Novel combinations and uses of antibodies

Abstract

The present invention describes the use of a first antibody molecule that specifically binds to FcγRIIB via its Fab region but lacks an Fc region or has reduced binding to Fcγ receptors via the Fc region, for use in combination with a second antibody molecule that specifically binds to a receptor present on tumor cells, in the treatment of FcγRIIB-negative cancer in a patient, where the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, as well as pharmaceutical compositions and kits comprising these two antibody molecules, and methods of treating cancer using these two antibodies.

Description

[Technical field]

[0001] The present invention relates to the combination of 1) an antibody molecule that specifically binds to FcγRIIB via its Fab region and that lacks an Fc region or has reduced binding to Fcγ receptors via the Fc region, and 2) an antibody molecule that specifically binds to a receptor present on tumor cells, wherein the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, in the treatment of FcγRIIB-negative cancers. [Background technology]

[0002] It has long been understood that the inhibitory Fc gamma receptor (FcγR) IIB, expressed by numerous cells of the immune system, negatively regulates both innate and adaptive immunity through the engagement of immune complexes (ICs). Similarly, the knowledge that FcγRIIB negatively regulates monoclonal antibody-mediated immunotherapy has been known for over a decade. Thus, when treated with therapeutic monoclonal antibodies (mAbs), FcγRIIB-deficient mice are able to eliminate tumors more efficiently than wild-type (WT) mice, indicating that expression of FcγRIIB on effector cells (i.e., macrophages and monocytes) results in suppression of their phagocytic and cytotoxic capabilities in vivo. Furthermore, FcγRIIB modulates the antigen-presenting capacity of dendritic cells (DCs), with FcγRIIB-negative DCs having an improved ability to activate naive T cells (van Montfoor et al., J Immunol. 2012 Jul 1;189(1):92-101). Recently, antagonistic antibodies have been developed that block FcγRIIB signaling and internalization in B cells. Such antibodies have shown efficient deletion of FcγRIIB-expressing B cells, efficiently enhanced rituximab-mediated deletion of normal and malignant B cells, and demonstrated their usefulness in hematological cancers (WO2012 / 022985). FcγRIIB blocking antibodies with wild-type IgG1 Fc with good FcγR binding function and Fc engineered for defective FcγR binding (IgG1 N297Q) showed similar ability to improve rituximab-mediated B cell depletion, indicating that the boosting effect of rituximab is independent of anti-FcγRIIB Fc. However, it has not been investigated or demonstrated whether such antibodies are also useful in enhancing the therapeutic activity of tumor-directed antibodies, such as anti-HER2 or anti-EGFR, in the treatment of FcγRIIB-negative cancers, such as most solid cancers.

[0003] Recently, the present inventors have demonstrated the distinct antitumor enhancing effects of Fc-FcγR dominant and deficient anti-FcγRIIB antibodies in the therapeutic activity of immunomodulatory antibodies against the immunosuppressive checkpoints CTLA-4 and PD-1 expressed by T cells. Specifically, in the context of anti-CTLA-4, the strongest antitumor enhancing effect was observed with Fc-FcγR deficient anti-FcγRIIB antibodies (WO2019 / 138005). Conversely, in the context of combination immunotherapy with Fc dominant but not Fc deficient anti-PD-1 antibodies, anti-FcγRIIB enhanced therapeutic antitumor activity (WO2021 / 009358). While studies in genetic knockout animals have shown the potential therapeutic enhancement effects of anti-FcγRIIB antibodies with tumor-directed targeting antibodies in both FcγRIIB+ (e.g., B cell lymphomas when combined with anti-CD20 antibodies) and FcγRIIB cancers (e.g., solid tumors in combination with anti-HER2 antibodies) (Clynes et al, Nat Med. 2000 Apr;6(4):443-6), it remained unclear whether anti-FcγRIIB antibodies would enhance the therapeutic activity of tumor-directed targeting antibodies, such as anti-HER2, in the treatment of FcγRIIB cancers, and if so, what type of anti-FcγRIIB antibody would do so. Summary of the Invention [Problem to be solved by the invention]

[0004] As used herein, an Fc region is absent or the Fc region is an FcγR, e.g., F(ab)′ 2We demonstrate that only anti-FcγRIIB antibodies that exhibit reduced or absent binding to antibodies or non-glycosylated antibodies are able to enhance the therapeutic activity of tumor-directed antibodies, such as anti-HER2 and anti-EGFR, used to treat FcγRIIB-negative cancers, including solid tumors. This is in contrast to the widespread use of Fc:FcγR-dominant, as well as Fc:FcγR-deficient, anti-FcγRIIB antibodies to boost activity and overcome resistance to B-cell-directed antibodies, such as anti-CD20 for the treatment of NHL (WO2012 / 022985), and to our previous patent applications, which describe the distinct Fc:Fcγ-dependency of anti-FcγRIIB to enhance the therapeutic activity of immunomodulatory anti-PD-1 and anti-CTLA-4 antibodies (as opposed to tumor cell-directed targets), as described in patent applications WO2021 / 009358 and WO2019 / 138005. Furthermore, the data of the present invention support the understanding that antibodies lacking an Fc region or in which the Fc region is linked to an FcγR, e.g., F(ab)′ 2 We demonstrate that combination treatment with anti-FcγRIIB antibodies that exhibit reduced or absent binding to FcγRIIB or nonglycosylated antibodies allows for anti-HER2 treatment of cancers with low HER2 expression, which has not been demonstrated for treatment with currently used clinically approved anti-HER2 regimens. [Means for solving the problem]

[0005] In treating FcγRIIB negative cancer in a patient, A second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that specifically binds to at least one activating Fcγ receptor. Disclosed herein is a first antibody molecule that specifically binds FcγRIIB via (or through) the Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via (or through) the Fc region for use in combination with

[0006] For use in treating FcγRIIB-negative cancer in a patient, (i) a first antibody molecule that specifically binds to FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor. Also disclosed herein is a pharmaceutical composition comprising:

[0007] (i) a first antibody molecule that specifically binds to FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor. Further disclosed herein is a kit for use in treating an FcγRIIB-negative cancer, comprising:

[0008] In the manufacture of a medicament for use in treating an FcγRIIB-negative cancer in a patient, (i) a first antibody molecule that specifically binds to FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor. Further disclosed herein is the use of

[0009] 1. A method of treating an FcγRIIB-negative cancer in a patient, comprising: (i) a first antibody molecule that specifically binds to FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region capable of activating at least one activating Fcγ receptor. Also disclosed herein are methods comprising administering

[0010] In the examples below, reference is made to the following figures: [Brief description of the drawings]

[0011] [Figure 1]Figure 1A and Figure 1B show the survival curves. Female BalbC mice (n=12) were subcutaneously injected with TUBO cells (1x106). Tumor growth was monitored (measured with calipers) and when tumors reached approximately 7x7mm, mice were randomized and treated twice weekly as indicated. Tumor growth was tracked twice weekly until tumors reached a predetermined size (ethical end point) at which time mice were euthanized. (Figure 1A) HER2-high cancer model. The therapeutic effect of anti-HER2 (10mg / kg) in combination with Fc null anti-FcγRIIB-NA (AT130-2NA) was compared to isotype control antibody (FITC IgG2a) and anti-HER2 (10mg / kg) single agent treatment. Mice were dosed three times (2-3 days between doses). In this model of HER2-high cancer, combination treatment with anti-FcγRIIB-NA (AT130-2NA) delayed tumor growth and increased the number of complete responders compared to anti-HER2 monotherapy. The study was repeated three times with comparable results, and the results of one representative experiment are shown in Figure 1A. (Figure 1B) HER2-low cancer model. This figure shows the therapeutic effect of anti-HER2 (1 mg / kg) in combination with Fc null anti-FcγRIIB-NA (AT130-2NA) compared to an isotype control antibody (FITC IgG2a) and 1 mg / kg anti-HER as monotherapy. Mice were dosed three times (2-3 days between doses). In this model of HER2-low cancer, combination treatment with anti-FcγRIIB-NA (AT130-2NA) delayed tumor growth and increased the number of complete responders compared to anti-HER2 treatment alone, making anti-HER2 treatment of HER2-low cancer as effective as (single agent anti-HER2) treatment of HER2-high cancer. In Figure 1C, the HER2 receptor was targeted in HER2-high and HER2-low experimental cancer models. Tumors were established as described in AB and mice (n=3) were treated with 1 mg / kg or 10 mg / kg of fluorescent dye (AF647)-labeled anti-HER2. Mice were dosed twice (2-3 days between doses), after which mice were euthanized and tumors were harvested. Tumors were enzymatically digested and fluorescent dye (AF647)-labeled anti-HER2 was quantified by FACS.Tumor-bearing mice in the HER2-high model (injected with 10 mg / kg anti-HER2) showed a 10-fold increase in targeted HER2 receptors compared to tumors from mice in the HER2-low model (injected with 1 mg / kg anti-HER2). [Diagram 2] Survival curves. Female BalbC mice (n=12) were injected subcutaneously with TUBO cells (1x106). Tumor growth was monitored (measured by caliper) and when tumors reached approximately 7x7mm, mice were randomized and treated with therapeutic mAbs twice a week. Tumor growth was followed twice a week until tumors reached a predefined size (ethical end point) at which time mice were euthanized. The therapeutic efficacy of anti-HER2 (1mg / kg) in combination with Fc null anti-FcγRIIB-NA (AT130-2NA) was compared to anti-HER2 in combination with wild-type anti-FcγRIIB (AT130-2wt), an isotype control antibody (FITC IgG2a), and anti-HER2 at 1mg / kg as a single agent treatment. Mice were dosed three times (2-3 days between doses). Anti-HER2 in combination with anti-FcγRIIB-NA (AT130-2NA) showed a delay in tumor growth compared to anti-HER2 treatment alone, which was not seen when anti-HER2 was combined with wild-type anti-FcγRIIB (AT130-2wt). [Diagram 3] Female BalbC mice were subcutaneously injected with TUBO cells (1x106). Tumor growth was monitored (measured by caliper) and when tumors reached approximately 7x7mm, mice were randomized and treated with therapeutic mAbs twice a week. Mice were sacrificed and tumors were harvested 24 hours after the third injection, i.e., 7-8 days after the start of treatment. Immune cell content of tumor single cell suspensions was analyzed by FACS. Fc null anti-FcγRIIB-NA is named AT-130-2NA in the figure. The number of myeloid cells, especially CD11b+F4 / 80+ / MHCIIlow, was significantly increased in the group treated with the combination of anti-HER2 and anti-FcγRIIB-NA. [Figure 4]Lung area covered with metastases. Female C57 mice were injected intravenously with B16 cells (5x105). Four days after tumor cell injection, mice were injected with antibodies (10mg / kg ip-isotype control, TA99, AT130-2-NA, and a combination of TA99 and AT130-2-NA). Five treatments were administered with 2-3 days interval. Twenty-one days after the start of treatment, mice were sacrificed and metastatic content in the lungs was quantified. A reduction in lung metastases was seen with TA99 alone, but the effect of TA99 was significantly increased when combined with anti-FcγRIIB-NA (AT130-2NA). Anti-FcγRIIB-NA has no therapeutic effect as monotherapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Thus, the present invention provides (i) a first antibody molecule that specifically binds to FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) The use of a second antibody molecule that specifically binds to a receptor present on a tumor cell and has an Fc region capable of activating at least one activating Fcγ receptor.

[0013] Therefore, the second antibody molecule is a tumor-directed targeting antibody, or also called a tumor-directed targeting antibody. The therapeutic activity of this antibody depends on the engagement of FcγR. The binding of the second antibody molecule to a receptor on the tumor cell and the subsequent engagement of FcγR on the immune effector cell causes redirected FcγR-dependent immune effector cell-mediated killing of the antibody-coated target tumor cell, for example, by macrophage-dependent ADCC or ADCP. The tumor-directed targeting antibody may or may not be able to kill tumor cells by additional mechanisms, for example, by blocking tumor growth factor signaling, as is believed to be the case for certain anti-HER2 antibodies. Nevertheless, the present invention is applicable to any tumor-directed targeting antibody, whose mechanism includes FcγR-dependent tumor cell killing. Therefore, the present invention is directed to maximizing therapeutic activity by optimizing FcγR-dependent tumor cell killing.

[0014] This combination is intended to be used in the treatment of FcγRIIB-negative cancers in patients to improve the therapeutic efficacy of the second antibody molecule by enhancing binding of the Fc portion to activating FcγRs while reducing binding / activation of inhibitory FcγRs.

