated probes. After washing, the plates are developed with pNPP substrate (1 mg/ml) and analyzed at OD of405-650.

In order to demonstrate presence of anti-CD20 antibodies in sera of immunized mice or binding of monoclonal antibodies to five cells expressing the CD20, 15 flow cytometry can be used. Briefly, cell lines expressing C.D20 (grown under standard growth conditions) are mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA and 0.02% sodium-azide, and incubated at 4 °C for 30 min. After washing, the cells are reacted with fluorescein-labeled anti-human IgG antibody under the same conditions as the primary antibody staining. The samples can be 20 analyzed by flow cytometry with a FACS instrument using fight and side scatter properties to gate on single, living cells. An alternative assay using fluorescence microscopy may be used (in addition to or instead of) the flow cytometry assay. Cells can be stained exactly as described above and examined by fluorescence microscopy. This method allows visualization of individual cells, but may have diminished 25 sensitivity depending on the density of the antigen.

Anti~CD20 human IgGs can be further tested for reactivity with CD20 antigen by Western blotting. Briefly, cell extracts from cells expressing CD20 can be prepared and subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens will fie transferred to 30 nitrocellulose membranes, blocked with 20% mouse serum, and probed with the monoclonal antibodies to be tested. Human IgG binding can be detected using anti-' human IgG alkaline phosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO). 35 Phagocytic and Cell Killing Activities of Human Monoclonal Antibodies to CD20

In addition to binding specifically to CD20, human monoclonal anti-CD20 antibodies can be tested for their ability to mediate phagocytosis and killing of cells expressing CD20. The testing of monoclonal antibody activity in vitro will provide -33- 2017204254 22Jun2017 10 15 20 25 30 an initial screening prior to testing in vivo models. Briefly, polymorphonuclear cells (PMNs), NK cells, monocytes or other effector cells, from healthy donors can be purified by Ficoll Hypaque density centrifugation, followed by lysis of contaminating erythrocytes. Washed PMNs, can be suspended in RPMI supplemented with 10% heat-inactivated fetal calf serum and mixed with 51Cr labeled cells expressing CD20, at various ratios of effector cells to tumor cells (-effector cells:tumor cells). Purified human anti-CD20 IgGs can then be added at various concentrations. Irrelevant human IgG can be used as negative control. Assays can be carried out for 4 to 20 hours at 37 °C depending on the effector cell type used. Samples can be assayed for cytolysis by measuring 51Cr release into the culture supernatant. Anti-CD20 monoclonal antibodies can also be tested in combinations with each other to determine whether cytolysis is enhanced with multiple monoclonal antibodies.

Human monoclonal antibodies which bind to CD20 also can be tested in an in vivo model (e.g., in mice) to determine their efficacy in controlling growth of CD20-expressing tumor cells. These antibodies can be selected, for example, based on the following criteria, which are not intended to be exclusive: 1. binding to live cells expressing CD20; 2. low dissociation rate from CD20; 3. high affinity of binding to CD20; 4. binding to a unique epitope on CD20; and/or binding in a specific orientation to CD20, and/or binding to a specific form of CD20; 5. opsonization of cells expressing CD20; 6. mediation of growth inhibition, phagocytosis and/or killing of cells expressing CD20 in the presence of human effector cells; 7. ability to induce CDC on either CD55/CD59 negative or positive cells; 8. ability to induce homotypic adhesion; 9. ability to induce translocation into lipid rafts upon binding to CD20; 10. ability to induce apoptosis; 11. ability to induce ADCC on cells expressing CD20; 12. ability to deplete cells which express CD20; and/or 13. ability to deplete cells which express low levels of CD20 (CD20low cells).

Preferred human monoclonal antibodies of the invention meet one or more of these criteria.

Human monoclonal anti-CD20 antibodies can be tested for their ability to mediate CDC using a variety of known techniques. For example, serum for complement can be obtained from the blood of healthy subjects which can be centrifuged and harvested. To determine the CDC activity of various mAbs, different methods can be used. 51 Cr release can for example be measured or elevated membrane penneability can -34- 2017204254 22Jun2017

be assessed using a propidium iodide (PI) exclusion assay. Briefly, target cells can be washed and resuspended in RPMI-1% BSA at 1 x 106/ml, Various concentrations of mAb can be added to the cells and allowed to bind for 10-15 min at room temperature. Serum can then be added to a final concentration of 20% (v/v) and the cells incubated at 5 37 °C for 45 min. All cells from each sample can be added to the PI solution in a FACS tube. The mixture can then be assessed immediately by flow cytometry using a FACScalibur flow cytometer and analysed using CellQuest pro software (BD Biosciences, Mountain view, CA).

To test for the ability to initiate apoptosis, human monoclonal anti~CD20 10 antibodies can, for example, be incubated with CD20 positive tumor cells, e.g., Daudi at 37 °C for about 20 hours. The cells can be harvested, washed in Annexin-V-FITC binding buffer (BD biosciences), and labeled with Annexin V-FITC (BD biosciences) for 15 min in the dark at 4 °C. All cells from each sample can he added to PI solution (10 iig/ml in PBS) in a FACS tube and assessed immediately by flow cytometry (as 15 above).

In a particular embodiment of the invention, the human monoclonal antibodies are used in combination, e.g., as a pharmaceutical composition comprising two or more anti-CD20 monoclonal antibodies. For example, human anti-CD20 monoclonal antibodies having different but complementary activities can be combined 20 in a single therapy to achieve a desired therapeutic or diagnostic effect. In a preferred embodiment, the composition includes an anti-CD20 human monoclonal antibody that mediates CDC combined with another human anti-CD20 monoclonal antibody that induces apoptosis. In another embodiment, the composition includes an anti-CD20 human monoclonal antibody that mediates highly effective killing of target cells in the 25 presence of effector cells, combined with another human anti-CD20 monoclonal antibody that inhibits the growth of cells expressing CD20. H. Production of Transgenic Non-human Animals Which Generate Human Monoclonal Anti-CD20 Antibodies 30 In yet another aspect, the invention provides transgenic and transchromosomal non-human animals, such as transgenic or transcbromosomal mice, which are capable of expressing human antibodies that specifically bind to CD20. In a particular embodiment, the invention provides a transgenic or transchromosomal mouse having a genome comprising a human heavy chain transgene, such that the mouse 35 produces human anti- CD20 antibodies when immunized with cells expressing CD20. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAb mice, as described in detail herein and exemplified. Alternatively, the human heavy chain transgene can be maintained -35- 2017204254 22 Jun2017 extracbromosomally, as is the case for transchromosomal {e.g., KM) mice as described in WO 02/43478. Such transgenic and transchromosomal animals are capable of producing multiple isotypes of human monoclonal antibodies to CD20 {e.g., IgG, IgA and/or IgE) by undergoing V-D-J/V-J recombination and isotype switching. The design 5 of a transgenic or transchromosomal non-human animal that responds to foreign antigen stimulation with a heterologous antibody repertoire, requires that the heterologous immunoglobulin transgenes contained within the transgenic animal function correctly throughout the pathway of B cell development. This includes, for example, isotype switching of the heterologous heavy chain transgene. Accordingly, transgenes are 10 constructed so as that isotype switching can be induced and one or more of the following characteristics of antibody genes: (1) high level and cell-type specific expression, (2) functional gene rearrangement, (3) activation of and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signal transduction, (6) somatic hypennutation, and (7) domination of the transgene antibody locus during the immune 15 response.

Not all of the foregoing criteria need be met. For example, in those embodiments wherein the endogenous immunoglobulin loci of the transgenic animal are functionally disrupted, the tons gene need not activate allelic exclusion. Further, in those embodiments wherein the tonsgene comprises a functionally rearranged heavy 20 and/or light chain immunoglobulin gene, the second criteria of functional gene rearrangement is unnecessary, at least for that transgene which is already rearranged.

For background on molecular· immunology, see, Fundamental Immunology, 2nd edition (1989), Paul William E., ed. Raven Press, N.Y.

In certain embodiments, the transgenic or transchromosomal non-human 25 animals used to generate the human monoclonal antibodies of the invention contain rearranged, unreairanged or a combination of rearranged and unrearranged heterologous immunoglobulin heavy and light chain tons genes in the germline of the transgenic animal. Each of the heavy chain transgenes comprises at least one Ch gene. In addition, the heavy chain transgene may contain functional isotype switch sequences, which are 30 capable of supporting isotype switching of a heterologous transgene encoding multiple

Ch genes in the B cells of the transgenic animal. Such switch sequences may be those which occur naturally in the germline immunoglobulin locus from the species that serves as the source of the transgene Ch genes, or such switch sequences may be derived from those which occur in the species that is to receive the transgene construct (the transgenic 35 animal). For example, a human transgene construct that is used to produce a transgenic mouse may produce a higher frequency of isotype switching events if it incorporates switch sequences similar to those that occur naturally in the mouse heavy chain locus, as presumably the mouse switch sequences are optimized to function with the mouse -36- 2017204254 22 Jun2017 switch recombinase enzyme system, whereas tire human switch sequences are not.

Switch sequences may be isolated and cloned by conventional cloning methods, or may be synthesized tie novo from overlapping synthetic oligonucleotides designed on the basis of published sequence information relating to immunoglobulin switch region 5 sequences (Mills et al., Nucl. Acids Res. 15:7305-7316 (1991); Sideras etal., Inti.

Immunol 1:631-642 (1989)). For each of the foregoing transgenic animals, functionally rearranged heterologous heavy and light chain immunoglobulin transgenes axe found in a significant fraction of the B cells of tire transgenic animal (at least 10 percent).

The transgenes used to generate the transgenic non-human animals of the 10 invention include a heavy chain transgene comprising DNA encoding at least one variable gene segment, one diversity gene segment, one joining gene segment and at least one constant region gene segment. The immunoglobulin light chain transgene comprises DNA encoding at least one variable gene segment, one joining gene segment and at least one constant region gene segment. The gene segments encoding the light 15 and heavy chain gene segments are heterologous to the transgenic animal in that they are derived from, or correspond to, DNA encoding immunoglobulin heavy and light chain gene segments from a species not consisting of the transgenic non-human animal. In one aspect of the invention, the transgene is constructed such that the individual gene segments are unreanranged, i.e,, not rearranged so as to encode a functional 20 immunoglobulin light or heavy chain. Such unrearranged transgenes support recombination of the V, D, and J gene segments (functional rearrangement) and preferably support incorporation of all or a portion of a D region gene segment in the resultant rearranged immunoglobulin heavy chain within the transgenic animal when exposed to CD20 antigen. 25 In an alternate embodiment, the transgenes comprise an unreatranged "mini-locus”. Such transgenes typically comprise a substantial portion of the C, D, and J segments as well as a subset of the V gene segments. In such transgene constructs, the various regulatory sequences, e.g. promoters, enhancers, class switch regions, splice-donor and splice-acceptor sequences for KNA processing, recombination signals and the 30 like, comprise corresponding sequences derived from the heterologous DNA. Such regulatory sequences may be incorporated into the transgene from the same or a related species of the non-human animal used in the invention. For example, human immunoglobulin gene segments may be combined in a transgene with a rodent immunoglobulin enhancer sequence for use in a transgenic mouse. Alternatively, 35 synthetic regulatory sequences may be incorporated into the transgene, wherein such synthetic regulatory sequences are not homologous to a functional DNA sequence that is known to occur naturally in the genomes of mammals. Synthetic regulatory sequences are designed according to consensus rules, such as, for example, those specifying the -37- 2017204254 22Jun2017 permissible sequences of a splice-acceptor site or a promoter/enhancer motif. For example, a mimlocus comprises a portion of the genomic immunoglobulin locus having at least one internal (z.e., not at a terminus of the portion) deletion of a non-essential DNA portion {e.g., intervening sequence] intron or portion thereof) as compared to the 5 naturally-occurring germline Ig locus.

Preferred transgenic and transchromosomal non-human animals, e.g., mice, will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a human after adjusting for volume.

The repertoire will ideally approximate that shown in a human when 10 adjusted for volume, usually with a diversity at least about 10% as great, preferably 25 to 50% or more. Generally, at least about a thousand different immunoglobulins (ideally IgG), preferably 104 to 106 or more, will be produced, depending on the number of different V, J and D regions introduced into the mouse genome and driven by the additional diversity generated by V(-D-)J gene segment rearrangements random 15 nucleotide additions at the joining regions. Typically, the immunoglobulins will exhibit an affinity (Kd) for preselected antigens of below 10'7 M, such as of below 10'8 Μ, 10'9 M or 10‘10 M or even lower.

Transgenic and transchromosomal non-human animals, e.g., mice, as described above can be immunized with, for example, cells expressing CD20. 20 Alternatively, the transgenic animals can be immunized with DNA encoding human CD20. The animals will then produce B cells winch undergo class-switching via switch recombination (cis-switching) and express immunoglobulins reactive with CD20. The immunoglobulins can be human antibodies (also referred to as “human sequence antibodies”), wherein the heavy and light chain polypeptides are encoded by human 25 transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences; these human antibodies can be referred to as being substantially identical to a polypeptide sequence encoded by a human Vf and Jl or VH, DH and JH gene segments, even though other nongermline sequences may be present as a result of somatic mutation and differential V-J 30 and V-D-J recombination joints. The variable regions of each antibody chain are typically at least 80 percent similar to human germline V, J, and, in the case of heavy chains, D, gene segments; frequently at least 85 percent similar to human geimline sequences present on the transgene; often 90 or 95 percent or more similar to human germline sequences present on the transgene. However, since non-gennline sequences 35 are introduced by somatic mutation and VJ and VDJ joining, the human sequence antibodies will frequently have some variable region sequences which are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germiine of the mice. Typically, such non-germline sequences (or individual nucleotide -38- 2017204254 22Jun2017 positions) will cluster in or near CDRs, or in regions where somatic mutations are known to cluster.

Another aspect of the invention includes B cells derived from transgenic or transchromosomal non-human animals as described herein. The B cells can be used 5 to generate hybridomas expressing human monoclonal antibodies which bind with high affinity (e.g., a dissociation equilibrium constant (1¾) of lower than 10"' M) to human CD20. Thus, in another embodiment, the invention provides a hybridoma which produces a human antibody having an affinity (1¾) of below 1 O'7 M, such as of below 10"8 Μ, 10‘9 M or 1 O'10 M or even lower when determined by scatchard analysis of CD20 10 expressing cells using a radio-actively labeled monoclonal antibody or by determination of the half-maximal binding concentration using FACS analysis.

Herein the monoclonal antibody comprises a human sequence light chain composed of (1) a light chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VL gene segment 15 and a human Jt segment, and (2) a light chain constant region encoded by a human Cl gene segment; and a human sequence heavy chain composed of a (1) a heavy chain variable region having a polypeptide sequence which is substantially identical to a polypeptide sequence encoded by a human VH gene segment, a D region, and a human Jh segment, 20 and (2) a constant region encoded by a human Ch gene segment.

The development of high affinity human monoclonal antibodies against CD20 can be facilitated by a method for expanding the repertoire of human variable region gene segments in a transgenic non-human animal having a genome comprising ail integrated human immunoglobulin transgene, said method comprising introducing into 25 the genome a V gene transgene comprising V region gene segments which are not present in said integrated human immunoglobulin transgene. Often, the V region transgene is a yeast artificial chromosome comprising a portion of a human Vh or Vl (Vk) gene segment array, as may naturally occur in a human genome or as may be spliced together separately by recombinant methods, which may include out-of-order or 30 omitted V gene segments. Often at least five or more functional V gene segments are contained on the YAC. In this variation, it is possible to make a transgenic animal produced by the V repertoire expansion method, wherein the animal expresses an immunoglobulin chain comprising a variable region sequence encoded by a V region gene segment present on the V region transgene and a C region encoded on the human 35 Ig transgene. By means of the V repertoire expansion method, transgenic animals having at least 5 distinct V genes can be generated; as can animals containing at least about 24 V genes or more. Some V gene segments may be non-functional (e.g., -39- 2017204254 22Jun2017 pseudogenes and the like); these segments may be retained or may be selectively deleted by recombinant methods available to the skilled artisan, if desired.

Once the mouse germline has been engineered to contain a functional YAC having an expanded V segment repertoire, substantially not present in the human 5 Ig transgene containing the J and C gene segments, fee trait can be propagated and bred into other genetic backgrounds, including backgrounds where the functional YAC having an expanded V segment repertoire is bred into a non-human animal germline having a different human Ig transgene. Multiple functional YACs having an expanded V segment repertoire may be bred into a germline to work with a human Ig transgene (or 10 multiple human Ig transgenes). Although referred to herein as YAC transgenes, such transgenes when integrated into the genome may substantially lack yeast sequences, such as sequences required for autonomous replication in yeast; such sequences may optionally be removed by genetic engineering (e.g., restriction digestion and pulsed-field gel electrophoresis or other suitable method) after replication in yeast is no longer 15 necessary (/, e., prior to introduction into a mouse ES cell or mouse prozygote). Methods of propagating the trait of human sequence immunoglobulin expression, include breeding a transgenic animal having the human Ig transgene(s), and optionally also having a functional Y AC having an expanded V segment repertoire. Both Vh and Vl gene segments may be present on the YAC. The transgenic animal may be bred into any 20 background desired by fee practitioner, including backgrounds harboring other human transgenes, including human Ig transgenes and/or transgenes encoding other human lymphocyte proteins. The invention also provides a high affinity human sequence immunoglobulin produced by a transgenic mouse having an expanded V region repertoire YAC transgene. Although the foregoing describes a preferred embodiment of 25 fee transgenic animal of the invention, other embodiments axe contemplated which have been classified in three categories: I. Transgenic animals containing an unrearranged heavy and rearranged light chain immunoglobulin transgene; H. Transgenic animals containing an unrearranged heavy and 30 unrearranged light chain immunoglobulin transgene; and 1Π. Transgenic animal containing rearranged heavy and an unrearranged light chain immunoglobulin transgene.

Of these categories of transgenic animal, the preferred order of preference is as follows II > I > III where the endogenous light chain genes (or at least the K gene) 35 have been knocked out by homologous recombination (or other method) and I > II > ΙΠ where the endogenous light chain genes have not been knocked out and must be dominated by allelic exclusion. -40- 2017204254 22 Jun2017

ΙΠ. Bispecific/ Multispeeific Molecules Which Bind to CD2Q

In yet another embodiment of the invention, human monoclonal antibodies to CD20 can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., air Fab’ fragment) to generate a bispeeific or 5 multispecific molecule which binds to multiple binding sites or target epitopes. For example, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, peptide or binding mimetic.

Accordingly, the present invention includes bispecific and multispecific 10 molecules comprising at least one first binding specificity for CD20 and a second binding specificity for a second target epitope, hr a particular embodiment of the invention, the second target epitope is anFc receptor, e.g., human FcyKJ (CD64) or a human Fca receptor (CD89), or a T cell receptor, e.g., CDS. Therefore, the invention includes bispecific and multispecific molecules capable of binding both to FcyR, Fca'R 15 or FcsR expressing effector cells (e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and to target cells expressing CD20. These bispecific and multispecific molecules target CD20 expressing cells to effector cell and, like the human monoclonal antibodies of the invention, trigger Fc receptor-mediated effector cell activities, such as phagocytosis of a CD2Q expressing cells, antibody dependent cellular cytotoxicity 20 (ADCC), cytokine release, or generation of superoxide anion.

Bispecific and multispeeific molecules of the invention can further - · include a third binding specificity, in addition to an anti-Fc binding specificity and an anti-CD2Q binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, e.g., a molecule which binds to a surface protein 25 involved in cytotoxic activity and thereby increases the immune response against the target cell. The "anti-enhancement factor portion" can be an antibody, fractional antibody fragment or a ligand that binds to a given molecule, e.g., an antigen or a receptor, and thereby results in an enhancement of the effect of the binding determinants for the Fc receptor or target cell antigen. The "anti-enhancement factor portion" can 30 bind an Fc receptor or a target cell antigen. Alternatively, the anti-enhancement factor portion can bind to an entity that is different from the entity to which the first and second binding specificities bind. For example, the anti-enhancement factor portion can bind a cytotoxic T cell (e.g., via CD2, CD3, CDS, CD28, CD4, CD40, ICAM-1 or other immune cell that results in an increased immune response against the target cell). 35 In one embodiment, the bispecific and multispecific molecules of the invention comprise as a binding specificity at least one antibody, including, e.g., an Fab, Fab’, F(ab')2, Fv, or a single chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof such as a Fv or a single chain construct as -41- 2017204254 22Jun2017 described in Ladner et al. US 4,946,778, The antibody may also be a binding-domain immunoglobulin fusion protein as disclosed in US 2003/0118592 and US 2003/0133939.

In one embodiment bispecific and multispecific molecules of the invention comprise a binding specificity for an FcyR or an FcaR present on the surface 5 of an effector cell, and a second binding specificity for a target cell antigen, e.g., CD20.

In one embodiment, the binding specificity for an Fc receptor is provided by a human monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG), As used herein, the term "IgG receptor" refers to any of the eight γ-chain genes located on chromosome 1. These genes encode a total of twelve 10 transmembrane or soluble receptor isoforms which are grouped into three Fey receptor classes: FcyRI (CD64), FcyRH(CD32), and FcyRIII (CD 16). In one preferred embodiment, the Fey receptor is a human high affinity FcyRI.

The production and characterization of these preferred monoclonal antibodies are described by Fanger et al. in WO 88/00052 and in US 4,954,617, These 15 antibodies bind to an epitope of FcyRI, FcyRII or FcyRIII at a site which is distinct from the Fey binding site of the receptor and, thus, their binding is not blocked substantially by physiological levels of IgG. Specific anti-FcyRI antibodies useful in this invention are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. In other embodiments, the anti-Fey receptor antibody is a humanized form of monoclonal antibody 22 (H22). The 20 production and characterization of the H22 antibody is described in Graziano, R.F. et al. (1995) J. Immunol 155 (10): 4996-5002 and WO 94/10332. The H22 antibody producing cell line was deposited at the American Type Culture Collection on November 4, 1992 under the designation HA022CL1 and has the accession No. CRL 11177. 25 In still other preferred embodiments, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., an Fc-alpha receptor (FeaRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of one α-gene (FeaRI) located on chromosome 19. This gene is known to 30 encode several alternatively spliced transmembrane isoforms of 55 to 110 IcDa. FeaRI (CD89) is constitutively expressed on monocytes/macrophages, eosinophilic and neutrophilic granulocytes, but not on non-effector cell populations. FeaRI has medium affinity for both IgAl and IgA2, which is increased upon exposure to cytokines such as G-CSF or GM-CSF (Morton, H.C. et al. (1996) Critical Reviews in Immunology 35 16:423-440). Four FcaRI-specific monoclonal antibodies, identified as A3, A59, A62 and A77, which bind FeaRI outside the IgA ligand binding domain, have been described (Monteiro, R.C. et al. (1992)/ Immunol. 148:1764). -42-

FcaRI and FcyRI are preferred trigger receptors for use in the invention because they are (1) expressed primarily on immune effector cells, e.g,, monocytes, PMNs, macrophages and dendritic cells; (2) expressed at high levels (e.g., 5,000- 100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC, phagocytosis); (4) 2017204254 22Jun2017 5 mediate enhanced antigen presentation of antigens, including self-antigens, targeted to them.

