Combination of fcγriib (cd32b) and cd20 specific antibodies

Abstract

The present invention relates to the combined use of FcyRIIb (CD32B)- and CD20-specific antibodies, in particular to a method of treating a patient having target cells expressing FcyRIIb, the method comprising the combined administration of: (i) an antibody molecule specifically binding to a surface antigen of said target cells, said antibody molecule having an Fc domain capable of binding to FcyRIIb; and (ii) an agent inhibiting or reducing the binding between the Fc domain of said antibody molecule and FcyRIIb, characterized in that the patient is selected on the basis of an elevated level of FcyRIIb expression by his target cells.

Description

[0001] This application is a divisional application of application number 201710789909.3, filed on August 19, 2011, and entitled “Combined use of FcγRIIB (CD32B) and CD20-specific antibodies.” Application number 201710789909.3 is a divisional application of application number 201180040351.X, filed on August 19, 2011, and entitled “Combined use of FcγRIIB (CD32B) and CD20-specific antibodies.” Background Art

[0002] The present invention relates to agents that prevent binding between antibody Fc domains and cell surface FcγRIIb. The present invention also relates to compositions comprising the agents for use in treating patients having target cells such as cancer cells using antibody-based compositions.

[0003] In addition, the present invention also relates to a method for predicting the response of a target cell to an antibody-based therapy, in particular, a method for predicting the response of a target cell to an antibody-based therapy when the antibody ligand is sensitive to FcγRIIb-mediated internalization, and in particular to the use of FcγRIIb expression levels and / or internalization of therapeutic antibodies mediated by FcγRIIb as prognostic markers of the response of the target cell to the therapy.

[0004] The mechanism by which monoclonal antibodies (mAbs) can produce therapeutic effects is by recruiting natural effector systems such as cytotoxic cells (e.g., macrophages) and enzymes (e.g., complement), which then target the cells to which the mAb binds, stimulating the removal of cancer cells and other unwanted cells.

[0005] For example, type I anti-CD20 mAbs (such as the current market leader, rituximab) work by binding to CD20 molecules on the surface of B cells via the mAb's antigen-binding domain and eliminating these target B cells. They do this by recruiting and activating these effector cells, which interact with the mAb's Fc domain through Fcγ receptors (FcγRs) expressed on their surface.

[0006] The anti-CD20 monoclonal antibody (mAb) rituximab improves overall survival (OS) in patients with follicular (FL) lymphoma and diffuse large B-cell lymphoma (DLBCL) (1-4). In mantle cell lymphoma (MCL), only modest responses have been observed (5), while in chronic lymphocytic leukemia (CLL), responses to initial single-agent rituximab trials have not been as dramatic as those seen in other non-Hodgkin lymphomas (NHL) (reviewed in (6)). A subset of lymphomas exhibits primary resistance to rituximab or eventually develops resistance to combination therapy containing rituximab (7). The molecular basis underlying this resistance to treatment and the observed sensitivity of different NHL subtypes to rituximab treatment is currently unknown but may include elevated levels of CD20 expression (8-10), elevated expression of complement defense molecules (CD55 and CD59) (11, 12), development of apoptosis resistance (13), and suboptimal Fcγ receptor (FcγR) interactions due to expression of low-affinity alleles (14).

[0007] It would be highly desirable to improve the effectiveness of these antibodies in settings where treatment is suboptimal or resistance is evident.

[0008] It is generally believed that the Fc:FcγR interaction is crucial for the efficacy of anti-CD-20 mAbs (15-18). Consistent with this, lymphoma patients carrying the higher affinity 158V allele in FcγRIIIa respond better to rituximab than lymphoma patients with the lower affinity 158F allele (14), leading many researchers to focus on improving the interaction of mAbs with FcγRIIIa, for example by defucosylation (19). In contrast, less attention has been paid to the inhibitory FcγRIIb, which acts as a negative regulator of stimulatory activity received by ITAM-bearing receptors such as the B cell antigen receptor (BCR) and activating FcγRs. In B cells, this interaction serves to limit B cell proliferation after immune complex binding, while in macrophages, engagement of FcγRIIb results in inhibition of cytotoxic activity (15).

[0009] In B-cell malignancies, FcγRIIb is expressed in CLL / SLL, MCL, and FL, the latter being particularly expressed during transformation. In DLBCL, FγRIIb expression is weak, explaining why no correlation has been shown between its expression and response to rituximab-CHOP (R-CHOP) chemotherapy (20, 21). Regarding activating FcγRs, polymorphisms that affect FcγRIIb activity have also been found, with the 232I allele more effectively inhibiting BCR-mediated calcium flux than the 232T allele (22, 23). However, Weng and Levy were unable to determine a correlation between these polymorphisms and response to rituximab treatment in FL patients.

[0010] An increasing number of anti-CD20 mAbs are available for clinical investigation. These different anti-CD20 mAbs are classified as type I (e.g., rituximab, ofatumumab) or type II (e.g., tositumomab (B1), GA101, 11B8) based on their ability to redistribute CD20 in the plasma membrane and their activity in different effector cell assays (25-27).

[0011] The inventors and others have demonstrated that type II mAbs are more efficient at eliminating B cell targets in a number of model systems (18, 19). For example, in a CD20 transgenic (Tg) model of human normal B cell depletion (25, 27), in which the higher ability of type II mAbs to trigger lysosomal cell death is not apparent, the inventors demonstrated that this potency correlated with their resistance to internalization (28). This is in contrast to type I mAbs such as rituximab, which are rapidly internalized from the cell surface along with CD20 in a process that is energy- and temperature-dependent and involves actin redistribution (28). The rates of modulation vary significantly in cells from different sources (primary tumors versus cell lines, CLL versus FL), but the molecular basis for this remains unexplained.

[0012] WO 2008 / 002933 describes antibodies specific for FcγRIIb (CD32B) and CD20, and methods of using a combination of these two antibodies to treat B cell-related diseases or disorders. However, there is no teaching or suggestion of identifying and / or treating patient subgroups, i.e., those patients whose target cell FcγRIIb expression levels are elevated, or whose antibody types are suitable for combination therapy with FcγRIIb antibodies.

[0013] Surprisingly, the inventors demonstrated that FcγRIIb surface expression was significantly correlated with cell subtype modulation, and that overexpression could convert Ramos cells from slow-modulating to fast-modulating cells. FcγRIIb internalization occurs alongside CD20 and precedes its activation. Together, these data provide a clear molecular rationale for the heterogeneity in modulation rates previously observed within and between different NHL subtypes.

[0014] Thus, the inventors have demonstrated that, surprisingly, a key factor determining the effectiveness of antibodies against antigens such as CD20 is interaction with inhibitory FcγRIIb (also known as and including CD32, CD32B, CD32B1, CD32B2, FcRII, FcγRII, or FcRIIB) on the surface of the same cell. This interaction leads to internalization of the antibody by the target cell, thereby removing its ability to interact with effector cell Fc receptors. The inventors have further demonstrated that agents such as anti-CD32 mAbs are capable of blocking this internalization. The inventors have also demonstrated that such agents can be used in combination with antibodies (e.g., rituximab) to target cell surface antigens and improve their activity in clearing normal B cells or tumor cells in vivo.

[0015] The present invention provides a composition comprising:

[0016] (i) an antibody molecule that specifically binds to a cell surface antigen of a target cell, the antibody molecule having an Fc domain capable of binding to FcγRIIb; and

[0017] (ii) an agent that inhibits or reduces the binding of FcγRIIb to the Fc domain of the antibody molecule,

[0018] Characterized in that the composition is used to treat patients having target cells with elevated FcγRIIb expression levels.

[0019] According to another aspect, the present invention provides the use of an agent for inhibiting or reducing the binding between the Fc domain of an antibody molecule and FcγRIIb on a target cell, wherein the antibody molecule specifically binds to a target cell surface antigen, characterized in that the use is for the preparation of a medicament for treating a patient having target cells with elevated FcγRIIb expression levels.

[0020] According to another aspect, the present invention provides a method for treating a patient having target cells expressing FcγRIIb, the method comprising the combined administration of: (i) an antibody molecule that specifically binds to a surface antigen of the target cell, the antibody molecule having an Fc domain capable of binding to FcγRIIb; and (ii) an agent that inhibits or reduces the binding between the Fc domain of the antibody molecule and FcγRIIb, wherein the patient is selected based on an elevated level of FcγRIIb expression on the target cells.

[0021] In certain embodiments of the compositions, uses or methods of the invention, the agent inhibits or reduces the binding of FcγRIIb present on the target cell to the Fc domain of the antibody molecule.

[0022] According to another aspect, the present invention provides the use of FcγRIIb expression on a target cell as a prognostic marker of the response of said target cell to treatment with an antibody molecule that specifically binds to an antigen on the surface of said target cell, said antibody molecule having an Fc domain capable of binding to FcγRIIb, whereby increased levels of FcγRIIb indicate a reduced response or no response to treatment with said antibody molecule.

[0023] In one embodiment, the use of FcyRIIb expression on target cells as a prognostic marker does not require the use of FcyRIIc expression on target cells as a prognostic marker.

[0024] According to another aspect, the present invention provides a method for predicting the response of a patient's target cells to treatment with an antibody molecule that specifically binds to a target cell surface antigen and has an Fc domain capable of binding to FcγRIIb, characterized in that the method comprises determining the level of FcγRIIb expression on the target cells, whereby an increased level of FcγRIIb indicates a reduced response or no response to treatment with the antibody molecule.

[0025] In one embodiment, the method for predicting a target cell response in a patient comprises determining the level of expression of FcyRIIb on said target cell and does not further comprise determining the level of expression of FcyRIIc on said target cell.

[0026] It has been demonstrated that although all antibodies possess an Fc-binding antibody constant domain and known Fcγ receptor binding ability, not all antibodies are internalized in an FcγRIIb-dependent manner, making the identification of suitable antibodies critical to the therapeutic success of combination therapies containing modulators of FcγRIIb function and to avoiding treatments that are not beneficial to the patient.

[0027] In certain embodiments of the compositions, uses or methods of the present invention, an antibody molecule that specifically binds to a cell surface antigen of a target cell can also be internalized into the cell in an FcγRIIb-dependent manner, wherein the antibody has an Fc domain that can bind to FcγRIIb.

[0028] In certain embodiments of the compositions, uses or methods of the invention, the agent that inhibits or reduces binding of FcγRIIb to the Fc domain of the antibody molecule also inhibits or reduces further internalization of the antibody molecule into the cell.

[0029] In certain embodiments of the compositions, uses or methods of the invention, the target cell is a cancer cell. Conveniently, the target cell is a B cell.

[0030] Advantageously, according to the present invention, the elevated expression of FcγRIIb on the target cells is relative to a control or reference. Preferably, the control is the normal expression level of FcγRIIb in cells of the same type as the target cells.

[0031] "Increased expression levels of FcγRIIb" are defined below in "Definitions." FcγRIIb expression levels can be calculated as the ratio of the geometric mean fluorescence intensity (geometric MFI or Geo MFI) of FcγRIIb to an isotype control. Alternatively, FcγRIIb expression levels can be calculated by immunohistochemistry of tumor biopsies. One skilled in the art will appreciate that there are a variety of techniques and methods for determining FcγRIIb expression levels.

[0032] The present invention also teaches how to identify antibodies suitable for combination therapy with FcyRIIb antibodies, ie, those antibodies that are internalized from the surface of target cells in an FcyRIIb-dependent manner. The present invention also provides for identifying patient subgroups suitable for combination therapy with FcyRIIb antibodies.

[0033] In another aspect, the present invention provides an assay for identifying an agent that reduces or inhibits binding between an Fc domain of an antibody directed against a target cell surface antigen and FcγRIIb on a target cell, the assay comprising determining the extent of binding between the Fc domain and FcγRIIb in the presence and absence of a test agent. If the test agent reduces or inhibits binding of the Fc domain to FcγRIIb, it is identified as a useful agent. Such an assay can also be used to identify which agents (e.g., antibody molecules) are suitable for combined therapy with anti-FcγRIIb antibodies.

[0034] In another preferred embodiment, the assay for identifying agents useful in practicing the uses and methods of the present invention comprises screening for agents that block FcγRIIb stimulation / signaling, as indicated by phosphorylation of tyrosine-293 in the ITIM motif within the cell by Western blot. For example, Raji cells are incubated with an antibody to a cell surface antigen (e.g., anti-CD20 mAb rituximab) in the presence or absence of an anti-FcγRIIb test agent prior to immunoblotting for phosphorylated FcγRIIb. The amount of phosphorylated FcγRIIb is elevated in cells stimulated with rituximab, and, in contrast to the amount of phosphorylated FcγRIIb, the amount of phosphorylated FcγRIIb is elevated in cells stimulated with rituximab. Figure 4 Similar to the blocker shown in A, AT10 should be inhibited by the addition of the test reagent. Figure 1 These reagents should also preferentially block the internalization of rituximab as shown in the quenching experiment in A. Figure 2 B shows a representative example of blocking by an anti-FcyRIIb blocker (in this case AT10). These assays can also be used to identify which agents (eg, antibody molecules) are suitable for combination therapy with anti-FcyRIIb antibodies.

[0035] In a preferred embodiment, an assay for identifying agents suitable for combination therapy with an FcγRIIb antibody calculates the percentage of an agent (e.g., antibody molecule) internalized into cells expressing FcγRIIb. The assay is characterized in that the method comprises determining the percentage of an agent (e.g., antibody molecule) remaining on the cell surface after incubation of cells expressing the agent (e.g., antibody molecule) target and FcγRIIb, whereby a decrease in the percentage of agent (e.g., antibody molecule) accessible to the cell surface is predictive of a response to treatment with the antibody molecule.

[0036] Neubig et al (2003) Pharmacol. Rev. 55, 597-606, incorporated herein by reference, describes various types of ligands that can be screened to identify agents of the invention that inhibit or reduce the binding of FcγRIIb to the Fc domain of an antibody molecule.

[0037] The above-mentioned ligands can be small organic or inorganic substances, but preferably they are peptides or polypeptides. Generally, when the ligand is a small organic or inorganic substance, its relative molecular weight (M r ) is 50 to 2000, for example, 100 to 1000, such as 100 to 500.

[0038] Typically, ligands are expressed with a K in the mM to pM range. d Binding to FcγRIIb, for example, in the μM (micromolar) to nM range. Generally, ligands with the lowest Kd are preferred.

[0039] The ligand may be a peptidomimetic, a nucleic acid, a peptide nucleic acid (PNA) or an aptamer. It may also be a lipid or a carbohydrate.

[0040] The ligand may be a polypeptide that binds to FcγRIIb. Such polypeptides (including oligopeptides) are generally M r 500 to M r 50,000, but can be larger.

[0041] The polypeptide may also be a binding protein based on a modular framework, such as ankyrin repeat proteins, armadillo repeat proteins, leucine-rich proteins, tetartriopeptide repeat proteins or designed ankyrin repeat proteins (DARPins) or proteins based on lipocalin or fibronectin domains or Affilin scaffolds.

[0042] Conveniently, the test agent is a library of test compounds, and preferably, the library is any peptide library, protein library, antibody library, recombinant combinatorial antibody library or scFV or Fab phage display library.

[0043] Preferably, in the composition, use or method of the invention, said agent (ii) is one or more antibody molecules that specifically bind to FcγRIIb. Conveniently, said one or more antibody molecules do not comprise a domain capable of recruiting effector cells.

[0044] Conveniently, said one or more antibody molecules are one or more monoclonal antibody molecules.

[0045] Preferably, the agent inhibits or reduces FcγRIIb signaling.Even more preferably, the agent inhibits or reduces internalization of the antibody molecule by a target cell.

[0046] In the following embodiments, SEQ ID NO refers to the sequence shown in clones 1-13 below.

[0047] Those skilled in the art will appreciate that there are three complementarity determining regions (CDRs) on the variable regions of the heavy and light chains of immunoglobulins. The assignment of amino acids to each CDR described herein is consistent with the definition in Kabat EA et al. 1991, "Sequences of Proteins of Immulogical Interest", 5th edition, NIH publication number 91-3242, pages xv-xvii.

[0048] Those skilled in the art will appreciate that there are other methods for assigning amino acids to CDRs. For example, the International ImMunoGeneTics Information System ( http: / / www.imgt.org / and Lefranc, “The Immunoglobulin Facts Book,” published by Academic Press, 2001).

