Novel combinations and uses of antibodies

Abstract

Described is the use of a first antibody molecule that specifically binds to Fc [gamma] Rllb through its Fab region but lacks an Fc region or through its Fc region has reduced binding to the Fc [gamma] receptor for use in the treatment of Fc [gamma] Rllb negative cancer in a patient in combination with a second antibody molecule, and a second antibody molecule that specifically binds to a receptor present on an immune cell that inhibits anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activated Fc [gamma] receptor, and wherein the binding of the second antibody molecule to the receptor on the immune cell causes depletion and / or deactivation of the immune cell; as well as pharmaceutical compositions and kits comprising these two antibody molecules; and methods of treating cancer using the two antibodies.

Description

[0001] This application is a divisional application of the Chinese invention patent application filed on January 10, 2019, with the invention name “Novel Combinations and Uses of Antibodies” and application number 201980007995.5. Technical Field

[0002] The present invention relates to the use of the following antibody molecules: 1) an antibody molecule that specifically binds to FcγRIIb through its Fab region but lacks an Fc region or has reduced binding to at least one Fcγ receptor through its Fc region; and 2) an immune cell-depleting or deactivating antibody molecule that specifically binds to a receptor present on immune cells that suppress anti-cancer immunity and has an Fc region that binds to at least one activating Fcγ receptor, wherein the antibody molecule and the immune cell-depleting or deactivating antibody molecule are used in combination to treat FcγRIIb-negative cancers. Background Art

[0003] It has long been recognized that inhibitory Fcγ receptors (FcγR) IIB expressed by numerous cells of the immune system negatively regulate both innate and adaptive immunity by engaging immune complexes (ICs). Similarly, for more than a decade, it has been known that FcγRIIB negatively regulates monoclonal antibody-mediated immunotherapy. Thus, when treated with therapeutic mAbs, FcγRIIB-deficient mice were able to clear tumors more effectively than wild-type (WT) mice, suggesting that expression of FcγRIIB on effector cells (i.e., macrophages and monocytes) leads to inhibition of their phagocytic and cytotoxic potential in vivo. In addition, FcγRIIB regulates the antigen presenting potential of dendritic cells (DCs) and FcγRIIB-ve (van Montfoor et al., J Immunol. 2012 Jul 1; 189(1): 92-101). DC has an improved ability to activate naive T cells. Recently, antagonist antibodies that block FcγRIIB signaling and internalization in B cells have been developed. These antibodies showed effective depletion of FcγRIIB-expressing B cells and effectively enhanced rituximab-mediated depletion of normal and malignant B cells, demonstrating utility in hematological cancers. However, it has not been examined or demonstrated whether these antibodies will also have utility in the treatment of FcγRIIB-negative cancers (such as solid cancers). Summary of the Invention

[0004] Surprisingly, it was shown herein that anti-FcγRIIB antibodies (e.g., F(ab)'2 antibodies or deglycosylated antibodies) that lack only the Fc region or whose Fc region exhibits reduced or impaired binding to FcγRs can enhance the therapeutic activity of antibodies used to treat FcγRIIB-negative cancers (including solid cancers). This finding was unexpected because previous studies have shown that wild-type IgG1 anti-FcγRIIB antibodies can also block FcγRIIB receptors in vitro and can also prevent rituximab internalization and rituximab-induced FcγRIIB phosphorylation.

[0005] According to the present invention, it is possible to enhance the therapeutic activity of immunomodulatory anti-cancer antibodies, whose therapeutic activity depends on the engagement of FcγR. Such antibodies include, but are not limited to, antibodies against so-called checkpoint inhibitor targets (e.g., CTLA-4), immune agonist targets (e.g., OX40, 4-1BB, and GITR), and interleukin-2 receptor (IL-2R).

[0006] Disclosed herein is a first antibody molecule that specifically binds to FcγRIIb via or through its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via or through its Fc region, wherein the first antibody molecule is

[0007] a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the second antibody molecule to the receptor on the immune cell causes depletion and / or deactivation of the immune cell;

[0008] The combination is used to treat FcγRIIb-negative cancer in patients.

[0009] Also disclosed herein is a pharmaceutical composition comprising:

[0010] (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and

[0011] (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the second antibody to the receptor on the immune cell causes depletion and / or deactivation of the immune cell;

[0012] The pharmaceutical composition is used to treat FcγRIIb-negative cancer in a patient.

[0013] Further disclosed herein is a kit for treating FcγRIIb-negative cancer, the kit comprising:

[0014] (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and

[0015] (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the second antibody molecule to the receptor on the immune cell causes depletion or deactivation of the immune cell.

[0016] Further disclosed herein are uses of the following antibody molecules:

[0017] (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and

[0018] (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the second antibody to the receptor on the immune cell causes depletion or deactivation of the immune cell;

[0019] It is used to manufacture a medicament for treating FcγRIIb-negative cancer in a patient.

[0020] Also disclosed herein is a method for treating an FcγRIIb-negative cancer in a patient, the method comprising administering:

[0021] (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and

[0022] (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that is capable of activating at least one activating Fcγ receptor, and wherein the binding of the second antibody to the receptor on the immune cell causes depletion or deactivation of the immune cell. DETAILED DESCRIPTION

[0023] Therefore, the present invention relates to the combined use of:

[0024] (i) an antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region (hereinafter generally referred to as a first antibody molecule or said first antibody molecule), and

[0025] (ii) an antibody molecule that specifically binds to a receptor present on an immune cell (hereinafter generally referred to as a second antibody molecule or the second antibody molecule), wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the antibody molecule to the receptor on the immune cell causes depletion or deactivation of the immune cell. Therefore, this second antibody molecule is an immune cell-depleting or deactivating antibody molecule.

[0026] This combination is intended for use in the treatment of FcγRIIb-negative cancers in patients, with the goal of improving the therapeutic efficacy of the second antibody molecule by enhancing the binding of the Fc portion of the second antibody molecule to activating FcγRs while reducing the binding / activation of inhibitory FcγRs.

[0027] Fc receptors are membrane proteins found on the cell surface of immune effector cells (such as macrophages). The name comes from its binding specificity to the Fc region of antibodies, which is the conventional way for antibodies to bind to receptors. However, in the case of antibodies specifically binding to one or more Fc receptors, some antibodies can also bind to Fc receptors through the CDR sequences of the antibody.

[0028] A subgroup of Fc receptors is Fcγ receptors (Fcγ receptors, FcγRs) that are specific for IgG antibodies. There are two types of Fcγ receptors: activated Fcγ receptors (also expressed as activated Fcγ receptors) and inhibitory Fcγ receptors. Activating receptors and inhibitory receptors transmit their signals through immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs), respectively. In humans, FcγRIIb (CD32b) is an inhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a) and FcγRIV are activated Fcγ receptors. FcγgRIIIb is a GPI-linked receptor expressed on neutrophils, which lacks the ITAM motif but is also considered to be activating by its ability to cross-link lipid rafts and bind to other receptors. In mice, the activating receptors are FcγRI, FcγRIII, and FcγRIV.

[0029] It is well known that antibodies regulate immune cell activity by interacting with Fcγ receptors. Specifically, how antibody immune complexes regulate immune cell activation is determined by the relative engagement of their activating and inhibitory Fcγ receptors. Different antibody isotypes bind to activating and inhibitory Fcγ receptors with different affinities, resulting in different A:I ratios (activation:inhibition ratios) (Nimmerjahn et al.; Science 2005 Dec 2;310(5753):1510-2).

[0030] By binding to inhibitory Fcγ receptors, antibodies can inhibit, block, and / or downregulate effector cell function.

[0031] By binding to activating Fcγ receptors, antibodies can activate effector cell function and thereby trigger mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine release and / or antibody-dependent endocytosis, and NETosis (i.e., activation and release of NETs, ​​neutrophil extracellular traps) in the case of neutrophils. Binding of antibodies to activating Fcγ receptors can also lead to an increase in certain activation markers (such as CD40, MHCII, CD38, CD80, and / or CD86).

[0032] The antibody molecule that specifically binds to FcγRIIb according to the present invention, i.e., the first antibody, binds to or interacts with this Fcγ receptor through the Fab region of the antibody, i.e., through the antigen binding region on the antibody that binds to the antigen, the antigen being composed of one constant domain and one variable domain in each of the heavy and light chains. Specifically, it binds to FcγRIIb present on immune effector cells, and specifically binds to FcγRIIb present on the surface of immune effector cells. If such an antibody would have a usual or ordinary Fc region, the antibody may also have bound to the activating Fcγ receptor through the normal interaction between the Fc region and the Fc receptor. However, according to the present invention, the antibody molecule that specifically binds to FcγRIIb completely lacks the Fc region or has reduced binding to the Fcγ receptor, which means that the antibody molecule that specifically binds to or interacts with FcγRIIb binds poorly to the Fcγ receptor or is unable to bind to or interact with it at all. This appears to have at least two therapeutically important consequences:

[0033] 1) The lack of Fc-mediated binding to activating FcγRs leaves a greater number of activating Fcγ receptors available for Fc binding of (other) therapeutic anti-cancer antibodies. This is important because the increasing number of activating FcγRs (vs. inhibitory FcγRs; Nimmerjahn et al.; Science 2005 Dec 2;310(5753):1510-2) is known to increase effector cell-mediated target cell depletion—a mechanism that underlies the activity of both checkpoint inhibitors and immune agonists, as well as other immunomodulatory antibodies (e.g., anti-IL-2R).

[0034] 2) Lack or reduction of Fc-mediated binding to inhibitory FcγRs has been shown to reduce inhibitory signaling in FcγR-expressing immune effector cells. Therefore, lack or reduction of Fc-mediated binding to FcγRs of FcγRIIB-targeting antibodies may improve therapeutic efficacy through at least two mechanisms involving both improved activating FcγRs and reduced inhibitory Fcγ signaling in immune effector cells in response to a second immunomodulatory anti-cancer antibody.

[0035] By "reduced binding" or "binding with reduced affinity" in this context is meant a reduction in Fc-mediated binding of the antibody molecule to an Fcγ receptor, or in other words, the Fc region of the antibody molecule that specifically binds to FcγRIIb binds to the activating Fcγ receptor with a lower affinity than the Fc region of a normal human IgG1. The reduction in binding can be assessed using techniques such as surface plasmon resonance. In this context, "normal IgG1" means a conventionally produced IgG1 with a non-mutated Fc region that has not been generated to alter its glycosylation. As a reference to this "normal IgG1", it is possible to use rituximab produced in CHO cells without any modification (Tipton et al., Blood 2015 125: 1901-1909; rituximab is described, inter alia, in EP 0 605 442).

[0036] By "reduced binding" is meant that binding of the Fc region of an antibody molecule that specifically binds to FcγRIIb to an activating Fcγ receptor is reduced by at least 10-fold for all Fc receptors compared to binding of the Fc region of normal human IgG1 to the same receptor. In some embodiments, it is reduced by at least 20-fold. In some embodiments, it is reduced by at least 30-fold. In some embodiments, it is reduced by at least 40-fold. In some embodiments, it is reduced by at least 50-fold. In some embodiments, it is reduced by at least 60-fold. In some embodiments, it is reduced by at least 70-fold.

[0037] In some embodiments of the invention, the antibody molecule that specifically binds FcγRIIb is not bound to its Fc region at all, and in some such cases, the antibody has no Fc region; it may then be a Fab, Fab'2, scFv, or a PEGYLATED version thereof.

