HK40118491A - COMBINATION OF A FCγRIIB- AND A TUMOR ANTIBODY FOR USE IN THE TREATMENT OF AN FCγRIIB-NEGATIVE CANCER - Google Patents

Description

The combination of FcγRIIB and tumor antibodies is used for the treatment of FcγRIIB-negative cancers.

Technical Field

The present invention relates to the combined use of the following in the treatment of FcγRIIB-negative cancers: 1) an 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, and 2) an antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor.

Background Technology

It has long been recognized that the inhibitory Fcγ receptor (FcγR) IIB, expressed by many cells of the immune system, negatively regulates innate and adaptive immunity through the binding of immune complexes (ICs). Similarly, the knowledge that FcγRIIB negatively regulates monoclonal antibody-mediated immunotherapy has been known for over a decade. Thus, FcγRIIB-deficient mice were able to clear tumors more effectively than wild-type (WT) mice when treated with therapeutic monoclonal antibodies (mAbs), suggesting that FcγRIIB expression on effector cells (i.e., macrophages and monocytes) causes a suppression of their phagocytic and cytotoxic potential in vivo. Furthermore, FcγRIIB modulates the antigen-presenting potential of dendritic cells (DCs), and FcγRIIB-negative DCs have an improved ability to activate primordial T cells (van Montfoor et al., J Immunol. 2012 July 1; 189(1):92-101). Recently, antagonistic antibodies that block FcγRIIB signaling and internalization in B cells have been developed. These antibodies have shown effective depletion of FcγRIIB-expressing B cells and have effectively promoted rituximab-mediated depletion in both normal and malignant B cells, demonstrating their utility in hematological malignancies (WO 2012/022985). FcγRIIB blocking antibodies containing wild-type IgG1 Fc proficient in FcγR binding, as well as FcγRIIB blocking antibodies containing Fc engineered for impaired FcγR binding (IgG1 N297Q), have shown similar ability to enhance rituximab-mediated B cell depletion, indicating that the rituximab-promoting effect is independent of the anti-FcγRIIB Fc. However, it has not been examined or demonstrated whether such antibodies will also be effective in enhancing the therapeutic activity of tumor-directly targeting antibodies (e.g., anti-HER2 or anti-EGFR) in the treatment of FcγRIIB-negative cancers, such as most solid cancers.

Recently, we demonstrated the differential antitumor enhancement of the therapeutic activity of immunomodulatory antibodies against the immunosuppressive checkpoints CTLA-4 and PD-1 expressed on T cells by Fc-FcγR proficient and impaired anti-FcγRIIB antibodies. Specifically, the strongest antitumor enhancement was observed with Fc-FcγR impaired anti-FcγRIIB antibodies in the context of anti-CTLA-4 (WO 2019/138005). Conversely, in the context of combination immunotherapy with Fc-proficient but not Fc-impaired anti-PD-1 antibodies, anti-FcγRIIB enhanced the therapeutic antitumor activity (WO 2021/009358). Although studies in gene knockout animals have shown that anti-FcγRIIB antibodies, in combination with tumor-directing antibodies, have potential therapeutic enhancement effects in both FcγRIIB+ (e.g., B-cell lymphoma when combined with anti-CD20 antibodies) and FcγRIIB- cancers (e.g., solid cancers when combined with anti-HER2 antibodies) (Clynes et al., Nature Medicine, April 2000; 6(4):443-6), it remains unclear whether anti-FcγRIIB antibodies (e.g., anti-HER2 in the treatment of FcγRIIB cancers) enhance the therapeutic activity of tumor-directing antibodies and (if so) what types of FcγRIIB antibodies enhance the therapeutic activity of tumor-directing antibodies.

Summary of the Invention

In this paper, we demonstrate that only anti-FcγRIIB antibodies (e.g., F(ab)' ₂ antibodies or deglycosylated antibodies) lacking the Fc region or whose Fc region exhibits binding to reduced or impaired FcγR can enhance the therapeutic activity of tumor-directly targeting antibodies (e.g., anti-HER2 and anti-EGFR antibodies for the treatment of FcγRIIB-negative cancers, including solid cancers). This contrasts with our previous patent applications, which describe the use of Fc:FcγR-enhanced and Fc:FcγR-impaired anti-FcγRIIB antibodies in enhancing activity and overcoming resistance to B-cell-directly targeting antibodies (e.g., anti-CD20 for the therapy of NHL (WO 2012/022985)), and the widespread use of differential Fc:FcγR-dependent anti-FcγRIIB antibodies described in patent applications WO 2021/009358 and WO 2019/138005 for enhancing the therapeutic activity of immunomodulatory (as opposed to tumor cell-directly targeting) anti-PD-1 and anti-CTLA-4 antibodies. Furthermore, our data demonstrate that combination therapy with anti-FcγRIIB antibodies (e.g., F(ab)' ₂ antibodies or deglycosylated antibodies) that lack an Fc region or whose Fc region shows reduced or impaired binding to FcγR enables anti-HER2 therapy for cancers with low HER2 expression that are not suitable for treatment with currently used clinically approved anti-HER2 regimens.

This article discloses a first antibody molecule that specifically binds to FcγRIIB via (or through) its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via (or through) its Fc region. This first antibody molecule is intended for use in combination with a second antibody molecule in the treatment of FcγRIIB-negative cancers in patients.

The second antibody molecule specifically binds to receptors present on tumor cells, and the second antibody molecule has an Fc region that binds to at least one activated Fcγ receptor.

This article also discloses a pharmaceutical composition comprising:

(i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and

(ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor;

This pharmaceutical composition is intended for use in the treatment of FcγRIIB-negative cancers in patients.

This article further discloses a kit for use in the treatment of FcγRIIB-negative cancers, 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 the Fcγ receptor via its Fc region, and

(ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor.

This article further discloses the following:

(i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and

(ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor;

Use in the manufacture of a medicine for the treatment of FcγRIIB-negative cancers in patients.

This article also discloses a method for treating FcγRIIB-negative cancers in patients, which includes administering:

(i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and

(ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region capable of activating at least one activated Fcγ receptor.

Detailed Implementation

Therefore, the present invention relates to the combined use of the following:

(i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and

(ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region capable of activating at least one activated Fcγ receptor.

Therefore, the second antibody molecule is a tumor-direct targeting antibody, or it is also referred to as a direct tumor-targeting antibody. The therapeutic activity of this antibody depends on FcγR binding. The binding of the second antibody molecule to a receptor on tumor cells, and subsequently to FcγR on immune effector cells, triggers FcγR-dependent immune effector cell-mediated killing (e.g., via macrophage-dependent ADCC or ADCP) of antibody-coated target tumor cells. Tumor-direct targeting antibodies may or may not provide tumor cell killing through additional mechanisms (e.g., by blocking tumor growth factor signaling, as is believed in with some anti-HER2 antibodies). In any case, the present invention applies to any tumor-direct targeting antibody whose mechanism encompasses FcγR-dependent tumor cell killing. Therefore, the present invention relates to maximizing therapeutic activity by optimizing FcγR-dependent tumor cell killing.

This combination is designed for the treatment of FcγRIIB-negative cancers in patients, with the aim of improving the therapeutic efficacy of the second antibody molecule through enhanced binding of its Fc moiety to activating FcγR and reduced binding/activation to inhibitory FcγR.

Fc receptors are membrane proteins located on the cell surface of immune effector cells, such as macrophages. The name comes from their specificity in binding to the Fc region of antibodies, which is the usual way antibodies bind to receptors. However, while some antibodies may bind specifically to one or more Fc receptors, others may also bind to Fc receptors via the antibody's CDR sequence.

The Fc receptor subset is the Fcγ receptor (Fc-gamma receptor, FcgammaR, FcgR), which is specific for IgG antibodies. Two types of Fcγ receptors exist: activated Fcγ receptors (also referred to as active Fcγ receptors) and inhibitory Fcγ receptors. Activated and inhibitory receptors transmit signals via the immunoreceptor tyrosine activation motif (ITAM) or immunoreceptor tyrosine inhibition motif (ITIM), respectively. In humans, FcγRIIB (FcγRIIb, FcgRIIB, CD32b) is an inhibitory Fcγ receptor, while FcγRI(CD64), FcγRIIA(CD32a), FcγRIIC(CD32c), FcγRIIIA(CD16a), and FcγRIV are activated Fcγ receptors. FcγgRIIIB is a GPI-linked receptor expressed on neutrophils, lacking the ITAM motif, but is also considered activating due to its ability to cross-link lipid rafts and bind to other receptors. In mice, the activated receptors are FcγRI, FcγRIII, and FcγRIV.

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

By binding to the inhibitory Fcγ receptor, antibodies can inhibit, block, and/or downregulate the function of effector cells.

By binding to activated Fcγ receptors, antibodies can activate effector cell functions and thereby trigger mechanisms such as antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cytokine release and/or antibody-dependent endocytosis, and in the case of neutrophils, NETosis (i.e., activation and release of NETs). Antibodies binding to activated Fcγ receptors can also lead to increases in certain activation markers, such as CD40, MHCII, CD38, CD80, and/or CD86.

The antibody molecule according to the invention, which specifically binds to FcγRIIB (i.e., the first antibody), binds to or interacts with the Fcγ receptor via the antibody's Fab region (i.e., via an antigen-binding region on the antibody that binds to the antigen, the antigen-binding region consisting of a constant domain and a variable domain of each of the heavy and light chains). Specifically, it binds to FcγRIIB present on immune effector cells, and particularly to FcγRIIB present on the surface of immune effector cells. If the antibody has a normal or common Fc region, it may also bind to the activated Fcγ receptor through the normal interaction between the Fc region and the Fc receptor. However, according to the invention, the antibody molecule that specifically binds to FcγRIIB completely lacks an Fc region or has reduced binding to the Fcγ receptor, meaning that the antibody molecule that specifically binds to FcγRIIB binds poorly to the Fcγ receptor or cannot bind to or interact with it at all. This appears to have at least two therapeutically important consequences:

1) The lack of Fc-mediated binding to activating FcγRs allows a greater number of activating Fcγ receptors to be available for Fc binding to (other) therapeutic anticancer antibodies. This is important because it is known that increasing the number of clusters of activating FcγRs (in contrast to inhibitory FcγRs; Nimmerjahn et al.; Science 2005 Dec 2; 310(5753):1510-2) increases effector cell-mediated target cell loss, which is a potential mechanism for the activity of both checkpoint inhibitors, immune agonists, and other immunomodulatory antibodies such as anti-IL-2R.

