Novel combinations of antibodies and uses thereof

Abstract

In the treatment of FcγRIIb-negative cancer in a patient, a first 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 its Fc region, and a second antibody molecule for use therewith that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, and the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, such that binding of the second antibody molecule to the receptor on the immune cell causes elimination and / or inactivation of the immune cell, are described, as well as pharmaceutical compositions and kits comprising these two antibody molecules, and methods of treating cancer using these two antibodies. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] The present invention relates to the combined use, in the treatment of FcγRIIb-negative cancers, of 1) an antibody molecule that specifically binds to FcγRIIb via its Fab region but lacks an Fc region or has reduced binding to at least one Fcγ receptor via its Fc region, and 2) an antibody molecule that suppresses anti-cancer immunity and depletes or inactivates immune cells by specifically binding to a receptor present on immune cells, wherein the immune cell-depleting or immune cell-inactivating antibody molecule has an Fc region that binds to at least one activating Fcγ receptor. [Background technology]

[0002] The inhibitory Fcγ receptor (FcγR) IIB, expressed by numerous cells of the immune system, has long been recognized to negatively regulate both innate and adaptive immunity through immune complex (IC) engagement. Similarly, knowledge that FcγRIIB negatively regulates monoclonal antibody-mediated immunotherapy has been known for over a decade. Thus, FcγRIIB-deficient mice can eliminate tumors more effectively than wild-type (WT) mice when treated with therapeutic monoclonal antibodies, indicating that FcγRIIB expression on effector cells (i.e., macrophages and monocytes) suppresses their phagocytic and cytotoxic potential in vivo. Furthermore, FcγRIIB regulates the antigen-presenting potential of dendritic cells (DCs) and FcγRIIB-positive DCs (van Montfoor et al., J Immunol. 2012 Jul 1;189(1):92-101). DCs have an enhanced ability to activate naive T cells. Recently, antagonistic antibodies that block FcγRIIB signaling and internalization in B cells have been developed. These antibodies have shown efficient depletion of FcγRIIB-expressing B cells, enhanced rituximab-mediated depletion of normal and malignant B cells, and demonstrated utility in hematological malignancies. However, the utility of these antibodies in the treatment of FcγRIIB-negative cancers, such as solid tumors, has not been explored or demonstrated. Summary of the Invention

[0003] Here, Applicants show that only anti-FcγRIIB antibodies that lack an Fc region or whose Fc region exhibits reduced or impaired binding to FcγRs, such as F(ab)'2 antibodies or aglycosylated antibodies, can unexpectedly enhance the therapeutic activity of antibodies used to treat FcγRIIB-negative cancers, including solid tumors. This finding was unexpected, as previous studies have shown that wild-type IgG1 anti-FcγRIIB antibodies can similarly block FcγRIIB receptors and prevent rituximab internalization and rituximab-induced FcγRIIB phosphorylation in vitro.

[0004] According to the present invention, it is possible to enhance the therapeutic activity of immunomodulatory anti-cancer antibodies, whose therapeutic activity depends on FcγR engagement, including, but not limited to, so-called checkpoint inhibitor targets such as CTLA-4, immune agonist targets such as OX40, 4-1BB, and GITR, and interleukin-2 receptor (IL-2R).

[0005] Disclosed herein is a method for treating FcγRIIb-negative cancer in a patient, comprising administering to the patient a therapeutically effective amount of an antibody that specifically binds FcγRIIb via (or through) its Fc region and that lacks an Fc region or has reduced binding to an Fcγ receptor via (or through) its Fc region, A second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and binding of the second antibody molecule to the receptor on the immune cell causes elimination and / or inactivation of the immune cell.

[0006] Also disclosed herein is a pharmaceutical composition for use in treating an FcγRIIb-negative cancer in a patient, comprising: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, and the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and binding of the second antibody to the receptor on the immune cell causes removal and / or inactivation of the immune cell.

[0007] Further disclosed herein is a kit for use in treating FcγRIIb-negative cancer, comprising: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, and the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and binding of the second antibody molecule to the receptor on the immune cell causes removal or inactivation of the immune cell.

[0008] Further disclosed herein is a method for treating an FcγRIIb-negative cancer in a patient, comprising: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) Use of a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, and the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and binding of the second antibody to the receptor on the immune cell causes removal or inactivation of the immune cell.

[0009] Also disclosed herein is a method for treating an FcγRIIb-negative cancer in a patient, comprising: (i) a first antibody molecule that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, and the second antibody molecule has an Fc region that is capable of activating at least one activating Fcγ receptor, and binding of the second antibody to the receptor on the immune cell causes removal or inactivation of the immune cell. [Brief explanation of the drawings]

[0010] In the examples below, reference is made to the following figures:

[0011] [Figure 1] A potential mechanism explaining why the present invention works is shown. An antibody molecule (in this case, an anti-CTLA-4 antibody) that eliminates or inactivates immune cells binds to a receptor present on immune cells (in this case, Tregs) that suppress anti-cancer immunity. The Fc region of the anti-CTLA-4 antibody binds to an activating Fcγ receptor (in this case, present on the surface of macrophages). Furthermore, an antibody molecule that specifically binds to FcγRIIb binds to FcγRIIb present on the surface of macrophages. Because this specific antibody has an Fc region with reduced binding to activating Fcγ receptors, it does not bind to activating Fcγ receptors present on macrophages and instead freely binds to the Fc region of the anti-CTLA-4 antibody. Figure 1A shows the use of only the anti-CTLA-4 antibody. The anti-CTLA-4 antibody binds to both activating and inhibitory Fcγ receptors, thereby activating them, resulting in attenuated activation and therefore reduced effectiveness compared to a situation in which the anti-CTLA-4 antibody only binds to activating Fcγ receptors. Figure 1B shows that the combination of an anti-CTLA-4 antibody and a WT (wild-type) FcγRIIb antibody reduces activation. In this case, the FcγRIIb antibody blocks activating Fcγ receptors and activates inhibitory Fcγ receptors, which is the opposite of the desired effect of the present invention. Figure 1C shows that the combination of an anti-CTLA-4 antibody and an aglycosylated FcγRIIb antibody of the present invention results in maximal activation. There is no blocking of activating Fcγ receptors and no activation of inhibitory Fcγ receptors, resulting in maximal target removal. Here, the aglycosylated FcγRIIb antibody may lack an Fc region or otherwise have reduced binding to FcRs. [Figure 2]These results demonstrate that FcγRIIB antibodies impaired Fc binding to activating Fc receptors, whereas wild-type antibodies retained binding to activating Fc receptors and promoted B cell depletion by CD20 monoclonal antibodies (Figure 2A-B). CFSE+ hCD20+ / - x mFcγRII- / - (targeting) and mFcγRII- / - (non-targeting) splenocytes were infused into hFcγRIIB+ / - x mFcγRII- / - recipient mice. As before, mice were administered wild-type or N297Q FcγRIIB monoclonal antibody (6G11) (2 x 20 mg / kg) followed by Rit (0.2-2 mg / kg), and the ratio of CFSE+ CD19+ cells in the blood (A) and spleen (B) was determined. Data are aggregated from at least two independent experiments. (Figure 2C) CFSE+ hCD20+ / - x mFcγRII- / - (targeting) and mFcγRII- / - (non-targeting) splenocytes were injected into hFcγRIIB+ / - x mFcγRII- / - recipient mice. Mice were treated with wild-type or N297Q FcγRIIB monoclonal antibody (6G11) (20 mg / kg) followed by Rit (2 mg / kg). Expression of activating mFcγRs was quantified on splenic F4 / 80+ effector cells using the indicated monoclonal antibodies. (Figure 2D-E) The ability of wild-type and N297Q (NQ) hFcγRIIB-specific monoclonal antibodies (6G11; 10 μg / mL for 15 min) to induce hFcγRIIB ITIM phosphorylation (pFcγRIIB) in (Figure 2D) mouse BMDMs and (Figure 2E) isolated primary peripheral blood monocytes after high-density culture is shown. α-Tubulin, GAPDH, and hFcγRIIB were used as loading controls, as indicated. Representative blots are shown. [Figure 3]We demonstrate that a combination of wild-type and FcγR-null FcγRIIB monoclonal antibodies can optimally eliminate target cells. CFSE+ hCD20+ / - (target) and mFcγRII- / - (non-target) splenocytes were injected into hFcγRIIB+ / - x mFcγRII- / - (Balb / c) recipient mice. As indicated on the x-axis, mice were administered wild-type (2 × 10–20 mg / kg) or F(ab')2 (2 × 20 mg / kg) mFcγRII (AT130-5) or wild-type (2 × 20 mg / kg) or F(ab')2 (2 × 40 mg / kg) hFcγRIIB monoclonal antibody (AT10) followed by Rit (0.2–2 mg / kg). The percentage of splenic CFSE+CD19+ cells was then determined as before. Data are aggregated from 1–3 independent experiments. Each point represents the result of an individual mouse, and the mean ratio is indicated by the horizontal line. Data were analyzed using one-way ANOVA. [Figure 4] Figure 4A shows the evaluation of Treg depletion by anti-IL2R monoclonal antibody + / - FcγRIIB blockade with an Fc-inactive NA mutant monoclonal antibody. Female Balb / c mice were administered 100 μg of AT130-2NA intraperitoneally (i.p.). Six hours later, 100 μg of PC61 was administered i.p. Four days later, Tregs (FoxP3+) in the blood, spleen, and lymph nodes were assessed by FAC. Mice were sacrificed, and single-cell suspensions obtained from the spleen, lymph nodes, and blood were stained with antibodies against CD4, CD8, and B220 prior to intracellular FoxP3 staining before analysis by FAC. Leukocyte counts were determined in each tissue. Tregs were defined as CD8-CD4+FoxP3+, and the number of Tregs was calculated using the leukocyte count. Figure 4B shows C57BL / 6 mice were treated as described above. Figure 4C) shows the CD8 / Treg ratio calculated from Figure 4B). [Figure 5]Figure 1 shows the evaluation of Treg depletion by anti-IL2R monoclonal antibody + / - FcγRIIB blockade with wild-type or NA mutant monoclonal antibodies. Wild-type AT130-2 did not show any improvement in depletion, whereas the NA mutant did. A) 100 μg of AT130-2NA or mIgG1 wild-type AT130-2 was administered i.p. to female Balb / c mice. 100 μg of PC61 was administered i.p. for 6 hours. Four days later, Tregs (FoxP3+) in the spleen were determined by FAC. Mice were sacrificed, and single-cell suspensions obtained from the spleen were stained with antibodies against CD4, CD8, and B220 prior to intracellular FoxP3 staining before analysis by FAC canto. Leukocyte counts in each tissue were determined. Tregs were defined as CD8-CD4+FoxP3+, and the number of Tregs was calculated using the leukocyte count. Compared with wild-type mIgG1 AT130-2, the number of Tregs was significantly lower in the spleens of mice treated with the N297A antibody in combination with PC61 for 4 days (unpaired T-test, P=0.044). [Figure 6]Combination therapy with anti-CTLA-4 and FcγRIIB blockade is shown. 5×10 CT26 cells were subcutaneously injected into female BALB / c mice. When tumor width × length was approximately 100 mm, mice were randomly assigned to treatment groups. Treatments were administered on days 0, 2, 4, and 11. 9H10 (hamster anti-mouse CTLA4) alone mice received daily ip injections of 200 μg of antibody in 200 μL of PBS. On day 0, combination mice received ip injections of 100 μg of AT130-2N297A (anti-mouse CD32) in 200 μL of PBS, followed 6 hours later by ip injections of 200 μg of 9H10 in 200 μL of PBS. On days 2, 4, and 11, mice receiving the combination received a single 200 μL ip injection of both antibodies (200 μg of 9H10 and 100 μg of AT130-2NA). Tumor width and length were measured, and mice were sacrificed when tumor length × width exceeded 400 mm. Figure 6A) shows the treatment schedule: Group 1: no antibody; Group 2: anti-mCD32 (AT130-2NA; 100 μg); Group 3: anti-CTLA-4 (9H10; 200 μg); Group 4: AT130-2 plus (PC61) 6 hours later. Tumors were allowed to establish and treated at 100 mm. An additional dose was administered on day 12. Figure 6B) shows the growth of individual tumors. Figure 6C) shows the mean tumor area + / - SD or SEM. Figure 6D) shows animal survival. Figure 6E) Composite from two separate experiments (n=10 / group) showing survival. The combination of 9H10 and AT130-2NA (NA combo) was significantly more potent at extending survival than 9H10 alone (p=0.0179). [Figure 7]Combination therapy of anti-CTLA-4 and FcγRIIB blockade is shown, comparing wild-type (denoted M1 combo) and Fc-inactive (denoted NA combo) AT130-2 monoclonal antibodies. 5 × 10 CT26 cells were injected subcutaneously into female BALB / c mice. When tumors were approximately 100 mm in width and length, mice were randomized into treatment groups. Treatments were administered on days 0, 2, 4, and 11. On day 0, combination mice received 100 μg of AT130-2N297A or AT130-2mIgG1 (anti-mouse CD32) ip in 200 μL of PBS, followed 6 hours later by 200 μg of 9H10 ip in 200 μL of PBS. On days 2, 4, and 11, mice treated with the combination received a single ip injection of 200 μL of both antibodies (200 μg of 9H10 and 100 μg of AT130-2NA / mIgG1). Tumor width and length were measured, and mice were sacrificed when tumor length × width exceeded 400 mm. Data are shown as survival curves below (N = 11–12). Survival time was significantly longer in the group treated with the NA combination compared to mice receiving the mIgG1 combination (log-rank test P = 0.0460). [Figure 8] To demonstrate the combined effect of anti-PD-L1 and FcγRIIB blockade on antibody NA format, experiments were performed using both wild-type mIgG1 and NA formats. Figure 8A shows the growth of individual tumors in each group (one graph per group). Numbers indicate the number of surviving mice in each group. Figure 8B shows the survival curves of the mice. [Figure 9] Figure 1 shows wild-type (top graph) and N297A (bottom graph) formats of the anti-mouse FcγRIIB monoclonal antibody AT130-2 binding to FcγR. This shows SPR analysis of AT130-2 in either format binding to the indicated mouse FcγR (100 nM). [Figure 10]Figure 10 shows wild-type 6G11 vs. N297A 6G11 binding to Fcγ receptors (mouse and human). This shows SPR analysis of native and N297A anti-huCD32b 6G11 hIgG1 binding to the indicated low-affinity human and mouse FcγRs. Figures 10A and B show results for human FcγR (100 nM). Figures 10C and D show results for mouse FcγR (100 nM). Figures 10E and E show results for human FcγR (added in successive increments) - FcγRIIb and hCD64. Figures 10G and H show results for mouse FcγR (100 nM). This is the same as Figure 10C, but the scale has been changed for a clearer view. DETAILED DESCRIPTION OF THE INVENTION