[0015] Fc receptors are membrane proteins found on the cell surface of immune effector cells such as macrophages. The name derives from their binding specificity to the Fc region of an antibody, which is the usual way that antibodies bind to receptors. However, certain antibodies can also bind to Fc receptors through the antibody's CDR sequences, where the antibody specifically binds to one or more Fc receptors.

[0016] A subgroup of Fc receptors includes Fcγ receptors (Fc-gamma receptors, Fc gamma R, FcgR), which are specific for IgG antibodies. There are two types of Fcγ receptors: activating Fcγ receptors (also referred to as activating Fcγ receptors) and inhibitory Fcγ receptors. Activating and inhibitory receptors transmit their signals via immunoreceptor tyrosine-based activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs), respectively. In humans, FcγRIIB (FcγRIIb, FcgRIIB, CD32b) is an inhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIA (CD32a), FcγRIIC (CD32c), FcγRIIIA (CD16a), and FcγRIV are activating Fcγ receptors. FcγRIIIB is a GPI-linked receptor expressed on neutrophils that lacks ITAM motifs but is also considered activating due to its ability to cross-link lipid rafts and bind other receptors. In mice, the activating receptors are FcγRI, FcγRIII and FcγRIV.

[0017] It is well known that antibodies regulate immune cell activity through interactions with Fcγ receptors. Specifically, how antibody immune complexes regulate immune cell activation is determined by the relative engagement of activating and inhibitory Fcγ receptors. Different antibody isotypes bind to activating and inhibitory Fcγ receptors with different affinities, resulting in different A:I ratios (Nimmerjahn et al; Science. 2005 Dec 2; 310(5753):1510-2).

[0018] By binding to inhibitory Fcγ receptors, antibodies can inhibit, block, and / or downregulate effector cell function.

[0019] Antibodies can activate effector cell function by binding to activating Fcγ receptors, thereby inducing mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine release, and / or antibody-dependent endocytosis, and in the case of neutrophils, NETosis (i.e., activation and release of NETs (neutrophil extracellular traps)). Antibodies that bind to activating Fcγ receptors can also lead to an increase in certain activation markers, such as CD40, MHCII, CD38, CD80, and / or CD86.

[0020] The antibody molecule according to the invention, i.e. the first antibody, that specifically binds to FcγRIIB binds to or interacts with this Fcγ receptor via the Fab region of the antibody, i.e. via the antigen-binding region on the antibody that binds to the antigen, composed of one constant region and one variable region of each of the heavy and light chains. In particular, it binds to FcγRIIB present on immune effector cells (in particular to FcγRIIB present on the surface of immune effector cells). If this antibody has a normal or normal Fc region, it will also be able to bind to activating Fcγ receptors, due to the normal interaction of the Fc region with the Fc receptor. However, according to the invention, the antibody molecule that specifically binds to FcγRIIB completely lacks an Fc region or has reduced binding to Fcγ receptors, which means that the antibody molecule that specifically binds to FcγRIIB does not bind well to Fcγ receptors or is not able to bind or interact with Fcγ receptors at all. This appears to have at least two important therapeutic consequences: 1) The lack of Fc-mediated binding to activating FcγRs leaves many more activating Fc gamma receptors available for binding to the Fc of (other) therapeutic anti-cancer antibodies. This is important because the clustering of an ever-increasing number of activating FcγRs (versus inhibitory FcγRs; Nimmerjahn et al; Science. 2005 Dec 2;310(5753):1510-2) is known to increase effector cell-mediated target cell deletion, the mechanism underlying the activity of both checkpoint inhibitors, immune agonists, and other immune-modulating antibodies (such as anti-IL-2R). 2) Absence or reduction of Fc-mediated binding to inhibitory FcγRs has been shown to reduce inhibitory signaling in FcγR-expressing immune effector cells. Thus, absence or reduction of Fc-mediated binding of FcγRIIB-targeted antibodies to FcγRs likely improves therapeutic efficacy through at least two mechanisms, including both improved activation of FcγRs and reduced inhibitory Fcγ signaling in immune effector cells in response to a second immunomodulatory anti-cancer antibody.

[0021] "Reduced binding" or "reduced affinity binding" in the present context means that an antibody molecule has reduced Fc-mediated binding to an Fcγ receptor, in other words, the Fc region of an antibody molecule that specifically binds to FcγRIIB binds to an activated Fcγ receptor with lower affinity than the Fc region of normal human IgG1. Reduced binding can be assessed using techniques such as surface plasmon resonance. In the present context, "normal IgG1" means a conventionally produced IgG1 with a non-mutated Fc region that has not been produced to alter its glycosylation. As a reference for this "normal IgG1", rituximab produced in CHO cells can be used without any modification (Tipton et al, Blood 2015 125:1901-1909; rituximab is described, for example, in EP0605442).

[0022] By "reduced binding" is meant that the binding of the Fc region of an antibody molecule that specifically binds to FcγRIIB, which binds to an activating Fcγ receptor, is reduced by at least 10-fold to all Fc receptors, as compared to the binding of the Fc region of normal human IgG1 to the same receptor. In some embodiments, binding is reduced by at least 20-fold. In some embodiments, binding is reduced by at least 30-fold. In some embodiments, binding is reduced by at least 40-fold. In some embodiments, binding is reduced by at least 50-fold. In some embodiments, binding is reduced by at least 60-fold. In some embodiments, binding is reduced by at least 70-fold.

[0023] In some embodiments of the invention, an antibody molecule that specifically binds to FcγRIIB does not bind to its Fc region at all, and in some such cases, the antibody does not have an Fc region, and the antibody molecule then comprises Fab, Fab' 2 , scFv, or a PEGylated version thereof.

[0024] In some embodiments, an antibody molecule that specifically binds to FcγRIIB may be a llama antibody, in particular a llama hcIgG. Like all mammals, camelids produce conventional antibodies (IgG) made of two heavy chains and two light chains linked in a Y-shape by disulfide bonds. 1 However, they also produce two unique subclasses of immunoglobulin G: IgG, also known as heavy-chain IgG (hcIgG). 2 and IgG 3 These antibodies are made with only two heavy chains that lack the CH1 region, but still have the V HThey have an antigen-binding domain at the N-terminus, designated H. Conventional Igs require the association of both heavy and light chain variable regions, which allows for a large variety of antigen-antibody interactions. Although isolated heavy and light chains still exhibit this ability, they exhibit much lower affinity compared to paired heavy and light chains. A unique feature of hcIgGs is the ability of their monomeric antigen-binding regions to bind antigens without the need for pairing with another region, with specificity, affinity, and especially diversity comparable to conventional antibodies.

[0025] In some embodiments, reduced binding means that the antibody has a 20-fold decrease in affinity for binding to FcγRI.

[0026] To reduce the binding of IgG1 antibodies, such as IgG1 antibodies, to Fc receptors, it is possible to modify the Fc region of an IgG antibody by aglycosylation. For example, such aglycosylation of an IgG1 antibody can be achieved by, for example, an amino acid substitution of asparagine at position 297 (N297X) in the antibody chain. The substitution can be by glutamine (N297Q), or by alanine (N297A), or by glycine (N297G), or by asparagine (N297D), or by serine (N297S).

[0027] The Fc region may be modified by further substitutions (see, for example, Table 1), for example as described in Jacobsen FW et al., JBC 2017, 292, 1865-1875. Such additional substitutions include L242C, V259C, A287C, R292C, V302C, L306C, V323C, I332C, and / or K334C. Such modifications also include the following combinations of substitutions in IgG1: L242C, N297G, K334C, A287C, N297G, L306C, R292C, N297G, V302C, N297G, V323C, I332C, and V259C, N297G, L306C.

[0028] Alternatively, the carbohydrate in the Fc region can be enzymatically cleaved, and / or the cells used for antibody production can be grown in medium that prevents carbohydrate addition, and / or cells engineered to lack glycosylation capacity can be used for antibody production, or, as described above, by production of the antibody in a host cell that does not glycosylate, or does not functionally glycosylate, e.g., a prokaryote, including E. coli.

[0029] Reduction of affinity for Fc gamma receptors can further be achieved by engineering amino acids in the antibody Fc region (such modifications have been previously described, for example, by Xencor, Macrogenics, and Genentech), or by production of the antibody in a host cell that does not glycosylate, or does not functionally glycosylate, such as a prokaryote, including E. coli.

[0030] In addition to reducing binding to Fc gamma receptors through the Fc region, it is preferred in some embodiments that antibody molecules that specifically bind to FcγRIIB do not result in phosphorylation of FcγRIIB upon target binding, an event that blocks activity within immune cells.

[0031] Immune effector cells expressing Fc gamma receptors herein primarily refer to innate effector cells, specifically including macrophages, neutrophils, monocytes, natural killer (NK) cells, basophils, eosinophils, mast cells, and platelets. Cytotoxic T cells and memory T cells typically do not express FcγR, but may do so in certain circumstances. In some embodiments, the immune effector cells are innate immune effector cells. In some embodiments, the immune effector cells are macrophages.

[0032] In contrast to antibody molecules that specifically bind to FcγRIIB, antibody molecules that specifically bind to or interact with receptors present on tumor cells, i.e., second antibody molecules or tumor-directly targeting antibodies, have Fc regions that bind to or interact with activating Fcγ receptors to an undiminished, or at least substantially undiminished, extent. Binding of the second antibody to tumor cells results in activation of Fc receptor-dependent anti-tumor activity, such as depletion, antibody-dependent cellular cytotoxicity (ADCC), and / or antibody-dependent cellular phagocytosis (ADCP). Depletion, as used herein, means depletion, loss, or removal of tumor cells via physical clearance of the cells, resulting in depletion of the tumor cells.

[0033] To determine whether an antibody molecule is a tumor-depleting antibody molecule in the sense of the present invention, it is possible to use an in vitro ADCC or ADCP assay. To determine whether an antibody molecule is a tumor cell-depleting antibody molecule, the same assay is carried out in the presence and absence of a depleting antibody, showing whether the depleting antibody tested is actually depleting.

[0034] ADCC assays can be performed by labeling target cells with calcein AM (acetyl methyl ester) followed by the addition of diluted concentrations of antibody. Target cells are then co-cultured with human peripheral blood mononuclear cells (PBMCs) at an effector:target (E:T) ratio of 50:1 for 4 hours at 37°C. Plates are centrifuged at 400 xg for 5 minutes to pellet cells and the supernatants are transferred to white 96-well plates. Calcein release is measured using a Varioskan (Thermo Scientific) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Maximum percent release is calculated as follows: Maximum percent release = (sample / Triton treatment) * 100.

[0035] ADCP assays can be performed by labeling target cells with 5 mM carboxyfluorescein succinimidyl ether (CFSE) for 10 min at room temperature, followed by washing with medium containing fetal bovine serum. CFSE-labeled targets were then opsonized with diluted concentrations of antibodies and then co-cultured with bone marrow-derived macrophages (BMDMs) at an E:T ratio of 1:5 in 96-well plates for 1 h at 37°C. BMDMs were then labeled with anti-F4 / 80-allophycocyanin for 15 min at room temperature and washed twice with PBS. Plates were kept on ice, BMDMs were harvested by scraping the wells, and phagocytosis was assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4 / 80+CFSE+ cells within the F4 / 80+ cell population.

[0036] It is also possible to use a method such as that described by Cleary et al. in J Immunol, April 12, 2017, 1601473.

[0037] The tumor cells to which the second antibody molecule binds are FcγRIIB negative cancer tumors, meaning that FcγRIIB negative cancer tumors are tumors that do not display any FcγRIIB receptors. This can be examined by a variety of methods, including immunohistochemistry and flow cytometry, as shown in Tutt et al J Immunol, 2015, 195(11) 5503-5516, using anti-FcγRIIB specific antibodies.

[0038] In addition to specifically binding to targets on tumor cells, the second antibody molecule binds via its Fc region to activating Fcγ receptors present on immune effector cells. To be able to bind to activating Fcγ receptors, the Fc region of the second antibody must be glycosylated at least at position 297 in some embodiments. Carbohydrate residues at this position assist in binding to Fcγ receptors. In some embodiments, these residues are preferably biantennary carbohydrates containing GlnNAc, mannose, with terminal galactose residues and sialic acid. This is achieved by combining the CH of the Fc molecule with the 297 position of the Fc molecule.2 It is necessary to contain the moiety.