2) The lack of Fc-mediated binding to inhibitory FcγR or the reduction of Fc-mediated binding to inhibitory FcγR shows reduced inhibitory signaling in immune effector cells expressing FcγR. Therefore, the lack of Fc-mediated binding to FcγR targeting antibodies or the reduction of Fc-mediated binding to FcγR targeting antibodies may improve therapeutic efficacy through at least two mechanisms involving both enhanced activating FcγR in immune effector cells in response to secondary immunomodulatory anticancer antibodies and reduced inhibitory Fcγ signaling.

In this context, “reduced binding” or “binding with reduced affinity” means that the antibody molecule has reduced Fc-mediated binding to the Fcγ receptor, or in other words, the Fc region of the antibody molecule that specifically binds to the Fcγ RIIB binds to the activated Fcγ receptor with a lower affinity than the Fc region of normal human IgG1. Reduced binding can be assessed using techniques such as surface plasmon resonance. In this paper, “normal IgG1” refers to routinely generated IgG1 with an unmutated Fc region whose production does not alter its glycosylation. As a reference for this “normal IgG1,” unmodified rituximab generated in CHO cells can be used (Tipton et al., Blood 2015 125:1901-1909; for example, rituximab is described in EP 0 605 442).

"Reduced binding" means that, compared to the binding of the Fc region of normal human IgG1 to the same receptor, for all Fc receptors, the binding of the Fc region of an antibody molecule that specifically binds to FcγRIIB to the activated Fcγ receptor is reduced by at least 10-fold. In some embodiments, it is reduced by at least 20-fold. In some embodiments, it is reduced by at least 30-fold. In some embodiments, it is reduced by at least 40-fold. In some embodiments, it is reduced by at least 50-fold. In some embodiments, it is reduced by at least 60-fold. In some embodiments, it is reduced by at least 70-fold.

In some embodiments of the invention, the antibody molecule that specifically binds to FcγRIIB does not bind to its Fc region at all, and in some such cases, the antibody does not have an Fc region; it may be Fab, _Fab'2 , scFv, or their polyethylene glycolated forms.

In some embodiments, the antibody molecule that specifically binds to FcγRIIB can be an alpaca antibody, and particularly alpaca hcIgG. Like all mammals, camels produce conventional antibodies ( _IgG1 ) consisting of two heavy chains and two light chains linked together in a Y-shape by disulfide bonds. However, they also produce two distinct subclasses of immunoglobulin G: _IgG2 and _IgG3 , also known as heavy chain IgG (hcIgG). These antibodies consist of only two heavy chains, lacking the CH1 region, but still carrying an antigen-binding domain at their N-terminus, referred to as _VHH . Conventional Ig requires association from variable regions of both the heavy and light chains to allow for a high degree of diversity in antigen-antibody interactions. While separated heavy and light chains still exhibit this ability, they show very low affinity when compared to paired heavy and light chains. hcIgG is uniquely characterized by the ability of their monomeric antigen-binding regions to bind antigens with specificity, affinity, and especially diversity comparable to conventional antibodies, without needing to pair with another region.

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

To achieve reduced binding of IgG1 antibodies (such as IgG1 antibody) to the Fc receptor, the Fc region of the IgG antibody can be modified by deglycosylation. For example, such deglycosylation of IgG1 antibodies can be achieved, for instance, by substitution of the amino acid asparagine (N297X) at position 297 of the antibody chain. The substitution can be made using glutamine (N297Q), alanine (N297A), glycine (N297G), asparagine (N297D), or serine (N297S).

The Fc region can be modified by further substitutions, for example, as described by 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 the following combinations of substitutions in IgG1:

L242C, N297G, K334C

A287C, N297G, L306C

R292C, N297G, V302C

N297G, V323C, I332C and

V259C, N297G, L306C.

Alternatively, the carbohydrates in the Fc region can be enzymatically cleaved, and/or the cells used to produce antibodies can be grown in a culture medium that impairs carbohydrate addition, and/or cells engineered to lack the ability to add sugars can be used for antibody production, or through the production of antibodies in host cells (e.g., prokaryotes, including Escherichia coli) that do not glycosylate or functionally glycosylate antibodies, as described above.

Reduced affinity for the Fcγ receptor can be further achieved through the engineering of amino acids in the Fc region of the antibody (such modifications have been previously described by, for example, Xencor, Macrogenics, and Genentech) or through the production of antibodies in host cells (e.g., prokaryotes, including Escherichia coli) that do not glycosylate or functionally glycosylate the antibody.

In addition to reduced binding to the Fcγ receptor via the Fc region, in some embodiments, it is also preferred that the antibody molecule specifically binding to FcγRIIB does not induce phosphorylation of FcγRIIB upon binding to the target. ITIM phosphorylation of FcγRIIB is an inhibitory event that blocks its activity in immune cells.

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

In contrast to antibody molecules that specifically bind to FcγRIIB, antibody molecules that specifically bind to or interact with receptors present on tumor cells (i.e., secondary antibody molecules or tumor-directly targeting antibodies) have an Fc region that binds to or interacts with activated Fcγ receptors to a degree that is not reduced or at least not significantly reduced. Binding of secondary antibodies to tumor cells induces activation of Fc receptor-dependent antitumor activities (such as depletion), antibody-dependent cytotoxicity (ADCC), and/or antibody-dependent phagocytosis (ADCP). When depletion is mentioned herein, we mean the depletion, absence, or elimination of tumor cells caused by the physical clearance of cells.

To determine whether an antibody molecule is a tumor-depleted antibody molecule as understood in this invention, an in vitro ADCC or ADCP assay can be used. To determine whether an antibody molecule is a tumor cell-depleted antibody molecule, the same assay will be performed with and without depleted antibodies, which will show whether the depleted antibody being tested is actually being depleted.

ADCC assay is performed as follows: Target cells are labeled with calcein AM (acetylated methyl ester), followed by the addition of a diluted antibody concentration. The target cells are then co-cultured with human peripheral blood mononuclear cells (PBMCs) at a 50:1 effector:target (E:T) ratio at 37°C for 4 hours. 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 maximum release percentage is calculated as follows: %maximum release = (treated sample/triton) * 100.

ADCP assays were performed as follows: Target cells were labeled with 5 mM carboxyfluorescein succinimide (CFSE) for 10 minutes at room temperature, followed by washing in culture medium containing fetal bovine serum. The CFSE-labeled targets were then conditional with a diluted antibody concentration and co-cultured with bone marrow-derived macrophages (BMDM) at a 1:5E:T ratio in 96-well plates at 37°C for 1 hour. BMDM were then labeled with anti-F4/80 allophycocyanin for 15 minutes at room temperature and washed twice with PBS. The plates were kept on ice, wells were scraped to collect BMDM, and phagocytosis was assessed by flow cytometry using FACSCalibur (BD) to determine the percentage of F4/80+CFSE+ cells in the F4/80+ cell population.

Alternatively, methods such as those described by Cleary et al. in the Journal of Immunology, April 12, 2017, 1601473 can be used.

The second antibody molecule binds to FcγRIIB-negative cancerous tumors, meaning that these tumors do not present any FcγRIIB receptors. This can be tested using anti-FcγRIIB specific antibodies in various methods, including immunohistochemistry and flow cytometry, as noted by Tutt et al. in the *Journal of Immunology*, 2015, 195(11): 5503-5516.

In addition to specifically binding to targets on tumor cells, the second antibody molecule also binds to activated Fcγ receptors present on immune effector cells via its Fc region. To enable binding to activated Fcγ receptors, in some embodiments, the Fc region of the second antibody should be glycosylated at least at position 297. Carbohydrate residues at this position facilitate binding to the Fcγ receptor. In some embodiments, these residues are preferably biantennary carbohydrates containing GlnNAc, mannose with a terminal galactose residue, and sialic acid. It should contain the _CH2 portion of the Fc molecule.

Antibodies are well-known to technicians in the fields of immunology and molecular biology. Typically, an antibody consists of two heavy (H) chains and two light (L) chains. In this article, we sometimes refer to this complete antibody molecule as a full-size or full-length antibody. The heavy chain of an antibody contains one variable domain (VH) and three constant domains (CH1, CH2, and CH3), while the light chain contains one variable domain (VL) and one constant domain (CL). The variable domain (sometimes collectively referred to as the _FV region) binds to the antibody's target or antigen. Each variable domain contains three loops called complementarity-determining regions (CDRs), which are responsible for target binding. The constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions. Antibodies or immunoglobulins can be classified into different categories based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and in humans, several of these classes are further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2.

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

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

A functional fragment of a full-size antibody has the same antigen-binding properties as the corresponding full-size antibody and includes the same variable domains (i.e., VH and VL sequences) and/or the same CDR sequences as the corresponding full-size antibody. Having the same antigen-binding properties as the corresponding full-size antibody means that it binds to the same epitopes on the target of the full-size antibody. Such functional fragments can correspond to the Fv portion of a full-size antibody. Alternatively, such fragments can be Fab (also denoted as F(ab), a monovalent antigen-binding fragment without an Fc portion) or F(ab') ₂ (also denoted as _Fab'2 or Fab ₂ , a bivalent antigen-binding fragment containing two antigen-binding Fab portions linked together by disulfide bonds) or F(ab') (i.e., a monovalent variant of F(ab') ₂ ). Such fragments can also be single-chain variable fragments (scFv).

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

Molecules containing two CDR regions, such as those described by Vaughan and Sollazzo 2001, *Combinatorial Chemistry & High Throughput Screening*, 4:417-430, on page 418 (right column - 3 Our Design Strategy), consist only of H1 and H2 CDR hypervariable regions scattered within the framework region. This microantibody is described as capable of binding to a target. Pessi et al., 1993, *Nature*, 362:367-9 and Bianchi et al., 1994, *Journal of Molecular Biology*, 236:649-59, cited by Vaughan and Sollazzo, describe the H1 and H2 microantibodies and their properties in more detail. Qiu et al., 2007, *Nature Biotechnology*, 25:921-9, demonstrated that molecules composed of two linked CDRs can bind antigens. Quiocho, 1993, *Nature*, 362:293-4, provided an overview of "microantibody" technology. Ladner, 2007, *Nature Biotechnology*, 25:875-7, commented that molecules containing two CDRs can retain antigen-binding activity.

For example, Laue et al., 1997, *Journal of Biochemistry (JBC)*, 272:30937-44, described antibody molecules containing a single CDR region, demonstrating that a series of hexapeptides derived from the CDR exhibited antigen-binding activity, and noting that synthetic peptides with a single intact CDR exhibited strong binding activity. Monnet et al., 1999, *Journal of Biochemistry (JBC)*, 274:3789-96, showed that a series of 12-mer peptides and associated framework regions possessed antigen-binding activity, and commented that a single CDR3-like peptide could bind to the antigen. Heap et al., 2005, *Journal of General Virology (J. Gen. Virol.)*, 86:1791-1800, reported that "microantibodies" (molecules containing a single CDR) could bind to antigens, and showed that cyclic peptides derived from anti-HIV antibodies possessed antigen-binding activity and function. In Nicaise et al., 2004, Protein Science, 13:1882-91, it was shown that a single CDR can confer antigen-binding activity and affinity against its lysozyme antigen.

