Antibody

Abstract

In one aspect, the invention relates to immunoglobulin E (IgE) for use in repolarizing macrophages from a first phenotype to an anti-tumor phenotype in treating cancer in a subject, wherein the first phenotype comprises a resting (M0) macrophage phenotype or an anti-inflammatory (M2a) macrophage phenotype, and the anti-tumor phenotype comprises a newly polarized macrophage phenotype characterized by expression of the following cytokines and chemokines: tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin-1 beta (IL-1β); interleukin-6 (IL-6); RANTES or CCL5 (regulated upon activation, expressed and secreted by normal T cells); and / or interleukin-10 (IL-10).

Description

Technical Field

[0001] The present invention relates to the field of cancer treatment, and in particular to antibodies for use in the treatment of a subject's cancer. More specifically, the present invention relates to antibodies and methods for use in the treatment of cancer by reprogramming macrophages to an anti-tumor phenotype.

Background Art

[0002] Therapeutic antibodies currently complement conventional treatments for many malignancies, but almost all substances currently in development rely on only one of the five major human antibody classes, namely IgG, the most abundant antibody class in the blood (Weiner LM et al (2010) Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10: 317-327). The human immune system naturally has nine antibody classes and subclasses (IgM, IgD, IgG1-4, IgA1, IgA2, and IgE) that perform immune surveillance in various anatomical compartments and mediate the destruction of pathogens. However, only IgG (most often IgG1) has been applied to cancer immunotherapy.

[0003] One reason may be that IgG antibodies, particularly IgG1, can account for the largest proportion of antibodies circulating in human blood. Also, the choice of antibody class is based on pioneering studies in the late 1980s comparing a panel of chimeric antibodies of the same specificity, each with an Fc region belonging to one of nine antibody classes and subclasses (Bruggemann M et al (1987) Comparison of the effector functions of human immunoglobulins using a matched set of chimeric antibodies. J Exp Med 166: 1351-1361). Antibodies were evaluated for their ability to bind complement on antigen-expressing target cells in the presence of complement and their ability to mediate hemolysis and cytotoxicity. IgG1 was the most effective IgG subclass in complement-dependent cell killing in vitro, while IgA and IgE antibodies were completely inactive.

[0004] Subsequent clinical trials with antibodies recognizing the B cell marker CD20 supported the inference that IgG1 is the most suitable subclass for immunotherapy of patients with B cell malignancies, such as non-Hodgkin lymphoma (Alduaij W, Illidge TM (2011) The future of anti-CD20 monoclonal antibodies: are we making progress? Blood 117: 2993-3001). Since this study, comparisons of antitumor effects by various antibody classes have been limited to IgG and IgM in both mouse models and patients with lymphoid malignancies, although IgA has been shown to mediate ADCC in vitro and in vivo in a mouse model of lymphoma (Dechant M, Valerius T (2001) IgA antibodies for cancer therapy. Crit Rev Oncol Hematol 39: 69-77).

[0005] Complement-mediated tumor cell death is now known to be just one of several mechanisms by which antibodies can mediate the limitation of tumor growth (Weiner GJ (2007) Monoclonal antibody mechanisms of action in cancer. Immunol Res 39: 271-278). Known mechanisms include engaging immune effector molecules via the Fc region to induce the destruction of target cells mediated by immune cells through antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis (ADCP). Antibodies can also act directly on tumor cells to inhibit growth signaling pathways, induce apoptosis, limit tumor cell growth and cell differentiation, or block tumor cell adhesion and migration. Some antibodies recognize targets associated with the tumor vasculature to prevent tumors from obtaining essential nutrients delivered by the blood supply, while other antibodies have been developed to attack immune checkpoint targets (e.g., CTLA-4 and PD-1) to enhance T cell activation and overcome immunosuppressive elements of the tumor microenvironment (Ascierto PA et al (2010) Clinical experiences with anti-CD137 and anti-PD1 therapeutic antibodies. Semin Oncol 37: 508-516, Cai J, Han S, Qing R, Liao D, Law B, Boulton ME (2011) In pursuit of new anti-angiogenic therapies for cancer treatment. Front Biosci 16: 803-814). Extensive efforts have also been focused on the design of antibody conjugates that deliver toxic payloads in the form of cytotoxins, drug-activating enzymes, cytokines, or radionuclides to tumors (Govindan SV, Goldenberg DM (2010) New antibody conjugates in cancer therapy. Scientific World Journal 10: 2070-2089).Also, for the main purpose of optimizing the antigen specificity / affinity and effector functions of IgG antibodies, a multiplex antibody modification method has been devised to improve proven therapeutic agents such as trastuzumab (Kubota T et al (2009) Engineered therapeutic antibodies with improved effector functions. Cancer Sci 100: 1566-1572).

[0006] Antibodies of the IgE class play a central role in allergic reactions and anti-parasitic activities and have many properties that can be advantageous for cancer treatment. Active and passive immunotherapeutic methods based on IgE have been shown to be effective in both in vitro and in vivo cancer models, suggesting the potential use of such methods in humans (Leoh et al (2015) Curr Top Microbiol Immunol.; 388: 109-149). Thus, IgE therapeutic antibodies can result in enhanced immune surveillance against cancer cells and excellent effector cell function.

[0007] A fully human anti-HER2 / neu IgE has been developed using the variable region of single-chain Fv C6MH3-B1 (Daniels TR et al (2012) Targeting HER2 / neu with a fully human IgE to harness the allergic reaction against cancer cells. Cancer Immunol Immunother. 61: 991-1003.). C6MH3-B1 induced degranulation of RBL SX-38 cells expressing human FcεRI in vitro in the presence of mouse mammary cancer cells (D2F2 / E2) expressing human HER2 / neu, but not in parental D2F2 cells lacking HER2 / neu expression or with the extracellular domain of HER2 / neu shed (soluble) (ECD HER2It was not induced in the presence of . Such results suggest that an acute inflammatory response (type I hypersensitivity) occurs in the tumor microenvironment where the HER2 / neu antigen is highly expressed at high levels on the surface of cancer cells, promoting FcεRI cross-linking and inducing effector cell degranulation (Pegram M, Ngo D. (2006) Application and potential limitations of animal models utilized in the development of trastuzumab (Herceptin): a case study. Adv Drug Deliv Rev. 58: 723-734.).

[0008] A mouse / human chimeric IgE specific for the human MUC1 antigen has been developed (Teo PZ et al (2102) Using the allergic immune system to target cancer: activity of IgE antibodies specific for human CD20 and MUC1. Cancer Immunol Immunother. 61: 2295-2309.). This antibody has been shown to reduce tumor size in a mouse breast cancer cell line (4T1.hMUC1) that expresses transmembrane human MUC1. However, perhaps the 4T1 tumors are highly avascular and grow densely in large numbers, resulting in a weak response, which may interfere with drug delivery or the recruitment of effector cells.

[0009] In addition, a novel mouse / human chimeric anti-PSA IgE consisting of the variable regions of AR47.47 derived from PSA has been investigated (Daniels-Wells TR et al (2013) A novel IgE antibody targeting the prostate-specific antigen as a potential prostate cancer therapy. BMC Cancer. 13: 195-207.). Stimulation of human dendritic cells with a complex of PSA and anti-PSA IgE resulted in the activation of CD4 and CD8 T cells in vitro. This suggests that anti-PSA IgE complexes with PSA in the patient's blood and may induce a second immune response involved in the activation of cytotoxic T lymphocytes.

[0010] An alternative strategy to the passive administration of IgE is to induce an endogenous IgE response. A novel method for establishing an active vaccination protocol is to induce tumor antigen-specific IgE via the oral route (Riemer AB et al (2007) Active induction of tumor-specific IgE antibodies by oral mimotope vaccination. Cancer Res. 67: 3406-3411). Synthetically produced epitope mimics (mimotopes) were generated for the epitope of human HER2 / neu recognized by trastuzumab. Induction of high-titer serum IgE targeting the HER2 / neu antigen was observed, and endogenous anti-HER2 / neu IgE recognized HER2 / neu-expressing human breast cancer cells (SK-BR-3), resulting in both degranulation and cell injury of rat basophilic cells (RBL-2H3) expressing rodent FcεRI.

[0011] A mouse / human chimeric IgE antibody (MOv18IgE) specific for the cancer-related antigen folate receptor α has been demonstrated to have superior antitumor activity against IgE compared to otherwise identical IgG in syngeneic immunocompetent animals (Karagiannis et al., Cancer Res. 2017 Jun 1;77(11):2779-2783). TNFα / MCP-1 signaling has been identified as an IgE-mediated mechanism for the activation and tumor recruitment of monocytes and macrophages.

[0012] However, the subjects who respond to IgE-based cancer therapy are currently unknown. Moreover, the method to most effectively promote the anti-cancer action of IgE antibodies is also unknown.

Prior Art Documents

Non-Patent Documents

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Summary of the Invention

Means for Solving the Problems

[0014] Accordingly, in one aspect, provided in the present invention is immunoglobulin E (IgE) for use in the reprogramming of macrophages associated with tumors in a subject, wherein the reprogramming results in the regulation of cytokine expression and the enhancement of anti-tumor activity in the tumor microenvironment.

[0015] In another aspect, provided in the present invention is an immunoglobulin E (IgE) antibody for use in the reprogramming of macrophages from a first phenotype to an anti-tumor phenotype in the treatment of cancer in a subject, wherein the first phenotype includes a quiescent (M0) macrophage phenotype or an anti-inflammatory (M2a) macrophage phenotype, and the anti-tumor phenotype includes a newly polarized macrophage phenotype characterized by the expression of the following cytokines and chemokines: tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin 1 beta (IL-1β); interleukin 6 (IL-6); RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted); and / or interleukin 10 (IL-10).

[0016] In another aspect, provided herein is an immunoglobulin E (IgE) antibody for use in the treatment of a subject's cancer, wherein macrophages associated with the tumor in the subject have a quiescent (M0) macrophage phenotype or an anti-inflammatory (M2a) macrophage phenotype, and the IgE treatment promotes repolarization of the macrophages associated with the tumor to a newly polarized macrophage phenotype characterized by the expression of the following cytokines: tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin 1 beta (IL-1β); interleukin 6 (IL-6); RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted); and / or interleukin 10 (IL-10).

[0017] In one embodiment, the newly polarized macrophage expresses tumor necrosis factor alpha (TNFα). In another embodiment, the newly polarized macrophage expresses interferon gamma (IFNγ). In another embodiment, the newly polarized macrophage expresses interleukin 1 beta (IL-1β). In another embodiment, the newly polarized macrophage expresses interleukin 6 (IL-6). In another embodiment, the newly polarized macrophage expresses RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted). In another embodiment, the newly polarized macrophage expresses interleukin 10 (IL-10). Generally, the newly polarized macrophage phenotype can be characterized by any combination of two or more such cytokines. Preferably, the newly polarized macrophage phenotype is characterized by the expression of any three, four, five, or all six of the following cytokines and chemokines: tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted), and interleukin 10 (IL-10).