[0012] Therefore, the present invention provides (i) an antibody molecule (hereinafter often referred to as a first antibody molecule or the first antibody molecule) that specifically binds to FcγRIIb via its Fab region and lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) An antibody molecule (hereinafter often referred to as a second antibody or the second antibody) that specifically binds to a receptor present on an immune cell, where the immune cell is an immune cell that suppresses anti-cancer immunity, and the antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and the binding of this antibody molecule to the receptor on the immune cell causes the elimination or inactivation of the immune cell, is used in combination with an antibody molecule. Thus, this second antibody molecule is an antibody molecule that eliminates or inactivates immune cells.

[0013] This combination is intended for use in treating FcγRIIb-negative cancers in patients and aims to improve the therapeutic effect of the second antibody molecule by enhancing the binding of its Fc portion to activating FcγRs and reducing the binding / activation of inhibitory FcγRs.

[0014] 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 via their CDR sequences, where the antibody specifically binds to one or more Fc receptors.

[0015] A subgroup of Fc receptors is the Fcγ receptor (FcγR), which is specific for IgG antibodies. There are two types of Fcγ receptors: activating Fcγ receptors (also called 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 (CD32b) is an inhibitory Fcγ receptor, while FcγRI (CD64), FcγRIIa (CD32a), FcγRIIc (CD32c), FcγRIIIa (CD16a), and FcγRIV are activating Fcγ receptors. FcγgRIIIb is a GPI-linked receptor expressed on neutrophils that lacks ITAM motifs but is also considered activating due to its ability to crosslink lipid rafts and bind to other receptors. In mice, activating receptors are FcγRI, FcγRIII, and FcγRIV.

[0016] It is known that antibodies regulate immune cell activity through interaction 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 (activating:inhibitory ratios) (Nimmerjahn et al., Science. 2005 Dec 2;310(5753):1510-2).

[0017] By binding to inhibitory Fcγ receptors, antibodies can inhibit, block, and / or downregulate effector cell function.

[0018] By binding to activating Fcγ receptors, antibodies can activate effector cell function, 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 cause an increase in certain activation markers, such as CD40, MHCII, CD38, CD80, and / or CD86.

[0019] An antibody molecule according to the present invention that specifically binds to FcγRIIb, i.e., a first antibody, binds to or interacts with this Fcγ receptor via the antibody's Fab region, i.e., via the antigen-binding region on the antibody that binds to the antigen, which is composed of one constant region and one variable region of the heavy and light chains, respectively. In particular, it binds to FcγRIIb present on immune effector cells, particularly FcγRIIb present on the surface of immune effector cells. If this antibody had a conventional or ordinary Fc region, it would also be able to bind to activating Fcγ receptors through normal interaction between the Fc region and the Fc receptor. However, according to the present invention, an antibody molecule that specifically binds to FcγRIIb either completely lacks an Fc region or has reduced binding to Fcγ receptors, meaning that an antibody molecule that specifically binds to or interacts with FcγRIIb does not bind well to Fcγ receptors or is completely unable to bind to or interact with Fcγ receptors. This is believed to have at least two important therapeutic consequences: 1) The lack of Fc-mediated binding to activating FcγRs leaves more activating Fcγ receptors available for Fc binding 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 elimination, a mechanism underlying the activity of checkpoint inhibitors as well as immune agonists and other immunomodulatory 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 enhancement of activating FcγRs and reduction of inhibitory Fcγ signaling in immune effector cells responding to a second immunomodulatory anti-cancer antibody.

[0020] "Reduced binding" or "reduced affinity binding" in this 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 activating 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 this context, "normal IgG1" refers to a conventionally produced IgG1 with an unmutated 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, inter alia, in EP 0605442).

[0021] "Reduced binding" means that binding of the Fc region of an antibody molecule that specifically binds to FcγRIIb to activating Fcγ receptors is reduced by at least 10-fold at all Fc receptors compared to the binding of the Fc region of normal human IgG1 to the same receptors. 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.

[0022] In some embodiments of the present invention, an antibody molecule that specifically binds to FcγRIIb does not bind its Fc region at all, and in some such cases, the antibody does not have an Fc region, in which case it may be a Fab, Fab'2, scFv, or a PEGylated version thereof.

[0023] In some embodiments, the antibody molecule that specifically binds to FcγRIIb may be a llama antibody, particularly a llama hcIgG. Like all mammals, camelids produce conventional antibodies consisting of two heavy chains and two light chains joined in a Y-shape by disulfide bonds (IgG1). However, they also produce two unique subclasses of immunoglobulin G, IgG2 and IgG3, also known as heavy-chain IgG (hcIgG). These antibodies are composed of only two heavy chains that lack the CH1 region, but contain a V residue at their N-terminus. H It retains the antigen-binding region called H. Conventional Ig requires the combination of variable regions from both the heavy and light chains to allow for a high degree of diversity in antigen-antibody interactions. Separated heavy and light chains still exhibit this ability, but they exhibit significantly lower affinity compared to paired heavy and light chains. 4 A unique feature of hcIgGs is the ability of their monomeric antigen-binding domains to bind antigens without the need for pairing with additional domains and with specificity, affinity, and especially diversity comparable to conventional antibodies.

[0024] In some embodiments, reduced binding means that the antibody has a 20-fold reduced affinity for binding to FcγRI.

[0025] To obtain an IgG1 antibody, such as an IgG1 antibody, with reduced binding to Fc receptors, the Fc region of an IgG antibody can be modified by aglycosylation. For example, such aglycosylation of an IgG1 antibody can be achieved by, for example, substituting an asparagine amino acid at position 297 (N297X) of the antibody chain. The substitution may be with glutamine (N297Q), or alanine (N297A), or glycine (N297G), or asparagine (N297D), or with serine (N297S).

[0026] The Fc region may be modified by additional substitutions, for example, as described in Jacobsen FW et al., JBC 2017, 292, 1865-1875 (see, for example, Table 1). Such additional substitutions include L242C, V259C, A287C, R292C, V302C, L306C, V323C, I332C, and / or K334C. Such modifications also include combinations of the following substitutions in IgG1: L242C, N297G, K334C, A287C, N297G, L306C, R292C, N297G, V302C, N297G, V323C, I332C, and V259C, N297G, L306C.

[0027] Alternatively, the carbohydrate in the Fc region can be enzymatically cleaved, and / or the cells used to produce the antibody can be grown in a medium that impairs carbohydrate addition, and / or cells engineered to lack the ability to add carbohydrates can be used for antibody production, or production of the antibody in a host cell that does not glycosylate or does not functionally glycosylate the antibody, such as, for example, a prokaryote including E. coli, as described above.

[0028] Reduced affinity for Fcγ receptors can further be achieved through manipulation of amino acids in the Fc region of the antibody (such modifications have been previously described, for example, by Xencor, Macrogenics, and Genentech), or by production of the antibody in host cells that do not glycosylate or do not functionally glycosylate the antibody, such as prokaryotes, including E. coli.

[0029] In addition to having reduced binding to Fcγ receptors via the Fc region, in some embodiments, it is preferred that antibody molecules that specifically bind to FcγRIIb do not cause phosphorylation of FcγRIIb upon target binding, which is an inhibitory phenomenon that blocks immune cell activity.

[0030] Fcγ receptor-expressing immune effector cells herein primarily refer to innate immune effector cells, specifically including macrophages, neutrophils, monocytes, natural killer (NK) cells, basophils, eosinophils, mast cells, and platelets. Cytotoxic T cells and memory T cells do not normally express FcγR, but may do so under certain circumstances. In some embodiments, the immune effector cells are innate immune effector cells. In some embodiments, the immune effector cells are macrophages.

[0031] In contrast to antibody molecules that specifically bind to FcγRIIb, antibody molecules that specifically bind to or interact with receptors present on target immune cells, i.e., second antibody molecules or antibody molecules that deplete or inactivate immune cells, have an Fc region that binds to or interacts with an activating Fcγ receptor to a degree that is not reduced, or at least not substantially reduced. The immune cells to which the second antibody molecules, i.e., antibody molecules that deplete or inactivate immune cells, bind are immune cells that suppress anti-cancer immunity, and binding of the second antibody to those cells causes the depletion or inactivation of those immune cells, which may belong to the innate immune arm (e.g., TAM, TAN, or MDSC) or the adaptive immune arm (e.g., T cells) of the immune system.