[0039] Antibodies are well known to those skilled in the art of immunology and molecular biology. Typically, an antibody comprises two heavy chains (H) and two light chains (L). This complete antibody molecule is sometimes referred to herein as a full-size antibody or full-length antibody. The heavy chain of an antibody comprises one variable region (VH) and three constant regions (CH1, CH2, and CH3), and the light chain of an antibody molecule comprises one variable region (VL) and one constant region (CL). The variable regions (sometimes referred to as F) are V The variable regions (collectively referred to as the constant regions) bind to the antibody's target, or antigen. Each variable region contains three loops, called complementarity determining regions (CDRs), which are involved in target binding. The constant regions are not directly involved in binding the antibody to the antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant regions of their heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five main immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2.

[0040] Another part of an antibody is the Fc region (also known as the fragment crystallizable region), which contains the two constant regions of each of the antibody's heavy chains. As mentioned above, the Fc region is involved in the interaction between the antibody and the Fc receptor.

[0041] 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.

[0042] A functional fragment of a full-sized antibody has the same antigen-binding characteristics as the corresponding full-sized antibody, and contains either the same variable regions (i.e., VH and VL sequences) and / or the same CDR sequences as the corresponding full-sized antibody. A functional fragment has the same antigen-binding characteristics as the corresponding full-sized antibody, meaning that it binds to the same epitope on the target as the full-sized antibody. Such a functional fragment may correspond to the Fv portion of a full-sized antibody. Alternatively, such a fragment may be a monovalent antigen-binding fragment, Fab (also designated F(ab)), that does not contain an Fc portion, or a bivalent antigen-binding fragment, F(ab'), that contains two antigen-binding Fab portions linked together by disulfide bonds. 2 (Fab' 2 Or Fab 2 F(ab') (i.e., F(ab') 2 Such a fragment may also be a single chain variable fragment (scFv).

[0043] Functional fragments do not always contain all six CDRs of the corresponding full-size antibody. It is understood that molecules containing three or fewer CDR regions (sometimes only a single CDR or a part thereof) can retain the antigen-binding activity of the antibody derived from that CDR(s). For example, Gao et al., 1994, J.Biol.Chem., 269:32389-93, describes that the entire VL chain (including all three CDRs) has high affinity for its substrate.

[0044] Molecules containing two CDR regions are described, for example, by Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4:417-430. On page 418 (right column-3 Our Strategy for Design), a minibody is described that contains only H1 and H2 CDR hypervariable regions interspersed within framework regions. The minibody is described as being capable of binding to a target. Pessi et al., 1993, Nature, 362:367-9 and Bianchi et al., 1994, J. Mol. Biol., 236:649-59 are referenced by Vaughan & Sollazzo and describe H1 and H2 minibodies and their properties in more detail. Qiu et al., 2007, Nature Biotechnology, 25:921-9 demonstrate that a molecule consisting of two linked CDRs is capable of binding to an antigen. Quiocho 1993, Nature, 362:293-4 provides an overview of "minibody" technology. Ladner 2007, Nature Biotechnology, 25:875-7 comments that molecules containing two CDRs can retain antigen-binding activity.

[0045] Antibody molecules containing a single CDR region are described, for example, in Laune et al., 1997, JBC, 272:30937-44. There, it is demonstrated that various hexapeptides derived from CDRs exhibit antigen-binding activity, and it is noted that synthetic peptides of complete single CDRs exhibit strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, show that various 12-mer peptides and related framework regions have antigen-binding activity, and comment that CDR3-like peptides alone can bind to antigens. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, report that "micro-antibodies" (molecules containing a single CDR) can bind to antigens, and show that cyclic peptides from anti-HIV antibodies have antigen-binding activity and function. Nicaise et al., 2004, Protein Science, 13:1882-91, show that a single CDR can confer antigen-binding activity and affinity for its lysozyme antigen.

[0046] Thus, antibody molecules having five, four, three or fewer CDRs can retain the antigen-binding properties of the full-length antibody from which they are derived.

[0047] The antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody. If a derivative is used, it should have the same antigen-binding characteristics as the corresponding full-length antibody, in the sense that it binds to the same epitope on the target as the full-length antibody.

[0048] Thus, as used herein, the term "antibody molecule" includes all types of antibody molecules, as well as their functional fragments and derivatives thereof, including monoclonal antibodies, polyclonal antibodies, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies, bispecific antibodies, human antibodies, human-derived antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, single chain Fv (scFv), Fab fragments, F(ab') 2These include antibody fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), antibody heavy chains, antibody light chains, antibody heavy chain homodimers, antibody light chain homodimers, antibody heavy chain heterodimers, antibody light chain heterodimers, and antigen-binding functional fragments of such homodimers and heterodimers.

[0049] Furthermore, as used herein, the term "antibody molecule", unless otherwise specified, includes all classes of antibody molecules and functional fragments, including IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, and IgE.

[0050] In some embodiments, the antibody is human IgG1. One of skill in the art will appreciate that mouse IgG2a and human IgG1 share the ability to bind activating Fc gamma receptors and activate target cell deletion by activation of immune cells bearing activating Fc gamma receptors, for example, by ADCP and ADCC. Thus, in embodiments where mouse IgG2a is the preferred isotype for deletion in mice, human IgG1 is the preferred isotype for deletion in humans in such embodiments.

[0051] As outlined above, different types and forms of antibody molecules are encompassed by the present invention and will be known to those skilled in the art of immunology. It is well known that antibodies used for therapeutic purposes are often modified with additional moieties that modify the properties of the antibody molecule.

[0052] Thus, we include that the antibody molecules of the invention or used in accordance with the invention (e.g. monoclonal and / or polyclonal and / or bispecific antibody molecules) comprise a detectable moiety and / or a cytotoxic moiety.

[0053] A "detectable moiety" includes one or more from the group consisting of: an enzyme; a radioactive atom; a fluorescent moiety; a chemiluminescent moiety; a bioluminescent moiety. A detectable moiety allows for the visualization of the antibody molecule in vitro, and / or in vivo, and / or ex vivo.

[0054] "Cytotoxic moieties" include radioactive moieties and / or enzymes, where the enzymes are caspases and / or toxins, where the toxins are bacterial toxins or venoms, where the cytotoxic moiety is capable of inducing cell lysis.

[0055] We further include that the antibody molecule may be in isolated and / or purified form and / or may be PEGylated, which is the method of adding polyethylene glycol polymers to a molecule such as an antibody molecule or derivative to modify its behavior, e.g., to increase its hydrodynamic size and prevent renal clearance, thereby increasing its half-life.

[0056] As discussed above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein are as defined by Kabat EA et al. 1991, In "Sequences of Proteins of Immunological Interest" Fifth Edition, NIH Publication No. 91-3242, pp xv-xvii.

[0057] As those skilled in the art will recognize, there are other methods for assigning amino acids to each CDR, for example, the International ImMunoGeneTics information system (IMGT®) (http: / / www.imgt.org / and Lefranc "The Immunoglobulin Facts Book" published by Lefranc and Academic Press, 2001).

[0058] In a further embodiment, an antibody molecule of or for use in accordance with the present invention is an antibody molecule capable of competing with a particular antibody provided herein, e.g., an antibody molecule comprising any of the amino acid sequences set out in SEQ ID NOs: 1-194, for binding to a particular target.

[0059] By "capable of competing with" it is meant that a competing antibody is capable of at least partially inhibiting or otherwise interfering with the binding of an antibody molecule as defined herein to a particular target.

[0060] For example, such a competing antibody molecule may be capable of inhibiting 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%, or about 100% and / or may be capable of inhibiting the ability of an antibody described herein to prevent or reduce binding to a particular target 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%, or about 100%.

[0061] Competitive binding can be measured by methods well known to those skilled in the art, such as enzyme linked immunosorbent assay (ELISA).

[0062] ELISA assays can be used to evaluate epitope-modifying or blocking antibodies. Additional methods suitable for identifying competing antibodies are disclosed in Antibodies: A Laboratory Manual, Harlow & Lane (see, e.g., pages 567-569, 574-576, 583, and 590-612, 1988, CSHL, NY, ISBN 0-87969-314-2), which is incorporated herein by reference.

[0063] The targets of the antibodies according to the invention or for use in the invention are expressed on the cell surface, i.e. they are cell surface antigens and comprise an epitope (otherwise known in the present context as a cell surface epitope) against which the antibody is directed. Cell surface antigens and epitopes are terms that will be readily understood by those skilled in the art of immunology or cell biology.

[0064] "Cell surface antigen" includes cell surface antigens that are exposed on the extracellular side of the cell membrane, but only transiently exposed on the extracellular side of the cell membrane. "Transiently exposed" includes cell surface antigens that may be internalized or released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens may be released from the extracellular side of the cell membrane by cleavage, which may be mediated by proteases.

[0065] Also, cell surface antigens may be bound to the cell membrane, but may only be temporarily bound to the cell membrane. "Temporarily bound" includes that cell surface antigens may be released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens may be released from the extracellular side of the cell membrane by cleavage, which may be mediated by proteases.

[0066] It is further contemplated that the cell surface antigen may be an epitope present on a peptide, or a polypeptide, or a carbohydrate, or an oligosaccharide chain, or a lipid; and / or a protein, or a glycoprotein, or a lipoprotein.

[0067] Methods for assessing protein binding are known to those skilled in the art of biochemistry and immunology. Those skilled in the art will understand that these methods can be used to assess the binding of an antibody to a target and / or the binding of an Fc region of an antibody to an Fc receptor; as well as the relative strength, or specificity, or inhibition, prevention, or reduction of their interactions. Examples of methods that can be used to assess protein binding include, for example, immunoassays, BIAcore, Western blots, radioimmunoassays (RIA), and enzyme-linked immunosorbent assays (ELISA) (for a discussion of antibody specificity, see Fundamental Immunology Second Edition, Raven Press, New York, pp. 332-336 (1989)).

[0068] It is well known what is meant by an antibody specifically binding or interacting with a defined target molecule or antigen, and means that the antibody binds preferentially and selectively to its target and does not bind to non-target molecules. In the present context, the term "bind" can be used interchangeably with "interact with." Thus, "specifically binding antibody molecules," or "targeting a particular antibody molecule," includes an antibody molecule that specifically binds to a target but does not bind to a non-target, or that binds weaker (e.g., with lower affinity) to a non-target than to the target.

[0069] It also includes the meaning that an antibody specifically binds to a target more than to a non-target at least 2 times stronger, or at least 5 times stronger, or at least 10 times stronger, or at least 20 times stronger, or at least 50 times stronger, or at least 100 times stronger, or at least 200 times stronger, or at least 500 times stronger, or at least about 1000 times stronger.

[0070] In addition, 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 K d , or at least about 10 -11 K d , or at least about 10 -12 K d , 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 When an antibody binds to a target, it is meant to mean that it specifically binds to the target.

[0071] In some embodiments, an antibody molecule that specifically binds to FcγRIIB is a human antibody.

[0072] In some embodiments, antibody molecules that specifically bind to FcγRIIB are human-derived antibodies, i.e., originally human antibodies that have been modified as described herein.

[0073] In some embodiments, an antibody molecule that specifically binds to FcγRIIB is a humanized antibody, i.e., an originally non-human antibody that has been modified to increase similarity to a human antibody. A humanized antibody can be, for example, a mouse antibody or a llama antibody.

[0074] In some embodiments, an antibody molecule that specifically binds to FcγRIIB comprises the following constant regions (CH and CL): IgG1-CH [SEQ ID NO: 1] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1-CL [SEQ ID NO: 2] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0075] 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 the Fc region. As mentioned herein, in some embodiments, it is preferred that SEQ ID NO: 1 is aglycosylated by a N297Q substitution, and the IgG1-CH then has the following CH sequence [SEQ ID NO: 195] (the 297Q residue is shown in bold): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0076] In some embodiments and / or examples, mouse antibody molecules are used. These can also be used for surrogate antibodies. These can then include the following constant regions (CH and CL): CH[SEQ ID NO:196] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNV EVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK CL [SEQ ID NO: 197] QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS

[0077] Thus, these constant regions (SEQ ID NO: 196 and SEQ ID NO: 197) are of mouse origin. SEQ ID NO: 196 contains an N297A mutation (residue 297A is shown in bold in the sequence above). This N297A mutation in the mouse sequence corresponds to an N297Q mutation in the human sequence.

