Combination of fcγriib (cd32b) and cd20 specific antibodies

Abstract

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

Description

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

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

[0003] In addition, the present invention also relates to a method for predicting the response of a target cell to an antibody-based therapy, in particular, a method for predicting the response of a target cell to an antibody-based therapy when the antibody ligand is sensitive to FcγRIIb-mediated internalization, and in particular to the use of FcγRIIb expression levels and / or internalization of therapeutic antibodies mediated by FcγRIIb as prognostic markers of the response of the target cell to the therapy. 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 (such as the current market leader, rituximab) work by binding to CD20 molecules on the surface of B cells via the mAb's antigen-binding domain and eliminating these target B cells. They do this by recruiting and activating these effector cells, which interact with the mAb's Fc domain through Fcγ receptors (FcγRs) expressed on their surface.

[0006] The anti-CD20 monoclonal antibody (mAb) rituximab improves overall survival (OS) in patients with follicular (FL) lymphoma and diffuse large B-cell lymphoma (DLBCL) (1-4). In mantle cell lymphoma (MCL), only a mild response has been observed (5), while in chronic lymphocytic leukemia (CLL), the responses generated by the initial single-agent rituximab trials were not as dramatic as those generated in other non-Hodgkin's lymphomas (NHL) (reviewed in (6)). A fraction of lymphomas shows primary resistance to rituximab, or eventually develops resistance to combination therapies containing rituximab (7). The molecular basis behind this therapeutic resistance and the observed sensitivity of different NHL subtypes to rituximab treatment is currently unknown, but can include CD20 expression levels (8-10), high expression of complement defense molecules (CD55 and CD59) (11, 12), formation of resistance to apoptosis (13), and suboptimal Fcγ receptor (FcγR) interaction due to expression of low-affinity alleles (14).

[0007] It is highly desirable to improve the effectiveness of these antibodies in cases of poor or apparent resistance to treatment.

[0008] It is generally accepted that the Fc:FcγR interaction is critical for the efficacy of anti-CD-20 mAbs (15-18). In line with this, lymphoma patients carrying the higher affinity 158V allele in FcγRIIIa respond better to rituximab than those with the low affinity 158F allotype (14), leading many researchers to focus on improving the mAb interaction with FcγRIIIa, for example by defucosylation (19). In contrast, less attention has been given to the inhibitory FcγRIIb, which acts as a negative regulator of stimulatory activities 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 leads to inhibition of cytotoxic activity (15).

[0009] In B cell malignancies, FcyRIIb is expressed on CLL / SLL, MCL and FL, the latter especially during transformation. In DLBCL, FcyRIIb expression is weak, explaining the lack of correlation between its expression and response to rituximab-CHOP (R-CHOP) chemotherapy (20, 21). With respect to activating FcyRs, polymorphisms affecting FcyRIIb activity were also found, with the 232I allele being more efficient than the 232T allele in inhibiting BCR-mediated calcium flux (22, 23). However, Weng and Levy failed to establish a correlation between these polymorphisms and response to rituximab therapy in FL patients.

[0010] The number of anti-CD-20 mAbs available for clinical studies is increasing. These different anti-CD20 mAbs are classified as type I (e.g. rituximab, ofatumumab) or type II (e.g. tositumomab (B1), GA101, 11B8) (25-27) according to their ability to redistribute CD20 in the plasma membrane and their activity in different effector cell assays.

[0011] The inventors and others have demonstrated that type II mAbs are more efficient in depleting B cell targets in many model systems (18, 19). For example, in a CD20 transgenic (Tg) model of human normal B cell depletion in which the higher capacity of type II mAbs to trigger lysosomal cell death is not apparent (25, 27), the inventors demonstrated that this efficiency is related to their resistance to internalization (28). This is in contrast to type I mAbs such as rituximab, which rapidly internalize from the cell surface with CD20 in a process that is energy- and temperature-dependent and involves actin redistribution (28). The rate of modulation on cells from different origins (primary tumors versus cell lines, CLL versus FL) varies significantly, but the molecular basis for this is still unexplained.

[0012] WO 2008 / 002933 describes FcyRIIb (CD32B) and CD20 specific antibodies and methods of using a combination of these two antibodies to treat a B cell related disease or disorder. However, there is no teaching or suggestion to identify and / or treat a subset of patients, i.e. those patients having elevated levels of FcyRIIb expression on target cells, or those patients for whom the type of antibody is suitable for combination therapy with a FcyRIIb antibody. SUMMARY

[0013] Surprisingly, the inventors demonstrate that modulation by cell subtype is significantly correlated with surface expression of FcγRIIb, and that overexpression can convert Ramos cells from slow to fast modulators. Internalization of FcγRIIb occurs with CD20 and precedes its activation. All of these data provide a clear molecular rationale for the heterogeneity in the rate of modulation previously observed within and between different NHL subtypes.

[0014] Thus, the inventors demonstrate that the key factor determining the effectiveness of an antibody against an antigen 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 same cell surface, which is surprising. This interaction leads to internalization of the target cell for the antibody, thereby removing its ability to interact with effector cell Fc receptors. The inventors further demonstrate that an agent such as an anti-CD32 mAb can block this internalization. The inventors also demonstrate that such an agent can be used in combination with an antibody (e.g. rituximab) for targeting a cell surface antigen and improving its activity in vivo for the clearance of normal B cells or tumour cells.

[0015] The present application provides a composition comprising:

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

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

[0018] characterized in that the composition is for use in the treatment of a patient having target cells with elevated levels of FcγRIIb expression.

[0019] According to another aspect, the present application provides the use of an agent that inhibits or reduces 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 in the manufacture of a medicament for the treatment of a patient having target cells with elevated levels of FcγRIIb expression.

[0020] According to a further aspect, the present application provides a method of treating a patient having target cells expressing FcyRIIb, the method comprising administering in combination: (i) an antibody molecule which specifically binds to a surface antigen of the target cells, the antibody molecule having an Fc domain capable of binding to FcyRIIb; and (ii) an agent which inhibits or reduces binding between the Fc domain of the antibody molecule and FcyRIIb, characterised in that the patient is selected on the basis of elevated FcyRIIb expression levels on the target cells.

[0021] In certain embodiments of the composition, use or method of the application, the agent inhibits or reduces binding of FcyRIIb present on the target cells to the Fc domain of the antibody molecule.

[0022] According to a further aspect, the present application provides the use of FcyRIIb expression on target cells as a prognostic marker for the response of the target cells to treatment with an antibody molecule which specifically binds to a surface antigen of the target cells and which has an Fc domain capable of binding to FcyRIIb, whereby elevated levels of FcyRIIb indicate a reduced or no response to treatment with the antibody molecule.

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

[0024] According to a further aspect, the present application provides a method for predicting the response of target cells of a patient to treatment with an antibody molecule which specifically binds to a surface antigen of the target cells and which has an Fc domain capable of binding to FcyRIIb, the method comprising determining the level of FcyRIIb expression on the target cells, whereby elevated levels of FcyRIIb indicate a reduced or no response to treatment with the antibody molecule.

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

[0026] It has been demonstrated that, although all antibodies have an Fc binding antibody constant domain and known Fcy receptor binding capacity, not all antibodies are internalised in an FcyRIIb dependent manner, which makes the identification of suitable antibodies crucial for the success of combination therapy comprising an FcyRIIb function modulator and for avoiding treatment which is not beneficial to the patient.

[0027] In certain embodiments of the composition, use or method of the application, the antibody molecule that specifically binds to a cell surface antigen of a target cell is also capable of being internalized into said cell in an FcγRIIb-dependent manner, wherein said antibody has an Fc domain capable of binding FcγRIIb.

[0028] In certain embodiments of the composition, use or method of the application, the agent that inhibits or reduces the binding of FcγRIIb to the Fc domain of the antibody molecule also inhibits or reduces the further internalization of the antibody molecule into said cell.

[0029] In certain embodiments of the composition, use or method of the application, the target cell is a cancer cell. Conveniently, the target cell is a B cell.

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

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

[0032] The application also teaches how to identify antibodies suitable for combination therapy with an FcγRIIb antibody, i.e. those antibodies that are internalized by the target cell surface in an FcγRIIb-dependent manner. The application also provides for identifying a patient subset suitable for combination therapy with an FcγRIIb antibody.

[0033] In another aspect, the application provides an assay method for identifying an agent that reduces or inhibits the binding between an Fc domain of an antibody directed against a target cell surface antigen and FcγRIIb on the target cell, said assay method comprising determining the extent of binding between the Fc domain and FcγRIIb in the presence and absence of the 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 method can also be used to identify which agents (e.g. antibody molecules) are suitable for combination therapy with an anti-FcγRIIb antibody.

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

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

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

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

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

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

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

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

[0042] Conveniently, the test reagent 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 application, the reagent (ii) is one or more antibody molecules that specifically bind FcyRIIb. Conveniently, the one or more antibody molecules do not comprise a domain capable of recruiting effector cells.

[0044] Conveniently, the one or more antibody molecules is one or more monoclonal antibody molecules.

[0045] Preferably, the reagent inhibits or reduces FcyRIIb signalling. Even more preferably, the reagent inhibits or reduces the internalisation of the antibody molecule by the target cell.

[0046] In the following embodiments, SEQ ID NO refers to the sequences shown in the following Clones 1-13.

[0047] It will be known to the skilled person that there are three complementarity determining regions (CDRs) on the variable region 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 Ed. NIH Publication 91-3242, pages xv-xvii.

[0048] Those skilled in the art will know that there are other methods for assigning amino acids to the various CDRs. For example, the International ImMunoGeneTics information system (IMGT®) (Lefranc, M.-P. et al. (2003) Dev. Comp. Immunol. 31 : 139-158) and Lefranc "The Immunoglobulin FactsBook" published by Academic Press, 2001. http: / / www.imgt.org /

[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 and SEQ ID NO: 102 and SEQ ID NO: 103.

[0063] Preferably, the agent comprises a light chain variable region (VL) comprising the 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 and SEQ ID NO: 105 and SEQ ID NO: 106.

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

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

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

[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 sequences:

[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 agent of the application further comprises a constant region (CH) and (CL) of SEQ ID NO 1 and SEQ ID NO 2.

[0108] In a further embodiment, the agent is capable of competing with an agent described herein, such as an agent of the application comprising an amino acid sequence listed in the above embodiments (e.g. SEQ ID NO: 1-106), for inhibiting or reducing the binding of FcyRIIb to the Fc domain of the antibody molecule.

[0109] By "capable of competing with an agent described herein, such as an antigenic molecule, for inhibiting or reducing the binding of FcyRIIb to the Fc domain of the antibody molecule" as described herein, it is meant that the tested agent is capable of at least partially inhibiting or interfering with the binding of an agent described herein to FcyRIIb and inhibiting or reducing the binding of FcyRIIb to the Fc domain of the antibody molecule.

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

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

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

[0113] The agent of the present application can comprise the following constant regions (CH and CL):

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

[0115] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0116] γ-CL [SEQ ID NO: 2]

[0117] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0118] The agent of the present application can comprise one or more sequences of Clones 1-14:

[0119] Clone 1

[0120] VH [SEQ ID NO: 3]

[0121]

[0122] FrH1 CDRH1 FrH2 CDRH2

[0123]

[0124] FrH CDRH3 FrH4

[0125] -VL [SEQ ID NO: 16]

[0126]

[0127] FrL1 CDRL1 FrL2 CDRL2 FrL3

[0128]

[0129] CDRL3 FrL4

[0130] CDR regions

[0131] CDRH1:NYGMH [SEQ ID NO: 29]

[0132] CDRH2:VISYDGSNKYYADSVKG [SEQ ID NO: 30]

[0133] CDRH3:EWRDAFDI [SEQ ID NO: 31]

[0134] CDRL1:TGSSSNIGAGYDVH [SEQ ID NO: 32]

[0135] CDRL2:SDNQRPS [SEQ ID NO: 33]

[0136] CDRL3:AAWDDSLSGSWV [SEQ ID NO: 34]

[0137] Clone 2

[0138] -VH [SEQ ID NO: 4]

[0139]

[0140] -VL [SEQ ID NO: 17]

[0141]

[0142] CDR regions

[0143] CDRH1:TYGMH [SEQ ID NO: 35]

[0144] CDRH2:VIAYDGSKKDYADSVKG [SEQ ID NO: 36]

[0145] CDRH3:EYRDAFDI [SEQ ID NO: 37]

[0146] CDRL1:TGSSSNIGAGYDVH [SEQ ID NO: 38]

[0147] CDRL2: GNSNRPS [SEQ ID NO: 39]

[0148] CDRL3: AAWDDSVSGWM [SEQ ID NO: 40]

[0149] Clone 3

[0150] -VH [SEQ ID NO: 5]

[0151] QGTLVTVSS

[0152] -VL [SEQ ID NO: 18]

[0153]

[0154] CDR regions

[0155] CDRH1: NYGMH [SEQ ID NO: 41]

[0156] CDRH2: VISYDGSNRYYADSVKG [SEQ ID NO: 42]

[0157] CDRH3: DRWNGMDV [SEQ ID NO: 43]

[0158] CDRL1: SGSSSNIGAGYDVH [SEQ ID NO: 44]

[0159] CDRL2: ANNQRPS [SEQ ID NO: 45]

[0160] CDRL3: AAWDDSLNGPWV [SEQ ID NO: 46]

[0161] Clone 4

[0162] -VH [SEQ ID NO: 6]

[0163]

[0164] -VL [SEQ ID NO: 19]

[0165]

[0166] CDR regions

[0167] CDRH1:SYGMH[SEQ ID NO: 47]

[0168] CDRH2:VISYDGSDTAYADSVKG[SEQ ID NO: 48]

[0169] CDRH3:DHSVIGAFDI[SEQ ID NO: 49]

[0170] CDRL1:SGSSSNIGSNTVN[SEQ ID NO: 50]

[0171] CDRL2:DNNKRPS[SEQ ID NO: 51]

[0172] CDRL3:SSYAGSNNVV[SEQ ID NO: 52]

[0173] Clone 5

[0174] -VH [SEQ ID NO: 7]

[0175]

[0176] -VL [SEQ ID NO: 20]

[0177]