[0032] Cell elimination, as used herein, refers to the removal, deletion, or elimination of immune cells via physical clearance of the cells, particularly the removal of intratumoral immune cells or tumor-associated immune cells present in tumor-draining lymph nodes.

[0033] In this specification, the inactivation of immune cells refers to the blocking or reduction of activity, for example, the reduction of cytokine, growth factor, arginase or nitric oxide production.In this context, the inactivation of immune cells also includes the skewing of immune cells, so that their tumorigenic phenotype changes to anti-tumor phenotype, for example, by reducing the release of anti-inflammatory cytokines, reducing the release of angiogenesis-promoting growth factors, and increasing the release of inflammatory cytokines, reactive oxygen species (ROS), phagocytosis or ADCC activity.

[0034] Methods for determining whether an antibody is an immune cell depleting or inactivating antibody are further described below.

[0035] The immune cells to which the second antibody molecule specifically binds are immune cells that suppress anti-cancer immunity, including, but not limited to, the induction of adaptive T cell-mediated anti-cancer immunity, including the generation of a memory recall response.

[0036] The immune cells to which the second antibody molecule specifically binds may be regulatory T cells. reg (formerly known as suppressor T cells and sometimes referred to as suppressive regulatory T cells) are a subpopulation of T cells that are capable of suppressing other immune cells under normal and pathological immune environments. Alternatively, the immune cells to which the second antibody molecule specifically binds can be myeloid cells, particularly tumor-associated myeloid cells. In some embodiments, the tumor-associated myeloid cells are tumor-associated macrophages, which are sometimes referred to as TAMs. In some embodiments, the tumor-associated myeloid cells are tumor-associated neutrophils, which are sometimes referred to as TANs. In some embodiments, the tumor-associated myeloid cells are dendritic cells. In some embodiments, the tumor-associated myeloid cells are myeloid-derived suppressor cells, which can be monocytic or granulocytic.

[0037] In addition to specifically binding to a target on an immune cell, the second antibody molecule binds via its Fc region to an activating Fcγ receptor present on the same immune effector cell as the FcγRIIb bound by the first antibody molecule, and / or to an activating Fcγ receptor present on another immune effector cell. To be able to bind to an activating Fcγ receptor, the Fc region of the second antibody, at least in some embodiments, must be glycosylated at position 297. The carbohydrate residues at this position are useful for binding to the Fcγ receptor. In some embodiments, these residues are preferably biantennary carbohydrates containing GlnNAc, mannose, as well as terminal galactose residues and sialic acid. This must comprise the CH2 portion of the Fc molecule.

[0038] The cancers treated or treatable according to the present invention are FcγRIIb-negative cancers, meaning that the cancer does not display any FcγRIIb receptors, which can be examined using anti-FcγRIIB-specific antibodies in a variety of ways, including immunohistochemistry and flow cytometry, as shown in Tutt et al. J Immunol 2015,195(11)5503-5516.

[0039] Antibodies are 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, 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 V The variable regions (collectively referred to as CDRs) bind to the antibody's target, i.e., 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 major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM; in humans, some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0040] Another part of an antibody is the Fc region (also known as the fragment crystallizable region), which contains two constant regions on 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 includes 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-size antibody has the same antigen-binding characteristics as the corresponding full-size antibody, and contains either the same variable region (i.e., VH sequence and VL sequence) and / or the same CDR sequence as the corresponding full-size antibody. Having the same antigen-binding characteristics as the corresponding full-size antibody means that the functional fragment binds to the same epitope on the target as the full-size antibody. Such a functional fragment may correspond to the Fv portion of a full-size antibody. Alternatively, such a fragment may be Fab, also referred to as F(ab), which is a monovalent antigen-binding fragment that does not contain the Fc portion, or F(ab')2, which is a bivalent antigen-binding fragment containing two antigen-binding Fab portions linked together by disulfide bonds, or a monovalent variant of 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 portion 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, has described that the entire VL chain (containing all three CDRs) has high affinity for its substrate.

[0044] Molecules containing two CDR regions are described, for example, in Vaughan & Sollazzo 2001, Combinatorial Chemistry & High Throughput Screening, 4:417-430. On page 418 (right column -3 (Our Strategy for Design)), a minibody containing only the H1 and H2 CDR hypervariable regions interspersed within framework regions is described. Minibodies are described as being capable of binding to targets. Pessi et al., 1993, Nature, 362:367-9, and Bianchi et al., 1994, J. Mol. Biol., 236:649-59, referenced by Vaughan & Sollazzo, provide more detailed descriptions of H1 and H2 minibodies and their properties. 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 observes 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, where a series of hexapeptides derived from the CDR were demonstrated to exhibit antigen-binding activity, and it was noted that synthetic peptides of a complete single CDR exhibited strong binding activity. Monnet et al., 1999, JBC, 274:3789-96, demonstrated that various 12-mer peptides and associated framework regions have antigen-binding activity, and stated that CDR3-like peptides alone can bind to antigens. Heap et al., 2005, J. Gen. Virol., 86:1791-1800, reported that "microantibodies" (molecules containing a single CDR) can bind to antigens, and demonstrated 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] An antibody molecule may be a derivative of a full-length antibody or a fragment of such an antibody, where the derivative has the same antigen-binding characteristics as the corresponding full-size antibody, meaning that it binds to the same epitope on the target as the full-size antibody.

[0048] Thus, as used herein, the term "antibody molecule" includes all types of antibody molecules, 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 Fvs (scFvs), Fab fragments, F(ab') 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, antigen-binding functional fragments of such homodimers and heterodimers, as well as functional fragments and derivatives thereof.

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

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

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

[0052] Thus, antibody molecules of the invention or used in accordance with the invention (e.g., monoclonal and / or polyclonal and / or bispecific antibody molecules) may 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, and a bioluminescent moiety. The 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, and where the cytotoxic moieties are capable of inducing cell lysis.

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

[0056] As described above, the CDRs of an antibody bind to the antibody target. The amino acid assignments for each CDR described herein are in accordance with the definitions in Kabat EA et al., 1991, "Sequences of Proteins of Immunological Interest," Fifth Edition, NIH Publication No. 91-3242, pp. 1v-1vii.

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

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

[0059] By "capable of competing" is meant that the 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 competitor 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 binding ability of an antibody described herein to prevent or reduce binding to a specific target by at least about 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100%.

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

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

[0063] Antibodies are known to specifically bind or interact with a defined target molecule or antigen, i.e., they bind preferentially and selectively to their target and not to non-target molecules.

[0064] Targets of antibodies according to the invention, or for use in accordance with the invention, are expressed on the surface of cells, i.e., they are cell surface antigens that comprise the epitope (alternatively known in this context as a cell surface epitope) for the antibody. Cell surface antigen and epitope are terms readily understood by those skilled in the art of immunology or cell biology.

[0065] The term "cell surface antigen" includes cases where the cell surface antigen is exposed on the extracellular side of the cell membrane, but only transiently exposed on the extracellular side of the cell membrane. "Transiently exposed" includes cases where the cell surface antigen is internalized into the cell or released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens can be released from the extracellular side of the cell membrane by cleavage, which can be mediated by proteases.

[0066] Also, cell surface antigens may bind to cell membranes, but may only be transiently associated with the cell membrane. "Transiently associated" includes cases where cell surface antigens are 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.

[0067] Furthermore, 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.

[0068] 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, and the relative strength, specificity, inhibition, prevention, or reduction of these interactions.Examples of methods that can be used to assess protein binding include, for example, immunoassay, BIAcore, Western blot, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) (for a discussion of antibody specificity, see Fundamental Immunology, 2nd ed., Raven Press, New York, pp. 332-336 (1989)).

[0069] Thus, a "specifically binding antibody molecule" or a "target-specific antibody molecule" includes cases where the antibody molecule specifically binds to a target but does not bind to a non-target, or binds to the non-target weaker than it does to the target (e.g., with lower affinity).

[0070] It also includes the meaning that the antibody specifically binds to a 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 to a target than to a non-target.

[0071] In addition, the antibody targets 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 By binding, it is meant that the antibody specifically binds to the target.

[0072] As used herein, the term immune cell-depleting or immune cell-inactivating antibody molecule refers to an antibody molecule that, upon administration to a patient, specifically binds to a target expressed on the surface of an immune cell, resulting in the depletion or inactivation of the immune cell. In some embodiments, the target is a target preferentially expressed on a tumor or in the tumor microenvironment.

[0073] To determine whether an antibody molecule is an immune cell-depleting antibody molecule in the sense of the present invention, an in vitro antibody-dependent cellular cytotoxicity (ADCC) assay or an antibody-dependent cellular phagocytosis (ADCP) assay can be used. To determine whether an antibody molecule is an immune cell-depleting antibody molecule, the same assay is performed in the presence and absence of a depleting antibody, indicating whether the depleting antibody being tested is actually depleting.

[0074] ADCC assays can be performed by labeling target cells with calcein AM (acetyl methyl ester) followed by the addition of diluted concentrations of antibody. The target cells are then co-cultured with human peripheral blood mononuclear cells (PBMCs) at a 50:1 effector:target (E:T) ratio for 4 hours at 37°C. The plate is centrifuged at 400 x g for 5 minutes to pellet the cells, and the supernatant is transferred to a white 96-well plate. Calcein release is measured using a Varioskan (Thermo Scientific) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The percentage of maximum release is calculated as follows: % maximum release = (sample / Triton-treated) * 100.

[0075] The ADCP assay can be performed by labeling target cells with 5 mM carboxyfluorescein succinimidyl ester (CFSE) for 10 minutes at room temperature, followed by washing in fetal bovine serum-containing medium. The CFSE-labeled targets are then opsonized with diluted antibodies and then co-cultured with bone marrow-derived macrophages (BMDMs) at a 1:5 E:T ratio in a 96-well plate at 37°C for 1 hour. BMDMs are then labeled with anti-F4 / 80-allophycocyanin for 15 minutes at room temperature and washed twice with PBS. The plate is kept on ice, and the wells are scraped to collect the BMDMs. Phagocytosis is assessed by flow cytometry using a FACSCalibur (BD) to determine the percentage of F4 / 80+CFSE+ cells within the F4 / 80+ cell population.

[0076] It is also possible to use the method described by Cleary et al. in J Immunol, April 12, 2017, 1601473.

[0077] In some embodiments, the antibody molecule that specifically binds to FcγRIIb is a human antibody.

[0078] In some embodiments, antibody molecules that specifically bind to FcγRIIb are human-derived antibodies, i.e., human-derived antibodies that have been modified as described herein.

[0079] In some embodiments, an antibody molecule that specifically binds to FcγRIIb is a humanized antibody, i.e., an antibody of non-human origin that has been modified to increase its similarity to a human antibody. The humanized antibody may be, for example, a murine antibody or a llama antibody.

[0080] 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] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL-QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP-APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNA-KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP-REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP-VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IgG1-CL [SEQ ID NO: 2] QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV-KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0081] 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 with an N297Q substitution, and then the IgG1-CH has the following CH sequence [SEQ ID NO: 195], with the 297Q residue marked in bold. JPEG0007800852000001.jpg43167

[0082] In some embodiments and / or examples, murine antibody molecules are used. These can also be used as surrogate antibodies. They contain the following constant regions (CH and CL): JPEG0007800852000002.jpg55167CL[SEQ ID NO: 197] QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMET-TQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS

[0083] Therefore, these constant regions (SEQ ID NO: 196 and SEQ ID NO: 197) are of mouse origin. SEQ ID NO: 196 contains an N297A mutation (the 297A residue is shown in bold in the sequence above). This N297A mutation in the mouse sequence corresponds to the N297Q mutation in the human sequence.