[0018] In one embodiment, the newly polarized macrophage phenotype is further characterized by an increase in the expression of monocyte chemoattractant protein 1 (MCP-1) compared to the anti-inflammatory (M2a) macrophage phenotype.

[0019] In another embodiment, the newly polarized macrophage phenotype is further characterized by an increase in the expression of interleukin 4 (IL-4) compared to the anti-inflammatory (M2a) macrophage phenotype.

[0020] In another embodiment, the newly polarized macrophage phenotype is further characterized by an increase in the expression of interleukin 12 (IL-12), interleukin 13 (IL-13), chemokine (C-X-C motif) ligand 9 (CXCL9) and / or chemokine (C-X-C motif) ligand 11 (CXCL11) compared to the quiescent (M0) macrophage phenotype or the anti-inflammatory (M2a) macrophage phenotype.

[0021] In another embodiment, IgE treatment further promotes an increase in the expression of IFNγ and / or IL-12 by pro-inflammatory (M1) macrophages associated with tumors in a subject.

[0022] In another embodiment, the quiescent (M0) macrophage phenotype or the anti-inflammatory (M2a) macrophage phenotype is characterized by the expression of IL-4, IL-13, MCP-1, CXCL9 and / or CXCL11.

[0023] In another embodiment, macrophages associated with tumors in a subject are distributed so as to surround the periphery of the tumor.

[0024] In another embodiment, the newly polarized macrophage phenotype promotes the recruitment of additional monocytes and / or macrophages to tumors in a subject.

[0025] In another embodiment, the cancer includes skin cancer, breast cancer, head and neck squamous cell carcinoma, prostate cancer, ovarian cancer, colon cancer, glioma, gastric cancer or pancreatic cancer.

[0026] In another embodiment, the IgE includes an anti-folate receptor alpha (FRα) antibody, an anti-high molecular weight melanoma associated antigen (HMW-MAA) antibody, an anti-human epidermal growth factor receptor 2 (HER2) antibody or an anti-SF-25 antibody.

[0027] In a further aspect, there is provided a method comprising administering to a subject in need thereof a therapeutically effective amount of immunoglobulin E (IgE) for treating cancer in the subject, wherein macrophages associated with the tumor in the subject have a resting state (M0) macrophage phenotype or an anti-inflammatory (M2a) macrophage phenotype, and the IgE treatment promotes the repolarization of macrophages associated with the tumor to a newly polarized macrophage phenotype characterized by the expression of the following cytokines: tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin 1 beta (IL-1β); interleukin 6 (IL-6); RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted); and interleukin 10 (IL-10), and the newly polarized macrophage phenotype has enhanced anti-tumor activity as compared to the resting state (M0) macrophage phenotype or the anti-inflammatory (M2a) macrophage phenotype, thereby treating cancer in the subject.

[0028] In one embodiment, the method includes detecting one or more phenotypes of macrophages present in a tumor sample obtained from the subject, and administering IgE to the subject if resting state (M0) and / or anti-inflammatory (M2a) macrophages are present in the sample above a predetermined level.

[0029] In another embodiment, the method includes detecting the expression by macrophages present in a sample of one or more of the following cytokines: TNFα, IFNγ, IL-1β, IL-6, RANTES, IL-10, MCP-1, IL-4, IL-13, MCP-1, CXCL9, IL-12, and / or CXCL11.

Brief Description of the Drawings

[0030]

Figure 1

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Mode for Carrying Out the Invention

[0031] Surprisingly, it has been found that IgE treatment results in a unique profile of cytokine and chemokine expression by various macrophage phenotypes. In particular, the quiescent (M0) and anti-inflammatory (M2a) macrophage phenotypes have been found to be repolarizable by IgE treatment into a new macrophage phenotype with enhanced antitumor activity. This new macrophage phenotype is referred to herein as the newly polarized macrophage phenotype. The newly polarized macrophage phenotype is characterized by a specific profile of cytokine and chemokine expression that is distinct from both classical pro-inflammatory (M1) and anti-inflammatory (M2a) macrophages.

[0032] Accordingly, the newly polarized macrophage phenotype can be utilized to enhance anti-cancer treatment using IgE antibodies. For example, based on the macrophage phenotype associated with a tumor in a subject, the subject can be selected for IgE antibody treatment. In particular, the detection of quiescent (M0) and anti-inflammatory (M2a) macrophages in a tumor, e.g., detection above a predetermined level or a predetermined percentage of the total macrophages, can be used as an indicator for using IgE therapy to repolarize macrophages into an anti-tumor phenotype.

[0033] Furthermore, the present invention can be used in clinical situations where it is desirable to treat cancer by an immunotherapeutic method, i.e., to promote the reprogramming of macrophages towards an anti-tumor phenotype (e.g., to enhance the expression of cytokines / chemokines characteristic of this phenotype), resulting in enhanced killing of cancer cells via immune cells by mechanisms such as ADCC. Also, the present invention can be used, for example, to promote the recruitment of additional monocytes and / or macrophages to the tumor tissue by increasing the expression of chemokines such as MCP-1 by the newly reprogrammed macrophage phenotype in addition to the tumor cells themselves. This can be particularly desirable, for example, when anti-tumor macrophages are absent from the tumor or are present only in the periphery of the tumor tissue. Thus, the present invention represents a new clinical paradigm for the use of IgE therapy in cancer treatment, involving the treatment of a new patient subgroup and new clinical situations that require the reprogramming of macrophages towards an anti-tumor phenotype.

[0034] Antibody An antibody is a polypeptide ligand that specifically recognizes and binds to an epitope or a fragment of an antigen and contains at least one light or heavy chain immunoglobulin variable region. Antibodies typically consist of heavy and light chains, each of which has a variable region called the heavy chain variable (VH) region and the light chain variable (VL) region. Collectively, the VH and VL regions are responsible for binding to the antigen recognized by the antibody.

[0035] Antibodies include intact immunoglobulins and variants and portions of antibodies well known in the art, provided that such fragments retain at least one function of IgE and are, for example, capable of binding to the Fcε receptor. Antibodies also include genetically modified forms, such as chimeric, humanized (e.g., humanized antibodies containing mouse sequences in the variable regions) or human antibodies, bispecific antibodies, e.g., antibodies described in Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0036] Typically, naturally occurring immunoglobulins have heavy (H) and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are nine main isotypes or classes that determine the functional activity of antibody molecules: IgA1-2, IgD, IgE, IgG1-4, and IgM, corresponding to the heavy chain types α, δ, ε, γ, and μ. Thus, the class of an antibody is defined by the type of heavy chain present. The specific heavy chains differ in size and composition, with α and γ containing approximately 450 amino acids, while μ and ε have approximately 550 amino acids. Due to the differences in the constant regions of each heavy chain type, which selectively bind to specific receptor types (e.g., Fc receptors), the various effector functions of each antibody isotype can be discriminated. Thus, in embodiments of the present invention, the antibody preferably comprises an epsilon (ε) heavy chain, i.e., the antibody is of the IgE isotype that binds to the Fcε receptor.

[0037] Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). Together, the variable regions of the heavy and light chains specifically bind to an antigen. The variable regions of the light and heavy chains contain "framework" regions interrupted by three hypervariable regions also called "complementary determining regions" or "CDRs". The ranges of the framework regions and CDRs are defined (see Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The Kabat database is currently maintained online. The sequences of the various light or heavy chain framework regions are relatively conserved among species, e.g., humans. The framework region of an antibody is the combination of the framework regions of the constituent light and heavy chains and acts to position and align the CDRs in three-dimensional space.

[0038] CDRs mainly play the role of binding to the epitopes of antigens. The CDRs of each chain are typically called CDR1, CDR2, and CDR3 numbered from the N-terminus, and are generally identified by the chain on which a particular CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, and VL CDR1 is CDR1 of the variable domain of the light chain of the antibody in which it is found.

[0039] Antibodies have specific VH and VL region sequences and thus can have specific CDR sequences. Antibodies with various specificities (i.e., various binding sites for various antigens) have various CDRs. Although CDRs vary depending on the antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. Such positions within the CDRs are called specificity-determining residues (SDRs). "VH" refers to the variable region of the immunoglobulin heavy chain. "VL" refers to the variable region of the immunoglobulin light chain.

[0040] "Monoclonal antibodies" are antibodies produced by a single clone of B lymphocytes or by cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are prepared by methods known to those skilled in the art, such as generating hybrid antibody-forming cells by fusion of immunized spleen cells with myeloma cells, for example. Monoclonal antibodies include humanized monoclonal antibodies.

[0041] "Chimeric antibodies" contain sequences derived from two different antibodies, which are typically from different species. For example, a chimeric antibody can contain human and mouse antibody domains, such as a human constant region and a mouse variable region (e.g., from a mouse antibody that specifically binds to a target antigen).

[0042] Chimeric antibodies are typically constructed by fusing the variable and constant regions of immunoglobulin genes of light and heavy chains belonging to different species, for example, by genetic engineering. For example, the variable segments of the genes of murine monoclonal antibodies can be linked to human constant segments, such as kappa and epsilon. Thus, in one example, a therapeutic chimeric antibody is a hybrid protein consisting of the variable domain or antigen-binding domain of a murine antibody and the constant domain or effector domain of a human antibody, for example, the Fc (effector) domain of a human IgE antibody, although other mammalian species can also be used, or the variable regions can also be generated by molecular techniques. Methods for generating chimeric antibodies are well known in the art; see, for example, U.S. Patent No. 5,807,715.

[0043] A "humanized" antibody is an antibody that contains human framework regions and one or more CDRs of a non-human (e.g., murine, rat, or synthetic) antibody. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor." In one embodiment, all of the CDRs are derived from the donor immunoglobulin of the humanized immunoglobulin. The constant region is typically substantially identical to the human immunoglobulin constant region, i.e., at least about 85-90%, for example, about 95% or more identical. Thus, except for the CDRs, all parts of the humanized immunoglobulin are substantially identical to the corresponding parts of the native human immunoglobulin sequence.

[0044] Humanized antibodies typically contain a humanized immunoglobulin light chain and a humanized immunoglobulin heavy chain. Humanized antibodies typically bind to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of the humanized immunoglobulin or antibody can have a limited number of substitutions with amino acids obtained from the donor framework. Humanized or other monoclonal antibodies can have additional conservative substitutions of amino acids that do not substantially affect antigen binding or other immunoglobulin functions.