In another embodiment the bispecific molecule is comprised by two human monoclonal antibodies according to the invention which have complementary functional activities, such as one antibody predominately working by inducing CDC and 10 the other antibody predominately working by inducing apoptosis, e.g., 2F2 in combination with 1 IBS.

In other embodiments, bispecific and multispecific molecules of the invention further comprise a binding specificity which recognizes, e.g., binds to, a target cell antigen, e.g., CD20. hi a preferred embodiment, the binding specificity is provided 15 by a human monoclonal antib ody of the present invention.

An "effector cell specific antibody" as used herein refers to an antibody or functional antibody fragment that binds the Fc receptor of effector cells. Preferred antibodies for use in the subject invention bind the Fc receptor of effector cells at a site which is not bound by endogenous immuno globulin, 20 As used herein, the term "effector cell" refers to an immune cell which is involved in the effector phase of an immune response, as opposed to the cognitive and activation phases of an immune response. Exemplary immune cells include a cell of a myeloid or lymphoid origin, e.g., lymphocytes (e.g., B cells and T cells including cytolytic T cells (C'TLs)), killer cells, natural killer cells, macrophages, monocytes, 25 eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and carry out specific immune functions. In preferred embodiments, an effector cell is capable of inducing antibody-dependent cellular cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. For example, monocytes, macrophages, which express FcR are involved in 30 specific killing of target cells and presenting antigens to other components of the immune system, or binding to cells that present antigens. In other embodiments, an effector cell can phagocytose a target antigen, target cell, or microorganism. The expression of a particular FcR on an effector cell can be regulated by humoral factors such as cytokines. For example, expression of FcyRl has been found to be up-regulated 35 by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of FcyRl-bearing cells against targets. An effector cell can phagocytose or lyse a target antigen or a target cell. -43- "Target cell" shall mean any undesirable cell in a subject (e.g,, a human or animal) that can be targeted by a composition (e.g., a human monoclonal antibody, a bispecific or a multispecific molecule) of the invention. In preferred embodiments, the target cell is a cell expressing or overexpressing CD20. Cells expressing CD20 typically 5 include B cells and B cell tumors. 2017204254 22Jun2017

While human monoclonal antibodies are preferred, other antibodies which can be employed in the bispecific or multispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies. Such murine, chimeric and humanized monoclonal antibodies can be prepared by methods known in the art. 10 Bispecific and multispecific molecules of the present invention can be made using chemical techniques (see e.g., D. M. Kxanz etal. (1981) Proc. Natl. Acad.

Sci. USA 78:5807), "polydoma" techniques (See US .4,474,893, to Reading), or recombinant D'NA techniques.·

In particular, bispecific and multispecific molecules of tire present 15 invention can be prepared by conjugating the constituent binding specificities, e.g., the anti-FcR and anti-CD20 binding specificities, using methods known in die art and described in the examples provided herein. For example, each binding specificity of the bispecific and muitispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of 20- coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S -acetyl-thioacetate (SATA), 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-ma!eimidomethyl)cyclohexane-l-carboxylate (sulfo-SMCC) 25 (see e.g., Karpovslcy et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78,118-132); Brennan et al. {Science (1985) 229:81-83), and Glennie el al. (,/ Immunol (1987) 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL). 30 When the binding specificities are antibodies, they can be conjugated via sulfhydryi bonding of the C-terminus hinge regions of tire two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryi residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the same 35 vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific and multispecific molecule is a nxAb x mAb, mAb x Fab,

Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific and multispecific molecule of the invention, e.g., a bispecific molecule can be a single chain molecule, such as a single -44- 2017204254 22 Jun2017 chain bispecific antibody, a single chain bispecific molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific and multispecific molecules can also be single chain molecules or may comprise at least two single chain molecules. Methods for 5 preparing bi- and multispecific molecules are described for example in US 5,260,203; US 5,455,030; US 4,881,175; US 5,132,405; US 5,091,513; US 5,476,786;US 5,013,653; US 5,258,498; and US 5,482,858.

Binding of the bispecific and multispecific molecules to their specific targets can be confirmed by enzyme-linked immunosorbent assay (ELISA), a 10 radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), or a Western Blot Assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g·., an antibody) specific fox the complex of interest. For example, the FcR-antibody complexes can be detected using e.g., an enzyme-linked antibody or antibody fragment 15 which recognizes and specifically binds to the antibody-FcR complexes. Alternatively, . the complexes can be detected using any of a variety of other immunoassays. For example, the antibody can be radioactively labeled and used in a radioimmuno assay (RIA) (see, for example, Weintraub, B., .Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 20 1986). The radioactive isotope can be detected by such means as the use of a γ counter or a scintillation counter or by autoradiography. IV. Iromunoconiugates

In another aspect, the present invention features a human anti-CD20 25 monoclonal antibody conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radioisotope. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates which include one or more cytotoxins are referred to as “immunotoxins.” A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin 30 D, etliidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. 35 Suitable therapeutic agents for forming immunoconjugates of the invention include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, caimustine -45- 2017204254 22 Jun2017 (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorabicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and antbramycin 5 (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent. In another embodiment, the therapeutic agent is an immunosuppressant. In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, 10 chlorambucil, cyclophosphamide or riein A.

Antibodies of the present invention also can be conjugated to a radioisotope, e.g., iodine-131, yttrium-90 or indium-111, to generate cytotoxic radiopharmaceuticals for treating a CD20-related disorder, such as a cancer. Tire antibody conjugates of the invention can be used to modify a given biological 15 response, and tire drug moiety is not to he construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or 20 interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (’’IL-2”), interleuldn-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies are 25 well known, see, e.g., Arnon ei al, "Monoclonal Antibodies For hnmunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom ei al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In 30 Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And 35 Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-5 8 (1982). -46- 2017204254 22Jun2017

In a further embodiment, the human monoclonal antibodies according to the invention are attached to a linker-chelator, e.g., tiuxetan, which allows for the antibody to be conjugated to a radioisotope. 5 V. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g-, a pharmaceutical composition, containing one or a combination of human monoclonal antibodies of the present invention. The pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other 10 known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington; The Science and Practice of Pharmacy, 19!n Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. In one embodiment, the compositions include a combination of multiple (e.g., two or more) isolated human antibodies of the invention which act by different mechanisms, e.g., one antibody 15 which predominately acts by inducing CDC in combination with another antibody which predominately acts by inducing apoptosis.

Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e., combined with other agents. For example, tire combination therapy can include a composition of the present invention with at least 20 one anti-inflammatory agent or at least one immunosuppressive agent. In one embodiment such therapeutic agents include one or more anti-inflammatory agents, such as a steroidal drug or aNSAID (nonsteroidal anti-inflammatory drug). Preferred agents include, for example, aspirin and other salicylates, Cox-2 inhibitors, such as rofecoxib (Vioxx) and celecoxib (Celebrex), NSAIDs such as ibuprofen (Motrin, 25 Advil), fenoprofen (Nalfon), naproxen (Naprosyn), sulindac (Clinoril), diclofenac (Voltaren), piroxicam (Feldene), ketoprofen (Orudis), diflunisal (Dolobid), nabumetone (Relafen), etodolac (Lodine), oxapi-ozin (Daypro), and indomethacin (Indocin).

In another embodiment, such therapeutic agents include one or more 30 DMARDs, such as methotrexate (Rheumatrex), hydroxychloroquine (Plaquenil), sulfasalazine (Asulfidine), pyrimidine synthesis inhibitors, e.g., leflunomide (Axava), IL-1 receptor blocking agents, e.g., analdnra (Kineret), and TNF-α blocking agents, e.g., etanercept (Enbrel), infliximab (Remicade) and adalimumab.

In another embodiment, such therapeutic agents include one or more 35 immunosuppressive agents, such as cyclosporine (Sandimmune, Neoral) and azathioprine (hnural). -47- 2017204254 22Jun2017

In yet another embodiment, such therapeutic agents include one or more chemotherapeutics, such as doxorubicin (Adriamycin), cisplatin (Platinol), bleomycin (Blenoxane), camiustine (Gliadel), cyclophosphamide (Cytoxan, Procytox, Neosar), and chlorambucil (Leukeran), 5 In another embodiment, human antibodies of the present invention may be administered in combination with chlorambucil and prednisolone; cyclophosphamide and prednisolone; cyclophosphamide, vincristine, and prednisone; cyclophosphamide, vincristine, doxorubicin, and prednisone; fludarabine and anthracycline; or in combination with other common multi-drugs regimens for NHL, such as disclosed, e.g., 10 in Non-Hodgkin’s Lymphomas; Making sense of Diagnosis, Treatment, and Options, Lorraine Johnston, 1999, O’Reilly and Associates, Inc.

In yet another embodiment, the human antibodies maybe administered in conjunction with radiotherapy and/or autologous peripheral stem cell or bone marrow transplantation. 15 In still another embodiment, the human antibodies may be administered in combination with one or more antibodies selected from anti~CD25 antibodies, anti-CD^ antibodies, anti-CD21 antibodies, anti-CD22 antibodies, anti-CD37 antibodies, anti-CD38 antibodies, anti-IL6R antibodies, anti-IL8 antibodies, anii-IL15 antibodies, anti-IL15R antibodies, anti-CD4 antibodies, anli-CDlla antibodies {e.g., efalizumab), 20 anti-alpka-4/beta-1 integrin (VLA4) antibodies (e.g., natalizumab), and CTLA4-Ig.

In. a particular embodiment, the human monoclonal antibodies are administered in combination with an anti-CD25 antibody for the treatment of bullous pemphigoid, e.g., in patients with graft-versus-host disease. fix another particular embodiment, the human monoclonal antibodies are 25 administered in combination with one or more antibodies selected from anti-CD 19 antibodies, anti-CD21 antibodies, anti-CD22 antibodies, anti-CD3 7 antibodies, and anti-CD38 antibodies for the treatment of malignant diseases.

In still another particular embodiment, the human antibodies are administered in combination with one or more antibodies selected from anti-EL6R 30 antibodies, anti-IL8 antibodies, anti-IL15 antibodies, anti~IL15R antibodies, anti-CD4 antibodies, anti-CDlla antibodies (e.g., efalizumab), anti-alpha-4/beta-l integrin (VLA4) antibodies (e.g natalizumab), and CTLA4-Ig for the treatment of inflammatory diseases.

In yet a further embodiment, Ore human antibodies may be administered 35 in combination with an anti-C3b(i) antibody in order to enhance complement activation.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, -48- tiie carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bispecific and multispecific molecule, may be coated in a material to protect the compound from the action of acids 5 and other natural conditions that may inactivate the compound. 2017204254 22 Jun2017 A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S.M., el al. (1977) J, Phami. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid 10 addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromie, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include 15 those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, and tire like, as well as from nontoxic organic amines, such as N,N‘-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like. A composition of the present invention can be administered by a variety 20 of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible 25 polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J,R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. 30 To administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer 35 solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27). -49- 2017204254 22 Jun2017

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is 5 incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a 10 solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the 15 maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, 20 monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile 25 vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. 30 Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for 35 ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical -50- earner. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals, 2017204254 22 Jun2017 5 Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisoie (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) 10 metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be 15 presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient winch can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will 20 generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, preferably from about 0.1 per cent to about 70 per cent, most preferably from about 1 per cent to about 30 per cent.

Formulations of the present invention which are suitable for vaginal 25 administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to he appropriate. Dosage forms for the topical or transdemial administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a 30 pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, 35 intramuscular, intraarterial, intrathecal, intracapsular, infraorbital, infracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. -51- 2017204254 22Jun2017

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic 5 esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of 10 micro organisms may b e ensured both by sterilization proc edures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions, hi addition, prolonged absorption of the injectable pharmaceutical form maybe brought 15 about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In one embodiment the human monoclonal antibodies of the invention are administered in crystalline form by subcutaneous injection, cf. Yang et al. (2003) PNAS, 100(12):6934-6939 20 When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (more preferably, 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. 25 Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill hi the art. 30 Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic 35 factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in -52- 2017204254 22 Jun2017 combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily 5 determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved, hi general, a suitable daily 10 dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of a 15 therapeutic composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition). 20 In one embodiment, the human monoclonal antibodies according to the invention may be administered by infusion in a weekly dosage of 10 to 500 mg/m2, such as 200 to 400 mg/m2. Such administration may be repeated, e.g., 1 to 8 times, such as 3 to 5 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours, 25 In another embodiment, the human monoclonal antibodies are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to reduce toxic side effects.

In still another embodiment tire human monoclonal antibodies are administered in a weekly dosage of from 250 mg to 2000 mg, such as for example 300 30 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4 to 6 times. The administration may be performed by continuous infusion over a period of from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen may be repeated one or more times as necessary, for example, after 6 months or 12 months. The dosage can be determined or adjusted by measuring the amount of circulating monoclonal 35 anti-CD20 antibodies upon administration in a biological sample by using anti-idiotypic antibodies which target the anti-CD20 antibodies. -53- 2017204254 22Jun2017

In yet another embodiment, the human monoclonal antibodies are administered by maintenance therapy, such as, e.g., once a week for a period of 6 months or more.

In still another embodiment, the human monoclonal antibodies 5 according to the invention may be administered by a regimen including one infusion of a human monoclonal antibody against CD20 followed by an infusion of a human monoclonal antibody against CD20 conjugated to a radioisotope. The regimen may be repeated, e.g., 7 to 9 days later.

Therapeutic compositions can be administered with medical devices 10 known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as tire devices disclosed in US 5,399,163; US 5,383,851; US 5,312,335; US 5,064,413; US 4,941,880; US 4,790,824; or US 4,596,556. Examples of well-known implants and modules useful in the present invention include: US 4,487,603, which 15 discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering medicants through the skin; US 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; 20 US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art. hi certain embodiments, the human monoclonal antibodies of the 25 invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., US 4,522,811; US 5,374,548; and US 5,399,331, The liposomes may comprise 30 one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery {see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., US 5,416,016 to Low et al.); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Cononun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBSLett. 357:140; M. Owais et al. 35 (1995) Antimicroh. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe etal. (1995) Am. J. Physiol. 1233:134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; pl20 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. -54- 2017204254 22Jun2017

Laukkanen (1994) FEES Lett. 346:123; J.J. Killion; IJ. Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic 5 compounds in the liposomes are delivered by bolus injection to a site proximal to the desired area, e.g., the site of inflammation or infection, or the site of a tumor. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. 10 In a further embodiment, human monoclonal antibodies of the invention can be formulated to prevent or reduce their transport across the placenta. This can be done by methods known in the art, e.g., by PEGylation of the antibodies or by use of F(ab)2' fragments. Further references can he made to "Cimningham-Rimdles C, Zhuo Z, Griffith B, Keenan J. (1992) Biological activities of polyethylene-glycol 15 immunoglobulin conjugates. Resistance to enzymatic degradation. J Immunol Methods. 152:177-190; and to "Landor M. (1995) Maternal-fetal transfer of immunoglobulins,

Ann Allergy Asthma Immunol 74:279-283, This is particularly relevant when the antibodies are used for treating or preventing recurrent spontaneous abortion. A "therapeutically effective dosage" for tumor therapy can be measured 20 by objective tumor responses which can either be complete or partial. A complete response (CR) is defined as no clinical, radiological or other evidence of disease. A partial response (PR) results from a reduction in aggregate tumor size of greater than 50%. Median time to progression is a measure that characterizes the durability of the objective tumor response. 25 A "therapeutically effective dosage" for tumor therapy can. also be measured by its ability to stabilize the progression of disease. The ability of a compound to inhibit cancer can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth or apoptosis 30 by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the ait would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. 35 A "therapeutically effective dosage" for rheumatoid arthritis preferably will result in an ACR20 Preliminary .Definition of Improvement in the patients, more preferred in an ACR50 Preliminary Definition of Improvement and even more preferred in an ARC70 Preliminary Definition of Improvement. -55- 2017204254 22 Jun2017 ACR20 Preliminary Definition of Improvement is defined as: £:20% improvement in: Tender Joint Count (TCJ) and Swollen Joint Count (SWJ) and > 20% improvement in 3 of following 5 assessments: Patient Pain Assessment (VAS), Patient Global assessment (VAS), Physician Global Assessment (VAS), Patent 5 Self-Assessed Disability (HAQ), Acute Phase Reactant (CRP or BSR). ACR50 and ACR70 are defined hi the same way with >50% and >70% improvements, respectively. For further details see Felson et at in American College of Rheumatology Preliminary Definition of Improvement in Rheumatoid Arthritis;

Arthritis Rheumatism (1995) 38: 727-735. 10 The composition must he sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the earner can he an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of 15 required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the inj ectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. 20 When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier. VI. Uses and Methods of the Invention 25 The human antibodies (including immunoconjugates, bispecifics/multispecifics, compositions and other derivatives described herein) of the present invention have numerous in vitro and in vivo diagnostic and therapeutic utilities involving the diagnosis and treatment of disorders involving cells expressing CD20. For example, the antibodies can be administered to cells in culture, e.g., in vitro or ex vivo, 30 or to human subjects, e.g., in vivo, to treat, prevent and to diagnose a variety of disorders. As used herein, the term "subject" is intended to include human and nonhuman animals which respond to the human antibodies against CD20. Preferred subjects include human patients having disorders that can he corrected or ameliorated by inhibiting or controlling B cells (normal or malignant). 35 For example, in one embodiment, human antibodies of the present invention can be used to treat a subject with a turaorigenic disorder, e.g., a disorder characterized by the presence of tumor cells expressing CD20 including, for example, B cell lymphoma, e.g., NHL. Examples of tumorigenic diseases which can be treated -56- 2017204254 22Jun2017 and/or prevented include B cell lymphoma, e.g., NHL, including precursor B cell lymphoblastic leukemia/lymphoma and mature B cell neoplasms, such as B cell chronic lymhocytic leukemia(CLL)/small lymphocytic lymphoma (SLL), B cell prolympliocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular 5 lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B cell lymphoma, Burldtt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma (ALCL). 10 Further examples of B cell non-Hodgkin’s lymphomas are lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B cell lymphoma, mediastinal large B cell lymphoma, heavy chain diseases (including % μ, and a disease), lymphomas induced by therapy with immunosuppressive agents, such as cyclosporine-induced lymphoma, and methotrexate-induced lymphoma. 15 In a further embodiment, the human antibodies of the present invention can be used to treat Hodgkin’s lymphoma.

Examples of immune disorders in which CD20 expressing B cells are involved which can be treated and/or prevented include autoimmune disorders, such as psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, 20 inflammatory bowel disease (3BD), Crohn’s disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud’s syndrome, Sjogren’s syndrome, juvenile onset diabetes, Reiter’s disease, Beliefs disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immune-mediated 25 thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves’ disease, Hashimoto’s thyroiditis, Wegener’s granulomatosis, Omenn’s syndrome, chronic renal failure, acute infectious mononucleosis, HIV, and 30 herpes virus associated diseases. Further examples are severe acute respiratory distress syndrome and choreoretinitis. Furthermore, other diseases and disorders include those caused by or mediated by infection of B-cells with virus, such as Epstein-Barr virus (EBV).

Further examples of inflammatory, immune and/or autoimmune disorders 35 in which autoantibodies and/or excessive B lymphocyte activity are prominent and which can be treated and/or prevented, include the following: -57- 2017204254 22 Jun2017 vasculitides and other vessel disorders, such as microscopic polyangiitis, Churg-Strauss syndrome, and other ANC A-associated vasculitides, polyarteritis nodosa, essential cryoglobulinaemic vasculitis, cutaneous leukocytoclastic angiitis, Kawasaki disease, Takayasu arteritis, giant cell arthritis, Henoch-Schonlein purpura, primary or 5 isolated cerebral angiitis, erythema nodosum, thrombangiitis obliterans, thrombotic thrombocytopenic purpura (including hemolytic uremic syndrome), and secondary vasculitides, including cutaneous leukocytoclastic vasculitis (e.g., secondary to hepatitis B, hepatitis C, Waldenstrom’s macroglobulinemia, B-cell neoplasias, rheumatoid arthritis, Sjogren’s syndrome, or systemic lupus erythematosus); further examples are 10 erythema nodosum, allergic vasculitis, panniculitis, Weber-Christian disease, purpura hyperglobulinaemica, and Buerger’s disease; skin disorders, such as contact dermatitis, linear IgA dermatosis, vitiligo, pyoderma gangrenosum, epidermolysis bullosa acquisita, pemphigus vulgaris (including cicatricial pemphigoid and bullous pemphigoid), alopecia areata (including alopecia 15 universalis and alopecia totalis), dermatitis herpetiformis, erythema multiforme, and chronic autoimmune urticaria (including angioneurotic edema and urticarial vasculitis); immune-mediated cytopenias, such as autoimmune neutropenia, and pure red cell aplasia; connective tissue disorders, such as CNS lupus, discoid lupus 20 erythematosus, CREST syndrome, mixed connective tissue disease, polymyositis/dermatomyositis, inclusion body myositis, secondary amyloidosis, cryoglobulinemia type I and type II, fibromyalgia, phospholipid antibody syndrome, secondary hemophilia, relapsing polychondritis, sarcoidosis, stiff man syndrome, and rheumatic fever; a further example is eosinophil fasciitis; 25 arthritides, such as ankylosing spondylitis, juvenile chronic arthritis, adult Still’s disease, and SAPHO syndrome; further examples are sacroileitis, reactive arthritis, Still’s disease, and gout; hematologic disorders, such as aplastic anemia, primary hemolytic anemia (including cold agglutinin syndrome), hemolytic anemia secondary to CLL or 30 systemic lupus erythematosus; POEMS syndrome, pernicious anemia, and

Waldemstrom’s purpura hyperglobulinaemica; further examples are agranulocytosis, autoimmune neutropenia, Franklin’s disease, Seligmann’s disease, μ-chain disease, paraneoplastic syndrome secondary to thymoma and lymphomas, and factor VIII inhibitor formation; 35 endocrinopathies, such as polyendocrinopathy, and Addison’s disease; further examples are autoimmune hypoglycemia, autoimmune hypothyroidism, autoimmune insulin syndrome, de Quervain’s thyroiditis, and insulin receptor antibody-mediated insulin resistance; -58- 2017204254 22Jun2017 hepato-gastrointestinal disorders, such as celiac disease, Whipple’s disease, primary biliary cirrhosis, chronic active hepatitis, and primary sclerosing cholangiitis; a further example is autoimmune gastritis; nephropathies, such as rapid progressive glomerulonephritis, post-5 streptococcal nephritis, Goodpasture’s syndrome, membranous glomerulonephritis, and cryoglobulinemic nephritis; a iurther example is minimal change disease; neurological disorders, such as autoimmune neuropathies, mononeuritis multiplex, Lambert-Eaton’s myasthenic syndrome, Sydenham’s chorea, tabes dorsalis, and Guillain-Barre’s syndrome; further examples are myelopathy/tropical spastic 10 paraparesis, myasthenia gravis, acute inflammatory demyelinating polyneuropathy, and chronic inflammatory demyelinating polyneuropathy; cardiac and pulmonary disorders, such as fibrosing alveolitis, bronchiolitis obliterans, allergic aspergillosis, cystic fibrosis, Lbffler’s syndrome, myocarditis, and pericarditis; further examples are hypersensitivity pneumonitis, and 15 paraneoplastic syndrome secondary to lung cancer; allergic disorders, such as bronchial asthma and hyper-IgE syndrome; a further example is amaurosis fugax; ophthalmologic disorders, such as idiopathic chorioretinitis; infectious diseases, such as parvovirus B infection (including hands-and-20 socks syndrome); and gynecological-obstretical disorders, such as recurrent abortion, recurrent fetal loss, and intrauterine growth retardation; a further example is paraneoplastic syndrome secondary to gynaecological neoplasms; male reproductive disorders, such as paraneoplastic syndrome secondary 25 to testicular neoplasms; and transplantation-derived disorders, such as allograft and xenograft rejection, and graft-versus-host disease.