[0084] 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQTPGKGLEWVSLIG-WDGGSTYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARAYSGYELDY- WGQGTLVTVSS 1A01-VL [SEQ ID NO: 27] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLI- YDNNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNASI- FGGGTKLTVLG CDR area CDRH1:DYYMN [SEQ ID NO:51] CDRH2:LIGWDGGSTYYADSVKG [SEQ ID NO: 52] CDRH3:AYSGYELDY [SEQ ID NO: 53] CDRL1:SGSSSNIGNNAVN [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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFTRYD-GSNKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARE- NIDAFDVWGQGTLVTVSS 1B07-VL [SEQ ID NO: 28] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLI-YDNQQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCE- AWDDRLFGPVFGGGTKLTVLG CDR area CDRH1:SYGMH [SEQ ID NO: 57] CDRH2:FTRYDGSNKYYADSVRG [SEQ ID NO: 58] CDRH3:ENIDAFDV [SEQ ID NO: 59] CDRL1:SGSSSNIGNNAVN [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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVR- QAPGKGLEWVSSISDSGAGRYYADSVEGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARTHDSGELLDAFDIWGQGTLVTVSS 1C04-VL [SEQ ID NO: 29] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNHHVLWYQQLPGTAPKLLI-YGNSNRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGWVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQVPGKGLEWVAVISYD-GSNKNYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNFDNSGYAIPDAFD-IWGQGTLVTVSS 1E05-VL [SEQ ID NO: 30] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI-YDNNSRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLGG- PVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVR-QAPGKGLEWVAYISRDADITHYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC-TTGFDYAGDDAFDIWGQGTLVTVSS 2A09-VL [SEQ ID NO: 31] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNAVNWYQQLPGTAPKLLI-YGNSDRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGRWVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVR-QAPGKGLEWVALIGHDGNNKYYLDSLEGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCARATDSGYDLLYWGQGTLVTVSS 2B08-VL [SEQ ID NO: 32] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAP-KLLIYYDDLLPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCTT- WDDSLSGVVFGGGTKLTVLG CDR area CDRH1:DYYMS [SEQ ID NO:81] CDRH2: LIGHDGNNKYYLDSLEG [SEQ ID NO: 82] CDRH3:ATDSGYDLLY [SEQ ID NO: 83] CDRL1:SGSSSNIGNNAVN [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] EVQLLESGGGLVQPGGSLRLS-CAASGFTFSDYYMSWIRQAPGKGLEWVSAIGFSDDNTYYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAGGDGSGWSFWGQGTLVTVSS 2E8-VL [SEQ ID NO: 33] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNN-KRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY- CATWDDSLRGWVFGGGTKLTVLG CDR area CDRH1:DYYMS [SEQ ID NO:87] CDRH2:AIGFSDDNTYYADSVKG [SEQ ID NO: 88] CDRH3: GDGSGWSF [SEQ ID NO: 89] CDRL1:SGSSSNIGNNAVN [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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREWRDAFD- IWGQGTLVTVSS 5C04-VL [SEQ ID NO: 34] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSDNQRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGSWVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISRD- NSKNTLYLQMNSLRAEDTAVYYCARENFDAFDVWGQGTLVTVSS 5C05-VL [SEQ ID NO: 35] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSNS-QRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGQVVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVR-QAPGKGLEWVAVIAYDGSKKDYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAREYRDAFDIWGQGTLVTVSS 5D07-VL [SEQ ID NO: 36] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI-YGNSNRPSGVPDRFSGSKSGTTASLAISGLR- SEDEADYYCAAWDDSVSGWMFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYD-GINKDYADSMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERKDAFD- IWGQGTLVTVSS 5E12-VL [SEQ ID NO: 37] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAP-KLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY- CATWDDSLNGLVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYD-GSNRYYADSVKGRFTMSRD- NSKNTLYLQMNSLRAEDTAVYYCARDRWNGMDVWGQGTLVTVSS 5G08-VL [SEQ ID NO: 38] QSVLTQPPSASGTPGQRVTISCSGSSSNIGAGYDVHWYQQLPGTAPKLLI-YANNQRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGPWVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYD-GSDTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHSVIGAFD- IWGQGTLVTVSS 5H06-VL [SEQ ID NO: 39] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYDNN-KRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGSNNVVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVTSYD-GNTKYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDCGG- DCFDYWGQGTLVTVSS 6A09-VL [SEQ ID NO: 40] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI-YGNSNRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLNEGVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQLGEAFD- IWGQGTLVTVSS 6B01-VL [SEQ ID NO: 41] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNN-KRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLSGPVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVR-QAPGKGLEWVSAISGSGSSTYYADSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAGGDIDYFDYWGQGTLVTVSS 6C11-VL [SEQ ID NO: 42] QSVLTQPPSASGTPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYENN-KRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG CDR area CDRH1:DYGMS [SEQ ID NO: 141] CDRH2:AISGSGSSTYYADSVKG [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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERRDAFD- IWGQGTLVTVSS 6C12-VL [SEQ ID NO: 43] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAP-KLLIYSDNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYY- CATWDSDTPVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARDHSAA- GYFDYWGQGTLVTVSS 6D01-VL [SEQ ID NO: 44] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLI-YGNSIRPSGGPDRFSGSKSGTSASLAISGLR- SEDEADYYCASWDDSLSSPVFGGGTKLTVLG 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:ASWDDSLSSPV [SEQ ID NO: 158] Antibody clone: ​​6G03 6G03-VH [SEQ ID NO: 21] EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYGMHWVR-QAPGKGLEWVSGISWDSAIIDYAGSVKGRFTISRD-NSKNTLYLQMNSLRAEDTAVYYCAKDEAAAGAFDIWGQGTLVTVSS 6G03-VL [SEQ ID NO: 45] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI-YGNTDRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLSGPVVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTLSSYGISWVRQAPGKGLEWVSGIS-GSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSVGAYANDAFD-IWGQGTLVTVSS 6G08-VL [SEQ ID NO: 46] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYG-DTNRPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCAAWDDSLNGPVFGGGTKLTVLG CDR area CDRH1:SYGIS [SEQ ID NO: 165] CDRH2:GISGSGGNTYYADSVKG [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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMAVISYD-GSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELYDAFD- IWGQGTLVTVSS 6G11-VL [SEQ ID NO: 47] QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLI-YADDHRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSQRAVI- FGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREYKDAFD- IWGQGTLVTVSS 6H08-VL [SEQ ID NO: 48] QSVLTQPPSASGTPGQRVTISCTGSSNIGSNTVNWYQQLPGTAPKLLIYDNN-KRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQAWGTGIRVFGGGTKLTVLG CDR area CDRH1:NYGMH [SEQ ID NO: 177] CDRH2:VISYDGSNKYYADSVKG [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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYD-GSNKYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCAREFGYIILDY- WGQGTLVTVSS 7C07-VL [SEQ ID NO: 49] QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLI-YRDYERPSGVPDRFSGSKSGTSASLAISGLR- SEDEADYYCMAWDDSLSGVVFGGGTKLTVLG 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] EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHGMHWVRQAPGKGLEWVAVISYD-GTNKYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETW- DAFDVWGQGTLVTVSS 4B02-VL [SEQ ID NO: 50] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNNANWYQQLPGTAPKLLIYDNN-KRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTVVFGGGTKLTVLG CDR area CDRH1:NHGMH [SEQ ID NO: 189] CDRH2:VISYDGTNKYYADSVRG [SEQ ID NO: 190] CDRH3:ETWDAFDV [SEQ ID NO: 191] CDRL1:SGSSSNIGSNNAN [SEQ ID NO: 192] CDRL2:DNNKRPS [SEQ ID NO: 193] CDRL3:QAWDSSTVV [SEQ ID NO: 194]

[0085] In some embodiments, and 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.

[0086] In some embodiments, and sometimes preferred embodiments, an antibody molecule that specifically binds to FcγRIIb comprises the following constant regions: SEQ ID NO:1 (CH) and SEQ ID NO:2 (CL), and the following variable regions: SEQ ID NO:23 (VL) and SEQ ID NO:47 (VH), i.e., the constant and variable regions of clone 6G11, which have been further modified to reduce binding to Fcγ receptors via its Fc region. In some embodiments, and 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 containing the N297Q mutation.

[0087] In some embodiments, the immune cell-depleting or immune cell-inactivating antibody molecule 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 or IgG2 antibody.

[0088] In some embodiments, the immune cell-depleting or immune cell-inactivating antibody molecule is a humanized antibody molecule.

[0089] In some embodiments, the immune cell-depleting or immune cell-inactivating antibody molecule is a chimeric antibody.

[0090] As mentioned above, the immune cell-depleting or immune cell-inactivating antibody must have the ability to bind to FcγR.

[0091] The target to which the immune cell-depleting or immune cell-inactivating antibody molecule according to the present invention binds may be selected from the group consisting of CTLA-4, 4-1BB, OX40, TNFR2, PD-L1, IL-2R, and GITR.

[0092] In some embodiments of the present invention, the target to which the immune cell-depleting or immune cell-inactivating antibody molecule of the present invention binds is CTLA-4. CTLA-4, an abbreviation for cytotoxic T-lymphocyte-associated protein 4, is also known as CD152. It is a protein receptor that functions as an immune checkpoint and down-regulates immune responses. CTLA4 is constitutively expressed on regulatory T cells but increases only on conventional T cells after activation—a notable phenomenon, particularly in cancer. In some such embodiments, the immune cell-depleting antibody molecule is ipilimumab (e.g., Yervoy® from Bristol-Myers Squibb). In some such embodiments, the immune cell-depleting antibody molecule is tremelimumab (CP-675,206, formerly known as ticilimumab), a fully human monoclonal antibody against CTLA-4 that was previously under development by Pfizer and is currently in clinical development by MedImmune.

[0093] In some embodiments of the present invention, at least one target is 4-1BB, also designated CD137 and tumor necrosis factor receptor superfamily member 9 (TNFRSF9). 4-1BB is expressed on Tregs after CD4+ and CD8+ T cell activation, and its ligation is required for optimal protective CD8+ T cell responses against viruses and B cell lymphoma in mice. Anti-4-1BB-specific antibodies enhance the proliferation and survival of antigen-stimulated T cells in vitro, and, like anti-CD40, anti-4-1BB monoclonal antibodies promote antitumor immunity in preclinical cancer models, primarily dependent on CD8+ T cells. In some such embodiments, the immune cell-depleting antibody molecule is urelumab, a humanized agonistic IgG4 monoclonal antibody developed by Bristol-Myers Squibb. In some such embodiments, the immune cell-depleting antibody molecule is utomilumab (also designated PF-05082566, PF-2566, and PF-5082566), a human HuCAL monoclonal antibody agonist of 4-1BB developed by Pfizer.

[0094] In some embodiments of the present invention, at least one target is OX40. OX40, also known as tumor necrosis factor receptor superfamily, member 4 (TNFRSF4) and CD134, is a secondary costimulatory immune checkpoint molecule. In some such embodiments, the immune cell depleting antibody molecule is MEDI6469 (9B12), MEDI0562, PF-04518600, INCAGN01949, BMS-986178, MOXR0916, GSK3174998, MEDI6383 (see, for example, Table 1 in Buchan et al., Blood 2018 131:39-48).

[0095] In some embodiments of the invention, at least one target is TNFR-2. Tumor necrosis factor receptor 2 (TNFR-2 or TNFR2), also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) and CD120b, is a membrane receptor that binds tumor necrosis factor-α (TNFα).