[0178] CDR regions

[0179] CDRH1:NYGMH[SEQ ID NO: 53]

[0180] CDRH2:VISYDGSNKYYADSVKG[SEQ ID NO: 54]

[0181] CDRH3:DQLGEAFDI[SEQ ID NO: 55]

[0182] CDRL1:TGSSSNIGAGYDVH[SEQ ID NO: 56]

[0183] CDRL2:DNNKRPS[SEQ ID NO: 57]

[0184] CDRL3:ATWDDSLSGPV[SEQ ID NO: 58]

[0185] Clone 6

[0186] -VH [SEQ ID NO: 8]

[0187]

[0188] -VL [SEQ ID NO: 21]

[0189]

[0190] CDR regions

[0191] CDRH1:DYGMS [SEQ ID NO: 59]

[0192] CDRH2:AISGSGSSTYYADSVKG [SEQ ID NO: 60]

[0193] CDRH3:GDIDYFDY [SEQ ID NO: 61]

[0194] CDRL1:TGSSSNFGAGYDVH [SEQ ID NO: 62]

[0195] CDRL2:ENNKRPS [SEQ ID NO: 63]

[0196] CDRL3:AAWDDSLNGPV [SEQ ID NO: 64]

[0197] Clone 7

[0198] -VH [SEQ ID NO: 9]

[0199]

[0200] -VL [SEQ ID NO: 22]

[0201]

[0202] CDR regions

[0203] CDRH1:SYGMH [SEQ ID NO: 65]

[0204] CDRH2:VISYDGSNKYYADSVKG [SEQ ID NO: 66]

[0205] CDRH3:ERRDAFDI [SEQ ID NO: 67]

[0206] CDRL1:TGSSSNIGAGYDVH [SEQ ID NO: 68]

[0207] CDRL2: SDNQRPS [SEQ ID NO: 69]

[0208] CDRL3: ATWDSDTPV [SEQ ID NO: 70]

[0209] Clone 8

[0210] -VH [SEQ ID NO: 10]

[0211]

[0212] -VL [SEQ ID NO: 23]

[0213]

[0214] CDR regions

[0215] CDRH1: SYGMH [SEQ ID NO: 71]

[0216] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 72]

[0217] CDRH3: DHSAAGYFDY [SEQ ID NO: 73]

[0218] CDRL1: SGSSSNIGSNTVN [SEQ ID NO: 74]

[0219] CDRL2: GNSIRPS [SEQ ID NO: 75]

[0220] CDRL3: ASWDDSLSSPV [SEQ ID NO: 76]

[0221] Clone 9

[0222] -VH [SEQ ID NO: 11]

[0223]

[0224] -VL [SEQ ID NO: 24]

[0225]

[0226] CDR regions

[0227] CDRH1: SYGMH [SEQ ID NO: 77]

[0228] CDRH2: GISWDSAIIDYAGSVKG [SEQ ID NO: 78]

[0229] CDRH3: DEAAAGAFDI [SEQ ID NO: 79]

[0230] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 80]

[0231] CDRL2: GNTDRPS [SEQ ID NO: 81]

[0232] CDRL3: AAWDDSLSGPVV [SEQ ID NO: 82]

[0233] Clone 13

[0234] - VL [SEQ ID NO: 28]

[0235]

[0236] - VL [SEQ ID NO: 28]

[0237]

[0238] CDR regions

[0239] CDRH1: SYGIS [SEQ ID NO: 101]

[0240] CDRH2: GISGSGGNTYYADSVKG [SEQ ID NO: 102]

[0241] CDRH3: SVGAYANDAFDI [SEQ ID NO: 103]

[0242] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 104]

[0243] CDRL2: GDTNRPS [SEQ ID NO: 105]

[0244] CDRL3: AAWDDSLNGPV [SEQ ID NO: 106]

[0245] Clone 10

[0246] - VL [SEQ ID NO: 28]

[0247]

[0248] VL [SEQ ID NO: 25]

[0249]

[0250] CDR regions

[0251] CDRH1: SYGMH [SEQ ID NO: 83]

[0252] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 84]

[0253] CDRH3: ELYDAFDI [SEQ ID NO: 85]

[0254] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 86]

[0255] CDRL2: ADDHRPS [SEQ ID NO: 87]

[0256] CDRL3: ASWDDSQRAVI [SEQ ID NO: 88]

[0257] Clone 11

[0258] VH [SEQ ID NO: 13]

[0259]

[0260] VL [SEQ ID NO: 26]

[0261]

[0262] CDR regions

[0263] CDRH1: SYGMH [SEQ ID NO: 89]

[0264] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 90]

[0265] CDRH3: EFGYIILDY [SEQ ID NO: 91]

[0266] CDRL1: SGSSSNIGSNTVN [SEQ ID NO: 92]

[0267] CDRL2: RDYERPS [SEQ ID NO: 93]

[0268] CDRL3: MAWDDSLSGVV [SEQ ID NO: 94]

[0269] Clone 12

[0270] - VL [SEQ ID NO: 27]

[0271]

[0272] - VL [SEQ ID NO: 27]

[0273]

[0274] CDR regions

[0275] CDRH1: NHGMH [SEQ ID NO: 95]

[0276] CDRH2: VISYDGTNKYYADSVRG [SEQ ID NO: 96]

[0277] CDRH3: ETWDAFDV [SEQ ID NO: 97]

[0278] CDRL1: SGSSSNIGSNNAN [SEQ ID NO: 98]

[0279] CDRL2: DNNKRPS [SEQ ID NO: 99]

[0280] CDRL3: QAWDSSTVV [SEQ ID NO: 100]

[0281] Preferred target cell surface antigens can be selected from the following antigens: CD20, Thy-1 (CD90, Cluster of Differentiation 90 (Biofactors. 2009 May-Jun; 35(3):258-65)); Ly-6 (Lymphocyte Antigen 6 (Mol Biol Rep. 2009 Apr; 36(4):697-703)); CD59 (Complement Regulatory Protein (Mol Immunol. 2007 Jan; 44(1-3):73-81)); Fas (FS7-Related Cell Surface Antigen, CD95, APO-1 or TNFRSF6 (Adv Exp Med Biol. 2009; 647:64-93)); EGFR (Epidermal Growth Factor Receptor (FEBS J. 2010 Jan; 277(2):301-8)); Her2 (Human Epidermal Growth Factor Receptor 2 (Clin Breast Cancer. 2008 Oct; 8(5):392-401)); CXCR4 (Chemokine Receptor 4 (Biochim Biophys Acta. 2007 Apr; 1768(4):952-63)); CD19 (Cluster of Differentiation 19 (Cell Immunol. 1989 Feb; 118(2):368-81)); CD40 (Cluster of Differentiation 40 (Basic Clin Pharmacol Toxicol. 2009 Feb; 104(2):87-92)); HLA molecules (Human Leukocyte Antigen molecules (Korean J Lab Med. 2010 Jun; 30(3):203)); GM1 (Ganglioside, Monosialyl Tetrahexosyl Ganglioside (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).

[0282] Preferably, in the composition, use or method of the application, the surface antigen is selected from CD19, CD20, or CD40, more preferably in human form. CD20, especially human CD20, is most preferred.

[0283] 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 and internalized into the target cell in an FcγRIIb-dependent manner when bound to the target cell. Preferably, the monoclonal antibody is an anti-CD 19 antibody, an anti-CD20 antibody, or an anti-CD40 antibody. Most preferably, the monoclonal antibody is an anti-CD20 monoclonal antibody.

[0284] 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.

[0285] In one embodiment, the cell surface antigen is CD20 and the antibody molecule that specifically binds to a cell surface antigen is a type I antibody.

[0286] As noted 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 two different groups (43 and 25), and later in 2004 as (26) type I and type II mAbs. Initially, the basis for this was that anti-CD20 mAbs belonged to two different types of agents based on their ability to eliminate lymphoma xenografts: type I (e.g., rituximab and 1F5) utilized complement, and type II (e.g., Bl) did not. Both types of mAbs produced a significant prolongation of survival time, but by administering CVF to destroy complement activity, the efficacy of rituximab and 1F5 was significantly reduced, but there was no effect on the activity of Bl. These results clearly demonstrated that different CD20 mAbs operate different effector mechanisms in vivo. In addition, and in complete agreement with previous work, these results showed that rituximab and 1F5 were able to efficiently activate complement due to their ability to translocate CD20 into the lipid rafts of the target cell membrane, whereas Bl type mAbs were not able to do this (43). There was a good correlation with the ability of mAbs to engage complement and induce CD20 to move into the lipid rafts (43, 26). Thus, type I and type II can be defined essentially by their ability to translocate CD20 into the lipid rafts. This can be determined as described below. This also correlates with type II mAbs being able to initiate more efficient homotypic adhesion and direct cell death, but these cannot be used alone to define type I or type II mAbs (unlike the Tx-100 raft assay, see below).

[0287] Thus, according to the ability of these different anti-CD20 mAbs to redistribute CD20 in the plasma membrane and their activity in different effector cell assays, they are classified as type I (e.g. rituximab, ofatumumab) or type II (e.g. tositumomab (Bl), GA101, 11B8) (25-27). Type I (e.g. rituximab, ofatumumab) anti-CD20 monoclonal antibodies induce CD20 redistribution to large detergent-resistant microdomains (pores), while type II (e.g. tositumomab) anti-CD20 monoclonal antibodies are not able to achieve this (50).

[0288] As mentioned above, anti-CD20 mAbs are defined as type I or type II according to whether they redistribute CD20 into lipidic pores. This is achieved by the Tx-100 insolubility assay or by sucrose density gradient separation and Western blot. Both methods are described in Cragg et al Blood 2003 (43) as follows:

[0289] 1. Evaluation of pore-associated antigens by Triton X-100 insolubility

[0290] A rapid evaluation of the presence of antigens in the pore microdomains is performed at low temperature using a flow cytometry method based on Triton X-100 insolubility. Briefly, cells are washed in RPMI / 1 % BSA and resuspended at 2.5 x 10 6 / ml. Then, cells are incubated with 10 pg / ml of FITC-conjugated mAb for 15 min at 37 o C, washed in cold PBS / 1 % BSA / 20 mM sodium azide and the sample is then divided in two. One half is maintained on ice to calculate the 100 % surface antigen level, while the other half is treated with 0.5 % Triton X-100 for 15 min on ice to determine the proportion of antigens remaining in the insoluble pore fraction. Then, cells are maintained at 4 oC, 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, cells were first treated with 0.5% Triton X-100 for 15 minutes on ice and washed in PBS / BSA / azide before binding of FITC-labelled mAb. To assess whether more antigen could be shifted into a Triton-X 100 insoluble fraction by additional cross-linking, cells were incubated with FITC-mAb, washed and then divided into four aliquots as before. Two of these samples were incubated with goat anti-mouse Ig F(ab')2 fragments for 15 minutes on ice. After washing, one of the cross-linked samples and one of the non-cross-linked samples were lysed in Triton X-100 and washed as detailed above prior to flow cytometry.

[0291] 2. Sucrose density gradient separation and Western blotting - Lipid valve fraction preparation and Western blotting

[0292] Monoclonal Ab (1 μg / 10 6 cells) was added to cells at 37 o C. After a 20 minute incubation, cells were pelleted and lysed in ice-cold MES buffered saline containing 1.0% Triton X-100 (25 mM MES, pH 6.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 5 µ g / ml aprotinin, 5 μg / ml leupeptin, 10 mM EDTA). Lipid valve fractions were then prepared by sucrose density gradient centrifugation. Briefly, lysates were mixed with an equal volume of lysis buffer containing 80% sucrose, overlaid with a discontinuous 5-30% sucrose density gradient and then centrifuged at 200,000 x g for 16 h. Fractions (0.5 ml) were collected and analysed by Western blotting. A 15 ml sample of each fraction was diluted 1 : 1 in 2 x loading buffer, heated to 95 o C for 5 minutes and separated on a 15% SDS-PAGE gel before transfer to a PVDF membrane, incubation with primary antibody (e.g. mouse anti-CD20 antibody, clone 7D1 to detect CD20 or anti-Lyn rabbit polyclonal serum to identify valve fractions; Serotec, UK) and then with HRP-conjugated secondary antibody (Amersham Biosciences UK Ltd). Blots were visualised using ECL+plus (Amersham Biosciences UK Ltd).

[0293] Anti-CD20 mAbs can require the AxP motif in the CD20 large loop. (Ofatumumab and other Genmab antibodies do not). However, (Niederfelner et al. (51)) showed that type II mAbs have a slightly different binding region in the CD20 loop compared to type I mAbs.

[0294] Preferably, in the composition, use or method of the application, the target cell is a cancer cell. More preferably, the cancer is selected from non-Hodgkin's lymphoma, including but not limited to follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

[0295] In one embodiment, the application provides a composition, use and method for treating cancer, particularly a B-cell malignancy, preferably selected from lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, multiple lymphoma, Hodgkin's disease and non-Hodgkin's disease, diffuse large B-cell lymphoma, follicular lymphoma with diffuse large B-cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, diffuse small cleaved cell lymphoma or a combination thereof. In certain embodiments, the B-cell malignancy is a lymphoma, such as non-Hodgkin's lymphoma (NHL).

[0296] In another embodiment, the application provides a composition, use and method for treating. The inflammatory disease can be an autoimmune disease, such as Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type I diabetes mellitus, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, autoimmune inner ear disease, myasthenia gravis, Reiter's syndrome, Guillain-Barre disease, autoimmune hepatitis, familial polyglandular

[0297] In preferred embodiments, the disease treated includes chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), B-cell malignancy, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, systemic sclerosis and autoimmune blistering disease.

[0298] In one preferred embodiment, the treatment enhanced by the use of the application is treatment with an anti-CD20 mAb, such as rituximab.

[0299] Definitions

[0300] "Up-regulated" includes the meaning that the cell in question expresses a higher level of FcyRIIb on its surface as compared to a control or reference cell that expresses a low or intermediate level of FcyRIIb on its surface. For example, if the cell in question is a B cell, its FcyRIIb expression level is considered "up-regulated" if it is higher than the normal (preferably intermediate) level of FcyRIIb expression of a B cell of the same cell type. Alternatively, the expression level of the cell in question is considered "up-regulated" if it is higher than the level of expression of a different cell type that expresses FcyRIIb at a low or intermediate level.