[0045] Humanized immunoglobulins can be constructed by genetic engineering means (see, e.g., U.S. Patent No. 5,585,089). Typically, humanized monoclonal antibodies are generated by transferring the donor antibody complementarity determining regions of the heavy and light variable chains of a murine immunoglobulin to a human variable domain and then substituting human residues into the framework regions of the donor counterparts. The use of antibody components derived from humanized monoclonal antibodies removes potential problems associated with the immunogenicity of the donor antibody constant region. Techniques for generating humanized monoclonal antibodies are described, for example, by Jones et al., Nature 321:522, 1986, Riechmann et al., Nature 332:323, 1988, Verhoeyen et al., Science 239:1534, 1988, Carter et al., Proc. Nat’l Acad. Sci. U.S.A. 89:4285, 1992, Sandhu, Crit. Rev. Biotech. 12:437, 1992 and Singer et al., J. Immunol. 150:2844, 1993.

[0046] A "human" antibody (also referred to as a "fully human" antibody) is an antibody that contains all of the human framework regions and CDRs derived from human immunoglobulins. In one example, the framework and CDRs are derived from the amino acid sequences of human heavy and / or light chains of the same origin. However, the framework of a human antibody can be modified to contain CDRs from different human antibodies.

[0047] In embodiments of the invention, the antibody may be a monoclonal or polyclonal antibody, including chimeric, humanized or fully human antibodies.

[0048] In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Mutations that can occur in the human framework region and the humanized antibody framework region are known in the art (see, for example, U.S. Patent No. 5,585,089).

[0049] Alternatively, additional antibodies against a particular antigen may be generated by well-established methods, and at least the variable region or CDR from such antibodies may be used in the antibodies of the present invention (for example, the generated antibodies may be used to donate CDR or variable region sequences to the IgE acceptor sequence). Methods for polypeptide synthesis and immunization of host animals are well known in the art. Typically, a host animal (e.g., a mouse) is inoculated intraperitoneally with a quantity of immunogen and, when generating monoclonal antibodies, hybridomas prepared from its lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497.

[0050] Hybridomas that produce suitable antibodies can be grown using procedures known in vitro or in vivo. Monoclonal antibodies can be isolated from the culture medium or body fluids, if desired, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If unwanted activities are present, for example, the preparation can be carried out on an adsorbent produced with an immunogen that binds to a solid phase, and the desired antibody can be removed by eluting or releasing it from the immunogen. If desired, the antibody of interest (monoclonal or polyclonal) can be sequenced and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the antibody can be maintained in a vector in a host cell, and then the host cell can be grown and frozen for future use.

[0051] Human antibodies and antibody fragments can be selected and generated in vitro from an immunoglobulin variable (V) domain gene repertoire derived from non-immunized donors (e.g., from human subjects including patients suffering from the relevant disorder) using phage display techniques such as those described in U.S. Patent No. 5,565,332 and other published literature. For example, existing antibody phage display libraries can be panned in parallel against an enormous collection of synthetic polypeptides. According to this technique, antibody V domain genes are cloned in frame into either the major or minor coat protein gene of a filamentous bacteriophage, such as either M13 or fd, and presented on the surface of phage particles as functional antibody fragments. Since the filamentous particles contain a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in the selection of genes encoding antibodies that exhibit such properties. Thus, antibody sequences selected using phage display with a human library may contain human CDR or variable region sequences that confer specific binding to a specific antigen, and these can be used to prepare fully human antibodies for use in the present invention.

[0052] Methods for obtaining heavy and light chain sequences from human B cells and plasma cell clones are also well known in the art and are typically carried out using polymerase chain reaction (PCR) technology. Examples of this method are described in Kuppers R, Methods Mol Biol. 2004;271:225-38, Yoshioka M et al., BMC Biotechnol. 2011 Jul 21;11:75, Scheeren FA et al., PLoS ONE 2011, 6(4): e17189. doi:10.1371 / journal.pone.0017189, Wrammert J et al., Nature 2008 453, 667-671, Kurosawa N et al., BMC Biotechnol. 2011 Apr 13;11:39, Tiller et al., J Immunol Methods. 2008 January 1; 329(1-2): 112-124. Thus, antibody sequences selected using B cell clones may contain human CDR or variable region sequences that confer specific binding to a target antigen, and these may be used to provide fully human antibodies for use in the present invention.

[0053] IgE antibody The antibody to be administered to the subject is an IgE antibody, i.e., an antibody of the IgE isotype. There are some fundamental structural differences between IgE and IgG, and these have functional effects. IgE shares the same basic molecular structure as other classes of antibodies, but the heavy chain of IgE contains one more domain than the heavy chain of IgG. The Cε3 and Cε4 domains of IgE are homologous in sequence and similar in structure to the Cγ2 and Cγ3 domains of IgG, and thus the Cε2 domain is the most distinct and prominent feature of IgE. The Cε2 domain is folded against the heavy chain IgE and has been found to be in close contact with the Cε3 domain. This bent structure of the IgE heavy chain allows it to adopt an open or closed structure. The unbound IgE dimer has one chain in the open structure and one chain in the closed structure. The binding of FcεRI to IgE is biphasic, and after the initial binding to the open Cε chain, it is thought to be involved in a large-scale structural rearrangement that allows binding to the closed Cε chain. The binding between the IgE dimer and FcεRI occurs in a 1:1 stoichiometry despite the presence of two identical Cε chains. This rearrangement results in a very close interaction between IgE and FcεRI and a much higher affinity of IgE for the Fc receptor than that found for IgG and FcγR (McDonnell, J. M., R. Calvert, et al. (2001) Nat Struct Biol 8(5): 437-441).

[0054] The antibody used in the present invention can typically bind to Fcε receptors, for example, to FcεRI and / or FcεRII receptors. Preferably, the antibody can at least bind to FcεRI (i.e., the high-affinity Fcε receptor) or can at least bind to FcεRII (CD23, the low-affinity Fcε receptor). Typically, the antibody can also activate Fcε receptors, for example, expressed on cells of the immune system to initiate effector functions mediated by IgE.

[0055] The epsilon (ε) heavy chain is unique to IgE antibodies and contains an N-terminal variable domain VH and four constant domains Cε1 - Cε4. Similar to other antibody isotypes, the variable domain confers antigen specificity and the constant domains recruit isotype-specific effector functions.

[0056] IgE differs from the more abundant IgG isotypes in that it cannot fix complement and does not bind to the Fc receptors FcγRI, RII, and RIII expressed on the surface of monocytes, NK cells, and neutrophils. However, IgE can engage in very specific interactions with the "high-affinity" IgE receptor (FcεRI, Ka ~ 10 11 M -1 ) on a variety of immune cells such as mast cells, basophils, monocytes / macrophages, eosinophils, as well as the "low-affinity" receptor, FcεRII (also known as CD23, Ka ~ 10 7 M -1 ) expressed on inflammatory cells and antigen-presenting cells (e.g., monocytes / macrophages, platelets, dendritic cells, T and B lymphocytes).

[0057] The sites on IgE that mediate such receptor interactions are located in peptide sequences on the Cε chain and are distinctive. The FcεRI site is located in a gap created by residues between Gln301 and Arg376 and includes the junction between the Cε2 and Cε3 domains (Helm, B. et al. (1988) Nature 331, 180 - 183). The FcεRII binding site is located around the residue Val370 within Cε3 (Vercelli, D. et al. (1989) Nature 338, 649 - 651). The main difference that discriminates between the two receptors is that FcεRI binds to monomeric Cε, while FcεRII binds only to dimeric Cε, i.e., two Cε chains must be associated. IgE is glycosylated in vivo, but this is not required for binding to FcεRI and FcεRII.

[0058] Thus, binding to the Fcε receptor and related effector functions are typically mediated by the heavy chain constant domain of the antibody, particularly by the domains that together form the Fc region of the antibody. The antibodies described herein typically include at least a portion of an IgE antibody, such as one or more constant domains derived from IgE, preferably human IgE. In certain embodiments, the antibody includes one or more domains (derived from IgE) selected from Cε1, Cε2, Cε3, and Cε4. In one embodiment, the antibody includes at least Cε2 and Cε3, more preferably at least Cε2, Cε3, and Cε4, and preferably, in this case, the domains are derived from human IgE. In one embodiment, the antibody includes an epsilon (ε) heavy chain, preferably a human ε heavy chain.

[0059] Nucleotide sequences encoding constant domains derived from human IgE, particularly the Cε1, Cε2, Cε3, and Cε4 domains, are disclosed, for example, in International Publication No. WO 2013 / 050725. Amino acid sequences of other human and mammalian IgEs and their domains, including the human Cε1, Cε2, Cε3, and Cε4 domains and the human ε heavy chain sequence, are known in the art and are available from publicly accessible databases. For example, a database of human immunoglobulin sequences is available from the website of the International ImMunoGeneTics Information System (IMGT®) at http: / / www.imgt.org. As an example, sequences of various human IgE heavy (ε) chain alleles and their individual constant domains (Cε1-4) are available at http: / / www.imgt.org / IMGT_GENE-DB / GENElect?query=2+IGHE&species=Homo+sapiens.

[0060] Preferred antibodies The IgE antibodies used in the present invention can bind to any target antigen. Some non-limiting examples of such target antigens include, for example, folate receptor alpha (FRα) antibodies, high molecular weight melanoma-associated antigen (also known as HMW-MAA, chondroitin sulfate proteoglycan 4 or CSPG4), human epidermal growth factor receptor 2 (HER2 / neu, also known as erbB2), CD20, mucin 1 (MUC1), and prostate-specific antigen (PSA). That is, the present invention encompasses antibodies that specifically bind to any of the above targets or any other arbitrary antigen. Examples of suitable IgE antibodies are described, for example, in International Publication No. WO 2013 / 050725, Karagiannis et al., J Immunol 2007; 179:2832-2843, Daniels et al (2012), Cancer Immunol Immunother. 61: 991-1003, Daniels-Wells et al (2013), BMC Cancer. 13: 195-207, Teo et al (2012), Cancer Immunol Immunother. 61: 2295-2309, Karagiannis et al., Cancer Immunol Immunother. 2009 Jun;58(6):915-30, and Karagiannis et al., Cancer Res; 77(11), 2017.

[0061] Generally, functional fragments of the antibodies described herein can be used in the present invention. The functional fragment may be of any length specified above (e.g., at least 50, 100, 300, or 500 nucleotides, or at least 50, 100, 200, or 300 amino acids), provided that it retains the activities required when present in the antibody (e.g., specific binding to the antigen and Fcε receptor).

[0062] In addition, variants of the above amino acid and nucleotide sequences can also be used in the present invention, provided that the resulting antibody binds to the Fcε receptor. Typically, such variants have a high degree of sequence identity with one of the sequences specified above.

[0063] Similarity between amino acid or nucleotide sequences is expressed in terms of sequence similarity units, or in other words, called sequence identity. Sequence identity is frequently measured in terms of percent identity (or similarity or homology), and the higher the percentage, the more similar the two sequences are. Homologs or variants of amino acid or nucleotide sequences have a relatively high degree of sequence identity when aligned using standard methods.

[0064] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2:482, 1981, Needleman and Wunsch, J. Mol. Biol. 48:443, 1970, Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988, Higgins and Sharp, Gene 73:237, 1988, Higgins and Sharp, CABIOS 5:151, 1989, Corpet et al., Nucleic Acids Research 16:10881, 1988 and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994 shows a detailed discussion of sequence alignment methods and homology calculations.