[0078] In some embodiments, an antibody molecule that specifically binds to FcγRIIB comprises the sequence of one or more of the following clones: Antibody clone: ​​1A01 1A01-VH [SEQ ID NO: 3] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQTPGKGLEWVSLIGWDGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARAYSGYELDYWGQGTLVTVSS 1A01-VL [SEQ ID NO: 27] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNASIFGGGTKLTVLG CDR area CDRH1: DYYMN [SEQ ID NO:51] CDRH2: LIGWDGGSTYYADSVKG [SEQ ID NO: 52] CDRH3: AYSGYELDY [SEQ ID NO:53] CDRL1: SGSSNIGNNAVN [SEQ ID NO:54] CDRL2: DNNNRPS [SEQ ID NO:55] CDRL3: AAWDDSLNASI [SEQ ID NO:56] Antibody clone: ​​1B07 1B07-VH [SEQ ID NO: 4] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFTRYDGSNKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENIDAFDVWGQGTLVTVSS 1B07-VL [SEQ ID NO: 28] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNQQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCEAWDDRLFGPVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO:57] CDRH2: FTRYDGSNKYYADSVRG [SEQ ID NO: 58] CDRH3: ENIDAFDV [SEQ ID NO:59] CDRL1: SGSSNIGNNAVN [SEQ ID NO: 60] CDRL2:DNQQRPS [SEQ ID NO:61] CDRL3: WDDRLFGPV [SEQ ID NO: 62] Antibody clone: ​​1C04 1C04-VH[SEQ ID NO:5] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISDSGAGRYYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTHDSGELLDAFDIWGQGTLVTVSS 1C04-VL [SEQ ID NO: 29] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNHVLWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG CDR area CDRH1: SYAMS [SEQ ID NO: 63] CDRH2: SISDSGAGRYYADSVEG [SEQ ID NO: 64] CDRH3: THDSGELLDAFDI [SEQ ID NO: 65] CDRL1: SGSSSNIGSNHVL [SEQ ID NO: 66] CDRL2: GNSNRPS [SEQ ID NO: 67] CDRL3: AAWDDSLNGWV [SEQ ID NO: 68] Antibody clone: ​​1E05 1E05-VH [SEQ ID NO: 6] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQVPGKGLEWVAVISYDGSNKNYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNFDNSGYAIPDAFDIWGQGTLVTVSS 1E05-VL [SEQ ID NO: 30] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNSRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLGGPVFGGGTKLTVLG CDR area CDRH1:TYAMN [SEQ ID NO:69] CDRH2: VISYDGSNKNYVDSVKG [SEQ ID NO: 70] CDRH3: NFDNSGYAIPDAFDI [SEQ ID NO: 71] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 72] CDRL2: DNNSRPS [SEQ ID NO: 73] CDRL3:AAWDDSLGGPV [SEQ ID NO:74] Antibody clone: ​​2A09 2A09-VH[SEQ ID NO:7] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVAYISRDADITHYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGFDYAGDDAFDIWGQGTLVTVSS 2A09-VL [SEQ ID NO: 31] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNAVNWYQQLPGTAPKLLIYGNSDRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRWVFGGGTKLTVLG CDR area CDRH1:NAWMS [SEQ ID NO:75] CDRH2: YISRDADITHYPASVKG [SEQ ID NO: 76] CDRH3: GFDYAGDDAFDI [SEQ ID NO: 77] CDRL1: SGSSSNIGSNAVN [SEQ ID NO: 78] CDRL2: GNSDRPS [SEQ ID NO: 79] CDRL3: AAWDDSLNGRWV [SEQ ID NO: 80] Antibody clone: ​​2B08 2B08-VH [SEQ ID NO: 8] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVALIGHDGNNKYYLDSLEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARATDSGYDLLYWGQGTLVTVSS 2B08-VL [SEQ ID NO: 32] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYYDDLLPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCTTWDDSLSGVVFGGGTKLTVLG CDR area CDRH1: DYYMS [SEQ ID NO: 81] CDRH2: LIGHDGNNKYYLDSLEG [SEQ ID NO: 82] CDRH3:ATDSGYDLLY [SEQ ID NO:83] CDRL1: SGSSNIGNNAVN [SEQ ID NO: 84] CDRL2: YDDLLPS [SEQ ID NO: 85] CDRL3:TTWDDSLSGVV [SEQ ID NO:86] Antibody clone: ​​2E8-VH 2E8-VH [SEQ ID NO: 9] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSAIGFSDDNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDGSGWSFWGQGTLVTVSS 2E8-VL [SEQ ID NO: 33] QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLRGWVFGGGTKLTVLG CDR area CDRH1: DYYMS [SEQ ID NO: 87] CDRH2: AIGFSDDNTYYADSVKG [SEQ ID NO: 88] CDRH3: GDGSGWSF [SEQ ID NO: 89] CDRL1: SGSSNIGNNAVN [SEQ ID NO: 90] CDRL2: DNNKRPS [SEQ ID NO: 91] CDRL3:ATWDDSLRGWV [SEQ ID NO: 92] Antibody clone: ​​5C04 5C04-VH [SEQ ID NO: 10] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREWRDAFDIWGQGTLVTVSS 5C04-VL [SEQ ID NO: 34] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGSWVFGGGTKLTVLG CDR area CDRH1: NYGMH [SEQ ID NO: 93] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 94] CDRH3:WRDAFDI [SEQ ID NO:95] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 96] CDRL2: SDNQRPS [SEQ ID NO: 97] CDRL3:AAWDDSLSGSWV [SEQ ID NO:98] Antibody clone: ​​5C05 5C05-VH [SEQ ID NO: 11] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENFDAFDVWGQGTLVTVSS 5C05-VL [SEQ ID NO: 35] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSNSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGQVVFGGGTKLTVLG CDR area CDRH1: TYGMH [SEQ ID NO: 99] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 100] CDRH3:ENFDAFDV [SEQ ID NO: 101] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 102] CDRL2: SNSQRPS [SEQ ID NO: 103] CDRL3: AAWDDSLNGQVV [SEQ ID NO: 104] Antibody clone: ​​5D07 5D07-VH [SEQ ID NO: 12] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHVRQAPGKGLEWVAVIAYDGSKKDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYRDAFDIWGQGTLVTVSS 5D07-VL [SEQ ID NO: 36] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTTASLAISGLRSEDEADYYCAAWDDSVSGWMFGGGTKLTVLG CDR area CDRH1: TYGMH [SEQ ID NO: 105] CDRH2: VIAYDGSKKDYADSVKG [SEQ ID NO: 106] CDRH3:EYRDAFDI [SEQ ID NO: 107] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 108] CDRL2: GNSNRPS [SEQ ID NO: 109] CDRL3:AAWDDSVSGWM [SEQ ID NO: 110] Antibody clone: ​​5E12 5E12-VH [SEQ ID NO: 13] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGINKDYADSMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERKDAFDIWGQGTLVTVSS 5E12-VL [SEQ ID NO: 37] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLNGLVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 111] CDRH2: VISYDGINKDYADSMKG [SEQ ID NO: 112] CDRH3: ERKDAFDI [SEQ ID NO: 113] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 114] CDRL2: SNNQRPS [SEQ ID NO: 115] CDRL3:ATWDDSLNGLV [SEQ ID NO: 116] Antibody clone: ​​5G08 5G08-VH [SEQ ID NO: 14] EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYDGSNRYYADSVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYYCARDRWNGMDVWGQGTLVTVSS 5G08-VL [SEQ ID NO: 38] QSVLTQPPSASGTPGQRVTISCSGSSSNIGAGYDVHWYQQLPGTAPKLLIYANNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPWVFGGGTKLTVLG CDR area CDRH1: NYGMH [SEQ ID NO: 117] CDRH2: VISYDGSNRYYADSVKG [SEQ ID NO: 118] CDRH3:DRWNGMDV [SEQ ID NO: 119] CDRL1: SGSSSNIGAGYDVH [SEQ ID NO: 120] CDRL2: ANNQRPS [SEQ ID NO: 121] CDRL3: AAWDDSLNGPWV [SEQ ID NO: 122] Antibody clone: ​​5H06 5H06-VH [SEQ ID NO: 15] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHSVIGAFDIWGQGTLVTVSS 5H06-VL [SEQ ID NO: 39] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGSNNVVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 123] CDRH2: VISYDGSDTAYADSVKG [SEQ ID NO: 124] CDRH3:DHSVIGAFDI [SEQ ID NO: 125] CDRL1: SGSSSNIGSNTVN [SEQ ID NO: 126] CDRL2: DNNKRPS [SEQ ID NO: 127] CDRL3: SSYAGSNNVV [SEQ ID NO: 128] Antibody clone: ​​6A09 6A09-VH[SEQ ID NO: 16] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVTSYDGNTKYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDCGGDCFDYWGQGTLVTVSS 6A09-VL [SEQ ID NO: 40] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNEGVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 129] CDRH2:VTSYDGNTKYYANSVKG [SEQ ID NO: 130] CDRH3: EDCGGDCFDY [SEQ ID NO: 131] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 132] CDRL2: GNSNRPS [SEQ ID NO: 133] CDRL3: AAWDDSLNEGV [SEQ ID NO: 134] Antibody clone: ​​6B01 6B01-VH [SEQ ID NO: 17] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQLGEAFDIWGQGTLVTVSS 6B01-VL [SEQ ID NO: 41] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLSGPVFGGGTKLTVLG CDR area CDRH1: NYGMH [SEQ ID NO: 135] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 136] CDRH3:DQLGEAFDI [SEQ ID NO: 137] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 138] CDRL2: DNNKRPS [SEQ ID NO: 139] CDRL3:ATWDDSLSGPV [SEQ ID NO: 140] Antibody clone: ​​6C11 6C11-VH [SEQ ID NO: 18] EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISGSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDIDYFDYWGQGTLVTVSS 6C11-VL [SEQ ID NO: 42] QSVLTQPPSASGTPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYENNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG CDR area CDRH1: DYGMS [SEQ ID NO: 141] CDRH2: AISGSGSTYYADSVKG [SEQ ID NO: 142] CDRH3: GDIDYFDY [SEQ ID NO: 143] CDRL1: TGSSSNFGAGYDVH [SEQ ID NO: 144] CDRL2: ENNKRPS [SEQ ID NO: 145] CDRL3: AAWDDSLNGPV [SEQ ID NO: 146] Antibody clone: ​​6C12 6C12-VH [SEQ ID NO: 19] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERRDAFDIWGQGTLVTVSS 6C12-VL [SEQ ID NO: 43] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDSDTPVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 147] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 148] CDRH3:ERRDAFDI [SEQ ID NO: 149] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 150] CDRL2: SDNQRPS [SEQ ID NO: 151] CDRL3:ATWDSDTPV [SEQ ID NO: 152] Antibody clone: ​​6D01 6D01-VH [SEQ ID NO: 20] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARDHSAAGYFDYWGQGTLVTVSS 6D01-VL [SEQ ID NO: 44] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNSIRPSGGPDRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSLSSPVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 153] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 154] CDRH3: DHSAAGYFDY [SEQ ID NO: 155] CDRL1: SGSSSNIGSNTVN [SEQ ID NO: 156] CDRL2: GNSIRPS [SEQ ID NO: 157] CDRL3: ASWDDSLSPV [SEQ ID NO: 158] Antibody clone: ​​6G03 6G03-VH [SEQ ID NO: 21] EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYGMHWVRQAPGKGLEWVSGISWDSAIIDYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEAAAGAFDIWGQGTLVTVSS 6G03-VL [SEQ ID NO: 45] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTDRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGPVVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 159] CDRH2: GISWDSAIIDYAGSVKG [SEQ ID NO: 160] CDRH3: DEAAAGAFDI [SEQ ID NO: 161] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 162] CDRL2: GNTDRPS [SEQ ID NO: 163] CDRL3:AAWDDSLSGPVV [SEQ ID NO: 164] Antibody clone: ​​6G08 6G08-VH [SEQ ID NO: 22] EVQLLESGGGLVQPGGSLRLSCAASGFTLSSYGISWVRQAPGKGLEWVSGISGSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSVGAYANDAFDIWGQGTLVTVSS 6G08-VL [SEQ ID NO: 46] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDTNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG CDR area CDRH1: SYGIS [SEQ ID NO: 165] CDRH2: GISGSGSGNTYYADSVKG [SEQ ID NO: 166] CDRH3:SVGAYANDAFDI [SEQ ID NO: 167] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 168] CDRL2: GDTNRPS [SEQ ID NO: 169] CDRL3: AAWDDSLNGPV [SEQ ID NO: 170] Antibody clone: ​​6G11 6G11-VH [SEQ ID NO: 23] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELYDAFDIWGQGTLVTVSS 6G11-VL [SEQ ID NO: 47] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADDHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSQRAVIFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 171] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 172] CDRH3: ELYDAFDI [SEQ ID NO: 173] CDRL1: TGSSSNIGAGYDVH [SEQ ID NO: 174] CDRL2: ADDHRPS [SEQ ID NO: 175] CDRL3: ASWDDSQRAVI [SEQ ID NO: 176] Antibody clone: ​​6H08 6H08-VH [SEQ ID NO: 24] EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREYKDAFDIWGQGTLVTVSS 6H08-VL [SEQ ID NO: 48] QSVLTQPPSASGTPGQRVTISCTGSSNIGSNTVNWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQAWGTGIRVFGGGTKLTVLG CDR area CDRH1: NYGMH [SEQ ID NO: 177] CDRH2: VISYDGSNKYYAD SVKG [SEQ ID NO: 178] CDRH3:EYKDAFDI [SEQ ID NO: 179] CDRL1: TGSSSNIGSNTVN [SEQ ID NO: 180] CDRL2: DNNKRPS [SEQ ID NO: 181] CDRL3: QAWGTGIRV [SEQ ID NO: 182] Antibody clone: ​​7C07 7C07-VH[SEQ ID NO:25] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCAREFGYIILDYWGQGTLVTVSS 7C07-VL [SEQ ID NO: 49] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYRDYERPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCMAWDDSLSGVVFGGGTKLTVLG CDR area CDRH1: SYGMH [SEQ ID NO: 183] CDRH2: VISYDGSNKYYADSVKG [SEQ ID NO: 184] CDRH3: EFGYIILDY [SEQ ID NO: 185] CDRL1: SGSSSNIGSNTVN [SEQ ID NO: 186] CDRL2: RDYERPS [SEQ ID NO: 187] CDRL3: MAWDDSLSGVV [SEQ ID NO: 188] Antibody clone: ​​4B02 4B02-VH [SEQ ID NO: 26] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHGMHWVRQAPGKGLEWVAVISYDGTNKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWDAFDVWGQGTLVTVSS 4B02-VL [SEQ ID NO: 50] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNNANWYQQLPGTAPKLLIYDNNKRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTVVFGGGTKLTVLG CDR area CDRH1: NHGMH [SEQ ID NO: 189] CDRH2: VISYDGTNKYYADSVRG [SEQ ID NO: 190] CDRH3: ETWDAFDV [SEQ ID NO: 191] CDRL1: SGSSSNIGSNNNAN [SEQ ID NO: 192] CDRL2: DNNKRPS [SEQ ID NO: 193] CDRL3: QAWDSSTVV [SEQ ID NO: 194]