[0038] In some embodiments, the antibody molecule that specifically binds to FcγRIIb can be a lama antibody, and specifically a lama hcIgG. Like all mammals, camelids produce conventional antibodies consisting of two heavy chains and two light chains, which are bound together by disulfide bonds in a Y shape (IgG1). However, they also produce two unique subclasses of immunoglobulin G, IgG2 and IgG3, also known as heavy chain IgG (hcIgG). These antibodies are composed of only two heavy chains, which lack the CH1 region but still have a V domain at their N-termini. H The antigen binding domain of H. Conventional Ig requires the association of variable regions from both heavy and light chains to allow for high diversity of antigen-antibody interactions. Although isolated heavy and light chains still exhibit this ability, they exhibit very low affinity when compared to paired heavy and light chains. 4A unique feature of hcIgG is the ability of its monomeric antigen-binding domain to bind antigen with specificity, affinity, and notably diversity, comparable to conventional antibodies without the need for pairing with another domain.

[0039] In some embodiments, reduced binding means that the affinity of the antibody for binding to FcyRI is reduced by 20-fold.

[0040] In order to obtain a reduction in the binding of IgG1 antibodies (such as IgG1 antibodies) to Fc receptors, it is possible to modify the Fc region of IgG antibodies by deglycosylation. This deglycosylation, for example the deglycosylation of IgG1 antibodies, can be achieved, for example, by amino acid substitution of the asparagine at position 297 (N297X) in the antibody chain. The substitution can be carried out with glutamine (N297Q), or with alanine (N297A), or with glycine (N297G), or with asparagine (N297D), or by serine (N297S).

[0041] The Fc region can be modified by further substitutions, for example, as described by Jacobsen FW et al., Journal of Biological Chemistry (JBC) 2017, 292, 1865-1875 (see, for example, Table 1). Such additional substitutions include L242C, V259C, A287C, R292C, V302C, L306C, V323C, I332C and / or K334C. Such modifications also include combinations of the following substitutions in IgG1:

[0042] L242C, N297G, K334C;

[0043] A287C, N297G, L306C;

[0044] R292C, N297G, V302C;

[0045] N297G, V323C, I332C; and

[0046] V259C, N297G, L306C.

[0047] Alternatively, cells that can enzymatically cleave carbohydrates in the Fc region and / or cells used to produce the antibody can be grown in media impaired for carbohydrate addition and / or engineered to lack the ability to add sugars can be used for antibody production, or by producing the antibody in host cells that do not glycosylate or functionally do not glycosylate the antibody, such as prokaryotes including E. coli, as explained above.

[0048] Reduction in affinity for Fcγ receptors can be further achieved by engineering amino acids in the Fc region of the antibody (such modifications have been previously described by Xencor, Macrogenics, and Genentech), or by producing the antibody in a host cell that does not glycosylate or functionally does not glycosylate the antibody, such as a prokaryotic organism including E. coli.

[0049] In addition to reduced binding to Fcγ receptors via the Fc region, in some embodiments, it is preferred that antibody molecules that specifically bind to FcγRIIb do not cause phosphorylation of FcγRIIb upon target binding. Phosphorylation of the ITIM of FcγRIIb is an inhibitory event that blocks activity in immune cells.

[0050] Fcγ receptor expression immune effector cells mainly refer to innate effector cells in this article, and specifically include macrophages, neutrophils, monocytes, natural killer (NK) cells, basophils, eosinophils (eiosinophil), mast cells and platelets. Cytotoxic T cells and memory T cells do not express FcγRs usually, but may do so in specific cases. In certain embodiments, immune effector cells are innate immune effector cells. In certain embodiments, immune effector cells are macrophages.

[0051] In contrast to antibody molecules that specifically bind to FcγRIIb, antibody molecules that specifically bind to or interact with receptors present on target immune cells, i.e., second antibody molecules or immune cell depleting or deactivating antibody molecules, have an Fc region that binds to or interacts with activating Fcγ receptors to an extent that is not reduced or at least not substantially reduced. The immune cells bound by the second antibody molecule, i.e., immune cell depleting or deactivating antibody molecules, are immune cells that suppress anti-cancer immunity, and binding of the second antibody to the cells causes depletion or deactivation of the immune cells, which may belong to the innate (e.g., TAM, TAN, or MDSC) or adaptive (e.g., T cell) arm of the immune system.

[0052] Depletion of cells herein refers to the depletion, deletion or elimination of immune cells by physical removal of cells. Specifically, the depletion of cells refers to the depletion of immune cells within the tumor, or the depletion of tumor-associated immune cells, such as those present in the tumor-draining lymph nodes.

[0053] Inactivation of immune cells herein refers to blocking or reducing activity, e.g., reducing cytokine, growth factor, arginase, or nitric oxide production. In this context, inactivation of immune cells also encompasses the skewing of immune cells such that their pro-tumor phenotype is altered to an anti-tumor phenotype, e.g., by reducing the release of anti-inflammatory cytokines, reducing the release of pro-angiogenic growth factors, and increasing the release of pro-inflammatory cytokines, as well as increasing reactive oxygen species (ROS), phagocytosis, or ADCC activity.

[0054] Further explanation below is provided on how to determine whether an antibody is an immune cell depleting or deactivating antibody.

[0055] The immune cell to which the second antibody molecule specifically binds is an immune cell that suppresses anti-cancer immunity. In this context, anti-cancer immunity includes but is not limited to inducing adaptive T cell-mediated anti-cancer immunity, including generating a memory recall response.

[0056] The immune cell that the second antibody molecule specifically binds to can be a regulatory T cell. regs (formerly known as suppressor T cells, sometimes also known as inhibitory regulatory T cells) are T cell subsets that can suppress other immune cells in normal and pathological immune environments. The immune cells to which the second antibody molecule specifically binds can alternatively be bone marrow cells, specifically tumor-associated bone marrow cells. In some embodiments, tumor-associated bone marrow cells are tumor-associated macrophages, which are sometimes represented as TAMs. In some embodiments, the tumor-associated bone marrow cells are tumor-associated neutrophils, which are sometimes represented as TANs. In some embodiments, the tumor-associated bone marrow cells are dendritic cells. In some embodiments, the tumor-associated bone marrow cells are bone marrow-derived suppressor cells, which can be monocytes or granulocyte types.

[0057] In addition to specifically binding to the target on the immune cell, the second antibody molecule binds to an activated Fcγ receptor present on the same immune effector cell to which the first antibody molecule binds via its Fc region and / or to an activated Fcγ receptor present on another immune effector cell. To be able to bind to the activated Fcγ receptor, at least in some embodiments, the Fc region of the second antibody should be glycosylated at position 297. Carbohydrate residues in this position contribute to binding to the Fcγ receptor. In some embodiments, it is preferred that these residues are biphasic carbohydrates containing GlnNAc, mannose, a terminal galactose residue, and sialic acid. It should contain the CH2 portion of the Fc molecule.

[0058] Cancers to be treated or treatable according to the present invention are FcγRIIb negative cancers, meaning that the cancer is one in which no FcγRIIb receptors are present. This can be tested using anti-FcγRIIB specific antibodies in various methods including immunohistochemistry and flow cytometry, as indicated in Tutt et al., J Immunol 2015, 195(11): 5503-5516.

[0059] Antibodies are well known to those skilled in the art of immunology and molecular biology. Typically, antibodies include two heavy (H) chains and two light (L) chains. In this article, this complete antibody molecule is sometimes referred to as a full-size or full-length antibody. The heavy chain of an antibody includes a variable domain (VH) and three constant domains (CH1, CH2, and CH3), and the light chain of the antibody includes a variable domain (VL) and a constant domain (CL). The variable domains (sometimes collectively referred to as Fv regions) bind to the target or antigen of the antibody. Each variable domain includes three loops, which are referred to as complementary determining regions (CDRs), which are responsible for target binding. The constant domains do not directly participate in the combination of the antibody and the antigen, but exhibit various effector functions. According to the amino acid sequence of the heavy chain constant region of an antibody or immunoglobulin, the antibody or immunoglobulin can be divided into different classes. Immunoglobulins are divided into five major classes: IgA, IgD, IgE, IgG, and IgM, and in humans, several of these classes are further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, and IgG4; IgAl and IgA2.

[0060] Another part of the antibody is the Fc region (also called the fragment crystallizable domain), which includes two constant domains of the constant domains of each of the antibody heavy chains. As mentioned above, the Fc region is responsible for the interaction between the antibody and the Fc receptor.

[0061] As used herein, the term antibody molecule encompasses full-length or full-size antibodies as well as functional fragments of full-length antibodies and derivatives of such antibody molecules.

[0062] The functional fragment of a full-size antibody has the same antigen-binding properties as the corresponding full-size antibody and contains the same variable domains (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-size antibody. The functional fragment having the same antigen-binding properties as the corresponding full-size antibody means that the functional fragment binds to the same epitope on the target as the full-size antibody. Such a functional fragment may correspond to the Fv portion of the full-size antibody. Alternatively, such a fragment may be a Fab, also denoted as a monovalent antigen-binding fragment F(ab), or a bivalent antigen-binding fragment F(ab')2, wherein the monovalent antigen-binding fragment does not contain the Fc portion and the bivalent antigen-binding fragment contains two antigen-binding Fab portions linked together by a disulfide bond or F(ab') (i.e., a monovalent variant of F(ab')2). Such a fragment may also be a single-chain variable fragment (scFv).

[0063] Functional fragments do not always contain all six CDRs of the corresponding full-size antibody. It should be understood that molecules containing three or fewer CDR regions (in some cases, even just a single CDR or a portion thereof) can retain the antigen-binding activity of the antibody from which one or more CDRs are derived. For example, Gao et al., 1994, Journal of Biological Chemistry, 269: 32389-93, describe that the entire VL chain (containing all three CDRs) has a high affinity for its substrate.

[0064] Molecules containing two CDR regions are described in the following literature: for example, Vaughan and Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4: 417-430. On page 418 (right column - 3 Our design strategy), miniantibodies containing only H1 and H2 CDR hypervariable regions interspersed within the framework region are described. The miniantibodies are described as being able to bind to the target. Vaughan and Sollazzo cite the following literature: Pessi et al., 1993, Nature, 362: 367-9 and Bianchi et al., 1994, J. Mol. Biol., 236: 649-59, which describe the H1 and H2 miniantibodies and their properties in more detail. In Qiu et al., 2007, Nature Biotechnology, 25: 921-9, it was demonstrated that molecules consisting of two linked CDRs are able to bind to antigens. Quiocho 1993, Nature, 362: 293-4, provides a summary of the “minibody” technology. Ladner 2007, Nature Biotechnology, 25: 875-7, indicates that molecules containing two CDRs can retain antigen binding activity.

[0065] Antibody molecules containing a single CDR region are described in the following literature: for example, Laune et al., 1997, J. Biol. Chem., 272: 30937-44, in which a series of hexapeptides derived from CDRs were shown to exhibit antigen binding activity, and it was noted that synthetic peptides of intact single CDRs exhibited strong binding activity. In Monnet et al., 1999, J. Biol. Chem., 274: 3789-96, a series of 12-mer peptides and associated framework regions were shown to have antigen binding activity, and it was noted that only CDR3-like peptides were able to bind antigen. In Heap et al., 2005, J. Gen. Virol., 86: 1791-1800, it was reported that "minibodies" (molecules containing a single CDR) were able to bind antigen, and a cyclic peptide from an anti-HIV antibody was shown to have antigen binding activity and function. In Nicaise et al., 2004, Protein Science, 13: 1882-91, it was shown that a single CDR can confer antigen binding activity and affinity for its lysozyme antigen.

[0066] Thus, antibody molecules with five, four, three, or fewer CDRs can retain the antigen-binding properties of the full-length antibody from which the CDRs are derived.

[0067] The antibody molecule can also be a derivative of a full-length antibody or a fragment of such an antibody. When a derivative is used, the derivative should have the same antigen-binding properties as the corresponding full-length antibody, in the sense that the derivative binds to the same epitope on the target as the full-length antibody.