Therefore, antibody molecules with five, four, three or fewer CDRs are able to retain the antigen-binding properties of their derived full-length antibodies.

Antibody molecules can also be derivatives of full-length antibodies or fragments of such antibodies. When a derivative is used, it should have the same antigen-binding characteristics as the corresponding full-length antibody in terms of binding to the same epitopes on the target as the full-length antibody.

Therefore, as used herein, when referring to the term "antibody molecule," we include all types of antibody molecules, their functional fragments, and their derivatives, including: monoclonal antibodies, polyclonal antibodies, synthetic antibodies, antibodies produced in a recombinant manner, multispecific antibodies, bispecific antibodies, human antibodies, human-derived antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies, single-chain Fv (scFv), Fab fragments, F(ab') ₂ fragments, F(ab') fragments, disulfide-linked Fv (sdFv), antibody heavy chains, antibody light chains, homodimers of antibody heavy chains, homodimers of antibody light chains, heterodimers of antibody heavy chains, heterodimers of antibody light chains, and antigen-binding functional fragments of such homodimers and heterodimers.

In addition, 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 stated.

In some embodiments, the antibody is human IgG1. Those skilled in the art will understand that mouse IgG2a and human IgG1 bind to activating Fcγ receptors and share the ability to activate target cells lost through activation, for example, ADCP and ADCC, by immune cells carrying activating Fcγ receptors. Therefore, in embodiments where mouse IgG2a is preferably isotyped for loss in mice, human IgG1 is preferably isotyped for loss in humans in such embodiments.

As described above, the present invention covers different types and forms of antibody molecules, and is known to those skilled in the art of immunology. It is known that antibodies for therapeutic purposes are typically modified with additional components that modify the properties of the antibody molecule.

Therefore, antibody molecules including those of the present invention or antibody molecules used according to the present invention (e.g., monoclonal antibody molecules, and/or polyclonal antibody molecules, and/or bispecific antibody molecules) contain detectable portions and/or cytotoxic portions.

When referring to the "detectable portion," we include one or more of the following groups: enzymes; radioactive atoms; fluorescent portions; chemiluminescent portions; and bioluminescent portions. The detectable portion allows antibody molecules to be visualized in vitro, and/or in vivo, and/or ex vivo.

When referring to the “cytotoxic fraction,” we include the radioactive fraction and/or enzyme, where the enzyme is caspase and/or toxin, where the toxin is bacterial toxin or venom; where the cytotoxic fraction is capable of inducing cell lysis.

Further, this includes antibody molecules that may be in isolated and/or purified form, and/or PEGylated antibody molecules. PEGylation is a method by which a polyethylene glycol polymer is added to a molecule such as an antibody molecule or derivative to modify its behavior, for example, by increasing its hydrodynamic size to prolong its half-life, thereby preventing renal clearance.

As described above, the antibody's CDR binds to the antibody target. The amino acid assignment for each CDR described herein is based on the definitions given in Kabat EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, pp. xv-xvii.

As those skilled in the art will know, other methods exist for assigning amino acids to each CDR. For example, the International Immunogenetics Information System (IMGT®) (http://www.imgt.org/ and Lefranc and Lefranc's The Immunoglobulin Facts Book, Academic Press, 2001).

In a further embodiment, the antibody molecule of the present invention or used according to the present invention is an antibody molecule capable of competing with the specific antibodies provided herein, such as an antibody molecule for binding to a specific target comprising, for example, any amino acid sequence listed in, for example, SEQ ID NO:1 to 194.

When we say “capable of competing against…”, we mean that the competing antibody can at least partially inhibit or otherwise interfere with the binding of an antibody molecule to a specific target as defined herein.

For example, such competitive antibody molecules may be able to inhibit the binding of the antibody molecules described herein by at least about 10%; for example, 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 inhibit the ability of the antibodies described herein to prevent or reduce binding to a specific target by at least about 10%; for example, 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%.

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

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

The targets of the antibodies according to the invention, or the antibodies used according to the invention, are expressed on the surface of cells; that is, they are cell surface antigens, which will include epitopes against the antibody (also referred to as cell surface epitopes in this context). Cell surface antigens and epitopes are terms that will be readily understood by those skilled in immunology or cell biology.

When referring to "cell surface antigens," we include the following: cell surface antigens are exposed on the extracellular side of the cell membrane, but may only be exposed transiently. When referring to "transient exposure," we include the following: cell surface antigens can be internalized into the cell or released from the extracellular side of the cell membrane into the extracellular space. Cell surface antigens can be released from the extracellular side of the cell membrane through lysis, which can be mediated by proteases.

We also include the following: cell surface antigens can bind to the cell membrane, but can only associate with it transiently. When referring to "transient association," we include the following: cell surface antigens can be released from the extracellular space of the cell membrane. Cell surface antigens can be released from the extracellular space of the cell membrane through lysis, which can be mediated by proteases.

We further include the following: cell surface antigens may be peptides, or polypeptides, or carbohydrates, or oligosaccharide chains or lipids; and/or epitopes present on proteins, or glycoproteins or lipoproteins.

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 such methods can be used to assess the binding of an antibody to a target and/or the binding of the antibody's Fc region to an Fc receptor; and the relative strength, specificity, inhibition, prevention, or reduction of those interactions. Examples of methods that can be used to assess protein binding are, for example, immunoassays, BIAcore, Western blotting, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) (see *Fundamental Immunology Second Edition*, Raven Press, New York, pp. 332–336 (1989) on antibody specificity).

The meaning of an antibody that specifically binds to or interacts with a defined target molecule or antigen is well-known and implies that the antibody preferentially and selectively binds to its target rather than non-target molecules. In this context, the term "binds to" is used interchangeably with "interacts with". Therefore, when referring to "specifically binding antibody molecules" or "target-specific antibody molecules", we include the following: antibody molecules that specifically bind to a target but not to non-target molecules, or bind to non-target molecules more weakly than the target (e.g., with lower affinity).

We also include the meaning that an antibody that binds specifically to a target is at least 2 times, or at least 5 times, or at least 10 times, or at least 20 times, or at least 50 times, or at least 100 times, or at least 200 times, or at least 500 times, or at least about 1000 times stronger than an antibody that binds specifically to a non-target.

Furthermore, we include the following meaning: if an antibody binds to ^{a target with a Kd of at least about 10⁻¹ Kd, or at least about 10⁻² Kd, or at least about 10⁻³ Kd, or at least about 10⁻⁴} _{Kd ,} ^{or at} _least ^about _10⁻⁵ ^Kd _, ^or _at least about ^10⁻⁶ _Kd , or at least about ^10⁻⁷ _Kd , or at least about ^10⁻⁸ _Kd , or at least about ^10⁻⁹ _Kd , or at least about ^10⁻¹⁰ _Kd , or at least about ^10⁻¹¹ _Kd , or at least about ^10⁻¹² _Kd , or at least about ^10⁻¹³ _Kd , or at least about ^10⁻¹⁴ _Kd , or at least about 10⁻¹⁵ _Kd , then the antibody binds specifically to the target.

In some embodiments, the antibody molecule that specifically binds to FcγRIIB is a human antibody.

In some embodiments, the antibody molecule that specifically binds to FcγRIIB is a human-derived antibody, i.e., a modified original human antibody as described herein.

In some embodiments, the antibody molecule that specifically binds to FcγRIIB is a humanized antibody, i.e., a primitive non-human antibody that has been modified to increase its similarity to human antibodies. Humanized antibodies can be, for example, mouse antibodies or alpaca antibodies.

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

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS

SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL

GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE

EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

IgG1-CL [SEQ ID NO:2]

QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSK

QSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

These constant regions (SEQ ID NO:1 and SEQ ID NO:2) are of human origin. The Fc region is further modified to target reduced binding to the Fcγ receptor via its Fc region. As described herein, in some embodiments, preferably, SEQ ID NO:1 has been deglycosylated by N297Q substitution, and IgG1-CH has the following CH sequence [SEQ ID NO:195], where 297Q residues are marked in bold:

In some embodiments and/or examples, mouse antibody molecules are used. These can also be used with alternative antibodies. These may then contain the following constant regions (CH and CL):

CH[SEQ ID NO:196]

CL[SEQ ID NO:197]

QPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSK

QSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS

Therefore, these constant regions (SEQ ID NO:196 and SEQ ID NO:197) are of mouse origin. SEQ ID NO:196 contains the N297A mutation (297A residues are marked in bold in the above sequence). This N297A mutation in the mouse sequence corresponds to the N297Q mutation in the human sequence.

In some embodiments, the antibody molecule that specifically binds to FcγRIIB comprises one or more sequences of the following clones:

Antibody clone: 1A01

1A01-VH [SEQ ID NO:3]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMNWIRQTPGKGLEWVSLIGWDGGS

TYYADSVKGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARAYSGYELDYWGQGTLV

TVSS

1A01-VL [SEQ ID NO:27]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNNNRPSGV

PDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNASIFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFTRYDGSN

KYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENIDAFDVWGQGTLVT

VSS

1B07-VL [SEQ ID NO:28]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNQQRPSGV

PDRFSGSKSGTSASLAISGLRSEDEADYYCEAWDDRLFGPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISDSGAGR

YYADSVEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTHDSGELLDAFDIWGQGT

LVTVSS

1C04-VL [SEQ ID NO:29]

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNHVLWYQQLPGTAPKLLIYGNSNRPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKLTVLG 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:AAWDDSNGWV [ SEQ ID NO:68]

Antibody clone: 1E05

1E05-VH [SEQ ID NO:6]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQVPGKGLEWVAVISYDGSN

KNYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNFDNSGYAIPDAFDIWG

QGTLVTVSS

1E05-VL [SEQ ID NO:30]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNSRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLGGPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVAYISRDADIT

HYPASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTTGFDYAGDDAFDIWGQGTL

VTVSS

2A09-VL [SEQ ID NO:31]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNAVNWYQQLPGTAPKLLIYGNSDRPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGRWVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVALIGHDGNN

KYYLDSLEGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARATDSGYDLLYWGQGTLV

TVSS

2B08-VL [SEQ ID NO:32]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYYDDLLPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCTTWDDSLSGVVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSAIGFSDDNT

YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDGSGWSFWGQGTLVTV

SS

2E8-VL [SEQ ID NO:33]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNAVNWYQQLPGTAPKLLIYDNNKRPSGV

PDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLRGWVFGGGTKLTVLG CDR area

CDRH1: DYYMS [ SEQ ID NO:87]