[0096] In some embodiments of the invention, the target to which the immune cell-depleting or immune cell-inactivating antibody molecules according to the invention bind is programmed death-ligand 1 (PD-L1), also known as CD274 or B7 homolog 1 (B7-H1).

[0097] In some embodiments of the invention, at least one target is IL-2R, also known as CD25, which is highly expressed primarily on regulatory T cells.

[0098] In some embodiments of the invention, at least one target is GITR, a member of the TNFSFR family that is also expressed primarily on regulatory T cells.

[0099] In some embodiments, the antibody molecule that specifically binds FcγRIIb and the immune cell-depleting or immune cell-inactivating antibody molecule are administered to a patient simultaneously, meaning that they are either administered at the same time or separately within a close time period of each other.

[0100] In some embodiments, the antibody molecule that specifically binds to FcγRIIb is administered to a patient before the administration of the immune cell-depleting antibody molecule or the immune cell-inactivating antibody molecule. Such sequential administration can be achieved by temporally separating the two antibodies. Alternatively, or in combination with the first option, sequential administration can also be achieved by spatially separating the two antibody molecules, such as by administering the antibody molecule that specifically binds to FcγRIIb by intratumoral administration, such that the antibody molecule reaches the cancer prior to the immune cell-depleting antibody molecule, and then administering the antibody molecule by systemic administration, such that the antibody molecule reaches the cancer after the antibody molecule that specifically binds to FcγRIIb.

[0101] In some embodiments, the immune cell depleting antibody is administered to the patient prior to administration of the antibody molecule that specifically binds FcγRIIb. Such sequential administration can be achieved as described above.

[0102] For example, it will be known to those skilled in the art of medicine that drugs can be modified with different additives to alter the rate at which they are absorbed by the body, and can be modified in different forms to allow, for example, specific routes of administration to the body.

[0103] Thus, the compositions of the present invention, and / or antibodies, and / or drugs include those combined with excipients and / or pharmaceutically acceptable carriers and / or pharmaceutically acceptable diluents and / or adjuvants.

[0104] 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 bacteriostats, and / or solutes that render the formulation isotonic with the blood of the intended recipient, and / or aqueous and / or non-aqueous sterile suspensions that 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.

[0105] Extemporaneous injection solutions and suspensions may be prepared from sterile powders and / or granules and / or tablets of the kind previously described.

[0106] For parenteral administration to human patients, daily dosage levels of antibody molecules that specifically bind to FcγRIIb and / or immune cell-depleting or immune cell-inactivating antibody molecules typically range from 1 mg to 20 mg / kg (patient body weight), or in some cases up to 100 mg / kg, administered in single or divided doses. Lower doses may be used under special circumstances, e.g., in combination with chronic administration. In any event, the physician will determine the actual dosage that will be most suitable for an individual patient, which will vary with the age, weight, and response of the particular patient. The dosages described above are exemplary of the average case. Of course, there may be individual cases in which higher or lower dosage ranges are merited, and these are within the scope of the present invention.

[0107] Typically, the compositions and / or drugs of the present invention contain antibody molecules that specifically bind to FcγRIIb and / or immune cell-depleting or immune cell-inactivating antibodies at a concentration of approximately 2 mg / mL to 150 mg / mL, or approximately 2 mg / mL to 200 mg / mL. In a preferred embodiment, the drugs and / or compositions of the present invention contain antibody molecules that specifically bind to FcγRIIb and / or immune cell-depleting or immune cell-inactivating antibody molecules at a concentration of 10 mg / mL.

[0108] Generally, in humans, oral or parenteral administration of the compositions, antibodies, agents, and / or drugs of the present invention is the preferred and most convenient route. For veterinary use, the compositions, antibodies, agents, and / or drugs of the present invention are administered in a suitably acceptable formulation in accordance with normal veterinary practice, and a veterinarian will determine the dosage regimen and route of administration that will be most appropriate for a particular animal. Accordingly, the present invention provides pharmaceutical formulations comprising an antibody and / or drug of the present invention in an amount effective to treat various conditions (described above and further below). Preferably, the compositions, antibodies, agents, and / or drugs are adapted for delivery by a route selected from the group consisting of intravenous (IV), subcutaneous (SC), intramuscular (IM), or intratumoral.

[0109] In some embodiments, either the first antibody molecule or the second antibody, or both, may be administered using a plasmid or virus. Such a plasmid then contains 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 cellular or viral genome or viral virome, and such a cell or virus then acts 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 containing a nucleotide sequence encoding at least one of the antibody molecules described herein. In some embodiments, such an oncolytic virus contains a nucleotide sequence encoding a full-length human IgG antibody. Oncolytic viruses are known to those skilled in the art of medicine and virology.

[0110] The present invention also includes compositions, antibodies, and / or pharmaceutical agents and / or drugs containing pharmaceutically acceptable acid or base addition salts of the polypeptide-binding moieties of the present invention. The acids used to prepare pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in the present invention are, among others, those that 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 may also be used to generate pharmaceutically acceptable salt forms of the agents of the present invention. Chemical bases that can be used as reagents to prepare pharmaceutically acceptable base salts of the present agents, which 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 pharmacologically acceptable cations such 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, as well as other pharmaceutically acceptable organic amine base salts. The agents and / or polypeptide binding moieties of the present invention may 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 may be used. Those skilled in the art will recognize that lyophilization and reconstitution may result in varying degrees of antibody activity loss (e.g., with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies), and that use levels may need to be adjusted upward to compensate.In one embodiment, a lyophilized (freeze-dried) polypeptide binding moiety loses less than about 20%, or less than about 25%, or less than about 30%, or less than about 35%, or less than about 40%, or less than about 45%, or less than about 50% of its activity (before lyophilization) when rehydrated.

[0111] A combination of an antibody molecule that specifically binds to FcγRIIb and an immune cell-depleting or immune cell-inactivating antibody molecule can be used in the treatment of cancer.

[0112] As used herein, the term "patient" refers to an animal, including a human, that has been diagnosed with an FcγRIIb-negative cancer, or that is considered likely to be an FcγRIIb-negative cancer and / or that exhibits symptoms of such a cancer.

[0113] The patient may be a mammal or a non-mammal. Preferably, the patient is a human, or a mammal such as a horse, cow, sheep, pig, camel, dog, or cat. Most preferably, the mammalian patient is a human.

[0114] "Exhibiting" includes when a subject exhibits cancer symptoms and / or cancer diagnostic markers and / or when cancer symptoms and / or cancer diagnostic markers can be measured and / or assessed and / or quantified.

[0115] It will be readily apparent to one skilled in the art of medicine what cancer symptoms and cancer diagnostic markers are, and how to measure and / or assess and / or quantify whether there is a reduction or increase in the severity of cancer symptoms or whether there is a reduction or increase in cancer diagnostic markers, and how cancer symptoms and / or cancer diagnostic markers can be used to form a prognosis for cancer.

[0116] Cancer treatments are often administered as a course of treatment, i.e., the therapeutic agents are administered over a period of time. The length of the course of treatment depends on several 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 factors.

[0117] "Currently undergoing treatment" includes when a patient is currently undergoing a course of treatment and / or is currently receiving a therapeutic agent and / or is currently receiving a course of therapeutic agent.

[0118] In some embodiments, the FcγRIIb-negative cancer treated according to the present invention is a solid tumor.

[0119] In some embodiments, the cancer is selected from the group consisting of carcinoma, sarcoma, and lymphoma.

[0120] In some embodiments, the cancer is a carcinoma selected from the group consisting of adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, poorly differentiated or undifferentiated carcinoma, large cell carcinoma, and small cell carcinoma.

[0121] In some embodiments, the cancer is a sarcoma selected from the group consisting of osteosarcoma, chondrosarcoma, liposarcoma, and leiomyosarcoma. The FcγRIIb-negative cancer is selected from the group consisting of melanoma, breast cancer, ovarian cancer, prostate cancer, metastatic hormone-refractory prostate cancer, colorectal cancer, lung cancer, small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), non-small cell lung cancer, urothelial carcinoma, bladder cancer, kidney cancer, mesothelioma, Merkel cell carcinoma, and head and neck cancer.

[0122] All of the above-mentioned cancers are known, and the symptoms and cancer diagnostic markers are well described, as are the therapeutic drugs used to treat those cancers. Therefore, the symptoms, cancer diagnostic markers and therapeutic drugs used to treat the above-mentioned types of cancer will be known to those skilled in the art of medicine.

[0123] The clinical definition of the diagnosis, prognosis, and progression of most cancers is based on a specific classification known as staging. These staging systems collate many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis, prognosis, and / or progression of cancer. Those skilled in the art of oncology will know how to use staging systems to assess the diagnosis, prognosis, and / or progression of cancer, and which cancer diagnostic markers and cancer symptoms to use for this purpose.

[0124] "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.

[0125] It is known that cancer can cause abnormalities in cell morphology.These abnormalities often occur reproducibly in certain cancers, which means that examining these changes in morphology (also known as histological examination) can be used for cancer diagnosis or prognosis.The techniques for visualizing and preparing samples for examining cell morphology are known in the art, such as light microscopy or confocal microscopy.

[0126] "Histological examination" includes the presence of small mature lymphocytes, and / or the presence of small mature lymphocytes with narrow cytoplasmic borders, the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli, and / or the presence of small mature lymphocytes with dense nuclei lacking discernible nucleoli, and / or the presence of atypical cells, and / or cleaved cells, and / or prolymphocytes.

[0127] It is known that cancer is the result of the mutation of cell DNA, which can lead to cell death avoidance or uncontrollable proliferation.Therefore, the examination of these mutations (also known as cytogenetic examination) can be a useful tool for assessing the diagnosis and / or prognosis of cancer.An example of this is the deletion of chromosome position 13q14.1, which is characteristic of chronic lymphocytic leukemia.The technique for examining mutations in cells is known in the art, for example, fluorescence in situ hybridization (FISH).

[0128] "Cytogenetic testing" includes testing of DNA in cells, specifically chromosomes. 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 of chromosome 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) translocations, and / or antigen gene receptor rearrangements, and / or BCL2 rearrangements, and / or BCL6 rearrangements, and / or t(14:18) translocations, and / or t(11:14) translocations, and / or (q13:q32) translocations, and / or (3:v) translocations, and / or (8:14) translocations, and / or (8:v) translocations, and / or t(11:14) and (q13:q32) translocations.

[0129] Cancer patients are known to exhibit certain physical symptoms, which are often the result of cancer's physical burden.These symptoms often recur with the same cancer, and can be diagnostic and / or prognostic and / or progression characteristics of the disease.Those skilled in the art of medicine will understand which physical symptoms are related to which cancer, and how the evaluation of these body systems can be correlated with the diagnosis and / or prognostic and / or progression of the disease."Physical symptoms" include hepatomegaly and / or splenomegaly. [Example]

[0130] Specific, non-limiting examples are now described that illustrate some aspects of the present invention. To be able to investigate the inhibitory effects of FcγRIIB in a complex in vivo system, two sets of surrogate antibodies were required. The mouse equivalent of 6G11 is called AT130-2. To mute the Fc of the human antibody (and thus significantly impair or even eliminate FcγR binding), amino acid position 297 was substituted from N to Q. To mute the Fc of the mouse antibody, the same position was substituted from N to Q. Thus, while the mouse system is referred to as AT-130, this patent application relates to the human counterpart 6G11. Thus, human 6G11 corresponds to the mouse surrogate antibody AT1302-2, and 6G11-N297Q corresponds to AT130-3-N297A.

[0131] Another method (and one known to those skilled in the art) for Fc-muting an antibody is to remove the Fc portion to form Fab or Fab2 fragments.