[0301] According to the present application, the higher the degree of up-regulation of FcyRIIb expression by a target cell, the poorer the response of these cells is expected to be to treatment with an antibody molecule that specifically binds to an antigen on the surface of the target cell and has an Fc domain capable of binding to FcyRIIb. Thus, for example, Figure 2 The more up-regulated FcyRIIb expression is, the greater the benefit from using an agent of the present application that inhibits or reduces the binding of the Fc domain to FcyRIIb, as shown in Figures D and 3A. The level of FcyRIIb expression was measured by IHC (Figures 1C and 10D) and FACS (Figures 2C and 9C). Figure 10 b) and after separating MCL samples into FcyRIIb positive and negative, a clear difference in clinical response was observed after treatment with rituximab (Figures 8B and 9B). Figure 10 c and 10d).

[0302] The skilled person can readily determine the level of FcyRIIb expression on a cell by a variety of known methods, for example by the flow cytometry and immunohistochemical staining methods described in the Examples and Figures.

[0303] The skilled person will understand that the levels of expression of FcyRIIb that are "normal" and "up-regulated" will differ between different cell types and different disease states, and that the skilled person is able to identify the levels of expression of FcyRIIb that are "normal" and "up-regulated" for a given target cell or disease state using methods known in the art and described herein. Figure 2Exemplary levels of "normal" (or intermediate) and "elevated" expression levels of FcyRIIb on certain cell types are provided in C. In these particular examples, in follicular lymphoma (FL), the "normal" level is about 50 (ratio of geometric MFI FcyRIIb 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.

[0304] Preferably, the elevated FcyRIIb expression level is increased at least 1.1 fold relative to the normal (preferably intermediate) expression level of cells of the same cell type (or cells of a different cell type), or at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 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 fold or more relative to the normal (or intermediate) expression level of cells of the same cell type or of a different cell type. Preferably, they are increased at least 1.8 fold relative to the normal (or intermediate) expression level of cells of the same cell type or of a different cell type.

[0305] As shown in the accompanying examples, the inventors have determined a correlation between FcyRIIb expression levels and increased modulation of mAb from the cell surface. The higher the FcyRIIb expression level, the greater the modulation produced, i.e. a correlation between the degree of expression increase and modulation, as shown in Figure 2 D and 3A. In Figure 2 D, the FcyRIIb expression level was plotted against the level of modulation observed. The correlation is that the highest levels of FcyRIIb (e.g. > 400) result in the lowest levels of surface CD20 (< 20%) i.e. the greatest effect on modulation of mAb from the cell surface. In Figure 3 A, low (18), medium (70) and high (124) levels of FcyRIIb were introduced into a FcyRIIb negative cell line (Ramos) and this directly correlates with a decrease in cell surface CD20 levels proportional to the expression of FcyRIIb (60%, 40%, 30% for low, medium and high levels). The FcyRIIb expression level is expressed as the ratio of the geometric mean fluorescence intensity (geoMFI) of FcyRIIb to isotype control.

[0306] Modulation reduces the amount of mAb left on the cell surface. mAb require Fc to engage immune effector cell mechanisms (ADCC, ADCP, CDC) to clear target cells. Therefore, using a FcyRIIb blocking mAb (mAb that blocks FcyRIIb) to reduce modulation will improve Fc dependent effector cell function. This is shown in Figure 8 as ADCP (phagocytosis). Phagocytosis with rituximab alone is 40% but increases to 55% when FcyRIIb is blocked by AT10.

[0307] "Antibody molecule" includes a monoclonal antibody, a synthetic antibody, a recombinantly produced antibody, a multispecific antibody, a human antibody, a chimeric antibody, a camelized antibody, a single chain Fv (scFv), a single chain antibody, a Fab fragment, a F(ab') fragment, a disulfide linked Fv (sdFv), an intrabody, or an epitope binding fragment of any of the above. Preferably, the antibody of the application is a monoclonal antibody, more preferably a humanized or human antibody.

[0308] Methods for preparing and characterizing antibody molecules useful in the compositions, uses, and methods of the application are well known to those of skill in the art. For example, WO 2008 / 002933 (Section 5.3 to 5.3.1, pages 74-91) describes the preparation and characterization of monoclonal antibodies that specifically bind to a target cell surface antigen (e.g., CD20 or FcyRIIb). Useful antibodies that specifically bind FcyRIIb and CD20, including monoclonal antibodies produced by hybridomas deposited under the Budapest Treaty, are also disclosed in WO 2008 / 002933, pages 15-21, the contents of which are incorporated herein by reference.

[0309] "Specifically binds" includes agents such as antibody molecules that bind to a target antigen but do not bind to (are not cross-reactive with) or bind with less affinity to other antigens, i.e., with lower affinity than to the target antigen. For example, an antibody that specifically binds FcyRIIb can bind other peptides or polypeptides with lower affinity, as determined by, e.g., immunoassays, BIAcore, or other assays known in the art. Preferably, an antibody or fragment thereof that specifically binds FcyRIIb does not cross-react with other antigens. An antibody that specifically binds FcyRIIb can be identified by, e.g., immunoassays, BIAcore, or other assays known to those of skill in the art. An antibody or fragment thereof specifically binds FcyRIIb when its affinity for FcyRIIb is higher than its affinity for binding to any cross-reactive antigens (see, Fundamental Immunology Second Edition, Raven Press, New York, pages 332-336 (1989) for a discussion of antibody specificity) and when its affinity for FcyRIIb, particularly human FcyRIIb, more particularly native human FcyRIIb, is greater than its affinity for FcyRIIA, particularly human FcyRIIA, more particularly native human FcyRIIA. Representative antibodies are disclosed in U.S. Patent Applications 2004-0185045, 2005-0260213, and 2006-0013810, which are incorporated by reference herein in their entireties.

[0310] Preferably, certain FcγRIIb antibodies used in combination with CD20 antibodies for the compositions and methods of the application bind to the extracellular domain of native human FcγRIIb. In some embodiments, the antibody or fragment thereof binds FcγRIIb with an affinity that is at least two-fold greater than the affinity with which the antibody or fragment thereof binds FcγRIIA. In other embodiments, the antibody or fragment thereof binds FcγRIIb with an affinity that is at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 100-fold, at least 1000-fold, at least 10 4 -fold, at least 10 5 -fold, at least 10 6 -fold, at least 10 7 -fold, or at least 10 8 -fold greater than the affinity with which the antibody or fragment thereof binds FcγRIIA. In a preferred embodiment, the antibody or fragment thereof binds FcγRIIb with an affinity that is 100-fold, 1000-fold, 10 4 -fold, 10 5 -fold, 10 6 -fold, 10 7 -fold, or 10 8 -fold greater than the affinity with which the antibody or fragment thereof binds FcγRIIA.

[0311] The present application provides the following:

[0312] 1. A composition comprising:

[0313] (i) an antibody molecule that specifically binds a cell surface antigen of a target cell and has an Fc domain capable of binding FcγRIIb; and

[0314] (ii) an agent that inhibits or reduces binding of FcγRIIb to the Fc domain of the antibody molecule,

[0315] characterized in that the composition is for use in treating a patient having a target cell with elevated levels of FcγRIIb expression.

[0316] 2. Use of an agent that inhibits or reduces binding between a domain of an Fc of an antibody molecule and FcγRIIb on a target cell, wherein the antibody molecule specifically binds a surface antigen of the target cell, and characterized in that the use is in the manufacture of a medicament for treating a patient having a target cell with elevated levels of FcγRIIb expression.

[0317] 3. A method of treating a patient having target cells expressing FcγRIIb, the method comprising administering in combination: (i) an antibody molecule that specifically binds to a surface antigen of the target cells, the antibody molecule having an Fc domain capable of binding to FcγRIIb; and (ii) an agent that inhibits or reduces binding between the Fc domain of the antibody molecule and FcγRIIb, characterised in that the patient is selected on the basis of elevated FcγRIIb expression by the target cells of the patient.

[0318] 4. Use of FcγRIIb expression on target cells as a prognostic marker for the response of the target cells to treatment using an antibody molecule that specifically binds to a surface antigen of the target cells and that has an Fc domain capable of binding to FcγRIIb, whereby elevated levels of FcγRIIb indicate a reduced or no response to treatment using the antibody molecule.

[0319] 5. A method for predicting the response of target cells of a patient to treatment using an antibody molecule that specifically binds to a surface antigen of the target cells and that has an Fc domain capable of binding to FcγRIIb, characterised in that the method comprises determining the level of FcγRIIb expression on the target cells, whereby elevated levels of FcγRIIb indicate a reduced or no response to treatment using the antibody molecule.

[0320] 6. The composition, use or method according to any one of 1 to 3, wherein the agent inhibits or reduces binding of FcγRIIb on the target cells to the Fc domain of the antibody molecule.

[0321] 7. The composition, use or method according to any one of the preceding items, wherein the antibody molecule that specifically binds to a surface antigen of the target cells and that has an Fc domain capable of binding to FcγRIIb is capable of being internalised into the target cells in an FcγRIIb dependent manner.

[0322] 8. The composition, use or method according to any one of 1 to 3 and 6 to 7, wherein the agent that inhibits or reduces binding of FcγRIIb to the Fc domain of the antibody molecule also inhibits or reduces internalisation of the antibody molecule into the target cells.

[0323] 9. The composition, use or method according to any one of the preceding items, wherein the target cells are cancer cells.

[0324] 10. The composition, use or method according to any one of the preceding items, wherein the target cells are B cells.

[0325] 11. The composition, use or method according to any one of the preceding items, wherein the patient to be treated is a cancer patient and the treatment is cancer treatment.

[0326] 12. The composition, use or method according to any one of 9-11, wherein the cancer is selected from non-Hodgkin lymphoma, such as follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or chronic lymphocytic leukemia.

[0327] 13. The composition, use or method according to any one of the preceding items, wherein the agent is any one of: a polypeptide; an anticalin; a peptide; an antibody; a chimeric antibody; a single chain antibody; an aptamer; a darpin; a Fab, F(ab')2, Fv, ScFv or dAb antibody fragment; a small molecule; a natural product; an affimer; a peptidomimetic; a nucleic acid; a peptide nucleic acid molecule; a lipid; a carbohydrate; a modular framework-based protein, including ankyrin repeat proteins, armadillo repeat proteins, leucine-rich proteins, tetratricopeptide repeat proteins or designed ankyrin repeat proteins (DARPins).

[0328] 14. The composition, use or method according to any one of the preceding items, wherein the agent is one or more antibody molecules that specifically bind FcyRIIb.

[0329] 15. The composition, use or method according to 14, wherein the one or more antibody molecules do not comprise a domain capable of recruiting effector cells.

[0330] 16. The composition, use or method according to 15, wherein the one or more antibody molecules are monoclonal antibody molecules.

[0331] 17. The composition, use or method according to any one of 1-3 and 6-16, wherein the agent comprises a heavy chain variable region (VH) comprising the following CDRs:

[0332] (i) SEQ ID NO: 29 and SEQ ID NO: 30 and SEQ ID NO: 31; or

[0333] (ii) SEQ ID NO: 35 and SEQ ID NO: 36 and SEQ ID NO: 37; or

[0334] (iii) SEQ ID NO: 41 and SEQ ID NO: 42 and SEQ ID NO: 43; or

[0335] (iv) SEQ ID NO: 47 and SEQ ID NO: 48 and SEQ ID NO: 49; or

[0336] (v) SEQ ID NO: 53 and SEQ ID NO: 54 and SEQ ID NO: 55; or

[0337] (vi) SEQ ID NO: 59 and SEQ ID NO: 60 and SEQ ID NO: 61 ; or

[0338] (vii) SEQ ID NO: 65 and SEQ ID NO: 66 and SEQ ID NO: 67; or

[0339] (viii) SEQ ID NO: 71 and SEQ ID NO: 72 and SEQ ID NO: 73; or

[0340] (ix) SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79; or

[0341] (x) SEQ ID NO: 83 and SEQ ID NO: 84 and SEQ ID NO: 85; or

[0342] (xi) SEQ ID NO: 89 and SEQ ID NO: 90 and SEQ ID NO: 91 ; or

[0343] (xii) SEQ ID NO: 95 and SEQ ID NO: 96 and SEQ ID NO: 97; or

[0344] (xiii) SEQ ID NO: 101 and SEQ ID NO: 102 and SEQ ID NO: 103.

[0345] 18. The composition, use or method according to any one of 1-3 and 6-17, wherein the agent comprises a light chain variable region (VL) comprising the following CDRs:

[0346] (i) SEQ ID NO: 32 and SEQ ID NO: 33 and SEQ ID NO: 34; or

[0347] (ii) SEQ ID NO: 38 and SEQ ID NO: 39 and SEQ ID NO: 40; or

[0348] (iii) SEQ ID NO: 44 and SEQ ID NO: 45 and SEQ ID NO: 46; or

[0349] (iv) SEQ ID NO: 50 and SEQ ID NO: 51 and SEQ ID NO: 52; or

[0350] (v) SEQ ID NO: 56 and SEQ ID NO: 57 and SEQ ID NO: 58; or

[0351] (vi) SEQ ID NO: 62 and SEQ ID NO: 63 and SEQ ID NO: 64; or

[0352] (vii) SEQ ID NO: 68 and SEQ ID NO: 69 and SEQ ID NO: 70; or

[0353] (viii) SEQ ID NO: 74 and SEQ ID NO: 75 and SEQ ID NO: 76; or

[0354] (ix) SEQ ID NO: 80 and SEQ ID NO: 81 and SEQ ID NO: 82; or

[0355] (x) SEQ ID NO: 86 and SEQ ID NO: 87 and SEQ ID NO: 88; or

[0356] (xi) SEQ ID NO: 92 and SEQ ID NO: 93 and SEQ ID NO: 94; or

[0357] (xii) SEQ ID NO: 98 and SEQ ID NO: 99 and SEQ ID NO: 100; or

[0358] (xiii) SEQ ID NO: 104 and SEQ ID NO: 105 and SEQ ID NO: 106.

[0359] 19. The 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 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.