[0068] Therefore, as used herein, the term "antibody molecule" includes all types of antibody molecules and functional fragments and derivatives thereof, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, antibodies of human origin, humanized antibodies, chimeric antibodies, single-chain antibodies, single-chain Fv (scFv), Fab fragments, F(ab')2 fragments, F(ab') fragments, disulfide-linked Fv (sdFv), antibody heavy chains, antibody light chains, homodimers of antibody heavy chains, homodimers of antibody light chains, heterodimers of antibody heavy chains, heterodimers of antibody light chains, and antigen-binding functional fragments of such homodimers and heterodimers.

[0069] Further, as used herein, the term "antibody molecule" includes all classes of antibody molecules and functional fragments, including: IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD and IgE, unless otherwise specified.

[0070] In some embodiments, the antibody is human IgG1. It will be appreciated by those skilled in the art that mouse IgG2a and human IgG1 bind to activating Fcγ receptors and share the ability to activate deletions of target cells by activating immune cells bearing activating Fcγ receptors, for example, through ADCP and ADCC. Thus, in embodiments where mouse IgG2a is the preferred isotype for deletions in mice, human IgG1 is the preferred isotype for deletions in humans in such embodiments.

[0071] As mentioned above, the present invention encompasses different types and forms of antibody molecules and are known to those skilled in the art of immunology.It is well known that antibodies used for therapeutic purposes are often modified with additional components that modify the properties of the antibody molecule.

[0072] Thus, it is encompassed that the antibody molecule of the invention or used according to the invention (eg, a monoclonal antibody molecule and / or a polyclonal antibody molecule and / or a bispecific antibody molecule) comprises a detectable moiety and / or a cytotoxic moiety.

[0073] A "detectable moiety" comprises one or more substances from the group consisting of: an enzyme; a radioactive atom; a fluorescent moiety; a chemiluminescent moiety; a bioluminescent moiety. A detectable moiety allows the antibody molecule to be visualized in vitro and / or in vivo and / or ex vivo.

[0074] A "cytotoxic moiety" comprises a radioactive moiety and / or an enzyme, wherein the enzyme is a caspase and / or a toxin, wherein the toxin is a bacterial toxin or venom; wherein the cytotoxic moiety is capable of inducing cell lysis.

[0075] It is further contemplated that the antibody molecule may be in isolated and / or purified form and / or may be pegylated. Pegylation is a process in which polyethylene glycol polymers are added to a molecule (e.g., an antibody molecule or derivative) to modify its behavior, for example, by increasing its hydrodynamic size to extend its half-life, thereby preventing renal clearance.

[0076] As described above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein conform to the definitions according to Kabat EA et al., 1991, "Sequences of Proteins of Immunological Interest," Fifth Edition, NIH Publication No. 91-3242, pp. xv-xvii.

[0077] As will be appreciated by the skilled artisan, other methods exist for assigning amino acids to each CDR, for example, the International Immunogenetics Information System (IMGT(R)) (http: / / www.imgt.org / , and Lefranc and Lefranc, "The Immunoglobulin Facts Book," published by Academic Press, 2001).

[0078] In another embodiment, the antibody molecule of the invention or the antibody molecule used according to the invention is an antibody molecule that can compete with a specific antibody provided herein, for example, an antibody molecule comprising any of the amino acid sequences set forth in, for example, SEQ ID NOs: 1-194 for binding to a specific target.

[0079] "Capable of competing" means that the competing antibody is able to at least partially inhibit or otherwise interfere with the binding of an antibody molecule as defined herein to a specific target.

[0080] For example, such a competing antibody molecule may be capable of inhibiting the binding of an antibody molecule described herein by at least about 10%; (e.g., at least about 20% or at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, about 100%) and / or inhibiting the ability of an antibody described herein to prevent or reduce binding to a specific target by at least about 10%; (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or about 100%).

[0081] Competitive binding can be determined by methods well known to those skilled in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0082] Epitope modifications or blocking antibodies can be assessed using ELISA assays. Additional methods suitable for identifying competing antibodies are described in Antibodies: A Laboratory Manual, Harlow and Lane, incorporated herein by reference (e.g., see pages 567 to 569, 574 to 576, 583, and 590 to 612, 1988, CSHL, New York, ISBN 0-87969-314-2).

[0083] It is well known that antibodies specifically bind to or interact with a defined target molecule or antigen. That is, antibodies preferentially and selectively bind to their target over non-target molecules.

[0084] The target of the antibody according to the invention or the target of the antibody used according to the invention is expressed on the surface of a cell, i.e. the target is a cell surface antigen, which will comprise the epitope of the antibody (also referred to in this context as a cell surface epitope). Cell surface antigens and epitopes are terms that are readily understood by those skilled in the art of immunology or cell biology.

[0085] "Cell surface antigens" include those that are exposed on the extracellular side of the cell membrane, but may only be exposed transiently. "Transient exposure" includes those that can be internalized into the cell or released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens can be released from the extracellular side of the cell membrane by cleavage, which may be mediated by a protease.

[0086] It also includes that the cell surface antigen may be attached to the cell membrane, but may only be transiently associated with the cell membrane. "Transient association" includes that the cell surface antigen can be released from the extracellular side of the cell membrane into the extracellular space. The cell surface antigen can be released from the extracellular side of the cell membrane by cleavage, which can be mediated by a protease.

[0087] It further comprises: the cell surface antigen may be a peptide, or a polypeptide, or a carbohydrate, or an oligosaccharide chain, or a lipid; and / or an epitope present on a protein, a glycoprotein, or a lipoprotein.

[0088] Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. Those skilled in the art will appreciate that those methods can be used to assess the binding of an antibody to a target and / or the binding of an antibody's Fc region to an Fc receptor; and relative strength or specificity, or the inhibition, prevention, or reduction of those interactions. Examples of methods that can be used to assess protein binding are, for example, immunoassays, BIAcore, Western blots, radioimmunoassays (RIA), and enzyme-linked immunosorbent assays (ELISAs) (for discussion of antibody specificity, see Fundamental Immunology, 2nd edition, Raven Press, New York; pp. 332-336 (1989).

[0089] Thus, a "specifically binding antibody molecule" or "target-specific antibody molecule" includes antibody molecules that specifically bind to a target but do not bind to non-targets, or bind to non-targets more weakly than to a target (e.g., with low affinity).

[0090] Also encompassed is the fact that the antibody specifically binds to a target at least two times, or at least five times, or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times, or at least 200 times, or at least 500 times, or at least about 1000 times as strongly as the antibody specifically binds to a non-target.

[0091] Additionally, the following meanings are included: if the antibody is expressed with at least about 10 -1 K d , or at least about 10 -2 K d , or at least about 10 - 3 K d , or at least about 10 -4 K d , or at least about 10 -5 K d , or at least about 10 -6 K d , or at least about 10 -7 K d, or at least about 10 -8 K d , or at least about 10 -9 K d , or at least about 10 -10 Kd, or at least about 10 -11 K d , or at least about 10 -12 Kd, or at least about 10 -13 K d , or at least about 10 -14 K d , or at least about 10 -15 K d K d If the antibody specifically binds to the target, the antibody binds to the target specifically.

[0092] As used herein, the term immune cell depleting antibody molecule or immune cell deactivating antibody molecule refers to an antibody molecule that specifically binds to a target expressed on the surface of an immune cell when administered to a patient, wherein such binding causes depletion or deactivation of the immune cell. In some embodiments, the target is a target that is preferentially expressed on a tumor or in a tumor microenvironment.

[0093] In order to decide whether an antibody molecule is an immune cell depleting antibody molecule in the sense of the present invention, it is possible to use an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay or an antibody-dependent cellular phagocytosis (ADCP) assay. In order to decide whether an antibody molecule is an immune cell depleting antibody molecule, the same assay will be performed in the presence and absence of a depleting antibody, which will show whether the depleting antibody to be tested is actually depleting.

[0094] ADCC assay can be completed by labeling target cells with calcein AM (acetyl methyl ester) and subsequently adding diluted concentrations of antibodies. The target cells are then co-cultured with human peripheral blood mononuclear cells (PBMC) at an effector: target (E: T) ratio of 50: 1 at 37 ° C for 4 hours. The plate is centrifuged at 400 x g for 5 minutes to precipitate the cells, and the supernatant is transferred to a white 96-well plate. The release of calcein is measured using Varioskan (Thermo Scientific) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The maximum release percentage is calculated as follows: maximum release %=(sample / treated triton)*100.

[0095] ADCP assays can be performed by labeling target cells with 5 mM succinimidyl ester (CFSE) for 10 minutes at room temperature before washing in medium containing fetal bovine serum. CFSE-labeled targets are then opsonized with diluted concentrations of antibodies before co-culturing with bone marrow-derived macrophages (BMDM) at an E:T ratio of 1:5 in 96-well plates at 37°C for 1 hour. BMDM are then labeled with anti-F4 / 80-allophycocyanin for 15 minutes at room temperature and washed twice with PBS. The plates are placed on ice, the wells are scraped to collect the BMDM, and phagocytosis is assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4 / 80+CFSE+ cells within the F4 / 80+ cell population.

[0096] It is also possible to use the method described in Cleary et al., J. Immunol., 12 Apr. 2017, 1601473.

[0097] In some embodiments, the antibody molecule that specifically binds FcγRIIb is a human antibody.

[0098] In some embodiments, the antibody molecule that specifically binds FcγRIIb is an antibody of human origin, ie, an antibody originally derived from a human that has been modified as described herein.

[0099] In some embodiments, the antibody molecule that specifically binds to FcγRIIb is a humanized antibody, ie, an antibody originally derived from a non-human that has been modified to increase its similarity to a human antibody. The humanized antibody can be, for example, a murine antibody or a lama antibody.

[0100] In some embodiments, an antibody molecule that specifically binds to FcγRIIb comprises the following constant regions (CH and CL):

[0101]

[0102] These constant regions (SEQ ID NO: 1 and SEQ ID NO: 2) are of human origin. The Fc region is further modified to reduce binding to Fcγ receptors via its Fc region. As described herein, in some embodiments, it is preferred that SEQ ID NO: 1 has been deglycosylated by an N297Q substitution, and the IgG1-CH then has the following CH sequence [SEQ ID NO: 195], wherein the 297Q residue is marked in bold:

[0103]

[0104] In some embodiments and / or examples, mouse antibody molecules are used. These mouse antibody molecules can also be used to replace antibodies. Then, these mouse antibody molecules can include the following constant regions (CH and CL):

[0105]

[0106] Thus, these constant regions (SEQ ID NO: 196 and SEQ ID NO: 197) are of murine origin. SEQ ID NO: 196 includes an N297A mutation (residue 297A is marked in bold in the sequence above). This N297A mutation in the murine sequence corresponds to the N297Q mutation in the human sequence.