[0065] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from multiple sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md) and the Internet, and is intended for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of the method for determining sequence identity using this program is available on the NCBI website on the Internet.

[0066] Typically, variants include one or more conservative substitutions of amino acids compared to the native amino acid or nucleotide sequence. Conservative substitutions are substitutions that do not substantially affect or decrease the affinity for the target antigen of the antibody and / or the Fcε receptor. For example, a human antibody that specifically binds to a target antigen may contain up to 1, up to 2, up to 5, up to 10, or up to 15 conservative substitutions compared to the native sequence (e.g., the sequence defined above) and still retain specific binding to the target polypeptide. Also, the term conservative mutation includes the use of a substituted amino acid at the position of the non-substituted parental amino acid, provided that the antibody specifically binds to the target antigen. Non-conservative substitutions are substitutions that decrease activity or binding to the target antigen and / or the Fcε receptor.

[0067] Functionally similar amino acids that can be exchanged by conservative substitutions are well known to those skilled in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0068] Cytokine In embodiments of the present invention, IgE therapy can be used to promote the reprogramming to a novel anti-tumor macrophage phenotype characterized by a specific profile of cytokine expression. In general, the term "cytokine", as used herein, includes chemokines, which are typically small cytokines involved in promoting the chemotaxis of cells such as monocytes, lymphocytes, and other immune system cells.

[0069] Interferon gamma (IFN-γ) is a cytokine whose biological activities are normally associated with cytostatic / cytotoxic and anti-tumor mechanisms during cell-mediated adaptive immune responses. Despite sufficient evidence relating the role of IFN-γ in tumor immunosurveillance, reports have been constantly made suggesting that this IFN-γ can also have a tumor-promoting effect under certain circumstances. The best-characterized function of IFN-γ is to upregulate major histocompatibility (MHC) class I molecules, which helps in the stimulation and presentation of antigens in professional antigen-presenting cells (Seliger B et al (2008) IFN inducibility of major histocompatibility antigens in tumors. Adv Cancer Res. 101: 249-76). IFN-γ controls the differentiation and function of many immune cell types and is essentially involved in all situations of Th1-mediated immune responses through the control of T cell differentiation, activation, and homeostasis, which inhibits the development of Th2 cells but promotes the development of regulatory T (Treg) cells (Agnello D et al (2003) Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights. J Clin Immunol. 23(3): 147-61). It also activates macrophages and induces the production of chemokines. Thus, IFN-γ can be important in the induction of tumor rejection.

[0070] Inflammatory cytokines, such as interleukin 1α (IL-1α), IL-1β, IL-6, and tumor necrosis factor α (TNFα), have been found to promote or suppress cancer. However, the cellular and molecular basis underlying such contradictory results remains enigmatic. Interleukin 1 (IL-1) is known to be upregulated in many tumor types and has been implicated as a factor in tumor progression through the expression of metastatic and angiogenic genes and growth factors. Cancer cells can either directly produce IL-1 or induce cells in the tumor microenvironment to produce IL-1 (Portier M et al (1993) Cytokine gene expression in human multiple myeloma. Br J Haematol. 85: 514-520). However, Haabeht et al (Oncoimmunology (2016) 5(1): e1039763) showed that IL-1 (IL-1α and IL-1β) synergizes with IFN-γ to induce antitumor activity in tumor-infiltrating macrophages. This synergistic action between IL-1 and IFN-γ can explain the mechanism by which inflammation suppresses rather than promotes cancer when it is driven by tumor-specific Th1 cells. Collectively, these data suggest a central role for the inflammatory cytokine IL-1, and more specifically, in the enhancement of Th1-mediated immunity against cancer, as a benchmark in inflammation.

[0071] The strong association between inflammation and cancer can be inferred from the high levels of IL-6 in the tumor microenvironment, which promotes tumorigenesis. IL-6 is often regarded as an inflammatory cytokine with functions comparable to those of TNFα and IL-1β in the context of inflammation.

[0072] Until recently, interleukin 10 (IL-10) was considered an immunosuppressive cytokine that hinders antitumor immunity. It has been revealed that IL-10 is essential for T helper 1 cell function and antitumor cell lysis activity. The strong anti-inflammatory function of IL-10 is mainly indirect and cell-mediated, and in cooperation with its immune-stimulating function, it improves tumor-specific immune surveillance (Dennis et al (2013) Curr Opin Oncol. 25(6): 637-645).

[0073] Interleukins (IL) 4 and 13 are structurally and functionally related. They regulate the immune response and immune microenvironment not only under normal physiological conditions but also in cancer. Both cytokines initiate signal transduction and mediate biological effects such as tumor growth, cell survival, cell adhesion, and metastasis. In certain cancers, the presence of such cytokine receptors can act as biomarkers of cancer malignancy. In addition, it has been found that both such cytokines and their receptors play important roles in the regulation of the immune system for tumor growth. IL-4 causes an increase in B lymphocytes and antibody production, and also increases the production of T lymphocytes. Since IL-13 cannot act directly on T cells, it is considered less critical for immune polarization than IL-4. However, recent studies on IL-13 have revealed that this cytokine plays a significant role in many situations of immune control. Studies by Terabe et al (Cancer Immunol Immunother. (2004) 53(2): 79-85), etc., have shown that IL-13 is at the center of a new immune control pathway in which tumor immune surveillance is suppressed by NKT cells.

[0074] Interleukin 12 (IL-12) has been considered an ideal candidate for cancer immunotherapy due to its ability to activate both innate (NK cells) and adaptive (cytotoxic T lymphocytes) immunity. IL-12 acts on diverse immune cells, and it is thought that the Th1-type immune response against specific pathogens is organized by the overall physiological role of IL-12. Although the potent antitumor effect of IL-12 is well established, this cytokine is considered unable to directly inhibit cancer growth, except possibly in some cases (Ferretti E et al (2010) Direct inhibition of human acute myeloid leukemia cell growth by IL-12. Immunol Lett. 133: 99-105). Rather, IL-12 acts as a major orchestrator of the Th1-type immune response against cancer. Another important concept is that IL-12 is thought to induce a more potent antitumor response when it is present directly at the tumor site rather than systemically.

[0075] RANTES (regulated on activation, normal T cell expressed and secreted), also known as chemokine (C-C motif) ligand 5 (CCL5), is a member of the beta family of chemokines and a potent chemoattractant for lymphocytes and monocytes to sites of inflammation. Also, with the help of specific cytokines released by T cells (e.g., IL-2 and IFN-γ), RANTES induces the proliferation and activation of specific natural killer (NK) cells to form CHAK (CC chemokine-activated killer) cells.

[0076] Chemokine CXCL9 plays an important role in the chemotaxis of immune cells, and accumulating evidence has shown that the therapeutic efficacy of strategies by tumor-specific T cells can be enhanced by manipulating the tumor microenvironment containing CXCL9 (Ding et al (2016) Cancer Med. 5(11): 3246-3259). CXCL9 can promote cancer metastasis by enhancing the migration and infiltration of tumor cells (Ding Q et al (2016) An alternatively spliced variant of CXCR3 mediates the metastasis of CD133+ liver cancer cells induced by CXCL9. Oncotarget. 7: 14405-14414) and the disruption of endothelial cell monolayers (Amatschek S et al (2011) CXCL9 induces chemotaxis, chemorepulsion and endothelial barrier disruption through CXCR3-mediated activation of melanoma cells. Br. J. Cancer. 104: 469-479). However, as a tumor suppressor, it mainly mobilizes tumor-infiltrating CD8+ T cells and NK cells (Clancy-Thompson E et al (2015) Melanoma induces, and adenosine suppresses, CXCR3-Cognate chemokine production and T-cell infiltration of lungs bearing metastatic-like disease. Cancer Immunol. Res. 3: 956-967) and inhibits tumor angiogenesis (Addison C. L et al (2000) The CXC chemokine, monokine induced by interferon-gamma, inhibits non-small cell lung carcinoma tumor growth and metastasis. Hum. Gene Ther. 11: 247-261).

[0077] Interferon-inducible T cell chemoattractant (I-TAC / CXCL11) is an IFN-inducible chemokine that mediates the recruitment of T cells, natural killer (NK) cells, and monocytes / macrophages at the site of infection.

[0078] Nakamura et al. (in the article) evaluated whether high intratumoral expression of immune mediators of the "classical" Th1, Th2, Th9, and Th17 responses could have any association with clinical outcomes. Examination of individual mediators showed that 9 out of 22 mediators: CXCL-9, CXCL-10, CXCL-11, IFNγ, TNFα, IL-4, MCP-1, IL-23, CXCL-13 were significantly associated with improved patient survival compared to patients with low genomic expression. The cytokines with the most prominent association with improved survival were CXCL-10 and IFNγ, with a 22% lower risk of death (CXCL-10: HR = 0.78, P = 0.0037; IFNγ: HR = 0.78, P = 0.0036). Moreover, subjects with high intratumoral expression of the Th2 cytokines, IL-4 and MCP-1, showed excellent survival, with a 19% and 17% reduction in the risk of death, respectively (IL-4: HR = 0.78, P = 0.012; MCP-1: HR = 0.83, P = 0.029). It has been found that a newly polarized macrophage phenotype resulting from IgE cross-linking of M2a macrophages shows increased expression of both IL-4 and MCP-1.

[0079] In addition, Nakamura et al. observed the greatest association with improved survival at high expression of a certain proportion of Th1 cytokines (IFNγ (HR = 0.78, P = 0.0036), CXCL-9 (HR = 0.81, P = 0.012), CXCL-10 (HR = 0.78, P = 0.0037), and CXCL-11 (HR = 0.8, P = 0.0078)). Also, when combining such mediators, the combination of CXCL-9 and CXCL-10, which is a combination of two mediators, had an HR of 0.74 (P = 0.0006), and the combination of CXCL-9, CXCL-10, and CXCL-11 had an HR of 0.73 (P = 0.00028). The highest improvement in patient survival was measured by high expression of all four mediators (IFNγ, CXCL-9, CXCL-10, and CXCL-11) (HR = 0.72, P = 0.00021). These may indicate the synergistic function of such mediators that can confer a survival benefit. Similarly, it has been found that the newly polarized macrophage phenotype derived from M2a macrophages shows a similar profile with the appearance of IFNγ and increased secretion of CXCL9 and CXCL11.

