Combination application of FcγRIIB (CD32B) and CD20 specific antibodies

Abstract

The present invention relates to the combined use of FcγRIIb (CD32B) and CD20 specific antibodies, and specifically provides a method for treating patients with 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, characterized in that the patient is selected based on the increased expression level of FcγRIIb in his target cells.

Description

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

[0002] The present invention relates to an agent that prevents binding between an antibody Fc domain and cell surface FcγRIIb. The present invention also relates to a composition comprising the agent for use in treating a patient having a target cell such as a cancer cell treated with an antibody-based composition.

[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 level and / or internalization of therapeutic antibodies mediated by FcγRIIb as a prognostic marker for the response of a target cell to said therapy. Background Art

[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 (e.g., 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 the effector cells' 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 produced in initial trials of single-agent rituximab have been less dramatic than those produced in other non-Hodgkin lymphomas (NHL) (reviewed in (6)). A subset of lymphomas show primary resistance to rituximab or eventually develop resistance to combination therapies containing rituximab (7). The molecular basis underlying this resistance and the observed sensitivity of different NHL subtypes to rituximab therapy is currently unknown but may include elevated levels of CD20 expression (8-10), high expression of complement defense molecules (CD55 and CD59) (11, 12), development of apoptotic 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 critical 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 those 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 following 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 being more effective than the 232T allele in inhibiting BCR-mediated calcium flux (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 induce 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 differ significantly on 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 FcγRIIb (CD32B) and CD20 specific antibodies and methods of using the combination of these two antibodies to treat B cell related diseases or disorders. However, there is no teaching or suggestion to identify and / or treat a subgroup of patients, i.e., those patients with elevated expression levels of FcγRIIb on target cells, or patients whose antibody types are suitable for combined treatment with FcγRIIb antibodies. Summary of the invention

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

[0014] Thus, the inventors have demonstrated that the key factor determining the effectiveness of antibodies against antigens such as CD20 is the interaction with the inhibitory FcγRIIb (also known as and including CD32, CD32B, CD32B1, CD32B2, FcRII, FcγRII or FcRIIB) on the surface of the same cell, which is surprising. 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 able to block 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 that can bind 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 increased 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 patients with target cells having 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 composition, use or method 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 the target cell to treatment with an antibody molecule that specifically binds to an antigen on the surface of the target cell, the antibody molecule having an Fc domain capable of binding to FcγRIIb, whereby an increased level of FcγRIIb indicates a reduced response or no response to treatment with the antibody molecule.

[0023] In one embodiment, the use of FcγRIIb expression on a target cell as a prognostic marker does not require the use of FcγRIIc expression on a target cell 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 expression level of FcγRIIb 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 FcγRIIb on said target cell and does not additionally comprise determining the level of expression of FcγRIIc 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 comprising FcγRIIb function modulators and to avoiding treatments that do not benefit the patient.

[0027] In certain embodiments of the composition, use or method 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 the 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 composition, use or method 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 level of FcγRIIb" is defined in "Definitions" below. The expression level of FcγRIIb can be calculated as the ratio of the geometric mean fluorescence intensity (geometric MFI or Geo MFI) of FcγRIIb to the isotype control. Alternatively, the expression level of FcγRIIb can be calculated by immunohistochemistry of tumor biopsies. It will be appreciated by those skilled in the art that there are a variety of techniques and methods for determining the expression level of FcγRIIb.

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

[0033] On the other hand, the present invention provides an assay for identifying an agent that reduces or inhibits binding between an Fc domain of an antibody 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 the binding of the Fc domain to FcγRIIb, it is identified as a useful agent. Such an assay can also be used to identify which agent (e.g., antibody molecule) is suitable for combined therapy with an anti-FcγRIIb antibody.

[0034] In another preferred embodiment, the assay for identifying agents useful in the practice of the uses and methods of the invention comprises screening for agents that block FcγRIIb stimulation / signaling, such as agents that block FcγRIIb stimulation / signaling as indicated by phosphorylation of tyrosine-293 in the ITIM motif in cells by Western blot. For example, Raji cells are incubated with antibodies to cell surface antigens (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, with Figure 4 Similar to the use of AT10 as a blocking agent as shown in A, it 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 typical example of blocking by an anti-FcγRIIb 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-FcγRIIb antibodies.

[0035] In a preferred embodiment, the assay method for identifying an agent suitable for combined therapy with an FcγRIIb antibody calculates the percentage of an agent (e.g., antibody molecule) internalized into a cell expressing FcγRIIb. The assay method 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 a target expressing the agent (e.g., antibody molecule) and a cell expressing FcγRIIb, whereby the percentage of a reduction in the accessible agent (e.g., antibody molecule) on the cell surface can predict 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 have K values ​​ranging from mM to pM. 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 complementary 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 each CDR. 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 the following amino acid sequences: 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 present invention described herein, such as comprising the amino acid sequence listed in the above embodiments (eg, 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 can at least partially inhibit or interfere with the binding of the agent described herein to FcγRIIb and inhibit or reduce the binding of FcγRIIb to the Fc domain of the antibody molecule.

[0110] For example, the agent can inhibit the binding of the agent described herein by at least 10%, for example at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 100%, and / or inhibit 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%. For example, 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), which is 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 reagent of the present invention may comprise one or more sequences of Clones 1 - 14:

[0123] Clone 1

[0124]

[0125] CDR Area

[0126] CDRH1: NYGMH[SEQ ID NO:29]

[0127] CDRH2: VISYDGSNKYYADSVKG[SEQ ID NO:30]

[0128] CDRH3: EWRDAFDI[SEQ ID NO:31]

[0129] CDRL1: TGSSSNIGAGYDVH[SEQ ID NO:32]

[0130] CDRL2: SDNQRPS[SEQ ID NO:33]

[0131] CDRL3: AAWDDSLSGSWV[SEQ ID NO:34]

[0132] Clone 2

[0133]

[0134] CDR Area

[0135] CDRH1:TYGMH[SEQ ID NO:35]

[0136] 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]

[0137] Clone 3

[0138]

[0139] CDR Area

[0140] CDRH1:NYGMH[SEQ ID NO:41]

[0141] CDRH2:VISYDGSNRYYADSVKG[SEQ ID NO:42]CDRH3:DRWNGMDV[SEQ ID NO:43]CDRL1:SGSSSNIGAGYDVH[SEQ ID NO:44]CDRL2:ANNQRPS[SEQ ID NO:45]

[0142] CDRL3:AAWDDSLNGPWV[SEQ ID NO:46]

[0143] Clone 4

[0144]

[0145] CDR Area

[0146] CDRH1:SYGMH[SEQ ID NO:47]

[0147] 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]

[0148] Clone 5

[0149]

[0150] CDR Area

[0151] CDRH1:NYGMH[SEQ ID NO:53]

[0152] 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]

[0153] Clone 6

[0154]

[0155] CDR Area

[0156] CDRH1:DYGMS[SEQ ID NO:59]

[0157] CDRH2:AISGSGSSTYYADSVKG[SEQ ID NO:60]CDRH3:GDIDYFDY[SEQ ID NO:61]CDRL1:TGSSSNFGAGYDVH[SEQ ID NO:62]CDRL2:ENNKRPS[SEQ ID NO:63]

[0158] CDRL3:AAWDDSLNGPV[SEQ ID NO:64]

[0159] Clone 7

[0160]

[0161] CDR Area

[0162] CDRH1:SYGMH[SEQ ID NO:65]

[0163] 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]

[0164] Clone 8

[0165]

[0166] CDR Area

[0167] CDRH1:SYGMH[SEQ ID NO:71]

[0168] 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]

[0169] Clone 9

[0170]

[0171] CDR Area

[0172] CDRH1:SYGMH[SEQ ID NO:77]

[0173] 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]

[0174] Clone 13

[0175]

[0176] CDR Area

[0177] CDRH1:SYGIS[SEQ ID NO:101]

[0178] 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]

[0179] Clone 10

[0180]

[0181]

[0182] CDR Area

[0183] CDRH1:SYGMH[SEQ ID NO:83]

[0184] 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]

[0185] Clone 11

[0186]

[0187] CDR Area

[0188] CDRH1:SYGMH[SEQ ID NO:89]

[0189] 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]

[0190] Clone 12

[0191]

[0192] CDR Area

[0193] CDRH1:NHGMH[SEQ ID NO:95]

[0194] CDRH2:VISYDGTNKYYADSVRG[SEQ ID NO:96]

[0195] CDRH3:ETWDAFDV[SEQ ID NO:97]

[0196] CDRL1:SGSSSNIGSNNAN[SEQ ID NO:98]

[0197] CDRL2:DNNKRPS[SEQ ID NO:99]

[0198] CDRL3:QAWDSSTVV[SEQ ID NO:100]

[0199] 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 Research Institute; 2010 Jan; 277 (2): 301-8)); Cancer. 2008 Oct; 8(5): 392-401); CXCR4 (chemokine receptor 4 (Biochim Biophys Acta. 2007 Apr; 1768(4): 952-63); CD19 (differentiation cluster 19 (Cell Immunol. 1989 Feb; 118(2): 368-81); CD40 (differentiation cluster 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).