[0079] In some embodiments, which are sometimes preferred embodiments, antibody molecules that specifically bind to FcγRIIB comprise 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.

[0080] In some embodiments, which are 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 are further modified to reduce binding to Fcγ receptors via the Fc region. In some embodiments, which are 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, which include the N297Q mutation.

[0081] In some embodiments, the antibody molecule that specifically binds to a receptor present on a tumor cell is a human antibody molecule or a human-derived antibody molecule. In some such embodiments, the human antibody molecule or human-derived antibody molecule is an IgG antibody. In some such embodiments, the human antibody molecule or human-derived antibody molecule is an IgG1 antibody or an IgG2 antibody.

[0082] In some embodiments, an antibody molecule that specifically binds to a receptor present on a tumor cell, the antibody molecule that specifically binds to a receptor present on a tumor cell is a humanized antibody molecule.

[0083] In some embodiments, the antibody molecule that specifically binds to a receptor present on a tumor cell is a chimeric antibody.

[0084] As mentioned above, an antibody molecule that specifically binds to a receptor present on a tumor cell must have the ability to bind to FcγR.

[0085] A combination of an antibody molecule that specifically binds to FcγRIIB and an antibody molecule that specifically binds to a receptor present on a tumor cell may be used in the treatment of cancer.

[0086] The term "patient" is used herein to mean an animal, including a human, that has been diagnosed as having an FcγRIIB-negative cancer or that is considered likely to have an FcγRIIB-negative cancer and / or that exhibits symptoms of such a cancer.

[0087] It is contemplated that the patient may be a mammal or a non-mammal. Preferably, the patient is a mammal such as a human, horse, cow, sheep, pig, camel, dog, or cat. Most preferably, the mammalian patient is a human.

[0088] "Exhibiting" includes that the subject exhibits cancer symptoms and / or cancer diagnostic markers and / or that the cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0089] It will be readily apparent to one of ordinary skill in the medical arts what cancer symptoms and cancer diagnostic markers are, and how to measure and / or assess and / or quantify whether there is a decrease or increase in the severity of a cancer symptom, or whether there is a decrease or increase in a cancer diagnostic marker, and how the cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0090] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agent is administered over a period of time. The length of time for the course of treatment depends on many factors, including the type of therapeutic agent being administered, the type of cancer being treated, the severity of the cancer being treated, and the age and health of the patient, among other reasons.

[0091] "During treatment" includes when the patient is currently undergoing a course of treatment and / or is currently receiving a therapeutic agent and / or is currently receiving a series of therapeutic agents.

[0092] In some embodiments, the FcγRIIB negative cancer treatable according to the present invention is a solid tumor.

[0093] The clinical definition of diagnosis, prognosis, and progression of many cancers depends on a specific classification known as staging. These staging systems work by collating several different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and / or prognosis and / or progression of cancer. Those skilled in the oncology field will know how to use the staging systems to assess the diagnosis and / or prognosis and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms to use to do so.

[0094] "Cancer staging" includes the Rai staging system, including stage 0, stage I, stage II, stage III, and stage IV; and / or the Binet staging system, including stage A, stage B, and stage C; and / or the Ann Arbour staging system, including stage I, stage II, stage III, and stage IV.

[0095] It is known that cancer can cause abnormalities in cell morphology. These abnormalities often occur reproducibly in certain cancers, meaning that examination of these changes in morphology (also known as histological examination) can be used to diagnose or prognose cancer. Techniques for visualizing and preparing samples for visualization to examine cell morphology, such as light microscopy or confocal microscopy, are well known in the art.

[0096] "Histological examination" includes the presence of small mature lymphocytes and / or small mature lymphocytes with narrow cytoplasmic borders, the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli and / or the presence of small mature lymphocytes with narrow cytoplasmic borders and dense nuclei lacking discernible nucleoli, and / or the presence of atypical cells and / or cleaved cells and / or prolymphocytes.

[0097] It is well known that cancer is the result of mutations in the DNA of cells, which allow cells to avoid cell death or grow uncontrollably. Therefore, testing for these mutations (also known as cytogenetic testing) can be a useful tool for evaluating the diagnosis and / or prognosis of cancer. An example of this is the deletion of chromosomal location 13q14.1, which is a hallmark of chronic lymphocytic leukemia. Techniques for investigating mutations in cells are well known in the art, such as fluorescent in situ hybridization (FISH).

[0098] "Cytogenetic testing" includes testing of the DNA (particularly chromosomes) of cells. Cytogenetic testing can be used to identify DNA alterations that may be associated with the presence of refractory and / or recurrent cancer. These include: deletion of the long arm of chromosome 13, and / or deletion at chromosomal location 13q14.1, and / or trisomy of chromosome 12, and / or deletion of the long arm of chromosome 12, and / or deletion of the long arm of chromosome 11, and / or deletion of 11q, and / or deletion of the long arm of chromosome 6, and / or deletion of 6q, and / or deletion of the short arm of chromosome 17, and / or deletion of 17p, and / or t(11:14) translocation, and 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.

[0099] Cancer patients are known to exhibit certain physical symptoms, which are often the result of the burden that cancer places on the body. These symptoms often recur with the same cancer, and may therefore be characteristic of the diagnosis and / or prognosis and / or progression of the disease. Those skilled in the art of medicine will understand which physical symptoms are associated with which cancers, and how the evaluation of these body systems may correlate with the diagnosis and / or prognosis and / or progression of the disease. "Physical symptoms" include hepatomegaly and / or splenomegaly.

[0100] In some embodiments, the target bound by the antibody molecule that specifically binds to a receptor present on tumor cells is human epidermal growth factor receptor 2 (HER2). In such embodiments, the FcγRIIB negative cancer being treated can be a cancer selected from the group consisting of breast cancer and gastric cancer.

[0101] In the present context, breast cancer includes metastatic breast cancer (MBC) and early stage breast cancer (EBC).

[0102] In this context, gastric cancer can also be referred to as gastric adenocarcinoma or stomach cancer, including gastroesophageal junction (GEJ) adenocarcinoma, and further includes metastatic gastric cancer (MGC) and metastatic GEJ adenocarcinoma.

[0103] Trastuzumab (Herceptin®) is currently used to treat HER2-expressing breast cancer, either alone or in combination with chemotherapy or other drugs, and such treatment has significantly improved overall survival. However, many patients remain incurable. Other patients develop trastuzumab resistance leading to disease recurrence, and in addition, it has been shown that some HER2-positive breast cancers may become HER2-negative or have low HER2 expression over time. Means of improving anti-HER2 therapy and overcoming resistance are therefore highly desirable in order to cure more patients.

[0104] In some embodiments, the FcγRIIB negative cancer treated by the present invention is a cancer with low expression of HER2. Patients with cancer with low expression of HER2 often do not respond or do not respond well to standard treatment, such as treatment with trastuzumab and / or trastuzumab biosimilars. However, as described herein, by combining with an antibody molecule that specifically binds to FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via the Fc region, it is possible to treat cancer with low expression of HER2.

[0105] To determine whether HER2 is low in a cancer, standard HER2 assays, such as those using immunohistochemistry (IHC) or fluorescent in situ hybridization (FISH), can be used, which are often performed on a biopsy taken from the patient.

[0106] IHC testing is based on staining of the HER2 protein. When used to measure the amount of HER2 on the surface of cells in breast cancer tissue samples, it gives a score of 0-3+. A score of 0-1+ is considered HER2 negative. A score of 2+ is considered borderline. A score of 3+ is considered HER2 positive. In this context, an IHC score of 0-1+ for a patient with breast cancer can classify the patient as having a cancer with low HER2 expression. In some embodiments, a score of 0-1+ is considered to represent a cancer with low HER2 expression. In some embodiments, a score of 3+ is considered to represent a cancer with high HER2 expression, i.e., not a cancer with low HER2 expression according to the present invention. In the following examples, a well-established HER2 expressing immune-competitive Balb / C TUBO breast cancer tumor model was used. This model was employed to allow the evaluation of the ability of Fc:Fcγ-deficient (Fc-muted) anti-FcγRIIB antibodies to enhance anti-HER2 antitumor activity against cancers with low HER2 expression (HER2-low) and, for comparison, against cancers with high HER2 expression (HER2-high). Thus, in the HER2-high tumor model, animals received a full therapeutic dose of anti-HER2 antibody, which resulted in a strong occupancy of HER2 expressed in the tumor. In the HER2-low model, animals received a lower dose of antibody, which resulted in approximately 10-fold fewer HER2 receptors in the cancer cells targeted by the anti-HER2 antibody, as demonstrated by flow cytometry analysis of tumors recovered from mice treated with fluorochrome-conjugated anti-HER2 antibody. Thus, all other factors are identical except for the antibody targeting the HER2 receptor, making this tumor model system ideal for evaluating and demonstrating anti-FcγRIIB-mediated enhancement of anti-HER2 efficacy against cancers that highly express HER2, and for evaluating and demonstrating anti-FcγRIIB-mediated enhancement of the therapeutically significant effect of anti-HER2 against cancers that lowly express HER2.

[0107] FISH, which is based on HER2 labeling, is more accurate than IHC, but is more expensive and takes longer to return results. This is why IHC testing is usually the first test done to see if a cancer is HER2 positive. With FISH testing, you get either a positive or negative score (some hospitals refer to a negative test result as a "zero").

[0108] The two tests can be combined. For example, if the IHC test result is borderline, it can be combined with a FISH test to provide a better basis for determining whether the cancer is HER2 positive or not. For example, IHC can be used.