[0080] Collectively, Nakamura et al. conclude that such findings identify the immune secretome mediators of the "classical" (Th1, Th2, and Th17) responses that provide some protection against bacteria and viruses, and further, that mediators involved in conditions such as allergy and autoimmunity are associated with a better prognosis in the context of a positive prognosis for ovarian cancer survival. Specifically, the properties of TNFα / MCP-1 may have a role in anti-cancer immunity. Such and other immune properties, which are normally deployed in infection clearance, are enhanced by specific immunotherapeutic methods, such as by anti-tumor specific IgE or by avirulent parasite vaccines (Baird, J.R. et al (2013) Avirulent Toxoplasma gondii generates therapeutic antitumor immunity by reversing immunosuppression in the ovarian cancer microenvironment. Cancer Res. 73(13): 3842-51, Fox, B.A. et al (2017) Cancer therapy in a microbial bottle: Uncorking the novel biology of the protozoan Toxoplasma gondii. PLoS Pathog. 13(9): e1006523), which can restore immunosuppression and confer therapeutic benefits in ovarian and other cancers.

[0081] The cytokines and chemokines described in this specification, as well as their amino acid sequences (and nucleotide sequences encoding the amino acid sequences), are well known to those skilled in the art, and these sequences are available from publicly accessible databases. The expression of cytokines and chemokines by macrophage populations can be detected by any suitable method (e.g., at the polypeptide or RNA level), for example, using antibodies (which may be labeled) against them. Such antibodies are known and commercially available. For example, cytokines and chemokines can be detected by standard methods, including, for example, ELISA, magnetic beads, and / or fluorescence labeling. In particular, a fluorescent label (i.e., a fluorophore) can be conjugated to an anti-cytokine antibody (or to magnetic microparticles that similarly bind an anti-cytokine antibody). In one embodiment, cytokines can be detected using a magnetic Luminex® assay as described in the examples. However, any other suitable alternative may be used instead, including, for example, RT-PCR to detect the expression of transcripts encoding cytokines.

[0082] Compositions and treatment methods Provided herein are compositions comprising a carrier and one or more therapeutic IgE antibodies or functional fragments thereof. The compositions can be prepared in unit dosage forms for administration to a subject. The amount and timing of administration are left to the discretion of the treating physician to achieve the desired purpose. The antibodies can be formulated for systemic or local (e.g., intratumoral) administration. In one example, the therapeutic IgE antibody is formulated for parenteral administration, such as intravenous administration.

[0083] The composition for administration may comprise a pharmaceutically acceptable carrier, for example, a solution of an antibody (or a functional fragment thereof) dissolved in an aqueous carrier. A variety of aqueous carriers, such as buffered saline, etc. can be used. Such solutions are sterile and generally free of undesirable substances. Such compositions can be sterilized by conventional well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc., if necessary to approximate physiological conditions. The concentration of the antibody in such formulations may vary widely and is selected mainly based on factors such as the volume of the liquid, viscosity, body weight, etc., according to the particular administration method selected and the needs of the subject.

[0084] Typical dosages of pharmaceutical compositions for intravenous administration include from about 1 μg to 1 g of antibody per kg of the subject's body weight per day, more preferably from 10 μg to 100 mg, and most preferably from 0.1 to 15 mg. In particular, when administering the drug to an isolated site other than the circulatory or lymphatic system, for example, the lumen of a body cavity or organ, a dosage of from 0.1 to up to about 100 mg per kg per day can be used. The actual methods for preparing the administrable compositions are known or apparent to those skilled in the art and are described in detail in published literature such as Remington’s Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa. (1995).

[0085] The antibodies are provided in lyophilized form and can be rehydrated with sterile water prior to administration, although they can also be provided in sterile solutions of known concentration. The antibody solution is then added to an infusion bag containing 0.9% sodium chloride, USP and is typically administered at a dose of 0.5 - 15 mg / kg body weight. Considerable technology based on experience in the art in the administration of antibody pharmaceuticals is available and these have been commercially available in the United States since the approval of RITUXAN® in 1997. The antibodies can be administered by slow infusion rather than by intravenous or rapid intravenous injection. In one example, after administration of a high loading dose, a low level maintenance dose is administered. For example, after infusion of a first loading dose of 4 mg / kg over a period of about 90 minutes, a weekly maintenance dose of 2 mg / kg is administered by infusion over a period of 30 minutes, if the prior dose has been well tolerated, for 4 - 8 weeks.

[0086] The antibody (or a functional fragment thereof) can be administered to delay or inhibit the growth of cells such as cancer cells. In such applications, a therapeutically effective amount of the antibody is administered to the subject in an amount sufficient to inhibit cancer cell growth, replication or metastasis, or to inhibit cancer signs or symptoms. In some embodiments, the antibody is administered to the subject to inhibit or prevent the progression of metastasis or to reduce the size or number of metastases such as micrometastases to regional lymph nodes (Goto et al., Clin. Cancer Res. 14(11):3401 - 3407, 2008).

[0087] Suitable subjects can include, but are not limited to, subjects diagnosed with cancer such as melanoma, prostate cancer, squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), breast cancer (including, but not limited to, basal breast cancer, ductal carcinoma, and lobular breast cancer), lung cancer (e.g., adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and mesothelioma, small cell lung cancer or non - small cell lung cancer), leukemia (e.g., acute myeloid leukemia and 11q23 - positive acute leukemia), lymphoma (e.g., cutaneous lymphoma), neuroectodermal tumors (e.g., astrocytoma, glioma, or neuroblastoma), ovarian cancer, colon cancer, gastric cancer, pancreatic cancer, bone cancer (e.g., chordoma), glioblastoma, or sarcoma (e.g., chondrosarcoma). Preferably, the antibody is administered to treat solid tumors. In another embodiment, the antibody is administered to treat hematological tumors (e.g., leukemia or lymphoma).

[0088] The therapeutically effective amount of the antibody depends on the severity of the disease and the general state of the patient's health. The therapeutically effective amount of the antibody is an amount that results in either subjective alleviation of symptoms(s) or objectively identifiable improvement as recognized by a clinician or other qualified observer. Such compositions can be administered in combination with, simultaneously or sequentially, another chemotherapeutic agent.

[0089] In embodiments of the present invention, the therapeutic IgE antibody can be used to treat patients suffering from diseases such as cancer, for example, a subgroup of subjects who can benefit from the re - polarization of macrophages to an anti - tumor phenotype. Thus, the subgroup of patients to be treated can be, for example, patients in whom quiescent (M0) macrophages or anti - inflammatory (M2a) macrophages are present within the tumor. The presence of such M0 or M2a macrophages within the tumor can be determined using known techniques, for example, by detection of the expression of characteristic profiles of cytokines and / or chemokines and / or cell - surface markers.

[0090] Here, the present invention will be further described with reference to the following non - limiting embodiments for illustrative purposes only. [Examples]

[0091] The role of IgE antibodies in macrophage activation and polarization was investigated. One of the downstream effects of the interaction of IgE with macrophages may be the release of soluble mediators by cells upon FcεRI cross-linking (Josephs et al (2017) Anti-Folate Receptor-α IgE but not IgG Recruits 330 Macrophages to Attack Tumors via TNFα / MCP-1 Signaling. Cancer Research 77(5): 1127-1141).

[0092] For this reason, cell cultures of M0, M1, and M2a macrophages were treated with SF-25 or NIP IgE and then incubated with anti-human IgE to induce cross-linking of the FcεRI-binding antibody. The controls for this test consisted of SF-25 or NIP IgE antibody treatment alone, anti-IgE alone, and untreated cells. The supernatants were collected 4 hours after treatment to allow for cytokine production and release.

[0093] To understand whether the interaction of IgE with macrophages results in the distinct production of pro-inflammatory and anti-inflammatory mediators, a broad population of cytokines and chemokines was analyzed. By performing a multiplex assay using magnetic beads, it was possible to obtain the cytokine profiles of M0, M1, and M2a macrophages. This enabled the simultaneous detection of many different soluble mediators in the same sample.

[0094] Methods and Materials Isolation and Culture of Primary Cells Peripheral venous blood (50 ml) was collected from healthy donors using a K2EDTA spray-coated collection tube. To isolate peripheral blood mononuclear cells (PBMCs), an equal volume of blood and 2% FCS / 2 mM EDTA were gently mixed to a final volume of 30 ml and gently pipetted onto 15 ml of Ficoll-Paque™ PLUS density gradient in a 50 ml conical tube. The tube was then centrifuged at 1200×g with gentle acceleration and no braking for 20 minutes at room temperature (RT). The plasma interface was collected using a plastic Pasteur pipette, transferred to a new vial, and washed at 4°C for 10 minutes by maximum acceleration and deceleration at 600×g using PBS. Red blood cells present in the sample were lysed by incubating the cells with 5 ml of RBC lysis buffer for 5 minutes at RT followed by washing with PBS + 2% FCS / 2 mM EDTA.

[0095] Monocytes were isolated from human blood using an indirect magnetic labeling system, the Pan Monocyte Isolation Kit (Miltenyi Biotec), to isolate native monocytes from human PBMCs. Using this technique, simultaneous enrichment of classical (CD14++CD16++), non-classical (CD14+CD16++) and intermediate (CD14++CD16+) monocyte populations was performed. A highly pure monocyte population was obtained by removing labeled and magnetically bound cells. After isolating PBMCs, the cells were passed through a 40 μm cell strainer to remove any aggregates, and the cell count in the sample was quantified using a hemocytometer. Next, 1×10 8 of PBMCs were transferred to a new vial and washed using PBS at 600×g for 5 minutes with maximum acceleration and deceleration. After discarding the supernatant, the cells were resuspended in 400 μl of MACS buffer (PBS supplemented with 0.5% fetal calf serum and 2 mM EDTA).

[0096] To isolate human monocytes by negative selection, the Pan Monocyte Isolation Kit (Miltenyi Biotec) protocol was followed. Briefly, 100 μl of FcR blocking reagent was added to the sample to block unwanted binding of antibodies to human Fc receptors, and the sample was mixed up and down with a pipette. Then, 100 μl of biotin antibody cocktail was added, and the sample was incubated at 4 °C for 5 minutes to promote binding of the antibodies to monocytes. Thereafter, 300 μl of MACS buffer and 200 μl of anti-biotin microbeads were added to the vial by thoroughly mixing them. Next, the cells were incubated again at 4 °C for 10 minutes. After incubation, the sample was subjected to magnetic cell separation by inserting the LS column into the MidiMACS™ separator placed on a MultiStand (Miltenyi Biotec). The column was first rinsed with 3 ml of MACS buffer, and when the reservoir was empty, the cell suspension was loaded onto the column. The flow-through, which is the enriched monocyte fraction, was collected. The column was washed three times by adding 3 ml of MACS buffer, and the unlabeled cells were collected and mixed with the effluent collected in the previous step. To collect the labeled cells, which are mainly lymphocytes, the column was removed from the separator and placed on a suitable collection tube. 5 ml of MACS buffer was added onto the column, and the magnetically labeled non-monocytes were flushed out using a plunger.