[0107] In some embodiments, the antibody molecule that specifically binds to FcγRIIb comprises one or more sequences from the following clones:

[0108] Antibody clone: ​​1A01

[0109]

[0110]

[0111] CDR area

[0112] CDRHl:DYYMN [ SEQ ID NO: 51]

[0113] CDRH2:LIGWDGGSTYYADSVKG [ SEQ ID NO:52]CDRH3:AYSGYELDY [ SEQ ID NO:53]CDRL1:SGSSSNIGNNAVN [ SEQ ID NO: 54] CDRL2: DNNNRPS [ SEQ ID NO: 55]

[0114] CDRL3:AAWDDSLNASI [ SEQ ID NO: 56]

[0115] Antibody clone: ​​1B07

[0116]

[0117] CDR area

[0118] CDRH1:SYGMH [ SEQ ID NO: 57]

[0119] CDRH2: FTRYDGSNKYYADSVRG [ SEQ ID NO: 58] CDRH3: ENIDAFDV [ SEQ ID NO: 59] CDRL1: SGSSSNIGNNAVN [ SEQ ID NO: 60] CDRL2: DNQQRPS [ SEQ ID NO: 61]

[0120] CDRL3: WDDRLFGPV [ SEQ ID NO: 62]

[0121] Antibody clone: 1C04

[0122]

[0123] CDR area

[0124] CDRH1: SYAMS [ SEQ ID NO: 63]

[0125] CDRH2: SISDSGAGRYYADSVEG [ SEQ ID NO: 64]

[0126] CDRH3: THDSGELLDAFDI [ SEQ ID NO: 65]

[0127] CDRL1: SGSSSNIGSNHVL [ SEQ ID NO: 66]

[0128] CDRL2: GNSNRPS [ SEQ ID NO: 67]

[0129] CDRL3: AAWDDSLNGWV [ SEQ ID NO: 68]

[0130] Antibody clone: 1E05

[0131]

[0132] CDR area

[0133] CDRH1: TYAMN [ SEQ ID NO: 69]

[0134] CDRH2: VISYDGSNKNYVDSVKG[ SEQ ID NO: 70]

[0135] CDRH3: NFDNSGYAIPDAFDI [ SEQ ID NO: 71]

[0136] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 72]

[0137] CDRL2: DNNSRPS [ SEQ ID NO: 73]

[0138] CDRL3: AAWDDSLGGPV [ SEQ ID NO: 74]

[0139] Antibody clone: 2A09

[0140]

[0141] CDR area

[0142] CDRH1: NAWMS [ SEQ ID NO: 75]

[0143] CDRH2: YISRDADITHYPASVKG [ SEQ ID NO: 76]

[0144] CDRH3: GFDYAGDDAFDI [ SEQ ID NO: 77]

[0145] CDRL1: SGSSSNIGSNAVN [ SEQ ID NO: 78]

[0146] CDRL2: GNSDRPS [ SEQ ID NO: 79]

[0147] CDRL3: AAWDDSLNGRWV [ SEQ ID NO: 80]

[0148] Antibody clone: 2B08

[0149]

[0150] CDR area

[0151] CDRH1: DYYMS [SEQ ID NO: 81

[0152] CDRH2: LIGHDGNNKYYLDSLEG [ SEQ ID NO: 82

[0153] CDRH3: ATDSGYDLLY [ SEQ ID NO: 83

[0154] CDRL1: SGSSSNIGNNAVN [ SEQ ID NO: 84

[0155] CDRL2: YDDLLPS [ SEQ ID NO: 85

[0156] CDRL3: TTWDDSLSGVV [ SEQ ID NO: 86

[0157] Antibody clone: 2E8-VH

[0158]

[0159] CDR area

[0160] CDRH1: DYYMS [ SEQ ID NO: 87

[0161] CDRH2: AIGFSDDNTYYADSVKG [ SEQ ID NO: 88]CDRH3: GDGSGWSF [ SEQ ID NO: 89]CDRL1: SGSSSNIGNNAVN [ SEQ ID NO: 90]CDRL2: DNNKRPS [ SEQ ID NO: 91

[0162] CDRL3: ATWDDSLRGWV [ SEQ ID NO: 92

[0163] Antibody clone: 5C04

[0164]

[0165] CDR area

[0166] CDRH1: NYGMH [ SEQ ID NO: 93

[0167] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 94]

[0168] CDRH3: WRDAFDI [ SEQ ID NO: 95]

[0169] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 96]

[0170] CDRL2: SDNQRPS [ SEQ ID NO: 97]

[0171] CDRL3: AAWDDSLSGSWV [ SEQ ID NO: 98]

[0172] Antibody Clone: 5C05

[0173]

[0174] CDR area

[0175] CDRH1: TYGMH [ SEQ ID NO: 99]

[0176] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 1 00]

[0177] CDRH3: ENFDAFDV [ SEQ ID NO: 101] [[ID='48]]

[0178] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 102]<00'00517>

[0179] CDRL2: SNSQRPS [ SEQ ID NO: 1 03]

[0180] CDRL3: AAWDDSLNGQVV [ SEQ ID NO: 104]

[0181] Antibody Clone: 5D07

[0182] ]」 <'

[0183] CDR area

[0184] CDRH1: TYGMH [ SEQ ID NO: 105]

[0185] CDRH2: VIAYDGSKKDYADSVKG [ SEQ ID NO: 106]

[0186] CDRH3: EYRDAFDI [ SEQ ID NO: 107]

[0187] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 108]

[0188] CDRL2: GNSNRPS [ SEQ ID NO: 109]

[0189] CDRL3: AAWDDSVSGWM [ SEQ ID NO: 110]

[0190] Antibody clone: 5E12 <00005​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0201]

[0202] CDR area

[0203] CDRH1: NYGMH [ SEQ ID NO: 117]

[0204] CDRH2: VISYDGSNRYYADSVKG [ SEQ ID NO: 118]

[0205] CDRH3: DRWNGMDV [ SEQ ID NO: 119]

[0206] CDRL1: SGSSSNIGAGYDVH [ SEQ ID NO: 120]

[0207] CDRL2: ANNQRPS [ SEQ ID NO: 121]

[0208] CDRL3: AAWDDSLNGPWV [ SEQ ID NO: 122]

[0209] Antibody clone: 5H06

[0210]

[0211] CDR area

[0212] CDRH1: SYGMH [ SEQ ID NO: 123]

[0213] CDRH2: VISYDGSDTAYADSVKG [ SEQ ID NO: 124]

[0214] CDRH3: DHSVIGAFDI [ SEQ ID NO: 125]

[0215] CDRL1: SGSSSNIGSNTVN [ SEQ ID NO: 126]

[0216] CDRL2: DNNKRPS [ SEQ ID NO: 127]

[0217] CDRL3: SSYAGSNNVV[ SEQ ID NO: 128]

[0218] Antibody Clone: 6A09

[0219]

[0220] CDR area

[0221] CDRH1: SYGMH [ SEQ ID NO: 129]

[0222] CDRH2: VTSYDGNTKYYANSVKG [ SEQ ID NO: 130]

[0223] CDRH3: EDCGGDCFDY [ SEQ ID NO: 131]

[0224] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 132]

[0225] CDRL2: GNSNRPS [ SEQ ID NO: 133]

[0226] CDRL3: AAWDDSLNEGV [ SEQ ID NO: 134]

[0227] Antibody Clone: 6B01

[0228]

[0229] CDR area

[0230] CDRH1: NYGMH [ SEQ ID NO: 135]

[0231] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 136]

[0232] CDRH3: DQLGEAFDI [ SEQ ID NO: 137] [[ID=6�]]

[0233] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 138]

[0234] ]>CDRL2: DNNKRPS [SEQ ID NO: 139

[0235] CDRL3: ATWDDSLSGPV [ SEQ ID NO: 140

[0236] Antibody clone: 6C11

[0237]

[0238] CDR area

[0239] CDRH1: DYGMS [ SEQ ID NO: 141

[0240] CDRH2: AISGSGSSTYYADSVKG [ SEQ ID NO: 142

[0241] CDRH3: GDIDYFDY [ SEQ ID NO: 143

[0242] CDRL1: TGSSSNFGAGYDVH [ SEQ ID NO: 144

[0243] CDRL2: ENNKRPS [ SEQ ID NO: 145

[0244] CDRL3: AAWDDSLNGPV [ SEQ ID NO: 146

[0245] ]>Antibody clone: 6C12

[0246]

[0247] CDR area

[0248] CDRHl: SYGMH [ SEQ ID NO: 147

[0249] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 148

[0250] CDRH3: ERRDAFDI [ SEQ ID NO: 149

[0251] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 150

[0252] CDRL2: SDNQRPS [ SEQ ID NO: 151

[0253] CDRL3: ATWDSDTPV [ SEQ ID NO: 152

[0254] Antibody clone: 6D01

[0255]

[0256] CDR area

[0257] CDRH1: SYGMH [ SEQ ID NO: 153

[0258] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 154

[0259] CDRH3: DHSAAGYFDY [ SEQ ID NO: 155

[0260] CDRL1: SGSSSNIGSNTVN [ SEQ ID NO: 156

[0261] CDRL2: GNSIRPS [ SEQ ID NO: 157

[0262] CDRL3: ASWDDSLSSPV [ SEQ ID NO: 158

[0263] Antibody clone: 6G03

[0264]

[0265] CDR area

[0266] CDRH1: SYGMH [ SEQ ID NO: 159

[0267] CDRH2: GISWDSAIIDYAGSVKG [ SEQ ID NO: 160

[0268] CDRH3: DEAAAGAFDI [ SEQ ID NO: 161

[0269] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 162]

[0270] CDRL2: GNTDRPS [ SEQ ID NO: 163]

[0271] CDRL3: AAWDDSLSGPVV [ SEQ ID NO: 164]

[0272] Antibody Clone: 6G08

[0273]

[0274] CDR area

[0275] CDRH1: SYGIS [ SEQ ID NO: 165]

[0276] CDRH2: GISGSGGNTYYADSVKG [ SEQ ID NO: 166]

[0277] CDRH3: SVGAYANDAFDI [ SEQ ID NO: 167]

[0278] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 168]

[0279] CDRL2: GDTNRPS [ SEQ ID NO: 169]

[0280] CDRL3: AAWDDSLNGPV [ SEQ ID NO: 170]

[0281] Antibody Clone: 6G11

[0282]

[0283] CDR area

[0284] ]>CDRH1: SYGMH [ SEQ ID NO: 171]

[0285] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 172]

[0286] CDRH3: ELYDAFDI [ SEQ ID NO: 173]

[0287] CDRL1: TGSSSNIGAGYDVH [ SEQ ID NO: 174]

[0288] CDRL2: ADDHRPS [ SEQ ID NO: 175]

[0289] CDRL3: ASWDDSQRAVI [ SEQ ID NO: 176]

[0290] Antibody clone: 6H08

[0291]

[0292] CDR area

[0293] CDRH1: NYGMH [ SEQ ID NO: 1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0303] CDRH2: VISYDGSNKYYADSVKG [ SEQ ID NO: 184]

[0304] CDRH3: EFGYIILDY [ SEQ ID NO: 185]

[0305] CDRL1: SGSSSNIGSNTVN [ SEQ ID NO: 186]

[0306] CDRL2: RDYERPS [ SEQ ID NO: 187]

[0307] CDRL3: MAWDDSLSGVV [ SEQ ID NO: 188]

[0308] Antibody Clone: 4B02

[0309]

[0310] CDR area

[0311] CDRH1: NHGMH [ SEQ ID NO: 189]

[0312] CDRH2: VISYDGTNKYYADSVRG [ SEQ ID NO: 190]

[0313] CDRH3: ETWDAFDV [ SEQ ID NO: 191]

[0314] CDRL1: SGSSSNIGSNNAN [ SEQ ID NO: 192]

[0315] CDRL2: DNNKRPS [ SEQ ID NO: 193]

[0316] CDRL3: QAWDSSTVV [ SEQ ID NO: 194]

[0317] In some embodiments, sometimes preferred embodiments, the antibody molecule that specifically binds to FcγRIIb includes the following CDR regions: SEQ ID NO: 171 (CDRH1), SEQ ID NO: 172 (CDRH2), SEQ ID NO: 173 (CDRH3), SEQ ID NO: 174 (CDRL1), SEQ ID NO: 175 (CDRL2) and SEQ ID NO: 176 (CDRL3), i.e., the CDR regions of clone 6G11.

[0318] In some embodiments, sometimes preferred embodiments, an antibody molecule that specifically binds to FcγRIIb comprises the following constant regions: SEQ ID NO: 1 (CH) and SEQ ID NO: 2 (CL); and the following variable regions: SEQ ID NO: 23 (VL) and SEQ ID NO: 47 (VH), i.e., the constant and variable regions of clone 6G11, which have been further modified to reduce binding to Fcγ receptors via its Fc region. In some embodiments, sometimes preferred embodiments, an antibody molecule that specifically binds to FcγRIIb comprises the following constant regions: SEQ ID NO: 195 (CH) and SEQ ID NO: 2 (CL); and the following variable regions: SEQ ID NO: 23 (VL) and SEQ ID NO: 47 (VH), i.e., the constant and variable regions of clone 6G11 comprising the N297Q mutation.