[0109] Generally, only cancers that test IHC3+ or FISH positive respond to standard treatment with agents that target HER2.

[0110] When the target to which the second antibody molecule binds is HER2, the second antibody molecule may be trastuzumab (Herceptin®) or a trastuzumab biosimilar, such as trastuzumab-anns (Kanjinti®), trastuzumab-qyyp (Trazimera®), trastuzumab-pkrb (Herzuma®), trastuzumab-dttb (Ontruzant®), or trastuzumab-dkst (Ogivri®). A trastuzumab biosimilar refers to an antibody molecule that is very similar to trastuzumab and has no clinically meaningful differences from trastuzumab. Alternatively, the second antibody molecule can be a toxin-conjugated enhanced variant of trastuzumab or a trastuzumab biosimilar, such as fam-trastuzumab-deruxtecan-nxki (Enhertu®), or T-DM1 or ado-trastuzumab emtansine (Kadcyla®), or other FcγR-binding anti-HER2 antibody drug conjugate.

[0111] In other cases, the second antibody can be used in conjunction with a third antibody, which can be tumor-directly targeted, such as the anti-HER2 antibody pertuzumab, or immunomodulatory, such as an anti-PD-1 / PD-L1 antibody. Additionally, the second antibody can be an anti-HER2 antibody used in any anti-HER2-containing therapeutic regimen.

[0112] In some embodiments, the FcγRIIB-negative cancers treated according to the present invention are cancers in patients who have previously been successfully treated with trastuzumab and / or trastuzumab biosimilars, but have subsequently developed resistance to trastuzumab or trastuzumab biosimilars and therefore no longer respond to such treatment.Cancers with low expression of HER2.As described herein, such resistance can be overcome by combining an antibody molecule that specifically binds FcγRIIB via the Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via the Fc region.

[0113] In some embodiments, the target bound by the antibody molecule that specifically binds to a receptor present on tumor cells is the human epidermal growth factor receptor (EGFR). In such embodiments, the FcγRIIB-negative cancer being treated may be a cancer selected from the group consisting of head and neck cancer and colorectal cancer.

[0114] In this context, head and neck cancer includes locally or regionally advanced squamous cell carcinoma of the head and neck, recurrent locoregional disease or metastatic squamous cell carcinoma of the head and neck, and recurrent or metastatic squamous cell carcinoma of the head and neck.

[0115] In the present context, colorectal cancer includes K-Ras wild-type, EGFR-expressing metastatic colorectal cancer.

[0116] When the target to which the second antibody molecule binds is EGFR, the second antibody molecule can be cetuximab (Erbitux®) or a cetuximab biosimilar, which means an antibody molecule that is so similar to cetuximab that it has no clinically meaningful differences from cetuximab.

[0117] Each of the above-mentioned cancers is well known and the symptoms and cancer diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Thus, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat the above-mentioned types of cancer will be known to those of ordinary skill in the medical arts.

[0118] In some embodiments, an antibody molecule that specifically binds to FcγRIIB and an antibody molecule that specifically binds to a receptor present on a tumor cell are administered to a patient simultaneously, meaning that they are either administered together at the same time or administered separately very close in time to each other.

[0119] In some embodiments, an antibody molecule that specifically binds to FcγRIIB is administered to a patient prior to administration of an antibody molecule that specifically binds to a receptor present on a tumor cell. Such sequential administration can be achieved by temporal separation of the two antibodies. Alternatively, or in combination with the first option, sequential administration can also be achieved by spatial separation of the two antibody molecules, such as by administering an antibody molecule that specifically binds to FcγRIIB in a manner that allows it to reach the cancer before the antibody molecule that specifically binds to a receptor present on a tumor cell, and then administering it in a manner that allows it to reach the cancer after the antibody molecule that specifically binds to FcγRIIB, such as by administering it systemically.

[0120] In some embodiments, an antibody molecule that specifically binds to a receptor present on a tumor cell is administered to a patient prior to administering an antibody molecule that specifically binds to FcγRIIB. Such sequential administration can be accomplished as described above.

[0121] Those skilled in the art of medicine will know that drugs can be modified with various additives, for example to change the rate at which the drug is absorbed by the body, and can be modified in various forms, for example to allow for particular routes of administration to the body.

[0122] It is therefore contemplated that the compositions, and / or antibodies, and / or drugs of the present invention may be combined with excipients, and / or pharma- ceutically acceptable carriers, and / or pharma-ceutically acceptable diluents, and / or adjuvants.

[0123] It is also included that the compositions and / or antibodies and / or drugs of the invention may be suitable for parenteral administration, including aqueous and / or non-aqueous sterile injection solutions which may contain antioxidants and / or buffers and / or bacteriostatic agents and / or solutes which 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 drugs of the invention may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (i.e., lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, water for injections, immediately prior to use.

[0124] Extemporaneous injection solutions and suspensions may be prepared from sterile powders and / or granules and / or tablets of the kind previously described.

[0125] For parenteral administration to human patients, the daily dosage level of antibody molecules that specifically bind to FcγRIIB and / or antibody molecules that specifically bind to receptors present on tumor cells is usually 1 mg / kg to 20 mg / kg of patient body weight, or even up to 100 mg / kg, administered in single or multiple doses. In special circumstances, it is also possible to use lower doses, for example in combination with chronic administration. In any case, the physician will determine the actual dosage that is most suitable for any individual patient, which will vary with the age, weight, and response of the particular patient. The dosages listed above are examples of the average case. Of course, there may be individual cases in which higher or lower dosage ranges are required, and such cases are within the scope of the present invention.

[0126] Generally, in humans, oral or parenteral administration of the compositions, and / or antibodies, and / or agents, and / or drugs of the invention is the preferred route, with parenteral administration being the most common used for antibodies. For veterinary use, the compositions, and / or antibodies, and / or agents, and / or drugs of the invention are administered in a suitably acceptable formulation according to normal veterinary practice, and the veterinarian will determine the administration regime and route of administration that will be most appropriate for the particular animal. Thus, the invention provides pharmaceutical formulations comprising the antibodies and / or agents of the invention in an amount effective to treat various conditions (mentioned above and further described below). Preferably, the compositions, and / or antibodies, and / or agents, and / or drugs are adapted for delivery by a route selected from the group including intravenous (IV), subcutaneous (SC), intramuscular (IM), or intratumoral.

[0127] In some embodiments, either the first antibody molecule or the second antibody, or both, may be administered through the use of a plasmid or a virus. Such a plasmid then comprises a nucleotide sequence encoding either the first antibody molecule or the second antibody, or both. In some embodiments, a nucleotide sequence encoding a portion or the complete sequence of either the first antibody molecule or the second antibody, or both, is incorporated into a cell or viral genome or into the viral virome, and such a cell or virus then serves as a delivery vehicle for either the first antibody molecule or the second antibody, or both (or a delivery vehicle for a nucleotide sequence encoding either the first antibody molecule or the second antibody, or both). For example, in some embodiments, such a virus may be in the form of a therapeutic oncolytic virus comprising a nucleotide sequence encoding at least one of the antibody molecules described herein. In some embodiments, such an oncolytic virus comprises a nucleotide sequence encoding a full-length human IgG antibody. Oncolytic viruses are known to those skilled in the art of medicine and virology.

[0128] The present invention also includes compositions and / or antibodies and / or agents and / or drugs comprising pharma- ceutically acceptable acid or base addition salts of the polypeptide binding moieties of the present invention. The acids used to prepare pharma- ceutically acceptable acid addition salts of the above-mentioned base compounds useful in the present invention are, inter alia, those which form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [i.e., 1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts. Pharmaceutically acceptable base addition salts can also be used to generate pharma-ceutically acceptable salt forms of the agents according to the invention. Chemical bases that can be used as reagents to prepare pharma-ceutically acceptable base salts of the agents that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharmacologically acceptable cations as 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 lower alkanolammonium, and other base salts of pharma-ceutically acceptable organic amines. The agents and / or polypeptide binding moieties of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, cake drying) and / or reconstitution technique can be used. Those of skill in the art will understand that lyophilization and reconstitution may lead to varying degrees of loss of antibody activity (e.g., in conventional immunoglobulins, IgM antibodies tend to experience greater loss of activity than IgG antibodies) and usage levels may need to be adjusted upward to compensate.In one embodiment, a lyophilized (freeze-dried) polypeptide binding moiety, when rehydrated, loses about 20% or less, or about 25% or less, or about 30% or less, or about 35% or less, or about 40% or less, or about 45% or less, or about 50% or less of its activity (before lyophilization). EXAMPLES

[0129] Here, specific non-limiting examples embodying certain aspects of the present invention are described. Two sets of surrogate antibodies were used to allow investigation of the blocking effect of FcγRIIB in a complex in vivo system. The mouse equivalent of Fc competitive antibody 6G11 is called AT130-2. To Fc-mute the human antibody (so that binding to FcγR is severely impaired or negligible), amino acid position 297 is substituted from N to Q. To make the mouse antibody Fc-mute, the same position is substituted from N to Q. Thus, in the mouse system, it is called AT-130, while the present patent application concerns the human equivalent 6G11. Briefly, human 6G11 corresponds to the mouse surrogate AT1302-2 (double Fc:FcγR dominant, also referred to herein as Fc-competitive), while 6G11-N297Q corresponds to AT130-3-N297A (double Fc:FcγR deficient, also referred to herein as Fc-mute). We have previously shown that these human and mouse Fc:FcγR dominant or Fc:FcγR deficient anti-FcγRIIB antibodies are functionally and biochemically equivalent (WO2019 / 138005 and WO2021 / 009358).

[0130] A different (and well known to those skilled in the art) method of Fc-muting an antibody is to remove the Fc portion and produce a Fab or F(ab') 2 It forms fragments.

[0131] The anti-HER2 mAb used below is clone 7.16.4 (mIgG2a) obtained from BioXcell.

[0132] Surrogate anti-mouse FcγRIIB mAb AT130-3-N297A improves the in vivo antitumor efficacy of anti-HER2 mAb and enables treatment of HER2-low expressing cancers Therapeutic effects in TUBO tumor model (HER2-high expressing cancer model) To evaluate the in vivo anti-tumor efficacy of anti-mouse FcγRIIB mAb AT130-3-N297A in combination with anti-HER2 mAb, the combination was investigated in vivo in the TUBO tumor model described below.

[0133] Mice were bred and maintained in a facility in Lund, Sweden, in accordance with applicable regulations and guidelines, including those of the Institution and the Swedish Board of Agriculture. Female BalbC mice, 6-8 weeks of age, were supplied by Taconic (Bomholt, Denmark) and maintained in a local animal facility. TUBO cells (University of Turin) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells reached semi-confluency, they were detached with trypsin and cultured at 10 × 10 6 The cells were resuspended in sterile PBS at 1 × 10 cells / mL. Glutamax-buffered RPMI (RPMI medium), FCS (fetal calf serum), and PBS (phosphate-buffered saline) were all purchased from Invitrogen and used below. Mice were resuspended in 1 × 10 6 100 μL of cell suspension corresponding to cells / mouse were injected subcutaneously. 12-13 days after injection, mice were treated ip with 10 mg / kg of antibody twice a week as indicated (isotype control, anti-HER2, AT130-3-N297A, and combination of anti-HER2 and AT130-3-N297A). Tumors were measured twice a week until they reached 15 mm in diameter, after which mice were terminated.

[0134] In this HER2-high experimental cancer model, anti-mouse FcγRIIB mAb AT130-3-N297A significantly improved anti-HER2-mediated survival compared to single-agent anti-HER2 therapy (Figure 1A).

[0135] Combined treatment with anti-Fcγ RIIB enables anti-HER2 therapeutic effects on HER2 low-expressing cancers

[0136] To evaluate the in vivo effect of anti-mouse FcγRIIB mAb AT130-3-N297A in combination with anti-HER2 mAb against HER2-low cancer, the same HER2-high TUBO mouse tumor model as described above was used, but with a lower dose of antibody, which results in fewer HER2 receptors on the cancer cells that are occupied and targeted by anti-HER2.Thus, all other factors are the same, except for the antibody targeting HER2 receptors, making this tumor model ideal for evaluating and demonstrating the anti-HER2 effect that anti-FcγRIIB can achieve against HER2-low expressing cancer.