[0132] (Experimental Procedure) (animal) hCD20 Tg (transgenic), hFcγRIIB + / - and mFcγRIIB - / -Mice were previously described with genotypes confirmed by PCR and / or flow cytometry (Beers et al., Blood 2008 Nov 15;112(10):4170-7; Roghanian et al., Cancer Cell 27, 473-488, April 13, 2015). Mice were bred and maintained in local facilities in accordance with UK Home Office guidelines or the local Swedish ethical committee.

[0133] (cell culture) Cell culture was performed in supplemented RPMI (RPMI 1640 containing 2 mM glutamine, 1 mM pyruvate, 100 IU / mL penicillin and streptomycin, and 10% FCS [Myoclone]) (GIBCO BRL, Paisley, Scotland). Mouse splenic B cells were purified by negative selection using a MACS B cell isolation kit (Miltenyi Biotec, UK) and then cultured in the same medium. Cell lines were obtained from ECACC and maintained in antibiotic-free RPMI medium.

[0134] Generation of human monocyte-derived macrophages (MDMs) and murine bone marrow-derived macrophages (BMDMs) Human MDMs were differentiated from peripheral blood obtained from the National Blood Service, either Southampton General Hospital (Southampton, UK) or Halmstad, or the blood centre at Skane University Hospital (Sweden). Briefly, adherent CD14 +Monocytes were cultured in RPMI supplemented with 25–100 ng / mL endotoxin-low recombinant human macrophage colony-stimulating factor (M-CSF; R&D Systems, US or produced in-house) as previously described (Roghanian et al., Cell Immunol. 2010;265(2):120–6). Half of the medium was replaced with fresh M-CSF every 2 days until harvest. On days 7–10 of culture, MDMs were harvested and subsequently briefly incubated with cold PBS. Murine BMDMs were generated from cells isolated from the bone marrow of mouse femurs and tibias as previously described (Williams et al., J Immunol. 2013 Oct 15;191(8):4130-40). Briefly, bone marrow cells were cultured in RPMI supplemented with 20% L929 cell-conditioned medium (containing M-CSF). Cells were cultured at 37°C in 5% CO2 for 10-12 days before use. Macrophage differentiation was routinely confirmed by morphological examination and / or flow cytometry for CD11b and F4 / 80 expression.

[0135] (Antibodies and Reagents) Monoclonal antibodies were typically produced from hybridoma culture supernatants or stably transfected CHO-k1 cells (obtained from ECACC). F(ab')2 fragments were generated as previously described (Glennie et al., 1987). The hFcγRII monoclonal antibody AT10 was previously described (Greenman et al., 1991). Antibodies included anti-CTLA4 (9H10; Bio X Cell, US), anti-IL2R (PC-61.5.3; Bio X Cell / in-house production), and anti-PDL-1 (10F.9G2; Bio X Cell, US). The hFcγRII monoclonal antibodies 6G11 hIgG1 and N297Q were produced at BioInvent (see Roghanian et al., Cancer Cell 27, 473–488, April 13, 2015). The mFcγRI monoclonal antibodies AT130-2, mIgG1, mIgG2a, and mIgG1 N297A were generated in-house. AT130-5 (Williams et al., Eur J Immunol. 2012;42(8):2109-20, and Tutt et al. J Immunol. 2015,195(11)5503-5516) is a mouse anti-mouse FcγRII antibody similar to the human antibody clone 6G11. Antibodies against hFcγRIIB (clone EP888Y; Abcam, UK), phosphorylated hFcγRIIB (clone EP926Y; Origene, US), GAPDH (Abcam, UK), and α-tubulin (Cell Signaling, US) were used for immunoblotting. For PBMC immunophenotyping, FcγRIIB monoclonal antibody labeled with PE using the zenon labeling kit (Molecular Probes) was used in combination with anti-CD3-FITC, anti-CD19-PerCP-Cy5.5, and anti-CD56-APC (antibodies obtained from Biolegend).

[0136] (Flow cytometry) Fluorescently conjugated monoclonal antibodies were purchased from BD Biosciences, eBiosciences, Biolegend, and AbD Serotec (all UK) or produced in-house. Flow cytometry was performed as previously described (Tutt et al., 1998). Samples were evaluated on a FACScan, FACSCalibur, or FACSCanto II, and data were analyzed with CellQuest Pro, FACSDiva (all BD Biosciences, UK), or FCS Express (De Novo Software, CA, US).

[0137] (Western blotting) As previously described (Roghanian et al, Cancer Cell 27, 473-488, April 13, 2015).

[0138] (In vivo immunotherapy) Adoptive transfer: As previously described (Beers et al., Blood. 2010 Jun 24;115(25):5191-201). B cell depletion: Mice were intravenously administered hCD20 or hFcγRIIB monoclonal antibodies, alone or in combination, and leukocyte counts were assessed as previously described (Beers et al., Blood. 2010 Jun 24;115(25):5191-201).

[0139] (CT26) CT26 cells were maintained in complete DMEM and harvested using trypsin-EDTA. Cells were washed, resuspended in PBS, and the concentration was adjusted to 5 × 10 using a hemocytometer. 6 The cell suspension (5 × 10 cells / mL) was adjusted to 100 μL. 5 Cells) were injected subcutaneously into BALB / c mice (house-bred from the original strain obtained from Charles River, UK). Tumors were allowed to establish and tumor size was measured three times weekly prior to randomization and treatment. Tumors measured 400 mm length x width. 2 If the tumor exceeded 100%, the tumor was considered to be in the late stage.

[0140] (MC38) MC38 cells were maintained in complete DMEM and harvested using trypsin-EDTA. Cells were washed, resuspended in PBS, and the concentration was adjusted to 5 × 10 using a hemocytometer. 6 The cell suspension (5 × 10 cells / mL) was adjusted to 100 μL. 5 Cells) were injected subcutaneously into C56 / B16 mice (obtained from Taconic, Denmark). Tumors were allowed to establish and tumor size was measured prior to randomization and treatment. Treatments were performed at a dose of 50-100 mm. 2 The tumor volume was initially 2000 mm , and tumors were subsequently measured twice weekly. Treatment was administered four times with a 3-4 day treatment interval. The dose of anti-PD-L1 was set at 10 mg / kg, and both AT130-2 variants were set at 20 mg / kg. 2 If the tumor exceeded 100%, the tumor was considered to be in the late stage.

[0141] (statistical analysis) To compare experimental groups, Wilcoxon, paired, or unpaired t-test analyses were performed. Kaplan-Meier survival curves were generated and analyzed by the log-rank test. For in vivo adoptive transfer assays involving two or more groups, one- or two-way analysis of variance was used.

[0142] For differences between OR and CR, a chi-squared test was used. Statistical analysis was performed using GraphPad Prism (version 5 or 6). Unless otherwise stated, asterisks represent the following significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001.

[0143] (result) (The effectiveness of B cell depletion depends on the format of the FcγRIIB monoclonal antibody.) FcγRIIB is expressed on both target B cells and effector monocytes / macrophages, making it difficult to interpret where FcγRIIB monoclonal antibodies affect deeper target cell elimination. To further analyze this, we take advantage of our various hFcγRIIBTg and KO mouse strains to provide a system in which either target cells, effector cells, or both can be targeted with FcγRIIB monoclonal antibodies. hCD20, which lacks hFcγRIIB, is a target cell line. + / - Targeting hFcγRIIB + / - x mFcγRIIB - / - In adoptive transfer assays of recipient mice, treatment with FcγR null and wild-type FcγRIIB monoclonal antibodies alone did not have the expected effect on B cell depletion (Figure 2A). When combined with rituximab, FcγR null FcγRIIB monoclonal antibodies enhanced the depletion of both circulating target cells (Figure 2A) and tissue-resident target cells (Figure 2B), whereas wild-type FcγRIIB monoclonal antibodies impaired depletion. This was highly unexpected to us, indicating that the use of a normal FcγRIIB monoclonal antibody impairs target cell depletion by the second antibody. Splenic F4 / 80 from treated mice + When FcγR expression in macrophages was assessed, it was evident that the detection of mFcγRIV (Figure 2C) was reduced when wild-type FcγRIIB monoclonal antibodies were used, but not when FcγR null antibodies were used. This may partially explain the inhibitory effect of wild-type FcγRIIB monoclonal antibodies. This indicates that conventional IgG FcγRIIB monoclonal antibodies block activating FcγRIV, thus exacerbating its clearance.

[0144] This so-called scorpion effect (reviewed by Hogarth) occurs when functional Fc regions from cell surface-binding monoclonal antibodies occupy the Fc-binding groove of FcγRs expressed on the same cells and is therefore not observed with FcγR-null FcγRIIB monoclonal antibodies. This has been previously described and explains the potential for overinterpretation of the relative importance of individual FcγRs when Fc regions are blocked with functional anti-FcγR monoclonal antibodies, such as the FcγRIV monoclonal antibody 9E9 (Tipton et al., Blood 2015 125:1901-1909).

[0145] In addition to physical inhibition, this scorpion effect may also result in receptor cross-linking and FcγR activation. Because ITIM-containing FcγRIIB is the only inhibitory FcγR on effector cells and its activation may contribute to suppression of effector cell function (Dahal et al., Immunol Rev. 2015 Nov;268(1):104-22), we assessed its activation after treatment with wild-type or FcγR-null FcγRIIB monoclonal antibodies. We previously showed that in B cells (which express only FcγRIIB), treatment with the antagonist 6G11 wild-type or NQ monoclonal antibodies does not activate FcγRIIB (Roghanian et al., Cancer Cell 27, 473-488, April 13, 2015). However, wild-type, but not FcγR-null, FcγRIIB monoclonal antibodies inhibited the expression of FcγRIIB in treated human monocyte-derived macrophages (MDMs) (Fig. 2D) and mouse hFcγRIIB. + / - x mFcγRIIB - / -This resulted in phosphorylation of FcγRIIB-ITIM in both BMDMs (Figure 2E), providing evidence for this phenomenon in our system and demonstrating that effector activation is suboptimal when wild-type FcγRIIB monoclonal antibodies are used. This indicates that normal FcγRIIB monoclonal antibodies activate inhibitory signals in immune effector cells. Taking this finding, combined with those described above, we considered the optimal FcγRIIB monoclonal antibody format for target cell depletion and then investigated how optimal depletion could be achieved.

[0146] (Wild-type and FcγR null hFcγRIIB monoclonal antibodies can be combined for optimal target cell depletion) Next, we examined the efficacy of various forms of hFcγRIIB and mFcγRII monoclonal antibodies in the presence or absence of rituximab in a system in which mFcγRIIB is expressed only on target B cells and hFcγRIIB is expressed only on effector cells. This system allowed for colocalization analysis of target-restricted and effector-restricted FcγRIIB targeting. First, we examined the effect of monoclonal antibodies on FcγRIIB binding only on the target. Treating mice with a suboptimal dose of single-agent rituximab minimized depletion of target B cells (Figure 3). Treating mice with an optimal dose of single-agent wild-type mFcγRII monoclonal antibody (targeting FcγRIIB present only on the target) eliminated approximately 50% of the target cells. Coadministration of wild-type mFcγRII monoclonal antibody and rituximab resulted in profound depletion (approximately 75% of resident splenic B cells), whereas addition of Fc-null F(ab')2mFcγRII monoclonal antibody, either alone or in the presence of rituximab, was ineffective, demonstrating that conventional FcγRIIB monoclonal antibodies can be used to deplete FcγRIIB-expressing targets in the absence of FcγRIIB on effectors.