[0360] 20. The composition, use or method of any of 1-3 and 6-19, wherein the agent comprises a variable light chain (VL) amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

[0361] 21. The composition, use or method of any of 1-3 and 6-20, wherein the agent comprises the following CDR amino acid sequences:

[0362] (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

[0363] (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

[0364] (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

[0365] (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

[0366] (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

[0367] (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

[0368] (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

[0369] (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

[0370] (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

[0371] (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

[0372] (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

[0373] (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

[0374] (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.

[0375] 22. The composition, use or method of any of 1-3 and 6-21, wherein the agent comprises the following amino acid sequence:

[0376] (i) SEQ ID NO: 3 and SEQ ID NO: 16; or

[0377] (ii) SEQ IS NO: 4 and SEQ ID NO: 17; or

[0378] (iii) SEQ IS NO: 5 and SEQ ID NO: 18; or

[0379] (iv) SEQ ID NO: 6 and SEQ ID NO: 19; or

[0380] (v) SEQ ID NO: 7 and SEQ ID NO: 20; or

[0381] (vi) SEQ ID NO: 8 and SEQ ID NO: 21 ; or

[0382] (vii) SEQ ID NO: 9 and SEQ ID NO: 22; or

[0383] (viii) SEQ ID NO: 10 and SEQ ID NO: 23; or

[0384] (ix) SEQ ID NO: 11 and SEQ ID NO: 24; or

[0385] (x) SEQ ID NO: 12 and SEQ ID NO: 25; or

[0386] (xi) SEQ ID NO: 13 and SEQ ID NO: 26; or

[0387] (xii) SEQ ID NO: 14 and SEQ ID NO: 27; or

[0388] (xiii) SEQ ID NO: 15 and SEQ ID NO: 28.

[0389] 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 of 17-22 for binding to the Fc domain of the antibody molecule, thereby for inhibiting or reducing the binding of FcyRIIb to the Fc domain of the antibody molecule.

[0390] 24. The composition, use or method according to any one of 1-3 and 6-23, wherein the agent inhibits or reduces FcyRIIb signaling.

[0391] 25. The composition, use or method of any of 1-3 and 6-24, wherein the agent inhibits or reduces internalization of the antibody molecule by the target cell.

[0392] 26. The composition, use or method of any of the preceding, wherein the cell surface antigen is selected from CD19, CD20 or CD40.

[0393] 27. The composition, use or method of any of the preceding, wherein the antibody molecule that specifically binds a cell surface antigen is a CD20 antibody.

[0394] 28. The composition, use or method of 27, wherein the CD20 antibody is a type I CD20 antibody.

[0395] 29. The composition, use or method of any of the preceding, wherein the cell surface antigen is CD20 and the antibody molecule that specifically binds a cell surface antigen is a type I antibody.

[0396] 30. The composition, use or method of any of the preceding, wherein the elevated expression of FcyRIIb on the target cell is determined relative to a control, preferably the normal level of expression of FcyRIIb in a cell of the same type as the target cell.

[0397] 31. A composition substantially as described herein with reference to the description and figures.

[0398] 32. A use substantially as described herein with reference to the description and figures.

[0399] 33. A method substantially as described herein with reference to the description and figures. BRIEF DESCRIPTION OF DRAWINGS

[0400] Specific aspects of the application will now be described by way of example with reference to the accompanying drawings in which:

[0401] Figure 1 A- Figure 1 B: Type I mAb internalization from the cell surface of normal and malignant human B cells.

[0402] A) Primary CLL cells were incubated with Tosit-488, GA101gly-488, Ritux-488 or Ofatum-488 (all at 5 μg / ml) for 2 or 6 hours. Then, cells were collected and washed twice before an anti-Alexa-488 antibody was added to half of the samples and incubated for 1 hour at 4°C. Cells were then washed twice and analysed by flow cytometry. oC. Incubate for 30 minutes to distinguish internalized from non-internalized mAb. Calculate surface accessible CD20 (%) 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 Wilcoxon paired test (**p value < 0.001) and mean values are shown. B) Then Tosit-488 or Ritux-488 were 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 Mann Whitney test and mean values are shown.

[0403] Figure 1 C- Figure 1 G: Lack of correlation between CD20 modulation and CLL phenotype / prognostic markers. C) Correlation between modulation and known CLL prognostic factors. CLL cases were phenotyped for IgVH gene mutation status, Zap-70 and CD38 expression and these samples were subjected to the internalization assay to evaluate modulation as in Figure 1 A). Correlation between each prognostic feature and CD20 modulation was analyzed by Spearman correlation analysis. No correlation was observed with each prognostic factor (p>0.05). D) Similarly, sIg status, ability of cells to initiate calcium flux and viability of CLL cells were evaluated and compared to CD20 modulation. Again, no correlation was observed. E) CD20 expression of CLL cells was evaluated by FACS using Ritux-488 and compared to CD20 modulation. A weak correlation was observed (Spearman r value -0.34, p=0.038). Subsequent analysis of multivariate regression for CD20 modulation using CD20 and FcγRIIb expression showed that the weak correlation with CD20 was not significant (p=0.638). F) sIg expression of IgM positive CLL cases was determined by FACS and expression levels were compared to CD20 modulation. No correlation was found (p>0.05). G) CLL cells were incubated with Rit m2a-488 for 2 hours and the internalization assay was performed as in Figure 1 A). In single CLL cases, differences in CD38 expression can be seen. FACS plots show samples before (left) and after (right) quenching. Corresponding histograms show CD38 +ve and CD38 -ve cells modulate at the same rate. CD38 +ve and CD38 -ve cells are represented by solid and open peaks, respectively. These results represent 3 different cases.

[0404] Figure 2 Modulation is an Fc-dependent process. A) Internalization assay as described in la) was repeated after incubating CLL cells with Alexa-488 labeled Rituximab fragments Fab', F(ab')2, and IgG for 6 hours. Data represent the mean modulation levels + / - SD of 3 different CLL samples. B) CLL cells were incubated with Tosit-488, Ritux-488 + / - AT10 and Rit m2a-488 for 2 and 6 hours as in la). Mean values + / - modulation from 6 different CLL samples are shown. Addition of the anti-FcyRII mAb AT10 to Rituximab reduced CD20 modulation to a similar level as Rit m2a, while addition of AT10 to Rit m2a did not significantly differ from modulation. C) Various normal and malignant B cell samples were stained for FcyRIIb expression using AT10-PE. Histograms show the difference in FcyRIIb expression in 3 different CLL cases. High (black line), medium (dark grey line) and low expressing cells are indicated. Scatter plot shows the difference in FcyRIIb expression of healthy B cells, CLL, SLL, MCL, FL and SL BCL. FcyRIIb expression is expressed as FcyRIIb: isotype control geometric MFI ratio. Median values are shown. D) CD20 modulation and FcyRIIb expression were plotted for all NHL subtypes and normal B cells (obtained from the internalization assay described in la) incubated with Ritux-488 for 6 hours). Analysis was performed using the Spearman correlation assuming a non-parametric distribution. Strong correlation is shown. Spearman r value = -0.74, 95% confidence interval between -0.83 and -0.61, p<0.0001.

[0405] Figure 3 FcyRIIb expression is the main determinant for CD20 modulation. A) Ramos cells transfected with FcyRIIb were sorted for low, medium and high levels of FcyRIIb expression and evaluated at the 6 hour time point of the internalization assay using Tosit-488 and Ritux-488 together with the empty control transfected cells. Error bars represent the mean + / - SD from individual experiments. Geometric MFI values of FcyRIIb expression of the sorted cells are listed on the right. B) Internalization assay was repeated using Tosit-488 and Ritux-488 on normal Ramos cells, Rx3 cells (lacking BCR expression), empty control transfected Rx3 cells and FcyRIIb transfected Rx3 cells after 6 hours of incubation. Data points from 5 individual experiments are shown together with the median values.

[0406] Figure 4 : Co-ligation of CD20 and FcγRIIb occurs primarily in cis and leads to FcγRIIb activation. A) Raji cells were incubated with specific mAbs (10 μg / ml) at 37 o C for 2 hours, after which the cells were harvested, lysed and immunoblotted for phosphorylated FcγRIIb. B) Left: PKH26-labeled Ramos cells (FcγRIIb -ve , R1) were mixed with the sorted Ramos transfectants with high FcγRIIb expression at a ratio of 1:1 ( Figure 3 Described in A). Right: FcγRIIb after 6 hours incubation with Ritux-488 +ve and FcγRIIb -ve CD20 modulation on cells. As a control, the two cells were also incubated alone. The data represent the mean modulation level + / - SD from 3 independent experiments. C) In a similar experiment, low FcγRIIb expressing CLL was PKH26 labeled and then mixed with higher FcγRIIb expressing CLL at a 1:1 ratio. The experiment was performed three times, each time using a different high FcγRIIb expressing CLL. 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 mean modulation level + / - SD. D) Different CLL samples were plated at 20 x 10 5 , 4 x 10 5 and 1 x 10 5 E) Raji cells were cultured at 20 x 10 5 , 4 x 10 5 and 1 x 10 5 cells / ml and specific mAb (10 μg / ml) at 37 o C for 2 hours. Images were captured using a bright field microscope to demonstrate differences in cell proximity. Cells were then collected and evaluated for phosphorylated FcγRIIb by immunoblotting. Figure 4 As described in A.

[0407] Figure 5: Rituximab, CD20 and FcyRIIb are internalized together into lysosomes. A) CLL cells were incubated with Tosit-488 or Ritux-488 for 2 hours, then stained with anti-CD19-APC and AT10-PE. Data is shown as the mean + / - 1 SD of FcyRIIb expression as a percentage of untreated (n+6 CLL samples). FcyRIIb 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 confocal microscopy. This image represents FcyRIIb staining in unstimulated cells. C) The same CLL samples were incubated with Ritux-488 for 30 minutes, then prepared for microscopy as in 5B. In addition, cells were stained with biotinylated LAMP-1 and streptavidin-546 (red) to stain for lysosomes. Tosit-488 was uniformly retained on the surface, AT10-647 staining did not change relative to baseline as in 5B. There was no colocalization with LAMP-1. D) CLL cells were treated with Ritux-488 for 6 hours, and evaluated as in 5B. Here, two representative cells are shown. The top cell shows clear colocalization between Ritux-488 and AT10-647, but no colocalization with LAMP-1. The bottom cell shows colocalization of all three fluorescent dyes. In each case, a brightfield (BF) image from the same cell is shown. Scale bar represents 5 μm. Figure 5 Figure 5

[0408] Figure 6 : Lack of inhibitory receptors enhances the clearing capacity of anti-CD20 mAbs. hCD20 Tg mice (WT) or hCD20 Tg mice lacking CD32 (CD32KO) were treated with Rituximab carrying mouse IgGl (ml) or mouse IgG2a (m2a), 250 mg intravenously (iv), and B cell clearance was monitored for 90 days by serial bleeds from the mice and flow cytometry using B220 and CD19 mAbs staining.

[0409] Figure 7 ​​: Blocking the inhibitory receptor CD32b (FcγRIIb) enhances the efficacy of anti-CD20 mAbs in a human xenograft system. CD20-positive human tumor cells (Daudi or Raji) were inoculated into SCID mice and then treated with rituximab, AT10, or both, and mouse survival or tumor growth was monitored. The dose of mAb used is shown in the figure legend. In A), Daudi cells were inoculated simultaneously and tumors were monitored every 30 days by caliper. In B) and C), Raji cells were inoculated intravenously.

[0410] Figure 8 Phagocytosis of CLL cells treated with rituximab was enhanced by co-incubation with an FcγRII blocking mAb. Monocytes were derived from healthy volunteers and differentiated into macrophages using M-CSF in 6-well plates for at least 7 days before use. Macrophages were then harvested and plated in 96-well plates at 5 x 10 5 Cells were allowed to adhere to the CFSE-labeled CLL cells per well before addition. CFSE-labeled CLL cells were left untreated or opsonized with rituximab (ritux) and the FcγRIIb blocking mAb AT10 (fab')2 at 10 μg / ml for 15 minutes or 6 hours, then washed twice and added to macrophages (1:1 ratio) for at least 30 minutes. Then, anti-CD16 f(ab)2-APC (5 μg / ml) was added to each well for 15 minutes at room temperature (RT) to stain macrophages, and the wells were washed once with FACS wash (PBS BSA azide) at room temperature. Further ice-cold FACS wash was added, and the plates were incubated on ice for 10 minutes before being collected for FACS analysis. The % double-positive macrophages represents the percentage of CD16+CFSE+ positive cells, expressed as % of the total number of CD16+ cells. N=3 replicates, and the line represents the mean. The data clearly show that phagocytosis of CLL cells was higher when rituximab was added only for 15 minutes than for 6 hours. This increase in efficacy was associated with modulation of rituximab from the cell surface and could be reversed by treatment with mAb AT10, which blocks FcγRIIb. Importantly, only FcγRIIb was The mAb was added to the CLL cells and therefore had no effect on the macrophages themselves. In addition, only the Fab2 fragment of AT10 was used, so increased phagocytosis could not occur due to more mAb binding to the CLL cell surface. This conclusion is also supported by the observation that no increase in phagocytosis was observed after only 15 minutes of incubation (at which time a small amount of rituximab modulation had already occurred).

[0411] Figure 9: FcγRIIb expression was detected by IHC. Paraffin-embedded tissue was stained using anti-CD32b specific mAb EP888Y. Images from four different FL patients are shown (x40 total magnification, and x150 magnification insets). FcγRIIb expression was detected by flow cytometry by staining matched live cells with AT10-PE. The upper left corner of each image shows the FcγRIIb expression obtained by flow cytometry.