[0137] Mice were bred and maintained as described above. Female BalbC mice aged 6-8 weeks were supplied by Taconic (Bomholt, Denmark) and maintained in the local animal facility. TUBO cells (University of Turin) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells reached semi-confluency, they were detached with trypsin and cultured at 10 × 10 6 Resuspended in sterile PBS at 1 × 10 cells / mL. 6 100 μL of cell suspension corresponding to cells / mouse were injected subcutaneously. 12-13 days after injection, mice were treated ip with 1 mg / kg of antibody twice a week as indicated (isotype control, anti-HER2, and anti-HER2 in combination with AT130-3-N297A). Tumors were measured twice a week until they reached a diameter of 15 mm, after which mice were terminated.

[0138] In this experimental HER2-low model, anti-HER2 single agent treatment showed severely impaired therapeutic efficacy, with only 1 of 10 treated mice cured compared to 3 of 10 in the HER2-high cancer model. After combination with anti-mouse FcγRIIB mAb AT130-3-N297A, which has no anti-tumor activity by itself, a complete therapeutic effect was observed in the HER2-low cancer model similar to that observed in the HER2-high model (3 of 10 mice cured) (Figure 1B). Next, an experiment was designed to compare the amount of HER2 targeted in the HER2-high and HER2-low models. Tumors were established as described in AB and mice (n=3) were treated with 1 mg / kg or 10 mg / kg Alexa Flour (AF) 647-labeled anti-HER2, allowing the determination of antibody targeting HER2 in the HER2-high and HER2-low cancer models. Mice were dosed twice (2-3 days between doses). Two days after the second injection, mice were euthanized and tumors were harvested. Tumors were minced into small pieces and enzymatically digested with a mixture of DNAse and Liberase at 37°C. The tumor solution was then filtered through a cell strainer to obtain a single-cell solution. Fluorescent dye (AF647)-labeled anti-HER2 in tumors was quantified by FACS. Tumor cells from HER2-high cancer models (mice injected with 10 mg / kg anti-HER2) showed a 10-fold increase in targeted HER2 receptors compared to tumor cells from HER2-low cancer models (injected with 1 mg / kg anti-HER2).

[0139] No therapeutic effect observed when Fc-competitive AT130-2 was combined with anti-HER2 mAb To assess whether Fc-competitive AT130-2 also improves the in vivo anti-tumor efficacy of anti-HER2 mAbs, the combination was investigated in vivo in the tumor models described below.

[0140] Mice were bred and maintained as described above. Female BalbC mice aged 6-8 weeks were supplied by Taconic (Bomholt, Denmark) and maintained in the local animal facility. TUBO cells (University of Turin) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells reached semi-confluency, they were detached with trypsin and cultured at 10 × 10 6 Resuspended in sterile PBS at 1 × 10 cells / mL. 6 100 μL of cell suspension corresponding to cells / mouse was injected subcutaneously. 12-13 days after injection, mice were treated ip with 10 mg / kg of antibody twice a week as indicated (isotype control, anti-HER2, or combination of anti-HER2 with AT130-2-N297A or AT130-2 wt). Tumors were measured twice a week until they reached 15 mm in diameter, after which mice were terminated.

[0141] Fc:FcγR dominant (wt) AT130-2 therefore shows no improvement in therapeutic antitumor efficacy when combined with anti-HER2 (FIG. 2).

[0142] The improved therapeutic efficacy when anti-HER2 therapy was combined with anti-mouse FcγRIIB mAb AT130-3-N297A is associated with increased influx of myeloid cells within the tumor. To assess the mechanism of action of the therapeutic effect of combining anti-HER2 therapy with anti-mouse FcγRIIB mAb AT130-2-N297A, immune profiling of treated tumors was performed as described below.

[0143] Mice were bred and maintained as described above. Female BalbC mice aged 6-8 weeks were supplied by Taconic (Bomholt, Denmark) and maintained in the local animal facility. TUBO cells (University of Turin) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells reached semi-confluency, they were detached with trypsin and cultured at 10 × 10 6 Resuspended in sterile PBS at 1 × 10 cells / mL.6 100 μL of cell suspension corresponding to cells / mouse was injected subcutaneously. When tumors reached approximately 7×7 mm in size, mice were injected with antibodies (10 mg / kg ip-isotype control, anti-HER2, AT130-2-N297A, and combination of anti-HER2 and AT130-2-N297A). Tumors were harvested 24 hours after the third injection, 7–8 days after treatment initiation (at this time, tumors in the combination group had clearly regressed).

[0144] Tumors were chopped into small pieces and enzymatically digested with a mixture of DNAse and Liberase at 37°C. The tumor solution was filtered through a cell strainer to obtain a single cell solution. Before staining, the cell solution was blocked with IVIg (human normal immunoglobulin for intravascular administration, Kiovig, Takeda). Immune cells were identified and quantified by FACS using the following markers: CD45, CD3, CD4, CD8, CD25, CD11b, Ly6C, Ly6G, MHCII, F4 / 80, CD49b, and NK 1.1 (all from BD Biosciences).

[0145] As seen in Figure 3, the anti-HER2 / anti-FcγRIIB-NA combination alters immune cell composition within tumors. Combined treatment with anti-HER2 and anti-FcγRIIB-NA results in an increase in the CD11b+ / F4 / 80+ population compared to single treatments, which is consistent with increased recruitment of effector cells and increased antibody-mediated depletion of HER2-targeted tumor cells. This increase is most pronounced in the HER2-high model (mice treated with an anti-HER2 dose of 10 mg / kg) (Figure 3).

[0146] B16 lung metastasis model To assess whether anti-FcγRIIB-NA can enhance the depletion activity and therapeutic efficacy of other tumor-directed therapeutic antibodies targeting solid tumors, the therapeutic efficacy of the combination of anti-FcγRIIB-NA with TA99, an antibody specific for the gp75 melanoma tumor antigen, in a B16 metastatic melanoma model was investigated.

[0147] Mice were bred and maintained as described above. Female C57 mice, 6-8 weeks old, were supplied by Taconic (Bomholt, Denmark) and maintained in the local animal facility. B16 cells (ATCC) were grown in Glutamax-buffered RPMI supplemented with 10% FCS. When cells reached semi-confluency, they were detached with trypsin and 2.5 × 10 6 Resuspended in sterile PBS at 5 × 10 cells / mL. 5 200 μL of cell suspension corresponding to cells / mouse were injected iv. Four days after tumor cell injection, mice were injected with antibodies (10 mg / kg ip - isotype control, TA99, AT130-2-N297A, and combination of TA99 and AT130-2-N297A). Five treatments were administered with 2-3 days interval. Twenty-one days after the start of treatment, mice were sacrificed and metastatic content in the lungs was quantified. Figure 4

[0148] A moderate reduction in lung metastases was observed after treatment with TA99 alone compared to untreated animals (Figure 4). After combination treatment with anti-FcγRIIB-NA, which by itself does not affect metastasis formation, the therapeutic effect of the tumor-directed targeting antibody TA99 was greatly enhanced, significantly reducing lung metastases compared to TA99 treatment alone. Thus, combination treatment with anti-FcγRIIB-NA enhances the therapeutic effect of various tumor-directed targeting antibodies specific for various tumor antigens associated with various FcγRIIB solid cancers.

Claims

1. A first antibody molecule that specifically binds FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, for use in treating an FcγRIIB-negative cancer in a patient, in combination with a second antibody molecule that specifically binds to a receptor present on a tumor cell, wherein the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor.

2. (i) a first antibody molecule that specifically binds FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor; A pharmaceutical composition for use in treating an FcγRIIB-negative cancer in a patient.

3. (i) a first antibody molecule that specifically binds FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) A kit for use in treating an FcγRIIB-negative cancer, comprising a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor.

4. In the manufacture of a medicament for use in treating an FcγRIIB-negative cancer in a patient, (i) a first antibody molecule that specifically binds FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region that binds to at least one activating Fcγ receptor. Use of.

5. 1. A method of treating an FcγRIIB negative cancer in a patient, comprising: (i) a first antibody molecule that specifically binds FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via the Fc region; and (ii) administering a second antibody molecule that specifically binds to a receptor present on a tumor cell, the second antibody molecule having an Fc region capable of activating at least one activating Fcγ receptor.

6. A first antibody molecule for use in combination with a second antibody molecule described in claim 1, a pharmaceutical composition for use according to claim 2, a kit for use according to claim 3, a use according to claim 4, or a method according to claim 5, wherein the FcγRIIB negative cancer is a solid cancer.

7. A first antibody molecule for use in combination with a second antibody molecule according to claim 1 or 6, a pharmaceutical composition for the use according to claim 2 or 6, a kit for the use according to claim 3 or 6, a use according to claim 4 or 6, or a method according to claim 5 or 6, wherein the binding of the second antibody molecule to the receptor on the tumor cell causes depletion of the tumor cell.

8. A first antibody molecule for use in combination with a second antibody molecule of claim 1, 6, or 7, wherein the first antibody lacks an Fc region; a pharmaceutical composition for the use of claim 2, 6, or 7; a kit for the use of claim 3, 6, or 7; a use of claim 4, 6, or 7; or a method of claim 5, 6, or 7.

9. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 8, wherein the second antibody molecule binds to human epidermal growth factor receptor 2 (HER2), a pharmaceutical composition for the use according to claim 21 or any one of claims 6 to 8, a kit for the use according to claim 3 or any one of claims 6 to 8, a use according to claim 4 or any one of claims 6 to 8, or a method according to any one of claims 5 or any one of claims 6 to 8.

10. A first antibody molecule for use in combination with a second antibody molecule according to claim 9, a pharmaceutical composition for the use according to claim 9, a kit for the use according to claim 8, a use according to claim 8, or a method according to claim 9, wherein the cancer is selected from the group consisting of breast cancer and gastric cancer.

11. A first antibody molecule for use in combination with a second antibody molecule of claim 9 or 10, a pharmaceutical composition for the use of claim 9 or 10, a kit for the use of claim 9 or 10, a use of claim 9 or 10, or a method of claim 9 or 10, wherein the cancer underexpresses HER2.

12. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 9 to 11, a pharmaceutical composition for the use according to any one of claims 9 to 11, a kit for the use according to any one of claims 9 to 11, a use according to any one of claims 9 to 11, or a method according to any one of claims 9 to 11, wherein the cancer is a cancer in a patient who has previously been treated with an antibody molecule that specifically binds to HER2 but has developed resistance to the antibody.

13. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 9 to 12, a pharmaceutical composition for the use according to any one of claims 9 to 12, a kit for the use according to any one of claims 9 to 12, a use according to any one of claims 9 to 12, or a method according to any one of claims 9 to 12, wherein the second antibody molecule is trastuzumab or a trastuzumab biosimilar.

14. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 8, wherein the second antibody molecule binds to human epidermal growth factor receptor (EGFR), a pharmaceutical composition for the use according to claim 21 or any one of claims 6 to 8, a kit for the use according to claim 3 or any one of claims 6 to 8, a use according to claim 4 or any one of claims 6 to 8, or a method according to any one of claims 5 or any one of claims 6 to 8.

15. 15. The method of claim 14, the pharmaceutical composition for the use of claim 14, the kit for the use of claim 14, the use of claim 14, or the method of claim 14, wherein the cancer is selected from the group consisting of head and neck cancer and colorectal cancer.

16. A first antibody molecule for use in combination with a second antibody molecule according to claim 14 or 15, a pharmaceutical composition for the use according to claim 14 or 15, a kit for the use according to claim 14 or 15, a use according to claim 14 or 15, or a method according to claim 14 or 15, wherein the second antibody molecule is cetuximab or a cetuximab biosimilar.

17. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 16, a pharmaceutical composition for the use according to any one of claims 2 or 6 to 16, a kit for the use according to any one of claims 3 or 6 to 16, a use according to any one of claims 4 or 6 to 16, or a method according to any one of claims 5 or 6 to 16, wherein the first antibody molecule is selected from the group consisting of a human antibody molecule, a humanized antibody molecule, and an antibody molecule of human origin.

18. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 17, wherein the first antibody molecule is a monoclonal antibody molecule or a monoclonal-derived antibody molecule, a pharmaceutical composition for the use according to any one of claims 2 or 6 to 17, a kit for the use according to any one of claims 3 or 6 to 17, a use according to any one of claims 4 or 6 to 17, or a method according to any one of claims 5 or 6 to 17.