[0097] To confirm the level of purity of the monocyte population, samples for flow cytometry (performed using a BD FACSCanto™ II at the Biomedical Research Centre Flow Cytometry Core, King’s College London) were prepared as follows. BV786-conjugated mAb (1×10 5The cells were stained with 1 μl per cell and BV711-conjugated mAb against CD16 at 4°C for 30 minutes. After incubation, the cells were washed once with FACS buffer (phosphate-buffered saline (PBS, Gibco) supplemented with 2% fetal bovine serum (Gibco)) and maintained in fresh FACS buffer until ready for data acquisition on the flow cytometer. Gates were set using single positive controls and FMO controls for each fluorophore.

[0098] Monocytes were isolated by the above magnetic separation and resuspended in RPMI 1640 medium GlutaMAX (Gibco), 2% FBS to promote adhesion, and the cells were seeded at a density of 1 - 1.5×10 6 cells / ml in 6-well plates, and 2 ml of the cell suspension was added to each well, and the plates were placed in a tissue culture incubator. After 2 hours of incubation, monocyte adhesion was confirmed under a microscope, and the medium was carefully removed by tilting the plate and aspirating with a pipette. To remove cells that did not adhere completely, each well was washed with 1 ml of sterile PBS, and RPMI 1640 supplemented with 10% FCS and penicillin / streptomycin (penicillin (100 U / ml) and streptomycin (100 U / ml) (Life Technologies)) with 20 ng / ml of M-CSF (monocyte colony-stimulating factor, Peprotech) was added and replenished with 2 ml to proliferate monocytes ex vivo. Thereafter, every 3 days, half of each fresh well was replaced with medium containing 40 ng / ml of M-CSF. After 7 days, differentiated macrophages were observed.

[0099] From this point on, various cytokines were added to the cell culture based on the desired cell phenotype: macrophages of M1 or M2. To polarize macrophages to the M1 phenotype, 20 ng / ml of interferon gamma (IFNγ, Life Technologies) and 100 ng / ml of lipopolysaccharide (LPS, Sigma) were added to the cells and left for 72 hours. On the other hand, to polarize macrophages to the M2 phenotype, 20 ng / ml of interleukin 4 (IL-4, Peprotech) was added and left for 72 hours.

[0100] Production of SF-25IgE and IgG1 antibodies Production of SF-25IgE by spinner flask Sp2 / 0 cells expressing the SF-25IgE antibody were seeded into a 1 L spinner flask with IMDM medium at a total initial volume of 180 ml at a cell density of 5 × 105 cells / ml. Then, fresh medium was added to the suspension up to 500 ml, and it was placed on a spinner plate in a humidified incubator at 37 °C and 5% CO2 at a constant speed of 7.5 rpm. After 3 days, fresh medium was added to the cell suspension up to a final volume of 1 L, and the cells were grown for 3 weeks without further addition of fresh medium. Every 3 days, 1 ml of sample was taken from each cell suspension to observe antibody production over time. The samples were collected into 1.5 ml tubes, centrifuged at 12,500 × g for 5 minutes, filter-sterilized through a 0.45 μm filter, and stored at -20 °C.

[0101] Production of SF-25IgE by shaking flask Sp2 / 0 cells expressing the SF-25IgE antibody were seeded into a 1 L Erlenmeyer flask with IMDM complete medium at an initial volume of 60 ml at a cell density of 5 × 105 cells / ml. Then, fresh complete medium was added to the suspension up to 150 ml, and it was placed in a humidified incubator at 37 °C and 5% CO2 on a shaking platform at a constant speed of 55 rpm. Every 3 days, 1 ml of sample was taken from the cell suspension to observe antibody production over time. The samples were collected into 1.5 ml tubes, centrifuged at 12,500 × g for 5 minutes, filter-sterilized through a 0.45 μm filter, and stored at -20 °C.

[0102] Production of SF-25IgE in Roller Bottles Sp2 / 0 cells expressing the SF-25IgE antibody were seeded at a cell density of 5×105 cells / ml into a 2 L roller bottle containing 250 ml of total IMDM complete medium. Subsequently, by inserting a 10 ml sterile pipette into the cell suspension, CO2 was injected into the cells, and then the pipette was connected to a CO2 tank to allow the gas to flow into the cell culture at a maximum pressure of 1.5 mBar for 2 minutes. After removing the pipette, the bottle lid was tightly closed, and the bottle was placed on a rotating cylinder at 37°C. Every 3 days, the cell density was monitored, and the cell culture was diluted 1:2 with fresh medium until the maximum working volume of 1200 ml was reached. As described previously, CO2 was supplied to the cells every 3 days or at the end of a 2-week culture, and at this point, the cell supernatant was collected.

[0103] Purification of SF-25IgE Antibody After antibody production in the Sp2 / 0 cell supernatant, to purify the SF-25IgE antibody, the cell supernatant was centrifuged at 400×g for 30 minutes to pellet the cells. After centrifugation, the supernatant was collected and filtered using a Stericup® 0.45 μm filter unit. 0.01% sodium azide was added to the supernatant, and then it was applied to affinity chromatography. Purification of human chimeric SF-25IgE was performed using a HiTrap KappaSelect column (GE Healthcare Life Sciences).

[0104] All affinity chromatography purifications of SF-25IgE were performed using an automated device equipped with a peristaltic pump (AKTA Prime), passing the liquid through the column at 1 ml / min. In the first step, the column was washed with PBS, and then the cell culture supernatant containing the secreted antibody was passed through the column and bound to the agarose beads. Subsequently, the captured antibody was eluted with elution buffer (0.2 M glycine, pH 2.3). The eluate was immediately neutralized with neutralization buffer (1 M Tris, pH 8.2). The column was then re-equilibrated by circulating the PBS solution. Size exclusion chromatography of high performance liquid chromatography (HPLC) was performed to exclude any aggregated and / or degraded antibody products. The entire process was carried out at 4 °C.

[0105] Activation of macrophages by IgE antibodies Cross-linking of IgE to monocyte-derived macrophages (MDM) To understand the role that IgE antibodies may play in macrophage polarization and activation, experiments on the involvement and cross-linking of IgE to the surface of monocyte-derived macrophages were performed using SF-25 and NIP IgE antibodies as described, for example, in European Patent No. 0397700 and Gould, H. J et al., Eur. J. Immunol. 1999. 29: 3527-3537. NIP IgE is a non-specific IgE produced against the hapten nitrohydroxyiodoacetic acid (4-hydroxy-3-iodo-5-nitrophenylacetic acid).

[0106] M0, M1 and M2a (MDM) macrophages were incubated with 5 μg / ml of IgE antibody (Dako) for 1 hour at 37 °C by directly adding the antibody solution onto the cell culture plates. After 1 hour, the supernatant was completely removed with a sterile pipette, 1 ml of sterile PBS was added to each well, and then any unbound antibody was washed away. Subsequently, if applicable, the cells were treated with 1 μg / ml of polyclonal anti-human IgE antibody to cross-link the IgE antibodies already bound to the cell surface. The cells were further incubated for 1 hour at 37 °C.

[0107] Cells were harvested 4 hours after stimulation to examine the expression of MDM cell surface markers, and the supernatant was collected 24 hours after stimulation to investigate cytokine release.

[0108] Magnetic Luminex assay for cytokine detection in macrophage supernatants Macrophage supernatants were collected 4 hours after stimulation, and cells were allowed to produce and secrete cytokines under various stimulation conditions. The supernatant was recovered by gently tilting the culture plate and removing it with a pipette without disturbing the adherent cells. The samples were then centrifuged at 600 × g to remove floating cells and debris, and then transferred to new tubes and stored at -20 °C until use.

[0109] On the day of the assay, the samples were thawed at room temperature, diluted 1:1 with sterile PBS, and maintained on ice until ready to be added to the assay plate.

[0110] The multiplex assay was performed using a magnetic Luminex kit according to the protocol instructions. Briefly, immediately after adding 50 μl of the microparticle cocktail to each well of the plate, 50 μl of the sample was added. The plate was sealed and incubated on an orbital shaker at room temperature for 2 hours. Washing was performed by adding and removing 100 μl of wash buffer to each well. The washing procedure was repeated 3 times. After removing the liquid from the final wash, 50 μl of the diluted biotin antibody cocktail was added to each well, the plate was sealed, and incubated on an orbital shaker at room temperature for 1 hour. The washing step was repeated as above, and 50 μl of streptavidin PE was added to the wells. The plate was sealed again and incubated for 30 minutes as above. After further washing steps were performed, 100 μl of wash buffer was added to each well to resuspend the microparticles, the plate was sealed, and left on a shaker for up to 90 minutes until ready to be analyzed on a Luminex™ analyzer.

[0111] Results Cytokines IL-12, IFNγ, TNFα involved in Th1 response Cross-linking of IgE caused a dramatic increase in TNFα secretion (100 - about 2,000 pg / ml) in both M0 and M2a macrophages, while in M1 cells, production of this mediator was very low or not detected at all under all experimental conditions tested (Figure 1).

[0112] Although to a lesser extent, cross-linking of IgE enhanced production of IFNγ in both M0 and M2a subsets. M1 macrophages showed a 4-fold higher baseline level of IFNγ compared to other subsets, but the concentration of IFNα remained stable upon activation by IgE.

[0113] When measuring IL-12 concentration, the same type and scale of regulation was observed. When cross-linked with both SF-25 and NIP IgE antibodies, IL-12 production increased from 100 to about 1500 - 2000 pg / ml in M0 and M2a macrophages. In M1 cells, a high baseline level of IL-12 (about 1,500 pg / ml) was characteristic and remained unaffected by IgE treatment.

[0114] Anti-inflammatory cytokines IL-4, IL-10, IL-13 Upon IgE cross-linking, IL-4 production slightly increased in both M0 and M2a subsets, while in M1 cells, it remained unaffected.

[0115] Regarding IL-10, the baseline levels were very similar between M0 and M1 macrophages (20 and 25 pg / ml), and much lower in M2a (about 7 pg / ml). However, cross-linking of IgE effectively upregulated the release of IL-10 in M0 and M2a subsets, while in M1 macrophages, the levels remained unchanged.

[0116] The background concentration of IL-13 was the same across all three macrophage populations. However, after cross-linking of SF-25 and NIP IgE on the cell surface, IL-13 secretion increased due to IgE cross-linking in M0 and M2a cells, but not in M1 macrophages (Figure 2).

[0117] Cytokines IL-1γ and IL-6 involved in the inflammatory response When analyzing the secretion of both IL-1β and IL-6, the responses to IgE activation were again similar between the M0 and M2a phenotypes. The baseline concentrations of both cytokines were very low across the three cell subsets and remained stable across all treatments in M1 macrophages. However, in M0 and M2a macrophages, cross-linking of cell-bound IgE resulted in a net increase in the secretion of IL-1β and IL-6 (Figure 3).

[0118] Macrophage chemoattractant chemokines MCP-1 (CCL2) and RANTES (CCL5) It is known that MCP-1 recruits monocytes, macrophages, and dendritic cells to the site of inflammation. This effect at the site of inflammation is promoted by RANTES, which is a regulator of many important macrophage functions such as chemotaxis and phagocytosis. Therefore, the concentrations of MCP-1 and RANTES upon IgE activation were tested here to assist in further analysis of the interaction of this antibody class with macrophages through any secretion regulation.