In one embodiment, the disease is an inflammatory, immune and/or autoimmune disorder selected from ulcerative colitis, Crohn’s disease, juvenile onset 30 diabetes, multiple sclerosis, immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia (including autoimmune hemolytic anemia), myasthenia gravis, systemic sclerosis, and pemphigus vulgaris.

In another embodiment, human antibodies of the invention can be used to 35 detect levels of CD20, or levels of cells which contain CD20 on their membrane surface, which levels can then be linked to certain disease symptoms. Alternatively, the antibodies can be used to deplete or interact with the function of CD20 expressing cells, thereby implicating these cells as important mediators of the disease. This can be -59» 2017204254 22 Jun2017 achieved by contacting a sample and a control sample with the anti-CD20 antibody under conditions that allow for the formation of a complex between the antibody and CD20. Any complexes formed between the antibody and CD20 are detected and compared in the sample and the control. 5 Human antibodies of the invention can be initially tested for binding activity associated with therapeutic or diagnostic use in vitro. For example, the antibodies can be tested using flow cytometric assays described in the Examples below. Moreover, activity of the antibodies in triggering at least one effector-mediated effector cell activity, including inhibiting the growth of and/or killing of cells expressing CD20, 10 can be assayed. For example, the ability of the antibodies to trigger CDC and/or apoptosis can be assayed. Protocols for assaying for CDC, homotypic adhesion, molecular clustering or apoptosis are described in the Examples below.

Human antibodies of the invention also have additional utility in therapy and diagnosis of a variety of CD20-related diseases. For example, the human 15 antibodies can be used to elicit in vivo or in vitro one or more of the following biological activities: to inhibit the growth of and/or differentiation of a cell expressing CD20; to kill a cell expressing CD20; to mediate phagocytosis or ADCC of a cell expressing CD20 in the presence of human effector cells; to mediate CDC of a cell expressing CD20 in the presence of complement; to mediate apoptosis of a cell 20 expressing CD20; to induce homotypic adhesion; and/or to induce translocation into lipid·rafts upon binding CD20,

In a particular embodiment, the human antibodies are used in vivo to treat, prevent or diagnose a variety of CD2Q-related diseases. Examples of CD20-related diseases include, among others, B cell lymphoma, e.g., NHL, and immune 25 diseases, e.g., autoimmune diseases, such as those listed above.

In a particular embodiment, the antibodies of the invention are used to treat or to prevent NHL, as the antibodies deplete the CD20 bearing tumor cells).

Non-Hodgkin’s lymphoma is a type of B cell lymphoma. Lymphomas, e.g., B cell lymphomas, are a group of related cancers that arise when a lymphocyte (a 30 blood cell) becomes malignant. The normal function of lymphocytes is to defend the body against invaders: germs, viruses, fungi, even cancer. There are many subtypes and maturation stages of lymphocytes and, therefore, there are many kinds of lymphomas. Like nonnal cells, malignant lymphocytes can move to many parts of the body. Typically, lymphoma cells form tumors in the lymphatic system: bone marrow, lymph 35 nodes, spleen, and blood. However, these cells can migrate to other organs. Certain types of lymphoma will tend to grow in locations in which the nonnal version of the cell resides. For example, it’s common for follicular NHL tumors to develop in the lymph nodes. -60- CD20 is usually expressed at elevated levels on neoplastic (i.e., tumorigenic) B cells associated with NHL. Accordingly, CD20 binding antibodies of the invention can be used to deplete CD20 bearing tumor cells which lead to NHL and, thus, can be used to prevent or treat this disease. 2017204254 22Jun2017 5 Human antibodies (e.g., human monoclonal antibodies, multispecific and bispecific molecules) of the present invention also can be used to block or inhibit other effects of CD20. For example, it is known that CD20 is expressed on B lymphocytes and is involved in the proliferation and/or differentiation of these cells. Since B lymphocytes function as immunomodulators, CD20 is an important target for antibody 10 mediated therapy to target B lymphocytes, e.g., to inactivate or kill B lymphocytes, involved in autoimmune disorders. Such autoimmune disorders include, for example, the above listed diseases

Suitable routes of administering the antibody compositions (e.g., human monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates ) 15 of the invention in vivo and in vitro are well known in the art and can be selected by those of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenous or subcutaneous). Suitable dosages of the molecules used will depend on the age and weight of the subject and the concentration and/or formulation of the antibody composition. Furthermore, tumor load can be determined 20 and used to calculate suitable dosages.

As previously described, human anti-CD20 antibodies of the invention can be co-administered with one or other more therapeutic agents, e.g, a cytotoxic agent, a radiotoxic agent or an immunosuppressive agent. The antibody can be linked to the agent (as an immunocomplex) or can be administered separate from the agent. In the 25 latter case (separate administration), the antibody can be administered before, after or concurrently with the agent or can be co-administered with other known therapies, e.g., an anti-cancer therapy, e.g., radiation. Such therapeutic agents include, among others, anti-neoplastic agents such as doxorubicin, cisplatin, bleomycin, carmustine, chlorambucil, and cyclophosphamide. Co-administration of the human anti-CD20 30 antibodies of the present invention with chemotherapeutic agents provides two anticancer agents which operate via different mechanisms which yield a cytotoxic effect to human tumor cells. Such co-administration can solve problems due to development of resistance to drugs or a change in the antigenicity of the tumor cells which would render them unreactive with the antibody. 35 Target-specific effector cells, e.g., effector cells linked to compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural -61- 2017204254 22 Jun2017 killer cells and other IgG- or IgA-receptor bearing cells. If desired, effector cells can be obtained from the subject to be treated. The target-specific effector cells, can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be in the order of 108 to 109 but will vary depending on 5 the therapeutic purpose. In general, the amount will be sufficient to obtain localization at the target cell, e.g., a tumor cell expressing CD20, and to effect cell killing by, e.g., phagocytosis. Routes of administration can also vary.

Therapy with target-specific effector cells can be performed in conjunction with other techniques for removal of targeted cells. For example, anti-10 tumor therapy using the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention and/or effector cells armed with these compositions can be used in conjunction with chemotherapy. Additionally, combination immunotherapy may be used to direct two distinct cytotoxic effector populations toward tumor cell rejection. For example, anti-CD20 antibodies linked to anti-Fc-yRI or anti-15 CD3 may be used in conjunction with IgG- or IgA-receptor specific binding agents.

Bispecific and multispecific molecules of the invention can also be used to modulate FcyR or FcaR levels on effector cells, such as by capping and elimination of receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose. 20 The compositions (e.g., human antibodies, multispecific and bispecific molecules and immunoconjugates) of the invention which have complement binding sites, such as portions from IgGl, -2, or -3 or IgM which bind complement, can also be used in the presence of complement. In one embodiment, ex vivo treatment of a population of cells comprising target cells with a binding agent of the invention and 25 appropriate effector cells can be supplemented by the addition of complement or senmi containing complement. Phagocytosis of target cells coated with a binding agent of the invention can be improved by binding of complement proteins. In another embodiment target cells coated with the compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be lysed by complement. In yet another 30 embodiment, the compositions of the invention do not activate complement.

The compositions (e.g., human antibodies, multispecific and bispecific molecules and immunoconjugates) of the invention can also be administered together with complement. Accordingly, within the scope of the invention are compositions comprising human antibodies, multispecific or bispecific molecules and serum or 35 complement. These compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies, multispecific or bispecific molecules of the invention and the complement or serum can be administered separately. Binding of the - 62 - 2017204254 22 Jun2017 compositions of the present invention to target cells causes translocation of the CD20 antigen-antibody complex into lipid rafts of the cell membrane. Such translocation creates a high density of antigen-antibody complexes which may efficiently activate and/or enhance CDC. S Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., human antibodies and ixnmunoconjugates) and instructions for use. The kit can further contain one or more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent, or one or more additional human antibodies of the invention (e.g., a human antibody having a 10 complementary activity).

Accordingly, patients treated with antibody compositions of the invention can be additionally administered (prior to, simultaneously with, or following administration of a human antibody of the invention) with another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic 15 effect of the human antibodies. hi other embodiments, the subject can be additionally treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fey or Fca receptors by, for example, treating the subject with a cytokine. Preferred cytokines for administration during treatment with the multispecific molecule include of granulocyte 20 colony-stimulating factor (G-CSF), granulocyte- macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TMF).

The compositions (e.g., human antibodies, multispecific and bispecific molecules) of the invention can also be used to target cells expressing FcyR or CD20-, for example for labeling such cells. For such use, the binding agent can be linked to a 25 molecule that can be detected. Thus, the invention provides methods for localizing ex vivo or in vitro cells expressing Fc receptors, such as FcyR, or CD20. The detectable label can be, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

In a particular embodiment, the invention provides methods for detecting 30 the presence of CD20 antigen in a sample, or measuring the amount of CD20 antigen, comprising contacting the sample, and a control sample, with a human monoclonal antibody which specifically binds to CD20, under conditions that allow for formation of a complex between the antibody or portion thereof and CD20. The formation of a complex is then detected, wherein a difference complex formation between the sample 35 compared to the control sample is indicative the presence of CD20 antigen in the sample. -63- 2017204254 22 Jun2017

In other embodiments, the invention provides methods for treating a disorder involving cells expressing CD20 in a subject, e.g., non-Hodgkin’s lymphoma or rheumatoid arthritis, by administering to the subject the human antibodies described above. Such antibodies and derivatives thereof axe used to inhibit CD20 induced 5 activities associated with certain disorders, e.g., proliferation and/or differentiation. By contacting the antibody with CD20 (e.g., by administering the antibody to a subject), the ability of CD20 to induce such activities is inhibited and, thus, the associated disorder is treated.

Accordingly, in another embodiment, the present invention provides a 10 method for treating or preventing a tumorigenic disorder involving CD20 expressing cells, e,g,, NHL. The method involves administering to a subject an antibody composition of the present invention in an amount effective to treat or prevent the disorder. The antibody composition can be administered alone or along with another therapeutic agent, such as a cytotoxic or a radiotoxic agent which acts in conjunction 15 with or synergistically with the antibody composition to heat or prevent the diseases involving CD20 expressing cells. In a particularly preferred embodiment, the present invention provides a method for treating non-Hodgkin’s lymphoma. hi another embodiment, the present invention provides a method for treating or preventing an autoimmune disorder involving human CD20 expressing cells, 20 e.g., those diseases as listed above. The method involves administering to a subject an antibody composition of the present invention in an amount effective to treat or prevent the disorder. The antibody composition can be administered alone or along with another therapeutic agent, such as an immunosuppressant which acts in conjunction with or synergistically with the antibody composition to treat or prevent the disease involving 25 cells expressing CD20.

In still another embodiment, the invention provides a method for detecting the presence or quantifying the amount of CD20-expressing cells in vivo or in vitro. The method comprises (i) administering to a subject a composition (e.g., a multi-or bispecific molecule) of the invention conjugated to a detectable marker; (ii) exposing 30 the subject to a means for detecting said detectable marker to identify areas containing CD20-expressing cells. hi yet another embodiment; immmioconjugates of the invention can be used to target compounds (e.g., therapeutic agents, labels, cy to toxins, radiotoxins immunosuppressants, etc.) to cells which have CD20 expressed on their surface by 35 linking such compounds to the antibody. Thus, the invention also provides methods for localizing ex vivo or in vitro cells expressing CD20, such as Reed-Sternbcrg cells (e.g., with a detectable label, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor). Alternatively, the immunoconjugates can be used to kill cells -64- 2017204254 22 Jun2017 which have CD20 expressed on their surface by targeting cytotoxins or radiotoxins to CD20.

The present invention is farther illustrated by the following examples which should not be construed as farther limiting. 5

EXAMPLES B-cell lines used in the examples

Cell line Origin Obtained from Daudi Negroid Burkitt’s Lymphoma ECACC (85011437) ARH-77 IgG plasma ceil leukemia DSMZ (ACC 512) DOHH Refractory immunoblastic B cell lymphoma DSMZ (ACC 47) Raji Negroid Burkitt’s Lymphoma ECACC (85011429) SU-DHL-4 B-NHL, diffuse histiocytic lymphoma DSMZ (ACC 495) Ramos-EHRB Burkitt’s Lymphoma ECACC (85030804) Tauoue Human B-cell leukemia DSMZ (ACC 399) 15 Daudi, ARH-77, DOHH, Raji, Ramos-EHRB, and Tanoue B-cell lines were cultured in RPMI1640 culture medium supplemented with 10% fetal calf serum (FCS) (Optimum C241, Wisent Inc., st. Bruno, Canada), 2 mM L-glutamine, 100 lU/ml penicillin, 100 pg/ml streptomycin, and 1 mM sodium pyruvate (all Gibco BRL, Life Technologies, Paisley, Scotland). 20 - SU-DHL-4 B-cell line was cultured in the same medium but without sodium pyruvate.

Cultures were maintained at 37 °C in a humidified 5% C02 incubator, split and harvested at 80-90% confluence. Medium was refreshed twice a week. At this time cells were split and seeded out to 1-1.5 x 10“ cells/ml to ensure viability and 25 optimal growth.

Example 1 Production of Human Antibodies Against CD20 HCo7 and KM Mice. Fully human monoclonal antibodies to CD20 were prepared using HCo7 and KM mice which express human antibody genes. In the KM 30 mouse strain, the endogenous mouse kappa light chain gene has been hornozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820 and the endogenous mouse heavy chain gene has been hornozygously disrupted as described in Example 1 of -65- 2017204254 22Jun2017 PCX Publication WO 01/09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild ei at (1996) Nature Biotechnolog)> 14:845-851. This mouse strain also carries a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478. 5 The HCo7 mice have a JKD disruption in their endogenous light chain (kappa) genes (as described in Chen etal. (1993) EMBOJ. 12: 821-830), a CMD disruption in their endogenous heavy chain genes (as described in Example 1 of WO 01/14424), a KCo5 human kappa light chain transgene (as described in Fishwild et at (1996) Nature Biotechnology 14:845-851), and a HCo7 human heavy chain transgene 10 (as described in US 5,770,429). UCo7 and KM Mice Immunizations'. HCo7 and KM mice were immunized with human CD20 transfected NS/0 cells. For the first immunization, per mouse, lx 107 cells in 150 μΐ PBS were mixed 1:1 with Complete Freunds Adjuvant and 15 injected intraperitoneally (ip.). Subsequent i.p. immunizations were done using a similar amount of cells without adjuvant. Three and two days prior to fusion the mice were intravenously boosted with 0.5 x 107 ceils suspended in PBS.

The presence of antibodies directed against human CD20 in the serum of the mice was monitored by flow cytometry using FACS analysis, using human CD20 20 transfected NS/0 cells as well as CD20 negative parental NS/0 cells.

Generation of Hyhridomas Producing Human Monoclonal Antibodies to CD2Q-. The mouse splenocytes were isolated from the HCo7 and KM mice and fused with PEG to a mouse myeloma cell line based upon standard protocols. The resulting 25 hybridomas were then screened for human IgG,K production by ELISA and for CD20 specificity using human CD20 transfected NS/0 and SKBR3 cells by FACS analysis. Single cell suspensions of splenic lymphocytes from immunized mice were fused to one-fourth the number of SP2/0 nonsecreting mouse myeloma cells (ATCC, CRL 1581) with 50% PEG (Sigma). Cells were plated at approximately 1 x 105/well in flat bottom 30 microtiter plate, followed by about two week incubation in selective medium containing 10% fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3-5% origen (IGEN) inDMEM (Mediated!, CRL 10013, with high glucose, L-glutamine and sodium pyruvate) plus 5 mM HEPES, 0.055 noM 2-mercaptoethanol, 50 mg/ml gentamycin and lx HAT (Sigma, CRL P-7185). After 1-2 weeks, cells were cultured in 35 medium in which the HAT was replaced with HT. Individual wells were then screened by flow cytometry for human anti~CD20 monoclonal IgG antibodies. Once extensive hybridoma growth occurred, medium was monitored usually after 10-14 days. The antibody secreting hybridomas were replated, screened again and, if still positive for - 66 - 2017204254 22 Jun2017 human IgG, anti-CD20 monoclonal antibodies were subcloned by limiting dilution. The stable subclones were then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization. One clone from each hybridoma, which retained the reactivity of parent cells (by FACS), was chosen. 5-10 vial cell banks were 5 generated for each clone and stored in liquid nitrogen.

Selection of Human Monoclonal Antibodies Binding to CD20/Primaiy Screens: To determine the isotype of antibodies, an isotype ELISA was performed. Wells of microtiter plates were coated with 1 pg/ml of mouse anti-human kappa light 10 chain, 50 μΐ/well in PBS incubated 4 °C overnight. After blocking with 5% chicken serum, the plates were reacted with supernatant and purified isotype control. Plates were then incubated at ambient temperature for 1-2 hours. The wells were then reacted with either human IgGl, IgG2, IgG3 or IgG4-specific Horseradish peroxidase - conjugated probes. Plates were developed and analyzed as described above.

15 Four hybridoma cell lines were generated, three from fusion of KM mouse and one from fusion of HCo7 mouse, expressing the following antibodies: 2F2: a human monoclonal IgGl,K antibody with the nucleotide sequences: SEQ ID NOs: 1 and 3- and the amino acid sequences: SEQ ID NOs: 2 and 4. 4C9: a human monoclonal IgGl.K antibody with exactly the same amino 20 acid sequences as 2F2: SEQ ID NOs: 2 and 4. 7D8: a human monoclonal IgGl ,κ antibody with the nucleotide sequences: SEQ ID NOs: 5 and 7 and the amino acid sequences: SEQ ID NOs: 6 and 8. 1 IBS: a human monoclonal IgG3, κ antibody with the nucleotide sequences: SEQ ID NOs: 9 and 11 and the amino acid sequences: SEQ ID NOs: 10 and 25 12.

The term “2F2” is used herein to designate both the antibody derived from hybridoma clone 2F2 and the identical antibody derived from hybridoma clone 4C9.

The antibodies of the invention can be switched to other isotypes as 30 determined by the transgenic or transchromosomal non-human animal from which they are derived. In one embodiment of the invention, the 11B8 human monoclonal IgG3,ic antibody can be switched to a human monoclonal IgGl,K isotype having exactly the same Vh and Vf sequences, hi another embodiment, the 2F2 IgGlqc antibody or 7D8 IgGl,K antibody can be switched to a human monoclonal IgG2, IgG4, IgAl, IgA2 or 35 IgE isotype having exactly the same Vh and Vl sequences. -67- 2017204254 22 Jun2017

Example 2 Antibody Sequencing of Human Antibodies Against CD20

Sequencing of the Vt_ and Vw Regions RNA preparation·. Total RNA was prepared from 5x106 cells of all 5 HuMAb CD20 liybridoma cell lines (2F2, 7D8 and 1 IBS) with RNeasy kit (Qiagen, Westburg, Lensden, Netherlands) according to the manufacturer’s protocol. cDNA preparation of2F2 and 7D8: 55~RACB-Ready Complementary DNA (cDNA) of RNA was prepared from 1 gg total RNA, using the SMART RACE 10 cDNA Amplification kit (Clonetech), following the manufacturer’s protocol.