CDRH2:AIGFSDDNTYYADSVKG [ SEQ ID NO:88]

CDRH3:GDGGSGWSF [ 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREWRDAFDIWGQGTLVT

VSS

5C04-VL [ SEQ ID NO:34]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSDNQRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGSWVFGGGTKLTVLG 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: AAWDSLSGSWV [ SEQ ID NO:98]

Antibody clone: 5C05

5C05-VH [SEQ ID NO:11]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARENFDAFDVWGQGTLVT

VSS

5C05-VL [ SEQ ID NO:35]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSNSQRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGQVVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIAYDGSK

KDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREYRDAFDIWGQGTLVT

VSS

5D07-VL [ SEQ ID NO:36]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG

VPDRFSGSKSGTTASLAISGLRSEDEADYYCAAWDDSVSGWMFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGINK

DYADSMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERKDAFDIWGQGTLVTV

SS

5E12-VL [ SEQ ID NO:37]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSNNQRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLNGLVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYDGSN

RYYADSVKGRFTMSRDNSKNTLYLQMNSLRAEDTAVYYCARDRWNGMDVWGQGTL

VTVSS

5G08-VL [ SEQ ID NO:38]

QSVLTQPPSASGTPGQRVTISCSGSSSNIGAGYDVHWYQQLPGTAPKLLIYANNQRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPWVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDT

AYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDHSVIGAFDIWGQGTLVT

VSS

5H06-VL [ SEQ ID NO:39]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYDNNKRPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCSSYAGSNNVVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVTSYDGNT

KYYANSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDCGGDCFDYWGQGTL

VTVSS

6A09-VL [ SEQ ID NO:40]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNEGVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQLGEAFDIWGQGTLV

TVSS

6B01-VL [ SEQ ID NO:41]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYDNNKRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDSLSGPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSAISGSGSST

YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDIDYFDYWGQGTLVTV

SS

6C11-VL [ SEQ ID NO:42]

QSVLTQPPSASGTPGQRVTISCTGSSSNFGAGYDVHWYQQLPGTAPKLLIYENNKRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARERRDAFDIWGQGTLVT

VSS

6C12-VL [ SEQ ID NO:43]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYSDNQRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDSDTPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARDHSAAGYFDYWGQGT

LVTVSS

6D01-VL [ SEQ ID NO:44]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYGNSIRPSGGP

DRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSLSSPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFGSYGMHWVRQAPGKGLEWVSGISWDSAII

DYAGSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDEAAAGAFDIWGQGTLV

TVSS

6G03-VL [ SEQ ID NO:45]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTDRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGPVVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTLSSYGISWVRQAPGKGLEWVSGISGSGGNT

YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSVGAYANDAFDIWGQGTL

VTVSS

6G08-VL [ SEQ ID NO:46]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGDTNRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGPVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWMAVISYDGSN

KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARELYDAFDIWGQGTLVTV

SS

6G11-VL [ SEQ ID NO:47]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADDHRPSG

VPDRFSGSKSGTSASLAISGLRSEDEADYYCASWDDSQRAVIFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAREYKDAFDIWGQGTLVT

VSS

6H08-VL [ SEQ ID NO:48]

QSVLTQPPSASGTPGQRVTISCTGSSSNIGSNTVNWYQQLPGTAPKLLIYDNNKRPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCQAWGTGIRVFGGGTKLTVLG CDR area

CDRH1:NYGMH [ SEQ ID NO:177]

CDRH2: VISYDGSNKYYAD SVKG [ SEQ ID NO:178]

CDRH3:EYKDAFDI [ SEQ ID NO:179]

CDRL1:TGSSSNIGSNTVN [ SEQ ID NO:180]

CDRL2:DNNKRPS [ SEQ ID NO:181]

CDRL3:QAWGTGIRV [ SEQ ID NO:182]

Antibody clone: 7C07

7C07-VH [SEQ ID NO:25]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSN

KYYADSVKGRFTISRDNSQNTLYLQMNSLRAEDTAVYYCAREFGYIILDYWGQGTLVT

VSS

7C07-VL [ SEQ ID NO:49]

QSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYRDYERPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCMAWDDSLSGVVFGGGTKLTVLG 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]

EVQLLESGGGLVQPGGSLRLSCAASGFTFSNHGMHWVRQAPGKGLEWVAVISYDGTN

KYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARETWDAFDVWGQGTLVT

VSS

4B02-VL [ SEQ ID NO:50]

QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNNANWYQQLPGTAPKLLIYDNNKRPSGVP

DRFSGSKSGTSASLAISGLRSEDEADYYCQAWDSSTVVFGGGTKLTVLG 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]

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

In some embodiments (sometimes preferred embodiments), the 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 has been further modified to have reduced binding to the Fcγ receptor via its Fc region. In some embodiments (sometimes preferred embodiments), the 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.

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

In some embodiments, antibody molecules that specifically bind to receptors present on tumor cells, or humanized antibody molecules that specifically bind to receptors present on tumor cells.

In some embodiments, antibody molecules that specifically bind to receptors present on tumor cells are chimeric antibodies.

As mentioned above, antibody molecules that specifically bind to receptors present on tumor cells must have the ability to bind to FcγR.

Combinations of antibody molecules that specifically bind to FcγRIIB and antibody molecules that specifically bind to receptors present on tumor cells can be used to treat cancer.

As used herein, the term "patient" refers to an animal, including humans, that has been diagnosed with FcγRIIB-negative cancer, or a cancer that is believed to be FcγRIIB-negative and/or exhibits symptoms of such cancer.

We include the following: the patient can 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.

When we refer to “exhibit”, we include the following: the subject exhibits cancer symptoms and/or cancer diagnostic markers, and/or the cancer symptoms and/or cancer diagnostic markers can be measured, and/or assessed, and/or quantified.

For medical professionals, it will be obvious what constitutes cancer symptoms and cancer diagnostic biomarkers, how to measure and/or assess and/or quantify whether there is a decrease or increase in the severity of cancer symptoms or cancer diagnostic biomarkers, and how those cancer symptoms and/or cancer diagnostic biomarkers can be used to formulate prognoses for cancer.

Cancer treatment is typically administered in courses, meaning that a treatment agent is applied over a period of time. The length of a course of treatment will depend on many factors, among others, including the type of treatment agent applied, the type of cancer being treated, the severity of the cancer, and the patient's age and health condition.

When we say “during treatment”, we include the following: the patient is currently receiving a course of treatment, and/or is receiving a therapeutic agent, and/or is receiving a course of therapeutic agents.

In some embodiments, the FcγRIIB-negative cancer to be treated according to the present invention is a solid cancer.

The clinical definition of diagnosis, prognosis, and progression of many cancers relies on certain classifications called staging. These staging systems are used to organize many different cancer diagnostic markers and cancer symptoms to provide an overview of the diagnosis and/or prognosis and/or progression of cancer. Technicians in the field of oncology will know how to use staging systems to assess the diagnosis, and/or prognosis, and/or progression of cancer, and which cancer diagnostic markers and cancer symptoms should be used for assessment.

"Cancer staging" includes Rai staging, which includes stages 0, I, II, III and IV, and/or Binet staging, which includes stages A, B and C, and/or Ann Arbour staging, which includes stages I, II, III and IV.

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

"Histological examination" includes the presence of small mature lymphocytes, and/or small mature lymphocytes with narrow cytoplasmic margins, small mature lymphocytes with dense nuclei lacking identifiable nucleoli, and/or small mature lymphocytes with narrow cytoplasmic margins and dense nuclei lacking identifiable nucleoli, and/or atypical cells, and/or lysed cells, and/or prelymphocytes.

It is well known that cancer results from mutations in cellular DNA that can cause cells to either avoid cell death or proliferate uncontrollably. Therefore, examining these mutations (also known as cytogenetic testing) can be a useful tool for assessing the diagnosis and/or prognosis of cancer. A real example is the deletion at chromosome 13q14.1, a characteristic feature of chronic lymphocytic leukemia. Techniques for examining mutations in cells are well known in the art; for example, fluorescence in situ hybridization (FISH).

"Cytogenetic testing" includes the examination of DNA within cells, particularly chromosomes. Cytogenetic testing can be used to identify DNA changes that may be associated with the presence of refractory and/or recurrent cancers. Such changes may include: deletions of the long arm of chromosome 13, and/or deletions at the 13q14.1 chromosome position, and/or trisomy of chromosome 12, and/or deletions of the long arm of chromosome 12, and/or deletions of the long arm of chromosome 11, and/or deletions of 11q, and/or deletions of the long arm of chromosome 6, and/or deletions of 6q, and/or deletions of the short arm of chromosome 17, and/or deletions of 17p, and/or t(11:14)... Translocations, 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.

It is known that patients with cancer exhibit certain physical symptoms, which are often a result of the burden the cancer places on the body. These symptoms frequently recur in the same cancer and can therefore be characteristic of the disease's diagnosis, and/or prognosis, and/or progression. Technicians in the medical field will understand which physical symptoms are associated with which cancers and how to assess which body systems may be relevant to the diagnosis, and/or prognosis, and/or progression of the disease. "Physical symptoms" include hepatomegaly and/or splenomegaly.

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

In this context, breast cancer includes metastatic breast cancer (MBC) and early-stage breast cancer (EBC).

In this context, gastric cancer can also refer to gastric adenocarcinoma or stomach cancer, and includes gastroesophageal junction (GEJ) adenocarcinoma. It further includes metastatic gastric cancer (MGC) and metastatic GEJ adenocarcinoma.

Trastuzumab is currently used alone or in combination with chemotherapy or other drugs to treat HER2-expressing breast cancer, and such treatment has significantly improved overall survival. However, many patients are still not cured. Other patients develop trastuzumab resistance, leading to disease recurrence, and it has also been shown that some HER2-positive breast cancers may become HER2-negative or low in HER2 expression over time. Therefore, there is an urgent need to improve anti-HER2 therapies and ways to overcome resistance in order to cure more patients.

In some embodiments, the FcγRIIB-negative cancer to be treated according to the present invention is cancer with low HER2 expression. Patients with cancer with low HER2 expression typically do not respond or respond poorly to standard care treatments, such as treatment with trastuzumab and/or trastuzumab biosimilars. However, treatment of cancer with low HER2 expression becomes possible, as described herein, by combining with antibody molecules that specifically bind to FcγRIIB via their Fab region and lack an Fc region or have reduced binding to the Fcγ receptor via their Fc region.

To determine whether cancer has low HER2 expression, standard HER2 assays that are typically performed on biopsies taken from patients can be used, such as assays using immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH).