[0412] Figure 10 : FcγRIIb levels predict clinical outcomes in rituximab-treated MCL patients. As proof of concept for the present in vitro findings, the inventors retrospectively examined FcγRIIb expression in a cohort of MCL patients who had received rituximab. Diagnostic paraffin-embedded tissue was stained by immunohistochemistry using an FcγRIIb-specific mAb ( Figure 11 Strong membrane staining was observed in FcγRIIb+ve but not in FcγRIIb-ve lymphoma samples. Figure 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 numerical value detected by flow cytometry in 2D is expressed as Figure 10 These results correlated with FcγRIIb expression of the corresponding DMSO-frozen samples obtained by flow cytometry ( Figure 2 C, inset values.) Although only a small cohort of 16 MCL patients was studied, patients with FcγRIIb-ve lymphoma had much better median progression-free survival than those with FcγRIIb+ve cells (median 852 days versus 189 days, respectively). Figure 10 c shows the survival difference in the FcγRIIb+ and - subgroups. The two groups were comparable in clinical characteristics (MCL International Prognostic Index, data not shown), but were heterogeneous in the type of chemotherapy used. To explain this, the inventors examined patients treated with either a single dose of rituximab or fludarabine, cyclophosphamide, and rituximab (FCR) for initial treatment and observed similar results. Figure 10 d shows the survival differences in FcγRIIb + and − subgroups after the patient cohort was further controlled as described.

[0413] Figure 11: Confirmation of the specificity of the anti-FcyRIIb mAbs used in the immunohistochemistry method. FcyRIIa and FcyRIIb transfected Ramos cells were cyto- centrifuged and paraffin-embedded. Immunohistochemistry with the mAb to human FcyRIIb confirmed a strong membrane staining in FcyRIIb transfected Ramos but no staining in FcyRIIa transfected cells.

[0414] Figure 12 : CD32B specific clones inhibit rituximab internalization. Y axis shows CD20 (%) accessible at the surface. Rituximab-alexa 488 was added to CD32B transfected Ramos cells in the presence or absence of different mAbs blocking CD32b (WT or 297Q (nq) mutant) and modulation was evaluated after 1, 2, 6 and 24 hours. As a control of the blocking capacity of CD32 mAbs, the inventors also used the CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragments (Fabs)) and a negative control isotype-matched irrelevant mAb (isotype wt or nq). Finally, B1 labeled with ALEXa 488 was included as a control mAb not rapidly modulating. Data clearly indicate that all 3 nCoDeR® mAbs (C1, C11 and C13) are able to block rituximab modulation in wt or 297q form. In particular, C11 mAb is extremely efficient.

[0415] Figure 13 : Anti-CD32b mAbs ability to block rituximab modulation. All 13 mAbs (nq) and C11 are in wt. Rituximab-alexa 488 was added to CD32B transfected Ramos cells in the presence or absence of different mAbs blocking CD32b (WT or 297Q mutant) and modulation was evaluated after 1, 2, 6 and 24 hours. Y axis shows CD20 (%) accessible at the surface. As a control of the ability to block CD32 mAbs, the inventors also used the CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragments (Fabs)) and a negative control isotype-matched irrelevant mAb (isotype wt or nq). Finally, B1 labeled with ALEXa 488 was included as a control mAb not rapidly modulating. In addition, control CD32 negative Ramos cells were used to evaluate the maximal effect of blocking CD32 mAbs. Data clearly show that most nCoDeR® mAbs are able to block rituximab modulation. In particular, C10 and C11 mAbs are extremely efficient, showing almost complete blockage of modulation even at 24 hours.

[0416] Figure 14 A: Correlation between affinity of anti-CD32b blocking mAbs and their ability to inhibit CD32b phosphorylation after rituximab binding. The relative affinity of mAbs was determined by a dose titration assay to detect mAb binding to CD32B transfected CHO cells. Briefly, CD32B transfected CHO K1 adherent cells were seeded in FMAT plates. IgG was titrated from 30 nM to about 0.015 nM in 1 :2 dilutions and allowed to bind for 1 hour at room temperature. After washing, bound IgG was detected using anti-human IgG-APC antibody. Finally, plates were washed and read in FMAT (Applied Biosystems). This gives EC50 values for mAb binding to cells expressing the target and can be converted to relative affinities. The relative affinities were then correlated to the ability of anti-CD32b blocking mAbs to inhibit CD32b phosphorylation after rituximab binding. This was determined by stimulating cells with rituximab in the presence or absence of anti-CD32b mAbs and then Western blotting for phospho-CD32b. The anti-CD32b mAbs were then ranked according to their ability to block CD32 phosphorylation, with 1 being the most effective. There was a strong correlation between the affinity of mAbs and their ability to block CD32b phosphorylation.

[0417] Figure 14 B: Correlation between affinity of anti-CD32b blocking mAbs and their ability to inhibit rituximab modulation. Correlation between affinity of anti-CD32b blocking mAbs and their ability to inhibit rituximab modulation. The relative affinity of mAbs was determined by a dose titration assay to detect mAb binding to CD32B transfected CHO cells. Briefly, CD32B transfected CHO K1 adherent cells were seeded in FMAT plates. IgG was titrated from 30 nM to about 0.015 nM in 1 :2 dilutions and allowed to bind for 1 hour at room temperature. After washing, bound IgG was detected using anti-human IgG-APC. Finally, plates were washed and read in FMAT (Applied Biosystems). This gives EC50 values for mAb binding to cells expressing the target and can be converted to relative affinities. The relative affinities were then correlated to the ability of anti-CD32b blocking mAbs to inhibit rituximab modulation on CD32b transfected Ramos cells (shown in previous figure). There was a strong significant correlation between the affinity of mAbs and their ability to block rituximab modulation. This data confirms the key role of CD32B in facilitating rituximab modulation at the surface of target cells.

[0418] Figure 15: Mediated by Clone 1. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 1. Cells were seeded into FMAT plates. Immune complexes were prepared by coating FITC to BSA, which was then mixed with a FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0419] Figure 16 : Mediated by Clone 2. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 2. Cells were seeded into FMAT plates. Immune complexes were prepared by coating FITC to BSA, which was then mixed with a FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0420] Figure 17 : Mediated by Clone 3. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 3. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with a FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0421] Figure 18: Mediated by Clone 4. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 4. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0422] Figure 19 : Mediated by Clone 5. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 5. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0423] Figure 20 : Mediated by Clone 6. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 6. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0424] Figure 21: Mediated by Clone 7. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 7. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0425] Figure 22 : Mediated by Clone 8. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 8. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0426] Figure 23 : Mediated by Clone 9. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by Clone 9. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody in a 10:1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either of immune complexes (IC) or mAb.

[0427] Figure 24: Mediated by clone 10. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by clone 10. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibodies in a 10: 1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is binding of immune complexes (IC) or mAb.

[0428] Figure 25 : Mediated by clone 11. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by clone 11. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibodies in a 10: 1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is binding of immune complexes (IC) or mAb.

[0429] Figure 26 : Mediated by clone 12. Dose dependent binding of hCD32B transfected cells and dose dependent binding and inhibition of immune complexes to hCD32B transfected cells. Circles show dose dependent binding of hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding of hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complexes to hCD32B transfected CHO K1 cells. Mediated by clone 12. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibodies in a 10: 1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is binding of immune complexes (IC) or mAb.

[0430] Figure 27: Mediated by clone 13. Dose dependent binding of hCD32B transfected cells and dose dependent inhibition of immune complex binding to hCD32B transfected cells. Circles show dose dependent binding to hCD32A transfected CHO K1 cells, black diamonds show dose dependent binding to hCD32B transfected CHO K1 cells. Crosses show dose dependent inhibition of immune complex binding to hCD32B transfected CHO K1 cells. Mediated by clone 13. Cells were seeded into FMAT plates, immune complexes were prepared by coating FITC to BSA, which was then mixed with FITC specific hlgGl antibody at a 10: 1 molar ratio. Total intensity reflects binding, the higher the intensity, the higher the binding. Binding is either immune complex (IC) or mAb binding.

[0431] Figure 28 : Shows cell specificity of anti-CD32B antibodies. PBMC were isolated from peripheral blood using Ficoll density gradient. Cells were stained using cell specific markers and binding of CD32B specific antibodies was evaluated. As shown in the figure, for clone 1-13 only B cells (CD19+ cells) were positively stained.

[0432] Figure 29 : Dose dependent staining of B cells by clone 1-13. PBMC were isolated from peripheral blood using Ficoll density gradient. Cells were stained as indicated using CD19, followed by staining with 10, 1 or 0.1 mg / ml of CD32B specific antibodies. In this figure, B cells (known to express CD32B) were gated out using a CD19 specific mAb. This gate is referred to as "Ml". As the concentration of CD32B mAb was lowered, the number of stained B cells dropped from nearly 100% to a much lower number, showing specific and dose dependent staining of B cells as expected from the CD32B specific mAb.

[0433] Figure 30 : Ability of each mAb to inhibit Fc-mediated CD32B phosphorylation. Raji cells (CD32B positive) were treated with Rituximab (Rit) which leads to CD32B phosphorylation. This was done in the presence or absence of CD32B specific mAbs 1-13, which figure indicates the ability of each mAb to inhibit Fc-mediated CD32B phosphorylation. "TUB" = tubulin control.

[0434] Figure 31Effect of CD32b on the rate of modulation of type I anti-CD20 mAbs. The ability of CD32b to promote the internalization of other type I anti-CD20 mAbs. Alexa-488-labeled versions of each mAb were incubated with pCDNA3-transfected Ramos or CD32B-transfected Ramos cells for 1 or 6 hours, and the degree of modulation was determined as previously described. The mAbs used were rituximab (RTX), ofatumumab (OFA) prepared in-house, and tositumomab (Tos). The data clearly show that OFA internalization rates are similar to those of RTX and are accelerated by the presence of CD32b.

[0435] Figure 32 Anti-CD19 mAbs are also internalized from the surface of malignant human B cells in a manner that is partially dependent on CD32B. Ramoshu CD32b transfectants. Internalization using other surface antigens can also be affected by CD32b expression. Modulation assays were performed using different mAbs in the presence (+) or absence (-) of CD32 blockade using AT10 as described above. Ramos CD32B transfectants were used in this 6-hour assay. * p < 0.05. f3.3 = MHC class II; RFB9 = CD19; RTX = rituximab. If the mAb remains on the cell surface, it can be quenched. If it is internalized, it cannot be quenched. The lower the percentage of quenching, the higher the level of internalization. The data clearly show that after incubation with CD32 blockade, surface modulation by RTX and RFB9 mAbs is significantly reduced, with a smaller reduction by F3.3 mAb. 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.

[0436] Figure 33 Internalization using other surface antigens can also be affected by CD32 expression. Alexa-488-labeled versions of each mAb were incubated with pCDNA3-transfected Ramos cells or CD32B-transfected Ramos cells for 24 hours, and the degree of modulation was determined as previously described. * = p < 0.05. The y-axis shows modulation / internalization or surface accessible antigen (%). This figure shows that this is due to an increase in internalization / modulation in the presence of huCD32b. Note that for the anti-CD19 mAb and anti-CD40 mAb, there was a statistically significant decrease in cell surface expression in the presence of CD32b (*, p < 0.05).

[0437] Figure 34 : Amino acid sequences of the constant regions of 14 antibody clones against human CD32B in the IgG1-λ format. The amino acid sequences of the IgG1-CH and λ-CL regions are shown.

[0438] Figure 35 Amino acid sequence of the variable region of antibody clone 1 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The CDR sequences are indicated as boxed sequences separated by the labeled framework regions.

[0439] Figure 36 Amino acid sequence of the variable region of antibody clone 2 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The CDR sequences are indicated as boxed sequences separated by the labeled framework regions.

[0440] Figure 37 Amino acid sequence of the variable region of antibody clone 3 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0441] Figure 38 Amino acid sequence of the variable region of antibody clone 4 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences. Figure 39 Amino acid sequence of the variable region of antibody clone 5 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0442] Figure 40 Amino acid sequence of the variable region of antibody clone 6 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0443] Figure 41 Amino acid sequence of the variable region of antibody clone 7 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0444] Figure 42 Amino acid sequence of the variable region of antibody clone 8 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0445] Figure 43 Amino acid sequence of the variable region of antibody clone 9 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0446] Figure 44 Amino acid sequence of the variable region of antibody clone 10 against human CD32B. Amino acid sequences of the VH and VL regions are shown. The boxed sequences represent CDR sequences.

[0447] Figure 45: Amino acid sequence of the variable region of antibody clone 11 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0448] Figure 46 : Amino acid sequence of the variable region of antibody clone 12 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences.

[0449] Figure 47 : Amino acid sequence of the variable region of antibody clone 13 against human CD32B. The amino acid sequences of the VH and VL regions are shown. The sequences in the box represent the CDR sequences. DETAILED DESCRIPTION

[0450] Example

[0451] Example 1: CD20 modulation in primary CLL and other NHL samples

[0452] The inventors previously observed heterogeneity in the rate and extent of rituximab modulation in a cohort of CLL samples (28). To validate and extend these findings, the inventors expanded the cohort to a total of 48 CLL samples ( Figure 1 As previously described, the inventors compared the ability of rituximab and tositumomab (clinically relevant prototypes of type I and type II mAbs, respectively) to reduce surface-accessible CD20 over 2, 6, and 24 hours ( Figure 1 A, data not shown). In addition, the inventors also evaluated the recently FDA-approved, in-house produced ofatumumab (type I) for relapsed CLL and the non-glycosylated form of GA101 (GA101 gly ; type II (19) were tested. Consistent with previous observations, there was considerable heterogeneity between samples, but type I mAbs resulted in significantly more modulation than type II mAbs. At 6 h, modulation was near maximal in the presence of rituximab, with 18% to 65% (median 37%) of the mAb still accessible in the quenching assay ( Figure 1 A). Consistent with its natural type I, ofatumumab also resulted in a high degree of modulation (median 26% accessible at 6 hours). In contrast, tositumomab and GA101 gly showed much less modulation, with a median of 80% (range 61-89%) and 70% (range 57-81%) accessible bound mAb at 6 hours.

[0453] In the CLL cohort, the inventors examined a number of factors known to be important in CLL prognosis, including ZAP-70 expression (29, 30), CD38 positivity (31, 32), and IgVHmutational status (31, 33, 34). Results Figure 1 C) showed no correlation with any of these disease markers.