19. The first antibody molecule may be a full-length antibody, a chimeric antibody, a single-chain antibody, a Fab fragment, (Fab') ２ Fragment, Fab' fragment, (Fab') ２ A first antibody molecule for use in combination with a second antibody molecule of claim 1 or any one of claims 6 to 18, the first antibody molecule being selected from the group consisting of a Fv fragment, an Fv fragment and an scFv fragment; a pharmaceutical composition for the use of claim 2 or any one of claims 6 to 18; a kit for the use of claim 3 or any one of claims 6 to 18; a use of claim 4 or any one of claims 6 to 18; or a method of claim 5 or any one of claims 6 to 18.

20. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 18, wherein the first antibody molecule is a human IgG antibody molecule having an aglycosylated Fc region or an IgG antibody molecule of human origin having an aglycosylated Fc region, a pharmaceutical composition for use according to any one of claims 2 or 6 to 18, a kit for use according to any one of claims 3 or 6 to 18, a use according to any one of claims 4 or 6 to 18, or a method according to any one of claims 5 or 6 to 18.

21. A first antibody molecule for use in combination with a second antibody molecule according to claim 20, a pharmaceutical composition for the use according to claim 20, a kit for the use according to claim 20, a use according to claim 20, or a method according to claim 20, wherein the IgG antibody molecule is an IgG1 antibody molecule or an IgG2 antibody molecule.

22. A first antibody molecule for use in combination with a second antibody molecule according to claim 21, wherein the IgG antibody molecule is a non-glycosylated human IgG1, or a non-glycosylated humanized mouse antibody, or a non-glycosylated humanized llama hcIgG antibody, or a non-glycosylated chimerized mouse IgG, a pharmaceutical composition for the use according to claim 21, a kit for the use according to claim 21, a use according to claim 21, or a method according to claim 21.

23. A first antibody molecule for use in combination with a second antibody molecule according to claim 22, which is aglycosylated by an amino acid substitution at position 297, a pharmaceutical composition for the use according to claim 22, a kit for the use according to claim 22, the use according to claim 22, or the method according to claim 22.

24. A first antibody molecule for use in combination with a second antibody molecule of claim 23, which is aglycosylated by an N297Q substitution, a pharmaceutical composition for the use of claim 23, a kit for the use of claim 23, the use of claim 23, or the method of claim 23.

25. The first antibody molecule comprises: (i) a variable heavy chain (VH) comprising the following CDRs: SEQ ID NO:51 and SEQ ID NO:52 and SEQ ID NO:53; and A variable light chain (VL) comprising the following CDRs: SEQ ID NO:54 and SEQ ID NO:55 and SEQ ID NO:56; (ii) a VH comprising the following CDRs: SEQ ID NO:57 and SEQ ID NO:58 and SEQ ID NO:59; and VL comprising the following CDRs: SEQ ID NO:60 and SEQ ID NO:61 and SEQ ID NO:62; (iii) a VH comprising the following CDRs: SEQ ID NO:63 and SEQ ID NO:64 and SEQ ID NO:65; and VL comprising the following CDRs: SEQ ID NO:66 and SEQ ID NO:67 and SEQ ID NO:68; (iv) a VH comprising the following CDRs: SEQ ID NO:69 and SEQ ID NO:70 and SEQ ID NO:71; and VL comprising the following CDRs: SEQ ID NO:72 and SEQ ID NO:73 and SEQ ID NO:74; (v) a VH comprising the following CDRs: SEQ ID NO:75 and SEQ ID NO:76 and SEQ ID NO:77; and VL comprising the following CDRs: SEQ ID NO:78 and SEQ ID NO:79 and SEQ ID NO:80; (vi) a VH comprising the following CDRs: SEQ ID NO: 81 and SEQ ID NO: 82 and SEQ ID NO: 83; and VL comprising the following CDRs: SEQ ID NO:84 and SEQ ID NO:85 and SEQ ID NO:86; (vii) a VH comprising the following CDRs: SEQ ID NO:87 and SEQ ID NO:88 and SEQ ID NO:89; and VL comprising the following CDRs: SEQ ID NO:90 and SEQ ID NO:91 and SEQ ID NO:92; (viii) a VH comprising the following CDRs: SEQ ID NO:93 and SEQ ID NO:94 and SEQ ID NO:95; and VL comprising the following CDRs: SEQ ID NO:96 and SEQ ID NO:97 and SEQ ID NO:98; (ix) a VH comprising the following CDRs: SEQ ID NO:99 and SEQ ID NO:100 and SEQ ID NO:101; and VL comprising the following CDRs: SEQ ID NO:102 and SEQ ID NO:103 and SEQ ID NO:104; (x) a VH comprising the following CDRs: SEQ ID NO: 105 and SEQ ID NO: 106 and SEQ ID NO: 107; and VL comprising the following CDRs: SEQ ID NO:108 and SEQ ID NO:109 and SEQ ID NO:110; (xi) a VH comprising the following CDRs: SEQ ID NO:111 and SEQ ID NO:112 and SEQ ID NO:113; and VL comprising the following CDRs: SEQ ID NO:114 and SEQ ID NO:115 and SEQ ID NO:116; (xii) a VH comprising the following CDRs: SEQ ID NO:117 and SEQ ID NO:118 and SEQ ID NO:119; and VL comprising the following CDRs: SEQ ID NO:120 and SEQ ID NO:121 and SEQ ID NO:122; (xiii) a VH comprising the following CDRs: SEQ ID NO: 123 and SEQ ID NO: 124 and SEQ ID NO: 125; and VL comprising the following CDRs: SEQ ID NO:126 and SEQ ID NO:127 and SEQ ID NO:128; (xiv) a VH comprising the following CDRs: SEQ ID NO: 129 and SEQ ID NO: 130 and SEQ ID NO: 131; and VL comprising the following CDRs: SEQ ID NO:132 and SEQ ID NO:133 and SEQ ID NO:134; (xv) a VH comprising the following CDRs: SEQ ID NO: 135 and SEQ ID NO: 136 and SEQ ID NO: 137; and VL comprising the following CDRs: SEQ ID NO:138 and SEQ ID NO:139 and SEQ ID NO:140; (xvi) a VH comprising the following CDRs: SEQ ID NO: 141 and SEQ ID NO: 142 and SEQ ID NO: 143; and VL comprising the following CDRs: SEQ ID NO:144 and SEQ ID NO:145 and SEQ ID NO:146; (xvii) a VH comprising the following CDRs: SEQ ID NO: 147 and SEQ ID NO: 148 and SEQ ID NO: 149; and VL comprising the following CDRs: SEQ ID NO:150 and SEQ ID NO:151 and SEQ ID NO:152; (xviii) a VH comprising the following CDRs: SEQ ID NO: 153 and SEQ ID NO: 154 and SEQ ID NO: 155; and VL comprising the following CDRs: SEQ ID NO:156 and SEQ ID NO:157 and SEQ ID NO:158; (xix) a VH comprising the following CDRs: SEQ ID NO: 159 and SEQ ID NO: 160 and SEQ ID NO: 161; and VL comprising the following CDRs: SEQ ID NO:162 and SEQ ID NO:163 and SEQ ID NO:164; (xx) a VH comprising the following CDRs: SEQ ID NO: 165 and SEQ ID NO: 166 and SEQ ID NO: 167; and VL comprising the following CDRs: SEQ ID NO:168 and SEQ ID NO:169 and SEQ ID NO:170; (xxi) a VH comprising the following CDRs: SEQ ID NO: 171 and SEQ ID NO: 172 and SEQ ID NO: 173; and VL comprising the following CDRs: SEQ ID NO:174 and SEQ ID NO:175 and SEQ ID NO:176; (xxii) a VH comprising the following CDRs: SEQ ID NO: 177 and SEQ ID NO: 178 and SEQ ID NO: 179; and VL comprising the following CDRs: SEQ ID NO:180 and SEQ ID NO:181 and SEQ ID NO:182; (xxiii) a VH comprising the following CDRs: SEQ ID NO: 183 and SEQ ID NO: 184 and SEQ ID NO: 185; and A VL comprising the following CDRs: SEQ ID NO:186 and SEQ ID NO:187 and SEQ ID NO:188; or (xxiv) a VH comprising the following CDRs: SEQ ID NO: 189 and SEQ ID NO: 190 and SEQ ID NO: 191; and VL comprising the following CDRs: SEQ ID NO:192 and SEQ ID NO:193 and SEQ ID NO:194; A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 24, a pharmaceutical composition for the use according to any one of claims 2 or 6 to 24, a kit for the use according to any one of claims 3 or 6 to 24, a use according to any one of claims 4 or 6 to 24, or a method according to any one of claims 5 or 6 to 24 comprising

26. The first antibody molecule comprises: (i) a VH having SEQ ID NO:3, and a VL having SEQ ID NO:27; (ii) a VH having SEQ ID NO:4, and a VL having SEQ ID NO:28; (iii) a VH having SEQ ID NO:5, and a VL having SEQ ID NO:29; (iv) a VH having SEQ ID NO:6, and a VL having SEQ ID NO:30; (v) a VH having SEQ ID NO:7, and a VL having SEQ ID NO:31; (vi) a VH having SEQ ID NO:8, and a VL having SEQ ID NO:32; (vii) a VH having SEQ ID NO:9, and a VL having SEQ ID NO:33; (viii) a VH having SEQ ID NO: 10, and a VL having SEQ ID NO: 34; (ix) a VH having SEQ ID NO: 11, and a VL having SEQ ID NO: 35; (x) a VH having SEQ ID NO: 12 and a VL having SEQ ID NO: 36; (xi) a VH having SEQ ID NO: 13, and a VL having SEQ ID NO: 37; (xii) a VH having SEQ ID NO: 14, and a VL having SEQ ID NO: 38; (xiii) a VH having SEQ ID NO: 15, and a VL having SEQ ID NO: 39; (xiv) a VH having SEQ ID NO: 16, and a VL having SEQ ID NO: 40; (xv) a VH having SEQ ID NO: 17, and a VL having SEQ ID NO: 41; (xvi) a VH having SEQ ID NO: 18, and a VL having SEQ ID NO: 42; (xvii) a VH having SEQ ID NO: 19, and a VL having SEQ ID NO: 43; (xviii) a VH having SEQ ID NO: 20, and a VL having SEQ ID NO: 44; (xix) a VH having SEQ ID NO:21, and a VL having SEQ ID NO:45; (xx) a VH having SEQ ID NO:22 and a VL having SEQ ID NO:46; (xxi) a VH having SEQ ID NO:23, and a VL having SEQ ID NO:47; (xxii) a VH having SEQ ID NO:24, and a VL having SEQ ID NO:48; (xxiii) a VH having SEQ ID NO: 25, and a VL having SEQ ID NO: 49; or (xxiv) a VH having SEQ ID NO:26, and a VL having SEQ ID NO:50; A first antibody molecule for use in combination with a second antibody molecule according to claim 1, a pharmaceutical composition for the use according to claim 2, a kit for the use according to claim 3, a use according to claim 4, or a method according to claim 5, comprising the steps of:

27. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 26, a pharmaceutical composition for use according to any one of claims 2 or 6 to 26, a kit for use according to any one of claims 3 or 6 to 26, a use according to any one of claims 4 or 6 to 26, or a method according to any one of claims 5 or 6 to 26, wherein the first antibody molecule comprises a VH comprising the following CDRs: SEQ ID NO:171 and SEQ ID NO:172 and SEQ ID NO:173, and a VL comprising the following CDRs: SEQ ID NO:174 and SEQ ID NO:175 and SEQ ID NO:

176.

28. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 27, wherein the first antibody molecule comprises a VH having SEQ ID NO: 23 and a VL having SEQ ID NO: 47, a pharmaceutical composition for the use according to any one of claims 2 or 6 to 27, a kit for the use according to any one of claims 3 or 6 to 27, a use according to any one of claims 4 or 6 to 27, or a method according to any one of claims 5 or 6 to 27.

29. A first antibody molecule for use in combination with a second antibody molecule according to claim 27 or 28, wherein the first antibody molecule has a constant heavy chain (CH) having SEQ ID NO: 195 and a constant light chain (CL) having SEQ ID NO: 2, a pharmaceutical composition for the use according to claim 27 or 28, a kit for the use according to claim 27 or 28, a use according to claim 27 or 28, or a method according to claim 27 or 28.

30. 20. An antibody molecule that specifically binds to FcγRIIB for use, a pharmaceutical composition for use, a kit, a use, or a method substantially as herein described in the accompanying claims, specification, examples, and / or drawings.

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