[0049] In one embodiment, the agent comprises a heavy chain variable region (VH) comprising the following CDRs:

[0050] (i) SEQ ID NO: 29 and SEQ ID NO: 30 and SEQ ID NO: 31; or

[0051] (ii) SEQ ID NO: 35 and SEQ ID NO: 36 and SEQ ID NO: 37; or

[0052] (iii) SEQ ID NO:41 and SEQ ID NO:42 and SEQ ID NO:43; or

[0053] (iv) SEQ ID NO:47 and SEQ ID NO:48 and SEQ ID NO:49; or

[0054] (v) SEQ ID NO: 53 and SEQ ID NO: 54 and SEQ ID NO: 55; or

[0055] (vi) SEQ ID NO: 59 and SEQ ID NO: 60 and SEQ ID NO: 61; or

[0056] (vii) SEQ ID NO: 65 and SEQ ID NO: 66 and SEQ ID NO: 67; or

[0057] (viii) SEQ ID NO:71 and SEQ ID NO:72 and SEQ ID NO:73; or

[0058] (ix) SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79; or

[0059] (x) SEQ ID NO:83 and SEQ ID NO:84 and SEQ ID NO:85; or

[0060] (xi) SEQ ID NO:89 and SEQ ID NO:90 and SEQ ID NO:91; or

[0061] (xii) SEQ ID NO:95 and SEQ ID NO:96 and SEQ ID NO:97; or

[0062] (xiii) SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103.

[0063] Preferably, the agent comprises a light chain variable region (VL) comprising the following CDRs:

[0064] (i) SEQ ID NO: 32 and SEQ ID NO: 33 and SEQ ID NO: 34; or

[0065] (ii) SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40; or

[0066] (iii) SEQ ID NO:44 and SEQ ID NO:45 and SEQ ID NO:46; or

[0067] (iv) SEQ ID NO: 50 and SEQ ID NO: 51 and SEQ ID NO: 52; or

[0068] (v) SEQ ID NO: 56 and SEQ ID NO: 57 and SEQ ID NO: 58; or

[0069] (vi) SEQ ID NO: 62 and SEQ ID NO: 63 and SEQ ID NO: 64; or

[0070] (vii) SEQ ID NO: 68 and SEQ ID NO: 69 and SEQ ID NO: 70; or

[0071] (viii) SEQ ID NO:74 and SEQ ID NO:75 and SEQ ID NO:76; or

[0072] (ix) SEQ ID NO:80 and SEQ ID NO:81 and SEQ ID NO:82; or

[0073] (x) SEQ ID NO:86 and SEQ ID NO:87 and SEQ ID NO:88; or

[0074] (xi) SEQ ID NO:92 and SEQ ID NO:93 and SEQ ID NO:94; or

[0075] (xii) SEQ ID NO:98 and SEQ ID NO:99 and SEQ ID NO:100; or

[0076] (xiii) SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106.

[0077] Optionally, the agent comprises a heavy chain variable region (VH) amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

[0078] Optionally, the agent comprises a light chain variable region (VL) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0079] Preferably, the agent comprises the following CDR amino acid sequence:

[0080] (i) SEQ ID NO: 29 and SEQ ID NO: 30 and SEQ ID NO: 31 and SEQ ID NO: 32 and SEQ ID NO: 33 and SEQ ID NO: 34; or

[0081] (ii) SEQ ID NO:35 and SEQ ID NO:36 and SEQ ID NO:37 and SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40; or

[0082] (iii) SEQ ID NO:41 and SEQ ID NO:42 and SEQ ID NO:43 and SEQ ID NO:44 and SEQ ID NO:45 and SEQ ID NO:46; or

[0083] (iv) SEQ ID NO:47 and SEQ ID NO:48 and SEQ ID NO:49 and SEQ ID NO:50 and SEQ ID NO:51 and SEQ ID NO:52; or

[0084] (v) SEQ ID NO:53 and SEQ ID NO:54 and SEQ ID NO:55 and SEQ ID NO:56 and SEQ ID NO:57 and SEQ ID NO:58; or

[0085] (vi) SEQ ID NO:59 and SEQ ID NO:60 and SEQ ID NO:61 and SEQ ID NO:62 and SEQ ID NO:63 and SEQ ID NO:64; or

[0086] (vii) SEQ ID NO:65 and SEQ ID NO:66 and SEQ ID NO:67 and SEQ ID NO:68 and SEQ ID NO:69 and SEQ ID NO:70; or

[0087] (viii) SEQ ID NO:71 and SEQ ID NO:72 and SEQ ID NO:73 and SEQ ID NO:74 and SEQ ID NO:75 and SEQ ID NO:76; or

[0088] (ix) SEQ ID NO:77 and SEQ ID NO:78 and SEQ ID NO:79 and SEQ ID NO:80 and SEQ ID NO:81 and SEQ ID NO:82; or

[0089] (x) SEQ ID NO:83 and SEQ ID NO:84 and SEQ ID NO:85 and SEQ ID NO:86 and SEQ ID NO:87 and SEQ ID NO:88; or

[0090] (xi) SEQ ID NO:89 and SEQ ID NO:90 and SEQ ID NO:91 and SEQ ID NO:92 and SEQ ID NO:93 and SEQ ID NO:94; or

[0091] (xii) SEQ ID NO:95 and SEQ ID NO:96 and SEQ ID NO:97 and SEQ ID NO:98 and SEQ ID NO:99 and SEQ ID NO:100; or

[0092] (xiii) SEQ ID NO: 101 and SEQ ID NO: 102 and SEQ ID NO: 103 and SEQ ID NO: 104 and SEQ ID NO: 105 and SEQ ID NO: 106.

[0093] Even more preferably, the agent comprises the following amino acid sequence:

[0094] (i) SEQ ID NO: 3 and SEQ ID NO: 16; or

[0095] (ii) SEQ IS NO: 4 and SEQ ID NO: 17; or

[0096] (iii) SEQ IS NO: 5 and SEQ ID NO: 18; or

[0097] (iv) SEQ ID NO: 6 and SEQ ID NO: 19; or

[0098] (v) SEQ ID NO: 7 and SEQ ID NO: 20; or

[0099] (vi) SEQ ID NO: 8 and SEQ ID NO: 21; or

[0100] (vii) SEQ ID NO: 9 and SEQ ID NO: 22; or

[0101] (viii) SEQ ID NO: 10 and SEQ ID NO: 23; or

[0102] (ix) SEQ ID NO: 11 and SEQ ID NO: 24; or

[0103] (x) SEQ ID NO: 12 and SEQ ID NO: 25; or

[0104] (xi) SEQ ID NO: 13 and SEQ ID NO: 26; or

[0105] (xii) SEQ ID NO: 14 and SEQ ID NO: 27; or

[0106] (xiii) SEQ ID NO:15 and SEQ ID NO:28.

[0107] The reagent of the present invention further comprises the constant regions (CH) and (CL) of SEQ ID NO 1 and SEQ ID NO 2.

[0108] In a further embodiment, the agent can compete with the agent of the invention described herein, such as comprising the amino acid sequence listed in the above embodiments (e.g., SEQ ID NO: 1-106), to inhibit or reduce the binding of FcγRIIb to the Fc domain of the antibody molecule.

[0109] As used herein, "capable of competing with an agent such as an antigen molecule described herein" for inhibiting or reducing the binding of FcγRIIb to the Fc domain of the antibody molecule means that the test agent is capable of at least partially inhibiting or interfering with the binding of an agent described herein to FcγRIIb and inhibiting or reducing the binding of FcγRIIb to the Fc domain of the antibody molecule.

[0110] For example, the agent is capable of inhibiting the binding of an agent described herein by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100%, and / or inhibiting the ability of the agent to inhibit or reduce the binding of FcγRIIb to the Fc domain of an antibody molecule by at least 10%, e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100%.

[0111] Competitive binding can be determined by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0112] ELISA assays can be used to evaluate epitope modifications or blocking antibodies. Other methods suitable for identifying competing antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane (e.g., see pages 567 to 569, 574 to 576, 583, and 590 to 612, 1988, CSHL, NY, ISBN 0-87969-314-2), incorporated herein by reference.

[0113] The reagents of the present invention may comprise the following constant regions (CH and CL):

[0114] IgG1-CH [SEQ ID NO: 1]

[0115] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

[0116] QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

[0117] ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

[0118] TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR

[0119] EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD

[0120] GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0121] γ-CL[SEQ ID NO:2]QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0122] The reagents of the present invention may comprise one or more sequences of clones 1-14:

[0123] Clone 1

[0124] VH [SEQ ID NO: 3]

[0125]

[0126] -VL[SEQ ID NO:16]

[0127]

[0128] CDR area

[0129] CDRH1:NYGMH [SEQ ID NO:29]

[0130] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO:30]

[0131] CDRH3:EWRDAFDI [SEQ ID NO:31]

[0132] CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:32]

[0133] CDRL2:SDNQRPS[SEQ ID NO:33]

[0134] CDRL3:AAWDDSLSGSWV[SEQ ID NO:34]

[0135] Clone 2

[0136] -VH[SEQ ID NO:4]

[0137]

[0138] -VL[[SEQ ID NO:17]

[0139]

[0140] CDR area CDRH1:TYGMH[SEQ ID NO:35]

[0141] CDRH2:VIAYDGSKKDYADSVKG[SEQ ID NO:36]CDRH3:EYRDAFDI[SEQ ID NO:37]CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:38]CDRL2:GNSNRPS[SEQ ID NO:39]CDRL3:AAWDDSVSGWM[SEQ ID NO:40]

[0142] Clone 3

[0143] -VH[SEQ ID NO:5]

[0144]

[0145] -VL[SEQ ID NO:18]

[0146]

[0147] CDR area

[0148] CDRH1:NYGMH[SEQ ID NO:41]

[0149] CDRH2:VISYDGSNRYYADSVKG[SEQ ID NO:42]CDRH3:DRWNGMDV[SEQ ID NO:43]CDRL1:SGSSSNIGAGYDVH[SEQ ID NO:44]CDRL2:ANNQRPS[SEQ ID NO:45]

[0150] CDRL3:AAWDDSLNGPWV[SEQ ID NO:46]

[0151] Clone 4

[0152] -VH[[SEQ ID NO:6]

[0153]

[0154] -VL[SEQ ID NO:19]

[0155]

[0156] CDR area

[0157] CDRH1:SYGMH[SEQ ID NO:47]

[0158] CDRH2:VISYDGSDTAYADSVKG[SEQ ID NO:48]CDRH3:DHSVIGAFDI[SEQ ID NO:49]CDRL1:SGSSSNIGSNTVN[SEQ ID NO:50]CDRL2:DNNKRPS[SEQ ID NO:51]CDRL3:SSYAGSNNVV[SEQ ID NO:52]

[0159] Clone 5

[0160] -VH[SEQ ID NO:7]

[0161]

[0162] -VL[SEQ ID NO:20]

[0163]

[0164] CDR area

[0165] CDRH1:NYGMH[SEQ ID NO:53]

[0166] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO:54]CDRH3:DQLGEAFDI[SEQ ID NO:55]CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:56]CDRL2:DNNKRPS[SEQ ID NO:57]CDRL3:ATWDDSLSGPV[SEQ ID NO:58]

[0167] Clone 6

[0168] -VH[SEQ ID NO:8]

[0169]

[0170] -VL[SEQ ID NO:21]

[0171]

[0172] CDR area

[0173] CDRH1:DYGMS[SEQ ID NO:59]

[0174] CDRH2:AISGSGSSTYYADSVKG[SEQ ID NO:60]CDRH3:GDIDYFDY[SEQ ID NO:61]CDRL1:TGSSSNFGAGYDVH[SEQ ID NO:62]CDRL2:ENNKRPS[SEQ ID NO:63]

[0175] CDRL3:AAWDDSLNGPV[SEQ ID NO:64]

[0176] Clone 7

[0177] -VH[SEQ ID NO:9]

[0178]

[0179] -VL[SEQ ID NO:22]

[0180]

[0181] CDR area

[0182] CDRH1:SYGMH[SEQ ID NO:65]

[0183] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO:66]CDRH3:ERRDAFDI[SEQ ID NO:67]CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:68]CDRL2:SDNQRPS[SEQ ID NO:69]CDRL3:ATWDSDTPV[SEQ ID NO:70]

[0184] Clone 8

[0185] -VH[SEQ ID NO:10]

[0186]

[0187] -VL[SEQ ID NO:23]

[0188]

[0189] CDR area

[0190] CDRH1:SYGMH[SEQ ID NO:71]

[0191] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO:72]CDRH3:DHSAAGYFDY[SEQ ID NO:73]CDRL1:SGSSSNIGSNTVN[SEQ ID NO:74]CDRL2:GNSIRPS[SEQ ID NO:75]CDRL3:ASWDDSLSSPV[SEQ ID NO:76]

[0192] Clone 9

[0193] -VH[SEQ ID NO:11]

[0194]

[0195] -VL[SEQ ID NO:24]

[0196]

[0197] CDR area

[0198] CDRH1:SYGMH[SEQ ID NO:77]

[0199] CDRH2:GISWDSAIIDYAGSVKG[SEQ ID NO:78]CDRH3:DEAAAGAFDI[SEQ ID NO:79]CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:80]CDRL2:GNTDRPS[SEQ ID NO:81]CDRL3:AAWDDSLSGPVV[SEQ ID NO:82]

[0200] Clone 13

[0201] -VH[SEQ ID NO:15]

[0202]

[0203] -VL[SEQ ID NO:28]

[0204]

[0205] CDR area

[0206] CDRH1:SYGIS[SEQ ID NO:101]

[0207] CDRH2:GISGSGGNTYYADSVKG[SEQ ID NO:102]CDRH3:SVGAYANDAFDI[SEQ ID NO:103]CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:104]CDRL2:GDTNRPS[SEQ ID NO:105]CDRL3:AAWDDSLNGPV[SEQ ID NO:106]

[0208] Clone 10

[0209] -VH[SEQ ID NO:12]

[0210]

[0211] -VL[SEQ ID NO:25]

[0212]

[0213] CDR area

[0214] CDRH1:SYGMH[SEQ ID NO:83]

[0215] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO:84]CDRH3:ELYDAFDI[SEQ ID NO:85]CDRL1:TGSSSNIGAGYDVH[SEQ ID NO:86]CDRL2:ADDHRPS[SEQ ID NO:87]CDRL3:ASWDDSQRAVI[SEQ ID NO:88]

[0216] Clone 11

[0217] -VH[SEQ ID NO:13]

[0218]

[0219] -VL[SEQ ID NO:26]

[0220]

[0221] CDR area

[0222] CDRH1:SYGMH[SEQ ID NO:89]

[0223] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO:90]CDRH3:EFGYIILDY[SEQ ID NO:91]CDRL1:SGSSSNIGSNTVN[SEQ ID NO:92]CDRL2:RDYERPS[SEQ ID NO:93]CDRL3:MAWDDSLSGVV[SEQ ID NO:94]

[0224] Clone 12

[0225] -VH[SEQ ID NO:14]

[0226]

[0227] -VL[SEQ ID NO:27]

[0228]

[0229] CDR area

[0230] CDRH1:NHGMH[SEQ ID NO:95]

[0231] CDRH2:VISYDGTNKYYADSVRG[SEQ ID NO:96]

[0232] CDRH3:ETWDAFDV[SEQ ID NO:97]

[0233] CDRL1:SGSSSNIGSNNAN[SEQ ID NO:98]

[0234] CDRL2:DNNKRPS[SEQ ID NO:99]

[0235] CDRL3:QAWDSSTVV[SEQ ID NO:100]

[0236] Preferred target cell surface antigens can be selected from the following antigens: CD20, Thy-1 (CD90, differentiation cluster 90 (Biofactors. 2009 May-Jun; 35(3):258-65)); Ly-6 (lymphocyte antigen 6 (Mol Biol Rep. 2009 Apr; 36(4):697-703)); CD59 (complement regulatory protein (Mol Immunol. 2007 Jan; 44(1-3):73-81)); Fas (FS7-related cell surface antigen, CD95, APO-1 or TNFRSF6 (Adv Exp Med Biol. 2009; 647:64-93)); EGFR (epidermal growth factor receptor (FEBS J. 2010 Jan; 277(2):301-8)); Her2 (human epidermal growth factor receptor 2 (Clin Breast Cancer. 2008 Oct; 8(5): 392-401); CXCR4 (chemokine receptor 4 (Biochim Biophys Acta. 2007 Apr; 1768(4): 952-63); CD19 (cluster of differentiation 19 (Cell Immunol. 1989 Feb; 118(2): 368-81); CD40 (cluster of differentiation 40 (Basic Clin Pharmacol Toxicol. 2009 Feb; 104(2): 87-92); HLA molecule (human leukocyte antigen molecule (Korean J Lab Med. 2010 Jun; 30(3): 203); GM1 (ganglioside, monosialotetrahexosylganglioside (J Lipid Res. 2010 Sep; 51(9): 2731-8); CD22 (Cheson (2008) NEJM 359(6):613-26); CD23 (Cheson, 2008); CD80 (Cheson, 2008); CD74 (Cheson, 2008); DRD (Cheson, 2008).