Vh and Vl regions were amplified using an advantage HF 2 PCR Kit (Clonetech, BD) and using the following primers: 15 Vjc RACB2 5! GCA GGC ACA CAA CAG AGG CAG TTC CAG ATT TC anneals in C-kappa VH RACE2 5’ GCT GTG CCC CCA GAG GTG CTC TTG GAG G anneals in CH, cDNA preparation of 11B8: Complementary DNA (cDNA) of RNA from 11B8 cells was prepared from 3 pg total RNA with AMV Reverse Transcriptase 20 with buffer (Roche Diagnostics GmbH, Mannheim, Germany), oligo d(T)[5 (Promega, Madison, WI, USA), dNTP (Roche Diagnostics GmbH, Mannheim, Germany) and RNAsin (Promega) according to the manufacturer’s protocol (2000, version 3). PCR primers used to amplify Vh and Vi regions for cloning: 25

Primer pairs used:

VH: FR1 5’ primers AB62 CAg gTK CAg CTg gTg CAg TC AB63 SAg gTg CAg CTg KTg gAg TC 30 AB65 gAg gTg CAg CTg gTg CAg TC Vh leader 5’ nrimers AB85 ATg gAC Tgg ACC Tgg AgC ATC AB86 ATg gA A TTg ggg CTg AgC Tg 35 AB87 ATg gAg TTT ggR CTg AgC Tg AB88 ATg AAA C AC CTg Tgg TTC TTC AB89 ATg ggg TC A ACC gCC ATC CT -68- 2017204254 22 Jun2017

Vh 3' primer AB90 TgC CAg ggg gAA gAC CgA Tgg VK: FR1 5! primers 5 AB8 RAC ATC CAg ATg AYC CAg TC AB9 gYC ATC YRg ATg ACC CAg TC AB10 gAT ATT gTg ATg ACC CAg AC AB11 gAA ATT gTg TTg ACR CAg TC AB12 gAA ATW gTR ATg ACA CAg TC 10 AB13 gAT gTT gTg ATg ACA CAG TC AB14 gAA ATT gTg CTg ACT CAg TC Vr leader 5’ primers AB123 CCC gCT Cag CTC CTg ggg CTC CTg 15 AB124 CCC TgC TCA gCT CCT ggg gCT gC AB125 CCC AgC gCA gCT TCT CTT CCT CCT gC AB126 ATg gAA CCA Tgg AAg CCC CAg CAC AgC Vr 3’ primer 20 AJB16 Cgg gAA gAT gAA gAC AgA Tg wherein K = T or G, S = C or G, R = A or G, Y = C or T, and W = A or T, PCR conditions used to amplify Vh and yL regions for cloning 2F2 and 25 7D8: Polymerase chain reactions (PCR) were performed with HF polymerase mix (Clonetech.) on a T1 cycler (Biometra, Westburg). -69- 2017204254 22 Jun2017 PCR conditions: 94 °C 30 sec 5 cycles 5 72 °C 1 min 94 °C 30 sec 70 °C 30 sec 5 cycles 72 °C 1 min 10 94 °C 30 sec 68 °C 30 sec 27-30 cycles 72 °C 1 min 15 PCR conditions used to amplify Va and Vi regions for cloning 11B8: Polymerase chain reactions (PCR) were performed with AmpliTaq polymerase (Perkin Elmer) on aTl Cycler (Biometra, Westburg, Leusden, Netherlands). 20 25

PCR cycling protocol; 94 °C 2 min 11 cycles 94 °C 30 sec 65 °C 30 sec, minus 1 °C per cycle 72 °C 30 sec 30 cycles 94 °C 30 sec 55 °C 30 sec 72 °C 30 sec 72 °C 10 min cool down to 4 °C 30 Cloning of VH and VL inpGEMT-Vector System II (2F2, 7D8, and 1 IBS): After analysing the PCR products on an agarose gel, the products were purified with the QIAEXII Gel Extraction Kit (Qiagen, Westburg, Leusden, Netherlands). Two independently amplified PCR products of each Vh and Vl region were cloned in pGEMT-Vector System II (Promega) according to manufacturer’s protocol (1999, 35 version 6). After transformation to E. coli JM1Q9, individual colonies were screened by colony PCR using T7 and SP6 primers, 30 annealing cycles at 55 °C. Plasmid DNA from colonies was purified using Qiaprep Spin miniprep lot (Qiagen). To further -70- 2017204254 22 Jun2017 analyse the Vh and Vl regions, aNcollNotl (NE Biolabs, Westburg, Leusden, Netherlands) digestion was performed and analysed on agarose gel.

Sequencing (2F2, 7D8 and 11B8): The V-regions regions were 5 sequenced after cloning in the pGEMT-Vector System Π. Sequencing was performed at Baseclear (Leiden, Netherlands). The sequences were analyzed by aligning germline ν'-gene sequences in Vbase fwww.mrc-cpe.cam.ac.uk/imt-doc/public/intro .htmUittp ://www.mrc-cpe. cam. ac.uto'vbase-ok.php?menu=901. 10 The sequences obtained are shown in Figures 53-58.

Example 3 Recombinant production of 2F2 and Ϊ1Β8 in GS-NS/0 cell line 2F2T: The heavy chain and light chain variable regions of the 2F2 antibody were amplified, using PCR, from a standard cloning vector, pGem-5Zf 15 (Promega), using primers which included an optimal Kozak sequence and suitable restriction sites to clone the fragments in the GS constant region vectors pCONylf and PCONk (Lonza).

After amplification, the fragments were purified and digested with the restriction enzymes for cloning and ligated in the two vectors. The heavy chain variable 20 fragment was digested with Hind HI and Bsi WI and ligated into the pCONylf vector which had been digested with Hind ΠΙ and Bsi WI, and dephosphorylated with alkaline phosphatase. The light chain variable fragment was digested with Hind HI and Apa I and ligated into the PCONjc vector which had been digested with Hind ΙΠ and Apa I, and dephosphorylated with alkaline phosphatase. The pCON7lf/variable~heavy and 25 PCON/c/variable-light vectors are shown in Figures 1 and 2, respectively. Transformed E. coli colonies were checked by colony PCR and 2 positive colonies of each the heavy chain (HC) and light chain (LC) construct were grown for plasmid isolation. Isolated plasmid of these 4 clones was sequenced to confirm the sequence. Both of the HC clones and one of the LC clones were found to have the correct sequences. 30 The two HC and one LC constructs were combined to give two combinations of LC-HC and transiently co-transfected in CHO-K1 cells to check the constructs for proper production of 2F2 antibody. Normal production levels were reached for all combinations in tins expression experiment and 1 clone of each of the HC and LC constructs were chosen for construction of a double-gene vector.

35 Standard cloning procedures were used to combine the HC and LC constructs in a double-gene cloning vector, designated ρΓ.ΟΝγ1£/κ2Ρ2, by ligating the complete expression cassette from the heavy chain vector, pCONyl&variable-heavy, -71- 2017204254 22 Jun2017 into the light chain vector, pCONx/variable-light. The pCON7lf7/i:2F2 vector is shown in Figure 3.

This construct was again functionally tested in a transient transfection in CHO-K1 cells and showed normal expression levels. 5 The variable regions of the pCONyl f/x2F2 plasmid were sequenced to reconfirm the correct sequences.

Linear plasmid was prepared for stable transfections by digesting pCONyli7K2F2 with a unique restriction enzyme, Pvu I, cutting outside regions vital for expression. Complete linearization was confirmed by agarose gel electrophoresis and 10 the DNA was purified and stored at -20 °C until use.

Six transfections of NS/0 host cells were performed, by electroporation with plasmid DNA, using the above linear DNA plasmid. Following transfection, the cells were distributed into 96-wells plates and incubated. Selective medium (containing 10% dialysed fetal calf serum (dFCS) and 10 μΜ of the GS-inhibitor L-methionine 15 sulphoximine but lacking glutamine) was added and the plates were monitored to determine when the non-transfected cells died to leave foci of transfected cells. For further details concerning GS vector systems, see WO 87/04462. The transfected plates were incubated for approximately three weeks to allow colony formation. The resulting colonies were examined microscopically to verify that the colonies were of a suitable 20 size for assay (covering greater than 60% of the bottom of the well), and that only one colony was present in each well. Cell supernatants from 436 transfectants were screened for assembled antibody by IgG,K-ELISA. Using this data, 111 transfectants were selected for progression and further assessment in static culture. Cultures of the selected cell lines were expanded and adapted to low-serum containing medium (containing 25 bovine serum albumin (BSA) and added 1% dFCS) and a further assessment of productivity in static culture was undertaken (ELISA and measurement of percentage confluence). The 65 highest ranking cell lines were selected for progression. A preliminary assessment of the productivity of the selected cell lines was made in batch shake flask suspension culture in low serum-containing medium (containing BSA and 30 added 1% dFCS). Based upon harvest antibody concentration (by ELISA) and acceptable growth characteristics, 30 cell lines were selected for further evaluation in serum-free medium using a batch shake flask suspension culture. The 10 cell lines that produced the highest antibody concentrations were further evaluated in duplicate fed-batch shake flask suspension cultures in serum-free medium. Product concentrations at 35 harvest were determined by protein A high performance liquid chromatography (HPLC), according to well-known standard methods. All cell lines produced 2F2 antibody (denoted 2F2T) in good yields in the range of from 671-1333 mg/L as determined by protein A HPLC. -72- 2017204254 22 Jun2017 11B8T: in a similar way a GS-NS/0 cell line was established for recombinant production of 11B8 (denoted 11B8T) modifying the transfection procedure slightly as follows. 5 Four transfections of NS/0 host cells were performed, by electroporation with plasmid DNA, using the above linear DNA plasmid. After examining the resulting colonies microscopically to verify that the colonies were of a suitable size for assay (covering greater than 60% of the bottom of the well) and that only one colony was present in each well, cell supernatants from 596 transfectants were screened for 10 assembled antibody by IgGpc-ELISA. Using this data, 100 transfectants were selected for progression and further assessment in static culture, Cultures of the selected cell lines were expanded and adapted to low-serum containing medium (containing bovine serum albumin (BSA) and added 1% dFCS) and a further assessment of productivity in static culture was undertaken (ELISA and measurement of percentage confluence). The 15 60 highest ranking cell lines were selected for progression, and an additional 13 cell lines for which productivity data was unavailable -were also progressed. A preliminary assessment of the productivity of the selected cell lines was made in batch shake flask suspension culture in low serum-containing medium (containing BSA and added 1% dFCS). Based upon harvest antibody concentration (by ELISA) and acceptable growth 20 characteristics, 10 cell lines were selected for further evaluation in duplicate fed-batch shake flask suspension cultures in low serum-containing medium (containing BSA and added 1% dFCS). Product concentrations at harvest were determined by protein A high performance liquid chromatography (HPLC), according to well-known standard methods. Based on this one of the cell lines was discarded. The resulting 9 cell lines all 25 produced 11B8 antibody (denoted “11B8T’5) in good yields in the range of from 354-771 mg/L as determined by protein A HPLC.

Example 4 Comparison of liybridoma-derived 2F2 and transfectoma-derived recombinant 2F2T 30 By use of gel electrophoresis (SDS-PAGE and native agarose gel electrophoresis) it was shown that 2F2 and 2F2T are of the same size, and only slightly differ in electric charge.

Furthermore, 2F2 and 2F2T bind to CD20-transfected NS/0 cells and Raji cells with similar affinity as measured by flow cytometry using FACScalibur™ (Becton 35 Dickinson, San Diego, CA, USA). No binding to non-transfected NS/0 cells was observed demonstrating the specificity of 2F2 and 2F2T. 2F2 and 2F2T also induce CDC in a concentration-dependent maimer to the same extent in ARFI-77 cells (IgG plasma cell leukemia), Daudi cells, DOHH cells (refractory immunoblastic B cell -73- 2017204254 22 Jun2017 25 30 lymphoma progressed from follicular centroblastic/cenfrolytic lymphoma, DSMZ, Braunschweig, Germany) and Raji cells, measuring cell lysis (number of Pi-positive cells) by flow cytometry (FACScalibur). In a second experiment, the concentrations of 2F2 and 2F2T were kept constant while serum was added in different concentrations. 5 No significant differences between 2F2 and 2F2T were observed.

Finally, 2F2 and 2F2T bound to cell-associated CD20 binds complement factor Clq strongly and to the same extent. The experiment was performed in Daudi cells, DOHH cells, and Raji cells using fluorescein-conjugated anti-Clq polyclonal antibodies for detecting binding of Clq. 10

Example S Binding Characteristics of Human Antibodies Against CD20 1 Binding to different cell lines·. NS/0, NS/0 transfected with human CD20, Daudi and Raji cells were incubated for 30 min at 4 °C with culture supernatant containing human antibodies 2F2,7D8, and 11B8 followed by incubation with FITC-15 conjugated anti-human IgG Ab. Binding was assessed by flow cytometry using a FACScalibur flow cytometer. Fluorescence intensities were compared with negative control isotype matched samples. As shown in Figure 4, all three antibodies bound to NS/0 cells transfected with human CD20, whereas no binding was observed to parental, non-transfected NS/0 cells. All three antibodies also bound to the two different Burkitt 20 lymphoma B cell lines (Raji and Daudi) indicating that 2F2, 7D8, and ΠB8 are CD20 specific. Supernatant containing 7D8 or 11B8 were tested under non-saturating conditions, therefore, lower mean fluorescence intensities compared to 2F2 are observed. EC so value of 2F2 as determined by flow cytometry: In order to determine the apparent affinity of 2F2 for CD20 expressed on human B cells, a binding curve was made of 2F2 using isolated PBMCs from three human donors and gating of CD3-negative cells. The isolated PBMCs were incubated for 1 hour with a concentration range of FITC labeled 2F2 and analysed on FACS, and the mean fluorescence intensity (MFI) determined. The MFI values are shown in Figures 5A and 5B as a function of the antibody concentration. The EC50 values were calculated by use of Graph Pad Prism 3.02 by non-linear regression. The EC50 value of 2F2 in humans was similar for all three donors with a mean (± s.e.m.) of287 ± 12.7 ng/ml ¢1.9 ± 0.1 11M). 35

Binding ofJ2jI~labeled mAbs to CD20 expressing cells: mAbs were iodinated using Iodobeads (Pierce Chemical Co., Rockford, IL). 125I-labelled mAbs were serially diluted and incubated with Ramos-EHRB (cells for 2 hours at 37 °C in the -74- presence of sodium azide and 2-deoxyglucose to prevent endocytosis. The cell bound and free 125I labeled mAbs were then separated by centrifugation at 14,000 x g for 2 min through a mixture ofphthalate oils, allowing rapid separation without disturbing the binding equilibrium. The pelleted cells together with bomid antibody were then counted 5 using a gamma counter (Wallac UK Ltd, Milton Keynes, UK). 2017204254 22 Jun2017

As shown in Figure 6, 2F2 and 1 IBS exhibit similar ICd (or similar saturation points) indicating that both antibodies bind with similar affinity. However, 1 IBS saturates at a lower level than 2F2 indicating that it recognized a different form of CD20. This is also in agreement with a further experiment showing that a similar 10 number of 2F2 and rituximab antibody molecules binds to CD20 on Ramos-EHRB cells and Daudi cells, as shown by the similar levels of binding saturation (approximately 2-3 x 10s antibody molecules per cell). 11B8 andBl, in contrast, saturate at half this level and only about 1-2 x 103 antibody molecules bind to the Ramos-EHRB cells (Figure 7A) and Daudi cells (Figure 7B). 15 To exclude the possibility that the iodinated antibodies bind via Fc- receptors, binding curves were confirmed by use of anti-CD20 F(ab3fr fragments.

Again, similar numbers of 2F2 and rituximab-F(ab’)2 fragments bound to both Ramos-EHRB and Daudi cells. Also in these experiments, the number of 2F2 or rituximab antibody molecules bound to Ramos-EHRB cells and Daudi cells saturates at 20 approximately twice the number of 11B8 and B1 molecules bound to the cells.

Dissociation rate: To determine the dissociation rate of the mAbs, Ramos-EHRB cells (final volume of 1 ml in the presence of azide/2DOG) were incubated for 2 hours at 37 °C with 2 pg/ml 125I mAbs to achieve maximum binding. 25 Following centrifugation in a microfuge (2000 rpm for 2 min), the supernatant was removed, the pellet quickly resuspended in 1 ml medium, and immediately transferred to 9 ml medium at 37 °C in a 15 ml conical tube. At various times over the next 2 hours, 0,4 ml samples were removed and separated on phthalate oils to determine the level of radiolabeled mAbs remaining on the cell surface. As shown in Figure 8, both 2F2 and 30 11B8 dissociated significantly more slowly from CD20 than rituximab or Bl.

Dissociation rates of anti-CD20 F(ab)2 fragments: Ramos-EHBR cells were saturated with 2 gg/ml of i25I-labeled F(ab)2 fragments of 2F2,11B8, and rituximab, respectively. The Ramos-EHBR cells were washed and incubated in the 35 presence of a high concentration of the unlabeled antibody. The maximal (initial) binding to Ramos-EHRB cells was set at 100%. At several time points over the next 3 hours following loading, 0.4 ml samples were removed and separated on phthalate oil to determine the levels of radiolabeled mAb remaining on the cell surface. As can be seen -75- 2017204254 22 Jun2017 from Figure 9,2F2 and 11B8 dissociated much more slowly from the surface of CD20 than rituximab. At 90 min, approximately 50% of the F(ab)2 rituximab molecules were bound to the cell, whereas half of the F(ab)2 2F2 molecules were dissociated after 3 hours. The kd (k0ff) values for 2F2,11B8T, and rituximab are calculated as follows: 5 F(ab)2 2F2: lcd = In 2!tVl (sec) = In 2/10800 (sec) = 6.4 x 1 O'5 sec4 F(ab)211B8T: kd - In 2HVl (sec) = In 2/9000 (sec) = 7.7 x 10'5 sec"1 10 F(ab)2 rituximab: kd = In 2/ty, (sec) = In 2/5400 (sec) = 1.3 x 10'4 sec'1

Anti-CD20 mAh functional off rates: The impact of the slow 2F2 dissociation rate compared to rituximab was assessed in a functional CDC assay. To this 15 end, Daudi or SU-DHL4 cells were pre-incubated with 10 pg/ml anti-CD20 mAb or an isotype control antibody, washed and incubated in medium for different time points. At these time points after start of the assay, samples were incubated with complement (normal human serum 20 vol/vol%) and then incubated for another 45 min at 37 °C. Thereafter, cell lysis was determined on FACS by using PI (propidium iodide) staining 20 method. The % lysed cells (Pi-positive cells) are shown in Figure 10A (Daudi cells) or Figure 10B (SU-DHL4 cells) as a function of incubation time. 2F2 induced high CDC in both cell lines, and still lysed up to 90% of the cells after 6 hours, indicating that the CD20 saturation of the cells remained sufficiently high to induce complement-mediated lysis of most of tire cells. Rituximab, in contrast and in agreement with the above 25 dissociation rate studies, dissociated rapidly from the cells and failed to induce specific lysis following the 6 hour incubation period. 1 IBS was used as a control and did not induce CDC.

Example 6 CDC of Human Antibodies Against CD20 30 Serum preparation: Serum for complement lysis was prepared by drawing blood from healthy volunteers into autosep gel and clot activator vacutainer tubes (BD biosciences, Rutherford, NJ) which were held at room temperature for 30-60 min and then centrifuged at 3000 rpm for 5 min. Serum was harvested and stored at -80 °C. 35 -76-

Flow cytometry: For flow cytometry a FACScalibur flow cytometer was used with CellQuest pro software (BD Biosciences, Mountain view, CA). At least 5000 events were collected for analysis with cell debris excluded by adjustment of the forward sideward scatter (FCS) threshold. 2017204254 22 Jun2017 5 10 15 CDC kinetics: In a first set of experiments (n-3) the kinetics of CDC of five different B-cell lines, i.e., Daudi, SU-DHL-4, Raji, DOHH and ARH-77, were determined by adding 10 pg/ml 2F2, rituximab and an IgG control antibody, respectively, for 10 min before human serum was added. At several time intervals (up to one hour) after induction of CDC, the cells were suspended in PI solution and ceil lysis (number of Pi-positive cells) was measured by flow cytometry. The results are depicted in Figures 11A (ARH-77 cells), 11 B (Daudi cells), 11C (Raji cells), UD (DOHH) and 11 E (SU-DHL-4). As seen, addition of antibodies induced cell lysis within 5 min. Interestingly, addition of 2F2 resulted in a marked cell lysis of more than 80% in all five B-cell lines. Rituximab induced more than 80% cell lysis only in the SU-DHL-4 and Daudi cell lines, whereas the cell lysis of the DOHH cell line was ~5G%, and less than 20% in the ARH-77 and Raji cell lines. No lysis was observed with the IgG control antibody (data only shown in Figure 1 IB).

20 CDC serum titration: In a separate set of experiments (n=5), NHS (normal human serum) was titrated at two different antibody concentrations of 0.5 ^g/ml and 5 jag/ml. Cells were pre-incubated with 2F2 or rituximab for 10 min, before a concentration range of NHS was added. At 45 min after induction of CDC, cells were resuspended in PI solution. Cell lysis (number of Pi-positive cells) was measured by 25 flow cytometry. Figures 12A-D show the percentage of lysed (Pi-positive) cells as a function of NHS concentration. Figure 12A shows cell lysis of Daudi cells, Figure 12B cell lysis of ARH-77 cells, Figure 12C cell lysis ofDOHFI cells, and Figure 12D cell lysis of Raji cells. Increased lysis of cells was observed with increased NHS concentration. Addition of 2F2 caused maximal lysis of Daudi cells at the highest NHS 30 and antibody concentration. Rituximab induced about 50% cell lysis of Daudi cells at the highest NHS concentration. in ARH-77 cells, only the highest concentration of NHS and 2F2 led to approximately 75% cell lysis. Lower antibody concentrations were insufficient to induce ARH-77 cell lysis. Rituximab was not able to induce cell lysis of ARH-77 cells 35 in this experiment. 2F2 was able to induce NHS-concentration dependent cell lysis of DOHH cells at both the high and the low concentration, whereas rituximab was not able to induce lysis under these conditions. -77- 2017204254 22 Jun2017

Finally, 2F2 induced NHS-concentration-dependent lysis of Raji cells, which was only apparent by use of 5 pg/ml mAb. No lysis was observed with rituximab.

In these experiments, no lysis was observed with the isotype control 5 antibody (data not shown). CDC antibody titration: To measure the ability of the anti-CD20 antibodies to induce CDC at low concentrations, an experiment was performed where the antibodies were titrated (n=6). Various cell lines were pre-incubated with a 10 concentration range of 2F2 and rituximab, respectively, for 10 min before NHS was added. After 45 min incubation at 37 °C (when maximal lysis occurs) the cells were resuspended in PI solution and cell lysis (number of Pi-positive cells) was measured by flow cytometry. Figures 13A (Daudi cells), 13B (DOHH cells), 13C (ARH-77 cells), and 13D (Raji cells) show the percentage of lysed (Pi-positive) cells as a function of 15 antibody concentration. Both 2F2 and rituximab induced a concentration-dependent increase in cell lysis. 2F2 induced more than 80% lysis of Daudi cells upon addition of 2 pg/ml, whereas with rituximab this level was not reached even after addition of 10 pg/ml, Furthermore, 2F2 induced more than 80% lysis of DOHH cells at 0.4 pg/ml, whereas minimal lysis was observed with rituximab at this concentration. The maximal 20 lysis of DOHH cells with rituximab (-30% of total cell analyzed) was reached at 10 pg/ml. Induction of lysis of ARH-77 and Raji cells by 2F2 was lower, but still -70% lysis was reached at an antibody concentration of 10 pg/ml. At its highest concentration, rituximab induced lysis in only -23% of ARH-77 cells, and in only -6% of Raji cells.

In a similar experiment, 2F2, 2F2T, 11B8T, and rituximab were 25 investigated for their ability to induce CDC of Daudi and Raji cell lines, see Figures 14A and 14B. Also in this experiment more than 80% lysis of Daudi cells was observed with (transfectoma-derived) 2F2T at 10 pg/ml, whereas rituximab reached only to 60% lysis even at 10 pg/ml, cf. Figure 14 A. Lysis of Daudi cells with 2F2T was identical to the lysis obtained with hybridoma-derived 2F2.