[0200] 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.

[0201] 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 when bound 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.

[0202] 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.

[0203] 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.

[0204] 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 type I and type II mAbs (26). 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. Both types of mAbs produced significant prolongation of survival, but 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 the different CD20 mAbs operate different effector mechanisms in vivo. In addition, and in full agreement with previous work, these results show that rituximab and 1F5 are able to effectively activate complement due to the transfer of 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 can essentially be defined by their ability to move CD20 into lipid rafts. This can be determined as described below. This is also correlated with type II mAbs that are able to induce more effective homogeneous 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).

[0205] Therefore, these different anti-CD20 mAbs are classified into type I (e.g., rituximab, ofatumumab) or type II (e.g., tositumomab (B1), GA101, 11B8) according to 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), while type II (e.g., tositumomab) anti-CD20 mAbs cannot achieve this (50).

[0206] As described above, anti-CD20 mAbs are defined as type I or type II depending on whether they redistribute CD20 into lipid valves. This is accomplished by 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:

[0207] 1. Evaluation of valve-associated antigens by Triton X-100 insolubility

[0208] Rapid assessment of the presence of antigens in valve microdomains was performed using a flow cytometry method based on Triton X-100 insolubility at low temperature. Briefly, cells were washed in RPMI / 1% BSA and plated at 2.5 x 10 6 / ml resuspended. The cells were then incubated with 10 μg / ml FITC-conjugated mAb for 15 minutes at 37°C, washed in cold PBS / 1% BSA / 20mM sodium azide, and the samples were then divided into two halves. One half was maintained 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 FITC-labeled mAb. To evaluate whether more antigen could be moved into the Triton-X 100-insoluble fraction by additional cross-linking, cells were incubated with FITC-mAb as before, washed, and then divided into four aliquots. Two of these samples were incubated with goat anti-mouse Ig F(ab') 2 The fragments were incubated together on ice for 15 minutes. After washing, one of the cross-linked and one of the non-cross-linked samples were lysed in Triton X-100 and washed as detailed above prior to flow cytometry.

[0209] 2. Sucrose Density Gradient Separation and Western Blotting - Lipid Valve Fraction Preparation and Western Blotting

[0210] 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 μg / ml aprotinin, 5μg / ml leupeptin, 10mM 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, covered with a discontinuous 5-30% sucrose density gradient, and then centrifuged at 200,000xg for 16h. Fractions (0.5ml) were collected and analyzed by Western blotting. 15ml samples of each fraction were 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, 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), and then incubated with an HRP-coupled secondary antibody (Amersham Biosciences UK Ltd). Blots were visualised using ECL+plus (Amersham Biosciences UK Ltd).

[0211] Anti-CD20 mAbs may require an AxP motif in the CD20 macroloop (which is not required for ofatumumab and other Genmab antibodies). However, (Niederfelner et al. (51)) showed that type II mAbs bind to slightly different regions in the CD20 loop compared to type I mAbs.

[0212] Preferably, in the composition, use or method of the present invention, the target cell is a cancer cell. More preferably, the target cell is selected from a cancer of non-Hodgkin's lymphoma, including but not limited to follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

[0213] In one embodiment, the present invention provides compositions, uses and methods for treating cancer, in particular B-cell malignancies, preferably selected from lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, multiple lymphomas, 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).

[0214] In another embodiment, the present invention provides compositions, uses and methods for treatment. 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.

[0215] In a preferred embodiment, 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 herpetic diseases.

[0216] In a preferred embodiment, the therapy enhanced by use of the present invention is treatment with an anti-CD20 mAb such as rituximab.

[0217] definition

[0218] "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 a low or medium level of FcγRIIb on its surface. For example, if the cell in question is a B-type cell, its FcγRIIb expression level is considered "elevated" if its FcγRIIb expression level is higher than the normal (preferably medium) expression level of FcγRIIb of a B cell of the same cell type. Alternatively, if the expression level of the cell in question is higher than a different cell type that expresses FcγRIIb at a low or medium level, its expression level is considered "elevated".

[0219] According to the present invention, the higher the degree of elevated FcγRIIb expression by target cells, the poorer the response of these 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 is expected to be. 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 the binding of the Fc domain to FcγRIIb. Fig.10 b) and after separation of MCL samples into FcγRIIb positive and negative, significant differences in clinical response were observed after rituximab-based treatment ( Fig.10 c and 10d).

[0220] 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 as described in the Figures and Examples.

[0221] It will be understood by those skilled in the art that "normal" and "elevated" expression levels of FcγRIIb will vary between different cell types and different disease states, and that 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 moderate) and "elevated" expression of FcγRIIb on certain cell types are provided in C. In these specific examples, in follicular lymphoma (FL), the "normal" level is about 50 (ratio of geometric MFI FcγRIIb to isotype control), and the "elevated" level is about 125 or 400 or more, while in diffuse large B-cell lymphoma (DLBCL), the "normal" level is about 20, and the "elevated" level is about 80 or more, while in mantle cell lymphoma (MCL), the "normal" level is about 60, and the "elevated" level is about 110 or 190 or more, and in chronic lymphoid leukemia (CLL), the "normal" level is about 100, and the "elevated" level is about 300 or more.

[0222] Preferably, the elevated FcγRIIb expression level is increased by at least 1.1 fold relative to the normal (preferably median) expression level of cells of the same cell type (or cells of a different cell type), or by 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, 4.6, 5.7, 5.8, 6.9, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 9. , 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 , 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0 or 25.0 times or more. Preferably, they increase by at least 1.8 times relative to the normal (or medium) expression level of cells of the same cell type or different cell types.

[0223] 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 produce higher modulation, i.e., there is a correlation between the degree of increased expression and modulation, such as 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 directly correlated with a reduction in cell surface CD20 levels proportional to the expression of FcγRIIb (60%, 40%, 30% for low, medium and high levels). FcγRIIb expression levels are expressed as the ratio of the geometric mean fluorescence intensity (geometric MFI) of FcγRIIb to the isotype control.

[0224] Modulation reduces the amount of mAb left on the cell surface. mAbs require Fc to engage 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 was 40% with rituximab alone, but increased to 55% when FcγRIIb was blocked by AT10.

[0225] "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), intracellular antibodies, or epitope-binding fragments of any of the above. Preferably, the antibody of the present invention is a monoclonal antibody, more preferably a humanized or human antibody.

[0226] Methods for preparing and characterizing antibody molecules that can be used 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.

[0227] "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 can 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. The antibody or fragment thereof specifically binds to FcγRIIb when the antibody or fragment thereof 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 the antibody or fragment thereof 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 Applications 2004-0185045, 2005-0260213, and 2006-0013810, which are incorporated herein by reference in their entirety.

[0228] Preferably, certain FcγRIIb antibodies used in combination with CD20 antibodies in the compositions and methods of the 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 bind to FcγRIIb is 100 times, 1000 times, 10 times, or more than the affinity of the antibody or fragment thereof to bind to FcγRIIA. 4 times, 10 5times, 10 6 times, 10 7 times, 10 8 times.

[0229] This application provides the following items:

[0230] 1. A composition comprising:

[0231] (i) an antibody molecule that specifically binds to a cell surface antigen of a target cell and has an Fc domain that can bind to FcγRIIb; and

[0232] (ii) an agent that inhibits or reduces the binding of FcγRIIb to the Fc domain of the antibody molecule,

[0233] Characterized in that the composition is used to treat patients having target cells with increased FcγRIIb expression levels.