[0147] Next, we investigated targeting FcγRIIB specifically on effector cells. Treatment of mice with wild-type or F(ab')2 hFcγRIIB (targeting FcγRIIB only on effector cells) did not result in B cell depletion, as expected. However, the combination of rituximab and wild-type or F(ab')2 hFcγRIIB increased target cell depletion compared to rituximab alone. Even more potent depletion was observed when B cells were targeted with wild-type mFcγRII monoclonal antibody and effectors with F(ab')2 hFcγRIIB. In contrast, treatment with wild-type mFcγRIIB monoclonal antibody together with wild-type hFcγRIIB monoclonal antibody inhibited depletion (Figure 3). This indicates that conventional blocking FcγRIIB monoclonal antibodies impair target depletion. Effectors were blocked using an Fc-modified Fab2 monoclonal antibody.

[0148] Further investigation of these combinations revealed that when wild-type hFcγRIIB monoclonal antibody was used to block effector cell hFcγRIIB, the combination of rituximab and wild-type mFcγRII monoclonal antibody only resulted in approximately 30% depletion. When rituximab and wild-type mFcγRII monoclonal antibody, which both opsonize target B cells, were combined with an Fc-null F(ab')2 hFcγRIIB monoclonal antibody, which blocks effector cell hFcγRIIB, a much more pronounced depletion was observed, resulting in approximately 90% depletion of target cells (Figure 3).

[0149] Optimal formulation of FcgRIIB-blocking monoclonal antibodies enhances Treg depletion Next, we assessed whether this ability to enhance target elimination by blocking FcγRIIB could be extended to other cellular targets, such as Tregs. This is similar to the previous example, but uses the IL2R to deplete Tregs. To address this, 100 μg of an Fc-inactive anti-FcγRIIb monoclonal antibody (AT130-2 mIgG1 NA, Figure 9) was administered intraperitoneally to Balb / c mice. Six hours later, 100 μg of anti-IL2R (PC61) was administered intraperitoneally to deplete FoxP3+ Treg cells. These were then assessed by FAC in the blood, spleen, and lymph nodes four days later. AT130-2NA was shown to enhance Treg elimination, particularly in the spleen (Figure 4A). To address the reproducibility of this effect, we repeated this experiment in C57BL / 6 mice. Again, AT130-2NA improved Treg depletion in B6 mice, particularly in the spleen and lymph nodes (Figure 4B), resulting in higher CD8:Treg ratios in the blood, spleen, and lymph nodes (Figure 4C). Blood ratios were significantly higher in combination with NA than in PC61 alone (PC61 vs. combination P = 0.0218). To confirm our initial results, we next evaluated the ability of wild-type AT130-2 versus the Fc-inactive AT130-2NA mutant to enhance IL2R monoclonal antibody-mediated Treg depletion. Wild-type AT130-2 did not demonstrate improved depletion, whereas the NA mutant did ( * Unpaired T-test, P=0.044) (FIG. 5). This therefore indicates that normal FcγRIIb blocking monoclonal antibodies do not improve clearance.

[0150] Monoclonal antibody-mediated FcgRIIB blockade enhances CTLA-4 immunotherapy. Again, this is the same concept as above, but using an antibody against an additional target that is highly expressed on tumor-associated Treg cells (CTLA-4), resulting in anti-tumor immunity. Therefore, to address whether this approach could enhance anti-cancer immunotherapy, CT26 cells were injected subcutaneously into female BALB / c mice. Tumors measuring approximately 100 mm in width and length were then injected subcutaneously into the BALB / c mice. 2At certain times, mice were randomly assigned to treatment groups. On days 0, 2, 4, and 11, mice were intraperitoneally administered 200 μg of 9H10 (hamster anti-mouse CTLA4) antibody in 200 μL of PBS. On day 0, mice were administered 100 μg of AT130-2 N297A. The width and length of the tumors were measured, and tumors with a length × width of 400 mm were then measured. 2 Mice were sacrificed when tumor growth exceeded 9H10 (Figure 6A). Figure 6B shows the growth of individual tumors in each group, and Figure 6C shows the median area (+ / - SEM or SD). Figure 6D shows the survival curves for these mice, and Figure 6E shows the combined survival curve from the second experiment. The difference in survival between the NA combination and 9H10 alone was statistically significant (log-rank test 0.0179). Finally, to address whether this effect was dependent on the NA format of the antibody, the experiment was repeated using wild-type mIgG1 and compared with the NA format as before. The NA variant was not significantly different from the 9H10 alone group, and the NA combination was significantly more effective than the combination with the wild-type monoclonal antibody (log-rank test P = 0.0460) (Figure 7). This indicates that the wild-type antibody, i.e., a conventional glycosylated monoclonal antibody, does not bind effectively and instead impairs the desired therapeutic effect.

[0151] Monoclonal antibody-mediated FcγRIIB blockade enhances PD-L1 immunotherapy Similar to CTLA4, the efficacy of antibodies targeting PD-L1 appears to depend on FcγR activation. However, distinct forms of CTLA-4 and PD-L1 are expressed on a variety of cells, particularly myeloid lineages and cancer cells. To address whether combining PD-L1 antibodies with FcγRIIB blockade could enhance anti-cancer immunotherapy, MC38 cells were injected subcutaneously into female C57 / Bl6 mice. Tumors measuring approximately 100 mm in width and length were injected subcutaneously into the PD-L1-targeted mice. 2Mice were randomly assigned to treatment groups when tumor volume reached 2000 mm. On days 0, 2, 4, and 11, mice were intraperitoneally injected with 10 mg / kg of 9H10 (hamster anti-mouse CTLA4) antibody in 200 μL of PBS. On day 0, combination mice were injected with 100 μg of AT130-2 N297A or wild-type AT130-2. Tumor width and length were measured, and tumor volumes were adjusted to 2000 mm. 2 Mice were sacrificed when tumor growth exceeded 100%. Figure 8A shows the growth of individual tumors in each group. The numbers indicate the number of surviving mice in each group. Figure 8B shows the survival curves of the mice. To address whether this effect was dependent on the NA format of the antibody, we performed experiments using both wild-type mIgG1 and the NA format as before. The NA variant was more effective than the wild-type monoclonal antibody, with more mice surviving (Figure 8B). This indicates that the most effective combination of PD-L1 antibodies, which primarily target cancer cells and monocytes / macrophages / myeloid-derived suppressor cells, is the non-glycosylated NA format.

[0152] Collectively, the above data demonstrate that inhibition of FcγRIIB as a means of enhancing the therapeutic efficacy of other antibodies is broad and applicable for antibodies against a variety of targets (CD20, CD25, CTLA4, and PD-L1) expressed on different cell types (B cells, Treg cells, and myeloid cells).

[0153] Comparison of wild-type 6G11 vs. N297A 6G11 binding to Fcγ receptors (mouse and human) SPR analysis was performed on a Biacore T200 (GE Healthcare). Samples were flowed at 30 mL / min in HBS-EP+ buffer at 25°C. Data were analyzed using BiaEvaluation software. The response of a blank control flow cell was automatically subtracted before data analysis. For comparison of FcγR binding, 6G11 wild-type or 6G11 N297Q hIgG1 was immobilized on a CM5 sensor chip by amine coupling at pH 5, and recombinant human or mouse FcγR (100 nM) (R&D Systems) was injected across both surfaces for 180 seconds. Alternatively, various concentrations of FcγR (0–500 nM) were added sequentially and the response measured. The results are shown in Figures 10A–H.

Claims

1. 1. A pharmaceutical composition for use in treating an FcγRIIb-negative cancer in a patient, comprising: (i) a first antibody molecule that specifically binds FcγRIIb via its Fab region and that lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and the binding of the second antibody to the receptor on the immune cell causes the elimination and / or inactivation of the immune cell; and Including, The pharmaceutical composition, wherein the second antibody molecule specifically binds to a receptor selected from the group consisting of CTLA-4 and PD-L1.

2. 1. A kit for use in treating an FcγRIIb-negative cancer, comprising: (i) a first antibody molecule that specifically binds FcγRIIb via its Fab region and that lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and the binding of the second antibody molecule to the receptor on the immune cell causes the elimination and / or inactivation of the immune cell; and Including, The kit, wherein the second antibody molecule specifically binds to a receptor selected from the group consisting of CTLA-4 and PD-L1.

3. 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 that lacks an Fc region or has reduced binding to Fcγ receptors via its Fc region; (ii) a second antibody molecule that specifically binds to a receptor present on an immune cell, wherein the immune cell is an immune cell that suppresses anti-cancer immunity, the second antibody molecule has an Fc region that binds to at least one activating Fcγ receptor, and binding of the second antibody to the receptor on the immune cell causes elimination and / or inactivation of the immune cell, The use, wherein the second antibody molecule specifically binds to a receptor selected from the group consisting of CTLA-4 and PD-L1.

4. The kit of claim 2 , wherein the first antibody lacks an Fc region.

5. The kit according to claim 2 or 4, wherein the immune cells that suppress anti-cancer immunity are regulatory T cells (Treg).

6. The kit according to claim 2 or 4, wherein the immune cells that suppress anti-cancer immunity are myeloid cells.

7. The kit of claim 6 , wherein the myeloid cells are tumor-associated macrophages.

8. The kit according to any one of claims 2, 4 to 7, wherein the FcγRIIb-negative cancer is a solid cancer.

9. 9. The kit of claim 8, wherein the solid tumor is selected from the group consisting of carcinoma, sarcoma, and lymphoma.

10. 10. The kit of claim 8 or 9, wherein the solid cancer is selected from the group consisting of melanoma, breast cancer, prostate cancer, colorectal cancer, lung cancer, bladder cancer, kidney cancer, mesothelioma, Merkel cell carcinoma, and head and neck cancer.

11. The kit according to any one of claims 2, 4 to 10, 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.

12. The kit according to any one of claims 2, 4 to 11, wherein the first antibody molecule is a monoclonal antibody molecule or an antibody molecule derived from a monoclonal antibody.

13. 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 The kit according to any one of claims 2, 4 to 12, wherein the antibody fragment is selected from the group consisting of an antibody fragment, an Fv fragment, and an scFv fragment.

14. The kit according to any one of claims 2, 4 to 13, wherein the first antibody molecule is a human IgG antibody molecule having an aglycosylated Fc region, or a human-derived IgG antibody molecule having an aglycosylated Fc region.

15. The kit of claim 14 , wherein the IgG antibody molecule is an IgG1 or IgG2 antibody molecule.

16. 16. The kit of claim 15, 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.

17. The kit of claim 16, which is non-glycosylated due to an amino acid substitution at position 297.

18. 18. The kit of claim 17, which is non-glycosylated by an N297Q substitution.