30 Lysis of Raji cells was more difficult, but again both 2F2 and 2F2T induced lysis of Raji cells to a similar extent (Figure 14B). Rituximab was not able to induce CDC of Raji cells which is in agreement with the experiment shown in Figure 13D.

As can be seen from Figures 14A and 14B neither Daudi nor Raji cells 35 were susceptible to CDC by 11B8T. B1 induced lysis of Daudi cells, hut only to a small extent, and was not able to induce lysis of Raji cells. -78- 2017204254 22 Jun2017 CDC activity of anti-CD20 in Daudi cells: To determine the CDC activity of each antibody, elevated membrane permeability was assessed using FACS analysis ofpropidium iodide (PI)-stained cells. Briefly, the Daudi cells were washed and resuspended in RPMI/1% BSA at 1 x 106 cells/ml. Various concentrations of 5 human monoclonal antibodies were added to the Daudi cells and allowed to bind to CD20 on the cells for 10-15 min at room temperature. Thereafter, serum as a source of complement was added to a final concentration of 20% (v/v) and the mixtures were incubated for 45 min at 37 °C. The ceils were then kept at 4 °C until analysis. Each sample (150 μΐ) was then added to 10 μΐ of PI solution (10 gg/ml in PBS) in aFACS 10 tube. The mixture was assessed immediately by flow cytometry. As shown in Figure 15A, 2F2 and 7D8 showed superior CDC activity compared to rituximab.

In a second experiment, cells were labeled with human monoclonal antibodies as above, then washed and incubated in PBS for 45 min at 37 °C prior to the addition of human serum. This ensured that only antibody bound to the cell at the time 15 of serum addition was available to activate complement for cell lysis. As shown in

Figure 15B, decreased CDC activity was found for rituximab compared to 2F2 and 7D8 indicating that the human antibodies (2F2 and 7D8) are not affected by washing the cells prior to the addition of serum. 20 CDC activity of anti-CD20 in Raji cells: CDC activity was assessed using Raji cells which have relatively high surface expression of CD55 and CD59 and, therefore, are more resistant to complement attack. Human antibodies were added to Raji cells and allowed to bind for 15 min. Human serum (20%) was added and the mixtures incubated for 45 min at 37 °C. As shown in Figure 16A, rituximab was 25 ineffective in mediating CDC of Raji cells whereas significant levels of cell lysis occurred in Raji cells opsonized with 2F2 or 7D8. Accordingly, 2F2 and 7D8 have a unique capacity to lyse CD55/59 positive target cells.

In a separate experiment, Raji ceils were pre-incubated with saturating concentrations of anti-CD55 mAb (final concentration of 5 gg/ml) and anti-CD59 mAh 30 (final concentration of 5 pg/ml) to block the effects of these complement defense molecules. Human anti-CD20 antibodies were then added along with serum (20%) as above for 45 min at 37 °C. As shown in Figure 16B, the blockade of CD55 and CD59 molecules resulted in almost 100% lysis of Raji cells with human antibodies 2F2 or 7D8 whereas only a 25% increase in cell lysis was observed using rituximab. 35

Role of complement inhibitors I - Expression of surface molecules:

Since complement inhibitors such as CD55 and CD59 appear to play an important role in susceptibility to rituximab-induced CDC, an experiment was performed to determine -79- 2017204254 22 Jun2017 the expression of these molecules on the B-cell lines under investigation (Raji, Daudi, DOHH, ARH-77, and SU-DHL-4).

The cells were stained with FITC-conjugated anti-CD55, anti-CD59 and anti-CD20 antibodies and molecules expression was analyzed by flow cytometry. The 5 results are shown in the below Table 1.

Table 1

Expression CD20 CD55 CD59 ARH-77 ++ ++++ ++ Raji ++ .1.,,1,,.1- tTT DOHH .r|T„L IT +++ ++ SU-DHL-4 +++ + ++ Daudi ++ + +

Role of complement inhibitors II — Blockade of CD55 and CD59: To 10 further study the roles of CD55 and CD59 in anti-CD20-induced CDC, both complement inhibitor molecules were blocked by specific antibodies prior to induction of CDC (n=3). Raji cells were used because only partial lysis was induced by 2F2 alone. Raji cells (1 x 105 cells/50 μΐ) were pre-incubated with a concentration range of 2F2 and rituximab together with anti-CD55 (5 pg/ml) or ant:i-CD59 (5 μg/ml) antibodies for 10 15 min, before pooled NHS (20%) was added. At 45 min after induction of CDC, cells were resuspended in PI solution. Cell lysis (number of Pi-positive cells) was measured by flow cytometry. Figures 17A-C show the percentage of lysed (Pi-positive) cells as a function of antibody concentration, and show one experiment which is exemplary of three experiments. Figure 17A shows incubation of Raji cells with anti-CD55 antibody, 20 Figure 17B incubation of Raji cells with anti-CD59 antibody, and Figure 17C incubation of Raji cells with anti-CD55 and anti-CD59 antibodies.

As can be seen in Figure 17A, addition of anti-CD55 antibody did not influence 2F2 or rifuximab-induced CDC. Addition of anti-CD59 antibody increased susceptibility of the cells to both 2F2 and to rituximab with -30% (Figure 17B). 25 Addition of both anti-CD55 and anti-CD59 further enhanced anti-CD20-induced lysis of cells with -30% (Figure 17C).

Role of complement factors, as determined by flow cytometry /- Cl q binding: Anti-CD20 antibodies (2F2 and rituximab) and an isotype control antibody 30 were added to various B-cell lines. After 10 min incubation, NHS (1 vol/vol%) was added. After further incubation for 10 min at 37 °C and washing of the cells, the supernatant was discarded and the cell pellet was incubated with FITC-conjugated anti- -80- 2017204254 22Jun2017

Clq antibody. Data show mean fluorescence intensity of cells stained with Clq and are depicted in Figures 18A (Daudi), 18B (ARH-77), ISC (DOHH), and 1SD (Raji) (n=6). The results indicated antibody concentration-dependent increase in binding of Clq by 2F2, irrespective of the E-cell line investigated. Moreover, Clq binding by 2F2 was 5 always higher than binding by rituximab, in all cell lines tested. No increase in mean fluorescence was observed with the isotype control antibody (data not shown).

Role of complement factors, as determined by flow cytometry II -Complement activation via the classical route: Fixation of C4c to antibody-coated cells 10 is an indication of activation of complement activation via the classical route, Anti-CD20 antibodies (2F2 and rituximab) and an isotype control antibody were added to various B-cell lines. After 10 min incubation at 37 °C, NHS (1 vol/vol%) was added. After further incubation and washing of the cells, the supernatant was discarded and the cell pellet was incubated with FITC-conjugated anti-C4c antibody. Data show mean 15 fluorescence intensity of cells stained with C4c and are depicted in Figures 19A (Daudi), 19B (ARH-77), 19C (DOHH), and 19D (Raji) (n=6). Complement factor C4c fixation to 2F2 was demonstrated in all B-cell lines tested (n=3), with a maximum reached at ~1 pg/ml of antibody. Fixation of C4c after 2F2 binding was much higher than after rituximab, irrespective of the cell line tested. No increase in mean fluorescence was 20 observed with the isotype control antibody (data not shown). CDC in heat-inactivated serum: Cells (Daudi cells, ARH-77 cells or Raji cells) and antibodies (rituximab, 2F2, 2F2T, 11B8, and isotype control antibody HuMab-KLH IgGl) were pre-incuhated in a concentration range of anti-CD20 25 antibodies for 10 min, before NHS (active or heat-inactivated in a water hath at 57 °C at 30 min) was added. At 45 min after induction of CDC, cells were resuspended in PI solution. Cell lysis (number ofPI-positive cells) was measured by flow cytometry. No lysis of the cells was observed in the presence of heat-inactivated serum, irrespective of the cell-line and CD20-antibody used, no CDC was observed in the presence of heat-30 inactivated serum.

Example 7 ADCC of Human Antibodies Against CD20 AJDCC Assay I

Enrichment of human neutrophils: Polymorphonuclear cells (neutrophils, 35 PMNs) were enriched from heparinized whole blood. Blood was diluted twice in RPMI 1640 and was layered on Ficoll (Lymphocyte Separation Medium 1077 g/ml, 710 g, RT, 20 min; Bio Whittaker, cat. 17-829E, lot no. 0148 32) and centrifuged at 2000 rpm for 20 min. The mononuclear cell layer was removed, and erythrocytes within the pellet -81- 2017204254 22 Jun2017 containing neutrophils were hypotonically lysed using ice-cold NB4CI solution (155 mM NH4CI, 10 mM NaHC03: 0.1 mM EDTA, pH 7.4). The remaining neutrophils were washed twice and resuspended in RPMI1640 supplemented with 10% FCS (RPMI-10). 5 Enrichment of human peripheral blood mononuclear cells: Human blood was diluted twice in RPMI 1640 and blood cells were layered on Ficoll (Lymphocyte Separation Medium 1077 g/ml, 710 g, RT, 20 min; Bio Whittaker,

Cambrex Bio Science Venders, Venders, Belgium, cat, 17-S29B, lot no. 0148 32). Peripheral blood mononuclear cells (MNCs) were collected from the interphase, washed 10 and resuspended in RPMI 1640 culture medium supplemented with 10% FCS, 2 mM L- glutamine, 5 U/ml penicillin, 50 pg/ml streptomycin (all derived from Bio Whittaker) to which 25 mM HEPES (BioWhittaker) was added. ADCC set up: Target B-cells (freshly isolated B-cells or from B-cell 15 lines) were labeled with 20 pCi 5ICr (Amersham Biosciences, Uppsala, Sweden) for 2 hours. After extensive washing in RPMI-10, the cells were adjusted to 1 x 105 cells/ml.

Whole blood or isolated effector cells (50μ1; MNCs, PMNs) or plasma (50 μΐ), sensitizing antibodies (50 μΐ), and RPMI-10 (50 μΐ) were added to round-bottom microtiter plates (Greiner Bio-One GmbH, Frickenhausen, Germany). Assays were 20 started by adding target cells (50 μΐ) giving a final volume of 200 μΐ. For isolated effector cells, an effector to target (E:T) ratio of 40:1 was used. For whole blood, an amount of 33 vol/vol% was used corresponding to an estimated effector to target ratio of 40:1. After incubation (3 hours, 37 °C), assays were stopped by centrifugation, and Cr - release from triplicates was measured in counts per minute (cpm) in a scintillation 25 counter. Percentage of cellular cytotoxicity was calculated using the following formula: % specific lysis = (experimental cpm - basal cpm)/(maximal cpm - basal cpm) x 100 with maximal 51Cr release determined by adding perchloric acid (3% final 30 concentration) to target cells, and basal release measured in the absence of sensitizing antibodies and effector cells.

Statistics: Data were analyzed by one-way ANOYA, followed by Tukey’s multi comparison post-hoc test. Analysis was performed using Graph Pad 35 Prism (version 3.02 for Windows, Graph Pad Software, San Diego, CA, USA). -82-

Lysis of ARE-77 cells: In a first set of experiments, ARB-77 cells were used as target cells (Figure 20). Addition of 2F2 (n=3), rituximab (n=3) or 11B8T (n=I) resulted in MNC-mediated lysis of ARH-77 cells of approximately 50%. No specific lysis was observed in the presence of neutrophils. Addition of plasma (to evaluate the 5 role of complement) induced lysis of ARH-77 cells after incubation with 2F2, but not 2017204254 22Jun2017 after incubation with rituximab (p<0.05,2F2 vs. no antibody, ANOVA) or 11B8T. In the presence of whole blood, lysis of ARH-77 cells increased after incubation with 2F2 (p<0.05,2F2 vs. rituximab and 2F2 vs. no antibody, ANOVA), but not with rituximab. Specific lysis induced by rituximab was hr fact very low in the presence of whole blood. 10 11B8T induced cell lysis of approximately 25% (n=l) in the presence of whole blood, hi file absence of antibody, non-specific lysis of 10-15% was observed.

Lysis of B-CLL cells: In a second set of experiments, chronic B-lymphocytic leukaemia (B-CLL) cells obtained from B-CLL patients (n=12) were 15 subcloned for 5 rounds and then nsed as target cells in the experiment (Figure 21). In the absence of antibody, no specific lysis was observed, but addition of 2F2, 11B8T or rituximab (10 pg/ml) increased MNC-mediated specific lysis to 10-20% (p<0.001, ANOVA). Incubation of target cells with plasma and 2F2 induced specific lysis of B-CLL cells, whereas no specific lysis was observed with 11B8T or rituximab (pO.001, 20 ANOVA). Moreover, 2F2 mediated specific lysis of B-CLL cells after incubation in whole blood. No specific lysis of B-CLL cells by whole blood was observed with 11B8T (pO.Ol, ANOVA) or rituximab (pO.001, ANOVA). No specific lysis was observed in the presence of neutrophils.

Because rituximab was able to mediate effective ADCC but not CDC of 25 the tumor cells tested, it is likely that whole blood-induced B-cell lysis by 2F2 is mediated via complement.

Lysis of hairy cell leulcaemia (HCL) cells: In a third set of experiments lysis of HCL cells by 2F2,11B8T, and rituximab by ADCC or in tire presence of plasma 30 or whole blood was determined. Data are shown in Figure 22. Whereas neutrophils could not mediate ADCC irrespective of the mAh used, 11B8T was able to induce MNC-mediated lysis of HCL cells more efficiently than 2F2 (p<0.001, ANOVA) or rituximab (p<0.05, ANOVA). 2F2 and rituximab were not able to induce MNC-mediated lysis of HCL cells. Plasma-mediated lysis of the cells was strongly enhanced 35 with 2F2, as compared to rituximab (p<0.05, ANOVA), 11B8T (p<0.01, ANOVA) or without antibody (pO.001, ANOVA). When lysis induced by anti-CD20 in the presence of whole blood was studied, 2F2 induced complete lysis of cells, and was -83- superior to rituximab (p<0.01, ANOVA), 11B8T or no antibody added (pO.GOl, ANOVA). 2017204254 22 Jun2017

Lysis of B-ALL cells: Using cells from two patients the ability of 2F2 5 and rituximab to induce lysis B-ALL cells by ADCC or complement was investigated (Figure 23). As was observed in the previous experiments, 2F2 aid rituximab induced MNC-mediated ADCC of B-ALL cells to a similar extent But again 2F2 was able to induce plasma- and whole blood-mediated lysis of B-ALL cells, whereas rituximab was not. 10

Lysis of follicular lymphoma cells: When lysis of follicular lymphoma cells (n=2) was investigated, a different picture emerged (Figure 24). A minor PMN-mediated lysis of cells with 2F2 was observed, and both 2F2 and rituximab were not able to induce MNC-mediated ADCC. 11B8T was still able to induce MNC-mediated 15 lysis of approximately 20%, Although a relatively high plasma-mediated lysis was induced by rituximab, complete plasma-mediated lysis was observed with 2F2. Also with whole blood, complete lysis was observed with 2F2, whereas 70% lysis with rituximab. Minimal plasma- or whole blood-mediated lysis by 11B8T was observed. 20 Lysis of primary’ mantle cell lymphoma cells: Specific lysis of mantle cell lymphoma cells was more difficult to induce (n=l, Figure 25). Minimal or no lysis by 2F2,11B8T or rituximab was observed after addition of PMN or MNC and CD20 mAbs. However, 2F2 was still able to induce approximately 40% lysis by plasma or whole blood, whereas with rituximab only 10-20% of the cells were lysed·. 1 IB ST was 25 not able to induce lysis of primary mantle cell lymphoma cells.

Antibody concentration-dependent lysis of ARH-77 cells in whole blood: hr a further experiment (n~4) dose-dependency regarding the induction of ADCC on ARH-77 cells in the presence of whole blood was analyzed. As can be seen in Figure 30 26, titration of 2F2 induced a dose-dependent increase in the percentage of specific lysis (p<0.05: treatment-effect, two-way ANOVA) of ARH-77 cells. No specific lysis of ARH-77 ceils was observed with rituximab.

ADCC assay II 35

Preparation slCr-labeled target cells: ARH-77 cells and Raji cells were collected (3 x 106 cells) in RPMI++, spun down (1500 rpm; 5 min), resuspended in 140 μΐ 51Cr (Chromium-51; CJSll-lmCi, batch 12; 140 μΐ is about 100 pCi) and incubated .84- 2017204254 22 Jun2017 (37 °C water bath; 1 hour); After washing cells (1500 rpm, 5 min, in PBS, 3x), cells were resuspended in KPMI-H- and counted by trypan blue exclusion. Cells were brought at concentration of 2 x 104 eells/ml. 5 Preparation of effector cells: Fresh peripheral blood mononuclear cells (MNC) were isolated from 40 ml of heparin blood by Ficoll (Bio Whittaker; lymphocyte separation medium, cat 17-829E) via manufacturer’s instructions. After resuspension of cells in RPMD-f, cells were counted by trypan blue exclusion and adjusted to a concentration of 1 x 10s cells/ml. 10 ADCC set np: 50 μΐ RPMI++ was pipetted into 96 wells plates, and 50 μΐ of 51Cr-labeled targets cells were added. Thereafter, 50 μΐ of antibody was added, diluted in RPM1++ (final concentrations 10,1,0.1,0.01 pg/ml). Cells were incubated (RT, 10 min), and 50 μΐ effector cells were added, resulting in an effector to target ratio 15 of 50:1 (for determination of maximal lysis, 50 μΐ 5% Triton-X-100 was added instead of effector cells). Cells were spun down (500 rpm, 5 min), and incubated (37 °C, 5% C02) 4 hours). After spinning down the cells (1500 rpm, 5 min), 100 μ! of supernatant was harvested into micronic tubes, and counted in a gamma counter. The percentage specific lysis was calculated as follows: 20 % specific lysis = (cpm sample- cpm target cells only)/(cpm maximal lysis -cpm target cells only) x 100

Statistics: Data were analyzed by one-way ANOVA, followed by 25 Tulcey’s multi comparison post-hoc test. Analysis was performed using Graph Pad Prism (version 3.02 for Windows, Graph Pad Software, San Diego, CA, USA).

Antibody concentration-dependent lysis of AJRH-77and Raji cells: 2F2T and 11B8T were tested for their ability to induce ADCC of ARH-77 and Raji cells (n=3) 30 in comparison with rituximab. A dose-effect relation with CD20 mAbs was observed in ADCC of ARH-77 cells using MNC as effector cells (Figure 27). Both 2F2T and 11B8T induced specific lysis of ARH-77 cells which was maximal (50%) at 10 pg/ml of mAb. Rituximab induced only 25% lysis of target cells. Addition of the isotype control 35 antibody (HuMab-KLH) did not induce ADCC. No specific lysis was observed without addition of MNCs (data not shown). -85- 2017204254 22Jun2017

When Raji cells were used as target cells, a similar picture as with ARH-77 cells emerged (Figure 28). Both 2F2T and 11B8T induced MNC-mediated lysis of Raji cells, albeit that 2F2T seemed more potent than 11B8T at low concentrations. The maximum lysis reached with 2F2T and 11B8T was approximately 35%. Rituximab 5 induced MNC-mediated lysis of Raji cells, although only 20% of target cells were susceptible to rituximab. Addition of the isotype control antibody (HuMab-KLH) did not induce ADCC. No specific lysis was observed without addition of MNCs (data not shown). 10 Example 8 FRET and Triton-X insolubility analysis

Preparation of Cy3- and Cy5-conjugated mAb for fluorescence resonance energy transfer (FRET): Monoclonal antibodies were directly conjugated to bifunctional NHS-ester derivatives of Cy3 and Cy5 (Amserham Biosciences UK Ltd) as described in the manufacturer’s instructions. Briefly, mAb were dialyzed against 0.1 M 15 carbonate/bicarbonate buffer (pH 9). Thereafter, dye was dissolved in H2O, immediately added to 1 mg of the mAb, and incubated at room temperature in the dark for 45 min. The labeled mAbs were separated from the unconjugated dye by gel chromatography using a PDIO-Sephadex G25 column equilibrated in PBS. Molar ratios of coupling were determined spectrophotometrically from 8552 = 150/mM/cm for Cy3, 20 8550 = 250/mM/cm for Cy5, and ε28ο = 170/mM/cm for protein, and ranged from 5- to δ ιό Id excess dye:protein. FRET analysis: Daudi cells were resuspended at 5 x 106 cells/ml in PBS/0.1% BSA, and equimolar donor (Cy3)-conjugated and acceptor (Cy5)-conjugated 25 mAb were combined and added to the cell suspension (final concentration 10 pg/ml). Cells were incubated for 30 min in the dark, at 4 °C or 37 °C. Each experiment included cells labeled with donor- and acceptor-conjugated mAb after pre-incubation with a 20-fold molar excess of unconjugated mAb, and cells labeled with donor-or acceptor-conjugated mAh in the presence of equimolar unlabeled mAb. To assess the association 30 of labeled antigens, flow cytrometric FRET measurement was carried out using a FACScalibur (BD Biosciences). The fluorescence intensities at 585 nm (FL2) and 650 mn (FL3), both excited at 488 nm, and the fluorescence intensities at 661 nm (FL4), excited at 635 nm, were detected and used to calculate FRET according to the equitation below, where A is acceptor (Cy5), and D is donor (Cy3). All values obtained were 35 corrected for autofluoresence using the following formula: FRET = FL3(D,A)-FL2(D,A)/a-FL4(D,A)/b where a = FL2(D)/FL3(D), and b = FL4(A)/FL3(A) -86- 2017204254 22 Jun2017

Correction parameters were obtained using data collected from single-labeled cells, and side angle light scattering was used to gate out debris and dead cells. FRET between donor and acceptor mAb derivatives on dually labeled cells is expressed in terms of 5 acceptor sensitized emission at 488 run. Larger FRET values indicate closer physical association of the donor- and acceptor labeled antibodies or a higher density of acceptor-labeled mAh in the vicinity of donor-labeled mAb.

Assessment of raft associated antigen by Triton X-100 (TX) insolubility: 10 As a rapid assessment of the presence of antigen in raft microdomains, a flow cytometry method based on Triton X-100 (TX) insolubility at low temperatures was used, as described previously. In brief, Daudi cells were washed in RPMI/1% BSA and resuspended at 2.5 x 106/ml. The cells (100 μΐ) were then incubated with 10 pg/ml of FITC conjugated mAb for 15 min at 37 °C, washed in cold PBS/1% BSA/ 20 mM 15 sodium azide (PBS-BS), and the sample was divided in half. All samples were kept on ice throughout the remainder of the assay. One half was maintained on ice to allow calculation of 100% surface antigen levels, whilst the other was treated with 0.5% TX for 15 min on ice to determine to proportion of antigens remaining in the insoluble raft fraction. Cells were then maintained at 4 °C throughout the remainder of the assay, 20 washed once in PBS-BS, resuspended in PBS-BS and assessed by flow cytometry. To determine the constitutive level of raft association of the target antigens, cells were first treated with 0.5% TX for 15 min on ice and washed in PBS-BS prior to binding of FITC-labeled mAb.