[0234] 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 is characterized in that the use is for the preparation of a medicament for treating patients with target cells expressing elevated levels of FcγRIIb.

[0235] 3. 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 in his or her target cells.

[0236] 4. Use of FcγRIIb expression on target cells as a prognostic marker for the response of the target cells to treatment with an antibody molecule, wherein the antibody molecule specifically binds to a surface antigen of the target cell and the antibody molecule has an Fc domain capable of binding to FcγRIIb, whereby an increased level of FcγRIIb indicates a reduced response or no response to treatment with the antibody molecule.

[0237] 5. A method for predicting the response of a patient's target cells to treatment with an antibody molecule, wherein the antibody molecule 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 expression level of FcγRIIb on the target cell, whereby an increased level of FcγRIIb indicates a reduced response or no response to treatment with the antibody molecule.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 9. A composition, use or method according to any preceding claim, wherein the target cell is a cancer cell.

[0242] 10. A composition, use or method according to any preceding claim, wherein the target cell is a B cell.

[0243] 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.

[0244] 12. The composition, use or method according to 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.

[0245] 13. The composition, use or method according to any of the preceding items, wherein the agent is any of the following: polypeptide; anticalin; peptide; antibody; chimeric antibody; single-chain antibody; aptamer; darpin; Fab, F(ab′) 2 , Fv, ScFv or dAb antibody fragments; small molecules; natural products; affibodies; peptidomimetics; nucleic acids; peptide nucleic acid molecules; lipids; carbohydrates; modular framework-based proteins, including ankyrin repeat proteins, armadillo repeat proteins, leucine-rich proteins, tetratricopeptide repeat proteins or designed ankyrin repeat proteins (DARPins).

[0246] 14. A composition, use or method according to any preceding claim, wherein the agent is one or more antibody molecules that specifically bind to FcγRIIb.

[0247] 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.

[0248] 16. The composition, use or method according to 15, wherein the one or more antibody molecules are monoclonal antibody molecules.

[0249] 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:

[0250] (i) SEQ ID NO:29 and SEQ ID NO:30 and SEQ ID NO:31; or

[0251] (ii) SEQ ID NO:35 and SEQ ID NO:36 and SEQ ID NO:37; or

[0252] (iii) SEQ ID NO:41 and SEQ ID NO:42 and SEQ ID NO:43; or

[0253] (iv) SEQ ID NO:47 and SEQ ID NO:48 and SEQ ID NO:49; or

[0254] (v) SEQ ID NO:53 and SEQ ID NO:54 and SEQ ID NO:55; or

[0255] (vi) SEQ ID NO:59 and SEQ ID NO:60 and SEQ ID NO:61; or

[0256] (vii) SEQ ID NO:65 and SEQ ID NO:66 and SEQ ID NO:67; or

[0257] (viii) SEQ ID NO:71 and SEQ ID NO:72 and SEQ ID NO:73; or

[0258] (ix) SEQ ID NO:77 and SEQ ID NO:78 and SEQ ID NO:79; or

[0259] (x) SEQ ID NO:83 and SEQ ID NO:84 and SEQ ID NO:85; or

[0260] (xi) SEQ ID NO:89 and SEQ ID NO:90 and SEQ ID NO:91; or

[0261] (xii) SEQ ID NO:95 and SEQ ID NO:96 and SEQ ID NO:97; or

[0262] (xiii) SEQ ID NO:101, SEQ ID NO:102, and SEQ ID NO:103.

[0263] 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:

[0264] (i) SEQ ID NO:32 and SEQ ID NO:33 and SEQ ID NO:34; or

[0265] (ii) SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40; or

[0266] (iii) SEQ ID NO:44 and SEQ ID NO:45 and SEQ ID NO:46; or

[0267] (iv) SEQ ID NO:50 and SEQ ID NO:51 and SEQ ID NO:52; or

[0268] (v) SEQ ID NO:56 and SEQ ID NO:57 and SEQ ID NO:58; or

[0269] (vi) SEQ ID NO:62 and SEQ ID NO:63 and SEQ ID NO:64; or

[0270] (vii) SEQ ID NO:68 and SEQ ID NO:69 and SEQ ID NO:70; or

[0271] (viii) SEQ ID NO:74 and SEQ ID NO:75 and SEQ ID NO:76; or

[0272] (ix) SEQ ID NO:80 and SEQ ID NO:81 and SEQ ID NO:82; or

[0273] (x) SEQ ID NO:86 and SEQ ID NO:87 and SEQ ID NO:88; or

[0274] (xi) SEQ ID NO:92 and SEQ ID NO:93 and SEQ ID NO:94; or

[0275] (xii) SEQ ID NO:98 and SEQ ID NO:99 and SEQ ID NO:100; or

[0276] (xiii) SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106.

[0277] 19. A composition, use or method according to any one of 1-3 and 6-18, wherein the agent comprises a heavy chain variable region (VH) amino acid sequence selected from the group consisting of the following amino acid sequences: 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.

[0278] 20. A composition, use or method according to any one of 1-3 and 6-19, wherein the agent comprises a light chain variable region (VL) amino acid sequence selected from the group consisting of the following amino acid sequences: 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.

[0279] 21. The composition, use or method according to any one of 1-3 and 6-20, wherein the agent comprises the following CDR amino acid sequence:

[0280] (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

[0281] (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

[0282] (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

[0283] (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

[0284] (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

[0285] (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

[0286] (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

[0287] (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

[0288] (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

[0289] (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

[0290] (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

[0291] (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

[0292] (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.

[0293] 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:

[0294] (i) SEQ ID NO:3 and SEQ ID NO:16; or

[0295] (ii) SEQ IS NO:4 and SEQ ID NO:17; or

[0296] (iii) SEQ IS NO:5 and SEQ ID NO:18; or

[0297] (iv) SEQ ID NO:6 and SEQ ID NO:19; or

[0298] (v) SEQ ID NO:7 and SEQ ID NO:20; or

[0299] (vi) SEQ ID NO:8 and SEQ ID NO:21; or

[0300] (vii) SEQ ID NO:9 and SEQ ID NO:22; or

[0301] (viii) SEQ ID NO:10 and SEQ ID NO:23; or

[0302] (ix) SEQ ID NO: 11 and SEQ ID NO: 24; or

[0303] (x) SEQ ID NO: 12 and SEQ ID NO: 25; or

[0304] (xi) SEQ ID NO: 13 and SEQ ID NO: 26; or

[0305] (xii) SEQ ID NO: 14 and SEQ ID NO: 27; or

[0306] (xiii) SEQ ID NO:15 and SEQ ID NO:28.

[0307] 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.

[0308] 24. The composition, use or method of any one of 1-3 and 6-23, wherein the agent inhibits or reduces FcγRIIb signaling.

[0309] 25. The composition, use or method according to any one of 1-3 and 6-24, wherein the agent inhibits or reduces internalization of the antibody molecule by the target cell.

[0310] 26. A composition, use or method according to any preceding claim, wherein the cell surface antigen is selected from CD19, CD20 or CD40.

[0311] 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.

[0312] 28. The composition, use or method of 27, wherein the CD20 antibody is a type I CD20 antibody.

[0313] 29. The composition, use or method according to any of the preceding items, wherein the cell surface antigen is CD20, and the antibody molecule that specifically binds to the cell surface antigen is a type I antibody.

[0314] 30. The composition, use or method according to any preceding claim, wherein the elevated expression of FcγRIIb on the target cell is determined relative to a control, preferably the control is the normal expression level of FcγRIIb in cells of the same type as the target cell.

[0315] 31. A composition substantially as herein described with reference to the accompanying drawings.

[0316] 32. A method for use substantially as herein described with reference to the accompanying drawings.

[0317] 33. A method substantially as herein described with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0318] Specific aspects of the present invention will now be described by way of the accompanying drawings:

[0319] Figure 1 A- Figure 1 B: Internalization of type I mAbs from the cell surface of normal and malignant human B cells.

[0320] A) Primary CLL cells were incubated with Tosit-488, GA101gly-488, Ritux-488 or Ofatum-488 (all at 5 μg / ml) for 2 hours or 6 hours. Then, the cells were collected and washed twice, after which anti-Alexa-488 antibody was added to half of the samples for 30 minutes at 4°C 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 value is shown. B) Tosit-488 or Ritux-488 was 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.