[0237] Preferably, in the composition, use or method of the present invention, the surface antigen is selected from CD19, CD20, or CD40, more preferably, a human form thereof. CD20, especially human CD20, is most preferred.

[0238] Advantageously, the antibody molecule that specifically binds to the cell surface antigen is a monoclonal antibody, preferably a monoclonal antibody that is removed from the cell surface upon binding to a target cell and internalized into the target cell in an FcγRIIb-dependent manner. Preferably, the monoclonal antibody is an anti-CD19 antibody, an anti-CD20 antibody, or an anti-CD40 antibody. Most preferably, the monoclonal antibody is an anti-CD20 monoclonal antibody.

[0239] In a preferred embodiment, the antibody molecule that specifically binds to a cell surface antigen is a type I anti-CD20 antibody. In another preferred embodiment, the antibody molecule that specifically binds to a cell surface antigen is not a type II anti-CD20 antibody.

[0240] In one embodiment, the cell surface antigen is CD20, and the antibody molecule that specifically binds to the cell surface antigen is a type I antibody.

[0241] As mentioned above, there are two types of anti-CD20 monoclonal antibodies (mAbs). Anti-CD20 mAbs were first defined by the inventors in 2003 as belonging to different groups (43 and 25), and then in 2004 as (26) type I and type II mAbs. Initially, the basis for this was that anti-CD20 mAbs belonged to two different types of agents based on their ability to eliminate lymphoma xenografts: type I (e.g., rituximab and 1F5) utilized complement, and type II (e.g., B1) did not utilize complement. Both types of mAbs produced a significant prolongation of survival, but the disruption of complement activity by administration of CVF significantly reduced the efficacy of rituximab and 1F5, but had no effect on the activity of B1. These results clearly demonstrate that different CD20 mAbs operate different effector mechanisms in vivo. Furthermore, and in complete agreement with previous work, these results show that rituximab and 1F5 are able to effectively activate complement due to their ability to transfer CD20 to lipid rafts in the target cell membrane, whereas type B1 mAbs are unable to do so (43). This correlates well with the ability of mAbs to engage complement and induce CD20 to move into lipid rafts (43, 26). Thus, type I and type II are essentially defined by their ability to move CD20 into lipid rafts. This can be determined as described below. This also correlates with type II mAbs being able to elicit more efficient homotypic adhesion and direct cell death, but these cannot be used alone to define type I or type II mAbs (unlike the Tx-100 valve assay, see below).

[0242] Therefore, these different anti-CD20 mAbs have been classified as type I (e.g., rituximab, ofatumumab) or type II (e.g., tositumomab (B1), GA101, 11B8) based on their ability to redistribute CD20 in the plasma membrane and their activity in different effector cell assays (25-27). Type I (e.g., rituximab, ofatumumab) anti-CD20 mAbs induce CD20 redistribution to large detergent-resistant microdomains (valves), whereas type II (e.g., tositumomab) anti-CD20 mAbs cannot achieve this (50).

[0243] As described above, anti-CD20 mAbs are defined as type I or type II based on whether they redistribute CD20 to the lipid valves. This is accomplished by either a Tx-100 insolubility assay or by sucrose density gradient separation and Western blotting. These two methods are described in Cragg et al Blood 2003 (43) as follows:

[0244] 1. Evaluation of Valve-associated Antigens by Triton X-100 Insolubility

[0245] For rapid assessment of the presence of antigens in valve microdomains, a flow cytometric method based on Triton X-100 insolubility was used at low temperature. Briefly, cells were washed in RPMI / 1% BSA and plated at 2.5 x 10 6 The cells were then incubated with 10 μg / ml FITC-conjugated mAb for 15 minutes at 37°C, washed in cold PBS / 1% BSA / 20 mM sodium azide, and the sample was split in half. One half was kept on ice to calculate 100% surface antigen levels, while the other half was treated with 0.5% Triton X-100 on ice for 15 minutes to determine the proportion of antigen remaining in the insoluble valve fraction. The cells were then maintained at 4°C for the remainder of the assay, washed once in PBS / BSA / azide, resuspended, and evaluated by flow cytometry as detailed above. Similar results were obtained using an indirect detection method. To determine the constitutive level of target antigen valve binding, the cells were first treated with 0.5% Triton X-100 on ice for 15 minutes and washed in PBS / BSA / azide before binding the FITC-labeled mAb. To evaluate whether additional antigen could be transferred to the Triton-X 100 insoluble fraction by additional cross-linking, cells were incubated with FITC-mAb as previously described, washed, and then divided into four aliquots. Two of these samples were incubated with goat anti-mouse Ig F(ab')2 fragments on ice for 15 minutes. After washing, one cross-linked sample and one non-cross-linked sample were lysed in Triton X-100 and washed as detailed above before flow cytometry.

[0246] 2. Sucrose Density Gradient Separation and Western Blotting - Lipid Valve Fraction Preparation and Western Blotting

[0247] Monoclonal Ab (1 μg / 10 6 cells) were added to the cells at 37°C. After a 20-minute incubation, the cells were pelleted and lysed in ice-cold MES-buffered saline (25 mM MES, pH 6.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 μThe cells were lysed in 5% 4% aprotinin (5 μg / ml aprotinin, 5 μg / ml leupeptin, 10 mM EDTA). The lipid valve fraction was then prepared by sucrose density gradient centrifugation. Briefly, the lysate was mixed with an equal volume of lysis buffer containing 80% sucrose, overlaid with a discontinuous 5-30% sucrose density gradient, and then centrifuged at 200,000 x g for 16 h. Fractions (0.5 ml) were collected and analyzed by Western blotting. A 15 ml sample of each fraction was diluted 1:1 in 2x loading buffer, heated to 95°C for 5 minutes, and separated on a 15% SDS-PAGE gel, then transferred to a PVDF membrane and incubated with a primary antibody (e.g., mouse anti-CD20 antibody, clone 7D1 for CD20 detection or anti-Lyn rabbit polyclonal serum for valve fraction identification; Serotec, UK), followed by incubation with an HRP-conjugated secondary antibody (Amersham Biosciences UK Ltd). Blots were visualised using ECL+plus (Amersham Biosciences UK Ltd).

[0248] Anti-CD20 mAbs may require an AxP motif in the CD20 macroloop. (Ofatumumab and other Genmab antibodies do not.) However, (Niederfelner et al. (51)) showed that type II mAbs bind to slightly different regions in the CD20 loop compared to type I mAbs.

[0249] Preferably, in the composition, use or method of the present invention, the target cell is a cancer cell. More preferably, the target cell is a cancer cell selected from non-Hodgkin's lymphoma, including but not limited to follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

[0250] In one embodiment, the present invention provides compositions, uses and methods for treating cancer, particularly B-cell malignancies, preferably selected from the group consisting of lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, multiple lymphoma, Hodgkin's disease and non-Hodgkin's disease, diffuse large B-cell lymphoma, follicular lymphoma with diffuse large B-cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, diffuse small cleaved cell lymphoma or a combination thereof. In certain embodiments, the B-cell malignancy is a lymphoma, such as non-Hodgkin's lymphoma (NHL).

[0251] In another embodiment, the present invention provides compositions, uses and methods for treating. The inflammatory disease can be an autoimmune disease, for example, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, autoimmune inner ear disease, myasthenia gravis, Reiter's syndrome, Graves' disease, autoimmune hepatitis, familial multiple adenocarcinoma and ulcerative colitis, or a combination thereof. In a specific embodiment, the autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus.

[0252] In preferred embodiments, the diseases treated include chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), B-cell malignancies, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, systemic sclerosis, and autoimmune blistering diseases.

[0253] In a preferred embodiment, the therapy enhanced by use of the present invention is treatment with an anti-CD20 mAb such as rituximab.

[0254] definition

[0255] "Elevated" includes the meaning that the cell in question expresses a higher level of FcγRIIb on its surface compared to a control or reference cell that expresses low or moderate levels of FcγRIIb on its surface. For example, if the cell in question is a B cell, its FcγRIIb expression level is considered "elevated" if its FcγRIIb expression level is higher than the normal (preferably moderate) expression level of FcγRIIb by B cells of the same cell type. Alternatively, the cell in question is considered "elevated" if its expression level is higher than that of a different cell type that expresses FcγRIIb at low or moderate levels.

[0256] According to the present invention, the more elevated the expression of FcγRIIb by target cells, the less responsive these cells are expected to be to treatment with an antibody molecule that specifically binds to a target cell surface antigen and has an Fc domain capable of binding to FcγRIIb. Figure 2 As shown in D and 3A, the more FcγRIIb expression is elevated, the greater the benefit from using an agent of the present invention that inhibits or reduces binding of the Fc domain to FcγRIIb. Figure 10 b) and after separation of MCL samples into FcγRIIb positive and negative, significant differences in clinical responses were observed after rituximab-based treatment ( Figure 10 c and 10d).

[0257] Those skilled in the art can easily determine the expression level of FcγRIIb on cells by various known methods, such as flow cytometry and immunohistochemical staining methods described in the Figures and Examples.

[0258] Those skilled in the art will understand that "normal" and "elevated" expression levels of FcγRIIb will vary between different cell types and different disease states, and those skilled in the art will be able to identify "normal" and "elevated" expression levels of FcγRIIb for a given target cell or disease state using methods known in the art and described herein. Figure 2 Exemplary levels of "normal" (or intermediate) and "elevated" expression of FcγRIIb on certain cell types are provided in Figure C. In these specific examples, in follicular lymphoma (FL), "normal" levels are about 50 (ratio of geometric MFI FcγRIIb to isotype control) and "elevated" levels are about 125 or 400 or more, while in diffuse large B-cell lymphoma (DLBCL), "normal" levels are about 20 and "elevated" levels are about 80 or more, while in mantle cell lymphoma (MCL), "normal" levels are about 60 and "elevated" levels are about 110 or 190 or more, and in chronic lymphoid leukemia (CLL), "normal" levels are about 100 and "elevated" levels are about 300 or more.

[0259] Preferably, the elevated FcγRIIb expression level is at least 1.1-fold relative to the normal (preferably median) expression level in cells of the same cell type (or cells of a different cell type), or at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0 , 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 , 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6 0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65.0, 66.0, 67.0, 68.0, 69.0, 70.0, 71.0, 72.0, 73.0, 74.0, 75.0, 76.0, 77.0

[0260] As shown in the accompanying examples, the inventors have determined that there is a correlation between the level of FcγRIIb expression and increased modulation of mAbs from the cell surface. Higher levels of FcγRIIb expression result in higher modulation, i.e., there is a correlation between the degree of increased expression and modulation, as shown in FIG. Figure 2 D and 3A. Figure 2 In D, the level of FcγRIIb expression is plotted against the level of modulation observed. The correlation is that the highest levels of FcγRIIb (e.g., >400) result in the lowest levels of surface CD20 (<20%), i.e., have the greatest effect on mAb modulation from the cell surface. Figure 3In A, low (18), medium (70), and high (124) levels of FcγRIIb were introduced into an FcγRIIb-negative cell line (Ramos), which was directly associated with a reduction in cell surface CD20 levels proportional to FcγRIIb expression (60%, 40%, and 30% for low, medium, and high levels, respectively). FcγRIIb expression levels were expressed as the ratio of the geometric mean fluorescence intensity (geometric MFI) of FcγRIIb to the isotype control.

[0261] Modulation reduces the amount of mAb remaining on the cell surface. mAbs require Fc to participate in immune effector cell mechanisms (ADCC, ADCP, CDC) to eliminate target cells. Therefore, using FcγRIIb blocking mAbs (mAbs that block FcγRIIb) to reduce modulation will improve Fc-dependent effector cell function. Figure 8 ADCP (phagocytosis) is shown in Figure 2. Phagocytosis with rituximab alone was 40%, but this increased to 55% when FcγRIIb was blocked by AT10.

[0262] "Antibody molecules" include monoclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, human antibodies, chimeric antibodies, camelized antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intrabodies, or epitope-binding fragments of any of the foregoing. Preferably, the antibodies of the present invention are monoclonal antibodies, more preferably humanized or human antibodies.

[0263] Methods for preparing and characterizing antibody molecules useful in the compositions, uses, and methods of the present invention are well known to those skilled in the art. For example, WO 2008 / 002933 (Sections 5.3 to 5.3.1, pages 74-91) describes the preparation and characterization of monoclonal antibodies that specifically bind to target cell surface antigens (e.g., CD20 or FcγRIIb). Useful antibodies that specifically bind to FcγRIIb and CD20, including monoclonal antibodies produced by hybridomas deposited under the Budapest Treaty, are also disclosed on pages 15 to 21 of WO 2008 / 002933, the contents of which are incorporated herein by reference.

[0264] "Specific binding" includes agents such as antibody molecules that bind to the target antigen but do not bind to other antigens (do not cross-react with other antigens) or bind to the other antigens weakly, i.e., with a lower affinity than the target antigen. For example, an antibody that specifically binds to FcγRIIb may bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIAcore, or other assays known in the art. Preferably, antibodies or fragments thereof that specifically bind to FcγRIIb do not cross-react with other antigens. Antibodies that specifically bind to FcγRIIb can be identified by, for example, immunoassays, BIAcore, or other assays known to those skilled in the art. An antibody or fragment thereof specifically binds FcγRIIb when it binds to FcγRIIb with a higher affinity than to any cross-reactive antigen as determined using assays such as Western blot, reflex immunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) (see Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336 (1989) for a discussion of antibody specificity) and when it binds to FcγRIIb, particularly human FcγRIIb, more particularly native human FcγRIIb, with a greater affinity than the antibody or fragment thereof binds to FcγRIIA, particularly human FcγRIIA, more particularly native human FcγRIIA. Representative antibodies are disclosed in U.S. Patent Application Nos. 2004-0185045, 2005-0260213, and 2006-0013810, which are incorporated herein by reference in their entireties.

[0265] Preferably, certain FcγRIIb antibodies used in combination with CD20 antibodies in the compositions and methods of the present invention bind to the extracellular domain of native human FcγRIIb. In some embodiments, the antibody or fragment thereof binds to FcγRIIb with an affinity that is at least two times greater than the affinity with which the antibody or fragment thereof binds to FcγRIIA. In other embodiments, the antibody or fragment thereof binds to FcγRIIb with an affinity that is at least 4 times, at least 6 times, at least 8 times, at least 10 ... 4 times, at least 10 5 times, at least 10 6 times, at least 10 7 times or at least 10 8 In a preferred embodiment, the affinity of the antibody or fragment thereof to FcγRIIb is 100-fold, 1000-fold, 1000-fold, or 1000-fold higher than the affinity of the antibody or fragment thereof to FcγRIIA. 4 times, 10 5times, 10 6 times, 10 7 times, 10 8 times.

[0266] This application provides the following:

[0267] 1. A composition comprising:

[0268] (i) an antibody molecule that specifically binds to a cell surface antigen of a target cell and has an Fc domain capable of binding to FcγRIIb; and

[0269] (ii) an agent that inhibits or reduces the binding of FcγRIIb to the Fc domain of the antibody molecule,

[0270] Characterized in that the composition is used to treat patients having target cells with elevated FcγRIIb expression levels.

[0271] 2. Use of an agent for inhibiting or reducing the binding between the Fc domain of an antibody molecule and FcγRIIb on a target cell, wherein the antibody molecule specifically binds to a surface antigen of the target cell, and wherein the use is for the preparation of a medicament for treating a patient having target cells with elevated FcγRIIb expression levels.