As shown in Figures 29A, 29B, and 29, fluorescence resonance energy 25 transfer (FRET) analysis indicates clustering of CD20 upon incubation with 2F2 or 7D8. No such clustering was observed upon incubation with 11B8. These results are consistent with the TX treatment data, of. Figure 29C, (/. e., 2F2 and 7D8, unlike 11B8 remain with die insoluble fraction of the cell following binding) and support the concept that 2F2 and 7D8, upon binding, translocate CD20 into lipid raft compartment of the B 30 cell membrane.

As shown if Figure 30 (FRET values and s.e.m. of three experiments using one-way ANOVA followed by Tukey’s multi comparison post-hoc test) the FRET analysis indicates clustering of rituximab and 2F2, whereas no clustering was observed with 11B8T. These data are in agreement with the data obtained after treatment with 35 0.5% TX prior to binding of FITC-labeled mAbs as shown in Figure 31 (n-2). -87- 2017204254 22 Jun2017

Preparation of lipid raft fractions and Western blotting: Another way to examine the association of CD20 with lipid rafts, is to investigate the distribution of CD20 between the raft and non-raft membrane fractions using the sucrose gradient fractionation method as disclosed by Deans, J.P., et al., J Biol. Chem., 1998. 273(1): pp 5 344-348, except that Optiprep (Sigma) was used instead of sucrose. Monoclonal antibodies directed against CD20 (10 pg/ml) were allowed to bind to Daudi cells (1 x I07) for 20 min at 37 °C. Following this incubation, the cells were pelleted, washed twice with PBS and lysed in ice-cold lysis buffer (1.0% TX in MES-buffered saline (25 mM MES, pH 6.5,150 mM NaCI, 1 mM phenylmethylsulfonyl fluoride, 5 yg/ral 10 aprotinm, 5 /ig/ml leupeptin, 10 mM EDTA)). The cell pellet was resuspended thoroughly and incubated for 20 min on ice. Thereafter, the lysate was mixed with 400 μΐ cold 60% Optiprep (Sigma). The sample was overlaid with a 600 μΐ step of each 35%, 30%, 25%, 20%, 0% Optiprep in lysis buffer. Tire gradients were spun at 40.000 ppm at 4 °C for 18 hours. Six fractions from the top were collected, resolved on a 4-15% 15 SDS-PAGE gel, transferred onto nitrocellulose membranes and incubated with primary antibody (mouse anti-CD20 polysera; Serotec, UK), followed by HRP-conjugated secondary antibody (rabbit anti mouse-HRP; Jackson, Bar Harbor, Maine, USA). Blots were visualised using Supersignal West Dura extended duration substrate (Pierce, Wobum, MA, USA). 20 The results arc shown in Figure 32. As it can be seen, CD20 molecules are confined to the high-density fraction 5 (untreated cells). Cells treated with rituximab showed a distinct shift in CD20 distribution with a significant proportion in the lower density membrane fractions 2 and 3, coincident with the fraction where membrane rafts are expected to sediment. Cells treated with 2F2 also showed this shift to fractions 2 and 25 3. In contrast, cells treated with. 11B8T for 20 min showed a similar distribution to untreated ceils, with CD20 molecules in fraction 5. In conclusion binding to 2F2 and rituximab induces a shift of CD20 molecules to the lower density membrane fractions, whereas binding to 11B8T does not. 30 Example 9 Apoptosis of Burltitt Cell Lines with Human Antibodies Against CD20

Apoptosis: Daudi cells, 0.5 x 106 in 1 ml tissue culture medium, were placed into 24-well flat-bottom plates with 1 or 10 pg/ml mAb or control antibodies, and incubated at 37 °C. After 20 hours, cells were harvested, washed in Annexin-V-FITC 35 binding buffer (BD biosciences) and labeled with Aimexin V-FITC (BD biosciences) for 15 min in the dark at 4 °C. The cells were kept at 4 °C until analysis. Each sample (150 μΐ) was added to 10 μΐ of PI solution (10 μg/ml in PBS) in aFACS tube. The mixture was assessed immediately by flow cytometry using a FACScalibur flow cytometer with -88- 2017204254 22 Jun2017

CellQuest pro software (BD Biosciences, Mountain view, CA). At least 10,000 events were collected for analysis.

Induction of apoptosis in Daudi cells: Daudi cells were incubated for 20 5 hours in the presence of human antibodies against CD20 (1 pg/ml) (without the addition of a secondary cross-linking antibody). Induction of apoptosis was assessed by AnnexinV/PI staining using flow cytometry.

As shown in Figures 33A-G, 11B8 shows clear evidence of inducing apoptosis (similar to that induced by an anti-IgM antibody). 2F2 and 7D8 did not induce 10 apoptosis of Daudi cells. An apoptosis-inducing mouse anti-CD20 antibody, AT80, was used as a control

Induction of apoptosis in Raji cells: Induction of apoptosis of Raj i cells was tested with a concentration range of CD20 mAbs. Figure 34 shows the percentage 15 of-annexm-V-positive cells. As can be seen from Figure 34, the positive control mouse anti-human CD20-mAb, Bl, induced a concentration-dependent increase in apoptosis of Raji cells with a maximum of approximately 70% at 10 pg/ml mAb. Also 11B8 was a strong inducer of apoptosis, resulting in apoptosis of Raji cells with a maximum of 53.4% at 10 pg/ml mAb. On the other hand, 2F2 and rituximab were very poor in 20 inducing apoptosis of Ra ji cells, with slightly elevated levels of apoptosis compared to negative control levels.

Induction of apoptosis in Daudi cells: The same picture emerged when Daudi cells were used as target cells, after addition of 1.0 pg/ml CD20 mAb (Figures 25 35A and 35B). Data in Figure 35 A show the total of annexin-V positive cells, and data in Figure 35B (the X-axis showing annexin-V, and the Y-axis showing PI) show the percentages of Daudi cells in early apoptosis (annexin-V positive and PI negative) and late apoptosis (annexin-V positive and PI positive). Again, BothBl (65.9%) and 11B8T (56.3%) were strong inducers of apoptosis (Figure 36), when used at a concentration of 30 1.0 /xg/nal. Addition of 2F2T resulted in a low level of apoptotic Daudi cells (17%).

Addition of rituximab resulted in approximately 29% apoptosis of Daudi cells. Addition of isotype control antibody HuMab-KLH did not induce apoptosis of Daudi cells (6%). -89- 2017204254 22Jun2017 5 10

Example 10 Homotypic Adhesion of Cells with Human Antibodies Against CD20

Homotypic aggregation correlates with induction of apoptosis.

Therefore, the ability of the anti-CD2G mAbs to induce homotypic aggregation of B cells was investigated

Homotypic aggregation of Ramos-EHRJB cells: Ramos-EHRB cells (0.5 x 106 in 1 ml tissue culture medium) were incubated at 37 °C for 4 hours in the presence of anti~CD20 antibodies 11B8, 2F2, or 7D8 (without cross-linking) and induction of homotypic adhesion was assessed by light microscopy (as described above).

As shown in Figures 36A-E, 1 IBS caused extensive aggregation of Ramos-EHRB cells (similar to the aggregation caused by murine anti-CD20 antibody, AT80). 2F2, and 7D8 did not induce homotypic aggregation of Ramos cells.

Homotypic aggregation of Daudi cells: Daudi cells were placed into 24-15 well flat-bottom plates with 1 or 10 pg/ml anti-CD20 mAbs or control antibody, and incubated at 37 °C for 4 hours. The extent of homotypic aggregation, was determined by light microscopy. As can be seen from Figure 37,2F2 hardly induced homotypic aggregation of Daudi cells, with 1.0 pg/ml (and 10 pg/ml, data not shown). Rituximab gave little homotypic aggregation of Daudi cells. In contrast, the B1 antibody was a 20 strong inducer of homotypic aggregation.

Example 11 Immunotherapy Using Human Antibodies Against CD20

Therapy with high dose (100 pg) 2F2 and 7D8 of SCID mice challenged with Daudi cells: The SCID mice were obtained from Harlan UK Ltd., Blackthorn, 25 Oxon, UK, and bred and maintained under pathogen free conditions. Daudi ceils (2.5 x 106) were injected i.v. into the tail vein of cohorts of 12-16 weeks old SCID mice, followed 7 days later by injection of 100 pg of 2F2 or 7D8 via the same route. Animals were sacrificed upon presentation of limb paralysis, according to the instructions of the animal ethics committee. As shown in Figure 38, survival of the mice is prolonged after 30 treatment with 2F2 or 7D8.

Therapy with high dose (100 pg) 2F2 and rituximab of SCID mice challenged with Tanoue cells: Tanone cells (2.5 x 106 in 200 μΐ PBS) were injected i.v. into the tail vein of cohorts of 12-16 week old SCID mice (Harlan UK Ltd., Blackthorn, 35 Oxon, UK) followed 7 days later by the injection of 100 pg (in 200 pi PBS) of anti- CD20 mAh via the same route. In this experiment, 2F2 was compared to rituximab and BL Animals were sacrified upon presentation of rear-limb paralysis. The results are shown in Figure 39. At day 39, die first two control mice died, and death within this -90- 2017204254 22Jun2017 group was complete at day 54. Only one mouse died within this time interval following 2F2 treatment and survival was considerably increased for the other mice in this group. One mouse died 81 days following injection of the tumor cells and the remaining mice (60% of the total number) survived beyond the termination of the experiment at 100 5 days post tumor challenge. Rituximab in contrast only increased survival for 2 out of 5 mice (dying at 66 and 83 days post challenge) and none of the mice survived until the end of the experiment. In the Bl-group, the survival of SCID mice was similar to that in the 2E2 group, with two mice dying on day 48, and one mouse on day 76. In this group, forty percent was alive at the time the experiment was terminated. 10

Dose response of2F2 and rituximab treatment of SCID mice challenged mth Daudi cells: To assess the efficacy of 2F2 in comparison to rituximab in protection against tumorigenesis, a dose titration was performed in therapy of SCED mice challenged with Daudi tumor cells. Daudi cells express more CD20 than Tanoue 15 cells and are more sensitive to killing in vitro. 10 groups of SCID mice (4 per group) and 1 control group (5 SCID mice) were injected with 2.5 x 10s' Daudi cells (in 200 μΐ PBS) i.v. on day 0, and then treated with 20,5, 2, 0.5 or 0.1 μg (in 200 μΐ PBS) rituximab, 2F2 or PBS (control) i.v. on day 7. Animals were sacrificed upon presentation of rear-limb paralysis. The results are shown in Figure 40. 20 In the control group, all mice died within the time interval of26-29 days.

However, a clear· dose-effect relation was observed with 2F2 (Figure 40, upper graph). Whereas no effect was observed with doses of 0.1 pg and 0.5 pg 2F2, as little as 2 pg 2F2 substantially extended survival until day 41, 5 pg 2F2 extended survival until day 47, and 20 pg 2F2 extended survival even until clay 50. 25 In contrast, rituximab even tested at the highest dose of 20 pg only slightly increased survival and no dose-effect relation was therefore observed at the lower concentrations tested (Figure 40, lower graph).

Therapy of SCID mice with Daudi tumors by 11B8T and SI: Daudi cells 30 (2.5 x 106) in 200 pi PBS were injected i.v. into the tail vein of cohorts of 12-16 week old SCID mice, followed 7 days later by the injection of 100 pg 11B8 or B1 in 200 pi PBS via the same route. Animals were sacrificed upon presentation of rear-limb paralysis. In control mice treated with PBS, all mice died within a time interval of 35-53 days (Figure 41). 1 IB ST treatment strongly protected the mice, with mice dying 35 between 72 and 98 days post tumor challenge, hi the B1-treatment group, most mice survived until day 98 and 40% of the mice survived beyond the end of the experiment, i.e., day 100. -91- 2017204254 22 Jun2017

Example 12 Evaluation of anti-CD20 antibodies in a Daudi-luc xenograft model using SCID mice

The therapeutic efficacy of anti-CD20 antibodies was evaluated in a mouse model in which disseminated outgrowth of human B-cell tumor cells is followed 5 using external optical imaging. In this model tumor cells are transfected with firefly luciferase. Upon administration of luciferin (Molecular Probes, Leiden, The Netherlands) to the mice the labeled cells can be detected in vivo by bioluminescent imaging using a highly sensitive CCD camera, cf. Wetterwald el al. (2002) American Journal of Pathology, 160(3):1143-1153. 10 Daudi cells were transfected with gWIZ luciferase from Gene Therapy

Systems (San Diego, CA) and cultured in RPMI with 10% FCS, Pen/Strep, Sodium Pyruvate and 1 ug/ml puromycin (Sigma). Cells were analysed for luciferase expression (expressed in RLU/1 x 105 cells) in a luminometer and for CD20 expression by FACS. 2.5 x 106 luciferase-transfected Daudi cells/mouse were injected i.v. into SCID mice. 15 Eight days after inoculation, the mice received a single dose (10 /xg) treatment of 2F2T, 11B8T, rituximab, B1 or isotype control antibody (huIgGl) (6 mice per treatment group). For imaging, mice were anesthetized by i.p. injection of a mixture of ketamine /xylazine /atropine. Synthetic D-Luciferin (sodium salt, Molecular Probes) was given i.p. at a dose of 25 mg/ml. Mice were then placed in a light tight box and after 3 min, 20 imaging was started using a VersArray 1300B liquid nitrogen cooled CCD detector (Roper Scientific). Photons emitted from the luciferase were counted over an exposure period of 5 min. Under illumination black and white images were made for reference. MetaVue software (Universal Imaging Corp) was used for data collection and image analysis. Statistical significance of differences between groups was established using 25 one-way analysis of variance with a Newman-Keuls post test using GraphPad PRISM version 3.02 (Graphpad Software Inc).

Imaging from the back side was performed at one-week intervals. On day 8, the day of treatment, light emission was only detected at the inoculation sites in the tail. Tumor formation at distant sites was detected on day 14 in all mice from the 30 isotype control group (huIgGl) and in one mouse from the rituximab group. In the following weeks light emission steadily increased. Figure 42 gives the images of all mice made on day 39 (31 days after treatment), in which biohuninescence is represented in red color (the dark areas in the mice) (light intensity >50 photons per 5 min) as overlay on the black and white body image of the mice. The tumor mass in each mouse 35 was quantified on day 25, 32, 39, and 46 by integrating the light signals over the body surface, cf. Figure 43. The fastest tumor growth was observed in the isotype control group. Treatment with rituximab gave significant inhibition of tumor growth. However, -92- tumor growth inhibition by 2F2T, 11B8T and B1 was significantly more potent (see below Table 2 for significance levels. 2017204254 22 Jun2017

Table 2

Significance levels of differences in integrated light intensity between groups at different time points

Day 25 Day32 Day 39 Day 46 B1 vs. rituximab P > 0.05 P < 0.05 P < 0.01 P< 0.001 B1 vs. 11B8T P > 0.05 P > 0.05 P > 0.05 P > 0.05 B1 vs. 2F2T P > 0.05 P > 0.05 P > 0.05 P > 0.05 Bl. vs. huIgGl P < 0.001 P< 0.001 P< 0.001 rituximab vs. 11B8T P > 0.05 P < 0.05 P<0.01 P< 0.001 rituximab vs. 2F2T P > 0.05 P > 0.05 P > 0.05 P < 0.001 rituximab vs. huIgGl P< 0,001 P > 0.05 P < 0.05 11B8T vs. 2F2T P > 0.05 P > 0.05 P > 0.05 P > 0.05 11B8T vs. huIgGl P < 0.001 P< 0.001 P < 0.001 2F2T vs. huIgGl P < 0.001 P<0.01 P<0.01 10 Example 13 Pilot and Pharmacokinetic study in cynomolgus monkeys

The objective was to detennine the pharmacokinetic pattern and pharmacological effects of 2F2 in cynomolgus monkeys (approximately 2 years old; weight range of 2.1-2.6 kg) following once daily intravenous in&amp;sion administrations (via the saphenous vein) for 4 consecutive days. The study also compared the 15 pharmacological effects of rituximab in order to determine its equivalent potential. For this purpose, 6 male and 6 female cynomolgus monkeys were assigned to 6 dose groups that received 2F2 or rituximab at dose levels of 1.25, 6.25 and 12.5 mg/kg/day at a constant dose volume of 10 ml/lcg for 4 consecutive days, in total 5,25 and 50 mg/kg, respectively. On completion of the last dose administration, the animals were retained 20 for a post dose observation period of 130 days. The practices and procedures adopted during this study were consistent with the OECD Principles of Good Laboratory Practice as set forth by the United Kingdom Department of Health. All animals were observed at regular intervals for signs of ill health or reaction to treatment and were subjected to a physical examination. Laboratory investigations of haematology, coagulation, clinical 25 chemistry and mine analysis were performed during the study. Blood samples and lymph node biopsies were obtained (from the superficial lymph nodes) for flow cytometry analysis throughout the dosing and post dose observation periods. The following cell phenotypes were analysed by flow cytometry: CD3, CD4, CDS, CD20 -93- 2017204254 22 Jun2017 and CD21. On completion of the post dose observation period tine animals were sacrificed and subjected to a detailed necropsy.

There were no adverse clinical signs or any findings that were considered to be related to treatment with 2F2 or rituximab. Figures 44 and 45 shows the flow 5 cytometry analysis of CD20 and CD21 expressing cells in peripheral blood of treated animals, respectively. Figure 46 shows the flow cytometry analysis of CD20 expressing cells in lymph nodes. Together, both phenotypes analysed during the study indicate a strong and efficient B cell depletion after administration of 2F2 and rituximab at 6.25 l mg/kg/day (25 mg/kg in total) and 12.5 mg/kg/day (50 mg/kg in total). In addition, data 10 shows that repopulation of CD20 expressing cells in the lymph nodes and peripheral blood of 2F2 treated animals restarted approximately at day 75 post dosing of 25 mg/kg and 50 mg/lcg, i.e.f markedly later than in rituximab treated animals.

Furthermore, Figures 47A-C show the flow cytometric analysis of CD20lowCD23~CD40htgh expressing cell subpopulations in the peripheral blood (Y. 15 Vugmeyster et al (2003) Cytometry 52A:101-109).

Peripheral blood cells obtained from either 2F2 or rituximab treated monkeys at dose levels of 1.25 mg/kg (Figure 47A), 6.25 mg/lcg (Figure 47B), and 12.5 mg/kg (Figure 47C) once daily by intravenous infusion administrations for 4 consecutive days were incubated with anti-human CD20 F1TC murine monoclonal antibody 20 (Coulter) at room temperature for 10 min. Afterwards, count beads were added together with PBS and the cells were washed twice (300 g for 10 min), followed by immediate analysis of CD20lowCD23+CD40high vs. CD20ili8tl CD23+CD40!ovv expressing cell subpopulation in a flow cytometer (Beckman Coulter). Results of CD2Qlow CD23''’CD40h,eh cells shown are expressed as cells per μΐ. As can be seen from the 25 Figure 47 2F2 was capable of inducing a complete and longer depletion of CD20lowCD23+CD40ll,gh expressing cells compared to rituximab.

Example 14 Epitope mapping using site-directed mutagenesis

Epitope mapping studies using a mutagenesis approach have indicated 30 that alanine at position 170 (A170) and proline at position 172 (P172) in the second extracellular loop are critical for the recognition of human CD20 by known anti-CD20 antibodies. In studies by Deans and colleagues (MJ. Polyak, et al, Blood, (2002) 99(9): pp 3256-3262; M.J. Polyak, et al,./. Immunol., (1998) 161(7): pp 3242-3248) the binding of all anti-CD2Q mAbs tested was abrogated by changing A170 and P172 into 35 the corresponding murine CD20 residues SI 70 and S172. Some heterogeneity in the recognition of the AxP epitope has been recognized however as most antibodies like rituximab recognize murine CD20 with S170 and SI72 mutated to the human A170xP172 sequence whereas some others require additional mutations immediately N~ -94- 2017204254 22 Jun2017 terminal of the AxP sequence. To verify whether the A170xP172 motive is also important for the binding of the antibodies according to the invention the AxP sequence was mutated into SxS using site-directed mutagenesis (AxP mutant = A170S, P172S), cells were transfected with the AxP mutant and wild-type (WT) CD20 DNA, and the 5 binding characteristics of the anti-CD20 rnAbs were compared.

Further mutants were prepared, P172S (proline at position 172 mutated to serine), N166D (asparagine at position 166 mutated to aspartic acid), and N163D (asparagine at position 163 mutated to aspartic acid), using site-directed mutagenesis to evaluate whether the mutated, amino acid residues are important for binding of the 10 antibodies of the invention.

To examine this, a CD20 expression vector was constructed by amplifying the CD20 coding sequence using suitable primers introducing restriction sites and an ideal Kozak sequence for optimal expression. The amplified fragment was digested and ligated in the expression vector pEE'13.4. After transformation in E. coli, colonies were screened 15 for inserts and two clones were selected for sequencing to confirm the correct sequence. The construct was named pEEl3.4CD20HS.

Mutagenesis was performed to introduce the AxP mutation and to introduce 20 mouse mutations in the extracellular loop regions of human CD20. Mutagenesis was checked by restriction enzyme digestion and sequencing. The 20 constructs were transiently transfected in CHO cells (for AxP mutations) or HEK293F cells and analyzed 24 or 48 hours post-transfection using flow cytometry.

Oligonucleotide PCR Primers: Oligonucleotide primers were synthesized and quantified by Isogen BV (Maarssen, The Netherlands). Primers were 25 reconstituted in water in a concentration of 100 pmol/μΐ and stored at -20 °C until required. A summary of PCR and sequencing primers is shown in Table 3.

Optical density determination of nucleic acids: Optical density was determined using air Ultrospec 2100 pro Classic (Amersham Biosciences, Uppsala, 30 Sweden) according to the manufacturer's instructions. The DNA concentration was measured by analysis of the ODjeonm, where one ODzeonm unit = 50 pg/ml. The reference solution was identical to the solution used to dissolve the nucleic acids.