The IHC test is based on staining for the HER2 protein. When used to determine the amount of HER2 on the surface of cells in a breast cancer tissue sample, it gives a score from 0 to 3+. A score of 0 to 1+ indicates HER2 negativity. A score of 2+ indicates borderline. A score of 3+ indicates HER2 positivity. In this context, an IHC score of 0 to 1+ for a patient with breast cancer can classify the patient as having cancer with low HER2 expression. In some embodiments, a score of 0 to 1+ is considered to indicate HER2-low expression cancer. In some embodiments, a score of 3+ is considered to indicate HER2-high expression cancer, i.e., not HER2-low expression cancer according to the invention. In the following examples, a well-developed HER2-expressing immunomodulatory Balb/C TUBO breast cancer tumor model was used. This model was adapted to allow assessment of the ability of Fc:FcγR-impaired (Fc silencing) anti-FcγRIIB antibody to enhance anti-HER2 antitumor activity against cancers with low HER2 expression (HER2 low) and (for comparison) also against cancers with high HER2 expression (HER2 high). Therefore, in HER2-overexpressing tumor models, animals received a full therapeutic dose of anti-HER2 antibodies, which resulted in strong occupancy of HER2 expressed in the tumor. In HER2-underexpressing models, animals received lower doses of antibodies, resulting in approximately 10-fold lower HER2 receptor occupancy on cancer cells targeted by the anti-HER2 antibodies, as demonstrated by flow cytometry analysis of tumors harvested from mice treated with fluorescently conjugated anti-HER2 antibodies. Thus, all other factors except antibody targeting of the HER2 receptor are identical, making this tumor model system ideal for: assessing and demonstrating enhanced anti-FcγRIIB-mediated efficacy against HER2-overexpressing cancers, and achieving anti-FcγRIIB-mediated efficacy against HER2-underexpressing cancers.

FISH, based on the HER2 marker, is more accurate than IHC, but it is more expensive and takes longer to return results. This is why IHC testing is often the first test performed to check if cancer is HER2-positive. With FISH testing, you get a positive or negative score (some hospitals refer to a negative test result as "zero").

These two tests can be combined; for example, if the IHC test result is borderline, it can be combined with the FISH test to provide a better basis for determining whether the cancer is HER2-positive. For example, IHC can be used...

Generally speaking, only cancers that test positive for IHC 3+ or FISH will respond to standard care treatment with HER2-targeting drugs.

When the target of the second antibody molecule is HER2, the second antibody molecule can be trastuzumab or a trastuzumab biosimilar, such as trastuzumab-ANNS, trastuzumab-QYYP, trastuzumab-PKRB, trastuzumab-DTTB, or trastuzumab-DKST. When referring to trastuzumab biosimilars, we mean here an antibody molecule that is highly similar to trastuzumab and has no clinically significant differences. Alternatively, the second antibody molecule can be an enhanced variant of trastuzumab or a trastuzumab biosimilar conjugated with a toxin, such as fam-trastuzumab-drutecan-NXKI or T-DM1, or ado-trastuzumab-emtansine or other FcγR-conjugated anti-HER2 antibody drug conjugates.

In other cases, the second antibody can be used in conjunction with a third antibody, which can be a tumor-directing agent (e.g., the anti-HER2 antibody pertuzumab) or an immunomodulatory agent (e.g., an anti-PD-1/PD-L1 antibody). Furthermore, the second antibody can be any anti-HER2 antibody used in any treatment regimen containing anti-HER2.

In some embodiments, the FcγRIIB-negative cancer to be treated according to the invention is cancer in patients who have previously been successfully treated with trastuzumab and/or trastuzumab biosimilars but have subsequently developed resistance to trastuzumab or trastuzumab biosimilars and therefore no longer respond to such treatments. Cancer with low HER2 expression. As described herein, overcoming such resistance is possible through combinations with antibody molecules that specifically bind to FcγRIIB via their Fab region and lack an Fc region or have reduced binding to the Fcγ receptor via their Fc region.

In some embodiments, the antibody molecule that specifically binds to a receptor present on tumor cells targets the human epidermal growth factor receptor (EGFR). In such embodiments, the FcγRIIB-negative cancer to be treated can be cancer selected from the group consisting of head and neck cancer and colorectal cancer.

In this context, head and neck cancer includes locally or regional advanced squamous cell carcinoma of the head and neck, recurrent locally or metastatic squamous cell carcinoma of the head and neck, and recurrent or metastatic squamous cell carcinoma of the head and neck.

In this context, colorectal cancer includes metastatic colorectal cancer that expresses EGFR in the K-Ras wild-type variant.

When the target of the second antibody molecule is EGFR, the second antibody molecule can be cetuximab or a cetuximab biosimilar. When referring to cetuximab biosimilars, we mean antibody molecules that are highly similar to cetuximab and have no clinically significant differences.

Each of the aforementioned cancers is known, and its symptoms and diagnostic markers are well described, as are the therapeutic agents used to treat those cancers. Therefore, the symptoms, cancer diagnostic markers, and therapeutic agents used to treat the aforementioned cancer types will be known to those skilled in the medical field.

In some embodiments, an antibody molecule that specifically binds to FcγRIIB and an antibody molecule that specifically binds to a receptor present on tumor cells are administered to the patient simultaneously, meaning that they are either administered together at once or separately at very close intervals.

In some embodiments, an antibody molecule specifically binding to FcγRIIB is administered to the patient prior to the administration of an antibody molecule that specifically binds to a receptor present on tumor cells. Such sequential administration can be achieved through temporal separation of the two antibodies. Alternatively, or in combination with the first option, sequential administration can also be achieved through spatial separation of the two antibody molecules by: administering the antibody molecule specifically binding to FcγRIIB in a manner such that it reaches the cancer prior to the antibody molecule specifically binding to a receptor present on tumor cells, and then administering the antibody molecule specifically binding to a receptor present on tumor cells in a manner such that it reaches the cancer after the antibody molecule specifically binding to FcγRIIB.

In some embodiments, an antibody molecule that specifically binds to a receptor present on tumor cells is administered to the patient prior to the administration of an antibody molecule that specifically binds to FcγRIIB. Such sequential administration can be achieved as described above.

Those skilled in the medical field will know that drugs can be modified with different additives, for example, to change the rate at which the drug is absorbed by the body; and can be modified in different ways, for example, to allow for specific routes of administration to the body.

Therefore, we include the following: the compositions, and/or antibodies, and/or pharmaceuticals of the present invention can be combined with excipients, and/or pharmaceutically acceptable carriers, and/or pharmaceutically acceptable diluents and/or adjuvants.

We also include the following: the compositions, and/or antibodies, and/or pharmaceuticals of the present invention are suitable for parenteral administration, including aqueous and/or non-aqueous sterile injectable solutions containing antioxidants, and/or buffers, and/or antibacterial agents, and/or solutes that make 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 thickeners. The compositions, and/or antibodies, and/or pharmaceuticals of the present invention may be present in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored under lyophilized (i.e., freeze-dried) conditions where only a sterile liquid carrier (e.g., water for injection) needs to be added immediately before use.

Temporary injection solutions and suspensions can be prepared from the aforementioned types of sterile powders, and/or granules, and/or tablets.

For parenteral administration to human patients, the daily dose level of antibody molecules that specifically bind to FcγRIIB and/or antibody molecules that specifically bind to receptors present on tumor cells is typically 1 mg/kg of patient body weight to 20 mg/kg, or in some cases even up to 100 mg/kg, administered as a single dose or in divided doses. Lower doses may be used in special cases, for example, in combination with long-acting administration. In any case, the physician will determine the actual dose that will be most appropriate for any individual patient, and this actual dose will vary depending on the specific patient's age, weight, and response. The above doses are exemplary general cases. Of course, there may be individual cases in which higher or lower dose ranges are required, and this is within the scope of the invention.

Generally, in humans, oral or parenteral administration of the compositions, and/or antibodies, and/or pharmaceuticals, and/or medicaments of the present invention is the preferred route, with parenteral administration being the most common for antibodies. For veterinary use, the compositions, and/or antibodies, and/or pharmaceuticals, and/or medicaments of the present invention are administered as suitably acceptable formulations according to normal veterinary practice, and the veterinarian will determine the dosing regimen and route of administration that will be most appropriate for a particular animal. Therefore, the present invention provides a pharmaceutical formulation comprising an amount of the antibodies and/or pharmaceuticals of the present invention that is effective in treating a variety of conditions (as described above and further below). Preferably, the compositions, and/or antibodies, and/or pharmaceuticals, and/or medicaments are suitable for delivery via a route selected from the group consisting of: intravenous (IV); subcutaneous (SC), intramuscular (IM), or intratumoral administration.

In some embodiments, a first antibody molecule, or a second antibody, or both, may be administered using a plasmid or a virus. Such a plasmid then contains a nucleotide sequence encoding the first antibody molecule, or the second antibody, or both. In some embodiments, the nucleotide sequence encoding a portion or the entire sequence of the first antibody molecule, or the second antibody, or both, is integrated into the genome of a cell or virus or into a virome within a virus; such a cell or virus then acts as a delivery medium for the first antibody molecule, or the second antibody, or both (or a delivery medium for the nucleotide sequence encoding the first antibody molecule, or the second antibody, or both). For example, in some embodiments, such viruses 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 oncolytic viruses contain a nucleotide sequence encoding a full-length human IgG antibody. Oncolytic viruses are known to those skilled in the art of medicine and virology.

This invention also includes compositions, and/or antibodies, and/or pharmaceutical preparations, and/or drugs comprising pharmaceutically acceptable acid or base addition salts of polypeptide-binding moieties of the invention. The acid used to prepare the pharmaceutically acceptable acid addition salt of the aforementioned base compounds usable in this invention is one that forms a non-toxic acid addition salt (i.e., a salt containing a pharmaceutically acceptable anion, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogen sulfate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, hydrogen tartrate, succinate, maleate, fumarate, gluconate, sucrose, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and bis(hydroxynaphthyl)ate [i.e., 1,1'-methylene-bis(2-hydroxy-3-naphthyl)ate] salt, etc.). Pharmaceutically acceptable base addition salts can also be used to produce pharmaceutically acceptable salt forms of pharmaceutical preparations according to the invention. The chemical bases of pharmaceutically acceptable base salts that can be used as reagents to prepare the inherently acidic pharmaceutical agents of the present invention are those chemical bases that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to, those derived from such pharmaceutically 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-methylglucosamine-(glucamine)), and other base salts of lower alkanol ammonium and pharmaceutically acceptable organic amines. The pharmaceutical agents and/or peptide-binding portions of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilization method (e.g., spray drying, filter cake drying) and/or reconstitution technique can be employed. Those skilled in the art will understand that lyophilization and reconstitution can cause varying degrees of loss of antibody activity (e.g., for conventional immunoglobulins, IgM antibodies tend to have a greater loss of activity than IgG antibodies), and the level of use may need to be upregulated to compensate for this. In one embodiment, the lyophilized (freeze-dried) polypeptide-binding moiety loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (before lyophilization) upon rehydration.