[0319] In some embodiments, the immune cell depletion or deactivation antibody molecule is a human antibody molecule or an antibody molecule of human origin. In some such embodiments, the human antibody molecule or the antibody molecule of human origin is an IgG antibody. In some such embodiments, the human antibody molecule or the antibody molecule of human origin is an IgG1 or IgG2 antibody.

[0320] In some embodiments, the immune cell-depleting or inactivating antibody molecule is a humanized antibody molecule.

[0321] In some embodiments, the immune cell-depleting or inactivating antibody molecule is a chimeric antibody.

[0322] As described above, the immune cell-depleting or -activating antibody molecule must have the ability to bind to FcγR.

[0323] The target bound by the immune cell-depleting or deactivating antibody molecule according to the present invention may be selected from the group consisting of CTLA-4, 4-1BB, OX40, TNFR2, PD-L1, IL-2R and GITR.

[0324] In some embodiments of the present invention, the target bound by the immune cell depletion or deactivation antibody molecule according to the present invention is CTLA-4. CTLA-4 or CTLA4, which stands for cytotoxic T lymphocyte-associated protein 4, is also known as CD152. It is a protein receptor that acts as an immune checkpoint that downregulates the immune response. CTLA4 is constitutively expressed in regulatory T cells, but is only upregulated in conventional T cells after activation - a phenomenon that is particularly significant in cancer. In some such embodiments, the immune cell depletion antibody molecule is ipilimumab (e.g., from Bristol-Myers Squibb) In some such embodiments, the immune cell-depleting antibody molecule is tremelimumab (formerly denoted ticilimumab and CP-675,206), a fully human monoclonal antibody against CTLA-4 previously developed by Pfizer and currently in clinical development by MedImmune.

[0325] In some embodiments of the present invention, at least one target is 4-1BB, which is also expressed as CD137 and tumor necrosis factor receptor superfamily member 9 (TNFRSF9). 4-1BB is expressed on Treg after CD4+ and CD8+ T cell activation, and its connection is necessary for the optimal protective CD8T cell response against mouse antiviral and B cell lymphoma. Anti-4-1BB specific antibodies enhance the proliferation and survival rate of antigen-stimulated T cells in vitro, and similar to anti-CD40, anti-4-1BB mAb relies heavily on promoting anti-tumor immunity in preclinical cancer models of CD8T cells. In some such embodiments, the immune cell depletion antibody molecule is urelumab (urelumab), which is a humanized agonist IgG4 monoclonal antibody developed by Bristol-Myers Squibb. In some such embodiments, the immune cell-depleting antibody molecule is utomilumab (also denoted PF-05082566, PF-2566, and PF-5082566), a human HuCAL mAb agonist of 4-1BB developed by Pfizer.

[0326] In some embodiments of the present invention, at least one target is OX40. OX40 (also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4) and CD134) is a secondary co-stimulatory immune checkpoint molecule. In some such embodiments, the immune cell depletion antibody molecule is MEDI6469 (9B12), MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383 (see, e.g., Buchan et al., Blood>>2018131: Table 1 of 39-48).

[0327] In some embodiments of the invention, at least one target is TNFR-2. Tumor necrosis factor receptor 2 (TNFR-2 or TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a membrane receptor that binds tumor necrosis factor alpha (TNFa).

[0328] In some embodiments of the present invention, the target bound by the immune cell depleting or deactivating antibody molecule according to the present invention is programmed death ligand 1 (PD-L1), also known as CD274 or B7 homolog 1 (B7-H1).

[0329] In some embodiments of the present invention, at least one target is IL-2R. IL-2R is also known as CD25 and is highly expressed primarily on regulatory T cells.

[0330] In some embodiments of the invention, at least one target is GITR. GITR is a member of the TNFSFR family and is also primarily expressed on regulatory T cells.

[0331] In some embodiments, the antibody molecule that specifically binds to FcγRIIb and the immune cell depleting or deactivating antibody molecule are administered to the patient simultaneously, which means that the two antibody molecules can be administered together at one time point or separately and very close in time to each other.

[0332] In some embodiments, an antibody molecule that specifically binds to FcγRIIb is administered to the patient prior to administering the immune cell depletion or deactivation antibody molecule. This sequential administration can be achieved by temporarily separating the two antibodies. Alternatively, or in combination with the first option, sequential administration can also be achieved by spatially separating the two antibody molecules; administering the antibody molecule that specifically binds to FcγRIIb in a manner (such as intratumorally) such that it reaches the cancer before the immune cell depletion antibody molecule, and then administering the immune cell depletion antibody molecule in a manner (such as systemically) such that it reaches the cancer after the antibody molecule that specifically binds to FcγRIIb.

[0333] In some embodiments, an immune cell depleting antibody is administered to the patient prior to administration of an antibody molecule that specifically binds FcγRIIb. Such sequential administration can be achieved as described above.

[0334] Those skilled in the medical arts will appreciate that drugs can be modified with various additives, for example to alter the rate at which the drug is absorbed by the body; and can be modified in various forms, for example to allow for specific routes of administration to the body.

[0335] Therefore, the composition and / or antibody and / or medicament comprising the present invention may be combined with an excipient and / or a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable diluent and / or an adjuvant.

[0336] It is also contemplated that the compositions and / or antibodies and / or drugs of the present invention may be suitable for parenteral administration, comprising aqueous and / or non-aqueous sterile injection solutions which may contain antioxidants and / or buffers and / or bacteriostats and / or solutes that render the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions which may contain suspending agents and / or thickening agents. The compositions and / or antibodies and / or agents and / or drugs of the present invention may be present in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately prior to use.

[0337] Extemporaneous injection solutions and suspensions may be prepared from sterile powders and / or granules and / or tablets of the kind previously described.

[0338] For parenteral administration to human patients, the daily dosage level of the antibody molecule that specifically binds to FcγRIIb and / or the immune cell depleting or deactivating antibody molecule, administered in a single or divided dose, will typically be from 1 mg / kg to 20 mg / kg of patient body weight, or in some cases even up to 100 mg / kg. In special cases, for example, in conjunction with long-term administration, lower doses may be used. In any case, the physician will determine the actual dosage that is most suitable for any individual patient, and the actual dosage will vary with the age, weight, and response of the particular patient. The above dosages are examples of general circumstances. Of course, in individual cases, higher or lower dosage ranges should be used, and these dosage ranges are within the scope of the present invention.

[0339] Typically, the compositions and / or medicaments of the present invention will contain an antibody molecule that specifically binds to FcγRIIb and / or an immune cell depleting or deactivating antibody at a concentration of between about 2 mg / ml and 150 mg / ml, or between about 2 mg / ml and 200 mg / ml. In a preferred embodiment, the medicaments and / or compositions of the present invention will contain an antibody molecule that specifically binds to FcγRIIb and / or an immune cell depleting or deactivating antibody at a concentration of 10 mg / ml.

[0340] Typically, in humans, oral or parenteral administration of the compositions and / or antibodies and / or medicaments and / or drugs of the present invention is the most convenient preferred route. For veterinary use, the compositions and / or antibodies and / or medicaments and / or drugs of the present invention are administered as appropriately acceptable formulations according to normal veterinary practice, and the veterinarian will determine the dosage regimen and route of administration that is most suitable for a particular animal. Therefore, the present invention provides a pharmaceutical formulation comprising an amount of antibodies and / or medicaments of the present invention (as described above and further described below) that are effective for treating various conditions. Preferably, the compositions and / or antibodies and / or medicaments and / or drugs are suitable for delivery by a route selected from the group consisting of: intravenous (IV); subcutaneous (SC); intramuscular (IM) or intratumoral.

[0341] In certain embodiments, the first antibody molecule or the second antibody or both can be administered by using a plasmid or a virus.Such plasmids then include the nucleotide sequence encoding the first antibody molecule or the second antibody or both.In certain embodiments, the nucleotide sequence encoding part or all of the sequence of the first antibody molecule or the second antibody or both is integrated into a cell or viral genome or integrated into a viral group (viriome) in a virus;Then, such cells or viruses serve as the first antibody molecule or the second antibody or both delivery vehicles (or the first antibody molecule or the second antibody or both are encoded by the nucleotide sequence delivery vehicle).For example, in certain embodiments, this virus can be in the form of a therapeutic oncolytic virus, and the therapeutic oncolytic virus includes a nucleotide sequence encoding at least one antibody molecule in an antibody molecule described herein.In certain embodiments, this oncolytic virus includes a nucleotide sequence encoding a full-length human IgG antibody.Oncolytic viruses are known to those skilled in the art of medicine and virology.

[0342] The present invention also encompasses compositions and / or antibodies and / or medicaments and / or drugs comprising a polypeptide binding portion of the present invention in the form of a pharmaceutically acceptable acid addition salt or base addition salt. Acids used to prepare pharmaceutically acceptable acid addition salts of the above-mentioned base compounds useful in the present invention are acids that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, sucrose, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts, among others. Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the agents according to the present invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the agents of the present invention are chemical bases that form non-toxic base salts with such compounds, which base salts are acidic in nature. Such non-toxic base salts include, but are not limited to, base salts derived from such pharmaceutically acceptable cations, such as base salts of alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium); ammonium or water-soluble amine addition salts, such as N-methylglucamine-(meglumine); and other base salts of lower alkanolammonium and pharmaceutically acceptable organic amines, etc. The agents and / or polypeptide binding moieties of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique can be used. Those skilled in the art will understand that lyophilization and reconstitution may result in varying degrees of antibody activity loss (e.g., for conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies), and that use levels may have to be adjusted upward to compensate. In one embodiment, the lyophilized (freeze-dried) polypeptide binding moiety loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (before lyophilization) when rehydrated.

[0343] The combination of an antibody molecule that specifically binds to FcγRIIb and an immune cell-depleting or inactivating antibody molecule can be used to treat cancer.

[0344] As used herein, the term "patient" refers to an animal, including a human, that has been diagnosed with an FcyRIIb-negative cancer or a cancer that is thought to be an FcyRIIb-negative cancer and / or exhibits symptoms of such a cancer.

[0345] It is contemplated that the patient may be a mammal or a non-mammal. Preferably, the patient is a human or a mammal, such as a horse, or a cow, or a sheep, or a pig, or a camel, or a dog or a cat. Most preferably, the mammalian patient is a human.

[0346] “Manifestation” includes: the subject manifests cancer symptoms and / or cancer diagnostic markers, and / or the cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0347] For those skilled in the medical field, it is easy to understand what the symptoms of cancer and cancer diagnostic markers will be and how to measure and / or evaluate and / or quantify whether the severity of cancer symptoms decreases or increases, or whether cancer diagnostic markers decrease or increase; and how those cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0348] Cancer treatment is typically administered as a course of treatment, that is, the therapeutic agent is administered over a period of time. The length of a course of treatment depends on many factors, including the type of therapeutic agent administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the patient, among other factors.

[0349] "During treatment" includes: the patient is currently receiving a course of treatment, and / or is receiving a therapeutic agent, and / or is in the process of receiving a therapeutic agent.

[0350] In some embodiments, the FcyRIIb-negative cancer to be treated according to the invention is a solid cancer.

[0351] In some embodiments, the cancer is selected from the group consisting of carcinoma, sarcoma, and lymphoma.

[0352] In some embodiments, the cancer is a carcinoma selected from the group consisting of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic or undifferentiated carcinoma, large cell carcinoma, and small cell carcinoma.

[0353] In some embodiments, the cancer is a sarcoma selected from the group consisting of osteosarcoma, chondrosarcoma, liposarcoma, and leiomyosarcoma.