[0321] Figure 1 C- Figure 1 G: Lack of correlation between CD20 modulation and CLL phenotypic / 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 prognosis of 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 induce 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 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.

[0322] Figure 2 : Modulation is an Fc-dependent process. A) When CLL cells were incubated with Alexa-488-labeled rituximab fragments Fab', F(ab') 2 The internalization assay described in 1a) was repeated after incubation with IgG for 6 hours. The data represent the average modulation level + / -SD of 3 different CLL samples. B) CLL cells were incubated with Tosit-488, Ritux-488+ / -AT10 and Rit m2a-488 as in 1a) for 2 hours and 6 hours. The average + / - modulation from 6 different CLL samples is shown. Adding 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 difference in modulation. C) AT10-PE was used to stain a variety of normal and malignant B cell samples for FcγRIIb expression. The histogram shows the difference 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 difference 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 variability. The median is shown. D) CD20 modulation and FcγRIIb expression are plotted for all NHL subtypes and normal B cells (obtained from the internalization assay described in 1A), 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.

[0323] Figure 3:FcγRIIb expression is the main determinant of CD20 modulation. A) Ramos cells transfected with FcγRIIb were sorted to express low, medium and high levels of FcγRIIb and evaluated together with blank-transfected cells at the 6-hour time point of internalization assays using Tosit-488 and Ritux-488. Error bars represent the mean + / -SD from independent experiments. The geometric MFI values ​​of FcγRIIb expression of the sorted cells are listed on the right. B) The internalization assay was repeated after 6 hours of incubation on normal Ramos cells, Rx3 cells (lack of BCR expression), blank-transfected Rx3 cells, and FcγRIIb-transfected Rx3 cells using Tosit-488 and Ritux-488. Data points from 5 independent experiments are shown together with the median.

[0324] Figure 4 : CD20 and FcγRIIb co-ligation occurs mainly in cis form and leads to FcγRIIb activation. A) Raji cells were incubated with specific mAbs (10 μg / ml) at 37°C for 2 hours, after which the cells were collected, lysed and subsequently immunoblotted for phosphorylated FcγRIIb. B) Left: PKH26-labeled Ramos cells (FcγRIIb -ve , R1) were mixed with the selected Ramos transfectants with high FcγRIIb expression at a ratio of 1:1 ( Figure 3 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 labeled with PKH26 and then mixed with CLL with higher FcγRIIb expression at 1:1. 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 incubated at 20x 10 5 , 4x 10 5 and 1x10 5 10 cells / ml were incubated with Ritux-488 for 6 hours, and the internalization assay was performed as described above at 6 hours. E) Raji cells were cultured 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 cellular phosphorylated FcγRIIb by immunoblotting, as shown in Figure 2. Figure 4 As described in A.

[0325] 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 shows FcγRIIb staining in unstimulated cells. C) The same CLL samples were incubated with Ritux-488 for 30 minutes and then as shown. Figure 5 b) Treated as shown. 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 microscopy 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 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 obvious 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.

[0326] Figure 6: 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 carrying mouse IgG1 (m1) or mouse IgG2a (m2a), 250 mg intravenously (iv), and B cell clearance was monitored by serial bleeding of mice and flow cytometry using B220 and CD19 mAb staining for 90 days.

[0327] 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, 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 legend. In A), Daudi cells were inoculated simultaneously and tumors were monitored every 305 days by calipers. In B) and C), Raji cells were inoculated intravenously.

[0328] Figure 8 : Phagocytosis of CLL cells treated with rituximab is enhanced by co-incubation with FcγRII blocking mAb. Monocytes were derived from healthy volunteers and differentiated into macrophages using M-CSF in 6-well plates for a minimum of 7 days before use. Macrophages were then collected and plated at 5 x 10 in 96-well plates for a minimum of 2 hours. 5 Cells / well were allowed to adhere, after which CFSE-labeled CLL cells were added. CFSE-labeled CLL cells were left untreated or treated with 10 μg / ml of rituximab (ritux) and FcγRIIb blocking mAb AT10 (fab') 2 After conditioning for 15 minutes or 6 hours, the cells were washed twice and added to macrophages (1:1 ratio) for at least 30 minutes. 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 RT. Further ice-cold FACS wash was added and the plates were incubated on ice for 10 minutes before collection 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, lines represent mean values. The data clearly show that phagocytosis of CLL cells was higher when rituximab was added only for 15 minutes than for 6 hours. This increase in efficacy was associated with modulation of rituximab from the cell surface, which could be reversed by treatment with mAb AT10 that blocks FcγRIIb. Importantly, only FcγRIIb was blocked 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 and therefore 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).

[0329] Fig. 9 : FcγRIIb expression was detected by IHC. Paraffin-embedded tissues were stained using anti-CD32b specific mAb EP888Y. Images from 4 different FL patients are shown (x40 total magnification, and x150 magnification insets). FcγRIIb expression was detected by flow cytometry by staining matched live cells using AT10-PE. The upper left corner of each image shows the FcγRIIb expression obtained by flow cytometry.

[0330] Fig.10 :FcγRIIb levels predict clinical outcomes in rituximab-treated MCL patients. As proof of concept for the in vitro findings of the present invention, the inventors retrospectively tested FcγRIIb expression in a cohort of MCL patients who had received rituximab. Diagnostic paraffin-embedded tissues were stained by immunohistochemistry using an FcγRIIb-specific mAb ( Fig.11 ). Strong membrane staining was observed in FcγRIIb+ve but not in FcγRIIb-ve lymphoma samples. Fig.10 FcγRIIb staining shown in a and 10b is consistent with Figure 2 The expression of FcγRIIb shown in D was correlated with that detected by flow cytometry (the values ​​detected by flow cytometry in 2D are expressed as Fig.10 These results correlated with FcγRIIb expression of the corresponding DMSO-frozen samples obtained by flow cytometry ( Figure 2C, inset values.) Although only a small cohort of 16 MCL patients was studied, patients with FcγRIIb-ve lymphoma had a much better median progression-free survival than those with FcγRIIb+ve cells (median 852 days vs. 189 days, respectively). Fig.10 c shows the survival difference in FcγRIIb+ and - subgroups. These two groups are comparable in clinical characteristics (MCL International Prognostic Index, data not shown), but are heterogeneous in the type of chemotherapy used. To explain this, the inventors examined those patients treated with a single agent of rituximab or fludarabine, cyclophosphamide and rituximab (FCR) for initial treatment and observed similar results. Fig.10 d shows the survival differences in FcγRIIb+ and − subgroups after the patient cohort was further controlled as described.

[0331] Fig.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 mAb for human FcγRIIb demonstrated strong membrane staining in FcγRIIb transfected Ramos but no staining in FcγRIIa transfected cells.

[0332] Fig.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 negative control isotype-matched irrelevant mAbs (isotype wt or nq). Finally, ALEXa 488-labeled B1 was used as a control mAb that did not modulate rapidly. The data clearly show that all 3 All mAbs (C1, C11 and C13) were able to block modulation of rituximab in either wt or 297q form. In particular, C11 mAb was extremely effective.

[0333] Fig.13: Ability of anti-CD32b mAbs to block modulation by rituximab. All 13 mAbs (nq) and C11 were 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 does not modulate rapidly. In addition, control CD32-negative Ramos cells were used to evaluate the maximal effect of the CD32 blocking mAbs. The data clearly show that most All mAbs were able to block rituximab modulation. In particular, C10 and C11 mAbs were extremely effective, showing almost complete blockade of modulation even at 24 hours.

[0334] Fig.14 A: Correlation between the affinity of anti-CD32b blocking mAbs and the ability to inhibit CD32b phosphorylation after rituximab binding. The relative affinity of mAbs was determined by a dose titration experiment detecting mAbs binding to CD32B-transfected CHO cells. Briefly, CD32B-transfected CHO K1 adherent cells were seeded in FMAT plates. IgG was titrated from 30nM to approximately 0.015nM at a 1:2 dilution and bound 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 FMAT (Applied Biosystems). This gives the EC50 value for mAb binding to cells expressing the target and can be converted into relative affinity. The relative affinity is then correlated with the ability of anti-CD32b blocking mAbs to inhibit CD32b phosphorylation after rituximab binding. The assay was performed by stimulating cells with rituximab in the presence or absence of an anti-CD32b mAb and then Western blotting for phospho-CD32b. CD32b mAbs were then classified according to their ability to block CD32 phosphorylation, with 1 being the most potent. There was a close correlation between the affinity of the mAb and its ability to block CD32b phosphorylation.