Attached Figure Description

The following figures are referenced in the following examples:

Figure 1. Figures 1A to 1B show the survival curves. Female BalbC mice (n = 12) were subcutaneously injected with TUBO cells (1 x ^10⁶ ). Tumor growth was monitored (measured by calipers), and mice were randomized and treated twice weekly as instructed when the tumor reached approximately 7 x 7 mm. Tumor growth was tracked twice weekly until the tumor reached a predetermined size (ethical endpoint), at which point the mice were euthanized. Figure 1A. HER2-positive cancer model. The efficacy of anti-HER2 (10 mg/kg) in combination with Fc null anti-FcγRIIB-NA (AT130-2NA) was compared with that of the isotype control antibody (FITC IgG2a) and with anti-HER2 (10 mg/kg) monotherapy. Mice were administered the drug three times (with intervals of 2 to 3 days between administrations). In this HER2-prone cancer model, combination treatment with anti-FcγRIIB-NA (AT130-2NA) delayed tumor growth and increased the number of complete responders compared to anti-HER2 monotherapy. The study was repeated three times, and the results are comparable; results from one representative experiment are shown in Figure 1A. Figure 1B shows the HER2-prone cancer model. This figure illustrates the therapeutic effect of anti-HER2 (1 mg/kg) in combination with FcγRIIB-NA (AT130-2NA) compared to the isotype control antibody (FITC IgG2a) and anti-HER2 at 1 mg/kg as a monotherapy. Mice were administered the drug three times (with intervals of 2 to 3 days between administrations). In this HER2-low cancer model, combined treatment with anti-FcγRIIB-NA (AT130-2NA) delayed tumor growth and increased the number of complete responders compared to anti-HER2 treatment alone, making anti-HER2 treatment in HER2-low cancers as effective as (single-agent anti-HER2) treatment in HER2-high cancers. Figure 1C shows the targeting of the HER2 receptor in experimental HER2-high and HER2-low cancer models. Tumors were established as described in A-B, and mice (n=3) were treated with 1 mg/kg or 10 mg/kg of fluorescently labeled (AF647) anti-HER2. Mice were administered the drug twice (with an interval of 2-3 days between administrations), and subsequently euthanized, and tumors were collected. Tumors were enzymatically digested, and the fluorescently labeled (AF647) anti-HER2 was quantified by FACS. Tumors from mice in a HER2-low model (injected with 1 mg/kg anti-HER2) showed a 10-fold increase in targeting the HER2 receptor compared to tumors from mice in a HER2-high model (injected with 10 mg/kg anti-HER2).

Figure 2. Survival curves. Female BalbC mice (n=12) were subcutaneously injected with TUBO cells ( ^1x10⁶ ). Tumor growth was monitored (measured with calipers), and mice were randomized to receive a therapeutic mAb twice weekly when the tumor reached approximately 7x7 mm. Tumor growth was tracked twice weekly until the tumor reached a predetermined size (ethical endpoint), at which point the mice were euthanized. The efficacy of anti-HER2 therapy in combination with Fc null anti-FcγRIIB-NA (AT130-2NA) (1 mg/kg) was compared with anti-HER2 therapy in combination with wild-type anti-FcγRIIB (AT130-2 wt), with an isotype control antibody (FITC IgG2a), and with 1 mg/kg anti-HER2 therapy as a single treatment. Mice were administered the drug three times (with intervals of 2 to 3 days between administrations). Compared with anti-HER2 treatment alone, anti-HER2 combined with anti-FcγRIIB-NA (AT130-2NA) showed delayed tumor growth. When anti-HER2 was combined with wild-type anti-FcγRIIB (AT130-2 wt), no delay in tumor growth was observed.

Figure 3. Subcutaneous injection of TUBO cells ( ^1x10⁶ ) into female BalbC mice. Tumor growth was monitored (measured with calipers), and mice were randomized to receive therapeutic mAbs twice weekly when the tumor reached approximately 7x7 mm. Mice were sacrificed and tumors harvested 24 hours after three injections, on days 7–8 following treatment. The immune cell content of tumor single-cell suspensions was analyzed by FACS. The Fc-null anti-FcγRIIB-NA was designated AT-130-2NA in the figure. In the group treated with the combination of anti-HER2 and anti-FcγRIIB-NA, the number of bone marrow cells, particularly those with ^low CD11b+F4/80+/MHCII, was significantly increased.

Figure 4. Lung region covered by metastases. Female C57 mice were intravenously injected with B16 cells (5 x ^10⁵ ). Four days after tumor cell injection, mice were injected with antibodies (10 mg/kg intraperitoneal-isotype control, TA99, AT130-2-NA, and a combination of TA99 and AT130-2-NA). Five treatments were administered at 2-3 day intervals. On day 21 after treatment initiation, mice were sacrificed and the amount of metastases in the lungs was quantified. A reduction in lung metastases was observed with TA99 alone; however, the effect of TA99 was greatly increased when combined with anti-FcγRIIB-NA (AT130-2NA). Anti-FcγRIIB-NA as a monotherapy had no therapeutic effect.

Example

Specific, non-limiting examples embodying certain aspects of the invention will now be described. To allow examination of the blocking effect of FcγRIIB in complex in vivo systems, two alternative antibody sets have been used. The mouse equivalent of the Fc-active antibody 6G11 is referred to as AT130-2. To silence the human antibody Fc (thus severely impairing or negligibly impairing binding to FcγR), amino acid position 297 has been replaced from N to Q. To silence the mouse antibody Fc, the same position has been replaced from N to Q. Thus, in the mouse system, we will refer to AT-130, while this patent application relates to the human counterpart 6G11. In short, human 6G11 corresponds to the mouse alternative AT1302-2 (both are Fc:FcγR proficient, also referred to herein as Fc active), while 6G11-N297Q corresponds to AT130-3-N297A (both are Fc:FcγR impaired, also referred to herein as Fc silent). We have previously shown that these human and mouse Fc:FcγR proficient or Fc:FcγR impaired anti-FcγRIIB antibodies are functionally and biochemically equivalent (WO 2019/138005 and WO 2021/009358).

Different methods for silencing antibody Fc (and are well known to those skilled in the art) would be to remove the Fc portion and form a Fab or F(ab') ₂ fragment.

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

The alternative anti-mouse FcγRIIB mAb AT130-3-N297A improves the in vivo anti-tumor effect of anti-HER2 mAb. And it enables the treatment of cancers with low HER2 expression.

Therapeutic effects in the TUBO tumor model (HER2-overexpressing cancer model)

To evaluate the in vivo antitumor effect of the combination with the anti-HER2 mAb against mouse FcγRIIB mAb AT130-3-N297A, the combination was studied in vivo in the TUBO tumor model as described below.

Mice were fed and maintained at a facility in Lund, Sweden, in accordance with applicable rules and guidelines (including those of the facility and the Swedish Agricultural Council). Six- to eight-week-old female BalbC mice were provided by Taconic (Bomholt, Denmark) and maintained at a local animal facility. TUBO cells (University of Turin) were grown in glutamax-buffered RPMI supplemented with 10% FCS. When the cells were semi-confluent, they were separated with trypsin and resuspended in sterile PBS at 10 x ^10⁶ cells/ml. Glutamax-buffered RPMI (RPMI medium), FCS (fetal bovine serum), and PBS (phosphate-buffered saline) were all from Invitrogen and are used below. Mice were subcutaneously injected with 100 μl of cell suspension corresponding to 1 x ^10⁶ cells/mouse. Twelve to thirteen days after injection, mice were treated twice weekly with intraperitoneal injections of 10 mg/kg antibody (isotype control, anti-HER2, AT130-3-N297A, and a combination of anti-HER2 and AT130-3-N297A), as shown in the figure. Tumors were measured twice weekly until they reached a diameter of 15 mm, after which the mice were sacrificed.

In this experimental HER2-prone cancer model, anti-HER2 FcγRIIB mAbAT130-3-N297A significantly improved anti-HER2-mediated survival compared with single-agent anti-HER2 therapy (Figure 1A).

Anti-FcγRIIB combination treatment achieves anti-HER2 therapeutic effects in cancers with low HER2 expression.

To evaluate the in vivo efficacy of the anti-mouse FcγRIIB mAb AT130-3-N297A in combination with an anti-HER2 mAb against HER2-low-expressing cancers, the same HER2-high TUBO mouse tumor model described above was used, but at a lower dose of the antibody, resulting in less HER2 receptor occupation and targeting on cancer cells by the anti-HER2 antibody. Thus, all other factors except antibody targeting of the HER2 receptor were identical, making this tumor model ideal for evaluating and demonstrating the anti-HER2 efficacy of anti-FcγRIIB against HER2-low-expressing cancers.

Mice were fed and maintained as described above. Six- to eight-week-old female BalbC mice were provided by Taconic (Bomholt, Denmark) and maintained in a local animal facility. TUBO cells (University of Turin) were grown in glutamax-buffered RPMI supplemented with 10% FCS. When the cells were semi-confluent, they were separated with trypsin and resuspended in sterile PBS at 10 x ^10⁶ cells/ml. Mice were subcutaneously injected with 100 μl of cell suspension corresponding to ¹ x 10⁶ cells/mouse. Mice were treated twice weekly with intraperitoneal injections of 1 mg/kg antibody (isotype control, anti-HER2, and a combination of anti-HER2 and AT130-3-N297A) for 12 to 13 days post-injection, as shown in the figure. Tumors were measured twice weekly until they reached a diameter of 15 mm, after which the mice were euthanized.

In this experimental HER2-low model, anti-HER2 monotherapy showed severely impaired therapeutic efficacy, with only 1/10 of treated animals cured compared to 3/10 in the HER2-high cancer model. Combining this with the anti-mouse FcγRIIB mAb AT130-3-N297A, which has no intrinsic antitumor activity, resulted in complete therapeutic efficacy in the HER2-low tumor model (3/10 mice cured) similar to that observed in the HER2-high model (Figure 1B). Next, experiments were designed to compare the amount of HER2 targeting in the HER2-high and HER2-low models. Tumors were established as described in A-B, and mice (n=3) were treated with 1 mg/kg or 10 mg/kg of Alexa Flour (AF)647-labeled anti-HER2 to determine antibody targeting of HER2 in the HER2-high and HER2-low cancer models. Mice were administered the drug twice (with an interval of 2 to 3 days between administrations). Two days after the second injection, mice were euthanized and tumors were collected. The tumors were cut into small pieces and enzymatically digested at 37°C using a mixture of DNase and Liberase. The tumor solution was further filtered through a cell filter to obtain a single-cell solution. Anti-HER2 markers labeled with the fluorescent dye (AF647) in the tumors were quantified by FACS. Tumor cells from a HER2-high cancer model (injected with 10 mg/kg anti-HER2 in mice) showed a 10-fold increase in targeting the HER2 receptor compared to tumor cells from a HER2-low cancer model (injected with 1 mg/kg anti-HER2 in mice).