Plasmid DNA isolation from E. coli culture: Plasmid DNA was isolated 35 from E. coli cultures using kits from Qiagen according to the manufacturer's instructions (Westburg BV, Leusden, The Netherlands). For 'bulk' plasmid preparation either a Hi-Speed plasmid Maxi ldt or a Ηί-Speed plasmid Midi kit were used (Qiagen). For a small scale plasmid preparation (i.e., 2 ml of E coli culture) a Qiaprep Spin Miniprep Kit -95- 2017204254 22Jun2017 (Qiagen) was used and the DNA eluted in 50 μΐ TE (Tris-HCl 10 mM pH 8.0, EDTA 1 mM). PCR amplification: PCR reactions were performed according to the 5 manufacturer's instructions for the Pfu-Turbo® Hotstart DNA polymerase (Stratagene, Amsterdam, The Netherlands). Each 20 μΐ reaction contained 1 x PCR reaction buffer, 200 μΜ mixed dNTPs, 6.7 praol of each fox-ward and reverse primer, approximately 1 ng template DNA and 1 unit of Pfu-Turbo® Hotstart DNA polymerase. PCR reactions were performed on a T-gradient Thermocycler 96 (Biometra GmbH, Goettingen, 10 Germany) using a 30 cycle program of: +95 °C for 2 min, followed by 30 cycles of: +95 °C for 30 sec, anneal: a gradient of45-65 °C for 30 sec and extension: +72 °C for 2 min, followed by a final extension step of 10 min at 72 °C and subsequent storage at 4 °C.

The completed reactions were analysed by agarose gel electrophoresis. 15 Agarose gel electi-ophoresis: Agarose gel electrophoresis was performed according to Sambroolc (Molecular Cloning Laboratory Manual, 3rd edition) using gels of 50 ml, in 1 x Tris/acetic acid/EDTA (TAE) buffer. DNA was visualized by the inclusion of ethidium bromide in the gel and observation under UV light. Gel images were recorded by a CCD camera and an image analysis system (GeneGnome; Syngene, 20 Cambridge, UK).

Restriction enzyme digestions: Restriction enzymes were supplied by New England Biolabs (Beverly, MA) and used according to the supplier's recommendations. In general, 100 ng was digested with 5 units of enzyme(s) in 25 appropriate buffer in a final volume of 10 μΐ, Reaction volumes were scaled up as appropriate. Digestions were incubated for a minimum of 60 min at the manufacturer's recommended temperature.

Eor fragments requiring double digestions with restriction enzymes which have incompatible buffer or temperature requirements, digestions were performed 30 sequentially so as to offer favourable conditions for each enzyme in turn.

Alkaline phosphatase treatment: Shrimp alkaline phosphatase (USB, Cleveland, OH) was used according to the supplier's recommendations. Alkaline phosphatase removes 5’-phosphate groups from the ends of DNA fragments thereby 35 preventing self-ligation. This is of particular relevance when self re-ligation of a DNA fragment could result in a replication-competent vector. The enzyme is active in most restriction enzyme buffers and was added as appropriate. After the digestion, the enzyme was inactivated by raising the temperature to 70 °C for 15 min. -96- 2017204254 22 Jun2017

Purification of PCR and restriction enzyme reaction products: Purification was carried out using the mini~elute PCR Purification kit (supplied by Qiagen), according to the manufacturer's instructions. Briefly, DNA samples were 5 diluted in 5 volumes of binding buffer I (Qiagen) and loaded onto a mini-elute column within an Eppendorf centrifuge tube. The assembly was centrifuged in a bench-top microcentrifuge. The column was washed twice with buffer Π (Qiagen): Following buffer application, the assembly was centrifuged and the flow-through was discarded. The column was dried by centrifugation in the absence of added buffer. DNA was 10 eluted by adding elution buffer to the column and the eluate collected by centrifugation. Isolated DNA was quantified by UV spectroscopy and quality assessed by agarose gel-electrophoresis.

Isolation of DNA fragments from agarose gel: Where appropriate (z.e., 15 when multiple fragments were present), digested DNA samples were separated by gel electrophoresis and the desired fragment excised from the gel and recovered using the QIAEXII gel extraction kit (Qiagen), according to the manufacturer's instructions. Briefly, DNA bands were excised from the agarose gel and melted in an appropriate buffer at +55 °C. QIAEX II resin was added and incubated for 5 min. QIAEX Π resin 20 was pelleted by a short centrifugation step (1 min, 14000 g, RT) and washed twice with 500 μΐ of wash buffer PE. The final pellet was dried in a hood and DNA was eluted with the appropriate volume of TE and temperature (depending on the size of the DNA).

Ligation of DNA fragments: Ligations were perfonned with the Quick 25 Ligation Kit (New England Biolabs) according to the manufacturer's instructions. For each ligation, the vector DNA was mixed with approximately 3-fold molar excess of insert DNA such that the total amount of DNA was lower than 200 ng in 10 μΐ, with volume adjusted with water as appropriate. To this was added 10 μΐ 2 x Quick Ligation Buffer and 1 μΐ Quick T4 DNA ligase and the ligation mix was incubated for 5-30 min 30 at room temperature.

Transformation of DNA into bacteria: Samples of DNA were used to transform One Shot DH5a-TlR competent E. coli cells (Invitrogen, Breda, The Netherlands) using the heat-shock method according to the manufacturer's instructions. 35 Briefly, 1-5 μΐ of DNA solution (typically 2 μΐ of DNA ligation mix) was added to an aliquot of transformation-competent bacterial cells and the mixture incubated on ice for 30 min. The cells were then heat-shocked by transferring to a waterbath at 42 °C for 30 sec followed by a further incubation on ice for 5 min. Cells were left to recover by -97- 2017204254 22 Jun2017 incubation in a non-selective culture medium (SOC) for 1 hour with agitation at 37 °C and were subsequently spread onto agar plates containing appropriate selective agent (ampicillin at 50 pg/ml). Plates were incubated for 16-18 hours at +37 °C or until colonies of bacteria became evident. 5

Screening of bacterial colonies by PCR: B acterial colonies were screened for the presence of vectors containing the desired sequences using the PCR colony screening technique. 20 μΐ of PCR reaction mix containing 0.5 volumes of HotStarTaq Master Mix (Qiagen), 4 pmol of the forward and reverse primers and 10 completed with water was added to a PCR tube. A colony was lightly touched with a 20 μΐ pipet tip, once touched in 2 ml LB in a culture tube (for growing bacteria containing the corresponding plasmid) and resuspended, in the 20 μΐ PCR mix. PCR was performed on a T-gradient Thermocycler 96 (Biometra) using a 35 cycle program of: +95 °C for 15 min, followed by 35 cycles of: +94 °C for 30 sec, anneal: 55 °C for 30 sec and 15 extension: +72 °C for 2 min, followed by a final extension step of 10 min at 72 °C and subsequent storage at 4°C. The completed reactions were analyzed by agarose gel electrophoresis. See Table 3 for details of primer pairs used for colony PCR. DNA sequencing: Plasmid DNA samples were send to AGOWA 20 (Berlin, Germany) for sequence analysis. Sequences were analyzed using the VectorNTI software package (Informax, Frederick, MD, USA).

Table 3

Name Application Length OHgo Sequence CD20P172S CD20 mutagenesis 36 tggggagtttttctcagaggaattcgatggttcacagttgta CD20N166D CD20 mutagenesis 39 TGTAACAGTATTGGGTAGATGGG CD20N163D CD20 mutagenesis 36 AATCATGGACATACTTAATATTA cd20exfor CD20 construction 41 TATAGCCCGGGGCCGCCACCATGACAACACCCAGAAATTCA cd20exrev CD20 construction 38 GCOTCTCATGTACATIAAGGAGAGCTGTCATTTTCTAT pecl3,4seqrcv2 Colony PCR 23 TCGGACATCTCATGACTTTCTfT pConKseql Colony PCR 23 GTAGTCTGAGCAGTACTCGTTGC cd20hsapmutr (AxP) CD20 mutagenesis 42 TGGGGAGTTTTTCTCAGAGGAATTCGATGGTTCACAGTTGTA cd20hsapmutf (AxP) CD20 mutagenesis 42 tacaactgtgaaccatcgaattcctctgagaaaaactcccca CD20seq2 CD20 sequencing 23 TGTAACAGTATTGGGTAGATGGG cd20seql CD20 sequencing 23 AATCATGGACATACTTAATATTA -98- 2017204254 22 Jun2017

Mutagenesis: The mutagenesis was performed, using either the QuikChange® XL Site-Directed Mutagenesis kit (Cat 200517-5, Lot 1120630,

Stratagene Europe) according to the manufacturer’s instructions.

Mutagenesis reactions were concentrated using ethanol precipitation and 5 transfonned into either oneshot DH5&amp;-T1R competent E. coli cells or electroporated into ElectroTen-Blue® Electroporation-Competent Cells. Colonies were checked by colony PCR and restriction digestion prior to transfection. HEK293F cell transfection: HEK293F cells were obtained from 10 Invitrogen and transfected according to the manufacturer’s instructions, using 293fectin. The HEK293F cells were used for all the single mutant sequences, CHO cell transfection: CHO cells grown to approximately 95% confluence were transiently transfected with CD20 wild-type, mutant cDNA or a 15 combination of both constructs, using lipofectamine 2000 (M668-019, Invitrogen, Breda, Netherlands). To this end, 24 pg precipitated DNA was diluted (1 pg/pl) in 500 μΐ optimem, in ratios of AxP 100% : WT 0%; AxP 33.3% ; WT 66.6%; AxP 66.6% : WT 3 3.3%; AxP 0% :WT 100%. For each transfection 24 μΐ lipofectamine was diluted in 500 μΐ optimem. Then, the diluted lipofectamine was incubated (RT, 5 min), and the 20 diluted DNA combined with the diluted lipofectamine. After gently mixing and incubating the solution (RT, 20 min), 1000 μΐ DNA/lipofectamine was added to the CHO cells, thoroughly mixed and incubated for 48 hours at 37 °C, 5% CO2. Two days after transfection of CHO cells, cells were washed twice with FACS buffer (PBS supplemented with 0.1% BSA and 0.002% NaN3). CHO cells were treated with 25 trypsin/EDTA (Gibco BRL, Life Technologies, Paisley, Scotland) and lifted off the culture plates.

Anti-CD20 Antibody binding: HEK293F cells and CHO cells were taken up in PBS in a concentration of 2 x 106/ml, and added to round bottom plates (1 x 30 105/well). Then, 50 μΐ CD20 mAh was added, in serial dilutions of 10, 5,2.5, or 0 pg per well (4 °C, 30 min). After washing in FACS buffer (PBS supplemented with 0.1 % BSA and 0.002% NaNa), the cells were analyzed on a flow cytometer (Becton Dickinson, San Diego, CA, USA), and 5,000 events per sample were acquired at high flow rate. 35 As can be seen from Figures 48A-E, all anti-CD2G mAbs bound efficiently to CHO cells expressing WT CD20. As expected, rituximab did not bind the AxP mutant (Figure 48A), and B1 bound this mutant poorly (Figure 48D). Both 2F2 and 11B8 in contrast bound to WT and AxP mutant CD20 equally well (Figure 48B and -99- 2017204254 22 Jun2017

Figure 48C). Titrating the amount of WT CD20 on the surface indeed titrated the binding of rituximab and B1. Both 2F2 and 11B8 again were insensitive to the absence or presence of the mutation.

This study indicates that the binding of 2F2 and 1 IB 8 to human CD20 is 5 insensitive to mutations at amino acid positions 170 and 172. 2F2 and 1 IBS therefore represent a new class of CD20 mAbs recognizing a novel CD20 epitope.

Figure 49A shows percentage binding of 2F2,11B8T, B1 or rituximab to mutant P172S vs. WT CD20, Figure 49B shows percentage binding of 2F2T, 11B8T,

Bl, CAT (CAT 13.6E12, a mouse monoclonal IgG2A anti-CD20 antibody, 10 Diatec.Com), a control isotype antibody (KLH), or rituximab to mutant CD20 (AxP) vs. WT CD20.

For the mutant wherein asparagine at position 166 has been replaced with aspartic acid (CD20N166D) 2F2 showed very low binding, whereas Bl, rituximab and 11B8T were able to bind, see Figure 49C. In a similar experiment CAT 13.6E12 and 15 rituximab were able to bind to CD20N166D, whereas 2F2T only showed very low binding, see Figure 49D. For the mutant wherein asparagine at position 163 has been replaced by aspartic acid (CD20N163D) again rituximab, 11B8T, and Bl were able to bind to CD20N163D, whereas 2F2 and 2F2T only showed very low binding, see Figure 49E. In a similar experiment CAT 13.6E12 and rituximab were able to bind to 20 CD20N163D, whereas 2F2T only showed very low binding, see Figure 49F.

These experiments indicate that 2F2 and 1 IBS bind to different epitopes.

Example 15 Epitope mapping using Pepscan method

Synthesis ofpeptides; 7-, 9-, and 15-mer peptides were synthesized 25 according to standard methods. In some cases chemical linkage of the legs of a 15-mer peptide helps to identify amino acid sequences of a potentially discontinuous epitope. According to known procedures (H.M. Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81:3998; J.W. Slootstra et al. (1996) Mol. Divers. 1:87; and WO 01/60769), 7-, 9-, and 15-mer peptides were synthesized that could be possible binding sites or epitopes 30 involved in binding of 2F2 or 11B8 to the human CD20 molecule. The 9- and 15-mers were synthesized as loops and screened using credit-card format mini-PEPSCAN cards (455 peptide foimat/card). In all looped peptides amino acids at varied positions were replaced by a cysteine (e.g., acetyl-XCXXXXXXXXXXXCX-minicard). The peptides were synthesized using standard Fmoc-chemistry and deprotected using TFA with 35 scavengers. Subsequently, the deprotected peptides were reacted on the microarray with an 0.5 mM solution of l,3-bis(bromomethyl)benzene in ammonium bicarbonate (20 mM, pH 7.9), supplemented with acetonitrile (1:1 (v/v)). The microairays were gently shaken in the solution for 30-60 min, while completely covered in the solution. Finally, -100- 2017204254 22 Jun2017 5 10 15 the micro arrays were washed extensively with excess of Millipore H2O and sonicated in disrupt-buffer containing 1% sodium dodecylsulfate, 0.1% j3-mercaptoethanol, in PBS (pH 7.2) at 70 °C for 30 min, followed by sonication in millipore H?P for another 45 min. Subsequently, the microwells were ready for screening in an ELISA-assay. Pepscan ELISA-assay: The 455-well credit card-format polyethylene cards, containing the covalently linked peptides, were incubated with serum (diluted 1:1000 unblocking solution which contains 5% horse serum (v/v) and 5% ovalbumin (w/v)) ¢4 °C, over night). After washing, the peptides were incubated with anti-human antibody peroxidase (dilution 1:1000, 1 hour, 25 °C), and after washing the peroxidase substrate, 2,2'-azino-di-3-ethylbenztMazoline sulfonate and 2 μΐ/ml 3% H2O2 were added. After one hour, the color development was measured. The color development of the ELISA was quantified with a CCD-camera and an image processing system. The set up consists of a CCD-camera and a 55 nun lens (Sony CCD Video Camera XC-77RR, Nikon micro-nikk or 55 mm f/2.8 lens), a camera adaptor (Sony Camera adaptor DC-77RR) and the Image Processing Software package Optimas, version 6.5 (Media Cybernetics, Silver Spring, MD 20910, U.S.A.). Optimas runs on a pentium Π computer system. I 20 The absorbances (OD values) for the peptides at different antibody concentrations are shown in below Table 4 and Table 5. -101- 2017204254 22 Jun2017

GMX-055PC

Table 4 102 ΠΕ8 10 jxg/ml 11B8 100 jttg/M 7D8 10 iig/ml 7D8 100 jig/ral rituximab 10 jig/ml 2F2 10 wg/ml 2F2 100 iig/ml Bl 10 ag/in! Bl 100 iig/ml EMECLNHKAHCPYI 763 2997 134 41 90 48 66 147 304 LKMECLNFIRCHTPY 165 738 160 41 120 49 87 179 216 KMESCNETRACTPYI 625 3090 142 52 123 39 78 170 308 MESLCFIRAHCPYIN 179 956 127 55 102 43 65 119 178 CFIRAIITPC 188 534 181 69 134 91 114 170 212 CffiAHTPYC 151 449 186 60 132 57 92 Ϊ51 195 CRAHTPYIC 427 1605 188 64 145 48 87 179 216 CAHTPYINC 179 452 174 65 125 42 106 161 172 ' - ; ... ,.ιΐ. «. ·’: '*·'·.·'/v.%y;;7- -/¾% ' - v · IPAGIYA 217 950 164 76 177 48 85 165 192 PAGIYAP 449 2501 170 64 111 43 85 165 300 AGIYAPI 251 2207 188 73 130 44 98 18? 143 GIYAPIC 99 251 152 64 141 34 93 177 147 r/A.'PICV 137 313 174 58 159 58 99 175 90 GIYAPIA 172 857 177 96 156 62 96 165 121 IYAPIAV 161 654 181 58 116 62 76 161 106

Table 5 11B8 10 ftg/ml 71)8 10 itg/rnl rituximab 10 ftg/ml 2F2 10 ftg/ml ·, ♦ " 4 T PCINIYNAEPANPCE 118 163 152 65 YCNIYNAEPANPSCK 287 181 2418 86 ICIYNAEPANPSECN 138 192 142 78 NCYNAEPANPSEKCS 93 121 2649 49 ICNAEPANPSEKN CP 115 165 3283 43 YCAEPANPSEICNSCS 106 188 3770 65 NCEPANPSEKNSPCT 159 183 3476 61 ACPANPSEKNSPSCQ 146 148 250 77 ECANPSEICNSPSTCY 134 179 188 68 2017204254 22 Jun2017 5 As appears from Table 4,11B8 showed binding to AGIYAP of the small first extracellular loop of human CD20 at both 10 /ig/ml and 100 /ig/ml, whereas the other antibodies tested did not show significant binding to AGIYAP.

Furthermore, 1 IBS showed binding to MESLNFIRAHTPYI of the second extracellular loop of human CD20 at both 10 /ig/ml and 100 /tg/ml, whereas the 10 other antibodies tested did not show significant binding to MESLNFIRAHTPYI.

As appears from Table 5, rituximab showed binding to EPANPSEK of the second extracellul ar loop of human CD20 at both 1 /ig/ml and 10 /ig/ml, whereas the other antibodies tested did not show significant binding to EPANPSEK. 15 Example 16 Anti-idiotypic antibodies

Generation of anti-idiotypic antibodies: Mouse anti-idiotypic antibodies were made by immunizing Balb/C mice with 2F2 or 11B8T, and generating hybridomas from spleens of these mice by fusion with NS1 myeloma cells using standard techniques. The following anti-idiotypic antibodies were generated: anti-2F2 sab 1.1, 20 anti-2F2 sab 1.2, anti-2F2 sab 1.3, anti-11B8T sab 2.2, anti-11B8T sab 2.3, anti-11B8T sab 2.4, anti-11B8T sab 2.5, and anti-11B8T sab 2.6. These were tested for specific binding to 2F2T, 7D8 and 11B8T. ELISA plates were coated with purified 2F2T, 7D8 or 11B8T (diluted in PBS to a final concentration of 1-2 /ig/ml, 37 °C, 2 hours), Plates were blocked with PBS containing 0.05% Tween-20 and 2% chicken serum (RT, 1 25 hour). Subsequently, the plates were incubated with supernatants from cultures of the anti-idiotypic antibodies (final concentration adjusted to 1-10 pg/ml, RT, 2 hours). Bound mouse anti-idiotypic antibodies were detected with rabbit-anti-mouse IgG-IiRP conjugated antibody (Jackson frnmunoResearoh). -103- 2017204254 22 Jun2017

As shown in Figure 50 anti-2F2 sab 1.1, anti-2F2 sab 1.2, and anti-2F2 sab 1.3 bind to 2F2T and 7D8, but not to 11B8T or an unrelated, isotype control human antibody. Since 2F2T and 7D8 are very homologous in Vl and VH sequence, reaction of anti-2F2 idiotypic antibodies with 7D8 was expected. 5 Figure 51 shows that anti-11B8T sab 2.2, anti-11B8T sab 2.3, anti- 11B8T sab 2.4, anti-11B8T sab 2.5, and anti-11B8T sab 2.6 all bind to 11B8T to a similar extent.

Anti-idiotypic antibodies as an immunodiagnoslic tool: The 2F2/7D8 10 and 11B8T specific anti-idiotypic antibodies can be used as an iminunodiagnostic tool to detect and quantify levels of human monoclonal antibodies against CD20 in laboratory or patient samples. This may be useful for examining pharmalcokinetics of the anti-CD20 antibody or for detennining and adjusting, the dosage of the anti~CD20 antibody and for monitoring the disease and the effect of treatment in a patient. As an example of 15 such an assay, ELISA plates were coated with 4 jitg/ml anti-2F2 sab 1.1, anti~2F2 sab 1.2 or anti-2F2 sab 1.3. Plates were blocked with PBS containing 0,05% Tween-20 and 2% chicken serum (RT, 1 hour). Subsequently, the plates were incubated with a serial dilution of 2F2T (10,000-9.77 ng/ml, RT, 2 hours). Bound 2F2T was detected with mouse-anti-human IgG HRP-conjugated antibody. As shown in Figures 52A-C a dose 20 dependent binding of 2F2T was observed.

Equivalents

Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the 25 invention described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the dependent claims are also contemplated to be within the scope of the invention.

Incorporation by Reference 30 All patents, pending patent applications and other publications cited herein are hereby incorporated by reference in their entirety. -104-

Claims (102)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. . An isolated, human monoclonal antibody which binds to human CD20.

2. The antibody of claim 1, selected from the group consisting of an IgGl, an IgG2, an IgG3, an IgG4, an IgM, an IgAl, an IgA2, a secretory IgA. an IgD, and an IgE antibody.

3. The antibody of claim 2, wherein the antibody is an IgGl antibody.

4. The antibody of claim 2, wherein the antibody is an IgG3 antibody.

5. The antibody of claim 2, wherein the antibody is an IgG4 antibody,

6. The antibody of claim 2, wherein the antibody is an IgAl or IgA2 antibody.

7. The antib ody of any one of the preceding claims, wherein the antibody dissociates horn human CD20 with a dissociation rate constant (¾) of about IQ'5 sec"1 or less.

S. The antibody of any one of the preceding claims, wherein the antibody binds to human CD20 with an affinity constant (Kp) of about 5 nM or less.