[0335] Fig.14B: Correlation between the affinity of anti-CD32b blocking mAbs and their ability to inhibit rituximab modulation. Correlation between the affinity of anti-CD32b blocking mAbs and their ability to inhibit rituximab modulation. The relative affinity of mAbs was determined by a dose titration experiment detecting mAb binding to CD32B-transfected CHO cells. Briefly, CD32B-transfected CHO K1 adherent cells were seeded in FMAT plates. IgG was titrated from 30nM to approximately 0.015nM 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 FMAT (Applied Biosystems). This gives the EC50 value for mAb binding to cells expressing the target and can be converted into relative affinity. The relative affinity then correlates with the ability of the anti-CD32b blocking mAbs to inhibit rituximab modulation on CD32b-transfected Ramos cells (shown in the previous figure). There was a clear and significant correlation between the affinity of the mAbs and their ability to block rituximab modulation. This data confirms the critical role of CD32B in promoting rituximab modulation on the surface of target cells.

[0336] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0337] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0338] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0339] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0340] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0341] Fig. 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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0342] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0343] Fig. 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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0344] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0345] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0346] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0347] Fig.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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0348] Fig. 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. The cross shows 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 to BSA, which was then mixed with FITC-specific hIgG1 antibodies in a 10:1 molar ratio. The total intensity reflects the binding, and the higher the intensity, the higher the binding. Binding is the binding of immune complexes (IC) or mAbs.

[0349] Fig.28: Showing the cell specificity of anti-CD32B antibodies. PBMCs isolated from peripheral blood were prepared using a Ficoll density gradient. Cells were stained with cell-specific markers and the binding of CD32B-specific antibodies was evaluated. As shown in the figure, for clone 1-13, only B cells (CD19+ cells) were positively stained.

[0350] Fig.29 : 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 out using a CD19-specific mAb. The gate is referred to as "M1". When the concentration of CD32B mAb was reduced, the number of B cells stained dropped from nearly 100% to a much lower value, as expected based on the CD32B-specific mAb, showing specific and dose-dependent staining of B cells.

[0351] Fig.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.

[0352] Fig.31 : Effect of CD32b on the modulation rate of type I anti-CD20 mAbs. The ability of CD32b to promote the internalization of other type I anti-CD20 mAbs. An Alexa-488 labeled version of each mAb was 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 show that the internalization rate of OFA is similar to that of RTX and is accelerated by the presence of CD32b.

[0353] Fig.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 using different mAbs in the presence (+) or absence (-) of CD32 blockade using AT10 as described previously. Ramos CD32B transfectants were used in this 6-hour assay. *p<0.05. f3.3=MHC class II; RFB9=CD19; RTX=rituximab. If 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 surface modulation of F3.3 mAb is reduced to a lesser extent. 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.

[0354] Fig.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 extent of modulation was determined as previously described. * = p < 0.05. The y-axis shows modulation / internalization or surface accessible antigen (%). The figure shows that this is due to an increase in internalization / modulation in the presence of huCD32b. Note that for anti-CD19 mAb and anti-CD40 mAb, there was a statistically significant reduction in cell surface in the presence of CD32b (*, p < 0.05).

[0355] Fig.34 : Amino acid sequences of the constant regions of 14 antibody clones against human CD32B in the form of IgG1-λ. The amino acid sequences of the IgG1-CH and λ-CL regions are shown.

[0356] Fig.35 : Amino acid sequences of the variable regions of antibody clone 1 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The marked CDR sequences are indicated as boxed sequences separated by marked framework regions.

[0357] Fig.36 : Amino acid sequences of the variable regions of antibody clone 2 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The marked CDR sequences are indicated as boxed sequences separated by marked framework regions.

[0358] Fig.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.

[0359] Fig.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. Fig.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.

[0360] Fig.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.

[0361] Fig.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.

[0362] Fig.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.

[0363] Fig.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.

[0364] Fig.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.

[0365] Fig.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.

[0366] Fig.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.

[0367] Fig.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. DETAILED DESCRIPTION

[0368] Example 1: CD20 modulation in primary CLL and other NHL samples

[0369] 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 A). 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 in-house produced ofatumumab (type I) recently approved by the FDA 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 nearly 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.

[0370] In the CLL cohort, the inventors examined a variety of 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.

[0371] The inventors have 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 modulated rapidly in the presence of rituximab and was more uniform than on malignant B cells, suggesting that factors associated with the malignancy contribute to the observed heterogeneity. The rate of rituximab-induced modulation of SLL and MCL cells was similar to that of CLL ( Figure 1 A), while for DLBCL and FL, the rates were slightly lower (when compared with Figure 1 However, in FL, the inventors did observe that 2 / 11 patient samples upregulated rituximab very rapidly, resulting in almost undetectable mAb or CD20 levels after 6 hours of incubation.

[0372] Example 2: Modulation of CD20 in B-NHL is an Fc-dependent process.

[0373] 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 may be Fc-dependent even in the absence of effector cells and used Fab' and F(ab') fragments of rituximab to characterize the Fc-dependent effector cell responses. 2 This was tested by repeated internalization assays of the fragments ( Figure 2 A). Rituximab Fab' showed only low levels of modulation as demonstrated by cell binding assays, which could be explained by the requirement 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 h of incubation, F(ab') 2 Fragments (40% surface accessible CD20) showed much less modulation 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 AT10, consistent with rituximab F(ab') 2 fragment or Rit m2a (which binds FcγRIIb with lower affinity than rituximab carrying human IgG1) Figure 2 B) As expected, co-incubation with AT10 resulted in very little effect on the modulation of Rit m2a.

[0374] Example 3: FcγRIIb expression on normal B cells and B cell tumors.

[0375] 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 was significant heterogeneity in FcγRIIb expression among the groups. Expression on CLL cells was high, 20 to 300 times higher than isotype control. DLBCL and most FL showed low FcγRIIb expression. Two FL cases showed very high FcγRIIb expression, which was surprisingly the case with the extremely rapid modulation previously observed. MCL and SLL expressed moderate but heterogeneous levels of FcγRIIb ( Figure 2 C), which is consistent with previous findings (21).

[0376] Example 4: FcγRIIb expression regulates CD20 modulation.

[0377] Taken 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 of all obtained 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 a logarithmic curve with most FL and DLBCL cases located in the upper part and CLL and MCL samples 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 emphasizing the ability of this receptor in regulating the clearance of anti-CD20:CD20 complexes from the cell surface.

[0378] To directly address the role of FcγRIIb in CD20 modulation, a plasmid encoding FcγRIIb was transfected into -ve Ramos cells. FcγRIIb produced +ve The 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 - Ramos cells. 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 Medium>FcγRIIb +ve high( Figure 3 A). Similarly, using B cells obtained 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 the regulation of CD20 modulation in this transgenic model.

[0379] 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 to unmanipulated Ramos cells and blank control Rx3-transfected cells ( Figure 3 B). These data clearly show 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 characteristically 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).

[0380] Example 5: FcγRIIb activation precedes modulation of CD20 and FcγRIIb

[0381] To further probe the relevant interaction between anti-CD20 mAb and FcγRIIb, the inventors studied antibody-mediated FcγRIIb stimulation as shown 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) and then immunoblotted for phosphorylated FcγRIIb. Phosphorylated FcγRIIb was increased 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).

[0382] Example 6: CD20 and FcγRIIb cross-linking occurs mainly in cis form

[0383] 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-incubated 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 not to the level of FcγRIIb +ve This result suggests that although trans interactions can occur, modulation of CD20 mAb by FcγRIIb is primarily driven in a cis manner.

[0384] 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 marker) 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 mAb 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).

[0385] In another experiment of this type, CLL cells were cultured at reduced concentrations to reduce the potential for cell:cell interactions, and 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 level of modulation was similar to that of the 2 x 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 events in the same cell, with neighboring FcγRIIb-expressing cells having only a minor effect.

[0386] Example 7: Internalization of FcγRIIb into lysosomes together with CD20

[0387] To determine the fate of FcγRIIb after rituximab occupies the cell surface, the inventors monitored its expression and localization by flow cytometry and confocal microscopy. The inventors evaluated the expression of FcγRIIb on the surface of B cells from 6 different CLL cases using flow cytometry 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).