[0272] 3. A method for treating a patient having target cells expressing FcγRIIb, the method comprising administering in combination: (i) an antibody molecule that specifically binds to a surface antigen of the target cell, the antibody molecule having an Fc domain capable of binding to FcγRIIb; and (ii) an agent that inhibits or reduces the binding between the Fc domain of the antibody molecule and FcγRIIb, wherein the patient is selected based on an elevated level of FcγRIIb expression on their target cells.

[0273] 4. Use of FcγRIIb expression on target cells as a prognostic marker of the response of said target cells to treatment with an antibody molecule, said antibody molecule specifically binding to a surface antigen of said target cell, and said antibody molecule having an Fc domain capable of binding to FcγRIIb, whereby increased levels of FcγRIIb indicate a reduced response or no response to treatment with said antibody molecule.

[0274] 5. A method for predicting the response of a patient's target cells to treatment with an antibody molecule that specifically binds to a target cell surface antigen and has an Fc domain capable of binding to FcγRIIb, characterized in that the method comprises determining the level of FcγRIIb expression on the target cells, whereby an increased level of FcγRIIb indicates a reduced response or no response to treatment with the antibody molecule.

[0275] 6. The composition, use or method according to any one of 1 to 3, wherein the agent inhibits or reduces the binding of FcγRIIb on the target cell to the Fc domain of the antibody molecule.

[0276] 7. The composition, use or method according to any preceding claim, wherein the antibody molecule that specifically binds to a target cell surface antigen and has an Fc domain capable of binding to FcγRIIb can be internalized into the target cell in an FcγRIIb-dependent manner.

[0277] 8. The composition, use or method according to any one of 1-3 and 6-7, wherein the agent that inhibits or reduces the binding of FcγRIIb to the Fc domain of the antibody molecule also inhibits or reduces the internalization of the antibody molecule into the target cell.

[0278] 9. The composition, use or method of any preceding claim, wherein the target cell is a cancer cell.

[0279] 10. The composition, use or method of any preceding claim, wherein the target cell is a B cell.

[0280] 11. The composition, use or method according to any preceding claim, wherein the patient to be treated is a cancer patient and the treatment is cancer treatment.

[0281] 12. The composition, use or method of any one of items 9-11, wherein the cancer is selected from non-Hodgkin's lymphoma, such as follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

[0282] 13. A composition, use or method according to any of the preceding items, wherein the agent is any of the following: a polypeptide; anticalin; a peptide; an antibody; a chimeric antibody; a single-chain antibody; an aptamer; a darpin; a Fab, F(ab′)2, Fv, ScFv or dAb antibody fragment; a small molecule; a natural product; an affibody; a peptidomimetic; a nucleic acid; a peptide nucleic acid molecule; a lipid; a carbohydrate; a modular framework-based protein, including ankyrin repeat proteins, armadillo repeat proteins, leucine-rich proteins, tetratricopeptide repeat proteins or designed ankyrin repeat proteins (DARPins).

[0283] 14. The composition, use or method of any preceding claim, wherein the agent is one or more antibody molecules that specifically bind to FcγRIIb.

[0284] 15. The composition, use or method of 14, wherein the one or more antibody molecules do not comprise a domain capable of recruiting effector cells.

[0285] 16. The composition, use or method of 15, wherein the one or more antibody molecules are monoclonal antibody molecules.

[0286] 17. The composition, use or method of any one of 1-3 and 6-16, wherein the agent comprises a heavy chain variable region (VH) comprising the following CDRs:

[0287] (i) SEQ ID NO: 29 and SEQ ID NO: 30 and SEQ ID NO: 31; or

[0288] (ii) SEQ ID NO: 35 and SEQ ID NO: 36 and SEQ ID NO: 37; or

[0289] (iii) SEQ ID NO:41 and SEQ ID NO:42 and SEQ ID NO:43; or

[0290] (iv) SEQ ID NO:47 and SEQ ID NO:48 and SEQ ID NO:49; or

[0291] (v) SEQ ID NO: 53 and SEQ ID NO: 54 and SEQ ID NO: 55; or

[0292] (vi) SEQ ID NO: 59 and SEQ ID NO: 60 and SEQ ID NO: 61; or

[0293] (vii) SEQ ID NO: 65 and SEQ ID NO: 66 and SEQ ID NO: 67; or

[0294] (viii) SEQ ID NO:71 and SEQ ID NO:72 and SEQ ID NO:73; or

[0295] (ix) SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79; or

[0296] (x) SEQ ID NO:83 and SEQ ID NO:84 and SEQ ID NO:85; or

[0297] (xi) SEQ ID NO:89 and SEQ ID NO:90 and SEQ ID NO:91; or

[0298] (xii) SEQ ID NO:95 and SEQ ID NO:96 and SEQ ID NO:97; or

[0299] (xiii) SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103.

[0300] 18. The composition, use or method of any one of 1-3 and 6-17, wherein the agent comprises a light chain variable region (VL) comprising the following CDRs:

[0301] (i) SEQ ID NO: 32 and SEQ ID NO: 33 and SEQ ID NO: 34; or

[0302] (ii) SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40; or

[0303] (iii) SEQ ID NO:44 and SEQ ID NO:45 and SEQ ID NO:46; or

[0304] (iv) SEQ ID NO: 50 and SEQ ID NO: 51 and SEQ ID NO: 52; or

[0305] (v) SEQ ID NO: 56 and SEQ ID NO: 57 and SEQ ID NO: 58; or

[0306] (vi) SEQ ID NO: 62 and SEQ ID NO: 63 and SEQ ID NO: 64; or

[0307] (vii) SEQ ID NO: 68 and SEQ ID NO: 69 and SEQ ID NO: 70; or

[0308] (viii) SEQ ID NO:74 and SEQ ID NO:75 and SEQ ID NO:76; or

[0309] (ix) SEQ ID NO:80 and SEQ ID NO:81 and SEQ ID NO:82; or

[0310] (x) SEQ ID NO:86 and SEQ ID NO:87 and SEQ ID NO:88; or

[0311] (xi) SEQ ID NO:92 and SEQ ID NO:93 and SEQ ID NO:94; or

[0312] (xii) SEQ ID NO:98 and SEQ ID NO:99 and SEQ ID NO:100; or

[0313] (xiii) SEQ ID NO: 104, SEQ ID NO: 105, and SEQ ID NO: 106.

[0314] 19. The composition, use or method of any one of items 1-3 and 6-18, wherein the agent comprises a heavy chain variable region (VH) amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0315] 20. The composition, use or method of any one of items 1-3 and 6-19, wherein the agent comprises a light chain variable region (VL) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0316] 21. The composition, use or method of any one of 1-3 and 6-20, wherein the agent comprises the following CDR amino acid sequence:

[0317] (i) SEQ ID NO: 29 and SEQ ID NO: 30 and SEQ ID NO: 31 and SEQ ID NO: 32 and SEQ ID NO: 33 and SEQ ID NO: 34; or

[0318] (ii) SEQ ID NO:35 and SEQ ID NO:36 and SEQ ID NO:37 and SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40; or

[0319] (iii) SEQ ID NO:41 and SEQ ID NO:42 and SEQ ID NO:43 and SEQ ID NO:44 and SEQ ID NO:45 and SEQ ID NO:46; or

[0320] (iv) SEQ ID NO:47 and SEQ ID NO:48 and SEQ ID NO:49 and SEQ ID NO:50 and SEQ ID NO:51 and SEQ ID NO:52; or

[0321] (v) SEQ ID NO:53 and SEQ ID NO:54 and SEQ ID NO:55 and SEQ ID NO:56 and SEQ ID NO:57 and SEQ ID NO:58; or

[0322] (vi) SEQ ID NO:59 and SEQ ID NO:60 and SEQ ID NO:61 and SEQ ID NO:62 and SEQ ID NO:63 and SEQ ID NO:64; or

[0323] (vii) SEQ ID NO:65 and SEQ ID NO:66 and SEQ ID NO:67 and SEQ ID NO:68 and SEQ ID NO:69 and SEQ ID NO:70; or

[0324] (viii) SEQ ID NO:71 and SEQ ID NO:72 and SEQ ID NO:73 and SEQ ID NO:74 and SEQ ID NO:75 and SEQ ID NO:76; or

[0325] (ix) SEQ ID NO:77 and SEQ ID NO:78 and SEQ ID NO:79 and SEQ ID NO:80 and SEQ ID NO:81 and SEQ ID NO:82; or

[0326] (x) SEQ ID NO:83 and SEQ ID NO:84 and SEQ ID NO:85 and SEQ ID NO:86 and SEQ ID NO:87 and SEQ ID NO:88; or

[0327] (xi) SEQ ID NO:89 and SEQ ID NO:90 and SEQ ID NO:91 and SEQ ID NO:92 and SEQ ID NO:93 and SEQ ID NO:94; or

[0328] (xii) SEQ ID NO:95 and SEQ ID NO:96 and SEQ ID NO:97 and SEQ ID NO:98 and SEQ ID NO:99 and SEQ ID NO:100; or

[0329] (xiii) SEQ ID NO: 101 and SEQ ID NO: 102 and SEQ ID NO: 103 and SEQ ID NO: 104 and SEQ ID NO: 105 and SEQ ID NO: 106.

[0330] 22. The composition, use or method according to any one of 1-3 and 6-21, wherein the agent comprises the following amino acid sequence:

[0331] (i) SEQ ID NO: 3 and SEQ ID NO: 16; or

[0332] (ii) SEQ IS NO: 4 and SEQ ID NO: 17; or

[0333] (iii) SEQ IS NO: 5 and SEQ ID NO: 18; or

[0334] (iv) SEQ ID NO: 6 and SEQ ID NO: 19; or

[0335] (v) SEQ ID NO: 7 and SEQ ID NO: 20; or

[0336] (vi) SEQ ID NO: 8 and SEQ ID NO: 21; or

[0337] (vii) SEQ ID NO: 9 and SEQ ID NO: 22; or

[0338] (viii) SEQ ID NO: 10 and SEQ ID NO: 23; or

[0339] (ix) SEQ ID NO: 11 and SEQ ID NO: 24; or

[0340] (x) SEQ ID NO: 12 and SEQ ID NO: 25; or

[0341] (xi) SEQ ID NO: 13 and SEQ ID NO: 26; or

[0342] (xii) SEQ ID NO: 14 and SEQ ID NO: 27; or

[0343] (xiii) SEQ ID NO:15 and SEQ ID NO:28.

[0344] 23. The composition, use or method according to any one of 1-3 and 6-16, wherein the agent is capable of competing with the agent according to 17-22, thereby being used to inhibit or reduce the binding of FcγRIIb to the Fc domain of the antibody molecule.

[0345] 24. The composition, use or method of any one of 1-3 and 6-23, wherein the agent inhibits or reduces FcγRIIb signaling.

[0346] 25. The composition, use or method of any one of 1-3 and 6-24, wherein the agent inhibits or reduces internalization of the antibody molecule by the target cell.

[0347] 26. A composition, use or method according to any preceding claim, wherein the cell surface antigen is selected from CD19, CD20 or CD40.

[0348] 27. The composition, use or method according to any preceding claim, wherein the antibody molecule that specifically binds to a cell surface antigen is a CD20 antibody.

[0349] 28. The composition, use or method of 27, wherein the CD20 antibody is a type I CD20 antibody.

[0350] 29. The composition, use or method according to any preceding claim, wherein the cell surface antigen is CD20, and the antibody molecule that specifically binds to the cell surface antigen is a type I antibody.

[0351] 30. The composition, use or method of any preceding claim, wherein the elevated expression of FcγRIIb on the target cell is determined relative to a control, preferably the normal expression level of FcγRIIb in cells of the same type as the target cell.

[0352] 31. A composition substantially as herein described with reference to the accompanying drawings.

[0353] 32. A method for use substantially as herein described with reference to the accompanying drawings.

[0354] 33. A method substantially as herein described with reference to the accompanying drawings. Example

[0355] Specific aspects of the present invention will now be described with reference to the accompanying drawings:

[0356] Figure 1 A- Figure 1 B: Internalization of type I mAbs from the cell surface of normal and malignant human B cells.

[0357] A) Primary CLL cells were incubated with Tosit-488, GA101gly-488, Ritux-488, or Ofatum-488 (all at 5 μg / ml) for 2 or 6 hours. The cells were then collected and washed twice, after which anti-Alexa-488 antibody was added to half of the samples and incubated at 4°C for 30 minutes to distinguish between internalized and non-internalized mAbs. Surface accessible CD20 (%) was calculated using the following formula: surface accessible CD20+ (geometric MFI before quenching – geometric MFI after quenching) / (geometric MFI before quenching) x 100. Each point represents a sample from a different CLL patient. Statistical analysis was performed using the Wilcoxon paired test (**p value < 0.001), and the mean values ​​are shown. B) Tosit-488 or Ritux-488 were then used to detect a variety of primary B cell tumors and normal B cells from healthy volunteers using the same assay. Statistical analysis was performed using the Mann Whitney test, and mean values ​​are shown.

[0358] Figure 1 C- Figure 1 G: Lack of correlation between CD20 modulation and CLL phenotype / prognostic markers. C) Correlation between modulation and known CLL prognostic factors. CLL cases were phenotyped for IgVH gene mutation status, Zap-70 and CD38 expression, and these samples were subjected to internalization assays to evaluate the effect of CD20 on CLL. Figure 1 A) Modulation was evaluated. The correlation between each prognostic feature and CD20 modulation was performed by Spearman correlation analysis. No correlation was observed with each prognostic factor (p>0.05). D) Similarly, sIg status, the ability of cells to trigger calcium flux, and the viability of CLL cells were evaluated and compared with CD20 modulation. Again, no correlation was observed. E) CD20 expression of CLL cells was evaluated by FACS using Ritux-488 and compared with CD20 modulation. A weak correlation was observed (Spearman r value -0.34, p=0.038). Subsequent analysis of multivariate regression of CD20 modulation using CD20 and FcγRIIb expression showed that the weak correlation with CD20 was not significant (p=0.638). F) sIg expression in IgM-positive CLL cases was determined by FACS, and expression levels were compared with CD20 modulation. No correlation was found (p>0.05). G) CLL cells were incubated with Rit m2a-488 for 2 hours and Figure 1 A) Internalization assay was performed. Differential CD38 expression was observed in individual CLL cases. FACS curves show samples before (left) and after (right) quenching. The corresponding histograms show CD38 expression in individual samples. +ve and CD38-ve Cells modulate at the same rate. +ve and CD38 -ve Cells are represented by solid and hollow peaks, respectively. These results are representative of 3 different cases.

[0359] Figure 2 Modulation is an Fc-dependent process. A) The internalization assay described in 1a) was repeated after CLL cells were incubated with Alexa-488-labeled rituximab fragments Fab', F(ab')2, and IgG for 6 hours. Data represent mean modulation levels + / - SD for three different CLL samples. B) CLL cells were incubated with Tosit-488, Ritux-488 + / - AT10, and Rit m2a-488 as in 1a) for 2 and 6 hours. Mean + / - modulation from six different CLL samples is shown. Adding the anti-FcγRII mAb AT10 to rituximab reduced CD20 modulation to a level similar to Rit m2a, while adding AT10 to Rit m2a had no significant effect on modulation. C) Various normal and malignant B cell samples were stained for FcγRIIb expression using AT10-PE. The histogram shows the differences in FcγRIIb expression in 3 different CLL cases. High (black line), medium (dark gray line) and low expressing cells (light gray line) are indicated. The dotted lines show the differences in FcγRIIb expression between healthy B cells, CLL, SLL, MCL, FL and SLBCL. FcγRIIb expression is expressed as the ratio of FcγRIIb:isotype control geometric MFI to control for small differences due to inter-assay differences. The median is shown. D) CD20 modulation and FcγRIIb expression are plotted for all NHL subtypes and normal B cells (obtained by the internalization assay described in 1A) and incubated with Ritux-488 for 6 hours). The analysis was performed using Spearman correlation assuming a non-parametric distribution. A strong correlation is shown. Spearman r value = -0.74, 95% confidence interval between -0.83 and -0.61, p < 0.0001.

[0360] Figure 3: FcγRIIb expression is the main determinant of CD20 modulation. A) Ramos cells transfected with FcγRIIb were sorted for expression of low, medium, and high levels of FcγRIIb and evaluated at the 6-hour time point of internalization assays using Tosit-488 and Ritux-488 along with blank-transfected cells. Error bars represent the mean + / - SD from independent experiments. Geometric MFI values ​​for FcγRIIb expression of the sorted cells are listed on the right. B) Internalization assays were repeated after 6 hours of incubation using Tosit-488 and Ritux-488 on normal Ramos cells, Rx3 cells (lacking BCR expression), blank-transfected Rx3 cells, and FcγRIIb-transfected Rx3 cells. Data points from 5 independent experiments are shown along with the median.