9. The antibody of any one of the preceding claims, wherein the antibody has one or more of the characteristics selected from the group consisting of: (i) capable of inducing complement dependent cytotoxicity (CDC) of cells expressing CD20 in the presence of complement; (ii) capable of inducing complement dependent cytotoxicity (CDC) of cells expressing CD20 and high levels of CDS 5 and/or CDS 9 in the presence of complement; (in) capable of inducing apoptosis of cells expressing CD2Q; (iv) capable of inducing antibody dependent cellular cytotoxicity (ADCC) of cells expressing CD20 hr the presence of effector cells; (v) capable of inducing bomotypic adhesion of cells which express CD20; (vi) capable of translocating into lipid rafts upon binding to CD20; (vii) capable of prolonging the survival of a subject having tumor cells which express CD20; (viii) capable of depleting cells expressing CD20; and . (ix) capable of depleting cells expressing low levels of CD20 (CD20|OW cells).

10. The antibody of claim 9, which has one or more of the following characteristics selected from the group consisting of: (i) capable of inducing at least 20%, and preferably at least 30% CDC mediated lysis ofB-CLL cells in the presence of 33 vol/vol% plasma within 3 hours at 37 °C at an antibody concentration of 10 /rg/ml; (ii) capable of inducing at least 20%, and preferably at least 30% lysis of B-CLL cells in the presence of 33 vol/vol% whole blood cells within 3 hours at 37 °C at an antibody concentration of 10 ag/rnl; (in) capable of prolonging the 50% survival rate of SCID mice injected with Daudi cells by more than 30% at a dose of 20 μ%; and (iv) capable of depleting peripheral B cells expressing low levels of CD20 (CD20low B cells) to undetectable levels for more than 50 days in cynomolgus monkeys at a dosage of 6.25 mg/kg per day for 4 consecutive days.

11. The antibody of claim 10, which is capable of capable of inducing at least 20%, and preferably at least 30% CDC mediated lysis ofB-CLL cells in the presence of 33 voi/vol% plasma within 3 hours at 37 DC at an anti-CD20 antibody concentration of 10 /tg/ml.

12. The antibody of any of the preceding claims 1-11 encoded by human heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SBQ ID NO:l and SEQ ID NO:3, respectively, and conservative sequence modifications thereof.

13. The antibody of any of the preceding claims 1-11 encoded by human heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO:5 and SEQ ID NO:7, respectively, and conservative sequence modifications thereof.

14. The antibody of any of the preceding claims 1-11 encoded by human heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NO:9 and SEQ ID NO: 11, respectively, and conservative sequence modifications thereof.

15. The antibody of any of the preceding claims 1-11 having a human heavy chain and human kappa light chain variable regions comprising the amino acid sequences as set forth in SEQ ID NO;2 and SEQ ID NO:4, respectively, and conservative sequence modifications thereof.

16. The antibody of any of the preceding claims 1-11 having human heavy chain and human kappa light chain variable regions which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to tire amino acid sequences as set forth in SEQ 3D NO:2 and SEQ ID NO:4, respectively.

17. The antibody of any of the preceding claims 1-11 having a human heavy chain and human kappa light chain variable regions comprising the amino acid sequences as set forth in SEQ ID NO:6 and SEQ ID NO;8, respectively, and conservative sequence modifications thereof.

18. The antibody of any of the preceding claims 1-11 having human heavy chain and human kappa light chain variable regions which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the amino acid sequences as set forth in SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

19. The antibody of any of the preceding claims 1-11 having a human heavy chain and human kappa light chain variable regions comprising the amino acid sequences as set forth in SEQ ID NO: 10 and SEQ ID NO: 12, respectively, and conservative sequence modifications thereof.

20. The antibody of any of the preceding claims 1-11 having human heavy chain and human kappa light chain variable regions which are at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to the amino acid sequences as set forth in SEQ ID NO: 10 and SEQ 3D NO: 12, respectively.

21. The antibody of any of the preceding claims 1-11 comprising at least one human variable region selected from the group consisting of: (i) SEQ ID NOs:2, 4, 6, 8, 10, or 12; and (ii) a sequence which is at least 90% homologous, preferably at least 95% homologous, and more preferably at least 98%, or at least 99% homologous to any one of the amino acid sequences as set forth in (i) above.

22. An isolated human monoclonal antibody which binds to an epitope on human CD20 defined by the antibody of any one of claims 12-21.

23. An isolated human monoclonal antibody which binds to an epitope on CD20 (i) which does not comprise or require tire amino acid residue proline at position 172; (ii) which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172; (iii) which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166; (iv) which does not comprise or require the amino acid residue proline at position 172, but which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166; or (v) which does not comprise or require the amino acid residues alanine at position 170 or proline at position 172, but which comprises or requires the amino acid residues asparagine at position 163 and asparagine at position 166.

24. An isolated human monoclonal antibody which binds to CD20, wherein the antibody has one or more of the following characteristics: (i) binds to mutant P172S CD20 (proline at position 172 mutated to serine) with at least the same affinity as to human CD20; (ii) binds to mutant AxP (alanine at position 170 mutated to serine, and proline at position 172 mutated to serine) with at least the same affinity as to human CD20; (iii) shows a reduced binding of 50% or more to mutant N166D (asparagine at position 166 mutated to aspartic acid) compared to human CD20 at an antibody concentration of 10 /tg/ml; or (iv) shows a reduced binding of 50% or more to mutant N163D (asparagine at position 163 mutated to aspartic acid) compared to human CD20 at an antibody concentration of 10 jtig/ml.

25. An isolated human monoclonal antibody which binds to an epitope in the small first extracellular loop of human CD20.

26. An isolated human monoclonal antibo dy which binds to a discontinuous epitope on CD20.

27. An isolated human monoclonal antibody which binds to a discontinuous epitope on CD20, wherein the epitope comprises part of the first small extracellular loop and part of the second extracellular loop.

28. An isolated human monoclonal antibody which binds to a discontinuous epitope on CD20, wherein the epitope has residues AGIYAP of the small first extracellular loop and residues MESLNFIRAHTPYI of the second extracellular loop.

29. An isolated human monoclonal antibody which has the binding characteristics of the antibody of any one of claims 11-28.

30. An isolated human monoclonal antibody which binds to human CD20 comprising at least one CDR sequence selected from the group consisting of: (i) SEQ ID NOs: 13,14,15, 16,17, or 18; (ii) conservative sequence modifications of the sequences listed in (i); and (iii) fragments of any one of the sequences defined in (i) or (ii) which retain the ability to bind to human CD20,

31. The antibody of claim 30, comprising SEQ ID NO:15.

32. The antibody of claim 30, comprising at least four CDR sequences selected from the group consisting of: (i) SEQ ID NOs: 13,14,15,16,17, or 18; (ii) conservative sequence modifications of the sequences listed in (i); and (iii) fragments of any one of the sequences defined in (i) or (ii) which retain the ability to bind to human CD20.

33. The antibody of claim 30, comprising SEQ ID NOs: 13,14, 15, 16,17, and 18.

34. An isolated human monoclonal antibody which binds to human CD20 comprising at least one CDR sequence selected from the group consisting of: (i) SEQ ID NOs: 19,20,21, 22, 23, or 24; (ii) conservative sequence modifications of the sequences listed in (i); and (iii) fragments of any one of tire sequences defined in (i) or (ii) which retain the ability to bind to human CD2Q.

35. The antibody of claim 34, comprising at least four CDR sequences selected from the group consisting of: (i) SEQ ID NOs: 19, 20,21, 22, 23, or 24; (ii) conservative sequence modifications of the sequences listed in (i); and (iii) fragments of any one of the sequences defined in (i) or (ii) which retain the ability to bind to human CD20.

36. The antibody of claim 34, comprising SEQ ID NOs: 19, 20,21, 22,23, and 24.

37. An isolated human monoclonal antibody which binds to human CD20 comprising at least one CDR sequence selected from the group consisting of: (i) SEQ ID NOs: 25,26, 27,28,29, or 30; \ (ii) conservative sequence modifications of the sequences listed in (i); and (iii) fragments of any one of the sequences defined in (i) or (ii) which retain the ability to hind to human CD20.

38. The antibody of claim 37, comprising SEQ ID NO:27.

39. The antibody of claim 37, comprising at least four CDR sequences selected from the group consisting of: (i) SEQ ID NOs: 25,26,27,28, 29, or 30; (ii) conservative sequence modifications of the sequences listed in (i); and (iii) fragments of any one of the sequences defined in (i) or (ii) which retain the ability to bind to human CD20.

40. The antibody of claim 39, comprising SEQ ID NOs: 25,26, 27, 28, 29, and 30.

41. The antibody of any of the preceding claims 1-11 comprising at least one CDR selected from the group consisting of (i) SEQ ED NOs: 13,19, or 25, or a sequence having 1 amino acid substitution, deletion or addition of the sequence of SEQ ID NOs: 13,19 or 25; (ii) SEQ ID NOs: 14,20, or 26, or a sequence having 1-4 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs: 14,20, or 26; (iii) SEQ ID NOs:15, or 27, or a sequence having 1-4 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs:l 5, or 27; (iv) SEQ ID NO: 16, or a sequence having 1-2 amino acid substitutions, deletions or additions of the sequence of SEQ ID NO: 16; (v) SEQ ID NO: 17, or a sequence having 1-2 amino acid substitutions, deletions or additions of the sequence of SEQ ID NO :17; and (vi) SEQ ID NOs: 18, or 30, or a sequence having 1-2 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs: 18, or 30.

42. The antibody of claim 41 comprising at least four CDRs selected from the group consisting of (i) SEQ ID NOs:13, 19, or 25, or a sequence having 1 amino acid substitution, deletion or addition of the sequence of SEQ ID NOs:13,19 or 25; (ii) SEQ ID NOs: 14, 20, or 26. or a sequence having 1-4 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs:14, 20, or 26; (iii) SEQ ID NOs:15, or 27, or a sequence having 1-4 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs:15, or 27; (iv) SEQ ID NO: 16, or a sequence having 1-2 amino acid substitutions, deletions or additions of the sequence of SEQ ID NO: 16; (v) SEQ ID NO: 17, or a sequence having 1-2 amino acid substitutions, deletions or additions of the sequence of SEQ ID NO: 17; and (vi) SEQ ID NOs: 18, or 30, or a sequence having 1-2 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs: 18, or 30.

43. The antibody of claim 41 comprising one CDR selected from the group consisting of SEQ ID NOs: 15, or 27, and a sequence having 1-4 amino acid substitutions, deletions or additions of the sequence of SEQ ID NOs: 15, or 27.

44. The antibody of any one of die preceding claims which is an intact antibody selected from the group consisting of: an intact IgGl antibody, an intact IgG2 antibody, an intact IgG3 antibody, an intact IgG4 antibody, an intact IgM antibody, an intact IgAl antibody, an intact IgA2 antibody, an intact secretory IgA antibody, an intact IgD antibody, and an intact IgE antibody, wherein the antibody is glycosylated in a eukaryotic cell.

45. The antibody of any one of the preceding claims which is an antibody fragment or a single chain antibody.

46. . The antibody of any one of the preceding claims which is a binding-domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide in the form of a heavy chain variable region or a light chain variable region as defined in claim 24 that is fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CHS constant region, fused to the CH2 constant region.

47. The antibody of any one of the preceding claims produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, in which V-(D)-J gene segment rearrangements have resulted in the formation of a functional human heavy chain transgene and a functional human light chain transgene, fused to an immortalized cell.

48. A hybridoma comprising a B cell obtained from a transgenic nonhuman animal in which V-(D)-J gene segment rearrangements have resulted in the formation of a functional human heavy chain transgene and a functional light chain transgene fused to an immortalized cell, wherein the hybridoma produces a detectable amount of the monoclonal antibody of any one of the preceding claims.

49. A hybridoma which produces a human monoclonal antibody encoded by human IgG heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ED NOs:l, 5, or 9 and SEQ ID MOs:3,7, or 11, respectively, and conservative sequence modifications thereof.

50. A hybridoma which produces a human monoclonal having IgG heavy chain and kappa light chain variable regions which comprise the amino acid sequences as set forth in SEQ ID NOs:2, 6, or 10 and SEQ ID NOs: 4, 8, or 12, respectively, and conservative sequence modifications thereof.

51. The antibody of any one of claims 1-45 produced by a transfectoma comprising nucleic acids encoding a human heavy ehain and a human light chain.

52. A transfectoma comprising nucleic acids encoding a human heavy chain and a human light chain, wherein the transfectoma produces a detectable amount of the antibody of any one of claims 1-45.

53. A transfectoma which produces a human monoclonal antibody encoded by human IgG heavy chain and human kappa light chain nucleic acids comprising nucleotide sequences in their variable regions as set forth in SEQ ID NOs:l, 5, or 9 and SEQ ID NOs*,3,7, or 11, respectively, and conservative sequence modifications thereof.

54, A transfectoma which produces a human monoclonal antibody having IgG heavy chain and kappa light chain variable regions which comprise the amino acid sequences as set forth in SEQ ED NOs:2, 6. or 10 and SEQ ED NOs;4, 8, or 12, respectively, and conservative sequence modifications thereof.

5 5. A eukaryotic or prokaryotic ho st cell which produces a human monoclonal antibody having heavy chain and light chain variable regions which comprise the amino acid sequences as set forth in SEQ ID NOs:2, 6, or 10 and SEQ ID NOs:4, 8, or 12, respectively, and conservative sequence modifications thereof.

56. A transgenic non-human animal or plant which produces a human monoclonal antibody having heavy chain and light chain variable regions which comprise the amino acid sequences as set forth in SEQ ID NOs:2. 6, or 10 and SEQ ED NOs:4, 8, or 12, respectively, and conservative sequence modifications thereof.

57. A method of producing a human monoclonal antibody which binds to human CD20, comprising: immunizing a transgenic non-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene with human CD20 or a cell expressing human CD20, such that antibodies are produced by B cells of the animal; isolating B cells of the animal; fusing the B cells with myeloma cells to form immortal, hybridoma cells that secrete human monoclonal antibodies specific for human CD20; and isolating the human monoclonal antibodies specific for CD20 from tire culture supernatant of the hybridoma, or the transfectoma derived from such hybridoma.

58. A method according to claim 57, wherein the immunization is performed with cells which have been transfected with human CD20.

59. An isolated human monoclonal antibody which binds to human CD20 obtained by: immunizing a transgenic noil-human animal having a genome comprising a human heavy chain transgene and a human light chain transgene with a cell which has been transfected with human CD20, such that antibodies are produced by B cells of the animal; isolating B cells of the animal; fusing the B cells with myeloma cells to form immortal, hybricloma cells that secrete human monoclonal antibodies specific for human CD20; and isolating the human monoclonal antibodies specific for CD20 from the culture supernatant of the hybridoma, or the transfectoma derived from such frybridoma.

60. An isolated human antibody comprising a heavy chain variable region amino acid sequence derived from a human Vh3-13/DP-44 germline sequence (SEQ ID NO: 54) and a light chain variable region amino acid sequence derived from a human L6/JK4-CK (SEQ ID NO:55) germline sequence, wherein the human antibody binds to human CD20.

61. An isolated human antib o dy comprising a heavy chain variable region amino acid sequence derived from a human VH3-09/JH6b germline sequence (SEQ ID NG:56) and a light chain variable region amino acid sequence derived from a human Vr,-L6/JK5 gerniline sequence (SEQ ID NO:57), wherein the human antibody binds to human CD20.

62. A composition comprising the human antibody of any one of claims 1-45 and a pharmaceutically acceptable carrier.

63. A composition comprising a combination of two or more human antibodies according to any one of claims 1-45, which have complementary functional activities.

64. A composition according to claim 61 comprising a first antibody according to claim 15 and a second antibody according to claim 19.

65. A composition according to any of claims 62-64 further comprising a therapeutic agent.

66. The antibody according to any one of claims 1-45, further comprising a chelator linker for attaching a radioisotope.

67. An immunoconjugate comprising an antibody according to any one of claims 1-45 linked to a cytotoxic agent, a radioisotope, or a drug.

68. A bispecific molecule comprising an antibody according to any one of claims 1-45 and a binding specificity for a human effector cell.

69. A bispecific molecule comprising an antibody according to any one of claims 1-45 and a binding specificity for a human Fc receptor or a binding specificity for a T cell receptor, such as CD3.

70. A method of inhibiting growth of a ceil expressing CD20, comprising contacting the cell with an effective amount of an antibody according to any one of claims 1-45 such that the growth of the cell is inhibited.

71. A method of killing a cell expressing CD20, comprising contacting the cell with the antibody of any one of claims 1-45, such that killing of the cell expressing CD20 occurs.

72. The method of any one of claims 70 or 71, wherein die cell is a B lymphocyte or a tumor cell.

73. A method of treating or preventing a disease or disorder involving cells expressing CD20, comprising administering to a subject a human antibody, composition, imimmoconjugate, or bispecific molecule according to any one of claims 1-45 or 60-69, an expression vector according to any one of the claims 94-97, in an amount effective to treat or prevent the disease.

74. The method of claim 73, wherein the disease is a B cell lymphoma.

75. The method of claim 73, wherein the disease is B cell non-Hodgkin’s lymphoma.

76. The method of claim 73, wherein the disease is selected from the group consisting of precursor B cell lymphoblastic leukemiadymphama and mature B cell neoplasms, such as B cell chronic lymhocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), cutaneous follicle center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma (ALCL).

77. The method of claim 76, wherein the disease is follicular lymphoma (FL).

78. The method of claim 76, wherein the disease is B cell chronic lymhocytic leukemia (CLL)/small lymphocytic lymphoma (SLL).

79. The method of claim 73, wherein the disease is selected from the group consisting of lymphomatoid granulomatosis, primary effusion lymphoma, intravascular large B cell lymphoma, mediastinal large B cell lymphoma, heavy chain diseases (including γ, μ, and a disease), lymphomas inducedby therapy with immunosuppressive agents, such as cyclosporine-induced lymphoma, and methotrexate-induced lymphoma.

80. A method of treating or preventing an immune disease involving CD20 expressing immune cells, comprising administering to a subject the antibody, composition, immunoeonjugate, or bispecific molecule according to any one of claims 1-45 or 60-67 or an expression vector according to any one of the claims 94-97, in an amount effective to treat or prevent the immune disease.

81. The method of claim 80, wherein treatment includes the killing of B cells which produce antibodies against auto antigens.

82. The method of claim 73, wherein the disease or disorder is selected from the group consisting of psoriasis, psoriatic arthritis, dermatitis, systemic scleroderma and sclerosis, inflammatory bowel disease (IBD), Crohn’s disease, ulcerative colitis, respiratory distress syndrome, meningitis, encephalitis, uveitis, glomerulonephritis, eczema, asthma, atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis, Raynaud’s syndrome, Sjogren’s syndrome, juvenile onset diabetes, Reiter’s disease, Behcet’s disease, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupus nephritis, systemic lupus erythematosus, rheumatoid arthritis (RA), atopic dermatitis, pemphigus, Graves’ disease, Hashimoto’s thyroiditis, Wegener’s granulomatosis, Omenn’s syndrome, chronic renal failure, acute infectious mononucleosis, HIV, and herpes virus associated diseases.

83. The method of claim 82, wherein the autoimmune disease is rheumatoid arthritis (RA).

84. The method of claim 73, wherein the disease is an inflammatory, immune and/or autoimmune disorder selected from ulcerative colitis, Crohn’s disease, juvenile onset diabetes, multiple sclerosis, immune-mediated thrombocytopenias, such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, hemolytic anemia, myasthenia gravis, systemic sclerosis, and pemphigus vulgaris.

85. The method of claim 73, wherein the disease is an inflammatory, immune and/or autoimmune disorder selected from inflammatory bowel disease (H3D), ulcerative colitis, Crohn’s disease, and multiple sclerosis.

86. The method of any one of claims 70-85, further comprising separately administering another therapeutic agent to the subject.

87. The method of claim 86, wherein the therapeutic agent is a cytotoxic agent or a radiotoxic agent.

88. The method of claim 86, wherein the therapeutic agent is an immunosuppressant.

89. The method of claim 86, wherein the therapeutic agent is an immunological modulating agent, such as a cytokine or a chemokine.

90. The method of claim 86, wherein the therapeutic agent is selected from the group consisting of doxorubicin, cisplatin, bleomycin, carmustine, chlorambucil, and cyclophosphamide.

91. The method of claim 86, wherein the therapeutic agent is selected from the group consisting of anti-CD25 antibodies, anti-CD 19 antibodies, anti~CD21 antibodies, anti-CD22 antibodies, anti-CD37 antibodies, anti-QD3S antibodies, anti-1L6R antibodies, anti-IL8 antibodies, anti-IL15 antibodies, anti-IL15R antibodies, anti-CD4 antibodies, anti-CD 11 a antibodies, anti-alpha-4/beta-1 integrin (VLA4) antibodies, CTLA4-Ig, and anti-C3b(i) antibodies.

92. An in vitro method for detecting the presence of CD20 antigen, or a cell expressing CD20, in a sample comprising: contacting the sample with the antibody of any one of claims 1-45 under conditions that allow for formation of a complex between the antibody and CD20; and detecting the formation of a complex.

93. A kit for detecting the presence of CD20 antigen, or a cell expressing CD20, in a sample comprising the antibody of any one of claims 1-45.

94. An in vivo method for detecting CD20 antigen, or a cell expressing CD20, in an subject comprising: administering the antibody of any one of claims 1-45 under conditions that allow for formation of a complex between the antibody and CD20; and detecting the formed complex.

95. An expression vector comprising a nucleotide sequence encoding the variable region of a light chain, heavy chain or both light and heavy chains of a human antibody which hinds human CD2G,

96. The expression vector of claim 95, further comprising a nucleotide sequence encoding the constant region of a light chain, heavy chain or both light and heavy chains of a human antibody which hinds human CD20.

97. An expression vector comprising a nucleotide sequence encoding a heavy chain variable region comprising a nucleotide selected from the group consisting of the nucleotide sequences as set forth in SEQ ID NOs: 1, 5, and 9, and a light variable region comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences as set forth in SEQ ID NOs: 3, 7, and. 11, and conservative modifications thereof.

98. An expression vector comprising a nucleotide sequence encoding a heavy chain variable region comprising an amino acid sequence selected from the group consisting of the amino acid sequences as set forth in SEQ ID NOs :2,6, and 10, and a light chain variable region comprising the amino acid sequence selected from the group consisting of the amino acid sequences as set forth in shown in SEQ ID NOs: 4, S, and 12, and conservative sequence modifications thereof.

99. A pharmaceutical composition comprising the expression vector of any one of claims 95-9S and a pharmaceutically acceptable carrier.

100. An anti-idiotypic antibody binding to an antibody of any one of · 5 claims 1-45. \

101. An anti-idiotypic antibody binding to 2F2, 1 IBS or 7D8.

102. Use of an anti-idiotypic antibody of claim 100 or 101 for 10 detecting the level of human monoclonal antibody against CD20 in a sample.