[0354] The FcγRIIb-negative cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, prostate cancer, metastatic hormone-refractory prostate cancer, colorectal cancer, lung cancer, small cell lung cancer (NSCLC), small cell lung cancer (SCLC), non-small cell lung cancer, urothelial carcinoma, bladder cancer, kidney cancer, mesothelioma, Merkel cell carcinoma, and head and neck cancer.

[0355] Each of the above cancers is well known, and symptoms and cancer diagnostic markers and therapeutic agents for treating the cancers are also well described. Thus, the symptoms, cancer diagnostic markers and therapeutic agents for treating the above cancer types are known to those skilled in the medical field.

[0356] Clinical definitions of the diagnosis, prognosis, and progression of a wide range of cancers rely on certain classifications known as staging. These staging systems are used to collate a wide range of different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of a cancer. Those skilled in the art of oncology will understand how to use staging systems to assess the diagnosis and / or prognosis and / or progression of a cancer, as well as which cancer diagnostic markers and cancer symptoms should be used to make that assessment.

[0357] "Cancer stage" includes: Rai staging, which includes stage 0, stage I, stage II, stage III, and stage IV; and / or Binet staging, which includes stage A, stage B, and stage C; and / or Ann Arbor staging, which includes stage I, stage II, stage III, and stage IV.

[0358] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur reproducibly in certain cancers, which means that examining these morphological changes (also referred to as histological examination) can be used for diagnosis or prognosis of cancer. Techniques for visualizing samples to examine cell morphology and preparing samples for visualization are well known in the art; for example, optical microscopy or confocal microscopy.

[0359] "Histological examination" includes: the presence of small mature lymphocytes; and / or the presence of small mature lymphocytes with narrow cytoplasmic borders; the presence of small mature lymphocytes with pyknotic nuclei lacking discernible nucleoli; and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and with pyknotic nuclei lacking discernible nucleoli; and / or the presence of atypical cells and / or lysed cells and / or prolymphocytes.

[0360] As is well known, cancer is the result of mutations in the cell's DNA, which may cause the cell to avoid cell death or to proliferate uncontrollably. Therefore, checking these mutations (also referred to as cytogenetic tests) may be a useful tool for assessing cancer diagnosis and / or prognosis. An example of this is the deletion of chromosome position 13q14.1, a characteristic of chronic lymphocytic leukemia. The techniques for checking mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

[0361] "Cytogenetic examination" includes examination of the DNA in cells, and specifically of the chromosomes. Cytogenetic examination can be used to identify changes in the DNA that may be associated with the presence of refractory cancer and / or recurrent cancer. Such changes may include: a deletion in the long arm of chromosome 13; and / or a deletion at chromosome position 13q14.1; and / or a trisomy of chromosome 12; and / or a deletion in the long arm of chromosome 12; and / or a deletion in the long arm of chromosome 11; and / or a deletion of 11q; and / or a deletion in the long arm of chromosome 6; and / or a deletion of 6q; and / or a deletion in the short arm of chromosome 17; and / or a deletion of 17p; and / or a t( 11:14) translocation; and / or (q13:q32) translocation; and / or antigen gene receptor rearrangement; and / or BCL2 rearrangement; and / or BCL6 rearrangement; and / or t(14:18) translocation; and / or t(11:14) translocation; and / or (q13:q32) translocation; and / or (3:v) translocation; and / or (8:14) translocation; and / or (8:v) translocation; and / or t(11:14) and (q13:q32) translocations.

[0362] It is known that patients with cancer exhibit certain physical symptoms, which are generally due to the burden of the cancer on the body. Those symptoms often recur in the same cancer and can therefore serve as diagnostic and / or prognostic and / or progression characteristics of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers and will assess how those physical symptoms relate to the diagnosis and / or prognosis and / or progression of the disease. "Physical symptoms" include hepatomegaly and / or splenomegaly. BRIEF DESCRIPTION OF THE DRAWINGS

[0363] In the examples below, reference is made to the following figures:

[0364] Figure 1 The potential mechanism that explains why the present invention works is presented. Immune cell depleting or deactivating antibody molecules (in this case, anti-CTLA-4 antibodies) bind to receptors present on immune cells (in this case, Tregs) that suppress anti-cancer immunity. The Fc region of the anti-CTLA-4 antibody binds to activated Fcγ receptors, which in this case are present on the surface of macrophages. In addition, antibody molecules that specifically bind to FcγRIIb bind to FcγRIIb present on the surface of macrophages. Because the Fc region of this specific antibody has reduced binding to activated Fcγ receptors, it does not bind to any activated Fcγ receptors present on macrophages. Instead, the activated Fcγ receptors are free to bind to the Fc region of the anti-CTLA-4 antibody.

[0365] Figure 1A shows the use of only anti-CTLA-4 antibodies. Anti-CTLA-4 antibodies bind to and activate both activating and inhibitory Fcγ receptors, resulting in weak activation and, therefore, a reduced effect compared to when anti-CTLA-4 antibodies bind only to activating Fcγ receptors.

[0366] Figure 1 B shows that the combination of anti-CTLA-4 antibody and WT (wild type) FcγRIIb antibody results in reduced activation. In this case, the FcγRIIb antibody blocks activating Fcγ receptors and activates inhibitory Fcγ receptors, which is the opposite of the desired effect according to the present invention.

[0367] Figure 1 C shows that the combination of an anti-CTLA-4 antibody and a deglycosylated FcγRIIb antibody according to the present invention results in maximum activation. There is no blockade of activating Fcγ receptors and no activation of inhibitory Fcγ receptors, resulting in maximum target depletion. Here, the deglycosylated FcγRIIb antibody can be an antibody tethered to an Fc domain or have additional reduced binding to FcRs.

[0368] Figure 2 It was demonstrated that FcγRIIB antibodies with impaired Fc binding to activating Fc receptors, but not wild-type antibodies with retained binding to activating Fc receptors, promoted B cell depletion by CD20 mAb.

[0369] ( Figure 2 AB) CFSE + hCD20 + / ×mFcγRII - / - (target) and mFcγRII - / - (Non-target) splenocytes injected into hFcγRIIB + / ×mFcγRII - / - Recipient mice. Mice received WT or N297Q FcγRIIB mAb (6G11) (2 x 20 mg / kg) followed by Rit (0.2-2 mg / kg), and blood (A) and spleen (B) CFSE were determined as described previously. + CD19 + The data from at least 2 independent experiments were combined. Figure 2 C) CFSE + hCD20 + / ×mFcγRII - / - (target) and mFcγRII - / - (Non-target) splenocytes injected into hFcγRIIB + / ×mFcγRII - / -Recipient mice. Mice received WT or N297Q FcγRIIB mAb (6G11) (20 mg / kg) followed by Rit (2 mg / kg) and were immunostained in spleen F4 / 80 with the indicated mAb. + Quantification of activating mFcγR expression on effector cells. Figure 2 DE) WT and N297Q (NQ) hFcγRIIB-specific mAbs (6G11; 10 μg / ml for 15 min) were cultured at high density in ( Figure 2 D) Mouse BMDM and ( Figure 2 E) Ability to induce hFcγRIIB ITIM phosphorylation (pFcγRIIB) on isolated primary peripheral blood mononuclear cells. α-Tubulin, GAPDH and hFcγRIIB were used as loading controls as indicated; representative blots are shown.

[0370] Figure 3 It was demonstrated that WT and FcyR-free FcyRIIB mAbs can be combined to achieve optimal depletion of target cells.

[0371] CFSE + hCD20 + / - (target) and mFcγRII - / - (Non-target) splenocytes injected into hFcγRIIB + / - ×mFcγRII - / - (Balb / c) recipient mice. Mice received WT (2x10-20 mg / kg) or F(ab')2 (2x20 mg / kg) mFcγRII (AT130-5) or WT (2x20 mg / kg) or F(ab')2 (2x40 mg / kg) hFcγRIIB mAb (AT10), followed by Rit (0.2-2 mg / kg) as indicated on the X-axis, and spleen CFSE was determined as previously described. + CD19 + Ratio of cells. Data from one to three independent experiments were combined. Each point depicts the results from a single mouse, with the mean ratio indicated by a horizontal line. Data were analyzed using one-way ANOVA.

[0372] Figure 4 shows the assessment of Treg depletion in the context of anti-IL2R mAb + / - FcγRIIB blocking with Fc-inert-NA mutant mAb. Figure 4A ) Female Balb / c mice were given 100 μg of AT130-2NA by intraperitoneal injection (ip). Six hours later, 100 μg of PC61 was given ip. Four days later, the Treg (FoxP3) in the blood, spleen, and lymph nodes was determined by FACs. +Mice were sacrificed and single-cell suspensions were obtained from spleen, LN, and blood. These suspensions were stained with antibodies against CD4, CD8, and B220 before intracellular FoxP3 staining, followed by analysis using FACS canto. Leukocyte counts were determined for each tissue. Tregs were defined as CD8-CD4+FoxP3+, and the number of Tregs was calculated using leukocyte counts. Figure 4B ) As above, but using C57BL / 6 mice. Figure 4C )Depend on Figure 4B ) Calculate the CD8 / Treg ratio.

[0373] Figure 5 Figure 2 shows the evaluation of Treg depletion in the presence of anti-IL2R mAb blocking with wild-type or NA mutant mAbs + / - FcγRIIB. WT AT130-2 did not appear to provide any improvement in depletion; whereas NA variants did. A) Female Balb / c mice were given 100 μg AT130-2NA or mIgG1 WT AT130-2 ip. Six hours later, 100 μg PC61 was given ip. Tregs in the spleen were determined 4 days later by FACs (FoxP3 + Mice were sacrificed and single cell suspensions were obtained from the spleen. The single cell suspensions were stained with antibodies against CD4, CD8, and B220 before intracellular FoxP3 staining and then analyzed using FACS canto. The white blood cell count of each tissue was determined. Tregs were defined as CD8-CD4+FoxP3+, and the number of Tregs was calculated using white blood cell counts. Compared to wild-type mIgG1 AT130-2, the number of Tregs in the spleens of mice that received the combination of N297A antibody and PC61 for 4 days was significantly reduced (unpaired T test, P=0.044).

[0374] Figure 6 shows the combination therapy using anti-CTLA-4 and FcγRIIB blockade. 5 CT26 cells were injected SC into female BALB / c mice. When the tumor width × length was approximately 100 mm 2The mice were randomized into treatment groups. Treatment was performed on days 0, 2, 4, and 11. Only 9H10 (hamster anti-mouse CTLA4) mice received 200 μl PBS containing 200 μg of antibody IP every day. On day 0, combination mice received 200 μl PBS containing 100 μg AT130-2 N297A (anti-mouse CD32) IP, and these combination mice received 200 μl PBS containing 200 μg 9H10 IP 6 hours later. On days 2, 4, and 11, combination mice received two antibodies (200 μg 9H10 and 100 μg AT130-2NA) in a single 200 μl IP injection. The width and length of the tumor were measured, and when the length × width of the tumor exceeded 400 mm, the tumor was injected with 200 μl of PBS. 2 When , the mice were killed. Figure 6A ) indicates the treatment regimen. Group 1: no Ab; Group 2: anti-mCD32 (AT130-2NA; 100 μg); Group 3: anti-CTLA-4 (9H10; 200 μg); Group 4: combination (PC61) injected 6 hours after AT130-2 injection. Tumors were allowed to establish and were placed at 100 mm 2 An additional dose was given on day 12. Figure 6B ) shows the growth of individual tumors. Figure 6C ) represents mean tumor area + / - SD or SEM. Figure 6D ) represents the animal survival rate. Figure 6E ) Complexes from 2 separate experiments (n=10 / group) show survival rates and demonstrate that the combination of 9H10 and AT130-2NA (NA combination) is significantly more effective in extending survival than 9H10 alone (p=0.0179).