[0388] 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 with FcγRIIb as part of an anti-CD20:CD20:FcγRIIb complex in CLL cells, the inventors incubated them with Tosit-488 or Ritux-488, then fixed and stained FcγRIIb (using Alexa 647-labeled F(ab') from AT10) with 5% paraformaldehyde. 2 ) and lysosomal marker LAMP-1. Before 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 hours of stimulation, Tosit-488 was still equally distributed on the cell surface, whereas AT10-647 showed no change from its baseline appearance at 30 minutes and did not colocalize with LAMP-1 ( Figure 5 D). In contrast, over the same time course, Ritux-488 showed a different dot pattern, with the majority of cells (58%) showing 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 between all three cell lines reflects that Ritux-488 and FcγRIIb are internalized together and may occupy other intracellular compartments before they appear in lysosomes.

[0389] Example 8: FcγRIIb inhibits type I anti-CD20 mAb in vivo

[0390] To elucidate whether FcγRIIb might 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 of mice and flow cytometry using B220 and CD19 mAb staining for 90 days ( Figure 6 ). These variants either strongly (ml) or weakly (m2a) bind to CD32b. The data clearly show that depletion is suboptimal when the ml isotype is used (compared to m2a), and loss of CD32 results in a significant improvement in depletion efficacy. In contrast, m2a is roughly similar in the presence or absence of CD32.

[0391] Example 9: FcγRIIb enhances and improves the activity of anti-CD20 mAb against human tumors in vivo

[0392] To examine the effects of CD32 on human tumor cells and the potential to enhance 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 tumor cells by blocking modulation rather than through any effect on host effector cells. In these experiments, CD20-positive, CD32-positive human tumor cells (Daudi or Raji) were inoculated into SCID mice, and then the cells were treated with rituximab, AT10, or both, and the mice were monitored for survival or tumor growth ( Figure 7 ). The doses of mAbs used are shown in the legend. In A), Daudi cells were inoculated subcutaneously and tumors were monitored by caliper measurements every 3-5 days. In B and C, Raji cells were injected intravenously and the survival of the animals was monitored. In both models, AT10 was shown to enhance and potentiate the activity of rituximab, demonstrating the potential of this combination in vivo.

[0393] Example 10: FcgRIIb levels predict clinical outcomes in MCL patients treated with rituximab

[0394] As proof of concept for our in vitro findings, we retrospectively examined the expression of FcγRIIb in a cohort of MCL patients receiving rituximab. Diagnostic paraffin-embedded tissue was stained by immunohistochemistry using an FcγRIIb-specific mAb ( Fig.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. Fig.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 Fig.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 a much better median progression-free survival than those with FcγRIIb+ve cells (median 852 days vs. 189 days, respectively). Fig.10 c shows the survival difference in FcγRIIb+ and - subgroups. These two groups are comparable in clinical characteristics (MCL International Prognostic Index, data not shown), but are heterogeneous in the type of chemotherapy used. To explain this, the inventors examined those patients treated with a single agent of rituximab or fludarabine, cyclophosphamide and rituximab (FCR) for initial treatment and observed similar results. Fig.10 d shows the difference in survival in FcγRIIb+ and − subgroups after the patient cohort was further controlled as described.

[0395] The experimental theory in Examples 10 and 11 is as follows. If a cell expresses high levels of CD32b (FcγRIIb), it will internalize rituximab more quickly (shown as a percentage reduction in surface accessible CD20). If there is less rituximab on the cell surface, there is less Fc-dependent effector activity (e.g., phagocytosis or ADCC), and therefore less killing of tumor cells and thus less therapeutic results. Therefore, the inventors examined the FcγRIIb expression of the patient cohort treated with MCL and determined whether they were high or low CD32b expressers. Clinical data has been obtained for this cohort, so the clinical results are classified according to whether they are high or low FcγRIIb expressing tumors. The reasoning is that tumors expressing low levels of FcγRIIb can be successfully treated with rituximab, while those expressing high levels of FcγRIIb are less effective. This is exactly what the clinical data shows. Fig.11The specificity of the mAb used for FcγRIIb staining is shown. It only stains cells expressing FcγRIIb, but not the closely related FcγRIIa.

[0396] In the measurement of FcγRIIb levels by IHC ( Fig.10 b) and after dividing the MCL samples into FcγRIIb positive and negative, the inventors observed clear differences in clinical responses after rituximab-based treatment ( Fig.10 c and 10d).

[0397] Example 11: Selection of anti-CD32b monoclonal antibodies

[0398] Figure 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) are identical. Fig.36 The constant regions are shown in .

[0399] use scFv phage display library was screened against CD32B (FcγRIIb). Human CD32A was used as a non-target. 3 -Fc fused extracellular domains of CD32A and CD32B and purified using protein A. Three consecutive protein screens were performed. Non-targets were used as competitors in all screens. The generated phages were 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 using coating proteins in ELISA and by transfecting CHO cells in FMAT. For the determination of IC inhibitory properties, IgG was bound to CD32B transfected CHO cells, followed by the addition of IC in the form of IgG1 coated bovine serum albumin. The bound IC was then detected and the inhibitory properties of IgG were evaluated.

[0400] Example 12: Ability of anti-CD32b mAb to block rituximab modulation

[0401] 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 blocking CD32 mAbs, the inventors also used CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragment (Fab)) and negative control isotype-matched irrelevant mAbs (isotype wt or nq) included. Fig.12The data in clearly demonstrate that all three nCoder mAbs (C1, C3 and C11) are able to block modulation by rituximab in either wt or 297q form.

[0402] Fig.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 the CD32 blocking mAbs. Fig.12 The data clearly demonstrate that all nCoder mAbs were able to block rituximab modulation.

[0403] Previous groups have demonstrated that FcγRIIb regulates rituximab internalization. Therefore, these experiments sought to examine whether blocking FcγRIIb using an anti-FcγRIIb mAb would reduce the amount of rituximab internalized.

[0404] Example 13: Affinity of anti-CD32b blocking mAbs and their correlation between inhibition of CD32b phosphorylation after rituximab binding and inhibition of rituximab modulation

[0405] The relative affinity of mAb was determined by measuring the dose titration test of mAb binding to CD32B transfected CHO cells. In brief, CD32B transfected CHO K1 adherent cells were inoculated in FMAT plates. IgG was titrated from 30nM to about 0.015nM at a dilution of 1:2 and combined for 1 hour at room temperature. After washing, anti-human IgG-APC was used to detect the bound IgG. Finally, the plate was washed and read in FMAT (Applied Biosystems). This gives the EC50 value of mAb binding to cells expressing the target and can be converted into relative affinity. The relative affinity is then related to the ability of anti-CD32b blocking mAb to inhibit CD32b phosphorylation after rituximab binding. This is determined by using rituximab to stimulate cells in the presence or absence of anti-CD32b mAb and then performing Western blotting for phospho-CD32b. Then, it is classified according to the ability of CD32b mAb to block CD32 phosphorylation, with 1 being the most effective. Fig.14 A shows that there is a clear and close correlation between the affinity of mAb and its ability to block CD32b phosphorylation. Fig.14 B shows a clear 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.

[0406] 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 Fig.14 The higher the affinity, the better it blocks rituximab binding-induced FcγRIIb activation (measured by the amount of phospho-FcγRIIb stained by Western blot) and the better it blocks modulation.

[0407] Example 14: Dose-dependent binding to hCD32B-transfected cells and immune complexes Dose-dependent binding and inhibition of hCD32B-transfected cells

[0408] Cells were seeded into FMAT plates.Immunocomplexes 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 to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells mediated by clone 1-13 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.

[0409] The assay was designed to: 1) determine the specificity of the mAb. CD32B and CD32A are very closely related molecules. However, while CD32B transmits an inhibitory signal, CD32A transmits a positive signal, so it is critical for the desired effect that the antibody only binds to CD32B; 2) In addition, in order to effectively block the signal through CD32B, the antibody must not only bind to CD32B, but also block the binding of its natural ligand immune complex (IC). Therefore, the figure shows binding to CD32A, CD32B, and inhibition of IC binding. The figure shows that all mAbs are specific for CD32B and do not bind to CD32A, and they all inhibit IC binding.

[0410] Example 15: Cell Specificity of Anti-CD32B Antibodies

[0411] 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. Fig.28 As shown, clone 1-13 stained positive only for B cells (CD19+ cells).