[0454] The inventors have previously demonstrated that, despite different modulation patterns for each NHL subtype, they also exhibit considerable CD20 modulation heterogeneity (28). To further explore this, the inventors extended the number of primary samples analyzed to include 8 healthy volunteers, 7 SLL, 7 MCL, 11 FL, and 7 DLBCL Figure 1 B). The difference in the ability of type I and type II mAbs to induce modulation persisted across all histological subtypes. The inventors also observed rapid modulation of CD20 on B cells from healthy volunteers in the presence of rituximab and also more uniform than malignant B cells, suggesting that factors associated with malignancy contribute to the heterogeneity observed. The rate of rituximab-induced modulation of SLL and MCL cells was similar to CLL Figure 1 A), while for DLBCL and FL, the rate was slightly lower (p < 0.0001 and 0.0027, respectively, when compared to CLL of Figure 1 A). However, in FL, the inventors did observe that 2 / 11 patient samples modulated rituximab very rapidly, such that the level of mAb or CD20 was barely detectable after 6 hours incubation.

[0455] Example 2: Modulation of CD20 in B-NHL is an Fc-dependent process.

[0456] 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 can also be Fc-dependent even in the absence of effector cells, and tested this by repeating the internalization assay using Fab' and F(ab')2 fragments of rituximab Figure 2A). Rituximab Fab' showed only low levels of modulation as demonstrated by cell binding assays, which can be explained by the need for bivalent cross-linking to CD20 or by its low affinity monovalent binding (data not shown). In contrast, F(ab')2and IgG showed similar binding profiles (results not shown), but after 6 hours incubation the F(ab')2fragment (40% surface accessible CD20) was much less modulated than the intact IgG (20% surface accessible CD20). Since the tests were performed using highly enriched (> 95% pure) B cells, only the abundantly present FcR, FcγRIIb, was inhibitory. The reduction of modulation with Rituximab in the presence of the blocking anti-FcγRII mAb antibody AT10 was comparable to the Rituximab F(ab')2fragment or Rit m2a, which binds FcγRIIb with lower affinity than Rituximab carrying human IgGl Figure 2 B). As expected, co-incubation with AT10 resulted in very little effect on modulation of Rit m2a.

[0457] Example 3: FcγRIIb expression on normal B cells and B cell tumors.

[0458] Given the possibility that FcγRIIb:Fc interaction can affect the rate of CD20 modulation, the inventors examined FcγRIIb expression on normal B cells and primary B cell tumor groups. As shown in Figure Figure 2 C, FcγRIIb expression was significantly heterogeneous among groups. Expression was higher on CLL cells, 20 to 300 fold over isotype control. DLBCL and most FLs exhibited low FcγRIIb expression. Two FL cases exhibited very high FcγRIIb expression, surprisingly, these were the same two cases previously observed to be extremely fast modulating. MCL and SLL expressed intermediate but heterogeneous levels of FcγRIIb Figure 2 C), which is also consistent with previous findings (21).

[0459] Example 4: FcγRIIb expression regulates CD20 modulation.

[0460] In summary, these findings suggest that FcγRIIb expression can be a major determinant of CD20 modulation from B cell targets. To test this hypothesis, the inventors compared FcγRIIb expression and CD20 modulation rate on all available healthy B cells and primary NHL samples Figure 2D). Spearman correlation analysis revealed a significant correlation between these parameters (Spearman r value -0.74, 95% confidence interval -0.83 to -0.61, p < 0.0001). The data show that most FL and DLBCL cases are located in the upper part and CLL and MCL samples form a broad log curve. This figure also demonstrates that small differences in FcγRIIb expression can lead to relatively large changes in CD20 modulation at low expression levels, thereby emphasizing the ability of this receptor to modulate the clearance of the anti-CD20:CD20 complex from the cell surface.

[0461] To directly address the role of FcγRIIb in CD20 modulation, FcγRIIb -ve Ramos cells. The FcγRIIb +ve cells exhibited variable FcγRIIb expression levels and were then sorted into subclones expressing low, intermediate and high FcγRIIb. These cells and 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 Intermediate > FcγRIIb +ve High ( Figure 3 A). Similarly, using B cells obtained from FcγRII knock-out (KO) mice expressing either wild-type or transgenic CD20, the modulation in FcγRIIb - / - mouse cells was less than in the 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.

[0462] Since FcγRIIb is a negative regulator of BCR activation on B cells (reviewed in (35)) and CD20 physically associates with the BCR after CD20 mAb engagement (36, 37), the inventors hypothesized that BCR expression or signaling activity could influence modulation. Therefore, to exclude that differences in BCR expression were responsible for these findings, BCR deficient Ramos cells (Rx3) were transfected with FcγRIIb and CD20 modulation was compared to unmanipulated Ramos cells and to cells transfected with the empty control (Rx3) Figure 3B). These data clearly indicate that the Rx3 cells, which lack BCR expression, modulate slower than Ramos cells, but that this defect can be overcome by expressing high levels of FcyRIIb (FcyRIIb +ve Rx3 cells). This superior role of FcyRIIb over BCR in mediating CD20 modulation is supported by the following evidence: 1) the inventors observed high levels of modulation in CLL cells that uniquely express low levels of BCR (38); and 2) the inventors did not observe any correlation between surface immunoglobulin (sIg) expression on CLL cells and CD20 modulation Figure 1 E).

[0463] Example 5: FcyRIIb activation precedes CD20 and FcyRIIb modulation

[0464] To further probe the interplay between anti-CD20 mAbs and FcyRIIb, the inventors investigated antibody-mediated FcyRIIb stimulation, as shown by tyrosine-293 phosphorylation in the ITIM motif of the cytoplasmic tail of FcyRIIb. Raji cells were incubated with tositumomab or rituximab in the presence or absence of the anti-FcyRIIb blocking mAb (AT10), and then immunoblotted for phosphorylated FcyRIIb. Phosphorylated FcyRIIb was elevated in rituximab-stimulated cells, but not in tositumomab-stimulated cells, and was inhibited by the addition of AT10 Figure 4 A). Similar results were observed with Daudi cells (data not shown).

[0465] Example 6: CD20 and FcyRIIb cross-linking occurs primarily in cis

[0466] Rituximab can be co-engaged by CD20 and FcyRIIb on the same (cis) or on adjacent cells (trans). To investigate this, the inventors labeled FcyRIIb - Ramos cells were co-incubated with Ramos transfectants expressing high levels of FcyRIIb Figure 4 B), and then compared the levels of modulation in each cell type, with both cell types cultured separately as controls. As shown previously, when cultured separately, FcyRIIb +ve cells showed greater modulation than cells lacking FcyRIIb Figure 4 B). In co-culture, FcyRIIb -ve modulation levels were slightly increased, but did not reach the levels of FcyRIIb +veobserved levels in the cells. This result indicates that although trans interactions can occur, the modulation of CD20 mAb by FcγRIIb is primarily driven in cis.

[0467] To confirm that this finding was not specific to the Ramos cell line, the inventors co-cultured CLL samples expressing low FcγRIIb (distinguished by PKH26 labeling) with cells from three different CLL cases expressing high levels of FcγRIIb (distinguished by lack of PKH26 labeling) and measured the modulation of CD20 mAb by FcγRIIb in the mixed population. Figure 4 C) As observed in the previous assay using Ramos cells, in the mixed population, the modulation in low FcγRIIb B cells did not approach the modulation observed in the high FcγRIIb population, again indicating that the modulation of CD20 mAb by FcγRIIb is primarily driven in cis. Interestingly, however, it was noted that co-culture with the most rapidly modulating CLL cells resulted in the greatest increase in modulation of CLL expressing low FcγRIIb, but this increase was only moderate (about 18%, data not shown).

[0468] In another experiment of this type, CLL cells were cultured at reduced concentrations to decrease the potential for cell:cell interactions, and as a result, there was a weak tendency for modulation to decrease with decreasing cell concentration Figure 4 D) The same experiment was repeated using different concentrations of Raji cells, and again there was little change in the level or extent of modulation observed (data not shown). Importantly, bright field micrographs taken during this experiment indicated that the cells were much more spread out at 1 x 10 6 cells / ml than at 2 x 10 5 cells / ml, suggesting that the potential for intercellular (trans) interactions was much lower. In addition, the inventors observed that there was no significant difference in the levels of phosphorylated FcγRIIb at different cell densities Figure 4 E) Taken together, these data indicate that FcγRIIb mediates its effect on CD20 mAb modulation primarily through events in the same cell, with neighboring FcγRIIb expressing cells having only a small effect.

[0469] Example 7: FcγRIIb internalizes into lysosomes with CD20

[0470] 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 used flow cytometry to evaluate the expression of FcγRIIb on the surface of B cells from 6 different CLL cases and found that it decreased within 2 hours of incubation with rituximab but not with tositumomab Figure 5A). These findings suggest that FcyRIIb can be internalized as part of a tripartite complex with CD20 and rituximab (but not tositumomab).

[0471] The inventors and others have previously reported that endocytosis of rituximab leads to its trafficking into early endosomes and subsequent degradation in lysosomes (9, 28). To explain whether the same process occurs in CLL cells with FcyRIIb as part of the anti-CD20:CD20:FcyRIIb complex, the inventors incubated them with Tosit-488 or Ritux-488, then fixed and stained for FcyRIIb (using F(ab')2 from AT10 labeled with Alexa 647) and the lysosomal marker LAMP-1. Prior to stimulation with mAb, FcyRIIb staining was diffuse and not localized to the plasma membrane (Fig. 4A). 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 uniquely remained at the surface, while Ritux-488 exhibited intracellular punctate staining, consistent with the inventors' previous observations (9, 28) (Fig. 4B). Figure 5 C, data not shown and (28)) After 6 hours of stimulation, Tosit-488 was still equally distributed on the cell surface, while AT10-647 showed no change from its baseline appearance at 30 minutes and no colocalization with LAMP-1 (Fig. 4C). Figure 5 D) In contrast, Ritux-488 showed a different punctate pattern over the same time course, with most cells (58%) exhibiting colocalization between AT10-647 and Ritux-488 (Fig. 4D). Figure 5 E) Colocalization of Ritux-488 with LAMP1 and AT10-647 was also observed in 33% of cells. It is postulated that the lower degree of colocalization observed between all three cell lines reflects internalization of Ritux-488 with FcyRIIb and occupation of other intracellular compartments before they appear in lysosomes.

[0472] Example 8: FcyRIIb inhibits type I anti-CD20 mAb in vivo

[0473] To explain whether FcyRIIb can 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 FcyRIIb (CD32 KO). In these experiments, mice were treated with a variant of rituximab carrying mouse IgGl (ml) or mouse IgG2a (m2a) (250 μg, iv) and B cell depletion was monitored by serial bleeds from the mice and flow cytometry using B220 and CD19 mAbs to stain for B cells for 90 days (Fig. 1). Figure 6 ) These variants bind strongly (ml) or weakly (m2a) to CD32b. The data clearly show that depletion is suboptimal when using the ml isotype (compared to m2a) and that the loss of CD32 leads to a significant improvement in the efficacy of depletion. In contrast, m2a is roughly similar in the presence or absence of CD32.

[0474] Example 9: FcyRIIb enhances and potentiates the activity of anti-CD20 mAbs against human tumors in vivo

[0475] To examine the role of CD32 on human tumor cells and the potential to enhance current therapeutic mAbs (e.g. rituximab), the inventors used a xenograft system. In this system, only the human tumor cells express hCD32, so any therapeutic effect is most likely to arise from an effect on the tumor cells through blockade 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, then the cells were treated with rituximab, AT10 or both and the mice were monitored for survival or tumor growth (Fig. 6). Figure 7 ) The doses of mAbs used are indicated in the figure legends. In A), Daudi cells were inoculated subcutaneously and the 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 enhanced and potentiated the activity of rituximab, demonstrating the potential of the combination in vivo.

[0476] Example 10: FcgRIIb levels predict clinical outcome of MCL patients treated with rituximab

[0477] As a proof of concept of the inventors' in vitro findings, the inventors retrospectively examined FcyRIIb expression in a cohort of MCL patients treated with rituximab. Diagnostic paraffin-embedded tissues were stained by immunohistochemistry using a FcyRIIb specific mAb (Fig. 7). Figure 11). Strong membrane staining was observed in FcyRIIb+ve but not in FcyRIIb-ve lymphoma samples. These results correlated with FcyRIIb expression of the corresponding DMSO frozen samples obtained by flow cytometry. The FcyRIIb staining by IHC correlated with FcyRIIb expression shown by flow cytometry (values detected by flow cytometry in 2D are shown as Figure 10 a and 10b. The FcyRIIb staining shown in a and 10b correlated with FcyRIIb expression shown by flow cytometry (values detected by flow cytometry in 2D are shown as Figure 2 D. The FcyRIIb staining shown in a and 10b correlated with FcyRIIb expression shown by flow cytometry (values detected by flow cytometry in 2D are shown as Figure 10 a and 10b. The FcyRIIb staining shown in a and 10b correlated with FcyRIIb expression shown by flow cytometry (values detected by flow cytometry in 2D are shown as Figure 10 c shows the survival difference in the FcyRIIb + and - subgroups. These two groups were comparable in clinical features (MCL International Prognostic Index, data not shown) but had heterogeneity in the type of chemotherapy used. To account for this, the inventors examined those patients treated with single agent rituximab or fludarabine, cyclophosphamide and rituximab (FCR) for initial treatment and observed similar results. Figure 10 d shows the difference in survival in the FcyRIIb + and - subgroups after further control of the patient cohort as described.

[0478] The rationale for the experiments in Examples 10 and 11 is described below. If cells express high levels of CD32b (FcyRIIb), they will internalize rituximab more rapidly (shown as the percent reduction in surface accessible CD20). If there is less rituximab on the surface of the cells, there is less Fc dependent effector activity (e.g. phagocytosis or ADCC) and therefore less killing of tumor cells and hence less therapeutic outcome. Thus, the inventors examined the FcyRIIb expression of a cohort of patients treated for MCL and determined whether they were high or low CD32b expressers. This cohort had clinical data available and so the clinical outcome was stratified according to whether they were high or low FcyRIIb expresser tumors. The rationale was that tumors expressing low levels of FcyRIIb could be successfully treated with rituximab and those expressing high levels of FcyRIIb would be less effective. This is exactly what the clinical data showed. Figure 11 The specificity of the mAb used for FcyRIIb staining is shown. It only stains cells expressing FcyRIIb and does not stain the closely related FcyRIIa.