[0119] Cross-linking of cell-bound IgE did not result in regulation of the MCP-1 supernatant concentration in either M0 or M1 macrophages. In contrast, the M2a subset responded to IgE activation by enhancing the release of MCP-1 under all conditions tested.

[0120] Regulation of RANTES secretion upon IgE treatment showed the same pattern in both M0 and M2a macrophages, where the inventors detected a dramatic increase in this production when cell-bound IgE was cross-linked. Interestingly, when RANTES levels increased in M0 and M2a macrophages, the peak concentration almost matched the baseline concentration in M1 cells, where instead, regulation of RANTES secretion was not detected across all tested conditions (Figure 3).

[0121] Interferon gamma-induced chemokines MIG (CXCL9) and I-TAC (CXCL11) The MIG concentration in M1 macrophages remained unchanged among the various conditions tested, indicating that IgE activation has no effect on this production in this macrophage subset. A similar effect was instead observed between M0 and M2a macrophages, where cross-linking of IgE resulted in a slight increase in MIG secretion.

[0122] Analysis of I-TAC concentration revealed that the background level of this chemokine was much higher in M1 macrophages compared to the other two subsets, with a baseline concentration of approximately 400 pg / ml being characteristic of M1, about four times higher than the baseline concentrations detected in M0 and M2a cells. Nevertheless, treatment of M0 and M2a cultures by IgE cross-linking dramatically increased the amount of I-TAC released into the supernatant, with this concentration rising from approximately 80 to approximately 160 pg / ml in M0 cells and from 50 to approximately 200 pg / ml in M2a macrophages (Figure 4).

[0123] In summary, it was observed that cross-linking of cell-bound IgE induced the same effect on M0 and M2a macrophages with all the soluble mediators tested, except those derived from MCP-1. Cross-linking of IgE in M2a macrophages, but not in M0 or M1 subsets, induced enhanced MCP-1 secretion. On the other hand, M1 macrophages were mostly unresponsive to IgE activation, but had two notable exceptions, where cross-linking of IgE induced upregulation of the pro-inflammatory mediators IFNγ and IL-12 (Table 3).

[0124]

Table 1

[0125] In Table 3, changes in cytokine and chemokine release in macrophages before and after IgE cross-linking were set as ↑ detection of increase and ↓ detection of decrease, and if the soluble mediator was not repeated from the "physiological state" column to the column named "IgE cross-linking" in the finding column, this production remained unchanged between the two conditions. Cross-linking of cell-bound IgE to M0 and M2a macrophages maintained or increased the production of all soluble mediators tested. Interestingly, MCP-1 was upregulated only upon cross-linking of M2a cells, suggesting a potential role of anti-tumor IgE antibodies in the induction of MCP-1 production and secretion by M2a macrophages. The polarization state of M1 macrophages was maintained upon stimulation of the cells with IgE, suggesting that IgE antibodies maintain a pro-inflammatory profile in the TME. In contrast, cross-linking of cell-bound IgE to M0 and M2a macrophages resulted in a newly polarized macrophage phenotype that secretes both pro-inflammatory and anti-inflammatory mediators, thereby explaining the remarkable role of IgE antibodies in the bias of the quiescent (M0) or tumor-promoting (M2a) phenotype towards a new phenotype characterized by new pro-inflammatory activity.

[0126] Macrophages are tissue-resident phagocytes and antigen-presenting cells (APCs) that differentiate from circulating peripheral blood mononuclear cells. They perform important activities and regulatory functions in both innate and adaptive immunity (Murray PJ, Wynn TA (2011). Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol, 11(11): 723-37). Activated macrophages of various phenotypes are usually classified into M1 macrophages (CAM) and M2 macrophages (AAM). Classically activated M1 macrophages include immune effector cells with an acute inflammatory phenotype. They are highly aggressive against bacteria and produce large amounts of lymphokines (Murray PJ, Wynn TA (2011). Obstacles and opportunities for understanding macrophage polarization. J Leukoc Biol, 89(4):557-63). Selectively activated anti-inflammatory M2 macrophages (alternatively activated, anti-inflammatory M2-macrophages) can be divided into at least three subgroups. Such subtypes have various functions including immune regulation, maintenance of tolerance, and tissue repair / wound healing. In fact, cells of the monocyte / macrophage lineage show remarkable flexibility in response to endogenous as well as exogenous stimuli, which enables the invalidation of the initial M1 / M2 polarization process, for example, M2-polarized macrophages can be converted to the M1 activation state under specific conditions.

[0127] Primary human macrophages are difficult to isolate in sufficient quantities from tissues and do not proliferate in culture. In addition, it is generally recognized that the resulting cells often show significant phenotypic heterogeneity. Since human blood monocytes are readily available in large numbers and can be differentiated into macrophages in vitro, monocyte-derived macrophages (MDM) are an excellent alternative.

[0128] Most studies on macrophage characterization have been performed on mouse cells with profiles quite different from human-derived macrophages. For example, CD206 is a good M2 marker in mice, while CD200R seems to work better in human cells. In addition, the differences between M1 and M2 markers have a quantitative tendency. For example, both M1 and M2 express MHCII, but M1 expresses it at a much higher intensity. Since this is true for most but not all of the surface markers used for M1 / M2 differentiation, it is highly desirable to have a "positive control". However, the generally recognized marker profile of M1 macrophages is CD68+ / CD80+ / CD163- or CD163low, while M2 macrophages are characterized as CD68+ / CD80- / CD163+. However, the upregulation of CD163 depends on the method of macrophage polarization (IL-4 / IL-10, RPMI medium or ex vivo). Ideally, multiple methods including morphology (M1: oval or circular, M2: meatball-shaped or with dendritic processes), gene expression (e.g., iNOS / Arg1 ratio, cytokine IL-1 beta, chemokine CCL2 and Ptgs2) and flow cytometry analysis are used for characterization.

[0129] Discussion Monoclonal antibodies (mAbs) can exert antitumor effects through multiple mechanisms. However, it has recently been understood how mAbs acting through Fc-mediated mechanisms play a key role in the involvement of the immune system and the regulation of the immune profile of the tumor microenvironment (Bakema et al (2014) Fc Receptor-Dependent Mechanisms of Monoclonal Antibody Therapy of Cancer. In Current topics in microbiology and immunology, 382: 373-392, Moore et al (2010) Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions. mAbs 2(2): 181-189).Currently, some mAbs in use, such as trastuzumab and ipilimumab, have provided evidence of a substantial contribution by Fc-mediated mechanisms to the clinical efficacy of such classes of anticancer agents (Peggs et al, J Exp Med 2009 Aug 3;206(8):1717-1725; Simpson et al. J Exp Med 2013 Aug 26;210(9):1695-1710; Romano et al. Proc Natl Acad Sci USA 2015 May 12;112(19):6140-6145; Arce Vargas et al (2018) Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies. Cancer Cell. 33(4): 649-663.e4; Shi et al (2014) Engagement of immune effector cells by trastuzumab induces HER2 / ERBB2 downregulation in cancer cells through STAT1 activation. Breast Cancer Research. 16(2): R33). Therefore, Fc modifications that not only regulate the cytotoxic function of mAbs but also overcome the immunosuppressive microenvironment in the majority of solid malignancies are of particular interest (Bakema et al (see above), Dahan et al (2015) FcγRs Modulate the Anti-tumor Activity of Antibodies Targeting the PD-1 / PD-L1 Axis. Cancer Cell. 28(3): 285-295, Moore et al (see above)).Among the strategies explored to modify the Fc domain to improve clinical efficacy, modification of the glycosylation profile and selection of various isotype subclasses are the most investigated strategies (Ferrara et al (2006) Modulation of therapeutic antibody effector functions by glycosylation engineering: Influence of Golgi enzyme localization domain and co-expression of heterologous β1, 4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Biotechnology and Bioengineering. 93(5): 851-861, Kellner et al (2017) Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy. Transfusion Medicine and Hemotherapy: Offizielles Organ Der Deutschen Gesellschaft Fur Transfusionsmedizin Und Immunhamatologie. 44(5): 327-336, Schlothauer et al (2016) Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions. Protein Engineering Design and Selection. 29(10): 457-466).

[0130] In contrast, one of the strategies that remains under-investigated is the use of antibodies with Fc regions of classes other than the commonly used IgG. Modification of antibodies of the IgE class is one such strategy. Indeed, because various Fc receptors are expressed by specific subsets of immune cells, the use of a new antibody isotype such as IgE serves as a bridge to the involvement of diverse populations of immune effector cells and ultimately to potential improvements in clinical outcomes.

[0131] The potential advantages of IgE antibodies in the treatment of solid malignancies rely on the unique biological properties of the immunoglobulin class, along with the demonstrated infiltration of many key IgE receptor-expressing immune effector cells in the tumor microenvironment.Based on such findings, several IgE-based immunotherapeutic methods, including recombinant IgE antibodies targeting tumor antigens, have been developed for the purpose of inducing IgE-mediated immune responses against tumor cells (Daniels et al (2012) Targeting HER2 / neu with a fully human IgE to harness the allergic reaction against cancer cells. Cancer Immunology, Immunotherapy. 61(7): 991-1003, Josephs et al (2017) Anti-Folate Receptor-α IgE but not IgG Recruits 330 Macrophages to Attack Tumors via TNFα / MCP-1 Signaling. Cancer Research. 77(5): 1127-1141, Josephs et al (2018) An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE. Allergy. 2018 Dec;73(12):2328-2341, Karagiannis et al (2012) Recombinant IgE antibodies for passive immunotherapy of solid tumours: from concept towards clinical application. Cancer Immunology, Immunotherapy: CII, 61(9), 1547-1564, Kershaw et al (1998) Tumor-specific IgE-mediated inhibition of human colorectal carcinoma xenograft growth. Oncology Research. 10(3): 133-142).

[0132] Analysis of cytokines and chemokines performed on supernatants collected from macrophage cell cultures stimulated with IgE enabled the identification of mediators involved with IgE or soluble mediators upon IgE crosslinking.

[0133] In M1 macrophages, production of pro-inflammatory cytokines such as IFNγ and IL-12 was maintained upon IgE crosslinking. Josephs et al. recently demonstrated upregulation of pro-inflammatory immune-related pathways including IL-12 and NK cell activation properties in the lungs of rats bearing tumors treated with MOv18IgE (see Josephs et al., 2018 above). Considering such findings, in line with the known pro-inflammatory and antigen-presenting functions of that subset, it is possible that at least M1 macrophages may be maintained by the involvement and crosslinking of IgE.