[0412] In resting PBMCs, CD32B is expressed only on B cells, whereas the closely related CD32A is expressed on monocytes and neutrophils. The previous figure showed specificity for transfected CHO cells. Fig.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 the specificity of the antibodies when the antigen is expressed in normal, non-transfected PBMCs.

[0413] Example 16: Dose-dependent staining of B cells by anti-FcγRIIb mAb clone 1-13

[0414] PBMCs isolated from peripheral blood were prepared using a Ficoll density gradient. Cells were stained with CD19 and then with 10, 1 or 0.1 mg / ml CD32B specific antibody. Fig.29 It is shown how the staining of B cells by each clone is dose-dependent.

[0415] exist Fig.29 In the , 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 dropped from nearly 100% to much lower values, demonstrating the specific and dose-dependent staining of B cells expected by the CD32B-specific mAb.

[0416] This is also evidence of antibody specificity. As mentioned, CD32A and CD32B are extremely closely related, and obtaining specific antibodies is not easy. Any specific antibody shows dose-dependent binding, which is exactly Fig.29 , i.e., 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.

[0417] Example 17: Ability of each mAb to inhibit Fc-mediated CD32B phosphorylation

[0418] Raji cells (CD32B positive) were treated with rituximab, which resulted in phosphorylation of CD32B. This was done in the presence or absence of 1-13 CD32B specific mAbs, Fig.30 The ability of each mAb to inhibit Fc-mediated phosphorylation of CD32B was demonstrated.

[0419] The assumption 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 ( Fig.30 ) and modulation ( Fig.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 has the same activity.

[0420] Example 18: Effect of CD32b on the modulation rate of type I anti-CD20 mAb

[0421] The ability of CD32b to promote internalization of other type I anti-CD20 mAbs is shown in Fig.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 described above. The mAbs used were rituximab (RTX), ofatumumab (OFA) prepared in-house, and tositumomab (Tos). The data clearly show that OFA internalizes at a similar rate to RTX and is accelerated by CD32b.

[0422] 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 extends to other anti-CD20 mAbs, the inventors tested another clinically relevant type I mAb, ofatumumab (Teeling, 2004 (52) ofatumumab), which, as expected, was rapidly internalized like rituximab.

[0423] 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

[0424] Ramos huCD32b transfectants. Internalization using other surface antigens was also affected by CD32b expression. Modulation assays were performed using different mAbs in the presence (+) or absence (-) of CD32 blockade using AT10 as previously described. Ramos CD32B transfectants were used in this 6 hour assay. *p<0.05. f3.3=MHC class II; RFB9=CD19; RTX=rituximab. Fig.32It is clearly shown that after incubation with CD32 blockade, surface modulation of RTX and RFB9 mAbs was significantly reduced, and that of F3.3 was less reduced. 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.

[0425] The inventors wished 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 MHCII) and mAbs that block FcγRIIb would reduce their internalization. The data showed that blocking FcγRIIb also reduced CD19 mAb modulation.

[0426] Example 20: Internalization using other surface antigens is also affected by CD32b expression

[0427] Ramos cells have no 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) will increase the level of internalization.

[0428] 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. Fig.33 It was shown that internalization using other antigens is also affected by CD32b expression.

[0429] Materials and Methods

[0430] cell

[0431] 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% CO 2. Rx3 Ramos cells lacking BCR expression were generated previously (36). Ramos FcγRIIb transfectants and control cells transfected with an empty vector (FcγRIIb negative) were 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 an 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 FcγRIIb transfectants expressing low, medium, or high levels of FcγRIIb were sorted using a FACS Aria flow cytometer (BD Biosciences, USA).

[0432] Blood Donor

[0433] Normal human B cells were obtained from healthy volunteers with informed consent. Peripheral blood was drawn at K2E or LiH, lymphocytes were isolated using Lymphoprep (Axis-Shield, UK) according to the manufacturer's protocol, and B cells were isolated by negative selection using the Human B Cell Isolation Kit II (Miltenyi Biotec, Germany).

[0434] Clinical samples

[0435] CLL / SLL, FL, DLBCL, and MCL samples were obtained with informed consent in accordance with the Declaration of Helsinki. Blood samples were collected in K2E or LiH using Lymphoprep, solid tissue was broken down by 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 Science Department at the University of Southampton under the permission of the Human Tissue Regulatory Authority. The University Hospital Southampton National Health Service Trust obtained ethical approval for the use of clinical samples from the Southampton and South West Hampshire Research Ethics Committee. For CLL cells, the mutation status of IgVH genes (33) and CD38 positivity (44) were determined as previously described in detail. Briefly, for IgVH analysis, heavy chain genes were amplified from cDNA using VH leader primer mixture and Cμ100 primers. All nucleotide sequences were aligned with the V-base directory and mutation 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).

[0436] Viability assay

[0437] Cell viability was assessed by flow cytometry after staining with FITC-labeled Annexin V and PI as described previously (25).

[0438] Antibodies and reagents

[0439] 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 described previously (48). Fab' fragments were generated by incubation with 20 mM 2-mercaptoethanol at 25°C for 30 min followed by addition of excess iodoacetamide. Western blot antibodies used were anti-actin (AC74, Sigma, UK) and anti-phospho-FcγRIIb (Cell Signaling Technology, UK).

[0440] Flow cytometry

[0441] Fluorochrome-labeled mAbs were obtained from BD Biosciences or prepared in-house. Alexa488 (Invitrogen) was used to couple mAbs according to the manufacturer's protocol. Flow cytometry was as described previously (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 the expression of FcγRIIb was determined using PE-labeled AT10 (prepared in-house and out-house). In order to control for inter-experimental differences, FcγRIIb expression was expressed as the ratio of FcγRIIb:isotype control geometric mean fluorescence intensity (MFI).

[0442] Internalization assay

[0443] Internalization assays were performed as described in detail previously (28). Briefly, 2-4 x 10 5 Cells / well were incubated with Alexa-488 labeled mAbs 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 antibodies at 4°C for 30 minutes with or without quenching antibody anti-Alexa-488 antibody (Invitrogen). The samples were then washed once and analyzed on a flow cytometer.

[0444] To investigate the interaction of the Fc region of cell-bound anti-CD20 mAbs with FcγRIIb on neighboring cells, FcγRIIb was labeled with PKH26 (Sigma Aldrich) according to the manufacturer's instructions. -ve Then, the PKH26-labeled cells were mixed with the same number (2.5 x 10 5 The co-incubation assay was performed using FcγRIIb transfected Ramos cells (100 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. Other variations of this co-incubation assay are described in the figure legend.

[0445] Western blotting

[0446] 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, then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Sigma Aldrich), visualized by enhanced chemiluminescence (ECL, GE Healthcare, UK or Pierce Biotechnology, UK), and exposed to light-sensitive film (Hyperfilm ECL, GE Healthcare, UK) or Biospectrum AC imaging system (UVP, UK).

[0447] Optical and confocal microscopy

[0448] To measure the intracellular trafficking of anti-CD20 mAbs and FcγRIIb, CLL cells were incubated with the appropriate Alexa 488-labeled mAbs for different times as described in the figure legends, and then harvested, washed, and fixed with 2% paraformaldehyde. For detection of FcγRIIb and LAMP-1, cells were then permeabilized with 0.3% saponin and stained with Alexa-647-labeled AT10 F(ab') 2 (labeled using Alexa Fluor-647 labeling kit (Invitrogen) according to the manufacturer's protocol) and / or biotin-conjugated anti-human CD107a (LAMP-1) antibody (eBioscience, UK). Then, the cells were washed, streptavidin-Alexa Fluor-547 (Invitrogen) was added, and then further washed. Afterwards, the cells were transferred to a slide and images were captured using LAS-AF v2 software on a TCS-SP5 laser scanning confocal microscope (Leica Microsystems, UK) (10x eyepiece, 100x objective lens).

[0449] To determine cell proximity at different cell dilutions, 1-20 x 10 5 Cells were inoculated with 100 μg / ml and stimulated with different mAbs for 2 and / or 6 hours, and then their relative proximity was evaluated 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).

[0450] Data analysis

[0451] 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.

[0452] Exemplary compositions, formulations, and modes of administration

[0453] 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.

[0454] 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.

[0455] 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 antibodies, and the like.