[0479] The FcyRIIb levels were measured by IHC in the MCL cohort (Example 10) and correlated with the FcyRIIb expression shown by flow cytometry (values detected by flow cytometry in 2D are shown as Figure 10b) and after separating the MCL samples in FcyRIIb positive and negative, the inventors observed a clear difference in clinical response after rituximab-based treatment Figure 10 c and 10 d).

[0480] Example 11: Selection of anti-CD32b monoclonal antibodies

[0481] Figures 37-47 The amino acid sequences of the variable regions (VH and VL) and CDR regions of the 14 antibody clones are shown in Table 1. In each case, the constant regions (CH and CL) are identical. Figure 36 The constant regions are shown in Table 2.

[0482] Using n-CoDeR ® Screening against CD32B (FcyRIIb) was performed using n-CoDeR scFv phage display library. Human CD32A was used as non-target. Extracellular domains of CD32A and CD32B fused to mlgG3-Fc were produced in HEK293E and purified using protein A. Three consecutive rounds of protein screening were performed. Non-target was used as competitor in all screenings. The phage produced was converted into scFv / Fab producing format and transformed into E. coli Top10 bacteria for screening of individual clones. The screening determined the specificity for human CD32B and CD32A and was analyzed in ELISA using coated proteins and by transfecting CHO cells in FMAT. For determination of IC inhibitory properties, IgG was allowed to bind to CD32B transfected CHO cells, after which IC in the form of IgGl coated bovine serum albumin was added. The bound IC was then detected and the inhibitory properties of the IgG were evaluated.

[0483] Example 12: Ability of anti-CD32b mAbs to block rituximab modulation

[0484] Rituximab-alexa 488 was added to CD32B transfected Ramos cells in the presence or absence of different CD32b blocking mAbs (wt or 297Q variants) and modulation was evaluated after 1, 2, 6 and 24 hours. As a control for the ability to block CD32 mAbs, the inventors also included CD32a and CD32b bispecific mAb AT10 (IgG and Fab2 fragments (Fabs)) and a negative control irrelevant mAb (isotype wt or nq) matched for isotype. Figure 12 The data in Table 3 clearly show that all three nCoder mAbs (C1, C3 and C11) are able to block rituximab modulation in both wt and 297Q formats.

[0485] Figure 13The ability of anti-CD32b mAbs to block rituximab modulation was shown using all 13 mAbs. In addition, control CD32 negative Ramos cells were used to assess the maximum effect of CD32 blocking mAbs. Figure 12 The data in Figure 8 clearly show that all nCoder mAbs are able to block rituximab modulation.

[0486] Previous experiments have shown that FcyRIIb modulates rituximab internalization. Therefore, these experiments sought to examine whether blocking FcyRIIb with anti-FcyRIIb mAbs would decrease the amount of rituximab internalization.

[0487] Example 13: Correlation of anti-CD32b blocking mAb affinity and its ability to inhibit CD32b phosphorylation after rituximab binding and its ability to inhibit rituximab modulation

[0488] The relative affinity of the mAbs was determined by a dose titration experiment measuring mAb binding to CD32B transfected CHO cells. Briefly, CD32B transfected CHO K1 adherent cells were seeded in FMAT plates. IgG was titrated from 30 nM to approximately 0.015 nM in 1 :2 dilutions 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 the FMAT (Applied Biosystems). This gave an EC50 value for the mAb binding to the target expressing cells and this could be converted to a relative affinity. This relative affinity was then correlated to the ability of the anti-CD32b blocking mAb to inhibit CD32b phosphorylation after rituximab binding. This was determined by stimulating the cells with rituximab in the presence or absence of anti-CD32b mAb and then Western blotting for phospho-CD32b. The mAbs were then ranked according to their ability to block CD32 phosphorylation, with 1 being the most effective. Figure 14 A shows a clear correlation between mAb affinity and its ability to block CD32b phosphorylation. Figure 14 B shows a clear correlation between mAb affinity and its ability to block rituximab modulation. This data demonstrates the important role of CD32B in facilitating rituximab modulation from the surface of target cells.

[0489] The theory is that higher mAb affinity will block FcyRIIb better. Therefore, the better the mAb blocks FcyRIIb, the better it will block rituximab modulation / internalization. This is exactly what is shown in Figure 9. Figure 14The higher the affinity, the better it blocks rituximab binding-induced FcγRIIb activation (as measured by the amount of phospho-FcγRIIb stained by Western blot) and the better it blocks modulation.

[0490] Example 14: Dose-dependent binding to hCD32B-transfected cells and immune complexes and inhibition of hCD32B-transfected cells

[0491] Cells were seeded onto FMAT plates. Immune complexes were prepared by coating FITC onto BSA, which was then mixed with FITC-specific hIgG1 antibody at a 10:1 molar ratio. Figures 15-27 Dose-dependent binding of clone 1-13 to hCD32B-transfected cells and dose-dependent binding and inhibition of immune complexes to hCD32B-transfected cells are shown, respectively. Circles show dose-dependent binding to hCD32A-transfected CHO K1 cells, and black diamonds show dose-dependent binding to hCD32B-transfected CHO K1 cells. Crosses show dose-dependent inhibition of immune complexes to hCD32B-transfected CHO K1 cells.

[0492] The assay was designed to: 1) determine the specificity of the mAb. CD32B and CD32A are closely related molecules. However, while CD32B transmits inhibitory signals, CD32A transmits positive signals, making it crucial for the antibody to bind only to CD32B for the desired effect. 2) Furthermore, to effectively block signaling through CD32B, the antibody must not only bind to CD32B but also block binding of its natural ligand, the immune complex (IC). Therefore, the figure shows binding to CD32A, CD32B, and inhibition of IC binding. The figure demonstrates that all mAbs are specific for CD32B and do not bind CD32A, and they all inhibit IC binding.

[0493] Example 15: Cell Specificity of Anti-CD32B Antibodies

[0494] PBMCs were isolated from peripheral blood using a Ficoll density gradient. Cells were stained with cell-specific markers and evaluated for binding of CD32B-specific antibodies. Figure 28 As shown, clone 1-13 stained positive only for B cells (CD19+ cells).

[0495] In resting PBMCs, CD32B is expressed only on B cells, whereas the closely related CD32A is expressed on monocytes and neutrophils. The previous figure shows specificity for transfected CHO cells. Figure 28The antibodies were also shown to bind B cells expressing CD32B in its native form, whereas they did 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 PBMC.

[0496] Example 16: Dose-dependent staining of B cells by anti-FcyRIIb mAb clone 1-13

[0497] PBMC were prepared from peripheral blood using Ficoll density gradient. Cells were stained with CD19, after which they were stained with 10, 1 or 0.1 mg / ml of CD32B specific antibodies. Figure 29 It is shown how the staining of B cells by the various clones is dose-dependent.

[0498] In Figure 29 , B cells (known to express CD32B) were gated using a CD19 specific mAb. This gate was called "Ml". When the concentration of the CD32B mAb was lowered, the number of stained B cells dropped from nearly 100% to a much lower value, demonstrating the specific and dose-dependent staining of B cells by the CD32B specific mAb.

[0499] This is also evidence of the specificity of the antibodies. As already mentioned, CD32A and CD32B are very closely related and it is not easy to obtain specific antibodies. Any specific antibody shows dose-dependent binding, which is exactly what is demonstrated in Figure 29 , i.e. lowering the dose of the antibody reduces the amount of B cell staining from nearly 100% (as observed in the highest dose). Thus, this figure demonstrates for the second time the specificity of the antibodies when the antigen is expressed in normal, non-transfected B cells.

[0500] Example 17: Ability of the various mAbs to inhibit Fc-mediated CD32B phosphorylation

[0501] Raji cells (CD32B positive) were treated with Rituximab, which resulted in CD32B phosphorylation. This was done in the presence or absence of CD32B specific mAb 1-13, Figure 30 demonstrating the ability of the various mAbs to inhibit Fc-mediated CD32B phosphorylation.

[0502] The conclusion after Examples 17 and 18 is that the Fc region of Rituximab binds to FcyRIIb and that this results in FcyRIIb activation. This is measured by phosphorylation of the ITIM region of FcyRIIb. Blocking this interaction with anti-FcyRIIb mAbs blocks phosphorylation Figure 30 ) and modulation Figure 31 and 32). The wt FcyRIIb IgGl also has the ability to bind FcyRIIb through its Fc region, whereby the inventors checked whether the N297Q mutant (with an Fc that does not bind FcyRIIb) also has similar activity. It does.

[0503] Example 18: Role of CD32b in the rate of modulation of type I anti-CD20 mAbs

[0504] The ability of CD32b to contribute to the internalization of other type I anti-CD20 mAbs is shown in Figure 31 . Alexa-488 labeled versions of each mAb were incubated with pCDNA3 transfected Ramos or CD32B transfected Ramos cells for 1 hour or 6 hours, and then the extent of modulation was determined as before. The mAbs used were rituximab (RTX), ofatumumab (OFA) made in-house, and tositumomab (Tos). The data clearly show that the rate of internalization of OFA is similar to that of RTX, and is accelerated by CD32b.

[0505] These modulation effects were observed using rituximab (a type I anti-CD20), but were less apparent using tositumomab (a type II anti-CD20 mAb). Therefore, to explain 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.

[0506] Example 19: Anti-CD19 mAbs are also internalized from the cell surface of malignant human B cells in a partially CD32B dependent manner

[0507] Ramos huCD32b transfectants. Internalization using other surface antigens is 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 = type II MHC; RFB9 = CD19; RTX = rituximab. Figure 32 It is clearly shown that surface modulation of the RTX and RFB9 mAbs is significantly reduced after incubation with CD32 blockade, with less reduction in surface modulation of F3.3. These data suggest that target antigens such as CD19 can also be internalized from the surface of malignant human B cells in a partially CD32B dependent manner, and can be blocked by anti-CD32b mAbs.

[0508] The inventors wished to determine whether non-CD20 target antigens were also affected by CD32b expression. Therefore, the inventors examined whether mAbs to other target antigens (CD19 and MHCII) and mAbs that block CD32b would reduce their internalization. Data showed that blocking CD32b also reduced CD19 mAb modulation.

[0509] Example 20: Internalization using other surface antigens is also affected by CD32b expression

[0510] Ramos cells were rendered CD32b negative, thereby demonstrating the level of internalization in the absence of CD32B. If an antigen is able to be internalized by CD32b, then expression of that antigen (on Ramos-CD32B cells) will increase the level of internalization.

[0511] Alexa-488 labelled versions of each mAb were incubated with pCDNA3 transfected Ramos cells or CD32B transfected Ramos cells for 24 hours and the degree of modulation determined as before. * = p<0.05. Figure 33 It is shown that internalization using other antigens is also affected by CD32b expression.

[0512] Materials and Methods

[0513] Cells

[0514] Human cell lines (Daudi, Raji, Ramos) were obtained from ECACC and maintained in RPMI supplemented with 10% foetal calf serum (FCS) (Lonza, UK), glutamine and pyruvate (both from Invitrogen) at 37°C, 5% CO2. BCR expression deficient Rx3 Ramos cells were generated previously (36). Ramos FcyRIIb transfectants and control cells transfected with empty vector (FcyRIIb negative) were as described previously (36) and maintained in supplemented RPMI as above with the addition of geneticin (Invitrogen, UK). Rx3 cells transfected with FcyRIIb and empty vector were prepared and maintained in the same way. FcyRIIb surface expression was determined by flow cytometry using PE labelled AT10 (described below). Cell populations of Ramos FcyRIIb transfectants expressing low, medium or high levels of FcyRIIb were sorted using a FACS Aria flow cytometer (BD Biosciences, USA).

[0515] Blood donors

[0516] Normal human B cells were obtained from healthy volunteers with informed consent. Peripheral blood was drawn in K2E or LiH and lymphocytes isolated using Lymphoprep (Axis-Shield, UK) according to the manufacturer's protocol and B cells isolated by negative selection using the human B cell isolation kit II (Miltenyi Biotec, Germany).

[0517] Clinical samples

[0518] 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 and solid tissues were disrupted through a sterile mesh and centrifuged. Cells were cryopreserved in RPMI supplemented with human AB serum and 10% DMSO and stored under the Human Tissue Authority licence at the Tumour Bank, Cancer Sciences Unit, University of Southampton. Ethical approval for use of clinical samples was obtained from Southampton and South West Hampshire Research Ethics Committee by the University Hospital Trust, Southampton. For CLL cells, the mutational status of IgVH genes was determined as previously detailed (33) and CD38 positivity (44). Briefly, for IgVH analysis, heavy chain genes were amplified from cDNA using a VH forward primer mix and a Cμ100 primer. All nucleotide sequences were aligned to the V-base catalogue and the mutational status determined using a 98% cut-off. For CD38 analysis, anti-CD38 PE (clone HB7, BD Biosciences) was used. Determination of ZAP-70 status was performed as described by Crespo et al. (30). Surface Ig expression of CLL cells was determined by flow cytometry as previously described (45, 46).

[0519] Viability assay

[0520] Cell viability was assessed by flow cytometry after staining with FITC-labelled annexin V and PI as previously described (25).

[0521] Antibodies and reagents

[0522] Rituximab was kindly donated by the Cancer Pharmacy Department, Southampton General Hospital. Rit m2a (rituximab with mouse IgG2a Fc region), WR17 (anti-CD37) and all mouse IgG2a were prepared as previously described (18). Anti-FcyRII mAb (AT10) was prepared in-house as previously described (47). Tositumomab was kindly donated by Prof Tim Illidge (Manchester, United Kingdom). Ofatumumab and GA101 were prepared in-house according to the sequences published in the patents glyAlexa-488 and anti-Alexa 488 reagents were purchased from Invitrogen. F(ab')2 fragments were prepared as previously described (48). Fab' fragments were generated by addition of excess iodoacetamide after incubation with 20 mM 2-mercaptoethanol for 30 minutes at 25°C. The Western blotting antibodies used were anti-actin antibody (AC74, Sigma, UK) and anti-phospho-FcyRIIb antibody (Cell Signaling Technology, UK).