[0361] Figure 4 : Co-ligation of CD20 and FcγRIIb occurs primarily in cis and leads to FcγRIIb activation. A) Raji cells were incubated with specific mAbs (10 μg / ml) at 37°C for 2 hours, after which they were harvested, lysed, and immunoblotted for phosphorylated FcγRIIb. B) Left: PKH26-labeled Ramos cells (FcγRIIb -ve , R1) were mixed with the sorted Ramos transfectants with high FcγRIIb expression at a ratio of 1:1 ( Figure 3 Described in A). Right: FcγRIIb after 6 hours incubation with Ritux-488 +ve and FcγRIIb -ve CD20 modulation on cells. As a control, the two cells were also incubated alone. The data represent the average modulation level + / - SD from 3 independent experiments. C) In a similar experiment, CLL with low FcγRIIb expression was PKH26 labeled and then mixed with CLL with higher FcγRIIb expression at a 1:1 ratio. The experiment was performed three times, each time using a different CLL with high FcγRIIb expression. The FcγRIIb level (geometric MFI) was 42 (low), 275, 306 and 165 for high-expressing cells. The internalization assay was then performed as shown in 4B. The data represent the average modulation level + / - SD. D) Different CLL samples were injected at 20 x 10 5 , 4x 10 5 and 1x10 5 10 cells / ml were incubated with Ritux-488 for 6 hours, and internalization assay was performed as described above at 6 hours. E) Raji cells were plated at 20 x 10 5 , 4x 10 5 and 1x 10 5Cells / ml were incubated with specific mAbs (10 μg / ml) at 37°C for 2 hours. Images were captured using bright field microscopy to demonstrate differences in cell proximity. Cells were then collected and evaluated for phosphorylated FcγRIIb by immunoblotting. Figure 4 As described in A.

[0362] Figure 5 : Rituximab, CD20, and FcγRIIb are internalized into lysosomes together. A) CLL cells were incubated with Tosit-488 or Ritux-488 for 2 hours, and then stained with anti-CD19-APC and AT10-PE. Data are shown as the mean + / - 1SD of FcγRIIb expression as a percentage of untreated (n+6 CLL samples). FcγRIIb expression after Ritux-488 was significantly lower than after Tosit-488, *p<0.05. B) CLL cells were washed, fixed, and permeabilized, then stained with AT10-647 (blue), washed, and analyzed using a confocal microscope. The image represents FcγRIIb staining in unstimulated cells. C) The same CLL samples were incubated with Ritux-488 for 30 minutes, and then as shown in FIG. Figure 5 b) were treated as shown. The cells showed clear colocalization between Ritux-488 (green) and AT10-647 (blue) at this time. D) CLL cells were incubated with Tosit-488 for 6 hours and then prepared for microscopic observation as shown in 5B. In addition, cells were stained with biotinylated LAMP-1 and streptavidin-546 (red) to stain for lysosomes. Tosit-488 remained uniformly on the surface, and AT10-647 staining did not change relative to the baseline as shown in 5B. There was no colocalization with LAMP-1. E) CLL cells were treated with Ritux-488 for 6 hours and as shown in Figure 5 D) were evaluated. Two representative cells are shown here. The top cell shows clear colocalization between Ritux-488 and AT10-647, but no colocalization with LAMP-1. The bottom cell shows colocalization of all three fluorescent dyes. Bright field (BF) images from the same cell are shown in each case. The scale bar represents 5 μm.

[0363] Figure 6: The lack of inhibitory receptors enhances the clearance of anti-CD20 mAbs. hCD20 Tg mice (WT) or hCD20 Tg mice lacking CD32 (CD32KO) were treated with rituximab containing mouse IgG1 (m1) or mouse IgG2a (m2a) at a dose of 250 mg intravenously (iv). B cell depletion was then monitored by serial bleeding and flow cytometry using B220 and CD19 mAb staining for 90 days.

[0364] Figure 7 Blocking the inhibitory receptor CD32b (FcγRIIb) enhances the efficacy of anti-CD20 mAbs in a human xenograft system. CD20-positive human tumor cells (Daudi or Raji) were inoculated into SCID mice and then treated with rituximab, AT10, or both, and the mice were monitored for survival or tumor growth. The doses of mAbs used are shown in the figure legends. In A), Daudi cells were inoculated simultaneously, and tumors were monitored every 30 days by caliper. In B) and C), Raji cells were inoculated intravenously.

[0365] Figure 8 Phagocytosis of CLL cells treated with rituximab was enhanced by co-incubation with an FcγRII blocking mAb. Monocytes were derived from healthy volunteers and differentiated into macrophages using M-CSF in 6-well plates for at least 7 days before use. Macrophages were then harvested and plated in 96-well plates at 5 x 10 5Cells were allowed to adhere to the plate before the addition of CFSE-labeled CLL cells. CFSE-labeled CLL cells were left untreated or opsonized with 10 μg / ml of rituximab and the FcγRIIb blocking mAb AT10(fab')2 for 15 minutes or 6 hours, then washed twice and added to macrophages (1:1 ratio) for at least 30 minutes. Then, anti-CD16f(ab)2-APC (5 μg / ml) was added to each well for 15 minutes at room temperature (RT) to stain macrophages, and then the wells were washed once with FACS wash (PBS BSA azide) at room temperature. Further ice-cold FACS wash was added and the plates were incubated on ice for 10 minutes before being collected for FACS analysis. % double positive macrophages represents the percentage of CD16+CFSE+ positive cells, expressed as % of the total number of CD16+ cells. N=3 replicates, and the line represents the mean. The data clearly show that phagocytosis of CLL cells was higher when rituximab was added for only 15 minutes than for 6 hours. This increase in efficacy was associated with modulation of rituximab from the cell surface and could be reversed by treatment with mAb AT10 that blocks FcγRIIb. Importantly, only FcγRIIb was The mAb was added to the CLL cells and therefore had no effect on the macrophages themselves. In addition, only the Fab2 fragment of AT10 was used, so increased phagocytosis could not occur due to more mAb binding to the CLL cell surface. This conclusion is also supported by the observation that no increase in phagocytosis was observed after only 15 minutes of incubation (at which time a small amount of rituximab modulation had already occurred).

[0366] Figure 9 : FcγRIIb expression was detected by IHC. Paraffin-embedded tissue was stained using anti-CD32b specific mAb EP888Y. Images from four different FL patients are shown (x40 total magnification, and x150 magnification insets). FcγRIIb expression was detected by flow cytometry by staining matched live cells with AT10-PE. The upper left corner of each image shows the FcγRIIb expression obtained by flow cytometry.

[0367] Figure 10 : FcγRIIb levels predict clinical outcomes in rituximab-treated MCL patients. As proof of concept for the present in vitro findings, the inventors retrospectively examined FcγRIIb expression in a cohort of MCL patients who had received rituximab. Diagnostic paraffin-embedded tissue was stained by immunohistochemistry using an FcγRIIb-specific mAb ( Figure 11 Strong membrane staining was observed in FcγRIIb+ve but not in FcγRIIb-ve lymphoma samples. Figure 10FcγRIIb staining shown in a and 10b is consistent with Figure 2 The expression of FcγRIIb by flow cytometry shown in D is related (the numerical value detected by flow cytometry in 2D is expressed as Figure 10 These results correlated with FcγRIIb expression of the corresponding DMSO-frozen samples obtained by flow cytometry ( Figure 2 C, inset values.) Although only a small cohort of 16 MCL patients was studied, patients with FcγRIIb-ve lymphoma had much better median progression-free survival than those with FcγRIIb+ve cells (median 852 days versus 189 days, respectively). Figure 10 c shows the survival difference in the FcγRIIb+ and - subgroups. The two groups were comparable in clinical characteristics (MCL International Prognostic Index, data not shown), but were heterogeneous in the type of chemotherapy used. To explain this, the inventors examined patients treated with either a single dose of rituximab or fludarabine, cyclophosphamide, and rituximab (FCR) for initial treatment and observed similar results. Figure 10 d shows the survival differences in FcγRIIb+ and − subgroups after the patient cohort was further controlled as described.

[0368] Figure 11 : Demonstration of the specificity of the anti-FcγRIIb mAb used in immunohistochemistry. Ramos cells transfected with FcγRIIa and FcγRIIb were cytospun and paraffin-embedded. Immunohistochemistry using a mAb against human FcγRIIb demonstrated strong membrane staining in FcγRIIb-transfected Ramos cells but absent in FcγRIIa-transfected cells.

[0369] Figure 12 : CD32B-specific clones inhibit rituximab internalization. The Y axis shows surface accessible CD20 (%). Rituximab-alexa488 was added to Ramos cells transfected with CD32B in the presence or absence of different CD32b-blocking mAbs (WT or 297Q (nq) mutant) and modulation was evaluated after 1, 2, 6 and 24 hours. As a control for the blocking ability of CD32mAbs, the inventors also used CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragment (Fab)) and a negative control isotype-matched irrelevant mAb (isotype wt or nq). Finally, ALEXa 488-labeled B1 was used as a control mAb that was not rapidly modulated. The data clearly show that all 3 All mAbs (C1, C11, and C13) were able to block rituximab modulation in either the wt or 297q form. In particular, the C11 mAb was extremely effective.

[0370] Figure 13 : The ability of anti-CD32b mAbs to block modulation by rituximab. All 13 mAbs (nq) and C11 are wt. Rituximab-alexa488 was added to Ramos cells transfected with CD32B in the presence or absence of different CD32b blocking mAbs (WT or 297Q mutant) and modulation was evaluated after 1, 2, 6 and 24 hours. The Y axis shows surface accessible CD20 (%). As a control for the ability of CD32 mAbs to block, the inventors also used the CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragment (Fab)) and a negative control isotype-matched irrelevant mAb (isotype wt or nq). Finally, ALEXa 488-labeled B1 was included as a control mAb that was not rapidly modulated. In addition, control CD32-negative Ramos cells were used to assess the maximal effect of the mAbs blocking CD32. The data clearly show that most All mAbs were able to block rituximab modulation. In particular, the C10 and C11 mAbs were extremely effective, showing almost complete blockade of modulation even at 24 hours.

[0371] Figure 14 A: Correlation between the affinity of anti-CD32b blocking mAbs and their ability to inhibit CD32b phosphorylation after rituximab binding. The relative affinity of mAbs was determined by dose titration assays testing mAb binding to CD32B-transfected CHO cells. Briefly, CD32B-transfected CHO K1 adherent cells were plated in FMAT plates. IgG was titrated from 30 nM to approximately 0.015 nM at a 1:2 dilution and allowed to bind for 1 hour at room temperature. After washing, bound IgG was detected using an anti-human IgG-APC antibody. Finally, the plates were washed and read in an FMAT (Applied Biosystems). This provides the EC50 value for mAb binding to target-expressing cells, which can be converted into relative affinity. This relative affinity correlates with the ability of the anti-CD32b blocking mAb to inhibit CD32b phosphorylation after rituximab binding. This is determined by stimulating cells with rituximab in the presence or absence of an anti-CD32b mAb and then performing a Western blot for phospho-CD32b. CD32b mAbs are then classified based on their ability to block CD32 phosphorylation, with 1 being the most potent. The affinity of the mAb is closely correlated with its ability to block CD32b phosphorylation.

[0372] Figure 14 B: Correlation between the affinity of anti-CD32b blocking mAbs and their ability to inhibit rituximab-mediated chemotaxis. Correlation between the affinity of anti-CD32b blocking mAbs and their ability to inhibit rituximab-mediated chemotaxis. The relative affinity of mAbs was determined by dose titration assays of mAb binding to CD32B-transfected CHO cells. Briefly, CD32B-transfected CHO K1 adherent cells were plated on FMAT plates. IgG was titrated from 30 nM to approximately 0.015 nM at a 1:2 dilution and allowed to bind for 1 hour at room temperature. After washing, bound IgG was detected using anti-human IgG-APC. Finally, the plates were washed and read in an FMAT (Applied Biosystems). This provides the EC50 value for mAb binding to target-expressing cells, which can be converted into relative affinity. This relative affinity correlates with the ability of the anti-CD32b blocking mAb to inhibit rituximab-mediated chemotaxis on CD32b-transfected Ramos cells (shown in the previous figure). There was a significant correlation between the affinity of the mAbs and their ability to block rituximab modulation. These data confirm the critical role of CD32B in promoting rituximab modulation on the target cell surface.

[0373] Figure 15 : Mediated by clone 1. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 1. Cells were seeded into FMAT plates. Immune complexes were prepared by coating FITC onto BSA, which was then mixed with a FITC-specific hIgG1 antibody at a 10:1 molar ratio. The total intensity reflects binding, with the higher the intensity, the higher the binding. Binding is either immune complex (IC) or mAb binding.

[0374] Figure 16: Mediated by clone 2. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 2. Cells were seeded into FMAT plates. Immune complexes were prepared by coating FITC onto BSA, which was then mixed with a FITC-specific hIgG1 antibody at a 10:1 molar ratio. The total intensity reflects binding, with the higher the intensity, the higher the binding. Binding is expressed as binding of the immune complex (IC) or mAb.

[0375] Figure 17 : Mediated by clone 3. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 3. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0376] Figure 18 : Mediated by clone 4. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 4. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of the immune complex (IC) or mAb.

[0377] Figure 19: Mediated by clone 5. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 5. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of the immune complex (IC) or mAb.

[0378] Figure 20 : Mediated by clone 6. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 6. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of the immune complex (IC) or mAb.

[0379] Figure 21 : Mediated by clone 7. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 7. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of the immune complex (IC) or mAb.

[0380] Figure 22: Mediated by clone 8. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 8. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of the immune complex (IC) or mAb.

[0381] Figure 23 : Mediated by clone 9. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 9. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is either immune complex (IC) or mAb binding.

[0382] Figure 24 : Mediated by clone 10. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 10. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is either immune complex (IC) or mAb binding.

[0383] Figure 25: Mediated by clone 11. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 11. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is either immune complex (IC) or mAb binding.

[0384] Figure 26 : Mediated by clone 12. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 12. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is either immune complex (IC) or mAb binding.

[0385] Figure 27 : Mediated by clone 13. Dose-dependent binding to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells. Mediated by clone 13. Cells were seeded into FMAT plates, and immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibodies at a 10:1 molar ratio. The total intensity reflects binding, and the higher the intensity, the higher the binding. Binding is the binding of the immune complex (IC) or mAb.

[0386] Figure 28: Demonstrating the cell specificity of anti-CD32B antibodies. PBMCs were isolated from peripheral blood using a Ficoll density gradient. Cells were stained with cell-specific markers and evaluated for binding of CD32B-specific antibodies. As shown in the figure, for clone 1-13, only B cells (CD19+ cells) were positively stained.

[0387] Figure 29 Figure 1: Dose-dependent staining of B cells of clone 1-13. PBMCs isolated from peripheral blood were prepared using a Ficoll density gradient. As shown, cells were stained with CD19 and then stained with 10, 1, or 0.1 mg / ml CD32B-specific antibodies. In this figure, B cells (known to express CD32B) were gated using a CD19-specific mAb. This gate is referred to as "M1." As the concentration of CD32B mAb decreased, the number of B cells stained decreased from nearly 100% to a much lower value, as expected from a CD32B-specific mAb, demonstrating specific and dose-dependent staining of B cells.

[0388] Figure 30 : The ability of each mAb to inhibit Fc-mediated CD32B phosphorylation. Raji cells (CD32B positive) were treated with rituximab (Rit), which causes CD32B phosphorylation. This was done in the presence or absence of CD32B-specific mAbs 1-13, and the figure shows the ability of each mAb to inhibit Fc-mediated CD32B phosphorylation. "TUB" = tubulin control.

[0389] Figure 31 Effect of CD32b on the rate of modulation of type I anti-CD20 mAbs. The ability of CD32b to promote the internalization of other type I anti-CD20 mAbs. Alexa-488-labeled versions of each mAb were incubated with pCDNA3-transfected Ramos or CD32B-transfected Ramos cells for 1 or 6 hours, and the degree of modulation was determined as previously described. The mAbs used were rituximab (RTX), ofatumumab (OFA) prepared in-house, and tositumomab (Tos). The data clearly show that OFA internalization rates are similar to those of RTX and are accelerated by the presence of CD32b.