There was no therapeutic effect when Fc-active AT130-2 was combined with anti-HER2 mAb.

To assess whether Fc activity AT130-2 also improves the in vivo antitumor effect of anti-HER2 mAb, in vivo studies of the combination were conducted in tumor models, as described below.

Mice were fed and maintained as described above. Six- to eight-week-old female BalbC mice were provided by Taconic (Bomholt, Denmark) and maintained in a local animal facility. TUBO cells (University of Turin) were grown in glutamax-buffered RPMI supplemented with 10% FCS. When the cells were semi-confluent, they were separated with trypsin and resuspended in sterile PBS at 10 x ^10⁶ cells/ml. Mice were subcutaneously injected with 100 μl of cell suspension corresponding to ¹ x 10⁶ cells/mouse. Mice were treated twice weekly with intraperitoneal injections of 10 mg/kg antibody (isotype control, anti-HER2, or a combination of anti-HER2 and AT130-2-N297A or AT130-2 wt) for 12 to 13 days post-injection, as shown in the figure. Tumors were measured twice weekly until they reached a diameter of 15 mm, after which the mice were euthanized.

Fc:FcγR Breast (wt) AT130-2 therefore did not show improved therapeutic antitumor effects when combined with anti-HER2 (Figure 2).

The improved therapeutic effect of antiHER2 therapy combined with anti-mouse FcγRIIB mAb AT130-3-N297A was associated with increased influx of myeloid cells into the tumor.

To evaluate the mechanism of action of the combination of anti-HER2 therapy and anti-mouse FcγRIIB mAb AT130-2-N297A, immunoanalysis was performed on the treated tumors, as described below.

Mice were fed and maintained as described above. Six- to eight-week-old female BalbC mice were provided by Taconic (Bomholt, Denmark) and maintained in a local animal facility. TUBO cells (University of Turin) were grown in glutamax-buffered RPMI supplemented with 10% FCS. When the cells were semi-confluent, they were separated with trypsin and resuspended in sterile PBS at 10 x ^10⁶ cells/ml. Mice were subcutaneously injected with 100 μl of cell suspension corresponding to ¹ x 10⁶ cells/mouse. Once the tumors reached approximately 7 x 7 mm in size, mice were injected with antibodies (10 mg/kg intraperitoneal—isotype control, anti-HER2, AT130-2-N297A, and a combination of anti-HER2 and AT130-2-N297A). Tumors were harvested 24 hours after three injections, on days 7 to 8 after the start of treatment (when tumors in the combination groups showed significant regression).

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

As shown in Figure 3, the anti-HER2/anti-FcγRIIB-NA combination altered the composition of immune cells in tumors. Compared to single treatment, the combination treatment with anti-HER2 and anti-FcγRIIB-NA resulted in an increased CD11b+/F4/80+ population, consistent with increased recruitment of effector cells and increased antibody-mediated depletion of HER2-targeting tumor cells. This increase was most pronounced in the HER2-high model (mice were administered a 10 mg/kg anti-HER2 dose) (Figure 3).

B16 lung metastasis model

To assess whether anti-FcγRIIB-NA can enhance the depletion activity and therapeutic efficacy of other tumor-direct-targeting therapeutic antibodies targeting solid tumors, we investigated the therapeutic effect of combining anti-FcγRIIB-NA with TA99 (an antibody specific to the gp75 melanoma tumor antigen) in a B16 metastatic melanoma model.

Mice were fed and maintained as described above. Six- to eight-week-old female C57 mice were provided by Taconic (Bomholt, Denmark) and maintained in a local animal facility. B16 cells (ATCC) were grown in glutamax-buffered RPMI supplemented with 10% FCS. When the cells were semi-confluent, they were separated with trypsin and resuspended in sterile PBS at 2.5 x ^10⁶ cells/ml. Mice were intravenously injected with 200 μl of cell suspension corresponding to ⁵ x 10⁵ cells/mouse. Four days after tumor cell injection, mice were injected with antibodies (10 mg/kg intraperitoneal-isotype control, TA99, AT130-2-N297A, and a combination of TA99 and AT130-2-N297A). Five treatments were administered at 2 to 3-day intervals. On day 21 after the start of treatment, mice were euthanized and the amount of metastatic tumors in the lungs was quantified. Figure 4

Compared to untreated animals, a moderate reduction in lung metastases was observed after treatment with TA99 alone (Figure 4). The therapeutic effect of the tumor-directing antibody TA99 was significantly enhanced after combination treatment with anti-FcγRIIB-NA (which itself has no effect on metastasis formation), resulting in a significant reduction in lung metastases compared to TA99 monotherapy. Therefore, combination treatment with anti-FcγRIIB-NA enhances the therapeutic efficacy of different tumor-directing antibodies specific to different tumor antigens associated with different FcγRIIB solid cancers.

Claims (30)