[0375] Figure 7 Combination therapy comparing anti-CTLA-4 and FcγRIIB blockade by WT (denoted as M1 combination) and Fc-inert (denoted as NA combination) AT130-2 mAb was presented. 5 CT26 cells were injected SC into female BALB / c mice. When the tumor width × length was approximately 100 mm 2The mice were randomized to the treatment groups at 0, 2, 4 and 11 days. Treatment was performed on day 0, 2, 4 and 11. On day 0, the combination mice received 200 μl PBS containing 100 μg AT130-2N297A or AT130-2mIgG1 (anti-mouse CD32) in IP, and 6 hours later these combination mice received 200 μl PBS containing 200 μg 9H10 in IP. On day 2, 4 and 11, the combination mice received two antibodies (200 μg 9H10 and 100 μg AT130-2 NA / mIgG1) in a single 200 μl IP injection. The width and length of the tumor were measured, and when the length × width of the tumor exceeded 400 mm, the tumor was injected with 200 μl of PBS. 2 The mice were sacrificed at 4 hr. The data are shown in the survival curve below for N = 11-12. There was a significantly longer survival in the NA combination treatment group compared to mice receiving the mlgG1 combination (log-rank test P = 0.0460).

[0376] Figure 8 demonstrates combination therapy with anti-PD-L1 and FcγRIIB blockade, and to address whether this effect depends on the NA form of the antibody, experiments were performed using both WT mIgG1 and NA forms. Figure 8A The growth of individual tumors in each group is shown (one graph per group). The numbers indicate the number of surviving mice in each group. Figure 8B Shown is the survival curve of mice.

[0377] Figure 9 Shown are wild type (top) and N297A (bottom) forms of the anti-mouse FcyRIIB mAb AT130-2 binding to FcyRs.SPR analysis of AT130-2 binding to the indicated mouse FcyRs (100 nM) in either form is shown.

[0378] Figure 10 shows binding of WT 6G11 versus N297A 6G11 to Fcγ receptors (mouse and human). It shows SPR analysis of binding of native and N297A anti-huCD32b 6G11 hIgG1 to the indicated low affinity human and mouse FcγRs. Figure 10A A and B show the results for human FcγR (100 nM). Figure 10C A and D show the results for mouse FcγR (100 nM). Figure 10E A and E show the results for human FcγR (added in increasing series)-FcγRIIb and hCD64. Figure 10G A and H show the results for mouse FcγR (100 nM), ie, the same as panels c and d, but rescaled for clearer viewing.

[0379] Examples

[0380] Specific, non-limiting examples embodying certain aspects of the present invention will now be described. In order to allow examination of the blocking effect of FcγRIIB in a complex in vivo system, two sets of surrogate antibodies must be used. The mouse equivalent of 6G11 is called AT130-2. In order to silence the human antibody Fc (and therefore to severely weaken or negligible binding to FcγR), amino acid position 297 has been substituted from N to Q. In order to silence the mouse antibody Fc, the same position is substituted from N to Q. Therefore, in the mouse system, reference will be made to AT-130, while this patent application relates to the human counterpart 6G11. In short, human 6G11 corresponds to the mouse surrogate AT1302-2, while 6G11-N297Q corresponds to AT130-3-N297A.

[0381] A different way to silence the Fc of an antibody (and well known to those skilled in the art) is to remove the Fc portion and form a Fab or Fab2 fragment.

[0382] Experimental procedures

[0383] animal

[0384] hCD20Tg (transgenic), hFcγRIIB + / - and mFcγRIIB - / - Mice have been previously described with genotypes confirmed by PCR and / or flow cytometry (Beers et al., Blood, 2008 Nov 15;112(10):4170-7; Roghanian et al., Cancer Cell 27, 473-488, 2015 Apr 13). Mice were bred and maintained in local facilities according to UK Home Office guidelines or the local Swedish Ethical committee.

[0385] Cell culture

[0386] Cell culture was performed in supplemented RPMI (RPMI 1640 containing 2 mM glutamine, 1 mM pyruvate, 100 IU / ml penicillin and streptomycin, and 10% FCS [Myoclone]) (GIBCO BRL, Paisley, Scotland). Mouse splenic B cells were purified by negative selection using the MACSB cell isolation kit (Miltenyi Biotec, UK) and cultured in the same medium. The cell line was obtained from ECACC and maintained in RPMI medium without antibiotic supplementation.

[0387] Generation of human monocyte-derived macrophages (MDM) and mouse bone marrow-derived macrophages (BMDM)

[0388] Human MDM were mixed with peripheral blood obtained from the National Blood Service, Southampton General Hospital (Southampton, UK) or from hospitals in Halmstad or Skåne University Hospital ( Peripheral blood differentiation was obtained from the blood center at the University Hospital (Sweden). Briefly, adherent CD14+ monocytes were cultured in supplemented RPMI containing 25-100 ng / mL endotoxin-low recombinant human macrophage-colony stimulating factor (M-CSF; R&D Systems, USA or in-house production) as previously described (Roghanian et al., Cell Immunol. 2010; 265(2): 120-6.). Half of the culture medium was replaced with fresh M-CSF every 2 days until harvest. On day 7-10 of culture, after a brief incubation with cold PBS, MDMs were harvested.

[0389] Mouse BMDMs were generated from cells isolated from the bone marrow of mouse femurs and tibias as previously reported (Williams et al., J Immunol. 2013 Oct 15;191(8):4130-40. Briefly, bone marrow cells were cultured in supplemented RPMI containing 20% ​​L929 cell-conditioned medium (containing M-CSF). Cells were cultured at 37°C, 5% CO2 for 10-12 hours prior to use. Macrophage differentiation was routinely confirmed by morphological examination and / or flow cytometry for CD11b and F4 / 80 expression.

[0390] Antibodies and reagents

[0391] mAbs are typically produced from culture supernatants of hybridomas or stably transfected CHO-k1 cells (obtained from ECACC). F(ab')2 fragments were generated as previously described (Glennie et al., 1987). hFcγRII mAb AT10 was previously described (Greenman et al., 1991). Anti-CTLA4 (9H10; Bio X Cell, USA), anti-IL2R (PC-61.5.3; Bio X Cell / in-house), anti-PDL-1 (10F.9G2 Bio X Cell, USA). hFcγRII mAb 6G11 hIgG1 and N297Q were produced by BioInvent (see Roghanian et al., Cancer Cell 27, 473-488, April 13, 2015). mFcγRII mAb AT130-2mIgG1, mIgG2a, and mIgG1 N297A were produced in-house. AT130-5 (Williams et al., Eur J Immunol. 2012; 42(8): 2109-20 and Tutt et al., J Immunol. 2015, 195(11): 5503-5516) is a murine anti-mouse FcγRII antibody similar to the human antibody clone 6G11. Antibodies against hFcγRIIB (clone EP888Y; Abcam, UK), phosphorylated hFcγRIIB (clone EP926Y; Origene, USA), GAPDH (Abcam, UK), and α-tubulin (Cell Signaling, USA) were used for immunoblotting. For PBMC immunophenotyping, PE-labeled FcγRIIB mAb was used in combination with anti-CD3-FITC, anti-CD19-PerCP-Cy5.5, and anti-CD56-APC (antibodies obtained from Biolegend) using the zenon labeling kit (Molecular Probes).

[0392] Flow cytometry

[0393] Fluorescently conjugated mAbs were purchased from BD Biosciences, eBiosciences, BioPharm, AbD Serotec (all UK) or manufactured in-house. Flow cytometry was performed as previously described (Tutt et al., 1998) using samples evaluated on a FACScan, FACSCalibur, or FACSCanto II, with data analyzed using CellQuestPro, FACSDiva (all BD Biosciences, UK), or FCS Express (De Novo Software, California, USA).

[0394] Western blotting

[0395] As previously described (Roghanian et al., Cancer Cell 27, 473-488, April 13, 2015).

[0396] In vivo immunotherapy

[0397] Adoptive transfer: As previously described (Beers et al. Blood 2010 Jun 24;115(25):5191-201).

[0398] B cell depletion: Mice were given hCD20 or hFcyRIIB mAb iv alone or in combination, and leukocytes were assessed as previously described (Beers et al. Blood 2010 Jun 24;115(25):5191-201).

[0399] CT26

[0400] CT26 cells were maintained in complete DMEM and harvested using trypsin-EDTA. Cells were washed, resuspended in PBS, and the concentration was adjusted to 5 × 10 cells / mL using a hemocytometer. 6 cells / ml. 100 μl of cell suspension (5×10 5 The cells were injected sc into BALB / c mice (bred in-house using original materials obtained from Charles River Laboratories, UK). Tumors were allowed to establish and tumor size was measured three times per week before randomization and treatment. 2 When the tumor is diagnosed, it is considered to be advanced.

[0401] MC38

[0402] MC38 cells were maintained in complete DMEM and harvested using trypsin-EDTA. Cells were washed, resuspended in PBS, and the concentration was adjusted to 5 × 10 using a hemocytometer. 6cells / ml. 100 μl of cell suspension (5×10 5 The cells were injected sc into C56 / B16 mice (obtained from Taconic, Denmark). Tumors were allowed to establish and tumor size was measured before randomization and treatment. 2 Tumor volumes were measured twice weekly thereafter. Four treatments were performed with 3-4 days between treatments, and the dose of anti-PD-L1 was set at 10 mg / kg and the dose of both AT130-2 variants was set at 20 mg / kg. When the tumor volume exceeded 2000 mm 2 When the tumor is diagnosed, it is considered to be advanced.

[0403] Statistical analysis

[0404] To compare experimental groups, Wilcoxon, paired or unpaired t-test analysis was performed; Kaplan Meier curves were generated and analyzed by log-rank test. For in vivo adaptive transfer assays containing >2 groups, one-way or two-way ANOVA was used.

[0405] For differences in OR and CR, chi-square tests were used. Statistical analysis was performed using GraphPad Prism (v5 or 6). Asterisks indicate the following significance: *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001, unless otherwise stated.

[0406] result

[0407] The efficiency of B cell depletion depends on the FcγRIIB mAb format

[0408] FcγRIIB is expressed on both target B cells and effector monocytes / macrophages, making it difficult to interpret where FcγRIIB mAbs contribute their effects to the more pronounced absence of target cells. To further dissect this, various hFcγRIIB Tg and KO mouse strains were utilized to provide a system in which target cells or effectors or both can be targeted with FcγRIIB mAbs. + / - Target adoptive transfer to hFcγRIIB + / - ×mFcγRIIB - / - In the assay with FcγR alone and WT FcγRIIB mAb treatment had no effect on B cell depletion, as expected ( Figure 2 A). No FcγR FcγRIIB mAb enhances circulation when combined with rituximab ( Figure 2 A) and tissue residency ( Figure 2 B) Depletion of both target cells, while WT FcγRIIB mAb attenuated the deletion. This demonstrates that target cell depletion by secondary antibody is attenuated using normal FcγRIIB mAb, which was quite unexpected for the inventors. When evaluating FcγR expression in spleen F4 / 80 from treated mice + When the expression on macrophages was investigated, it was evident that mFcγRIV ( Figure 2 C) was detected at a lower level, which could partly explain the inhibitory effect of the WT FcyRIIB mAb. This suggests that the normal IgG FcyRIIB mAb worsens the deletion by blocking activation of FcyRIV.

[0409] This so-called scorpion effect (reviewed by Hogarth) occurs when a functional Fc domain from a cell surface-binding mAb occupies the Fc binding cleft of an FcγR expressed on the same cell and is therefore not observed in the absence of FcγR FcγRIIB mAb. The potential over-interpretation of the relative importance of FcγRs when blocking individual FcγRs with Fc-functional anti-FcγR mAbs (such as FcγRIV mAb 9E9) has been previously described and demonstrated (Tipton et al., Blood 2015125:1901-1909).