[0390] Figure 32: Anti-CD19 mAbs are also internalized from the surface of malignant human B cells in a manner that is partially dependent on CD32B. Ramoshu CD32b transfectants. Internalization using other surface antigens can also be affected by CD32b expression. Modulation assays were performed as described above using different mAbs in the presence (+) or absence (-) of CD32 blockade using AT10. Ramos CD32B transfectants were used in this 6-hour assay. *p<0.05. f3.3 = MHC class II; RFB9 = CD19; RTX = Rituximab. If the mAb remains on the cell surface, it can be quenched. If it is internalized, it cannot be quenched. The lower the percentage of quenching, the higher the level of internalization. The data clearly show that surface modulation of RTX and RFB9 mAbs is significantly reduced after incubation with CD32 blockade, and the reduction of surface modulation of F3.3 mAb is less. These data suggest that target antigens such as CD19 can also be internalized from the surface of malignant human B cells in a manner that is partially dependent on CD32b and can be blocked by anti-CD32b mAbs.

[0391] Figure 33 Internalization using other surface antigens can also be affected by CD32 expression. Alexa-488-labeled versions of each mAb were incubated with pCDNA3-transfected Ramos cells or CD32B-transfected Ramos cells for 24 hours, and the degree of modulation was determined as previously described. * = p < 0.05. The y-axis shows modulation / internalization or surface accessible antigen (%). This figure shows that this is due to an increase in internalization / modulation in the presence of huCD32b. Note that for the anti-CD19 mAb and anti-CD40 mAb, there was a statistically significant decrease in cell surface in the presence of CD32b (*, p < 0.05).

[0392] Figure 34 : Amino acid sequences of the constant regions of 14 antibody clones against human CD32B in the IgG1-λ format. The amino acid sequences of the IgG1-CH and λ-CL regions are shown.

[0393] Figure 35 : Amino acid sequence of the variable region of antibody clone 1 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The labeled CDR sequences are indicated as boxed sequences separated by labeled framework regions.

[0394] Figure 36 : Amino acid sequence of the variable region of antibody clone 2 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The labeled CDR sequences are indicated as boxed sequences separated by labeled framework regions.

[0395] Figure 37: Amino acid sequence of the variable region of antibody clone 3 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0396] Figure 38 : Amino acid sequence of the variable region of antibody clone 4 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences. Figure 39 : Amino acid sequence of the variable region of antibody clone 5 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0397] Figure 40 : Amino acid sequence of the variable region of antibody clone 6 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0398] Figure 41 : Amino acid sequence of the variable region of antibody clone 7 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0399] Figure 42 : Amino acid sequence of the variable region of antibody clone 8 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0400] Figure 43 : Amino acid sequence of the variable region of antibody clone 9 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0401] Figure 44 : Amino acid sequence of the variable region of antibody clone 10 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0402] Figure 45 : Amino acid sequence of the variable region of antibody clone 11 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0403] Figure 46 : Amino acid sequence of the variable region of antibody clone 12 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0404] Figure 47 : Amino acid sequence of the variable region of antibody clone 13 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0405] Example 1: CD20 modulation in primary CLL and other NHL samples

[0406] The inventors previously observed heterogeneity in the rate and extent of rituximab modulation in a cohort of CLL samples (28). To validate and extend these findings, the inventors expanded the cohort to a total of 48 CLL samples ( Figure 1 As previously described, the inventors compared the ability of rituximab and tositumomab (clinically relevant prototypes of type I and type II mAbs, respectively) to reduce surface-accessible CD20 over 2, 6, and 24 hours ( Figure 1 A, data not shown). In addition, the inventors also compared the recently FDA-approved in-house produced ofatumumab (type I) for relapsed CLL and the non-glycosylated form of GA101 (GA101 gly ; type II) (19) were tested. Consistent with previous observations, there was considerable heterogeneity between samples, but type I mAbs resulted in significantly more modulation than type II mAbs. At 6 h, modulation was near maximal in the presence of rituximab, with 18% to 65% (median 37%) of the mAb still accessible in the quenching assay ( Figure 1 A). Consistent with its natural type I, ofatumumab also resulted in a high degree of modulation (median 26% accessible at 6 hours). In contrast, tositumomab and GA101 gly showed much less modulation, with a median of 80% (range 61-89%) and 70% (range 57-81%) accessible bound mAb at 6 hours.

[0407] In the CLL cohort, the inventors examined several factors known to be important in CLL prognosis, including ZAP-70 expression (29, 30), CD38 positivity (31, 32), and IgVH gene mutation status (31, 33, 34). Figure 1 C) showed no correlation with any of these disease markers.

[0408] The inventors previously demonstrated that although each NHL subtype has different modulation patterns, they also exhibit considerable heterogeneity in CD20 modulation (28). To further explore this, the inventors expanded the number of primary samples analyzed to include 8 healthy volunteers, 7 SLL, 7 MCL, 11 FL, and 7 DLBCL ( Figure 1B). The difference in the ability of type I and type II mAbs to induce modulation persisted across all histological subtypes. The inventors also observed that CD20 on B cells from healthy volunteers was rapidly modulated in the presence of rituximab and was more uniform than on malignant B cells, suggesting that factors related to the malignancy contribute to the observed heterogeneity. The rate of rituximab-induced modulation in SLL and MCL cells was similar to that in CLL ( Figure 1 A), while for DLBCL and FL, the rates were slightly lower (when compared with Figure 1 However, in FL, we did observe that 2 / 11 patient samples upregulated rituximab very rapidly, resulting in almost undetectable mAb or CD20 levels after 6 hours of incubation.

[0409] Example 2: CD20 modulation in B-NHL is an Fc-dependent process.

[0410] The inventors and others have previously found that the in vivo efficacy of anti-CD20 mAbs is Fc-dependent (28). The inventors hypothesized that modulation might be Fc-dependent even in the absence of effector cells and tested this by repeating internalization assays using Fab' and F(ab')2 fragments of rituximab ( Figure 2 A). Rituximab Fab' showed only low levels of modulation, as confirmed by cell binding assays, which could be explained by the need for bivalent cross-linking of CD20 or by its low affinity monovalent binding (data not shown). In contrast, F(ab')2 and IgG showed similar binding profiles (results not shown), but after 6 hours of incubation, F(ab')2 fragments (40% surface accessible CD20) regulated much less than intact IgG (20% surface accessible CD20). Since highly enriched (>95% pure) B cells were used for testing, the only abundant FcR present was the inhibitory FcγRIIb. Modulation was reduced with rituximab in the presence of the blocking anti-FcγRII mAb antibody AT10, comparable to that of rituximab F(ab')2 fragments or Rit m2a, which bind FcγRIIb with lower affinity than rituximab bearing human IgG1 ( Figure 2 B) As expected, co-incubation with AT10 resulted in very little effect on the modulation of Rit m2a.

[0411] Example 3: FcyRIIb expression on normal B cells and B cell tumors.

[0412] Considering the possibility that FcγRIIb:Fc interactions could affect the rate of CD20 modulation, the inventors examined FcγRIIb expression on normal B cells and primary B cell tumor panels. Figure 2 As shown in Figure 3, there is significant heterogeneity in FcγRIIb expression across the groups. Expression on CLL cells is high, ranging from 20 to 300-fold compared to isotype controls. DLBCL and most FL cells exhibit low FcγRIIb expression. Two FL cases exhibit very high FcγRIIb expression, which, surprisingly, is the case with the previously observed extremely rapid modulation. MCL and SLL cells express moderate but heterogeneous levels of FcγRIIb ( Figure 2 C), which is also consistent with previous findings (21).

[0413] Example 4: FcγRIIb expression regulates CD20 modulation.

[0414] Together, these findings suggest that FcγRIIb expression may be a major determinant of CD20 modulation from B cell targets. To test this hypothesis, the inventors compared FcγRIIb expression and CD20 modulation rates in all available healthy B cells and primary NHL samples ( Figure 2 D). Spearman correlation analysis revealed a significant correlation between these parameters (Spearman r value -0.74, 95% confidence interval -0.83 to -0.61, p < 0.0001). The data show that the majority of FL and DLBCL cases are located in the upper part of the logarithmic curve, while CLL and MCL samples are widely distributed. This figure also demonstrates that at low expression levels, small differences in FcγRIIb expression can lead to relatively large changes in CD20 modulation, thereby highlighting the ability of this receptor to regulate the clearance of anti-CD20:CD20 complexes from the cell surface.

[0415] To directly address the role of FcγRIIb in CD20 modulation, a plasmid encoding FcγRIIb was transfected into -ve Ramos cells. FcγRIIb produced +ve Cells exhibited variable levels of FcγRIIb expression and were subsequently sorted into subclones expressing low, medium, and high FcγRIIb. These cells were then evaluated in an internalization assay along with the parental FcγRIIb. - In the presence of rituximab, the rate of CD20 modulation at 6 hours correlated with FcγRIIb expression, with the order of increasing modulation being FcγRIIb. -ve >FcγRIIb +ve Low>FcγRIIb +ve FcγRIIb +ve high( Figure 3A). Similarly, using B cells derived from wild-type and FcγRII knockout (KO) mice expressing transgenic CD20, modulation was less in FcγRIIb- / - mouse cells than in wild-type counterparts (data not shown), but it should be noted that appreciable modulation was still observed in the absence of FcγRII, suggesting that factors other than FcγRII are involved in regulating CD20 modulation in this transgenic model.

[0416] Since FcγRIIb is a negative regulator of BCR activation on B cells (reviewed in (35)) and CD20 physically associates with the BCR following CD20 mAb engagement (36, 37), we hypothesized that BCR expression or signaling activity could affect modulation. Therefore, to exclude differences in BCR expression as the cause of these findings, BCR-deficient Ramos cells (Rx3) were transfected with FcγRIIb and CD20 modulation was compared with unmanipulated Ramos cells and cells transfected with a blank control Rx3 ( Figure 3 B). These data clearly demonstrate that Rx3 cells lacking BCR expression modulate more slowly than Ramos cells, but this defect can be overcome by expressing high levels of FcγRIIb (FcγRIIb +ve Rx3 cells). This dominant role of FcγRIIb relative to BCR in regulating CD20 modulation is supported by the following evidence: 1) the inventors observed high levels of modulation in CLL cells that typically express low levels of BCR (38); and 2) the inventors did not observe any correlation between surface immunoglobulin (sIg) expression and CD20 modulation on CLL cells ( Figure 1 E).

[0417] Example 5: FcγRIIb activation precedes modulation of CD20 and FcγRIIb

[0418] To further explore the interaction between anti-CD20 mAb and FcγRIIb, the inventors studied antibody-mediated FcγRIIb stimulation, as indicated by phosphorylation of tyrosine-293 in the ITIM motif within the cells. Raji cells were incubated with tositumomab or rituximab in the presence or absence of an anti-FcγRIIb blocking mAb (AT10), followed by immunoblotting for phosphorylated FcγRIIb. Phosphorylated FcγRIIb was elevated in rituximab-stimulated cells but not in tositumomab-stimulated cells and was inhibited by the addition of AT10 ( Figure 4 A) Similar results were observed with Daudi cells (data not shown).

[0419] Example 6: CD20 and FcγRIIb cross-linking occurs primarily in cis

[0420] Rituximab can be co-ligated by CD20 and FcγRIIb on the same (cis) or adjacent cells (trans). To investigate this, the inventors tagged FcγRIIb with PKH26. - Ramos cells were co-cultured with Ramos transfectants expressing high FcγRIIb ( Figure 4 B) The levels of modulation in each cell type were then compared, with the two cell types cultured alone as controls. As previously shown, when cultured alone, FcγRIIb +ve cells showed greater modulation than cells lacking FcγRIIb ( Figure 4 B) In co-culture, FcγRIIb -ve The level of regulation in FcγRIIb was slightly increased, but did not reach that of FcγRIIb +ve This result suggests that although trans interactions can occur, FcγRIIb modulation of CD20 mAb is primarily driven in cis.

[0421] To confirm that this finding was not specific to the Ramos cell line, the inventors co-cultured CLL samples expressing low FcγRIIb (distinguished by PKH26 labeling) with cells from three different CLL cases expressing high levels of FcγRIIb ( Figure 4 C). As observed in previous assays using Ramos cells, in mixed populations, modulation in low FcγRIIb B cells did not approach that observed in the high FcγRIIb population, again suggesting that FcγRIIb modulation of CD20 mAbs is primarily driven in cis. However, it is interesting to note that co-culture with the most rapidly modulating CLL cells resulted in the greatest increase in modulation in low FcγRIIb-expressing CLL, but this increase was only modest (approximately 18%, data not shown).

[0422] In another experiment of this type, CLL cells were cultured at reduced concentrations to reduce the potential for cell:cell interactions, and as a result, there was a weak trend toward less modulation with decreasing cell concentration ( Figure 4 D) The same experiment was repeated using different concentrations of Raji cells and again little variation in the level or extent of modulation was observed (data not shown). Importantly, bright field microscopy images taken during this experiment showed that the 2x 10 6 Compared with 1x 10 cells / ml, 5 In addition, the inventors observed no significant difference in the level of phosphorylated FcγRIIb at different cell densities ( Figure 4E) Taken together, these data suggest that FcγRIIb mediates its effects on CD20 mAb modulation primarily through intracellular events, with adjacent FcγRIIb-expressing cells having only a minor effect.

[0423] Example 7: FcγRIIb is internalized into lysosomes together with CD20

[0424] To determine the fate of FcγRIIb after rituximab takes up residence on the cell surface, the inventors monitored its expression and localization by flow cytometry and confocal microscopy. The inventors used flow cytometry to evaluate the expression of FcγRIIb on the surface of B cells from six different CLL cases and found that it decreased within 2 hours of incubation with rituximab but not with tositumomab ( Figure 5 A) These findings suggest that FcγRIIb may be internalized as part of a tripartite complex with CD20 and rituximab (but not tositumomab).

[0425] The inventors and others have previously reported that endocytosis of rituximab leads to its trafficking to early endosomes and subsequent degradation in lysosomes (9, 28). To elucidate whether the same process occurs in CLL cells with FcγRIIb as part of an anti-CD20:CD20:FcγRIIb complex, the inventors incubated them with Tosit-488 or Ritux-488, then fixed and stained for FcγRIIb (using Alexa 647-labeled F(ab')2 from AT10) and the lysosomal marker LAMP-1. Prior to mAb stimulation, FcγRIIb staining was diffuse and not localized in the plasma membrane ( Figure 5 B). After 30 minutes of incubation with anti-CD20 mAb, the inventors observed a clear difference in staining between Ritux-488 and Tosit-488, whereby Tosit-488 remained exclusively on the surface, while Ritux-488 exhibited intracellular punctate staining, consistent with the inventors' previous observations ( Figure 5 C, data not shown and (28). After 6 h of stimulation, Tosit-488 remained equally distributed on the cell surface, whereas AT10-647 showed no change from its baseline appearance at 30 min and did not colocalize with LAMP-1 ( Figure 5 D). In contrast, Ritux-488 showed a different dot pattern over the same time course, with the majority of cells (58%) exhibiting colocalization between AT10-647 and Ritux-488 ( Figure 5E) Colocalization of Ritux-488 with LAMP1 and AT10-647 was also observed in 33% of cells. It is speculated that the lower degree of colocalization observed across all three cell lines reflects that Ritux-488 and FcγRIIb are internalized together and may occupy other intracellular compartments before appearing in lysosomes.

[0426] Example 8: FcγRIIb inhibits type I anti-CD20 mAb in vivo

[0427] To elucidate whether FcγRIIb could inhibit the efficacy of type I anti-CD20 mAbs in vivo, the inventors performed B cell depletion experiments in hCD20 Tg wild-type mice and hCD20 Tg mice lacking FcγRIIb (CD32 KO). In these experiments, mice were treated with rituximab variants carrying mouse IgG1 (m1) or mouse IgG2a (m2a) (250 μg, iv), and then B cell depletion was monitored by serial bleeding and flow cytometry using B220 and CD19 mAb staining for 90 days ( Figure 6 These variants either strongly bound (ml) or weakly bound (m2a) to CD32b. The data clearly show that depletion was suboptimal when the ml isoform was used (compared to m2a), with loss of CD32 leading to a significant improvement in depletion efficacy. In contrast, m2a was largely similar in the presence or absence of CD32.