[0456] In some embodiments, the composition of the present invention is formulated in liposomes for targeted delivery of the antibodies of the present invention. Liposomes are vesicles containing 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 included 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.

[0457] The method of using the composition of the present invention includes but is not limited to parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural and mucosal (e.g., intranasal and oral routes). In a specific embodiment, the composition of the present invention is administered intramuscularly, intravenously or subcutaneously. The composition can be administered by any convenient route, for example, by infusion or bolus injection, by epithelial or mucocutaneous lining absorption (e.g., oral mucosa, rectal or intestinal mucosa, etc.), and can be administered with other bioactive agents. Administration can be systemic or local. In addition, pulmonary administration can also be adopted, for example, by using an inhaler or atomizer, and prepared together with an aerosol. See, e.g., U.S. Patents 6,019,968; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publications WO 92 / 19244; WO 97 / 32572; WO 97 / 44013; WO 98 / 31346; and WO 99 / 66903, each of which is incorporated herein by reference in its entirety.

[0458] The amount of the composition of the present invention that is effective in treating, preventing or ameliorating one or more symptoms associated with a disorder can be determined by standard clinical techniques. The exact 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 circumstances of each patient. The effective dose can be inferred from a dose response curve from an in vitro or animal model test system.

[0459] For the antibodies included in the present invention, the dosage applied to the patient is usually 0.0001 mg to 100 mg per kg of patient body weight for each antibody in the composition independently. Preferably, the dosage of each antibody applied to the patient is 0.0001 mg to 20 mg per kg of patient body weight, 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 02.5 mg, or 0.01 mg to 0.10 mg. Generally, due to the immune response to exogenous polypeptides, human antibodies have a longer half-life in the human body relative to antibodies from other species. Thus, lower dosages and less frequent administration of human antibodies are generally possible. In addition, the dosage and frequency of administration of the antibodies or fragments thereof of the invention can be reduced by enhancing the uptake and tissue penetration of the antibodies through modification (eg, lipidation).

[0460] 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.

[0461] 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.

[0462] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias 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 carrier for administering a therapeutic agent. Such a pharmaceutical carrier can be a sterile liquid, such as water and oil, 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 necessary, 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.

[0463] 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, about 2 hours apart, about 3 hours apart, about 4 hours apart, about 5 hours apart, about 6 hours apart, about 7 hours apart, about 8 hours apart, about 9 hours apart, about 10 hours apart, about 10 hours apart, about 100 hours apart, about 11 hours apart, about 12 hours apart, less than 24 hours apart, or less than 48 hours apart. In a preferred embodiment, two or more components are administered in the same patient visit.

[0464] Treatment is effective and prevention is effective to encompass the dosage and frequency of administration provided by the invention. Dosage and frequency are usually further different according to the factors specific to each patient, depending 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 regimens by considering these factors and, for example, reporting in the literature and recommending dosages in the physician's desk reference (the 56th edition, 2002).

[0465] Summarize

[0466] 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 consider possible mechanisms to explain the limited therapeutic activity of rituximab and other type I CD20 mAbs, and provide opportunities to block or circumvent this process and thereby develop more potent agents, which is particularly important. The present invention provides a molecular theory for the 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 related to known prognostic factors for CLL. Analysis of other B-NHL subtypes showed that MCL exhibited heterogeneous modulation similar to CLL, but FL and particularly DLBCL showed significantly less modulation. Based on these results, the inventors report here the correlation between the expression levels of FcγRIIb in these malignancies and their modulation during a 6-hour incubation. In addition, the inventors propose that this modulation may explain some of the heterogeneity observed in these diseases in response to rituximab. 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 prove that the use of rituximab improves overall survival in CLL, and its benefit in MCL is even smaller. Thus, it is generally found that B-cell malignancies expressing FcγRIIb are more likely to modulate CD20 and often result in reduced benefits 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. Although it is a single case discovery, the inventors believe that this may potentially provide an important way to address rituximab resistance.

[0467] 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 in vitro studies of the present invention showed that rituximab predominantly 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.

[0468] The present invention also shows that co-incubation with blocking anti-FcγRIIb mAb is able to inhibit FcγRIIb activation and rapid internalization of rituximab. Taken together, these data demonstrate a positive correlation between FcγRIIb activation and internalization of mAb from the cell surface.

[0469] 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 indicate that FcγRIIb is a key regulator of CD20 modulation.

[0470] 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, only 18% (42 / 234 cases) were FcγRIIb positive by immunohistochemistry in an earlier series (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 potent than 232T for BCR-mediated calcium regulation in autoimmune disease (22, 23)) were associated with rituximab efficacy but were unable to demonstrate any correlation between this polymorphism and single-agent response in FL patients. The main concern of these authors themselves was that only 17 patients had the 232T allele, again limiting the statistical power of the study. Additionally, 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 internalization rate, 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 mAb treatment is performed.

[0471] In addition, in different in vivo models, the inventors of the present invention 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 treatment.

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Claims

1. Use of the reagent in the preparation of a medicament for treating non-Hodgkin's lymphoma in a patient in combination with an antibody molecule, a) The antibody molecule i) comprises an Fc domain capable of binding to FcγRIIb, and ii) specifically binds to a cell surface antigen of a target cell in the patient; b) the target cells have an elevated expression level of FcγRIIb, c) the agent specifically binds to FcγRIIb and prevents or reduces the binding between the Fc domain of the antibody molecule and FcγRIIb on the target cell, and d) the cell surface antigen is selected from CD19, CD20 or CD40; The agent is selected from an antibody or an antigen-binding fragment thereof that binds to human FcγRIIb, and the antibody or the antigen-binding fragment thereof comprises: (a) a heavy chain variable region VH comprising VH CDR1, VH CDR2 and VH CDR3 identical to those in SEQ ID NO: 12; and (b) a light chain variable region, VL, comprising VL CDR1, VL CDR2 and VL CDR3 identical to those in SEQ ID NO: 25; 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 use according to claim 1, wherein the antibody molecule is a CD20 antibody.

3. The use according to claim 2, wherein the CD20 antibody is a type I CD20 antibody. The use according to claim 1 , wherein the target cell is a cancer cell. The use according to claim 1 , wherein the target cell is a B cell.

6. The use according to claim 1, wherein the non-Hodgkin's lymphoma is selected from follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

7. The use according to claim 1, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:

12.

8. The use according to claim 1, wherein the VL consists of the amino acid sequence shown in SEQ ID NO:

25.

9. The use according to claim 1, further comprising a heavy chain constant region CH as shown in SEQ ID NO:

1.

10. The use according to claim 1, further comprising a light chain constant region CL as shown in SEQ ID NO:

2.

11. Use of a composition in the preparation of a medicament for treating non-Hodgkin's lymphoma in a patient, the composition comprising: (i) an antibody molecule that specifically binds to a cell surface antigen of a target cell and has an Fc domain that is capable of binding to FcγRIIb; as well as (ii) an agent that prevents or reduces the binding of FcγRIIb to the Fc domain of the antibody molecule; Furthermore, the cell surface antigen is selected from CD19, CD20 or CD40, The composition is used to treat a patient having target cells with elevated levels of FcγRIIb expression; The agent is selected from an antibody or an antigen-binding fragment thereof that binds to human FcγRIIb, and the antibody or the antigen-binding fragment thereof comprises: (a) a heavy chain variable region VH comprising VH CDR1, VH CDR2 and VH CDR3 identical to those in SEQ ID NO: 12; and (b) a light chain variable region, VL, comprising VL CDR1, VL CDR2 and VL CDR3 identical to those in SEQ ID NO: 25; 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.

12. The use according to claim 11, wherein the antibody molecule is a CD20 antibody.

13. The use according to claim 12, wherein the CD20 antibody is a type I CD20 antibody. The use according to claim 11 , wherein the target cell is a cancer cell.

15. The use according to claim 11, wherein the target cell is a B cell.

16. The use according to claim 11, wherein the non-Hodgkin's lymphoma is selected from follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

17. The use according to claim 11, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:

12.

18. The use according to claim 11, wherein the VL consists of the amino acid sequence shown in SEQ ID NO:

25.

19. The use according to claim 11, further comprising a heavy chain constant region CH as shown in SEQ ID NO:

1.

20. The use according to claim 11, further comprising a light chain constant region CL as shown in SEQ ID NO:2.

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