[0410] In addition to physical blocking, this scorpion effect has the potential to deliver receptor cross-linking and FcγR activation. Since ITIM-containing FcγRIIB is the only inhibitory FcγR on effector cells and its activation may contribute to the inhibition of effector cell function (Dahal et al., Immunol Rev. 2015 Nov; 268(1): 104-22), its activation was assessed after treatment with WT or FcγR-free FcγRIIB mAb. It was previously shown that on B cells (which only express FcγRIIB), treatment with antagonist 6G11 WT or NQ mAb did not activate FcγRIIB (Roghanian et al., Cancer Cell 27, 473-488, April 13, 2015). However, WT but not FcγR-free FcγRIIB mAb caused activation of treated human monocyte-derived macrophages (MDM) ( Figure 2 D) and mouse hFcγRIIB + / - ×mFcγRIIB - / - BMDM( Figure 2E) Phosphorylation of FcγRIIB-ITIM in both provides evidence for this phenomenon in the system and indicates that effector activation may not be optimal when using WT FcγRIIB mAb. This suggests that normal FcγRIIB mAb activates inhibitory signaling in immune effector cells. Taking this observation into consideration along with the above considerations for the optimal FcγRIIB mAb format for depletion of target cells, we next explored how optimal depletion could be achieved.

[0411] WT and FcγR-free hFcγRIIB mAbs can be combined for optimal target cell depletion

[0412] In a system where mFcγRIIB is expressed only on target B cells and hFcγRIIB is expressed only on effector cells, the efficacy of different hFcγRIIB and mFcγRII mAb formats was examined in the presence or absence of rituximab. This system enables the simultaneous analysis of target-directed and effector-restricted FcγRIIB. Initially, only the effects of mAbs that bind FcγRIIB on the target were examined. Treatment of mice with suboptimal doses of single-agent rituximab resulted in minimal depletion of target B cells ( Figure 3 Treatment of mice with an optimal dose of a single-agent WT mFcγRII mAb (targeting FcγRIIB present only on the target) resulted in approximately 50% depletion of target cells. Co-administration of the WT mFcγRII mAb with rituximab resulted in significant depletion (approximately 75% of resident splenic B cells); whereas addition of the Fc-free F(ab')2mFcγRII mAb had no effect with rituximab alone or in the presence of rituximab. This suggests that to deplete FcγRIIB-expressing targets in the absence of FcγRIIB on effectors, you would use a normal FcγRIIB mAb.

[0413] Subsequently, the specific targeting of FcγRIIB to effector cells was examined; treatment of mice with WT or F(ab')2hFcγRIIB (targeting FcγRIIB on effectors only) resulted in no deletion of B cells, as expected. However, the combination of rituximab and WT or F(ab')2hFcγRIIB caused increased target cell depletion compared to rituximab alone. Even more effective deletion was observed when WT mFcγRII mAb was used to target B cells and F(ab')2hFcγRIIB was used to target effectors. In contrast, treatment with WT mFcγRIIB mAb together with WT hFcγRIIB mAb abolished the deletion ( Figure 3 ). This indicates that normal blocking FcγRIIB mAb weaken Depletion of target: Effector blocking using Fc-modified Fab2 mAbs.

[0414] Exploring these combinations further, when WT hFcγRIIB mAb was used to block effector cell hFcγRIIB, depletion using the combination of rituximab and WT mFcγRII mAb was only about 30%. Much more significant depletion was seen when rituximab and WT mFcγRII mAb (both of which opsonize target B cells) were combined with Fc-free F(ab')2 hFcγRIIB mAb (which blocks effector cell hFcγRIIB), resulting in about 90% depletion of target cells ( Figure 3 ).

[0415] Optimal format of FcgRIIB-blocking mAb enhances Treg depletion

[0416] Next, we evaluated whether the ability to enhance target depletion by blocking FcgRIIB could be translated to other cellular targets, such as Tregs. This was similar to the above example, but Tregs were depleted using IL2R. To address this question, 100 μg of Fc-inert anti-FcγRIIb mAb (AT130-2 mIgG1 NA, Figure 9 ) ip to Balb / c mice. Subsequently, 6 hours later, 100 μg of anti-IL2R (PC61) was given ip to deplete FoxP3+ Treg cells. These were then assessed by FACs in blood, spleen, and lymph nodes 4 days later. AT130-2NA was shown to ameliorate Treg depletion, particularly in the spleen ( Figure 4A To address the reproducibility of this effect, the experiment was repeated in C57BL / 6 mice. Again, it was seen that AT130-2NA could ameliorate Treg deficiency in B6 mice, particularly in the spleen and LNs ( Figure 4B ), leading to higher CD8:Treg ratios in blood, spleen, and LN ( Figure 4C ). In the case of NA combination, the ratio in blood was significantly higher than that of PC61 alone (PC61 v combination P = 0.0218). To confirm the earlier findings, the ability of WT AT130-2 to Fc-inert AT130-2 NA mutant to improve IL2R mAb-mediated Treg deletion was next evaluated. WT AT130-2 did not give any improvement in deletion; whereas the NA variant could give an improvement (*unpaired T test, P = 0.044) ( Figure 5 ). Thus, this suggests that normal FcγRIIb blocking mAb does not improve depletion.

[0417] mAb-mediated FcgRIIB blockade enhances CTLA-4 immunotherapy

[0418] Again, this is the same concept as above, but using an antibody against yet another target that is strongly expressed on tumor-associated Treg cells (CTLA-4), leading to anti-tumor immunity. Therefore, to address whether this approach might enhance anti-cancer immunotherapy, CT26 cells were inoculated SC into female BALB / c mice. When the tumor width × length was approximately 100 mm 2 Mice were randomized to treatment groups at 4 hr. On days 0, 2, 4, and 11, mice received 200 μg of 9H10 (hamster anti-mouse CTLA4) antibody in 200 μl of PBS IP. On day 0, combination mice received 100 μg of AT130-2N297A. Tumor width and length were measured, and tumors were resected when the length × width of the tumor exceeded 400 mm. 2 The mice were killed ( Figure 6A ). Figure 6B The growth of individual tumors in each group is shown, and Figure 6C The median area is represented by . The survival curve of these mice is shown in Figure 6D , where the composite survival curve from the second experiment is shown in Figure 6E The difference in survival between the NA combination and 9H10 alone was statistically significant (log-rank test 0.0179). Finally, to address whether this effect depended on the NA form of the antibody, the experiment was repeated using WT mIgG1 and compared with the NA form as described above. The NA variants were not significantly different from the 9H10 alone group, and the NA combination was significantly more effective than the combination with the WT mAb (log-rank test P = 0.0460) ( Figure 7 ). This suggests that WT Ab, i.e., normally glycosylated mAb, cannot combine effectively and instead weakens the desired therapeutic effect.

[0419] mAb-mediated FcγRIIB blockade enhances PD-L1 immunotherapy

[0420] As with CTLA4, the effects of antibodies targeting PD-L1 are thought to depend on activating FcγRs. However, different forms of CTLA-4 and PD-L1 are expressed on a variety of cells, most notably myeloid and cancer cells. To address whether the combination of PD-L1 antibodies and FcγRIIB blockade enhances anticancer immunotherapy, MC38 cells were inoculated SC into female C57 / Bl6 mice. When the tumor width × length is approximately 100 mm 2Mice were randomized to treatment groups at 4 hr. On days 0, 2, 4, and 11, mice received 10 mg / kg 9H10 (hamster anti-mouse CTLA4) antibody in 200 μl PBS ip. On day 0, combination mice received 100 μg AT130-2N297A or WT AT130-2. Tumor width and length were measured, and tumor size was increased when tumor volume exceeded 2000 mm. 2 When , the mice were killed. Figure 8A The growth of individual tumors in each group is shown. The numbers indicate the number of surviving mice in each group. Figure 8B The survival curves of mice are shown. To address whether this effect depends on the NA form of the antibody, experiments were performed using both WT mIgG1 and the NA form as described above. The NA variants were more effective than the combination with WT mAb and resulted in more mice surviving ( Figure 8B This suggests that the most effective combination of PD-L1 antibodies, which primarily target cancer cells and monocytes / macrophages / myeloid-derived suppressor cells, is the deglycosylated NA form.

[0421] In summary, the above data show that blockade of FcγRIIB as a means to enhance the therapeutic efficacy of other antibodies is broad and applicable to antibodies against a variety of targets (CD20, CD25, CTLA4 and PD-L1) expressed on various cell types (B cells, Treg cells and myeloid cells).

[0422] Comparison of WT 6G11 versus N297A 6G11 binding to Fcγ receptors (mouse and human)

[0423] SPR analysis was performed on a Biacore T200 (GE Healthcare). Samples were run at 30 ml / min in HBS-EP+ buffer at 25°C. Data were analyzed using BiaEvaluation software. The response of the blank control flow cell was automatically subtracted before data analysis. To compare FcγR binding, 6G11WT or 6G11N297Q hIgG1 was immobilized on a CM5 sensor chip at pH 5 by amine coupling, and recombinant human or mouse FcγR (100 nM) (R&D systems) was injected across both surfaces for 180 s. Alternatively, a series of concentrations of FcγR (0-500 nM) were added sequentially, and the responses were measured. The results are shown in Figure 10A -H.

Claims

1. A first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, wherein the first antibody molecule is combined with: a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the second antibody molecule to the receptor on the immune cell causes depletion and / or deactivation of the immune cell; The combination is used to treat FcγRIIb-negative cancer in patients.

2. A pharmaceutical composition comprising: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor, and wherein binding of the second antibody to the receptor on the immune cell causes depletion and / or deactivation of the immune cell; The pharmaceutical composition is used to treat FcγRIIb-negative cancer in a patient.

3. A kit for treating FcγRIIb-negative cancer, comprising: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and wherein binding of the second antibody molecule to the receptor on the immune cell causes depletion and / or deactivation of the immune cell.

4. Use of the following antibody molecules, (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor, and wherein the binding of the second antibody to the receptor on the immune cell causes depletion and / or deactivation of the immune cell; It is used to manufacture a medicament for treating FcγRIIb-negative cancer in a patient.

5. A method for treating an FcγRIIb-negative cancer in a patient comprising administering: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, and (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that is capable of activating at least one activating Fcγ receptor, and wherein the binding of the second antibody to the receptor on the immune cell causes depletion and / or deactivation of the immune cell.

6. The first antibody molecule for use in combination with a second antibody molecule according to claim 1, the pharmaceutical composition for use according to claim 2, the kit for use according to claim 3, the use according to claim 4 or the method according to claim 5, wherein the first antibody lacks an Fc region.

7. The first antibody molecule for use in combination with the second antibody molecule according to claim 1 or 6, the pharmaceutical composition for use according to claim 2 or 6, the kit for use according to claim 3 or 6, the use according to claim 4 or 6, or the method according to claim 5 or 6, wherein the immune cells that suppress anti-cancer immunity are regulatory T cells (Tregs).

8. The first antibody molecule for use in combination with the second antibody molecule according to claim 1 or 6, the pharmaceutical composition for use according to claim 2 or 6, the kit for use according to claim 3 or 6, the use according to claim 4 or 6, or the method according to claim 5 or 6, wherein the immune cells that suppress anti-cancer immunity are bone marrow cells.

9. The first antibody molecule for use in combination with a second antibody molecule according to claim 8, the pharmaceutical composition for use according to claim 8, the kit for use according to claim 8, the use according to claim 8, or the method according to claim 8, wherein the bone marrow cells are tumor-associated macrophages.

10. The first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 9, the pharmaceutical composition for use according to any one of claims 2 or 6 to 9, the kit for use according to any one of claims 3 or 6 to 9, the use according to any one of claims 4 or 6 to 9, or the method according to any one of claims 5 or 6 to 9, wherein the FcγRIIb-negative cancer is a solid cancer.

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