[0428] Example 9: FcγRIIb enhances and improves the activity of anti-CD20 mAb against human tumors in vivo

[0429] To examine the effects of CD32 on human tumor cells and the potential to enhance the efficacy of current therapeutic mAbs (e.g., rituximab), the inventors employed a xenograft system. In this system, only human tumor cells express hCD32, so any therapeutic effect is most likely derived from an effect on the tumor cells by blocking modulation rather than from any effect on host effector cells. In these experiments, CD20-positive, CD32-positive human tumor cells (Daudi or Raji) were inoculated into SCID mice, which were then treated with rituximab, AT10, or both, and the mice were monitored for survival or tumor growth ( Figure 7 The mAb dosages used are shown in the figure legends. In A), Daudi cells were inoculated subcutaneously, and tumors were monitored every 3-5 days by caliper measurement. In B and C, Raji cells were injected intravenously, and animal survival was monitored. In both models, AT10 was shown to enhance and potentiate the activity of rituximab, demonstrating the potential of this combination in vivo.

[0430] Example 10: FcgRIIb levels predict clinical outcomes in rituximab-treated MCL patients

[0431] As proof of concept for our in vitro findings, we retrospectively examined FcγRIIb expression in a cohort of MCL patients receiving rituximab. Diagnostic paraffin-embedded tissue was stained by immunohistochemistry using an FcγRIIb-specific mAb ( Figure 11 Strong membrane staining was observed in FcγRIIb+ve but not in FcγRIIb-ve lymphoma samples. These results correlated with FcγRIIb expression in the corresponding DMSO-frozen samples obtained by flow cytometry. Figure 10 FcγRIIb staining shown in a and 10b is consistent with Figure 2 The expression of FcγRIIb by flow cytometry shown in D is related (the value detected by flow cytometry in 2D is Figure 10 Figure 10a and digital insets in 10b). Although only a small cohort of 16 MCL patients was studied, patients with FcγRIIb-ve lymphoma had much better median progression-free survival than those with FcγRIIb+ve cells (median 852 days vs. 189 days, respectively). Figure 10 c shows the survival difference in the FcγRIIb+ and - subgroups. The two groups were comparable in clinical characteristics (MCL International Prognostic Index, data not shown), but were heterogeneous in the type of chemotherapy used. To explain this, the inventors examined patients treated with either a single dose of rituximab or fludarabine, cyclophosphamide, and rituximab (FCR) for initial treatment and observed similar results. Figure 10 d shows the difference in survival in FcγRIIb+ and − subgroups after the patient cohort was further controlled as described.

[0432] The rationale for the experiments in Examples 10 and 11 is as follows. If cells express high levels of CD32b (FcγRIIb), they will internalize rituximab more rapidly (shown as a percentage reduction in surface accessible CD20). If there is less rituximab on the cell surface, there will be less Fc-dependent effector activity (e.g., phagocytosis or ADCC), and therefore less tumor cell killing and, therefore, less therapeutic outcome. Therefore, the inventors examined the expression of FcγRIIb in a cohort of patients with MCL being treated and determined whether they were high or low CD32b expressers. Clinical data were already available for this cohort, so the clinical outcomes were categorized based on whether they were high or low FcγRIIb expressing tumors. The reasoning was that tumors expressing low levels of FcγRIIb would be successfully treated with rituximab, while those expressing high levels of FcγRIIb would be less effective. This is exactly what the clinical data showed. Figure 11The specificity of the mAb used for FcγRIIb staining is demonstrated. It only stains cells expressing FcγRIIb, but not the closely related FcγRIIa.

[0433] In the case of FcγRIIb levels measured by IHC ( Figure 10 b) and after dividing MCL samples into FcγRIIb positive and negative, the inventors observed clear differences in clinical responses after rituximab-based treatment ( Figure 10 c and 10d).

[0434] Example 11: Selection of anti-CD32b monoclonal antibodies

[0435] Figures 37-47 The amino acid sequences of the variable regions (VH and VL) and CDR regions of 14 antibody clones are shown in . In each case, the constant regions (CH and CL) were identical. Figure 36 The constant regions are shown in .

[0436] use The scFv phage display library was screened for CD32B (FcγRIIb). Human CD32A was used as a non-target. The extracellular domains of CD32A and CD32B fused to mIgG3-Fc were prepared in HEK293E and purified using protein A. Three consecutive protein screens were performed. The non-target was used as a competitor in all screens. The generated phage was converted into a format for producing scFv / Fab and transformed into E. coli Top10 bacteria for screening of individual clones. The screening determined the specificity for human CD32B and CD32A and was analyzed in ELISA using coating proteins and by transfecting CHO cells in FMAT. For the determination of IC inhibition properties, IgG was bound to CD32B-transfected CHO cells, after which IC in the form of IgG1-coated bovine serum albumin was added. The bound IC was then detected and the inhibitory properties of the IgG were evaluated.

[0437] Example 12: Ability of anti-CD32b mAb to block rituximab modulation

[0438] Rituximab-alexa 488 was added to Ramos cells transfected with CD32B in the presence or absence of different CD32b blocking mAbs (WT or 297Q variant), and modulation was evaluated after 1, 2, 6, and 24 hours. As a control for the ability of CD32 mAbs to block CD32, the inventors also used the CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragment (Fab)) and a negative control isotype-matched irrelevant mAb (isotype wt or nq). Figure 12The data clearly demonstrate that all three nCoder mAbs (C1, C3, and C11) are able to block rituximab modulation in either the wt or 297q form.

[0439] Figure 13 The ability of anti-CD32b mAbs to block rituximab modulation is shown using all 13 mAbs. In addition, control CD32-negative Ramos cells were used to assess the maximal effect of CD32-blocking mAbs. Figure 12 The data clearly demonstrate that all nCoder mAbs are able to block rituximab modulation.

[0440] Previous groups have demonstrated that FcγRIIb regulates rituximab internalization. Therefore, these studies sought to examine whether blocking FcγRIIb with an anti-FcγRIIb mAb would reduce the amount of rituximab internalized.

[0441] Example 13: Affinity of anti-CD32b blocking mAbs and their correlation between inhibition of CD32b phosphorylation after rituximab binding and inhibition of rituximab modulation

[0442] The relative affinity of the mAb was determined by a dose titration assay measuring mAb binding to CD32B-transfected CHO cells. Briefly, CD32B-transfected CHO K1 adherent cells were seeded on FMAT plates. IgG was titrated from 30 nM to approximately 0.015 nM at a 1:2 dilution and allowed to bind for 1 hour at room temperature. After washing, bound IgG was detected using anti-human IgG-APC. Finally, the plates were washed and read in an FMAT (Applied Biosystems). This gives the EC50 value for mAb binding to target-expressing cells, which can be converted into relative affinity. This relative affinity is then correlated with the ability of the anti-CD32b blocking mAb to inhibit CD32b phosphorylation after rituximab binding. This was determined by stimulating cells with rituximab in the presence or absence of an anti-CD32b mAb and then performing a Western blot for phospho-CD32b. CD32b mAbs were then classified according to their ability to block CD32 phosphorylation, with 1 being the most effective. Figure 14 A shows that there is a clear correlation between the affinity of mAb and its ability to block CD32b phosphorylation. Figure 14 Figure B shows a clear and significant correlation between mAb affinity and its ability to block rituximab modulation. This data demonstrates the important role of CD32B in promoting rituximab modulation from the target cell surface.

[0443] The theory is that higher mAb affinity will better block FcγRIIb. Therefore, the better the mAb blocks FcγRIIb, the better it will block rituximab modulation / internalization. This is exactly Figure 14 The higher the affinity, the better it blocks rituximab binding-induced FcγRIIb activation (as measured by the amount of phospho-FcγRIIb stained by Western blot) and the better it blocks modulation.

[0444] Example 14: Dose-dependent binding to hCD32B-transfected cells and immune complexes and inhibition of hCD32B-transfected cells

[0445] Cells were seeded onto FMAT plates. Immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibody at a 10:1 molar ratio. Figure 15-27 Dose-dependent binding of clone 1-13 to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells are shown, respectively. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells.

[0446] The assay was designed to: 1) determine the specificity of the mAb. CD32B and CD32A are closely related molecules. However, while CD32B transmits inhibitory signals, CD32A transmits positive signals, so it is crucial for the antibody to bind only to CD32B for the desired effect. 2) Furthermore, to effectively block signaling through CD32B, the antibody must not only bind to CD32B but also block binding of its natural ligand, the immune complex (IC). Therefore, the figure shows binding to CD32A, CD32B, and inhibition of IC binding. The figure demonstrates that all mAbs are specific for CD32B and do not bind CD32A, and they all inhibit IC binding.

[0447] Example 15: Cell Specificity of Anti-CD32B Antibodies

[0448] PBMCs were isolated from peripheral blood using a Ficoll density gradient. Cells were stained with cell-specific markers and evaluated for binding of CD32B-specific antibodies. Figure 28 As shown, clone 1-13 stained positive only for B cells (CD19+ cells).

[0449] In resting PBMCs, CD32B is expressed only on B cells, whereas the closely related CD32A is expressed on monocytes and neutrophils. The previous figure shows specificity for transfected CHO cells. Figure 28 It is shown that the antibodies also bind to B cells expressing the native form of CD32B, whereas they do not stain neutrophils or monocytes expressing CD32A. Thus, this figure is evidence of antibody specificity when the antigen is expressed in normal, non-transfected PBMCs.

[0450] Example 16: Dose-dependent staining of B cells by anti-FcγRIIb mAb clone 1-13

[0451] PBMCs were isolated from peripheral blood using a Ficoll density gradient. Cells were stained with CD19 and then with 10, 1, or 0.1 mg / ml of a CD32B-specific antibody. Figure 29 It is shown how the staining of B cells by each clone is dose-dependent.

[0452] exist Figure 29 In the ELISA, B cells (known to express CD32B) were gated using a CD19-specific mAb. This gate is referred to as "M1." As the concentration of the CD32B mAb was decreased, the number of stained B cells decreased from nearly 100% to much lower values, demonstrating the specific and dose-dependent staining of B cells expected from the CD32B-specific mAb.

[0453] This is also evidence of antibody specificity. As mentioned, CD32A and CD32B are very closely related, and obtaining specific antibodies is not easy. Any specific antibody shows dose-dependent binding, which is exactly Figure 29 As demonstrated in Figure 1 , lowering the antibody dose reduced the amount of B cell staining by nearly 100% (as observed at the highest dose). Thus, this figure demonstrates for the second time the specificity of the antibody when the antigen is expressed in normal, non-transfected B cells.

[0454] Example 17: Ability of each mAb to inhibit Fc-mediated CD32B phosphorylation

[0455] Raji cells (CD32B positive) were treated with rituximab, which resulted in phosphorylation of CD32B. This was done in the presence or absence of CD32B-specific mAbs 1-13. Figure 30 The ability of each mAb to inhibit Fc-mediated CD32B phosphorylation was demonstrated.

[0456] The hypothesis behind Examples 17 and 18 is that the Fc region of rituximab binds to FcγRIIb and this results in activation of FcγRIIb. This is measured by phosphorylation of the ITIM region of FcγRIIb. Blocking this interaction with anti-FcγRIIb mAb blocks phosphorylation ( Figure 30 ) and modulation ( Figure 31 and 32 ). wt FcγRIIb IgG1 also has the ability to bind to FcγRIIb through its Fc region, so the inventors examined whether the N297Q mutant (having an Fc that does not bind to FcγRIIb) also has similar activity. It had the same activity.

[0457] Example 18: Effect of CD32b on the modulation rate of type I anti-CD20 mAb

[0458] The ability of CD32b to promote the internalization of other type I anti-CD20 mAbs was shown in Figure 31 Alexa-488-labeled versions of each mAb were incubated with pCDNA3-transfected Ramos or CD32b-transfected Ramos cells for 1 or 6 hours, and the extent of modulation was determined as previously described. The mAbs used were rituximab (RTX), ofatumumab (OFA) prepared in-house, and tositumomab (Tos). The data clearly showed that OFA internalized at a similar rate to RTX and was accelerated by CD32b.

[0459] These modulations were observed with rituximab (a type I anti-CD20) but were less pronounced with tositumomab (a type II anti-CD20 mAb). Therefore, to elucidate whether this extended to other anti-CD20 mAbs, we tested another clinically relevant type I mAb, ofatumumab (Teeling, 2004 (52)), which, as expected, was rapidly internalized like rituximab.

[0460] Example 19: Anti-CD19 mAb is also internalized from the cell surface of malignant human B cells in a manner that is partially dependent on CD32B

[0461] Ramos huCD32b transfectants. Internalization of other surface antigens is also affected by CD32b expression. Modulation assays using various mAbs were performed as previously described, in the presence (+) or absence (-) of CD32 blockade using AT10. Ramos CD32B transfectants were used in this 6-hour assay. *p < 0.05. f3.3 = MHC class II; RFB9 = CD19; RTX = rituximab. Figure 32It is clearly shown that surface modulation by RTX and RFB9 mAbs was significantly reduced, and by a lesser extent by F3.3, after incubation with CD32 blockade. These data suggest that target antigens such as CD19 can also be internalized from the surface of malignant human B cells in a manner that is partially dependent on CD32b and can be blocked by anti-CD32b mAbs.

[0462] The inventors wanted to determine whether target antigens other than CD20 were also affected by FcγRIIb expression. Therefore, the inventors examined whether mAbs against other target antigens (CD19 and MHC II) and mAbs that block FcγRIIb would reduce their internalization. The data showed that blocking FcγRIIb also reduced CD19 mAb modulation.

[0463] Example 20: Internalization using other surface antigens is also affected by CD32b expression

[0464] Ramos cells lack CD32b, thus demonstrating the level of internalization in the absence of CD32b. If an antigen can be internalized by CD32b, then expressing that antigen (on Ramos-CD32b cells) should increase the level of internalization.

[0465] Alexa-488 labeled versions of each mAb were incubated with pCDNA3-transfected Ramos cells or CD32B-transfected Ramos cells for 24 hours, and the extent of modulation was determined as previously described. * = p < 0.05. Figure 33 It was shown that internalization using other antigens is also affected by CD32b expression.

[0466] Materials and Methods

[0467] cell

[0468] Human cell lines (Daudi, Raji, Ramos) were obtained from ECACC and maintained in RPMI supplemented with 10% fetal calf serum (FCS) (Lonza, UK), glutamine, and pyruvate (both from Invitrogen) at 37°C, 5% CO2. Rx3 Ramos cells lacking BCR expression were generated previously (36). Ramos FcγRIIb transfectants and control cells transfected with empty vector (FcγRIIb negative) were generated as previously described (36) and maintained in supplemented RPMI as described above with the addition of geneticin (Invitrogen, UK). Rx3 cells transfected with FcγRIIb and empty vector were prepared and maintained in the same manner. FcγRIIb surface expression was determined by flow cytometry using PE-labeled AT10 (described below). Cell populations of Ramos FcyRIIb transfectants expressing low, medium or high levels of FcyRIIb were sorted using a FACS Aria flow cytometer (BD Biosciences, USA).

[0469] blood donor

[0470] Normal human B cells were obtained from healthy volunteers who gave informed consent. Peripheral blood was drawn at K2E or LiH, and lymphocytes were isolated using Lymphoprep (Axis-Shield, UK) according to the manufacturer's protocol. B cells were isolated by negative selection using the Human B Cell Isolation Kit II (Miltenyi Biotec, Germany).

[0471] Clinical samples

[0472] CLL / SLL, FL, DLBCL, and MCL samples were obtained with informed consent in accordance with the Declaration of Helsinki. Blood samples were collected using Lymphoprep in K2E or LiH, solid tissue was disaggregated by passing through a sterile mesh and centrifuged. Cells were cryopreserved in RPMI supplemented with human AB serum and 10% DMSO and stored in the Tumor Bank of the Cancer Sciences Department at the University of Southampton under a Human Tissue Regulatory Authority license. Southampton University Hospital National Health Service Foundation Trust obtained ethical approval for the use of clinical samples from the Southampton and South West Hampshire Research Ethics Committee. For CLL cells, the mutational status of the IgVH genes (33) and CD38 positivity (44) were determined as previously described. Briefly, for IgVH analysis, heavy chain genes were amplified from cDNA using a VH leader primer mix and Cμ100 primers. All nucleotide sequences were aligned to the V-base catalog, and mutational status was determined using a 98% cutoff. For CD38 analysis, anti-CD38 PE (clone HB7, BD Biosciences) was used. ZAP-70 status was determined as described by Crespo et al. (30). Surface Ig expression on CLL cells was determined by flow cytometry as previously described (45, 46).