[0134] The secretion of all tested mediators except one (IL-1β, IL-4, IL-6, IL-10, IL-12, IL-13, IFNγ, TNFα, RANTES, MIG and I-TAC) was regulated in the same way by cross-linking of cell-bound IgE to non-activated M0 and M2a macrophages. This suggests that the two subsets may be characterized by similar molecular mechanisms in their ability to control the activation of the FcεRI pathway. More specifically, the production of all tested soluble mediators was either maintained at the same level or increased with IgE cross-linking. The population of cytokines secreted by M0 and M2a after cross-linking with SF-25IgE did not coincide with the defined macrophage subtypes. However, based on the mediator characteristics presented by IgE-stimulated cells, this corresponded to a newly polarized macrophage subset that secretes both pro-inflammatory and anti-inflammatory mediators, as well as chemotactic factors. Cross-linking of IgE to both induced M0 and M2a macrophages enhanced the level of the pro-inflammatory M1 cytokine TNFα. On the other hand, IgE cross-linking showed different effects on the production of the macrophage chemotactic substance MCP-1 in M0 and M2 macrophages. In M2a macrophages, treatment with IgE enhanced the production and release of MCP-1.

[0135] Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), bind to specific cell surface transmembrane receptors that are coupled to heterotrimeric G proteins, and activation of this heterotrimeric G protein results in activation of an intracellular signaling cascade that promotes migration toward the chemokine source. Migration and infiltration of monocytes, memory T lymphocytes, and natural killer (NK) cells are controlled by MCP-1, also known as CCL2. Monocytes are important for the initiation of tumor arteriogenesis because they adhere to and invade endothelium activated by increased shear stress resulting from large pressure differences between perfused regions (Scholz et al (2001) Arteriogenesis, a new concept of vascular adaptation in occlusive disease. Angiogenesis. 4: 247-257). MCP-1 is implicated in this process because it not only attracts monocytes but also promotes adhesion by inducing upregulation of MAC-1, a receptor for intracellular adhesion molecule-1 (ICAM-1) expressed on activated endothelium. In addition, MCP-1 has been shown to increase the antiproliferative activity against tumor cells when added to macrophages in tissue culture (Zachariae et al (1990) Properties of monocyte chemotactic and activating factor (MCAF) purified from a human fibrosarcoma cell line. J Exp Med. 171: 2177-2182), thereby driving apoptosis of the cells.

[0136] Increasing epidemiological and clinical data support the concept that chronic inflammation promotes tumor growth and progression. As a major pro-inflammatory cytokine, tumor necrosis factor (TNFα) can act as an endogenous tumor promoter that mediates inflammation and carcinogenesis. It has been shown that TNFα stimulates proliferation, survival, migration, and angiogenesis in most cancer cells that are resistant to TNF-induced cytotoxicity, leading to tumor promotion. However, TNFα also has the ability to suppress tumor cell proliferation and induce tumor regression. Therefore, TNFα is a double-edged sword and can be either tumorigenic or antitumorigenic (Wang and Lin (2008) Tumor necrosis factor and cancer, buddies or foes? Acta Pharmacol Sin. 29(11): 1275-1288). A number of agents, including natural and synthetic compounds, have been shown to sensitize tumor cells to TNFα-induced cell death by inhibiting NF-κB activation. When such compounds are combined with TNFα, synergistic cytotoxicity occurs in tumor cells (Wang X et al (2006) 17-allylamino-17-demethoxygeldanamycin synergistically potentiates tumor necrosis factor-induced lung cancer cell death by blocking the nuclear factor-kappaB pathway. Cancer Res. 66(2): 1089-95, Zhang S et al (2004) Suppressed NF-kappaB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNF-alpha-induced apoptosis in human cancer cells. Carcinogenesis.25(11): 2191-9, Fas SC et al (2006) Wogonin sensitizes resistant malignant cells to TNFalpha- and TRAIL-induced apoptosis. Blood. 108(12): 3700-6, Ju W et al (2007) A critical role of luteolin-induced reactive oxygen species in blockage of tumor necrosis factor-activated nuclear factor-kappaB pathway and sensitization of apoptosis in lung cancer cells. Mol Pharmacol. 71(5): 1381-8, Rae C et al (2007) Elevated NF-kappaB responses and FLIP levels in leukemic but not normal lymphocytes: reduction by salicylate allows TNF-induced apoptosis. Proc Natl Acad Sci U S A. 104(31): 12790-5, Shukla S, Gupta S (2004) Suppression of constitutive and tumor necrosis factor alpha-induced nuclear factor (NF)-kappaB activation and induction of apoptosis by apigenin in human prostate carcinoma PC-3 cells: correlation with down-regulation of NF-kappaB-responsive genes. Clin Cancer Res.10(9): 3169-78, Shishodia S et al (2006) A synthetic triterpenoid, CDDO-Me, inhibits IkappaBalpha kinase and enhances apoptosis induced by TNF and chemotherapeutic agents through down-regulation of expression of nuclear factor kappaB-regulated gene products in human leukemic cells. Clin Cancer Res. 12(6): 1828-38). In addition, TNFα has been used as an adjuvant reagent to enhance the anti-cancer effects of chemotherapeutic agents such as doxorubicin (Cao W et al (2005) TNF-alpha promotes Doxorubicin-induced cell apoptosis and anti-cancer effect through downregulation of p21 in p53-deficient tumor cells. Biochem Biophys Res Commun. 330(4): 1034-40), to sensitize carcinomas with low epidermal growth factor receptor (EGFR) expression to anti-EGFR therapy (Hambek M et al (2001) Tumor necrosis factor alpha sensitizes low epidermal growth factor receptor (EGFR)-expressing carcinomas for anti-EGFR therapy. Cancer Res. 61(3): 1045-9), or to overcome acquired resistance to EGFR tyrosine kinase inhibitors in non-small cell lung cancer cells (Ando K et al (2005) Enhancement of sensitivity to tumor necrosis factor alpha in non-small cell lung cancer cells with acquired resistance to gefitinib.Clin Cancer Res. 11(24 Pt 1): 8872-9). The combination of TNFα and chemotherapeutic agents has been shown to be an effective treatment strategy for many tumors by increasing tumor sensitivity to treatment. In addition, TNFα can activate T cells and dendritic cells to enhance the host's anti-tumor adaptive immune response.

[0137] The findings presented herein demonstrate that it is possible to stimulate M0 and M2a macrophages to secrete TNFα, and that alternatively activated M2a macrophages are the only cell type that upregulates MCP-1 upon crosslinking with IgE. Thus, IgE has the ability to specifically stimulate this normally anti-inflammatory subset of alternatively activated macrophages towards a more mature and activated phenotype that typically shares some characteristics associated with classically activated M1 macrophages. Such findings suggest the role of IgE-mediated polarization of alternatively activated macrophages in cancer treatment by targeting macrophages, for example, the use of macrophage-centered immunotherapies to directly re-educate macrophages to an active phenotype in tumors.

[0138] All published documents referred to in the above specification are hereby incorporated by reference herein. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

**Claim 1** A pharmaceutical composition for use in the reprogramming of macrophages from a first phenotype to an anti-tumor phenotype in the treatment of a subject's cancer, comprising immunoglobulin E (IgE), wherein the subject is one in which, when one or more phenotypes of macrophages present in a tumor sample obtained from the subject are detected, resting (M0) and / or anti-inflammatory (M2a) macrophages are present in the sample at levels exceeding a predetermined level, the first phenotype comprising a resting (M0) macrophage phenotype or an anti-inflammatory (M2a) macrophage phenotype, and the anti-tumor phenotype comprising a newly polarized macrophage phenotype characterized by the expression of the following cytokines and chemokines: tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin 1 beta (IL-1β); interleukin 6 (IL-6); RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted); and interleukin 10 (IL-10). **Claim 2** A pharmaceutical composition for use in the treatment of a subject's cancer, comprising immunoglobulin E (IgE), wherein the subject is one in which, when one or more phenotypes of macrophages present in a tumor sample obtained from the subject are detected, resting (M0) and / or anti-inflammatory (M2a) macrophages are present in the sample at levels exceeding a predetermined level, and the macrophages associated with the tumor in the subject have a resting (M0) macrophage phenotype or an anti-inflammatory (M2a) macrophage phenotype, and the treatment promotes the reprogramming of the macrophages associated with the tumor to a newly polarized macrophage phenotype characterized by the expression of the following cytokines: tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin 1 beta (IL-1β); interleukin 6 (IL-6); RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted); and interleukin 10 (IL-10). **Claim 3** The pharmaceutical composition according to claim 1 or 2, wherein the newly polarized macrophage phenotype is further characterized by an increase in the expression of monocyte chemoattractant protein 1 (MCP-1) compared to the anti-inflammatory (M2a) macrophage phenotype. **Claim 4** The pharmaceutical composition according to any one of claims 1 to 3, wherein the newly polarized macrophage phenotype is further characterized by an increase in the expression of interleukin 12 (IL-12), interleukin 13 (IL-13), chemokine (C-X-C motif) ligand 9 (CXCL9) and / or chemokine (C-X-C motif) ligand 11 (CXCL11) as compared to the resting (M0) macrophage phenotype or the anti-inflammatory (M2a) macrophage phenotype.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the treatment further promotes an increase in the expression of IFNγ and / or IL-12 by inflammation-promoting (M1) macrophages associated with a tumor in a subject.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein macrophages associated with a tumor in a subject are distributed so as to surround the periphery of the tumor.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the newly polarized macrophage phenotype promotes the recruitment of additional monocytes and / or macrophages to a tumor in a subject.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the cancer comprises skin cancer, breast cancer, head and neck squamous cell carcinoma, prostate cancer, ovarian cancer, colon cancer, glioma, gastric cancer, lung cancer or pancreatic cancer.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the IgE comprises an anti-folate receptor alpha (FRα) antibody, an anti-high molecular weight melanoma-associated antigen (HMW-MAA) antibody, an anti-human epidermal growth factor receptor 2 (HER2) antibody or an anti-SF-25 antibody.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the newly polarized macrophage phenotype is further characterized by the expression of one or more of the following cytokines: MCP-1, IL-4, IL-13, CXCL9, IL-12 and / or CXCL11.

11. A pharmaceutical composition for use in the reprogramming of macrophages associated with tumors in a subject, comprising immunoglobulin E (IgE), wherein the subject is a subject in which resting state (M0) and / or anti-inflammatory (M2a) macrophages are present in a tumor sample obtained from the subject at levels exceeding a predetermined level when detecting one or more phenotypes of macrophages present in the tumor sample, and wherein said reprogramming results in modulation of cytokine expression and enhancement of anti-tumor activity in the tumor microenvironment, and wherein said reprogrammed macrophages express tumor necrosis factor alpha (TNFα); interferon gamma (IFNγ); interleukin 1 beta (IL-1β); interleukin 6 (IL-6); RANTES or CCL5 (regulated upon activation, normal T cell expressed and secreted); and interleukin 10 (IL-10), said pharmaceutical composition.

12. The pharmaceutical composition according to claim 11, wherein the reprogrammed macrophages express monocyte chemoattractant protein 1 (MCP-1).

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