1. A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to an Fcγ receptor via its Fc region, the first antibody molecule being used in combination with a second antibody molecule in the treatment of FcγRIIB-negative cancer in a patient, the second antibody molecule specifically binding to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor. 2. A pharmaceutical composition comprising: (i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and (ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor; The pharmaceutical composition is intended for use in the treatment of FcγRIIB-negative cancers in patients. 3. A kit for use in the treatment of FcγRIIB-negative cancers, 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 the Fcγ receptor via its Fc region, and (ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor. 4. The following items: (i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and (ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region that binds to at least one activated Fcγ receptor; Use in the manufacture of a medicine for the treatment of FcγRIIB-negative cancers in patients. 5. A method for treating FcγRIIB-negative cancer in a patient, comprising administering: (i) A first antibody molecule that specifically binds to FcγRIIB via its Fab region and lacks an Fc region or has reduced binding to the Fcγ receptor via its Fc region, and (ii) A second antibody molecule that specifically binds to a receptor present on tumor cells, the second antibody molecule having an Fc region capable of activating at least one activated Fcγ receptor. 6. The first antibody molecule for use in combination with the second antibody molecule according to claim 1, the pharmaceutical composition for use according to claim 2, the kit for use according to claim 3, the use according to claim 4, or the method according to claim 5, wherein the FcγRIIB-negative cancer is a solid cancer. 7. The first antibody molecule for use in combination with the second antibody molecule according to claim 1 or 6, the pharmaceutical composition for use according to claim 2 or 6, the kit for use according to claim 3 or 6, the use according to claim 4 or 6, or the method according to claim 5 or 6, wherein the binding of the second antibody molecule to the receptor on the tumor cells causes depletion of the tumor cells. 8. The first antibody molecule for use in combination with a second antibody molecule according to claim 1, 6 or 7, the pharmaceutical composition for use according to claim 2, 6 or 7, the kit for use according to claim 3, 6 or 7, the use according to claim 4, 6 or 7, or the method according to claim 5, 6 or 7, wherein the first antibody lacks an Fc region. 9. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 8, a pharmaceutical composition for use according to any one of claims 21 or 6 to 8, a kit for use according to any one of claims 3 or 6 to 8, use according to any one of claims 4 or 6 to 8, or a method according to any one of claims 5 or 6 to 8, wherein the second antibody molecule binds to human epidermal growth factor receptor 2 (HER2). 10. The first antibody molecule for use in combination with the second antibody molecule according to claim 9, the pharmaceutical composition for use according to claim 9, the kit for use according to claim 8, the use according to claim 8, or the method according to claim 9, wherein the cancer is selected from the group consisting of breast cancer and gastric cancer. 11. The first antibody molecule for use in combination with a second antibody molecule according to claim 9 or 10, the pharmaceutical composition for use according to claim 9 or 10, the kit for use according to claim 9 or 10, the use according to claim 9 or 10, or the method according to claim 9 or 10, wherein the cancer has low HER2 expression. 12. The first antibody molecule for use in combination with the second antibody molecule according to any one of claims 9 to 11, the pharmaceutical composition for use according to any one of claims 9 to 11, the kit for use according to any one of claims 9 to 11, the use according to any one of claims 9 to 11, or the method according to any one of claims 9 to 11, wherein the cancer is cancer in a patient who has previously been treated with an antibody molecule that specifically binds to HER2 but has developed resistance to that antibody. 13. The first antibody molecule for use in combination with the second antibody molecule according to any one of claims 9 to 12, the pharmaceutical composition for use according to any one of claims 9 to 12, the kit for use according to any one of claims 9 to 12, the use according to any one of claims 9 to 12, or the method according to any one of claims 9 to 12, wherein the second antibody molecule is trastuzumab or a trastuzumab biosimilar. 14. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 8, a pharmaceutical composition for use according to any one of claims 21 or 6 to 8, a kit for use according to any one of claims 3 or 6 to 8, use according to any one of claims 4 or 6 to 8, or a method according to any one of claims 5 or 6 to 8, wherein the second antibody molecule binds to human epidermal growth factor receptor (EGFR). 15. The first antibody molecule for use in combination with the second antibody molecule according to claim 14, the pharmaceutical composition for use according to claim 14, the kit for use according to claim 14, the use according to claim 14, or the method according to claim 14, wherein the cancer is selected from the group consisting of head and neck cancer and colorectal cancer. 16. The first antibody molecule for use in combination with a second antibody molecule according to claim 14 or 15, the pharmaceutical composition for use according to claim 14 or 15, the kit for use according to claim 14 or 15, the use according to claim 14 or 15, or the method according to claim 14 or 15, wherein the second antibody molecule is cetuximab or a cetuximab biosimilar. 17. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 16, a pharmaceutical composition for use according to any one of claims 2 or 6 to 16, a kit for use according to any one of claims 3 or 6 to 16, use according to any one of claims 4 or 6 to 16, or a method according to any one of claims 5 or 6 to 16, wherein the first antibody molecule is selected from the group consisting of: human antibody molecules, humanized antibody molecules, and human-derived antibody molecules. 18. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 17, a pharmaceutical composition for use according to any one of claims 2 or 6 to 17, a kit for use according to any one of claims 3 or 6 to 17, use according to any one of claims 4 or 6 to 17, or a method according to any one of claims 5 or 6 to 17, wherein the first antibody molecule is a monoclonal antibody molecule or an antibody molecule of monoclonal origin. 19. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 18, a pharmaceutical composition for use according to any one of claims 2 or 6 to 18, a kit for use according to any one of claims 3 or 6 to 18, use according to any one of claims 4 or 6 to 18, or a method according to any one of claims 5 or 6 to 18, wherein the first antibody molecule is selected from the group consisting of: full-length antibody, chimeric antibody, single-chain antibody, Fab fragment, (Fab') ₂ fragment, Fab' fragment, (Fab') ₂ fragment, Fv fragment, and scFv fragment. 20. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 18, a pharmaceutical composition for use according to any one of claims 2 or 6 to 18, a kit for use according to any one of claims 3 or 6 to 18, use according to any one of claims 4 or 6 to 18, or a method according to any one of claims 5 or 6 to 18, wherein the first antibody molecule is a human IgG antibody molecule having a deglycosylated Fc region or a human-derived IgG antibody molecule having a deglycosylated Fc region. 21. The first antibody molecule for use in combination with a second antibody molecule according to claim 20, the pharmaceutical composition for use according to claim 20, the kit for use according to claim 20, the use according to claim 20, or the method according to claim 20, wherein the IgG antibody molecule is an IgG1 or IgG2 antibody molecule. 22. The first antibody molecule for use in combination with a second antibody molecule according to claim 21, the pharmaceutical composition for use according to claim 21, the kit for use according to claim 21, the use according to claim 21, or the method according to claim 21, wherein the IgG antibody molecule is deglycosylated human IgG1, or deglycosylated humanized mouse antibody, or deglycosylated humanized alpaca hcIgG antibody, or deglycosylated chimeric mouse IgG. 23. The first antibody molecule for use in combination with a second antibody molecule according to claim 22, the pharmaceutical composition for use according to claim 22, the kit for use according to claim 22, the use according to claim 22, or the method according to claim 22, wherein it has been deglycosylated by amino acid substitution at position 297. 24. The first antibody molecule for use in combination with a second antibody molecule according to claim 23, the pharmaceutical composition for use according to claim 23, the kit for use according to claim 23, the use according to claim 23, or the method according to claim 23, wherein it has been deglycosylated by N297Q substitution. 25. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 24, a pharmaceutical composition for use according to any one of claims 2 or 6 to 24, a kit for use according to any one of claims 3 or 6 to 24, use according to any one of claims 4 or 6 to 24, or a method according to any one of claims 5 or 6 to 24, wherein the first antibody molecule comprises: (i) Variable heavy chains (VH) containing the following CDRs: SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53, and Variable light chains (VLs) containing the following CDRs: SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; (ii) VHs containing the following CDRs: SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, and VLs containing the following CDRs: SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:62; (iii) VHs containing the following CDRs: SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65, and VLs containing the following CDRs: SEQ ID NO:66, SEQ ID NO:67, and SEQ ID NO:68; (iv) VHs containing the following CDRs: SEQ ID NO:69, SEQ ID NO:70, and SEQ ID NO:71, and VLs containing the following CDRs: SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74; (v) VHs containing the following CDRs: SEQ ID NO:75, SEQ ID NO:76, and SEQ ID NO:77, and VLs containing the following CDRs: SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80; (vi) VHs containing the following CDRs: SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83, and VLs containing the following CDRs: SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86; (vii) VHs containing the following CDRs: SEQ ID NO:87, SEQ ID NO:88, and SEQ ID NO:89, and VLs containing the following CDRs: SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:92; (viii) VHs containing the following CDRs: SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95, and VLs containing the following CDRs: SEQ ID NO:96, SEQ ID NO:97, and SEQ ID NO:98; (ix) VHs containing the following CDRs: SEQ ID NO:99 and SEQ ID NO:100 and SEQ ID NO:101, and VLs containing the following CDRs: SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104; (x) VHs containing the following CDRs: SEQ ID NO:105, SEQ ID NO:106, and SEQ ID NO:107, and VLs containing the following CDRs: SEQ ID NO:108, SEQ ID NO:109, and SEQ ID NO:110; (xi) VHs containing the following CDRs: SEQ ID NO:111, SEQ ID NO:112, and SEQ ID NO:113, and VLs containing the following CDRs: SEQ ID NO:114, SEQ ID NO:115, and SEQ ID NO:116; (xii) VHs containing the following CDRs: SEQ ID NO:117, SEQ ID NO:118, and SEQ ID NO:119, and VLs containing the following CDRs: SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122; (xiii) VHs containing the following CDRs: SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125, and VLs containing the following CDRs: SEQ ID NO:126, SEQ ID NO:127, and SEQ ID NO:128; (xiv) contains VHs with the following CDRs: SEQ ID NO:129, SEQ ID NO:130, and SEQ ID NO:131, and VLs containing the following CDRs: SEQ ID NO:132, SEQ ID NO:133, and SEQ ID NO:134; (xv) VHs containing the following CDRs: SEQ ID NO:135, SEQ ID NO:136, and SEQ ID NO:137, and VLs containing the following CDRs: SEQ ID NO:138, SEQ ID NO:139, and SEQ ID NO:140; (xvi) VHs containing the following CDRs: SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, and VLs containing the following CDRs: SEQ ID NO:144, SEQ ID NO:145, and SEQ ID NO:146; (xvii) contains VHs with the following CDRs: SEQ ID NO:147, SEQ ID NO:148, and SEQ ID NO:149, and VLs containing the following CDRs: SEQ ID NO:150, SEQ ID NO:151, and SEQ ID NO:152; (xviii) VHs containing the following CDRs: SEQ ID NO:153, SEQ ID NO:154, and SEQ ID NO:155, and VLs containing the following CDRs: SEQ ID NO:156, SEQ ID NO:157, and SEQ ID NO:158; (xix) contains VHs with the following CDRs: SEQ ID NO:159, SEQ ID NO:160, and SEQ ID NO:161, and VLs containing the following CDRs: SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164; (xx) VHs containing the following CDRs: SEQ ID NO:165, SEQ ID NO:166, and SEQ ID NO:167, and VLs containing the following CDRs: SEQ ID NO:168, SEQ ID NO:169, and SEQ ID NO:170; (xxi) contains VHs with the following CDRs: SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173, and VLs containing the following CDRs: SEQ ID NO:174, SEQ ID NO:175, and SEQ ID NO:176; (xxii) VHs containing the following CDRs: SEQ ID NO:177, SEQ ID NO:178, and SEQ ID NO:179, and VLs containing the following CDRs: SEQ ID NO:180, SEQ ID NO:181, and SEQ ID NO:182; (xxiii) VHs containing the following CDRs: SEQ ID NO:183, SEQ ID NO:184, and SEQ ID NO:185, and VLs containing the following CDRs: SEQ ID NO:186, SEQ ID NO:187, and SEQ ID NO:188; or (xxiv) VHs containing the following CDRs: SEQ ID NO:189, SEQ ID NO:190, and SEQ ID NO:191, and VLs containing the following CDRs: SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194. 26. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 to 25, a pharmaceutical composition for use according to any one of claims 2 to 25, a kit for use according to any one of claims 3 to 25, use according to any one of claims 4 to 25, or a method according to any one of claims 5 to 25, wherein the first antibody molecule comprises: (i) VH having SEQ ID NO:3 and VL having SEQ ID NO:27; (ii) VH having SEQ ID NO:4 and VL having SEQ ID NO:28; (iii) VH having SEQ ID NO:5 and VL having SEQ ID NO:29; (iv) VH having SEQ ID NO:6 and VL having SEQ ID NO:30; (v) VH having SEQ ID NO:7 and VL having SEQ ID NO:31; (vi) VH having SEQ ID NO:8 and VL having SEQ ID NO:32; (vii) VH having SEQ ID NO:9 and VL having SEQ ID NO:33; (viii) VH having SEQ ID NO:10 and VL having SEQ ID NO:34; (ix) VH having SEQ ID NO:11 and VL having SEQ ID NO:35; (x) VH having SEQ ID NO:12 and VL having SEQ ID NO:36; (xi) VH having SEQ ID NO:13 and VL having SEQ ID NO:37; (xii) VH having SEQ ID NO:14 and VL having SEQ ID NO:38; (xiii) VH having SEQ ID NO:15 and VL having SEQ ID NO:39; (xiv) VH having SEQ ID NO:16 and VL having SEQ ID NO:40; (xv) VH having SEQ ID NO:17 and VL having SEQ ID NO:41; (xvi) VH having SEQ ID NO:18 and VL having SEQ ID NO:42; (xvii) has VH with SEQ ID NO:19 and VL with SEQ ID NO:43; (xviii) VH having SEQ ID NO:20 and VL having SEQ ID NO:44; (xix) has VH with SEQ ID NO:21 and VL with SEQ ID NO:45; (xx) has VH with SEQ ID NO:22 and VL with SEQ ID NO:46; (xxi) has VH with SEQ ID NO:23 and VL with SEQ ID NO:47; (xxii) having VH with SEQ ID NO:24 and VL with SEQ ID NO:48; (xxiii) VH having SEQ ID NO:25 and VL having SEQ ID NO:49; or (xxiv) VH having SEQ ID NO:26 and VL having SEQ ID NO:50; 27. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 26; a pharmaceutical composition for use according to any one of claims 2 or 6 to 26; a kit for use according to any one of claims 3 or 6 to 26; use according to any one of claims 4 or 6 to 26; or a method according to any one of claims 5 or 6 to 26. The first antibody molecule comprises: a VH containing the following CDRs: SEQ ID NO:171, SEQ ID NO:172, and SEQ ID NO:173, and a VL containing the following CDRs: SEQ ID NO:174, SEQ ID NO:175, and SEQ ID NO:176. 28. A first antibody molecule for use in combination with a second antibody molecule according to any one of claims 1 or 6 to 27, a pharmaceutical composition for use according to any one of claims 2 or 6 to 27, a kit for use according to any one of claims 3 or 6 to 27, use according to any one of claims 4 or 6 to 27, or a method according to any one of claims 5 or 6 to 27, wherein the first antibody molecule comprises: VH having SEQ ID NO: 23 and VL having SEQ ID NO: 47. 29. The first antibody molecule for use in combination with a second antibody molecule according to claim 27 or 28, the pharmaceutical composition for use according to claim 27 or 28, the kit for use according to claim 27 or 28, the use according to claim 27 or 28, or the method according to claim 27 or 28, wherein the first antibody molecule has: a constant heavy chain (CH) having SEQ ID NO: 195 and a constant light chain (CL) having SEQ ID NO: 2. 30. An antibody molecule specifically binding to FcγRIIB for use, a pharmaceutical composition for use, a kit, an application, or a method thereof, substantially as described herein in the appended claims, specification, examples, and/or drawings.