[0523] Flow cytometry

[0524] Fluorochrome-labelled mAbs were obtained from BD Biosciences or were prepared in-house. mAbs were conjugated with Alexa 488 (Invitrogen) according to the manufacturer's protocol. Flow cytometry was performed as previously described (49). Samples were evaluated on a FACScan (BD Biosciences) or a FACSCalibur (BD Biosciences) and data were analysed using CellQuest Pro (BD Biosciences) or FCS Express (DeNovo Software, USA). B cells were identified using APC-labelled anti-human CD19 antibody (prepared in-house) and FcyRIIb expression was determined using PE-labelled AT10 (prepared in-house and ex-house). To control for inter-assay variability, FcyRIIb expression was expressed as the ratio of FcyRIIb: isotype control geometric mean fluorescence intensity (MFI).

[0525] Internalization assay

[0526] Internalization assays were performed as previously detailed (28). Briefly, 2-4 x 10 5 cells / well were incubated with Alexa-488 labelled 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-labelled anti-CD19 antibody for 30 minutes at 4°C with or without quenching antibody anti-Alexa-488 antibody (Invitrogen). Samples were then washed once and analysed on a flow cytometer.

[0527] To investigate the interaction of the Fc region of cell-bound anti-CD20 mAb with FcγRIIb on neighboring cells, FcγRIIb was labeled with PKH26 (Sigma Aldrich) according to the manufacturer's instructions. -ve Then, the PKH26-labeled cells were mixed with the same number (2.5 x 10 5 10 cells) were co-incubated with FcγRIIb-transfected Ramos cells. Both cell types were incubated separately as controls. Internalization assays were then performed as described above and compared to the modulation of PKH26-labeled and unlabeled cell populations. Additional variations of this co-incubation assay are described in the figure legends.

[0528] Western blotting

[0529] The protocol was as previously described (36). Briefly, approximately 2 x 10 6 Cells / well were incubated with mAb (5-10 μg / ml). Samples were then separated by SDS-PAGE, and proteins were immediately transferred to PVDF membranes. The membranes were blocked with 5% w / v skim milk powder, incubated with appropriately diluted primary antibodies, washed, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Sigma Aldrich). The membranes were visualized by enhanced chemiluminescence (ECL, GE Healthcare, UK or Pierce Biotechnology, UK) and exposed to light-sensitive film (Hyperfilm ECL, GE Healthcare, UK) or a Biospectrum AC imaging system (UVP, UK).

[0530] Optical and confocal microscopy

[0531] To measure the intracellular trafficking of anti-CD20 mAbs and FcγRIIb, CLL cells were incubated with the appropriate Alexa 488-labeled mAbs for various times as described in the figure legends, then harvested, washed, and fixed with 2% paraformaldehyde. For detection of FcγRIIb and LAMP-1, cells were permeabilized with 0.3% saponin and incubated with Alexa-647-labeled AT10 F(ab')2 (labeled using the Alexa Fluor-647 Labeling Kit (Invitrogen) according to the manufacturer's protocol) and / or biotin-conjugated anti-human CD107a (LAMP-1) antibody (eBioscience, UK). Cells were then washed, and streptavidin-Alexa Fluor-547 (Invitrogen) was added, followed by further washing. Afterwards, the cells were transferred to glass slides, and images were captured using LAS-AF v2 software on a TCS-SP5 laser scanning confocal microscope (Leica Microsystems, UK) (10x eyepiece, 100x objective).

[0532] To determine the proximity of cells at different cell dilutions, 1-20 x 10 5 Cells were seeded at 400 μg / ml and stimulated with various mAbs for 2 and / or 6 hours, and then their relative proximity was assessed by light microscopy. Cells were observed using an Olympus CKX21 inverted microscope (Olympus, UK) using a 10x or 20x / 0.25 PH lens. Images were acquired with a CCL2 cooled digital camera (Olympus) and processed using Cell B (Olympus Soft Imaging Solution) and Adobe Photoshop CS2 software (Adobe, San Jose, CA).

[0533] Data Analysis

[0534] Data were analyzed using GraphPad Prism (GraphPad Software, USA). Paired nonparametric data were analyzed using the Wilcoxon matched-pairs test, while unpaired data were analyzed using the Mann-Whitney test.

[0535] Example compositions, formulations, and modes of administration

[0536] 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.

[0537] In a specific embodiment, the subject or patient is an animal, preferably a mammal, such as a non-primate (e.g., a cow, pig, horse, cat, dog, rat, etc.) and a primate (e.g., a monkey such as a cynomolgus monkey and a human). In a preferred embodiment, the subject is a human.

[0538] A variety of delivery systems are known and can be used to administer the compositions of the application, e.g., liposomes, microparticles, microcapsules, recombinant cells capable of expressing antibodies, etc.

[0539] In some embodiments, the compositions of the application are formulated in liposomes for targeted delivery of the antibodies of the application. Liposomes are vesicles comprising concentrically arranged phospholipid bilayers that enclose an aqueous phase. Liposomes typically comprise multiple 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 due in part to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art and are encompassed within the scope of the application, see, e.g., 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.

[0540] Methods of administering the compositions of the application include, but are not limited to, parenteral (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and buccal routes). In a specific embodiment, the compositions of the application are administered intramuscularly, intravenously, or subcutaneously. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can be used, e.g., by use of an inhaler or nebulizer, and formulation 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.

[0541] The amount of the composition of the present application that will be effective in the treatment, prevention or amelioration of one or more symptoms of the disorder can be determined by standard clinical techniques. In addition, in vitro assays can optionally be employed to help identify optimal dosage ranges. The precise amount will depend on the severity of the disease, the age, weight and general health condition of the patient, and the frequency and mode of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0542] For the antibodies encompassed by the present application, the dosage administered to a patient is typically 0.0001 mg to 100 mg of each antibody in the composition per kg of patient body weight. Preferably, the dosage of each antibody administered to a patient is 0.0001 mg to 20 mg, 0.0001 mg to 10 mg, 0.0001 mg to 5 mg, 0.0001 mg to 2 mg, 0.0001 mg to 1 mg, 0.0001 mg / kg to 0.75 mg, 0.0001 mg to 0.5 mg, 0.0001 mg to 0.25 mg, 0.0001 mg to 0.15 mg, 0.0001 mg to 0.10 mg, 0.001 mg to 0.5 mg to 02.5 mg, or 0.01 mg to 0.10 mg per kg of patient body weight. Typically, human antibodies have a longer half-life in humans relative to antibodies from other species due to the immune response to the foreign polypeptide. As such, lower dosages and less frequent administration of human antibodies are typically possible. In addition, the dosage and frequency of administration of the antibodies or fragments thereof of the present application can be reduced by modifications, such as lipidization, that enhance uptake and tissue penetration of the antibodies.

[0543] In one embodiment, the dosage of each antibody of the composition of the present application administered to a patient is 0.01 mg to 1000 mg per day.

[0544] The compositions of the present application comprise a prophylactically or therapeutically effective amount of the agents and antibodies described herein and a pharmaceutically acceptable carrier.

[0545] In a particular embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvant), excipient, or vehicle with which the therapeutic is administered. Such a pharmaceutical carrier can be a sterile liquid, such as water and oils, including those of

[0546] In various embodiments, the administration of the antibodies and agents can be simultaneous or spaced less than 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 100 hours apart, about 11 hours to about 12 hours apart, less than 24 hours apart, or less than 48 hours apart. In preferred embodiments, the two or more components are administered in the same patient visit.

[0547] Therapeutically and prophylactically effective encompasses the dosages and frequencies of administration provided by the present application. The dosages and frequencies will generally further vary according to factors specific to each patient, depending on the particular therapeutic or prophylactic agent administered, the severity and type of the disease, the route of administration, and the age, body weight, response, and past medical history of the patient. Those of skill in the art can select an appropriate regimen by considering these factors and the dosages reported in the literature and recommended in the Physician's Desk Reference (56th ed., 2002), for example.

[0548] SUMMARY

[0549] Recently, the inventors discovered that type I mAbs, such as rituximab, are modulated by human CD20 Tg mouse B cells (in vitro and in vivo) and by certain primary tumor cells derived from patients with NHL, thereby limiting their ability to recruit effector cells and deplete target cells (28). The inventors considered possible mechanisms to explain the limited therapeutic activity of rituximab and other type I CD20 mAbs, and provided an opportunity to block or circumvent this process and thereby develop more potent agents, which is particularly important. The present invention provides a molecular theory of CD20 modulation induced by rituximab and ofatumumab. FcyRIIb expression would 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, important but heterogeneous CD20 modulation was observed, and this heterogeneity can not be associated with known prognostic factors for CLL. Analysis of other B-NHL subtypes indicated that MCL exhibited similar heterogeneous modulation as CLL, but FL and particularly DLBCL showed significantly less modulation. Based on these results, the inventors report here a correlation between FcyRIIb expression levels in these malignancies and their modulation upon 6 hour incubation. In addition, the inventors suggest that this modulation can explain some of the heterogeneity observed in response to rituximab in these diseases. In DLBCL and FL, where rituximab is established as a first-line treatment in combination with chemotherapy, rituximab proved 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 less. Thus, it was found in general that B cell malignancies expressing FcyRIIb are more likely to modulate CD20 and tend to result in a reduced benefit from rituximab treatment. However, even in DLBCL and FL, certain cases do not respond to rituximab. As an example, transformed FL cases are generally poorly responsive to therapy and express FcyRIIb (21), an observation that is consistent with the inventors' own observation that one of the high FcyRIIb expressing samples (FL- 1) was identified as FL and exhibited a corresponding high rate of modulation. Figure 2 C) was identified as FL and exhibited a corresponding high rate of modulation, in agreement. Although a single case finding, the inventors believe that this can potentially provide an important way to address rituximab resistance.

[0550] CD20 modulation showed a strong correlation with FcyRIIb expression levels, regardless of the B-NHL disease subtype. It has been previously suggested that FcyRIIb can inhibit therapeutic mAb efficacy by competing with activating Fc receptors on effector cells, thereby inhibiting cytotoxic signaling (40). The in vitro studies of the present application show that rituximab primarily crosslinks CD20 and FcyRIIb on the same cell, leading to FcyRIIb activation, and both surface antigens are rapidly paired for internalization into the lysosome with the bound mAb for degradation. FcyRIIb expression leads to a decrease in effector cell recruitment through its ability to downregulate surface expression of the mAb on target cells.

[0551] The present application also shows that co-incubation with a blocking anti-FcyRIIb mAb is able to inhibit FcyRIIb activation and rituximab rapid internalization. Altogether, these data confirm a positive correlation between FcyRIIb activation and mAb internalization from the cell surface.

[0552] The strong correlation between type I anti-CD20 mAb-induced CD20 modulation and FcyRIIb expression in different B-NHL subtypes, together with the transfection studies of the present application, show that FcyRIIb is a key modulator of CD20 modulation.

[0553] Other groups have investigated the role of FcyRIIb in lymphoma. Camilleri-Broet et al (20) failed to show a significant correlation between R-CHOP response and FcyRIIb expression in DLBCL, however, only 18% (42 / 234 cases) were considered FcyRIIb positive by immunohistochemistry in the study of the 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 FcyRIIb alleles (232I allele, more efficient than 232T for BCR-mediated calcium regulation in autoimmune diseases (22, 23)) were associated with rituximab efficacy, but failed to demonstrate any correlation between this polymorphism and single-agent treatment 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. In addition, the polymorphisms investigated reflect the efficiency of BCR inhibition in autoimmune diseases, none of the published observations suggest that these polymorphisms are relevant or affect Fc binding of human IgGl in lymphoma. FcyRIIb expression will be an important prognostic marker of the success of immunotherapy using type I mAbs (including rituximab and ofatumumab) through its ability to modulate the rate of internalization. The effect is less pronounced when treated with type II mAbs.

[0554] In addition, the inventors demonstrate in different in vivo models the ability of CD32 to limit mAb efficacy and the capacity of anti-CD32b mAb to overcome this limitation and enhance rituximab treatment capacity.

[0555] References

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Claims

1. A monoclonal antibody or antigen-binding fragment thereof that binds to human FcγRIIb, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 identical to those in SEQ ID NO: 12; and (b) a light chain variable region 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 identified according to the Kabat numbering system or the IMGT numbering system, and wherein the antibody is a recombinantly produced antibody.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:

12.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

25.

4. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain constant region as shown in SEQ ID NO:

1.

5. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a light chain constant region as shown in SEQ ID NO:

2.

6. A composition comprising: (i) antibody molecules, (a) it comprises an Fc domain capable of binding to FcγRIIb, and (b) specifically bind to cell surface antigens of target cells; (ii) reagents, (a) it specifically binds to FcγRIIb, and (b) preventing or reducing the binding between the Fc domain of the antibody molecule and FcγRIIb on the target cell, The agent is the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The composition of claim 6, wherein the cell surface antigen is selected from CD19, CD20 or CD40.

8. The composition of claim 6, wherein the antibody or antigen-binding fragment thereof comprises: (a) A heavy chain variable region comprising: i. VH CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 83; ii. VH CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 84; and iii. VH CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 85; and (b) a light chain variable region comprising: i VL CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 86; ii. VL CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 87; and iii. A VL CDR3 consisting of the amino acid sequence shown in SEQ ID NO:

88.

9. The composition of claim 6, wherein the target cell is a cancer cell.

10. The composition of claim 6, wherein the target cell is a B cell.

11. The composition of claim 6, wherein the antibody molecule is a CD20 antibody.

12. The composition of claim 11, wherein the CD20 antibody is a type I CD20 antibody.

13. The composition of claim 6, wherein the composition is encapsulated in a liposome, microparticle, microcapsule, or recombinant cell capable of expressing the antibody molecule or the agent.

14. The composition of claim 6, wherein the composition is formulated for parenteral, epidural, or mucosal administration.

15. The composition of claim 14, wherein the parenteral administration is selected from intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous administration, and the mucosal administration is selected from intranasal and oral routes.

16. The composition of claim 6, wherein the composition is formulated for infusion or bolus injection, or for absorption through epithelial or mucocutaneous linings.

17. The composition of claim 6, wherein the composition is formulated for absorption through the oral mucosa, rectal and intestinal mucosa.

18. The composition of claim 6, wherein the composition is used to treat a patient with elevated expression levels of FcγRIIb.

19. The composition of claim 18, wherein the patient is a cancer patient.

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