[0473] Viability assay

[0474] Cell viability was assessed by flow cytometry after staining with FITC-labeled Annexin V and PI as described previously (25).

[0475] Antibodies and reagents

[0476] Rituximab was a gift from the Department of Oncology Pharmacy, Southampton General Hospital. Rit m2a (rituximab with a mouse IgG2a Fc region), WR17 (anti-CD37), and all mouse IgG2a were prepared as previously described (18). Anti-FcγRII mAb (AT10) was prepared in-house as previously described (47). Tositumomab was a gift from Prof Tim Illidge (Manchester, United Kingdom). Ofatumumab and GA101 were prepared in-house according to published sequences. gly(Glycosylated GA101 with unmodified Fc region). NB: These mAbs were produced in CHO or 293F cells and thus may differ from mAbs produced for clinical use (e.g., in their carbohydrate structure). Alexa-488 and anti-Alexa 488 reagents were purchased from Invitrogen. F(ab')2 fragments were prepared as previously described (48). Fab' fragments were generated by incubation with 20 mM 2-mercaptoethanol at 25°C for 30 minutes followed by addition of excess iodoacetamide. Western blot antibodies used were anti-actin antibody (AC74, Sigma, UK) and anti-phospho-FcγRIIb antibody (Cell Signaling Technology, UK).

[0477] Flow cytometry

[0478] Fluorochrome-labeled mAbs were obtained from BD Biosciences or prepared in-house. Alexa488 (Invitrogen)-coupled mAbs were used according to the manufacturer's protocol. Flow cytometry was performed as previously described (49). Samples were evaluated on a FACScan (BD Biosciences) or FACSCalibur (BD Biosciences), and data were analyzed using CellQuest Pro (BD Biosciences) or FCS Express (DeNovo Software, USA). B cells were identified using an APC-labeled anti-human CD19 antibody (prepared in-house), and FcγRIIb expression was determined using PE-labeled AT10 (prepared in-house). To control for inter-assay variability, FcγRIIb expression was expressed as the ratio of FcγRIIb:isotype control geometric mean fluorescence intensity (MFI).

[0479] Internalization assay

[0480] Internalization assays were performed as previously described (28). Briefly, 2-4 x 10 5 Cells / well were incubated with Alexa-488-labeled mAb at a final concentration of 5 μg / ml. Samples were collected after 1, 2, 6, and / or 24 hours, washed twice, resuspended, and incubated with APC-labeled anti-CD19 antibody for 30 minutes at 4°C with or without quenching antibody anti-Alexa-488 antibody (Invitrogen). Samples were then washed once and analyzed on a flow cytometer.

[0481] To investigate the interaction of the Fc region of cell-bound anti-CD20 mAb with FcγRIIb on neighboring cells, FcγRIIb was labeled with PKH26 (Sigma Aldrich) according to the manufacturer's instructions. -veThen, the PKH26-labeled cells were mixed with the same number (2.5 x 10 5 10 cells) were co-incubated with FcγRIIb-transfected Ramos cells. Both cell types were incubated separately as controls. Internalization assays were then performed as described above and compared to the modulation of PKH26-labeled and unlabeled cell populations. Additional variations of this co-incubation assay are described in the figure legends.

[0482] Western blotting

[0483] The protocol was as described previously (36). Briefly, approximately 2 x 10 6 Cells / well were incubated with mAb (5-10 μg / ml). Samples were then separated by SDS PAGE, and proteins were immediately transferred to PVDF membranes. The membranes were blocked with 5% w / v skim milk powder, incubated with appropriately diluted primary antibodies, washed, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Sigma Aldrich). Images were visualized by enhanced chemiluminescence (ECL, GE Healthcare, UK or Pierce Biotechnology, UK) and exposed to light-sensitive film (Hyperfilm ECL, GE Healthcare, UK) or a Biospectrum AC imaging system (UVP, UK).

[0484] Optical and confocal microscopy

[0485] To determine the intracellular trafficking of anti-CD20 mAbs and FcγRIIb, CLL cells were incubated with the appropriate Alexa 488-labeled mAbs for various times as described in the figure legends, then collected, washed, and fixed using 2% paraformaldehyde. For detection of FcγRIIb and LAMP-1, cells were permeabilized with 0.3% saponin and incubated with Alexa-647-labeled AT10 F(ab')2 (labeled using the Alexa Fluor-647 labeling kit (Invitrogen) according to the manufacturer's protocol) and / or biotin-conjugated anti-human CD107a (LAMP-1) antibody (eBioscience, UK). Cells were then washed, and streptavidin-Alexa Fluor-547 (Invitrogen) was added, followed by further washing. Afterwards, the cells were transferred to glass slides and images were captured on a TCS-SP5 laser scanning confocal microscope (Leica Microsystems, UK) (10x eyepiece, 100x objective) using LAS-AF v2 software.

[0486] To determine the proximity of cells at different cell dilutions, 1-20 x 10 5 Cells were seeded with 400 μg / ml of culture medium and stimulated with different mAbs for 2 hours and / or 6 hours, and then their relative proximity was assessed by optical microscopy. Cells were observed using an Olympus CKX21 inverted microscope (Olympus, UK) using a 10x or 20x / 0.25 PH lens. Images were acquired using a CCL2 cooled digital camera (Olympus) and processed using Cell B (Olympus Soft imaging solution) and Adobe Photoshop CS2 software (Adobe, San Jose, CA).

[0487] Data Analysis

[0488] Data analysis was performed using GraphPad Prism (GraphPad Software, USA). Paired nonparametric data were analyzed using the Wilcoxon paired test, while unpaired data were analyzed using the Mann-Whitney test.

[0489] Example compositions, formulations, and modes of administration

[0490] The present invention provides methods for treating, preventing, and ameliorating one or more symptoms associated with a disease, disorder, or infection by administering to a subject or patient an effective amount of a pharmaceutical composition of the present invention.

[0491] In a specific embodiment, the subject or patient is an animal, preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkeys such as cynomolgus monkeys and humans). In a preferred embodiment, the subject is a human.

[0492] A variety of delivery systems are known and can be used to administer the compositions of the invention, for example, liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing the antibody, and the like.

[0493] In some embodiments, the compositions of the present invention are formulated in liposomes for targeted delivery of the antibodies of the present invention. Liposomes are vesicles comprising concentrically arranged phospholipid bilayers that encapsulate an aqueous phase. Liposomes typically contain various types of lipids, phospholipids, and / or surfactants. The components of the liposomes are arranged in a bilayer structure, similar to the lipid arrangement of biological membranes. Liposomes are particularly preferred delivery vehicles, in part due to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and are within the scope of the present invention, see, for example, Epstein et al, 1985, Proc. Natl. Acad. Sci. USA, 82: 3688; Hwang et al, 1980 Proc. Natl. Acad. Sci. USA, 77: 4030-4; U.S. Patents 4,485,045 and 4,544,545, all of which are incorporated herein by reference in their entirety.

[0494] The method of using the composition of the present invention includes but is not limited to parenteral administration (for example, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural and mucosal (for example, intranasal and oral route). In a specific embodiment, the composition of the present invention is by intramuscular, intravenous or subcutaneous administration. The composition can be used by any convenient approach, for example, by infusion or bolus injection, by epithelial or mucocutaneous lining absorption (for example, oral mucosa, rectum or intestinal mucosa etc.), and can be used together with other bioactivators. Use can be whole body or local. In addition, lung administration can also be adopted, for example, by using inhaler or nebulizer, and prepare together with aerosol. 98 / 31346; and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0495] The amount of the composition of the present invention effective to treat, prevent, or ameliorate one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise amount used in the formulation also depends on the route of administration, the severity of the disease, and is determined according to the physician's diagnosis and the individual patient's circumstances. The effective dose can be inferred from dose-response curves derived from in vitro or animal model test systems.

[0496] For the antibodies encompassed by the present invention, the dosage administered to a patient is typically 0.0001 mg to 100 mg of each antibody independently per kg of patient body weight in the composition. Preferably, the dosage administered to a patient is 0.0001 mg to 20 mg, 0.0001 mg to 10 mg, 0.0001 mg to 5 mg, 0.0001 mg to 2 mg, 0.0001 mg to 1 mg, 0.0001 mg / kg to 0.75 mg, 0.0001 mg to 0.5 mg, 0.0001 mg to 0.25 mg, 0.0001 mg to 0.15 mg, 0.0001 mg to 0.10 mg, 0.001 mg to 0.5 mg to 0.2.5 mg, or 0.01 mg to 0.10 mg. Typically, human antibodies have a longer half-life in the human body relative to antibodies from other species due to the immune response to foreign polypeptides. Thus, lower dosages and less frequent administration of human antibodies are generally possible. Additionally, the dosage and frequency of administration of an antibody or fragment thereof of the invention can be reduced by enhancing the uptake and tissue penetration of the antibody through modification (eg, lipidation).

[0497] In one embodiment, the dosage of each antibody of the composition of the invention administered to a patient is 0.01 mg to 1000 mg per day.

[0498] The compositions of the present invention comprise a prophylactically or therapeutically effective amount of an agent and an antibody as described herein and a pharmaceutically acceptable carrier.

[0499] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle for administering the therapeutic agent. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injections. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain a small amount of a wetting agent or emulsifier or a pH buffer. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0500] In various embodiments, administration of the antibody and reagent can be performed simultaneously or less than 1 hour apart, about 1 hour apart, about 1 hour apart to about 2 hours apart, about 2 hours apart to about 3 hours apart, about 3 hours apart to about 4 hours apart, about 4 hours apart to about 5 hours apart, about 5 hours apart to about 6 hours apart, about 6 hours apart to about 7 hours apart, about 7 hours apart to about 8 hours apart, about 8 hours apart to about 9 hours apart, about 9 hours apart to about 10 hours apart, about 10 hours apart to about 100 hours apart, about 11 hours apart to about 12 hours apart, less than 24 hours apart, or less than 48 hours apart. In preferred embodiments, two or more components are administered in the same patient visit.

[0501] The present invention provides the dosage and frequency that treatment is effective and prevention effectively encompasses.Dosage and frequency are usually further different according to each patient's unique factor, and this depends on the specific therapeutic agent or preventive agent used, disease severity and type, route of administration and patient age, body weight, response and previous medical history. Those skilled in the art can select suitable scheme by considering these factors and for example reporting in the literature and recommending in the physician's desk reference (the 56th edition, 2002).

[0502] Summarize

[0503] Recently, the inventors discovered that type I mAbs like rituximab are modulated by human CD20 Tg mouse B cells (in vitro and in vivo) and by certain primary tumor cells derived from patients with NHL, thereby limiting their ability to recruit effector cells and deplete target cells (28). The inventors considered possible mechanisms explaining the limited therapeutic activity of rituximab and other type I CD20 mAbs and provided opportunities to block or circumvent this process and thereby develop more potent agents. This invention provides a molecular theory of CD20 modulation induced by rituximab and ofatumumab. FcγRIIb expression will provide an important prognostic marker for response to type I anti-CD20 mAbs. When primary CLL / SLL cells were incubated with type I anti-CD20 mAbs, significant but heterogeneous CD20 modulation was observed, and this heterogeneity may not be associated with known prognostic factors for CLL. Analysis of other B-NHL subtypes showed that MCL exhibited similar heterogeneous modulation to CLL, but FL and particularly DLBCL showed significantly less modulation. Based on these results, the inventors report here a correlation between FcγRIIb expression levels in these malignancies and their modulation during a 6-hour incubation. Furthermore, the inventors propose that this modulation may explain some of the heterogeneity in response to rituximab observed in these diseases. In DLBCL and FL, where rituximab is established as a first-line treatment in combination with chemotherapy, rituximab has been shown to be most beneficial. In contrast, it is more difficult to demonstrate an improvement in overall survival with rituximab in CLL, and its benefit in MCL is even smaller. Thus, overall, it was found that B-cell malignancies expressing FcγRIIb are more likely to modulate CD20, often resulting in a reduced benefit from rituximab treatment. However, even in DLBCL and FL, some cases do not respond to rituximab. As an example, transformed FL cases are often poorly responsive to treatment and express FcγRIIb (21), an observation consistent with the inventors' own observation that one of the high FcγRIIb expressing samples ( Figure 2 C) was identified as FL and showed a corresponding high rate of modulation, which is consistent with the previous finding. Although this is a single case, the inventors believe that this may potentially provide an important way to address rituximab resistance.

[0504] CD20 modulation showed a strong correlation with FcγRIIb expression levels, regardless of B-NHL disease subtype. It has been previously proposed that FcγRIIb may inhibit cytotoxic signaling by competing with activating Fc receptors on effector cells, thereby inhibiting therapeutic mAb efficacy (40). The present in vitro studies showed that rituximab primarily cross-links CD20 and FcγRIIb on the same cells, resulting in FcγRIIb activation and rapid paired internalization of both surface antigens along with the bound mAb into lysosomes for degradation. Expression of FcγRIIb leads to reduced effector cell recruitment through its ability to downregulate surface expression of mAbs on target cells.

[0505] The present invention also shows that co-incubation with a blocking anti-FcyRIIb mAb can inhibit FcyRIIb activation and rapid internalization of rituximab. Taken together, these data demonstrate a positive correlation between FcyRIIb activation and mAb internalization from the cell surface.

[0506] The strong correlation between type I anti-CD20 mAb-induced CD20 modulation and FcγRIIb expression in different B-NHL subtypes, together with the transfection studies of the present invention, suggests that FcγRIIb is a key regulator of CD20 modulation.

[0507] Other groups have investigated the role of FcγRIIb in lymphoma. Camilleri-Broet et al (20) failed to show a significant correlation between R-CHOP response and FcγRIIb expression in DLBCL, however, in an earlier series only 18% (42 / 234 cases) were FcγRIIb positive by immunohistochemistry (21). Given the relatively low positivity rate, the number of positive cases was likely insufficient to detect a difference. Rather than overexpression, Weng and Levy (24) investigated whether two FcγRIIb alleles (the 232I allele, which is more effective than 232T for BCR-mediated calcium regulation in autoimmune diseases (22, 23)) were associated with rituximab efficacy but were unable to demonstrate any correlation between this polymorphism and single-agent response in FL patients. A major concern for these authors themselves was that only 17 patients had the 232T allele, again limiting the statistical power of the study. Furthermore, the polymorphisms studied reflect the efficiency of BCR inhibition in autoimmune diseases, and none of the published observations suggest that these polymorphisms are relevant in lymphoma or affect Fc binding of human IgG1. Through its ability to modulate the rate of internalization, FcγRIIb expression will become an important prognostic marker for the success of immunotherapy with type I mAbs, including rituximab and ofatumumab. The effect is less pronounced when type II mAbs are used.

[0508] Furthermore, in different in vivo models, the inventors demonstrated the ability of CD32 to limit the efficacy of mAbs and the ability of anti-CD32b mAbs to overcome this limitation and enhance rituximab therapy.

[0509] References

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Claims

1. A monoclonal antibody that binds to FcγRIIb and comprises: (a) a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 identical to those in SEQ ID NO: 12; (b) a light chain variable region comprising a VL CDR1, VL CDR2, and VL CDR3 identical to those in SEQ ID NO: 25; and (c) a heavy chain constant region comprising a mutated heavy chain constant region, wherein the mutated heavy chain constant region has a mutation relative to the wild-type heavy chain constant region set forth in SEQ ID NO: 1, wherein the mutation is an N297Q mutation at amino acid position 297 of the human IgG1 heavy chain as determined according to the EU index in Kabat, Wherein VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 are determined according to the Kabat numbering system or the IMGT numbering system.

2. The antibody of claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:

12.

3. The antibody of claim 1, wherein the VL consists of the amino acid sequence shown in SEQ ID NO:

25.

4. The antibody of claim 1, further comprising a light chain constant region (CL) as shown in SEQ ID NO:

2. A composition comprising the monoclonal antibody according to any one of claims 1 to 4.

6. The composition of claim 5, wherein the composition is formulated for parenteral administration.

7. The composition of claim 6, wherein the parenteral administration is selected from intravenous administration and subcutaneous administration.

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