19. said first antibody molecule comprising the following CDRs: (i) SEQ ID NO: 51 and SEQ ID NO: 52 and SEQ ID NO: 53, or (ii) SEQ ID NO: 57 and SEQ ID NO: 58 and SEQ ID NO: 59, or (iii) SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, or (iv) SEQ ID NO: 69 and SEQ ID NO: 70 and SEQ ID NO: 71, or (v) SEQ ID NO: 75 and SEQ ID NO: 76 and SEQ ID NO: 77, or (vi) SEQ ID NO: 81 and SEQ ID NO: 82 and SEQ ID NO: 83, or (vii) SEQ ID NO: 87 and SEQ ID NO: 88 and SEQ ID NO: 89, or (viii) SEQ ID NO: 93 and SEQ ID NO: 94 and SEQ ID NO: 95, or (ix) SEQ ID NO: 99 and SEQ ID NO: 100 and SEQ ID NO: 101, or (x) SEQ ID NO: 105 and SEQ ID NO: 106 and SEQ ID NO: 107, or (xi) SEQ ID NO: 111 and SEQ ID NO: 112 and SEQ ID NO: 113, or (xii) SEQ ID NO: 117 and SEQ ID NO: 118 and SEQ ID NO: 119, or (xiii) SEQ ID NO: 123, SEQ ID NO: 124, and SEQ ID NO: 125, or (xiv) SEQ ID NO: 129 and SEQ ID NO: 130 and SEQ ID NO: 131, or (xv) SEQ ID NO: 135, SEQ ID NO: 136, and SEQ ID NO: 137, or (xvi) SEQ ID NO: 141 and SEQ ID NO: 142 and SEQ ID NO: 143, or (xvii) SEQ ID NO: 147 and SEQ ID NO: 148 and SEQ ID NO: 149, or (xviii) SEQ ID NO: 153 and SEQ ID NO: 154 and SEQ ID NO: 155, or (xix) SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, or (xx) SEQ ID NO: 165 and SEQ ID NO: 166 and SEQ ID NO: 167, or (xxi) SEQ ID NO: 171 and SEQ ID NO: 172 and SEQ ID NO: 173, or (xxii) SEQ ID NO: 177 and SEQ ID NO: 178 and SEQ ID NO: 179, or (xxiii) SEQ ID NO: 183 and SEQ ID NO: 184 and SEQ ID NO: 185, or (xxiv) The kit of any one of claims 2, 4 to 18, comprising a heavy chain variable region (VH) comprising SEQ ID NO: 189, SEQ ID NO: 190 and SEQ ID NO:

191.

20. said first antibody molecule comprising the following CDRs: (i) SEQ ID NO: 54 and SEQ ID NO: 55 and SEQ ID NO: 56, or (ii) SEQ ID NO: 60 and SEQ ID NO: 61 and SEQ ID NO: 62, or (iii) SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, or (iv) SEQ ID NO: 72 and SEQ ID NO: 73 and SEQ ID NO: 74, or (v) SEQ ID NO: 78 and SEQ ID NO: 79 and SEQ ID NO: 80, or (vi) SEQ ID NO: 84 and SEQ ID NO: 85 and SEQ ID NO: 86, or (vii) SEQ ID NO: 90 and SEQ ID NO: 91 and SEQ ID NO: 92, or (viii) SEQ ID NO: 96 and SEQ ID NO: 97 and SEQ ID NO: 98, or (ix) SEQ ID NO: 102 and SEQ ID NO: 103 and SEQ ID NO: 104, or (x) SEQ ID NO: 108 and SEQ ID NO: 109 and SEQ ID NO: 110, or (xi) SEQ ID NO: 114 and SEQ ID NO: 115 and SEQ ID NO: 116, or (xii) SEQ ID NO: 120 and SEQ ID NO: 121 and SEQ ID NO: 122, or (xiii) SEQ ID NO: 126, SEQ ID NO: 127, and SEQ ID NO: 128, or (xiv) SEQ ID NO: 132 and SEQ ID NO: 133 and SEQ ID NO: 134, or (xv) SEQ ID NO: 138 and SEQ ID NO: 139 and SEQ ID NO: 140, or (xvi) SEQ ID NO: 144 and SEQ ID NO: 145 and SEQ ID NO: 146, or (xvii) SEQ ID NO: 150 and SEQ ID NO: 151 and SEQ ID NO: 152, or (xviii) SEQ ID NO: 156 and SEQ ID NO: 157 and SEQ ID NO: 158, or (xix) SEQ ID NO: 162 and SEQ ID NO: 163 and SEQ ID NO: 164, or (xx) SEQ ID NO: 168 and SEQ ID NO: 169 and SEQ ID NO: 170, or (xxi) SEQ ID NO: 174 and SEQ ID NO: 175 and SEQ ID NO: 176, or (xxii) SEQ ID NO: 180 and SEQ ID NO: 181 and SEQ ID NO: 182, or (xxiii) SEQ ID NO: 186 and SEQ ID NO: 187 and SEQ ID NO: 188, or (xxiv) The kit of any one of claims 2, 4 to 19, comprising a light chain variable region (VL) comprising SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO:

194.

21. The kit of any one of claims 2, 4 to 20, wherein the first antibody molecule comprises a heavy chain variable region (VH) amino acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:

26.

22. the first antibody molecule comprises a light chain variable region selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:

50. The kit according to any one of claims 2, 4 to 21, comprising a (VL) amino acid sequence.

23. the first antibody molecule having the following CDR amino acid sequences: (i) SEQ ID NO: 51 and SEQ ID NO: 52 and SEQ ID NO: 53 and SEQ ID NO: 54 and SEQ ID NO: 55 and SEQ ID NO: 56, or (ii) SEQ ID NO: 57 and SEQ ID NO: 58 and SEQ ID NO: 59 and SEQ ID NO: 60 and SEQ ID NO: 61 and SEQ ID NO: 62, or (iii) SEQ ID NO: 63 and SEQ ID NO: 64 and SEQ ID NO: 65 and SEQ ID NO: 66 and SEQ ID NO: 67 and SEQ ID NO: 68, or (iv) SEQ ID NO: 69 and SEQ ID NO: 70 and SEQ ID NO: 71 and SEQ ID NO: 72 and SEQ ID NO: 73 and SEQ ID NO: 74, or (v) SEQ ID NO: 75 and SEQ ID NO: 76 and SEQ ID NO: 77 and SEQ ID NO: 78 and SEQ ID NO: 79 and SEQ ID NO: 80, or (vi) SEQ ID NO: 81 and SEQ ID NO: 82 and SEQ ID NO: 83 and SEQ ID NO: 84 and SEQ ID NO: 85 and SEQ ID NO: 86, or (vii) SEQ ID NO: 87 and SEQ ID NO: 88 and SEQ ID NO: 89 and SEQ ID NO: 90 and SEQ ID NO: 91 and SEQ ID NO: 92, or (viii) SEQ ID NO:93 and SEQ ID NO:94 and SEQ ID NO:95 and SEQ ID NO:96 and SEQ ID NO:97 and SEQ ID NO:98, or (ix) SEQ ID NO: 99 and SEQ ID NO: 100 and SEQ ID NO: 101 and SEQ ID NO: 102 and SEQ ID NO: 103 and SEQ ID NO: 104, or (x) SEQ ID NO: 105 and SEQ ID NO: 106 and SEQ ID NO: 107 and SEQ ID NO: 108 and SEQ ID NO: 109 and SEQ ID NO: 110; or (xi) SEQ ID NO: 111 and SEQ ID NO: 112 and SEQ ID NO: 113 and SEQ ID NO: 114 and SEQ ID NO: 115 and SEQ ID NO: 116, or (xii) SEQ ID NO: 117 and SEQ ID NO: 118 and SEQ ID NO: 119 and SEQ ID NO: 120 and SEQ ID NO: 121 and SEQ ID NO: 122; or (xiii) SEQ ID NO: 123 and SEQ ID NO: 124 and SEQ ID NO: 125 and SEQ ID NO: 126 and SEQ ID NO: 127 and SEQ ID NO: 128, or (xiv) SEQ ID NO: 129 and SEQ ID NO: 130 and SEQ ID NO: 131 and SEQ ID NO: 132 and SEQ ID NO: 133 and SEQ ID NO: 134, or (xv) SEQ ID NO: 135 and SEQ ID NO: 136 and SEQ ID NO: 137 and SEQ ID NO: 138 and SEQ ID NO: 139 and SEQ ID NO: 140, or (xvi) SEQ ID NO: 141 and SEQ ID NO: 142 and SEQ ID NO: 143 and SEQ ID NO: 144 and SEQ ID NO: 145 and SEQ ID NO: 146, or (xvii) SEQ ID NO: 147 and SEQ ID NO: 148 and SEQ ID NO: 149 and SEQ ID NO: 150 and SEQ ID NO: 151 and SEQ ID NO: 152; or (xviii) SEQ ID NO: 153 and SEQ ID NO: 154 and SEQ ID NO: 155 and SEQ ID NO: 156 and SEQ ID NO: 157 and SEQ ID NO: 158, or (xix) SEQ ID NO: 159 and SEQ ID NO: 160 and SEQ ID NO: 161 and SEQ ID NO: 162 and SEQ ID NO: 163 and SEQ ID NO: 164, or (xx) SEQ ID NO: 165 and SEQ ID NO: 166 and SEQ ID NO: 167 and SEQ ID NO: 168 and SEQ ID NO: 169 and SEQ ID NO: 170, or (xxi) SEQ ID NO: 171 and SEQ ID NO: 172 and SEQ ID NO: 173 and SEQ ID NO: 174 and SEQ ID NO: 175 and SEQ ID NO: 176, or (xxii) SEQ ID NO: 177 and SEQ ID NO: 178 and SEQ ID NO: 179 and SEQ ID NO: 180 and SEQ ID NO: 181 and SEQ ID NO: 182, or (xxiii) SEQ ID NO: 183 and SEQ ID NO: 184 and SEQ ID NO: 185 and SEQ ID NO: 186 and SEQ ID NO: 187 and SEQ ID NO: 188, or (xxiv) The kit according to any one of claims 2, 4 to 22, comprising SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193 and SEQ ID NO:

194.

24. the first antibody molecule having the following amino acid sequence: (i) SEQ ID NO: 3 and SEQ ID NO: 27, or (ii) SEQ ID NO: 4 and SEQ ID NO: 28, or (iii) SEQ ID NO: 5 and SEQ ID NO: 29, or (iv) SEQ ID NO: 6 and SEQ ID NO: 30, or (v) SEQ ID NO: 7 and SEQ ID NO: 31, or (vi) SEQ ID NO: 8 and SEQ ID NO: 32, or (vii) SEQ ID NO: 9 and SEQ ID NO: 33, or (viii) SEQ ID NO: 10 and SEQ ID NO: 34, or (ix) SEQ ID NO: 11 and SEQ ID NO: 35, or (x) SEQ ID NO: 12 and SEQ ID NO: 36, or (xi) SEQ ID NO: 13 and SEQ ID NO: 37, or (xii) SEQ ID NO: 14 and SEQ ID NO: 38, or (xiii) SEQ ID NO: 15 and SEQ ID NO: 39, or (xiv) SEQ ID NO: 16 and SEQ ID NO: 40, or (xv) SEQ ID NO: 17 and SEQ ID NO: 41, or (xvi) SEQ ID NO: 18 and SEQ ID NO: 42, or (xvii) SEQ ID NO: 19 and SEQ ID NO: 43, or (xviii) SEQ ID NO: 20 and SEQ ID NO: 44, or (xix) SEQ ID NO: 21 and SEQ ID NO: 45, or (xx) SEQ ID NO: 22 and SEQ ID NO: 46, or (xxi) SEQ ID NO: 23 and SEQ ID NO: 47, or (xxii) SEQ ID NO: 24 and SEQ ID NO: 48, or (xxiii) SEQ ID NO: 25 and SEQ ID NO: 49, or (xxiv) The kit according to any one of claims 2, 4 to 23, comprising SEQ ID NO: 26 and SEQ ID NO:

50.

25. The kit according to any one of claims 2, 4 to 18, wherein the first antibody molecule is an antibody molecule capable of competing with the antibody molecule defined in any one of claims 19 to 23 for binding to FcγRIIb.

26. The kit according to any one of claims 2, 4 to 25, wherein the second antibody molecule is selected from the group consisting of a human antibody molecule, a humanized antibody molecule, and an antibody molecule of human origin.

27. The kit of any one of claims 2, 4 to 26, wherein the second antibody molecule is a human IgG antibody.

28. The kit of any one of claims 2, 4 to 27, wherein the second antibody molecule specifically binds to CTLA-4.

29. The kit of any one of claims 2, 4 to 27, wherein the second antibody molecule specifically binds to PD-L1.

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